Calcium Channelopathies: Structural Insights into Disorders of the Muscle Excitation-Contraction Complex.

Ion channels are membrane proteins responsible for the passage of ions down their electrochemical gradients and across biological membranes. In this, they generate and shape action potentials and provide secondary messengers for various signaling pathways. They are often part of larger complexes containing auxiliary subunits and regulatory proteins. Channelopathies arise from mutations in the genes encoding ion channels or their associated proteins. Recent advances in cryo-electron microscopy have resulted in an explosion of ion channel structures in multiple states, generating a wealth of new information on channelopathies. Disease-associated mutations fall into different categories, interfering with ion permeation, protein folding, voltage sensing, ligand and protein binding, and allosteric modulation of channel gating. Prime examples of these are Ca2+-selective channels expressed in myocytes, for which multiple structures in distinct conformational states have recently been uncovered. We discuss the latest insights into these calcium channelopathies from a structural viewpoint.

BK channel density is regulated by endoplasmic reticulum associated degradation and influenced by the SKN-1A/NRF1 transcription factor.

Ion channels are present at specific levels within subcellular compartments of excitable cells. The regulation of ion channel trafficking and targeting is an effective way to control cell excitability. The BK channel is a calcium-activated potassium channel that serves as a negative feedback mechanism at presynaptic axon terminals and sites of muscle excitation. The C. elegans BK channel ortholog, SLO-1, requires an endoplasmic reticulum (ER) membrane protein for efficient anterograde transport to these locations. Here, we found that, in the absence of this ER membrane protein, SLO-1 channels that are seemingly normally folded and expressed at physiological levels undergo SEL-11/HRD1-mediated ER-associated degradation (ERAD). This SLO-1 degradation is also indirectly regulated by a SKN-1A/NRF1-mediated transcriptional mechanism that controls proteasome levels. Therefore, our data indicate that SLO-1 channel density is regulated by the competitive balance between the efficiency of ER trafficking machinery and the capacity of ERAD.

Loss of Mitochondrial Ca2+ Uniporter Limits Inotropic Reserve and Provides Trigger and Substrate for Arrhythmias in Barth Syndrome Cardiomyopathy.

BACKGROUND: Barth syndrome (BTHS) is caused by mutations of the gene encoding tafazzin, which catalyzes maturation of mitochondrial cardiolipin and often manifests with systolic dysfunction during early infancy. Beyond the first months of life, BTHS cardiomyopathy typically transitions to a phenotype of diastolic dysfunction with preserved ejection fraction, blunted contractile reserve during exercise, and arrhythmic vulnerability. Previous studies traced BTHS cardiomyopathy to mitochondrial formation of reactive oxygen species (ROS). Because mitochondrial function and ROS formation are regulated by excitation-contraction coupling, integrated analysis of mechano-energetic coupling is required to delineate the pathomechanisms of BTHS cardiomyopathy. METHODS: We analyzed cardiac function and structure in a mouse model with global knockdown of tafazzin (Taz-KD) compared with wild-type littermates. Respiratory chain assembly and function, ROS emission, and Ca2+ uptake were determined in isolated mitochondria. Excitation-contraction coupling was integrated with mitochondrial redox state, ROS, and Ca2+ uptake in isolated, unloaded or preloaded cardiac myocytes, and cardiac hemodynamics analyzed in vivo. RESULTS: Taz-KD mice develop heart failure with preserved ejection fraction (>50%) and age-dependent progression of diastolic dysfunction in the absence of fibrosis. Increased myofilament Ca2+ affinity and slowed cross-bridge cycling caused diastolic dysfunction, in part, compensated by accelerated diastolic Ca2+ decay through preactivated sarcoplasmic reticulum Ca2+-ATPase. Taz deficiency provoked heart-specific loss of mitochondrial Ca2+ uniporter protein that prevented Ca2+-induced activation of the Krebs cycle during ß-adrenergic stimulation, oxidizing pyridine nucleotides and triggering arrhythmias in cardiac myocytes. In vivo, Taz-KD mice displayed prolonged QRS duration as a substrate for arrhythmias, and a lack of inotropic response to ß-adrenergic stimulation. Cellular arrhythmias and QRS prolongation, but not the defective inotropic reserve, were restored by inhibiting Ca2+ export through the mitochondrial Na+/Ca2+ exchanger. All alterations occurred in the absence of excess mitochondrial ROS in vitro or in vivo. CONCLUSIONS: Downregulation of mitochondrial Ca2+ uniporter, increased myofilament Ca2+ affinity, and preactivated sarcoplasmic reticulum Ca2+-ATPase provoke mechano-energetic uncoupling that explains diastolic dysfunction and the lack of inotropic reserve in BTHS cardiomyopathy. Furthermore, defective mitochondrial Ca2+ uptake provides a trigger and a substrate for ventricular arrhythmias. These insights can guide the ongoing search for a cure of this orphaned disease.

