Alterations In Excitation-contraction Coupling Research Articles

0089 : Echocardiography and right ventricular function: validation of functional criteria compared to in-vivo and ex-vivo contractility parameters

Right ventricular (RV) dysfunction is a major determinant of long-term survival in congenital heart diseases. Early echocardiographic detection of RV failure is mandatory, but recent parameters need to be validated. Objectives were to: (1) validate standard and strain echocardiographic parameters for evaluation of RV systolic function, compared to hemodynamic parameters; (2) assess the accuracy of these parameters for early detection of RV failure. Combined RV overload as observed in repaired tetralogy of Fallot was surgically reproduced in 2-month-old piglets (n=6). Age-matched piglets were used as controls (n=4). RV function was evaluated at baseline and 4 months of follow-up by standard and strain echocardiographic parameters, compared to hemodynamic (conductance catheter). Sarcomere shortening and calcium transients were recorded in RV isolated myocytes (IonOptix). Contractile reserve was assessed by in-vivo (dobutamine 5奯kg) and ex-vivo (isoprenaline 100nM) ?-adrenergic stimulation. 4 months after surgery, hemodynamic RV ejection fraction (FEVD) was significantly decreased (29.7% [26.2-34] vs 42.9% [40.7-48.6], p<0.01), and inotropic responses to dobutamine were attenuated (contractile reserve ΔEmax = 51% vs 193% for controls). On echocardiography FAC, TAPSE, S’ peak and RV free wall longitudinal strain rate were significantly decreased and correlated with FEVD. Strain rate and S’ peak were correlated with ?Emax (r=0.75 and 0.78, p<0.05). Isolated RV myocytes from operated animals exhibited hypertrophy, decreased sarcomere shortening peak in response to isporenaline (ΔL= 7.8 ± 2.8% vs 10.7 ± 2.9%, p<0.05), and increased spontaneous calcium waves suggesting perturbations of calcium homeostasis. In this model, both standard and strain echocardiographic parameters allowed the detection of early impairments of RV function and cardiac reserve, which are associated with cardiac excitation-contraction coupling alterations.

Direct and indirect measurement of neuromuscular fatigue in Canadian football players.

This study assessed the effects of a fatiguing game simulation (G-Sim) on the balance of collegiate Canadian football players. The purpose of the study was to evaluate postural control as a potential tool for monitoring neuromuscular fatigue (NMF) in collision-based team sports. Fifteen male Canadian football players were recruited (mean±SD: age 21.8±1.6 years, weight 97.6±14.7 kg). Indirect NMF measures (postural sway and countermovement jump (CMJ)) were performed 24 h before (TBase), immediately before (TPre) and after (TPost), and 24 h (T24) and 48 h after (T48) a Canadian football G-Sim. Peak isometric knee extensor torque of a maximal voluntary contraction (MVC) and electrically evoked tetani at 20 Hz (P20) and 80 Hz (P80) were also recorded as direct NMF measures at TBase, TPre, TPost, and T48. At TPost, we observed significant declines in MVC, P20, and the MVC/P80 ratio (-15.3%, -15.7%, and -12.1%, respectively; n=12) along with reductions in CMJ takeoff velocity and peak power (-6.9% and -6.5%, respectively; n=12) and larger area of the center of pressure trajectory (95.2%; n=10) during a 60-s postural sway task. All variables were no longer different than baseline by T48. Acute neuromuscular impairment in this cohort is likely attributable to alterations in excitation-contraction coupling due to structural damage and central activation failure. Congruency between the direct and indirect measures of NMF suggests monitoring postural sway has the potential to identify both neuromuscular and somatosensory alterations induced by acute game-induced fatigue in collision-based team sports players.

