Correlation between cytosolic free calcium, contracture, ATP, and irreversible ischemic injury in perfused rat heart

The relations between ATP depletion, increased cytosolic free calcium concentration [( Cai]), contracture development, and lethal myocardial ischemic injury, as evaluated by enzyme release, were examined using 19F nuclear magnetic resonance to measure [Cai] in 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA)-loaded perfused rat hearts. Total ischemia at 37 degrees C was induced in beating hearts, potassium-arrested hearts, magnesium-arrested hearts, and hearts pretreated with 0.9 microM diltiazem to reduce but not abolish contractility. In the beating hearts, time-averaged [Cai], which is intermediate between the systolic and the basal [Cai], was 544 +/- 74 nM. In contrast, in the potassium- and magnesium-arrested hearts, the time-averaged values are lower than in beating hearts (352 +/- 88 nM for potassium arrest, 143 +/- 22 nM for magnesium arrest). During ischemia, ATP depletion, contracture, and a rise in [Cai] are delayed by cardiac arrest, but all occur more rapidly in the potassium-arrested hearts than in the magnesium-arrested hearts. The diltiazem-treated hearts were generally similar to the magnesium-arrested hearts in their response to ischemia. Under all conditions, contracture development was initiated after tissue ATP had fallen to less than 50% of control; invariably, there was a progressive rise in [Cai] during and following contracture development. Reperfusion with oxygenated perfusate shortly after peak contracture development resulted in a return of [Cai] to its preischemic level, resynthesis of creatine phosphate, no significant enzyme release, and no substantial loss of 5F-BAPTA from the heart. The data demonstrate that an increase in [Cai] precedes lethal myocardial ischemic injury. This rise in [Cai] may accelerate the depletion of cellular ATP and may directly contribute to the development of lethal ischemic cell injury.     

Amiloride in ouabain-induced acidification, inotropy and arrhythmia: 23Na & 31P NMR in perfused hearts.

The increase in intracellular sodium (Nai), resulting from inhibition of the Na/K ATPase by cardiac glycosides, is known to increase calcium influx via Na(+)-Ca2+ exchange, and thereby increase contractility. This increase in intracellular Ca2+ has been related to the development of intracellular acidification and enhanced activity of the Na(+)-H+ exchanger as a measure by the cell to prevent further acidification. Thus, the efflux of the H+ ions results in an additional increase in Nai. This may subsequently lead to an increased rate of Ca2+ influx and therefore to the potentiation of the effects of cardiac glycosides. To assess the role of Na(+)-H+ exchange in the mechanism of ouabain action in the beating heart we used amiloride, a known inhibitor of Na(+)-H+ exchange. Isolated rat hearts were perfused with either ouabain (50 microM) alone (n = 8, Group I), amiloride (1.0 mM) + ouabain (50 microM) (n = 8, Group II), or amiloride (1.0 mM) alone as a control group (n = 4, Group III). 23Na and 31P NMR spectroscopy were used to assess the changes in Nai and intracellular pH (pHi), respectively, while simultaneous and continuous monitoring of left ventricular pressure was carried out. Perfusion with both ouabain alone (Group I) or ouabain + amiloride (Group II), resulted in a time dependent increase in Nai levels, reaching (within 25 mins) a maximum of 200 +/- 7% of control in Group I, and 170 +/- 10% of control in Group II. Concurrently, a mild but significant decrease in pHi was observed in both groups. This decrease, however, was significantly higher in Group II compared to Group I (0.34 pH units vs. 0.19 pH units, respectively; P less than 0.05), suggesting that inhibition of Na(+)-H+ exchange by amiloride limits the recovery from ouabain-induced intracellular acidification. While developed pressure gradually increased in Group I to a maximum of 268 +/- 52% of control, the addition of amiloride in Group II substantially reduced the positive inotropic effect. Ventricular fibrillation (VF) developed in three of the eight hearts in Group I within 10-13 mins after the addition of ouabain. Interestingly, the rate of Nai increase in hearts that sustained VF was significantly higher compared to those without VF (mean slope 10.1 +/- 2.11 vs. 3.9 +/- 1.0, respectively; P less than 0.0001). Ventricular fibrillation did not develop in Group II or III.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of Na+/H+ exchange inhibitors in cardiac ischemia.

