Length-dependent Activation Research Articles

Development of an Integrated Human Model of Electrophysiology and Actin-Myosin Binding to Describe Sarcomere Force Generation in Cardiomyocytes

We have developed an integrated human model which links detailed sub-models of sarcomere electrophysiology and actin-myosin kinetics to describe sarcomere force generation. While many published quantitative models connect cardiac electrophysiology to force generation or full heart mechanics, these models typically include a lumped representation of actin-myosin mechanics without sufficient granularity. In order to mechanistically represent both heart failure conditions and treatment strategies which operate at the actin-myosin level, we have developed an integrated multiscale model of sarcomere force generation. Our platform connects published electrophysiology and lumped force generation models to an actin-myosin (AM) model developed based on published data on cardiac myosin modulators. Specifically, the AM model was calibrated to published in vitro data of phosphate release and ATP turnover, and can successfully reproduce the molecular effect of cardiac myosin modulation. We connected this in vitro model with healthy and heart failure phenotypes to describe cardiac force generation before and after therapy. With this multiscale approach we can also simulate sarcomere length-dependent activation, calcium-force relationships, and determine how these simulations are affected in failing myocardium after treatment with cardiac modulators. We demonstrate that models calibrated to data at one scale often need to be adjusted in the context of a multiscale model. For example, the calcium transient produced from a published heart failure electrophysiology model generates little force in the integrated multiscale model, underscoring the continuing challenge of linking in vitro experimental efforts to clinical outputs. We discuss our methods to connect information across scales into one coherent mathematical framework. The final model describes how specific therapeutic interventions at different stages of the actin-myosin cycle will alter sarcomere force generation, which will propagate to changes in clinical outcomes like ejection fraction.

Developmental increase in β-MHC enhances sarcomere length-dependent activation in the myocardium.

Shifts in myosin heavy chain (MHC) isoforms in cardiac myocytes have been shown to alter cardiac muscle function not only in healthy developing hearts but also in diseased hearts. In guinea pig hearts, there is a large age-dependent shift in MHC isoforms from 80% α-MHC/20% β-MHC at 3 wk to 14% α-MHC/86% β-MHC at 11 wk. Because kinetic differences in α- and β-MHC cross-bridges (XBs) are known to impart different cooperative effects on thin filaments, we hypothesize here that differences in α- and β-MHC expression in guinea pig cardiac muscle impact sarcomere length (SL)-dependent contractile function. We therefore measure steady state and dynamic contractile parameters in detergent-skinned cardiac muscle preparations isolated from the left ventricles of young (3 wk old) or adult (11 wk old) guinea pigs at two different SLs: short (1.9 µm) and long (2.3 µm). Our data show that SL-dependent effects on contractile parameters are augmented in adult guinea pig cardiac muscle preparations. Notably, the SL-mediated increase in myofilament Ca2+ sensitivity (ΔpCa50) is twofold greater in adult guinea pig muscle preparations (ΔpCa50 being 0.11 units in adult preparations but only 0.05 units in young preparations). Furthermore, adult guinea pig cardiac muscle preparations display greater SL-dependent changes than young muscle preparations in (1) the magnitude of length-mediated increase in the recruitment of new force-bearing XBs, (2) XB detachment rate, (3) XB strain-mediated effects on other force-bearing XBs, and (4) the rate constant of force redevelopment. Our findings suggest that increased β-MHC expression enhances length-dependent activation in the adult guinea pig cardiac myocardium.

Regulatory light chain phosphorylation augments length-dependent contraction in PTU-treated rats.

