Loss of MCL-1 leads to impaired autophagy and rapid development of heart failure

Abstract

Myeloid cell leukemia-1 (MCL-1) is an anti-apoptotic BCL-2 protein that is up-regulated in several human cancers. MCL-1 is also highly expressed in myocardium, but its function in myocytes has not been investigated. We generated inducible, cardiomyocyte-specific Mcl-1 knockout mice and found that ablation of Mcl-1 in the adult heart led to rapid cardiomyopathy and death. Although MCL-1 is known to inhibit apoptosis, this process was not activated in MCL-1-deficient hearts. Ultrastructural analysis revealed disorganized sarcomeres and swollen mitochondria in myocytes. Mitochondria isolated from MCL-1-deficient hearts exhibited reduced respiration and limited Ca(2+)-mediated swelling, consistent with opening of the mitochondrial permeability transition pore (mPTP). Double-knockout mice lacking MCL-1 and cyclophilin D, an essential regulator of the mPTP, exhibited delayed progression to heart failure and extended survival. Autophagy is normally induced by myocardial stress, but induction of autophagy was impaired in MCL-1-deficient hearts. These data demonstrate that MCL-1 is essential for mitochondrial homeostasis and induction of autophagy in the heart. This study also raises concerns about potential cardiotoxicity for chemotherapeutics that target MCL-1.

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

Figure 1.

Cardiomyocyte-specific ablation of Mcl-1 in the adult heart leads to contractile dysfunction and death. (A) Western blot analysis of MCL-1 in control hearts and in the border zone of the infarct. (B) Quantitation of outer mitochondrial membrane (OMM) and matrix MCL-1 isoforms (n = 5–6). (C) Western blot analysis for MCL-1 in heart and skeletal muscle after 2 wk. (D) Representative M-Mode echocardiograms at 2 wk. (E) Echocardiographic analysis revealed reduced ejection fraction (EF) and fractional shortening (FS) as well as enlarged left ventricular diastolic dimension (LVDd) in Mcl-1^f/fCre+ mice. n = 5–14; (*) P < 0.05. (F) Kaplan-Meyer survival curve (P < 0.05). Data are mean ± standard error of the mean (SEM).

Figure 2.

Ablation of Mcl-1 in the adult heart leads to cardiac hypertrophy. (A) Mcl-1^f/fCre⁺ hearts are enlarged on gross morphology by 1 wk. (B) Ventricle weight and lung weight to body weight ratios from Mcl-1^f/f and Mcl-1^f/fCre⁺ mice 2 wk after initiating tamoxifen. n = 15–17. (C) Representative images of wheat germ agglutinin (WGA) staining at 2 wk. Bar, 35 μm. (D) Quantitation of myocyte cross-sectional area (n = 4) at 2 wk. (E) Analysis of hypertrophy markers by qPCR at 2 wk. n = 9–10. (F) MHC isotype switching in Mcl-1^flox/floxCre⁺ hearts at 2 wk shown by qPCR. n = 9–10. Data are mean ± SEM.

Figure 3.

Ablation of Mcl-1 leads to cardiomyocyte disorganization, fibrosis, and inflammation. (A) H&E staining shows thinning of the left ventricular wall in Mcl-1^f/fCre⁺ hearts 2 wk after tamoxifen injection. Bar, 5 mm. (B) Left atrial thrombus in the Mcl-1^f/fCre⁺ heart. Bars: left, 500 μm; right, 100 μm. (C) Representative H&E staining shows marked myocyte disorganization and degeneration in Mcl-1^f/fCre+ hearts at 2 wk. Bars: left, 500 μm; right, 100 μm. (D) Masson's trichrome staining reveals extensive fibrosis in the left ventricle (LV) of Mcl-1^f/fCre⁺ hearts at 2 wk. Bars: left, 500 μm; right, 100 μm. Quantitation of fibrosis (n = 3) (E) and analysis of Col1A1 mRNA levels at 2 wk (n = 9–10) (F). (G) Representative CD45 staining at 2 wk. Bar, 50 μm. (H) Analysis of inflammatory markers by qPCR at 2 wk (n = 5–8). Data are mean ± SEM.

