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Isolated cardiomyocytes from adult rats were incubated in anoxic bicarbonate-buffered media at extracellular pH (pH(o)) 6.4 until a cytosolic Ca(2+) overload and intracellular pH (pH(i)) of 6.4 were reached. On reoxygenation, the pH of the medium was changed to 7.4 to activate the Na(+)/H(+)exchanger (NHE) and the Na(+)-HCO(-)(3) symporter (NBS). The reoxygenation was performed in the absence or presence of the NHE inhibitor HOE-642 (3 micromol/l) and/or the NBS inhibitor DIDS (0.5 mmol/l), as in bicarbonate-free media. In reoxygenated control cells pH(i) rapidly recovered to the preanoxic level, and a burst of spontaneous oscillations of cytosolic Ca(2+) occurred, accompanied by the development of hypercontracture. When NBS and NHE were simultaneously inhibited during reoxygenation, pH(i) recovery was prevented, Ca(2+) oscillations were attenuated, and hypercontracture was abolished. Sole inhibition of NBS or NHE showed no protection against hypercontracture. In the absence of cytosolic acidosis, HOE-642 or DIDS did not prevent hypercontracture induced by Ca(2+) overload. The results demonstrate that simultaneous inhibition of NHE and NBS is needed to protect myocardial cells against reoxygenation-induced hypercontracture.

The objective of this study was to investigate the effect of Na+-H+ exchange (NHE) and HCO3--Na+ symport inhibition on the development of rigor contracture. Freshly isolated adult rat cardiomyocytes were subjected to 60 min metabolic inhibition (MI) and 5 min re-energization (Rx). The effects of perfusion of HCO3- or HCO3--free buffer with or without the NHE inhibitor HOE642 (7 microM) were investigated during MI and Rx. In HCO3--free conditions, HOE642 reduced the percentage of cells developing rigor during MI from 79 +/- 1% to 40 +/- 4% (P < 0.001) without modifying the time at which rigor appeared. This resulted in a 30% reduction of hypercontracture during Rx (P < 0.01). The presence of HCO3- abolished the protective effect of HOE642 against rigor. Cells that had developed rigor underwent hypercontracture during Rx independently of treatment allocation. Ratiofluorescence measurement demonstrated that the rise in cytosolic Ca2+ (fura-2) occurred only after the onset of rigor, and was not influenced by HOE642. NHE inhibition did not modify Na+ rise (SBFI) during MI, but exaggerated the initial fall of intracellular pH (BCEFC). In conclusion, HOE642 has a protective effect against rigor during energy deprivation, but only when HCO3--dependent transporters are inhibited. This effect is independent of changes in cytosolic Na+ or Ca2+ concentrations.

The question was investigated whether cardiomyocytes can be resuscitated after extreme energy depletion, i.e. after loss of ATP >70%. Isolated ventricular cardiomyocytes of the adult rat were exposed to metabolic inhibition with dinitrophenol and cyanide (DNP 0.2 mm; KCN 2 mm). After rapid energy depletion, cells were "reoxygenated" by wash-out of DNP and KCN. Intracellular calcium, cell length, ATP and creatine phosphate (CrP) of the cardiomyocytes were monitored. Metabolic inhibition resulted in a depletion of the stores of ATP and CrP by more than 95% of the normoxic values and caused a cytosolic Ca2+ overload. Parameters of metabolic recovery were: (i) resynthesis of CrP; (ii) recovery of a normal cytosolic Ca2+ control; and (iii) the elicitation of energy-dependent hypercontracture. "Reoxygenation", i.e. wash-out of metabolic inhibitors, reactivated oxidative phosphorylation. Consecutively, CrP levels recovered to 76.0+/-7.3%, ATP levels recovered to 10. 4+/-2.3% (means+/-s.dn=10) of the initial normoxic values, a normoxic intracellular calcium level was re-established and hypercontracture was elicited. Prolongation of metabolic inhibition with KCN (2 mm) or inhibition of the Na+/K+ pump with ouabain (0.5 mm) disabled the cardiomyocytes to recover from cytosolic Ca2+ overload and prevented hypercontracture. It is concluded that even after extensive energy depletion metabolic resuscitation of the myocardial cell remains possible and a critical range of ATP for recovery, i.e. a "threshold" of a 70% loss of ATP, does not exist., (Copyright 1998 Academic Press)

