Rate Of Tension Redevelopment Research Articles

Abnormal kinetics of contraction caused by an α-actinin 2 missense variant in the EF-3-4 hand domain in human myocardium.

ACTN2 gene encodes α-actinin 2 protein, which binds actin. In cardiac muscle, α-actinin 2 is located in the Z-disc of the sarcomere, where it anchors myofibrillar actin filaments. A 48-years old female presented with out-of-hospital ventricular fibrillation arrest. Echocardiogram showed stage II diastolic dysfunction, elevated RVSP, and abnormal global longitudinal strain consistent with early LV systolic dysfunction. Cardiopulmonary testing indicated AHA functional class IV HF. Genetic testing identified a missense heterozygous variant of unknown significance in ACTN2 (gene previously associated with HCM and DCM), and SCNB2 (β-2 subunit of type II voltage-gated sodium channel). Histopathology demonstrated mild interstitial fibrosis of the LV free wall. We investigated the effects of the heterozygous A868T variant in α-actinin 2 on cardiac muscle mechanics. LV free wall samples were obtained from the patient's explanted heart at the time of cardiac transplantation. Permeabilized cardiac muscle preparations (CMPs) containing A868T and WT proteins displayed increased myofilament Ca2+-sensitivity of isometric force and lower maximal isometric specific force when compared to human donor samples. Sinusoidal stiffness was decreased at all levels of Ca2+-activation in A8686T CMPs. The rate of tension redevelopment (kTR) was faster at all levels of Ca2+-activation in A8686T CMPs. Computational modeling suggests that faster kTR in A868T CMPs is mainly due to faster cross-bridge detachment (g). The kinetic changes are consistent with decreased force generation by A868T CMPs being due to abnormalities in the cross-bridge interaction. Molecular dynamics simulation did not find significant differences in the interaction of A868T variant with titin and α-actinin. This project establishes the importance of α-actinin 2 variant in the Z-disc mechanosensitive interaction of thin- and thick-filament distal from its structural role in the sarcomere.

Abstract P432: Alpha-actinin 2 Missense Variant And Its Role In Cardiac Muscle Force Production And Diastolic Dysfunction

ACTN2 gene encodes for the alpha-actinin 2 protein, which has an actin-binding role. In cardiac muscle, alpha-actinin 2 is located in the Z-disc of the sarcomere, where it anchors myofibrillar actin filaments. A 48-years old female patient presented with ventricular fibrillation arrest was successfully resuscitated. Coronary angiography exhibited normal coronary arteries. Cardiac MRI did not show myocardial scar tissue or arrhythmogenic right ventricular dysplasia (ARVD). Echocardiogram showed normal left ventricular ejection fraction, stage II diastolic dysfunction and elevated right ventricular systolic pressure. Genetic testing identified a missense variant of unknown significance in ACTN2 (a gene previously associated with dilated cardiomyopathy/ARVD), and in SCNB2 (encodes sodium voltage-gated channel beta subunit 2, a gene previously associated with long QT syndrome). We investigated the effects of the ACTN2 variant on cardiac muscle contraction, providing an inside view of the importance of ACTN2 in sarcomere function. Left ventricular free wall samples were obtained from the patient’s explanted heart at the time of cardiac transplantation. Mechanics of contraction were measured in permeabilized cardiac muscle preparations (CMPs) in the presence of 3% dextran T-500 (to restore the myofilament lattice to physiological dimensions) at 30°C. The experiments were performed at sarcomere lengths 2.1 μm and 2.3 μm achieved by HeNe laser diffraction. CMPs containing ACTN2 mutant protein displayed increased myofilament Ca 2+ sensitivity of isometric force and lower maximal isometric force compared to human donor samples (control). Sinusoidal stiffness was decreased at all levels of Ca 2+ activation and isometric force in ACTN2 variant CMPs. The rate of tension redevelopment was faster at all levels of Ca 2+ activation and isometric force. Alpha-actinin 2 is an important sarcomeric protein that regulates the kinetics of cardiac muscle contraction. This work establishes the importance of alpha-actinin 2 in the Z-disc mechanotransduction to thin and thick filament interactions.