Figure 4.

Ablation of Mcl-1 does not result in activation of apoptosis. (A) Quantitation of TUNEL positive nuclei in heart sections. n = 3–4. (B) Quantitation of myocytes positive for cleaved caspase-3 in heart sections. n = 3–4. (C) Caspase-3 activity assay at 2 wk. Positive control consists of HL-60 cells treated with 0.5 μg/mL actinomycin D for 24 h. n = 3–6; (*) P < 0.001 vs. Mcl-1^f/f. (D) Western blot for cleaved caspase-3 at 2 wk. Positive control consists of HL-1 cells treated with 1 μM staurosporine for 8 h. (E) Western blot analysis for PARP cleavage. (F) Western blot analysis for AIF in cytosolic, mitochondrial, and nuclear fractions at 2 wk. (G) Western blot analysis for BCL-2 family proteins at 2 wk in Mcl-1^f/f and Mcl-1^f/fCre⁺ hearts. (H) Quantitation of BCL-2 protein levels. n = 7; (*) P < 0.05 vs. Mcl-1^f/f. (I) Western blot analysis for BH3-only proteins in mitochondrial fractions at 2 wk. Data are mean ± SEM.

Figure 5.

Ablation of Mcl-1 leads to degeneration of mitochondria. (A) Ultrastructural images reveal the presence of swollen mitochondria (white arrows) and lipid accumulation (black arrows) in MCL-1-deficient hearts 1 wk after tamoxifen injection. Bars: top panels, 2 μm; bottom left, 0.5 μm; bottom center, 1 μm; bottom right, 0.5 μm. (B) LDH activity in blood at 1 wk. n = 5–6. (C) Swelling of isolated mitochondria in the presence of 250 μM calcium with or without 1 μM CsA 1 wk after tamoxifen injection. (D) Complex I and complex II activities in isolated mitochondria at 1 wk. n = 4.

Figure 6.

Ablation of Mcl-1 in Ppif^−/− mice delays development of heart failure and death. (A) Representative M-Mode echocardiograms at 2 wk. (B) Echocardiographic analysis of Ppif^−/−Mcl-1^f/f and Ppif^−/−Mcl-1^f/fCre⁺ mice. n = 5–19; (*) P < 0.05. (C) LDH activity in blood at 1 wk. n = 5–6. (D) Ventricle weight and lung weight to body weight ratios at 2 wk after initiating tamoxifen. n = 17–21. (E) Kaplan-Meyer survival curve. P < 0.05. (F) Representative H&E staining shows myocyte disorganization and degeneration in Ppif^−/−Mcl-1^f/fCre⁺ hearts at 2 wk. Bars: left, 500 μm; right, 100 μm. (G) Swelling of isolated mitochondria in the presence of 0, 0.5, or 1 mM calcium at 1 wk. (H) Quantitation of TUNEL-positive nuclei in heart sections. n = 3. (I) Quantitation of myocytes positive for cleaved caspase-3 in heart sections. n = 3. Data are mean ± SEM.

Figure 7.

Ablation of Mcl-1 in the adult heart leads to impaired autophagy. (A) Representative Western blots of heart lysates prepared at 2 wk after initiation of tamoxifen administration. (B) Quantitation analyses of LC3II/I, p62, and total LC3 levels. n = 6–13. (C) Western blots for Parkin in cytosolic and mitochondrial fractions at 1 wk. (D) Western blot for PINK1 in the mitochondrial fraction at 1 wk. (E) Quantitation of PINK1 levels in mitochondria. n = 3. (F) Analysis of LAMP2 levels at 2 wk in heart lysates. (G) Assessment of autophagic flux in vivo using chloroquine 1 wk after tamoxifen treatment. (H) Quantitation of LC3II/I levels in a chloroquine study. n = 4–6. (I) Representative Western blot of heart lysates after resting or acute swimming. (J) Quantitation of LC3II/I levels in swimming study. n = 4–8. (K) Quantitation of p62 levels in a swimming study. n = 3–4. Data are mean ± SEM.