Cardioprotection

Chapter 28

Cardioprotection David J. Lefer1 and Roberto Bolli2 1

Department of Surgery, Division of Cardiothoracic Surgery, Emory University School of Medicine, and the Carlyle Fraser Heart Center, Atlanta,

GA; 2Department of Medicine, Division of Cardiovascular Medicine, University of Louisville, Louisville, KY

MECHANISMS OF MUSCLE INJURY AND CELL DEATH Forms of Cell Death There are three proposed types of cell death thought to participate in myocardial ischemia/reperfusion (I/R) injury that include: apoptosis, necrosis, and autophagy. Apoptosis is programmed cell death, a process in which an intricate series of events ultimately results in the death of the cell and degradation of its intracellular components without stimulating an inflammatory response. Autophagy also results in a programmed series of events; however, it is generally thought to result in the destruction of individual cell components instead of the entire cell itself. Necrosis is cell death resulting in uncontrolled lysis of the cardiac myocyte cell membrane and release of inflammatory molecules. The following sections will focus primarily on the pathogenesis of myocardial cell injury resulting in necrosis during myocardial I/R as apoptosis and autophagy are covered extensively in other chapters.

Myocyte Injury during Ischemia The major biological changes occurring during myocardial ischemia that are produced by sudden occlusion of a major coronary artery resulting in both reversible and irreversible myocardial cell injury have been extensively investigated by Jennings and Reimer and very elegantly summarized in a series of papers published during the late 1970s and 1980s (1
6). The vast majority of studies that investigated myocyte injury during ischemia focused on canine models of coronary occlusion. In the canine heart there is a clear transmural gradient of arterial collateral blood flow, with the most significant reductions of myocardial blood flow observed in the subendocardium (96
98% reduction vs. control) and the greatest blood flow in the subepicardial regions (30
60% reduction vs. control). Far less is known regarding myocardial cell injury in the zones of high flow ischemia or in the zones of moderate ischemia

Muscle. DOI: http://dx.doi.org/10.1016/B978-0-12-381510-1.00028-4 © 2012 Elsevier Inc. All rights reserved.

due to a number of technical difficulties. Reimer and colleagues (3) first described the “wavefront” of irreversible myocardial cell injury that begins in the subendocardium and progresses toward the subepicardium. The “wavefront” phenomenon occurs between 40 minutes and 6 hours of ischemia. The vast majority of classical studies of myocardial cell injury and death have focused on the subendocardium. As early as 15
30 seconds following the induction of severe ischemia aerobic metabolism ceases and anaerobic glycolysis becomes the principal source of new highenergy phosphate (6). During this transition lactate levels increase by four-fold and the reserve supply of creatine phosphate is essentially depleted (Figure 28.1). The lactate that is produced accumulates since it can longer be metabolized due to lack of oxygen. Anaerobic glycolysis proceeds very rapidly for the first minute and then continues at a much slower rate. At 15 minutes following ischemia when myocardial injury is reversible glycolysis is manifest by reduced glycogen levels and increased lactate in the myocardial tissue (3,5
7). Anaerobic glycolysis does not produce adequate quantities of high-energy phosphates to prevent the progressive depletion of ATP and by 15 minutes of ischemia ATP is reduced to 35% of control levels. Adenosine monophosphate (AMP) accumulates and is subsequently dephosphorylated to adenosine. By 40
60 minutes of ischemia anaerobic glycolysis has slowed further or stopped and 94% of normal ATP is depleted (5,6). At this time the myocardial cell demonstrated clear structural signs of irreversible injury that include: (i) the presence of amorphous matrix densities in the mitochondria; (ii) swollen mitochondria; and (iii) defects in the myocardial cell membranes. Reperfusion of the ischemic region at this time is followed by massive swelling, the development of contraction bands, calcium accumulation in the myocardium, and further cell membrane disruption (3,6). At 60 minutes of severe ischemia approximately 95% of the myocytes in the ischemic zone are irreversibly injured. If the ischemia persists for a period of up to 24

369

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(b) 100

te

cta

La

4 3 2

ATP

1

Ischemic myocardium potentially salvageable by timely intervention

80 60 40

Irreversible phase

5

Reversible phase

Onset of irreversible injury

6

Fraction of at-risk myocardium

ATP and lactate (arbitrary units)

(a)

Cardiac Muscle

20

Cumulative dead myocardium

0 0

30 5 10 15 20 Minutes

40

50

0 Time

1

2

3

4

5 Hours

6

12

18

FIGURE 28.1 Time course of myocardial cell injury. (a) Adenosine triphosphate (ATP) levels significantly decrease in minutes following the onset of coronary ischemia and this reduction in ATP significantly contributes to irreversible myocardial cell injury. Concomitant with the decrease in ATP is an increase in myocardial lactate levels that occurs at 5
40 minutes following myocardial ischemia which also contributes to myocardial cell death. (b) Relationship between the duration of myocardial ischemia and the extent of myocardial necrosis. As ischemia persists beyond 30
40 minutes myocardial cell death is initiated. At 6 hours following the onset of myocardial ischemia nearly all of the myocardium at risk undergoes cell death. Interventions including revascularization procedures must be instituted within 4
6 hours to result in any significant myocardial salvage. (From Kumar V, Abbas AK, Fausto N, Aster J, editors. Robbins & Cotran Pathologic Basis of Disease, 8th edition. Philadelphia, PA: Saunders, 2009, with permission of Elsevier.)

Permanent occlusion

(a) 100%

(b) Viability

Temporary occlusion with reperfusion

100%

Restoration of flow

0 % of original

Function Reperfusion injury Salvage Post-ischemic ventricular dysfunction

0%

0% 0 2 min 20 min

120 min Time

6 hr

0 2 min 20 min

120 min Time

6 hr

Days

FIGURE 28.2 Permanent occlusion vs. reperfusion. (a) Permanent coronary artery occlusion results in significant reductions in left ventricular function (green line) within 2 minutes of the cessation of blood flow. The transition from reversible injury to irreversible cell death occurs following 20
30 minutes of myocardial ischemia and myocardial viability decreases (red line). (b) Transient coronary artery occlusion and reperfusion results in myocardial salvage if blood flow is restored in a timely manner. Post-ischemic left ventricular dysfunction (green line) occurs within 2 minutes and is maintained until coronary blood flow is restored. If coronary reperfusion occurs within 120 minutes ventricular dysfunction can be alleviated. Myocardial viability (red line) is directly dependent on coronary blood flow and restoration of blood flow within 2 hours following ischemia results in additional reperfusion injury, but ultimately results in myocardial salvage when compared to permanent occlusion. (From Kumar V, Abbas AK, Fausto N, Aster J, editors. Robbins & Cotran Pathologic Basis of Disease, 8th edition. Philadelphia, PA: Saunders, 2009, with permission of Elsevier.)

hours the characteristic mitochondrial and sarcolemmal changes progress very little. Thus, the hallmark ultrastructural features indicative of the transition from reversible to irreversible injury during severe myocardial ischemia are: (i) cell membrane defects; (ii) mitochondrial swelling and calcium accumulation; and (iii) nuclear chromatin clumping (3,5,6). While permanent occlusion ultimately results in a significant degree of irreversible injury and cell death with severe impairments in left ventricular

function and heart failure; restoration of blood flow to the ischemic myocardium induces additional unwanted myocardial injury (Figure 28.2) that has been termed “reperfusion injury” (8).

