Publication Date

2008-07-10

Availability

Open access

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Physiology and Biophysics (Medicine)

Date of Defense

2008-06-24

First Committee Member

Gerhard Dahl - Committee Chair

Second Committee Member

James D. Potter - Committee Member

Third Committee Member

Nanette H. Bishopric - Committee Member

Fourth Committee Member

W. Glenn L. Kerrick. - Mentor

Fifth Committee Member

R. John Solaro - Outside Committee Member

Abstract

An arginine (R) to a glycine (G) mutation at position 145 in the highly reserved inhibitory domain of cardiac troponin I (cTnI) is associated with hypertrophic cardiomyopathy (HCM), an autosomal dominant disease characterized by left ventricular hypertrophy. An arginine (R) to tryptophan (W) mutation at the same position in cTnI is associated with restrictive cardiomyopathy (RCM), a disease characterized by diastolic dysfunction with normal left ventricular size and normal systolic function. In this study we addressed the functional consequences of the human cardiac troponin I (hcTnI) HCM R145G mutation and hcTnI RCM R145W mutation in transgenic mice. Simultaneous measurements of the ATPase activity and force in skinned papillary fibers from hcTnI R145G transgenic mice (Tg-R145G) versus hcTnI wild type transgenic mice (Tg-WT) showed a significant decrease in the maximal Ca2+ activated force without changes in the maximal ATPase activity and an increase in the Ca2+ sensitivity by both ATPase activity and force development. No difference in the cross-bridge turnover rate was observed at the same level of cross-bridge attachment (activation state) showing that changes in Ca2+ sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared to Tg-WT fibers. The addition of 3mM BDM at pCa 9.0 showed that there was approximately 2~4 percent of force generating cross-bridges attached in Tg-R145G fibers compared to less than 1.0 percent in Tg-WT fibers, suggesting the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca2+] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared to Tg-WT. These results suggest that the phenotype of HCM is most likely caused by the compensatory mechanisms in the cardiovascular system which are activated by: 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge; 2) incomplete relaxation (diastolic dysfunction) caused by prolonged [Ca2+] and force transients; and 3) an inability of the cardiac TnI to completely inhibit activation at low levels of diastolic Ca2+ in Tg-R145G. Simultaneous measurements of the ATPase activity and force in transgenic skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) versus Tg-WT showed that there was a ~13 to ~16 percent increase in the maximal Ca2+ activated force and ATPase activity, respectively. The rate of dissociation of force generating cross-bridges (g) and energy cost (ATPase/force) was the same in all groups of fibers. These results suggest that the increase in force and ATPase activity is associated with an increase in the number of force generating cross-bridges attached at all activation levels. Additionally, there was a large increase in the Ca2+ sensitivity of force development and ATPase activity. In intact fibers, the mutation caused prolonged force and intracellular [Ca2+] transients, as expected due to the increased Ca2+ sensitivity (slower dissociation rate of Ca2+ from cTnC). The above cited results suggest that: 1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca2+ transients, especially at high heart rates; 2) there would be a decrease in ventricular filling (diastolic dysfunction); and 3) an increase in contractility during systole that would off-set the negative effect of a decrease in diastolic filling on ventricle stroke volume thus allowing the heart to maintain normal stroke volume despite the compromise in RCM (Tg-R145W) heart.

Keywords

Troponin Complex; Force And Calcium Transient

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