Effects of N-(2-mercaptopropionyl)-glycine on mitochondrial function in ischemic-reperfused heart

OBJECTIVE: A possible mechanism for N-(2-mercaptopropionyl)-glycine (MPG) underlying the improvement of contractile function and mitochondrial activity of ischemic-reperfused rat hearts was examined. METHODS: Isolated, perfused hearts were subjected to 35 min ischemia-60 min reperfusion. At the end of ischemia or reperfusion, myocardial Na(+) content and mitochondrial oxygen consumption rate (OCR) were examined. The perfused heart was treated with 0.1-1 mM MPG for 30 min prior to ischemia or for the first 30 min of reperfusion. RESULTS: Ischemia increased myocardial Na(+) content (sodium overload) and decreased mitochondrial OCR. The left ventricular developed pressure (LVDP) of the untreated heart recovered to 19.8+/-3.8% of the preischemic value and the infarct area amounted to 23.3+/-1.7% of the left ventricle. The thiobarbiturate-reacting substance (TRS) was also increased in the reperfused, but not ischemic, myocardium. Pretreatment of the perfused heart with 0.3-1 mM MPG attenuated the ischemia-induced sodium overload and decrease in the OCR. Pretreatment with the agent also enhanced the postischemic recovery of LVDP, attenuated reperfusion-induced increase in TRS, and reduced the infarct area. Although the postischemic treatment with MPG suppressed the increase in TRS in the reperfused myocardium, a LVDP recovery of reperfused hearts was not observed. Cardiac mitochondria were isolated and examined for the direct effect of MPG on their function. Incubation with either 12.5 mM sodium lactate or 1 microM phenylarsine oxide neither altered the mitochondrial membrane potential nor induced mitochondrial swelling, whereas incubation with a combination of these agents elicited the membrane potential depolarization and swelling. Incubation of mitochondria with 1 mM MPG attenuated these events. CONCLUSION: These results suggest that both attenuation of sodium overload and preservation of the mitochondrial function may largely contribute to cardioprotection of MPG in the ischemic-reperfused heart.     

Mitochondrial function in myocardial stunning

Mitochondrial respiration parameters were studied in mitochondria isolated from normal, ischemic and post-ischemic rabbit hearts. Mitochondrial function was related to tissue content of high energy phosphates (HEP) and cardiac function in the isolated working rabbit heart preparation. It was found that after 10 and 20 mins of global normothermic ischemia followed by 20 mins of Langendorff reperfusion, mitochondrial function and HEP content of the myocardium were not significantly diminished. Myocardial creatine phosphate even showed a significant overshoot as compared to the pre-ischemic condition. When these hearts were allowed to perform work, recovery of cardiac function was incomplete while mitochondrial function and HEP content remained in the normal range. Prolonged ischemia (30 mins) resulted in a significant depression of mitochondrial function and myocardial ATP content during and after ischemia. Recovery of contractile function was severely depressed. These results show that impaired cardiac function after a mild ischemic insult (myocardial stunning) can be associated with near normal mitochondrial function and HEP contents.     

Sodium accumulation during ischemia induces mitochondrial damage in perfused rat hearts

OBJECTIVE: The present study aimed to elucidate the involvement of sodium overload and following damage to mitochondria during ischemia in the genesis of ischemia/reperfusion injury of perfused rat hearts. METHODS: Isolated, perfused hearts were exposed to different durations (15-35 min) of ischemia followed by 60-min reperfusion. At the end of ischemia or reperfusion, myocardial sodium and calcium contents and myocardial high-energy phosphates were determined. The cardiac mitochondrial ability to produce ATP was measured using saponin-skinned bundles. The effects of sodium on the mitochondrial membrane potential and the oxidative phosphorylation rate were examined using isolated mitochondria from normal hearts. RESULTS: Post-ischemic recovery of left ventricular developed pressure decreased in an ischemic duration-dependent manner. Ischemia induced an increase in myocardial sodium, but not calcium. This increase was dependent on the duration of ischemia. The oxygen consumption rate of skinned bundles from the ischemic heart decreased at the end of ischemia. Incubation of mitochondria with various concentrations of sodium chloride or sodium lactate in vitro resulted in a depolarization of mitochondrial membrane potential and a decrease in ATP-generating activity. This decrease was not restored after elimination of sodium compounds. CONCLUSIONS: The present findings suggest that ischemia induces an increase in sodium influx from the extracellular space and that accumulated sodium may induce irreversible damage to mitochondria during ischemia. This mitochondrial dysfunction may be one of the most important determinants for the genesis of ischemia/reperfusion injury in perfused rat hearts.     