Myocardial Reperfusion Injury The development of novel therapeutic strategies such as thrombolytic agents and primary percutaneous transluminal

Chapter | 28

Cardioprotection

coronary angioplasty (PTCA) in the 1980s ushered in the “era of reperfusion” for the treatment of acute myocardial infarction (9
13). While reperfusion relieves ischemia it also results in a complex series of pathological phenomena that significantly amplify reversible and irreversible myocardial injury. Reperfusion of the ischemic myocardium induces a number of important changes in cardiac electrophysiology and contractility that result in significant impairment in left ventricular function (i.e. “myocardial stunning” and permanent impairments) (8). Myocardial reperfusion injury is highly complex and at present is known to involve pathophysiological effects of: (i) reactive oxygen species (ROS); (ii) inappropriate immune responses and inflammation; (iii) mitochondrial injury; and (iv) myocardial “no-reflow”. These highly important components of reperfusion injury will be briefly discussed. While the existence of reperfusion injury in man remains somewhat controversial it is clear that timely reperfusion is the most effective treatment for myocardial ischemia and that “time is muscle” with longer periods of ischemia resulting in greater myocardial injury. Reactive Oxygen Species Reactive oxygen species (ROS) are molecules with unpaired electrons in their outer orbit that are highly reactive, and initiate chain reactions that result in irreversible chemical changes in lipids or proteins. These deleterious reactions result in profound cellular dysfunction and cytotoxicity. It is estimated that approximately 5% of the oxygen consumed by normal tissues are transformed into ROS. These basally generated ROS are efficiently detoxified by endogenous enzymatic free radical scavengers, such as superoxide dismutase, glutathione peroxidase, and catalase (14,15). However, during myocardial reperfusion following ischemia, the flux of ROS generated by the heart and coronary circulation exceeds the capacity of endogenous oxidant defense mechanisms to detoxify ROS and prevent deleterious radical-mediated reactions. There are three major lines of evidence that implicate ROS in the pathogenesis of myocardial reperfusion injury: (i) ROS are detected in post-ischemic myocardium; (ii) exposure of myocardium to exogenous ROS results in myocyte and myocardial tissue dysfunction that is comparable to that elicited by I/R injury; and (iii) pretreatment of animals with antioxidant enzymes (e.g. superoxide dismutase) or genetic overexpression of these enzymes in experimental animals affords protection against reperfusion injury. A number of different experimental approaches have been used to detect ROS production in the post-ischemic myocardium. Electron paramagnetic resonance (EPR) spectroscopy is one of the most widely used methods for monitoring ROS generation in the heart. Studies based on this technique have

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clearly demonstrated a rapid and profound increase in ROS production after reperfusion of ischemic myocardium (16
19). Zweier and colleagues (18) were among the first to measure oxygen free radical formation directly in isolated, buffer-perfused rabbit hearts under baseline conditions, during ischemia, and after reperfusion. These early studies suggested that superoxide anion is the predominant ROS produced after reperfusion and that endothelial cells represent an important source of the ROS. EPR spectroscopy has also been used to demonstrate enhanced ROS production in experimental models of myocardial stunning (20
22). The EPR signals detected in venous blood draining the ischemic
reperfused heart suggest that secondary lipid radicals, such as alkyl and alkoxy radicals, are produced for as long as 3 hours after reflow, with maximal levels detected at 2
4 minutes after reperfusion. Studies performed by the Bolli group in 1990 (20
22) clearly demonstrated a role for ROS in the pathogenesis of myocardial stunning. Enhanced production of ROS after myocardial I/R in humans is also supported by different indirect measures of oxidant stress. For example, it has been shown that serum levels of vitamin E are depleted, whereas serum conjugated dienes and thiobarbituric acid reactive substances (TBARS) are elevated in patients undergoing coronary artery bypass graft (CABG) surgery (23). Inflammatory Response The association of a profound inflammatory response during the pathogenesis of myocardial infarction was first described in 1939 by Mallory and colleagues in seminal studies in the human heart (24). Pathologists have observed significant inflammatory lesions with robust neutrophil and leukocytic infiltration in autopsy specimens obtained at various times (i.e. hours to days) following the onset of myocardial infarction. These findings suggest that myocardial inflammation may contribute to the pathogenesis of acute myocardial infarction and/or is involved in myocardial healing. Clearly, this inflammatory response is required to promote healing of the myocardium following infarction as early attempts to completely abolish this immune response with high-dose corticosteroids (25
27) in patients produced disastrous results in the 1976 despite promising preclinical data (28). The controversy surrounding the precise role of the neutrophil and inflammatory response in the development of myocardial injury in the setting of I/R continued as scientists and clinicians investigated novel, highly targeted anti-neutrophil and anti-inflammatory agents for the treatment of myocardial reperfusion injury for over a decade in the 1980s and 1990s. We will provide a brief review of the preclinical and clinical studies that shape our current understanding of the role of neutrophils and

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inflammation in the sequelae of myocardial reperfusion injury. Following the development of thrombolytic therapy and PTCA to initiate reperfusion of the ischemic myocardium numerous investigations were focused on the physical removal or pharmacological inhibition of infiltrating leukocytes and neutrophils (29). Experiments were performed with leukocyte-specific filters (30
33), monoclonal antibodies directed against specific endothelial and leukocyte adhesion glycoproteins (28,34
43), and numerous anti-leukocyte and anti-inflammatory agents (26,29) in a number of in vivo and in vitro models and animal species. Extensive studies of novel agents targeting various components of the complement cascade as a means to attenuate myocardial inflammation and reperfusion injury (44). Overall, the results of these studies revealed mixed results with some studies demonstrating reductions in myocardial infarct size and improved left ventricular function while others failed to demonstrate significant cardioprotective actions. Despite variable experimental results and continued controversy regarding the role of inflammation and neutrophils in myocardial reperfusion injury several clinical trials were performed testing the potential efficacy of anti-leukocyte monoclonal antibodies in patients undergoing either thrombolysis or PTCA for acute myocardial infarction (MI) (45
47) between 2001 and 2002. These trials included: FESTIVAL Study, HALT-MI Study, and the LIMIT AMI study (Table 28.1). The FESTIVAL study (45) was a small study of only 60 patients that was primarily designed to study safety of the CD-18 monoclonal antibody, Hu23FG (LeukArrest), in patients treated with PTCA. SPECT imaging failed to demonstrate any significant difference in infarct size in the FESTIVAL study. The HALT-MI study (46) also investigated