Striated Preferentially Expressed Protein Kinase (SPEG) in Muscle Development, Function, and Disease.

Mutations in striated preferentially expressed protein kinase (SPEG), a member of the myosin light chain kinase protein family, are associated with centronuclear myopathy (CNM), cardiomyopathy, or a combination of both. Burgeoning evidence suggests that SPEG plays critical roles in the development, maintenance, and function of skeletal and cardiac muscles. Here we review the genotype-phenotype relationships and the molecular mechanisms of SPEG-related diseases. This review will focus on the progress made toward characterizing SPEG and its interacting partners, and its multifaceted functions in muscle regeneration, triad development and maintenance, and excitation-contraction coupling. We will also discuss future directions that are yet to be investigated including understanding of its tissue-specific roles, finding additional interacting proteins and their relationships. Understanding the basic mechanisms by which SPEG regulates muscle development and function will provide critical insights into these essential processes and help identify therapeutic targets in SPEG-related disorders.

Evolution of Excitation-Contraction Coupling.

In mammalian cardiomyocytes, Ca2+ influx through L-type voltage-gated Ca2+ channels (VGCCs) is amplified by release of Ca2+ via type 2 ryanodine receptors (RyR2) in the sarcoplasmic reticulum (SR): a process termed Ca2+-induced Ca2+-release (CICR). In mammalian skeletal muscles, VGCCs play a distinct role as voltage-sensors, physically interacting with RyR1 channels to initiate Ca2+ release in a mechanism termed depolarisation-induced Ca2+-release (DICR). In the current study, we surveyed the genomes of animals and their close relatives, to explore the evolutionary history of genes encoding three proteins pivotal for ECC: L-type VGCCs; RyRs; and a protein family that anchors intracellular organelles to plasma membranes, namely junctophilins (JPHs). In agreement with earlier studies, we find that non-vertebrate eukaryotes either lack VGCCs, RyRs and JPHs; or contain a single homologue of each protein. Furthermore, the molecular features of these proteins thought to be essential for DICR are only detectable within vertebrates and not in any other taxonomic group. Consistent with earlier physiological and ultrastructural observations, this suggests that CICR is the most basal form of ECC and that DICR is a vertebrate innovation. This development was accompanied by the appearance of multiple homologues of RyRs, VGCCs and junctophilins in vertebrates, thought to have arisen by 'whole genome replication' mechanisms. Subsequent gene duplications and losses have resulted in distinct assemblies of ECC components in different vertebrate clades, with striking examples being the apparent absence of RyR2 from amphibians, and additional duplication events for all three ECC proteins in teleost fish. This is consistent with teleosts possessing the most derived mode of DICR, with their Cav1.1 VGCCs completely lacking in Ca2+ channel activity.

Na+ microdomains and sparks: Role in cardiac excitation-contraction coupling and arrhythmias in ankyrin-B deficiency.

