The Calpain-Calpastatin System and the Calcium Paradox in the Isolated Perfused Pigeon Heart

Abstract

To examine whether the calpain-calpastatin system is activated during the calcium paradox in the isolated perfused pigeon heart, we separated the protease from its inhibitor calpastatin and studied its kinetic properties. The protease exhibits kinetic properties similar to those of mammalian m-calpains. Ca²⁺ requirements for half and maximum activities are 220 µM and 2 mM, respectively. In the absence of Ca²⁺ the protease is strongly activated by Mn²⁺ or Sr²⁺. In the presence of Ca²⁺, Mn²⁺ and Sr²⁺ exhibit a synergistic effect; Mg²⁺ and Ba²⁺ have no effect, whereas Co²⁺, Ni²⁺ and Cd²⁺ completely inhibit its activation. Furthermore, we measured the activity of calpain and calpastatin under either conditions inducing a calcium paradox, or protecting the heart against this phenomenon. Although the calpain/calpastatin ratio is lowered during Ca²⁺ depletion, during Ca²⁺ repletion it is markedly inverted. Calpain activation during reperfusion is inhibited by the presence of 200 µM Mn²⁺ or Ba²⁺, in the Ca²⁺-free medium. Gel filtration of calpastatin, isolated from either untreated hearts or during Ca²⁺ depletion, produces two main peaks of ñ150 and 40 kDa of molecular mass, respectively, whereas calpastatin isolated during the 2^nd min of reperfusion appears to be shifted to the 150 kDa form. All the above data suggest that this system may be involved in the induction of the calcium paradox in pigeon heart.

Calpain inhibitor MDL 28170 protects against the Ca2+ paradox in rat hearts

“…In the present study, we found increased calpain‐mediated degradation of α‐fodrin during Ca 2+ repletion, which was attenuated by the selective calpain inhibitor MDL 28170. These findings are indicative of calpain activation during the Ca 2+ paradox and are consistent with those of a previous study that reported increased in vitro cleavage products of denatured casein by extracts from Ca 2+ ‐paradoxic hearts . Moreover, confocal image analysis showed the translocation of both μ‐ and m‐calpain to the sarcolemmal membrane during Ca 2+ repletion, suggesting that both isoforms contribute to Ca 2+ paradox‐induced heart injury.…”

“…In addition, other proteins, including Na + /K + ‐ATPase and nitric oxide synthase, have been shown to be targets of calpain activation in the heart . A previous study in pigeon hearts demonstrated that the Ca 2+ paradox may trigger a transient increase in calpain activity . However, it has not been determined whether calpain mediates Ca 2+ paradox‐induced heart injury and, if so, which isoform of calpain is involved.…”

“…13,24 A previous study in pigeon hearts demonstrated that the Ca 2+ paradox may trigger a transient increase in calpain activity. 25 However, it has not been determined whether calpain mediates Ca 2+ paradoxinduced heart injury and, if so, which isoform of calpain is involved. In addition, the underlying mechanism needs to be clarified.…”

Modulation of nuclear factor-kappaB improves cardiac dysfunction associated with cardiopulmonary bypass and deep hypothermic circulatory arrest*

“…Calcium overload associated with ischemia and reperfusion activates calpains and reduces calpastatin levels (17, 18). Until recently, no in vivo studies have examined calpain regulation of the NF-κB pathway during reperfusion of immature myocardium.…”

Different Roles for Contracture and Calpain in Calcium Paradox-Induced Heart Injury

“…Although the amount of Ca 2+ required for the in vitro activation of μ-calpain and m-calpain was different and m-calpain was regulated by binding to phosphatidylinositol 4,5-bisphosphate [12], [13], ample studies have shown that calpains are activated during ischemia/reperfusion, resulting in the cleavage of its substrates, such as Na + /K + -ATPase, α-fodrin, a prominent component of the membrane skeleton, and consequently heart injury [16], [17], [18], [19]. We and others have previously found that after Ca 2+ repletion, calpains are activated and both μ- and m-calpain are anchored to the sarcolemmal membrane [20], [21]; moreover, the addition of MDL28170, an inhibitor of calpain, reduced the cleavage of α-fodrin and rescued myocardial dysfunction and cell death [20]. However, MDL28170 did not substantially reduce the level of LVEDP and the degradation of troponin I [20], a regulatory protein involved in maintaining the diastolic state.…”