Background: Resupply of oxygen to the myocardium after extended periods of ischemia or hypoxia can rapidly aggravate the already existing injury by provoking hypercontracture of cardiomyocytes (acute reperfusion injury). Previous studies indicated that halothane can protect ischemic-reperfused myocardium. The aim of the present study was to analyze on the cellular level the mechanism by which halothane may protect against reoxygenation-induced hypercontracture., Methods and Results: To simulate ischemia-reperfusion, isolated adult rat cardiomyocytes were incubated at pH 6.4 under anoxia and reoxygenated at pH 7.4 in the presence or absence of 0.4 mmol/L halothane. Reoxygenation was started when intracellular Ca2+ (measured with fura 2) had increased to > or = 10(-5) mol/L and pHi (BCECF) had decreased to 6.5. Development of hypercontracture was determined microscopically. In the control group, reoxygenation provoked oscillations of cytosolic Ca2+ (72+/-9 per minute at fourth minute of reoxygenation) accompanied by development of hypercontracture (to 65+/-3% of end-ischemic cell length). When halothane was added on reoxygenation, Ca2+ oscillations were markedly reduced (4+/-2 per minute, P<.001) and hypercontracture was virtually abolished (90+/-4% of end-ischemic cell length, P<.001). Halothane did not influence the recovery of pHi during reoxygenation. Similar effects on Ca2+ oscillations and hypercontracture were observed when ryanodine (3 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ release, or cyclopiazonic acid (10 micromol/L), an inhibitor of the sarcoplasmic reticulum Ca2+ pump, were applied instead of halothane., Conclusions: Halothane protects cardiomyocytes against reoxygenation-induced hypercontracture by preventing oscillations of intracellular Ca2+ during the early phase of reoxygenation.

After prolonged periods of energy depletion, myocardial cells may rapidly deteriorate during the early stage of reperfusion. It has now been clearly demonstrated that this kind of acute lethal reperfusion injury is due to specific processes elicited by cellular re-energization. The most prominent single cause of acute harm to the reoxygenated myocardial cells is myofibrillar hypercontraction. Hypercontraction is caused by a resupply of energy of the myofibrils at excessive cytosolic Ca2+ concentrations. Additionally, the ability of the cytoskeleton to withstand large mechanical forces seems to be weakened after a prolonged period of energy depletion. Intracellular acidosis during the early stage of reperfusion represents a natural mechanism of protection against acute reperfusion injury. The reperfused myocardial cell may also suffer from uncontrolled water uptake and increased sarcolemmal fragility, favoring osmotic damage of cell membranes. As yet therapeutical interventions trying to specifically interfere with these pathomechanisms of reperfusion injury have only been tested experimentally. It seems promising to evaluate their utility for myocardial protection in cardio-surgical operations.

Effects of Na+/H+ exchange inhibition and cytosolic acidosis on reoxygenated adult rat ventricular cardiomyocytes were investigated. Cells were incubated in anoxic media at pH 6.4 until pCa of < or = 5, intracellular pH (pHi) of 6.5, and cytosolic [Na+] of 50 mM were reached. On reoxygenation, medium pH was changed to 7.4 to activate Na+/H+ exchange. In one group, 20 microM HOE-694, an inhibitor of Na+/H+ exchange, was added. With or without HOE-694, cytosolic Ca2+ and Na+ returned to control levels within 10 min of reoxygenation. In the absence of HOE-694, the pHi renormalized (to 7.2) within 8 min, but irreversible hypercontracture and transient Ca2+ oscillations were observed. In the presence of HOE-694, pHi stayed acidotic (at 6.5), hypercontracture was prevented, and Ca2+ oscillations were attenuated. When the Na+ pump was inhibited with 0.1 mM ouabain, even partial recovery of Ca2+ control became impossible unless HOE-694 was added. Our conclusions are 1) activation of Na+/H+ exchange does not impair recovery of cytosolic Na+ and Ca2+ control unless activity of the sarcolemmal Na+ pump is critically reduced, and 2) due to prolongation of cytosolic acidosis, inhibition of Na+/H+ exchange protects against reoxygenation-induced hypercontracture and cytosolic Ca2+ oscillations.