Cardiac Muscle

Hu23F2G versus placebo, but in 420 patients with STsegment elevation just prior to PTCA. This multi-center, double-blinded, placebo-controlled clinical trial clearly demonstrated that Hu23F2G did not ameliorate infarct size in patients who underwent primary PTCA. Finally, a recombinant human CD18 monoclonal antibody (rhuMAb CD18) was tested in patients treated with a thrombolytic agent for acute MI. Despite a good safety profile, RhuMAb CD18 failed to modify coronary blood flow, infarct size, or the rate of ECG increase in this study. While these clinical studies have only tested a single antineutrophil agent (i.e. anti-CD18 antibody) the negative results from these studies significantly reduced the enthusiasm for further clinical investigation of highly specific anti-neutrophil agents in patients with acute MI. No-Reflow Phenomenon The condition whereby tissue perfusion does not fully occur or progressively declines in the presence of a patent epicardial coronary artery is referred to as the “no-reflow” phenomenon. Kloner and colleagues were the first to describe this important phenomenon in seminal experiments performed in 1974 in ischemic canine hearts (48,49). Dogs were subjected to various durations of coronary ischemia (either 40 or 90 minutes) and blood flow restoration to the ischemic region was observed following 40 minutes of ischemia but not with 90 minutes. Following 90 minutes of ischemia there was only partial blood flow restoration despite complete elimination of the coronary occlusion. Myocardial perfusion defects as measured by thioflavin S and carbon black were most prominent in the sub-endocardium. Electron microscopy studies revealed significant capillary damage in the form of swollen endothelial cells, intraluminal endothelial protrusions, and platelet plugging and fibrin thrombi. Furthermore,

TABLE 28.1 Potential Cardioprotective Agents That Have Been Tested in Clinical Trials to Reduce Myocardial Infarct Size or Improve LV Function and Have Failed Type of Agent

Study

Outcome

WBC inhibitors (anti-CD18 mAbs) Calcium channel blockers (nifedipine) h-SOD (free radical scavenger) Rheoth RX (Poloxamer 188) Trimetazidine (anti-oxidant) Fluosol Hyaluronidase Complement inhibitors

FESTIVAL Rusnak et al. (2001) SPRINT II Goldbourt et al. (1993) Flaherty et al. (1994). EMIP-FR The EMIP-FR Group (2000) ESPRIM The ESPRIM Group (1994) TAMI-9 Wall et al. (1994) Pre-thrombolytic era COMPLY Trial Mahaffey et al. (2003) APEX Trial Armstrong et al. (2007) ESCAMI Zeymer et al. (2001) CASTEMI Bar et al. (2006) MAGIC MAGIC Trial investigators (2002) Kitakaze et al. (2007)

No k infarct size Increased mortality No m in LV function No effect on death, shock, or reinfarction No effect on mortality or clinical outcomes No k infarct size or m LV function No effect on infarct size No k infarct size or k in mortality (large APEX)

Na1/H1 exchange inhibitor Magnesium Nicorandil

No effect on infarct size, clinical outcomes, LVEF No effect on mortality, CHF, or VT No effect on mortality, infarct size, LVEF

Table compiled by Robert Kloner. MD, NIH Workshop on New Horizons in Cardioprotection, 2011.

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interstitial and myocardial cellular edema was observed in these and other experiments and was thought to contribute to no-reflow via significant compression of the capillary network to further exacerbate this condition. It has become very clear that the longer ischemia persists, the more likely the no-reflow phenomenon is to occur. The development of PTCA for the treatment of acute myocardial infarction brought the no-reflow phenomenon to light since this technique resulted in complete coronary artery patency yet a myocardial perfusion defect persisted. The no-reflow phenomenon is highly variable in patients that undergo PTCA, but can be very sudden and dramatic. Contrast dye will stagnate in the coronary arteries, the patient will suffer from chest pain, and hemodynamic instability will occur. Similar observations have also been made following thrombolytic therapy in patients evidenced by reperfusion chest pain, ST-segment elevation, and hemodynamic deterioration. The no-reflow phenomenon can also evolve as a gradual decrease in myocardial perfusion that persists. A clinical study (50) of microvascular obstruction following treatment with thrombolytics or PTCA for acute myocardial infarction determined that microvascular obstruction predicts more frequent cardiovascular complications (Figure 28.3). Furthermore this study also provides very strong evidence that microvascular status is a very strong prognostic marker for event-free survival even after control for myocardial infarct size (50). Myocardial no-reflow remains a

100

% event-free survival

No microvascular obstruction 80 60 Microvascular obstruction 40 20

p < 0.01 between groups

0 0

5

10

15

20

25

Months FIGURE 28.3 Myocardial “no-reflow” and event-free survival in patients following acute myocardial infarction. Microvascular obstruction was assessed using magnetic resonance imaging at 10 days following myocardial infarction. Patients with demonstrated microvascular obstruction exhibit 50% event-free survival as compared to patients with no demonstrated microvascular obstruction that experience 90% eventfree survival (p , 0.01 between groups) (From Wu K, et al. Prognostic significance of microvascular obstruction by magnetic resonance imaging in patients with acute myocardial infarction. Circulation 1998;97:765– 772, with permission from the American Heart Association.)

significant clinical problem and provides very strong evidence for the existence of myocardial reperfusion injury in man.

CARDIOPROTECTION INDUCED BY PRECONDITIONING AND POSTCONDITIONING Preconditioning Preconditioning refers to myocardial protection against I/R injury by physiological or pharmacological intervention prior to a prolonged ischemic event. Ischemic preconditioning (IPC) was discovered over 20 years ago by Murry et al. (51). In this pivotal study, brief periods of ischemia (four cycles of 5 minutes of non-lethal coronary artery occlusion followed by 5 minutes of reperfusion) administered before a prolonged ischemic event, significantly attenuated myocardial cell death (51). Following this work, IPC has been shown to be cardioprotective in all animal species investigated, and is currently considered to be the most protective intervention against myocardial I/R injury (52,53). Some key signaling mediators of IPC are: phosphatidylinositol-3-kinase (PI3K), Akt, nitric oxide (NO), protein kinase G (PKG), mitochondrial ATP-sensitive potassium channels (KATP), adenosine, oxygen derived free radicals, and the mitochondrial permeability transition pore (MPTP) (54). Pharmacological preconditioning is similar to IPC; however, no ischemia is induced to obtain the cardioprotective benefits. The cardioprotective agents mimic the signaling induced by IPC. Adenosine, bradykinin, nitric oxide, and opioids all serve to protect the myocardium when pharmacologically administered before an ischemic event (55
57). Preconditioning has two phases of protection. An early phase, 1
3 hours following the onset of preconditioning (51), and a late phase, 18
24 hours following the onset, which may persist for up to 72 hours (58). The early phase results from a modification of proteins already present in the tissue, while the late phase results from an induction of new cytoprotective proteins (52). However, while the early phase protects against myocardial infarction, it fails to decrease the degree of myocardial contractile dysfunction or stunning (52). The late phase, however, protects against myocardial infarction and preserves left ventricular function (52). The late phase may then be more beneficial, as it confers greater cardioprotection and has a longer duration (59). While preconditioning is the most potent and reproducible cardioprotective intervention, it has not yet been fully translated to the clinic (60). One main reason for this is that the timing of myocardial ischemia must be known for preconditioning to be effectively applied.

