Oxidative stress in early metabolic syndrome impairs cardiac RyR2 and SERCA2a activity and modifies the interplay of these proteins during Ca2+ waves.

We investigated how oxidative stress (OS) alters Ca2+ handling in ventricular myocytes in early metabolic syndrome (MetS) in sucrose-fed rats. The effects of N-acetyl cysteine (NAC) or dl-Dithiothreitol (DTT) on systolic Ca2+ transients (SCaTs), diastolic Ca2+ sparks (CaS) and Ca2+ waves (CaW), recorded by confocal techniques, and L-type Ca2+ current (ICa), assessed by whole-cell patch clamp, were evaluated in MetS and Control cells. MetS myocytes exhibited decreased SCaTs and CaS frequency but unaffected CaW propagation. In Control cells, NAC/DTT reduced RyR2/SERCA2a activity blunting SCaTs, CaS frequency and CaW propagation, suggesting that basal ROS optimised Ca2+ signalling by maintaining RyR2/SERCA2a function and that these proteins facilitate CaW propagation. Conversely, NAC/DTT in MetS recovered RyR2/SERCA2a function, improving SCaTs and CaS frequency, but unexpectedly decreasing CaW propagation. We hypothesised that OS decreases RyR2/SERCA2a activity at early MetS, and while decreased SERCA2a favours CaW propagation, diminished RyR2 restrains it.

Dendritic cell-specific transmembrane protein is required for synovitis and bone resorption in inflammatory arthritis.

Objective: Dendritic Cell-Specific Transmembrane Protein (DC-STAMP) is essential for the formation of fully functional multinucleated osteoclasts. DC-STAMP deficient mice, under physiological conditions, exhibit osteopetrosis and develop systemic autoimmunity with age. However, the function of DC-STAMP in inflammation is currently unknown. We examined whether genetic ablation of DC-STAMP attenuates synovitis and bone erosion in TNF transgenic (Tg) and K/BxN serum-induced murine rheumatoid arthritis. Methods: We evaluated arthritis onset in Tg(hTNF) mice lacking DC-STAMP and 50:50 chimeric mice by visual examination, measurement of ankle width, micro-CT-scan analysis and quantitation of the area occupied by osteoclasts in bone sections. To further investigate the cellular and molecular events modulated by DC-STAMP, we measured serum cytokines, determined changes in cytokine mRNA expression by monocytes activated with IL4 or LPS/IFNγ and enumerated immune cells in inflamed mouse joints. Results: Synovitis, bone loss and matrix destruction are markedly reduced in Dcstamp-/-;Tg(hTNF) mice. These mice had significantly lower CCL2 and murine TNF serum levels and exhibited impaired monocyte joint migration compared to Tg(hTNF) mice. The reduced arthritic severity in Dcstamp deficient mice was associated with compromised monocyte chemotaxis, cytokine production, and M2 polarization. Conclusion: These results reveal that DC-STAMP modulates both bone resorption and inflammation and may serve as an activity biomarker and therapeutic target in inflammatory arthritis and metabolic bone disease.

