Pathophysiology of heart disease: a collaborative project of medical students and faculty. Internet Archive Language. Sabatine, and Leonard S. Lilly - Valvular heart disease / Mia M. Edwards, Patrick T. O'Gara, and Leonard S. Lilly - Heart failure / Ravi Vikram Shah and Michael A. Fifer - The cardiomyopathies / Marc N. Why Your Heart Is Not A Pump (& What Most Doctors Don’t Know About The True Cause Of Heart Disease). Both drawn to the art of healing and repelled by the way medicine was―and continues to be―practiced in the United States, Cowan returned from Swaziland, went to medical school, and established a practice in New Hampshire and, later, San Francisco. Pathophysiology of Heart Disease: A Collaborative Project of Medical Students and Faculty PDF Tags. Pathophysiology of Heart Disease: A Collaborative Project of Medical Students and Faculty pdf download, Pathophysiology of Heart Disease: A Collaborative Project of Medical Students and Faculty pdf, Pathophysiology.

1. Pathophysiology Of Cardiovascular Disease Pdf
2. Pathophysiology Of Heart Disease Pdf

Myocardial infarction (MI), also known as a heart attack, occurs when blood flow decreases or stops to a part of the heart, causing damage to the heart muscle.^[1] The most common symptom is chest pain or discomfort which may travel into the shoulder, arm, back, neck or jaw.^[1] Often it occurs in the center or left side of the chest and lasts for more than a few minutes.^[1] The discomfort may occasionally feel like heartburn.^[1] Other symptoms may include shortness of breath, nausea, feeling faint, a cold sweat or feeling tired.^[1] About 30% of people have atypical symptoms.^[8] Women more often present without chest pain and instead have neck pain, arm pain or feel tired.^[11] Among those over 75 years old, about 5% have had an MI with little or no history of symptoms.^[12] An MI may cause heart failure, an irregular heartbeat, cardiogenic shock or cardiac arrest.^[3][4]

Most MIs occur due to coronary artery disease.^[3] Risk factors include high blood pressure, smoking, diabetes, lack of exercise, obesity, high blood cholesterol, poor diet and excessive alcohol intake, among others.^[5][6] The complete blockage of a coronary artery caused by a rupture of an atherosclerotic plaque is usually the underlying mechanism of an MI.^[3] MIs are less commonly caused by coronary artery spasms, which may be due to cocaine, significant emotional stress and extreme cold, among others.^[13][14] A number of tests are useful to help with diagnosis, including electrocardiograms (ECGs), blood tests and coronary angiography.^[7] An ECG, which is a recording of the heart's electrical activity, may confirm an ST elevation MI (STEMI), if ST elevation is present.^[8][15] Commonly used blood tests include troponin and less often creatine kinase MB.^[7]

Treatment of an MI is time-critical.^[16]Aspirin is an appropriate immediate treatment for a suspected MI.^[9]Nitroglycerin or opioids may be used to help with chest pain; however, they do not improve overall outcomes.^[8][9]Supplemental oxygen is recommended in those with low oxygen levels or shortness of breath.^[9] In a STEMI, treatments attempt to restore blood flow to the heart and include percutaneous coronary intervention (PCI), where the arteries are pushed open and may be stented, or thrombolysis, where the blockage is removed using medications.^[8] People who have a non-ST elevation myocardial infarction (NSTEMI) are often managed with the blood thinner heparin, with the additional use of PCI in those at high risk.^[9] In people with blockages of multiple coronary arteries and diabetes, coronary artery bypass surgery (CABG) may be recommended rather than angioplasty.^[17] After an MI, lifestyle modifications, along with long term treatment with aspirin, beta blockers and statins, are typically recommended.^[8]

Worldwide, about 15.9 million myocardial infarctions occurred in 2015.^[10] More than 3 million people had an ST elevation MI, and more than 4 million had an NSTEMI.^[18] STEMIs occur about twice as often in men as women.^[19] About one million people have an MI each year in the United States.^[3] In the developed world, the risk of death in those who have had an STEMI is about 10%.^[8] Rates of MI for a given age have decreased globally between 1990 and 2010.^[20] In 2011, a MI was one of the top five most expensive conditions during inpatient hospitalizations in the US, with a cost of about $11.5 billion for 612,000 hospital stays.^[21]

• 2Signs and symptoms
• 3Causes
• 4Mechanism
• 5Diagnosis
• 6Management
• 7Prevention
  • 7.1Primary prevention
  • 7.2Secondary prevention
• 8Prognosis
• 10Society and culture

Terminology[edit]

Myocardial infarction (MI) refers to tissue death (infarction) of the heart muscle (myocardium). It is a type of acute coronary syndrome, which describes a sudden or short-term change in symptoms related to blood flow to the heart.^[22] Unlike other causes of acute coronary syndromes, such as unstable angina, a myocardial infarction occurs when there is cell death, as measured by a blood test for biomarkers (the cardiac protein troponin or the cardiac enzyme CK-MB).^[16] When there is evidence of an MI, it may be classified as an ST elevation myocardial infarction (STEMI) or Non-ST elevation myocardial infarction (NSTEMI) based on the results of an ECG.^[23]

The phrase 'heart attack' is often used non-specifically to refer to a myocardial infarction and to sudden cardiac death. An MI is different from—but can cause—cardiac arrest, where the heart is not contracting at all or so poorly that all vital organs cease to function, thus causing death. It is also distinct from heart failure, in which the pumping action of the heart is impaired. However, an MI may lead to heart failure.^[24]

Signs and symptoms[edit]

Pain[edit]

Chest pain is the most common symptom of acute myocardial infarction and is often described as a sensation of tightness, pressure, or squeezing. Pain radiates most often to the left arm, but may also radiate to the lower jaw, neck, right arm, back, and upper abdomen.^[25] The pain most suggestive of an acute MI, with the highest likelihood ratio, is pain radiating to the right arm and shoulder.^[26] Similarly, chest pain similar to a previous heart attack is also suggestive.^[27] The pain associated with MI is usually diffuse, does not change with position, and lasts for more than 20 minutes.^[23]Levine's sign, in which a person localizes the chest pain by clenching one or both fists over their sternum, has classically been thought to be predictive of cardiac chest pain, although a prospective observational study showed it had a poor positive predictive value.^[28] Pain that responds to nitroglycerin does not indicate the presence or absence of a myocardial infarction.^[9]

Other symptoms[edit]

Chest pain may be accompanied by sweating, nausea or vomiting, and fainting,^[23][26] and these symptoms may also occur without any pain at all.^[25] In women, the most common symptoms of myocardial infarction include shortness of breath, weakness, and fatigue.^[29]Shortness of breath is a common, and sometimes the only symptom, occurring when damage to the heart limits the output of the left ventricle, with breathlessness arising either from low oxygen in the blood, or pulmonary edema.^[25][30] Other less common symptoms include weakness, light-headedness, palpitations, and abnormalities in heart rate or blood pressure.^[16] These symptoms are likely induced by a massive surge of catecholamines from the sympathetic nervous system, which occurs in response to pain and, where present, low blood pressure.^[31]Loss of consciousness due to inadequate blood flow to the brain and cardiogenic shock, and sudden death, frequently due to the development of ventricular fibrillation, can occur in myocardial infarctions.^[24] Cardiac arrest, and atypical symptoms such as palpitations, occur more frequently in women, the elderly, those with diabetes, in people who have just had surgery, and in critically ill patients.^[23]

'Silent' myocardial infarctions can happen without any symptoms at all.^[12] These cases can be discovered later on electrocardiograms, using blood enzyme tests, or at autopsy after a person has died. Such silent myocardial infarctions represent between 22 and 64% of all infarctions,^[12] and are more common in the elderly,^[12] in those with diabetes mellitus^[16] and after heart transplantation. In people with diabetes, differences in pain threshold, autonomic neuropathy, and psychological factors have been cited as possible explanations for the lack of symptoms.^[32] In heart transplantation, the donor heart is not fully innervated by the nervous system of the recipient.^[33]

Women[edit]

In women, myocardial infarctions can present with different symptoms. The classic presentation of chest pain occurs in about 50% of women. Women can also commonly experience back or neck pain, indigestion, heartburn, lightheadedness, shortness of breath, fatigue, nausea, or pain in the back of the jaw. These symptoms are often overlooked or mistaken for another condition.^[34][35]

Causes[edit]

The most prominent risk factors for myocardial infarction are older age, actively smoking, high blood pressure, diabetes mellitus, and total cholesterol and high-density lipoprotein levels.^[19] Many risk factors of myocardial infarction are shared with coronary artery disease, the primary cause of myocardial infarction,^[16] with other risk factors including male sex, low levels of physical activity, a past family history, obesity, and alcohol use.^[16] Risk factors for myocardial disease are often included in risk factor stratification scores, such as the Framingham Risk Score.^[19] At any given age, men are more at risk than women for the development of cardiovascular disease.^[36]High levels of blood cholesterol is a known risk factor, particularly high low-density lipoprotein, low high-density lipoprotein, and high triglycerides.^[37]

Many risk factors for myocardial infarction are potentially modifiable, with the most important being tobacco smoking (including secondhand smoke).^[16] Smoking appears to be the cause of about 36% and obesity the cause of 20% of coronary artery disease.^[38] Lack of physical activity has been linked to 7–12% of cases.^[38][39] Less common causes include stress-related causes such as job stress, which accounts for about 3% of cases,^[38] and chronic high stress levels.^[40]

Diet[edit]

There is varying evidence about the importance of saturated fat in the development of myocardial infarctions. Eating polyunsaturated fat instead of saturated fats has been shown in studies to be associated with a decreased risk of myocardial infarction,^[41] while other studies find little evidence that reducing dietary saturated fat or increasing polyunsaturated fat intake affects heart attack risk.^[42][43] Dietary cholesterol does not appear to have a significant effect on blood cholesterol and thus recommendations about its consumption may not be needed.^[44]Trans fats do appear to increase risk.^[42] Acute and prolonged intake of high quantities of alcoholic drinks (3–4 or more daily) increases the risk of a heart attack.^[45]

Genetics[edit]

Family history of ischemic heart disease or MI, particularly if one has a male first-degree relative (father, brother) who had a myocardial infarction before age 55 years, or a female first-degree relative (mother, sister) less than age 65 increases a person's risk of MI.^[36]

Genome-wide association studies have found 27 genetic variants that are associated with an increased risk of myocardial infarction.^[46] The strongest association of MI has been found with chromosome 9 on the short arm p at locus 21, which contains genes CDKN2A and 2B, although the single nucleotide polymorphisms that are implicated are within a non-coding region.^[46] The majority of these variants are in regions that have not been previously implicated in coronary artery disease. The following genes have an association with MI: PCSK9, SORT1, MIA3, WDR12, MRAS, PHACTR1, LPA, TCF21, MTHFDSL, ZC3HC1, CDKN2A, 2B, ABO, PDGF0, APOA5, MNF1ASM283, COL4A1, HHIPC1, SMAD3, ADAMTS7, RAS1, SMG6, SNF8, LDLR, SLC5A3, MRPS6, KCNE2.^[46]

