Caffeine-induced Ca2 Research Articles

SERCA-1 conformational change exerted by the Ca2+-channel blocker diltiazem affects mammalian skeletal muscle function

In skeletal muscle (SM), inward Ca2+-currents have no apparent role in excitation-contraction coupling (e-c coupling), however the Ca2+-channel blocker can affect twitch and tetanic muscle in mammalian SM. Experiments were conducted to study how diltiazem (DLZ) facilitates e-c coupling and inhibits contraction. 1) In complete Extensor Digitorum Longus (EDL) muscle and single intact fibres, 0.03 mM DLZ causes twitch potentiation and decreases force during tetanic activity, with increased fatigue. 2) In split open fibres isolated from EDL fibres, DLZ inhibits sarcoplasmic reticulum (SR) Ca2+-loading in a dose-dependent manner and has a potentiating effect on caffeine-induced SR Ca2+-release. 3) In isolated light SR (LSR) vesicles, SERCA1 hydrolytic activity is not affected by DLZ up to 0.2 mM. However, ATP-dependent Ca2+-uptake was inhibited in a dose-dependent manner at a concentration where e-c coupling is changed. 4) The passive Ca2+-efflux from LSR was reduced by half with 0.03 mM diltiazem, indicating that SR leaking does not account for the decreased Ca2+-uptake. 5) The denaturation profile of the SERCA Ca2+-binding domain has lower thermal stability in the presence of DLZ in a concentration-dependent manner, having no effect on the nucleotide-binding domain. We conclude that the effect of DLZ on SM is exerted by crossing the sarcolemma and interacting directly with the SERCA Ca2+-binding domain, affecting SR Ca2+-loading during relaxation, which has a consequence on SM contractility. Diltiazem effect on SM could be utilized as a tool to understand SM e-c coupling and muscle fatigue.

Muscle fibre mitochondrial [Ca2+ ] dynamics during Ca2+ waves in RYR1 gain-of-function mouse.

A fraction of the Ca2+ released from the sarcoplasmic reticulum (SR) enters mitochondria to transiently increase its [Ca2+ ] ([Ca2+ ]mito ). This transient [Ca2+ ]mito increase may be important in the resynthesis of ATP and other processes. The resynthesis of ATP in the mitochondria generates heat that can lead to hypermetabolic reactions in muscle with ryanodine receptor 1 (RyR1) variants during the cyclic releasing of SR Ca2+ in the presence of a RyR1 agonist. We aimed to analyse whether the mitochondria of RYR1 variant muscle handles Ca2+ differently from healthy muscle. We used confocal microscopy to track mitochondrial and cytoplasmic Ca2+ with fluorescent dyes simultaneously during caffeine-induced Ca2+ waves in extensor digitorum longus muscle fibres from healthy mice and mice heterozygous (HET) for a malignant hyperthermia-causative RYR1 variant. Mitochondrial Ca2+ -transient peaks trailed the peak of cytoplasmic Ca2+ transients by many seconds with [Ca2+ ]mito not increasing by more than 250 nM. A strong linear relationship between cytoplasmic Ca2+ and [Ca2+ ]mito amplitudes was observed in HET RYR1 KI fibres but not wild type (WT). Our results indicate that [Ca2+ ]mito change within the nM range during SR Ca2+ release. HET fibre mitochondria are more sensitive to SR Ca2+ release flux than WT. This may indicate post-translation modification differences of the mitochondrial Ca2+ uniporter between the genotypes.

Chronic repetitive cooling and caffeine-induced intracellular Ca2+ elevation differentially impact adaptations in slow- and fast-twitch rat skeletal muscles.

