James D. McCully

Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury

Mitochondrial damage and dysfunction occur during ischemia and modulate cardiac function and cell survival significantly during reperfusion. We hypothesized that transplantation of autologously derived mitochondria immediately prior to reperfusion would ameliorate these effects. New Zealand White rabbits were used for regional ischemia (RI), which was achieved by temporarily snaring the left anterior descending artery for 30 min. Following 29 min of RI, autologously derived mitochondria (RI-mitochondria; 9.7 ± 1.7 × 10(6)/ml) or vehicle alone (RI-vehicle) were injected directly into the RI zone, and the hearts were allowed to recover for 4 wk. Mitochondrial transplantation decreased (P < 0.05) creatine kinase MB, cardiac troponin-I, and apoptosis significantly in the RI zone. Infarct size following 4 wk of recovery was decreased significantly in RI-mitochondria (7.9 ± 2.9%) compared with RI-vehicle (34.2 ± 3.3%, P < 0.05). Serial echocardiograms showed that RI-mitochondria hearts returned to normal contraction within 10 min after reperfusion was started; however, RI-vehicle hearts showed persistent hypokinesia in the RI zone at 4 wk of recovery. Electrocardiogram and optical mapping studies showed that no arrhythmia was associated with autologously derived mitochondrial transplantation. In vivo and in vitro studies show that the transplanted mitochondria are evident in the interstitial spaces and are internalized by cardiomyocytes 2-8 h after transplantation. The transplanted mitochondria enhanced oxygen consumption, high-energy phosphate synthesis, and the induction of cytokine mediators and proteomic pathways that are important in preserving myocardial energetics, cell viability, and enhanced post-infarct cardiac function. Transplantation of autologously derived mitochondria provides a novel technique to protect the heart from ischemia-reperfusion injury.

Myocardial protection by PJ34, a novel potent poly (ADP-ribose) synthetase inhibitor.

BACKGROUND The activation of poly (ADP-ribose) synthetase plays an important role in the pathogenesis leading to myocardial ischemia-reperfusion injury. The aim of this study was to determine if a novel potent inhibitor of poly (ADP-ribose) synthetase, PJ34, provides myocardial protection. METHODS Pigs were subjected to 60 minutes of regional ischemia followed by 180 minutes of reperfusion. Ten mg/kg of PJ34 (PJ34; n = 6) was administrated intravenously (treated group) from 15 to 5 minutes before reperfusion followed by 3 mg/kg/hour of PJ34 from 5 minutes before reperfusion to the end of 180 minutes reperfusion. Control pigs (n = 7) received vehicle only. Arterial and left ventricular pressure and coronary flow were monitored. RESULTS The PJ34 showed significant reduction on infarct size (37.5%+/-4.5% and 50.5%+/-4.8% of the area at risk) for PJ34 and control pigs groups, respectively, (p < 0.05). Significant reduction in postsystolic shortening, as well as improvement on segment shortening, and positive first derivative of pressure over time (+dP/dt) maximum were also observed in PJ34 versus control pigs (p < 0.05). CONCLUSIONS Our results suggest that PJ34 provides cardioprotection by decreasing myocardial infarct size and enhancing postischemic regional and global functional recovery.

Developmental differences in cytosolic calcium accumulation associated with surgically induced global ischemia: Optimization of cardioplegic protection and mechanism of action ☆ ☆☆ ★ ★★ ♢

