Michael S. Parmacek - US grants

Cardiac contractile function is determined by the expression of distinct set of tissue-specific genes. These include, myofibrilla isoforms, cell surface receptors, as well as lineage-specific enzymes and structural proteins. The tissue specific expression of these genes is controlled at the level of transcription. However, relatively little is understood about the molecular mechanisms that control cardiac-specific transcription. Because of its restricted pattern of expression, we have used the murine slow/cardiac troponin C (cTnC) gene as a model system to define the molecular mechanisms that regulate gene expression in cardiac myocytes. We have shown both in vitro, and by the direct injection of plasmid DNA into the adult rat heart in vivo, that cTnC gene expression in cardiac myocytes is controlled by a cardiac-specific promoter/enhancer located in the immediate 5' flanking region of the gene (bp-124-+1). This transcriptional enhancer contains previously undescribed nuclear protein binding sites that bind novel cardiac-specific nuclear protein complexes. The demonstration of cardiac-specific transacting factors suggests that a distinct set of cardiac-specific transcription factor may play a central role in cardiac myocyte-specific gene expression. The proposed studies are designed to elucidate the molecular mechanism that control cTnC gene expression during murine development in vivo with particular attention placed on examining the role of these novel cardiac- specific nuclear protein complexes. Specifically, we propose to i) examine the tissue-and developmental-specificity of the cTnC 5' flanking promoter/enhancer in transgenic mice, ii) examine how protein-protein interactions between factors that bind to the cTnC enhancer and promoter control the transcriptional activation of the cTnC gene in cardiac myocytes, and iii) biochemically characterize and clone the transcription factors that regulate cTnC gene expression, with particular emphasis on purification of cardiac-specific nuclear protein complexes. Elucidation of the transcriptional program that controls cTnC gene expression in cardiac myocytes should fundamentally increase our understanding of the molecular mechanisms that control gene expression in the heart. The proposed studies are relevant both to normal cardiac development and to the molecular pathogenesis of cardiac hypertrophy.

DESCRIPTION (Adapted from Investigator's Abstract): The phenotypic plasticity of vascular smooth muscle cells (SMCs) permits this muscle cell lineage to subserve diverse functions including the maintenance of arterial tone via contraction- relaxation and vessel wall integrity by proliferation and synthesis of extracellular matrix. By differentially regulating the expression of distinct sets of SMC lineage-specific genes, SMCs can modulate their phenotype from primarily contractile to primarily synthetic. However, relatively little is currently understood about the molecular mechanisms that control SMC-specific gene expression. One approach to understanding the molecular mechanisms that regulate SMC differentiation is to identify and characterize the cis-acting sequences and trans-acting factors that control SMC-specific transcription. Because of its SMC lineage-restricted pattern of expression, we have used the murine SM22alpha gene as a model system to examine the mechanisms that control SMC-specific gene expression. Preliminary studies demonstrated that SM22alpha is one of the earliest developmental markers of the SMC lineage. Moreover, the 280-bp SM22alpha promoter directs arterial SMC lineage-restricted gene expression in transgenic mice. This transcriptional regulatory element contains previously undescribed nuclear protein binding sites that bind lineage-restricted trans-acting factors. These data support the hypothesis that novel SMC lineage- restricted transcription factors control the expression of the SM22alpha gene in SMCs. The proposed studies are designed to elucidate the molecular mechanisms that control the SMC-specific pattern of SM22alpha gene. The specific aims of these studies are to: (i) identify the cis-acting elements that control activity for the arterial SMC-specific SM22alpha promoter during embryonic and postnatal development, (ii) characterize the trans-acting factors that regulate expression of the SM22alpha gene, (iii) examine the molecular mechanisms underlying activity of positive and negative regulatory factors on SM22alpha promoter activity, and (iv) clone and characterize SMC-specific transcription factors that regulate activity of the SM22alpha gene in SMCs should fundamentally increase understanding of SMC development and differentiation. As such, the proposed studies are relevant to understanding the pathogenesis of atherosclerosis and restenosis following balloon angioplasty.

This is an exciting and dynamic time for the study of cardiac muscle. The intersections of structural biology, molecular biology and biochemistry hold the promise of rapid advancement in delineating the atomic and molecular details of muscle contraction. Developmental biology, genetics and molecular biology are yielding rapid advancements in the understanding of the program of muscle specification and development. The molecular defects of a number of inherited muscle diseases have recently been delineated and gene therapies are being explored. This training program is based in the School of Medicine of the University of Pennsylvania which provides a unique environment for research and clinical training in the area of cardiac muscle. The basic research aspects of this program draw upon the expertise of the Pennsylvania Muscle Institute, Institute for Human Gene Therapy, and the recently created Training Program in Muscle and Motility. The distribution of trainers in this application includes important representation in the faculty of the School of Medicine in both clinical and basic science departments. This training grant is designed to develop clinician/scientists, clinicians and research scientists who will be leaders in the emerging areas of molecular cardiology, with particular emphasis on cardiac muscle development, function and disease. We are requesting modest support: seven trainees per year. As the range of interests and expertise of the trainers listed on this grant will attest, virtually all of the important areas of current research in cardiac muscle are covered by the trainers listed on this grant. The disciplines utilized by the trainers include molecular biology, genetics, biochemistry, structural biology, cell biology, developmental biology, and physiology. This provides the potential for an unparalleled training environment in the area of cardiac muscle biology.

