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Putative Sarcomeric Targets of Doxorubicin Induced Matrix Metalloproteinase‐2 Activity in Cardiomyocyctes

Anthracyclines, such as doxorubicin, are used to treat a variety of cancers. However, doxorubicin's therapeutic utility is limited because it causes dose‐dependent myocardial injury, which may lead to heart failure in up to 11% of patients. Doxorubicin triggers this injury, in part, by stimulating cellular oxidative stress. Oxidative stress is known to activate matrix metalloproteinase‐2 (MMP‐2), an extra‐ and intra‐cellular protease, which has been implicated in many heart pathologies. Activated intracellular MMP‐2 has been shown to proteolyze sarcomeric proteins α‐actinin, troponin I, and the regulatory protein glycogen synthase kinase‐3β (GSK‐3β) in cardiac tissue or cells under oxidative stress, impairing contractile function. We hypothesized that intracellular MMP‐2 plays a role in doxorubicin cardiotoxicity by proteolyzing these substrates. Neonatal rat ventricular myocytes (NRVM) and human fibrosarcoma (HT1080) cells were treated with doxorubicin (0.5 μM) ± selective MMP‐2 inhibitors ARP‐100 (1 μM) or ONO‐4817 (1 μM) for 2–48 hr. Cell death was evaluated by lactate dehydrogenase release, MMP‐2 activity was measured by gelatin zymography, and MMP‐2 protein and its protein target levels were analyzed by immunoblot. Doxorubicin was used at a concentration which caused 15% cell death in neoplastic HT1080 cells but none in NRVM after 24 hr. In NRVMs, 24 hr doxorubicin increased intracellular MMP‐2 activity up to 311% relative to vehicle control, and this was attenuated by 60% with ARP‐100 or ONO‐4817. 24 hr doxorubicin‐induced MMP‐2 activation is caused, in part, by a 213% increase in MMP‐2 protein levels. Doxorubicin reduced troponin I levels by 40%, however, this was not normalized by ARP‐100 or ONO‐4817. There were no significant changes in α‐actinin or GSK‐3β levels. Doxorubicin at an antitumour concentration activates myocardial MMP‐2 in part by increasing MMP‐2 protein levels. The role of doxorubicin‐induced MMP‐2 activation on the reduction of cellular troponin I levels needs further investigation. Other potential MMP‐2 targets in the sarcomere (eg. titin, myosin light chain‐1) and in other cellular compartments need to be analyzed to better understand the pathophysiological actions of doxorubicin in cardiac myocytes.