Abstract P417: Cardiomyopathy-associated Variant In Troponin T Tail Domain Promotes Disruption Of Both Frank-starling Mechanism And Cardiac Myofilament Performance

Missense variant Ile79Asn in human cardiac troponin T (HcTnT-I79N) tail region has been linked to familial hypertrophic cardiomyopathy (HCM), arrhythmia, and sudden cardiac death. It has been reported that inotropic stimulation with high extracellular Ca 2+ or isoproterenol led to diastolic dysfunction in both isolated and in vivo HcTnT-I79N mice hearts. Although HcTnT-I79N effects are acknowledged to be dependent on the inotropic state of the cardiac muscle, little is known about how this pathogenic variant affects the Frank-Starling law of the heart. To further investigate the functional and structural consequences of this deadly variant in a stretch-dependent manner, cardiac tissues were harvested from non-transgenic (NTg) control mice and transgenic mice bearing HcTnT-I79N. Left ventricular papillary muscle bundles were permeabilized and mounted for mechanical measurements. Sarcomere length (SL 1.9, 2.1 or 2.3 μm) was set at pCa 8 using HeNe laser diffraction and then Ca 2+ -dependence of isometric force, sinusoidal stiffness (SS, 0.2% PTP length oscillation) and rate of tension redevelopment ( k TR ) were measured. We observed that HcTnT-I79N tissue exhibited increased myofilament Ca 2+ -sensitivity of force, increased SS, slower k TR at all levels of Ca 2+ -activation, and diminished length-dependent activation (LDA). Small-angle X-ray diffraction revealed that HcTnT-I79N permeabilized cardiac muscles exhibit smaller myofilament lattice spacing at longer SLs (2.1 μm and 2.3 μm) compared to NTg. Using 3% Dextran T500 to osmotically compress the myofilament lattice (SL 2.1 μm), HcTnT-I79N showed no change in myofilament lattice spacing and little change in contractile indices associated with LDA. Interestingly, upon osmotic compression, HcTnT-I79N displayed a decrease in disordered relaxed state (DRX, ON state) of myosin and an increase in super-relaxed state (SRX, OFF state) of myosin. We conclude that altered cardiac myofilament performance, lack of responsiveness to osmotic compression, and reduced LDA observed with HcTnT-I79N are partially due to a combination of smaller myofilament lattice and disturbed ON and OFF states of myosin.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N′)-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N′-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N′-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

High efficiency preparation of skinned mouse cardiac muscle strips from cryosections for contractility studies.

What is the central question of this study? Can frozen cardiac papillary muscles and cryosectioning be used to reliably obtain uniform cardiac muscle strips with high yields? What is the main finding and its importance? A new method was developed using frozen cardiac papillary muscles and cryosectioning to reliably obtain uniform cardiac muscle strips with high yields. Experimental results demonstrate that this new methodology significantly increases the efficiency and application of quantitative biomechanical studies using skinned muscle fibres with an additional advantage of no need for transferring live animals. Skinned cardiac muscle preparations are widely used to study contractile function of myofilament proteins and pathophysiological changes. The current methods applied in these biomechanical studies include detergent permeabilization of freshly isolated papillary muscle, ventricular trabeculae, surgically dissected ventricular muscle strips, mechanically blended cardiac muscle bundles or myocytes, and enzymatically isolated single cardiomyocytes. To facilitate and expand the skinned cardiac muscle approach, we have developed an efficient and readily practical method for mechanical studies of skinned mouse cardiac papillary muscle strips prepared from cryosections. Longitudinal papillary muscle strips of 120-150µm width cut from 35-70µm-thick cryosections are mounted to a force transducer and chemically skinned for the studies of force-pCa and sarcomere length-tension relationship and rate of tension redevelopment. In addition to more effective skinning and perfusion than with whole papillary muscle and much higher yield of useful preparations than that from trabeculae, this new methodology has two more major advantages. One is to allow for the use of frozen cardiac muscle in storage to maximize the value of muscle samples, facilitating resource sharing among research institutions without the need of transferring live animals or fresh biopsies. The other is that the integrity of the muscle strips is well preserved during the preparation and mechanical studies, allowing coupled characterization of myofilament proteins. The combined power of biomechanics and protein biochemistry can provide novel insights into integrative physiological and pathophysiological mechanisms of cardiac muscle contraction while the high yield of high-quality muscle strips also provides an efficient platform for development of therapeutic reagents.