Intracellular Ca2+ concentration ([Ca2+]i) is considered important in the regulation of skeletal muscle mass. This study tested the hypothesis that chronic repeated cooling and/or caffeine ingestion would acutely increase [Ca2+]i and hypertrophy muscles potentially in a fiber-type-dependent manner. Control rats and those fed caffeine were subjected to repeated bidiurnal treatments of percutaneous icing, under anesthesia, to reduce the muscle temperature below ∼5°C. The predominantly fast-twitch tibialis anterior (TA) and slow-twitch soleus (SOL) muscles were evaluated after 28 days of intervention. The [Ca2+]i elevating response to icing was enhanced by caffeine loading only in the SOL muscle, with the response present across a significantly higher temperature range than in the TA muscle under caffeine-loading conditions. In both the TA and SOL muscles, myofiber cross-sectional area (CSA) was decreased by chronic caffeine treatment (mean reductions of 10.5% and 20.4%, respectively). However, in the TA, but not the SOL, CSA was restored by icing (+15.4 ± 4.3% vs. noniced, P < 0.01). In the SOL, but not TA, icing + caffeine increased myofiber number (20.5 ± 6.7%, P < 0.05) and satellite cell density (2.5 ± 0.3-fold) in cross sections. These contrasting muscle responses to cooling and caffeine may reflect fiber-type-specific [Ca2+]i responses and/or differential responses to elevated [Ca2+]i.

BS-452682-2 THE IMPAIRED INTRACELLULAR CALCIUM BUFFERING IN ATRIAL MYOCYTES OF PATIENTS WITH LONG-STANDING PERSISTENT ATRIAL FIBRILLATION IS MEDIATED BY REDUCED CARDIAC TROPONIN C LEVELS

Decreased levels of myofilament proteins and impaired atrial Ca2+ homeostasis contribute to atrial hypocontractility and thromboembolic events in long-standing persistent atrial fibrillation (AF). The myofilament protein cardiac troponin C (cTnC) is known to be the major cytosolic Ca2+ buffer, but its role in AF-associated Ca2+ handling abnormalities remains poorly investigated. This study aimed to clarify the influence of cTnC on impaired intracellular Ca2+ signalling in AF. Right atrial myocytes from sinus rhythm (Ctrl) and AF patients, as well as atrial induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) with knocked down cTnC levels (si-cTnC) were investigated by simultaneously measuring membrane currents (patch-clamp technique) and cytosolic Ca2+ (Fluo-3 AM and Fluo-4 AM). Myofilament protein levels were determined using immunoblotting. Cardiac troponin C protein levels were reduced in atrial tissues of AF patients. By integrating Na+-Ca2+-exchange current (INCX) during caffeine-induced Ca2+ release, SR Ca2+ content was determined and similar in both groups. Buffering parameters (maximum buffer capacity, Bmax, and dissociation constant of buffers, Kd) were obtained by fitting a hyperbolic function on buffering curves generated from INCX and the caffeine-induced Ca2+ transient. However, intracellular calcium buffering was reduced in AF, presumably due to the reduced cardiac troponin C. (Figures A and B). Interestingly, the reduction of cTnC levels using small interfering RNA (siRNA) caused the same alterations in intracellular Ca2+ buffering in atrial iPSC-CM. (Figure C). In these modelled cells, a higher Ca2+ spark frequency was observed as opposed to the control group (Figure D). Diminished cTnC levels underlie the reduced intracellular Ca2+ buffering observed in AF. By promoting spontaneous SR Ca2+ release events that could trigger delayed afterdepolarizations, reduced cTnC levels could contribute to the arrhythmogenic substrate promoting AF.

Glu-rich N-terminal variable region of troponin T functions as a myofilament Ca2+-reservoir to reduce SR Ca2+-cycling.

