Changes In Excitation-contraction Coupling Research Articles

A mouse model of Huntington's disease shows altered ultrastructure of transverse tubules in skeletal muscle fibers.

Huntington's disease (HD) is a fatal and progressive condition with severe debilitating motor defects and muscle weakness. Although classically recognized as a neurodegenerative disorder, there is increasing evidence of cell autonomous toxicity in skeletal muscle. We recently demonstrated that skeletal muscle fibers from the R6/2 model mouse of HD have a decrease in specific membrane capacitance, suggesting a loss of transverse tubule (t-tubule) membrane in R6/2 muscle. A previous report also indicated that Cav1.1 current was reduced in R6/2 skeletal muscle, suggesting defects in excitation-contraction (EC) coupling. Thus, we hypothesized that a loss and/or disruption of the skeletal muscle t-tubule system contributes to changes in EC coupling in R6/2 skeletal muscle. We used live-cell imaging with multiphoton confocal microscopy and transmission electron microscopy to assess the t-tubule architecture in late-stage R6/2 muscle and found no significant differences in the t-tubule system density, regularity, or integrity. However, electron microscopy images revealed that the cross-sectional area of t-tubules at the triad were 25% smaller in R6/2 compared with age-matched control skeletal muscle. Computer simulation revealed that the resulting decrease in the R6/2 t-tubule luminal conductance contributed to, but did not fully explain, the reduced R6/2 membrane capacitance. Analyses of bridging integrator-1 (Bin1), which plays a primary role in t-tubule formation, revealed decreased Bin1 protein levels and aberrant splicing of Bin1 mRNA in R6/2 muscle. Additionally, the distance between the t-tubule and sarcoplasmic reticulum was wider in R6/2 compared with control muscle, which was associated with a decrease in junctophilin 1 and 2 mRNA levels. Altogether, these findings can help explain dysregulated EC coupling and motor impairment in Huntington's disease.

SAH-Induced Electrophysiological Changes of Ventricular Myocytes and Role of N-acetylcysteine Protection.

Electrocardiogram (ECG) changes in patients with subarachnoid hemorrhage (SAH) are frequent. ST- and/or T-wave changes in ECG seem to predominate. To investigate the ion channel mechanisms of SAH-induced ventricular excitation-contraction coupling changes and the possible protective effect of N-acetylcysteine (NAC). Three groups of rabbits were used for the experiments. In two groups, SAH was induced by replacing the cerebrospinal fluid (CSF) with fresh autologous blood. In the control group, CSF was replaced with isotonic saline. In one SAH group, NAC was administered daily beginning at SAH induction. On day 5, ventricular action potentials, ionic currents, contractions, and intracellular free ion concentrations were recorded from the myocytes. In the SAH group, no change was found in the sodium currents, but the transient outward potassium currents were depressed, rapid repolarizing currents were increased, and t-type calcium currents were increased. Contractions and the intracellular free calcium concentration were depressed. NAC treatment, in contrast, not only restores these electrical remodeling changes but also the contractile abnormalities in the cardiac myocytes. The changes in the action potential duration can be attributed to the measured ionic current changes. However, the exact mechanism, other than the oxidative stress, by which the NAC treatment protects the cardiac muscle needs additional investigations.

Synchronized electromechanical integration recording of cardiomyocytes

Cardiac issues are always one of major health problems that attract wide attention by the public. It is urgent to explore a preclinical strategy to efficiently prevent the life-threatening arrhythmias by precisely assessing the cardiac excitation-contraction behavior. Conventional label-free asynchronous strategies are difficult to synchronously record and precisely match the excitation and contraction signals in vitro, while label-based strategies generally present pharmacological adverse effects and phototoxicity that significantly interfere the natural excitation and contraction signals. Both types of strategies preclude to exactly understand how cardiac excitation-contraction coupling changes in quantitative and coherent detail when dysfunctions occur. Here, we show a label-free synchronized electromechanical integration detection strategy that can synchronously monitor electrical and mechanical signals of cardiomyocytes over a long period of time by an integrated microelectrode-interdigitated electrode (ME-IDE). ME-IDE can detect subtle changes in electromechanical integration signals induced by drugs that target excitation-contraction coupling. Moreover, electromechanical integration delay is explored to specifically recognize the sodium channel inhibition. Furthermore, biomimetic electronic pacemaker function provides an alternative way to efficiently assess the drug-induced arrhythmia using refractory period of cardiomyocytes.