Osmotic Compression Influences Cross-Bridge Detachment Rate in Transgenic Hypertrophic Cardiomyopathy Variant Hctnt-I79N and Non-Transgenic Mouse Cardiac Muscle

Missense variant Ile79Asn in human cardiac troponin T (HcTnT-I79N) has been linked to adverse phenotypic outcomes of hypertrophic cardiomyopathy (HCM) including arrhythmia and sudden cardiac death. This study investigates cross-bridge kinetics of left ventricular papillary muscle bundles (Ca2+-sensitivity of isometric force, sinusoidal stiffness and rate of tension redevelopment (kTR)) in a (1) length-dependent activation manner and (2) under osmotic compression. Cardiac tissues were harvested from both non-transgenic wild-type (NTg) mice and transgenic mice bearing the HcTnT-I79N variant. Sarcomere lengths (1.9, 2.1 or 2.3 μm) were set at pCa 8.0 using HeNe laser diffraction. Sinusoidal stiffness (0.2% PTP length oscillation) and kTR were obtained during Ca2+ activation when force was at least 20% of maximum. Increased Ca2+-sensitivity of isometric force and maximum force were observed in NTg cardiac preparations upon an increase in SL (1.9 vs 2.1 μm, and 2.1 vs 2.3 μm). Interestingly, HcTnT-I79N exhibited increased Ca2+-sensitivity and maximum force at SL 1.9 vs 2.1 μm, while only maximum force increased upon further stretch (SL 2.1 vs 2.3 μm). This blunted effect at SL 2.1 vs 2.3 μm cannot be explained by differences in thin filament length in HcTnT-I79N vs NTg. Although HcTnT-I79N exhibited significantly lower maximum kTR, neither NTg nor HcTnT-I79N showed significant changes in maximum kTR over the entire range of SL (1.9-2.3 μm). After osmotically compressing the myofilament lattice using 3% Dextran T500 (SL 2.1 μm), (1) only NTg cardiac preparations displayed increased myofilament Ca2+-sensitivity; (2) kTR increased at most activation levels for both NTg and HcTnTI79N; (3) no changes in maximum kTR were observed; (4) both NTG and HcTnT-I79N displayed increased cross-bridge detachment rates (g); (5) HcTnT-I79N showed slower g when compared to NTG with and without Dextran. NIH-HL128683.

Annulment of Cardiac Muscle Length-Dependent Force Activation in Transgenic Mice Bearing the HcTnT-I79N Mutation