Effect of Hypertrophic Calcium Signals and Altered Excitation-Contraction Coupling on the Calcineurin-NFAT Pathway

Many questions remain unanswered as to how calcium (Ca2+) signals regulate the Calcineurin (CaN)- Nuclear Factor of Activated T-cells (NFAT) signaling pathway. The CaN-NFAT cascade is one of the key pathways regulating the development of cardiac hypertrophy, a condition which is recognized as a leading risk factor for heart failure. In addition, the effect of certain functional modifications in proteins involved in excitation-contraction coupling (ECC) on cardiac hypertrophy is still not well understood. In this work, we develop a mathematical model for the CaN-NFAT pathway and incorporate it into an integrative computational model of the ventricular myocyte to better understand the relationship between Ca2+ signals, CaN-NFAT, and hypertrophy. Model results suggest the frequency of calcium transients (i.e. heart rate) may be a primary determinant of hypertrophic signaling, whereas diastolic Ca2+ level and inositol trisphosphate (IP3)-mediated changes in nuclear Ca2+ level appear to play less of a role. Furthermore, alterations in the function of ECC proteins as may occur under conditions of stress, heart disease, and/or therapeutic intervention were investigated. Results indicate L-type Ca2+ channel overexpression increases NFAT activity, while overexpression of the sarcoplasmic reticulum Ca2+ ATPase, which mimics the functional effect of therapeutic SERCA gene transfer, has a relatively minor effect on NFAT activity. Therefore, an important prediction of this model is that enhancement of SERCA function is not predicted to promote the hypertrophic response, and it therefore may be possible to treat both pathological hypertrophy and SR dysfunction simultaneously.

Protective effect of distillated Nerium oleander on heart of type 2 diabetic rats.

The current study aims to investigate the possible role of NO distillate either for therapeutic or for protective potential in diabetic cardiomyopathy. Protective and restorative effects of distillated Nerium oleander (NO) on the diabetes-induced electrophysiological and structural alterations were investigated. Type 2 diabetes was induced by combination of single dose streptozotocin injection and high fat diet for four weeks. Experimental groups were designed as follows: control, diabetic, restorative-NO treated diabetic and protective-NO treated diabetic. Intracellular action potentials (AP) and contractile activities were measured form left ventricular papillary muscle strips as well as histopathological examination of heart tissue and biochemical examinations of serum were performed. Type 2 diabetes induced AP prolongation was prevented with both ways of NO treatments. Moreover, treatments produced nearly complete restorations of diabetes-induced depressed amplitude and altered kinetics of contractile activities. In parallel to electrophysiological parameters, both histopathological and biochemical results indicates the NO induced beneficial effects on the diabetes related alterations. Distillated Nerium oleander (NO) can be a highly potential therapeutic or preventive agent on the diabetes induced excitation-contraction coupling alterations (Tab. 3, Fig. 3, Ref. 23).

Why isn't endogenous ouabain more widely accepted?

A new idea is the most quickly acting antigen known to science . —Wilfred Trotter During the past few years, several distinguished senior colleagues who visited our department and inquired about the status of endogenous ouabain (EO) have asked, “How come I didn't know about these [published]

The DHPR Calcium Current in Mammalian Skeletal Muscle: Physiological Necessity or Tolerated Evolutionary Remnant?

In contrast to cardiac excitation-contraction coupling (ECC), skeletal-muscle ECC is based on Ca2+-influx-independent inter-channel communications between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1). The role of the small Ca2+ influx through the DHPR in mammalian skeletal muscle, which is not (immediately) required for ECC, is still enigmatic. Previously, we discovered that zebrafish, as well as all higher teleost fish, lack DHPR Ca2+ conductivity in skeletal muscle (Schredelseker et al., PNAS, 2010). Point mutation N617D in pore loop II of zebrafish DHPRα1S-b explained non-conductivity in fast muscle. To investigate the fascinating skeletal muscle DHPR Ca2+-conductivity / non-conductivity phenomenon we generated a non-conducting-DHPR knock-in mouse (n.c.DHPR) by introducing the N→D mutation into gene CACNA1S. Interestingly, homozygous n.c.DHPR mice are viable, fertile, visually indistinguishable, and identical in body-weight development to wild-type (WT) siblings. Myotubes isolated from newborn n.c.DHPR mice display complete lack of DHPR Ca2+ influx without altered ECC. No difference in locomotor activity (home cage activity), motor coordination (rotarod, beam walking), and muscle strength (endurance test, wire hang test) is observed in 3-7 months-old homozygous n.c.DHPR mice compared to WT. Identical results were obtained from forced frequency and fatigue tests on isolated EDL (fast twitch) and soleus (slow twitch) muscle fibers. As soon feasible, tests will be repeated on aged (18 months-old) mice to test for putative age-related accumulative effects on muscle performance, in order to understand if DHPR Ca2+ influx in mammalian skeletal muscle is a physiological necessity or just a tolerated evolutionary remnant of the ancestral pure Ca2+-influx dependent ECC of early chordates and phylogenetic branches below.Supported: FWF P-23229-B09; DK-W1101-B12.

