Gene-Targeted Mice with the Human Troponin T R141W Mutation Develop Dilated Cardiomyopathy with Calcium Desensitization

Abstract

Most studies of the mechanisms leading to hereditary dilated cardiomyopathy (DCM) have been performed in reconstituted in vitro systems. Genetically engineered murine models offer the opportunity to dissect these mechanisms in vivo. We generated a gene-targeted knock-in murine model of the autosomal dominant Arg141Trp (R141W) mutation in Tnnt2, which was first described in a human family with DCM. Mice heterozygous for the mutation (Tnnt2R141W/+) recapitulated the human phenotype, developing left ventricular dilation and reduced contractility. There was a gene dosage effect, so that the phenotype in Tnnt2R141W/+mice was attenuated by transgenic overexpression of wildtype Tnnt2 mRNA transcript. Male mice exhibited poorer survival than females. Biomechanical studies on skinned fibers from Tnnt2R141W/+ hearts showed a significant decrease in pCa50 (-log[Ca2+] required for generation of 50% of maximal force) relative to wildtype hearts, indicating Ca2+ desensitization. Optical mapping studies of Langendorff-perfused Tnnt2R141W/+ hearts showed marked increases in diastolic and peak systolic intracellular Ca2+ ([Ca2+]i), and prolonged systolic rise and diastolic fall of [Ca2+]i. Perfused Tnnt2R141W/+ hearts had slower intrinsic rates in sinus rhythm and reduced peak heart rates in response to isoproterenol. Tnnt2R141W/+ hearts exhibited a reduction in phosphorylated phospholamban relative to wildtype mice. However, crossing Tnnt2R141W/+ mice with phospholamban knockout (Pln-/-) mice, which exhibit increased Ca2+ transients and contractility, had no effect on the DCM phenotype. We conclude that the Tnnt2 R141W mutation causes a Ca2+ desensitization and mice adapt by increasing Ca2+-transient amplitudes, which impairs Ca2+ handling dynamics, metabolism and responses to β-adrenergic activation.

Pathogenic troponin T mutants with opposing effects on myofilament Ca2+ sensitivity attenuate cardiomyopathy phenotypes in mice

“…To note, the TnT R141W mutation showed significantly Ca 2+ desensitization and also displayed an increase in Ca 2+ transient amplitudes as a consequence [12]. Our data are consistent with prior studies that corrected myofilament Ca 2+ sensitivity in mice rescued the cardiomyopathy phenotype.…”

“…The I79N/HET is therefore a hybrid background strain. The generation and characterization of the I79N and HET mouse models have been previously published [9, 12]. …”

“…Ventricular arrhythmia and sudden death are also a primary disease manifestation in HCM patients with TnT mutations [11]. Conversely, the missense TnT mutation R141W is associated with DCM in humans and mice [12–14]. This DCM associated mutation has been shown to induce Ca 2+ desensitization of the myofilament [15–18].…”

Calcium Signaling and Cardiac Arrhythmias

“…204 The remaining genes encode proteins that play various roles within cardiomyocytes to ensure proper contractile function. Various studies suggest that mutations in sarcomeric genes 205–207 as well as non-sarcomeric genes 208, 209 can alter Ca 2+ homeostasis, although the affected proteins are not directly involved in Ca 2+ -handling. On the other hand, there is a clear role of defective Ca 2+ -handling in DCM pathogenesis in patients with inherited mutations in phospholamban (PLN) and histidine-rich Ca 2+ -binding protein (HRC).…”

Immunological and pathological consequences of coxsackievirus RNA persistence in the heart

“…In these mice, functional analyses are normal at 2 weeks of age but, thereafter, chamber dilation and contractile dysfunction develop very rapidly (within ~2 weeks, see Knoll et al, 2002). DCM also develops quickly (in ~6 weeks) in knock-in mice expressing an autosomal dominant cardiac troponin mutation (Ramratnam et al, 2016). What of enterovirus-related DCM: how quickly can it develop?…”