The molecular programs regulating development and morphogenesis of the heart are dependent upon the capacity of cardiomyocytes to transduce and respond to developmental cues and environmental signals. Analyses of genetically engineered mice harboring null and conditional loss-of-function mutations in the Myocd
gene have shown that myocardin plays a critical role in regulating vascular SMC differentiation and patterning of the vascular system (13
). However, despite the finding that myocardin is expressed early and abundantly in the embryonic heart, its role, if any, in promoting cardiomyocyte differentiation and cardiac morphogenesis has remained unclear. The data presented in this report demonstrate unequivocally that in the embryonic heart, myocardin is not required for cardiac myocyte specification and/or looping morphogenesis, but is required for cardiac growth and chamber maturation, maintenance of cardiac function, and ultimately, embryonic survival. Surprisingly, the basis of myocardin function is not predicted by its activity in vascular SMC differentiation, but rather via the capacity to regulate cardiomyocyte proliferation and apoptosis in the embryonic heart, acting at least in part via activation of the Bmp10
As schematically shown in Figure , in the embryonic heart, myocardin is poised to respond to developmental cues, growth factors, and hemodynamic signals influencing the balance of cardiomyocyte proliferation and apoptosis required for atrial and ventricular chamber maturation. After completion of looping morphogenesis, coincident with the onset of rapid growth associated with chamber maturation, myocardin acting via induction of Bmp10
gene expression represses activity of the cell cycle inhibitor p57kip2
, stimulating cardiomyocyte proliferation. During this developmental window, myocardin is also required for maintenance of a subset of key cardiogenic factors, including Nkx2-5 and Mef2c (20
). BMP-induced activation of Nkx2-5
gene transcription occurs via binding of Smad1/4 to the Nkx2-5
transcriptional enhancer (32
). Consistent with these observations, conditional ablation of the Nkx2-5
gene at E12.5 leads to a block in chamber maturation, recapitulating the phenotype observed in Myocd
conditional mutant embryos (33
). Moreover, in the primitive heart, at least as early as E8.5, myocardin represses cardiomyocyte apoptosis, preserving cardiac function. Taken together, these data demonstrate that as a transcriptional coactivator, myocardin transduces signals influencing cardiomyocyte proliferation, structural organization of the cardiomyocyte, and programmed cell death required for the late stages of cardiac morphogenesis needed for atrial and ventricular chamber maturation.
Myocardin transduces signals regulating Bmp10 signaling required for cardiac chamber maturation.
The demonstration that cardiogenic factors including Nkx2-5 and Mef2c are downregulated in the hearts of E9.5 Myocd–/–
embryos was initially perplexing because Myocd–/–
embryos progress through the early stages of cardiogenesis, including formation of the primitive heart tube and looping morphogenesis, while Nkx2-5–/–
embryos exhibit a block in looping morphogenesis (25
). What explains the divergent phenotype of Myocd–/–
mutant embryos? Surprisingly, at E8.0–E8.5, prior to initiation of cardiac looping, Nkx-2.5 and Mef2c are expressed at comparable levels in control and Myocd–/–
embryos (Supplemental Figure 2). Similarly, comparable levels of Nkx-2.5 and Mef2c were observed at E8.5 in control and Bmp10–/–
). These data define a developmental window when myocardin (and Bmp10) regulates expression of the cardiogenic transcription factors Nkx-2-5 and Mef2c. This window occurs after formation of the primitive heart tube and looping morphogenesis (E8.0–E8.5 in the mouse) coincident with chamber maturation of the embryonic heart (E9.5). This observation highlights the role of myocardin as a transcriptional coactivator that is capable of transducing and responding to specific developmental cues in the embryonic heart.
Surprisingly, some, but not all, of myocardin-regulated functions in the embryonic heart are conserved in the heart during postnatal development. Myocd
gene ablation in the adult heart is accompanied by dissolution of sarcomeric organization, disruption of the intercalated disc, and cell-autonomous loss of cardiomyocytes via apoptosis (16
). The structural defects are attributable, at least in part, to a block in SRF-dependent genes encoding a subset of myofibrillar and structural proteins, including α-cardiac actin, MLC2v, desmin, and connexin 43 (16
). Interestingly, each of these proteins is observed in the hearts of E9.5 Myocd–/–
embryos (J. Huang, unpublished observations), suggesting that MRTF-A/MKL1 and/or MRTF-B/MKL2 may subserve a partially redundant function with myocardin in the embryonic heart: a capacity that is lost during postnatal development. In this regard, it is noteworthy that the structure of the sarcomere and the repertoire of genes encoding myofibrillar proteins are distinct in the embryonic and adult heart. In contrast, throughout development, myocardin represses programmed cell death in the cardiomyocyte.