The differential patterning of smooth muscle cells (SMCs) and pericytes within specific blood vessels ultimately defines and distinguishes the functional properties of the arteries, veins and capillaries. SMC phenotype and patterning, in turn, are determined via transcriptional programs that respond to developmental and environmental signals and cues. We have used transgenic and gene targeting strategies to elucidate the transcriptional programs that regulate vascular SMC differentiation. Our group and others have reported recently that the SAP domain transcription factor, myocardin, plays a critical role in SMC differentiation. Preliminary studies presented herein demonstrate that: i) myocardin is expressed in a precise developmentally regulated pattern in vascular and visceral SMCs, ii) forced expression of myocardin in non-SMCs activates multiple SMC-specific transcriptional regulatory elements, iii) forced expression of myocardin activates SMC-restricted genes in undifferentiated embryonic stem (ES) cells, and iv) expression of adominant-negative myocardin mutant protein or myocardin siRNA in SMCs represses activity of the SMC-specific SM22alpha-promoter. Together these studies suggest the central hypothesis that will be examined in the proposed studies: myocardin plays a critical role in the SRF-dependent transcriptional program that regulates SMC differentiation and phenotype. The overall goat of this project is to elucidate the molecular basis of myocardin-induced SMC differentiation. The specific aims are to: 1) Examine the cell autonomous functions of myocardin in SMCs during embryonic development, and on maintenance of the SMC phenotype, 2) Examine the molecular mechanisms underlying the activity and specificity of the myocardin-SRF dependent transcriptional program that regulates SMC differentiation, and 3) Generate and characterize mice containing null and conditioned mutations in the myocardin gene. At a basic level these studies will provide new insights into the transcriptional programs regulating SMC differentiation and modulation of SMC phenotype. As such, these studies are relevant to understanding the pathogenesis of atherosclerosis and other vascular proliferative syndromes.

DESCRIPTION (provided by applicant): The purpose of this program is to continue to provide rigorous multidisciplinary basic and translational research training for physician scientists committed to careers in academic medicine and PhD postdoctoral fellows in cardiovascular biology and medicine. The rationale underlying the training program is that basic translational and clinical cardiovascular research requires investigators with a strong foundation in molecular and cellular biology and ultimately formation of multi-disciplinary research collaborations composed of MD and PhD investigators. The program is centered in the University of Pennsylvania Cardiovascular Institute (CVI) which includes 180 members in 16 academic departments performing basic, translational and clinical cardiovascular investigation. Considerable infrastructural support from the School of Medicine is committed to the program including integrated basic and translational research space and core laboratory facilities in the recently constructed Penn Translational Research Center. The grant renewal will support 6 MD, MD/PhD and PhD postdoctoral fellows per year performing 2-3 years of dedicated research training. 29 NIH-funded Penn CVI faculty members in the Departments of Medicine (Cardiology and Endo/Diabetes), Surgery, Cell and Developmental Biology, Genetics, Physiology, Pharmacology and Biostatistics and Epidemiology serve as trainers and mentors. Trainees may enroll in either a Basic Research or Translational/Patient-Oriented Research Track. The core curriculum will be a well-supervised research preceptorship involving hypothesis-driven and design-driven discovery approaches to CV research. Practical research training will be supplemented by graduate and medical school class work, lectures, seminars, skill classes (medical writing, obtaining extramural support), postdoctoral career advising and courses in the ethical conduct of research. A successful strategy to attract individuals from under-represented minorities will be maintained. Internal and External Advisory Committees review trainee progress and programmatic direction. Over the past decade strong metrics for the training program, include: 1) successful recruitment of outstanding MD, MD/PhD and PhD trainees to the program, 2) > 95% of trainees completing the training program (several have obtained advanced degrees, 3) an average of 5.2 manuscripts published per trainee (many in high-impact journals), 4) the vast majority of trainees obtaining academic positions or pursuing additional postdoctoral studies. Programmatic enhancements described in this application include the physical aggregation of the Penn CVI faculty trainers and trainees in the newly constructed Penn CVI and the incorporation and integration of faculty mentors in Penn's Institute for Diabetes, Obesity and Metabolism as participating faculty.

