Thin filament incorporation of an engineered cardiac troponin C variant (L48Q) enhances contractility in intact cardiomyocytes from healthy and infarcted hearts.
ABSTRACT: Many current pharmaceutical therapies for systolic heart failure target intracellular [Ca(2+)] ([Ca(2+)]i) metabolism, or cardiac troponin C (cTnC) on thin filaments, and can have significant side-effects, including arrhythmias or adverse effects on diastolic function. In this study, we tested the feasibility of directly increasing the Ca(2+) binding properties of cTnC to enhance contraction independent of [Ca(2+)]i in intact cardiomyocytes from healthy and myocardial infarcted (MI) hearts. Specifically, cardiac thin filament activation was enhanced through adenovirus-mediated over-expression of a cardiac troponin C (cTnC) variant designed to have increased Ca(2+) binding affinity conferred by single amino acid substitution (L48Q). In skinned cardiac trabeculae and myofibrils we and others have shown that substitution of L48Q cTnC for native cTnC increases Ca(2+) sensitivity of force and the maximal rate of force development. Here we introduced L48Q cTnC into myofilaments of intact cardiomyocytes via adeno-viral transduction to deliver cDNA for the mutant or wild type (WT) cTnC protein. Using video-microscopy to monitor cell contraction, relaxation, and intracellular Ca(2+) transients (Fura-2), we report that incorporation of L48Q cTnC significantly increased contractility of cardiomyocytes from healthy and MI hearts without adversely affecting Ca(2+) transient properties or relaxation. The improvements in contractility from L48Q cTnC expression are likely the result of enhanced contractile efficiency, as intracellular Ca(2+) transient amplitudes were not affected. Expression and incorporation of L48Q cTnC into myofilaments was confirmed by Western blot analysis of myofibrils from transduced cardiomyocytes, which indicated replacement of 18±2% of native cTnC with L48Q cTnC. These experiments demonstrate the feasibility of directly targeting cardiac thin filament proteins to enhance cardiomyocyte contractility that is impaired following MI.
Project description:Tension development and relaxation in cardiac muscle are regulated at the thin filament via Ca(2+) binding to cardiac troponin C (cTnC) and strong cross-bridge binding. However, the influence of cTnC Ca(2+)-binding properties on these processes in the organized structure of cardiac sarcomeres is not well-understood and likely differs from skeletal muscle. To study this we generated single amino acid variants of cTnC with altered Ca(2+) dissociation rates (k(off)), as measured in whole troponin (cTn) complex by stopped-flow spectroscopy (I61Q cTn>WT cTn>L48Q cTn), and exchanged them into cardiac myofibrils and demembranated trabeculae. In myofibrils at saturating Ca(2+), L48Q cTnC did not affect maximum tension (T(max)), thin filament activation (k(ACT)) and tension development (k(TR)) rates, or the rates of relaxation, but increased duration of slow phase relaxation. In contrast, I61Q cTnC reduced T(max), k(ACT) and k(TR) by 40-65% with little change in relaxation. Interestingly, k(ACT) was less than k(TR) with I61Q cTnC, and this difference increased with addition of inorganic phosphate, suggesting that reduced cTnC Ca(2+)-affinity can limit thin filament activation kinetics. Trabeculae exchanged with I61Q cTn had reduced T(max), Ca(2+) sensitivity of tension (pCa(50)), and slope (n(H)) of tension-pCa, while L48Q cTn increased pCa(50) and reduced n(H). Increased cross-bridge cycling with 2-deoxy-ATP increased pCa(50) with WT or L48Q cTn, but not I61Q cTn. We discuss the implications of these results for understanding the role of cTn Ca(2+)-binding properties on the magnitude and rate of tension development and relaxation in cardiac muscle.
Project description:Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca(2+) signal. Promisingly, our smartly formulated Ca(2+)-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease.