The molecular basis of myocardin function in the embryonic heart was not anticipated by a decade of studies that defined its functions in SMCs and the vasculature (13
). Ectopic expression of myocardin in X. laevis
embryos activates some, but not all, SRF-regulated genes encoding contractile proteins expressed in the fetal heart (15
). The fetal heart program includes the transient expression of a select group of SRF-regulated genes encoding SMC-restricted contractile proteins, including SMA and SM22α, which are transiently expressed in the embryonic heart and reexpressed in the adult heart in response to hemodynamic stress. At E9.5, SM22α and SMA are not expressed in vascular SMCs populating the dorsal aorta of Myocd–/–
). This led us to postulate that the defects observed in the hearts of Myocd–/–
embryos were caused, at least in part, by a block in expression of the subset of SRF-dependent genes encoding SMC-restricted contractile proteins. Interestingly, this is not the case, as comparable levels of SM22α and SMA were observed in the hearts of Myocd–/–
embryos and control littermates. Taken together, these data demonstrate that distinct transcriptional mechanisms have evolved to control the expression of SRF-dependent genes encoding SMC contractile proteins in the embryonic heart and vasculature, respectively.
For many years it has been recognized that a subset of BMP growth factors plays critical roles in the morphogenetic program regulating development of the heart (17
). However, the mechanisms regulating expression of BMPs in the heart remain poorly understood. The findings that (a) Bmp10
gene expression is markedly downregulated in the hearts of Myocd–/–
and cardiomyocyte-restricted conditional mutant embryos, (b) forced expression of myocardin transactivates the Bmp10
promoter via binding of a myocardin/SRF protein complex to a conserved CArG box, (c) expression of phosphorylated Smad1/5/8 is repressed in Myocd–/–
hearts at E9.5, (d) Bmp10-regulated genes including Nkx2-5 and Mef2c are suppressed in the hearts of Myocd–/–
embryos, (e) p57kip2
is induced in the hearts of Myocd–/–
embryos, (f) Bmp10-conditioned medium rescues the proliferative defect in Myocd–/–
mutant hearts and suppresses p57kip2
, and most importantly, (g) shared morphogenetic defects are observed in the hearts of Myocd–/–
, and Bmp10–/–
embryos strongly support the conclusion that myocardin (and SRF) regulates transcription of the Bmp10
gene in the embryonic heart. This conclusion was not anticipated by a decade of research into the function of myocardin, which had not suggested a role for myocardin in the regulation of BMP-related growth factors.
The demonstration of apoptosis in the hearts of Myocd–/–
mutant embryos reveals a conserved function for myocardin throughout development of the heart. Consistent with this observation, conditional ablation of Srf
in the embryonic heart is associated with increased cardiomyocyte apoptosis and heart failure (8
). In this regard, it is noteworthy that Bmp10 did not rescue apoptosis in Myocd–/–
mutant hearts, suggesting strongly that myocardin-induced repression of programmed cell death is not linked to cell proliferation and is independent of BMP10 signaling. This primal function of myocardin (and SRF) appears to be cell autonomous and not dependent upon hemodynamic signals because the loss of myocardin in primary neonatal cardiomyocytes in cell culture rapidly induces apoptosis, acting via the intrinsic and extrinsic apoptotic pathways (16
). This observation raises a fundamental question: what is the evolutionary advantage of a transcriptional coactivator required for cardiomyocyte survival? Are there circumstances in the embryonic heart and/or when the heart adapts to hemodynamic stress in which cardiomyocyte apoptosis is beneficial? Does myocardin play a unique role in the embryonic heart in response to hemodynamic stress? The answers to these questions will provide new insights into understanding the molecular basis of cardiac morphogenesis and may be relevant to understanding the pathogenesis of heart failure and cardiomyopathy.