Missense mutation Ile79Asn in human cardiac troponin T (HcTnT-I79N) has been linked to phenotypic outcomes of hypertrophic cardiomyopathy (HCM) followed by adverse consequences including arrhythmias and sudden cardiac death. Little else is known about changes in cardiac muscle mechanics caused by the mutation. This study investigates cross-bridges kinetics of left ventricular papillary muscle bundles (Ca2+-sensitivity of isometric force, sinusoidal stiffness and rate of tension redevelopment (kTR)). Cardiac tissues were harvested from both non-transgenic wild-type (NTg) mice and transgenic mice bearing the HcTnT-I79N mutation. Sarcomere lengths (1.9, 2.1, and 2.3 μm) were set at pCa 8.0 using HeNe laser. Sinusoidal stiffness (0.2% PTP length oscillation) and kTR were obtained during Ca2+ activation when force was at least 20% of maximum. Increased Ca2+-sensitivity of isometric force was observed in NTg cardiac preparations upon an increase in SL (1.9 vs 2.1 μm, and 2.1 vs 2.3 μm). Interestingly, HcTnT-I79N exhibited an increased Ca2+-sensitivity at SL 1.9 vs 2.1 μm only. No further increase was observed at SL 2.1 vs 2.3 μm. This blunt effect indicates an abolishment of length-dependent activation at SL 2.1 vs 2.3 μm. Although HcTnT-I79N mutation exhibited a significant lower maximal kTR, neither NTg nor HcTnT-I79N showed significant changes in maximal kTR regarding length-dependent activation. After compressing the myofilament lattice spacing using 3% Dextran T500 (SL 2.1 μm), only NTg cardiac preparations displayed increased myofilament Ca2+ sensitivity. No changes in maximal kTR were observed for both NTg and HcTnT-I79N in the presence of dextran. In addition, no significant changes in passive force, maximal force and cooperativity of thin filament activation were observed between groups. Experiments were carried out at ∼21°C. Analysis of mathematical modeling of force and sinusoidal stiffness are in progress. NIH-HL128683.

Predicting Effects of Tropomyosin Stiffness on Cardiac Muscle Contraction Using Coarse-Grained Stochastic Modeling

During the cardiac thin filament activation process, Tropomyosin (Tm) oscillates over the surface of actin filament in the azimuthal direction to regulate the access of Myosin heads to actin binding sites. These dynamical modes of oscillation and the resultant cooperative activation effects depend critically on the stiffness characteristics and the mobility of Tm molecules. In this study, we developed a stochastic coarse-grained computational model to describe the Tm motions over the surface of actin filament using Langevin-Brownian dynamics. The model represents the structural arrangement of Tm molecules as a flexible chain with variable torsional stiffness using a crystal elastic network approach. The model accounts for the spatial interactions among nearest-neighbor regulatory units (RUs), which are thought to appear from the structural coupling of adjacent Tropomyosins. This elastic coupling between RUs is accomplished by assigning a multi-well potential energy for each RU. The model is then used to study the effects of Tm torsional stiffness variations on the cooperative activation of the thin filament. The results suggest that small perturbations in Tm torsional stiffness can lead to a significant effect on force-Ca2+ sensitivity, the rate of tension redevelopment, relaxation rate, and other contraction characteristics. The present stochastic computational model draws for the first time a more detailed molecular connection between Tm torsional stiffness, Tm modes of oscillations over actin surface, cooperativity among RUs, and dynamic muscle twitches. Thus, this coarse-graining approach may be useful in explaining many cardiomyopathies induced by structural remodeling and stiffening in Tm molecules as a result of point mutations in the human gene TPM1.

Structural and functional impact of troponin C-mediated Ca2+ sensitization on myofilament lattice spacing and cross-bridge mechanics in mouse cardiac muscle

Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca2+ sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca2+-sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca2+ sensitizer. Our findings suggest that both methods of increasing myofilament Ca2+ sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (kTR); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I1,1/I1,0 ratio from X-ray diffraction. Computational modeling suggests that Ca2+ sensitization through this cTnC mutation or bepridil accelerates kTR primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in kTR, we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca2+ activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca2+ sensitization may act through stabilization of cTnC's D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca2+ activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM.

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.

