Deletion of MCL-1 causes lethal cardiac failure and mitochondrial dysfunction

Abstract

MCL-1 is an essential BCL-2 family member that promotes the survival of multiple cellular lineages, but its role in cardiac muscle has remained unclear. Here, we report that cardiac-specific ablation of Mcl-1 results in a rapidly fatal, dilated cardiomyopathy manifested by a loss of cardiac contractility, abnormal mitochondria ultrastructure, and defective mitochondrial respiration. Strikingly, genetic ablation of both proapoptotic effectors (Bax and Bak) could largely rescue the lethality and impaired cardiac function induced by Mcl-1 deletion. However, while the overt consequences of Mcl-1 loss were obviated by combining with the loss of Bax and Bak, mitochondria from the Mcl-1-, Bax-, and Bak-deficient hearts still revealed mitochondrial ultrastructural abnormalities and displayed deficient mitochondrial respiration. Together, these data indicate that merely blocking cell death is insufficient to completely overcome the need for MCL-1 function in cardiomyocytes and suggest that in cardiac muscle, MCL-1 also facilitates normal mitochondrial function. These findings are important, as specific MCL-1-inhibiting therapeutics are being proposed to treat cancer cells and may result in unexpected cardiac toxicity.

Keywords: BCL-2; MCL-1; apoptosis; heart failure; mitochondria.

Figure 1.

Cardiomyopathy is induced upon constitutive Mcl-1 deletion in cardiac and skeletal muscle. (A) Survival of muscle-specific Mcl-1-deleted mice. (**) P < 0.01 by log-rank test when compared with controls. (B) Constitutive Mcl-1 deletion in heart and skeletal muscle. All tissues were harvested at P1 and immunoblotted with MCL-1 and Actin. (C) Hematoxylin and eosin (H&E)-stained longitudinal sections of the heart indicate dilated cardiomyopathy and a loss of myofibrils. Connective tissue indicative of fibrosis stained blue with the Masson's trichrome stain. Hearts were harvested at P7. Magnifications: top, 2×; middle and bottom, 40×. (D) H&E-stained longitudinal sections of the tongue skeletal muscle indicate swollen fragmented fibers with hyaline necrosis. Magnification, 40×. (E) Cardiac-specific deletion of Mcl-1 induced cell death. Caspase-3- and Caspase-7-stained sections of the heart indicate positive cells in constitutively Mcl-1-deleted hearts (harvested at P7). Magnification, 40×.

Figure 2.

Mcl-1 deletion in adult cardiac muscle results in rapid, fatal cardiomyopathy. (A) Tamoxifen-induced Mcl-1 deletion in the heart. All tissues were harvested 2 wk after tamoxifen initiation and immunoblotted with MCL-1 and Actin. (B) Cardiac function of a Mcl-1-deleted heart. Transthoracic ECHO studies were performed 3 wk after tamoxifen initiation. M-Mode images were recorded through IVS and LVPW at the papillary muscle level to get the LV dimensions and IVS and LVPW thickness. (C) Contractility of a Mcl-1-deleted heart as measured by ECHO for LV EF and FS. The data are presented as percent of baseline (before tamoxifen treatment). (**) P < 0.01. The number of mice analyzed is indicated as n. Note: Only two Mcl-1-deleted mice were analyzed at 6 wk due to the death of most animals by this time point. Error bars represent the standard error of the mean. (D) Survival of cardiac-specific Mcl-1-deleted mice. (**) P < 0.01 by log-rank test. (E) H&E-stained cross sections of the heart indicate dilated cardiomyopathy and a loss of myofibrils. Magnifications: left, 2×; right, 40×. (F) Fibrosis in the cross-sections of the heart stained blue with the Masson's trichrome stain. Magnifications: left, 2×; right, 40×. (G) Number of TUNEL- and Caspase-3-positive cardiomyocytes upon Mcl-1 deletion. The number of mice analyzed is indicated as n. Error bars represent the standard error of the mean. The P-values are indicated comparing wild-type with Mcl-1-deleted mice by two-tailed Student's t-test.

Figure 3.

Genetic ablation of Bax and Bak improves cardiac function and survival of cardiac-specific Mcl-1 deletion. (A) Survival of Ckmm-TKO mice and littermate control mice. (**) P < 0.01 by log-rank test when compared with Mcl-1^flox/flox Ckmm-Cre mice. (B) Contractility of Mcl-1^wt/wt, Mcl-1^flox/flox, Bax^flox/wt Bak^+/− Ckmm-Cre, Ckmm-DKO, and Ckmm-TKO hearts. The data are presented as percent of baseline (before tamoxifen treatment). (*) P < 0.05 versus littermate control mice. Error bars represent the standard error of the mean. (C) H&E-stained cross-sections of the heart indicate dilated cardiomyopathy and a loss of myofibrils, and Masson's trichrome staining indicates fibrosis in the Mcl-1^flox/flox Bax^flox/wt Bak^+/− Ckmm-Cre heart. Magnifications are indicated for each panel. All hearts were harvested at P84.

Figure 4.

Genetic ablation of Bax and Bak improves cardiac function and survival of cardiac-specific Mcl-1 deletion. (A) Tamoxifen-induced Mcl-1, Bax, and Bak deletion in heart. All tissues were harvested 8 wk after tamoxifen initiation and immunoblotted with MCL-1, BAX, and BAK. Manganese superoxide dismutase (MnSOD) served as the mitochondrial marker and Actin served as the loading control. (B) Survival of Myh-TKO mice and littermate controls. (**) P < 0.01 as determined by log-rank test when compared with Mcl-1^flox/flox Myh-Cre^ER. (C) Contractility of Mcl-1^wt/wt, Mcl-1^flox/flox Myh-Cre^ER, Myh-DKO, and Myh-TKO hearts. The data are presented as percent of baseline (before tamoxifen treatment). (*) P < 0.05; (**) P < 0.01. Error bars represent the standard error of the mean. (D) H&E-stained cross-sections of the heart indicate no evidence of cardiomyopathy in the Myh-TKO hearts. Masson's staining indicates no evidence of fibrosis in Myh-TKO hearts. All hearts were harvested 8 wk after tamoxifen initiation. Magnifications are as indicated.

Figure 5.

Mitochondrial ultrastructural abnormalities in Mcl-1-deleted hearts cannot be prevented by genetic ablation of Bax and Bak. Transmission electron micrographs indicate disrupted myocardium and disorganized mitochondria with abnormal cristae structure in A. Constitutively Mcl-1-deleted hearts (harvested at P9). Bar, 500 nm. (B) Tamoxifen-induced Mcl-1-deleted hearts (3 wk after tamoxifen initiation). Bar, 500 nm. (C) Constitutively Mcl-1-deleted Ckmm-TKO hearts (harvested at P84). (D) Tamoxifen induced Mcl-1-deleted Myh-TKO hearts (8 wk after tamoxifen initiation). For the Ckmm-TKO and Myh-TKO samples, regions with relatively normal morphology are indicated by i and iii, whereas the regions with abnormal ultrastructure are indicated by ii and iv. Bars: top panels (lower magnification), 2 μm; bottom panels (higher magnification), 500 nm.