Male/female differences in intracellular Na+ regulation during ischemia/reperfusion in mouse heart

We previously showed that beta-adrenergic stimulation revealed male/female differences in susceptibility to ischemia/reperfusion (I/R) injury. To explore whether altered [Na(+)](i) regulation is involved in the mechanism of this sex difference, we measured [Na(+)](i) by (23)Na NMR spectroscopy in isolated perfused mouse hearts. [Na(+)](i) increased to 195 +/- 3% (mean +/- S.E.) of the pre-ischemic level at 20 min of ischemia in male hearts, whereas [Na(+)](i) accumulation was slightly less in female hearts (176 +/- 2%, P < 0.05). There was no significant difference in the recovery of contractile function after reperfusion (male: 30.6 +/- 3.8%; female: 35.0 +/- 1.9%; P > 0.05). If hearts were treated with isoproterenol (ISO, 10 nmol/l), males exhibited significantly poorer recovery of post-ischemic contractile function than females (male: 13.0 +/- 1.9%; female: 28.1 +/- 1.2%; P < 0.05), and a significantly higher [Na(+)](i) accumulation during ischemia (male: 218 +/- 8%; female: 171 +/- 2%; P < 0.05). This ISO-induced male/female difference in [Na(+)](i) accumulation or contractile function was blocked by the nitric oxide synthase inhibitor, N(omega)-nitro-l-arginine methyl ester (1 micromol/l). Furthermore, in ISO-treated hearts, the Na(+)/K(+)-ATPase inhibitor, ouabain (200 micromol/l) did not abolish the male/female difference in [Na(+)](i) accumulation during I/R or functional protection. Thus the data show that the sex difference in the [Na(+)](i) regulation is mediated through a NO-dependent mechanism, and the difference in susceptibility to I/R injury appears to result from a difference in Na(+) influx.     

Increased mitochondrial K(ATP) channel activity during chronic myocardial hypoxia: is cardioprotection mediated by improved bioenergetics?

Increased resistance to myocardial ischemia in chronically hypoxic immature rabbit hearts is associated with activation of ATP-sensitive K(+) (K(ATP)) channels. We determined whether chronic hypoxia from birth alters the function of the mitochondrial K(ATP) channel. The K(ATP) channel opener bimakalim (1 micromol/L) increased postischemic recovery of left ventricular developed pressure in isolated normoxic (FIO(2)=0.21) hearts to values (42+/-4% to 67+/-5% ) not different from those of hypoxic controls but did not alter postischemic recovery of developed pressure in isolated chronically hypoxic (FIO(2)=0.12) hearts (69+/-5% to 72+/-5%). Conversely, the K(ATP) channel blockers glibenclamide (1 micromol/L) and 5-hydroxydecanoate (5-HD, 300 micromol/L) attenuated the cardioprotective effect of hypoxia but had no effect on postischemic recovery of function in normoxic hearts. ATP synthesis rates in hypoxic heart mitochondria (3.92+/-0.23 micromol ATP. min(-1). mg mitochondrial protein(-1)) were significantly greater than rates in normoxic hearts (2.95+/-0.08 micromol ATP. min(-1). mg mitochondrial protein(-1)). Bimakalim (1 micromol/L) decreased the rate of ATP synthesis in normoxic heart mitochondria consistent with mitochondrial K(ATP) channel activation and mitochondrial depolarization. The effect of bimakalim on ATP synthesis was antagonized by the K(ATP) channel blockers glibenclamide (1 micromol/L) and 5-HD (300 micromol/L) in normoxic heart mitochondria, whereas glibenclamide and 5-HD alone had no effect. In hypoxic heart mitochondria, the rate of ATP synthesis was not affected by bimakalim but was attenuated by glibenclamide and 5-HD. We conclude that mitochondrial K(ATP) channels are activated in chronically hypoxic rabbit hearts and implicate activation of this channel in the improved mitochondrial bioenergetics and cardioprotection observed.     