DESCRIPTION (provided by applicant): Transcriptional co-activators expand information encoded within the genome required for adaptation to environmental perturbation and stress. Myocardin is a remarkably potent transcriptional expressed exclusively in the heart and smooth muscle cells (SMCs). We have used transgenic and gene targeting strategies in mice to elucidate the molecular mechanisms that regulate heart and vascular development. Preliminary studies reveal that: i) myocardin is expressed in a precise developmentally regulated pattern in cardiomyocytes and SMCs, ii) forced expression of myocardin in embryonic stem cells activates cardiac-restricted genes associated with the hypertrophic gene program, iii) myocardin and MRTF-A activate overlapping, but distinct, sets of genes and most importantly iv) mice harboring a cardiac-specific conditional ablation of the myocardin gene exhibit hypertrophic cardiomyopathy. Together these studies suggest the central hypothesis that will be examined in the proposed studies: myocardin functions as a central regulator of cardiomyocyte growth and adaptation to stress via both feed-forward mechanisms, including activation of genes associated with the fetal /hypertrophic gene program, and feedback mechanisms regulating cell proliferation. The overall goal of this proposal is to elucidate the role of myocardin in the heart with particular focus on defining its function in cardiac myocyte differentiation and adaptation of the heart. The specific aims are to characterize: 1) the SRF- and MEF2-dependent transcriptional and morphogenetic programs regulated by myocardin in the heart, 2) mechanisms that distinguish activity and specificity of myocardin, MRTF-A and MRTF-B in the heart, and 3) molecular mechanisms modulating myocardin-dependent transcriptional activation in the adult heart and response to hypertrophic stimuli. The experimental strategy deployed emphasizes the translation and validation of molecular and cellular data in genetically engineered mice. At a basic level these studies will provide new insights into the transcriptional programs regulating cardiac myocyte differentiation and morphogenetic development of the heart. These studies also provide insights into the molecular mechanisms regulating adaptation of the heart to stress and the pathogenesis of cardiomyopathy. PUBLIC HEALTH RELEVANCE: This application will investigate how the transcriptional coactivator, Myocardin, regulates the differentiated state of the heart and the capacity of the heart to respond to hemodynamic stress. We have generated genetically altered mice with cardiac-restricted ablation of the Myocardin gene. Analysis of these mice will permit us to determine if myocardin controls the early differentiation of cardiac myocytes, as well as their role in re-programming gene expression in the adult heart when it is subjected to stress. These studies will provide new understanding into cardiac development as well as the underlying causes of heart failure and cardiomyopathy.

DESCRIPTION (provided by applicant): Transcriptional co-activators expand information encoded within the genome in response to developmental cues and environmental stress. Myocardin is remarkably potent transcriptional coactivator expressed exclusively in smooth muscle cells (SMCs) and cardiac myocytes. Our group and others have shown that myocardin, plays a critical role in regulating differentiation of vascular SMCs. The overall goal of the proposed studies is to elucidate the role of myocardin and two related transcriptional co-activators, MRTF-A and MRTF-B, in the embryonic and adult vasculature. During the last cycle of this award, we reported that: i) myocardin, MRTF-A and MRTF-B are expressed in distinct developmentally-regulated patterns in mesodermally- and neural crest-derived SMCs, ii) forced expression of myocardin, MRTF-A or MRTF-B in embryonic stem (ES) cells activates endogenous SMC-restricted genes, iii) mice in which the myocardin gene is ablated in neural crest-derived SMCs exhibit patent ductus arteriosus (PDA) resulting from a block in SMC differentiation, iv) myocardin-deficient primary aortic SMCs assume a synthetic phenotype, and v) MRTF-B null mice exhibit patterning defects of the cardiac outflow tract and great arteries attributable, in part, to a cell autonomous block in differentiation of neural crest-derived SMCs. Together these studies suggest the central hypothesis that will be examined in the proposed studies: Myocardin related transcription factors (MRTFs) transduce cell autonomous and non-cell autonomous signals required for SMC differentiation, vascular patterning and maintenance and adaptation of the vasculature during postnatal development. The specific aims are to examine: 1) the cell autonomous function(s) of myocardin, MRTF-A and MRTF-B that promote SMC differentiation and the contractile SMC phenotype;2) the role of MRTF-A and MRTF-B in differentiation of neural crest-derived SMCs and patterning of the cardiac outflow tract and great arteries;and 3) myocardin null and conditional mutant mice to elucidate the function of myocardin during embryonic angiogenesis and in maintenance and adaptation of the postnatal vasculature. The experimental strategies deployed emphasize the translation of molecular and cellular data to the intact vascular system utilizing genetically engineered mice. At a basic level, these studies will provide new insights into the molecular programs regulating smooth muscle cell differentiation and morphogenetic patterning of the vasculature. Moreover, these studies are directly relevant to understanding the molecular and genetic basis of vascular proliferative syndromes, congenital heart disease and diseases of the aorta. PUBLIC HEALTH RELEVANCE: Compelling evidence suggests that myocardin related transcription factors (MRTFs) play critical roles in regulating development of the cardiovascular system and maintenance of cardiovascular homeostasis. The proposed studies examine the molecular mechanisms regulating activity of MRTFs during embryonic and postnatal development. These studies are directly relevant to understanding vascular proliferative syndromes including atherosclerosis, common forms of congenital heart disease and aortic diseases.