title: 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 [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.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle. WA 98195, USA.
authors.forename: Erik R
authors.initials: ER
authors.lastname: Feest
authors.affiliation: Department of Bioengineering, University of Washington, Seattle. WA 98195, USA; Centers for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109, USA.
authors.forename: F
authors.initials: F
authors.lastname: Steven Korte
authors.affiliation: Department of Bioengineering, University of Washington, Seattle. WA 98195, USA.
authors.forename: An-Yue
authors.initials: AY
authors.lastname: Tu
authors.affiliation: Department of Bioengineering, University of Washington, Seattle. WA 98195, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Dai
authors.affiliation: Department of Bioengineering, University of Washington, Seattle. WA 98195, USA.
authors.forename: Maria V
authors.initials: MV
authors.lastname: Razumova
authors.affiliation: Department of Bioengineering, University of Washington, Seattle. WA 98195, USA; Department of Pathology, University of Washington, Seattle, WA 98195, USA; Centers for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109, USA.
authors.forename: Charles E
authors.initials: CE
authors.lastname: Murry
authors.affiliation: Department of Bioengineering, University of Washington, Seattle. WA 98195, USA; Centers for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98109, USA. Electronic address: mregnier@uw.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Regnier
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000256:Adenoviridae / Q000235:genetics; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors; D009200:Myocardial Contraction / Q000502:physiology; D009203:Myocardial Infarction / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D009210:Myofibrils / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D061251:Primary Cell Culture; D015202:Protein Engineering; D051381:Rats; D011916:Rats, Inbred F344; D014161:Transduction, Genetic; D019209:Troponin C / Q000235:genetics* / Q000378:metabolism; D014741:Video Recording
chemical_list: D019209:Troponin C; D002118:Calcium
keywords: Adenovirus; Ca(2+) sensitivity; Cardiomyocyte contractility; Cardiomyocytes; Gene therapy; Myocardial infarction