Postdevelopmental knockout of Orai1 improves muscle pathology in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD), an X-linked disorder caused by loss-of-function mutations in the dystrophin gene, is characterized by progressive muscle degeneration and weakness. Enhanced store-operated Ca2+ entry (SOCE), a Ca2+ influx mechanism coordinated by STIM1 sensors of luminal Ca2+ within the sarcoplasmic reticulum (SR) and Ca2+-permeable Orai1 channels in the sarcolemma, is proposed to contribute to Ca2+-mediated muscle damage in DMD. To directly determine the impact of Orai1-dependent SOCE on the dystrophic phenotype, we crossed mdx mice with tamoxifen-inducible, muscle-specific Orai1 knockout mice (mdx-Orai1 KO mice). Both constitutive and SOCE were significantly increased in flexor digitorum brevis fibers from mdx mice, while SOCE was absent in fibers from both Orai1 KO and mdx-Orai1 KO mice. Compared with WT mice, fibers from mdx mice exhibited (1) increased resting myoplasmic Ca2+ levels, (2) reduced total releasable Ca2+ store content, and (3) a prolonged rate of electrically evoked Ca2+ transient decay. These effects were partially normalized in fibers from mdx-Orai1 KO mice. Intact extensor digitorum longus muscles from mdx mice exhibited a significant reduction of maximal specific force, which was rescued in muscles from mdx-Orai1 KO mice. Finally, during exposure to consecutive eccentric contractions, muscles from mdx mice displayed a more pronounced decline in specific force compared with that of WT mice, which was also significantly attenuated by Orai1 ablation. Together, these results indicate that enhanced Orai1-dependent SOCE exacerbates the dystrophic phenotype and that Orai1 deficiency improves muscle pathology by both normalizing Ca2+ homeostasis and promoting sarcolemmal integrity/stability.

Interaction of MDIMP with the Voltage-Gated Calcium Channels.

Amino acid-derived isoindolines are synthetic compounds that were created with the idea of investigating their biological actions. The amino acid moiety was included on the grounds that it may help to avoid toxic effects. Recently, the isoindoline MDIMP was shown to inhibit both cardiac excitation-contraction coupling and voltage-dependent calcium channels. Here, we revealed that MDIMP binds preferentially to low-voltage-activated (LVA) channels. Using a holding potential of -90 mV, the following IC50 values were found (in micromolars): >1000 (CaV2.3), 957 (CaV1.3), 656 (CaV1.2), 219 (CaV3.2), and 132 (CaV3.1). Moreover, the isoindoline also promoted both accelerated inactivation kinetics of high-voltage-activated Ca2+ channels and a modest upregulation of CaV1.3 and CaV2.3. Additional data indicate that although MDIMP binds to the closed state of the channels, it has more preference for the inactivated one. Concerning CaV3.1, the compound did not alter the shape of the instantaneous current-voltage curve, and substituting one or two residues in the selectivity filter drastically increased the IC50 value, suggesting that MDIMP binds to the extracellular side of the pore. However, an outward current failed in removing the inhibition, which implies an alternative mechanism may be involved. The enantiomer (R)-MDIMP [methyl (R)-2-(1,3-dihydroisoindol-2-yl)-4-methylpentanoate], on the other hand, was synthesized and evaluated, but it did not improve the affinity to LVA channels. Implications of these findings are discussed in terms of the possible underlying mechanisms and pharmacological relevance. SIGNIFICANCE STATEMENT: We have studied the regulation of voltage-gated calcium channels by MDIMP, which disrupts excitation-contraction coupling in cardiac myocytes. The latter effect is more potent in atrial than ventricular myocytes, and this could be explained by our results showing that MDIMP preferentially blocks low-voltage-activated channels. Our data also provide mechanistic insights about the blockade and suggest that MDIMP is a promising member of the family of Ca2+ channel blockers, with possible application to the inhibition of subthreshold membrane depolarizations.

Transverse tubule remodeling enhances Orai1-dependent Ca2+ entry in skeletal muscle.

Exercise promotes the formation of intracellular junctions in skeletal muscle between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of transverse-tubules (TT) that increase co-localization of proteins required for store-operated Ca2+ entry (SOCE). Here, we report that SOCE, peak Ca2+ transient amplitude and muscle force production during repetitive stimulation are increased after exercise in parallel with the time course of TT association with SR-stacks. Unexpectedly, exercise also activated constitutive Ca2+ entry coincident with a modest decrease in total releasable Ca2+ store content. Importantly, this decrease in releasable Ca2+ store content observed after exercise was reversed by repetitive high-frequency stimulation, consistent with enhanced SOCE. The functional benefits of exercise on SOCE, constitutive Ca2+ entry and muscle force production were lost in mice with muscle-specific loss of Orai1 function. These results indicate that TT association with SR-stacks enhances Orai1-dependent SOCE to optimize Ca2+ dynamics and muscle contractile function during acute exercise.