Chlorpromazine inhibits loss of contractile function, compliance and ATP in ischemic rabbit heart

Using a modified Langendorff preparation, rabbit hearts were either continuously perfused at 37 degrees C for 150 min, in the presence of O2 and substrate, or after a 30 min equilibration period exposed to global ischemia followed by 30 min of reperfusion. Ischemia, for 90 min at 27 decrees C or for 60 min at 37 degrees C was compared. Perfusion pressure, heart rate, and ventricular volume were maintained constant. Contractile function and metabolic status were assessed. The effect of chlorpromazine, administered (30 mg/kg IP) 30 min prior to sacrifice, was compared to the untreated animal. (1) Chlorpromazine had little effect on the contractile function or metabolic status of hearts continuously perfused for 150 min in the presence of O2 and substrate. (2) The chloropromazine-treated hearts maintained contractile function and metabolic status at preischemic levels following exposure to 90 min of global ischemia at 27 degrees C and reperfusion at 37 degrees C. Untreated hearts exhibited a severe deterioration in both contractile and metabolic parameters under the same conditions. Both untreated and chlorpromazine-treated hearts exhibited loss of contractile function after 60 min ischemia at 37 degrees C; untreated hearts had undetectable ATP levels while chlorpromazine-treated hearts exhibited low levels of ATP. (3) Untreated hearts, exposed to 90 min of ischemia at 27 degrees C, exhibited a significant loss in compliance, while the compliance of chlorpromazine-treated hearts was unchanged from pre-ischemic levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Na+-H+ exchanger protects diabetic and non-diabetic hearts from ischemic injury: insight into altered susceptibility of diabetic hearts to ischemic injury.

It has been previously suggested that alterations in sodium homeostasis, leading to calcium overload may play a part in the mediation of cardiac ischemic injury. It has been demonstrated that the Na+-H+ exchanger plays an important role with regard to the regulation of intracellular sodium during ischemia and reperfusion and that inhibition of the Na+-H+ exchanger during ischemia protects hearts from ischemic injury. Studies using chemically-induced diabetic animals have suggested that the cardiac Na+-H+ exchanger in the diabetic heart is impaired and is responsible for limiting the increase in sodium during ischemia. The extent to which the Na+-H+ exchanger contributes to increases in intracellular sodium during ischemia in diabetic hearts is unclear as direct measurements of exchanger activity have not been made in genetically diabetic hearts. Therefore, this paper aims to address the following issues: (a) is the Na+-H+ exchanger impaired in a genetically diabetic rat heart: (b) does this impairment result in lower [Na]i or [Ca]i during ischemia; and (c) does Na+-H+ exchanger inhibition limit injury and functional impairment in diabetic hearts during ischemia and reperfusion? These issues were examined by inhibiting the Na+-H+ exchanger with ethylisopropylamiloride (EIPA) in isolated perfused hearts from both genetically diabetic (BB/W) and non-diabetic rats. Levels of intracellular sodium, intracellular calcium, intracellular pH and high energy phosphates (using 23Na,19F, 31P NMR spectroscopies, respectively) during global ischemia and reperfusion were also measured. The impact of diabetes on Na+-H+ exchanger activity was assessed by measuring pH recovery of these hearts after an acid load. Creatine kinase release during reperfusion was used as a measure of ischemic injury. This study demonstrated that the Na+-H+ exchanger is impaired in diabetic hearts. Despite this impaired activity, inhibition of Na+-H+ exchanger protected diabetic hearts from ischemic injury and was associated with attenuation of the rise in sodium and calcium, and limitation of acidosis and preservation of ATP during ischemia. The data presented here favor the use of Na+-H+ exchanger inhibitors to protect ischemic myocardium in diabetics. Also, the data provides possible mechanisms for the altered susceptibility of diabetic hearts to ischemic injury.     

Nicorandil improves post-ischemic myocardial dysfunction in association with opening the mitochondrial K(ATP) channels and decreasing hydroxyl radicals in isolated rat hearts.

BACKGROUND: Nicorandil has been reported to induce cardioprotection by opening the mitochondrial K(ATP) channels. However, whether nicorandil affects reactive oxygen species is unclear. METHODS AND RESULTS: The hearts of male Sprague-Dawley rats were excised and perfused on a Langendorff apparatus with Krebs-Henseleit solution with a gas mixture of 95% O(2) and 5% CO(2). 1 mmol/L of nicorandil was given 10 min before ischemia. Left ventricular developed pressure (LVDP, mmHg), +/-dP/dt (mmHg/s) and coronary flow (ml/min) were continuously monitored. All hearts were perfused for a total of 120 min consisting of a 30 min pre-ischemic period, followed by a 30 min global ischemia and 60 min reperfusion with and without 5-hydroxydecanoic acid sodium salt (5-HD), a mitochondrial K(ATP) channel blocker. The concentrations of 2,3-dihydroxybenzoic acid (2,3-DHBA), an indicator of hydroxyl radicals, in the perfusate during reperfusion period were also measured. Nicorandil significantly improved LVDP and +/-dP/dt, and increased coronary flow during reperfusion. Pretreatment with 5-HD abolished the improvement of LVDP and +/-dP/dt, and the increase in coronary flow induced by nicorandil. Nicorandil significantly attenuated the concentrations of 2,3-DHBA during reperfusion, which were restored by 5-HD. CONCLUSION: Nicorandil is protective against post-ischemic left ventricular dysfunction in association with opening the mitochondrial K(ATP) channels, decreasing hydroxyl radicals and increasing coronary flow in the isolated rat heart.     