Cardiac sodium (Na+) potassium ATPase (NaK) pumps, neuronal sodium channels (INa), and sodium calcium (Ca2+) exchangers (NCX1) may co-localize to form a Na+ microdomain. It remains controversial as to whether neuronal INa contributes to local Na+ accumulation, resulting in reversal of nearby NCX1 and influx of Ca2+ into the cell. Therefore, there has been great interest in the possible roles of a Na+ microdomain in cardiac Ca2+-induced Ca2+ release (CICR). In addition, the important role of co-localization of NaK and NCX1 in regulating localized Na+ and Ca2+ levels and CICR in ankyrin-B deficient (ankyrin-B+/-) cardiomyocytes has been examined in many recent studies. Altered Na+ dynamics may contribute to the appearance of arrhythmias, but the mechanisms underlying this relationship remain unclear. In order to investigate this, we present a mechanistic canine cardiomyocyte model which reproduces independent local dyadic junctional SR (JSR) Ca2+ release events underlying cell-wide excitation-contraction coupling, as well as a three-dimensional super-resolution model of the Ca2+ spark that describes local Na+ dynamics as governed by NaK pumps, neuronal INa, and NCX1. The model predicts the existence of Na+ sparks, which are generated by NCX1 and exhibit significantly slower dynamics as compared to Ca2+ sparks. Moreover, whole-cell simulations indicate that neuronal INa in the cardiac dyad plays a key role during the systolic phase. Rapid inward neuronal INa can elevate dyadic [Na+] to 35-40 mM, which drives reverse-mode NCX1 transport, and therefore promotes Ca2+ entry into the dyad, enhancing the trigger for JSR Ca2+ release. The specific role of decreased co-localization of NaK and NCX1 in ankyrin-B+/- cardiomyocytes was examined. Model results demonstrate that a reduction in the local NCX1- and NaK-mediated regulation of dyadic [Ca2+] and [Na+] results in an increase in Ca2+ spark activity during isoproterenol stimulation, which in turn stochastically activates NCX1 in the dyad. This alteration in NCX1/NaK co-localization interrupts the balance between NCX1 and NaK currents in a way that leads to enhanced depolarizing inward current during the action potential plateau, which ultimately leads to a higher probability of L-type Ca2+ channel reopening and arrhythmogenic early-afterdepolarizations.

Synergic PDE3 and PDE4 control intracellular cAMP and cardiac excitation-contraction coupling in a porcine model.

AIMS: Cyclic AMP phosphodiesterases (PDEs) are important modulators of the cardiac response to ß-adrenergic receptor (ß-AR) stimulation. PDE3 is classically considered as the major cardiac PDE in large mammals and human, while PDE4 is preponderant in rodents. However, it remains unclear whether PDE4 also plays a functional role in large mammals. Our purpose was to understand the role of PDE4 in cAMP hydrolysis and excitation-contraction coupling (ECC) in the pig heart, a relevant pre-clinical model. METHODS AND RESULTS: Real-time cAMP variations were measured in isolated adult pig right ventricular myocytes (APVMs) using a Förster resonance energy transfer (FRET) biosensor. ECC was investigated in APVMs loaded with Fura-2 and paced at 1 Hz allowing simultaneous measurement of intracellular Ca2+ and sarcomere shortening. The expression of the different PDE4 subfamilies was assessed by Western blot in pig right ventricles and APVMs. Similarly to PDE3 inhibition with cilostamide (Cil), PDE4 inhibition with Ro 20-1724 (Ro) increased cAMP levels and inotropy under basal conditions. PDE4 inhibition enhanced the effects of the non-selective ß-AR agonist isoprenaline (Iso) and the effects of Cil, and increased spontaneous diastolic Ca2+ waves (SCWs) in these conditions. PDE3A, PDE4A, PDE4B and PDE4D subfamilies are expressed in pig ventricles. In APVMs isolated from a porcine model of repaired tetralogy of Fallot which leads to right ventricular failure, PDE4 inhibition also exerts inotropic and pro-arrhythmic effects. CONCLUSIONS: Our results show that PDE4 controls ECC in APVMs and suggest that PDE4 inhibitors exert inotropic and pro-arrhythmic effects upon PDE3 inhibition or ß-AR stimulation in our pre-clinical model. Thus, PDE4 inhibitors should be used with caution in clinics as they may lead to arrhythmogenic events upon stress.