Excitation-Contraction coupling and intracellular calcium cycling in failing hearts

Alterations in Excitation-Contraction coupling have recently been shown to play a crucial role in the pathogenesis of heart failure (HF). In failing hearts, abnormalities of neurohormonal mechanisms, which were included chronic activation of the sympathetic nervous system or of the renin-angiotensin system result in structural and functional changes in the calcium (Ca(2 +) ) regulatory proteins. These changes include the decreased sarcoplasmic reticulum (SR) load, which could be caused by reduced SR Ca(2 +) -ATPase (SERCA2A) function and increased SR Ca(2 +) leak via cardiac ryanodine receptor (RyR2) , a functional defect in L-type Ca(2 +) channel and activation of the reversal mode of Na (+) /Ca(2 +) exchanger. The abnormal regulation of intracellular Ca(2 +) affects troponin C binding and actin-myosin cross bridging and modulates post signaling pathway, which in turn contributes to the contractile dysfunction of hearts and hence to the progression of HF. Moreover, diastolic Ca(2 +) leak may develop delayed after depolarization and triggered activity as a substrate for lethal arrhythmia and sudden cardiac death. In this review, we focus on the underlying mechanism of defective Ca(2 +) regulation in HF and on the possibility of proceeding to clinical application as a new treatment for HF by targeting Ca(2 +) regulatory proteins.

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance*

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1(-/-)) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca(2+) cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1(-/-) compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca(2+) transient (Δ[Ca(2+)]i) responses in NOS1(-/-) cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca(2+)]i across the range of pacing frequencies and increased myofilament Ca(2+) sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca(2+) reuptake, and restored SR Ca(2+) content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca(2+) leak, HYD improves Ca(2+) cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.

Structural and functional evaluation of branched myofibers lacking intermediate filaments

Intermediate filaments (IFs), composed of desmin and keratins, link myofibrils to each other and to the sarcolemma in skeletal muscle. Fast-twitch muscle of mice lacking the IF proteins, desmin and keratin 19 (K19), showed reduced specific force and increased susceptibility to injury in earlier studies. Here we tested the hypothesis that the number of malformed myofibers in mice lacking desmin (Des(-/-)), keratin 19 (K19(-/-)), or both IF proteins (double knockout, DKO) is increased and is coincident with altered excitation-contraction (EC) coupling Ca(2+) kinetics, as reported for mdx mice. We quantified the number of branched myofibers, characterized their organization with confocal and electron microscopy (EM), and compared the Ca(2+) kinetics of EC coupling in flexor digitorum brevis myofibers from adult Des(-/-), K19(-/-), or DKO mice and compared them to age-matched wild type (WT) and mdx myofibers. Consistent with our previous findings, 9.9% of mdx myofibers had visible malformations. Des(-/-) myofibers had more malformations (4.7%) than K19(-/-) (0.9%) or DKO (1.3%) myofibers. Confocal and EM imaging revealed no obvious changes in sarcomere misalignment at the branch points, and the neuromuscular junctions in the mutant mice, while more variably located, were limited to one per myofiber. Global, electrically evoked Ca(2+) signals showed a decrease in the rate of Ca(2+) uptake (decay rate) into the sarcoplasmic reticulum after Ca(2+) release, with the most profound effect in branched DKO myofibers (44% increase in uptake relative to WT). Although branched DKO myofibers showed significantly faster rates of Ca(2+) clearance, the milder branching phenotype observed in DKO muscle suggests that the absence of K19 corrects the defect created by the absence of desmin alone. Thus, there are complex roles for desmin-based and K19-based IFs in skeletal muscle, with the null and DKO mutations having different effects on Ca(2+) reuptake and myofiber branching.