The long Glu-rich N-terminal segment of avian pectoral muscle troponin T (TnT) and C-terminal extension of insect TnT are multi-valent Ca2+-binding sites with physiologically relevant affinities. Genetic deletion of the Glu-rich C-terminal extension of insect TnT decreases muscle and heart functions but increases the tolerance of cardiomyocytes to Ca2+ overloading, indicating decreased [Ca2+]i. In contrast, cardiomyocytes from gene-edited mice expressing modified cardiac TnT with N-terminal variable region replaced by a C-terminal segment of insect TnT (51E-cTnT) show higher shortening amplitude and contraction and relaxation velocities than that of wild type (WT) control. Isolated 51E-cTnT cardiomyocytes exhibited shorter resting sarcomeres, which can be corrected by inhibiting myosin ATPase, indicating increased resting [Ca2+]i. A similar gain-of-function model,fast skeletal muscle TnTgene-edited mice with the N-terminal variable region replaced by the N-terminal segment of adult turkey pectoral muscle TnT (Tx9-fsTnT), shows normal baseline muscle functions but slower and less rising of contractile force during caffeine-induced Ca2+ release from sarcoplasmic reticulum (SR) than that of WT control, indicating a decreased SR Ca2+ store. Consistently, 51E-cTnT mouse hearts respond to beta-adrenergic stimulation or Ca2+ agonist with higher than WT ventricular peak pressure and diastolic velocity. The data support a hypothesis that the Glu-rich segments of insect and avian flight muscle TnT function as myofilament Ca2+ reservoirs to tune muscle kinetics and reduce the amount of Ca2+ cycled during contraction and relaxation. The functional effects are prominent in heterozygotes of the genetically modified flies and mice whereas the loss-of-function in Drosophila TnT and gain-of-function in 51E-cTnT and Tx9-fsTnT mice both produced homozygote lethality, indicating sensitive dose effects of the myofilament Ca2+ reservoir for use as a potent mechanism to improve energetic efficiency of cardiac and skeletal muscles.

Pkd1 is necessary for full cardiac AngII-induced hypertrophy but not for full acute AngII-induced calcium handling changes.

The renin-angiotensin system (RAS) regulates blood pressure, extracellular fluid and electrolyte balance, cardiac inotropy, and vascular resistance. In the heart, angiotensin II (AngII) can induce hypertrophy directly by activating Protein Kinase D (PKD), a signaling kinase known to contribute to cardiac remodeling. Little is known, however, about the acute and chronic functional effects of AngII-induced PKD activation on calcium (Ca) handling. To investigate this, we used hearts from cardiac-specific PKD1 knockout (cKO) mice and their WT littermates. We measured Ca transients (CaT) and Ca spark (CaSp) frequencies in cardiomyocytes at 0.5 and 1 Hz pacing under acute and chronic AngII stimulation (100 nM in vitro and 3 mg/kg/day in osmotic minipump, respectively). Sarcoplasmic reticulum (SR) Ca load was assessed by rapid caffeine-induced Ca release. At baseline and after acute AngII treatment, CaT amplitude and tau decay, CaSp frequency and SR Ca load were similar in WT vs. PKD1 cKO. This was also the case for chronic exposure to AngII except that the tau decay was significantly slower in PKD1 cKO myocytes. In agreement with previous reports, echocardiographic measurements revealed that PKD1 cKO mice were also more resistant to chronic AngII-induced hypertrophy (LVPW increase 24.69 ± 2.74% vs. 54.73 ± 7.73 %) and ejection fraction reduction (4.08 ± 1.5% vs. 12.67 ± 0.52%). We conclude that despite PKD being necessary for the remodeling changes induced by chronic AngII, it does not alter the amplitude and kinetics of CaT and spontaneous Ca release, except for the slowed decay kinetics in the PKD1 cKO after chronic AngII treatment and decreased acute SR Ca load.

CaMKII-Dependent Contractile Dysfunction and Pro-Arrhythmic Activity in a Mouse Model of Obstructive Sleep Apnea.

Left ventricular contractile dysfunction and arrhythmias frequently occur in patients with sleep-disordered breathing (SDB). The CaMKII-dependent dysregulation of cellular Ca homeostasis has recently been described in SDB patients, but these studies only partly explain the mechanism and are limited by the patients' heterogeneity. Here, we analyzed contractile function and Ca homeostasis in a mouse model of obstructive sleep apnea (OSA) that is not limited by confounding comorbidities. OSA was induced by artificial tongue enlargement with polytetrafluorethylene (PTFE) injection into the tongue of wildtype mice and mice with a genetic ablation of the oxidative activation sites of CaMKII (MMVV knock-in). After eight weeks, cardiac function was assessed with echocardiography. Reactive oxygen species (ROS) and Ca transients were measured using confocal and epifluorescence microscopy, respectively. Wildtype PTFE mice exhibited an impaired ejection fraction, while MMVV PTFE mice were fully protected. As expected, isolated cardiomyocytes from PTFE mice showed increased ROS production. We further observed decreased levels of steady-state Ca transients, decreased levels of caffeine-induced Ca transients, and increased pro-arrhythmic activity (defined as deviations from the diastolic Ca baseline) only in wildtype but not in MMVV PTFE mice. In summary, in the absence of any comorbidities, OSA was associated with contractile dysfunction and pro-arrhythmic activity and the inhibition of the oxidative activation of CaMKII conveyed cardioprotection, which may have therapeutic implications.