Restricting calcium currents is required for correct fiber type specification in skeletal muscle.

Skeletal muscle excitation-contraction (EC) coupling is independent of calcium influx. In fact, alternative splicing of the voltage-gated calcium channel CaV1.1 actively suppresses calcium currents in mature muscle. Whether this is necessary for normal development and function of muscle is not known. However, splicing defects that cause aberrant expression of the calcium-conducting developmental CaV1.1e splice variant correlate with muscle weakness in myotonic dystrophy. Here, we deleted CaV1.1 (Cacna1s) exon 29 in mice. These mice displayed normal overall motor performance, although grip force and voluntary running were reduced. Continued expression of the developmental CaV1.1e splice variant in adult mice caused increased calcium influx during EC coupling, altered calcium homeostasis, and spontaneous calcium sparklets in isolated muscle fibers. Contractile force was reduced and endurance enhanced. Key regulators of fiber type specification were dysregulated and the fiber type composition was shifted toward slower fibers. However, oxidative enzyme activity and mitochondrial content declined. These findings indicate that limiting calcium influx during skeletal muscle EC coupling is important for the secondary function of the calcium signal in the activity-dependent regulation of fiber type composition and to prevent muscle disease.

STAC3 incorporation into skeletal muscle triads occurs independent of the dihydropyridine receptor.

Excitation-contraction (EC) coupling in skeletal muscles operates through a physical interaction between the dihydropyridine receptor (DHPR), acting as a voltage sensor, and the ryanodine receptor (RyR1), acting as a calcium release channel. Recently, the adaptor protein SH3 and cysteine-rich containing protein 3 (STAC3) has been identified as a myopathy disease gene and as an additional essential EC coupling component. STAC3 interacts with DHPR sequences including the critical EC coupling domain and has been proposed to function in linking the DHPR and RyR1. However, we and others demonstrated that incorporation of recombinant STAC3 into skeletal muscle triads critically depends only on the DHPR but not the RyR1. On the contrary, here, we provide evidence that endogenous STAC3 incorporates into triads in the absence of the DHPR in myotubes and muscle fibers of dysgenic mice. This finding demonstrates that STAC3 interacts with additional triad proteins and is consistent with its proposed role in directly or indirectly linking the DHPR with the RyR1.

Ablation of the cardiac ryanodine receptor phospho-site Ser2808 does not alter the adrenergic response or the progression to heart failure in mice. Elimination of the genetic background as critical variable.

BACKGROUND: Phosphorylation of the cardiac ryanodine receptor (RyR2) phospho-site S2808 has been touted by the Marks group as a hallmark of heart failure (HF) and a critical mediator of the physiological fight-or-flight response of the heart. In support of this hypothesis, mice unable to undergo phosphorylation at RyR2-S2808 (S2808A) were significantly protected against HF and displayed a blunted response to adrenergic stimulation. However, the issue remains highly controversial because several groups have been unable to reproduce these findings. An important variable not considered before is the genetic background of the mice used to obtain these divergent results. METHODS AND RESULTS: We backcrossed a RyR2-S2808A mouse into a congenic C57Bl/6 strain, the same strain used by the Marks group to conduct their experiments. We then performed several key experiments to confirm or discard the genetic background of the mouse as a relevant variable, including induction of HF by myocardial infarction and tests of integrity of adrenergic response. Congenic C57Bl/6 harboring the S2808A mutation showed similar echocardiographic parameters that indicated identical progression towards HF compared to wild type controls, and had a normal response to adrenergic stimulation in whole animal and cellular experiments. CONCLUSIONS: The genetic background of the different mouse models is unlikely to be the source of the divergent results obtained by the Marks group in comparison to several other groups. Cardiac adrenergic response and progression towards HF proceed unaltered in mice harboring the RyR2-S2808A mutation. Preventing RyR2-S2808 phosphorylation does not preclude a normal sympathetic response nor mitigates the pathophysiological consequences of MI.