Regulation of cardiac excitation-contraction coupling by fibroblasts in health and disease

Introduction: Cardiomyocyte structure and function are influenced directly via cell-cell communication, the ubiquitous extra-cellular matrix (ECM), and paracrine interactions. These interactions modulate cardiomyocytes in health and the changes that occur in disease. Clear mechanisms in these interactions are still lacking, particularly how fibroblasts may affect myocyte excitation-contraction coupling in a human model. Methods: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were co-cultured with human cardiac fibroblasts, isolated from heart failure patients, at a ratio of 2:1 fibroblasts:hiPSC-CMs for 24 hours in three different conditions: (1) hiPSC-CMs in fibroblast-conditioned medium, (2) co-culture with fibroblasts in tissue culture inserts, and (3) fibroblasts in direct contact with hiPSC-CMs. These conditions differentiate the various aspects of cardiomyocyte-fibroblast interaction. Optical mapping experiments were conducted to visualise changes in excitation-contraction coupling. Results: Human fibroblasts-conditioned medium altered excitation-contraction coupling by decreasing calcium transient amplitude and prolonging time to 80% decay vs baseline (p<0.001, -30±2.6% and +6±2.6%). Direct contact with fibroblasts further decreased calcium transient amplitude (p<0.001 vs. baseline, -46±4.3%) and further prolonged time to 80% decay (p<0.001 vs. baseline, +47±8.7%). Soluble Arginine-Glycine-Aspartic acid (RGD) motif-containing peptides present in the attachment site of many ECM proteins reduce the duration of transients and cause hiPSC-CM clustering and detachment from the glass surface substrate. These effects are not attenuated in the presence of anti-β1 or anti-β3-integrin receptor antibodies, but are also likely to prevent integrin receptor activation. Discussion:Human fibroblasts isolated from the ventricles of cardiomyopathy patients modulate calcium transients in hiPSC-CMs. The effects are dependent on whether the interaction is paracrine or direct, and unidirectional or bidirectional. Although it is unclear which interactions play the greatest role in modulating cardiomyocytes, it is accepted that multiple pathways and factors are involved. Further work is needed to identify mechanisms and understand their subsequent consequences in health and in disease.

Neuromuscular Fatigue Is Not Different between Constant and Variable Frequency Stimulation

This study compared fatigue development of the triceps surae induced by two electrical stimulation protocols composed of constant and variable frequency trains (CFTs, VFTs, 450 trains, 30 Hz, 167 ms ON, 500 ms OFF and 146 ms ON, 500 ms OFF respectively). For the VFTs protocol a doublet (100 Hz) was used at the beginning of each train. The intensity used evoked 30% of a maximal voluntary contraction (MVC) and was defined using CFTs. Neuromuscular tests were performed before and after each protocol. Changes in excitation-contraction coupling were assessed by analysing the M-wave [at rest (Mmax) and during MVC (Msup)] and associated peak twitch (Pt). H-reflex [at rest (Hmax) and during MVC (Hsup)] and the motor evoked potential (MEP) during MVC were studied to assess spinal and corticospinal excitability of the soleus muscle. MVC decrease was similar between the protocols (−8%, P<0.05). Mmax, Msup and Pt decreased after both protocols (P<0.01). Hmax/Mmax was decreased (P<0.05), whereas Hsup/Msup and MEP/Msup remained unchanged after both protocols. The results indicate that CFTs and VFTs gave rise to equivalent neuromuscular fatigue. This fatigue resulted from alterations taking place at the muscular level. The finding that cortical and spinal excitability remained unchanged during MVC indicates that spinal and/or supraspinal mechanisms were activated to compensate for the loss of spinal excitability at rest.

