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.     

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.     

Fasting in vivo delays myocardial cell damage after brief periods of ischemia in the isolated working rat heart

To assess the effects of fasting on recovery of function and exogenous glucose metabolism after 15 minutes of total ischemia, we perfused isolated working rat hearts from fed and fasted animals. Hearts were perfused in a recirculating system with bicarbonate buffer containing glucose (10 mM). Mechanical performance, release of marker proteins for ischemic membrane damage (lactate dehydrogenase, myoglobin, citrate synthase), and the concentrations of lactate and glucose in the perfusion medium were measured serially. Tissue metabolites were also measured. Fasting raised the myocardial glycogen content by 25%. Cardiac performance of perfused hearts from fed and fasted animals was the same during the preischemic and the post-ischemic period. The time of return of function to preischemic values was significantly less in hearts from fasted rats (2.3 versus 7.8 minutes, p less than 0.025). The release of cytosolic and mitochondrial marker proteins was significantly lower in hearts from fasted rats than in hearts from fed rats. Glucose metabolic rates during control and reperfusion were unchanged for hearts from fasted rats, but decreased for hearts from fed rats during reperfusion. The adenine nucleotide content at the end of ischemia was higher in hearts from fasted animals than in hearts from fed animals. We conclude that increasing glycogen levels prior to ischemia improves recovery of function, lessens membrane damage, and prevents loss of adenine nucleotides.     

Effects of propofol on ischemia and reperfusion in the isolated rat heart compared with thiamylal

The aim of the present study was to investigate whether clinical doses of propofol and thiamylal affect oxygen free radical production and intracellular calcium concentration ([Ca2+]i) in the post-ischemic reperfused heart. Forty-eight rat hearts were perfused with a Langendorff system and loaded with Fura-2 / AM as a [Ca2+]i marker. The hearts were divided into 6 groups as follows (each group: n = 8); Group S (saline), Group TL (thiamylal 100 microM), Group TH (thiamylal 300 microM), Group I (Intralipid), Group PL (propofol 3 microM), and Group PH (propofol 10 microM). All hearts were initially perfused for 5 min as control aerobic perfusion. Afterwards, no-flow ischemia was induced for 15 min, followed by reperfusion for 20 min. The formation of hydroxyl radicals in the coronary effluent was measured with high performance liquid chromatography using salicylic acid. At the beginning of the ischemia and reperfusion periods, increases in systolic and diastolic [Ca2+]i were observed in all groups except Group TH. The high dose of thiamylal significantly suppressed this initial increase in cytosolic [Ca2+]i (Group S 1.30+/-0.15; Group TL 0.99+/-0.17; Group TH 0.70+/-0.09, at 1 min after reperfusion; systolic [Ca2+]i : p < 0.05). Total DHBAs in the coronary effluent of all groups increased significantly 1 min after reperfusion, however, there were no significant differences among the groups. Clinical doses of propofol had no significant effect on myocardial function and [Ca2+]i before and after ischemia, whereas thiamylal suppressed the increase in [Ca2+]i during ischemia and reperfusion. However, free radical formation during reperfusion was unaffected by thiamylal and propofol.     

Protective effect of pretreatment with the calcium antagonist anipamil on the ischemic-reperfused rat myocardium: a phosphorus-31 nuclear magnetic resonance study

