Heart and Metabolism

Metabolic changes in stunning and hibernation

Roberto Ferrari
Dipartimento di Medicina Clinica e Sperimentale, Ospedale S. Anna, Corso Giovecca,
203, 44100 Ferrara, Italy (fri@dns.unife.it)

Correspondence: Prof. Roberto Ferrari, Dipartimento di Medicina Clinica e Sperimentale,
Ospedale S. Anna, Corso Giovecca, 203, 44100 Ferrara (fri@dns.unife.it)

Myocardial ischemia is very complex and, unless interrupted by early reperfusion, will culminate in cell death. Although a prerequisite for survival, reperfusion is not without hazard, however. Paradoxically, although ischemia is ‘bad’ and reperfusion is ‘good’, ischemia can be protective (preconditioning and a trigger for hibernation) and reperfusion can be ‘bad’ (reperfusion injury). Understanding, manipulating and exploiting these processes requires a detailed knowledge of the molecular mechanisms of ischemia and reperfusion. Much has already been learnt but much more remains to be discovered. With that knowledge we may be able to make a major impact on the devastating consequence of coronary heart disease.

Different facets of myocardial ischemia
There is no simple definition of ischemia. In an attempt to address the problem, Hearse invited 31 eminent cardiologists, all experts in the field, to provide a brief definitive definition.[1] The result was a multitude of differing and sometimes conflicting suggestions, ranging from just a few to several hundred words in length. Hearse then proposed that a fundamental distinction should be made between ‘physiological’ and ‘biochemical’ ischemia.[1] Although this is an oversimplification of a complex issue, a region of the heart may be considered as physiologically ischemic when,
as a consequence of flow reduction, it is unable to maintain normal contractile func-
tion.[1] This is how a clinical cardiologist perceives ischemia. He or she knows that it is linked, at best, with regional left ventricular dysfunction of both a systolic and diastolic nature (Figure 1).

Figure 1. Schematic representation of the possible outcomes of myocardial ischemia.

If the period of ischemia is short, there is no major molecular damage and functional impairment is reversible on reperfusion. However, if the ischemia is more severe or more prolonged, irreversible molecular damage could occur, recovery on reperfusion becomes impossible, and necrosis inevitably develops. This key transition might occur within minutes of the onset of ischemia, or take up to several hours depending on a multitude of factors (the underlying metabolic rate probably being the most important). For the clinical cardiologist this is, in turn, determined by the extent of residual flow, the underlying heart rate, the degree of hemodynamic change (such as an increase in pre- and after- load, wall stress), and by the effects of any accompanying neuroendocrine activation. This physiological ischemia, characterized by down-regulation of contraction in the absence of molecular changes, can also be considered a conservative adaptive response by the myocyte that down-regulates its contraction independently of extracardiac signals and, in so doing, reduces its energy needs in an attempt to maintain viability. A reserve reperfusion injury – the so-called myocardial stunning – has recently been discovered.
In contrast, in biochemical ischemia,[1] possibly in response to a series of complex and predominantly extracardiac neurohormonal signals (activated to ensure the maintenance of pump function and cardiac output), the myocyte will, at a high cost, succumb to a series of cellular mechanisms that will attempt to maintain contractile function despite impairment to the oxygen supply. In consequence, the supply of energy fails to match consumption, and intracellular equilibrium (steady-state metabolism) is sacrificed, initiating a cascade of increasingly severe metabolic perturbations. The cell will then become ‘metabolically distressed’[1] and, unless interrupted by early reperfusion, biochemical ischemia will inevitably progress towards cell death. As indicated in Figure 1, the mitochondria are the organelles most likely to be involved in the transition of reversible ischemia to definitive cell death. This is, perhaps, not surprising since these organelles play a fundamental role in cellular energy production (the ATP turnover of the human heart exceeding 30 kg per day), and in maintaining intracellular ionic homeostasis – the other key process which is threatened by ischemia.

Reperfusion of ischemic myocardium: an important determinant in the transition from ischemia to cell death
Our understanding of the complexities of ischemia and tissue injury is further complicated by the need to reperfuse the tissue in order to determine whether ischemic damage is reversible or irreversible. Some, but not all, investigators believe that reperfusion itself might be detrimental and able to inflict injury over and above that attributable to the ischemia it is expected to remedy.[2] Other investigators, however, question the existence of ‘reperfusion-induced injury’.[3] This question will not be discussed here. Instead, the concept that ischemia is not a static condition and that reperfusion is a part of the continuum of coronary artery disease will be explored. Such reperfusion might, however, occur at different times during the transition from angina to myocardial infarction, and have several different outcomes such as early or delayed recovery (stunning), no recovery or some recovery (hibernation).

