Myocardial hypothermia induced after reperfusion does not prevent adverse left ventricular remodeling nor improve cardiac function.

AIMS: The purpose of the study was to determine whether late therapeutic hypothermia (LTH), administered after reperfusion, could prevent adverse left ventricular (LV) remodeling and improve cardiac function in the rat myocardial ischemia/reperfusion model. MAIN METHODS: Rats were randomized to normothermia (n = 10) or LTH (initiated at 1 min after coronary artery reperfusion, n = 10) and subjected to 30 min of coronary occlusion followed by 6 weeks of reperfusion. Hypothermia was induced by pumping cold saline over the anterior surface of the LV until the temperature cooled to <32 °C. In the normothermic group, the heart was bathed in saline at 38 °C. KEY FINDINGS: After 6 weeks of recovery, fractional shortening of the LV was comparable in the LTH (20.2 ±â€¯0.6%) and normothermic group (20.0 ±â€¯2.1%; p = 0.918). Postmortem LV volume (0.47 ±â€¯0.04 ml in LTH and 0.44 ±â€¯0.05 ml in normothermic group) and lung wet/dry weight ratio were similar in both groups. There were no significant differences in scar size, scar thickness, infarct expansion index, LV cavity or transmurality (%) between groups. This data contrasts with our previous study showing that hypothermia administered during the ischemic phase significantly reduced the scar size; decreased LV cavity, infarct expansion index and transmurality (%), and improved the scar thickness. SIGNIFICANCE: LTH did not prevent adverse LV remodeling nor improve cardiac function in the rat myocardial ischemia/reperfusion model. To have a long term benefit on remodeling, hypothermia must be administered during the ischemic phase and not just the reperfusion phase.

Improved Long-term Survival with Remote Limb Ischemic Preconditioning in a Rat Fixed-Pressure Hemorrhagic Shock Model.

PURPOSE: We investigated whether bilateral, lower limb remote ischemic preconditioning (RIPC) improved long-term survival using a rat model of hemorrhagic shock/resuscitation. METHODS: Rats were anesthetized, intubated and ventilated, and randomly assigned to RIPC, induced by inflating bilateral pressure cuffs around the femoral arteries to 200 mmHg for 5 min, followed by 5-min release of the cuffs (repeated for 4 cycles), or control group (cuffs were inflated to 30 mmHg). Hemorrhagic shock was induced by withdrawing blood to a fixed mean blood pressure of 30 mmHg for 30 min, followed by 30 min of resuscitation with shed blood. Rats remained anesthetized for 1 h during which hemodynamics were monitored then they were allowed to survive for 6 weeks. RESULTS: The percentage of estimated total blood volume withdrawn to maintain a level of 30 mmHg was similar in both groups. RIPC significantly increased survival at 6 weeks: 5 of 27 (19%) rats in the control group and 13 of 26 (50%; p = 0.02) rats in the RIPC group survived. Blood pressure was higher in the RIPC group. The diastolic internal dimension of the left ventricle, an indicator of circulating intravascular blood volume, was significantly larger in the RIPC group at 1 h after initiation of resuscitation compared to the control group (p = 0.04). Left ventricular function assessed by fractional shortening was comparable in both groups at 1 h after initiation of resuscitation. Blood urea nitrogen (BUN) was within normal range in the RIPC group (17.3 ± 1.2 mg/dl) but elevated in the control group (22.0 ± 1.7 mg/dl) at 48 h after shock. CONCLUSIONS: RIPC significantly improved short-term survival in rats that were subjected to hemorrhagic shock, and this benefit was maintained long term. RIPC led to greater circulating intravascular blood volume in the early phase of resuscitation and improved BUN.

New and revisited approaches to preserving the reperfused myocardium.