To investigate a possible protective role of Na+/H+ exchange inhibition under ischemic conditions isolated rat hearts were subjected to regional ischemia and reperfusion. In these experiments all 6 untreated hearts suffered ventricular fibrillation on reperfusion. Addition of 1 x 10(-5) mol/l amiloride or 3 x 10(-7) mol/l 5-(N-ethyl-N-isopropyl)amiloride (EIPA) markedly decreased the incidence and duration of ventricular fibrillation or even suppressed fibrillation completely as in the case of 1 x 10(-6) mol/l EIPA. Both compounds diminished the activities of lactate dehydrogenase and creatine kinase in the venous effluent of the hearts during ischemia. At the end of the experiments tissue contents of glycogen, ATP and creatine phosphate were increased in the treated hearts as compared to control hearts. In an additional experiment the beneficial effects of Na+/H+ exchange inhibition during ischemia was confirmed in vivo with anaesthetized rats undergoing coronary artery ligation. In these animals amiloride or EIPA pretreatment caused a marked reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation. The concentration dependent inhibition of Na+ influx via Na+/H+ exchange by amiloride and EIPA was investigated in erythrocytes from hypercholesterolemic rabbits with Na+/H+ exchange activated by exposure to hyperosmotic medium. Furthermore the inhibition of Na+ influx by EIPA after intracellular acidification was studied in cardiac myocytes of neonatal rats. Both agents were effective in the same order of potency in the ischemic isolated working rat heart as in the erythrocyte model in which they inhibited Na+/H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of myocardial protection with potassium arrest in isolated ischemic rat hearts]

Isolated working rat hearts were exposed to 25 min ischemia, and functional recovery was assessed by aortic flow (AoF) and rate-pressure product (RPP) to evaluate the beneficial effects of potassium (20 mM) induced arrest (K-arrest) prior to ischemia. K-arrest improved the recovery of function after 30 min of reperfusion compared with the control group (%AoF: 68 +/- 6 vs 0%, %RPP: 90 +/- 3% vs 60 +/- 3%, p less than 0.01). The accumulation of Ca++ at the end of reperfusion was less in hearts with K-arrest (2.2 +/- 0.1 vs 4.5 +/- 0.3 mumol/g dry, p less than 0.01). There was no difference between the two groups in high energy phosphate content at the end of ischemia. The increase in intracellular Na+ (Nai) during ischemia was reduced in hearts with K-arrest (delta: 19 vs 46 mumol/g dry), and the level of intracellular K+ (Ki) was higher at the end of ischemia in hearts with K-arrest (341 +/- 4 vs 318 +/- 2 mumol/g dry, p less than 0.01). During the first 5 min of reperfusion, the level of Ki in K-arrested hearts jumped to a higher level than in the control group (delta: 15 vs 2 mumol/g dry, p less than 0.01). The level of Nai was lower in hearts with K-arrest after 5 min of reperfusion. These data suggested that K-arrest might preserve the activity of Na+/K+ ATPase during ischemia and early reperfusion, and that it attenuated the increase in Nai during ischemia and reperfusion, which resulted in less Ca++ overload during reperfusion via the Na+/Ca++ exchange mechanism and led to improved recovery.     

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.     

Na+ accumulation increases Ca2+ overload and impairs function in anoxic rat heart

Maintenance of low coronary flow (1 ml/min) during 40 or 70 min of anoxia maintained function and prevented Ca2+ overload during reoxygenation in isolated rat hearts. In comparison, recovery from 40 min of global ischemia resulted in only 20% of preischemic function and an increase in end-diastolic pressure (LVEDP) to 39 mmHg. Reperfusion Ca2+ uptake rose from 0.6 to 10.2 mumol/g dry tissue. Intracellular Na+ (Nai+) increased from 13 to 61 mumol/g dry tissue after 40 min of global ischemia, but was unchanged in hearts with low flow anoxia. When glucose and pyruvate were omitted from buffer used for anoxic perfusion, recovery was only 15% of preanoxic values, LVEDP rose to 32 mmHg, and reperfusion Ca2+ uptake was 7.2 mumol/g dry. In addition, Nai+ increased (47.4 mumol/g dry tissue) and ATP was depleted (1.0 mumol/g dry tissue) in the absence of substrate. In anoxic hearts supplied substrate, Nai+ stayed low (12 mumol/g dry tissue) and ATP was preserved (11.6 mumol/g dry tissue). Addition of ouabain (100 or 200 microM) and provision of zero-K+ buffer increased Nai+ and resulted in impaired functional recovery, increased LVEDP, and greater reperfusion Ca2+ uptake. These interventions also decreased energy availability in anoxic hearts. To distinguish between effects of Na+ accumulation and ATP depletion, monensin, a Na+ ionophore, was added during low flow anoxia. Monensin increased Nai+, decreased functional recovery and increased reperfusion Ca2+ uptake in a dose-dependent manner (1-10 microM) without changing ATP content. These results suggested that reduction of Nai+ accumulation by maintenance of Na+, K+ pump activity was the major mechanism of the beneficial effects of low coronary flow on reperfusion injury.     

