The overall goal of the proposed studies is to elucidate the molecular mechanisms involved in the regulation of cardiac muscle contraction by troponin (Tn) in health and disease. The current proposal will determine the effect of mutations in cTnT, cTnI and cTnC, known to cause familial hypertrophic cardiomyopathy (FHC or HCM), dilated cardiomyopathy (DCM) and restrictive cardiomyopathy (RCM) on the biochemical, contractile and electrophysiological properties of cardiac muscle. Knock-in mice will be generated expressing cTn subunits that contain mutations known to cause HCM, DCM and RCM in man and the morphological and in vitro and in vivo properties of these cardiac disease states will be investigated. The following Specific Aims will be pursued:
SPECIFIC AIM 1 : PHYSIOLOGICAL CONSEQUENCES OF TROPONIN - MEDIATED GENETIC DISORDERS STUDIED IN THE HCM, DCM, AND RCM MOUSE MODELS. We propose to utilize the following knock-in mice: (A) HCM: cTnI-R21C and cTnT-R92W;(B) DCM: cTnI- K183 and cTnT-R141W and (C) RCM: cTnI-K178E and cTnI-R145W. We will perform: i) Biochemical characterization;ii) Fiber studies (to establish Ca2+ sensitivity of ATPase/force and gapp);iii) Force and intracellular [Ca2+] transients;iv) Tissue analysis;and v) Physiological and electrophysiological characterization.
SPECIFIC AIM 2 : ELUCIDATE THE ROLE THAT TROPONIN C, A MOLECULAR Ca2+ SWITCH PLAYS IN HCM, DCM AND RCM. The following mutant cTnC knock-in mice are proposed for this study: cTnC-S37G (HCM), cTnC-F20Q (DCM) and cTnC-V44Q (RCM). If, as we hypothesize, cTnC is ultimately responsible for the calcium dependent phenotypic properties underlying HCM, DCM and RCM that are caused by the mutations in either cTnC or TnT and/or TnI, one would expect that making these knock-in mutations in cTnC that alter its Ca2+ binding affinity and/or other contractile properties of muscle, would produce phenotypes in proposed knock-in mice that are similar to those seen in man.
SPECIFIC AIM 3 : ANALYSIS OF THE PHYSIOLOGICAL MEASUREMENTS IN AIM 1 and 2 WILL BE UTILIZED TO PROPOSE UNIFYING THEORIES OF MECHANISMS RESPONSIBLE FOR HCM, RCM AND DCM. A comprehensive theory or theories regarding the mechanisms responsible for HCM, DCM and RCM will be proposed. We will evaluate the data and group the results according to various cardiomyopathies. The results will be further analyzed for correlations that define characteristics of HCM, RCM and DCM. Our multidimensional approach will allow elucidation of the mechanisms that are responsible for specific myopathies and the determination of the severity of specific mutations that cause malignant phenotypes and SCD in man. These studies will be critical in understanding the effect these genetic structural changes have on cardiac muscle and how they might lead to the three distinctive disease states.
Showing the most recent 10 out of 53 publications