Early coronary artery reperfusion improves outcomes for patients with ST-segment elevation myocardial infarction (STEMI), but morbidity and mortality after STEMI remain unacceptably high. The primary deficits seen in these patients include inadequate pump function, owing to rapid infarction of muscle in the first few hours of treatment, and adverse remodelling of the heart in the months that follow. Given that attempts to further reduce myocardial infarct size beyond early reperfusion in clinical trials have so far been disappointing, effective therapies are still needed to protect the reperfused myocardium. In this Review, we discuss several approaches to preserving the reperfused heart, such as therapies that target the mechanisms involved in mitochondrial bioenergetics, pyroptosis, and autophagy, as well as treatments that harness the cardioprotective properties of inhaled anaesthetic agents. We also discuss potential therapies focused on correcting the no-reflow phenomenon and its effect on healing and adverse left ventricular remodelling.

Cardioprotection: Where to from here?

The size of the myocardial infarction remains an important therapeutic target, because heart attack size correlates with mortality and heart failure. In this era, myocardial infarct size is reduced primarily by timely reperfusion of the infarct related coronary artery. Whereas numerous pre-clinical studies have shown that certain pharmacologic agents and therapeutic maneuvers reduce myocardial infarction size greater than reperfusion alone, very few of these therapies have translated to successful clinical trials or standard clinical use. In this review we discuss both the recent successes as well as recent disappointments, and describe some of the newer potential therapies from the preclinical literature that have not yet been tested in clinical trials.

Approaches to Improving Cardiac Structure and Function During and After an Acute Myocardial Infarction: Acute and Chronic Phases.

While progress has been made in improving survival following myocardial infarction, this injury remains a major source of mortality and morbidity despite modern reperfusion therapy. While one approach has been to develop therapies to reduce lethal myocardial cell reperfusion injury, this concept has not translated to the clinics, and several recent negative clinical trials raise the question of whether reperfusion injury is important in humans undergoing reperfusion for acute ST segment elevation myocardial infarction. Therapy aimed at reducing myocardial cell death while the myocytes are still ischemic is more likely to further reduce myocardial infarct size. Developing new therapies to further reduce left ventricular remodeling after the acute event is another approach to preserving structure and function of the heart after infarction. Such therapy may include chronic administration of pharmacologic agents and/or therapies developed from the field of regenerative cardiology, including cellular or non-cellular materials such as extracellular matrix. The optimal therapy will be to administer agents that both reduce myocardial infarct size in the acute phase of infarction as well as reduce adverse left ventricular remodeling during the chronic or healing phase of myocardial infarction. Such a dual approach will help optimize the preservation of both cardiac structure and function.

Bendavia restores mitochondrial energy metabolism gene expression and suppresses cardiac fibrosis in the border zone of the infarcted heart.

AIMS: We have observed that Bendavia, a mitochondrial-targeting peptide that binds the phospholipid cardiolipin and stabilizes the components of electron transport and ATP generation, improves cardiac function and prevents left ventricular remodeling in a 6week rat myocardial infarction (MI) model. We hypothesized that Bendavia restores mitochondrial biogenesis and gene expression, suppresses cardiac fibrosis, and preserves sarco/endoplasmic reticulum (SERCA2a) level in the noninfarcted border zone of infarcted hearts. MAIN METHODS: Starting 2h after left coronary artery ligation, rats were randomized to receive Bendavia (3mg/kg/day), water or sham operation. At 6weeks, PCR array and qRT-PCR was performed to detect gene expression. Picrosirius red staining was used to analyze collagen deposition. KEY FINDINGS: There was decreased expression of 70 out of 84 genes related to mitochondrial energy metabolism in the border zone of untreated hearts. This down-regulation was largely reversed by Bendavia treatment. Downregulated mitochondrial biogenesis and glucose & fatty acid (FA) oxidation related genes were restored by administration of Bendavia. Matrix metalloproteinase (MMP9) and tissue inhibitor of metalloproteinase (TIMP1) gene expression were significantly increased in the border zone of untreated hearts. Bendavia completely prevented up-regulation of MMP9, but maintained TIMP1 gene expression. Picrosirius red staining demonstrated that Bendavia suppressed collagen deposition within border zone. In addition, Bendavia showed a trend toward restoring SERCA2a expression. SIGNIFICANCE: Bendavia restored expression of mitochondrial energy metabolism related genes, prevented myocardial matrix remodeling and preserved SERCA2a expression in the noninfarcted border, which may have contributed to the preservation of cardiac structure and function.

