Ischemia–reperfusion associated myocardial contractile dysfunction may depend on Ca2+-activated cytoskeleton protein degradation

See article by Papp et al. [9] (pages 981–993) in this issue. Postischemic dysfunction, or myocardial stunning, is defined as ‘the mechanical dysfunction that persists after reperfusion despite the absence of irreversible damage and restoration of normal or near normal coronary flow’ (for a review, see Ref. [1]). Myocardial stunning may be considered as a relatively mild, sublethal injury that must be distinguished from myocardial infarction. It is generally admitted that myocardial stunning is a multifactorial process, that mostly depends on two major mechanisms: (1) generation of oxygen radicals, (2) calcium overload, both being probably involved in the process (for review, see Ref. [1]). The calcium hypothesis may explain many key features of the stunned myocardium. It has been postulated that calcium overload during reflow triggers myofilament dysfunction which uncouples excitation from contraction, so that for any given calcium transient, the myocardium generates less force [2]. A variety of biochemical abnormalities have been reported to explain the sustained, but reversible, myocardial contractile dysfunction that occurs after reperfusion. Immunonoblotting and SDS–PAGE analysis have permitted the identification of subtle Ca2+-dependent changes in the amount of myofibrillar proteins. Elevated [Ca2+]i during ischemia and during the initial reperfusion period have long-lasting after effects by activating Ca2+-dependent proteases, which could partially degrade contractile proteins [3]. Myofilament dysfunction would depend on protein degradation, the extent to which increases with the severity of injury, the major functional damage occurring on reperfusion [2]. Indeed, in stunned rat myocardium depression of myofilament function has been associated with a Ca2+-dependent degradation of α-actinin, a myofilament-associated scaffolding protein distributed at the level of the Z line [4] and of a thin filament regulatory protein, troponin I (TnI) [5]. The exact role of these changes in force generation is still unknown. Loss of α-actinin from myofilaments may inhibit the myosin ATPase activity and result in decreased contractility [6]. According to Gao et al. [7] alterations in the myofilaments themselves are entirely responsible for decrease in Ca2+ sensitivity. However, other myofibrillar proteins, such as TNT, myosin light chain-1, MLC1, are also degraded as a result of longer ischemia followed by reperfusion [2]. Moreover, calcium overload induces cross-linking between troponins and other cardiac proteins [8]. Finally, neither light nor electron microscopy examination have revealed clear histological damage and the factors responsible for the decrease in myofilament Ca2+ responsiveness in stunned myocardium are not clear yet. Taken together, there still exist discrepancies in the literature as to the precise nature of myofilament dysfunction which may be due to proteolytic injury to the contractile proteins and/or to oxyradical-mediated covalent modifications. Papp et al. [9] have analysed in parallel the role of a calcium-dependent neutral protease, calpain, on cardiomyocyte protein content and myofilament function. They describe that degradation of a cytoskeletal protein, desmin, is associated to the myofibril lost of Ca2+ sensitivity. Calpain I, a calcium-activated neutral protease, is expressed in cardiac cells and appears to be a physiologically relevant candidate for intracellular myofilament proteolysis following ischemia and reperfusion (for review, see Ref. [1]). Indeed, the short-lived elevation of [Ca2+]i which is induced in these latter conditions is within the range (around 1–20 μM) of calcium concentration that activates calpain I in vitro. Moreover, direct exposure of cardiac myofilaments to the activated enzyme reproduces the phenotype of the stunned myocardium: maximal force is depressed and sensitivity is blunted. Finally, in isolated heart models, the activity of calpain I is increased after ischemia–reperfusion and various enzyme inhibitors blunt the stunning [7]. Ultrastructural alterations may play an important role in the calpain-I mediated cardiac dysfunction but the nature of the altered contractile regulation in postischemic myocardial preparations is still controversial. In vitro calpain I is very effective in digesting TnI and TnT [10]. According to Gao et al. [5] calpain has a selective effect on troponin-I degradation, that is responsible of the decrease in Ca2+ sensitivity of the myofibrillar system. However, contractile dysfunction depending on Ca2+ activated intracellular proteases during reperfusion in stunned guinea-pig heart, induces a decrease in the content of Z line associated cytoskeletal proteins, i.e. desmin, α-actinin and spectrin [4]. In this issue, Papp et al. [9] have analysed whether cellular regulatory or structural changes are the primary causes of calpain-I induced myocardial dysfunction. They used an experimental model of permeabilized cardiomyocytes that have been submitted to ischemia/reperfusion in the presence of 2,3-butanedionemonoxime (BDM). BDM inhibits the myofibrillar machinery during the early ‘vulnerable phase’ of reoxygenation. Calpain