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Diastolic dysfunction and thin filament dysregulation resulting from excitation-contraction uncoupling in a mouse model of restrictive cardiomyopathy.

ABSTRACT: Restrictive cardiomyopathy (RCM) has been linked to mutations in the thin filament regulatory protein cardiac troponin I (cTnI). As the pathogenesis of RCM from genotype to clinical phenotype is not fully understood, transgenic (Tg) mice were generated with cardiac specific expression of an RCM-linked missense mutation (R193H) in cTnI. R193H Tg mouse hearts with 15% stoichiometric replacement had smaller hearts and significantly elevated end diastolic pressures (EDP) in vivo. Using a unique carbon microfiber-based assay, membrane intact R193H adult cardiac myocytes generated higher passive tensions across a range of physiologic sarcomere lengths resulting in significant Ca(2+) independent cellular diastolic tone that was manifest in vivo as elevated organ-level EDP. Sarcomere relaxation and Ca(2+) decay was uncoupled in isolated R193H Tg adult myocytes due to the increase in myofilament Ca(2+) sensitivity of tension, decreased passive compliance of the sarcomere, and adaptive in vivo changes in which phospholamban (PLN) content was decreased. Further evidence of Ca(2+) and mechanical uncoupling in R193H Tg myocytes was demonstrated by the biphasic response of relaxation to increased pacing frequency versus the negative staircase seen with Ca(2+) decay. In comparison, non-transgenic myocyte relaxation closely paralleled the accelerated Ca(2+) decay. Ca(2+) transient amplitude was also significantly blunted in R193H Tg myocytes despite normal mechanical shortening resulting in myocyte hypercontractility when compared to non-transgenics. These results identify for the first time that a single point mutation in cTnI, R193H, directly causes elevated EDP due to a myocyte intrinsic loss of compliance independent of Ca(2+) cycling or altered cardiac morphology. The compound influence of impaired relaxation and elevated EDP represents a clinically severe form of diastolic dysfunction similar to the hemodynamic state documented in RCM patients.

SUBMITTER: Davis J

PROVIDER: S-EPMC3443869 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Diastolic dysfunction and thin filament dysregulation resulting from excitation-contraction uncoupling in a mouse model of restrictive cardiomyopathy.

Davis Jennifer J Yasuda Soichiro S Palpant Nathan J NJ Martindale Joshua J Stevenson Tamara T Converso Kimber K Metzger Joseph M JM

Journal of molecular and cellular cardiology 20120606 3

Restrictive cardiomyopathy (RCM) has been linked to mutations in the thin filament regulatory protein cardiac troponin I (cTnI). As the pathogenesis of RCM from genotype to clinical phenotype is not fully understood, transgenic (Tg) mice were generated with cardiac specific expression of an RCM-linked missense mutation (R193H) in cTnI. R193H Tg mouse hearts with 15% stoichiometric replacement had smaller hearts and significantly elevated end diastolic pressures (EDP) in vivo. Using a unique carb ...[more]

PMID: 22683325

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Project description:BackgroundWe have previously shown that the deletion of the superoxide scavenger, CuZn superoxide dismutase, in mice (Sod1-/- mice) results in increased oxidative stress and an accelerated loss of skeletal muscle mass and force that mirror the changes seen in old control mice. The goal of this study is to define the effect of oxidative stress and ageing on muscle weakness and the Excitation Contraction (EC) coupling machinery in age-matched adult (8-10 months) wild-type (WT) and Sod1-/- mice in comparison with old (25-28 months) WT mice.MethodsIn vitro contractile assays were used to measure muscle contractile parameters. The activity of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) pump was measured using an NADH-linked enzyme assay. Immunoblotting and immunofluorescence techniques were used to measure protein expression, and real-time reverse transcription PCR was used to measure gene expression.ResultsThe specific force generated by the extensor digitorum longus muscle was reduced in the Sod1-/- and old WT mice compared with young WT mice along with significant prolongation of time to peak force, increased half relaxation time, and disruption of intracellular calcium handling. The maximal activity of the SERCA calcium uptake pump was significantly reduced in gastrocnemius muscle from both old WT (≈14%) and adult Sod1-/- (≈33%) mice compared with young WT mice along with increased expression of sarcolipin, a known inhibitor of SERCA activity. Protein levels of the voltage sensor and calcium uptake channel proteins dihydropyridine receptor α1 and SERCA2 were significantly elevated (≈45% and ≈57%, respectively), while the ratio of calstabin, a channel stabilizing protein, to ryanodine receptor was significantly reduced (≈21%) in Sod1-/- mice compared with young WT mice. The changes in calcium handling were accompanied by substantially elevated levels of global protein carbonylation and lipid peroxidation.ConclusionsOur data suggest that the muscle weakness in Sod1-/- and old WT mice is in part driven by reactive oxygen species-mediated EC uncoupling and supports a role for reduced SERCA pump activity in compromised muscle function. The novel quantitative mechanistic data provided here can lead to potential therapeutic interventions of SERCA dysfunction for sarcopenia and muscle diseases.

Dataset Information

Diastolic dysfunction and thin filament dysregulation resulting from excitation-contraction uncoupling in a mouse model of restrictive cardiomyopathy.

Publications

Diastolic dysfunction and thin filament dysregulation resulting from excitation-contraction uncoupling in a mouse model of restrictive cardiomyopathy.

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