Increase In Myofilament Ca2 Research Articles

Cardiomyopathy-Related Mutations (E244D, K247R, D270N, and K273E) in the H2-Helix of Cardiac Troponin T Have Varied Effects on Myofilament Responsiveness to Calcium and Crossbridge Recruitment Dynamics

Two hypertrophic cardiomyopathy (HCM)-related mutations, E244D and K247R, and two dilated cardiomyopathy (DCM)-related mutations, D270N and K273E, have been identified along a centralized helix of cardiac troponin T (cTnT). This helix, termed H2(T), is centrally located in the core domain of cardiac troponin, interacting with cardiac troponin I and troponin C. This indicates a functional role of H2(T) in translating conformational changes sensed in troponin C and troponin I to the rest of the thin filament, but this structure-function relationship is not well understood. To determine the significance that disease-related alterations in the structure of H2(T) have in altering contractile function, we measured the contractile properties of rat cardiac muscle fibers containing rat cTnT variants cTnTE245D, cTnTK248R, cTnTD271N, or cTnTK274E corresponding to human E244D, K247R, D270N, or K273E mutations, respectively. We measured simultaneous force production and ATPase activity, as well as force responses (F(t)) to step-length perturbations in demembranated cardiac muscle fibers activated at various [Ca2+], at both sarcomere lengths 2.0 and 2.3 μm. Fibers containing cTnTK274E exhibited an increase in myofilament Ca2+ sensitivity when compared to those containing wild-type (WT)-cTnT. In addition, crossbridge recruitment dynamics, as estimated by model-predictions of F(t) and ktr measurements, were slower in fibers containing cTnTK274E, a trend that was also seen in fibers containing cTnTD271N. Ca2+ sensitivity of fibers containing cTnTK248R or cTnTD271N was less than that of fibers containing WT-cTnT. Furthermore, maximal ATPase activity was slightly but significantly increased in fibers containing cTnTE245D or cTnTK248R. These findings suggest that mutations along H2(T) influence the cTnT-modulated mechanisms of myofilament activation and crossbridge recruitment dynamics, and may contribute in part to cardiac dysfunction associated with HCM and DCM diseases.

Regional Connexin43 De-Phosphorylation and AMP-Kinase Activation after Rapid Pacing in Myofilament Ca Sensitized Hearts

Increased myofilament Ca2+ sensitivity, a common finding in hypertrophic cardiomyopathy (HCM), is associated with an increased susceptibility for arrhythmias. In hearts expressing a Ca sensitizing troponin T (I79N) HCM mutation reentry-type arrhythmias are induced by fast pacing of isolated perfused hearts. Fast pacing also triggers arrhythmias in control hearts pre-treated with the Ca2+ sensitizer EMD57033. Regional conduction velocity (CV) slowing may contribute to formation of the arrhythmogenic substrate, but the underlying mechanism has not been elucidated.Hypothesis: We hypothesized that a change in intercellular coupling via gap junctions (GJ) is responsible.Methods: We analyzed connexin 43 (Cx43) by Western blot in total and soluble heart tissue fractions (three phosphoisoforms could be separated: P0,P1,P2), and via confocal imaging of immunostained frozen heart sections.Results: Under baseline conditions, no difference was detectable in Cx43 expression level, soluble/insoluble ratio and isoform distribution in all assays (all n≥7). However, after hearts were rapidly paced (12-18 Hz for 16 min), Cx43 expression was lower in I79N compared to control (64.8 ± 6.6%, n ≥ 7, p = 0.03) and the P0/P2 ratio increased (p = 0.01). Strikingly, immunostaining revealed areas with high accumulation of Cx43-P0 isoform (clusters) in I79N as well as EMD-treated hearts, while there was low homogeneous Cx43-P0 staining in controls. GJs are metabolically sensitive, so we next investigated AMP-kinase activation via phosphorylation at T172. Phosphorylation level was increased after pacing in I79N (to 225±47% of control). The activated kinase accumulated in the same regions where Cx43-P0 staining was observed.Conclusion: We conclude that during rapid pacing some regions become energy depleted, as evidenced by AMP-kinase activation. This triggers Cx43-P0 accumulation in the same regions and may contribute to the regional slowing of CV and thus formation of the arrhythmogenic substrate in Ca sensitized hearts.