Gene Therapy in Heart Failure.

Heart failure is a significant burden to the global healthcare system and represents an underserved market for new pharmacologic strategies, especially therapies which can address root cause myocyte dysfunction. Modern drugs, surgeries, and state-of-the-art interventions are costly and do not improve survival outcome measures. Gene therapy is an attractive strategy, whereby selected gene targets and their associated regulatory mechanisms can be permanently managed therapeutically in a single treatment. This in theory could be sustainable for the patient's life. Despite the promise, however, gene therapy has numerous challenges that must be addressed together as a treatment plan comprising these key elements: myocyte physiologic target validation, gene target manipulation strategy, vector selection for the correct level of manipulation, and carefully utilizing an efficient delivery route that can be implemented in the clinic to efficiently transfer the therapy within safety limits. This chapter summarizes the key developments in cardiac gene therapy from the perspective of understanding each of these components of the treatment plan. The latest pharmacologic gene targets, gene therapy vectors, delivery routes, and strategies are reviewed.

Decreased polycystin 2 expression alters calcium-contraction coupling and changes ß-adrenergic signaling pathways.

Cardiac disorders are the main cause of mortality in autosomal-dominant polycystic kidney disease (ADPKD). However, how mutated polycystins predispose patients with ADPKD to cardiac pathologies before development of renal dysfunction is unknown. We investigate the effect of decreased levels of polycystin 2 (PC2), a calcium channel that interacts with the ryanodine receptor, on myocardial function. We hypothesize that heterozygous PC2 mice (Pkd2(+/-)) undergo cardiac remodeling as a result of changes in calcium handling, separate from renal complications. We found that Pkd2(+/-) cardiomyocytes have altered calcium handling, independent of desensitized calcium-contraction coupling. Paradoxically, in Pkd2(+/-) mice, protein kinase A (PKA) phosphorylation of phospholamban (PLB) was decreased, whereas PKA phosphorylation of troponin I was increased, explaining the decoupling between calcium signaling and contractility. In silico modeling supported this relationship. Echocardiography measurements showed that Pkd2(+/-) mice have increased left ventricular ejection fraction after stimulation with isoproterenol (ISO), a ß-adrenergic receptor (ßAR) agonist. Blockers of ßAR-1 and ßAR-2 inhibited the ISO response in Pkd2(+/-) mice, suggesting that the dephosphorylated state of PLB is primarily by ßAR-2 signaling. Importantly, the Pkd2(+/-) mice were normotensive and had no evidence of renal cysts. Our results showed that decreased PC2 levels shifted the ßAR pathway balance and changed expression of calcium handling proteins, which resulted in altered cardiac contractility. We propose that PC2 levels in the heart may directly contribute to cardiac remodeling in patients with ADPKD in the absence of renal dysfunction.

Ryanodine receptor sensitivity governs the stability and synchrony of local calcium release during cardiac excitation-contraction coupling.