Force production by actin-myosin cross-bridges in cardiac muscle is regulated by thin-filament proteins and sarcomere length (SL) throughout the heartbeat. Prior work has shown that myosin regulatory light chain (RLC), which binds to the neck of myosin heavy chain, increases cardiac contractility when phosphorylated. We recently showed that cross-bridge kinetics slow with increasing SLs, and that RLC phosphorylation amplifies this effect, using skinned rat myocardial strips predominantly composed of the faster α-cardiac myosin heavy chain isoform. In the present study, to assess how RLC phosphorylation influences length-dependent myosin function as myosin motor speed varies, we used a propylthiouracil (PTU) diet to induce >95% expression of the slower β-myosin heavy chain isoform in rat cardiac ventricles. We measured the effect of RLC phosphorylation on Ca2+-activated isometric contraction and myosin cross-bridge kinetics (via stochastic length perturbation analysis) in skinned rat papillary muscle strips at 1.9- and 2.2-µm SL. Maximum tension and Ca2+ sensitivity increased with SL, and RLC phosphorylation augmented this response at 2.2-µm SL. Subtle increases in viscoelastic myocardial stiffness occurred with RLC phosphorylation at 2.2-µm SL, but not at 1.9-µm SL, thereby suggesting that RLC phosphorylation increases β-myosin heavy chain binding or stiffness at longer SLs. The cross-bridge detachment rate slowed as SL increased, providing a potential mechanism for prolonged cross-bridge attachment to augment length-dependent activation of contraction at longer SLs. Length-dependent slowing of β-myosin heavy chain detachment rate was not affected by RLC phosphorylation. Together with our previous studies, these data suggest that both α- and β-myosin heavy chain isoforms show a length-dependent activation response and prolonged myosin attachment as SL increases in rat myocardial strips, and that RLC phosphorylation augments length-dependent activation at longer SLs. In comparing cardiac isoforms, however, we found that β-myosin heavy chain consistently showed greater length-dependent sensitivity than α-myosin heavy chain. Our work suggests that RLC phosphorylation is a vital contributor to the regulation of myocardial contractility in both cardiac myosin heavy chain isoforms.

Sarcomere length-dependent effects on Ca2+-troponin regulation in myocardium expressing compliant titin.

Cardiac performance is tightly regulated at the cardiomyocyte level by sarcomere length, such that increases in sarcomere length lead to sharply enhanced force generation at the same Ca2+ concentration. Length-dependent activation of myofilaments involves dynamic and complex interactions between a multitude of thick- and thin-filament components. Among these components, troponin, myosin, and the giant protein titin are likely to be key players, but the mechanism by which these proteins are functionally linked has been elusive. Here, we investigate this link in the mouse myocardium using in situ FRET techniques. Our objective was to monitor how length-dependent Ca2+-induced conformational changes in the N domain of cardiac troponin C (cTnC) are modulated by myosin-actin cross-bridge (XB) interactions and increased titin compliance. We reconstitute FRET donor- and acceptor-modified cTnC(13C/51C)AEDANS-DDPM into chemically skinned myocardial fibers from wild-type and RBM20-deletion mice. The Ca2+-induced conformational changes in cTnC are quantified and characterized using time-resolved FRET measurements as XB state and sarcomere length are varied. The RBM20-deficient mouse expresses a more compliant N2BA titin isoform, leading to reduced passive tension in the myocardium. This provides a molecular tool to investigate how altered titin-based passive tension affects Ca2+-troponin regulation in response to mechanical stretch. In wild-type myocardium, we observe a direct association of sarcomere length-dependent enhancement of troponin regulation with both Ca2+ activation and strongly bound XB states. In comparison, measurements from titin RBM20-deficient animals show blunted sarcomere length-dependent effects. These results suggest that titin-based passive tension contributes to sarcomere length-dependent Ca2+-troponin regulation. We also conclude that strong XB binding plays an important role in linking the modulatory effect of titin compliance to Ca2+-troponin regulation of the myocardium.

The allosteric activation of cGAS underpins its dynamic signaling landscape.