Project description:Calcium binding to the regulatory domain of cardiac troponin C (cNTnC) causes a conformational change that exposes a hydrophobic surface to which troponin I (cTnI) binds, prompting a series of protein-protein interactions that culminate in muscle contraction. A number of cTnC variants that alter the Ca(2+) sensitivity of the thin filament have been linked to disease. Tikunova and Davis engineered a series of cNTnC mutations that altered Ca(2+) binding properties and studied the effects on the Ca(2+) sensitivity of the thin filament and contraction [Tikunova, S. B., and Davis, J. P. (2004) J. Biol. Chem. 279, 35341-35352]. One of the mutations they engineered, the L48Q variant, resulted in a pronounced increase in the cNTnC Ca(2+) binding affinity and Ca(2+) sensitivity of cardiac muscle force development. In this work, we sought structural and mechanistic explanations for the increased Ca(2+) sensitivity of contraction for the L48Q cNTnC variant, using an array of biophysical techniques. We found that the L48Q mutation enhanced binding of both Ca(2+) and cTnI to cTnC. Nuclear magnetic resonance chemical shift and relaxation data provided evidence that the cNTnC hydrophobic core is more exposed with the L48Q variant. Molecular dynamics simulations suggest that the mutation disrupts a network of crucial hydrophobic interactions so that the closed form of cNTnC is destabilized. The findings emphasize the importance of cNTnC's conformation in the regulation of contraction and suggest that mutations in cNTnC that alter myofilament Ca(2+) sensitivity can do so by modulating Ca(2+) and cTnI binding.
Project description:The Ca(2+) binding properties of the FHC-associated cardiac troponin C (cTnC) mutation L29Q were examined in isolated cTnC, troponin complexes, reconstituted thin filament preparations, and skinned cardiomyocytes. While higher Ca(2+) binding affinity was apparent for the L29Q mutant in isolated cTnC, this phenomenon was not observed in the cTn complex. At the level of the thin filament in the presence of phosphomimetic TnI, L29Q cTnC further reduced the Ca(2+) affinity by 27% in the steady-state measurement and increased the Ca(2+) dissociation rate by 20% in the kinetic studies. Molecular dynamics simulations suggest that L29Q destabilizes the conformation of cNTnC in the presence of phosphomimetic cTnI and potentially modulates the Ca(2+) sensitivity due to the changes of the opening/closing equilibrium of cNTnC. In the skinned cardiomyocyte preparation, L29Q cTnC increased Ca(2+) sensitivity in a highly sarcomere length (SL)-dependent manner. The well-established reduction of Ca(2+) sensitivity by phosphomimetic cTnI was diminished by 68% in the presence of the mutation and it also depressed the SL-dependent increase in myofilament Ca(2+) sensitivity. This might result from its modified interaction with cTnI which altered the feedback effects of cross-bridges on the L29Q cTnC-cTnI-Tm complex. This study demonstrates that the L29Q mutation alters the contractility and the functional effects of the phosphomimetic cTnI in both thin filament and single skinned cardiomyocytes and importantly that this effect is highly sarcomere length dependent.
Project description:Mutations in cardiac troponin I (cTnI) that cause hypertrophic cardiomyopathy (HCM) have been reported to change the contractility of cardiac myofilaments, but the underlying molecular mechanism remains elusive. In this study, Förster resonance energy transfer (FRET) was used to investigate the specific structural and kinetic effects that HCM related rat cTnI mutations R146G/Q and R163W exert on Ca(2+) and myosin S1 dependent conformational transitions in rat cTn structure. Ca(2+)-induced changes in interactions between cTnC and cTnI were individually monitored in reconstituted thin filaments using steady state and time resolved FRET, and kinetics were determined using stopped flow. R146G/Q and R163W all changed the FRET distances between cTnC and cTnI in unique and various ways. However, kinetic rates of conformational transitions induced by Ca(2+)-dissociation were universally slowed when R146G/Q and R163W were present. Interestingly, the kinetic rates of changes in the inhibitory region of cTnI were always slower than that of the regulatory region, suggesting that the fly casting mechanism that normally underlies deactivation is preserved in spite of mutation. In situ rat myocardial fiber studies also revealed that FRET distance changes indicating mutation specific disruption of the cTnIIR-actin interaction were consistent with increased passive tension.