Other[edit]

The risk of having a myocardial infarction increases with older age, low physical activity, and low socioeconomic status.^[36] Heart attacks appear to occur more commonly in the morning hours, especially between 6AM and noon.^[47] Evidence suggests that heart attacks are at least three times more likely to occur in the morning than in the late evening.^[48]Shift work is also associated with a higher risk of MI.^[49] And one analysis has found an increase in heart attacks immediately following the start of daylight saving time.^[50]

Women who use combined oral contraceptive pills have a modestly increased risk of myocardial infarction, especially in the presence of other risk factors.^[51] The use of non-steroidal anti inflammatory drugs (NSAIDs), even for as short as a week, increases risk.^[52]

Endometriosis in women under the age of 40 is an identified risk factor.^[53]

Air pollution is also an important modifiable risk. Short-term exposure to air pollution such as carbon monoxide, nitrogen dioxide, and sulfur dioxide (but not ozone) have been associated with MI and other acute cardiovascular events.^[54] For sudden cardiac deaths, every increment of 30 units in Pollutant Standards Index correlated with an 8% increased risk of out-of-hospital cardiac arrest on the day of exposure.^[55] Extremes of temperature are also associated.^[56]

A number of acute and chronic infections including Chlamydophila pneumoniae, influenza, Helicobacter pylori, and Porphyromonas gingivalis among others have been linked to atherosclerosis and myocardial infarction.^[57] As of 2013, there is no evidence of benefit from antibiotics or vaccination, however, calling the association into question.^[57][58] Myocardial infarction can also occur as a late consequence of Kawasaki disease.^[59]

Calcium deposits in the coronary arteries can be detected with CT scans. Calcium seen in coronary arteries can provide predictive information beyond that of classical risk factors.^[60]High blood levels of the amino acid homocysteine is associated with premature atherosclerosis;^[61] whether elevated homocysteine in the normal range is causal is controversial.^[62]

In people without evident coronary artery disease, possible causes for the myocardial infarction are coronary spasm or coronary dissection.^[63]

Mechanism[edit]

Atherosclerosis[edit]

The most common cause of a myocardial infarction is the rupture of an atherosclerotic plaque on an artery supplying heart muscle.^[24][64] Plaques can become unstable, rupture, and additionally promote the formation of a blood clot that blocks the artery; this can occur in minutes. Blockage of an artery can lead to tissue death in tissue being supplied by that artery.^[65] Atherosclerotic plaques are often present for decades before they result in symptoms.^[65]

The gradual buildup of cholesterol and fibrous tissue in plaques in the wall of the coronary arteries or other arteries, typically over decades, is termed atherosclerosis.^[66] Atherosclerosis is characterized by progressive inflammation of the walls of the arteries.^[65] Inflammatory cells, particularly macrophages, move into affected arterial walls. Over time, they become laden with cholesterol products, particularly LDL, and become foam cells. A cholesterol core forms as foam cells die. In response to growth factors secreted by macrophages, smooth muscle and other cells move into the plaque and act to stabilize it. A stable plaque may have a thick fibrous cap with calcification. If there is ongoing inflammation, the cap may be thin or ulcerate. Exposed to the pressure associated with blood flow, plaques, especially those with a thin lining, may rupture and trigger the formation of a blood clot (thrombus).^[65] The cholesterol crystals have been associated with plaque rupture through mechanical injury and inflammation.^[67]

Other causes[edit]

Atherosclerotic disease is not the only cause of myocardial infarction, and it may exacerbate or contribute to other causes. A myocardial infarction may result from a heart with a limited blood supply subject to increased oxygen demands, such as in fever, a fast heart rate, hyperthyroidism, too few red blood cells in the bloodstream, or low blood pressure. Damage or failure of procedures such as percutaneous coronary intervention or coronary artery bypass grafts may cause a myocardial infarction. Spasm of coronary arteries, such as Prinzmetal's angina may cause blockage.^[23][25]

Tissue death[edit]

If impaired blood flow to the heart lasts long enough, it triggers a process called the ischemic cascade; the heart cells in the territory of the blocked coronary artery die (infarction), chiefly through necrosis, and do not grow back. A collagenscar forms in their place.^[65] When an artery is blocked, cells lack oxygen, needed to produce ATP in mitochondria. ATP is required for the maintenance of electrolyte balance, particularly through the Na/K ATPase. This leads to an ischemic cascade of intracellular changes, necrosis and apoptosis of affected cells.^[68]

Cells in the area with the worst blood supply, just below the inner surface of the heart (endocardium), are most susceptible to damage.^[69] Ischemia first affects this region, the subendocardial region, and tissue begins to die within 15–30 minutes of loss of blood supply.^[70] The dead tissue is surrounded by a zone of potentially reversible ischemia that progresses to become a full-thickness transmural infarct.^[68][70] The initial 'wave' of infarction can take place over 3–4 hours.^[65][68] These changes are seen on gross pathology and cannot be predicted by the presence or absence of Q waves on an ECG.^[69] The position, size and extent of an infarct depends on the affected artery, totality of the blockage, duration of the blockage, the presence of collateral blood vessels, oxygen demand, and success of interventional procedures.^[25][64]

Tissue death and myocardial scarring alter the normal conduction pathways of the heart, and weaken affected areas. The size and location puts a person at risk of abnormal heart rhythms (arrhythmias) or heart block, aneurysm of the heart ventricles, inflammation of the heart wall following infarction, and rupture of the heart wall that can have catastrophic consequences.^[64][71]

Diagnosis[edit]

Criteria[edit]

An acute myocardial infarction, according to current consensus, is defined by elevated cardiac biomarkers with a rising or falling trend and at least one of the following:^[23][72]

• Symptoms relating to ischemia
• Changes on an electrocardiogram (ECG), such as ST segment changes, new left bundle branch block, or Q waves
• Changes in the motion of the heart wall on imaging
• Demonstration of a thrombus on angiogram or at autopsy.

Types[edit]

Myocardial infarctions are generally clinically classified into ST elevation MI (STEMI) and non-ST elevation MI (NSTEMI). These are based on changes to an ECG.^[23] STEMIs make up about 25 – 40% of myocardial infarctions.^[19] A more explicit classification system, based on international consensus in 2012, also exists. This classifies myocardial infarctions into five types:^[23]

1. Spontaneous MI related to plaque erosion and/or rupture, fissuring, or dissection
2. MI related to ischemia, such as from increased oxygen demand or decreased supply, e.g. coronary artery spasm, coronary embolism, anemia, arrhythmias, high blood pressure or low blood pressure
3. Sudden unexpected cardiac death, including cardiac arrest, where symptoms may suggest MI, an ECG may be taken with suggestive changes, or a blood clot is found in a coronary artery by angiography and/or at autopsy, but where blood samples could not be obtained, or at a time before the appearance of cardiac biomarkers in the blood
4. Associated with coronary angioplasty or stents
   • Associated with percutaneous coronary intervention (PCI)
   • Associated with stent thrombosis as documented by angiography or at autopsy
5. Associated with CABG
6. Associated with spontaneous coronary artery dissection in young, fit women

Cardiac biomarkers[edit]

There are a number of different biomarkers used to determine the presence of cardiac muscle damage. Troponins, measured through a blood test, are considered to be the best,^[19] and are preferred because they have greater sensitivity and specificity for measuring injury to the heart muscle than other tests.^[64] A rise in troponin occurs within 2–3 hours of injury to the heart muscle, and peaks within 1–2 days. The level of the troponin, as well as a change over time, are useful in measuring and diagnosing or excluding myocardial infarctions, and the diagnostic accuracy of troponin testing is improving over time.^[64] One high-sensitivity cardiac troponin is able to rule out a heart attack as long as the ECG is normal.^[73][74]

Other tests, such as CK-MB or myoglobin, are discouraged.^[75] CK-MB is not as specific as troponins for acute myocardial injury, and may be elevated with past cardiac surgery, inflammation or electrical cardioversion; it rises within 4–8 hours and returns to normal within 2–3 days.^[25]Copeptin may be useful to rule out MI rapidly when used along with troponin.^[76]

Electrocardiogram[edit]

Electrocardiograms (ECGs) are a series of leads placed on a person's chest that measure electrical activity associated with contraction of heart muscle.^[77] The taking of an ECG is an important part in the workup of an AMI,^[23] and ECGs are often not just taken once, but may be repeated over minutes to hours, or in response to changes in signs or symptoms.^[23]

ECG readouts product a waveform with different labelled features.^[77] In addition to a rise in biomarkers, a rise in the ST segment, changes in the shape or flipping of T waves, new Q waves, or a new left bundle branch block can be used to diagnose an AMI.^[23] In addition, ST elevation can be used to diagnose an ST segment myocardial infarction (STEMI). A rise must be new in V2 and V3 ≥2 mm (0,2 mV) for males or ≥1.5 mm (0.15 mV) for females or ≥1 mm (0.1 mV) in two other adjacent chest or limb leads.^[19][23] ST elevation is associated with infarction, and may be preceded by changes indicating ischemia, such as ST depression or inversion of the T waves.^[77] Abnormalities can help differentiate the location of an infarct, based on the leads that are affected by changes.^[16] Early STEMIs may be preceded by peaked T waves.^[19] Other ECG abnormalities relating to complications of acute myocardial infarctions may also be evident, such as atrial or ventricular fibrillation.^[78]

Imaging[edit]

Noninvasive imaging plays an important role in the diagnosis and characterisation of myocardial infarction.^[23] Tests such as chest X-rays can be used to explore and exclude alternate causes of a person's symptoms.^[23] Tests such as stress echocardiography and myocardial perfusion imaging can confirm a diagnosis when a person's history, physical examination (including cardiac examination) ECG, and cardiac biomarkers suggest the likelihood of a problem.^[79]

Echocardiography, an ultrasound scan of the heart, is able to visualize the heart, its size, shape, and any abnormal motion of the heart walls as they beat that may indicate a myocardial infarction. The flow of blood can be imaged, and contrast dyes may be given to improve image.^[23] Other scans using radioactive contrast include SPECTCT-scans using thallium, sestamibi (MIBI scans) or tetrofosmin; or a PET scan using Fludeoxyglucose or rubidium-82.^[23] These nuclear medicine scans can visualize the perfusion of heart muscle.^[23] SPECT may also be used to determine viability of tissue, and whether areas of ischemia are inducible.^[23][80]

Medical societies and professional guidelines recommend that the physician confirm a person is at high risk for myocardial infarction before conducting imaging tests to make a diagnosis,^[79][81] as such tests are unlikely to change management and result in increased costs.^[79] Patients who have a normal ECG and who are able to exercise, for example, do not merit routine imaging.^[79]

