investigator's application): The applicant's objective is to know how and when altered myofilament response to Ca2+ regulates dynamics and level of mechanical and biochemical activity of cardiac muscle. In vitro observations and studies on the phospholamban knock-out mouse indicate an important role, but as yet no experiments have specifically determined the impact of altered myofilament response to Ca2+ on activity and dynamics of myocytes and hearts. To approach this problem transgenic mice have been generated which over express isoforms and mutants of troponin I (TnI) and tropomyosin (Tm)-regulatory proteins that trigger and activate the actin-myosin reaction. Overexpressed proteins, shown to exchange with native counterparts, include beta-Tm and slow skeletal TnI- the fetal isoforms. Other transgenic mice are proposed that over-express mutant TnI's lacking protein kinase C (PKC) and PKA sites and mutant Tm's with different charge. The objectives address these questions:
Aims 1 and 2: What is the impact of Tm isoform switching, missense mutations and phosphorylation on heart muscle contraction and relaxation? Are differences in binding of Tn to thin filaments involved? Is the size of the functional unit of thin filaments altered? Aim 3: Is TnI the key element determining differences between cardiac and fast/slow skeletal muscle in effects of pH and sarcomere length on myofilament response to Ca2+? Aims 4 and 5: What is the significance of phosphorylation of TnI at its unique PKA and PKC sites on heart muscle contraction and relaxation? Mechanical and intracellular measurements are made at several levels of organization from single and multi-cellular preparations to hearts beating in situ. The investigators' experiments provide: 1) insight into the importance of altered myofilament response to Ca2+ in situ in the physiological context and 2) clearer understanding of the importance of this regulatory mechanism in cardiac pathology especially familial hypertrophic myopathies.
Showing the most recent 10 out of 99 publications