Project description:<h4>Aims</h4>SR33805, a potent Ca(2+) channel blocker, increases cardiac myofilament Ca(2+) sensitivity in healthy rat cardiomyocytes. Therefore, the aim of the present study was to evaluate the effects of SR33805 on contractile properties in ischaemic failing hearts after myocardial infarction (MI) in vivo and in vitro at the cellular level.<h4>Methods and results</h4>The effect of SR33805 (10 µM) was tested on the excitation-contraction coupling of cardiomyocytes isolated from rat with end-stage heart failure. Cell shortening and Ca(2+) transients were measured in intact cardiomyocytes, while contractile properties were determined in Triton X-100 permeabilized myocytes. Acute treatment with SR33805 restored the MI-altered cell shortening without affecting the Ca(2+) transient amplitude, suggesting an increase of myofilament Ca(2+) sensitivity in MI myocytes. Indeed, a SR33805-induced sensitization of myofilament activation was found to be associated with a slight increase in myosin light chain-2 phosphorylation and a more significant decrease on troponin I (TnI) phosphorylation. Decreased TnI phosphorylation was related to inhibition of protein kinase A activity by SR33805. Finally, administration of a single intra-peritoneal bolus of SR33805 (20 mg/kg) improved end-systolic strain and fractional shortening of MI hearts.<h4>Conclusion</h4>The present study indicates that treatment with SR33805 improved contractility of ischaemic failing hearts after MI in the rat by selectively modulating the phosphorylation status of sarcomeric regulatory proteins, which then sensitized the myofilaments to Ca(2+). Our results gave a proof of concept that manipulation of the Ca(2+) sensitivity of sarcomeric regulatory proteins can be used to improve contractility of a failing heart.
Project description:Protein kinase A (PKA) phosphorylation of myofibril proteins constitutes an important pathway for ?-adrenergic modulation of cardiac contractility and relaxation. PKA targets the N-terminus (Ser-23/24) of cardiac troponin I (cTnI), cardiac myosin-binding protein C (cMyBP-C) and titin. The effect of PKA-mediated phosphorylation on the magnitude of contraction has been studied in some detail, but little is known about how it modulates the kinetics of thin filament activation and myofibril relaxation as Ca(2+) levels vary. Troponin C (cTnC) interaction with cTnI (C-I interaction) is a critical step in contractile activation that can be modulated by cTnI phosphorylation. We tested the hypothesis that altering C-I interactions by PKA, or by cTnI phosphomimetic mutations (S23D/S24D-cTnI), directly affects thin filament activation and myofilament relaxation kinetics. Rat ventricular myofibrils were isolated and endogenous cTn was exchanged with either wild-type cTnI, or S23D/S24D-cTnI recombinant cTn. Contractile mechanics were monitored at maximum and submaximal Ca(2+) concentrations. PKA treatment of wild-type cTn or exchange of cTn containing S23D/S24D-cTnI resulted in an increase in the rate of early, slow phase of relaxation (kREL,slow) and a decrease in its duration (tREL,slow). These effects were greater for submaximal Ca(2+) activated contractions. PKA treatment also reduced the rate of contractile activation (kACT) at maximal, but not submaximal Ca(2+), and reduced the Ca(2+) sensitivity of contraction. Using a fluorescent probe coupled to cTnC (C35S-IANBD), the Ca(2+)-cTn binding affinity and C-I interaction were monitored. Ca(2+) binding to cTn (pCa50) was significantly decreased when cTnI was phosphorylated by PKA (?pCa50 = 0.31). PKA phosphorylation of cTnI also weakened C-I interaction in the presence of Ca(2+). These data suggest that weakened C-I interaction, via PKA phosphorylation of cTnI, may slow thin filament activation and result in increased myofilament relaxation kinetics, the latter of which could enhance early phase diastolic relaxation during ?-adrenergic stimulation.
