Department of Pediatrics, University of Alberta, Mazankowski Alberta Heart Institute, 462 Heritage Medical Research Centre, Edmonton, AB T6G 2S2, Canada.
Department of Pharmacology, University of Alberta, Edmonton, AB, Canada.
Cardiovasc Res. 2020 Apr 1;116(5):1021-1031. doi: 10.1093/cvr/cvz207.
Matrix metalloproteinase-2 (MMP-2) is a zinc-dependent protease which contributes to cardiac contractile dysfunction when activated during myocardial ischaemia-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.
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.
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.
基质金属蛋白酶-2（MMP-2）是一种锌依赖性蛋白酶，在心肌缺血再灌注（IR）损伤过程中被激活时，有助于心肌收缩功能障碍。MMP-2 定位于心肌细胞内的几个亚细胞部位；然而，其在肌浆网（SR）中的作用尚不清楚。将胞质溶胶中的 Ca2+泵入 SR 以促进肌肉松弛的 Ca2+-ATP 酶 SERCA2a 在心肌 IR 损伤中被降解；然而，负责这种情况的蛋白酶尚不清楚。我们假设 MMP-2 通过蛋白水解 SERCA2a 导致心脏收缩功能障碍，从而损害其在 IR 损伤中的活性。
在存在或不存在选择性 MMP 抑制剂 ARP-100 的情况下，将分离的大鼠心脏进行 IR 损伤，或作为对照进行有氧灌注。用 ARP-100 抑制 MMP 活性可显著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 和磷蛋白水平似乎没有变化。IR 心脏样本的电泳后通过 LC-MS/MS 证实存在约 70 kDa 的 SERCA2a 片段。从分离的大鼠心脏中制备的富含 SR 的微粒体中 MMP-2 活性与 SR 一起纯化。在体外与外源性 MMP-2 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 还在体外切割纯化的猪 SERCA2a。IR 损伤使富含 SR 的微粒体中的 SERCA 活性降低；然而，用 ARP-100 并不能防止这种情况。
本研究表明，MMP-2 活性存在于心肌富含 SR 的微粒体中，并且 SERCA2a 被 MMP-2 蛋白水解。在心肌 IR 损伤中抑制 MMP 的心脏保护作用可能包括防止 SERCA2a 降解。
