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A Drosophila melanogaster model of diastolic dysfunction and cardiomyopathy based on impaired troponin-T function.

ABSTRACT: RATIONALE:Regulation of striated muscle contraction is achieved by Ca2+ -dependent steric modulation of myosin cross-bridge cycling on actin by the thin filament troponin-tropomyosin complex. Alterations in the complex can induce contractile dysregulation and disease. For example, mutations between or near residues 112 to 136 of cardiac troponin-T, the crucial TnT1 (N-terminal domain of troponin-T)-tropomyosin-binding region, cause cardiomyopathy. The Drosophila upheld(101) Glu/Lys amino acid substitution lies C-terminally adjacent to this phylogenetically conserved sequence. OBJECTIVE:Using a highly integrative approach, we sought to determine the molecular trigger of upheld(101) myofibrillar degeneration, to evaluate contractile performance in the mutant cardiomyocytes, and to examine the effects of the mutation on the entire Drosophila heart to elucidate regulatory roles for conserved TnT1 regions and provide possible mechanistic insight into cardiac dysfunction. METHODS AND RESULTS:Live video imaging of Drosophila cardiac tubes revealed that the troponin-T mutation prolongs systole and restricts diastolic dimensions of the heart, because of increased numbers of actively cycling myosin cross-bridges. Elevated resting myocardial stiffness, consistent with upheld(101) diastolic dysfunction, was confirmed by an atomic force microscopy-based nanoindentation approach. Direct visualization of mutant thin filaments via electron microscopy and 3-dimensional reconstruction resolved destabilized tropomyosin positioning and aberrantly exposed myosin-binding sites under low Ca2+ conditions. CONCLUSIONS:As a result of troponin-tropomyosin dysinhibition, upheld(101) hearts exhibited cardiac dysfunction and remodeling comparable to that observed during human restrictive cardiomyopathy. Thus, reversal of charged residues about the conserved tropomyosin-binding region of TnT1 may perturb critical intermolecular associations required for proper steric regulation, which likely elicits myopathy in our Drosophila model.

SUBMITTER: Viswanathan MC

PROVIDER: S-EPMC4526186 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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A Drosophila melanogaster model of diastolic dysfunction and cardiomyopathy based on impaired troponin-T function.

Viswanathan Meera Cozhimuttam MC Kaushik Gaurav G Engler Adam J AJ Lehman William W Cammarato Anthony A

Circulation research 20131112 2

<h4>Rationale</h4>Regulation of striated muscle contraction is achieved by Ca2+ -dependent steric modulation of myosin cross-bridge cycling on actin by the thin filament troponin-tropomyosin complex. Alterations in the complex can induce contractile dysregulation and disease. For example, mutations between or near residues 112 to 136 of cardiac troponin-T, the crucial TnT1 (N-terminal domain of troponin-T)-tropomyosin-binding region, cause cardiomyopathy. The Drosophila upheld(101) Glu/Lys amino ...[more]

PMID: 24221941

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Project description:AIMS:Several mutations in the ventricular myosin regulatory light chain (RLC) were identified to cause familial hypertrophic cardiomyopathy (FHC). Based on our previous cellular findings showing delayed calcium transients in electrically stimulated intact papillary muscle fibres from transgenic Tg-R58Q and Tg-N47K mice and, in addition, prolonged force transients in Tg-R58Q fibres, we hypothesized that the malignant FHC phenotype associated with the R58Q mutation is most likely related to diastolic dysfunction. METHODS AND RESULTS:Cardiac morphology and in vivo haemodynamics by echocardiography as well as cardiac function in isolated perfused working hearts were assessed in transgenic (Tg) mutant mice. The ATPase-pCa relationship was determined in myofibrils isolated from Tg mouse hearts. In addition, the effect of both mutations on RLC phosphorylation was examined in rapidly frozen ventricular samples from Tg mice. Significantly, decreased cardiac function was observed in isolated perfused working hearts from both Tg-R58Q and Tg-N47K mice. However, echocardiographic examination showed significant alterations in diastolic transmitral velocities and deceleration time only in Tg-R58Q myocardium. Likewise, changes in Ca(2+) sensitivity, cooperativity, and an elevated level of ATPase activity at low [Ca(2+)] were only observed in myofibrils from Tg-R58Q mice. In addition, the R58Q mutation and not the N47K led to reduced RLC phosphorylation in Tg ventricles. CONCLUSION:Our results suggest that the N47K and R58Q mutations may act through similar mechanisms, leading to compensatory hypertrophy of the functionally compromised myocardium, but the malignant R58Q phenotype is most likely associated with more severe alterations in cardiac performance manifested as impaired relaxation and global diastolic dysfunction. At the molecular level, we suggest that by reducing the phosphorylation of RLC, the R58Q mutation decreases the kinetics of myosin cross-bridges, leading to an increased myofilament calcium sensitivity and to overall changes in intracellular Ca(2+) homeostasis.

Project description:AimsDiabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes.Methods and resultsStreptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed (i) marked DD not associated to cardiac fibrosis, (ii) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, (iii) reduced LV SERCA2 protein level and activity and (iv) slower SR Ca2+ uptake rate, (v) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, (vi) increased diastolic Ca2+, and (vii) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent.ConclusionsSERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment.

Dataset Information

A Drosophila melanogaster model of diastolic dysfunction and cardiomyopathy based on impaired troponin-T function.

Publications

A Drosophila melanogaster model of diastolic dysfunction and cardiomyopathy based on impaired troponin-T function.

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