Increased cross-bridge recruitment contributes to transient increase in force generation beyond maximal capacity in human myocardium

Cross-bridge attachment allows force generation to occur, and rate of tension redevelopment (ktr) is a commonly used index of cross-bridge cycling rate. Tension overshoots have been observed briefly after a slack-restretch ktr maneuver in various species of animal models and humans. In this study, we set out to determine the properties of these overshoots and their possible underlying mechanism. Utilizing human cardiac trabeculae, we have found that tension overshoots are temperature-dependent and that they do not occur at resting states. In addition, we have found that myosin cross-bridge cycle is vital to these overshoots as inhibition of the cycle results in the blunting of the overshoots and the magnitude of the overshoots are dependent on the level of myofilament activation. Lastly, we show that the number of cross-bridges transiently increase during tension overshoots. These findings lead us to conclude that tension overshoots are likely due to a transient enhancement of the recruitment of myosin heads into the cross-bridge cycling, regulated by the myocardium, and with potential physiological significance in determining cardiac output. News and noteworthyWe show that isolated human myocardium is capable of transiently increasing its maximal force generation capability by increasing cross-bridge recruitment following slack-restretch maneuver. This process can potentially have important implications and significance in cardiac contraction in vivo.

Engineered Troponins Modulate the Ca2+ Sensitivity of the Failing Human Myocardium

The amount of force generated by cardiac sarcomeres depends in part on the sensitivity of the myofilaments to Ca2+. We tested the hypothesis that force development of the failing human heart can also be increased or decreased by Ca2+ sensitized and/or desensitized troponin (Tn). All procedures were approved by the University of Kentucky Institutional Review Board. Utilizing permeabilized multicellular preparations isolated from the hearts of patients with heart failure, we exchanged the native troponin with Tn containing either human recombinant wild-type subunits, engineered TnCs or phosphomimetic TnIs that increase or decrease myofilament Ca2+ sensitivity. The results show that compared to the wild-type Tn, our Ca2+ sensitizing L48Q TnC and S150D TnI Tn exchanges both increased (p < 0.001) pCa50 from ∼5.48 to ∼5.84 and ∼5.68, respectively. Conversely, the Ca2+ desensitizing D73N TnC and Y26E TnI Tn exchanges both decreased (p < 0.001) pCa50 to ∼5.05 and ∼5.29, respectively. In the presence of saturating Ca2+, the maximal force per cross-sectional area, shortening velocity, rate of tension redevelopment (ktr) and power were all similar for the exchanged Tns suggesting the modified Tns have a negligible direct impact on myosin function (except for Y26E TnI, which slowed ktr by ∼25%). This study demonstrates that the myofilament responsiveness of the failing human heart to Ca2+ can be modulated by engineering either TnC or TnI. These findings open the door to potential new Tn targeted therapies for heart disease.

Abnormal Cardiac Cross-Bridge Kinetics in a Troponin T ILE79ASN Transgenic Mouse Model

The missense mutation Ile79Asn in human cardiac troponin T (HcTnT-I79N) has been associated with phenotypic outcomes of familial hypertrophic cardiomyopathy, arrhythmias, and sudden cardiac death. Previous characterization of a transgenic mouse model bearing the HcTnT-I79N mutation, which expressed 52% HcTnT-I79N of total troponin T, demonstrated increased Ca2+ sensitivity of isometric force. However, little is known about the changes in cross-bridge kinetics, if any, that result from the mutation. This study investigates isometric force, sinusoidal stiffness and rate of tension redevelopment (kTR) of skinned papillary muscle fiber bundles at two sarcomere lengths (1.9 µm and 2.1 µm); 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 and kTR were obtained after recording steady-state isometric tension by oscillating the fiber bundle 0.2% of its relaxed length or by applying a ramped shortening followed by a quick re-stretch to original fiber bundle length, respectively. As previously reported, the HcTnT-I79N mutation resulted in increased Ca2+-sensitivity of isometric force although no difference in maximum force per cross-sectional area was observed. Length-dependent activation at the longer SL resulted in increased Ca2+-sensitivity of isometric force and sinusoidal stiffness, and decreased kTR for both WT and HcTnT-I79N. However, HcTnT-I79N exhibited faster kTR and greater sinusoidal stiffness at sub-maximum pCa (-log[Ca2+]) solutions compared to WT regardless of SL. These data indicate that HcTnT-I79N mutation, and SL, markedly influence not only the dynamics of individual regulatory units, but also the kinetics of cross-bridge cycling.