Effects of inosine on glycolysis and contracture during myocardial ischemia

The effects of inosine (INO) on substrate metabolism and rigor formation in ischemic myocardium were examined in isolated rabbit hearts. Metabolite content was assessed in tissue extracts by chemical analysis and in the whole heart by 13C and 31P nuclear magnetic resonance spectroscopy. In ischemic hearts metabolizing either [3-13C]pyruvate or [1-13C]glucose, 1 mM INO increased both total and 13C-labeled alanine content; lactate content was unaffected. At 3 minutes of ischemia, tissue alanine was 1.81 +/- 0.11 microM/g wet wt (mean +/- SEM) in hearts perfused with pyruvate+INO versus 1.23 +/- 0.15 microM/g wet wt in hearts perfused with pyruvate alone (p less than 0.05). INO reduced tissue glycogen during ischemia in pyruvate-perfused hearts. Tissue alanine content in ischemic hearts that were supplied glucose+INO (1.29 +/- 0.13 microM/g wet wt) was greater than in ischemic hearts supplied glucose alone (0.65 +/- 0.14 microM/g wet wt). Alanine was found to originate from pyruvate and was a glycolytic end product in glucose-perfused hearts. INO raised the [3-13C]alanine/[3-13C]lactate ratio in ischemic, intact hearts (glucose = 0.24 +/- 0.07 versus glucose+INO = 0.60 +/- 0.09; pyruvate = 0.49 +/- 0.08 versus pyruvate+INO = 0.89 +/- 0.08). At 7 minutes of ischemia, ATP content fell to 70 +/- 3% with glucose+INO versus 58 +/- 5% with glucose alone. Rigor (stone heart) was delayed from 14.7 +/- 1.3 to 23.2 +/- 1.6 minutes with INO. INO did not change ATP content in ischemic hearts that were supplied pyruvate but delayed rigor (pyruvate = 9.9 +/- 1.2 minutes; pyruvate+INO = 15.6 +/- 1.0 minutes), possibly at the expense of glycogen. Supplemental glucose improved the effectiveness of INO with pyruvate to preserve ATP (pyruvate+glucose = 42 +/- 6%; pyruvate+glucose+INO = 72 +/- 6%) and further delayed rigor (pyruvate+glucose = 13.3 +/- 1.5 minutes; pyruvate+glucose+INO = 20.3 +/- 1.8 minutes). Glucose metabolism supported improved energetic and contractile states in ischemic hearts treated with INO. Thus, cardioprotection of the ischemic heart by INO was associated with preservation of functional integrity and improved energy production due to increased glycolytic activity. Activation of glycolysis in the presence of INO was accommodated by augmented alanine production without the additional accumulation of lactate.     

Intermittent perfusion of ischemic myocardium. Possible mechanisms of protective effects on mechanical function in isolated rat heart

Intermittent restoration of coronary flow during ischemia reduced myocardial damage and improved recovery of function. The mechanisms of the protective effects of intermittent perfusion were investigated in isolated rat hearts. Ventricular function was assessed as the product of developed pressure (left ventricular systolic pressure minus end-diastolic pressure) and heart rate. Recovery of function was calculated by division of the product at the end of reperfusion by that before ischemia. After 40 minutes of sustained global ischemia, intracellular Na+ (Nai) increased from 11 to 74 mumol/g dry wt. During 30 minutes of reperfusion, these hearts took up a large amount of 45Ca2+ (10 mumol/g dry wt), recovered only 24% of preischemic function, and had an increased left ventricular end-diastolic pressure (48 mm Hg). When the 40-minute period of ischemia was interrupted at 10-minute intervals by intermittent perfusion (three periods of 3 minutes) with either oxygenated or hypoxemic buffer, Nai increased to only 12 or 17 mumol/g dry wt, and reperfusion resulted in much lower 45Ca2+ uptake (0.5 and 0.5 mumol/g dry wt, respectively). Recovery of function was 100% of the preischemic value. When hypoxemic buffer without glucose was used for intermittent perfusion, Nai increased to 50 mumol/g dry wt, ATP was depleted, and reperfusion resulted in reduced recovery of function (76%) and moderately increased 45Ca2+ uptake (2.1 mumol/g dry wt). The role of Na(+)-K+ pump activity in maintaining low Nai was assessed by removing K+ from oxygenated or hypoxemic buffers used during intermittent perfusion. Under these conditions, Nai rose to 64 or 102 mumol/g dry wt, 45Ca2+ uptake increased to 4.4 or 9.4 mumol/g dry wt, and recovery of function was poor. There was a highly significant correlation between Nai during ischemia and reperfusion Ca2+ overload (r = 0.87) or impaired recovery of function (r = 0.96). These results indicate that prevention of an increase in Nai by maintenance of Na(+)-K+ pump activity is associated with a reduction of Ca2+ overload through Na+/Ca2+ exchange.     