Relationships between pre-ischemic ATP and glycogen content and post-ischemic recovery of rat heart

The effect of depletion of energy stores of rat hearts on their resistance to a total of 25 min ischemia was investigated by using a 31P-NMR method. Three experimental groups were compared: (1) pyruvate-perfused hearts depleted of adenine nucleotides (35% of normal) by 2-deoxyglucose (DG) treatment and containing deoxyglucose-6-phosphate (c. 40 mumol/g dry wt); (2) hearts partially depleted of glycogen stores (40 to 50% of initial) by long-term (2h) perfusion with pyruvate; (3) glucose perfused (11 nM) hearts with normal ATP and glycogen contents. By the end of ischemia the intracellular pH was decreased by 0.33, 0.90 and 1.40 units, respectively. Time to peak of ischemic contracture increased in this series from 3 to 18 and 24 min, respectively. At the peak of ischemic contracture ATP content was c. 30 to 40% (6 to 8 mumol/g dry wt) of normal value in all three groups. Reperfusion of hearts resulted in development of significant reperfusion contracture in glucose-perfused hearts and minor contracture in other series. Recovery of high energy phosphates and cardiac work index in DG-treated, glycogen-depleted and glucose-perfused hearts were: for phosphocreatine (PCr), 72, 102 and 83%; for ATP, 29, 47 and 56% and for cardiac work, 66, 78 and 24%, respectively. Recovery of cardiac work did not correlate linearly with tissue ATP. These data demonstrate that post-ischemic recovery of the contractile function of isovolumic heart may be dissociated from pre-ischemic myocardial ATP and glycogen contents. This dissociation can be explained by the two major factors: (1) the contribution of ischemic acidosis and catabolites accumulation to the cell damage and (2) by ATP compartmentation.     

Contribution of Na+/H+ exchange to Na+ overload in the ischemic hypertrophied hyperthyroid rat heart

OBJECTIVE: The mechanisms responsible for intracellular ion homeostasis in ischemic hypertrophied myocardium are not fully known. Moderately hypertrophied hyperthyroid hearts (T3) are characterized by the bioenergetic changes and increased Na+/H+ exchange (NHE) activity comparable with those observed in humans and experimental models of hypertrophy. Here we test the hypothesis whether NHE inhibition in T3 heart improves ion homeostasis during ischemia and contractile function during recovery. METHODS: We compared intracellular H+ (H+i) and Na+ (Na+i) accumulations during 28 min global ischemia in isolated perfused T3 and euthyroid (EUT) rat hearts with and without NHE inhibition by using 31P and 23Na NMR. Heart function was measured during control perfusion and 30 min following ischemic insult. RESULTS: In T3 hearts ischemia caused: (1) faster and greater Na+i accumulation (534+/-25% of preischemic level versus 316+/-22% in EUT, P<0.001); (2) lower acidification (pH(i) 6.66+/-0.66 versus 6.12+/-0.12 in EUT, P<0.001); and (3) faster hydrolysis of ATP. NHE inhibition (amiloride 1 mM) in T3 hearts lead to: (1) delayed and lower Na+i accumulation by 35+/-5%; (2) faster and greater acidification (pH(i) 6.45+/-0.15, P<0.05); (3) delayed ATP degradation; and (4) improved heart function during recovery. When NHE was inhibited, all T3 hearts (n=11) recovered 68+/-10% of their preischemic rate pressure product (RPP), while only two untreated T3 hearts (from 11) recovered approximately 40% of preischemic RPP. CONCLUSIONS: These data suggest that NHE inhibition could be useful intervention for the prevention of ischemic/reperfusion cell injury and could improve the function of the hypertrophied heart after acute ischemia.     

