Functional Consequences of the Human Cardiac Troponin I Hypertrophic Cardiomyopathy Mutation R145G in Transgenic Mice

Yuhui Wen,Jose Renato Pinto,Ying Wang,Yingcai Wang,Aldrin V Gomes,Yuanyuan Xu,James D Potter,W Glenn L Kerrick

doi:10.1074/jbc.m801661200

Abstract

In this study, we addressed the functional consequences of the human cardiac troponin I (hcTnI) hypertrophic cardiomyopathy R145G mutation in transgenic mice. Simultaneous measurements of 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 Ca(2+)-activated force without changes in the maximal ATPase activity and an increase in the Ca(2+) sensitivity of both ATPase 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 Ca(2+) sensitivity were not due to changes in cross-bridge kinetics. Energy cost calculations demonstrated higher energy consumption in Tg-R145G fibers compared with Tg-WT fibers. The addition of 3 mm 2,3-butanedione monoxime at pCa 9.0 showed that there was approximately 2-4% of force generating cross-bridges attached in Tg-R145G fibers compared with less than 1.0% in Tg-WT fibers, suggesting that the mutation impairs the ability of the cardiac troponin complex to fully inhibit cross-bridge attachment under relaxing conditions. Prolonged force and intracellular [Ca(2+)] transients in electrically stimulated intact papillary muscles were observed in Tg-R145G compared with Tg-WT. These results suggest that the phenotype of hypertrophic cardiomyopathy is most likely caused by the compensatory mechanisms in the cardiovascular system that are activated by 1) higher energy cost in the heart resulting from a significant decrease in average force per cross-bridge, 2) slowed relaxation (diastolic dysfunction) caused by prolonged [Ca(2+)] and force transients, and 3) an inability of the cardiac TnI to completely inhibit activation in the absence of Ca(2+) in Tg-R145G mice.

Highlights

The cTn complex is responsible for regulating cardiac muscle contraction, and it contains three subunits: cardiac troponin C, the Ca2ϩ-binding subunit; cardiac troponin I, the inhibitory subunit that binds to actin preventing the formation of cross-bridges; and cardiac troponin T, which anchors the cTn complex to the thin filament by binding to cTnC, cTnI, and tropomyosin
The human cardiac TnI (hcTnI) R145G mutation had no effect on the rate of dissociation of force-generating cross-bridge as a function of the activation state but caused a significant decrease in average force/cross-bridge, an increase in Ca2ϩ sensitivity of force development and ATPase activity, and a decrease in cooperativity of thin filament activation
The expression of hcTnI wild type (WT) and R145G proteins were driven by the ␣-MHC promoter in the mouse heart (Fig. 1a)

Summary

Introduction

The cTn complex is responsible for regulating cardiac muscle contraction, and it contains three subunits: cardiac troponin C (cTnC), the Ca2ϩ-binding subunit; cardiac troponin I (cTnI), the inhibitory subunit that binds to actin preventing the formation of cross-bridges; and cardiac troponin T (cTnT), which anchors the cTn complex to the thin filament by binding to cTnC, cTnI, and tropomyosin. Kruger et al [13] found a slightly decreased Ca2ϩ sensitivity of force development in myofibrils from mouse cardiac TnI R146G (mcTnI R146G) transgenic mice but no change in hcTnI R145G-exchanged murine cardiac myofibrils They observed a slightly significant upward shift of the passive forcesarcomere length curve at pCa 7.5, which was reversed by the addition of 2,3-butanedione monoxime (BDM) in both reconstituted myofibrils and transgenic mice myofibrils. The hcTnI R145G mutation had no effect on the rate of dissociation of force-generating cross-bridge as a function of the activation state (number of cross-bridges attached in the presence of Ca2ϩ) but caused a significant decrease in average force/cross-bridge, an increase in Ca2ϩ sensitivity of force development and ATPase activity, and a decrease in cooperativity of thin filament activation (decrease in the slope of the force-pCa relationship). All of these physiological findings suggest that the hcTnI R145G mutation would cause both systolic and diastolic dysfunction and may explain the poor prognosis for patients with this mutation

Methods

Results

Conclusion