Cyclic G/AMP synthase (cGAS) initiates type-1 interferon responses against cytosolic double-stranded (ds)DNA, which range from antiviral gene expression to apoptosis. The mechanism by which cGAS shapes this diverse signaling landscape remains poorly defined. We find that substrate-binding and dsDNA length-dependent binding are coupled to the intrinsic dimerization equilibrium of cGAS, with its N-terminal domain potentiating dimerization. Notably, increasing the dimeric fraction by raising cGAS and substrate concentrations diminishes duplex length-dependent activation, but does not negate the requirement for dsDNA. These results demonstrate that reaction context dictates the duplex length dependence, reconciling competing claims on the role of dsDNA length in cGAS activation. Overall, our study reveals how ligand-mediated allostery positions cGAS in standby, ready to tune its signaling pathway in a switch-like fashion.

Cardiac-specific knockout of Lmod2 results in a severe reduction in myofilament force production and rapid cardiac failure

Leiomodin-2 (Lmod2) is a striated muscle-specific actin binding protein that is implicated in assembly of thin filaments. The necessity of Lmod2 in the adult mouse and role it plays in the mechanics of contraction are unknown. To answer these questions, we generated cardiac-specific conditional Lmod2 knockout mice (cKO). These mice die within a week of induction of the knockout with severe left ventricular systolic dysfunction and little change in cardiac morphology. Cardiac trabeculae isolated from cKO mice have a significant decrease in maximum force production and a blunting of myofilament length-dependent activation. Thin filaments are non-uniform and substantially reduced in length in cKO hearts, affecting the functional overlap of the thick and thin filaments. Remarkably, we also found that Lmod2 levels are directly linked to thin filament length and cardiac function in vivo, with a low amount (<20%) of Lmod2 necessary to maintain cardiac function. Thus, Lmod2 plays an essential role in maintaining proper cardiac thin filament length in adult mice, which in turn is necessary for proper generation of contractile force. Dysregulation of thin filament length in the absence of Lmod2 contributes to heart failure.

Abstract 439: Elevating 2-deoxy Adenosine Triphosphate Improves Contraction in a Mouse Model of Genetic Dilated Cardiomyopathy

Several studies have identified reduced tension and Ca 2+ sensitivity as key regulators that precipitate dilated cardiomyopathy (DCM). We are developing a novel therapy to increase tension using adeno-associated viral (AAV) vectors that increase intracellular 2 deoxy-ATP (dATP) via overexpression of the enzyme ribonucleotide reductase (RNR). We have reported that dATP substitution for ATP increases the magnitude and rate of contraction and that RNR overexpression increases intracellular dATP and enhances cardiac function. Here we use a multiscale approach to test the effect of dATP in a genetic model of dilated cardiomyopathy (DCM) with a missense mutation (D230N) in alpha-tropomyosin (Tm). Using recombinant proteins in the in vitro motility assay, we found that filament velocity was lower with D230N compared to WT Tm (2.03±0.04 vs. 2.44±0.04 μm/s). This deficit was corrected when dATP was substituted for ATP (2.90±0.04 μm/s). Next, we used transgenic D230N mice that show progressive ventricular dilation and systolic dysfunction. Intact and demembranated trabeculae from these mice have decreased tension and calcium sensitivity of force, and loss of length-dependent activation (LDA) compared to WT mice. Substitution of dATP for ATP, increased Ca 2+ sensitivity in D230N Tm myocardium (pCa 50 =5.48±0.02 vs. 5.57±0.02) and restored LDA. Complete replacement of ATP with dATP resulted in an ~70% increase in force at submaximal [Ca 2+ ] and force at submaximal Ca 2+ was significantly increased by replacement of only 5% of the nucleotide pool with dATP. For isolated myofibrils from D230N Tm and WT mice, tension at submaximal Ca 2+ (pCa=5.6) was lower in D230N mice compared to WT mice (51±4 vs. 75±8 mN/mm 2 ) and this deficit was improved with dATP (68±5 mN/mm 2 ). Similarly, the rate of activation in D230N myofibrils was slower than in WT mice (2.1±0.2 vs.3.3±0.2 s -1 ) and partially corrected with dATP (2.7±0.2 s -1 ). These results demonstrate that enhanced cross bridge binding, via increasing dATP, can recover lost contractile function in a thin filament mutation model of DCM. In ongoing studies, we are testing the ability of elevated dATP, via AAV- RNR, to protect against reduced systolic function and progressive ventricular dilation in 2-3 week old D230N mice .