Effects of exercise training on excitation–contraction coupling and related mRNA expression in hearts of Goto-Kakizaki type 2 diabetic rats

Although, several novel forms of intervention aiming at newly identified therapeutic targets are currently being developed for diabetes mellitus (DM), it is well established that physical exercise continues to be one of the most valuable forms of non-pharmacological therapy. The aim of the study was to investigate the effects of exercise training on excitation-contraction coupling and related gene expression in the Goto-Kakizaki (GK) type 2 diabetic rat heart and whether exercise is able to reverse diabetes-induced changes in excitation-contraction coupling and gene expression. Experiments were performed in GK and control rats aged 10-11 months following 2-3 months of treadmill exercise training. Shortening, [Ca(2+)]i and L-type Ca(2+) current were measured in ventricular myocytes with video edge detection, fluorescence photometry and whole cell patch clamp techniques, respectively. Expression of mRNA was assessed in ventricular muscle with real-time RT-PCR. Amplitude of shortening, Ca(2+) transients and L-type Ca(2+) current were not significantly altered in ventricular myocytes from GK sedentary compared to control sedentary rats or by exercise training. Expression of mRNA encoding Tpm2, Gja4, Atp1b1, Cacna1g, Cacnb2, Hcn2, Kcna3 and Kcne1 were up-regulated and Gja1, Kcnj2 and Kcnk3 were down-regulated in hearts of sedentary GK rats compared to sedentary controls. Gja1, Cav3 and Kcnk3 were up-regulated and Hcn2 was down-regulated in hearts of exercise trained GK compared to sedentary GK controls. Ventricular myocyte shortening and Ca(2+) transport were generally well preserved despite alterations in the profile of expression of mRNA encoding a variety of cardiac muscle proteins in the adult exercise trained GK diabetic rat heart.

Role of Calsequestrin and Sorcin in the Regulation of Cardiac Excitation Contraction Coupling: A Transcriptional and Physiological Study

In the heart, Ca2+ release through the Ryanodine Receptors (RyR2) is a high integrated process that can be regulated by several accessory proteins. Sorcin (SOR) and Calsequestrin (CSQ2) are two Ca2+ binding proteins that can interact with RyR2 modulating the kinetic properties of this channel. However, at the cellular level the molecular mechanism that underlies this possible regulation is still unclear. In order to address this question we implemented an integrated experimental approach to assess the hypothesis that SOR and CSQ2 regulate the SR Ca2+ release process under physiological conditions. Specifically, we generated transgenic mice by breeding SOR-/- and CSQ2-/- animals. A completed qPCR transcriptional study was carried out in the entire population in order to address if the mRNA levels for other genes were altered during the generation of the murine strains. Interestingly, we found that the mRNA's level for RyR2 and Phospholamban were significantly decreased in CSQ-/- animals. Additionally, subcellular Ca2+ dynamics were evaluated by measuring Ca2+ sparks, Ca2+ transients and restitution experiments in isolated myocytes by means of confocal microscopy under physiological temperatures. Animals in which SOR and CSQ2 genes were simultaneously ablated presented a dramatic increase in the time to peak of Ca2+ sparks (41.2±0.30 vs. 18.9±0.30 ms) and Ca2+ transients (37.8±0.09 vs. 19.5±0.12 ms). Moreover, the double ablation of SOR and CSQ2 significantly changed the refractoriness of Ca2+ release. Summarizing, the data presented here indicates that although the SOR-/- itself cannot produce any changes in excitation contraction coupling, in combination with the CSQ2-/- can promote significant changes in the Ca2+. However, most of the alterations observed in the double KOs’ were not significantly different than those observed for the CSQ2-/- itself. Supported by R01-HL-084487 to AE.

