Visnagin protects against doxorubicin-induced cardiomyopathy through modulation of mitochondrial malate dehydrogenase

Abstract

Doxorubicin is a highly effective anti-cancer chemotherapy agent, but its usage is limited by its cardiotoxicity. To develop a drug that prevents the cardiac toxicity of doxorubicin while preserving its anti-tumor potency, we established a doxorubicin-induced cardiomyopathy model in zebrafish that recapitulated the cardiomyocyte apoptosis and contractility decline observed in patients. Using this model, we screened 3000 compounds and discovered that visnagin (VIS) and diphenylurea (DPU) rescue cardiac performance and circulatory defects caused by doxorubicin treatment in zebrafish. VIS and DPU reduced doxorubicin-induced apoptosis in cultured cardiomyocytes and in vivo in zebrafish and mouse hearts. Furthermore, VIS treatment improved cardiac contractility in doxorubicin-treated mice. Importantly, VIS and DPU caused no reduction in the chemotherapeutic efficacy of doxorubicin in several cultured tumor lines or in zebrafish and mouse xenograft models. Using affinity chromatography, we discovered that VIS binds to mitochondrial malate dehydrogenase (MDH2), one of the key enzymes in the tricarboxylic acid cycle. As with VIS, treatment with the MDH2 inhibitors mebendazole, thyroxine, and iodine prevented doxorubicin cardiotoxicity, as did treatment with malate itself, suggesting that modulation of MDH2 activity is responsible for VIS’s cardioprotective effects. Taken together, this study identified VIS and DPU as potent cardioprotective compounds and implicates MDH2 as a previously undescribed, druggable target for doxorubicin-induced cardiomyopathy.

Highly potent visnagin derivatives inhibit Cyp1 and prevent doxorubicin cardiotoxicity

“…We previously reported that C1 binds to mitochondrial malate dehydrogenase (MDH2) and postulated that modulation of MDH2 may be responsible for the cardioprotective effect of C1 (12). However, we have found manipulation of this pathway for therapeutic purposes to be challenging, given the importance of MDH2 in maintaining normal mitochondrial metabolism and bioenergetics.…”

“…As oxidative stress has been reported as a key mechanism of DOX-induced cardiotoxicity, we previously assessed hydrogen peroxide (H 2 O 2 ) levels in cultured cardiomyocytes (12). DOX treatment was associated with an increase in H 2 O 2 levels that was not affected by the addition of C1.…”

“…DOX uptake in the heart was, thus, quantifiable and allowed a threshold to be applied so that therapeutic impact was examined only in the hearts in which DOX retention, and hence the potential for toxicity, was confirmed ( Figure 4B). To assess for cardiac apoptosis in mice, we performed ex vivo imaging of the myocardium using an annexin V conjugate with a near-infrared fluorochrome, Annexin-Vivo 750 (13), a technique with which we had previously shown suppression of DOX-induced apoptosis with C1 at a dose of 25 mg/kg (12). C23 significantly reduced Annexin-Vivo fluorescence in the mouse heart at concentrations as low as 0.1 mg/kg by i.p.…”

“…TuAB zebrafish embryos at 30 hours after fertilization were treated with DOX 100 μM in 96-well plates as previously described (12). Visnagin analogs were dissolved in DMSO and added at concentrations ranging from 3 nM-10 μM (n = 6 fish per dose; < 1% DMSO v/v).…”

“…To tackle this problem, our laboratory established an embryonic zebrafish model of DOX cardiomyopathy characterized by pericardial edema, impaired cardiac contractility, and decreased blood flow through the vasculature (12). Through a large chemical screen in this model, we identified visnagin (compound 1 [C1]) as the most potent and least toxic antidote to DOX cardiotoxicity in zebrafish (half maximal effective concentration [EC 50 ] 1 μM; median toxic dose [TD 50 ] > 10 μM; Figure 1).…”