Research Interests
In simple terms, I am interested in how muscles form, how they can be repaired and how these processes evolved. The projects described below attempt to answer each of these questions individually and are defining novel actions for myostatin; a myokine that helps coordinate the growth and development of skeletal and cardiac muscle. They utilize both biomedical and comparative model systems and together provide a comprehensive understanding of the individual processes involved.
Functional Divergence of the Myostatin Gene Family
Myostatin is generally described as an extremely potent negative regulator of skeletal muscle growth (mstn-/- to left, B & D are wild-type), although its actions are required for normal muscle development. Recent studies also indicate that alleviating muscle from the negative influences of myostatin may have significant therapeutic potential. Thus, controlling its function and/or bioavailability could significantly impact the treatment of many different disorders of skeletal muscle growth including muscular dystrophy. Myostatin’s actions in non-mammalian vertebrates, however, have not been well described, but are suspected to regulate the development of many different fish tissues. Thus, the gene’s actions have diverged significantly as vertebrates similarly diversified. We have led the field in identifying distinct myostatin paralogs in the fishes and have defined their phylogenetic relationships in great detail. We ultimately identified two distinct monophyletic fish clades (MSTN-1 & -2) and two sub-clades specifically in the tetraploid salmonids (MSTN-1a, -1b, -2a, & -2b). These genes are differentially expressed in different adult and developing tissues, which is consistent with their divergent promoter sequences. Their transcripts are also alternatively processed in a tissue-specific manner that actually contributes to the pseudogenization of MSTN-2b and the preservation of MSTN-2a transcripts only in the brain. Recent computational studies have also identified evidence for positive selection in mammalian and salmonid homologs and suggest that differences in myostatin gene promoters may be equally if not more responsible for the evolution of this gene family. Our studies are therefore highly novel as the combination of computational and molecular analyses of salmonid myostatin genes provides a unique opportunity to investigate the evolutionary mechanisms responsible for gene duplicate divergence, a process long known to disproportionately influence evolutionary rate. We have also determined that myostatin inhibits some muscle growth processes in fish as it does in mammals and are currently exploring its non-muscle actions as well.
Myostatin Regulation of Cardiac Muscle Growth, Development and Function
Our recent efforts to address more biomedical issues include investigating myostatin’s actions in developing cardiac muscle. Skeletal and cardiac muscle are structurally and functionally very similar, especially compared to smooth muscle. It is therefore surprising that myostatin’s actions in cardiac muscle have not been investigated to date. The myokine is expressed in developing cardiac muscle and its expression increases with injury (i.e. myocardial infarction), which is similar to what occurs in skeletal muscle. We therefore determined that myostatin inhibits several growth processes in cardiac muscle and more importantly, that hearts of myostatin null mice are hypertrophied and functionally superior to those of wild-type mice. In fact, the structural and functional capacity of these hearts are similar to those of elite athletes and include increased cardiac output with stress and a heightened response to catecholamines. We have also begun characterizing myostatin signaling in mature cardiac muscle and in response to a myocardial infarction. Indeed, Ca2+ transients and cellular load as well as contractility, both basal and isoproterenol-stimulated, are greatly enhanced in primary cardiomyocytes isolated from myostatin null mice (see above). These studies further suggest that myostatin is actively involved in the tissue remodeling that occurs after an infarct, actions that ultimately contribute to heart failure, and that blocking the myokine’s actions could theoretically replace the detrimental pathological hypertrophy with the beneficial physiological hypertrophy. This in turn would improve cardiac performance and patient recovery. Future studies will not only investigate myostatin’s ability to inhibit physiological hypertrophy and excitation-contraction coupling, but will attempt to modulate myostatin’s actions using gene therapy protocols that attempt to disrupt myostatin’s intracellular actions.
Endocrine Myostatin; Systemic Regulator of the IGF/IGFBP Axis.
The “double muscling” that occurs in a myostatin null environment is believed to result from the loss of myostatin’s negative influences on local growth control. Several studies, however, suggest that myostatin signaling opposes the mitogenic action of insulin-like growth factors (IGF-I & -II), which stimulate every known aspect of skeletal muscle growth and development. We have replicated these results with proliferating cardiomyoblasts and have additionally determined that myostatin, whose mammalian expression is limited to striated muscle, also regulates the hepatic expression and circulating levels of the IGFs and some of their high affinity binding proteins. This suggests that the hypermuscularity of myostatin null animals results from the combined loss of myostatin’s local inhibitory effects as well as increased IGF production and systemic bioavailability. Myostatin, therefore, is a true endocrine factor and may indirectly help coordinate the growth of muscle, bone and other related tissues by inhibiting hepatic IGF-I production. We have also determined that myostatin’s inhibitory effects, at least on skeletal muscle, are mediated in part by the local production of IGFBP-3. Our studies additionally indicate that exogenous and extracellular IGFBP-3 rapidly localizes to the nuclei of myoblasts (D, dapi-stained nuclei; F, FITC-stained IGFBP-3) and inhibits cellular proliferation independent of IGF-binding. These results suggest that IGFBP-3 itself signals directly to the nucleus without mediation from second messengers. The long-term goal of these studies is to define the mechanisms and implications of nuclear IGFBP-3 in regulating skeletal muscle growth and in mediating myostatin’s actions. We have already identified several putative binding partners for IGFBP-3 that include extracellular matrix proteins, cytoskeletal proteins and transcription factors. Most importantly, however, we identified a regulatory subunit of the RNA polymerase II complex, Rpb3, that interacts specifically with myogenic transcription factors. These results were independently confirmed using molecular, cellular and biochemical methods and are the first to suggest a nuclear role for a secreted protein; the regulation of gene transcription. The rapid translocation of IGFBP-3 into muscle cell nuclei itself represents a dynamic and extremely novel paradigm shift in the understanding of peptide hormone/growth factor signaling. Its association with Rpb3 may also explain how the nuclear actions of IGFBP-3 differ between cell types where Rpb3 associates with different proteins. These studies will therefore improve our understanding of skeletal muscle growth and development, of the IGF/IGFBP axis and of fundamental signal transduction mechanisms. The combined administration of IGF-I and IGFBP-3 (Iplex) stimulates muscle growth far better than IGF-I alone and avoids the negative side effects and potential risks associated with IGF therapeutics. Thus, these studies could potentially impact the treatment of many muscle growth disorders as Iplex is currently being used in clinical trials.