The role of Na+ in the recovery from severe anoxic Ca2+ overload was investigated in isolated quiescent ventricular cardiomyocytes from adult rat. Changes of cytosolic Ca2+ and Na+ concentrations were followed by the fura 2 and Na(+)-binding benzofuran isophthalate techniques, respectively. When the fura 2 ratio (340/380 nm) reached saturation in anoxic cells, indicating a severe cytosolic Ca2+ overload, the cells were reoxygenated. This caused a rapid initial drop of cytosolic Ca2+ to a lower but still elevated level (phase I), followed by oscillatory Ca2+ transients at this level (phase II) and, within 10 min, the reestablishment of a stable cytosolic Ca2+ concentration at the normal resting level (phase III). As previously shown [B. Siegmund, R. Zude, and H. M. Piper. Am. J. Physiol. 263 (Heart Circ. Physiol. 32): H1262-H1269, 1992], Ca2+ shifts in phase I and II are mainly due to uptake and release of Ca2+ by the sarcoplasmic reticulum. Phase I was unchanged, and phase II was much prolonged (> 60 min) in cells reoxygenated under Na+ pump inhibition (0.2 mM ouabain) or Na+ depletion. Phase III could only be reestablished (< 10 min) when ouabain was eluted or external Na+ replenished, respectively. The results show that full recovery of cytosolic Ca2+ control (phase III) requires an active sarcolemmal Na+ pump and the availability of external Na+. This indicates that phase III is determined by the transsarcolemmal extrusion of Ca2+ by a tandem mechanism consisting of 1) the Na+ pump, generating an extracellular-to-intracellular Na+ gradient, and 2) the sarcolemmal Na+/Ca2+ exchange, driven by that gradient to extrude Ca2+.

When oxygen-deprived cardiomyocytes become energy depleted, they accumulate Na+ and Ca2+ in the cytosol. Influx of Ca2+ via the Na+/Ca2+ exchange mechanism seems to contribute to the development of Ca2+ overload, but Ca2+ overload may eventually also occur when this route is blocked. Hypoxic-reoxygenated cardiomyocytes in a state of severe overload of Na+ and Ca2+ can rapidly re-establish a normal cation control when oxidative energy production is re-initiated. The recovery of cellular Ca2+ control may be divided into three stages: first, sequestration of large amounts of Ca2+ into the sarcoplasmic reticulum; second, oscillatory movement of Ca2+ from and back into the sarcoplasmic reticulum and gradual extrusion across the sarcolemma; third, re-establishment of constant low cytosolic Ca2+ concentrations. When the Na+/Ca2+ exchanger is inhibited, extrusion of Ca2+ from the cells' interior is impaired and oscillatory Ca2+ movements between cytosol and sarcoplasmic reticulum continue for long time. Thus, the functions of the sarcoplasmic reticulum and the Na+/Ca2+ exchanger are of crucial importance for the recovery of Ca2+ control in reoxygenated cardiomyocytes. In re-energized cardiomyocytes, a persistent elevation of the cytosolic Ca2+ concentration provokes maximal force development and consecutive mechanical cell injury ("oxygen paradox"). This injury can be prevented when the contractile machinery is inhibited during the initial phase of reoxygenation as long as necessary for the re-establishment of a normal cytosolic Ca2+ control.