Effects of mitochondrial K(ATP) modulators on cardioprotection induced by chronic high altitude hypoxia in rats

OBJECTIVES: Adaptation of rats to intermittent high altitude hypoxia increases the tolerance of their hearts to acute ischemia/reperfusion injury. Our aim was to examine the role of mitochondrial ATP-sensitive potassium channels (K(ATP)) in this form of protection. METHODS: Adult male Wistar rats were exposed to hypoxia of 5000 m in a barochamber for 8 h/day, 5 days a week; the total number of exposures was 24-32. A control group was kept under normoxic conditions (200 m). Infarct size (tetrazolium staining) was measured in anesthetized open-chest animals subjected to 20-min regional ischemia (coronary artery occlusion) and 4-h reperfusion. Isolated perfused hearts were used to assess the recovery of contractile function following 20-min global ischemia and 40-min reperfusion. In the open-chest study, a selective mitochondrial K(ATP) blocker, 5-hydroxydecanoate (5 mg/kg), or openers, diazoxide (10 mg/kg) or BMS-191095 (10 mg/kg), were administered into the jugular vein 5 and 10 min before occlusion, respectively. In the isolated heart study, 5-hydroxydecanoate (250 micromol/l) or diazoxide (50 micromol/l) were added to the perfusion medium 5 or 10 min before ischemia, respectively. RESULTS: In the control normoxic group, infarct size occupied 62.2+/-2.0% of the area at risk as compared with 52.7+/-2.5% in the chronically hypoxic group (P<0.05). Post-ischemic recovery of contractile function (dP/dt) reached 60.0+/-3.9% of the pre-ischemic value and it was improved to 72.4+/-1.2% by adaptation to hypoxia (P<0.05). While 5-hydroxydecanoate completely abolished these protective effects of chronic hypoxia, it had no appreciable influence in normoxic groups. In contrast, diazoxide significantly increased the recovery of contractile function and reduced infarct size in normoxic groups only. The later effect was also observed following treatment with BMS-191095. CONCLUSION: The results suggest that opening of mitochondrial K(ATP) channels is involved in the cardioprotective mechanism conferred by long-term adaptation to intermittent high altitude hypoxia.     

Phentolamine prevents the adverse effects of adenosine on glycolysis and mechanical function in isolated working rat hearts subjected to antecedent ischemia

Adenosine inhibits glycolysis from exogenous glucose, reduces proton production and enhances post-ischemic left ventricular minute work (LV work) following ischemia in isolated working rat hearts perfused with glucose and fatty acids. In hearts partially depleted of glycogen by antecedent ischemic stress (AIS)--two cycles of ischemia (10 min) and reperfusion (5 min)--adenosine stimulates rather than inhibits glycolysis, increases proton production and worsens recovery of post-ischemic LV work. We determined if the switch in adenosine effect on glycolysis and recovery of LV work following ischemia in hearts subject to AIS was due to the reduction in glycogen content per se or because of alpha-adrenoceptor stimulation. One series of hearts underwent a 35-min period of substrate-free Langendorff perfusion (substrate-free glycogen depletion; SFGD) and a second series of hearts was subjected to AIS. Both series of hearts had a similar glycogen content (approximately 70 micromol/g dry wt) prior to drug treatment. In SFGD hearts perfused aerobically, adenosine (500 microM) inhibited glycolysis from exogenous glucose and reduced proton production. In SFGD hearts reperfused after prolonged ischemia, adenosine exerted similar effects on glucose metabolism and enhanced recovery of post-ischemic LV work (87.2 +/- 2.2% of preischemic values) relative to untreated hearts (25.9 +/- 13.3% of preischemic values). In AIS hearts perfused aerobically or subject to ischemia and reperfusion, phentolamine (1 microM) given in combination with adenosine, prevented adenosine-induced stimulation of glycolysis from exogenous glucose and reduced calculated proton production from glucose. Recoveries of post-ischemic LV work in AIS hearts for untreated, adenosine, phentolamine and adenosine/phentolamine groups were 34.4 +/- 11.4%, 8.6 +/- 3.9%, 16.3 +/- 13.5% and 73.2 +/- 13.1% respectively, of preischemic values. Glycogen depletion in the absence of ischemia does not switch the effect of adenosine from inhibition to stimulation of glycolysis or alter the cardioprotective properties of adenosine in hearts subject to ischemia and reperfusion. The detrimental switch in the metabolic and cardioprotective effects of adenosine, in hearts subject to AIS, can be prevented by phentolamine, an alpha-adrenoceptor antagonist. These data support the concept that modulation of glucose metabolism is an important factor in the mechanical functional recovery of the post-ischemic heart.     