Force-Dependent Recruitment from the Myosin Off State Contributes to Length-Dependent Activation

Cardiac muscle develops more force when it is activated at longer lengths. The concentration of Ca2+ required to develop half-maximal force also decreases. These effects are known as length-dependent activation and are thought to play critical roles in the Frank-Starling relationship and cardiovascular homeostasis. The molecular mechanisms underpinning length-dependent activation remain unclear, but recent experiments suggest that they may include recruitment of myosin heads from the off (sometimes called super-relaxed) state. This manuscript presents a mathematical model of muscle contraction that was developed to investigate this hypothesis. Myosin heads in the model transitioned between an off state (that could not interact with actin), an on state (that could bind to actin), and a single attached state. Simulations were fitted to experimental data using multidimensional parameter optimization. Statistical analysis showed that a model in which the rate of the off-to-on transition increased linearly with force reproduced the length-dependent behavior of chemically permeabilized myocardium better than a model with a constant off-to-on transition rate (F-test, p < 0.001). This result suggests that the thick-filament transitions are modulated by force. Additional calculations showed that the model incorporating a mechanosensitive thick filament could also reproduce twitch responses measured in a trabecula stretched to different lengths. A final set of simulations was then used to test the model. These calculations predicted how reducing passive stiffness would impact the length dependence of the calcium sensitivity of contractile force. The prediction (a 60% reduction in ΔpCa50) mimicked the 58% reduction in ΔpCa50 in myocardium from rats that expressed a giant isoform of titin and had low resting tension. Together, these computational results suggest that force-dependent recruitment of myosin heads from the thick-filament off state contributes to length-dependent activation and the Frank-Starling relationship.

Etiology-dependent impairment of relaxation kinetics in right ventricular end-stage failing human myocardium

BackgroundIn patients with end-stage heart failure, the primary etiology often originates in the left ventricle, and eventually the contractile function of the right ventricle (RV) also becomes compromised. RV tissue-level deficits in contractile force and/or kinetics need quantification to understand involvement in ischemic and non-ischemic failing human myocardium. Methods and resultsThe human population suffering from heart failure is diverse, requiring many subjects to be studied in order to perform an adequately powered statistical analysis. From 2009-present we assessed live tissue-level contractile force and kinetics in isolated myocardial RV trabeculae from 44 non-failing and 41 failing human hearts. At 1 Hz stimulation rate (in vivo resting state) the developed active force was not different in non-failing compared to failing ischemic nor non-ischemic failing trabeculae. In sharp contrast, the kinetics of relaxation were significantly impacted by disease, with 50% relaxation time being significantly shorter in non-failing vs. non-ischemic failing, while the latter was still significantly shorter than ischemic failing. Gender did not significantly impact kinetics. Length-dependent activation was not impacted. Although baseline force was not impacted, contractile reserve was critically blunted. The force-frequency relation was positive in non-failing myocardium, but negative in both ischemic and non-ischemic myocardium, while the β-adrenergic response to isoproterenol was depressed in both pathologies. ConclusionsForce development at resting heart rate is not impacted by cardiac pathology, but kinetics are impaired and the magnitude of the impairment depends on the underlying etiology. Focusing on restoration of myocardial kinetics will likely have greater therapeutic potential than targeting force of contraction.

Cardiomyopathy mutation (F88L) in troponin T abolishes length dependency of myofilament Ca2+ sensitivity.