A perfect match between physiological and biochemical ischemia maintains viability
During short periods of ischemia, for example in angina, there is a perfect match between biochemical and mechanical activity; this allows viability to be maintained. Restriction of coronary flow results in a rapid down-regulation of contraction and eventually quiescence. This is due to the effects of intracellular acidosis, which develops within seconds of the induction of ischemia and reduces calcium movements within the sarcolemma, sarcoplasmic reticulum and myofilaments.[4] Shortly after, the energy charge of the myocyte is reduced: creatine phosphate declines faster and to a greater extent than ATP. Anaerobic metabolism, as shown by lactate release in the coronary effluent, develops and contributes to the formation of limited amounts of ATP by oxygen-independent, substrate-level phosphorylation. Taken together, these findings suggest the occurrence of biochemical as well as physiological ischemia. Both down-regulation in contraction (and therefore in ATP consumption) and increased anaerobic ATP production explain why the decline in tissue ATP after the onset of ischemia is not immediate. The availability of this residual energy supply is essential to maintain cellular viability. Reperfusion at this stage results in a recovery of high-energy phosphate production, which, in turn, indicates that the mitochondria are still functionally intact and capable of normal aerobic metabolism; this is linked to a recovery of mechanical function which may be immediate or somewhat delayed.
This sequence of metabolic and functional events is not restricted to experimental models, but also occurs at the clinical level, for example, during angina induced by atrial pacing. Figure 2 shows that in coronary artery disease patients with angina, increasing heart rate (and, therefore, increasing the heart’s energy requirement to the extent that it is no longer met by the supply) results in a reduction of coronary sinus pH, which indicates the occurrence of myocardial acidosis.

Figure 2. Metabolic changes occurring during early phases of ischemia in coronary artery disease patients.

This is then followed by an increase in coronary sinus lactate (which is indicative of the development of anaerobic metabolism), and in a down-regulation of regional contraction (revealed by a reduction of ejection fraction, which is suggestive of systolic dysfunction). All these biochemical and mechanical events precede the occurrence of angina. Once the heart rate has returned to its basal level and the ischemia, therefore, no longer persists, coronary sinus pH and lactate return to normal values and left ventricular systolic function improves. However, the functional recovery is not immediate because of the presence of stunning.[5] Under such circumstances, viability is maintained but evidence of the ischemic insult persists for as long as the recovery of function does not match that of metabolism.

Figure 3. Effects of 90 min of ischemia followed by 30 min of reperfusion on mitochondrial function. Paced, isolated perfused rabbit hearts were used for these experiments. Under control and reperfusion conditions the hearts were perfused at a mean coronary flow of 25 ml/min. Ischemia was induced by reducing coronary flow to 1 ml/min. A: Typical example of a left ventricular pressure tracing from a whole heart subjected to ischemia and reperfusion. B and C: Typical examples of isolated mitochondrial tracing for oxygen consumption and ATP production. The mitochondria were isolated from hearts which had been aerobic for 30 min, ischemic for 30, 60 or 90 min and reperfused for 30 min. The numerical values reported in the oxygen consumption tracing represents rates (nmol oxygen/mg protein/min) consumed by the isolated mitochondria during states III and IV of respiration. Glutamate was used as respiratory substrate.