Perfusate sodium during ischemia modifies post-ischemic functional and metabolic recovery in the rabbit heart

Metabolic and functional recovery following 60 minutes of low flow (0.1 ml/min) ischemia were compared in rabbit hearts perfused with normal sodium and potassium, low sodium (120 mM NaCl replaced by 120 mM LiCl), or zero potassium perfusate during ischemia. During the control, pre-ischemic, and reperfusion periods, all hearts were perfused identically with normal sodium and potassium. 31P NMR was used to monitor intracellular pH (pHi), ATP, and phosphocreatine (PGr). Developed pressure, end diastolic pressure, pHi, and the integrated areas of ATP and PCr were equivalent in the three groups in the pre-ischemic period. The fall in pHi, PCr, ATP, and developed pressure and the rise in end diastolic pressure during 60 min ischemia also did not differ among the three groups. In contrast to the lack of an effect of perfusate sodium and potassium on the decline in parameters of metabolism and function during ischemia, there was a marked difference in the recovery of these indices during reperfusion. Hearts perfused with low sodium during ischemia exhibited the best recovery (expressed as percent of control) of developed pressure (95 +/- 4%), PCr (106 +/- 6%), and ATP (51 +/- 2%) and the smallest rise in end diastolic pressure (229 +/- 50%); hearts perfused with normal sodium and potassium during ischemia had intermediate recovery values for developed pressure (53 +/- 10%), PCr (78 +/- 9%), ATP (45 +/- 4%) and end diastolic pressure (487 +/- 73%) and the hearts perfused with zero potassium solution during ischemia exhibited the poorest recovery of developed pressure (23 +/- 6%), PCr (49 +/- 6%), ATP (39 +/- 5%) and end diastolic pressure (968 +/- 185%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of ischemic myocardial cell damage assessed by phosphorus-31 nuclear magnetic resonance

Phosphorus-31 nuclear magnetic resonance (31P NMR) can estimate tissue intracellular pH as well as the content of high-energy phosphate metabolites in isolated perfused hearts. We used 31P NMR to examine mechanisms associated with the recovery of ventricular function in hearts subjected to global ischemia and reperfusion, with special emphasis on intracellular pH, a previously unreported variable. Single-dose and multiple-dose administration of a hyperkalemic cardioplegic solution were compared with hypothermia alone in 18 isolated perfused rabbit hearts. Hearts in group 1 were subjected to 24 degrees C hypothermia during 60 minutes of global ischemia; group 2 hearts received a single injection of 37-mM KCL cardioplegic solution at 10 degrees C at the onset of ischemia; and group 3 hearts received a similar initial cardioplegic injection followed by two subsequent 24 degrees C injections at 20-minute intervals during the ischemic period. Using an intraventricular balloon, maximal dP/dt provided a quantitative index of left ventricular performance before and after ischemia. Return of ventricular function expressed as a percentage of control was 54 +/- 11% for group 1, 84 +/- 6% for group 2, and 101 +/- 18% for group 3. Differences in the rate of development of intracellular acidosis were noted during the 60-minute ischemic period. Intracellular pH fell to 6.09 +/- 0.12 in group 1, 6.31 +/- 0.09 in group 2, an 6.79 +/- 0.03 in group 3. In all three groups intracellular pH returned to control (pH 7.20) within 10 minutes of reflow. The metabolic correlates of functional recovery appeared to be the tissue content of ATP at the end of ischemia and after reflow. ATP content at the end of ischemia was 22 +/- 2% of control in group 1 hearts, 31 +/- 4% in group 2 and 64 +/- 2% in group 3. After 45 minutes of reperfusion, ATP levels recovered to 33 +/- 9% of control in group 1, to 71 +/- 9% in group 2 and to 86 +/- 6% in group 3. Although there were no differences between groups in the content of creatine phosphate after 60 minutes of ischemia, the rates of creatine phosphate decline were dissimilar. Further, during the early reflow period, a marked overshoot in tissue creatine phosphate was detected, especially in groups 1 and 2. Histologic damage assessed by light microscopy correlated with the metabolic data, confirming that multidose cardioplegia provided the best preservation of cellular morphology. These results demonstrate that the magnitude of intracellular acidosis and the associated increase in inorganic phosphate correlate inversely with recovery of postischemic ventricular structure and function. ATP, but not creatine phosphate, content correlates with return of contractile performance after reperfusion. The overshoot in creatine phosphate during early reperfusion might impede optimal restoration of ATP content and, as a result, optimal recovery of cell functions.     

Activation of ATP-sensitive K+ channels by cromakalim. Effects on cellular K+ loss and cardiac function in ischemic and reperfused mammalian ventricle.