Impact of mitochondria on local calcium release in murine sinoatrial nodal cells

Roles of mitochondria in sinoatrial nodal cells (SANCs) have not been fully clarified. We have previously demonstrated that mitochondrial Ca2+ efflux through the Na+-Ca2+ exchanger, NCXm, modulates sarcoplasmic reticulum (SR) Ca2+ content and automaticity of HL-1 cardiomyocytes. In this study, we extended this line of investigation to clarify the spatial and functional association between mitochondria and local calcium release (LCR) from the SR in murine SANCs. High-speed two dimensional (2D) and confocal line-scan imaging of SANCs revealed that LCRs in the early phase of the Ca2+ transient cycle length (CL) appeared with a higher probability near mitochondria. Although LCR increased toward the late phase of CL, no significant difference was noted in the occurrence of late LCRs near and distant from mitochondria. LCRs, especially in the late phase of CL, induced temporal and spatial heterogeneity of the Ca2+ transient amplitude. Attenuating mitochondrial Ca2+ efflux using an NCXm inhibitor, CGP-37157 (1 μM), reduced the amplitude, duration and size of LCR. It also attenuated early LCR occurrence, and simultaneously prolonged LCR period and CL. Additionally, CGP-37157 reduced caffeine-induced Ca2+ transient. Therefore, the inhibitory effect on LCR was attributable to the reduction of the SR Ca2+ content through NCXm inhibition. No obvious off-target effects of 1 μM CGP-37157 were found on T- and L-type voltage-gated Ca2+ currents and hyperpolarization-activated inward current. Taken together, these results suggest that mitochondria are involved in LCR generation by modulating the SR Ca2+ content through NCXm-mediated Ca2+ efflux in murine SANCs.

Human RyR2 (Ryanodine Receptor 2) Loss-of-Function Mutations: Clinical Phenotypes and In Vitro Characterization.

[Figure: see text].

Depressed β-adrenergic inotropic responsiveness and intracellular calcium handling abnormalities in Duchenne Muscular Dystrophy patients' induced pluripotent stem cell-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X‐linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca2+‐handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC‐CMs generated from DMD patients feature blunted positive inotropic response to β‐adrenergic stimulation. To test the hypothesis, [Ca2+]i transients and contractions were recorded from healthy and DMD‐CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD‐CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine‐induced Ca2+ release. In contrast to HC, DMD‐CMs exhibited reduced caffeine‐induced Ca2+ signal amplitude and recovery time. In support of the depleted SR Ca2+ stores hypothesis, in DMD‐CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA‐seq analyses demonstrated that in DMD CMs the RNA‐expression levels of specific subunits of the L‐type calcium channel, the β1‐adrenergic receptor (ADRβ1) and adenylate cyclase were down‐regulated by 3.5‐, 2.8‐ and 3‐fold, respectively, which collectively contribute to the depressed β‐adrenergic responsiveness.

Mitochondrial Calcium Uniporter Overexpression Exacerbates Arrhythmogenic Calcium Mishandling in Diseased Rat Ventricular Myocytes