Early reperfusion causes stunning: lingering evidence of preceding physiological and biochemical ischemia
There is now convincing evidence that the myocardium that has been reperfused after a short period of ischemia is characterized by a variety of unfavourable (but non-lethal) cellular changes that, given sufficient time, will revert to normal. The most prominent of these changes is myocardial stunning, which is the prolonged contractile dysfunction that occurs during reperfusion despite the absence of irreversible injury.[5,6] The duration of the dysfunction greatly exceeds that of the antecedent ischemia. For example, after 15 min of ischemia in dogs, myocardial function remains depressed for 24 hours.[7] However, by definition this form of injury is fully reversible, provided sufficient time is allowed. Interventions such as isotropic agents can override stunning and other interventions (such as anti-oxidants) can prevent its occurrence.[6]
A number of candidate mechanisms for stunning have been investigated; these include: an impaired ability to resynthesize high-energy phosphates, functional sympathetic denervation, heterogenous impairment of regional perfusion, abnormal electrical activation, loss of creatine kinase activity, damage to the collagen matrix, leukocyte activation, and decreased sensitivity of myofilaments to calcium. However, the two most plausible mechanisms relate to free radical induced injury during the early moments of reperfusion and impaired calcium homeostasis.
Numerous studies suggest that oxygen-derived free radicals contribute to post-ischemic dysfunction.[9] In dogs subjected to 15 min of coronary occlusion, stunning is reduced by drugs that scavenge oxygen radicals or prevent their generation. The generation of free radicals in the stunned myocardium has been directly demonstrated with electron paramagnetic resonance spectroscopy, and the attenuation of radical generation has been shown to result in the attenuation of contractile dysfunction.[8]. Although there is strong evidence that reactive oxygen intermediates play a major role in the pathogenesis of myocardial stunning, there is also evidence that this phenomenon is related to abnormalities of calcium homeostasis.[6] It is important to emphasize that calcium and free radical mechanisms are not mutually exclusive but may represent two facets of the same phenomenon. Thus, Bolli has suggested that oxygen free radicals may cause sarcolemmal and sarcoplasmic reticulum dysfunction and perturbations of calcium distribution. The latter, in turn, could exacerbate the damage initiated by the radicals and indeed could promote the production of further radicals.[6]

The transition from ischemia to cell death: when biochemical ischemia overrides physiological ischemia
If coronary flow remains severely reduced, the myocardium will remain quiescent but nonetheless biochemical ischemia intensifies and proceeds towards irreversible damage. From the metabolic point of view, prolongation of ischemia results in further decrease in intracellular pH and in a progressive increase of resting pressure and myocardial stiffness. The early increase in lactate is followed by a decline together with a further decrease in tissue content of ATP and CP. This supports the view that, after an initial stimulation, anaerobic glycolysis is inhibited by the more severe intracellular acidosis. At this stage profound ionic changes occur with a deletion of intracellular K⁺ and Mg2⁺ and an increase of Na⁺ and of cytosolic Ca2⁺. Interestingly, even after prolonged ischemia, total tissue calcium concentration is unchanged but mitochondrial calcium is increased, indicating an intracellular redistribution of the ion. Isolated mitochondrial function, however, is then maintained since only a slight reduction in the initial rate of ATP production is observed. In spite of this, reperfusion does not restore mitochondrial or myocardial function. On the contrary, it produces a further increase of stiffness and non-recovery of contractility or of tissue ATP and CP concentrations. During reperfusion there is a significant and sustained release of lactate, ions and CPK, massive influx of calcium and severe mitochondrial damage, suggesting that late reperfusion causes not only a wash-out of these substances but also an exacerbation of their release[.6] These findings indicate that a lesion of the cell membrane has occurred, leading to a breakdown of the permeability barrier to ions such as Ca2⁺ and Mg2⁺, as well as to larger molecules such as CPK, and that mitochondria are using the restored oxygen for buffering cytosolic Ca2⁺ rather than for ATP production. For this reason, mitochondria are supposed to play a central role in reperfusion damage. It appears that these organelles are quite resistant to ischemic damage; however, the presence of residual phosphorylation capacity in mitochondria during ischemia is associated with irreversible damage during reperfusion such that, paradoxically, mitochondrial uncouplers can afford cardiac protection.[9]
From this it follows that residual mitochondria function during ischemia might be interpreted as good or bad. This apparently contradictory concept arises from the finding that, on the one hand, intact, normally functioning mitochondria are essential for the recovery of mechanical function during reperfusion but, on the other hand, the inhibition of the respiratory chain or the addition of uncouplers of oxidative phosphorylation are able to limit the extent of enzyme release in various models of myocardial damage.[9] These findings suggest the complex scenario that the restoration of ATP production by mitochondrial oxidative phosphorylation is essential for myocardial recovery, but, at the same time, this mitochondrial activity can also contribute to those processes which produce cell necrosis. Understanding these mechanisms is important as, in an in vivo condition such as during evolving myocardial infarction, a continuous sequence of ischemia and reperfusion is likely to occur as collateral flow develops.

