Cardiovascular Research

Cardiovascular Research 2006 70(2):170-173; doi:10.1016/j.cardiores.2006.03.010

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Copyright © 2006, European Society of Cardiology

The sarcoplasmic reticulum as the primary target of reperfusion protection

Hans Michael Piper^*, Sascha Kasseckert and Yaser Abdallah

Institute of Physiology, Justus Liebig University, D-35392 Giessen, Germany

^* Corresponding author. Physiologisches Institut, Justus-Liebig-Universität, Aulweg 129, D-35392 Giessen, Germany. Tel.: +49 6419947240; fax: +49 6419947219. Email address: Michael.Piper{at}physiologie.med.uni-giessen.de

Received 10 March 2006; accepted 14 March 2006

Prolonged myocardial ischemia leads to the loss of viable tissue in the heart, even if the ischemic episode is eventually ended by reperfusion. As we now know today, the extent of infarct is only partially due to the ischemic injury. Another part of tissue loss is due to death signals generated at the time of and by the specific circumstances of reperfusion, a phenomenon termed "reperfusion injury" [1,2]. The existence of reperfusion injury has been revealed by successful attempts to interfere with such causes at the time of reperfusion and limit, thereby, the extent of final tissue loss. Reperfusion injury is initiated within the first minutes of reperfusion. This is why these minutes represent a valuable "window of opportunity" for myocardial protection. One of the most recent strategies to interfere within that time window is postconditioning, i.e. the repetitive application of brief bouts of ischemia within the first minutes of reperfusion [3]. This protocol is so promising because it has brought reperfusion protection to the clinical arena and reperfusion injury to the broad attention of clinical cardiologists.

The nature of the death signals activated during the first minutes of reperfusion is still a matter of debate. Opinions are also divided on whether the ensuing cell loss is due to necrosis or apoptosis. Cell damage in reperfused myocardium exhibits a peculiar histological appearance. It is characterized by hypercontracted sarcomeres ("contraction bands") and frequent sarcolemmal ruptures that gave rise to the term "contraction band necrosis" [4,5]. The histological picture has led to a hypothesis on the pathogenesis of acute reperfusion injury. According to this hypothesis, it is an uncontrolled activation of the contractile machinery in myocardial cells upon reperfusion that produces the final, decisive blow to cardiomyocytes already weakened by the ischemic history and ends their fate by disruption of the cytoskeleton and the sarcolemma. The unique histological appearance of reperfusion-damaged myocardium does not exclude another widely discussed possibility that apoptotic signalling is involved in the pathogenesis of reperfusion injury. Indeed, several studies found that apoptotic pathways are activated in reperfused myocardium [6,7].

What, then, are the primary end-points of reperfusion protection? To answer this question and to search for new strategies of reperfusion protection are largely the same endeavour, since it is the successful interference with the development of reperfusion injury that identifies the nature of the underlying causes. Presently, there are two main hypotheses: we have favoured the sarcoplasmic reticulum (SR), others the mitochondria as the primary target organelle of protection. Here we will put forward a unifying hypothesis, explaining that the SR likely represents the primary and mitochondria an important secondary target for reperfusion therapy (Fig. 1).

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Scheme of the pathogenesis of acute reperfusion injury. Reperfusion reactivates ATP production in mitochondria (Mito). Recovering energy production (High ATP) activates the Ca2+ pump (SERCA) of the sarcoplasmic reticulum (SR), which clears the cytosol from Ca2+ overload accumulated during ischemia. Repetitive release of Ca2+ through the ryanodine receptor Ca2+ release channel (RyR) and reuptake into the SR leads to Ca2+ oscillations with high cytosolic peak Ca2+ concentrations. This high Ca2+ together with ATP provokes myofibrillar hypercontracture (Ca2+ contracture) and subsequent disruption of cells (Necrosis). Ca2+ uptake through the uniporter into mitochondria causes the opening of mitochondrial permeability transition pores (mPTP) and cytochrome c (Cyt c) release. The former leads to failure of energy production (low ATP), and the latter activates apoptosis. Low ATP induces rigor contracture of the myofibrils, again leading to cell disruption. Protection by reperfusion injury salvage kinase pathways (RISK) may interfere favourably at the SR or at mitochondria.

 
In a series of studies using isolated cardiomyocytes, we have analysed on the cellular level the basic mechanisms by which reperfusion conditions induce hypercontracture of the contractile machinery, which is the most characteristic feature of myocardial reperfusion injury. We found that two different mechanisms can lead to the same final result of hypercontracture: first, a mechanism driven by Ca²⁺ overload and rapid crossbridge cycling (Ca²⁺ contracture) and, second, a mechanism caused by the slow turnover of crossbridges at very low ATP concentrations (rigor contracture) [8,9]. Ca²⁺ contracture is elicited if cardiomyocytes are reoxygenated in the presence of an ischemically accumulated cytosolic overload of Ca²⁺, and mitochondrial ATP production rapidly recovers. It is the high cytosolic Ca²⁺ concentration in conjunction with a sufficient ATP supply that activates a normal crossbridge cycling mechanism, but does so excessively because of the persistence of high cytosolic Ca²⁺ levels. The second mechanism is not dependent on cytosolic Ca²⁺ overload. Rigor contracture occurs at sub-millimolar, but non-zero ATP concentrations (<100µmol/l) [10]. It is thought to be caused by a Ca²⁺-independent activation of actin–myosin interactions along actin filaments at which rigor bonds are already formed at some of their many crossbridge sites. Rigor contracture is a regular feature of the ischemic myocardium, but may also occur during reperfusion, depending on the rapidity with which mitochondrial ATP production recovers [9].

