Perspective. Each year, over 900,000 adults in
the United States experience an acute myocardial infarction, and
~500,000 succumb to the sequelae of heart failure. In addition, ~2% of live born infants are born with a significant congenital heart defect, and many develop heart failure because of their altered hemodynamics or as a result of palliative surgery. For many of these patients, cardiac transplantation remains their only long-term therapeutic option.
At the cellular level, myocardial insufficiency results from the
cumulative death of cardiac myocytes and the inability of remaining
cells to effectively regenerate. Molecular strategies for
restoring damaged myocardium would be an important contribution
to therapy for this disease.
Overview. Striated muscle cells cease division
within weeks of birth. While skeletal muscle retains limited
capacity for regeneration through recruitment of satellite cells,
resident populations of adult myocardial stem cells have not definitively
been identified. Therefore, damaged myocardium has limited
or no ability to regenerate. Our laboratory’s
goal is to understand mechanisms regulating cell division, and
how such processes play a role in cardiovascular biology and disease. Our
current work is focused on: 1) characterization and manipulation of myogenic precursor
cells; 2) mechanisms of cell cycle withdrawal during differentiation; 3) the role of cell cycle machinery in post-mitotic cellular
responses;
and, 4) CDC5-mediated control of cell
division.
Characterization and manipulation of myogenic precursor cells.
Over the
past several years, the identification of heterogeneous populations
of muscle stem cells in the heart and bone marrow has generated great
enthusiasm for new approaches to muscle repair and regeneration.
These studies also have exposed the limitations of current strategies. Direct cell replacement has been hindered by the functional isolation observed in transplanted cells. Myogenic differentiation of stem cells has been checked by a low rate of engraftment. Attempts to directly manipulate the myocyte cell cycle have been restrained by the inability of cycling myocytes to complete mitosis. Further understanding of mechanisms that govern cell cycle withdrawal and myoblast fusion during myogenesis is needed to develop methods for expanding populations of muscle precursor cells to treat heart failure and skeletal muscle disorders. To address this need, we are studying murine fetal, neonatal, and adult muscle stem cells, as well as human embryonic stem cells, to determine their origins, mechanisms of self-renewal, and regulation of homing to developing and injured cardiac and skeletal muscle.
(Beating cardiac muscle cells derived from human embryonic stem cells)
Mechanisms of cell cycle withdrawal during differentiation.
Because
of our interest in muscle regeneration, we specifically are studying
the cell cycle withdrawal program in skeletal and cardiac myocytes. We have developed methods for differentiating embryonic stem cells in culture, and selecting ES cell-derived cardiac myocytes. We also have established conditions for differentiating rodent and avian myoblasts into myocytes with spontaneous contractile activity, and for establishing and manipulating primary myoblast cultures. We are using these systems to identify regulatory proteins that are differentially expressed in proliferating myoblasts versus post-mitotic myotubes. Our primary approaches have been subtractive hybridization and expression profiling by microarray analysis. These studies have led to the identification of several proteins that appear to regulate cell cycle withdrawal during muscle differentiation, and current studies focus on elucidating the mechanisms by which these candidate regulators act.
The role of cell cycle machinery in post-mitotic cellular responses.
Hypertrophy occurs in post-mitotic striated muscle as a response to various stresses in both physiological and pathological situations. While regulation of cyclin-Cdk activities is essential to cell size control in lower eukaryotes, their role in the hypertrophic response in striated muscle is incompletely understood. We have demonstrated that hypertrophic stimuli cause a transient burst of Cdk4 activity, remodeling of the retinoblastoma protein complex, and activation of a subset of E2F-1 target genes in murine myoblasts. This has led us to identify a physiological role for both E2F-1-mediated activation and repression of genes involved in cell growth versus division, respectively. Currently, we are investigating the mechanism(s) by which Cip1/Kip1 and INK4 classes of Cdk-inhibitors facilitate this burst of Cdk4 activity, evaluating the role of chromatin remodeling in the hypertrophic response, and identifying other components of the retinoblastoma protein complex that regulate hypertrophy.
CDC5-mediated control of cell division.
Efforts
toward myocardial regeneration have been limited by the inability
of cardiac myocytes to enter mitosis. We cloned human CDC5, and demonstrated its functional role in G2/M. More recently, we have defined its site-specific DNA binding properties. Currently, we are investigating the role of phosphorylation in regulating CDC5 function. As an adjunct to these studies, we are collaborating on the structural characterization of CDC5 proteins in complex with other macromolecules. These studies will define how CDC5 proteins are regulated and, in turn, regulate the cell cycle.
A related line of inquiry focuses on the recent finding that CDC5 family members associate with the pre-mRNA splicing complex, although how this association affects cell cycle progression is not completely understood. We are investigating how human CDC5 participates in pre-mRNA splicing, and how this in turn regulates G2/M transit. We recently have reported the domains of CDC5 responsible for nuclear localization and spliceosome association. Future goals of this project will include defining the roles of CDC5 proteins during development and tissue differentiation in higher eukaryotes.
Sources of Support.
The Bernstein Lab and its members have been supported by ~
University of California, San Francisco
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We are using these systems to identify regulatory proteins that are differentially expressed in proliferating myoblasts versus post-mitotic myotubes. Our primary approaches have been subtractive hybridization and expression profiling by microarray analysis. These studies have led to the identification of several proteins that appear to regulate cell cycle withdrawal during muscle differentiation, and current studies focus on elucidating the mechanisms by which these candidate regulators act.
