Ca2+ Sensitizing Troponin T Mutations Linked To Hypertrophic Cardiomyopathy Increase Apparent Cytosolic Ca2+ Binding

Abstract

In myocytes, free [Ca2+] is determined by the rate of Ca2+ influx and the Ca2+ buffering properties of the cytosol. Ca2+ binding to myofilaments (primarily Troponin C) represents a major portion of cytosolic Ca2+ buffering. To test the hypothesis that increased myofilament Ca2+ sensitivity decreases cytosolic free [Ca2+], we studied ventricular myocytes from transgenic mice overexpressing wild-type (WT) and Troponin T mutants (R278C, F110I and I79N). Myofilament Ca2+ sensitivity was altered in the following order: R278C < WT < F110I < I79N. Intracellular Ca2+ was released by rapid Caffeine application and quantified using the Na-Ca exchanger current intergral. The rise in free cytosolic Ca2+ was smaller in myocytes expressing I79N compared to WT despite the fact that the amount of Ca2+ released was the same. Both Ca2+ sensitizing mutants significantly decreased average Kd, but did not change Bmax. Independently, we quantified Ca2+ influx by integrating L-type Ca2+ current (in 0 Na+ and thapsigargin). Again, the rise in cytosolic free Ca2+ was smaller in I79N than WT. Taken together, these data demonstrate that Ca2+ sensitizing TnT mutants increase cytosolic free Ca2+ binding (by lowering the Kd).View Large Image | View Hi-Res Image | Download PowerPoint Slide