• Poor movement of the heart due to an MI as seen on ultrasound^[82]

• Pulmonary edema due to an MI as seen on ultrasound^[82]

Differential diagnosis[edit]

There are many causes of chest pain, which can originate from the heart, lungs, gastrointestinal tract, aorta, and other muscles, bones and nerves surrounding the chest.^[83] In addition to myocardial infarction, other causes include angina, insufficient blood supply (ischemia) to the heart muscles without evidence of cell death, gastroesophageal reflux disease; pulmonary embolism, tumors of the lungs, pneumonia, rib fracture, costochondritis, heart failure and other musculoskeletal injuries.^[83][84] Rarer severe differential diagnoses include aortic dissection, esophageal rupture, tension pneumothorax, and pericardial effusion causing cardiac tamponade.^[85] The chest pain in an MI may mimic heartburn.^[24] Causes of sudden-onset breathlessness generally involve the lungs or heart – including pulmonary edema, pneumonia, allergic reactions and asthma, and pulmonary embolus, acute respiratory distress syndrome and metabolic acidosis.^[83] There are many different causes of fatigue, and myocardial infarction is not a common cause.^[86]

Management[edit]

Pathophysiology Of Cardiovascular Disease Pdf

A myocardial infarction requires immediate medical attention. Treatment aims to preserve as much heart muscle as possible, and to prevent further complications.^[25] Treatment depends on whether the myocardial infarction is a STEMI or NSTEMI.^[64] Treatment in general aims to unblock blood vessels, reduce blot clot enlargement, reduce ischemia, and modify risk factors with the aim of preventing future MIs.^[25] In addition, the main treatment for myocardial infarctions with ECG evidence of ST elevation (STEMI) include thrombolysis or percutaneous coronary intervention, although PCI is also ideally conducted within 1–3 days for NSTEMI.^[64] In addition to clinical judgement, risk stratification may be used to guide treatment, such as with the TIMI and GRACE scoring systems.^[16][64][87]

Pain[edit]

The pain associated with myocardial infarction may be treated with nitroglycerin or morphine.^[25] Nitroglycerin (given under the tongue or intravenously) may improve the blood supply to the heart, and decrease the work the heart must do.^[25] It is an important part of therapy for its pain relief, despite there being no benefit to overall mortality.^[25][88] Morphine may also be used, and is effective for the pain associated with STEMI.^[25] The evidence for benefit from morphine on overall outcomes, however, is poor and there is some evidence of potential harm.^[89][90]

Anticoagulation[edit]

Aspirin, an antiplateletanticoagulant, is given as a loading dose with the goal of reducing the clot size and reduce further clotting in the affected artery.^[25][64] It is known to decrease mortality associated with acute myocardial infarction by at least 50%.^[64]P2Y12 inhibitors such as clopidogrel, prasugrel and ticagrelor are given concurrently, also as a loading dose, with the dose depending on whether further surgical management or fibrinolysis is planned.^[64] Prasugrel and ticagrelor are recommended in European and American guidelines, as they are active more quickly and consistently than clopidogrel.^[64] P2Y12 inhibitors are recommended in both NSTEMI and STEMI, including in PCI, with evidence also to suggest improved mortality.^[64]Heparins, particularly in the unfractionated form, act at several points in the clotting cascade, help to prevent the enlargement of a clot, and are also given in myocardial infarction, owing to evidence suggesting improved mortality rates.^[64] In very high-risk scenarios, inhibitors of the platelet glycoprotein α_IIbβ_3a receptor such as eptifibatide or tirofiban may be used.^[64]

There is varying evidence on the mortality benefits in NSTEMI. A 2014 review of P2Y12 inhibitors such as clopidogrel found they do not change the risk of death when given to people with a suspected NSTEMI prior to PCI,^[91] nor do heparins change the risk of death.^[92] They do decrease the risk of having a further myocardial infarction.^[64][92]

Angiogram[edit]

Primary percutaneous coronary intervention (PCI) is the treatment of choice for STEMI if it can be performed in a timely manner, ideally within 90–120 minutes of contact with a medical provider.^[64][93] Some recommend it is also done in NSTEMI within 1–3 days, particularly when considered high-risk.^[64] A 2017 review, however, did not find a difference between early versus later PCI in NSTEMI.^[94]

PCI involves small probes, inserted through peripheral blood vessels such as the femoral artery or radial artery into the blood vessels of the heart. The probes are then used to identify and clear blockages using small balloons, which are dragged through the blocked segment, dragging away the clot, or the insertion of stents.^[25][64]Coronary artery bypass grafting is only considered when the affected area of heart muscle large, and PCI is unsuitable, for example with difficult cardiac anatomy.^[95] After PCI, people are generally placed on aspirin indefinitely and on dual antiplatelet therapy (generally aspirin and clopidogrel) for at least a year.^[19][64][96]

Fibrinolysis[edit]

If PCI cannot be performed within 90 to 120 minutes in STEMI then fibrinolysis, preferably within 30 minutes of arrival to hospital, is recommended.^[64][97] If a person has had symptoms for 12 to 24 hours evidence for effectiveness of thrombolysis is less and if they have had symptoms for more than 24 hours it is not recommended.^[98] Thrombolysis involves the administration of medication that activates the enzymes that normally dissolve blood clots. These medications include tissue plasminogen activator, reteplase, streptokinase, and tenecteplase.^[25] Thrombolysis is not recommended in a number of situations, particularly when associated with a high risk of bleeding or the potential for problematic bleeding, such as active bleeding, past strokes or bleeds into the brain, or severe hypertension. Situations in which thrombolysis may be considered, but with caution, include recent surgery, use of anticoagulants, pregnancy, and proclivity to bleeding.^[25] Major risks of thrombolysis are major bleeding and intracranial bleeding.^[25] Pre-hospital thrombolysis reduces time to thrombolytic treatment, based on studies conducted in higher income countries, however it is unclear whether this has an impact on mortality rates.^[99]

Other[edit]

In the past, high flow oxygen was recommended for everyone with a possible myocardial infarction.^[75] More recently, no evidence was found for routine use in those with normal oxygen levels and there is potential harm from the intervention.^{[100][101][102][103]} Therefore, oxygen is currently only recommended if oxygen levels are found to be low or if someone is in respiratory distress.^[25][75]

If despite thrombolysis there is significant cardiogenic shock, continued severe chest pain, or less than a 50% improvement in ST elevation on the ECG recording after 90 minutes, then rescue PCI is indicated emergently.^[104][105]

Those who have had cardiac arrest may benefit from targeted temperature management with evaluation for implementation of hypothermia protocols. Furthermore, those with cardiac arrest, and ST elevation at any time, should usually have angiography.^[19]Aldosterone antagonists appear to be useful in people who have had an STEMI and do not have heart failure.^[106]

Rehabilitation[edit]

Cardiac rehabilitation benefits many who have experienced myocardial infarction,^[64] even if there has been substantial heart damage and resultant left ventricular failure. It should start soon after discharge from the hospital. The program may include lifestyle advice, exercise, social support, as well as recommendations about driving, flying, sport participation, stress management, and sexual intercourse.^[107]

Prevention[edit]

There is a large crossover between the lifestyle and activity recommendations to prevent a myocardial infarction, and those that may be adopted as secondary prevention after an initial myocardial infarction,^[64] because of shared risk factors and an aim to reduce atherosclerosis affecting heart vessels.^[25]

Primary prevention[edit]

Lifestyle[edit]

Physical activity can reduce the risk of cardiovascular disease, and people at risk are advised to engage in 150 minutes of moderate or 75 minutes of vigorous intensity aerobic exercise a week.^[108] Keeping a healthy weight, drinking alcohol within the recommended limits, and quitting smoking reduce the risk of cardiovascular disease.^[108]

Substituting polyunsaturated fats such as olive oil and rapeseed oil instead of saturated fats may reduce the risk of myocardial infarction,^[41] although there is not universal agreement.^[42] Dietary modifications are recommended by some national authorities, with recommendations including increasing the intake of wholegrain starch, reducing sugar intake (particularly of refined sugar), consuming five portions of fruit and vegetables daily, consuming two or more portions of fish per week, and consuming 4–5 portions of unsalted nuts, seeds, or legumes per week.^[108] The dietary pattern with the greatest support is the Mediterranean diet.^[109]Vitamins and mineral supplements are of no proven benefit,^[110] and neither are plant stanols or sterols.^[108]

Public health measures may also act at a population level to reduce the risk of myocardial infarction, for example by reduce unhealthy diets (excessive salt, saturated fat and trans fat) including food labeling and marketing requirements as well as requirements for catering and restaurants, and stimulating physical activity. This may be part of regional cardiovascular disease prevention programs, or through the health impact assessment of regional and local plans and policies.^[111]

Most guidelines recommend combining different preventive strategies. A 2015 Cochrane Review found some evidence that such an approach might help with blood pressure, body mass index and waist circumference. However, there was insufficient evidence to show an effect on mortality or actual cardio-vascular events.^[112]

Medication[edit]

Statins, drugs that act to lower blood cholesterol, decrease the incidence and mortality rates of myocardial infarctions.^[113] They are often recommended in those at an elevated risk of cardiovascular diseases.^[108]

Aspirin has been studied extensively in people considered at increased risk of myocardial infarction. Based on numerous studies in different groups (e.g. people with or without diabetes), there does not appear to be a benefit strong enough to outweigh the risk of excessive bleeding.^[114][115] Nevertheless, many clinical practice guidelines continue to recommend aspirin for primary prevention,^[116] and some researchers feel that those with very high cardiovascular risk but low risk of bleeding should continue to receive aspirin.^[117]

Secondary prevention[edit]

There is a large crossover between the lifestyle and activity recommendations to prevent a myocardial infarction, and those that may be adopted as secondary prevention after an initial myocardial infarct.^[64] Recommendations include stopping smoking, a gradual return to exercise, eating a healthy diet, low in saturated fat and low in cholesterol, and drinking alcohol within recommended limits, exercising, and trying to achieve a healthy weight.^[64][107] Exercise is both safe and effective even if people have had stents or heart failure,^[118] and is recommended to start gradually after 1–2 weeks.^[64] Counselling should be provided relating to medications used, and for warning signs of depression.^[64] Previous studies suggested a benefit from omega-3 fatty acid supplementation but this has not been confirmed.^[107]

Medications[edit]

Following a heart attack, nitrates, when taken for two days, and ACE-inhibitors decrease the risk of death.^[119] Other medications include:

Aspirin is continued indefinitely, as well as another antiplatelet agent such as clopidogrel or ticagrelor ('dual antiplatelet therapy' or DAPT) for up to twelve months.^[107] If someone has another medical condition that requires anticoagulation (e.g. with warfarin) this may need to be adjusted based on risk of further cardiac events as well as bleeding risk.^[107] In those who have had a stent, more than 12 months of clopidogrel plus aspirin does not affect the risk of death.^[120]