EPAC Activation of Cardiac Muscle

Exchange protein-directly activated by cAMP (EPAC) has been shown to be involved in the classical cAMP-activated protein kinase A (PKA) pathway in many different cell types. However, in the cardiac muscle the effects of EPAC have yielded contradictory results with reports of increased (Oestrich, 2007) and decreased (Pereira, 2007 & Cazorla, 2009) Ca2+ transient amplitude in mice and rats, respectively. The aim of this study was to examine the role of EPAC activation in excitation-contraction (EC) coupling in isolated rat ventricular muscle preparations. To activate the EPAC pathway, we used 8-(4-chlorophenylthio)-2’-O-methyladenosine-3’, 5’-cyclic monophosphate (cpTOME), an analogue of cAMP. Simultaneous measurements of intracellular calcium ([Ca2+]i) with Fura-2 and isometric force were made in RV trabeculae before and during cpTOME application. At 1.5 mM [Ca2+]o, 10 μM cpTOME had no effect on the amplitude of [Ca2+]i transients (0.7 ± 0.1 to 0.7 ± 0.1, P = 0.35) or on twitch force (24.1 ± 5.2 mN mm−2 to 22.80 ± 4.62 mN mm−2, P = 0.20) (n = 7). However, at 0.5 mM [Ca2+]o, cpTOME increased peak stress from 10.5 ± 2.8 mN mm−2 to 15.0 ± 2.7 mN mm−2, P = 0.01 (n = 6), but without any change in [Ca2+]i transients (P = 0.16). The force-[Ca2+]i relationship of intact trabeculae exhibited increased myofilament Ca2+ sensitivity with cpTOME at low [Ca2+]o but not at 1.5 mM [Ca2+]o. In isolated cells, cpTOME increased Ca2+ spark frequency (Fluo-4) from 6.6 per 100 μm3 s (n = 3) to 32.3 per 100 μm3 s (n = 9), P = 0.05, with a reduction in the peak amplitude of the sparks. The latter result recapitulates the idea that changes in RyR sensitivity do not alter the amplitude of Ca2+ transients (Eisner, 2009).

Genetically De-Sensitizing Myofilaments to Ca in a Mouse Model for Hypertrophic Cardiomyopathy Decreases Arrhythmia Incidence

Increased myofilament Ca sensitivity, a common finding in hypertrophic cardiomyopathy (HCM), is associated with an increased susceptibility for arrhythmias. In isolated hearts expressing a Ca sensitizing troponin T (I79N) HCM mutation fast pacing induces reentry-type tachycardia (VT) in 60% of the hearts. We hypothesized that reducing myofilament Ca sensitivity will decrease arrhythmia incidence.Methods: We generated knock-in mice with a TnT-R141W (HET) mutation, which is associated with dilated cardiomyopathy and reduced myofilament Ca sensitivity, and crossed them with I79N mice (CROSS). We determined heart weight-to-body weight ratio (HW/BW), cell diameter and fibrosis content and we monitored arrhythmia incidence in isolated hearts.Results: The HW/BW ratio was 6.6±1.1 in the WT, with 5.6±0.9 lower in the I79N, with 7.6±1.0 higher in the HET and similar to I79N in the CROSS (5.8±1.0). In the CROSS cell diameter was smaller than in the HET and fibrosis content attenuated compared to I79N. Importantly, VT incidence in the CROSS was similar to control (23.5% vs 11.1% in WT).Conclusions: Correcting myofilament Ca sensitivity reduces arrhythmia incidence of I79N mice and attenuates pathological remodeling observed in mice expressing each mutation alone.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Protein kinases participate in the contraction in response to levobupivacaine in the rat aorta