Mouse model of severe recessive RYR1-related myopathy.

Ryanodine receptor type I (RYR1)-related myopathies (RYR1 RM) are a clinically and histopathologically heterogeneous group of conditions that represent the most common subtype of childhood onset non-dystrophic muscle disorders. There are no treatments for this severe group of diseases. A major barrier to therapy development is the lack of an animal model that mirrors the clinical severity of pediatric cases of the disease. To address this, we used CRISPR/Cas9 gene editing to generate a novel recessive mouse model of RYR1 RM. This mouse (Ryr1TM/Indel) possesses a patient-relevant point mutation (T4706M) engineered into 1 allele and a 16 base pair frameshift deletion engineered into the second allele. Ryr1TM/Indel mice exhibit an overt phenotype beginning at 14 days of age that consists of reduced body/muscle mass and myofibre hypotrophy. Ryr1TM/Indel mice become progressively inactive from that point onward and die at a median age of 42 days. Histopathological assessment shows myofibre hypotrophy, increased central nuclei and decreased triad number but no clear evidence of metabolic cores. Biochemical analysis reveals a marked decrease in RYR1 protein levels (20% of normal) as compared to only a 50% decrease in transcript. Functional studies at end stage show significantly reduced electrically evoked Ca2+ release and force production. In summary, Ryr1TM/Indel mice exhibit a post-natal lethal recessive form of RYR1 RM that pheno-copies the severe congenital clinical presentation seen in a subgroup of RYR1 RM children. Thus, Ryr1TM/Indel mice represent a powerful model for both establishing the pathomechanisms of recessive RYR1 RM and pre-clinical testing of therapies for efficacy.

Role of STIM1/ORAI1-mediated store-operated Ca2+ entry in skeletal muscle physiology and disease.

Store-operated Ca2+ entry (SOCE) is a Ca2+ entry mechanism activated by depletion of intracellular Ca2+ stores. In skeletal muscle, SOCE is mediated by an interaction between stromal-interacting molecule-1 (STIM1), the Ca2+ sensor of the sarcoplasmic reticulum, and ORAI1, the Ca2+-release-activated-Ca2+ (CRAC) channel located in the transverse tubule membrane. This review focuses on the molecular mechanisms and physiological role of SOCE in skeletal muscle, as well as how alterations in STIM1/ORAI1-mediated SOCE contribute to muscle disease. Recent evidence indicates that SOCE plays an important role in both muscle development/growth and fatigue. The importance of SOCE in muscle is further underscored by the discovery that loss- and gain-of-function mutations in STIM1 and ORAI1 result in an eclectic array of disorders with clinical myopathy as central defining component. Despite differences in clinical phenotype, all STIM1/ORAI1 gain-of-function mutations-linked myopathies are characterized by the abnormal accumulation of intracellular membranes, known as tubular aggregates. Finally, dysfunctional STIM1/ORAI1-mediated SOCE also contributes to the pathogenesis of muscular dystrophy, malignant hyperthermia, and sarcopenia. The picture to emerge is that tight regulation of STIM1/ORAI1-dependent Ca2+ signaling is critical for optimal skeletal muscle development/function such that either aberrant increases or decreases in SOCE activity result in muscle dysfunction.

Functional impact of an oculopharyngeal muscular dystrophy mutation in PABPN1.