Poor Spontaneous and Oxytocin-Stimulated Contractility in Human Myometrium from Postdates Pregnancies

Prolongation of pregnancy i.e. going more than 10 days over the estimated due date, complicates up to 10% of all pregnancies and is associated with increased risk to both mother and fetus. Despite the obvious need for contractions of the uterus to end pregnancy, there have been no studies directly examining the role of uterine smooth muscle, myometrium, in the aetiology of prolonged pregnancy. This study tested the hypothesis that the intrinsic contractile characteristics of myometrium taken from women with prolonged pregnancy (>41 weeks and 3 days) was reduced compared to those delivering at term (39–41 weeks). We recruited women undergoing Caesarean Section (CS) delivery either pre-labour (n = 27) or in labour (n = 66) at term or postdates. The contractile ability of the postdates myometrium, whether spontaneous or elicited by oxytocin or high-K solution, was significantly reduced compared to term myometrium. These differences remained when adjusted for parity and other maternal characteristics. The findings remained significant when expressed per cross sectional area. Histological examination revealed no differences between the two groups. The contractile differences were however related to intracellular Ca transients suggesting an effect of [Ca] on reduced force production in the postdates group. In summary, myometrium from prolonged pregnancies contracts poorly in vitro even when stimulated with oxytocin and in active labour. Responses to high K+ and measurements of Ca suggest that alterations in excitation contraction coupling, rather than any histological changes of the myometrium, may underlie the differences between term and postdates myometrium. We show that postdates pregnancy is associated with poor myometrial activity and suggest that this may contribute to increased myometrial quiescence and hence, prolonged gestation.

Altered Excitation-Contraction Coupling in Human Chronic Atrial Fibrillation.

This review focuses on the (mal)adaptive processes in atrial excitation-contraction coupling occurring in patients with chronic atrial fibrillation. Cellular remodeling includes shortening of the atrial action potential duration and effective refractory period, depressed intracellular Ca2+ transient, and reduced myocyte contractility. Here we summarize the current knowledge of the ionic bases underlying these changes. Understanding the molecular mechanisms of excitation-contraction-coupling remodeling in the fibrillating human atria is important to identify new potential targets for AF therapy.

PKA phosphorylation of cardiac ryanodine receptor modulates SR luminal Ca2+ sensitivity

During physical exercise and stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor activation, resulting in protein kinase A (PKA)-mediated phosphorylation of the cardiac ryanodine receptor, RyR2, at Ser2808. Hyperphosphorylation of RyR2-S2808 has been proposed as a mechanism contributing to arrhythmogenesis and heart failure. However, the role of RyR2 phosphorylation during β-adrenergic stimulation remains controversial. We examined the contribution of RyR2-S2808 phosphorylation to altered excitation-contraction coupling and Ca(2+) signaling using an experimental approach at the interface of molecular and cellular levels and a transgenic mouse with ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Experimentally challenging the communication between L-type Ca(2+) channels and RyR2 led to a spatiotemporal de-synchronization of RyR2 openings, as visualized using confocal Ca(2+) imaging. β-Adrenergic stimulation re-synchronized RyR2s, but less efficiently in RyR2-S2808A than in control cardiomyocytes, as indicated by comprehensive analysis of RyR2 activation. In addition, spontaneous Ca(2+) waves in RyR2-S2808A myocytes showed significantly slowed propagation and complete absence of acceleration during β-adrenergic stress, unlike wild type cells. Single channel recordings revealed an attenuation of luminal Ca(2+) sensitivity in RyR2-S2808A channels upon addition of PKA. This suggests that phosphorylation of RyR2-S2808 may be involved in RyR2 modulation by luminal (intra-SR) Ca(2+) ([Ca(2+)](SR)). We show here by three independent experimental approaches that PKA-dependent RyR2-S2808 phosphorylation plays significant functional roles at the subcellular level, namely, Ca(2+) release synchronization, Ca(2+) wave propagation and functional adaptation of RyR2 to variable [Ca(2+)](SR). These results indicate a direct mechanistic link between RyR2 phosphorylation and SR luminal Ca(2+) sensing.

