Thin filament incorporation of an engineered cardiac troponin C variant (L48Q) enhances contractility in intact cardiomyocytes from healthy and infarcted hearts

J Mol Cell Cardiol. 2014 Jul:72:219-27. doi: 10.1016/j.yjmcc.2014.03.015. Epub 2014 Mar 29.

Abstract

Many current pharmaceutical therapies for systolic heart failure target intracellular [Ca(2+)] ([Ca(2+)]i) metabolism, or cardiac troponin C (cTnC) on thin filaments, and can have significant side-effects, including arrhythmias or adverse effects on diastolic function. In this study, we tested the feasibility of directly increasing the Ca(2+) binding properties of cTnC to enhance contraction independent of [Ca(2+)]i in intact cardiomyocytes from healthy and myocardial infarcted (MI) hearts. Specifically, cardiac thin filament activation was enhanced through adenovirus-mediated over-expression of a cardiac troponin C (cTnC) variant designed to have increased Ca(2+) binding affinity conferred by single amino acid substitution (L48Q). In skinned cardiac trabeculae and myofibrils we and others have shown that substitution of L48Q cTnC for native cTnC increases Ca(2+) sensitivity of force and the maximal rate of force development. Here we introduced L48Q cTnC into myofilaments of intact cardiomyocytes via adeno-viral transduction to deliver cDNA for the mutant or wild type (WT) cTnC protein. Using video-microscopy to monitor cell contraction, relaxation, and intracellular Ca(2+) transients (Fura-2), we report that incorporation of L48Q cTnC significantly increased contractility of cardiomyocytes from healthy and MI hearts without adversely affecting Ca(2+) transient properties or relaxation. The improvements in contractility from L48Q cTnC expression are likely the result of enhanced contractile efficiency, as intracellular Ca(2+) transient amplitudes were not affected. Expression and incorporation of L48Q cTnC into myofilaments was confirmed by Western blot analysis of myofibrils from transduced cardiomyocytes, which indicated replacement of 18±2% of native cTnC with L48Q cTnC. These experiments demonstrate the feasibility of directly targeting cardiac thin filament proteins to enhance cardiomyocyte contractility that is impaired following MI.

Keywords: Adenovirus; Ca(2+) sensitivity; Cardiomyocyte contractility; Cardiomyocytes; Gene therapy; Myocardial infarction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Action Potentials / physiology
  • Adenoviridae / genetics
  • Amino Acid Substitution
  • Animals
  • Calcium / metabolism
  • Female
  • Gene Expression
  • Genetic Therapy
  • Genetic Vectors
  • Myocardial Contraction / physiology
  • Myocardial Infarction / genetics
  • Myocardial Infarction / metabolism*
  • Myocardial Infarction / pathology
  • Myocardium / metabolism*
  • Myocardium / pathology
  • Myocytes, Cardiac / metabolism*
  • Myocytes, Cardiac / pathology
  • Myofibrils / genetics
  • Myofibrils / metabolism*
  • Myofibrils / pathology
  • Primary Cell Culture
  • Protein Engineering
  • Rats
  • Rats, Inbred F344
  • Transduction, Genetic
  • Troponin C / genetics*
  • Troponin C / metabolism
  • Video Recording

Substances

  • Troponin C
  • Calcium