Insights into length-dependent regulation of cardiac cross-bridge cycling kinetics in human myocardium

Cross-bridge cycling kinetics play an essential role in the heart's ability to contract and relax. The rate of tension redevelopment (ktr) slows down as a muscle length is increased in intact human myocardium. We set out to determine the effect of rapid length step changes and protein kinase A (PKA) and protein kinase C-βII (PKC-βII) inhibitors on the ktr in ultra-thin non-failing and failing human right ventricular trabeculae. After stabilizing the muscle either at L90 (90% of optimal length) or at Lopt (optimal length), we rapidly changed the length to either Lopt or L90 and measured ktr. We report that length-dependent changes in ktr occur very rapidly (in the order of seconds or faster) in both non-failing and failing muscles and that the length at which a muscle had been stabilized prior to the length change does not significantly affect ktr. In addition, at L90 and at Lopt, PKA and PKC-βII inhibitors did not significantly change ktr. Our results reveal that length-dependent regulation of cross-bridge cycling kinetics predominantly occurs rapidly and involves the intrinsic properties of the myofilament rather than post-translational modifications that are known to occur in the cardiac muscle as a result of a change in muscle/sarcomere length.

Datp Restores the Contractile Function of Cardiac Myofibril from Adult Dogs with Naturally Occurring Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is a major type of heart failure that usually results in loss of systolic function. Naturally-occurring canine DCM is a widely-accepted experimental paradigm for studying human DCM. 2-Deoxy-adenosine triphosphate (dATP) has been demonstrated as a superior energy-substrate over ATP for myosin cross-bridge formation and increased systolic function. Here, we report the first observations on the contractile properties of isolated cardiac myofibrils from both non-failing (NF) and DCM canine samples, and evaluated the beneficial effects of dATP on contractile performance of DCM canine samples.Compared to NF samples, tension was lower for DCM myofibrils at all measured Ca2+ levels, and pCa50 was right shifted by 0.22 pCa units. NTPase assays indicated replacement of ATP with dATP significantly increased myofilament activity in both NF and DCM samples. With substitution of dATP for ATP on DCM canine myofibrils, maximal tension (64.9±2.0 vs. 50.9±1.5 mN/mm2, p<0.005) and the Ca2+ sensitivity of tension was significantly improved for DCM myofibrils, restoring them to NF levels. dATP significantly increased the kinetics of activation (kACT), but had little impact on the rate of tension redevelopment (kTR), thus restoring the ratio of kACT/kTR (0.67±0.03, vs. 0.50±0.02, p<0.005), to the NF level (0.73±0.04). This suggests thin filament activation kinetics is impaired with DCM and restored when dATP is the contractile substrate. Additionally, there were no effects on the time to 50% (0.34±0.01 vs. 0.32±0.01, p=0.2565) and 90% (2.66±0.09 vs. 2.50±0.11, p=0.2515) relaxation, suggesting that dATP increases contraction in naturally-occurring canine DCM myofibrils, without impairing relaxation. Our findings suggest increasing cardiomyocyte dATP has potential as a therapeutic strategy to recover reduced systolic ventricular performance of DCM myocardium and is worth further evaluation for the treatment of DCM.

Effects of pseudo-phosphorylated rat cardiac troponin T are differently modulated by α- and β-myosin heavy chain isoforms.