Calcium-induced calcium release is the principal mechanism that triggers the cell-wide [Ca(2+)]i transient that activates muscle contraction during cardiac excitation-contraction coupling (ECC). Here, we characterize this process in mouse cardiac myocytes with a novel mathematical action potential (AP) model that incorporates realistic stochastic gating of voltage-dependent L-type calcium (Ca(2+)) channels (LCCs) and sarcoplasmic reticulum (SR) Ca(2+) release channels (the ryanodine receptors, RyR2s). Depolarization of the sarcolemma during an AP stochastically activates the LCCs elevating subspace [Ca(2+)] within each of the cell's 20,000 independent calcium release units (CRUs) to trigger local RyR2 opening and initiate Ca(2+) sparks, the fundamental unit of triggered Ca(2+) release. Synchronization of Ca(2+) sparks during systole depends on the nearly uniform cellular activation of LCCs and the likelihood of local LCC openings triggering local Ca(2+) sparks (ECC fidelity). The detailed design and true SR Ca(2+) pump/leak balance displayed by our model permits investigation of ECC fidelity and Ca(2+) spark fidelity, the balance between visible (Ca(2+) spark) and invisible (Ca(2+) quark/sub-spark) SR Ca(2+) release events. Excess SR Ca(2+) leak is examined as a disease mechanism in the context of "catecholaminergic polymorphic ventricular tachycardia (CPVT)", a Ca(2+)-dependent arrhythmia. We find that that RyR2s (and therefore Ca(2+) sparks) are relatively insensitive to LCC openings across a wide range of membrane potentials; and that key differences exist between Ca(2+) sparks evoked during quiescence, diastole, and systole. The enhanced RyR2 [Ca(2+)]i sensitivity during CPVT leads to increased Ca(2+) spark fidelity resulting in asynchronous systolic Ca(2+) spark activity. It also produces increased diastolic SR Ca(2+) leak with some prolonged Ca(2+) sparks that at times become "metastable" and fail to efficiently terminate. There is a huge margin of safety for stable Ca(2+) handling within the cell and this novel mechanistic model provides insight into the molecular signaling characteristics that help maintain overall Ca(2+) stability even under the conditions of high SR Ca(2+) leak during CPVT. Finally, this model should provide tools for investigators to examine normal and pathological Ca(2+) signaling characteristics in the heart.

A critical role for the chromatin remodeller CHD7 in anterior mesoderm during cardiovascular development.

CHARGE syndrome is caused by spontaneous loss-of-function mutations to the ATP-dependant chromatin remodeller chromodomain-helicase-DNA-binding protein 7 (CHD7). It is characterised by a distinct pattern of congenital anomalies, including cardiovascular malformations. Disruption to the neural crest lineage has previously been emphasised in the aetiology of this developmental disorder. We present evidence for an additional requirement for CHD7 activity in the Mesp1-expressing anterior mesoderm during heart development. Conditional ablation of Chd7 in this lineage results in major structural cardiovascular defects akin to those seen in CHARGE patients, as well as a striking loss of cardiac innervation and embryonic lethality. Genome-wide transcriptional analysis identified aberrant expression of key components of the Class 3 Semaphorin and Slit-Robo signalling pathways in Chd7(fl/fl);Mesp1-Cre mutant hearts. CHD7 localises at the Sema3c promoter in vivo, with alteration of the local chromatin structure seen following Chd7 ablation, suggestive of direct transcriptional regulation. Furthermore, we uncover a novel role for CHD7 activity upstream of critical calcium handling genes, and demonstrate an associated functional defect in the ability of cardiomyocytes to undergo excitation-contraction coupling. This work therefore reveals the importance of CHD7 in the cardiogenic mesoderm for multiple processes during cardiovascular development.

Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor.

Malignant hyperthermia (MH) susceptibility is a dominantly inherited disorder in which volatile anesthetics trigger aberrant Ca(2+) release in skeletal muscle and a potentially fatal rise in perioperative body temperature. Mutations causing MH susceptibility have been identified in two proteins critical for excitation-contraction (EC) coupling, the type 1 ryanodine receptor (RyR1) and Ca(V)1.1, the principal subunit of the L-type Ca(2+) channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca(2+) entry. The Ca(V)1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all Ca(V) channels [Carpenter D, et al. (2009) BMC Med Genet 10:104-115.]. To define the functional consequences of this mutation, we expressed it in dysgenic (Ca(V)1.1 null) myotubes. Unlike previously described MH-linked mutations in Ca(V)1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca(2+) release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in Ca(V)1.1 R174W-expressing myotubes, resting myoplasmic Ca(2+) levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type Ca(V)1.1. Our results indicate that Ca(V)1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca(2+) leak from the SR, and that perturbation of Ca(V)1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers.