Stochastic Simulation Assessing the Functional Changes Occurring during Heart Failure

Heart failure has a high prevalence in the general population and mortality rates that remain excessive. Heart failure is characterized by a the loss of the ability of the heart to pump blood and an increased propensity for fatal arrhythmias. Changes in excitation-contraction coupling occur in heart failure and involve the down regulation of sarcolemmal potassium channels, increased expression of the sodium-calcium exchanger, decreased SR calcium ATPase expression, as well are remodeling of the T-tubule network. These changes are aggravated by chronically increased beta-adrenergic stimulation which has been characterized as a maladaptive mechanism. In HF, a chronic hyperadrenerigic state results in chronic hyperphosphorlyation of the cardiac ryanodine receptor leading to increased sensitivity for calcium dependent channel activation. We have developed a computational model of excitation-contraction coupling (that improves upon Williams et al., 2011) based on the rat ventricular myocyte that includes the 20,000 calcium release units, each containing a cluster of 49 ryanodine receptors and 7 L-type calcium channels both of which gate stochastically. The model also includes the membrane currents that govern the action potential and the remaining components of calcium dynamics. The model is solved using our Ultra-fast Monte Carlo Algorithm and GPGPUs that enable this massive calculation to be solved efficiently. By implement changes in ion channel and transporter behavior in the model while also including the spatial re-organization of the TT system, we are able to examine how action potentials and Ca2+ signaling changes in HF. This work reveals how cellular Ca2+ instability arises and how late diastolic calcium release events (i.e. Ca2+ sparks) and increased diastolic leak underlie the propensity for development of arrhythmogenic calcium waves at the cellular level.

Role of STIM1 in Skeletal Excitation-Contraction Coupling

STIM1, a Ca2+-sensing protein on ER/SR membrane, mediates a store-operated Ca2+-entry (SOCE) by activating Orai1 Ca2+-entry channel on plasma membrane. E136X mutant of STIM1, truncated STIM1 missing binding abilities to Orai1, has been found in patients with immunodeficiency accompanying muscular hypotonia. To identify causes of the muscular hypotonia, E136X was expressed in mouse skeletal myotubes and dominant-negative effects of E136X were examined. Myotubes expressing E136X showed increases in both KCl (a membrane depolarizer resulting excitation-contraction coupling) and caffeine (a direct RyR1 agonist) responses. On the other hand, SOCE, resting cytosolic Ca2+ level, SR Ca2+ level were not significantly changed by the expression of E136X. In addition, E136X did not interfere with puncta formations by endogenous STIM1 and Orai1. These data mean that muscular hypotonia found in patients with E136X is due to changes in excitation-contraction coupling. Additionally, we suggest that C-terminus of STIM1 that is missing in E136X participates in the regulation of skeletal EC coupling.

Calcitonin gene‐related peptide restores disrupted excitation–contraction coupling in myotubes expressing central core disease mutations in RyR1

Central core disease (CCD) is a congenital human myopathy associated with mutations in the gene encoding the skeletal muscle ryanodine receptor (RyR1), resulting in skeletal muscle weakness and lower limb deformities. The muscle weakness can be at least partially explained by a reduced magnitude of voltage-gated Ca(2+) release (VGCR). To date, only a few studies have focused on identifying potential therapeutic agents for CCD. Therefore, in this work we investigated the potential use of the calcitonin gene related peptide (CGRP) to restore VGCR in myotubes expressing CCD RyR1 mutants. We also examined the influence of CCD mutants on Ca(2+)-dependent processes involved in myogenesis (myoblast fusion and sarcoendoplasmic reticulum Ca(2+)-ATPase isoform 2 (SERCA2) gene expression). C2C12 cells were transfected with cDNAs encoding either wild-type RyR1 or CCD mutants, and then exposed to CGRP (100 nm, 1-4 h). Expression of the I4897T mutant significantly inhibited SERCA2 gene expression and myoblast fusion, whereas the Y523S mutant exerted the opposite effect. Interestingly, both mutants clearly inhibited VGCR (50%), due to a reduction in SR Ca(2+) content. However, no major changes due to CGRP or CCD mutants were observed in I(CaL). Our data suggest that the Y523S mutant results in store depletion via decompensated SR Ca(2+) leak, while the I4897T mutant inhibits SERCA2 gene expression. Remarkably, in both cases CGRP restored VGCR, likely to have been by enhancing phospholamban (PLB) phosphorylation, SERCA activity and SR Ca(2+) content. Taken together, our data show that in the C2C12 model system, changes in excitation-contraction coupling induced by the expression of RyR1 channels bearing CCD mutations Y523S or I4897T can be reversed by CGRP.