Rapid Surface Cooling by ThermoSuit System Dramatically Reduces Scar Size, Prevents Post-Infarction Adverse Left Ventricular Remodeling, and Improves Cardiac Function in Rats.

BACKGROUND: The long-term effects of transient hypothermia by the non-invasive ThermoSuit apparatus on myocardial infarct (MI) scar size, left ventricular (LV) remodeling, and LV function were assessed in rat MI model. METHODS AND RESULTS: Rats were randomized to normothermic or hypothermic groups (n=14 in each group) and subjected to 30 minutes coronary artery occlusion and 6 weeks of reperfusion. For hypothermia therapy, rats were placed into the ThermoSuit apparatus at 2 minutes after the onset of coronary artery occlusion, were taken out of the apparatus when the core body temperature reached 32°C (in ≈8 minutes), and were then allowed to rewarm. After 6 weeks of recovery, rats treated with hypothermia demonstrated markedly reduced scar size (expressed as % of left ventricular area: hypothermia, 6.5±1.1%; normothermia, 19.4±1.7%; P=1.3×10(-6)); and thicker anterior LV wall (hypothermia, 1.57±0.09 mm; normothermia, 1.07±0.05 mm; P=3.4×10(-5)); decreased postmortem left ventricular volume (hypothermia, 0.45±0.04 mL; normothermia, 0.6±0.03 mL; P=0.028); and better LV fractional shortening by echocardiography (hypothermia, 37.2±2.8%; normothermia, 18.9±2.3%; P=0.0002) and LV ejection fraction by LV contrast ventriculography (hypothermia, 66.8±2.3%; normothermia, 56.0±2.0%; P=0.0014). CONCLUSIONS: Rapid, transient non-invasive surface cooling with the ThermoSuit apparatus in the acute phase of MI decreased scar size by 66.5%, attenuated adverse post-infarct left ventricular dilation and remodeling, and improved cardiac function in the chronic phase of experimental MI.

Does sex influence the incidence or severity of reperfusion-induced cardiac arrhythmias?

UNLABELLED: Whether sex affects the acute phase of myocardial ischemia in experimental animal models is currently being debated. Our purpose was to determine if sex influences either the incidence or severity of reperfusion-induced arrhythmias resulting from a brief coronary occlusion. Male and female Sprague-Dawley rats were assigned to the study. Anesthetized animals were subjected to a 5-minute coronary artery occlusion followed by 5 minutes of reperfusion. Mortality differed by sex: 10/27 (37%) of males died due to VT/VF while only 1/16 females (6%) died due to VT/VF (p = 0.033). Quantitative analysis of the electrocardiogram was performed on data acquired from 17 male and 15 female survivors. Analysis showed no other significant differences in ventricular arrhythmias between the two groups. CONCLUSION: Lethal reperfusion-induced arrhythmias led to a higher mortality in male rats versus female rats. Among survivors there was no difference in any other arrhythmic parameters measured.

Rapid Induction of Hypothermia by the ThermoSuit System Profoundly Reduces Infarct Size and Anatomic Zone of No Reflow Following Ischemia-Reperfusion in Rabbit and Rat Hearts.

INTRODUCTION: Although hypothermia reduces myocardial infarct size, noninvasive and rapid cooling methods are lacking. This study tests the effectiveness of a novel cooling apparatus on myocardial infarct size and no-reflow area in 2 models of coronary artery occlusion (CAO). METHODS AND RESULTS: Animals were randomized to normothermic (N) or hypothermic (H) groups after isolation of a proximal coronary artery. Animals were subjected to 30 minutes CAO and 3 hours reperfusion. In protocol 1 (rabbit, n = 8 per group), hypothermia was initiated, using the ThermoSuit apparatus (convective-immersion cooling), 5 minutes after the onset of CAO to a goal temperature of ∼32°C. In protocol 2 (rat, n = 5 per group), hypothermia was initiated 2 minutes after the onset of CAO to a goal temperature of ∼30°C. Goal temperature was reached in ∼20 minutes. In protocol 1, hypothermia caused an 82% reduction in infarct size as a percentage of the ischemic risk zone (N, 44% ± 5%; H; 8% ± 2%, P < 0.001) and an 89% reduction in the no-reflow area (N, 44% ± 4%; H, 5% ± 1%, P < 0.001). In protocol 2, hypothermia caused a 73% infarct size reduction (N, 51% ± 5%; H, 14% ± 6%, P < 0.01) and a 99% reduction in the no-reflow area (N, 33% ± 5%; H, 0.4% ± 0.3%, P < 0.01). CONCLUSION: The ThermoSuit device induced rapid hypothermia and limited infarct size and no reflow to the greatest extent ever observed in this laboratory with a single intervention.