Project description:Dilated cardiomyopathy (DCM) is often associated with sarcomere protein mutations that confer reduced myofilament tension-generating capacity. We demonstrated that cardiac twitch tension-time integrals can be targeted and tuned to prevent DCM remodeling in hearts with contractile dysfunction. We employed a transgenic murine model of DCM caused by the D230N-tropomyosin (Tm) mutation and designed a sarcomere-based intervention specifically targeting the twitch tension-time integral of D230N-Tm hearts using multiscale computational models of intramolecular and intermolecular interactions in the thin filament and cell-level contractile simulations. Our models predicted that increasing the calcium sensitivity of thin filament activation using the cardiac troponin C (cTnC) variant L48Q can sufficiently augment twitch tension-time integrals of D230N-Tm hearts. Indeed, cardiac muscle isolated from double-transgenic hearts expressing D230N-Tm and L48Q cTnC had increased calcium sensitivity of tension development and increased twitch tension-time integrals compared with preparations from hearts with D230N-Tm alone. Longitudinal echocardiographic measurements revealed that DTG hearts retained normal cardiac morphology and function, whereas D230N-Tm hearts developed progressive DCM. We present a computational and experimental framework for targeting molecular mechanisms governing the twitch tension of cardiomyopathic hearts to counteract putative mechanical drivers of adverse remodeling and open possibilities for tension-based treatments of genetic cardiomyopathies.
Project description:The cardiac thin filament regulates actomyosin interactions through calcium-dependent alterations in the dynamics of cardiac troponin and tropomyosin. Over the past several decades, many details of the structure and function of the cardiac thin filament and its components have been elucidated. We propose a dynamic, complete model of the thin filament that encompasses known structures of cardiac troponin, tropomyosin, and actin and show that it is able to capture key experimental findings. By performing molecular dynamics simulations under two conditions, one with calcium bound and the other without calcium bound to site II of cardiac troponin C (cTnC), we found that subtle changes in structure and protein contacts within cardiac troponin resulted in sweeping changes throughout the complex that alter tropomyosin (Tm) dynamics and cardiac troponin--actin interactions. Significant calcium-dependent changes in dynamics occur throughout the cardiac troponin complex, resulting from the combination of the following: structural changes in the N-lobe of cTnC at and adjacent to sites I and II and the link between them; secondary structural changes of the cardiac troponin I (cTnI) switch peptide, of the mobile domain, and in the vicinity of residue 25 of the N-terminus; secondary structural changes in the cardiac troponin T (cTnT) linker and Tm-binding regions; and small changes in cTnC-cTnI and cTnT-Tm contacts. As a result of these changes, we observe large changes in the dynamics of the following regions: the N-lobe of cTnC, the mobile domain of cTnI, the I-T arm, the cTnT linker, and overlapping Tm. Our model demonstrates a comprehensive mechanism for calcium activation of the cardiac thin filament consistent with previous, independent experimental findings. This model provides a valuable tool for research into the normal physiology of cardiac myofilaments and a template for studying cardiac thin filament mutations that cause human cardiomyopathies.
Project description:Defined as clinically unexplained hypertrophy of the left ventricle, hypertrophic cardiomyopathy (HCM) is traditionally understood as a disease of the cardiac sarcomere. Mutations in TNNC1-encoded cardiac troponin C (cTnC) are a relatively rare cause of HCM. Here, we report clinical and functional characterization of a novel TNNC1 mutation, A31S, identified in a pediatric HCM proband with multiple episodes of ventricular fibrillation and aborted sudden cardiac death. Diagnosed at age 5, the proband is family history-negative for HCM or sudden cardiac death, suggesting a de novo mutation. TnC-extracted cardiac skinned fibers were reconstituted with the cTnC-A31S mutant, which increased Ca(2+) sensitivity with no effect on the maximal contractile force generation. Reconstituted actomyosin ATPase assays with 50% cTnC-A31S:50% cTnC-WT demonstrated Ca(2+) sensitivity that was intermediate between 100% cTnC-A31S and 100% cTnC-WT, whereas the mutant increased the activation of the actomyosin ATPase without affecting the inhibitory qualities of the ATPase. The secondary structure of the cTnC mutant was evaluated by circular dichroism, which did not indicate global changes in structure. Fluorescence studies demonstrated increased Ca(2+) affinity in isolated cTnC, the troponin complex, thin filament, and to a lesser degree, thin filament with myosin subfragment 1. These results suggest that this mutation has a direct effect on the Ca(2+) sensitivity of the myofilament, which may alter Ca(2+) handling and contribute to the arrhythmogenesis observed in the proband. In summary, we report a novel mutation in the TNNC1 gene that is associated with HCM pathogenesis and may predispose to the pathogenesis of a fatal arrhythmogenic subtype of HCM.