Interplay between the protein kinase C (PKC)-mediated phosphorylation of troponin T (TnT)- and myosin heavy chain (MHC)-mediated effects on thin filaments takes on a new significance because: (1) there is significant interaction between the TnT- and MHC-mediated effects on cardiac thin filaments; (2) although the phosphorylation of TnT by PKC isoforms is common to both human and rodent hearts, human hearts predominantly express β-MHC while rodent hearts predominantly express α-MHC. Therefore, we tested how α- and β-MHC isoforms differently affected the functional effects of phosphorylated TnT. Contractile measurements were made on cardiac muscle fibers from normal rats (α-MHC) and propylthiouracil-treated rats (β-MHC), reconstituted with the recombinant phosphomimetic-TnT (T204E; threonine 204 replaced by glutamate). Ca2+ -activated maximal tension decreased differently in α-MHC + T204E (~68%) and β-MHC + T204E (~35%). However, myofilament Ca2+ sensitivity decreased similarly in α-MHC + T204E and β-MHC + T204E, demonstrating that a decrease in Ca2+ sensitivity alone cannot explain the greater attenuation of tension in α-MHC + T204E. Interestingly, dynamic contractile parameters (rates of tension redevelopment, crossbridge (XB) recruitment dynamics, XB distortion dynamics, and XB detachment kinetics) decreased only in α-MHC + T204E. Thus, the transition of thin filaments from the blocked- to closed-state was attenuated in α-MHC + T204E and β-MHC + T204E, but the closed- to open-state transition was attenuated only in α-MHC + T204E. Our study demonstrates that the effects of phosphorylated TnT and MHC isoforms interact to bring about different functional states of cardiac thin filaments.

Existence of Length-Dependent Modulation of Cross-Bridge Cycling Kinetics in Right Ventricles of Non-Failing and Failing Human Myocardium

Heart failure affects more than 5 million patients in the United States. The mechanisms by which the heart regulates cross-bridge cycling rate and if and how such regulation is altered in heart failure are poorly understood. One possible modulator of this cycling rate is muscle length. In a previously published study, we showed that the rate of tension redevelopment (ktr) can be reproducibly measured in intact cardiac trabeculae using a novel approach. Furthermore, we showed that slacking trabeculae from Lopt corresponding to end-diastolic volume in vivo) to L90 (corresponding to end-systolic volume in vivo) increases ktr by about 60%. While this regulation is present in a small animal model, its role in human myocardium in both health and disease remains unanswered. Therefore, we extended our studies using our novel method to right ventricular trabeculae isolated from non-failing donor and patients with heart failure. The failing samples had preserved length-tension relationship, reduced force-frequency relationship, and β-adrenergic desensitization as compared to non-failing donors. Contractile and relaxation kinetics of twitch of both non-failing and failing trabeculae were slowed down with increasing muscle length from L90 to Lopt. We were able to reproducibly measure ktr in intact human cardiac trabeculae; ktr during maximal myofilament activation in both non-failing and failing samples decreased with increasing muscle length. This effect of muscle length on ktr was also present at sub-maximal activation. These effects can be potentially due to post-translational modifications. We have preliminary ktr data after rapid length changes during maximal activation and PKA/PKCBII inhibition at maximal and sub-maximal activation levels. The overall data provides evidence that muscle length can regulate cross-bridge cycling kinetics in both non-failing and failing human hearts.

The Role of Cardiac Myosin Light Chain 2V Phosphorylation in the Healthy and Failing Myocardium