Recent clinical studies have revealed a new hypertrophic cardiomyopathy-associated mutation (F87L) in the central region of human cardiac troponin T (TnT). However, despite its implication in several incidences of sudden cardiac death in young and old adults, whether F87L is associated with cardiac contractile dysfunction is unknown. Because the central region of TnT is important for modulating the muscle length-mediated recruitment of new force-bearing cross-bridges (XBs), we hypothesize that the F87L mutation causes molecular changes that are linked to the length-dependent activation of cardiac myofilaments. Length-dependent activation is important because it contributes significantly to the Frank-Starling mechanism, which enables the heart to vary stroke volume as a function of changes in venous return. We measured steady-state and dynamic contractile parameters in detergent-skinned guinea pig cardiac muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or the guinea pig analogue (TnTF88L) of the human mutation at two different sarcomere lengths (SLs): short (1.9 µm) and long (2.3 µm). TnTF88L increases pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at short SL than at long SL; for example, pCa50 increases by 0.25 pCa units at short SL and 0.17 pCa units at long SL. The greater increase in pCa50 at short SL leads to the abolishment of the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTF88L fibers, ΔpCa50 being 0.10 units in TnTWT fibers but only 0.02 units in TnTF88L fibers. Furthermore, at short SL, TnTF88L attenuates the negative impact of strained XBs on force-bearing XBs and augments the magnitude of muscle length-mediated recruitment of new force-bearing XBs. Our findings suggest that the TnTF88L-mediated effects on cardiac thin filaments may lead to a negative impact on the Frank-Starling mechanism.

Deficiency of Length-Dependent Activation of Contraction in the Cardiac Muscle of Rats with Heart Failure: Assessment of the Muscle Strip and Single Cell Levels

This paper reports on a comparison of the extent of length-dependent activation of contraction in the right ventricle myocardium in healthy rats and rats with monocrotaline-induced heart failure on two levels of heart-tissue organization, that is, muscle stripes and isolated cardiomyocytes, within the framework of a single study. It has been shown that a deficiency in the length-dependent increase in the contractile force produced by failing myocardium when expressed in quantitative terms is similar at both levels of organization of myocardial tissue. These findings indicate that the mechanisms of length-dependent regulation of myocardial contractility in the failing heart are suppressed mainly at the cellular level. In muscle strips, the deficiency of the length–tension relationship appears to be more pronounced, most likely because the spatial organization of myocytes affects the integral contractile response of the muscle.

Role of the C-terminus mobile domain of cardiac troponin I in the regulation of thin filament activation in skinned papillary muscle strips

The C-terminus mobile domain of cTnI (cTnI-MD) is a highly conserved region which stabilizes the actin-cTnI interaction during the diastole. Upon Ca2+-binding to cTnC, cTnI-MD participates in a regulatory switching that involves cTnI to switch from interacting with actin toward interacting with the Ca2+-regulatory domain of cTnC. Despite many studies targeting the cTnI-MD, the role of this region in the length-dependent activation of cardiac contractility is yet to be determined. The present study investigated the functional consequences of losing the entire cTnI-MD in cTnI(1–167) truncation mutant, as it was exchanged for endogenous cTnI in skinned rat papillary muscle fibers. The influence of cTnI-MD truncation on the extent of the N-domain of cTnC hydrophobic cleft opening and the steady-state force as a function of sarcomere length (SL), cross-bridge state, and [Ca2+] was assessed using the simultaneous in situ time-resolved FRET and force measurements at short (1.8 μm) and long (2.2 μm) SLs. Our results show the significant role of cTnI-MD in the length dependent thin filament activation and the coupling between thin and thick filament regulations affected by SL. Our results also suggest that cTnI-MD transmits the effects of SL change to the core of troponin complex.