Systolic dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with diminished Ca2+ transient amplitude and impaired mitochondrial function due to reduced mitochondrial [Ca2+]. Pharmacological or genetic facilitation of mito-Ca2+ uptake was shown to restore Ca2+ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that acute pharmacological enhancement of mito-Ca2+ uptake can exacerbate spontaneous Ca2+ release in myocytes from diseased animals, increasing propensity to stress-induced arrhythmias. To test whether chronic recovery of mito-[Ca2+] restores systolic Ca2+ release without adverse effects in diastole, we overexpressed mitochondrial Ca2+ uniporter (MCU) in ventricular myocytes (VMs) from rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca2+] using genetic probe mtRCamp1h revealed that mito-[Ca2+] in TAB VMs paced at 1 Hz under β-adrenergic stimulation is significantly lower than in Shams. Adenoviral MCU overexpression in TAB VMs restored mito-[Ca2+]. Confocal imaging of VMs loaded with fluorescent indicator Rhod-2 revealed that MCU overexpression in TAB VMs reversed the decrease in Ca2+ transient amplitude. Importantly, the decay of caffeine-induced Ca2+ transient in MCU-overexpressing TAB VMs was prolonged, indicative of decreased NCX activity. Moreover, MCU overexpression in TAB VMs was accompanied by a further increase in incidence of proarrhythmic spontaneous Ca2+ waves, suggesting exacerbation of RyR2 dysfunction. Experiments using genetic probe OMM-HyPer showed a 10-fold increase in mito-ROS emission in TAB VMs overexpressing MCU. Importantly, application of mito-ROS scavenger mitoTEMPO (20 µM, 10 min) restored Ca2+ transient amplitude, reduced spontaneous Ca2+ waves and restored mito-[Ca2+] in TAB VMs, rendering them normal. These findings suggest that restoration of mito-[Ca2+] in VMs from diseased hearts by MCU overexpression improves systolic Ca2+ release possibly via reduced NCX-mediated Ca2+ efflux, but exacerbates increased mito-ROS and RyR2 activity, increasing arrhythmic potential.

Altered atrial cytosolic calcium handling contributes to the development of postoperative atrial fibrillation.

Aims Atrial fibrillation (AF) is a commonly occurring arrhythmia after cardiac surgery (postoperative AF, poAF) and is associated with poorer outcomes. Considering that reduced atrial contractile function is a predictor of poAF and that Ca2+ plays an important role in both excitation–contraction coupling and atrial arrhythmogenesis, this study aims to test whether alterations of intracellular Ca2+ handling contribute to impaired atrial contractility and to the arrhythmogenic substrate predisposing patients to poAF.Methods and results Right atrial appendages were obtained from patients in sinus rhythm undergoing open-heart surgery. Cardiomyocytes were investigated by simultaneous measurement of [Ca2+]i and action potentials (APs, patch-clamp). Patients were followed-up for 6 days to identify those with and without poAF. Speckle-tracking analysis of preoperative echocardiography revealed reduced left atrial contraction strain in poAF patients. At the time of surgery, cellular Ca2+ transients (CaTs) and the sarcoplasmic reticulum (SR) Ca2+ content were smaller in the poAF group. CaT decay was slower in poAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting preserved sodium-calcium exchanger function. In agreement, western blots revealed reduced SERCA2a expression in poAF patients but unaltered phospholamban expression/phosphorylation. Computational modelling indicated that reduced SERCA activity promotes occurrence of CaT and AP alternans. Indeed, alternans of CaT and AP occurred more often and at lower stimulation frequencies in atrial myocytes from poAF patients. Resting membrane potential and AP duration were comparable between both groups at various pacing frequencies (0.25–8 Hz).Conclusions Biochemical, functional, and modelling data implicate reduced SERCA-mediated Ca2+ reuptake into the SR as a major contributor to impaired preoperative atrial contractile function and to the pre-existing arrhythmogenic substrate in patients developing poAF.

Identification of drug modifiers for RYR1-related myopathy using a multi-species discovery pipeline.

Ryanodine receptor type I-related myopathies (RYR1-RMs) are a common group of childhood muscle diseases associated with severe disabilities and early mortality for which there are no available treatments. The goal of this study is to identify new therapeutic targets for RYR1-RMs. To accomplish this, we developed a discovery pipeline using nematode, zebrafish, and mammalian cell models. We first performed large-scale drug screens in C. elegans which uncovered 74 hits. Targeted testing in zebrafish yielded positive results for two p38 inhibitors. Using mouse myotubes, we found that either pharmacological inhibition or siRNA silencing of p38 impaired caffeine-induced Ca2+ release from wild type cells while promoting intracellular Ca2+ release in Ryr1 knockout cells. Lastly, we demonstrated that p38 inhibition blunts the aberrant temperature-dependent increase in resting Ca2+ in myotubes from an RYR1-RM mouse model. This unique platform for RYR1-RM therapy development is potentially applicable to a broad range of neuromuscular disorders.