Levobupivacaine is a long-acting amide local anesthetic that intrinsically produces vasoconstriction both in vivo and in vitro. Levobupivacaine increases intracellular calcium concentrations ([Ca2+]i) in vascular smooth muscle cells. The goals of this in vitro study were to investigate whether levobupivacaine-induced contraction is associated with increased Ca2+ sensitivity and to identify the protein kinases involved in mediating contraction in response to levobupivacaine in isolated rat aortic smooth muscle. The effect of levobupivacaine and potassium chloride (KCl) on the [Ca2+]i and tension was measured simultaneously with acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative levobupivacaine concentration–response curves were generated in the presence or absence of the following antagonists: GF 109203X; Y-27632; genistein; SP600125; PD 98059; and SB 203580. Levobupivacaine-induced protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and c-Jun NH2-terminal kinase (JNK) phosphorylation and Rho-kinase (ROCK-2) membrane translocation were detected in rat aortic vascular smooth muscle cells using Western blotting. The slope of the [Ca2+]i-tension curve for levobupivacaine was higher than that for KCl. Y-27632, GF 109203X, and SP600125 attenuated levobupivacaine-induced contraction in a concentration-dependent manner. Genistein, PD 98059, and SB 203580 attenuated levobupivacaine-induced contraction. Pretreatment with GF 109203X and Y-27632 inhibited levobupivacaine-induced PKC phosphorylation and Rho-kinase (ROCK-2) membrane translocation, respectively. Pretreatment with SP600125 or PD 98059 attenuated the levobupivacaine-induced phosphorylation of JNK and ERK, respectively. These results indicate that levobupivacaine-induced contraction involving an increase in myofilament Ca2+ sensitivity involves the primary activation of Rho-kinase-, PKC-, and JNK-mediated pathways of rat aortic smooth muscle.

Abstract P079: Cardiac Contractility in Response to Isoproterenol from Gravin Knockout (AKAP12) Mice

Gravin (AKAP12), one of the A-Kinase-Binding-Proteins (AKAPs), serves as a scaffold protein linking β 2 -adrenergic receptor (β 2 -AR), phosphatase 2B and several kinases including Protein Kinase A (PKA) and Protein Kinase C (PKC). The presence of gravin facilitates signal transduction of β 2 -AR and thus affects cardiac excitation-contraction coupling. We previously showed decreased cardiac hypertrophy, increased contractility and decreased arrhythmias in gravin-KO mice, following chronic β-AR stimulation. This response was associated with enhanced [Ca 2+ ] i homeostasis. Here, we test whether cardiac contraction is also affected in gravin knock-out (gravin-KO) mice. Trabeculae or small papillary muscles from the right ventricles were mounted between a force transducer and a motor arm, and superfused with K-H solution (pH 7.4) at room temperature. Developed force increased as external Ca 2+ ([Ca 2+ ] o ) was raised from 1 to 10 mM in both gravin-KO and wide-type (WT) muscles. Developed force and intracellular Ca 2+ transient (Ca 2+ i ) increased in a dose dependent manner as [Ca 2+ ] o was raised. Gravin-KO muscles had lower Ca 2+ i at any given [Ca 2+ ] o , but had similar force at [Ca 2+ ] o >4.0 mM; at higher [Ca 2+ ] o s, developed forces was significantly lower. Isoproterenol (ISO, 0.05-200nM) increased force and Ca 2+ i in both groups of muscles. However, force and Ca 2+ i started to blunt in gravin-KO muscle at higher ISO doses and became significantly lower at ISO does >50 nM. Both force and Ca 2+ i were lower at a given rest interval in gravin-KO muscles with no changes in recirculation fraction (RF). These results show that gravin-KO muscles maintain their response to both Ca 2+ and ISO with reduced capacity at higher doses, and to rest potentiations with lower magnitudes, but with no changes in recirculation fraction of Ca 2+ through the sarcoplasmic reticulum (SR). These findings suggest: (1) gravin-KO muscles may likely have smaller Ca 2+ content in the SR; (2) gravin plays an important modulatory role in the argumentation of force by Ca 2+ and β 2 -AR stimulation; (3) at physiological [Ca 2+ ] o , gravin-KO increases myofilament Ca 2+ responsiveness. Ongoing experiments are focused on changes myofilament Ca 2+ responsiveness in gravin-KO mice.

Abstract 9065: Dose-Dependent Rescue Effects by a Desensitizing TnI Molecule in a RCM Mouse Model

