A 5-year research plan is proposed with the broad, long-term objective of elucidating the intracellular mechanisms responsible for transducing mechanical stimuli into alterations in cardiomyocyte structure and function. The proposed investigations are a logical continuation of whole animal and cellular research conducted by the PI and co workers over the past 14 years. The investigator's previous studies have identified a critical relationship between cardiomyocyte workload, and the assembly of myofibrillar proteins into sarcomeres. This assembly process, in turn, represents the rate-limiting step in regulating myofibrillar protein turnover. Previously published studies and preliminary data indicate that focal adhesion/costmere assembly is critical to the assembly of newly synthesized contractile proteins into sarcomeres. In addition, the nonreceptor protein tyrosine kinase pp125fak (FAK) which localizes to cardiomyocyte focal adhesions, is activated in response to neurohormonal and mechanical stimuli that produce cardiomyocyte hypertrophy in vitro. Therefore, a series of experiments are first proposed to examine the role of specific components of the focal adhesion/costamere in regulating sarcomeric assembly. Second, available evidence indicates that neurohomonal and mechanical stimuli that induce sarcomeric assembly activate the novel PKC isoenzymes PKC-delta and PKC-epsilon. PKC activation may also be necessary for FAK activation and focal adhesion formation. Using replication-defective adenoviruses to over-express dominant-negative and constitutively active PKCs, the P.I. will critically analyze the role of specific PKC isoenzymes in regulating FAK phosphorylation, sarcomeric assembly, and myofibrillar protein degradation. Third, the myristolyated, alanine-rich protein kinase C substrate (MARCKS) is a 85kD membrane protein that may regulate cell spreading, stress fiber formation, and focal adhesion formation in nonmuscle cells. The P.I. and others have shown that MARCKS is expressed by cardiomyocytes, and is phosphorylated in response to PKC activation. The investigator hypothesizes that PKC-dependent phosphorylation and exit of MARCKS from the plasma membrane are necessary to initiate integrin clustering, FAK activation, and focal adhesion formation. Therefore, using a replication-defective adenovirus to over-express a nonphosphorylatable, dominant-negative mutant of MARCKS, the P.I. will examine whether this protein is a critical component of the signaling pathway involved in sarcomeric assembly during cardiomyocyte hypertrophy. The proposed investigation addresses focused questions in the context of specific, testable experimental hypotheses. The study will answer a fundamental question with important, practical significance. The future development of therapeutic strategies specifically targeted to metabolic pathways involved in cardiomyocyte growth regulation should have considerable utility in the future treatment of patients with left ventricular hypertrophy and heart failure.
Showing the most recent 10 out of 60 publications
