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

Abstract

Many current pharmaceutical therapies for systolic heart failure target intracellular [Ca2+] ([Ca2+]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 Ca2+ binding properties of cTnC to enhance contraction independent of [Ca2+]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 Ca2+ 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 Ca2+ 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 Ca2+ transients (Fura-2), we report that incorporation of L48Q cTnC significantly increased contractility of cardiomyocytes from healthy and MI hearts without adversely affecting Ca2+ transient properties or relaxation. The improvements in contractility from L48Q cTnC expression are likely the result of enhanced contractile efficiency, as intracellular Ca2+ 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.

A Tension-Based Model Distinguishes Hypertrophic versus Dilated Cardiomyopathy

“…In myocyte contractile assays, L48Q cTnC significantly increased cellular fractional shortening in the absence of changes in Ca 2+ transient amplitude ( data not shown ), as previously reported (Feest et al, 2014). Acute expression of I61Q cTnC caused a modest reduction in fractional shortening with no changes in Ca 2+ transient amplitude reflecting the decreased sensitivity of the myofilaments to Ca 2+ ( data not shown ).…”

“…We also used mutant cTnCs because they represent the proximal Ca 2+ binding element of the sarcomere, hence allowing for a more direct modulation of the Ca 2+ -tension relationship without potential confounding effects. The L48Q and I61Q variants used here have been rigorously quantified for their effects on tension and Ca 2+ handling dynamics (Kreutziger et al, 2011; Parvatiyar et al, 2010; Tikunova and Davis, 2004; Wang et al, 2012; Feest et al, 2014). …”

Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility

“…Based on the current computational models, we and others have designed engineered cTnC variants with altered Ca 2+ binding affinities (to cTn) and demonstrated that those variants alter the contractile function in cardiomyocytes and correct or prevent disease-related aberrant Ca 2+ binding and contractile function [163, 187, 188, 203-209]. These studies also provide important implications with respect to the design of Ca 2+ sensitizing or desensitizing small molecules or drugs.…”

Translation of Cardiac Myosin Activation With 2-Deoxy-ATP to Treat Heart Failure Via an Experimental Ribonucleotide Reductase-Based Gene Therapy

“…To evaluate the therapeutic potential of this delivery system, cardiomyocytes isolated from infarcted adult rat hearts were transduced. Contractility of the treated infarcted cardiomyocytes was significantly improved compared to untreated infarct cells, with the magnitude and rate of contraction restored to levels comparable to healthy (uninfarcted) cardiomyocytes(38). …”