To assess whether the prophylactic administration of anipamil, a new calcium antagonist, protects the heart against the effects of ischemia and reperfusion, rats were injected intraperitoneally twice daily for 5 days with 5 mg/kg body weight of this drug. The heart was then isolated and perfused by the Langendorff technique. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to monitor myocardial energy metabolism and intracellular pH during control perfusion and 30 min of total ischemia (37 degrees C), followed by 30 min of reperfusion. Pretreatment with anipamil altered neither left ventricular developed pressure under normoxic conditions nor the rate and extent of depletion of adenosine triphosphate (ATP) and creatine phosphate during ischemia. Intracellular acidification, however, was attenuated. On reperfusion, hearts from anipamil-pretreated animals recovered significantly better than untreated hearts with respect to replenishment of ATP and creatine phosphate stores, restitution of low levels of intracellular inorganic phosphate and recovery of left ventricular function and coronary flow. Intracellular pH recovered rapidly to preischemic levels, whereas in untreated hearts a complex intracellular inorganic phosphate peak indicated the existence of areas of different pH within the myocardium. It is concluded that anipamil pretreatment protects the heart against some of the deleterious effects of ischemia and reperfusion. Because this protection occurred in the absence of a negative inotropic effect during normoxia, it cannot be attributed to an energy-sparing effect during ischemia. Therefore, alternative mechanisms of action are to be considered.     

Pathophysiology and pathogenesis of contractile failure in stunned myocardium

To investigate excitation-contraction coupling in stunned myocardium, intracellular free calcium concentration [( Ca2+]i) was measured before and after ischemia in perfused hearts using gated 19F NMR and the Ca2+ indicator 5F-BAPTA. Maximal Ca(2+)-activated force was also measured in parallel experiments. Stunned myocardium was created by reperfusion after 15 min global ischemia at 37 degrees C in isolated ferret hearts. In stunned myocardium, peak [Ca2+]i was paradoxically higher than that in control, but maximal Ca(2+)-activated pressure was lower in stunned hearts. These results indicate that contractile failure in stunned myocardium is due to a decrease in the myofilament sensitivity to Ca2+ as well as to a decrease in maximal Ca(2+)-activated force; failure of activator Ca2+ delivery cannot be implicated. The role of intracellular calcium overload in the pathogenesis of stunned myocardium was also investigated. Time-averaged 19F NMR measurements directly revealed the increase in [Ca2+]i during ischemia and in the early phase of reperfusion. The strategies to prevent Ca overload during reperfusion with modified reperfusate succeeded in preserving contractile function. Transient Ca overload without ischemia induced by different causes, i.e., high [Ca]0 perfusion, ventricular fibrillation or treatment with adriamycin, also produced contractile dysfunction that outlasted the interventions themselves. Thus, we propose that transient Ca overload during ischemia and early reperfusion initiates long-lasting contractile dysfunction in stunned myocardium.     

Prevention of transcoronary macromolecular leakage after ischemia-reperfusion by the calcium entry blocker nisoldipine. Direct observations in isolated rat hearts

Coronary microvascular damage appears to play a role in reperfusion injury after myocardial ischemia. This study was designed to afford direct viewing of the effects of myocardial ischemia-reperfusion on the coronary microcirculation and to determine whether pretreatment with the calcium blocker nisoldipine would attenuate any microvascular damage during reperfusion. Four groups of isolated rat hearts were perfused with a solution that contained red cells and fluorescent albumin, but was essentially free of platelets and leukocytes. Group I served as a nonischemic control. Group II hearts were subjected to 30 minutes of no-flow ischemia followed by reperfusion. Group III hearts were pretreated with nisoldipine (1 microgram/min) for 5 minutes before ischemia, and group IV hearts were treated with nitroglycerin (93 micrograms/min) before and after ischemia to mimic the vasodilation caused by nisoldipine. Perfused coronary capillarity and transcoronary extravasation of plasma albumin were measured by direct visualization techniques before and after ischemia. For group I, there was no significant change in coronary resistance, perfused capillarity, or transcoronary extravasation with time. For both groups II and IV, ischemia-reperfusion caused no increase in coronary resistance, but a significant decrease in perfused capillarity and a marked increase in transcoronary extravasation of fluorescent albumin (P less than 0.05). The nisoldipine group (group III) demonstrated a similar decrease in perfused capillarity but no increase in protein extravasation during reperfusion. These results indicate that, in the heart, platelets and/or leukocytes are not absolutely necessary to induce either the no-reflow phenomenon or the permeability damage observed during reperfusion after ischemia. The protective effect of treatment with nisoldipine appeared to be independent of vasodilation. We speculate that this calcium blocker reduced endothelial uptake of calcium during reperfusion, preventing endothelial deformation and formation of interendothelial gaps.     