High-mobility group box 1 (HMGB1) impaired cardiac excitation-contraction coupling by enhancing the sarcoplasmic reticulum (SR) Ca(2+) leak through TLR4-ROS signaling in cardiomyocytes.

High-mobility group box 1 (HMGB1) is a proinflammatory mediator playing an important role in the pathogenesis of cardiac dysfunction in many diseases. In this study, we explored the effects of HMGB1 on Ca(2+) handling and cellular contractility in cardiomyocytes to seek for the mechanisms underlying HMGB1-induced cardiac dysfunction. Our results show that HMGB1 increased the frequency of Ca(2+) sparks, reduced the sarcoplasmic reticulum (SR) Ca(2+) content, and decreased the amplitude of systolic Ca(2+) transient and myocyte contractility in dose-dependent manners in adult rat ventricular myocytes. Inhibiting high-frequent Ca(2+) sparks with tetracaine largely inhibited the alterations of SR load and Ca(2+) transient. Blocking Toll-like receptor 4 (TLR4) with TAK-242 or knockdown of TLR4 by RNA interference remarkably inhibited HMGB1 induced high-frequent Ca(2+) sparks and restored the SR Ca(2+) content. Concomitantly, the amplitude of systolic Ca(2+) transient and myocyte contractility had significantly increased. Furthermore, HMGB1 increased the level of intracellular reactive oxygen species (ROS) and consequently enhanced oxidative stress and CaMKII-activated phosphorylation (pSer2814) in ryanodine receptor 2 (RyR2). TAK-242 pretreatment significantly decreased intracellular ROS levels and oxidative stress and hyperphosphorylation in RyR2, similar to the effects of antioxidant MnTBAP. Consistently, MnTBAP normalized HMGB1-impaired Ca(2+) handling and myocyte contractility. Taken together, our findings suggest that HMGB1 enhances Ca(2+) spark-mediated SR Ca(2+) leak through TLR4-ROS signaling pathway, which causes partial depletion of SR Ca(2+) content and hence decreases systolic Ca(2+) transient and myocyte contractility. Prevention of SR Ca(2+) leak may be an effective therapeutic strategy for the treatment of cardiac dysfunction related to HMGB1 overproduction.

Hypermuscular mice with mutation in the myostatin gene display altered calcium signalling.

Myostatin, a member of the transforming growth factor ß family, is a potent negative regulator of skeletal muscle growth, as myostatin-deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation-contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (Mstn(Cmpt-dl1Abc) or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the pCa-force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca(2+) concentration ([Ca(2+)]i) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura-2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 nM, n = 38 and 45), the amplitude of KCl-evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 nM, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod-2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 ΔF/F0, n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mM s(-1)) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 mM s(-1)) with no change in the peak-to-steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals.

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.

A differential pattern of gene expression in skeletal muscle of tumor-bearing rats reveals dysregulation of excitation­contraction coupling together with additional muscle alterations.

INTRODUCTION: Cachexia is a wasting condition that manifests in several types of cancer. The main characteristic of this condition is a profound loss of muscle mass. METHODS: By using a microarray system, expression of several hundred genes was screened in skeletal muscle of rats bearing a cachexia-inducing tumor, the AH-130 Yoshida ascites hepatoma. This model induced a strong decrease in muscle mass in the tumor-bearing animals, as compared with their healthy counterparts. RESULTS: The results show important differences in gene expression in EDL skeletal muscle between tumor-bearing animals with cachexia and control animals. CONCLUSIONS: The differences observed pertain to genes related to intracellular calcium homeostasis and genes involved in the control of mitochondrial oxidative phosphorylation and protein turnover, both at the level of protein synthesis and proteolysis. Assessment of these differences may be a useful tool for the design of novel therapeutic strategies to fight this devastating syndrome.