We have previously demonstrated that the R193H mutation of cardiac troponin I (cTnI) caused restrictive cardiomyopathy (RCM) and early death in transgenic mice (Du et al., 2006). RCM mutants are also characterized by increased myofilament Ca 2+ sensitivity and impaired cardiac relaxation (Gomes et al., 2005; Du et al., 2008). Restrictive cleavage of the N-terminal extension of cTnI has been shown to desensitize myofilament to Ca 2+ and enhance diastolic function in transgenic mice (Yu et al., 2001; Feng et al., 2008; McConnell et al., 2009). N-terminal truncated cTnI (cTnI-ND) also had the ability to correct the diastolic dysfunction and increase the lifespan of RCM mice expressing the cTnI R193H mutant in a wild type cTnI null background (Li et al, 2010). In the present study, we produced double transgenic mice (Double-TG) expressing different levels of mutant cTnI R193H and cTnI-ND to investigate the dose-dependent rescue effect of cTnI-ND and determine the mechanisms behind its protective role in young and aged RCM mice. In 2-month-old Double-TG mice, the rescue of these animals is secondary to the desensitization of their myofilament to Ca 2+ and the fixing of their cardiac relaxation impairment by cTnI-ND. The correction of the diastolic dysfunction by cTnI-ND occurs in a dose-dependent manner as evidenced by cell-based assays, measuring cardiac cell contractility of the various Double-TG mouse lines, and by echocardiography. In addition, in 8-10-month-old Double-TG mice, the rescue of RCM mice by cTnI-ND is characterized by both the restoration of their diastolic function and the amelioration of their systolic function, since both diastolic and systolic functions deteriorate in aged RCM mice. Thus, this study allows determining the efficiency of cTnI-ND in the treatment of RCM and evaluating how the dosage of RCM mutant proteins may influence the cardiac function. Also, consistent with the beneficial effect of cTnI-ND on the function of non-myopathic aging hearts (Biesiadescki et al., 2010), these data demonstrate the ability of cTnI-ND to rescue the RCM phenotype not only by correcting diastolic dysfunction in young RCM mice, but also by improving systolic function in aged RCM mice.

P21-activated kinase improves cardiac contractility during ischemia-reperfusion concomitant with changes in troponin-T and myosin light chain 2 phosphorylation

p21-activated kinase 1 (Pak1) is a serine/threonine kinase that activates protein phosphatase 2a, resulting in the dephosphorylation of cardiac proteins and increased myofilament Ca(2+) sensitivity. Emerging evidence indirectly indicates a role for Pak1 in ischemia-reperfusion (I/R), but direct evidence is lacking. We hypothesize that activation of the Pak1 signaling pathway is a cardioprotective mechanism that prevents or reverses the detrimental effects of ischemic injury by inducing posttranslational modifications in myofilament proteins that ultimately improve cardiac contractility following ischemic insult. In the present study, we subjected ex vivo hearts from wild-type (WT) and Pak1-knockout (KO) mice to 20 min of global cardiac ischemia followed by 30 min of reperfusion. In the absence of Pak1, there was an exacerbation of the increased end-diastolic pressure and reduced left ventricular developed pressure occurring after I/R injury. ProQ analysis revealed an increase in troponin-T phosphorylation at baseline in Pak1-KO hearts compared with WT. Significantly decreased myosin light chain 2 (MLC2) phosphorylation in Pak1-KO hearts compared with WT after I/R injury was confirmed by Western immunoblotting. These data indicate that Pak1-KO hearts have reduced recovery of myocardial performance after global I/R injury concomitant with changes in troponin-T and MLC2 phosphorylation. Finally, a protein-protein association between Pak1 and MLC2, and Pak1 and troponin-T, was determined by coimmunoprecipitation. Thus, results of our study provide a basis for targeting a novel pathway, including Pak1, in the therapies for patients with ischemic events.

Protein phosphatase 2A affects myofilament contractility in non-failing but not in failing human myocardium

Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation–contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (ΔpCa50 = 0.05 ± 0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ΔpCa50 = 0.10 ± 0.03) and dilated (DCM, ΔpCa50 = 0.06 ± 0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56α was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts.

Berbamine Increases Myocardial Contractility via a Ca2+-independent Mechanism

Berbamine (BM), a natural compound derived from Berberis vulgaris L, has been reported to inhibit cardiac contractile function at higher concentrations. Here, we report that BM had concentration-dependent biphasic effects on myocardial contraction in Langendorff-perfused rat hearts, that is, at lower concentrations (30-100 nM), it displayed positive inotropic and lusitropic effects, whereas at a higher concentration of 1 μM, it caused a negative inotropic effect after an initially weak increase. These effects were further confirmed in cardiomyocytes isolated from the left ventricles of rats. Moreover, the increased cell shortening by BM at concentrations from 0.1 to 100 nM was not associated with an alteration of intracellular Ca transients. Consistently, at 30 nM, BM shifted the cell shortening--Ca transient relationship curve induced by cumulative elevation of extracellular Ca concentration to the left. Furthermore, BM significantly increased membrane-bound but not filament-bound protein kinase C epsilon (PKCε) in the isolated hearts and cardiomyocytes. Such a translocation was inhibited by PKCε-specific inhibitor PKCε V1-2 concomitant with the abolishment of the BM-induced increase in contraction. These findings reveal the positive inotropic effect of BM in the myocardium and demonstrate that BM increases myocardial contractility by increasing myofilament Ca sensitivity via a PKCε-dependent signaling pathway.