Effect of D-600 on ischemic and reperfused rabbit myocardium: relation with timing and modality of administration

Summary In this study we have investigated the possibility that D-600, a phenylalkylamine calcium antagonist, protects the isolated rabbit heart against ischemia and reperfusion-induced damage.D-600 was either subcutaneously injected (2 mg/kg, twice daily for 5 to 6 days) in the rabbit before isolation of the heart, or delivered to the isolated hearts in the perfusate (10^–7 M), either at the onset of ischemia and during reperfusion, or only during post-ischemic reperfusion.Ischemia (90 min) was induced by reducing coronary flow from 25 to 1 ml/min, followed by 30 min of reperfusion. Myocardial damage was determined in terms of mechanical function, release of creatine phosphokinase (CPK) and noradrenaline, mitochondrial function, calcium homeostasis, and endogenous stores of ATP and creatine phosphate (CP). Administration of D-600 to the rabbits or to the isolated hearts at the time of ischemia exerted protection. There are four groups of evidence in support of this conclusion: 1) the rise in diastolic pressure during ischemia was diminished with greater recovery of developed pressure during reperfusion; 2) CPK and noradrenaline release during reperfusion were reduced; 3) the oxygen consumption and ATP generating capacities of mitochondria were better maintained; and 4) associated with this preservation of mitochondrial function was the maintenance of near normal calcium homcostasis and of endogenous ATP and CP stores. The two different modalities of administration did not produce substantially different results.When administered to the isolated hearts after the ischemic period, D-600 failed to improve mechanical recovery and release of endogenous substances. However, it reduced mitochondrial calcium overload and improved ATP production. The mechanism of the protective effect of D-600 seems to be multiple: energy-sparing effect, reduction of the toxicity mediated by endogenous catecholamines, and direct inhibition of mitochondrial calcium transport.     

Increased particulate partitioning of PKC epsilon reverses susceptibility of phospholamban knockout hearts to ischemic injury

Cytosolic Ca(2+) overload is a critical mediator of myocardial damage following cardiac ischemia-reperfusion. It has therefore been proposed that normalization of sarcoplasmic reticulum Ca(2+) cycling through inhibition or ablation of the Ca(2+) ATP-ase inhibitor phospholamban (PLN), which shows promise as a treatment for heart failure, could be beneficial in ischemic heart disease. However, a recent study has shown that globally ischemic PLN-deficient hearts exhibit increased ischemic injury, with impaired contractile, ATP, and phosphocreatine recoveries, compared to wild-type hearts. Since protein kinase C (PKC) family members are widely recognized as mediators of both post-ischemic injury and ischemic preconditioning, we assessed PKC levels in PLN-deficient hearts. Compared to genetically normal hearts, PLN-deficient hearts exhibited diminished particulate partitioning of PKC, a known cardioprotective PKC isoform, without alterations in the levels of membrane-associated PKC delta nor PKC alpha. To determine if decreased particulate partitioning of cardioprotective PKC epsilon was a cause of increased ischemic injury in PLN-deficient hearts, PLN-deficient mice were mated with mice expressing a myocardial-specific PKC epsilon translocation activator peptide, pseudo-epsilon receptor for activated kinase C (psi epsilon RACK). In psi epsilon RACK/PLN knockout (KO) hearts, PKC epsilon translocation to membranous cellular structures was augmented and this was associated with a significant acceleration of post-ischemic contraction and relaxation rates, as well as reduction of creatine phosphokinase release, compared to PLN-deficient hearts. Importantly, post-ischemic functional recovery reached pre-ischemic hyperdynamic values in psi epsilon RACK/PLN KO hearts, indicating super-rescue by the combination of PLN ablation and psi epsilon RACK expression. These findings suggest that diminished PKC epsilon particulate partitioning in PLN-deficient hearts is associated with attenuated contractile recovery upon ischemia-reperfusion and that increased translocation of PKC to membranous cellular structures confers full cardioprotection.     

Repetitive acidosis protects the ischemic heart: implications for mechanisms in preconditioned hearts.