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Postconditioning Postconditioning refers to when the myocardium is protected from ischemia
reperfusion damage by physiological or pharmacological interventions following reperfusion. In 1992, Zhao and colleagues conducted studies on postconditioning in the Vinten
Johansen laboratory using 5-minute perfusion
ischemia intervals repeated at the onset of reperfusion in anesthetized rabbits (59). However, there was a lack of efficacy of the postconditioning treatment which, at the time, paralleled the preconditioning treatment (59). In 1996 Na et al. showed that transient ischemia at the onset of arrhythmias following reperfusion reduced the incidence of ventricular fibrillation in anesthetized cats (66). In 2001, the Vinten
Johansen laboratory resumed postconditioning studies, this time using 30-second cycles (59), and in 2003 Zhao et al. reported that postconditioning significantly reduced infarct size in a dog model (67). Ischemic postconditioning, by a mechanical manipulation of blood flow, was initially performed by sequentially releasing and reapplying a ligature around the coronary artery (67,68). Recent studies have since used fluoroscopically guided angioplasty balloon catheters in a closed chest model (69). Cell culture models have also been developed to simulate the intervals of ischemia and reperfusion by using cycles of adding culture media that is either hypoxic or acidic and then changing back to normal media (70). Postconditioning reduces infarct size by decreasing cardiac myocyte apoptosis and necrosis (71,72). Postconditioning attenuates superoxide generation in the myocardial area-at-risk after 3 hours of reperfusion (70,71), induces antioxidants, and inhibits neutrophil adherence and accumulation (59). Studies have also

Control

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Post-cond

5000 4000 CK release (AU)

However, if the ischemic event is known, such as elective PTCA for ischemia, coronary bypass surgery, or heart transplantation, preconditioning could be effectively employed. Invasive measures can be used in these situations to induce IPC via balloon inflation or cross clamping. Yellon et al. were the first to effectively use IPC in the human heart during cardiac surgery (61). Remote ischemic preconditioning (RIP) is an alternative form of IPC. RIP utilizes transient ischemia of one vascular bed to precondition another vascular bed. Przyklenk et al. show that preconditioning the opposite coronary bed reduced myocardial infarct size to the same extent as preconditioning in the same coronary bed in a canine model (62). Birnbaum et al. show in a rabbit model that transient limb ischemia could be used for RIP of the heart (63), and subsequent studies in humans show that RIP, via transient limb ischemia, reduces myocardial I/R damage during coronary artery bypass surgery (64) and pediatric cardiac surgery (65).

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3000 2000 1000 0 4

8

24 48 Reperfusion time (h)

72

FIGURE 28.4 Myocardial postconditioning in patients. Effects of postconditioning on circulating levels of creatine kinase (CK) at 2
72 hours following acute myocardial infarction in control (yellow circles) and patients undergoing postconditioning. Circulating CK levels were attenuated in patients that underwent postconditioning. (From Staat P. Postconditioning the human heart. Circulation 2005;112:2143–8, with permission from the American Heart Association.)

demonstrated that hypoxic postconditioning results in less intracellular and mitochondrial Ca21 accumulation, potentially reducing the opening of the MPTP. Postconditioning has been demonstrated in several small proof-of-concept trials in the clinic (59). Seven prospective, randomized trials have used postconditioning in percutaneous coronary intervention (PCI), totaling 353 patients (73
79). There have been varying intervals of postconditioning applied, ranging from two to four cycles with each cycle consisting of 30
90 s per cycle (73
79). All studies performed thus far suggest a protective effect of postconditioning (73
79). Infarct size was attenuated (76) and ejection fraction was improved following postconditioning (76,77). Serum markers of cardiac damage, such as creatine kinase and troponin-I decreased with postconditioning (74
77). A recent study by Staat et al. (74) provides evidence that the human heart can be postconditioned. This study reveals reductions in circulating creatine kinase (CK) levels in patients treated with myocardial postconditioning (Figure 28.4). Resolution of ST-segment elevation provides further evidence for the beneficial effects of postconditioning in humans (73,78).

CARDIOPROTECTIVE AGENTS AND STRATEGIES Adenosine Adenosine is a purine nucleoside that is widely distributed in the body. Under basal conditions extracellular concentrations reside between 30 and 300 nM, but this

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375

Nitric oxide

N

O

Carbon monoxide

C

Hydrogen sulfide S

O H

H

Enzymatic production

nNOS iNOS eNOS

HO-1 HO-2 HO-3

CBS CSE (CGL) 3MST

Blood concentration

low nM

nM

pM – low nM

Half-life (in vivo)

seconds

minutes

seconds

1987

1991

1996

sGC-cGMP

sGC-cGMP

KATP Channel

S-nitrosylation

??

S-sulfhydration

Year of discovery as a physiological modulator Second messenger signal Chemical modification

can increase to as much as 10 μM during periods of hypoxia and ischemia (80). Under basal and critical situations, adenosine plays a major role in the cardiovascular system, and is thought to contribute to the reactive hyperemia observed in cardiac and skeletal muscle (81). Adenosine suppresses atrial automaticity and conduction, and has been used to treat supraventricular arrhythmias. Adenosine interacts with four receptor subtypes to exert its physiological functions: A1, A2A, A2B, and A3. At least three of the receptor subtypes are expressed in cardiomyocytes (A1, A2B, and A3), while A2A receptors are prevalent in the coronary vessels (82,83). A2A receptors are potent dilators of the coronary arteries, and A1 receptors in the atria slow rate and conduction in nodal tissue by coupling with KATP channels (84). At present the precise roles of each of the myocardial adenosine receptors is unclear; however, several adenosine receptor subtypes have been implicated in IPC and postconditioning (84). It has been proposed that adenosine released from cardiomyocytes during IPC binds to and stimulate A1 and A3 receptors, which in turn activates protein kinase C (PKC) (84). PKC can then interact with A2B early in reperfusion to increase the sensitivity of this receptor to adenosine (which is normally low compared to the other receptor subtypes). This then allows the adenosine that is present to activate protective signaling in the heart, including various protective kinases and prevention of MPTP formation (84). Use of adenosine as a cardioprotective molecule in vivo has some limitation, as it has a short half-life and hypotension results before adenosine can trigger a protected state (84). However, adenosine receptor agonists

FIGURE 28.5 Endogenously produced gases that modulate myocardial protection. Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) all produced by enzymes located in the myocardium and within the blood vessel wall resulting in nanomolar (nM) concentrations of these gases. These gases exhibit very short half-lives and signal via a number of second messenger signaling pathways. Both NO and H2S are know to chemically modify various protein residues to modulate cell signaling as well. nNOS is neuronal nitric oxide synthase; iNOS is inducible nitric oxide synthase; eNOS is endothelial nitric oxide synthase; HO-1 is heme oxygenase-1; HO-2 is heme oxygenase-2; HO-3 is heme oxygenase-3; CBS is cystathionine beta synthase; CSE(CGL) is cystathionine gamma lyase; 3MST is 3-mercaptopyruvate sulfur transferase.

have been used with success, such as one that targets the A2B receptors and does not lead to hypotension because these receptors are not expressed in most tissue (85). Use of adenosine in human clinical trials adjunct to reperfusion therapy, such as in the Amistad I and II trials, resulted in equivocal results at best (86,87). However, administration of high-dose adenosine has demonstrated improved clinical outcome in several small-scale clinical studies (88,89).

Gasotransmitters Nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) currently form the class of gas signaling molecules termed gasotransmitters that are endogenously produced by a number of enzymes (90) (Figure 28.5). These endogenous gases have recently been termed “gasotransmitters” and via complex signaling mechanisms exert a number of critical physiological actions essential for cardiovascular homeostasis. These molecules can modify proteins by reacting with amino acid residues such as the case of nitrosylation or sulfhydration reactions of NO and H2S, respectively. At physiological levels (nM
μM concentrations) these molecules are cardioprotective; however, at suprapharmacological levels they can all be deadly toxins.