Diltiazem in myocardial recovery from global ischemia and reperfusion

Effects of diltiazem on global myocardial ischemia and reperfusion have been examined in the isolated perfused guinea-pig heart. Diltiazem (8 × 10^?7m to 2.5 × 10^?5m) produced negative inotropic effects in nonischemic hearts. Hearts treated with diltiazem during low-flow ischemia which lasted 45 min followed by a 30 min reperfusion period showed significantly greater recovery of contractility. However, left ventricular end diastolic pressure remained elevated when compared to preischemic measurements. Diltiazem treatment resulted in significant amelioration of the increased wet wt/dry wt ratio and increased ATP and creatine phosphate levels during reperfusion. However, these parameters remained altered compared to nonischemic hearts. Pyruvate dehydrogenase in its active form (PDH_a) was significantly decreased by ischemia. Diltiazem treatment partially alleviated the decrease in PDH_a. These results suggest that diltiazem provides significant protection of myocardial function during ischemia and reperfusion.     

Effects of calcium antagonists and free radical scavengers on myocardial ischemia and reperfusion injury: evaluation by 31P-NMR spectroscopy

The Langendorff perfused rat heart was used to investigate whether myocardial damage during ischemia and reperfusion could be protected by free radical scavengers, calcium antagonist and adenosine. Myocardial high energy phosphates were measured by phosphorus-31 NMR spectroscopy during normal perfusion, 20 min of ischemia and 20 min of reperfusion. In hearts, which were treated both with free radical scavengers (FRS) (Superoxide dismutase): 24 IU/ml and catalase 22 IU/ml) and verapamil (10(-7) M), beta-ATP was significantly higher than that of FRS at the end of ischemia. However, beta-ATP recovered only to 83% of baseline value at the end of reperfusion. In view of myocardial metabolism, verapamil treated hearts were good for recovery of creatine phosphate (PCr) but not ATP at the end of reperfusion. Hearts which were treated with only adenosine did not differ from control hearts. However, when hearts were treated with both verapamil and adenosine (10(-4) M), recovery of both ATP and PCr content was significantly greater than that of control hearts. These results suggested that pretreatment with both verapamil and adenosine before and after global ischemia could protect ischemic myocardium, but, further studies are necessary to clarify the precise mechanism of protection.     

Cardiac-specific ablation of the Na+-Ca2+ exchanger confers protection against ischemia/reperfusion injury

During ischemia and reperfusion, with an increase in intracellular Na+ and a depolarized membrane potential, Ca2+ may enter the myocyte in exchange for intracellular Na+ via reverse-mode Na+-Ca2+ exchange (NCX). To test the role of Ca2+ entry via NCX during ischemia and reperfusion, we studied mice with cardiac-specific ablation of NCX (NCX-KO) and demonstrated that reverse-mode Ca2+ influx is absent in the NCX-KO myocytes. Langendorff perfused hearts were subjected to 20 minutes of global ischemia followed by 2 hours of reperfusion, during which time we monitored high-energy phosphates using 31P-NMR and left-ventricular developed pressure. In another group of hearts, we monitored intracellular Na+ using 23Na-NMR. Consistent with Ca2+ entry via NCX during ischemia, we found that hearts lacking NCX exhibited less of a decline in ATP during ischemia, delayed ischemic contracture, and reduced maximum contracture. Furthermore, on reperfusion following ischemia, NCX-KO hearts had much less necrosis, better recovery of left-ventricular developed pressure, improved phosphocreatine recovery, and reduced Na+ overload. The improved recovery of function following ischemia in NCX-KO hearts was not attributable to the reduced preischemic contractility in NCX-KO hearts, because when the preischemic workload was matched by treatment with isoproterenol, NCX-KO hearts still exhibited improved postischemic function compared with wild-type hearts. Thus, NCX-KO hearts were significantly protected against ischemia-reperfusion injury, suggesting that Ca2+ entry via reverse-mode NCX is a major cause of ischemia/reperfusion injury.     

