Myocardin is required for cardiomyocyte survival and maintenance of heart function

Abstract

Despite intense investigation over the past century, the molecular mechanisms that regulate maintenance and adaptation of the heart during postnatal development are poorly understood. Myocardin is a remarkably potent transcriptional coactivator expressed exclusively in cardiac myocytes and smooth muscle cells during postnatal development. Here we show that myocardin is required for maintenance of cardiomyocyte structure and sarcomeric organization and that cell-autonomous loss of myocardin in cardiac myocytes triggers programmed cell death. Mice harboring a cardiomyocyte-restricted null mutation in the myocardin gene (Myocd) develop dilated cardiomyopathy and succumb from heart failure within a year. Remarkably, ablation of the Myocd gene in the adult heart leads to the rapid-onset of heart failure, dilated cardiomyopathy, and death within a week. Myocd gene ablation is accompanied by dissolution of sarcomeric organization, disruption of the intercalated disc, and cell-autonomous loss of cardiomyocytes via apoptosis. Expression of myocardin/serum response factor-regulated myofibrillar genes is extinguished, or profoundly attenuated, in myocardin-deficient hearts. Conversely, proapoptotic factors are induced and activated in myocardin-deficient hearts. We conclude that the transcriptional coactivator myocardin is required for maintenance of heart function and ultimately cardiomyocyte survival.

Fig. 1.

MyHC-Cre/Myocd^F/F-mutant mice develop lethal dilated cardiomyopathy. (A) Kaplan-Meier survival curves for control Myocd^F/F mice (black line) (n = 60) and MyHC-Cre/Myocd^F/F-conditional mutant mice (red dashed line) (n = 62). (B) Hearts harvested from P300 MyHC-Cre/Myocd^F/F-mutant (n = 12) mice exhibit four-chamber enlargement compared to Myocd^F/F-control (n = 12) hearts. (C). An M-mode echocardiogram demonstrating that P300 MyHC-Cre/Myocd^F/F-mutant mice (n = 10) exhibit increased LV systolic and diastolic dimensions and decreased systolic function compared to Myocd^F/F-control mice (n = 10). (D, E) A Masson's trichrome-stained section of LV myocardium demonstrating that P300 MyHC-Cre/Myocd^F/F-mutant (E) hearts (n = 10) exhibit cardiomyocyte disarray with fibrosis (blue stain) compared to Myocd^F/F-control (D) hearts (n = 10). (F–H, K–M) LV myocardium harvested from P300 MyHC-Cre/Myocd^F/F-mutant mice (n = 6) and Myocd^F/F controls (n = 3) immunostained with antibodies that recognize α-cardiac actin (F, K), MLC2v (G, L), and tropomyosin (H, M). Note the loss of myofibrillar striations in the MyHC-Cre/Myocd^F/F hearts (mutant) (K–M) compared to Myocd^F/F (control) hearts (F–H). (I, J, N, O) Electron microscopy of hearts harvested from P300 MyHC-Cre/Myocd^F/F-mutant mice (n = 4) and Myocd^F/F controls (n = 4) revealed shortening and contracture of sarcomeres (arrows) in MyHC-Cre/Myocd^F/F-mutant hearts (N, O) compared to Myocd^F/F-control hearts (I, J). (P–R, U–W) Immunohistochemical analyses revealed that expression of Desmin (P, U) and Cx43 (Q, V) are markedly repressed in hearts harvested from MyHC-Cre/Myocd^F/F-mutant mice compared to Myocd^F/F controls, while comparable levels of N-Cadherin (R, W) immunostaining are observed. (S, T, X, Y) Electron microscopy of Myocd^F/F-control hearts (S, T) revealed distinct adherens junctions and desmosomes (arrows). By contrast, lightly-stained serpiginous intercalated discs with indistinct desmosomes and adherens junctions (arrows) were observed in MyHC-Cre/Myocd^F/F-mutant hearts (X, Y). Original magnification, ×200 (D, E); ×800 (F–H, K–M, P, U); ×600 (Q, R, V, W); ×10,000 (I, N); ×40,000 (J, O, S, X); ×100,000 (T, Y).

Fig. 2.