Dabigatran treatment: effects on infarct size and the no-reflow phenomenon in a model of acute myocardial ischemia/reperfusion.

The no-reflow phenomenon occurs when an epicardial coronary artery is reopened following myocardial infarction, but portions of the intramural microvasculature fail to reperfuse. One potential mechanism for this is the presence of fibrin tactoids. In addition, some recent studies have suggested that dabigatran treatment may be associated with increased incidence of myocardial infarction. Our aim was to investigate the effect on myocardial infarct size and no-reflow in an acute model of ischemia/reperfusion. Anesthetized, open-chest rabbits were randomly assigned to receive dabigatran (Dab, 0.5 mg/kg bolus + infusion, 0.15 mg/kg/h, IV, n = 11) or vehicle (Veh, n = 11) 15 m before a 30-m coronary artery occlusion and during 2.5 h of the 3 h reperfusion procedure. At the end of the reperfusion period, infarct size (% risk zone) and no-reflow defect were measured. The ischemic risk zone (% of left ventricle) was similar in groups, 24 % in Dab and 25 % in Veh. Necrosis was neither reduced nor increased by Dab treatment; expressed as a percentage of the risk region, infarct size was 30 ± 4 % in Dab and 28 ± 5 % in Veh, p = 0.76. The extent of no-reflow was comparable, expressed either as a percent of the risk region (19 ± 3 %, Dab and 18 ± 3 %, Veh) or as a percent of the necrotic zone (67 ± 8 % Dab and 65 ± 10 % Veh). Dab treatment had no effect on heart rate or blood pressure. Dabigatran treatment did not prevent or ameliorate the no-reflow phenomenon, suggesting that fibrin does not play a major role in the development of microvascular obstruction. Dabigatran did not exacerbate myocardial infarct size.

Hypothermia in the setting of experimental acute myocardial infarction: a comprehensive review.

A door-to-balloon time of less than 90 minutes is the gold standard for reperfusion therapy to treat acute myocardial infarction (MI). Because 30-day mortality remains ∼ 10%, new methods must be cultivated to limit myocardial injury. Therapeutic hypothermia has long been experimentally used to attenuate myocardial necrosis during MI with promising results, but the treatment has yet to gain popularity among most clinicians. Hypothermia, in the basic science setting, has been achieved using many techniques. In our review, we examine past and current methods of inducing hypothermia, benefits and setbacks of such methods, current and future clinical trials, and potential mechanisms.

Ranolazine treatment for myocardial infarction? Effects on the development of necrosis, left ventricular function and arrhythmias in experimental models.

Ranolazine, an inhibitor of the late current of the cardiac action potential (late I(Na)), is a well established clinical treatment for chronic angina. The late INa in cardiac myocytes also plays an important role in the pathophysiology of acute myocardial ischemia and reperfusion, and thus is a potential therapeutic target to ameliorate consequences of myocardial infarction. In experimental animal models, ranolazine has been shown to reduce myocardial infarct size, improve left ventricular function, decrease ischemia/reperfusion-induced arrhythmias and improve outcome in heart failure. Here we focus specifically on data from in vivo animal studies of myocardial ischemia and reperfusion.

Bendavia, a mitochondria-targeting peptide, improves postinfarction cardiac function, prevents adverse left ventricular remodeling, and restores mitochondria-related gene expression in rats.