Hydrogen Sulfide (H2S) H2S is primarily produced from L-cysteine by two PLP (pyridoxal-50 -phosphate)-dependent enzymes cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS) (91,92), and the combined actions of cystine amino

376

transferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3-MST) (93). All of these enzymes are present in the heart and cardiovascular system (94
96) and their dysfunction most likely contributes to the onset and progression of cardiovascular disease (97
100) underscoring the importance of H2S in cardiovascular function. H2S is a cardioprotective molecule used to both precondition the heart against I/R injury and to treat acute MI/R injury. Exogenous treatment with H2S results in significant infarct size reduction in both murine and rat models of I/R injury (94,101
105). Elrod et al. show a 72% reduction in infarct size, in vivo in mice, when a bolus of Na2S (50 μg/kg) was given at reperfusion (94). Calvert et al. show a 46% reduction in infarct size, in vivo in mice, when a bolus of Na2S (100 μg/kg) was administered 24 hours prior to the ischemic event (104). The cardioprotective effect of H2S results from stimulation of many cytoprotective signaling cascades. One of the most-reported mechanisms is activation of ATP-sensitive K1 channels (KATP). In smooth muscle cells, KATP activation causes hyperpolarization and relaxation (106). In the heart, KATP activation results in cardioprotection (103,107,108). However, sarcolemmal (107,108) and mitochondrial KATP channels (103) in the heart are only part of the mechanisms of H2S-mediated cardioprotection. H2S can activate ERK 1/2, PI3K/Akt, PKC, and Nrf-2 to promote cytoprotection (104,109). H2S also regulates leukocyte adhesion and leukocyte-mediated inflammation (110). Furthermore, H2S reduces several pro-inflammatory cytokines including: nuclear factor-κB, interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α) (111,112). Finally, H2S can readily scavenge hydrogen peroxide and increase intracellular levels of reduced glutathione (GSH) (113,114). Evidence of the cardioprotective action of H2S has been more recently tested in large animal models. Sodha et al. subjected Yorkshire pigs to 60 min of mid-left anterior descending coronary artery occlusion, followed by 2 hours of reperfusion (111). Na2S was administered 10 min before, and during reperfusion (100 μg/kg initial bolus, followed by 1 mg/kg/hr). H2S treatment significantly reduced infarct size, and improved left ventricular fractional shortening, and improved microvascular reactivity (111). Furthermore, levels of IL-6, IL-8, and TNF-α, and myeloperoxidase (MPO) activity decreased following H2S treatment (111). Osipov et al. investigated whether a bolus or infusion of H2S at reperfusion after 1 hour of left anterior descending coronary artery occlusion followed by 2 hours of reperfusion in Yorkshire pigs had an advantage over the other (115). Infusion was significantly more effective; however, both strategies augmented antiapoptotic signaling mechanisms in the myocardium (115). In

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another study, Osipov et al. investigated H2S in the setting of cardioplegia and cardiopulmonary bypass in Yorkshire pigs (116). Here again they investigated bolus and infusion strategies of H2S administration (bolus/infusion combo or infusion alone), this time during 1 hr of cardioplegia/cardiopulmonary bypass (116). Both strategies increased the pro-cell survival signaling of hemeoxygenase-1, phospho-heat shock protein 27, and phosphoERK 1/2 and decreased pro-apoptotic signaling by AIF and Bcl 2/adenovirus E1B 19 kDa-interacting protein (Bnip-3) (116).

Nitric Oxide (NO) Nitric oxide is produced by the three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS or NOS I), inducible NOS (iNOS or NOS II), and endothelial NOS (eNOS or NOS III). Both eNOS and nNOS are constitutively expressed, while iNOS is inducible. The constitutive NOSs can be activated or inhibited by phosphorylation at different Ser or Thr residues and/or by changes in Ca21– calmodulin binding. While there has been some controversy over the years as to the cardioprotective nature of NO, the evidence overwhelmingly supports NO as a highly cardioprotective molecule when administered at physiological levels in vivo in the setting of myocardial I/R injury (117). The cardioprotective actions of NO include antagonism of β-adrenergic stimulation, inhibition of influx of calcium through L-type calcium channels, activation of sarcolemmal and mitochondrial KATP channels, antioxidant effects, anti-inflammatory actions, activation of cyclooxygenase-2 and production of cytoprotective prostanoids, and inhibition of proapoptotic proteins and MPTP opening (117). NO can also bind the oxygenbinding center of cytochrome c oxidase with high affinity and compete with oxygen binding at this site (118,119). NO can also inhibit complex I and Fe-S centers, with a lower affinity (118). These suppressive interactions could occur during ischemia, when oxygen is limited, and effectively limit mitochondrial respiration during reperfusion of the ischemic myocardium, preventing and abrupt production of reactive oxygen species as electron transport resumes (117). Studies with genetically altered mice reveal that endogenous eNOS derived NO is very cardioprotective in the setting of myocardial I/R injury. When subjected to an ischemia
reperfusion protocol, eNOS-deficient mice (120) developed larger infarcts than their wildtype controls (121). Conversely, when using two different lines of genetically altered mice to overexpress eNOS (particularly in the endothelial cell) in the ischemia
reperfusion protocol, infarct size was reduced compared to the wildtype controls (122).

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Endogenous iNOS is very cardioprotective when overexpressed in the myocardium. Li et al. overexpressed Human iNOS in the mouse heart and observed profound protection when these mice were subjected to myocardial ischemia
reperfusion injury (123). Furthermore, iNOS transgenic mice developed lower infarct size when subjected to 30 min of coronary occlusion and 24 hour of reperfusion, compared to control (124). As previously mentioned, NO is intimately involved in IPC. Endogenous NO may not be required for the protective effects in the early phase of IPC (125,126); however, exogenous NO can induce early IPC-like protection (125,127). In the late phase of IPC, NO is involved in developing the preconditioned phenotype (128). NO derived from eNOS in the trigger phase of late IPC results in a cascade of signaling events that results in the activation of iNOS, which then confers protection (129). The early activation of eNOS and late activation of iNOS appear to be necessary for late phase protection (129). Postconditioning also seems to be dependent on NO to exert its protective phenotype. Postconditioning leads to PI3K, Akt and eNOS activation, and inhibition of PI3K or blockade of NOS with L-NAME reverses the protective effect of postconditioning (130,131). Nitrite is the oxidation product of NO, and was considered biologically inactive in the cardiovascular system until recent years (132). It is now clear that nitrite can be reduced to regenerate NO. This occurs in acidic, ischemic or hypoxic tissues by non-enzymatic disproportionation, or by nitrite reductases such as xanthine oxidase and heme-proteins such as deoxyhemoglobin or myoglobin (133
135). Nitrite administration during myocardial ischemia
reperfusion injury results in profound cardioprotection (133,136
138). Furthermore, in a recent study by Calvert et al. it was demonstrated that the cardioprotective effects of exercise may, in fact, be a result of increased eNOS activation during exercise and storage of the resulting NO by nitrite (139).