The ability of hypoxic-reoxygenated cardiomyocytes to recover from severe cytosolic Ca2+ overload was investigated using the fluorescent Ca2+ indicator fura-2 in ventricular cardiomyocytes from adult rats. When the fura-2 ratio (340/380 nm) reached saturation in hypoxic cardiomyocytes, indicating severe Ca2+ overload, they were reoxygenated. The cell then suddenly hypercontracted but reestablished, after a phase of Ca2+ oscillations, a normal Ca2+ control. Because these oscillations could be abolished by ryanodine (50 nM), they seem to depend on the function of the sarcoplasmic reticulum (SR). In the presence of caffeine (5 mM) and thapsigargin (100 nM), i.e., agents impairing Ca2+ sequestration in the SR, reoxygenation did not lead to Ca2+ oscillations or to a stable recovery of cytosolic Ca2+ control. The additional presence of ruthenium red (5 microM), an inhibitor of mitochondrial Ca2+ uptake, restored the ability of cells treated with caffeine or thapsigargin to reestablish a normal cytosolic Ca2+ control. The results show that cardiomyocytes are able to recover from severe hypoxic Ca2+ overload if, first, a closed sarcolemma is retained (as in isolated cardiomyocytes) and, second, the SR is available for rapid Ca2+ storage (impaired by caffeine and thapsigargin). The results also suggest that, in the case of an impairment of SR function, the inhibition of mitochondrial Ca2+ uptake (as by ruthenium red) has a protective effect.

Reoxygenation after 60 min substrate-free hypoxic perfusion (modified Tyrode solution, 37 degrees C) caused isolated Langendorff hearts (from rats) to rapidly develop hypercontracture and sarcolemmal disruptions indicated by massive and sudden loss of enzymes ("oxygen paradox"). Reoxygenation (30 min) caused an augmented loss of creatine kinase by 25.8% (lactate dehydrogenase by 40.1%) of the initial total tissue activity. It was investigated whether a temporary contractile blockade by 2,3-butanedione monoxime (BDM; 20 mM) can prevent reoxygenation-induced injury. In the presence of BDM, reoxygenation no longer caused hypercontracture or increased enzyme release. Instead, ultrastructure recovered, and contents of creatine phosphate (CrP) were partially restored (60 min hypoxia: 0.4 mumol CrP/g dry wt; after subsequent 60 min reoxygenation in presence of BDM: 7.8 mumol CrP/g dry wt). When BDM was eluted after first 20 min of reoxygenation, an attenuated but distinct increase in enzyme release was still observed. When BDM was eluted after 60 min of reoxygenation, ultrastructure did not deteriorate and increase of enzyme release remained virtually absent. During first 30 min after removal of BDM, the increased loss of creatine kinase amounted to only 5.7% (lactate dehydrogenase to 6.9%) of the initial total tissue activity. The results demonstrate that the oxygen paradox can be prevented in the hypoxic-reoxygenated heart when the contractile apparatus is temporarily paralyzed during the initial phase of reoxygenation.

Reoxygenation after 120-min substrate-free anoxia causes sudden hypercontracture in isolated rat cardiomyocytes. Reoxygenated-hypercontracted cardiomyocytes maintain their sarcolemmal integrity as indicated by the absence of enzyme release and reestablish a nearly normal free energy change of ATP hydrolysis within 15 min [Siegmund, B., A. Koop, T. Klietz, P. Schwartz, and H. M. Piper.Am J. Physiol. 258 (Heart Circ. Physiol. 27): H285-H291, 1990]. In the same model, it was now investigated whether a temporary contractile blockade by 20 mM 2,3-butanedione monoxime (BDM) can prevent reoxygenation-induced hypercontracture. When BDM was present during 120-min anoxia and the subsequent 15-min reoxygenation, hypercontracture could be prevented. The anoxic changes of high-energy phosphate contents, the free energy change of ATP hydrolysis, and the ultrastructure of the cells remained unaffected by the presence of BDM. When BDM was applied anoxically immediately before reoxygenation, it also prevented hypercontracture. Contracture still remained absent when BDM was washed out after the first 15 min of reoxygenation. These results demonstrate that a temporary contractile blockade (15 min) at the onset of reoxygenation prevents hypercontracture in anoxic-reoxygenated cardiomyocytes. This result, the energetic recovery, and the sarcolemmal integrity of cardiomyocytes in anoxia-reoxygenation demonstrate that reoxygenation-induced hypercontracture is not based on an already irreversible cell damage.