Deletions in the N-Terminus of Troponin T Reveal a Region Important for Ca 2+- and Length-Dependent Cardiac Myofilament Activation

Interactions between the N-terminus of cardiac troponin T (cT1), a region rich in negatively charged amino acids, and the head-to-tail overlapping region of tropomyosin are responsible for localizing the troponin complex on the thin filament. Thus, cT1 is uniquely positioned on the thin filament to modulate cardiac contractile activity. Previous studies have demonstrated that truncations of cT1 result in severe inhibition of Ca2+-activated force development and ATPase activity of cardiac myofilaments, indicating that cT1 is essential for cardiac myofilament activation. However, the specific region of cT1 that plays an important role in cardiac contractile activation remains unknown. To determine the specific region of cT1 that plays an important role in cardiac contractile activation, we progressively deleted cT1 in rat cardiac troponin T (RcTnT) to generate four deletion mutants, RcTnT 1-29 deletion, 1-43 deletion, 30-43 deletion, and 44-73 deletion. Cardiac contractile activity and crossbridge recruitment dynamics were studied in detergent-skinned rat cardiac papillary bundles reconstituted with either wild-type (control) or the deletion proteins. Our preliminary studies show that the Ca2+-dependent force development, ATPase activity, myofilament Ca2+ sensitivity, and rate of crossbridge recruitment were significantly depressed in reconstituted fibers containing RcTnT 1-43 deletion. Fiber bundles reconstituted with RcTnT 44-73 deletion exhibited an increase in myofilament Ca2+ sensitivity. Another significant finding is that both RcTnT 1-43 and RcTnT 1-29 deletions resulted in an ablation of length-dependent increase in myofilament Ca2+ sensitivity, an important component of the Frank-Starling mechanism of healthy heart function. Thus, our data indicates that the region of cT1 comprising 1-43 amino acids plays an important role in Ca2+- and length-dependent cardiac myofilament activation. Furthermore, our findings suggest that other regions of cT1 have specific roles in mediating cardiac myofilament activation.

Effects of R92 Mutations on Cardiac Contractile Function are Influenced by Changes in Myosin Heavy Chain Isoform

Mutations of cardiac troponin T (cTnT), many of which are found at its N-terminus (cT1), are associated with various forms of human heart disease. Within cT1, an amino acid at residue 92 (Arginine, R92) is a hotspot for point mutations. Previous studies using transgenic mouse models have shown that disease-related mutations R92 to Leucine (R92L) or Glutamine (R92Q) each influence contractile behavior of cardiac myofilaments. Because such cTnT mutations are often accompanied by an increase β-myosin heavy chain (MHC) expression in the failing human heart, we sought to determine whether the R92 mutation effects on cardiac contractile function are further influenced by a shift in MHC isoform content. Detergent-skinned papillary muscle fiber bundles were harvested from transgenic mice expressing R92L or R92Q against native α-MHC (R92L/α-MHC or R92Q/α-MHC) and transgenic mice expressing R92L or R92Q against predominantly β-MHC (R92L/β-MHC or R92Q/β-MHC). Constantly-activated fiber bundles from R92 transgenic and nontransgenic controls (α-MHC and β-MHC) were used to study how differences in MHC influence the effects of R92 mutations on Ca2+- and length-dependent contractile activation. Our study shows that, concomitant with previous findings, R92 mutations against α-MHC result in a decrease in ATPase activity, increase in myofilament Ca2+-sensitivity, and a decrease in cooperativity of myofilament force production. Furthermore, our study suggest that mutant R92 effects cardiac contractile dynamics in such a way that R92Q/α-MHC slows rates of crossbridge recruitment and crossbridge detachment and blunts the nonlinear effect that crossbridge distortion has on crossbridge recruitment. Interestingly, many of these R92 mutant effects were significantly influenced by expression of β-MHC. Collectively, these novel findings indicate a strong interaction effect and suggest that MHC structure further influences how cTnT regulates functional and dynamical aspects of cardiac contractile activation.