Beta blocker therapy such as metoprolol or carvedilol is recommended to be started within 24 hours, provided there is no acute heart failure or heart block.^[19][75] The dose should be increased to the highest tolerated.^[107] Contrary to what was long believed, the use of beta blockers does not appear to affect the risk of death, possibly because other treatments for MI have improved.^[121] When beta blocker medication is given within the first 24–72 hours of a STEMI no lives are saved. However, 1 in 200 people were prevented from a repeat heart attack, and another 1 in 200 from having an abnormal heart rhythm. Additionally, for 1 in 91 the medication causes a temporary decrease in the heart's ability to pump blood.^[122]

ACE inhibitor therapy should be started within 24 hours, and continued indefinitely at the highest tolerated dose. This is provided there is no evidence of worsening kidney failure, high potassium, low blood pressure, or known narrowing of the renal arteries.^[64] Those who cannot tolerate ACE inhibitors may be treated with an angiotensin II receptor antagonist.^[107]

Statin therapy has been shown to reduce mortality and subsequent cardiac events, and should be commenced with the aim of lowering LDL cholesterol. Other medications, such as ezetimibe, may also be added with this goal in mind.^[64]

Aldosterone antagonists (spironolactone or eplerenone) may be used if there is evidence of left ventricular dysfunction after an MI, ideally after beginning treatment with an ACE inhibitor.^[107][123]

Other[edit]

A defibrillator, an electric device connected to the heart and surgically inserted under the skin, may be recommended. This is particularly if there are any ongoing signs of heart failure, with a low left ventricular ejection fraction and a New York Heart Association grade II or III after 40 days of the infarction.^[64] Defibrillators detect potentially fatal arrhythmia and deliver an electrical shock to the person to depolarize a critical mass of the heart muscle.^[124]

Prognosis[edit]

The prognosis after myocardial infarction varies greatly depending on the extent and location of the affected heart muscle, and the development and management of complications.^[16] Prognosis is worse with older age, and social isolation.^[16] Anterior infarcts, persistent ventricular tachycardia or fibrillation, development of heart blocks, and left ventricular impairment are all associated with poorer prognosis.^[16] Without treatment, about a quarter of those affected by MI die within minutes, and about forty percent within the first month.^[16] Morbidity and mortality from myocardial infarction has however improved over the years due to earlier and better treatment:^[26] in those who have an STEMI in the United States, between 5 and 6 percent die before leaving the hospital and 7 to 18 percent die within a year.^[19]

It is unusual for babies to experience a myocardial infarction, but when they do, about half die.^[125] In the short-term, neonatal survivors seem to have a normal quality of life.^[125]

Complications[edit]

Complications may occur immediately following the myocardial infarction or may take time to develop. Disturbances of heart rhythms, including atrial fibrillation, ventricular tachycardia and fibrillation and heart block can arise as a result of ischemia, cardiac scarring, and infarct location.^[16][64]Stroke is also a risk, either as a result of clots transmitted from the heart during PCI, as a result of bleeding following anticoagulation, or as a result of disturbances in the heart's ability to pump effectively as a result of the infarction.^[64]Regurgitation of blood through the mitral valve is possible, particularly if the infarction causes dysfunction of the papillary muscle.^[64]Cardiogenic shock as a result of the heart being unable to adequately pump blood may develop, dependent on infarct size, and is most likely to occur within the days following an acute myocardial infarction. Cardiogenic shock is the largest cause of in-hospital mortality.^[26][64] Rupture of the ventricular dividing wall or left ventricular wall may occur within the initial weeks.^[64]Dressler's syndrome, a reaction following larger infarcts and a cause of pericarditis is also possible.^[64]

Heart failure may develop as a long-term consequence, with an impaired ability of heart muscle to pump, scarring, and increase in size of the existing muscle. Aneurysm of the left ventricle myocardium develops in about 10% of MI and is itself a risk factor for heart failure, ventricular arrhythmia and the development of clots.^[16]

Risk factors for complications and death include age, hemodynamic parameters (such as heart failure, cardiac arrest on admission, systolicblood pressure, or Killip class of two or greater), ST-segment deviation, diabetes, serum creatinine, peripheral vascular disease, and elevation of cardiac markers.^{[126][127][128]}

Epidemiology[edit]

Myocardial infarction is a common presentation of coronary artery disease. The World Health Organization estimated in 2004, that 12.2% of worldwide deaths were from ischemic heart disease;^[129] with it being the leading cause of death in high- or middle-income countries and second only to lower respiratory infections in lower-income countries.^[129] Worldwide, more than 3 million people have STEMIs and 4 million have NSTEMIs a year.^[18] STEMIs occur about twice as often in men as women.^[19]

Rates of death from ischemic heart disease (IHD) have slowed or declined in most high-income countries, although cardiovascular disease still accounted for one in three of all deaths in the US in 2008.^[130] For example, rates of death from cardiovascular disease have decreased almost a third between 2001 and 2011 in the United States.^[131]

In contrast, IHD is becoming a more common cause of death in the developing world. For example, in India, IHD had become the leading cause of death by 2004, accounting for 1.46 million deaths (14% of total deaths) and deaths due to IHD were expected to double during 1985–2015.^[132] Globally, disability adjusted life years (DALYs) lost to ischemic heart disease are predicted to account for 5.5% of total DALYs in 2030, making it the second-most-important cause of disability (after unipolar depressive disorder), as well as the leading cause of death by this date.^[129]

Society and culture[edit]

Depictions of heart attacks in popular media often include collapsing or loss of consciousness which are not common symptoms; these depictions contribute to widespread misunderstanding about the symptoms of myocardial infarctions, which in turn contributes to people not getting care when they should.^[133]

Legal implications[edit]

At common law, in general, a myocardial infarction is a disease, but may sometimes be an injury. This can create coverage issues in the administration of no-fault insurance schemes such as workers' compensation. In general, a heart attack is not covered;^[134] however, it may be a work-related injury if it results, for example, from unusual emotional stress or unusual exertion.^[135] In addition, in some jurisdictions, heart attacks suffered by persons in particular occupations such as police officers may be classified as line-of-duty injuries by statute or policy. In some countries or states, a person having suffered from an MI may be prevented from participating in activity that puts other people's lives at risk, for example driving a car or flying an airplane.^[136]

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Further reading[edit]

• Levine GN, Bates ER, Blankenship JC, Bailey SR, Bittl JA, Cercek B, et al. (March 2016). '2015 ACC/AHA/SCAI Focused Update on Primary Percutaneous Coronary Intervention for Patients With ST-Elevation Myocardial Infarction: An Update of the 2011 ACCF/AHA/SCAI Guideline for Percutaneous Coronary Intervention and the 2013 ACCF/AHA Guideline for the Management of ST-Elevation Myocardial Infarction: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Society for Cardiovascular Angiography and Interventions'. Circulation. 133 (11): 1135–47. doi:10.1161/CIR.0000000000000336. PMID26490017.

External links[edit]

• Myocardial infarction at Curlie
• American Heart Association's Heart Attack web site — Information and resources for preventing, recognizing and treating a heart attack.
• TIMI Score for UA/NSTEMI and STEMI

Abstract

Congestive heart failure accounts for half a million deaths per year in the US. Despite its place among the leading causes of morbidity, pharmcalogical and mechanic remedies have been able to slow the progression of the disease, today’s science has yet to provide a cure and there are few therapeutic modalities available for patients with advanced heart failure. There is a critical need to explore new therapeutic approaches in heart failure and gene therapy has emerged as a viable alternative. Recent advances in understanding of the molecular basis of myocardial dysfunction, together with the evolution of increasingly efficient gene transfer technology, has placed heart failure within reach of gene-based therapy. The recent successful and safe completion of a phase 2 trial targeting the sarcoplasmic reticulum calcium ATPase pump (SERCA2a) along with the start of more recent phase 1 trials opens a new era for gene therapy for the treatment of heart failure.

Introduction

Ongoing preclinical studies are providing a sound scientific basis for the evaluation of gene therapy strategies. Furthermore, the anatomical compartmentalization of the heart and its accessibility by surgical and percutaneous approaches, render the myocardium a highly amenable target system for gene therapy.

Over the last decade, novel molecular mechanisms associated with heart failure have been discovered creating new targets for therapeutic interventions. A number of these promising targets cannot be manipulated through pharmacological means but are ideal for gene therapy approaches.

In this review, we will highlight new strategies for the treatment of heart failure by gene transfer, focusing on the vectors, targets, and delivery methods along with the recent clinical results from early clinical trials.

Gene Delivery

Gene delivery vehicles fall into one of two categories: non-viral or viral gene delivery vectors. The most attractive features of non-viral gene delivery vectors are their safety profile and minimal immunogenicity. The size of the transgene in viruses is limited by the packaging constraints while with plasmid there are no packaging limitations that restricts the transgene size. However, despite extensive efforts to improve transfection efficiencies by combining “naked” DNA with delivery agents such as cationic lipids or by using plasmid DNA in combination with physical methods to enhance gene transfer, insufficient transfection efficiencies remain the Achilles heel of non-viral gene delivery methods.

Given these limitations of non-viral delivery vehicles, intense research has taken place over the past years aimed at harnessing the inherent capacity of viruses to deliver genes to cells. Today, the virus most widely used for cardiovascular gene transfer is adenovirus with a total of 54 trials, 24 of which are ongoing. Other viral vectors that have reached the clinical trial phase are derived from retroviruses, Sendai virus and adeno-associated virus. Given the non-proliferative nature of cardiomyocytes, lentiviruses could be promising vehicles for cardiovascular gene therapy. In contrast to AAV, lenti- and retroviral vectors integrate their genomes into the host genome. Whereas pseudo-random vector genome integration raises the risk of oncogenic transformation, genome integration is essential for stem cell therapy because stem cell therapy, by its very nature, requires an expansion of the stem cells to be effective. This expansion requires multiple rounds of cell division, during which the vector DNA of non-integrating vectors will be lost due to “dilution” of the vector genomes. In regards, to the potential for oncogenic transformation by γ-retro- and lentiviral vectors, it should be pointed that while both γ–retroviral vectors and lentiviral vectors preferentially integrate their genome into transcriptional units, lentiviral vector genome integration is, in contrast to γ-retroviral genome integration, usually more distant from transcription start sites. Together with the development of self-inactivating (SIN) lentiviral vectors, the resulting improved safety profile likely contributed to the approval of clinical trials using lentiviral vectors.