AB We evaluated the post-myocardial infarction (MI) therapeutic effects of Bendavia. Two hours after coronary artery ligation, rats were randomized to receive chronic Bendavia treatment (n = 28) or water (n = 26). Six weeks later, Bendavia significantly reduced scar circumference (39.7% +/- 2.2%) compared with water treatment (47.4% +/- 0.03%, P = 0.024) and reduced left ventricular (LV) volume by 8.9% (P = 0.019). LV fractional shortening was significantly improved by Bendavia (28.8% +/- 1.7%) compared with water treatment (23.8% +/- 1.8%, P = 0.047). LV ejection fraction was higher with Bendavia (55.3% +/- 1.4%) than water treatment (49.3% +/- 1.4%, P = 0.005). Apoptosis, within the MI border zone, was significantly less in the Bendavia group (32% +/- 3%, n = 12) compared with the water group (41% +/- 2%, n = 12; P = 0.029). Bendavia reversed mitochondrial function-related gene expression in the MI border, which was largely reduced in water-treated rats. Bendavia improved complex-I and -IV activity, and reduced production of reactive oxygen species and cytosolic cytochrome c level in the peri-infarcted region. Bendavia improved post-MI cardiac function, prevented infarct expansion and adverse LV remodeling, and restored mitochondria-related gene expression, complex-I and -IV activity, and reduced reactive oxygen species and cardiomyocyte apoptosis in the noninfarcted MI border.

Capsaicin-induced cardioprotection. Is hypothermia or the salvage kinase pathway involved?

PURPOSE: Topical capsaicin application was shown to reduce infarct size in experimental animal models. We hypothesized that cardioprotective properties of topical capsaicin application could be related to its hypothermic effect. METHODS: In the first arm of the study, anesthetized rats received capsaicin cream (Caps group) or vehicle (Control group, Ctrl) applied either 15 or 30 min prior to a 30-min coronary artery occlusion followed by 2-h reperfusion. Core body temperature was allowed to run its course, and was monitored via rectal probe. At the end of the protocol, hearts were excised and risk zone and infarct size were measured. In an additional set of animals, hearts were excised immediately after a 15-min application of capsaicin/vehicle, and were used to measure phosphorylated Akt and Erk1/2 with western blots. In the second arm of the study Ctrl (n = 6) and Caps-treated (n = 5) animals were subjected to the same protocol as rats in the first arm, but core body temperature was maintained at 36 °C. RESULTS: In the first arm of the study, capsaicin produced a rapid decrease in rectal temperature ranging from 0.22 to 1.78 °C at pre-occlusion, with a median level of 0.97 °C. A capsaicin-induced temperature decrease of >0.97 °C was associated with a 31.2 % smaller infarct compared to the control group. Capsaicin treatment induced an increase in the levels of phosphorylated Akt and Erk1/2 at the end of capsaicin cream application. No increase in the phosphorylation of downstream p70S6 was observed. Levels of phosphorylated Akt- and Erk1/2 did not correlate with temperature changes after treatment. In the second arm of the study, in which body core temperature was maintained at 36 °C, no change in the infarct size was observed in the capsaicin vs. control group. CONCLUSION: In the current study we for the first time demonstrated that the capsaicin induced cardioprotective effect might be related to mild hypothermia, caused by capsaicin topical application. The salvage kinase pathway appears not to be critical for capsaicin-induced cardioprotection.

Reduction of early reperfusion injury with the mitochondria-targeting peptide bendavia.

We recently showed that Bendavia, a novel mitochondria-targeting peptide, reduced infarction and no-reflow across several experimental models. The purpose of this study was to determine the therapeutic timing and mechanism of action that underlie Bendavia's cytoprotective property. In rabbits exposed to in vivo ischemia/reperfusion (30/180 min), Bendavia administered 20 minutes prior to reperfusion (0.05 mg/kg/h, intravenously) reduced myocardial infarct size by ∼50% when administered for either 1 or 3 hours of reperfusion. However, when Bendavia perfusion began just 10 minutes after the onset of reperfusion, the protection against infarction and no-reflow was completely lost, indicating that the mechanism of protection is occurring early in reperfusion. Experiments in isolated mouse liver mitochondria found no discernible effect of Bendavia on blocking the permeability transition pore, and studies in isolated heart mitochondria showed no effect of Bendavia on respiratory rates. As Bendavia significantly lowered reactive oxygen species (ROS) levels in isolated heart mitochondria, the ROS-scavenging capacity of Bendavia was compared to well-known ROS scavengers using in vitro (cell-free) systems that enzymatically generate ROS. Across doses ranging from 1 nmol/L to 1 mmol/L, Bendavia showed no discernible ROS-scavenging properties, clearly differentiating itself from prototypical scavengers. In conclusion, Bendavia is a promising candidate to reduce cardiac injury when present at the onset of reperfusion but not after reperfusion has already commenced. Given that both infarction and no-reflow are related to increased cellular ROS, Bendavia's protective mechanism of action likely involves reduced ROS generation (as opposed to augmented scavenging) by endothelial and myocyte mitochondria.