In cardiomyocytes with a rapidly recovering mitochondrial energy production under reperfusion conditions, elevated cytosolic Ca²⁺ levels do not just stay high, but are rapidly changed by various Ca²⁺ transporting mechanisms. Uptake of large quantities of Ca²⁺ into the SR by a re-energized SR Ca²⁺ pump (SERCA) can clear the cytosol very rapidly. Normally the SR will release the excessive Ca²⁺ load again very rapidly through the opening of its ryanodine receptor release channel, and this, by repetition, leads to oscillatory shifts of Ca²⁺ between the cytosol and the SR. It is the repetitively high cytosolic calcium peaks that cause the uncontrolled contractile activation in reperfusion-induced Ca²⁺ contracture [8,11]. A number of studies have demonstrated that manoeuvres which either attenuate these oscillatory Ca²⁺ elevations or reduce the Ca²⁺ sensitivity of the myofibrils in the first minutes of reoxygenation reduce hypercontracture development in reoxygenated isolated cells and infarct size in reperfused hearts in vivo [8,11–16].

A slow recovery of mitochondrial energy production under reperfusion conditions may have various causes. One reason is a continuing Ca²⁺ overload in the cytosol under conditions in which the driving force of Ca²⁺ uptake by the mitochondrial uniporter is high, as is the case when reoxygenated mitochondria become polarized by respiratory electron transport. The ensuing mitochondrial Ca²⁺ overload can then lead to opening of the mitochondrial permeability transition pore (mPTP), which causes energy production to cease. We showed previously by gradual inhibition of oxidative phosphorylation in reoxygenated cardiomyocytes that a Ca²⁺ contracture can be turned gradually into a rigor contracture in reoxygenated cardiomyocytes [9]. Since Ca²⁺ overload in metabolically surviving, reoxygenated cardiomyocytes takes on the form of SR-dependent Ca²⁺ oscillations, it can be expected that measures reducing these Ca²⁺ oscillations at the level of the SR also protect mitochondria against mPTP opening. This has not yet been demonstrated directly for cardiomyocytes with a Ca²⁺ overload originating from ischemic conditions, but for cardiomyocytes with cytosolic Ca²⁺ overload due to other causes [17].

If the SR plays a central role in acute reperfusion injury, one would expect that protective pathways interfering with reperfusion injury would also converge on SR functions. Among the protective signalling elements identified in whole hearts are PI 3-kinase, Akt kinase, and eNOS [3,18]. We showed recently [13,14] that activation of a pathway composed of some or all of these elements indeed protects cardiomyocytes against reperfusion-induced hypercontracture by reducing the oscillatory elevations of cytosolic Ca²⁺. The underlying mechanism seems to be that activation of SERCA (and possibly inactivation of the ryanodine receptor) through this protective signalling enables the SR to sequester larger amounts of Ca²⁺ from the cytosol and thereby keep it away from the contractile machinery [13,14]. These results do not necessarily contradict the hypothesis of other studies that this protective signalling affects mitochondrial functions, as effects on SR and mitochondria may be sequential (see above). It is also not excluded that the protective signalling influences mitochondrial functions more directly. It has been suggested that "reperfusion injury salvage kinases" (RISK) converge on the inhibition of glycogen synthase kinase-3β (GSK-3β), which results in inhibition of mPTP opening [19].

It has been argued that protective signals favourably affecting mitochondrial functions in cardiomyocytes upon reperfusion inhibit an otherwise occurring activation of a pro-apoptotic pathway, and that it is through such anti-apoptotic effects that these signals can be used to limit infarct size [18]. While the first hypothesis seems true, the second is questionable. Several studies have shown that activators of these signals and RISK [3,19] reduce signs of apoptosis in reperfused myocardial tissue. They seem to inhibit directly or indirectly the mitochondria-dependent activation of apoptosis initiated by the release of cytochrome c. This mitochondrial route to apoptosis may be triggered through SR-dependent Ca²⁺ oscillations as shown by Chen et al. [17]. What makes this apoptotic mechanism questionable in explaining the protective effect at the level of infarct size reduction is that, first, quantification of apoptotic cell death in tissue is notoriously imprecise and, second, animals lacking the mPTP regulating protein cyclophilin D show resistance to necrotic myocardial cell death induced by Ca²⁺ overload, reactive oxygen species, and ischemia–reperfusion [20].

In summary, the SR plays a central role in the pathogenesis of acute reperfusion injury. The SR controls the fate of cytosolic Ca²⁺ overload in the first minutes of reperfusion. In the most favourable situation, the SR serves as an intracellular sink for excess cytosolic Ca²⁺, but normally the repetitive uptake and release of Ca²⁺ through this organelle causes deleterious oscillations of cytosolic Ca²⁺ with high peak concentrations. These Ca²⁺ oscillations elicit a hypercontracture of the contractile machinery with deleterious consequences for the cells' structure. On a more delayed time scale, the oscillating Ca²⁺ overload can also lead to Ca²⁺-induced opening of mPTP and the various consequences of mitochondrial failure. Among these, reduction of ATP production, which favours development of rigor contracture, seems to be the most important.

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