Hypertrophic cardiomyopathy mutation R58Q in the myosin regulatory light chain perturbs thick filament-based regulation in cardiac muscle

Hypertrophic cardiomyopathy (HCM) is frequently linked to mutations in the protein components of the myosin-containing thick filaments leading to contractile dysfunction and ultimately heart failure. However, the molecular structure-function relationships that underlie these pathological effects remain largely obscure. Here we chose an example mutation (R58Q) in the myosin regulatory light chain (RLC) that is associated with a severe HCM phenotype and combined the results from a wide range of in vitro and in situ structural and functional studies on isolated protein components, myofibrils and ventricular trabeculae to create an extensive map of structure-function relationships. The results can be understood in terms of a unifying hypothesis that illuminates both the effects of the mutation and physiological signaling pathways. R58Q promotes an OFF state of the thick filaments that reduces the number of myosin head domains that are available for actin interaction and ATP utilization. Moreover this mutation uncouples two aspects of length-dependent activation (LDA), the cellular basis of the Frank-Starling relation that couples cardiac output to venous return; R58Q reduces maximum calcium-activated force with no significant effect on myofilament calcium sensitivity. Finally, phosphorylation of R58Q-RLC to levels that may be relevant both physiologically and pathologically restores the regulatory state of the thick filament and the effect of sarcomere length on maximum calcium-activated force and thick filament structure, as well as increasing calcium sensitivity. We conclude that perturbation of thick filament-based regulation may be a common mechanism in the etiology of missense mutation-associated HCM, and that this signaling pathway offers a promising target for the development of novel therapeutics.

Length-Dependent Activation is Reduced in Myocardium from Patients with Non-ischemic Heart Failure

Heart failure contributes to 1 in 9 deaths in the United States and often reflects impaired cellular-level contractile function. In healthy hearts, the myofilaments become more sensitive to Ca2+ as the myocardium is stretched. This effect is known as length-dependent activation and is the cellular-level basis of the Frank-Starling mechanism. Few studies have measured the magnitude of length-dependent activation in myocardium from failing human hearts. Multicellular preparations from the left ventricular free wall were chemically permeabilized and Ca2+-activated at sarcomere lengths of 1.9 and 2.3 µm, at physiological temperature (37°C). 12 hearts were explanted from patients receiving cardiac transplants; 6 patients had ischemic heart failure and 6 had non-ischemic heart failure. Another 6 hearts were obtained from organ donors. Calcium-sensitivity of contraction (defined as the pCa50 value) increased by 0.07 pCa units as the preparations were stretched from 1.9 to 2.3 µm in samples from organ donors (p<0.001) and ischemic failing hearts (p=0.003). Calcium-sensitivity did not change with sarcomere length in preparations from non-ischemic failing hearts. These data show for the first time that length-dependent activation of contraction is preserved in myocardium from patients with ischemic heart failure, but eliminated in myocardium from patients with non-ischemic heart failure. Because length-dependent activation is an important component of the Frank-Starling mechanism, patients who have non-ischemic disease may exhibit a diminished contractile response to increased ventricular filling. Different therapeutic strategies may be more effective in patients with ischemic and non-ischemic heart failure.

Pre-Activation of Cardiomyocytes Determines Contractile Force and Speed of Contraction; Role of Titin and Calcium

Cardiomyocytes have, through splicing, an exquisite control over the length of titin, enabling it to regulate passive stiffness. As titin sets the preload of the cardiomyocyte, we hypothesized that together with diastolic Ca2+ it pre-activates the cardiomyocyte during diastole and that this is a major determinant of force production in the subsequent systolic phase. Through this mechanism titin would play an important role in active force development and length-dependent activation. Mutations in the splicing factor RNA binding motif protein (RBM20) results in the expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work-loops that mimic the cardiac cycle in wildtype (WT) and heterozygous (HET) RBM20 deficient rats, with long compliant titin. In addition we studied membrane-permeabilized cardiomyocytes with different amounts of diastolic calcium. At low pacing frequencies, myocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT), but this difference disappeared when diastolic calcium increased at high pacing frequencies. HET myocytes operated at higher SL to achieve the same level of work (2.10 µm vs. 1.94 µm at 6 Hz). To delineate the effects of diastolic Ca2+ and titin pre-activation on force generation, measurements were performed in detergent-permeabilized cardiomyocytes isolated from rat hearts. In these cells cardiac twitches were simulated by transiently (1 s) exposing the cell to a Ca2+-concentration of 2 µM. Increasing pre-activation by pre-stretching the myocyte increased the kinetics of force development and total force development. Increasing diastolic Ca2+ did increase force development, but only within a small concentration range. These findings are consistent with our hypothesis that pre-activation can increase force development. Highly compliant titin allows cells to function at higher diastolic Ca2+, but this is expected to result in limited contractile reserve.