OBJECTIVE The effect of cardioplegic solutions with high concentrations of potassium or magnesium (or both) on cytosolic calcium accumulation was investigated with fura-2 in isolated perfused mature (n = 24) and aged (n = 24) rabbit hearts. METHODS We compared cytosolic calcium accumulation before ischemia (control), during 30 minutes of ischemia and 30 minutes of reperfusion under global ischemia, or after treatment with potassium (20 mmol/L), magnesium (20 mmol/L), or both. RESULTS Cytosolic calcium accumulation was increased during global ischemia in the mature heart (from 178.7 +/- 24.2 in the control group to 393.6 +/- 25.5 nmol/L; p < 0.005) and in the aged heart (from 187.4 +/- 18.7 in the control group to 501.0 +/- 46.1 nmol/L; p < 0.005). Potassium reduced cytosolic calcium accumulation during ischemia in both the mature and aged hearts (300.9 +/- 23.2 and 365.2 +/- 27.7 nmol/L, respectively; p < 0.05 vs global ischemia). Magnesium and potassium/magnesium completely controlled cytosolic calcium accumulation in the mature heart (198.7 +/- 27.5 nmol/L; p < 0.01 vs global ischemia and p < 0.05 vs potassium: 182.3 +/- 22.7 nmol/L; p < 0.05 vs global ischemia and potassium, respectively). Magnesium and potassium/magnesium attenuated cytosolic calcium accumulation in the aged heart (261.3 +/- 26.7, 262.3 +/- 25.2 nmol/L, respectively; p < 0.01 vs global ischemia). These changes in cytosolic calcium accumulation correlated with improved post-ischemic ventricular function. To investigate the mechanism(s) of magnesium-supplemented cardioplegic inhibition of cytosolic calcium accumulation, we performed parallel studies (n = 43) using nifedipine, ryanodine, and dimethylthiourea. Nifedipine with or without ryanodine reduced cytosolic calcium accumulation. Dimethylthiourea did not alter cytosolic calcium accumulation during global ischemia. Our results suggest that cytosolic calcium accumulation during global ischemia was mainly increased via the sarcolemmal 1-type calcium channel and the sarcoplasmic reticulum calcium-release channel. The modulating action of potassium/magnesium cardioplegia on cytosolic calcium accumulation during ischemia would appear to act through the inhibition of the myocardial 1-type calcium channel and the sarcoplasmic reticulum calcium-release channel. CONCLUSION Senescent cardiac dysfunction correlates with increased ischemia-induced cytosolic calcium accumulation. Magnesium-supplemented potassium cardioplegia ameliorates this age-related phenomenon at normothermia and may have important implications in myocardial protection in the elderly population.

Injection of isolated mitochondria during early reperfusion for cardioprotection

Previously, we demonstrated that ischemia induces mitochondrial damage and dysfunction that persist throughout reperfusion and impact negatively on postischemic functional recovery and cellular viability. We hypothesized that viable respiration-competent mitochondria, isolated from tissue unaffected by ischemia and then injected into the ischemic zone just before reperfusion, would enhance postischemic functional recovery and limit infarct size. New Zealand White rabbits (n = 52) were subjected to 30 min of equilibrium and 30 min of regional ischemia (RI) induced by snaring the left anterior descending coronary artery. At 29 min of RI, the RI zone was injected with vehicle (sham control and RI vehicle) or vehicle containing mitochondria (7.7 x 10(6) +/- 1.5 x 10(6)/ml) isolated from donor rabbit left ventricular tissue (RI-Mito). The snare was released at 30 min of RI, and the hearts were reperfused for 120 min. Our results show that left ventricular peak developed pressure and systolic shortening in RI-Mito hearts were significantly enhanced (P < 0.05 vs. RI-vehicle) to 75% and 83% of equilibrium value, respectively, at 120 min of reperfusion compared with 57% and 62%, respectively, in RI-vehicle hearts. Creatine kinase-MB, cardiac troponin I, and infarct size relative to area at risk were significantly decreased in RI-Mito compared with RI-vehicle hearts (P < 0.05). Confocal microscopy showed that injected mitochondria were present and viable after 120 min of reperfusion and were distributed from the epicardium to the subendocardium. These results demonstrate that viable respiration-competent mitochondria, isolated from tissue unaffected by ischemia and then injected into the ischemic zone just before reperfusion, significantly enhance postischemic functional recovery and cellular viability.

A novel peroxynitrite decomposer catalyst (FP-15) reduces myocardial infarct size in an in vivo peroxynitrite decomposer and acute ischemia-reperfusion in pigs

BACKGROUND Reactive oxygen and nitrogen species generated after reperfusion injury result in organ dysfunction. Peroxynitrite, a reactive nitrogen molecule produced from the reaction of superoxide anions and nitric oxide, is thought to be a causative agent in oxidative reperfusion injury. The aim of this study was to investigate the effects of a novel peroxynitrite decomposition catalyst (FP-15) in an acute myocardial ischemia/reperfusion model. METHODS Pigs were subjected to 60 minutes of regional ischemia by reversibly ligating the left anterior descending coronary artery followed by 180 minutes of reperfusion. In the treatment group (n = 6), an FP-15 (1 mg/kg) bolus was infused through the jugular vein after 30 minutes of ischemia followed by a continuous infusion (1 mg x kg(-1) x h(-1)) during reperfusion. Vehicle was infused in the control group (n = 6). Coronary flow was recorded by an ultrasonic flow probe and infarct size determined by tetrazolium staining. Arterial and left ventricular pressures were monitored continuously and regional myocardial function determined by sonomicrometry. RESULTS No significant differences were observed in either hemodynamics or ischemic area at risk. However, the infarct size was significantly reduced (35.3% +/- 3.5% versus 21.6% +/- 2.6% of the ischemic area, control versus FP-15-treated groups, respectively, p < 0.05). +dP/dt was transiently improved in the FP-15-treated groups while during most of the reperfusion period coronary flow, and was significantly lower in the FP-15-treated group as compared to the control group (p < 0.01). CONCLUSIONS FP-15 administration reduces myocardial infarct size and reactive hyperemia. These data support the pathogenic role of endogenously produced peroxynitrite and that FP-15 is effective in preventing myocardial reperfusion injury.