Carbon Monoxide (CO) Carbon monoxide is produced by heme oxygenase (HO) during the breakdown of heme, yielding iron, CO, and biliverdin, which is further converted to bilirubin by biliverdin reductase (140). There are two isoforms of HO: HO-1, and HO-2. HO-1 is inducible while HO-2 is constitutively expressed. CO/HO-1 appears to be critical for normal homeostasis. CO is cardioprotective in the setting of myocardial ischemia
reperfusion injury by several mechanisms. Like NO and H2S, CO can inhibit mitochondrial respiration by binding to cytochrome c oxidase which may attenuate reactive oxygen species (141). In a rat model of ischemia
reperfusion injury, pre-exposure to CO

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significantly reduced infarct size (142). In CO-treated hearts TNF-alpha was reduced, and p38 MAPK, Akt, eNOS, and cGMP were activated (142). In a porcine model of cardiopulmonary bypass surgery, treatment with CO resulted in an improvement in cardiac energetics and facilitated recovery, as well as prevention of edema formation and reduction in apoptosis (143). A CO-releasing molecule, CORM-3, was protective in an isolated rat heart model of ischemia reperfusion, reducing infarct size, as well as making isolated rat cardiomyocytes more resistant to hypoxia-reoxygenation injury (144). Furthermore, in an in vivo murine model of ischemia
reperfusion injury, CORM-3 significantly reduced infarct size compared to control (145) and treatment of mice with CORM-3 results in a late preconditioning phenotype, with infarct-reducing effects persisting up to 72 hours after CORM-3 administration (146). Experimental models of heart transplantation have investigated the cytoprotective effects of CO. CO reduced I/R injury and rejection of a mouse to rat cardiac transplantation (147). Inhalation of CO by the donor, and storage of the heart graft in a CO-saturated solution protected against I/R injury by antiapoptotic mechanisms (148). Inhalation of low-dose CO by the recipient after transplantation effectively reduced heart allograft rejection by downregulating pro-inflammatory molecules (149). Finally, in a murine allograft rejection model CORM-3 considerably prolonged the survival of transplanted hearts (144).

Antioxidants The majority of the evidence implicating ROS in the pathogenesis of myocardial I/R injury is based on experiments that examine the ability of free radical scavengers to alter the injury response. Superoxide dismutase and catalase have received the most attention in this regard. The first assessment of antioxidant enzyme therapy in myocardial reperfusion injury was performed by Jolly et al. (150), who studied a combination of SOD and catalase. This study (150) revealed that the combination of antioxidant enzymes significantly reduced myocardial infarct size in dogs after 90 minutes of coronary artery ischemia and 24 hours of reflow. Since this seminal report, there have been a large number of studies from different laboratories (150
156) that have similarly demonstrated a beneficial effect of SOD and/or catalase in experimental models of myocardial I/R injury. There is a large and nearly equal number of reports that either describe a failure of SOD treatment to exert cardioprotection or demonstrate an early protective effect that waned with increasing duration of reperfusion (157
162). It has been suggested that the great disparity in results that was observed with SOD therapy is related to either the dose

378

of the enzyme tested or the experimental conditions of the study (163). The variable results observed with SOD and/or catalase in the post-ischemic myocardium have resulted in considerable skepticism about the therapeutic potential of antioxidant enzymes for acute myocardial infarction in humans. Low-molecular-weight SOD mimetics (164) have similarly yielded positive results in animal models of myocardial I/R. However, it remains unclear whether these agents act merely to delay, rather than to prevent, myocardial necrosis. Another experimental strategy that has been used to assess the role of ROS in myocardial I/R injury is to assess the injury response in mutant mice in which a gene encoding antioxidant enzymes are deleted or overexpressed. Transgenic mice that overexpress various isoforms of SOD have also been developed (165
168), as well as mice that either overexpress or lack the peroxide detoxifying enzymes, catalase, or glutathione peroxidase (169); it has been shown that myocardial contractility is preserved in isolated, perfused hearts derived from mice that genetically overexpress endothelial cell superoxide dismutase (EC-SOD) (170). Similarly, mice that overexpress mitochondrial manganese-superoxide dismutase (MnSOD) are also protected against myocardial I/R (171). Glutathione peroxidase (GSHPx) is an important antioxidant enzyme that performs several vital functions, including the detoxification of lipid and nonlipid hydroperoxides, as well as hydrogen peroxide. Transgenic mice that overexpress GSHPx appear to be resistant to myocardial I/R injury (169), whereas GSHPx knockout mice are more susceptible to myocardial reperfusion injury compared with their wildtype counterparts (172). Overall, the studies of myocardial reperfusion injury performed to date in mice with genetically altered levels of antioxidant enzymes consistently yield results that support a role of ROS in myocardial I/R. Recombinant human superoxide dismutase (h-SOD) has been tested in two clinical trials of patients undergoing thrombolysis (173) or coronary angioplasty (174) for acute myocardial infarction. In a pilot clinical trial of 34 patients with acute anterior myocardial infarction receiving thrombolytic agents, h-SOD was randomly allocated to patients just before reperfusion (173). Arrhythmias and left ventricular function were monitored for a period of 3–4 weeks after thrombolytic therapy. SOD treatment failed to demonstrate any significant improvement in left ventricular regional ejection fraction. A subsequent multicenter, randomized, placebo-controlled clinical trial was designed to test the hypothesis that oxidant-mediated myocardial reperfusion injury is attenuated by treatment with h-SOD before balloon angioplasty (174). Left ventricular function was analyzed using paired contrast ventriculograms and paired radionuclide ventriculograms at 6 and 10 days after angioplasty. Both h-SOD and placebo-

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treated patients showed significant improvements in ventricular function after reperfusion, with no additional protection provided by treatment (174). Hence, the limited efforts to test the oxygen radical hypothesis of myocardial reperfusion injury in the clinical setting do not support the use of antioxidant enzymes in the treatment of patients who have a myocardial infarction.

CENTRAL ROLE OF MITOCHONDRIA IN MYOCARDIAL CELL SURVIVAL To date there have been three major salvage pathways that modulate cell survival by acting on mitochondrial targets: the reperfusion injury salvage kinase (RISK), protein kinase C (PKC), and janus-activated kinase (JAK)/ signal transducer and activator of transcription (STAT) pathways. While these pathways share some overlap in their signaling components, all appear distinct in their activation or end targets and their role in ischemic preconditioning and postconditioning. The RISK pathway describes a group of survival kinases that confer powerful cardioprotection when activated prior to, or at the time of reperfusion (Figure 28.6). Many of these kinases are also involved in IPC and postconditioning as well (175). Therefore pharmacological activation of these components would be a useful strategy to harness the benefits of IPC and postconditioning. The RISK pathway includes: PI3K, Akt, H11k, Erk 1/2, eNOS, PKC, PKG, p70s6K, and GSK-3β (176). In addition to IPC and postconditioning, activation of the RISK pathway can be accomplished by many agents activating G-protein-coupled receptors (GPCR) or growth factor receptors (GFR) including growth factors (TGF-β1, IGF1, FGF-2), opioids, adenosine, cytokines, insulin, bradykinin, corticotrophin-1, atrial natriuretic peptide (ANP), metformin, isoflurane, angiotensin II, and many others (175). Activation of the RISK pathway may occur prior to, or at the time of reperfusion for the cardioprotective benefit (175). This is because many of the downstream effectors of the RISK pathway inhibit the opening of the MPTP (177
179). The MPTP opens in the first few minutes of reperfusion due to the influx of ROS and increase in mitochondrial Ca21 resulting from reperfusion (180,181). MPTP opening causes mitochondrial membrane potential (ΔΨm) to depolarize (182), resulting in a rapid impairment of mitochondrial function and swelling of the mitochondria, ending in cell death (183). The main component necessary for formation of the MPTP is cyclophilin D (CypD); although the voltage-dependent anion channel (VDAC) and adenine nucleotide translocase (ANT) can also contribute to the formation of the MPTP, they do not seem to be obligatory (184).