The mitochondrial Na+/Ca2+ exchanger enables mitochondrial calcium balance in placental vessel from preeclampsia

Placental ischaemia and hypoxia accelerate the development of preeclampsia. Although mitochondria could actively regulate Ca2+ signals and involves in sarcoendoplasmic reticulum (SR) Ca2+ release in smooth muscle. AIM: To investigate mitochondrial calcium responses to caffeine-induced Ca2+ release via ryanodine receptor (RyR) in placental vessels from preeclampsia. METHODS: Placenta was collected from normal pregnancy (N = 40) or preeclampsia (N = 30). Vascular tension was recorded using Power-Lab system. Total internal reflection fluorescent microscope (TIRFM) was used to recorded and analyzed the [Ca2+]m uptake and efflux. RESULTS: Caffeine-induced contraction was decreased both in placental artery and placental vascular smooth muscle cells (VSMCs) from preeclampsia, which was related to the mitochondrial dysfunction. Mitochondria in placental vessels from preeclampsia showed impaired [Ca2+]m efflux function. To understand the role of NCLX in preeclampsia, studies about on vascular smooth muscle cells under ischaemia and hypoxia was performed in the absence of overexpression NCLX or not. Cells under ischaemia and hypoxia showed mitochondrial dyfunction similar to placental vessel from preeclampsia.

Role of Orai1 and L-type CaV1.2 channels in Endothelin-1 mediated coronary contraction under ischemia and reperfusion

Ischemia and Reperfusion (I/R) injuries are associated with coronary artery hypercontracture. They are mainly originated by an exacerbated response to agonists released by endothelium such as Endothelin (ET-1), involving the alteration in intracellular calcium handling. Recent evidences have highlighted the implication of Store-Operated Calcium Channels (SOCC) in intracellular calcium homeostasis in coronary artery. However, little is known about the role of SOCC in the regulation of coronary vascular tone under I/R.The aim of this study was to evaluate the role of SOCC and l-type Ca2+ channels (LTCC) in coronary artery vasoconstriction originated by ET-1 in I/R. We used Left Anterior Descendent coronary artery (LAD) rings, isolated from Wistar rats, to study the contractility and intracellular Ca2+ concentration ([Ca2+]i) under a simulated I/R protocol. We observed that responses to high-KCL induced depolarization and caffeine-induced Ca2+ release are attenuated in coronary artery under I/R. Furthermore, ET-1 addition in ischemia promotes transient and small rise of [Ca2+]i and coronary vascular tone. Meanwhile, these effects are significantly potentiated during reperfusion. The resulting ET-1-induced vasoconstrictions and [Ca2+]i increase were abolished by; GSK-7975A and gadolinium, inhibitors of SOCC; and nifedipine a widely used inhibitor of LTCC. Interestingly, using in situ Proximity Ligation Assay (PLA) in isolated coronary smooth muscle cells we found significant colocalization of LTCC CaV1.2 isoform with Orai1, the pore forming subunit of SOCC, and TRPC1 under I/R.Our data suggest that hypercontraction of coronary artery induced by ET-1 after I/R involves the co-activation of LTCC and SOCC, which colocalize significantly in the sarcolemma of coronary smooth muscle cells.

Fibroblast growth factor 9 (FGF9) inhibits myogenic differentiation of C2C12 and human muscle cells