I-dependent changes in the maximal force-generating capacity of the myocytes at saturating levels of Ca2+, the sensitivity of the cardiomyocytes to Ca2+ and cross-bridge cycling kinetics were characterized in parallel with the protein content. They demonstrate that calpain I induces both a major reduction in maximal isometric force, and a degradation mostly of desmin [9]. Desmin is the constitutive protein of the intermediate filaments of cardiac myocytes, usually described as closely associated to α-actinin and actin filaments at the level of the Z bands. Intermediate filaments of desmin link Z-bands with plasmalemma and the nucleus. As a consequence, these filaments were presumed to contribute to the organization of myofibrils and maintenance of cell shape [11]. Interestingly, the mechanical role of intermediate filaments has been recently analysed in smooth muscle cells of mice with a null mutation introduced in the desmin gene. While desmin appeared dispensable for normal development and viability, force generation and maintenance of passive tension, its absence had significant consequences for the mechanical properties of muscle tissue; the visceral smooth muscles developed only 40% of the normal contractile force and the maximal shortening velocity was reduced by 25–40% [12]. Other components of the cytoskeleton such as the myocyte microtubule network can be disrupted by reperfusion after brief ischemia [13]. Because of the sensitivity of microtubule stability to Ca2+ concentration, disruption could depend on Ca2+ overload associated to stunning. However, disruption was reversible and microtubular depolymerisation was not causally linked to myocardial dysfunction. Finally, abnormalities of many cytoskeleton proteins have been shown to develop, but to a larger extent, as a result of global ischemia [14]. Intracellular acidosis during the early stage of reperfusion represents a natural mechanism of protection against acute reperfusion injury. It induces alterations of the intracellular milieu and among others the binding of cytosolic proteins such as GAPDH and 23 kDa αB-crystallin to membrane, cytoskeletal, myofibrils proteins [15,16]. Such binding could be responsible for the alterations of contractility associated to myocardial stunning. Indeed, it is interesting to note that αB-crystallin, is a molecular stress chaperone, very abundant in cardiac muscle cells that interacts directly with actin and mostly desmin. Binding increasing considerably at slightly acidic pH (6.5) [15], the 23-kDa αB-crystallin could prevent disorganization of filaments. Indeed, a defect in expression of the peptide has been associated to the disorganization of desmin filaments that characterizes a well defined familial skeletal muscle disease [17]. The eventual role of the binding of the 23-kDa αB-crystallin to myofilament and cytoskeletal proteins in myofibrillar dysfunction should be further analysed. The exact nature of the degraded proteins that underlies the impaired calcium responsiveness of myofilaments, the relative importance of degradation of myofibrillar and cytoskeletal proteins and/or new binding of cytosolic associated proteins in stunned myocardium, as well as the mechanisms responsible for them are elusive. The factors that determine the severity of stunning after regional ischemia include, among others, the severity and duration of flow deprivation, the myocardial temperature, the size of the ischemic region, the loading conditions of the heart. Myofibrillar degradation probably depends on all these factors. Unfortunately, most studies were performed in different experimental settings (in situ or isolated heart models, skinned fibers, isolated myocytes etc.) and with variable durations of ischemia and reperfusion. The standard biochemical procedures (SDS–PAGE, HPLC, Western blot) did not always allow the detection of peptides with very high or very low kDa. Finally, the identification of the degraded proteins may depend on the sensitivity of antibodies. It is not even known whether the calcium hypothesis is applicable to myocardial stunning in vivo. Anyway, the recent findings, including those of Papp et al. [9], have emphasize new structural aspects of myocardial stunning, that is probably a multifactorial process and involves complex sequences of cellular perturbations. At present, it is important to determine to what extent calcium overload and impaired calcium responsiveness play a major role in the genesis of myofilament lesion responsible for stunning, the mechanism that produces such lesions, and how contractile dysfunction is associated to impaired calcium responsiveness. The organization of the contractile apparatus appears unchanged but subtle abnormalities of cytoskeletal proteins and their associated proteins may, as those concerning the myofibrillar proteins, be important factors of myofibril dysfunction. They need to be further analysed for a better comprehension of the pathophysiology of myocardial stunning. Moreover, changes of the myofibrillar, cytoskeleton and associated proteins expression could be used as markers in the evolution of ischemic damage. The author acknowledges the assistance of G. Butler-Brown for English editing and the support from the Centre National de la Recherche Scientifique (CNRS).