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

Abstract

Doxorubicin is a highly effective anticancer chemotherapy agent, but its use is limited by its cardiotoxicity. To develop a drug that prevents this toxicity, we established a doxorubicin-induced cardiomyopathy model in zebrafish that recapitulates the cardiomyocyte apoptosis and contractility decline observed in patients. Using this model, we screened 3000 compounds and found that visnagin (VIS) and diphenylurea (DPU) rescue the cardiac performance and circulatory defects caused by doxorubicin in zebrafish. VIS and DPU reduced doxorubicin-induced apoptosis in cultured cardiomyocytes and in vivo in zebrafish and mouse hearts. VIS treatment improved cardiac contractility in doxorubicin-treated mice. Further, VIS and DPU did not reduce the chemotherapeutic efficacy of doxorubicin in several cultured tumor lines or in zebrafish and mouse xenograft models. Using affinity chromatography, we found that VIS binds to mitochondrial malate dehydrogenase (MDH2), a key enzyme 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' cardioprotective effects. Thus, VIS and DPU are potent cardioprotective compounds, and MDH2 is a previously undescribed, druggable target for doxorubicin-induced cardiomyopathy.

Fig 1. Discovery of doxorubicin suppressors VIS and DPU in a doxorubicin-induced cardiomyopathy model in zebrafish

(A) Experimental scheme for inducing cardiomyopathy in zebrafish. (dpf, days post fertilization; Dox, doxorubicin) (B) Representative images of a normal heart (blue arrow) and doxorubicin-treated heart (red arrow).(DM, DMSO control) (C, M) Images reflecting heart contraction over time, derived from a region of interest drawn across the ventricle in sequential frames of high speed video of the beating heart. (D) Representative images of cardiomyocytes genetically labeled with nuclear dsRed driven by a cardiomyocyte-specific myl7 promoter. (E) Detection of apoptotic cardiomyocyte by TUNEL staining. Green, TUNEL, red, cardiomyocytes, yellow, apoptotic cardiomyocytes. (F,N) Quantification of fractional shortening (FS) as follows (VID_d-VID_s)/VID_d ×100. VID_d, diastolic ventricular internal dimension; VID_s, systolic ventricular internal dimension. n = 13–19 fish in each group, compared using Student’s T-test. (G) Quantification of cardiomyocyte number. n = 5 fish in each group, compared using Student’s T-test. (H) Quantification of apoptotic cardiomyocytes (CM). n = 5 fish in each group, compared using Student’s T-test. (I,J) VIS and DPU rescued heart contraction and blood flow in a dose dependent manner, as manifested by the percentage of normal looking fish out of total number of doxorubicin treated fish (K,L) Representative images showing grossly normal heart morphology in VIS or DPU rescued zebrafish. DM, DMSO treated control samples; Dox, doxorubicin treated samples; Dox + VIS, doxorubicin and VIS co-treated samples; Dox + DPU, doxorubicin and DPU co-treated samples. Statistics (compared to doxorubicin treated samples): * p< 0.05, ** p<0.01, and *** p<0.001.

Fig 2. VIS and DPU effects on doxorubicin-induced cardiac cell death in vitro and in vivo

Doxorubicin treated neonatal rat cardiomyocytes were treated with VIS and DPU and assessed for apoptosis with TUNEL (A) or AnnexinV (B) staining. (C) Doxorubicin treated HL1 cells were treated with VIS or DPU and assessed for apoptosis with Annexin V staining. (D) Viability of HL1 cells treated with doxorubicin and VIS or DPU. Effect of VIS or DPU on cardiac cell death in doxorubicin treated zebrafish (E) and mice (F) as measured by TUNEL assay. n = 4–5 mice per group. (G) Effect of VIS on doxorubicin-induced apoptosis in heart as assessed by ex vivo imaging of the whole heart with the dye AnnexinV-750. n = 5 mice per group, compared using one-way ANOVA and the Tukey post-test. DM – DMSO treated control samples, Dox – doxorubicin treated samples, Dox + VIS – doxorubicin and VIS co-treated samples, Dox + DPU – doxorubicin and DPU co-treated samples. Statistics (compared to doxorubicin treated samples): * p< 0.05, ** p<0.01, and *** p<0.001.

Fig 3. VIS effect on heart function in doxorubicin treated mice

Representative M-mode echocardiogram images of acute (A) and chronic (D) models: yellow line denotes diastolic left ventricle internal dimension (LVIDd) and red line denotes systolic left ventricle internal dimension (LVIDs). Effects of doxorubicin with and without VIS on fractional shortening (B,E) and strain rate (C,F) in both acute (B,C) and chronic (E,F) models. For acute models (B,C), groups were compared using one-way ANOVA followed by the Tukey post-test. For chronic models (E,F), groups were compared using one-way ANOVA followed by a one-tailed Student’s T-test. Cont – vehicle treated control mice, Dox – doxorubicin treated mice, Dox + VIS – doxorubicin and VIS co-treated mice. Statistics (compared to doxorubicin treated samples): * p< 0.05, ** p<0.01, and *** p<0.001.

Fig 4. VIS protects against doxorubicin cardiotoxicity via inhibition of MDH2

(A) Bergapten, a potent VIS derivative, with an alkyl linker that could be attached to a solid support (Compound 1). (B) Affinity chromatography using a bergapten-linked matrix and competitive elution with VIS. BGP, bergapten; DM, dimethyl sulfoxide. (C) Western blot of gel in (B) incubated with an anti-MDH2 antibody. (D) Viability was measured in HL-1 cardiomyocytes treated with doxorubicin and MDH2 inhibitors. (E) Rescue from doxorubicin-induced cardiotoxicity and death in zebrafish co-treated with MDH2 inhibitors.. MBD, mebendazole; T4, L-thyroxine. Data were collected over 5 independent experiments, with a total of 150 zebrafish larvae treated for each condition. (F) L-malic acid protects against doxorubicin-induced cardiotoxicity and death in a dose-dependent fashion. (G) The malate-aspartate shuttle inhibitor AOA protects against doxorubicin-induced cardiotoxicity and death in a dose-dependent fashion. AOA, aminooxyacetate. For panels (D)-(G), data were collected from a total of 150 zebrafish larvae treated for each condition, as measured over 5 independent experiments. % Viability and % Rescue are depicted as mean +/− standard error of the mean. * p< 0.05, ** p<0.01, and *** p<0.001 using a two-tailed Student’s T-test.

Fig 5. Lack of protective effect of VIS nor DPU on tumor cells in vitro or in vivo

Cell viability in multiple tumor lines including DU145 (A), LNCaP (B), MCF7 (C) and MDA-MB-231 (D) treated with VIS or DPU.. (E) Fold change in size of xenografted human tumor derived from Jurkat cell injection in zebrafish in vivo treated with either VIS or DPU. (F) Tumor volume in human tumor xenograft derived from injected MDA-MB-231 cells in mice treated with doxorubicin and VIS. Tumor size was assessed by caliper measurement of long axis a, short axis b and was calculated with equation V=1/2*a*b². n = 8 mice in each group. (G) Representative images of postmortem tumors from the three groups. (H) Effect of VIS on Dox inhibition of tumor growth.Cont – vehicle treated control samples, Dox – doxorubicin treated samples, Dox + VIS – doxorubicin and VIS co-treated samples, Dox + DPU – doxorubicin and DPU co-treated samples. NS, non-significant. Statistics (compared to doxorubicin treated samples): * p< 0.05, ** p<0.01, and *** p<0.001.