Repetitive brief ischemic episodes (ischemic preconditioning, PC) result in transient intracellular acidosis and protect the heart from subsequent ischemic injury, potentially through a protein kinase C (PKC)-dependent mechanism. We hypothesized that repetitive brief acidification of the heart without concomitant ischemia would also protect the heart from ischemic injury via a PKC-dependent mechanism. Isolated rat hearts underwent 30 min of global ischemia following control perfusion (CTL), or after PC or repetitive acidosis (RA), in the presence of absence of chelerythrine, a specific PKC inhibitor. Intracellular pH, PCr and ATP were measured using 31P NMR spectroscopy, while intracellular sodium [Na]i was measured using 23Na spectroscopy. Na,K-ATPase activity was measured prior to ischemia and on reperfusion. Both PC and RA resulted in transient acidification prior to ischemia. Ischemic injury, as assessed by creatinine kinase (CK) release on reperfusion, was reduced in both the PC and RA hearts [63+/-14 and 16+/-4 IU/g dry weight (dw) respectively, v 705+/-72 IU/gdw for control P<0.001], and was associated with improved functional recovery on reperfusion. PC and RA each significantly reduced Na,K-ATPase activity prior to ischemia (8.18+/-0.47 and 7.76+/-0.54 micromol ADP/h/mg protein) when compared to control (11.05+/-0.54 micromol ADP/h/mg protein P<0.05), limited the rate of ATP depletion during ischemia, and resulted in more rapid normalization of [Na]i on reperfusion. Chelerythrine resulted in intermediate CK release in PC and RA hearts (443+/-48 and 375+/-72 IU/gdw, P<0.001 v PC, P<0.01 v control), but did not alter the rate of ATP depletion or [Na]i kinetics in either PC or RA hearts. PC and RA each protect the ischemic heart, having in common ATP preservation during ischemia and more rapid normalization of [Na]i on reperfusion. These effects, not modulated by protein kinase C, are consistent with the hypothesis that ATP preservation during ischemia provides enhanced substrate for sodium efflux via the Na,K-ATPase on reperfusion.     

31P NMR spectroscopy of hypertrophied rat heart: effect of graded global ischemia

To investigate the cause for the greater susceptibility of hypertrophied hearts to ischemic injury, we determined the interrelations of total work output, contractile function and energy metabolism in isolated, perfused normal and hypertrophied rat hearts subjected to graded global ischemia. Cardiac hypertrophy was induced by giving rats seven daily injections of either triiodothyronine (0.2 mg/kg) or isoproterenol (5 mg/kg). All hearts were perfused at an aortic pressure of 100 mmHg in the isovolumic mode in an NMR spectrometer (7.05 Tesla). Heart rate, developed pressure, and coronary flow were monitored simultaneously with changes in pH, creatine phosphate, ATP and inorganic phosphate. During pre-ischemic perfusion, the total work output (rate-pressure product) of hyperthyroid hearts was 28% higher than that of control hearts, whereas hearts from isoproterenol-treated animals showed no difference. However, when related to unit muscle mass, work was normal in hyperthyroid hearts and 26% lower after isoproterenol. Contractile function per unit myocardium (developed pressure/g wet weight) was lower in the hypertrophied hearts. ATP content was the same in all groups. Creatine phosphate decreased 41% after triiodothyronine and 25% after isoproterenol. Inorganic phosphate levels and intracellular pH were similar in control and isoproterenol-treated rat hearts, but were higher in the hyperthyroid rat hearts. The phosphorylation potential and the free energy change of ATP hydrolysis were lowered by hypertrophy, the levels correlating with the depressed contractile function. At each ischemic flow rate, both work and contractile function per unit myocardium were the same for all hearts, but the relations between flow and phosphorylation potential were different for each type of heart. Thus, at low flow rates, hypertrophied hearts perform the same amount of work and have the same contractile function as control hearts, but with abnormal changes in energy metabolism, indicating that the relations of energy status to coronary flow, total work output and contractile function are altered during the process of hypertrophy.     

Expression of activated PKC epsilon (PKC epsilon) protects the ischemic heart, without attenuating ischemic H(+) production

PKC epsilon is a PKC isoform that translocates during preconditioning and may mediate cardioprotection. To investigate whether PKC epsilon activation is cardioprotective, Langendorff-perfused hearts from wild-type (WT) mice and from mice expressing constitutively active mutant PKC epsilon were subjected to 20 min ischemia and 40 min reperfusion while(31)P NMR spectra were acquired. Pre-ischemic glycogen levels were similar in WT and PKC epsilon hearts. During ischemia, ATP fell less in PKC epsilon than in WT hearts. Ischemic intracellular pH, however, was similar in WT and PKC epsilon hearts. During reperfusion, recovery of contractile function and ATP were greater in PKC epsilon than WT hearts. In conclusion, expression of activated PKC epsilon protected hearts from post-ischemic energetic and contractile dysfunction, consistent with the proposed cardioprotective role of PKC epsilon. Protection occurred in the PKC epsilon hearts without attenuation of ischemic H(+) production, implying that, at least in this ischemic model, reduced acidification during ischemia is not necessary for cardioprotection.     