Transcription factor CHF1/Hey2 regulates EC coupling and heart failure in mice through regulation of FKBP12.6

Heart failure is a leading cause of morbidity and mortality in Western society. The cardiovascular transcription factor CHF1/Hey2 has been linked to experimental heart failure in mice, but the mechanisms by which it regulates myocardial function remain incompletely understood. The objective of this study was to determine how CHF1/Hey2 affects development of heart failure through examination of contractility in a myocardial knockout mouse model. We generated myocardial-specific knockout mice. At baseline, cardiac function was normal, but, after aortic banding, the conditional knockout mice demonstrated a greater increase in ventricular weight-to-body weight ratio compared with control mice (5.526 vs. 4.664 mg/g) and a significantly decreased ejection fraction (47.8 vs. 72.0% control). Isolated cardiac myocytes from these mice showed decreased calcium transients and fractional shortening after electrical stimulation. To determine the molecular basis for these alterations in excitation-contraction coupling, we first measured total sarcoplasmic reticulum calcium stores and calcium-dependent force generation in isolated muscle fibers, which were normal, suggesting a defect in calcium cycling. Analysis of gene expression demonstrated normal expression of most genes known to be involved in myocardial calcium cycling, with the exception of the ryanodine receptor binding protein FKBP12.6, which was expressed at increased levels in the conditional knockout hearts. Treatment of the isolated knockout myocytes with FK506, which inhibits the association of FKBP12.6 with the ryanodine receptor, restored contractile function. These findings demonstrate that conditional deletion of CHF1/Hey2 in the myocardium leads to abnormalities in calcium handling mediated by FKBP12.6 that predispose to pressure overload-induced heart failure.

Advanced glycation end product cross-link breaker attenuates diabetes-induced cardiac dysfunction by improving sarcoplasmic reticulum calcium handling

Diabetic heart disease is a distinct clinical entity that can progress to heart failure and sudden death. However, the mechanisms responsible for the alterations in excitation-contraction coupling leading to cardiac dysfunction during diabetes are not well known. Hyperglycemia, the landmark of diabetes, leads to the formation of advanced glycation end products (AGEs) on long-lived proteins, including sarcoplasmic reticulum (SR) Ca2+ regulatory proteins. However, their pathogenic role on SR Ca2+ handling in cardiac myocytes is unknown. Therefore, we investigated whether an AGE cross-link breaker could prevent the alterations in SR Ca2+ cycling that lead to in vivo cardiac dysfunction during diabetes. Streptozotocin-induced diabetic rats were treated with alagebrium chloride (ALT-711) for 8 weeks and compared to age-matched placebo-treated diabetic rats and healthy rats. Cardiac function was assessed by echocardiographic examination. Ventricular myocytes were isolated to assess SR Ca2+ cycling by confocal imaging and quantitative Western blots. Diabetes resulted in in vivo cardiac dysfunction and ALT-711 therapy partially alleviated diastolic dysfunction by decreasing isovolumetric relaxation time and myocardial performance index (MPI) (by 27 and 41% vs. untreated diabetic rats, respectively, P < 0.05). In cardiac myocytes, diabetes-induced prolongation of cytosolic Ca2+ transient clearance by 43% and decreased SR Ca2+ load by 25% (P < 0.05); these parameters were partially improved after ALT-711 therapy. SERCA2a and RyR2 protein expression was significantly decreased in the myocardium of untreated diabetic rats (by 64 and 36% vs. controls, respectively, P < 0.05), but preserved in the treated diabetic group compared to controls. Collectively, our results suggest that, in a model of type 1 diabetes, AGE accumulation primarily impairs SR Ca2+ reuptake in cardiac myocytes and that long-term treatment with an AGE cross-link breaker partially normalized SR Ca2+ handling and improved diabetic cardiomyopathy.