While a number of factors underlie cardiac dysfunction in HF, recent attention has fallen on phosphorylation changes in myofilament proteins, notably the regulatory proteins. Cardiac myosin regulatory light chain (cMLC2) is a 166 amino acid protein located on the hinge region of myosin heavy chain and optimally positioned to modulate contraction. Recent studies have shown that cMLC2v contributes to regulating cardiac pump function and allows for beat-to-beat tuning of the myosin motor arm. Humans, as well as large rodents such as rabbits, have high sequence homology and a single phosphorylation site at ser15. We hypothesize that human MLC2v phosphorylation is a critical regulator of myocyte contraction that is dysregulated in HF and that increasing MLC2v phosphorylation with myosin light chain kinase (MLCK) can partially reverse the dysfunction seen with HF. Human donor or HF permeabilized cardiac tissue were incubated with either alkaline phosphatase to reduce phosphorylation or PKA to induce maximal phosphorylation of the other myofilament proteins. Maximal cMLC2v phosphorylation was then achieved by incubation with MLCK. Force-calcium relationship, rate of tension redevelopment, tension cost and length dependent activation was determined. To assess the role of cMLC2v phosphorylation on dynamic regulation of length dependent activation, intact rabbit cardiomyocytes were isolated from rabbit hearts and transfected with MLCK to provide maximal cMLC2v phosphorylation. Cells were then attached using a force transducer and length controller and a simulated length-force relationship, which approximate the pressure-volume relationship in vivo, was evaluated at different diastolic sarcomere lengths. We show that MLC2v phosphorylation is a critical regulator of myocyte contraction that is dysregulated in the failing heart and treatment with MLCK can partially reverse the dysfunction seen with HF.

The T1 Region of Cardiac Troponin T is Important for Tuning the Crossbridge Recruitment Dynamics in the Cardiac Muscle

The sequence heterogeneity in the T1 and T2 regions of cardiac TnT (cTnT) and fast skeletal TnT (fsTnT) suggests an important tissue-specific functional role. However, the physiological significance of such protein sequence heterogeneity remains elusive. We replaced T1 or T2 region of rat cTnT (RcT1 or RcT2) with its counterpart from rat fsTnT (RfsT1or RfsT2) to generate RfsT1-RcT2 and RcT1-RfsT2 recombinant chimeras. These chimeras were reconstituted into detergent-skinned rat cardiac myofibers for functional characterization. Dynamic contractile features of RfsT1-RcT2 and RcT1-RfsT2 reconstituted fibers were assessed by fitting the recruitment-distortion model to the force response of small amplitude (0.5%) muscle length changes. RfsT1-RcT2 fibers caused a significant decrease in both maximal tension (∼40%) and ATPase activity (∼44%), whereas, RcT1-RfsT2 fibers showed no effect. While the magnitude of crossbridge recruitment, E0, was significantly lower by ∼33%, the speed of crossbridge recruitment, b, was dramatically higher by ∼100% in RfsT1-RcT2 fibers. The rate of tension redevelopment, ktr, also increased by ∼20% in RfsT1-RcT2 reconstituted fibers. Furthermore, an increase in b shifted the frequency of minimum stiffness, fmin, to higher values in RfsT1-RcT2 fibers. On the other hand, b, ktr, and fmin were unaffected in RcT1-RfsT2 fibers. Our data suggests that cardiac-specific T1 plays an important role not only in the maximal activation of cardiac thin filament, but also plays a role in tuning the myocardial contraction such that the crossbridge recruitment dynamics matches with the heart rate.

Impact of site-specific phosphorylation of protein kinase A sites Ser23and Ser24of cardiac troponin I in human cardiomyocytes

PKA-mediated phosphorylation of contractile proteins upon β-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser(23)/Ser(24)) of cardiac troponin (cTn)I results in a decrease in myofilament Ca(2+) sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in the human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser(23) and Ser(24) of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated, cTnI-AD mimics Ser(23) unphosphorylated and Ser(24) phosphorylated, cTnI-DA mimics Ser(23) phosphorylated and Ser(24) unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca(2+) concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca(2+) sensitivity (pCa(50)) was significantly lower in cTnI-DD (pCa(50): 5.39 ± 0.01) compared with cTnI-AA (pCa(50): 5.50 ± 0.01), cTnI-AD (pCa(50): 5.48 ± 0.01), and cTnI-DA (pCa(50): 5.51 ± 0.01) at ~70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa(50) with cTnI-DD content was observed, saturating at ~55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca(2+) sensitivity, which is maximal at ~55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the DISCUSSION in this article.