KEY POINTS: Mutations in the gene encoding poly(A)-binding protein nuclear 1 (PABPN1) result in oculopharyngeal muscular dystrophy (OPMD). This disease is of late-onset, but the underlying mechanism is unclear. Ca2+ stimulates muscle growth and contraction and, because OPMD courses with muscle atrophy and weakness, we hypothesized that the homeostasis of Ca2+ is altered in this disorder. C2C12 myotubes were transfected with cDNAs encoding either PABPN1 or the PABPN1-17A OPMD mutation. Subsequently, they were investigated concerning not only excitation-contraction coupling (ECC) and intracellular levels of Ca2+ , but also differentiation stage and nuclear structure. PABPN1-17A gave rise to: inhibition of Ca2+ release during ECC, depletion of sarcoplasmic reticulum Ca2+ content, reduced expression of ryanodine receptors, altered nuclear morphology and incapability to stimulate myoblast fusion. PABPN1-17A failed to inhibit ECC in adult muscle fibres, suggesting that its effects are primarily related to muscle regeneration. ABSTRACT: Oculopharyngeal muscular dystrophy (OPMD) is linked to mutations in the gene encoding poly(A)-binding protein nuclear 1 (PABPN1). OPMD mutations consist of an expansion of a tract that contains 10 alanines (to 12-17). This disease courses with muscle weakness that begins in adulthood, but the underlying mechanism is unclear. In the present study, we investigated the functional effects of PABPN1 and an OPMD mutation (PABPN1-17A) using myotubes transfected with cDNAs encoding these proteins (GFP-tagged). PABPN1 stimulated myoblast fusion (100%), whereas PABPN1-17A failed to mimic this effect. Additionally, the OPMD mutation markedly altered nuclear morphology; specifically, it led to nuclei with a more convoluted and ovoid shape. Although PABPN1 and PABPN1-17A modified the expression of sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase and calsequestrin, the corresponding changes did not have a clear impact on [Ca2+ ]. Interestingly, neither L-type Ca2+ channels, nor voltage-gated sarcoplasmic reticulum (SR) Ca2+ release (VGCR) was altered by PABPN1. However, PABPN1-17A produced a selective inhibition of VGCR (50%). This effect probably arises from both lower expression of RyR1 and depletion of SR Ca2+ . The latter, however, was not related to inhibition of store-operated Ca2+ entry. Both PABPN1 constructs promoted a moderated decrease in cytosolic [Ca2+ ], which apparently results from down-regulation of excitation-coupled Ca2+ entry. On the other hand, PABPN1-17A did not alter ECC in muscle fibres, suggesting that adult muscle is less prone to developing deleterious effects. These results demonstrate that PABPN1 proteins regulate essential processes during myotube formation and support the notion that OPMD involves disruption of myogenesis, nuclear structure and homeostasis of Ca2+ .

Chronic atrial ionic remodeling by aldosterone: potentiation of L-type Ca2+ channels and its arrhythmogenic significance.

It is widely accepted that aldosterone induces atrial fibrillation (AF) by promoting structural changes, but its effects on the function of primary atrial myocytes remain unknown. We have investigated this point in adult rat atrial myocytes, chronically exposed to the hormone. This treatment produced larger amplitude of Ca2+ transients, longer action potential (AP) duration, and higher incidence of unsynchronized Ca2+ oscillations. Moreover, it also gave rise to increases in both cell membrane capacitance (Cm, 30 %) and activity of L-type Ca2+ channels (LTCCs, 100 %). Concerning K+ currents, a twofold increase was also observed, but only in a delayed rectifier component (IKsus). Interestingly, the maximal conductance (Gmax) of Na+ channels was also enhanced, but it occurred in the face of a negative shift in the voltage dependence of inactivation. Thus, at physiological potentials, a decreased fraction of available channels neutralized the effect on GNa-max. With regard to the effects on both Cm and LTCCs, they involved activation of mineralocorticoid receptors (MRs), were dose-dependent (EC50 ∼20-130 nM), and developed and recovered in days. Neither gating currents nor protein levels of LTCCs were altered. Instead, the effect on LTCCs was mimicked by cAMP, reverted by a PKA inhibitor, and attenuated by a nitric oxide donor (short-term exposures). Both EGTA and the antioxidant NAC prevented the increase in Cm, without significantly interfering with the upregulation of LTCCs. Overall, these results show that chronic exposures to aldosterone result in dire functional changes at the single myocyte level, which may explain the link between aldosteronism and AF.

