Cardiovascular Research

Cardiovascular Research 1999 43(2):285-287; doi:10.1016/S0008-6363(99)00164-9
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Understanding the temporal relationship of ATP loss, calcium loading, and rigor contracture during anoxia, and hypercontracture after anoxia in cardiac myocytes

David F. Stowe, M.D., Ph.D.

Professor of Anesthesiology and Physiology, Cardiovascular Research Center, The Medical College of Wisconsin, Milwaukee, Wisconsin, USA

Received 27 April 1999; accepted 27 April 1999

See article by Ladilov et al. [9] (pages 408–416) in this issue.

Much research effort has been directed toward understanding the pathogenesis of cardiac ischemia reperfusion injury and developing methods to ameliorate the degree of damage. The interesting discover by Murry et al., reported 13 years ago, that a brief period of ischemia and reperfusion can partially protect the heart from reperfusion damage after a subsequent period of ischemia has sparked intense interest in the area of cardioprotection [1]. Hans Michael Piper and his collaborators, working mostly at the University of Dusseldorf and Justus-Liebig University, have contributed substantially to our knowledge of the pathophysiology of ischemia and reperfusion. They are mostly using a model of simulated ischemia (anoxia, acidosis, and substrate depletion) in which cell shortening of isolated cardiomyocytes during anoxia (rigor contracture) and during re-oxygenation (hypercontracture) are measured temporally with indices of metabolism and myocyte ion concentrations. They have proposed that the increase in contracture on re-oxygenation (hypercontracture) leading to cell injury (oxygen paradox) is due to the occurrence of Ca²⁺ overloading at the time of ATP repletion (calcium paradox) [2,3].

More recently, Yury V. Ladilov, working with Dr. Piper and others, have begun to better understand the mechanisms of myocyte dysfunction during and after simulated ischemia and the pathways by which dysfunction can be reduced by treatments applied before simulated ischemia. In recent studies they have examined carefully the importance of Na⁺ H⁺ exchange and reverse Na⁺ Ca²⁺ exchange in causing Na⁺ and Ca²⁺ loading as contributing to myocyte contracture and its reduction by acidosis and Na⁺ H⁺ exchange inhibition [4,5].

Protein kinase C (PKC) activation and translocation occur during ischemic preconditioning and activation of specific PKC isoforms has been found to be an important link between activated sarcolemmal receptors and ATP regulated K channels [6]. In the beating heart, opening of K_ATP channels allows K efflux which leads to more rapid repolarization of the action potential and a subsequently shortened period of Ca²⁺ influx during the plateau phase of the action potential [7]. Reduced sarcolemmal Ca²⁺ influx could aid in reducing myoplasmic Ca²⁺ loading, which, with the formation of free radicals, are believed to be the most important contributors to ischemia reperfusion injury [8]. However, other regulators of Na⁺ and Ca²⁺ homeostasis, the ion exchangers and the Na⁺ and Ca²⁺ ATPase dependent pumps, undoubtedly exert a major role with depletion and restoration of ATP during ischemia and reperfusion, respectively.

The article appearing on pages 408–416 of this volume [9] of Cardiovascular Research is a report that extends on work the authors recently published on the effects of PKC activation in their model [10]. They showed that the PKC agonist 1,2-dioctanoyl-sn-glycerol (1,2 DOG), given for 10 min and discontinued for 10 min before 80 min of anoxia in isolated rat adult cardiomyocytes, (a) attenuated the time to anoxic rigor contracture, but (b) did not reduce the accumulation of Ca²⁺ (estimated by the fluorescent probe fura 2), and (c) did not retard the development of hypercontracture on re-oxygenation compared to non-treated cells. Hypercontracture, as applied to non-beating cardiomyocytes, is defined as an additional increase in cell shortening on re-oxygenation thought to be due to the magnitude of Ca²⁺ loading at the end of anoxia when ATP generation is renewed on supply of oxygen. Excessive Ca²⁺ load causes membrane disruption and cell death [11].

In the present study they tested additionally if PKC activation (a) is protective because it causes a delay in total ATP depletion (ATP sparing effect) during anoxia to retard the onset of contracture, (b) if PKC activation alters intracellular pH or the release of lactate, and (c) if PKC activation prior to simulated ischemia reduces Ca²⁺ overloading and reduces the development of hypercontracture on re-oxygenation. To assess the time point at which ATP depletion occurs they measured the ratio of fluorescence intensities at 340 and 375 nm wavelengths in individual rat cardiomyocytes loaded with the fluorescent dye Mg²⁺ fura 2. An abrupt increase in the ratio was interpreted as the time of complete hydrolysis of ATP coupled to the release and detection of Mg²⁺. They then coincided the onset of hydrolysis with the onset of rigor contracture that occurred at different times in different cells during the 80 min anoxic period. It is important to remember that ATP is required for the actinomyosin bond to break so that myofilament relaxation occurs, and that Ca²⁺ is required to bind with troponin C to break the inhibition of actin myosin binding.

