AIMS Matrix metalloproteinase-2 (MMP-2) is a zinc dependent protease which contributes to cardiac contractile dysfunction when activated during myocardial ischemia-reperfusion (IR) injury. MMP-2 is localized to several subcellular sites inside cardiac myocytes, however, its role in the sarcoplasmic reticulum (SR) is unknown. The Ca2+ ATPase SERCA2a, which pumps cytosolic Ca2+ into the SR to facilitate muscle relaxation, is degraded in cardiac IR injury, however, the protease responsible for this is unclear. We hypothesized that MMP-2 contributes to cardiac contractile dysfunction by proteolyzing SERCA2a, thereby impairing its activity in IR injury. METHODS AND RESULTS Isolated rat hearts were subjected to IR injury in the presence or absence of the selective MMP inhibitor ARP-100, or perfused aerobically as a control. Inhibition of MMP activity with ARP-100 significantly improved the recovery of cardiac mechanical function and prevented the increase of a 70 kDa SERCA2a degradation fragment following IR injury, although 110 kDa SERCA2a and phospholamban levels appeared unchanged. Electrophoresis of IR heart samples followed by LC-MS/MS confirmed the presence of a SERCA2a fragment of ∼70 kDa. MMP-2 activity co-purified with SR enriched microsomes prepared from the isolated rat hearts. Endogenous SERCA2a in SR enriched microsomes was proteolyzed to ∼70 kDa products when incubated in vitro with exogenous MMP-2. MMP-2 also cleaved purified porcine SERCA2a in vitro. SERCA activity in SR enriched microsomes was decreased by IR injury, however, this was not prevented with ARP-100. CONCLUSIONS This study shows that MMP-2 activity is found in SR enriched microsomes from heart muscle and that SERCA2a is proteolyzed by MMP-2. The cardioprotective actions of MMP inhibition in myocardial IR injury may include the prevention of SERCA2a degradation.

中文翻译：

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心肌MMP-2有助于心肌缺血再灌注损伤期间SERCA2a的蛋白水解。

AIMS基质金属蛋白酶2（MMP-2）是一种锌依赖性蛋白酶，当在心肌缺血再灌注（IR）损伤期间被激活时，会导致心脏收缩功能障碍。MMP-2位于心肌细胞内部的几个亚细胞部位，但是，其在肌浆网（SR）中的作用尚不清楚。Ca2 + ATPase SERCA2a可将胞液中的Ca2 +泵入SR以促进肌肉松弛，但在心脏IR损伤中会降解，但是尚不清楚引起这种蛋白酶的原因。我们假设MMP-2通过蛋白水解SERCA2a导致心脏收缩功能障碍，从而削弱其在IR损伤中的活性。方法和结果在存在或不存在选择性MMP抑制剂ARP-100的情况下，对离体大鼠的心脏进行IR损伤，或以有氧灌注作为对照。用ARP-100抑制MMP活性可显着改善心脏机械功能的恢复，并防止IR损伤后70 kDa SERCA2a降解片段的增加，尽管110 kDa SERCA2a和phosphorlamban水平似乎没有变化。对IR心脏样品进行电泳，然后进行LC-MS / MS证实，存在约70 kDa的SERCA2a片段。MMP-2活性与从分离的大鼠心脏制备的富含SR的微粒体共同纯化。当与外源性MMP-2体外孵育时，富含SR的微粒体中的内源性SERCA2a被蛋白水解为约70 kDa的产物。MMP-2还可以在体外切割纯化的猪SERCA2a。富含IR的微粒体中的SERCA活性会因IR损伤而降低，但是ARP-100不能阻止这一点。结论这项研究表明，在心肌中富含SR的微粒体中发现了MMP-2活性，并且SERCA2a被MMP-2蛋白水解。MMP抑制在心肌IR损伤中的心脏保护作用可能包括防止SERCA2a降解。