Increased Myofilament Ca 2+ Sensitivity Decreases Sarcomere Length and Increases Spark-Spark Interactions

People with familial hypertrophic cardiomyopathy (FHC) harboring mutations of cardiac troponin T (cTnT) are often at a high risk of sudden cardiac death. Transgenic mice harboring some of these cTnT mutations show increased myofilament sensitivity to Ca2+ and also shortened diastolic sarcomere length (SL). Our computational studies predicted that decreasing the distances between Ca2+ release units (CRUs) of the sarcoplasmic reticulum (SR) by decreasing SL can destabilize the Ca2+ control system and increase the probability of spontaneous Ca2+ waves. Destabilization results from enhanced crosstalk between neighboring CRUs. In this study we mimic the greater myofilament Ca2+ sensitivity conferred by cTnT mutations using the myofilament Ca2+ sensitizer EMD 57033 (EMD). At concentrations up to 3 μM, EMD had no effect on either the peak Ca2+ transient or the diastolic Ca2+ levels and did not alter the SR Ca2+ load. To test the prediction that SL shortening increases the coupling between CRUs, we loaded myocytes with Di8-ANEPPS and Fluo-4 and simultaneously measured SL and Ca2+ sparks in 2 spatial dimensions using the Zeiss 5 Live high-speed 2-D scanning confocal microscope. EMD (1.5 μM) decreased SL significantly compared to the control cells in normal Tyrode (1.58 μm vs.1.69 μm, p<0.05). The spark coupling strength measures the influence of one CRU on another and is derived from an analysis of the spatio-temporal distribution of Ca2+ sparks. EMD treatment significantly increased the spark coupling strength 2.5 fold. The enhanced spark-spark coupling as diastolic SL decreases may contribute to increased frequency of spontaneous Ca2+ waves during diastole that can lead to triggered arrhythmias and sudden cardiac death in FHC.

Myofilament calcium sensitization delays decompensated hypertrophy differently between the sexes following myocardial infarction

Contractile dysfunction is common to many forms of cardiovascular disease. Approaches directed at enhancing cardiac contractility at the level of the myofilaments during heart failure (HF) may provide a means to improve overall cardiovascular function. We are interested in gender-based differences in cardiac function and the effect of sarcomere activation agents that increase contractility. Thus, we studied the effect of gender and time on integrated arterial-ventricular function (A-V relationship) following myocardial infarction (MI). In addition, transgenic mice that overexpress the slow skeletal troponin I isoform were used to determine the impact of increased myofilament Ca(2+) sensitivity following MI. Based on pressure-volume (P-V) loop measurements, we used derived parameters of cardiovascular function to reveal the effects of sex, time, and increased myofilament Ca(2+) sensitivity among groups of post-MI mice. Analysis of the A-V relationship revealed that the initial increase was similar between the sexes, but the vascular unloading of the heart served to delay the decompensated stage in females. Conversely, the vascular response at 6 and 10 wk post-MI in males contributed to the continuous decline in cardiovascular function. Increasing the myofilament Ca(2+) sensitivity appeared to provide sufficient contractile support to improve contractile function in both male and female transgenic mice. However, the improved contractile function was more beneficial in males as the concurrent vascular response contributed to a delayed decompensated stage in female transgenic mice post-MI. This study represents a quantitative approach to integrating the vascular-ventricular relationship to provide meaningful and diagnostic value following MI. Consequently, the data provide a basis for understanding how the A-V relationship is coupled between males and females and the enhanced ability of the cardiovascular system to tolerate pathophysiological stresses associated with HF in females.

Functional effects of a tropomyosin mutation linked to FHC contribute to maladaptation during acidosis

Familial hypertrophic cardiomyopathy (FHC) is a leading cause of sudden cardiac death among young athletes but the functional effects of the myofilament mutations during FHC-associated ischemia and acidosis, due in part to increased extravascular compressive forces and microvascular dysfunction, are not well characterized. We tested the hypothesis that the FHC-linked tropomyosin (Tm) mutation Tm-E180G alters the contractile response to acidosis via increased myofilament Ca2+ sensitivity. Intact papillary muscles from transgenic (TG) mice expressing Tm-E180G and exposed to acidic conditions (pH 6.9) exhibited a significantly smaller decrease in normalized isometric tension compared to non-transgenic (NTG) preparations. Times to peak tension and to 90% of twitch force relaxation in TG papillary muscles were significantly prolonged. Intact single ventricular TG myocytes demonstrated significantly less inhibition of unloaded shortening during moderate acidosis (pH 7.1) than NTG myocytes. The peak Ca2+ transients were not different for TG or NTG at any pH tested. The time constant of re-lengthening was slower in TG myocytes, but not the rate of Ca2+ decline. TG detergent-extracted fibers demonstrated increased Ca2+ sensitivity of force and maximal tension compared to NTG at both normal and acidic pH (pH 6.5). Tm phosphorylation was not different between TG and NTG muscles at either pH. Our data indicate that acidic pH diminished developed force in hearts of TG mice less than in NTG due to their inherently increased myofilament Ca2+ sensitivity, thus potentially contributing to altered energy demands and increased propensity for contractile dysfunction.