Magnesium cardioplegia reduces cytosolic and nuclear calcium and DNA fragmentation in the senescent myocardium.

Previous reports have indicated that the senescent myocardium is less tolerant to surgically induced ischemia and that diminished functional recovery is associated with alterations in cytosolic calcium ([Ca2+]i) accumulation. Recently, increased [Ca2+]i has been suggested to alter nuclear calcium ([Ca2+]n) accumulation. To investigate the relation between [Ca2+]i and [Ca2+]n, we subjected mature and aged rabbit hearts to normothermic global ischemia, either without treatment or after treatment with potassium cardioplegia, magnesium cardioplegia, or a combination of potassium and magnesium cardioplegia. The relation between altered [Ca2+]n and DNA fragmentation was also investigated. Our results indicate that [Ca2+]i was increased during 30 minutes of normothermic global ischemia without treatment in both the mature and aged hearts (p < 0.05). Accumulation of [Ca2+]i during global ischemia was reduced with the use of potassium, magnesium, and a combination of potassium and magnesium cardioplegia (p < 0.05 versus untreated ischemia) in both the mature and aged hearts. Levels of [Ca2+]n were unaffected by global ischemia or cardioplegia in the mature myocardium; however, in the aged myocardium, [Ca2+]n was increased during global ischemia and with potassium cardioplegia and was associated with increased nuclear DNA fragmentation (p < 0.05). The use of magnesium and a combination of potassium and magnesium cardioplegia attenuated [Ca2+]n accumulation and nuclear DNA fragmentation (p < 0.05). Control of [Ca2+]i and [Ca2+]n was associated with enhanced functional recovery during reperfusion. These results indicate that during normothermic ischemia, there is increased [Ca2+]i and [Ca2+]n in the aged myocardium, and increased [Ca2+]n is associated with increased nuclear DNA fragmentation.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective opening of mitochondrial ATP-sensitive potassium channels during surgically induced myocardial ischemia decreases necrosis and apoptosis

OBJECTIVE Mitochondrial ATP-sensitive potassium channels have been proposed to be myoprotective. The relevance and specificity of this mechanism in cardiac surgery was unknown. The purpose of this study was to examine the effects of the mitochondrial potassium ATP-sensitive channel opener diazoxide on regional and global myocardial protection using a model of acute myocardial infarction. METHODS Pigs (n=19) were placed on total cardiopulmonary bypass and then subjected to 30 min normothermic regional ischemia by snaring the left anterior descending coronary artery (LAD). The aorta was then crossclamped and cold blood Deaconess Surgical Associates cardioplegia (DSA; n=6) or DSA containing 50 microM diazoxide (DZX; n=6) was delivered via the aortic root and the hearts subjected to 30 min hypothermic global ischemia. The crossclamp and snare were removed and the hearts reperfused for 120 min. RESULTS No significant differences in preload recruitable stroke work relationship, Tau, proximal, distal or proximal/distal coronary flow, regional or global segmental shortening, systolic bulging or post-systolic shortening were observed within or between DSA and DZX hearts during reperfusion. Infarct was present only in the region of LAD occlusion in both DSA and DZX hearts. Infarct size (% of area at risk) was 33.6+/-2.9% in DSA and was 16.8+/-2.4% in DZX hearts (P<0.01 versus DSA). Apoptosis as estimated by TUNEL positive nuclei was 120.3+/-48.8 in DSA and was significantly decreased to 21.4+/-5.3 in DZX hearts. Myocardial infarct was located centrally within the area at risk in both DSA and DZX hearts but was significantly increased at borderline zones within the area at risk in DSA hearts. CONCLUSIONS The addition of diazoxide to cardioplegia significantly decreases regional myocardial cell necrosis and apoptosis in a model of acute myocardial infarction and represents an additional modality for achieving myocardial protection.

The mitochondrial KATP channel and cardioprotection

Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels allow coupling of membrane potential to cellular metabolic status. Two K(ATP) channel subtypes coexist in the myocardium, with one subtype located in the sarcolemma (sarcK(ATP)) membrane and the other in the inner membrane of the mitochondria (mitoK(ATP)). The K(ATP) channels can be pharmacologically modulated by a family of structurally diverse agents of varied potency and selectivity, collectively known as potassium channel openers and blockers. Sufficient evidence exists to indicate that the K(ATP) channels and, in particular, the mitoK(ATP) channels play an important role both as a trigger and an effector in surgical cardioprotection. In this review, the biochemistry and surgical specificity of the K(ATP) channels are examined.