As can be seen from Table 1, each of the vector systems has its unique properties and vector specific advantages and limitations. One of the potential limitations of adenoviral vectors is that they can only trigger short term gene expression, although it can be argued that for certain applications—e.g., the expression of factors that stimulate cell division—this might be advantageous. The far bigger problem of adenoviral vectors is that they are highly immunogenic. While in cardiovascular gene therapy trials no adverse effect as devastating as the tragic death of Jesse Gelsinger in a trial for the treatment of ornithine transcarbamylase deficiency with an adenoviral vector encoding the deficient enzyme has been observed, the inflammation caused by adenoviral vectors is of significant concern and probably one of the main reasons for the declining the use of this vector system in clinical gene transfer for cardiovascular diseases.

Table 1

Vectors Systems Used in Cardiovascular Gene Transfer

NV: non-viral; Ad: adenovirus; AAV: adeno-associated virus; RV: retrovirus; LV: lentivirus; CTL: cytotoxic T lymphocyte. a as of June 2011

AAV Vectors in Cardiovascular Gene Transfer

AAV is a small, non-enveloped virus with a linear, single-stranded DNA that belongs to the family Parvoviridae, the subfamily Parvovirinae and the genus Dependovirus. As its name indicates, AAV depends on a helper virus such as adenovirus or herpes virus for productive replication. Contributing to its beneficial safety profile, AAV is not known to be associated with any human disease. Furthermore, the only cis-elements required for AAV vectors are the inverted terminal repeats (ITRs) that flank the (recombinant) viral genome. As a consequence of this, transduction with AAV vectors does not result in the expression of any viral genes, which likely contributes to their low immunogenicity when compared to most other viral vectors (see below).

As for all viruses, infection/transduction starts with the binding of the virus to its receptor(s) (Fig. 1). The receptors for some of the AAV serotypes have been described in the literature (see for a description of the AAV receptors) Receptor-binding is then followed by endocytosis, which for AAV2 is via the so-called CLIC/GEEC (Clathrin Independent Carriers/GPI-anchored protein Enriched Endocytic Compartment) pathway whereas for AAV5 it has been reported that endocytosis can occur both by clathrin coated vesicles or caveolae.

1: receptor binding & endocytosis; 2: escape into cytoplasm; 3: nuclear import; 4: capsid disassembly; 5: double strand synthesis

At least for the prototypical AAV serotype, AAV2, and AAV5 endocytosis is followed by transport to the Golgi (Fig. 1). Evidence suggests that once in the cytoplasm AAV is imported into the nucleus as an intact particle, presumably through the nuclear pore (Fig. 1). Import of viral particle is then followed by capsid uncoating and the release of the viral genome (Fig. 1). For transcription of the transgene, the single-stranded AAV genome must then first be converted into a double strand genome (Fig. 1).

Tropism of AAV Serotypes

To date, 13 AAV serotypes and more than 100 AAV variants have been described in the literature, although most gene transfer experiments are performed with vectors based on AAV1-9. Together, the AAV serotypes display broad, yet serotype-specific tissue and cell tropism (Table 2), which is likely, at least to a certain extent, a result of their diverse receptors (for a compilation see). Nonetheless—species differences notwithstanding—overall, AAV1, AAV6, AAV8 and AAV9 show strong cardiac transduction, with AAV9 appearing to be the most cardiotropic, at least in rodents. In light of the observed species differences it is unfortunate that our knowledge of the cardiotropism of the AAV serotypes in non-human primates is very limited, although it has been shown that AAV2 is able to transduce cardiomyocytes after intramyocardial injection and in Rhesus Macaques AAV6 is superior to AAV8 and AAV9 in transducing cardiac tissue when delivered by percutaneous, transendocardial injection. Importantly, the results from the recently completed phase II of the so-called CUPID (Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease) indicate that AAV1 can successfully transduce human cardiac tissue.

Table 2

Tropism of AAV Serotypes in Animal Models of Cardiac Diseases

Transductional Targeting

Despite the substantial cardiotropism of AAV1, AAV6 and AAV9, all of these serotypes transduce additional tissues, at least to some extent. It is, therefore, not surprising that substantial efforts have been made to develop AAV variants with further enhanced cardiac tropism.

One general approach to alter AAV tropism, including the isolation of AAV variants with cardiotropism, that recently has gained considerable attention is so-called directed evolution (Figure 2). All studies using this method are based on the initial creation of AAV libraries with diverse AAV capsids. One method uses the insertion of a (semi)-random peptide sequence into the AAV capsid. A variation of this method replaces a short segment of an AAV capsid protein, usually located in the major capsid protein VP3, with a (semi)random peptide sequence.

Generation of mutant AAV Library and Directed Evolution to Identify Cardiotropic AAVs. A) Creation of a library of AAVs through DNA shuffling. B) Selection of cardiotropic AAVs through directed evolution.

In a different approach to create AAV libraries composed of viruses with diverse capsids, viruses with chimeric capsid proteins, i.e. capsid proteins with stretches of amino acids from different serotypes within the same capsid protein, are generated. The method for the generation of these so-called shuffled capsid libraries is outlined in Fig. 2A.

Regardless on how the AAV libraries are generated, in the next step the most cardiotropic variants are then selected by either in vitro or in vivo evolution. Whereas in vitro selection might be technically simpler, it fails to select for variants that can efficiently overcome barriers, such as escape from the vasculature, that virions only encounter in an in vivo setting. A typical in vivo selection procedure is shown schematically in Fig. 2B. Briefly, the viral library is injected into the tail vein of mice, 24-72 hours later the heart is harvested and the capsid DNA amplified by PCR. After subcloning into a wild-type AAV backbone, a secondary viral library is created and the selection procedure repeated. After one or more additional rounds of selection, individual clones are isolated and the capsid genes sequenced. Recombinant viruses encoding reporter genes are then produced and used for a detailed characterization of the new cardiotropic AAV variants. While the method just described resulted in the successful identification of AAV variants with enhanced cardiac tropism, it enriches AAV variants that successfully deliver their DNA to the heart, regardless whether they successfully infect cardiac tissue. To overcome this potential limitation, Kleinschmidt and colleagues, after the harvesting of the heart, kept heart slices in organotypic culture and super-infected these slices with adenovirus. This should allow the replication and enrichment of AAV variants that successfully infected cardiac tissue. AAV variants that, for instance, only delivered their genomes to the cytoplasm of cardiomyocytes, on the other hand, will not be enriched.

Another successful way to enhance cardiac tropism has been used by Samulski and colleagues . This group replaced a hexapeptide sequence in the receptor-binding region of the AAV2 capsid with the corresponding peptides of other AAV serotypes and variants. One of the novel AAV2 variants, AAV2i8, which is a chimera between the AAV2 capsid and the AAV8 hexapeptide, showed significant skeletal and cardiac muscle tropism with significantly reduced transduction efficiencies of the liver. Clearly, these novel AAV variants are promising candidates for cardiac gene transfer. It has to be pointed out, however, that in cases where transduction of cardiomyocytes were compared to transduction by AAV9 the latter always showed the highest transduction efficiency.

Transcriptional Targeting

Ideally, transductional targeting alone is sufficient to drive the expression of the transgene exclusively in the heart. Unfortunately, however, AAV vectors that do not transduce non-cardiac tissues to some extent are thus far unavailable. An alternative and complementary approach to restrict transgene expression to the heart is transcriptional targeting, i.e. the use of cardiac specific promoters. An additional advantage of cardiac specific promoters is that, in contrast to the most commonly used promoter, the CMV promoter, their expression is not expected to be downregulated and, hence, should result in long-term gene expression.

One promoter that was used in both adenoviral and AAV vectors is the ventricle-specific Myosin Light Chain-2 (MLC-2v) promoter. While this promoter is more than 10-fold less efficient than the already weak RSV promoter, this inadequacy can be partially over come by the inclusion of 4 copies of the 250bp enhancer fragment of the MLC-v2 gene. This promoter construct drives expression that is only 3.8-fold lower than expression from the strong CMV promoter. For AAV vectors this comes, however, at the price of reducing the allowable size of the transgene by 1,000bp, which is not insignificant in a vector system that has a packaging capacity of ~5kb. The Myosin Heavy Chain Promoter, which has been used extensively in the generation of transgenic mice that express transgenes specifically in cardiomyocytes, is not suitable for AAV vectors because it is 5.5kb long. Unfortunately, expression from a minimal version of the same promoter is much lower than expression from RSV or CMV promoters. Maybe the most promising promoter for cardiac-specific expression that is suitable for AAV vectors is a 418 bp fragment from the chicken cardiac Troponin T (cTnT) promoter. The strength of this promoter is only approximately 2-fold lower when compared to the CMV promoter.

Regulation of Transgene Expression

Both the viral and heart-specific promoters mentioned in the previous section are constitutively active, i.e. the expression levels of the transgene cannot be regulated. For certain applications, however, it is important that the transgene is either expressed only under certain physiological conditions or that expression levels or the time-frame of expression can be regulated pharmacologically. The most commonly systems that allow the manipulation of expression levels with small molecules are the so-called Tet-on and Tet-off and the rapamycin/FK506 inducible systems. Both these systems as well as a mifepristone (RU486)-inducible system have been used in context of AAV vectors.

In addition it can be useful to express the transgene under specific pathologic conditions. One system that allows the induction of transgene expression under hypoxic conditions is based on the inclusion of a hypoxia response element into the promoter region. Hypoxia inducible system have been based on the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor 1α (HIF1α).

Gene Silencing with shRNAs and Artificial MicroRNAs

For many disorders, including cardiac diseases, it is necessary to overexpress a protein because the endogenous protein is either non-functional or expressed at insufficient levels. It is equally possible, however, that a disorder is caused by a mutated protein that has dominant effects or that an endogenous protein is expressed at pathologically high levels. Furthermore, in certain instances, it is therapeutically beneficial to suppress protein expression of protein that is expressed at physiological levels. A little more than a decade ago, Mello and colleagues made the seminal discovery that in Caenorhabditis elegans double-stranded RNA could prompt the degradation of complementary mRNA. Analysis of the mechanism of this process (reviewed in) revealed that the double-stranded RNA is converted by the RNAse Dicer into small 21-25 nucleotide-long RNA fragments. These fragments are then exported from nucleus in an exportin 5-dependent manner into the cytoplasm where the RNA strand that is complementary to the mRNA, the guide strand, is loaded into the RNA Induced Silencing Complex (RISC). A second class of small RNA molecules termed microRNAs (miRNAs) is encoded by long double-stranded regions usually found in introns of primary transcripts from polymerase II promoters. Whereas the process that leads to the production of miRNAs shares important aspects with the production of siRNAs, their mode of action is different. Presumably because miRNAs contain mismatches to their targets they do not trigger the degradation of the target mRNA but rather they suppress translation of the transcript. It was also shown that RNA interference also occurs in mammalian cells. This discovery revolutionized how protein expression can be silenced in cells grown in culture. It became rapidly clear, however, that small interfering RNAs (siRNAs) are poor drugs. Not only is siRNA delivery in vivo inefficient but the siRNAs are also unstable resulting in a limited and transient suppression of gene expression.

