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).

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.

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).

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).