ABSTRACT Osteoporosis and sarcopenia (osteosarcopenia (OS)) are twin-aging diseases. The biochemical crosstalk between muscle and bone seems to play a role in OS. We have previously shown that osteocytes produce soluble factors with beneficial effects on muscle and vice versa. Recently, enhanced FGF9 production was observed in the OmGFP66 osteogenic cell line. To test its role in myogenic differentiation, C2C12 myoblasts were treated with recombinant FGF9. FGF9 as low as 10 ng/mL inhibited myogenic differentiation, suggesting that FGF9 might be a potential inhibitory factor produced from bone cells with effects on muscle cells. FGF9 (10–50 ng/mL) significantly decreased mRNA expression of MyoG and Mhc while increasing the expression of Myostatin. Consistent with the phenotype, RT-qPCR array revealed that FGF9 (10 ng/mL) increased the expression of Icam1 while decreased the expression of Wnt1 and Wnt6 decreased, respectively. FGF9 decreased caffeine-induced Ca2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and reduced the expression of genes (i.e. Cacna1s, RyR2, Naftc3) directly associated with intracellular Ca2+ homeostasis. Myogenic differentiation in human skeletal muscle cells was similarly inhibited by FGF9 but required higher doses of 200 ng/mL FGF9. FGF9 was also shown to stimulate C2C12 myoblast proliferation. FGF2 and the FGF9 subfamily members FGF16 and FGF20 also inhibited C2C12 myoblast differentiation and enhanced proliferation. Intriguingly, the differentiation inhibition was independent of proliferation enhancement. These findings suggest that FGF9 may modulate myogenesis via a complex signaling mechanism.

A central core disease mutation in the Ca2+-binding site of skeletal muscle ryanodine receptor impairs single-channel regulation.

Cryoelectron microscopy and mutational analyses have shown that type 1 ryanodine receptor (RyR1) amino acid residues RyR1-E3893, -E3967, and -T5001 are critical for Ca2+-mediated activation of skeletal muscle Ca2+ release channel. De novo missense mutation RyR1-Q3970K in the secondary binding sphere of Ca2+ was reported in association with central core disease (CCD) in a 2-yr-old boy. Here, we characterized recombinant RyR1-Q3970K mutant by cellular Ca2+ release measurements, single-channel recordings, and computational methods. Caffeine-induced Ca2+ release studies indicated that RyR1-Q3970K formed caffeine-sensitive, Ca2+-conducting channel in HEK293 cells. However, in single-channel recordings, RyR1-Q3970K displayed low Ca2+-dependent channel activity and greatly reduced activation by caffeine or ATP. A RyR1-Q3970E mutant corresponds to missense mutation RyR2-Q3925E associated with arrhythmogenic syndrome in cardiac muscle. RyR1-Q3970E also formed caffeine-induced Ca2+ release in HEK293 cells and exhibited low activity in the presence of the activating ligand Ca2+ but, in contrast to RyR1-Q3970K, was activated by ATP and caffeine in single-channel recordings. Computational analyses suggested distinct structural rearrangements in the secondary binding sphere of Ca2+ of the two mutants, whereas the interaction of Ca2+ with directly interacting RyR1 amino acid residues Glu3893, Glu3967, and Thr5001 was only minimally affected. We conclude that RyR1-Q3970 has a critical role in Ca2+-dependent activation of RyR1 and that a missense RyR1-Q3970K mutant may give rise to myopathy in skeletal muscle.

Dantrolene sodium increases calcium binding by human recombinant cardiac calsequestrin and calcium loading by sheep cardiac sarcoplasmic reticulum.

Dantrolene interacts with ryanodine receptors in skeletal and cardiac muscle affecting sarcoplasmic reticulum calcium release. Since dantrolene is lipophilic it could also affect intra-sarcoplasmic reticulum calcium handling proteins such as calsequestrin. This study investigated whether dantrolene (1-30µmol/L) alters the polymerization state and the calcium binding capacity of recombinant cardiac calsequestrin and cardiac sarcoplasmic reticulum calcium handling. Human recombinant cardiac calsequestrin was used to make simultaneous measurements of turbidity (to indicate calsequestrin polymerization) and calcium binding to calsequestrin in the presence and absence of dantrolene. Caffeine-induced Ca2+ transients were used to investigate the effects of dantrolene on sarcoplasmic reticulum calcium loading and release in saponin-permeabilized cardiomyocytes laid down in monolayers in 96-well array plates. Dantrolene (1-30µmol/L) increased the polymerization state of calsequestrin and its calcium binding capacity. In the presence of dantrolene, calsequestrin-dependent turbidity increased 2.5-11.5-fold at 1.0mmol/L calcium added to unbuffered Ca2+ solutions and 3-10-fold when calcium was raised from 0.06 to 30µmol/L. The dantrolene-dependent increase in turbidity at 30µmol/L dantrolene was associated with a 3-fold increase in the number of calcium ions bound per calsequestrin molecule at 30 and 100µmol/L calcium. The caffeine-induced releasable calcium loaded by the sarcoplasmic reticulum at 0.63µmol/L free calcium in permeabilized cardiomyocytes was also increased in the presence of 30µmol/L dantrolene. Dantrolene alters the polymerization state and the calcium binding properties of cardiac calsequestrin and increases sarcoplasmic reticulum calcium loading in permeabilized cardiomyocytes.