More recently, it was shown that siRNAs can also be generated by expression of so-called short hairpin RNAs (shRNAs) from plasmids. Transcription of shRNAs is in general driven by pol III promoters. After an export to the cytoplasm they are converted into siRNAs by Dicer and the guide strand is loaded into the RISC, which then results in the degradation of the target mRNA. shRNA expression cassettes can also be incorporated into viral vectors, including AAV vectors. Owing to their ability to drive long-term expression, AAV vector-based shRNA mediated gene silencing results in a lasting suppression of target gene expression. It became soon clear, however, that AAV vector-based gene silencing with shRNAs is not without potential complications. Mark Kay’s group used the strongly hepatotropic AAV serotype 8 to deliver shRNAs directed against luciferase, human α1-antitrypsin or the Hepatitis B Virus (HBV) genome. The large number of diverse shRNAs made off-target effects unlikely and further analysis revealed that the toxicity is most likely due to saturation of shared components of the shRNA/miRNA pathway, in particular exportin 5. Importantly, however, the toxicity was both dependent on viral dose and the precise nature of the shRNA sequence suggesting that a careful selection of the shRNA sequence, promoter strength and vector dose should allow to minimize toxicity, at least for certain applications.

Immune Responses Against the Viral Vector and Transgenes

Adenoviral Vectors

As mentioned earlier, adenoviral vectors are highly immunogenic, which is likely a major reason for their declining use in human cardiovascular gene therapy. This is especially pronounced in first- and second generation adenoviral vectors that express several viral genes as well the transgene. This immunogenicity is partly alleviated with helper-dependent, so- called gutless, adenoviral vectors because they do not express any viral genes. Nonetheless, even though the adaptive, especially cellular, immune response with gutless adenoviral vectors is attenuated, they still elicit a strong innate immune response. That an innate immune response against adenoviral vectors can have catastrophic consequences was demonstrated in a trial with a second generation adenoviral vector that resulted in the death of a trial participant. An additional complication for the use of adenoviral vectors is the high prevalence of neutralizing antibodies against adenoviruses. Approximately 97% of the population harbor neutralizing antibodies against type C adenoviruses, which include the commonly used Ad2 and Ad5.

Furthermore, although helper-dependent adenoviral vectors show reduced myocardial inflammation after intramyocardial injection when compared to first generation adenoviral vectors, the adenoviral capsid also seems to modulate the immune response against the transgene. Thus, while AAV-mediated GFP expression is long-lasting, both after intramyocardial and intrapericardial injection, expression from both first generation and helper-dependent, i.e. gutless, adenoviral vectors was transient^, . In the future, it might be possible to devise strategies to overcome immune responses against either the adenoviral capsid or transgene but, at present, these immune reactions clearly pose a serious obstacle to the use of adenoviral vectors in cardiovascular gene therapy.

Lentiviral Vectors

Due to their ability to transduce non-dividing cells and their potential to trigger long-term transgene expression lentiviral vectors represent a possibly attractive gene delivery system for cardiovascular gene therapy. In contrast to adenoviral and AAV-based vectors, neutralizing antibodies against lentiviral vectors are usually not of concern. However, depending on the origin of the envelope protein used to create the pseudotyped lentiviral vectors, as well as the route of vector administration, a strong cytotoxic immune response can be generated. In general, this immune response is directed against the transgene, although a potential immune reaction against the envelope proteins can not be dismissed. The CTL-mediated destruction of transduced cells is likely due to fact that lentiviral vectors efficiently transduce antigen presenting cells such as Kupffer cell, splenic macrophages and dendritic cells. While in one report pseudotyping a feline immunodeficiency viral vector with the baculovirus envelope protein gp64 has been shown to overcome this limitation resulting in long-term transgene expression, in another mouse strain gene transfer with a gp64-psudotyped HIV-derived vector resulted only in transient (6 months) transgene expression. Whereas the use of tissue-specific promoters can mitigate the expression of the transgene in antigen presenting cells, at least under certain circumstances, these promoters appear not to be sufficiently tissue specific to prevent transgene expression in all antigen presenting cells. A clever approach to overcome this problem was developed by Naldini colleagues^, . In this system, several copies of an miRNA target sequence that is perfectly complementary to an miRNA that is highly and specifically expressed in hematopoietic cells are included into the 3′-untranslated region of the transgene mRNA. . These miRNA target sequences are recognized by the endogenous miRNA in antigen presenting cells resulting in the degradation of the transgene mRNA in these cells but not in target cells. That this is a promising approach has been shown in a mouse model of factor IX deficiency. In this system, a lentiviral vector triggered long-term factor IX expression^, .

AAV Vectors: Pre-Existing Neutralizing Antibodies

Regarding the effect of the immune system on transduction by AAV vectors, the comparatively high incidence of pre-existing neutralizing antibodies in the general population is probably the most concerning. While the prevalence of neutralizing antibodies varies with serotypes and geographic region, most studies report the presence of neutralizing titers of ≥1:20 in 20-40% of the population^-. The cutoff of titers ≥1:20, however, is not only arbitrary it is also likely clinically insufficient as an exclusion criteria. Both studies in humans^{, ,} and animals^- demonstrated that titers as low as 1:4 or even 1:2 can significantly influence or eliminate transgene expression or affect clinical results. For instance, in a clinical trial aimed at treating factor IX deficiency, in the high dose group with a liver-directed AAV vector encoding factor IX one individual with a neutralizing titer of 1:2 showed factor IX expression, albeit it only transiently. A second patient with a titer of 1:17, on the other hand, showed no factor IX expression. In the CUPID trial one exclusion criterion was neutralizing titers of >1:2^, . In this trial, many patients in the high-dose group of AAV1.SERCA2a showed clinical improvements. However, two individuals in the high-dose group that were originally judged to harbor no neutralizing antibodies later tested positive for neutralizing antibodies, and these patients did not show any improvement^, . Importantly, because patients with antibody titers as low as 1:4 were excluded from the CUPID trial, nearly half the prospective patients could not be enrolled because their antibody titers were too high^, . This illustrates that future work is needed to allow the inclusion of most if not all potential patients into clinical trials based on AAV vectors.

Innate Immune Response

In contrast to adenoviral vectors, AAV does not induce a strong immune response, although systemic injection can give rise to a transient increase in inflammatory cytokines. Furthermore, it has been demonstrated that AAV can interact with factors of the complement cascade and that these interactions are essential for the endocytosis of AAV by macrophages. Nonetheless, while it is clear that the innate immune system can play a role in the host response to AAV infection, this response is not nearly as strong as the innate immune response against adenoviral vectors.

Cellular Immune Response Against the Transgene and the AAV Capsid

In gene therapy applications where a missing protein is replaced a significant risk for an immune response against the transgene exists. Fortunately, however, in cardiovascular gene transfer the transgenes to be expressed are almost exclusively proteins that are also expressed under physiological conditions but the level of expression is up- or down-regulated during disease progression. Because of this, the host developed tolerance against these proteins and an immune response against the transgene is highly unlikely.

A priori, a cellular immune response against the AAV capsid would seem unlikely because AAV vectors do not express any viral genes. However, it became clear, in an otherwise successful clinical trial to treat hemophilia B, that cellular immune responses against the AAV capsid can occur and that such cellular immune responses can result in the destruction of transduced cells^, . More recently, however, it was demonstrated that such an immune response can be successfully suppressed by a short course of prednisolone. Furthermore, for cardiovascular gene therapy it is important to point out, however, that in the only cardiovascular gene therapy trial using AAV vectors, the CUPID trial, such a cytolytic T–cell response was not observed^, . In this trial, only one patient showed a transient positive signal in an ELISPOT assay indicating a cellular immune response against the AAV1 capsid and this was concurrent with an influenza infection. Nonetheless, it seems advisable to monitor such responses carefully in cardiovascular or other gene therapy trials with AAV vectors.

Gene Delivery

Antegrade Arterial Infusion

Percutaneous coronary artery catheterization is a minimally invasive and well-established procedure that allows homogenous gene delivery to each territory of the heart. The major advantages of this approach are that it is minimally invasive and relatively safe. Thus, it is especially attractive for patients with end-stage heart failure. However, gene delivery can be impeded in patients with severe coronary artery disease. More importantly, there is some variability regarding the efficiency of antegrade coronary gene transfer, partly related to the relatively fast transit of the vector through the vasculature. In a large animal model of volume-overload induced heart failure, antegrade coronary gene transfer was shown to significantly restore cardiac function (Figure 3a). Infusion through the lumen of an inflated angioplasty catheter with temporary occlusion of the coronary artery may increase myocardial gene expression but remains controversial (Figure 3b). In a study by Boekstegers et al. ischemia during coronary artery infusion did not significantly increase myocardial transduction . In contrast, infusion with temporary coronary occlusion at a considerably higher flow rate and consequently elevated coronary pressures resulted in higher gene expression, but was also associated with more myocardial injury .

Antegrade coronary artery infusion a) Coronary artery infusion: The vector is injected through a catheter without interruption of the coronary flow b) Coronary artery infusion with occlusion of a coronary artery: The vector is injected through the lumen of an inflated angioplasty catheter c) Coronary artery infusion with simultaneous blocking of a coronary artery and a coronary vein: The vector is injected through an inflated angioplasty catheter and resides in the coronary circulation until both balloons are deflated.

An approach that focuses on enhancing the vector residence time in the coronary circulation is coronary venous blockade. Antegrade coronary infusion with a short occlusion of both a coronary artery and a coronary vein enhanced myocardial gene expression^, (Figure 3c). This method preserved left ventricular function and inhibited ventricular remodeling in a large animal model of heart failure . However, even a short ischemia period should be considered as carrying a risk in patients with advanced heart failure.

To maximize the duration of vector exposure to the endothelium and at the same time minimize systemic distribution, Kaye et al. developed an extra-corporal device that drains blood from the coronary sinus using an occlusion catheter and returns the oxygenated coronary venous blood to the left main coronary artery via a peristaltic pump (V-Focus, Osprey Medical Inc, St Paul, MN, USA) ^, . In an ovine model of tachycardia induced heart failure, the closed loop recirculation method was more efficient in the transduction of cardiomyocytes than antegrade coronary infusion, which also translated into a greater improvement of left ventricular function^, . Most of the approaches to improve the efficiency of coronary artery infusion are based on increasing the exposure time of the vector to the endothelium. However, transduction efficacy correlates with coronary flow as well as exposure time and vector concentration . Based on the former, antegrade coronary artery infusion supported by means of an increased coronary flow, for example an intra-aortic balloon pump, might further enhance cardiac gene transfer ⁶³. Antegrade coronary artery infusion might be the safest percutaneous gene delivery method, but at the cost of efficiency relative to other techniques. However, to date, all optimization strategies implicate potential damage to the myocardium, limiting their clinical application.

