Matrix metalloproteinase inhibition protects against junctophilin‐2 proteolysis during doxorubicin‐induced cardiotoxicity

Treatment of cancer patients with anthracyclines is known to cause dose-dependent cardiotoxicity through several mechanisms including enhanced oxidative stress, ultimately resulting in defective excitation-contraction coupling. Loss of junctophilin-2 (JPH-2), which tethers transverse tubules (T-tubules) to the sarcoplasmic reticulum, is a feature of doxorubicin-induced cardiotoxicity, yet the protease involved in unclear. As activation of matrix metalloproteinase-2 (MMP-2) is known to contribute to doxorubicin-induced cardiotoxicity, we investigated here the role of MMP-2 in JPH-2 proteolysis and defective calcium transients in it. C57BL/6J mice were treated with doxorubicin for 4 weeks with or without the MMP inhibitor (doxycycline), MMP-2 preferring inhibitor (ONO-4817) or vehicle, and cardiac function was assessed using echocardiography. JPH-2 levels in ventricular extracts were measured. Calcium transients and JPH-2 levels were measured in neonatal rat ventricular cardiomyocytes treated with doxorubicin and ONO-4817. Both MMP inhibitors attenuated doxorubicin-induced cardiac systolic and diastolic dysfunction. Doxorubicin treatment resulted in JPH-2 cleavage in mouse hearts as evidenced by the appearance of lower molecular weight products of 63 and 25 kDa, which was prevented by MMP inhibitors. Loss of JPH-2 and impaired calcium transients were observed in neonatal rat ventricular cardiomyocytes treated with doxorubicin, while ONO-4817 attenuated these changes. In silico analysis predicted cleavage sites between JPH-2 MORN repeats and within its unstructured region. These results reveal that JPH-2 proteolysis is a consequence of MMP-2 activation and highlight the beneficial prophylactic action of two orally available MMP inhibitors in preventing doxorubicin-induced cardiotoxicity.