Ouabain is a Pharmacomimic of Mutant RyR2 Ca2+ Release Dysfunction but is not a Serum-Borne Trigger of CPVT

Mutation-linked RyR2 Ca2+ handling dysfunction is an intracellular driver for stress-evoked arrhythmias. Disease pathogenesis links augmented β-adrenergic stimulation to the acute dysfunction of mutant RyR2. However, the incomplete efficacy of β-blockade in clinical and experimental scenarios suggests that other serum-borne factors, in addition to catecholamines, contribute to Ca2+ release dysfunction. Cardiac electrical dysfunction in CPVT is remarkably similar to that elicited by digitalis toxicity and a related glycoside, ouabain, has been identified as an endogenous Ca2+-mobilising ‘hormone’. Circulating levels of adrenally-derived ouabain are reportedly elevated in heart failure and post-exercise. Here we investigated whether physiologic levels of ouabain could provoke mutant RyR2 channel dysfunction. In single HL-1 cardiomyocytes, ouabain (EC50 = 14.3nM) perturbed Ca2+ homeostasis in a manner strikingly similar to that determined in cells expressing CPVT-linked R4497C mutation. Moreover, dose-dependent ouabain-evoked Ca2+ dysfunction resulted in a left-shifted caffeine activation profile typical of mutant RyR2 channels. LC/MS/MS analysis determined equivalent levels of ouabain in serum obtained from CPVT- and normal subjects (500-800pM) and exercise stress-testing did not acutely elevate ouabain levels in either cohort. In cells expressing the R4497C mutation, which exhibited basal Ca2+ dysfunction, addition of ouabain at physiologic (1nM) and supra-physiologic (10-100nM) concentrations did not further exacerbate cellular Ca2+ abnormalities. In contrast, the addition of serum obtained from exercised normal and CPVT subjects (100ug protein equivalents; ∼2pM ouabain) evoked hallmark mutation-linked RyR2 dysfunction that was not completely abolished by β-blockade. These findings confirm that non-catecholaminergic factors contribute to RyR2 mutant dysfunction, but preclude the possibility that endogenous ouabain is a physiologically relevant trigger of CPVT. However, the hypothesis that endogenous ouabain may induce underlying changes in excitation-contraction coupling that increase the propensity for mutation-linked RyR2 dysfunction is being investigated.

Age-associated changes in excitation-contraction coupling are more prominent in ventricular myocytes from male rats than in myocytes from female rats

We evaluated effects of age on components of excitation-contraction (EC) coupling in ventricular myocytes from male and female rats to examine sex differences in mechanisms responsible for age-related contractile dysfunction. Myocytes were isolated from anesthetized young adult (approximately 3 mo) and aged (approximately 24 mo) Fischer 344 rats. Ca(2+) concentrations and contractions were measured simultaneously (37 degrees C, 2 Hz). Fractional shortening declined with age in males (6.7 +/- 0.6% to 2.4 +/- 0.4%; P < 0.05), as did peak Ca(2+) transients (47.7 +/- 4.6 to 28.1 +/- 2.1 nM; P < 0.05) and Ca(2+) current densities (-7.7 +/- 0.7 to -6.2 +/- 0.5 pA/pF; P < 0.05). Although sarcoplasmic reticulum (SR) Ca(2+) content was similar regardless of age in males, EC coupling gain declined significantly with age to 55.8 +/- 7.8% of values in younger males. In contrast with results in males, contraction and Ca(2+) transient amplitudes were unaffected by age in females. Ca(2+) current density declined with age in females (-7.5 +/- 0.5 to -5.1 +/- 0.7 pA/pF; P < 0.05), but SR Ca(2+) content actually increased dramatically (49.0 +/- 7.5 to 147.3 +/- 28.5 nM; P < 0.05). Even so, EC coupling gain was not affected by age in female myocytes. Age also promoted hypertrophy of male myocytes more than female myocytes. Age and sex differences in EC coupling were largely maintained when conditioning pulse frequency was increased to 4 Hz. Contractions, Ca(2+) transients, and EC coupling gain were also smaller in young females than in young males. Thus age-dependent changes are more prominent in myocytes from males than females. Increased SR Ca(2+) content may compensate for reduced Ca(2+) current to preserve contractile function in aged females, which may limit the detrimental effects of age on cardiac contractile function.