Retrograde Venous Infusion

The coronary venous system provides an alternate route for the percutaneous delivery of therapeutic agents to the myocardium. In a clinical setting, this approach is attractive for patients with impaired coronary artery circulation and limited potential for revascularization. Access to the myocardium can be achieved with this approach regardless of atherosclerosis severity or coronary artery obstruction.

Numerous studies showed that retrograde coronary venous infusion is an alternate myocardial delivery method for cardioprotective drugs^, . Shortly thereafter, retrograde venous infusion was also explored as a method for gene transfer. The rationale for a high transduction efficiency is based on controlling the exposure the time of vector to the endothelium, and on increasing the pressure gradient of capillary filtration^, .

Indeed, studies in large animal models demonstrated that an efficient and homogenous myocardial transduction can be achieved by retroinfusion into the coronary venous system^{, ,} . Boeksteger et al. showed that gene expression after pressure-regulated retrograde venous infusion was significantly higher than after antegrade coronary delivery if the retroinfusion was accompanied by simultaneous induced ischemia (Figure 4b). Compared to percutaneous or surgical direct myocardial injection, retrograde venous infusion also achieved a more homogeneous and efficient reporter gene expression.

V-Focus system and retrograde coronary venous infusion a) Recirculating antegrade coronary artery infusion: The vector is injected into a coronary artery, collected from the coronary sinus and after oxygenation readministered into the coronary artery b) Retrograde coronary venous infusion with simultaneous blocking of a coronary artery and a coronary vein: The vector is injected into a coronary vein and resides in the coronary circulation until both balloons are deflated.

Similar to antegrade coronary artery infusion, a closed loop recirculation retrograde venous infusion approach is also feasible. Recently, Dr. Charles Bridges group demonstrated an extremely high transduction efficiency in the majority of cardiac cardiomyocytes in sheep while minimizing collateral organ exposure, using a retrograde recirculation method during cardio-pulmonary bypass (CPB) surgery .

Retrograde coronary infusion has been shown to be safe, when performed by a trained and experienced surgeon. Trauma to the coronary veins, a potential complication, could be minimized with modifications and improvements in catheter design. However, the procedure is not completely without risks. Highly elevated coronary venous pressure can result in myocardial edema or hemorrhage. Careful pressure monitoring should always be used to avoid this complication. Though with the growing demand for cardiac resynchronization therapy, cannulation of the coronary veins has become a routine procedure, monitoring of the pressure regulation adds to the complexity of this procedure. More importantly, the ischemia during the infusion might not be well tolerated in patients with advanced heart failure.

In summary, percutaneous retrograde coronary infusion is an effective, minimally invasive procedure, but relatively complex approach to gene delivery specifically to the myocardium. This approach requires some expertise and meticulous pressure monitoring to be performed safely.

Aortic cross clamping

In rodents, the vector is injected into the aortic root while the aorta and the pulmonary artery are cross-clamped for a few heart beats . While cross clamping the beating heart is only relatively simple and routine in rodents, a modified version of this technique has been developed for large animals. In comparison to rodent models, large animals studies using aortic cross clamping are complex surgical procedures that require cardiac arrest and a complete surgical isolation of the heart in situ with CPB^, .

Virus injection into the aortic root during CPB with a cross-clamped aorta resulted in selective myocardial uptake and expression in piglets . To further increase the efficacy of this approach, Bridges et al. developed and refined a CBP platform with selective retrograde coronary sinus infusion and recirculation of the vector through the heart. This technique yielded extensive transgene expression in a significant percentage of cardiac myocytes^, .

Notwithstanding the very high efficiency of the advanced aortic cross clamping techniques in large animals, their highly invasive nature and the associated potential morbidity limits a possible translation into clinical studies to patients undergoing cardiac surgery for other reasons.

Intravenous infusion

Intravenous injection is the simplest and less invasive method among current available methods of cardiac gene delivery. In rodents, injection into the tail vein results in successful cardiac gene expression. However, as of today, application of this method is feasible only in small animals, mostly due to limited cardiac specificity. While dilution by the systemic blood circulation compromises the vector concentration in the cardiac circulation, uptake by other organs such as liver, lung, and spleen before the vectors reach the heart is another issue. Bypassing the liver and the spleen by injecting the vectors into the pulmonary artery did not improve the efficacy. Development of highly cardiotropic vectors without any harmful effects on non-targeted organs, together with methods to augment cardiac uptake, such as ultrasound targeted microbubble destruction^, may expand the application of intravenous injections to larger animals and eventually to human.

Direct intramyocardial injection

Intramyocardial injection is one of the most widely used gene transfer methods, ranging from small animal studies to clinical trials focusing on cardiac angiogenesis. The vectors are injected either epicardially (Figure 5a) or endocardially (Figure 5b) into the target area with a small gauge needle. The primary advantage of this method is that vector delivery bypasses the endothelial barrier. This results in a high local concentration at the injection site. In addition, by avoiding exposure to the blood, deactivation of the vectors by circulating DNases or neutralizing antibodies can be prevented. Furthermore, there is minimal exposure of the vector to off target organs, although local administration cannot completely avoid some systemic vector distribution^, .

Direct myocardial injection and pericardial injection a) Percutaneous myocardial injection: The vector is injected with an injection catheter via an endocardial approach b) Surgical myocardial injection: The vector is injected via an epicardial approach c) Percutaneous pericardial injection: The vector is injected via a substernal approach.

The simplest approach, however invasive, is the injection during the thoracotomy. Surgical delivery offers direct visual confirmation, which allows precise control of the injection site, including an infarct border.

Another method of intramyocardial injection is based on a composite catheter system (TransAccess) . This delivery technique utilizes an intravascular ultrasound device for catheter guidance to the injection site before the vector is injected transvenously with an extendable needle. However, coronary sinus accessibility limits the cardiac regions impacted by this approach.

The endocardial approach requires a catheter with a retractable injection needle, and imaging guidance modality for determining the injection site. This includes electrical mapping systems , fluoroscopy , echocardiography , and magnetic resonance imaging . Currently, the NOGA electromechanical mapping system is the most commonly used guiding system that is also used in clinical angiogenesis trials.

Intramyocardial injection is attractive for local gene delivery. However, the application of this method for heart failure, where cardiomyocytes are globally impaired might be limited by a circumscribed target area and inhomogeneous expression profiles^, .

Pericardial injection

Intrapericardial delivery is performed surgically in rodents , whereas for larger animals a percutaneous approach is available as well (Figure 5c). The percutaneous pericardial puncture has been proven to be feasible and safe, when guided by imaging techniques like fluoroscopy and intravascular ultrasound. The percutaneous access to the pericardial space can be achieved minimally invasive via a substernal/xhiphoidal puncture.

The pericardial space faces most of the cardiac wall except the septum. In heart failure, where widespread cardiac gene transfer with little systemic distribution is desired, this large-scale interface combined with the concept of a closed compartment can be a major advantage. These features potentially enable prolonged vector persistence, a slow release over time, a high vector concentration and minimal leakage to non-target organs. However, tightly joined pericardial cells restrict transfection to superficial myocardial layers^-. This limitation can be partly overcome by the co-administration of various pharmacological agents. Proteolytic enzymes and polyethyleneimine have been shown to increase the penetration depth of the vectors and to allow progressive release, often at the cost of cardiac toxicity^, . Although vectors are injected into a closed space, some studies reported extra-cardiac gene expression, probably due to the rapid turnover of the pericardium fluid through the lymphatic absorption.

Other delivery methods

Most cardiac gene transfer studies focus on the left ventricle. In contrast, a recently developed surgical gene “painting” method achieved transmural gene transfer in both atria without affecting ventricular cardiomyocytes . Although potentially interesting for targeting common atrial diseases like atrial fibrillation, this approach might be difficult to translate into a less invasive delivery method.

TARGETS

The last twenty years witnessed significant evolution in our understanding of the pathophysiology of heart failure in its molecular and cellular dimensions which broadened the scope of interventions available for gene therapy. We will discuss in this part some of the most important systems targeted to restore the function of failing cardiomyocytes. For targets to be validated, it is important that they rescue function in animal models when heart failure has been already established, that the rescue is not associated with arrhythmogenesis and that a gene-dose effect is established, ie with increasing expression of the gene of interest there is a concomitant improvement in function. Excitation-contraction coupling is dysregulated at multiple levels in the development of heart failure. For this reason, the various channels, transporters, and critical proteins have been targeted pharmacologically and by genetic editing to restore contractile function. In Figure 5, the various targets in excitation-contraction coupling are presented.

1. Targeting the β-adrenergic system

The β-adrenergic signaling is adversely affected by multiple changes which lead to β-ARs downregulation and de-sensitization. Up-regulation of the critical protein GRK2 seems to precipitate the abnormalities in β-AR signaling abnormalities. Several gene-based experiments tested the hypothesis that genetic manipulation of the myocardial β-AR system can enhance cardiac function.

a. Overexpression of β-AR

Overexpression of β-AR was initially tested as a simple way to overcome β-AR downregulation. Transgenic mice overexpressing the human β1-ARs suffered from severe cardiomyopathy . In contrast, mice with cardiac overexpression of β2-AR demonstrated increased basal myocardial adenylyl cyclase activity with increased left ventricular function . Both direct and intracoronary myocardial delivery of Adenovirus containing the human β2-AR transgene has resulted in enhanced cardiac performance in rodents and mammalian models ^,.

b. Inhibition of G protein-coupled receptor kinases (GRKs)

The interaction between activated β-ARs and G proteins is regulated by kinases that modulate the receptor activity by phosphorylation of its carboxyl terminus. Agonist-dependent desensitization is mediated by a family of GRKs which phosphorylate the agonist-occupied receptors resulting in functional uncoupling. GRK2 binds to the Gbg subunit of activated G proteins phosphorylating β-ARs which then attach to an inhibitory protein β–arrestin. GRK2 is the most expressed GRK in the heart. It has been implicated in the pathogenesis of dysfunctional cardiac β-AR signaling accounting for a deleterious activity in the failing heart . Studies in mice in which HF was induced by a myocardial infarction, showed that selective GRK2 ablation 10 days postinfarction resulted in increased survival, halted ventricular remodeling and enhanced cardiac contractile performance . A peptide termed βARKct capable of inhibiting GRK2 mediated β-AR desensitization has been evaluated in vivo in animals. Using intracoronary adenovirus-mediated βARKct transgene delivery to rabbits 3 weeks after induced myocardial infarction demonstrated a marked reversal of ventricular dysfunction . More recent studies have focused on overexpressing βARKct in large animal models .

c. Activation of cardiac AC expression

Although detrimental outcomes were demonstrated with multiple elements of the β-adrenergic system used to improve the expression of cAMP, activation of AC type VI (AC VI) seems to have a unique favorable profile. Overexpression of AC VI in transgenic mice resulted in improved cardiac function in response to adrenergic stimulation along with increased cAMP production in isolated cardiac myocytes. Importantly, AC VI had a neutral effect on basal heart function and was not associated with any structural heart abnormalities . In a pacing model of HF in pigs, intracoronary delivery of adenovirus encoding AC VI resulted in improved LV function and remodeling, associated with increased cAMP generating capacity . The favorable effects of AC VI in preclinical studies are encouraging and this approach is currently under investigation for initiation of clinical trials in patients with HF .

