The long-term goal of my research program is to understand the gene regulatory and upstream signaling programs involved in myocyte energy metabolism and growth. The ATP generating capacity and energy substrate preference of the myocyte are largely determined at the level of the mitochondria but also involve regulation of substrate uptake. In cardiac and skeletal muscle these energy metabolic pathways undergo dramatic shifts during development and maturation, and are altered in response to physiologic stimuli, such as exercise and fasting. Inappropriate regulation of energy metabolism (“metabolic dysregulation”) is an important contributor to the pathophysiology of human disease. Understanding the molecular mechanisms regulating energy metabolism in heart and skeletal muscle will advance our understanding of the etiologic role and the potential benefits of modulating metabolic dysregulation that is associated with obesity, diabetes, heart failure and aging.
Aspects of energy metabolic regulation are being probed at multiple levels:
1) Identification and characterization of the transcription factors/cofactors involved in pathways regulating myocyte energy metabolism. Characterizing the molecular pathway components and their regulation by physiologic changes provides mechanistic understanding of cardiac and skeletal muscle energy metabolism. These studies employ interaction screening (yeast two-hybrid and mammalian cell co-immunoprecipitation) and mapping of functional interfaces between interacting proteins. These latter studies inform the design of molecular tools (i.e. dominant negative or selective mutants and potential for ligand modulation) and the discovery of novel interacting proteins in silico using identified interaction domains.
2) Cell-based analysis of pathways involved in myocyte energy regulation. Cardiac and skeletal myocyte culture models are used to characterize downstream effects of modulating transcriptional pathways on target gene expression and metabolism. Essential to determining the specific role of transcriptional pathways is the comprehensive identification of regulated genes. The complementary approaches of chromatin immunoprecipitation (ChIP) to analyze factor binding to candidate and arrayed promoters and gene expression microarray analyses are being employed to characterize target genes. (ChIP array + global gene expression profiling = target gene identification).
3) Transgenic models to determine in vivo function and relevance of regulatory pathways. Mouse models, in which important regulatory proteins are overexpressed or “knocked out” in a tissue-specific manner, are employed to study the in vivo function of the identified factors and their effects on cardiac and skeletal muscle physiology.
My research involving the estrogen related receptor (ERR) family of orphan nuclear receptors presents a paradigm for the laboratory’s approaches. Many orphan members of the nuclear receptor (NR) superfamily have been recognized as important regulators of cellular metabolism. As a group, they present an exciting frontier for the study and therapeutic targeting of metabolism in health and disease. Using a yeast two-hybrid screen, the ERRa isoform was cloned as a novel interacting partner of the coactivator PPARg coactivator-1a (PGC-1a). ERRa was previously implicated in the development/differentiation-associated regulation of fatty acid oxidation in brown adipose, heart and slow-twitch (oxidative) skeletal muscle. The relative binding affinity of ERRa compared to other NRs for PGC-1a (by in vitro and co-IP assays) suggests that ERRs are likely the primary PGC-1a interacting partners in tissues where these factors are co-expressed. Thus, the identification of ERRa target genes will provide insight into the PGC-1a regulatory circuit in oxidative muscle, including cardiac and slow-twitch skeletal muscle.
The role of ERRs in cardiac and skeletal muscle energy metabolism is currently a major focus of studies in the laboratory. ERRs likely regulate metabolic gene targets by direct binding and transactivation or repression of important energy metabolic genes as well as by indirect mechanisms involving modulation of other transcriptional regulators (Figure). Recent evidence from studies employing the approaches outlined above have led to the hypotheses that ERRs are involved in regulating myocyte metabolism at 2 levels: 1) overall mitochondrial capacity (biogenesis and electron transport/oxphos enzyme gene expression); and 2) glucose versus fatty acid substrate selection. In addition to its PGC-1a-dependent activating functions in certain tissues and developmental stages, ERRs have independent regulatory roles in myocytes.
These hypotheses are being tested in cardiac and skeletal myocytes in culture using adenoviral systems to activate or inhibit components of the ERR pathway. Particular emphasis is placed on probing changes in myocyte gene expression (DNA microarray), gene promoter binding (ChIP), energy substrate (fatty acid or glucose) uptake and metabolism, and mitochondrial number and mitochondrial respiration analysis. Specific molecular manipulations include direct overexpression of wild-type (wt) ERR isoforms (ERRa, ERRb, ERRg); the expression of wt PGC-1a and PGC-1a mutants, which are selectively inactive for ERRs; and stable or transient expression of short-hairpin RNAs to induce siRNA inhibition of endogenous ERR isoform expression.
Animal models in which ERRa and ERRg are overexpressed or deleted are being developed to determine in vivo regulatory function in heart and skeletal muscle. The ERRa whole-body knockout (ERRa-/-) is a current model being investigated. In addition, the models for cardiac-specific ERR expression, aMHC-ERRa and aMHC-ERRg, are being studied to demonstrate the in vivo relevance of the regulation observed in cell culture and to assess the independent and overlapping regulatory roles of ERR isoforms in the heart. Alterations in glucose and fatty acid substrate utilization have been demonstrated in the ERRa-/- mice using the isolated working heart model. These studies as well as the analysis of aMHC models are performed in collaboration with faculty at the Washington University School of Medicine’s Center for Cardiovascular Research.
Projects being developed in the laboratory are focused on defining the role of ERRs in myocyte differentiation toward the long-term goal of delineating a link between myocyte metabolism and growth. Recent studies in rat neonatal cardiac myocytes revealed that ERR induces maturation in these relatively immature myocytes. Changes include myocyte elongation, cytoskeletal and myofibrillar protein reorganization, and mitochondrial redistribution along myofibrils. These changes parallel ERRa activation of fatty acid uptake and oxidation, indicative of a shift toward the adult cardiac metabolic profile. In skeletal myocyte differentiation models (C2C12 and L6 myoblasts-myotubes), the ERRa expression pattern and the effects of ERRa on expression of contractile protein isoforms also supports its pro-differentiation role. The primary near-term focus is to establish an embryonic stem cell-based cardiogenic model to investigate the role that the ERRa signaling pathways plays in cardiac myocyte differentiation. In collaboration with Loren Field at Indiana University School of Medicine, a transgenic ES model (MHC-neor/PGK-hygror), which allows selection of the cardiogenic lineage, will be employed for these studies. Using this model, the temporal pattern of ERRa (and ERRg) expression will be explored. At these defined critical time points, gain- and loss-of-function will be employed to identify genes downstream of the ERR signaling pathways that are essential for myocyte differentiation.
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