Excitation–contraction coupling changes during postnatal cardiac development

Cardiac contraction is initiated by the release of Ca 2+ from intracellular stores in response to an action potential, in a process known as “excitation–contraction coupling” (ECC). Here we investigate the maturation of ECC in the rat heart during postnatal development. We provide new information on how proteins of the sarcoplasmic reticulum (SR) and the t-tubules (TTs) assemble to form the structures that support EC coupling during postnatal development. We show that the surface membrane protein, caveolin-3 (Cav3), is a good protein marker for TTs in ventricular myocytes and compared it quantitatively to junctophilin-2 (JP2), a protein found on the SR at sites of SR-TT junctions, or couplons. Although JP2 and Cav3 associate primarily with the SR and TTs, respectively, we found that they occupy the appropriate sites at maturing structures in synchrony, as visualized with high resolution, quantitative 3-dimensional imaging. We also found the surprising result that while both ryanodine receptor type 2, (RyR2) and JP2 proteins are localized to the same membrane and sub-compartments, they assume their positions at very different rates: RyR2 moves to the SR membrane at the Z-disc very early in development while JP2 only appears in the SR membrane as the TTs mature. Our data suggest that, although RyR2 appears to be prepositioned at the sites ultimately occupied by dyad junctions, JP2 arrives at these sites in synchrony with the development of the TTs at the Z-discs. Finally, we report that EC coupling efficiency changes with development, in concert with these structural changes. Thus we provide the first well-integrated information that links the developing organization of proteins underlying EC coupling (RyR2, DHPR, Cav3 and JP2) to the developing efficacy of EC coupling.

Interval Training Normalizes Cardiomyocyte Function, Diastolic Ca 2+ Control, and SR Ca 2+ Release Synchronicity in a Mouse Model of Diabetic Cardiomyopathy

In the present study we explored the mechanisms behind excitation-contraction (EC) coupling defects in cardiomyocytes from mice with type-2 diabetes (db/db). We determined whether 13 weeks of aerobic interval training could restore cardiomyocyte Ca(2+) cycling and EC coupling. Reduced contractility in cardiomyocytes isolated from sedentary db/db was associated with increased diastolic sarcoplasmic reticulum (SR)-Ca(2+) leak, reduced synchrony of Ca(2+) release, reduced transverse (T)-tubule density, and lower peak systolic and diastolic Ca(2+) and caffeine-induced Ca(2+) release. Additionally, the rate of SR Ca(2+) ATPase-mediated Ca(2+) uptake during diastole was reduced, whereas a faster recovery from caffeine-induced Ca(2+) release indicated increased Na(+)/Ca(2+)-exchanger activity. The increased SR-Ca(2+) leak was attributed to increased Ca(2+)-calmodulin-dependent protein kinase (CaMKIIdelta) phosphorylation, supported by the normalization of SR-Ca(2+) leak on inhibition of CaMKIIdelta (AIP). Exercise training restored contractile function associated with restored SR Ca(2+) release synchronicity, T-tubule density, twitch Ca(2+) amplitude, SR Ca(2+) ATPase and Na(+)/Ca(2+)-exchanger activities, and SR-Ca(2+) leak. The latter was associated with reduced phosphorylation of cytosolic CaMKIIdelta. Despite normal contractile function and Ca(2+) handling after the training period, phospholamban was hyperphosphorylated at Serine-16. Protein kinase A inhibition (H-89) in cardiomyocytes from the exercised db/db group abolished the differences in SR-Ca(2+) load when compared with the sedentary db/db mice. EC coupling changes were observed without changes in serum insulin or glucose levels, suggesting that the exercise training-induced effects are not via normalization of the diabetic condition. These data demonstrate that aerobic interval training almost completely restored the contractile function of the diabetic cardiomyocyte to levels close to sedentary wild type.