Regulated Model of Steady-State Cardiac Length-Dependent Activation

Recent data suggests length-dependent activation derives from titin interacting with super-relaxed myosin crossbridges on the thick filament. This feature was incorporated into an existing model of crossbridge dynamics (Caremani et al, 2015) by replacing the detached post-ATP-hydrolysis state with an equilibrium mixture of rapidly interacting super-relaxed, detached, and weakly bound crossbridges. The model also included: (1) cooperative activation of the thin filament by both Ca++ and strongly bound crossbridges, (2) loosely coupled interaction that allows Ca++ to dissociate from troponin C (TnC) before strong crossbridges detach, (3) Ca++ bound more strongly to TnC when a strong crossbridge is also bound nearby. As suggested by data (Sun & Irving, 2015), the equilibrium between super-relaxed and detached crossbridges equaled 3:1 for a long sarcomere length. We supposed that the reduced forces exerted by titin on the thick filament at low sarcomere length made it more likely to form a super-relaxed crossbridge. As the force exerted by titin increases, the super-relaxed myosin transitions to an unfettered detached state and becomes available to form weak crossbridges. We altered the Gibbs free energy to form a super-relaxed crossbridge by less than 1 kT as titin's force decreased over sarcomere lengths from 2.25 to 1.9 µm. This caused the maximal steady-state force for isometric contraction at saturating Ca++ to reduce by ∼ 25%, as data suggests (Dobesh et al, 2002). The model thus unites existing data and provides a more quantitative understanding of how titin interacts with the thick filament at different sarcomere lengths. We anticipate that this model insight and more extensive data will lead to deeper understanding of cardiac length-dependent activation. (Supported by the Center for Engineering MechanoBiology through a grant from NSF's STC program (CMMI):15-48571).

Aberrant Cardiac Muscle Mechanics in a Hypertrophic Cardiomyopathy Troponin T ILE79ASN Transgenic Mouse

The missense mutation Ile79Asn in human cardiac troponin T (HcTnT-I79N) has been associated with phenotypic outcomes of hypertrophic cardiomyopathy (HCM), arrhythmias, and sudden cardiac death. Previous characterization of an HcTnT-I79N transgenic mouse, which expresses 52% of the mutant protein, demonstrated increased Ca2+-sensitivity of isometric force. Little else is known about any changes in cardiac muscle mechanics caused by the mutation. This study investigates cross-bridge kinetics of skinned papillary muscle bundles (isometric force, sinusoidal stiffness and rate of tension redevelopment, kTR), post-translational modifications (PTM) in sarcomeric proteins, and in vivo cardiac function. For fiber mechanics and PTM analysis, cardiac muscle was isolated from the left ventricle of non-transgenic wild-type (WT) mice or transgenic mice bearing the HcTnT-I79N mutation. Sarcomere length (SL) was set at pCa8 using HeNe laser diffraction after the attachment points of each skinned fiber bundle were fixed with glutaraldehyde to minimize end compliance. Sinusoidal stiffness (0.2% PTP length oscillation) and kTR were obtained after recording steady-state isometric tension. As previously reported, the HcTnT-I79N mutation led to increased Ca2+-sensitivity of isometric force. Length-dependent activation at the longer SL (2.1 vs 1.9 µm) resulted in increased Ca2+-sensitivity of isometric force and sinusoidal stiffness and slower kTR for both WT and HcTnT-I79N. However, HcTnT-I79N exhibited slightly faster kTR and greater sinusoidal stiffness at sub-maximum Ca2+-activation (∼40% of maximal isometric force) compared to WT regardless of SL. These data indicate that the HCM HcTnT-I79N mutation, and SL, markedly influence the dynamics of individual regulatory units as well as the kinetics of cross-bridge cycling. Next steps include echocardiographic, sarcomeric protein phosphorylation and further fiber mechanic analyses. NIH-HL128683.