Intracellular free calcium concentration measured with 19F NMR spectroscopy in intact ferret hearts.

Changes in the intracellular free Ca2+ concentration, [Ca2+]i, mediate excitation-contraction coupling in the heart and contribute to cellular injury during ischemia and reperfusion. To study these processes directly, we measured [Ca2+]i in perfused ferret (Mustela putorius furo) hearts using 19F NMR spectroscopy to detect the 5,5'-difluoro derivative of the Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). To load cells, hearts were perfused with the acetoxymethyl ester derivative of 5,5'-F2-BAPTA. We measured 19F NMR spectra and left ventricular pressure simultaneously, at rest and during pacing at various external Ca concentrations [( Ca]o). Although contractile force was attenuated by the Ca2+ buffering properties of 5,5'-F2-BAPTA, the decrease in pressure could be overcome by raising [Ca]o. Our mean value of 104 nM for [Ca2+]i at rest in the perfused heart agrees well with previous measurements in isolated ventricular muscle. During pacing at 0.6-4 Hz, time-averaged [Ca2+]i increased; the effect of pacing was augmented by increasing [Ca]o. [Ca2+]i more than tripled during 10-20 min of global ischemia, and returned toward control levels upon reperfusion. This approach promises to be particularly useful in investigating the physiology of intact hearts and the pathophysiology of alterations in the coronary circulation.     

Amiloride delays the ischemia-induced rise in cytosolic free calcium

An increase in cytosolic free calcium (Cai) has been shown to occur early during ischemia in perfused rat, ferret, and rabbit hearts. It has been proposed that this increase in Cai may occur as a result of exchange of Nai for Cao, which occurs as a result of an increase in Nai arising from exchange of Nao for H+i. The latter exchange is stimulated by the intracellular acidification that occurs during ischemia. To test this hypothesis, we examined Cai, Nai, ATP, and pHi during ischemia in rats in the presence and absence of 1 mM amiloride, a Na-H exchange inhibitor. Cai was measured using 19F nuclear magnetic resonance (NMR) of 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetra-acetic acid (5F-BAPTA)-loaded rat hearts. Nai was measured using 23Na NMR, and the shift reagent 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetramethylenephosph onate (Tm[DOTP]-5) was used to separate Nai and Nao. ATP and pH were determined from 31P NMR measurements. During 20 minutes of ischemia, amiloride did not significantly alter the ATP decline but did significantly attenuate the rise in Nai and Cai. After 20 minutes of ischemia, time-averaged Cai was 1.0 +/- 0.2 microM (mean +/- SEM) in amiloride-treated hearts compared with 2.3 +/- 0.9 microM in nontreated hearts. After 20 minutes of ischemia, Nai in the untreated heart was threefold greater than control, whereas in the amiloride-treated heart, Nai was not significantly different from control. These data are consistent with the involvement of Na-Ca exchange in the rise in Cai during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rates of glycolysis and glycogenolysis during ischemia in glucose-insulin-potassium-treated perfused hearts: A 13C, 31P nuclear magnetic resonance study

The effects of 11.7 mM glucose, insulin, and potassium (GIK) on metabolism during ischemia were investigated in the perfused guinea pig heart using magnetic resonance spectroscopy. Intracellular metabolites, primarily glycogen and glutamate, were labeled with 13C by addition of [1-13C]glucose to the perfusate during a normoxic, preischemic period. 13C and 31P NMR spectroscopy was used to observe the metabolism of 13C-labeled metabolites simultaneously with high-energy phosphorus metabolites and pH. The extent of acidosis and the rate and amount of labeled lactate accumulation during ischemia were the same in control (3 mM glucose + insulin) and GIK-treated hearts. In contrast, the rate of labeled glycogen mobilization during ischemia in GIK-treated hearts was one third the rate observed in control hearts. These observations suggest that GIK decreased the rate of glycogenolysis during ischemia without affecting the rate of glycolysis. We propose that glucose contributed as a glycolytic substrate to a greater extent during ischemia in GIK-treated hearts than in hearts perfused with 3 mM glucose and insulin. The glycogen-sparing effect of GIK demonstrated in these studies could delay the onset of ischemic damage in a clinical setting by prolonging the availability of glycolytic substrate necessary for production of high-energy phosphate.     