In tissue, mechanical cell-to-cell interactions may contribute to cardiomyocyte injury in anoxia-reoxygenation. In the present study, the disturbance of energy metabolism and cell injury were investigated in isolated cardiomyocytes, free of external mechanical constraints. Cardiomyocytes from adult rat, attached to culture dishes, were exposed to 120 min of anoxia and 15 min of reoxygenation in a substrate-free modified Tyrode solution. The energetic state of the cells in anoxia-reoxygenation was characterized by the free-energy change of ATP hydrolysis (delta GATP), amounting to 57 kJ/mol ATP in normoxia. After 120 min of anoxia, all cells were contracted to 65% of their length and delta GATP decreased to 41 kJ/mol. No lactate dehydrogenase was released. Reoxygenation caused a partial oxygen paradox: immediate hypercontracture of the cells, but no release of lactate dehydrogenase. delta GATP recovered to 51 kJ/mol within 15 min. The results demonstrate that anoxic cardiomyocytes can be energy depleted without losing sarcolemmal integrity. They can undergo hypercontracture, elicited by reoxygenation, and yet an almost normal delta GATP can be reestablished.

In isolated cardiomyocytes from adult rat heart the free energy change of ATP hydrolysis (dG) was determined under conditions of substrate-free anoxia. Changes of free cytosolic ADP concentrations, needed for the calculation of dG, were determined by two indirect methods since a direct measurement is not feasible: (i) via the mass action ratio of the creatine kinase reaction (CK) assuming near equilibrium conditions, and (ii) via quantification of the net hydrolysis of ATP to ADP by a detailed balancing of possible contribution to Pi production. Both approaches gave virtually identical results, showing that in anoxia only 6% of the ATP hydrolysed are hydrolysed to ADP and 94% completely to adenosine and further degradation products. The convergence of both methods also indicates that in this model the CK reaction is indeed catalysed near its equilibrium. Therefore estimations of free ADP and dG using its mass action ratio are valid. In anoxic cardiomyocytes dG values fell from 57 kJ/mol in normoxia to 42 kJ/mol after 120 min anoxia, corresponding to a decrease of ATP contents from 24 to 4 nmol/mg protein.

In medium 199 plus 20% fetal calf serum adult rat cardiomyocytes establish a long-term culture (25 days). During the first 10 days they change their gross morphology from the typical elongated in vivo shape (day 1), to a smooth spherical intermediate form (days 2 to 5), to a spread cell type beating spontaneously (days 10 to 15). During the first 10 days in culture, protein content per cell increases and the cell population decreases. By the tenth day, protein content has doubled, and about half of the cells originally plated remain. Thereafter both the protein content and the number of cells are essentially constant for the remainder of the 25-day period investigated. On days 1, 15 and 25 adenine nucleotide contents (213, 216 and 225 nmol/10(6) cells) and values of adenylate energy charge (0.91, 0.87 and 0.88) were similar. At all times in culture, palmitate (0.1 mM) is oxidized at higher rates than lactate (1 mM) and glucose (5 mM). At all times in culture glycolytic flux is sensitive to insulin with half maximal effect seen around 10(-9) M. Oxidation rates for all exogenous substrates are maximal at 15 days in culture, indicating maximal energy demand at this time. The conversion of glucose to lactate, however, progressively increases, so that at 25 days in culture, 70% of ATP derived from degradation of exogenous glucose is glycolytic. The results of this study demonstrate that oxidative metabolism of cardiomyocytes in long-term culture resembles, in its basic characteristics, that of the intact heart. In their increased glycolytic activity, however, they are clearly different.