Kinetic Implications from a Model of Cardiac Length-dependent Activation

Kinetic Implications from a Model of Cardiac Length-dependent Activation

Functional significance of C-terminal mobile domain of cardiac troponin I

Ca2+-regulation of cardiac contractility is mediated through the troponin complex, which comprises three subunits: cTnC, cTnI, and cTnT. As intracellular [Ca2+] increases, cTnI reduces its binding interactions with actin to primarily interact with cTnC, thereby enabling contraction. A portion of this regulatory switching involves the mobile domain of cTnI (cTnI-MD), the role of which in muscle contractility is still elusive. To study the functional significance of cTnI-MD, we engineered two cTnI constructs in which the MD was truncated to various extents: cTnI(1–167) and cTnI(1–193). These truncations were exchanged for endogenous cTnI in skinned rat papillary muscle fibers, and their influence on Ca2+-activated contraction and cross-bridge cycling kinetics was assessed at short (1.9 μm) and long (2.2 μm) sarcomere lengths (SLs). Our results show that the cTnI(1–167) truncation diminished the SL-induced increase in Ca2+-sensitivity of contraction, but not the SL-dependent increase in maximal tension, suggesting an uncoupling between the thin and thick filament contributions to length dependent activation. Compared to cTnI(WT), both truncations displayed greater Ca2+-sensitivity and faster cross-bridge attachment rates at both SLs. Furthermore, cTnI(1–167) slowed MgADP release rate and enhanced cross-bridge binding. Our findings imply that cTnI-MD truncations affect the blocked-to closed-state transition(s) and destabilize the closed-state position of tropomyosin.

Myofilament Remodeling and Function Is More Impaired in Peripartum Cardiomyopathy Compared with Dilated Cardiomyopathy and Ischemic Heart Disease

Peripartum cardiomyopathy (PPCM) and dilated cardiomyopathy (DCM) show similarities in clinical presentation. However, although DCM patients do not recover and slowly deteriorate further, PPCM patients show either a fast cardiac deterioration or complete recovery. The aim of this study was to assess if underlying cellular changes can explain the clinical similarities and differences in the two diseases. We, therefore, assessed sarcomeric protein expression, modification, titin isoform shift, and contractile behavior of cardiomyocytes in heart tissue of PPCM and DCM patients and compared these with nonfailing controls. Heart samples from ischemic heart disease (ISHD) patients served as heart failure control samples. Passive force was only increased in PPCM samples compared with controls, whereas PPCM, DCM, and ISHD samples all showed increased myofilament Ca2+ sensitivity. Length-dependent activation was significantly impaired in PPCM compared with controls, no impairment was observed in ISHD samples, and DCM samples showed an intermediate response. Contractile impairments were caused by impaired protein kinase A (PKA)-mediated phosphorylation because exogenous PKA restored all parameters to control levels. Although DCM samples showed reexpression of EH-myomesin, an isoform usually only expressed in the heart before birth, PPCM and ISHD did not. The lack of EH-myomesin, combined with low PKA-mediated phosphorylation of myofilament proteins and increased compliant titin isoform, may explain the increase in passive force and blunted length-dependent activation of myofilaments in PPCM samples.