Sildenafil Prevents and Reverses Transverse-Tubule Remodeling and Ca 2+ Handling Dysfunction in Right Ventricle Failure Induced by Pulmonary Artery Hypertension

Right ventricular (RV) failure (RVF) is the main cause of death in patients with pulmonary artery hypertension (PAH). Sildenafil, a phosphodiesterase type 5 inhibitor, was approved recently for treatment of PAH patients. However, the mechanisms underlying RV contractile malfunction and the benefits of sildenafil on RV function are not well understood. We aimed to investigate the following: (1) the ultrastructural and excitation-contraction coupling alterations underlying PAH-induced RVF; (2) whether the ultrastructural changes are reversible; and (3) the mechanisms underlying the therapeutic benefits of sildenafil in PAH-RVF. We used a single injection of monocrotaline in Wistar rats to induce pulmonary vascular proliferation, which led to PAH and RVF. RV myocytes displayed severe transverse (T)-tubule loss and disorganization, as well as blunted and dys-synchronous sarcoplasmic reticulum Ca(2+) release. Sildenafil prevented and reversed the monocrotaline-induced PAH and LV filling impairment. Early intervention with sildenafil prevented RV hypertrophy and the development of RVF, T-tubule remodeling, and Ca(2+) handling dysfunction. Although late treatment with sildenafil did not reverse RV hypertrophy in animals with established RVF, RV systolic function was improved. Furthermore, late intervention partially reversed both the impairment of myocyte T-tubule integrity and Ca(2+) handling protein and sarcoplasmic reticulum Ca(2+) release function in monocrotaline-treated rats. In conclusion, PAH-induced increase in RV afterload causes severe T-tubule remodeling and Ca(2+) handling dysfunction in RV myocytes, leading to RV contractile failure. Sildenafil prevents and partially reverses ultrastructural, molecular, and functional remodeling of failing RV myocytes. Reversal of pathological T-tubule remodeling, although incomplete, is achievable without the regression of RV hypertrophy.

New insights on sarcoplasmic reticulum calcium regulation in muscle fatigue

a consistent observation with fatigue in skeletal muscle is a decline in the amplitude of the myoplasmic Ca2+ transient, which is thought to result primarily from a reduced Ca2+ flux through the ryanodine receptor (RyR1) of the sarcoplasmic reticulum (SR) ([Fig. 1][1]). This in turn is thought to

Alterations of excitation-contraction coupling and excitation coupled Ca2+ entry in human myotubes carrying CAV3 mutations linked to rippling muscle

Rippling muscle disease is caused by mutations in the gene encoding caveolin-3 (CAV3), the muscle-specific isoform of the scaffolding protein caveolin, a protein involved in the formation of caveolae. In healthy muscle, caveolin-3 is responsible for the formation of caveolae, which are highly organized sarcolemmal clusters influencing early muscle differentiation, signalling and Ca2+ homeostasis. In the present study we examined Ca2+ homeostasis and excitation–contraction (E-C) coupling in cultured myotubes derived from two patients with Rippling muscle disease with severe reduction in caveolin-3 expression; one patient harboured the heterozygous c.84C>A mutation while the other patient harbored a homozygous splice-site mutation (c.102+ 2T>C) affecting the splice donor site of intron 1 of the CAV3 gene. Our results show that cells from control and rippling muscle disease patients had similar resting [Ca2+]i and 4-chloro-m-cresol-induced Ca2+ release but reduced KCl-induced Ca2+ influx. Detailed analysis of the voltage-dependence of Ca2+ transients revealed a significant shift of Ca2+ release activation to higher depolarization levels in CAV3 mutated cells. High resolution immunofluorescence analysis by Total Internal Fluorescence microscopy supports the hypothesis that loss of caveolin-3 leads to microscopic disarrays in the colocalization of the voltage-sensing dihydropyridine receptor and the ryanodine receptor, thereby reducing the efficiency of excitation–contraction coupling. Hum Mutat 32:309–317, 2011. © 2011 Wiley-Liss, Inc.