MDIMP, a novel cardiac Ca(2+) channel blocker with atrial selectivity.

In cardiac muscle cells both T-and L-type Ca(2+) channels (TTCCs and LTCCs, respectively) are expressed, and the latter are relevant to a process known as excitation-contraction coupling (ECC). Evidence obtained from docking studies suggests that isoindolines derived from α-amino acids bind to the LTCC CaV1.2. In the present study, we investigated whether methyl (S)-2-(1,3-dihydroisoindol-2-yl)-4-methylpentanoate (MDIMP), which is derived from L-leucine, modulates both Ca(2+) channels and ECC. To this end, mechanical properties, as well as Ca(2+) transients and currents, were all investigated in isolated cardiac myocytes. The effects of MDIMP on CaV1.2 (transiently expressed in 293T/17 cells) were also studied. In this system, evidence was found for an inhibitory action that develops and recovers in min, with an IC50 of 450µM. With respect to myocytes: atrial-TTCCs, atrial-LTCCs, and ventricular-LTCCs were also inhibited, in that order of potency. Accordingly, Ca(2+) transients, contractions, and window currents of LTCCs were all reduced more strongly in atrial cells. Interestingly, while the modulation of LTCCs was state-independent in these cells, it was state-dependent, and dual, on the ventricular ones. Furthermore, practically all of the ventricular LTCCs were closed at resting membrane potentials. This could explain their resistance to MDIMP, as they were affected in only open or inactivated states. All these features in turn explain the preferential down-regulation of the atrial ECC. Thus, our results support the view that isoindolines bind to Ca(2+) channels, improve our knowledge of the corresponding structure-function relationship, and may be relevant for conditions where decreased atrial activity is desired.

Chronic potentiation of cardiac L-type Ca(2+) channels by pirfenidone.

AIMS: On the basis of its ability to inhibit fibrosis, pirfenidone has drawn the attention as an intriguing candidate for treating cardiac disease. However, its precise electrophysiological effects have yet to be elucidated. Here, we have investigated its potential to modulate ion channels. METHODS AND RESULTS: Adult rat cardiac myocytes were investigated using whole-cell patch-clamp, western-blot and qRT-PCR techniques. Pirfenidone increased the density of L-type Ca(2+) current (I(CaL,) 50-100%), without significantly altering Na(+), K(+), or T-type Ca(2+) currents. The effect was dose-dependent, with an EC(50) of 2.8 µM. Its onset was slow, with a lag period larger than 1 h and time to maximum of 24-48 h. Concomitant changes were observed in the voltage-dependent activation of I(CaL) (-5 mV shift in both V(1/2) and k). In contrast, the following properties of I(CaL) remained normal: steady-state inactivation, Ca(V)1.2 levels (mRNA and protein), and intramembrane charge movement. Indeed, the conductance-to-charge ratio, or G(max)/Q(max), was increased by 80%. The effect on I(CaL) was mimicked by an inhibitor of nitric oxide (NO) synthase (NOS), and attenuated by both cyclic adenosine monophosphate (cAMP) and cAMP-dependent protein kinase (PKA) inhibitors. Conversely, cytokines, reactive oxygen species, and Ca(2+) were all ruled out as possible intermediaries. Additional experiments suggest that pirfenidone increases action potential duration by ∼50%. CONCLUSION: Pirfenidone augments I(CaL), not through higher expression of L-type channels, but through promoting their Ca(2+)-conducting activity. A possible inhibition of NOS expression is likely involved, with subsequent reduced NO production and stimulated cAMP/PKA signalling. These findings may be relevant to the cardioprotective effect of pirfenidone.