(849.9) Loss of Mitochondrial Dynamics Proteins Mitofusin-2 and Drp-1 in Myocardial Ischemia-Reperfusion Injury Is Prevented by Matrix Metalloproteinase-2 Preferring Inhibitors

Poster Board Number: E9

Wesam Bassiouni (Faculty of Medicine and Dentistry, University of Alberta), John Seubert (Faculty of Medicine and Dentistry, University of Alberta, Faculty of Medicine and Dentistry, University of Alberta), Richard Schulz (Faculty of Medicine and Dentistry, University of Alberta, Faculty of Medicine and Dentistry, University of Alberta)

Objective:

 Matrix metalloproteinase-2 (MMP-2) is a ubiquitous protease that cleaves several extracellular and intracellular proteins. MMP-2 is activated intracellularly in response to enhanced oxidative stress resulting from myocardial ischemia/reperfusion (I/R) injury. Oxidative stress impairs mitochondrial function which is regulated by different proteins including mitofusin-2 (Mfn-2) and dynamin-related protein-1 (Drp-1) which control mitochondrial dynamics involving fusion and fission, respectively. As both proteins are localized at the mitochondrial outer membrane and MMP-2 is localized at the mitochondrial-endoplasmic reticulum associated membrane we hypothesized that MMP-2 may proteolyze Mfn-2 and Drp-1. We therefore investigated whether inhibition of MMP-2 could protect against the loss of mitochondrial dynamics proteins during I/R injury.

Methods:

Hearts isolated from 3 month old C57BL/6J mice were perfused according to Langendorff at constant pressure and subjected to I/R injury (30 min ischemia and 40 min reperfusion) in absence or presence of MMP-2 preferring inhibitors ARP-100 (10 µM) or ONO-4817 (50 µM) or their vehicle (n=5 hearts per group). Effects on mechanical function were recorded.  At the end of reperfusion, the hearts were homogenized and subcellular fractions were prepared. The degradation of troponin I (TnI), an intracellular MMP-2 target, was measured in the cytosolic fraction as a marker of MMP-2 activity. Levels of Mfn-2 and Drp-1 in the mitochondrial fraction were also measured using western blot. In silico analysis of potential MMP-2 cleavage sites was performed using Procleave.

Results:

ARP-100 or ONO-4817 significantly increased left ventricular developed pressure compared to vehicle-treated I/R hearts (% of pre-ischemic baseline value at R40: IR+vehicle 25.5±3.2, IR+ARP 50.1±3.2, IR+ONO 57.3±1.6%, plt;0.05). Similarly, both inhibitors significantly improved the rates of contraction and relaxation (+/-dP/dt). TnI loss was increased in I/R hearts as shown by a significant reduction in TnI (~30 kDa) and the appearance of an ~22 kDa band. ARP-100 or ONO-4817 attenuated TnI loss, indicating MMP-2 activation. Levels of Mfn-2 and Drp-1 in the mitochondrial fraction were significantly reduced in the I/R group and ARP-100 or ONO-4817 attenuated this reduction (plt;0.05). Similar results were obtained using a different Mfn-2 antibody that binds to its C-terminus. In silico analysis of both mouse Mfn-2 and Drp-1 sequences showed several potential sites that could be targeted by MMP-2.

Conclusions:

During myocardial I/R injury, MMP-2 may affect mitochondrial dynamics by proteolysis of Mfn-2 and Drp-1. Inhibition of MMP-2 activity could protect against cardiac contractile dysfunction in part by preserving these proteins affecting mitochondrial fusion and fission.

Canadian Institutes of Health Research (to RS and JMS); Alberta Innovates Graduate Student Scholarship (to WB).