2. Targeting Ca²⁺ cycling proteins

HF is characterized by multiple defects in Ca²-handling proteins involved in excitation-contraction coupling (Figure 6). Reversal of those defects by gene therapy techniques has shown very promising results. We will review the main aspects of those novel therapies in this section.

Excitation-Contraction Coupling in cardiac myocytes provides multiple targets for gene therapy.

a. Overexpression of SERCA2a

More than twenty years ago, Gwathmey et al first reported that calcium cycling is abnormal in human heart failure and was found to be partially due to decreased SERCA2a activity regardless of the etiology of the heart failure ^-,. Improvement in cardiac contractility after gene transfer of SERCA2a has been demonstrated in a large number of experimental models of heart failure ^,. More importantly, long-term overexpression of SERCA2a by intracoronary delivery of AAV carrying SERCA2a has been associated with preserved systolic function and improved ventricular remodeling in a swine volume-overload model of HF . Beyond their effects on enhancing contractility, SERCA2a gene transfer has been shown to restore the energetics state of the heart ^, both in terms of energy supply and utilization, decrease ventricular arrhythmias ^, , and enhance coronary flow through activation of eNOS in endothelial cells .

b. PLN inhibition

Another approach to improve Ca²- handling involves inhibition of PLN. Decreasing PLN in human cardiac myocytes showed an improvement in contraction and relaxation velocities similar to the benefit seen with gene transfer of SERCA2a . Silencing of PLN expression in a sheep HF model resulted in improved SERCA activity along with improved systolic and diastolic LV function . In addition to the above conventional gene therapy strategies, RNAi therapy was used for the first time in a model of cardiac disease, specifically in rats with HF, in an attempt to suppress phospholamban expression. A AAV9-RNAi vector generated stable cardiac production of a regulatory RNA sequence, which in turn suppressed phospholamban expression. SERCA2a protein was subsequently increased accompanied by restoration of systolic and diastolic cardiac function .

c. Active I-1 and Inhibition of PP1

HF is associated with elevated PP1 activity in humans resulting in dephosphorylation of PLN. Overexpression of PP1 or ablation of I-1 in murine hearts has been associated with decreased β-AR-mediated contractile responses, depressed cardiac function and premature death consistent with HF ^-. Expression of a constitutively active I-1 in transgenic mice led to PP1 inhibition with increased phosphorylation of PLN and improved cardiac contractility. A recent study on transgenic mice expressing active I-1 confirmed the relationship between phosphorylation of PLN and SERCA2a activity. I-1 expression ameliorated ischemia/reperfusion-induced injury by reducing the infarct size and improving contractile recovery in addition to decreasing biomarkers of apoptosis and ER stress response ^-.

d. S100A1

S100 is part of a family of Ca²-modulated proteins implicated in intracellular regulatory activities. S100A1 is the most abundant S100 protein isoform in the heart. It promotes cardiac contractile and relaxation function through enhancing the activity of both RYRs and SERCA2a . In a rat model of HF, AAV6-mediated long-term expression of S100A1 resulted in a sustained in vivo reversal of LV dysfunction and remodeling ^, . More recently AAV9 gene transfer of S100A1 in a pre-clinical model of ischemic cardiomyopathy induced dramatic improvements in contractile function reinforcing the rationale that a clinical trial of S100A1 gene therapy for human heart failure should be forthcoming.

e. SUMO1

Recently Kho et al reported that the levels and activity of SERCA2a in cardiomyocytes are modulated in parallel with the levels of a cytoplasmic protein, small ubiquitin-like modifier type 1 (SUMO1) . SUMOs are a family of peptides that alter the function of other proteins in cells through a post-translational modification described as sumoylation. Sumoylation is involved in the modulation of various intracellular processes.

Kho et al. found that sumoylation enhanced the stability of SERCA2a in the cell as well as increase its activity. SUMO1 levels were reduced in murine and pig models of heart failure and in failing human ventricles. Increasing SUMO1 levels by AAV9 gene transfer led to a restoration of SERCA2a levels, improved hemodynamic performance, and reduced mortality among the animals with heart failure.

3. Homing of stem cells

The SDF1/CXCR4 complex has emerged as a therapeutic target in ischemic heart failure due to the ability of the SDF-1-CXCR4 system to promote the homing of stem cells to infracted myocardium. A clinical trial is underway to investigate the therapeutic benefit of SDF-1 overexpression in ischemic cardiomyopathy. In parallel, existing literature highlights the direct effects of CXCR4 on the myocardium and the cardiac myocyte. SDF-1 was shown to decrease myocardial contractility ex vivo and on cardiac myocytes. One recent report has shown increased ischemia-reperfusion injury in rat hearts overexpressing CXCR4, while another report investigated the modulation of beta-adrenergic receptor signaling by SDF-1 and CXCR4, raising interrogations over the potential complex interaction between these chemokines and the cardiovascular system. Pim-1 kinase has also been shown to enhance survival, proliferation, trafficking, lineage commitment, and functional engraftment of cardiac progenitor cells ^, . Pim-1 is unique as it mediates not only proliferation, but also lineage commitment, and functional engraftment in hearts ^, .

4.Targeting cell death

Apoptosis is a process of programmed cell death that is involved in normal organ development. In models of acute and subacute ischemia/reperfusion, overexpression of the anti-apoptotic protein Bcl-2, Akt or PI3 kinase reduces the rate of cardiomyocyte apoptosis and improved heart function . In ischemia/reperfusion injury where apoptosis plays an important part of myocardial damage gene therapy with pro-survival factors appears to be amenable to intervention, it is less clear if other forms of cardiac injury such as hypertrophy and HF can benefit from anti-apoptotic strategies. For these situations, sophisticated promoters with oxygen sensing and modified HIF1α promoters have been designed to induce survival factors in the setting of ischemia .

Clinical trials

Despite early failures, gene therapy trials for various diseases, most notably inherited blindness (whereby gene transfer by AAV vectors partially restored vision in a pediatric patient with Leber’s Congenital Amaurocis, a major cause of congenital blindness ), cancer, infectious diseases, monogenic diseases, and cardiovascular diseases are underway.

In heart failure, there are currently a number of trials ongoing or in the planning stages targeting various pathways for rescuing the failing myocardium. The targets that have been taken forward towards clinical trials include SERCA2a, adenylyl cyclase type 6, and SDF-1.

The first clinical trial of gene therapy in patients with HF was launched in the United States in 2007 ^, . CUPID was a multicenter trial designed to evaluate the safety profile and the biological effects of gene transfer of the SERCA2a cDNA by delivering a recombinant AAV1 (AAV1.SERCA2a) in patients with advanced HF. Participants in this trial were administered a single intracoronary infusion of AAV1.SERCA2a in an open-label approach ^, . Twelve month follow-up of these patients showed an acceptable safety profile ^, . Improvement was detected in several patients, reflected by symptomatic (5 patients), functional (4 patients), biomarker (2 patients) and LV function/remodeling (6 patients) parameters. In the phase 2 trial, 39 patients with advanced HF were randomized to receive intracoronary adeno-associated virus 1 (AAV1) mediated SERCA2a gene delivery (in one of 3 doses (low dose - 6 × 10¹¹ DRP, middle dose - 3 × 10¹² DRP and high dose - 1 × 10¹³ DRP) versus placebo. Patient’s symptoms (NYHA class, Minnesota Living With Heart Failure Questionnaire [MLWHFQ]), functional status (6 minute walk test [6MWT] and VO2 max), NT-proBNP levels and echocardiographic measures were evaluated over 6 months. Treatment success was determined by examining concordant trends in the above endpoints for group- and patient-based comparisons, as well as clinical outcomes. The AAV1.SERCA2a high-dose group met the pre-specified criteria for success at the group and individual patient levels. AAV1.SERCA2a treated patients, versus placebo, demonstrated improvement or stabilization in NYHA class, MLWHFQ, 6MWT, VO2 max, NT-proBNP levels, and LV end-systolic volumes. Significant increases in time to adjudicated CV events, and a decreased frequency of CV events per patient were observed in all patients receiving AAV1.SERCA2a. No increases in adverse events, disease-related events, or laboratory abnormalities were observed in AAV1.SERCA2a treated patients compared to placebo. Arrhythmias have been a concern with the overexpression of SERCA2a however in the CUPID trials there were no increases in arrhythmias as measured by ICD. Two other clinical trials targeting SERCA2a are currently enrolling patients. The first trial is in patients with advanced heart failure having received left ventricular assist devices at least one month prior to treatment and who will receive either AAV1.SERCA2a or saline. This trial is being conducted in the United Kingdom. A second Phase 2 monocenter double blind randomized placebo-controled, parallel study will be held in the Institut of Cardiology Pitié-Salpêtrière, Paris, France with the primary objective to investigate the impact of AAV1.SERCA2a on cardiac remodeling parameters in patients with severe heart failure.

In a separate clinical study adenovirus-5 encoding human adenylyl cyclase type 6 (is being delivered through intracoronary injection to patients with congestive heart failure. Intracoronary delivery of Ad5.hAC6 or PBS in 3:1 randomization with dose escalation, The patients will be randomized in a dose dependent fashion starting at 3.2 × 10⁹ viral particles to 3.2 × 10^12 viral particles in 6 dose groups using a 3:1 randomization fashion with PBS (buffered saline being used for control). The trial is currently enrolling patients.

An additional trial is examining the effects of injecting SDF-1 directly into the myocardium of patients with ischemic heart disease. An open label dose escalation study to evaluate the safety of a single escalating dose of SDF-1 administered by endomyocardial injection to cohorts of adults with ischemic heart failure is currently enrolling patients. SDF-1 naked DNA will be injected directly into the myocardium at multiple sites through a percutaneous, left ventricular approach.

Conclusion

With a better understanding of the molecular mechanisms associated with heart failure and improved vectors with cardiotropic properties, gene therapy can now be considered as a viable adjuntcive treatment to mechanical and pharmacological therapies for heart failure. In the coming years, more targets will emerge that are amenable to genetic manipulations along with more advanced vector systems which will undoubtedly lead to safer and more effective clinical trials in gene therapy for heart failure.

Acknowledgments

Sources of Funding

This work is supported by NIH R01 HL093183, HL088434, and P20HL100396.

Non Standard Abreviations

Footnotes

Disclosures

Roger Hajjar is a scientific founder of Celladon Co. which plans to commercilaize AAV1.SERCA2a for the treatment of heart failure.

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References

Pathophysiology Of Heart Disease Pdf