Doxorubicin Exposure Causes Subacute Cardiac Atrophy Dependent on the Striated Muscle-Specific Ubiquitin Ligase MuRF1

Abstract

Background Anthracycline chemotherapeutics, such as doxorubicin, are used widely in the treatment of numerous malignancies. The primary dose-limiting adverse effect of anthracyclines is cardiotoxicity that often presents as heart failure due to dilated cardiomyopathy years after anthracycline exposure. Recent data from animal studies indicate that anthracyclines cause cardiac atrophy. The timing of onset and underlying mechanisms are not well defined, and the relevance of these findings to human disease is unclear. Methods and Results Wild-type mice were sacrificed 1 week after intraperitoneal administration of doxorubicin (1-25 mg/kg), revealing a dose-dependent decrease in cardiac mass ( R²=0.64; P<0.0001) and a significant decrease in cardiomyocyte cross-sectional area (336±29 versus 188±14 µm²; P<0.0001). Myocardial tissue analysis identified a dose-dependent upregulation of the ubiquitin ligase, MuRF1 (muscle ring finger-1; R²=0.91; P=0.003) and a molecular profile of muscle atrophy. To investigate the determinants of doxorubicin-induced cardiac atrophy, we administered doxorubicin 20 mg/kg to mice lacking MuRF1 (MuRF1^-/-) and wild-type littermates. MuRF1^-/- mice were protected from cardiac atrophy and exhibited no reduction in contractile function. To explore the clinical relevance of these findings, we analyzed cardiac magnetic resonance imaging data from 70 patients in the DETECT-1 cohort and found that anthracycline exposure was associated with decreased cardiac mass evident within 1 month and persisting to 6 months after initiation. Conclusions Doxorubicin causes a subacute decrease in cardiac mass in both mice and humans. In mice, doxorubicin-induced cardiac atrophy is dependent on MuRF1. These findings suggest that therapies directed at preventing or reversing cardiac atrophy might preserve the cardiac function of cancer patients receiving anthracyclines.

Keywords: anthracycline; atrophy; cardiotoxicity; doxorubicin; heart failure; mice; ubiquitin.

Figure 1.. Doxorubicin induces a dose-dependent decrease in cardiac mass.

C57Bl6J mice were given a single dose of intraperitoneal doxorubicin. (A) Heart weight indexed to body weight at each dose (n=9 for 20 mg/kg, n=6 for all other doses) (B) Heart weight indexed to tibia length; (C) Heart weight indexed to body weight for male and female mice that received doxorubicin 20 mg/kg.

Figure 2.. Doxorubicin causes cardiomyocyte atrophy in vivo.

Mice were sacrificed 7 days after receiving doxorubicin 20 mg/kg i.p. (A) Representative wheat germ agglutinin-Alexa Fluor 488 conjugate stained heart sections. Analysis of myocyte cross-sectional areas from 4 hearts (800 cross-sectional areas total, 200 cross sections per mouse over 4–6 histological sections). A Student’s t-test was used to determine significance between groups. (B) Serum Troponin I was measured by ELISA. (C) Summary densitometry of immunoblots for markers of apoptosis. n= 6 for VC, n = 3–4 for DOX-treated. Results were compared using a one-way ANOVA. c-Caspase 3 = cleaved caspase 3; c-PARP = cleaved poly ADP ribose polymerase, LAD = left anterior descending coronary artery.

Figure 3.. Doxorubicin causes a dose-dependent decrease in contractile function on conscious echocardiography.

Mice were injected with intraperitoneal doxorubicin. Conscious echocardiography was performed at baseline and 7 days after injection. (A) Contractile function (fractional shortening) as a function of doxorubicin dose (n=15 for vehicle, n=9 for 20mg/kg, n=6 for all other doses) by logistic regression; (B) Left ventricular volumes at baseline and Day 7 by Student’s t-test; (C) Calculated left ventricular mass at baseline and on Day 7.

Figure 4.. Doxorubicin increases cardiac expression of MuRF-1 and a molecular signature of atrophy in vivo.

Mice were sacrificed 7 days after intraperitoneal injection of doxorubicin or vehicle control. (A) Quantitative RT-PCR assayed molecular markers of atrophy at various DOX doses. Logistic regression was applied to MuRF1 results. (B) Heart lysates were immunoblotted and (C) summary densitometry was compared (vehicle control vs. DOX 25 mg/kg) by Student’s t-test.

Figure 5.. MuRF1^−/− mice are protected from doxorubicin-induced cardiac atrophy.

MuRF1^−/− and littermate MuRF1^+/+ mice were given doxorubicin 20 mg/kg i.p. and sacrificed after 7 days. (A) Body weight before doxorubicin (Day 0) and on the day of sacrifice (Day 7); (B) Heart mass on Day 7; (C) Heart mass indexed to tibia length on Day 7 (inset: tibia length). (D) Contractile function (ejection fraction) was measured using (E) conscious echocardiography. #p<0.05 by Student’s T-test. One-way Analysis of Variance followed by an all pairwise multiple comparison procedure (Holm-Sidak method) compared multiple groups. *p<0.05 vs. Column 1 and Column 2, **p<0.05 vs. All other columns, ***p<0.05 vs. Column 1.

Figure 6.. Anthracyclines are associated with a subacute decrease in cardiac mass detected by cardiac MRI in cancer patients.

70 subjects from the DETECT-1 cohort analysis underwent cardiac MRI at baseline, then 1, 3, and 6 months after initiation of anthracycline-based chemotherapy. The rate of change in left ventricular mass by CMR was analyzed by constructing a linear mixed model with random intercepts and time effects using SAS 9.4. Inset shows full range of values.