Opening of mitochondrial ATP-sensitive potassium channels enhances cardioplegic protection

BACKGROUND Mitochondrial and sarcolemmal ATP-sensitive potassium channels have been implicated in cardioprotection; however, the role of these channels in magnesium-supplemented potassium (K/Mg) cardioplegia during ischemia or reperfusion is unknown. METHODS Rabbit hearts (n = 76) were used for Langendorff perfusion. Sham hearts were perfused for 180 minutes. Global ischemia hearts received 30 minutes of global ischemia and 120 minutes of reperfusion. K/Mg hearts received cardioplegia before ischemia. The role of ATP-sensitive potassium channels in K/Mg cardioprotection during ischemia and reperfusion was investigated, separately using the selective mitochondrial ATP sensitive potassium and channel blocker, 5-hydroxydecanoate, and the selective sarcolemmal ATP-sensitive potassium channel blocker HMR1883. Separate studies were performed using the selective mitochondrial ATP-sensitive potassium channel opener, diazoxide, and the nonselective ATP-sensitive potassium channel opener pinacidil. RESULTS Infarct size was 1.9%+/-0.4% in sham, 3.7%+/-0.5% in K/Mg, and 27.8%+/-2.4% in global ischemia hearts (p < 0.05 versus K/Mg). Left ventricular peak-developed pressure (percent of equilibrium) at the end of 120 minutes of reperfusion was 91%+/-6% in sham, 92% +/-2% in K/Mg, and 47%+/-6% in global ischemia (p < 0.05 versus K/Mg). Blockade of sarcolemmal ATP-sensitive potassium channels in K/Mg hearts had no effect on infarct size or left ventricular peak-developed pressure. However, blockade of mitochondrial ATP-sensitive potassium channels before ischemia significantly increased infarct size to 23%+/-2% in K/Mg hearts (p < 0.05 versus K/Mg; no statistical significance [NS] as compared to global ischemia) and significantly decreased left ventricular peak-developed pressure to 69%+/-4% (p < 0.05 versus K/Mg). Diazoxide when added to K/Mg cardioplegia significantly decreased infarct size to 1.5%+/-0.4% (p < 0.05 versus K/Mg). CONCLUSIONS The cardioprotection afforded by K/Mg cardioplegia is modulated by mitochondrial ATP-sensitive potassium channels. Diazoxide when added to K/Mg cardioplegia significantly reduces infarct size, suggesting that the opening of mitochondrial ATP-sensitive potassium channels with K/Mg cardioplegic protection would allow for enhanced myocardial protection in cardiac operations.

Adenosine-enhanced ischemic preconditioning modulates necrosis and apoptosis: effects of stunning and ischemia–reperfusion

BACKGROUND Adenosine-enhanced ischemic preconditioning extends the protection of ischemic preconditioning by both significantly decreasing infarct size and significantly enhancing postischemic functional recovery. METHODS The effects of adenosine-enhanced ischemic preconditioning on necrosis and apoptosis were investigated in the sheep heart using models of stunning (15 minutes regional ischemia, 120 minutes reperfusion) and ischemia-reperfusion (30 and 60 minutes regional ischemia, 120 minutes reperfusion). Ischemic preconditioned hearts received 5 minutes regional ischemia, 5 minutes reperfusion before ischemia. Adenosine-enhanced ischemic preconditioned hearts received a 10 mmol/L adenosine bolus (10 mL) through the left atrium coincident with ischemic preconditioning. Adenosine hearts received a 10 mmol/L bolus (10 mL) of adenosine. Regional ischemic hearts received no pretreatment. RESULTS Minimal apoptosis (< 45 per 3,000 myocytes) was observed in the stunning models but was significantly increased with ischemia-reperfusion in regional ischemic hearts after 30 minutes (p < 0.05 versus ischemic preconditioning, adenosine, or adenosine-enhanced ischemic preconditioning) and in adenosine and ischemic preconditioned hearts after 60 minutes ischemia (p < 0.05 versus adenosine-enhanced ischemic preconditioning). DNA laddering was apparent after 60 minutes ischemia in regional ischemia, adenosine, and ischemic preconditioning but not in adenosine-enhanced ischemic preconditioned hearts. CONCLUSIONS Adenosine-enhanced ischemic preconditioning significantly ameliorates necrosis and apoptosis in the regional ischemic blood-perfused heart.