Abstract 58: Differential Ventricular Remodeling Induced by Thin Filament Mutational Effects on the Tropomyosin Overlap Structure

Inherited mutations in cardiac thin filament proteins create primary alterations in structure. These changes then lead to pathologic remodeling of the heart. We have proposed that two mutations, tropomyosin (Tm) D230N which leads to dilated cardiomyopathy (DCM) and cardiac troponin T (cTnT) R92L, which leads to hypertrophic cardiomyopathy (HCM), directly affect the Tm overlap, a crucial structure regulating myofilament activation. These mutations lead to divergent ventricular remodeling, which we hypothesize is caused by differential effects on the Tm overlap, an inherently dynamic system. In order to investigate this hypothesis, we used time-resolved Forster resonance energy transfer (FRET) to measure discrete distances across the proteins of the overlap, and differential scanning calorimetry (DSC) in order to characterize the baseline structure of this region as well as the effects of mutants. We found that the cTnT R92L mutant increased interprotein distance at the proximal N-terminal end of this region. In contrast, the Tm D230N mutant decreases interprotein distance at the proximal C-terminal end of the overlap. DSC studies showed a significant increase in the association of Tm with actin in the presence of Tm D230N, predicted to increase cooperativity of thin filament activation. These finding are in agreement both with the human population who carry the disease, as well as the mouse lines in our lab, which phenocopy the human disease. Overall, the study further characterized the wild type structure of the Tm overlap, and revealed the differential structural effects of the mutants, thereby, for the first time linking local changes in structure to phenotypic changes and ventricular remodeling leading to HCM and DCM. Further studies will investigate the role of isoform and phosphorylation modifiers, elucidating how these modify human disease in the presence and absence of cardiomyopathy linked mutations.

Length-dependent activation is modulated by cardiac troponin I bisphosphorylation at Ser23 and Ser24 but not by Thr143 phosphorylation

Frank-Starling's law reflects the ability of the heart to adjust the force of its contraction to changes in ventricular filling, a property based on length-dependent myofilament activation (LDA). The threonine at amino acid 143 of cardiac troponin I (cTnI) is prerequisite for the length-dependent increase in Ca2+sensitivity. Thr143 is a known target of protein kinase C (PKC) whose activity is increased in cardiac disease. Thr143 phosphorylation may modulate length-dependent myofilament activation in failing hearts. Therefore, we investigated if pseudo-phosphorylation at Thr143 modulates length dependence of force using troponin exchange experiments in human cardiomyocytes. In addition, we studied effects of protein kinase A (PKA)-mediated cTnI phosphorylation at Ser23/24, which has been reported to modulate LDA. Isometric force was measured at various Ca2+concentrations in membrane-permeabilized cardiomyocytes exchanged with recombinant wild-type (WT) troponin or troponin mutated at the PKC site Thr143 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after incubation with exogenous PKA. Pseudo-phosphorylation of Thr143 increased myofilament Ca2+sensitivity compared with WT without affecting LDA in failing and donor cardiomyocytes. Subsequent PKA treatment enhanced the length-dependent shift in Ca2+sensitivity after WT and 143D exchange. Exchange with Ser23/24 variants demonstrated that pseudo-phosphorylation of both Ser23 and Ser24 is needed to enhance the length-dependent increase in Ca2+sensitivity. cTnI pseudo-phosphorylation did not alter length-dependent changes in maximal force. Thus phosphorylation at Thr143 enhances myofilament Ca2+sensitivity without affecting LDA, while Ser23/24 bisphosphorylation is needed to enhance the length-dependent increase in myofilament Ca2+sensitivity.

Comparison of myoplasmic calcium movements during excitation–contraction coupling in frog twitch and mouse fast-twitch muscle fibers

Single twitch fibers from frog leg muscles were isolated by dissection and micro-injected with furaptra, a rapidly responding fluorescent Ca2+ indicator. Indicator resting fluorescence (FR) and the change evoked by an action potential (ΔF) were measured at long sarcomere length (16°C); ΔF/FR was scaled to units of ΔfCaD, the change in fraction of the indicator in the Ca2+-bound form. ΔfCaD was simulated with a multicompartment model of the underlying myoplasmic Ca2+ movements, and the results were compared with previous measurements and analyses in mouse fast-twitch fibers. In frog fibers, sarcoplasmic reticulum (SR) Ca2+ release evoked by an action potential appears to be the sum of two components. The time course of the first component is similar to that of the entire Ca2+ release waveform in mouse fibers, whereas that of the second component is severalfold slower; the fractional release amounts are ∼0.8 (first component) and ∼0.2 (second component). Similar results were obtained in frog simulations with a modified model that permitted competition between Mg2+ and Ca2+ for occupancy of the regulatory sites on troponin. An anatomical basis for two release components in frog fibers is the presence of both junctional and parajunctional SR Ca2+ release channels (ryanodine receptors [RyRs]), whereas mouse fibers (usually) have only junctional RyRs. Also, frog fibers have two RyR isoforms, RyRα and RyRβ, whereas the mouse fibers (usually) have only one, RyR1. Our simulations suggest that the second release component in frog fibers functions to supply extra Ca2+ to activate troponin, which, in mouse fibers, is not needed because of the more favorable location of their triadic junctions (near the middle of the thin filament). We speculate that, in general, parajunctional RyRs permit increased myofilament activation in fibers whose triadic junctions are located at the z-line.

Coexistence of cardiac troponin T variants reduces heart efficiency

Corresponding to the synchronized contraction of the myocardium and rhythmic pumping function of the heart, a single form of cardiac troponin T (cTnT) is present in the adult cardiac muscle of humans and most other vertebrate species. Alternative splicing variants of cTnT are found in failing human hearts and animal dilated cardiomyopathies. Biochemical analyses have shown that these cTnT variants are functional and produce shifted myofilament Ca2+ sensitivity. We proposed a hypothesis that the coexistence of two or more functionally distinct TnT variants in the adult ventricular muscle that is normally activated as a syncytium may decrease heart function and cause cardiomyopathy (Huang et al., Am J Physiol Cell Physiol 294: C213–C222, 2008). In the present study, we studied transgenic mouse hearts expressing one or two cTnT variants in addition to normal adult cTnT to investigate whether desynchronized myofilament activation decreases ventricular efficiency. The function of ex vivo working hearts was examined in the absence of systemic neurohumoral influence. The results showed that the transgenic mouse hearts produced lower maximum left ventricular pressure, slower contractile and relaxation velocities, and decreased stroke volume compared with wild-type controls. Ventricular pumping efficiency, calculated by the ejection integral versus total systolic integral and cardiac work versus oxygen consumption, was significantly lower in transgenic mouse hearts and corresponded to the number of cTnT variants present. The results indicated a pathogenic mechanism in which the coexistence of functionally different cTnT variants in cardiac muscle reduces myocardial efficiency due to desynchronized thin filament activation.