Chapter | 28

Cardioprotection

379

Agonist

Agonist

GPCR

GFR

PI3K ERK 1/2

Cardiac myocyte

Akt P

H11K

P7OS6K

eNOS P

NO• GSK3β MPTP

Cx43

Mitochondrion

Pharmacologically inhibiting MPTP opening after the first few minutes of reperfusion is ineffective, confirming this early “window” of protection (185). However, the use of cyclosporine A, a direct inhibitor of CypD, before reperfusion has demonstrated successful outcomes in ST-segment elevation myocardial infarction (STEMI) patients (186). There have been several clinical trials investigating activators of the RISK pathway with some demonstration of clinical outcomes in reducing myocardial I/R injury; however, this is not the case with all trials (175). The Japanese clinical trial J-WIND-ANP found that 72 hours, infusion of a recombinant form of human ANP conferred cardioprotection in over 600 patients, reducing infarct size and increasing ejection fraction following an acute myocardial infarction (187). Administration of high-dose statins to patients with acute coronary syndromes more than 24 hours following reperfusion has demonstrated improved clinical outcomes, but it is unclear if administration at reperfusion would have any further beneficial effect (175,188,189). Also, as mentioned previously, trials investigating postconditioning strategies and adenosine, an activator of the RISK pathway, have also had success in improving outcomes following acute myocardial infarction. Protein kinase C (PKC) can be activated during ischemic preconditioning, or by adenosine directly (Figure 28.7). Adenosine, opioids, bradykinin, etc. activate GCPRs, which in turn activate PI3K/Akt leading to eNOS activation (190,191). NO then signals through the soluble guanylyl cyclase, leading to protein kinase G

FIGURE 28.6 Reperfusion injury salvage kinase (RISK) pathway. Various agents binding either G-protein-coupled receptor (GPCR) or growth factor receptor (i.e. fibroblast growth factor or insulin-like growth factor-1) activate phosphoinositide 3-kinase (PI3K), serine/threonine protein kinase (Akt), and finally endothelial nitric oxide synthase (eNOS) to increase nitric oxide (NO) levels. In addition, extracellular regulated kinase 1/2 (ERK 1/2) is also activated which in turn activates p70 ribosomal S6 protein kinase (P70S6K) to activate glycogen synthase kinase 3β (GSK3β). AKt can also activate P70S6K. Nitric oxide and GSK3b promote mitochondrial survival via the connexin43 (Cx43) and the mitochondrial permeability transition pore (MPTP). Mitochondrial preservation results in improved myocardial cell survival and reductions in myocardial infarct size. (From Heusch G, Boengler K, Schulz R. Cardioprotection: nitric oxide, protein kinases and mitochondria. Circulation 2008;118:1915
19, with permission from the American Heart Association.)

(PKG) activation, and finally PKC activation (191,192). This then activates mitochondrial KATP, regulated by connexin43 (Cx43) (193). As previously mentioned, adenosine can directly activate PKC, bypassing PI3K/Akt, eNOS, sGC, PKG (190,191). Additionally, natriuretic peptides such as ANP and brain natriuretic peptide (BNP) can bind natriuretic peptide receptors (NPR) activating particulate guanylyl cyclase (pGC), which leads to activation of PKG, and then PKC (190,194). The JAK/STAT pathway is involved in both early and late phase ischemic preconditioning as well as ischemic postconditioning (Figure 28.8). JAK can be activated by sarcolemmal glycoprotein 130 (gp130) receptors, which are stimulated by IL-6 type cytokines, or by TNFα receptors (190). JAK activates STAT, which can then translocate to the nucleus, activating gene transcription, or the mitochondria, where it can potentially effect both mitochondrial respiration and swelling (195,196). In the nucleus STAT can induce transcription for iNOS, cyclooxygenase-2 (COX-2), manganese superoxide dismutase (mnSOD) (190,197).

TRANSLATION TO THE CLINIC Limitation of myocardial infarct size has undoubtedly been one of the greatest failures of translation in modern times. Launched more than 40 years ago by Braunwald and colleagues (198,199), the concept that a therapeutic intervention can limit the size of a myocardial infarction has not yet been applied to patients (except for the

380

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Adenosine opioids bradykinin

Cardiac Muscle

ANP BNP

PI3K NPR GPCR Akt

ADENOSINE ONLY

pGC

P

eNOS P Cardiac myocyte NO•

SGC Activation

PKG

PKC Cx43

KATP

Mitochondrion

FIGURE 28.7 Protein kinase C (PKC) cardioprotective signaling pathway. Various agonists such as adenosine, opioids, bradykinin, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) interacting with G-protein-coupled receptors (GPCR) or natriuretic peptide receptor (NPR) result in activation of protein kinase G (PKG) and/or protein kinase C (PKC) activation resulting in the opening of mitochondrial KATP channels to attenuate myocardial injury during reperfusion of the ischemic myocardium. PI3K 5 phosphoinositide 3-kinase; Akt 5 serine/threonine protein kinase; eNOS 5 endothelial nitric oxide synthase; NO 5 nitric oxide; SGC 5 soluble quanylate cyclase; cx43 5 connexin 43. (From Heusch G, Boengler K, Schulz R. Cardioprotection: nitric oxide, protein kinases and mitochondria. Circulation 2008;118:1915
19, with permission from the American Heart Association.)

NO• IL-6 type cytokines

TNF-α

gp130

TNF-R

JAK Cardiac myocyte STAT 3

NO• Cx43

COX2

Mitochondrion

iNOS MnSOD Bcl-xL Nucleus

FIGURE 28.8 JAK-STAT pathway of cardioprotection. Activation of the glycoprotein 130 (gp130) receptor by cytokines as well as activation of the tumor necrosis factor receptor (TNF-R) by nitric oxide (NO) results in signal transducer and activator of transcription 3 (STAT 3) activation. STAT3 exerts protective actions on the mitochondria and also induces nuclear activation of cyclooxgygenase-2 (COX2), inducible nitric oxide synthase (iNOS), manganese superoxide dismutase (MnSOD), and Bcl-xL. Cx43 5 connexin43. (From Boengler K, Schulz R, Heusch G. Loss of cardioprotection with ageing. Cardiovasc Res 2009;83:247–61, with permission from Oxford University Press and the European Society of Cardiology.)