Biological actions of green tea catechins on cardiac troponin C

Catechins, biologically active polyphenols in green tea, are known to have a protective effect against cardiovascular diseases. In this study, we investigated direct actions of green tea catechins on cardiac muscle function to explore their uses as potential drugs for cardiac muscle disease. The effects of catechins were systematically investigated on the force-pCa relationship in skinned cardiac muscle fibres to determine their direct effects on cardiac myofilament contractility. The mechanisms of action of effective catechins were investigated using troponin exchange techniques, quartz crystal microbalance, nuclear magnetic resonance and a transgenic mouse model. (-)-Epicatechin-3-gallate (ECg) and (-)-epigallocatechin-3-gallate (EGCg), but not their stereoismers (-)-catechin-3-gallate and (-)-gallocatechin-3-gallate, decreased cardiac myofilament Ca(2+) sensitivity probably through its interaction with cardiac troponin C. EGCg restored cardiac output in isolated working hearts by improving diastolic dysfunction caused by increased myofilament Ca(2+) sensitivity in a mouse model of hypertrophic cardiomyopathy. The green tea catechins, ECg and EGCg, are Ca(2+) desensitizers acting through binding to cardiac troponin C. These compounds might be useful compounds for the development of therapeutic agents to treat the hypertrophic cardiomyopathy caused by increased Ca(2+) sensitivity of cardiac myofilaments.

Effects of Chronic Atrial Fibrillation on Active and Passive Force Generation in Human Atrial Myofibrils

Chronic atrial fibrillation (cAF) is associated with atrial contractile dysfunction. Sarcomere remodeling may contribute to this contractile disorder. Here, we use single atrial myofibrils and fast solution switching techniques to directly investigate the impact of cAF on myofilament mechanical function eliminating changes induced by the arrhythmia in atrial myocytes membranes and extracellular components. Remodeling of sarcomere proteins potentially related to the observed mechanical changes is also investigated. Myofibrils were isolated from atrial samples of 15 patients in sinus rhythm and 16 patients with cAF. Active tension changes following fast increase and decrease in [Ca(2+)] and the sarcomere length-passive tension relation were determined in the 2 groups of myofibrils. Compared to sinus rhythm myofibrils, cAF myofibrils showed (1) a reduction in maximum tension and in the rates of tension activation and relaxation; (2) an increase in myofilament Ca(2+) sensitivity; (3) a reduction in myofibril passive tension. The slow beta-myosin heavy chain isoform and the more compliant titin isoform N2BA were up regulated in cAF myofibrils. Phosphorylation of multiple myofilament proteins was increased in cAF as compared to sinus rhythm atrial myocardium. Alterations in active and passive tension generation at the sarcomere level, explained by translational and post-translational changes of multiple myofilament proteins, are part of the contractile dysfunction of human cAF and may contribute to the self-perpetuation of the arrhythmia and the development of atrial dilatation.

Does the DCM Functional Phenotype Predominate over that of HCM and RCM?