Excitation–contraction coupling gain in ventricular myocytes: insights from a parsimonious model

We present a minimal mathematical model of Ca(2+) spark triggering under voltage-clamp conditions in ventricular myocytes. The model predicts changes in excitation-contraction coupling 'gain' that result from diverse experimental interventions. We compare model results to several sets of data, and, in so doing, place apparent constraints on physiologically relevant model parameters. Specifically, the analysis suggests that many L-type Ca(2+) channel openings can potentially trigger each Ca(2+) spark, but the probability that an individual opening will trigger a spark is low. This procedure helps to reconcile contradictory results obtained in recent studies; moreover, this new model should be a useful tool for understanding changes in gain that occur physiologically and in disease.

Intracellular devastation in heart failure

End-stage heart failure is characterized by a number of abnormalities at the cellular level, which include changes in excitation-contraction coupling, alterations in contractile proteins and activation/deactivation of signaling pathways. Even though many of these changes are adaptive to the high workload and stress in heart failure, a significant number of these alterations are deeply deleterious to the cardiac cell. In this article, we will review the changes in calcium cycling that occur in myopathic hearts and how they can be effectively targeted. We will also focus on protein misfolding in the setting of cardiac dysfunction.

Calmodulin kinase II inhibition protects against structural heart disease

Beta-adrenergic receptor (betaAR) stimulation increases cytosolic Ca(2+) to physiologically augment cardiac contraction, whereas excessive betaAR activation causes adverse cardiac remodeling, including myocardial hypertrophy, dilation and dysfunction, in individuals with myocardial infarction. The Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) is a recently identified downstream element of the betaAR-initiated signaling cascade that is linked to pathological myocardial remodeling and to regulation of key proteins involved in cardiac excitation-contraction coupling. We developed a genetic mouse model of cardiac CaMKII inhibition to test the role of CaMKII in betaAR signaling in vivo. Here we show CaMKII inhibition substantially prevented maladaptive remodeling from excessive betaAR stimulation and myocardial infarction, and induced balanced changes in excitation-contraction coupling that preserved baseline and betaAR-stimulated physiological increases in cardiac function. These findings mark CaMKII as a determinant of clinically important heart disease phenotypes, and suggest CaMKII inhibition can be a highly selective approach for targeting adverse myocardial remodeling linked to betaAR signaling.

Cardiomyocyte calcium and calcium/calmodulin-dependent protein kinase II: friends or foes?

