title: Pathogenesis associated with a restrictive cardiomyopathy mutant in cardiac troponin T is due to reduced protein stability and greatly increased myofilament Ca2+ sensitivity.
abstract: BACKGROUND
  Dilated and hypertrophic cardiomyopathy mutations in troponin can blunt effects of protein kinase A (PKA) phosphorylation of cardiac troponin I (cTnI), decreasing myofilament Ca2+-sensitivity; however this effect has never been tested for restrictive cardiomyopathy (RCM) mutants. This study explores whether an RCM cardiac troponin T mutant (cTnT-ΔE96) interferes with convergent PKA regulation and if TnT instability contributes to greatly enhanced Ca2+-sensitivity in skinned fibers.
  
  
  METHODS
  Force of contraction in skinned cardiac porcine fiber and spectroscopic studies were performed.
  
  
  RESULTS
  A decrease of -0.26 and -0.25 pCa units in Ca2+-sensitivity of contraction after PKA incubation was observed for skinned fibers incorporated with WT or cTnT-ΔE96, respectively. To further assess whether cTnT-ΔE96 interferes solely with transmission of cTnI phosphorylation effects, skinned fibers were reconstituted with PKA pseudo-phosphorylated cTnI (cTnI-SS/DD.cTnC). Fibers displaced with cTnT-WT, reconstituted with cTnI-SS/DD.cTnC decreased Ca2+-sensitivity of force (pCa50=5.61) compared to control cTnI-WT.cTnC (pCa50=5.75), similarly affecting cTnT-ΔE96 (pCa50=6.03) compared to control \cTnI-WT.cTnC (pCa50=6.14). Fluorescence studies measuring cTnC(IAANS) Ca2+-affinity changes due to cTnT-ΔE96 indicated that higher complexity (thin filament) better recapitulates skinned fiber Ca2+ sensitive changes. Circular dichroism revealed reduced α-helicity and earlier thermal unfolding for cTnT-ΔE96 compared to WT.
  
  
  CONCLUSIONS
  Although ineffective in decreasing myofilament Ca2+-sensitivity to normal levels, cTnT-ΔE96 does not interfere with PKA cTnI phosphorylation mediated effects; 2) cTnT-ΔE96 requires actin to increase cTnC Ca2+-affinity; and 3) deletion of E96 reduces cTnT stability, likely disrupting crucial thin filament interactions.
  
  
  CONCLUSIONS
  The pathological effect of cTnT-ΔE96 is largely manifested by dramatic myofilament Ca2+-sensitization which still persists even after PKA phosphorylation mediated Ca2+-desensitization.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, FL 33136, USA.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Parvatiyar
authors.affiliation: Department of Biomedical Sciences, Florida State University, Tallahassee, FL 32306, USA. Electronic address: jose.pinto@med.fsu.edu.
authors.forename: Jose Renato
authors.initials: JR
authors.lastname: Pinto
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002311:Cardiomyopathy, Dilated / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D030342:Genetic Diseases, Inborn / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D009154:Mutation; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D009210:Myofibrils / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000235:genetics; D055550:Protein Stability; D013552:Swine; D020107:Troponin T / Q000235:genetics / Q000378:metabolism*
chemical_list: D020107:Troponin T; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium
keywords: Cardiac troponin T; Circular dichroism; Fluorescence; Restrictive cardiomyopathy; Skinned fiber; Troponin I phosphorylation