Early Alteration of the Control of Mitochondrial Function in Myocardial Ischemia

This study was aimed at evaluating the nature of the early mitochondrial alterations in isolated perfused rat hearts subjected to ischemia (37°C, 0.1 ml/min, 15 or 30 min) and reperfusion (10 min). The functional variables of isolated perfused hearts were continuously evaluated, and the mitochondrial respiration variables were determined at the end of protocol on cardiac permeabilized fibers. In a parallel series of experiments, the myocardial contents of inorganic phosphate (Pi), phosphocreatine (PC) and ATP were monitored by means of P-31 NMR spectroscopy for the 15-min ischemia group. Severe mitochondrial alterations were detected in the 30-min ischemia group: decrease of maximal respiration rate and apparent Km for ADP, loss of the stimulatory effect of creatine (Cr) and disruption of the outer membrane. The functional recovery was no more than 10% of the pre-ischemic value. In contrast, in the 15-min ischemia group, only the stimulatory effect of Cr on respiration was significantly decreased. On reperfusion, a restoration of pre-ischemic levels of Pi and PC and a stabilization of the ATP content were observed, demonstrating the establishment of an energy balance steady state. The functional recovery was 76% of pre-ischemic value. We conclude that the alterations related to energy production control (by ADP and Cr) and to energy transfer are the earliest damages to mitochondrial function during ischemia. In spite of a preserved capacity for ATP production, these alterations, which persist on reperfusion, could be responsible for an altered responsiveness of the mitochondrial function to energy demand.     

Energy metabolism and contractile function in rat heart during graded, isovolumic perfusion using 31P nuclear magnetic resonance spectroscopy

The isolated, perfused heart is known to exhibit a linear relation between aortic pressure, coronary flow rate, oxygen consumption and contractile function (rate-pressure product) over a wide range of aortic pressures. Our study sought to determine whether the cytosolic phosphorylation potential [( ATP]/[ADP][Pi]) is the link between mitochondrial respiration and contractile function in this preparation. 31P NMR spectroscopy was used to measure phosphate metabolite levels in isovolumic rat hearts during graded perfusion from 1.6 to 12.8 ml/min/g. It was found that an increase in contractile function paralleled the increase in flow rate, but that marked changes in creatine phosphate, inorganic phosphate and hydrogen ion concentration occurred only at lower flow rates. The cytosolic phosphorylation potential showed a high, positive correlation with contractile function at flow rates below 7.2 ml/min/g, which suggested that mitochondrial respiration was oxygen-limited and that the heart was ischemic. Thus, when oxygen limits myocardial oxidative phosphorylation, cytosolic energy metabolite levels may limit contractile function. At the higher flow rates studied, other metabolic controls may operate to link mitochondrial respiration and workload.     

Chronic exposure to morphine produces a marked cardioprotective phenotype in aged mouse hearts

Aging is often associated with decreased myocardial ischemic tolerance. We recently reported that chronic preconditioning produced by continuous exposure to morphine affords a profound cardioprotective phenotype in young mice. In this study, we determined if chronic exposure to morphine retained its ability to precondition the myocardium in the young or aged heart. Young (10-14 weeks) or aged (24-26 months) C57/BL6 mice were untreated, administered morphine acutely (30 microM), or implanted with a morphine pellet (75 mg) for 5 days prior to heart isolation and perfusion. Following equilibration, perfused hearts were subjected to 25 min ischemia and 45 min reperfusion. Untreated hearts from both young and aged mice displayed marked contractile dysfunction and LDH release following reperfusion. Acute infusion of morphine improved recovery of end-diastolic pressure and developed pressure in young (P < 0.05 vs. untreated) but not senescent hearts. Hearts from mice exposed to morphine for 5 days displayed a further improvement in post-ischemic contractile function (P < 0.05 vs. acute treatment), and a marked reduction in post-ischemic LDH efflux (P < 0.05 vs. untreated) in both young and senescent hearts. These data demonstrate that aged hearts maintain the ability to be preconditioned by chronic exposure to morphine in the absence of acute protection.