Calcium (Ca(2+)) is a critical second messenger in cell signaling. Elevated intracellular Ca(2+) can activate numerous Ca(2+)-regulated enzymes. These enzymes have different subcellular localizations and may respond to distinct modes of Ca(2+) mobilization. In cardiac muscle, Ca(2+) plays a central role in regulating contractility, gene expression, hypertrophy, and apoptosis. Many cellular responses to Ca(2+) signals are mediated by Ca(2+)/calmodulin-dependent enzymes, among which is the Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Putative substrates for CaMKII include proteins involved in regulating Ca(2+) storage and release, transcription factors, and ion channels. The major isoform of CaMKII in the heart is CaMKIIdelta. Two cardiac splice variants, CaMKIIdelta(B) and delta(C), differ in whether they contain a nuclear localization sequence. Our laboratory has examined the hypothesis that the nuclear delta(B) and the cytoplasmic delta(C) isoforms respond to different Ca(2+) stimuli and have distinct effects on hypertrophic cardiac growth and Ca(2+) handling. We have shown that pressure overload-induced hypertrophy differentially affects the nuclear delta(B) and the cytoplasmic delta(C) isoforms of CaMKII. Additionally, using isolated myocytes and transgenic mouse models, we demonstrated that the nuclear CaMKIIdelta(B) isoform plays a key role in cardiac gene expression associated with cardiac hypertrophy. The cytoplasmic CaMKIIdelta(C) isoform phosphorylates substrates involved in Ca(2+) handling. Dysregulation of intracellular Ca(2+) and resulting changes in excitation-contraction coupling characterize heart failure and can be induced by in vivo overexpression of CaMKIIdelta(C) and phosphorylation of its substrates. The differential location of CaMKII isoforms and their relative activation by physiological vs. pathological stimuli may provide a paradigm for exploring and elucidating how Ca(2+)/CaMKII pathways can serve as both friends and foes in the heart.

Exercise-induced muscle damage in humans.

Exercise-induced muscle injury in humans frequently occurs after unaccustomed exercise, particularly if the exercise involves a large amount of eccentric (muscle lengthening) contractions. Direct measures of exercise-induced muscle damage include cellular and subcellular disturbances, particularly Z-line streaming. Several indirectly assessed markers of muscle damage after exercise include increases in T2 signal intensity via magnetic resonance imaging techniques, prolonged decreases in force production measured during both voluntary and electrically stimulated contractions (particularly at low stimulation frequencies), increases in inflammatory markers both within the injured muscle and in the blood, increased appearance of muscle proteins in the blood, and muscular soreness. Although the exact mechanisms to explain these changes have not been delineated, the initial injury is ascribed to mechanical disruption of the fiber, and subsequent damage is linked to inflammatory processes and to changes in excitation-contraction coupling within the muscle. Performance of one bout of eccentric exercise induces an adaptation such that the muscle is less vulnerable to a subsequent bout of eccentric exercise. Although several theories have been proposed to explain this "repeated bout effect," including altered motor unit recruitment, an increase in sarcomeres in series, a blunted inflammatory response, and a reduction in stress-susceptible fibers, there is no general agreement as to its cause. In addition, there is controversy concerning the presence of sex differences in the response of muscle to damage-inducing exercise. In contrast to the animal literature, which clearly shows that females experience less damage than males, research using human studies suggests that there is either no difference between men and women or that women are more prone to exercise-induced muscle damage than are men.

Changes in excitation-contraction coupling in an isolated ventricular myocyte model of cardiac stunning.

To investigate cardiac stunning, we recorded intracellular [Ca(2+)], contractions, and electrical activity in isolated guinea pig ventricular myocytes exposed to simulated ischemia and reperfusion. After equilibration, ischemia was simulated by exposing myocytes to hypoxia, acidosis, hyperkalemia, hypercapnia, lactate accumulation, and substrate deprivation for 30 min at 37 degrees C. Reperfusion was simulated by exposure to Tyrode solution. Field-stimulated myocytes exhibited stunning upon reperfusion. By 10 min of reperfusion, contraction amplitude decreased to 43.0 +/- 5.5% of preischemic values (n = 15, P < 0.05), although action potential configuration and sarcoplasmic reticulum Ca(2+) stores, assessed with caffeine, were normal. Diastolic [Ca(2+)] and Ca(2+) transients (fura 2) were also normal in stunned myocytes. In voltage-clamped cells, peak L-type Ca(2+) current was reduced to 47.4 +/- 4.5% of preischemic values at 10 min of reperfusion (n = 21, P < 0.05). Contractions elicited by Ca(2+)-induced Ca(2+) release and the voltage-sensitive release mechanism were both depressed in reperfusion. Our observations suggest that stunning is associated with reduced L-type Ca(2+) current but that alterations in Ca(2+) homeostasis and release are not directly responsible for stunning.