Pharmacological modulation of [K+]o accumulation and action potential changes during acute myocardial ischemia is under evaluation as a promising new antiarrhythmic and cardioprotective strategy during myocardial ischemia and reperfusion. We studied the effects of cromakalim, a K+ channel opener that activates ATP-sensitive K+ channels, in isolated arterially perfused rabbit interventricular septa subjected to ischemia and reperfusion and, through use of the patch clamp technique, in inside-out membrane patches excised from guinea pig ventricular myocytes. During aerobic perfusion, 5 microM cromakalim shortened action potential duration (APD) from 217 +/- 7 to 201 +/- 10 msec, had no effect on [K+]o, and reduced tension by 17 +/- 3% (n = 11). During ischemia, pretreatment with 5 microM cromakalim resulted in 1) more rapid APD shortening (71 +/- 9 versus 166 +/- 7 msec at 10 minutes and 63 +/- 12 versus 122 +/- 8 msec at 30 minutes), 2) similar [K+]o accumulation after 10 minutes (8.9 +/- 0.3 versus 9.6 +/- 0.5 mM) but a trend toward increased [K+]o accumulation after 30 minutes (11.0 +/- 1.7 versus 9.6 +/- 1.0 mM), and 3) similar times for tension to decline to 50% of control (2.14 +/- 0.16 versus 2.14 +/- 0.19 minutes) but shorter time to fall to 20% of control (4.34 +/- 0.33 versus 4.90 +/- 0.22 minutes; p = 0.003). After 60 minutes of reperfusion following 30 minutes of ischemia, recovery of function was similar, with a trend toward better recovery of developed tension (to 58 +/- 9% versus 39 +/- 10% of control; p = 0.18) and tissue ATP levels in cromakalim-treated hearts but no differences in APD or rest tension. Thus, 5 microM cromakalim had mild effects in normal heart but greatly accelerated APD shortening during ischemia without markedly increasing [K+]o accumulation, possibly because the more rapid APD shortening reduced the time-averaged driving force for K+ efflux through ATP-sensitive K+ channels. A significant cardioprotective effect during 30 minutes of ischemia plus 60 minutes of reperfusion could not be demonstrated in this model. In excised membrane patches studied at room temperature, the ability of cromakalim to activate ATP-sensitive K+ channels was significantly potentiated by 100 microM but not 15 microM cytosolic ADP, suggesting that in addition to the modest fall in cytosolic ATP during early ischemia, the rapid increases in cytosolic ADP may further sensitize cardiac ATP-sensitive K+ channels to activation by cromakalim.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular pH and role of Na+/H+ exchange during ischaemia and reperfusion of normal and diabetic rat hearts

STUDY OBJECTIVE--The aim was to investigate the role of intracellular pH (pHi) and ion exchange on the functional recovery of perfused hearts isolated from normal (N) rats either receiving or not receiving amiloride (an Na+/H+ exchange inhibitor), and from STZ induced diabetic (D) rats with decreased Na+/H+ exchange activity. DESIGN--Working heart preparations were submitted to a zero flow ischaemic period of 30 min at 37 degrees C and then reperfused for 30 min. The time course of pHi decline during ischaemia and of recovery on reperfusion was followed by means of 31P-NMR. MEASUREMENTS AND MAIN RESULTS--In N hearts without amiloride, ischaemia caused a progressive decrease in pHi. This was slightly, although not significantly, more abrupt in N hearts receiving amiloride. D hearts showed a slower fall in pHi, but the mean value reached after 30 min did not differ significantly from that of normal hearts. pHi recovery on reperfusion was markedly slower in the D hearts compared to N hearts. The mean value reached after 30 min did not differ significantly from that of N hearts. pHi recovery was also markedly slower in N hearts exposed to amiloride during both ischaemia and reperfusion. The higher functional recovery on reperfusion, as assessed by the recoveries of aortic flow and stroke volume, was observed for those hearts with slower pHi recovery. Improved recoveries of aortic flow and stroke volume as compared to normal non-treated hearts were 34% and 21% for the diabetic hearts, and 22% and 40% for the normal hearts receiving amiloride. CONCLUSIONS--The comparison of data from diabetic rat hearts with reduced activity of the Na+/H+ exchange process v normal hearts with pharmacological block of the exchanger provide support for a critical role of the Na+/H+ exchanger in the initial stage of reperfusion.     

Relation between glycolysis and calcium homeostasis in postischemic myocardium.