Chapter | 28

Cardioprotection

381

TABLE 28.2 Potential Cardioprotective Agents That Have Been Tested in Clinical Trials to Reduce Myocardial Infarct Size or Improve LV Function and Have Shown Promise Type of Agent

Study

Outcome

Adenosine

k Anterior Infarct size with early reperfusion (#3 h) and improved clincial outcomes (AMISTAD II)

Therapeutic hyopthermia

AMISTAD I and II Mahaffey et al. (1999) Ross et al. (2005) COOL MI Hale, Kloner (1999)

Hyperoxemic reperfusion

AMIHOT O’Neill (2007)

GIK (glucose, insulin, K1)

Large number (IMMEDIATE TRIAL) Staat et al. (2005)

Ischemic postconditioning Cyclosporine A (pharmacological postconditioning) Atrial natriuretic peptide (ANP)

Piot et al. (2008) Kitakaze et al. (2007)

Protein kinase Cδ Inhibitor

DELTA MI Bates et al. (2008)

No overall differences in infarct size. Patients with anterior AMI cooled to ,35 C before PCI had k Infarcts No overall difference. Patients with anterior AMI reperfused ,6 h had m LVEF, k infarct size, and ST resolution Variable results but avoidance of hyperglycemia and early therapy may be critical Four 60 sec inflations and deflations of angioplasty balloon after stenting k infarct size and m reperfusion k Infarct size by enzyme assays k Infarct size by small amount and small m LVEF with no effect on mortality k Infarct size, improved ST segment resolution

Table compiled by Robert Kloner. MD, NIH Workshop on New Horizons in Cardioprotection, 2011. LVEF 5 left ventricular ejection fraction; PCI 5 percutaneous coronary intervention.

implementation of early coronary artery reperfusion). Thousands of studies and hundreds of millions of dollars have been spent over the past 40 years, to no avail. It is remarkable that despite this extraordinary record of failure, efforts continue to identify drugs/interventions that are efficacious in limiting infarct size in patients. Perhaps this is the most cogent evidence of the importance of finding such drugs/interventions. The reasons for the failure to translate cardioprotective therapies to the clinic are multifarious and have been addressed in detail in recent articles (200,201). A discussion of the causes of this translational failure is beyond the scope of the present chapter, but the most important reason is that the vast majority of experimental studies of infarct size limitation conducted to date have used methodologically inadequate approaches and/or inadequate animal models. Furthermore, all too often clinical trials of infarct size limitation have been launched prematurely and/or irrationally, without solid preclinical evidence of efficacy. This has resulted in a predictably negative outcome. Such premature/irrational clinical studies are not only dangerous for patients but also potentially harmful to the very concept of cardioprotection, as they inevitably lead to the conclusion that nothing works in patients. If progress is to be made, it is essential to avoid these mistakes, which have been repeated over and over again by many different clinical trial sponsors over the past 40 years. These sobering considerations, however, should not lead to therapeutic nihilism. There are a number of promising cardioprotective interventions that may well turn out

to be efficacious in humans if properly tested at a preclinical and clinical levels. Some of these interventions are listed in Table 28.2.

SUMMARY AND FUTURE DIRECTIONS The most urgent imperative for the future is the translation of cardioprotection to humans. As mentioned above, it will be crucial to learn from past mistakes. There are promising interventions, but solid preclinical evidence must be obtained before these interventions are studied in patients. That is, these interventions should be evaluated with the same level of rigor that is routinely used for the clinical evaluation of therapies in humans, i.e., with multicenter, randomized, blinded studies that use independent Cores to analyze the end-points and an independent Data Coordinating Center and Biostatistical Core. It is also essential that the efficacy of an intervention be evaluated in more than one species and in more than one laboratory, to ensure reproducibility. Lack of reproducibility has bedeviled the field of cardioprotection for 40 years. Obviously, if the efficacy of an intervention cannot be reproduced from one laboratory to another in relatively well-controlled preclinical models, it is implausible that it will be reproducible in the variable clinical setting. To address these issues, the NIH has recently launched a Consortium for PreclinicAl assESsment of CARdioprotective Therapies (CAESAR). The goal of this Consortium is to evaluate cardioprotective therapies with a level of rigor and an approach comparable to that used in clinical trials. The rationale, structure, purpose, and

382

implications of CAESAR have been described recently (200). In the abstract of this article, we state: An estimated 935,000 Americans suffer a myocardial infarction every year; because their prognosis is determined by the size of the infarct, reducing infarct size is of paramount importance to alleviate morbidity and mortality. For 40 years, the National Heart, Lung, and Blood Institute (NHLBI) has invested enormous resources (at least several hundred million dollars) in preclinical studies aimed at developing infarct-sparing therapies, and several hundred (if not thousand) therapies have been claimed to limit infarct size in preclinical models. Unfortunately, due largely to methodological problems, this enormous investment has not produced any notable clinical application, and no cardioprotective therapy is currently available for clinical use. Clearly, after 40 years of futile efforts, a new approach is needed to overcome the problems that have impeded the translation of cardioprotective therapies. The time has come to apply to preclinical research on cardioprotection the same standards of scientific rigor that are applied to clinical trials. In compliance with the recommendations of an National Heart, Lung, and Blood Institute (NHLBI)-sponsored workshop held in June 2003 and using the clinical trial networks established by the NHLBI as a model for developing a collaborative infrastructure for research sharing, a preclinical consortium has been organized that will operate in a manner analogous to a clinical trial network. This infrastructure has been named CAESAR (Consortium for PreclinicAl AssESsment of CARdioprotective therapies). Four Institutions (University of Louisville, Johns Hopkins, Emory University, and Medical College of Virginia) will work together to conduct blinded, randomized, and adequately powered studies using a rigorous design, dose-response analyses, optimal statistical methods, independent data analysis Cores, an independent statistical Core, verification of tetrazolium data with histology and plasma biomarkers, and relevant animal models (including conscious animals and models of comorbidities). Therapies will be tested in three species (anesthetized mouse, conscious rabbit, and conscious pig). A major goal is to ensure reproducibility; to this end, each study in each species will be performed in two centers using identical protocols. The structure of CAESAR will ensure that the consortium will be a true public resource available to all interested investigators and that all proposed studies will be evaluated in an equitable fashion. Proposals for studying cardioprotective therapies will be solicited from the entire scientific community. The consortium will be available at no cost to all NIH-funded investigators. This unique infrastructure will enable rigorous preclinical evaluation of promising cardioprotective therapies and will serve the entire scientific community (both in the academia and in the biomedical industry), thereby constituting a public resource. CAESAR will be a major paradigm shift in cardioprotection. By screening promising therapies and identifying those that are truly effective in relevant experimental models and, thus, most likely to be effective in patients,

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CAESAR will dramatically advance our ability to rationally translate basic findings into clinical use.

It is important to stress that CAESAR is a public resource available to any investigator who is interested in studying cardioprotective interventions. Information regarding the operations of this consortium and the opportunity for external investigators to use it can be obtained at http://nihcaesar.org/. In conclusion, past translational failure should not lead investigators to abandon their efforts. We believe that with a rigorous preclinical evaluation, it will be possible to identify truly efficacious therapies/interventions that will be likely to be effective in humans. Finding a therapy/intervention that does indeed reduce infarct size in patients would be a momentous achievement and a major development in cardiovascular medicine, for it would affect the prognosis of millions of patients with ischemic heart disease. As indicated above, approximately one million Americans suffer an acute myocardial infarction every year. Because the prognosis of those who survive is determined by the amount of damage suffered by the heart (202,203), reducing infarct size would diminish the prevalence of heart failure and lower mortality. The search for cardioprotective manipulations must continue.

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