MMP inhibitors attenuate doxorubicin-induced heart failure by preventing cardiac titin proteolysis

B. Y. Chan¹, A. Roczkowsky¹, N. Moser¹, M. Poirier¹, R. Ilarraza¹, H. Granzier², R. Schulz¹. ¹Pharmacology, University of Alberta, Edmonton, Canada, ²Cellular and Molecular Medicine, University of Arizona, Tucson.

Introduction: Heart failure remains a major, long-term complication with doxorubicin (DXR) chemotherapy (1). We investigated the mechanism of DXR cardiotoxicity in order to develop new strategies to prevent heart injury when treating cancer patients. DXR enhances oxidative stress and activates two intracellular isoforms of matrix metalloproteinase-2 (MMP-2) in cardiac myocytes (2). Intracellular MMP-2 is known to exacerbate myocardial oxidative stress injury by cleaving specific sarcomeric proteins including titin (3). We determined whether MMP-2 mediated titin proteolysis contributes to DXR-induced heart failure in vivo.

Method: 8-week old male C57BL/6J mice were treated with DXR (6 mg/kg/wk, i.p.) or saline vehicle (control) ± MMP inhibitors doxycycline (15 mg/kg/day, gavage) or ONO-4817 (60 mg/kg/day, gavage) for 4 weeks. Cardiac function was assessed by M-mode echocardiography before and after treatment (n=10 per group). The hearts and plasma were collected following post-treatment echocardiography and MMP-2 protein levels and activity were measured by western blot and gelatin zymography, respectively. Titin (T1) and its degradation product (T2) levels were measured by agarose gel electrophoresis. Data are represented as mean ± SEM (n animals) and statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test.

Results: DXR caused systolic and diastolic dysfunction marked by a significant reduction in left ventricular ejection fraction (64.0±2.3% vs. 45.5±2.4%, p<0.0001), fractional shortening (34.5±1.7% vs. 22.3±1.4%, p<0.0001), and E/A ratio (1.07±0.04 vs. 0.85±0.06, p<0.05) compared to control. Doxycycline or ONO-4817 prevented these changes. DXR trended to increase MMP-2 activity by two-fold in hearts (p=0.062, n=9) and this was not observed in doxycycline or ONO-4817 hearts (n=10 each). In plasma, DXR increased MMP-2 activity by two-fold (p<0.05, n=10) and protein levels by 225% (p<0.01, n=8) relative to control. ONO-4817, but not doxycycline, attenuated DXR-induced MMP-2 activity and protein levels in plasma (p<0.05, n=8-10). T2/T1 titin ratio was increased by 258±7% (n=8) in DXR hearts and this was reduced by ONO-4817 (169±10%, n=8), but not doxycycline (203±16%, n=8), relative to control.

Conclusions: MMP-2 contributes to DXR-induced heart failure in mice by proteolyzing cardiac titin and this was blocked by ONO-4817, which is the more potent MMP inhibitor compared to doxycycline. Prophylactic use of orally available MMP inhibitors may be a potential therapeutic strategy to prevent heart failure in cancer patients undergoing chemotherapy.

References:

1. Bloom MW et al. (2016). Circ Heart Fail 9: e002661.

2. Chan BY et al. (2016). FASEB J 30: 742.6.

3. Ali MA et al. (2010). Circulation 122: 2039-2047.