Free radicals and calcium: simultaneous interacting triggers as determinants of vulnerability to reperfusion-induced arrhythmias in the rat heart

Using the isolated perfused rat heart with regional ischemia and reperfusion, we have two antiarrhythmic interventions (the spin trap agent PBN [N-tert-butyl-alpha-phenylnitrone] and perfusate calcium reduction), administered just before reperfusion, to investigate mechanisms determining the vulnerability of the heart to reperfusion-induced ventricular fibrillation. Hearts were subjected to regional ischemia (5, 10, 20, 30 or 40 min) followed by reperfusion. Four groups were studied for each ischemic time: (i) control hearts with no antiarrhythmic intervention; (ii) hearts perfused with PBN (30 mumol/l) during the final 1 min of ischemia and throughout reperfusion, (iii) hearts perfused with low-calcium buffer (0.4 mmol/l) during the final 1 min of ischemia and throughout reperfusion and (iv) hearts perfused with PBN (30 mumol/l) and low-calcium (0.4 mmol/l) during the final 1 min of ischemia and throughout reperfusion. In control hearts, a bell-shaped time-vulnerability curve was obtained with 0, 91, 67, 33 and 17% of the hearts exhibiting irreversible fibrillation during reperfusion after 5, 10, 20, 30 and 40 min of ischemia, respectively. In the PBN group, the values were 8, 41 (P less than 0.05), 41, 33 and 8%, respectively. In the calcium reduction group the values were 17, 50, 8 (P less than 0.05), 8 and 0, respectively. Thus, PBN caused a significant reduction in reperfusion-induced ventricular fibrillation after 10 min of ischemia but had no significant effect with reperfusion after 20 min of ischemia. In contrast, calcium reduction had no significant effect after 10 min of ischemia but caused a significant reduction after 20 min of ischemia. When PBN treatment with calcium reduction were combined we obtained significant anti-arrhythmic effects after both 10 min (P less than 0.05) and 20 min (P less than 0.05) of ischemia. The additive effects of these two interventions, and the different ischemic-times after which they are most effective, has led us to propose that multiple triggers, each with different underlying mechanisms may be capable of initiating events which lead to ventricular fibrillation.     

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.     

Improved functional recovery of the atherosclerotic rabbit heart subjected to normothermic global ischemia

Contractile function before, during and after a period of ischemia was evaluated in perfused hearts from rabbits fed either 2% cholesterol or a control diet for two months. Rabbits were sacrificed and the hearts were perfused by the Langendorff normothermic perfusion technique. After a 30-min baseline period, the hearts were subjected to a 60-min period of low flow ischemia (0.2 mL/min) with Krebs-Henseleit solution containing either 2.5 mM (normocalcemic) or 0.5 mM (hypocalcemic) calcium. Subsequently the hearts were reperfused for 30 mins. No significant differences in baseline contractile function (expressed by developed pressure and dP/dtmax) were observed between hearts from cholesterol-fed and control rabbits. Although, initially the degree of contracture, as measured by an increase in end diastolic pressure from baseline, was less in cholesterol-fed rabbit hearts, this difference did not persist beyond 40 mins of ischemic perfusion. Hypocalcemic ischemic perfusion was associated with a delay in development of contracture relative to normocalcemic perfusion in the hearts from cholesterol-fed rabbits. These results suggest an early resistance to ischemia by hearts from cholesterol-fed rabbits. Upon reperfusion, the hearts from control rabbits exhibited a sudden increase in contracture following ischemic perfusion with 0.5 mM calcium which was not observed in the hearts from rabbits fed a cholesterol diet. There was improved functional recovery and less contracture development post reperfusion in the hearts from cholesterol-fed rabbits, independent of the concentration of calcium used during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+ transient, Mg2+, and pH measurements in the cardiac cycle by 19F NMR.