This study examined the hypothesis that glycolysis is required for functional recovery of the myocardium during reperfusion by facilitating restoration of calcium homeostasis. [Ca2+]i was measured in isolated perfused rabbit hearts by using the Ca2+ indicator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA) and 19F nuclear magnetic resonance spectroscopy. In nonischemic control hearts, inhibition of glycolysis with iodoacetate did not alter [Ca2+]i. In hearts subjected to 20 minutes of global zero-flow ischemia, [Ca2+]i increased from 260 +/- 80 nM before ischemia to 556 +/- 44 nM after 15 minutes of ischemia (p less than 0.05). After reperfusion with 5 mM pyruvate as a carbon substrate, [Ca2+]i increased further in hearts with intact glycolysis to 851 +/- 134 nM (p less than 0.05 versus ischemia) during the first 10 minutes of reperfusion, before returning to preischemic levels. In contrast, inhibition of glycolysis during the reperfusion period resulted in persistent severe calcium overload ([Ca2+]i, 1,380 +/- 260 nM after 15 minutes of reperfusion, p less than 0.02 versus intact glycolysis group). Furthermore, despite the presence of pyruvate and oxygen, inhibition of glycolysis during early reperfusion resulted in greater impairment of functional recovery (rate/pressure product, 3,722 +/- 738 mm Hg/min) than did reperfusion with pyruvate and intact glycolysis (rate/pressure product, 9,851 +/- 590 mm Hg/min, p less than 0.01). Inhibition of glycolysis during early reperfusion was also associated with a marked increase in left ventricular end-diastolic pressure during reperfusion (41 +/- 5 mm Hg) compared with hearts with intact glycolysis (16 +/- 2 mm Hg, p less than 0.01). The detrimental effects of glycolytic inhibition during early reperfusion were, however, prevented by initial reperfusion with a low calcium solution ([Ca]o, 0.63 mM for 30 minutes, then 2.50 mM for 30 minutes). In these hearts, the rate/pressure product after 60 minutes of reperfusion was 12,492 +/- 1,561 mm Hg/min (p less than 0.01 versus initial reflow with [Ca]o of 2.50 mM). These findings indicate that the functional impairment observed in postischemic myocardium is related to cellular Ca2+ overload. Glycolysis appears to play an important role in restoration of Ca2+ homeostasis and recovery of function of postischemic myocardium.     

Pre-treatment with the Na+/H+ exchange inhibitor cariporide delays cell-to-cell electrical uncoupling during myocardial ischemia

OBJECTIVE: Inhibition of Na(+)-H(+) exchange (NHE) delays the onset of myocardial rigor contracture during ischemia. The aim of this study was to analyse the effects of NHE inhibition on cell-to-cell electrical uncoupling during myocardial ischemia/reperfusion. METHODS: Twenty-six isolated rat hearts and 23 in situ porcine hearts were submitted to no-flow ischemia followed by reperfusion, with or without pre-treatment with cariporide (7 microM in rats and 3 mg/kg in pigs). Ischemic rigor and hypercontracture, conduction velocity and myocardial electrical impedance were monitored. RESULTS: Pre-treatment with cariporide delayed ATP depletion (luminescence assay in rat myocardium) and onset of rigor contracture (tension recordings or ultrasonic crystals) during ischemia both in rat and pig hearts (P<0.05). In addition, cariporide delayed the onset of sharp changes in tissue resistivity and phase angle in impedance recordings (four-electrode probes) from 10+/-1 to 13+/-1 min (P<0.001) in rat hearts, and from 22+/-1 to 38+/-2 min (P<0.001) in pigs. Blockade of impulse propagation (transmembrane action potentials in rat hearts) was also markedly delayed by cariporide (from 14+/-1 to 20+/-1 min, P<0.001). Reperfusion-induced LDH release in rat hearts and infarct size in pigs were markedly reduced by pre-treatment with cariporide. CONCLUSIONS: Inhibition of NHE with cariporide slows the progression of ischemic injury during myocardial ischemia, and delays the onset of cell-to-cell electrical uncoupling.     

Effects of amiloride and an analogue on ventricular arrhythmias, contracture and cellular injury during reperfusion in isolated and perfused guinea pig heart

The present study was designed to examine whether activation of Na+/H+ exchange and subsequent massive Ca2+ influx via Na+/Ca2+ exchange are involved in the pathogenesis of myocardial reperfusion injury. We tested the effects of 1 mM amiloride, which is known to inhibit both Na+/H+ and Na+/Ca2+ exchange, and 3 microM 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which is known to act as a specific inhibitor against Na+/H+ exchange, on the incidence of ventricular arrhythmias, isovolumic left ventricular function and creatine kinase (CK) release during reperfusion after 15 or 30 min of global ischemia in the isolated and perfused guinea pig heart. Treatment of a normally perfused heart with amiloride decreased heart rate significantly and tended to increase coronary flow and left ventricular developed pressure (LVDP), whereas treatment with EIPA decreased all of these 3 measurements significantly. Treatment with amiloride or EIPA for 15 min before ischemia, and during reperfusion after 15 min of ischemia, under electrical pacing at 240 rpm to eliminate a negative chronotropic effect abolished ventricular tachycardia (VT) and ventricular fibrillation (VF) during reperfusion associated with highly significant inhibition of increases in left ventricular end-diastolic pressure (LVEDP) and CK release. Amiloride or EIPA pretreatment also inhibited the incidence of VF and increases in LVEDP and CK release significantly during reperfusion after 30 min of ischemia. However, amiloride was more effective in preventing these events than EIPA. The treatment with amiloride or EIPA only during reperfusion after 15 or 30 min of ischemia also decreased the incidence of VF and inhibited the increases in LVEDP and CK release significantly, though less effectively than the pretreatment modality. These results suggest that EIPA prevents ventricular arrhythmias, contracture and myocardial cellular injury during reperfusion after 15 min of ischemia by inhibiting Na+/H+ exchange, while amiloride exerts more powerful protection against these events than EIPA during reperfusion after 30 min of ischemia by inhibiting both Na+/H+ and Na+/Ca2+ exchange.     