In vitro investigations into Hypertrophic Cardiomyopathy (HCM) and Restrictive Cardiomyopathy (RCM) show that mutations in cardiac Troponin T (cTnT) produce a pathogenic state via increase in myofilament Ca2+ sensitivity, whereas mutations in cTnT that cause Dilated Cardiomyopathy (DCM) decrease Ca2+ sensitivity and maximal force. Our aim was to determine whether combinatory mutations of an HCM, RCM and a DCM in cTnT yield an intermediate or dominant functional phenotype that could be correlated with the clinical condition seen in patients. Standard laboratory methods were used for cloning, expression and purification of the WT and mutants: ΔK210 (DCM), I79N (HCM), ΔE96 (RCM), ΔK210/I79N, ΔK210/ΔE96 and ΔE96/I79N. Porcine papillary skinned fibers were displaced with cTnT WT or mutant and reconstituted with HCTnI-TnC. The Ca2+ sensitivity of force development, maximal force and basal force were evaluated. The extent of TnT displacement was analyzed by measuring the unregulated tension at pCa 8.0 after cTnT treatment and none of the mutants showed an inability to displace the native cTn complex. Both double mutants (ΔK210/I79N and ΔK210/ΔE96) containing the DCM mutant showed a rightward shift in the Ca2+ sensitivity with a decrease in maximal force. In addition, the ΔK210 mutation rescued the impaired relaxation produced by the RCM mutation (ΔE96). From the skinned fiber data, ΔK210 has a dominant effect over I79N and DE96 mutations in cTnT. Circular dichroism measurements demonstrated that all three double mutants had lower alpha helical content than WT. In contrast, single mutants I79N (significantly) and DK210 (showed a tendency) to increase alpha helical content. These results suggest that cTnT can exist in multiple conformations that may be responsible for these distinct functional phenotypes. NIH HL-67415 and HL-42325 (JDP), AHA 0825368E (JRP).

Decreased Phosphorylation of the Gap Junction Protein Connexin43 and Increased Anisotropy of Conduction as a Consequence of Myofilament Ca2+ Sensitization

Increased myofilament Ca2+ sensitivity is associated with an increased susceptibility for arrhythmias, as previously demonstrated in mice expressing Ca2+ sensitizing troponin T (TnT-I79N) mutations and after acute application of the Ca2+ sensitizer EMD57033. We hypothesized that the arrhythmia risk is increased due to altered regulation of connexin43 (Cx43), subsequently leading to a slowing of cardiac conduction.Methods: Fast (longitudinal) and slow (lateral) conduction velocity (CV) was calculated using epi-fluorescence maps from isolated hearts by plotting local CVs against orientation. Phosphorylated Cx43 (P1, P2) from these hearts migrated slower in SDS PAGE and at least three distinct bands could be separated (P0,P1,P2).Results: The lateral CV, but not the longitudinal CV, was significantly reduced in TnT-I79N mice compared to control (16.9±0.8 cm/s (n = 11) vs. 21.5±1.4 cm/s (n = 11) respectively, p<0.05). As a direct consequence the anisotropy of conduction (fast/slow) was increased in TnT-I79N hearts (to 3.2±0.2 vs 2.3±0.1 in control). This change in CV was associated with decreased Cx43 phosphorylation (un-phosphorylated P0-Cx43 increased to 146±20% of control, p<0.05). Blebbistatin, a Ca2+ desensitizer and contractile uncoupler, prevented ventricular arrhythmias in TnT-I79N hearts. Strikingly, blebbistatin also prevented the increase in anisotropy and the decrease in Cx43 phosphorylation, while it had no effect in control hearts. Conversely, acutely increasing Ca2+ sensitivity with EMD decreased Cx43 phosphorylation (P0-Cx43 491± 147%, n = 4, p< 0.05) and rapidly slowed lateral conduction velocity (to 18.2±1.3 vs 22.9±1.1 cm/s in control, p<0.05) thereby increasing anisotropy (to 2.64±0.1 vs 2.17±0.07 in control) and rendered control hearts susceptible to arrhythmia induction.Conclusion: These data suggest that decreased Cx43 phosphorylation and increased conduction anisotropy is at least in part responsible for the increased arrhythmia susceptibility associated with myofilament Ca2+ sensitization.

Cardiac Troponin Mutations and Restrictive Cardiomyopathy

Mutations in sarcomeric proteins have recently been established as heritable causes of Restrictive Cardiomyopathy (RCM). RCM is clinically characterized as a defect in cardiac diastolic function, such as, impaired ventricular relaxation, reduced diastolic volume and increased end-diastolic pressure. To date, mutations have been identified in the cardiac genes for desmin, α-actin, troponin I and troponin T. Functional studies in skinned muscle fibers reconstituted with troponin mutants have established phenotypes consistent with the clinical findings which include an increase in myofilament Ca2+ sensitivity and basal force. Moreover, when RCM mutants are incorporated into reconstituted myofilaments, the ability to inhibit the ATPase activity is reduced. A majority of the mutations cluster in specific regions of cardiac troponin and appear to be mutational “hot spots”. This paper highlights the functional and clinical characteristics of RCM linked mutations within the troponin complex.