Late reperfusion of hibernating myocardium and recovery: physiological ischemia without biochemical ischemia
The term ‘hibernation’ has been borrowed from zoology and implies an adaptive reduction of energy utilization through reduced activity under conditions of a reduced energy supply. In the context of coronary artery disease, myocardial hibernation was originally seen as a chronic, adaptive reduction of myocardial contractile function in response to a reduction of myocardial blood flow. It was also viewed as a condition where there would be a complete recovery of contractile function upon the restoration of flow. Thus, in the concept of myocardial hibernation, the observed chronic reduction of myocardial contractile function is not regarded as the result of a persistent energetic deficit, but instead as a regulatory event which acts to avoid an ongoing energy deficit and thereby maintain myocardial integrity and viability.
Interestingly, the concept of myocardial hibernation does not originate in the laboratory instead it is entirely founded on clinical experience when, in the early 1980s, Rahimtoola reviewed the results of coronary bypass surgery trials and identified a subset of patients with coronary artery disease and chronic left ventricular dysfunction that improved upon revascularization.[10] Whereas originally the idea of an adaptive reduction of contractile function in response to a reduction in blood flow was straightforward and simple, the situation of chronic, yet reversible contractile dysfunction in the setting of coronary artery disease was not recognized and was seen as enormously complex and controversial.
The introduction of the concept of hibernation has challenged the traditional view that the extent of chronic contractile dysfunction necessarily reflects the amount of infarcted tissue. In hibernation, preservation of viability rather than the occurrence of necrosis accounts for the observed reduction in function. In view of the preserved viability of the tissue, hibernation is a key factor in assessing the potential benefit that might be expected from reperfusion/revascularization. Hibernating myocardium must be recognized and identified by appropriate diagnostic procedures and requires decisions by the responsible cardiologist for the selection of patients who will benefit from interventional reperfusion or surgical revascularization. Of course, hibernation is only one of several important aspects which must be considered in the selection of patients who will benefit from reperfusion or revascularization, and many patients with coronary artery disease and no evidence of hibernating myocardium will also benefit.
A hibernation-like metabolic adaptation to a severe sustained low-flow ischemia has recently been reported in studies with isolated perfused rabbit hearts in which there was a preceding short episode (10 min) of zero-flow ischemia. In these hearts, the early decline in contractile function was more pronounced and significantly faster than in control hearts that did not have the brief episode of zero-flow ischemia. The rapid decline in contractile function (physiological ischemia) during the brief episode of no-flow ischemia was accompanied by a greater decrease in interstitial[4] and intracellular[11] pH, and the contractile quiescence was attributed to a faster development of myocardial acidosis. Interestingly, interstitial and intracellular pH during the subsequent low-flow ischemia remained mildly reduced whereas these pH values were markedly decreased when low-flow ischemia was not preceded by zero-flow ischemia. During low-flow perfusion there was no lactate release, suggesting that biochemical ischemia did not occur. During reperfusion following the sustained ischemia, only a transient creatine kinase leakage occurred in the hearts with preceding zero-flow ischemia. Thus, the establishment of the experimental form of myocardial hibernation requires an initial period of zero-flow ischemia, during which time the rapid decrease in interstitial and intracellular pH trigger the decrease in contractile function and thereby facilitates the restoration of the balance between energy supply and energy demand. In other studies, in anaesthetized swine hearts in situ, the size of infarcts arising as a consequence of sustained (90 min) zero-flow ischemia was reduced by a short (10 min) period of no-flow ischemia immediately before the sustained ischemia.[12] A reduction in infarct size was also achieved by a 70% reduction in flow for 30 min preceding 60 min of total coronary artery occlusion.[13] These experimental studies attribute a potentially important role to an initial stimulus of severe ischemia as being critical to ‘triggering’ the development of a protective state with preserved viability during a subsequent period of sustained ischemia. Whether or not such an initial stimulus/trigger of severe ischemia represents a mandatory link between hibernation and ischemic preconditioning is unclear at present,[14] but it would support the hypothesis that hibernating myocardium, at least most of the time, might not be biochemically ischemic but will be physiologically ischemic.[15]

Acknowledgement
This work was supported by the National Research Council (C.N.R.), targeted project ‘Prevention and Control Disease Factors’ no. 93.00656 PF 41/115 and ‘The New Ischemic Syndromes’ European Commission project no. PL 95/0838. The authors thank Roberta Bonetti for the secretarial assistance in preparing the manuscript and Sandra Marini for editing the text.

REFERENCES

1: Circulation 1997 Dec 2;96(11):4065-82