As might be expected, anoxic rigor contracture occurred just after complete ATP depletion in all cells, independent of the time that rigor developed. Their other findings are quite interesting in that they show that after PKC activation with 1,2 DOG, those cells that developed contracture between 20–40 min and 40–60 min into anoxia actually accumulated Ca²⁺ faster than did the cells that developed contracture but were not pre-treated with 1,2 DOG. But 1,2 DOG also reduced the incidence of hypercontracture in those cell that developed anoxic contracture between 40 and 60 min of anoxia compared with the control groups of cells at the same time periods. They suggests that PKC activation, while it markedly reduces the number of cells that develop anoxic contracture and delays the onset of contracture, also induces greater Ca²⁺ loading, possibly because of the longer delay to contracture resulting from a delay in the extreme depletion of myocyte ATP.

Thus, accelerated Ca²⁺ loading appears to be a consequence of the delay in ATP depletion and rigor brought on by 1,2 DOG. A longer period of contracture, when it occurs, increases the time for Ca²⁺ to overload the cell. Interestingly, increased Ca²⁺ loading did not retard the fall of Ca²⁺ on re-oxygenation, but the delay in Ca²⁺ loading by 1,2 DOG decreased the incidence of hypercontracture on re-oxygenation. Overall, the investigators conclude that the ATP sparing effect by PKC activation is responsible for the decrease in hypercontracture after anoxia, but that the association between post anoxic hypercontracture and Ca²⁺ loading during anoxia does not apply after PKC activation. They postulate that the acceleration of Ca²⁺ loading during anoxia in the presence of 1,2 DOG is due to enhanced phosphorylation of the sarcolemmal Na⁺ Ca²⁺ exchanger.

Of course the results of their study only begs more research into these interesting findings. What is the cause and effect relationship between ATP depletion and cell contracture? Does this coincide with reduced hydrolysis of actinomyosin ATPase or Na⁺ K⁺ pump failure (or Ca²⁺ ATPase pump failure in beating hearts)? What is the temporal link between abrupt rigor contracture and the slower accumulation of Ca²⁺ over the next 15–20 min? Why don’t cells develop rigor contracture more uniformly under the same suffusate conditions? Why is hypercontracture on-re-oxygenation not necessarily linked to Ca²⁺ loading during anoxia in the 1,2 DOG treated group? How is this evidence linked to the ATP sparing effect of PKC activation? What is the mechanism of ATP sparing by PKC activation? It is important that other research groups corroborate the present studies and that the investigators continue their interesting work.

For researchers who examine ischemia reperfusion injury using whole animal or isolated heart models, the cardiomyocyte model may appear to have several shortcomings. It may be difficult to understand or to extrapolate these findings to the intact ischemic heart. But one obvious advantage of the isolated myocyte model is that the effects of simulated ischemia on the myocyte itself can be observed without the confounding effects of other cardiac cell types. Measurements that can be obtained include a rough index of abnormal contracture, changes in metabolism, and importantly, changes in intracellular cation concentrations.

There are, however, several disadvantages of the single myocyte model. If the cells are not stimulated to contract, the membranes do not depolarize and initiate the phasic changes in voltage gated concentration gradients, ATPase dependent ion pumps, and ion exchangers that normally occur. The interaction among the various cell types (vascular, endothelial, nervous, working myocytes) that together contribute to ischemia reperfusion injury can not be demonstrated. There is no index of normal contractility or how it changes after ischemia in the isolated myocyte model. The isolated myocytes are continuously suffused with anoxic solution to simulate ischemia rather than subjected to a cessation of perfusion (true ischemia) which produces metabolites that accumulate (e.g., adenosine, lactate, K⁺, peroxynitrite) and may modify the ischemic responses. Nevertheless, isolated rat hearts (and other models) develop diastolic contracture during ischemia and a failure of complete relaxation (elevated diastolic pressure) on reperfusion, and isolated heart models in which beat-to-beat myoplasmic Ca²⁺ and Na⁺ concentration are measured corroborate the isolated myocyte studies showing that Na⁺ and Ca²⁺ become elevated during simulated ischemia. However, in true ischemia, Ca²⁺ and Na⁺ also accumulate during initial reperfusion. Moreover, ischemic preconditioning may produce a decrease in ATP depletion during sustained ischemia as a cardioprotective mechanism [12,13].

Finally, one cannot stress enough the scientific importance of undertaking studies at different levels, subcellular, cellular, tissue, organ, and animal. To obtain an overall understanding of the pathophysiological mechanisms of ischemia reperfusion injury, the complementary (or contradictory) findings obtained form the various models and protocols can only assist investigators to synthesize a mechanistic scenario that explains the derangements caused by ischemic reperfusion injury and then, hopefully, how to ameliorate the damage. Thus in this journal’s article, Ladilov and collaborators succeed in addressing an important issue that contributes to a better understanding of the pathophysiology of cardiac ischemia reperfusion. Their work leads to additional fundamental questions that will need to be addressed.

	References

Top References

 

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