Delayed treatment with hypothermia protects against the no-reflow phenomenon despite failure to reduce infarct size.

BACKGROUND: Many studies have shown that when hypothermia is started after coronary artery reperfusion (CAR), it is ineffective at reducing necrosis. However, some suggest that hypothermia may preferentially reduce no-reflow. Our aim was to test the effects of hypothermia on no-reflow when initiated close to reperfusion and 30 minutes after reperfusion, times not associated with a protective effect on myocardial infarct size. METHODS AND RESULTS: Rabbits received 30 minutes coronary artery occlusion/3 hours CAR. In protocol 1, hearts were treated for 1 hour with topical hypothermia (myocardial temperature ≈32°C) initiated at 5 minutes before or 5 minutes after CAR, and the results were compared with a normothermic group. In protocol 2, hypothermia was delayed until 30 minutes after CAR and control hearts remained normothermic. In protocol 1, risk zones were similar and infarct size was not significantly reduced by hypothermia initiated close to CAR. However, the no-reflow defect was significantly reduced by 43% (5 minutes before CAR) and 38% (5 minutes after CAR) in hypothermic compared with normothermic hearts (P=0.004, ANOVA, P=ns between the 2 treated groups). In protocol 2, risk zones and infarct sizes were similar, but delayed hypothermia significantly reduced no-reflow in hypothermic hearts by 30% (55±6% of the necrotic region in hypothermia group versus 79±6% with normothermia, P=0.008). CONCLUSION: These studies suggest that treatment with hypothermia reduces no-reflow even when initiated too late to reduce infarct size and that the microvasculature is especially receptive to the protective properties of hypothermia and confirm that microvascular damage is in large part a form of true reperfusion injury.

Continuous heliox breathing and the extent of anatomic zone of noreflow and necrosis following ischemia/reperfusion in the rabbit heart.

BACKGROUND: Nitrogen may contribute to reperfusion injury. Some studies have shown that helium as a replacement for nitrogen in breathing gas (heliox) reduces cell necrosis after ischemia/reperfusion when used in a preconditioning fashion (intermittent heliox exposure). Our aim was to test whether heliox, breathed continuously throughout the ischemic and reperfusion periods, reduced necrosis and a marker of reperfusion injury, the no-reflow phenomenon. METHODS AND RESULTS: Anesthetized, open-chest rabbits received 30 min coronary artery occlusion/3 hrs reperfusion. Before CAO rabbits were randomized to heliox (30% oxygen + 70% helium, n=8) or air supplemented with oxygen to achieve blood gas values within physiologic range (n = 8). Rabbits received the appropriate mix during ischemic and reperfusion periods. Infarct size (% risk zone) and no-reflow defect were measured at the end of the reperfusion period. The ischemic risk zone was similar in both groups (28% of left ventricle in heliox and 29% in control). Heliox breathing did not reduce necrosis; infarct size, expressed as a percentage of the risk region was 44±4% in the heliox group and 49±5% in controls, p = 0.68. The extent of the no-reflow defect was not altered by heliox, either expressed as a percent of the risk region (29±4% in heliox and 28±3% in control) or as a percent of the necrotic zone (65±5% in heliox and 59±8% in control).Heliox treatment had no effect on hemodynamic parameters or arterial blood gas values. CONCLUSION: Continuous heliox breathing does not appear to be cardioprotective in the setting of acute myocardial infarction in the rabbit model. Heliox respiration administered during 30 minutes of ischemia and 180 minutes of reperfusion did not alter infarct size or the extent of no-reflow.