Posttetanic potentiation in mdx muscle

X-linked muscular dystrophy of the mouse (mdx) has been reported to progressively remodel skeletal muscle to preferentially reduce fast fiber composition. Despite this, mdx muscle displays normal levels of posttetanic potentiation (PTP). Since PTP may primarily depend on phosphorylation of the myosin regulatory light chain (RLC) in fast muscle fibers, maintenance of PTP with mdx disease progression is paradoxical and may represent an adaptation of the diseased muscle. This study assesses the role of RLC phosphorylation during PTP of mdx muscle. Extensor digitorum longus muscles were isolated from mdx and from C57BL/10 (control) mice at ~50 (young) and ~300 (adult) days and stimulated in vitro (25°C) to induce PTP. During potentiation, muscles were harvested for subsequent determination of RLC phosphorylation levels. Immunofluorescence was used to assess muscle fiber type composition and no age effects were found. The magnitude of PTP was higher (P<0.05) in mdx than control muscles at both young (mdx: 21.9±1.6%; control: 17.7±1.2%) and adult (mdx: 30.4±1.8%; control: 23.2±2.2%) ages. However, RLC phosphate content was similar between all groups both at rest and following stimulation. Our results are consistent with a model where the sensitivity of mdx muscle to RLC phosphorylation-induced force potentiation is increased by disease- and age-dependent alterations in excitation-contraction coupling noted for mdx and aging muscle.

Regulation of the Transient Outward Potassium Current Itof in Cardiac Hypertrophy by Sphingosine-1-Phosphate Signaling

The fast transient outward potassium current (Ito,f), which is carried by voltage-gated Kv4.2 and Kv4.3 potassium channels and auxiliary subunit KChIP2, plays a critical role in early repolarization of the cardiac action potential. Ito,f and its gene products are strongly down-regulated in cardiac hypertrophy and disease, leading to altered excitation-contraction coupling and electrical activity as well as hypertrophy. Despite the importance of Ito,f in normal and diseased hearts, the regulation of Ito,f remains poorly understood. Studies have shown that the biologically active sphingolipid, sphingosine-1-phosphate (S1P), induces cardiac hypertrophy. In addition, the inflammatory pro-hypertrophic cytokine TNF-alpha, which decreases Ito,f, activates sphingosine kinase 1, the highly regulated enzyme that produces S1P. Therefore, we investigated the role of TNF-alpha and S1P signaling in mediating the down-regulation of Ito,f. In cultured neonatal myocytes, the TNF-alpha inhibitor etanercept attenuated reductions in Ito,f current density that were caused by the hypertrophic agonist phenylephrine (PE). Inhibition of sphingosine kinases by dimethylsphingosine prevented reductions in Ito,f that were caused by PE. Furthermore, application of S1P reduced Ito,f current density and caused hypertrophy. To interrogate the down-stream events involved in TNF-alpha/S1P- induced reductions in Ito,f, we focused on NF-kappaB since it is one of TNF-alpha's principal down-stream effectors and a major player in cardiac hypertrophy. Inhibition of NF-kappaB signaling, via an adenoviral expression of an IkappaB-alpha dominant negative mutant, prevented Ito,f reductions caused by S1P, TNF-alpha and PE. Taken together, the data suggests a linear pathway whereby PE promotes the shedding of TNF-alpha, which activates sphingosine kinase and elevates S1P, leading to activation of NF-kappaB and down-regulation of Ito,f.We will test this model in future experiments by assessing the effects of cardiac disease on Ito,f in knockout mice lacking sphingosine kinase and TNF-alpha.

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca2+ going local?

Commentaries on Viewpoint: The cardiac contraction cycle: Is Ca²⁺ going local?