Myocardin is required for maintenance of cardiac structure and function. (A) Hearts harvested 6 days following the initiation of tamoxifen treatment from MerCreMer/Myocd^F/F mutants (n = 6) exhibited massive enlargement compared to hearts of tamoxifen-treated Myocd^F/F controls (n = 6). (B, C) Masson's trichrome-stained section of LV myocardium harvested 6 days after tamoxifen exposure of Myocd^F/F-control mice (n = 6) (B) and MerCreMer/Myocd^F/F-mutant mice (n = 6) (C) revealed myocyte disarray and fibrosis (blue stain) in the tamoxifen-treated mutant hearts. (D–G, I–L) Sections of LV myocardium immunostained with antibodies that recognize α-cardiac actin (D, I), MLC2v (E, J), tropomyosin (F, K), and α-actinin (G, L) demonstrated the loss of myofibrillar striations in tamoxifen-treated MerCreMer/Myocd^F/F (Cre+/Myocd^F/F)-mutant hearts (I–L) compared to Myocd^F/F-control hearts (D–G). EM revealed the loss of sarcomeres with loosely organized and randomly-oriented myofibers in the hearts of tamoxifen-treated MerCreMer/Myocd^F/F mutants (M) (n = 3) compared to Myocd^F/F-control mice (H) (n = 3). (N–P and R–T) Disruption of intercalated disc structure including loss of desmin (arrows) and Cx43-immunostaining was observed in tamoxifen-treated MerCreMer/Myocd^F/F-mutant hearts (R–T) compared to Myocd^F/F-control hearts (N–P). By contrast, comparable levels of N-Cadherin (N-Cad) are observed. (Q, U) EM revealed abnormal intercalated disc structure in tamoxifen-treated MerCreMer/Myocd^F/F-mutant (U) compared to Myocd^F/F-control hearts (Q). Original magnification, ×200 (B, C); ×800 (D–G, I–L, N, P, R, T); ×600 (O, S); ×40,000 (H, M, Q, U). (V) A graphic representation of cardiac gene expression as assessed by qRT-PCR 4 days after tamoxifen exposure in Myocd^F/F-control (n = 3) and MerCreMer/Myocd^F/F-mutant (n = 3) mice. The light blue bars (controls) and dark blue bars (mutants) show the relative level of GAPDH, myocardin (Myocd), α-MyHC (a-MyHC), α-cardiac actin (a-actin), MLC2v, tropomyosin (tropmyo), α-actinin, desmin, β-actin (b-actin), and Mrtf-b mRNA. Data are expressed as mean gene expression (arbitrary units) ± SEM.

Fig. 3.

Loss of myocardin triggers programmed cell death in the adult heart. Six days following the initiation of tamoxifen-treatment, hearts harvested from Myocd^F/F-control (n = 6) and MerCreMer/Myocd^F/F (Cre+/Myocd^F/F)-mutant (n = 6) mice were fixed, sectioned, and immunostained. (A, B) This representative photomicrograph of the LV freewall of a tamoxifen-treated MerCreMer/Myocd^F/F-mutant mouse (B) reveals widespread TUNEL-staining (green dots). By contrast, only rare TUNEL-positive myocytes are observed in this tamoxifen-treated Myocd^F/F-control mouse (A). (C–J) Dramatic induction of activated caspase 3 (brown stain), caspase 9 (brown stain), and Bcl-xS/L (brown stain) is observed in tamoxifen-treated MerCreMer/Myocd^F/F-mutant hearts compared to tamoxifen-treated Myocd^F/F controls. (K, L) EM of tamoxifen-treated MerCreMer/Myocd^F/F-mutant hearts reveals evidence of apoptosis, including nuclear chromatin aggregation, nuclear fragmentation, and cytoplasmic apoptotic body formation. Original magnification, ×400 (A–J); ×25,000 (K, L).

Fig. 4.

Cell-autonomous loss of myocardin in cardiomyocytes causes disruption of the contractile apparatus and apoptosis. (A–P) Replicate cultures of primary neonatal cardiomyocytes isolated from Myocd^F/F mice were infected with Ad-LacZ (A, C, E, G, I, K, M, O) or Ad-Cre (B, D, F, H, J, L, N, P). Seventy-two hours after infection, cells were harvested and immunostained with antibodies that recognize Cre (A, B), myocardin (C, D), α-cardiac actin (E, F), tropomyosin (G, H), caspase 9 (I, J), Bcl-x_S/L (K, L), p53 (M, N), and GATA-4 (O, P), respectively. (A–D) High-efficiency gene transduction was demonstrated by Cre-expression (red stain) accompanied by loss of myocardin immunostaining (green stain) in Ad-Cre transduced myocytes. (E–H) Confocal microscopy revealed loss myofibrils in Ad-Cre-transduced cells including loss of cardiac actin- (green stain, in E, F) and tropomyosin- (red stain in G, H). (I–N) Induction of apoptosis was observed in Ad-Cre transduced cells demonstrated by induction of caspase 9- (I, J) and Bcl-x_S/L-immunostaining (K, L). p53 was induced in Ad-Cre-transduced myocytes and translocated from the nucleus to the cytoplasm (M, N). GATA-4 is observed in Ad-LacZ and Ad-Cre-transduced myocytes (O, P). (Q) Cardiomyocyte gene expression was quantified by qRT-PCR performed 72 h after transduction of Myocd^F/F neonatal cardiomyocytes with Ad-Cre- (n = 3) (dark blue bars) or Ad-LacZ- (n = 3) (light blue bars). The relative levels of GAPDH, myocardin (Myocd), α-MyHC, α-cardiac actin, MLC2v, tropomyosin (tropmyo), α-actinin, desmin, β-actin, Mrtf-b, and SRF gene expression are expressed as mean gene expression (arbitrary units) ± SEM. Original magnification, ×400 (A–P).