Adenosine-mediated early preconditioning in mouse: protective signaling and concentration dependent effects

OBJECTIVES: Signaling in adenosine-mediated preconditioning is controversial. We examined roles of mitochondrial (mito) K(ATP) channels, protein kinase C (PKC) and nitric oxide (NO). METHODS: Langendorff perfused C57/Bl6 mouse hearts were subjected to 20 min ischemia and 45 min reperfusion. Effects of adenosine-mediated preconditioning were assessed in the absence and presence of signaling inhibitors. RESULTS: Control hearts recovered 70+/-2 mmHg ventricular pressure, and released 18.1+/-2.0 IU/g lactate dehydrogenase (LDH). Preconditioning with 10 microM adenosine limited necrosis (10.6+/-1.4 IU/g) without modifying contractility (72+/-2 mmHg) whereas 50 microM adenosine reduced necrosis (10.3+/-1.6 IU/g) and contractile dysfunction (91+/-2 mmHg). All protective effects of 10 and 50 microM adenosine were abrogated by mito K(ATP) channel blockade with 100 microM 5-hydroxydecanoate (5-HD) during the 'trigger' phase, but unaltered by PKC or NO synthase inhibition with 3 microM chelerythrine or 100 microM N(G)-nitro-L-arginine methyl ester (L-NAME), respectively. Protection against necrosis was eliminated by 5-HD but unaltered by chelerythrine or L-NAME during the 'mediation' phase (ischemia-reperfusion). Reduced contractile dysfunction with 50 microM adenosine was partially sensitive to 5-HD and chelerythrine, and only eliminated by co-infusion of the inhibitors. CONCLUSIONS: Adenosine-mediated preconditioning is dose-dependent with high level stimulation reducing contractile dysfunction in addition to necrosis. Preconditioning is triggered by a mito K(ATP) channel dependent process independently of PKC and NO. Subsequent protection against necrosis is also mediated by a mito K(ATP) channel dependent process independent of PKC and NO. In contrast, functional protection may be mediated by parallel mito K(ATP) and PKC dependent paths.     

Enhancement of susceptibility to ouabain in ischemic rat heart]

During 10 mins of reperfusion after 25 mins global ischemia, subtoxic doses of ouabain (50, 100 microM) were used and followed by 20 mins reperfusion with standard buffer. At these doses ouabain had no harmful effects with 29% and 45% increase in developed pressure in aerobic hearts. Intracellular Na+ (Nai), 45Ca2+ uptake and recovery of ventricular function were measured. Nai increased from 15 to 64 mumol/g dw with no increase in 45Ca2+ uptake during ischemia. Upon reperfusion with standard buffer, additional gain in Nai at 2 mins (73 mumol/g dw) was followed by a rapid decline (at 10 mins: 48 mumol/g dw). 45Ca2+ uptake increased from 0.8 to 7.5 mumol/g dw after 30 mins reperfusion with decreased recovery of function (45%) and increased LVEDP (29 mmHg). Reperfusion with ouabain accelerated initial rise in Nai (2 mins: 79 and 83 mumol/g dw) and decline of Nai was retarded (10 mins: 65 and 83 mumol/g dw). Consequently, 45Ca2+ uptake and depression of function were augmented (Ca: 10.0, 11.5 mumol/g dw; function: 27%, 18%; LVEDP: 47, 48 mmHg) even when hearts were switched back to standard buffer. Combination of high K+ (20mM) reversed the effect of ouabain. The results suggested increased susceptibility to ouabain was caused by inhibited outward Na+ transport resulting in enhanced Ca2+ influx through Na+/Ca2+ exchange.     

Decreased myofilament responsiveness in myocardial stunning follows transient calcium overload during ischemia and reperfusion.

