Investigating the role of MMP-2 in sarcoplasmic reticulum dysfunction during cardiac ischemia-reperfusion injury

A. Roczkowsky¹, G. Armanious², B. Y. Chan¹, R. Ilarraza¹, H. Young², R. Schulz¹. ¹Pharmacology, University of Alberta, Edmonton, Canada, ²Biochemistry, University of Alberta, Edmonton, Canada.

Introduction: Ischemic heart disease is a leading cause of global mortality (1). Reperfusion of ischemic hearts causes further injury and results in cardiac contractile dysfunction. Ischemia-reperfusion (IR) injury is associated with impaired sarcoplasmic reticulum (SR) calcium transport and activation of matrix metalloproteinase-2 (MMP-2) (2, 3). MMP-2 is also an intracellular protease which targets proteins within cardiomyocytes such as troponin I and titin (3). Our preliminary results show that MMP-2 can degrade SERCA2a, a protein channel which transports calcium into the SR to facilitate muscle relaxation during diastole, in vitro. We hypothesize that MMP-2 proteolyzes SERCA2a during IR injury.

Method: Three groups of isolated hearts from male Sprague Dawley rats were perfused in working mode: a) aerobic perfusion (n=12), b) 20 min ischemia followed by 30 min reperfusion (IR, n=7), or c) IR+ARP-100, an MMP inhibitor (10μM, n=6). Mechanical function was measured as cardiac work, the product of cardiac output and peak systolic pressure. SR enriched fractions were obtained from hearts through differential centrifugation. These fractions were incubated with MMP-2 (37°C, 2 hr) and following electrophoresis SERCA2a and its degradation measured by immunoblot. SERCA2a levels in ventricular homogenates were measured by immunoblot. For statistical analysis either two-way or one-way ANOVA followed by Tukey’s test were used.

Results: In hearts subjected to IR injury, ARP-100 significantly improved contractile function during reperfusion from 2.6±1.0 to 5.1±0.5 mmHg•mL•min^-1•0.001 in IR versus IR+ARP-100 groups, respectively (p<0.05). SR enriched fractions incubated with MMP-2 resulted in SERCA2a proteolysis to a prominent ∼70kDa band and less abundant smaller products all which were prevented by ARP-100. MMP-2 also caused a reduction in SERCA2a oligomers (≥200kDa). The protein levels of 100kDa SERCA2a in ventricular homogenates was unchanged (p=0.70, n=6-7), however, a ∼70kDa degradation band trended to increase in IR two-fold which was normalized by ARP-100 (p=0.07, n=4-5).

Conclusions: We demonstrated a cardioprotective effect of the MMP inhibitor ARP-100 in myocardial IR injury. We also show evidence of SERCA2a proteolysis during IR injury which may be caused by MMP-2. This study aims to provide insight into the mechanisms of IR injury and may aid in the development of new MMP inhibitors to treat ischemic heart disease.

References:

1. Hausenloy DJ and Yellon DM (2013). J Clin Invest 123: 93-100

2. Osada M et al. (1998). Am J Physiol 274: H2025-H2034

3. Hughes BG and Schulz R (2014). Basic Res Cardiol 109: 424-443