19F NMR indicators have been used to measure the free cytosolic cation concentrations ([Mn+]i, where M is the atomic symbol and n is the value of the charge) of Ca2+, H+, and Mg2+ in perfused ferret hearts. The [Ca2+]i transient, cytosolic pH (pHi), and [Mg2+]i have also been followed at 16 phases in the cardiac cycle in hearts paced at 1.25 Hz at 30 degrees C. The initial [Ca2+]i rose rapidly after a 50-ms delay, was maximal at greater than 1.5 microM after 150 ms, and declined thereafter to the initial concentration. In contrast, no significant changes in pHi (pH 7.03 +/- 0.08) or [Mg2+]i (1.2 +/- 0.1 mM) were detected in the cycle. A decrease in developed pressure when the [Ca2+]i indicator (but not the pHi or [Mg2+]i indicator) was loaded into hearts was substantially reversed by the addition of 50 microM ZnCl2 to the perfusion medium. The Zn2+ was taken up into the myoplasm and displaced Ca2+ bound to the indicator, a symmetrically substituted difluoro derivative of 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), as evidenced by the appearance of the Zn-5FBAPTA resonance. The decrease in developed pressure caused by 5FBAPTA, therefore, may be due to its Ca2+ buffering effect on the myoplasm. By coloading hearts with the [Ca2+]i and pHi indicators, simultaneous measurement of several [Mn+]i was demonstrated, which should provide a useful addition to the methods available to monitor cardiac function and pharmacology.     

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.     

Evidence that cytosolic and ecto 5'-nucleotidases contribute equally to increased interstitial adenosine concentration during porcine myocardial ischemia

The purpose of this study was to determine the roles of cytosolic and ecto 5'-nucleotidase in myocardial ischemia-induced increases in interstitial fluid (ISF) adenosine. Pentobarbital anesthetized, open chest pigs were instrumented with two microdialysis fibers in the distally perfused bed of the left anterior descending (LAD) coronary artery to estimate ISF metabolites. Fibers in control hearts were perfused with standard Krebs buffer. In two additional groups, after collecting one dialysate sample with normal Krebs, fibers were perfused with buffer supplemented with either L-homocysteine thiolactone (5 mM) or the ecto 5'-nucleotidase inhibitor α, β-methylene adenosine 5'-diphosphate (AOPCP, 5 mM). Hearts were then submitted to 60 minutes LAD occlusion and two hours reperfusion. Dialysate nucleosides and AMP were measured by high performance liquid chromatography. The local delivery of homocysteine did not alter preischemic dialysate adenosine concentration (0.30 ± 0.04 μM) compared to pre-homocysteine infusion (0.39 ± 0.04 μM) or control hearts (0.36 ± 0.04 μM), but AOPCP significantly decreased preischemic dialysate adenosine levels (from 0.36 ± 0.02 to 0.14 ± 0.03 μM). During LAD occlusion both homocysteine and AOPCP reduced dialysate levels by approximately 50 %. At 30 minutes ischemia dialysate adenosine concentrations were 19.47 ± 2.72, 11.41 ± 2.44, and 7.93 ± 1.01 μ:M in control, homocysteine, and AOPCP hearts, respectively. AOPCP significantly increased dialysate AMP levels; at 60 minutes ischemia AMP levels were 6.22 ± 2.97 μM in control hearts and 38.60 ± 5.69 μM in AOPCP treated hearts. These results suggest that both cytosolic and ecto 5'-nucleotidase contribute to ischemia-induced increases in ISF adenosine in porcine myocardium. Received: 13 October 1998, Returned for revision: 5 November 1998, Revision received: 17 December 1998, Accepted: 4 January 1999     

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.