The purpose of this study was to test the hypothesis that abnormal intracellular calcium handling characterizes myocardial stunning. Isolated, isovolumic, buffer-perfused ferret hearts were loaded with the bioluminescent calcium indicator aequorin for simultaneous measurement of individual calcium transients and left ventricular pressure. After 15 minutes of global ischemia and 20 minutes of reperfusion, left ventricular developed pressure was significantly reduced (75 +/- 7 versus 93 +/- 6 mm Hg, p < 0.05). During ischemia, [Ca2+]i levels were significantly elevated compared with preischemic levels, both during systole (1.38 +/- 0.31 versus 0.88 +/- 0.2 microM, p < 0.05) and end diastole (0.85 +/- 0.16 versus 0.38 +/- 0.13 microM, p < 0.05). Early during reperfusion, [Ca2+]i was also significantly elevated during systole (1.63 +/- 0.44 versus 0.88 +/- 0.20 microM, p < 0.05) and end diastole (0.75 +/- 0.15 versus 0.38 +/- 0.13 microM, p < 0.05). After 20 minutes of reperfusion, myocardial stunning occurred, but [Ca2+]i was not significantly different from preischemic levels. Thus, myocardial stunning does not result from decreased levels of activator calcium. The force-pCa relation generated by the stunned hearts was shifted downward compared with that generated by the control hearts, consistent with a decrease in maximum calcium-activated force (Fmax). At steady state during tetanus, the decrease in Fmax was confirmed, but there was no significant difference in the slope of the force-pCa relation of the stunned hearts versus controls. Thus, we conclude that stunned myocardium is characterized by decreased Fmax without desensitization of the myofilaments to [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)     

The protective effect of enriched branched chain amino acid formulation in the ischemic heart: a phosphorous-31 nuclear magnetic resonance study

Branched chain amino acids (BCAA) have been found to have a protective effect on the ischemic myocardium. Isolated rat hearts were perfused with phosphate-free Krebs-Henseleit (KH) solution with or without BCAA. A Latex balloon-tipped catheter was inserted into the left ventricle to measure intracavitary pressures. Hearts were subjected to 18 minutes of 'no flow' global ischemia and then reperfused for 30 mins at 37 degrees C. Metabolism of high energy phosphates during ischemia and recovery was studied by P-31 NMR. Intracellular pH was calculated from the chemical shift of Pi. Pressure recovery was better with KH + BCAA (89 +/- 16%) than with KH (41 +/- 26%) (P = 0.0001); dP/dt recovery was also improved with BCAA (84 +/- 19% vs 27 +/- 27% for KH) (P = 0.0003). After 18 mins of ischemia, ATP levels in the BCAA group were higher than in the KH perfused hearts (33 +/- 20 vs 17 +/- 10% of pre-ischemic value) (P = 0.02). No significant difference was found in the intracellular pH at the end of the ischemic period. Following reperfusion the recovery of pH was better in the BCAA group (7.09 +/- 0.06 vs 7.04 +/- 0.06) (P = 0.03). These results show that BCAA protect the heart from myocardial ischemic injury, decrease depletion of ATP during ischemia, and enhance post-ischemic hemodynamic function.     

The genesis of arrhythmias during myocardial ischemia. Dissociation between changes in cyclic adenosine monophosphate and electrical instability in the rat

It has been proposed that increases in tissue cyclic adenosine monophosphate during ischemia may be responsible for the induction of arrhythmias that occur during the early minutes of ischemia. We have tested this hypothesis using the isolated perfused rat heart with coronary artery occlusion for 30 minutes. In control hearts, after a transient small rise, cyclic adenosine monophosphate content remained close to its preischemic value (3.0 +/- 0.1 nM/g dry weight) throughout the period of occlusion. Eight percent (1/12) of the hearts fibrillated. Ninety-two percent (11/12) of the hearts exhibited ventricular tachycardia, and the mean total number of premature ventricular complexes was 528 +/- 121. Inclusion of epinephrine (1.0 microM) in the perfusion fluid elevated cyclic adenosine monophosphate prior to coronary occlusion (to 10.7 +/- 0.6 nM/g dry weight) and also throughout the ischemic period. It also increased arrhythmias such that 83% (20/24) of hearts fibrillated, 100% exhibited ventricular tachycardia, and the mean number of premature ventricular complexes increased to 747 +/- 86. Inclusion of forskolin (0.2 microM), which stimulates adenyl cyclase independently of the beta-receptor, increased cyclic adenosine monophosphate content to a greater extent than epinephrine, to 14.1 +/- 0.9 nM/g dry weight before the onset of ischemia and to 8.2 +/- 0.4 nM/g dry weight after 30 minutes of ischemia. Despite the large increase in cyclic adenosine monophosphate, there was no increase in rhythm disturbances which were less than those seen in controls.(ABSTRACT TRUNCATED AT 250 WORDS)