Cardiotoxicity of environmental contaminant tributyltin involves myocyte oxidative stress and abnormal Ca2+ handling

Tributyltin (TBT) is an organotin environmental pollutant widely used as an agricultural and wood biocide and in antifouling paints. Countries began restricting TBT use in the 2000s, but their use continues in some agroindustrial processes. We studied the acute effect of TBT on cardiac function by analyzing myocardial contractility and Ca2+ handling. Cardiac contractility was evaluated in isolated papillary muscle and whole heart upon TBT exposure. Isolated ventricular myocytes were used to measure calcium (Ca2+) transients, sarcoplasmic reticulum (SR) Ca2+ content and SR Ca2+ leak (as Ca2+ sparks). Reactive oxygen species (ROS), as superoxide anion (O2•-) was detected at intracellular and mitochondrial myocardium. TBT depressed cardiac contractility and relaxation in papillary muscle and intact whole heart. TBT increased cytosolic, mitochondrial ROS production and decreased mitochondrial membrane potential. In isolated cardiomyocytes TBT decreased both Ca2+ transients and SR Ca2+ content and increased diastolic SR Ca2+ leak. Decay of twitch and caffeine-induced Ca2+ transients were slowed by the presence of TBT. Dantrolene prevented and Tiron limited the reduction in SR Ca2+ content and transients. The environmental contaminant TBT causes cardiotoxicity within minutes, and may be considered hazardous to the mammalian heart. TBT acutely induced a negative inotropic effect in isolated papillary muscle and whole heart, increased arrhythmogenic SR Ca2+ leak leading to reduced SR Ca2+ content and reduced Ca2+ transients. TBT-induced myocardial ROS production, may destabilize the SR Ca2+ release channel RyR2 and reduce SR Ca2+ pump activity as key factors in the TBT-induced negative inotropic and lusitropic effects.

Long-Term Regulation of Excitation-Contraction Coupling and Oxidative Stress in Cardiac Myocytes by Pirfenidone.

Pirfenidone (PFD) is used to treat human pulmonary fibrosis. Its administration to animals with distinct forms of cardiovascular disease results in striking improvement in cardiac performance. Here, its functional impact on cardiac myocytes was investigated. Cells were kept 1–2 days under either control culture conditions or the presence of PFD (1 mM). Subsequently, they were subjected to electrical stimulation to assess the levels of contractility and intracellular Ca2+. The PFD treatment promoted an increase in both peak contraction and kinetics of shortening and relaxation. Moreover, the amplitude and kinetics of Ca2+ transients were enhanced as well. Excitation–contraction coupling (ECC) was also investigated, under whole-cell patch-clamp conditions. In keeping with a previous report, PFD increased twofold the density of Ca2+ current (ICa). Notably, a similar increase in the magnitude of Ca2+ transients was also observed. Thus, the gain of ECC was unaltered. Likewise, PFD did not alter the peak amplitude of caffeine-induced Ca2+ release, indicating stimulation of Ca2+-induced–Ca2+-release (CICR) at constant sarcoplasmic reticulum Ca2+ load. A phase-plane analysis indicated that PFD promotes myofilament Ca2+ desensitization, which is being compensated by higher levels of Ca2+ to promote contraction. Interestingly, although the expression of the Na+/Ca2+ exchanger (NCX) was unaffected, the decay of Ca2+ signal in the presence of caffeine was 50% slower in PFD-treated cells (compared with controls), suggesting that PFD downregulates the activity of the exchanger. PFD also inhibited the production of reactive oxygen species, under both, basal conditions and the presence of oxidative insults (acetaldehyde and peroxide hydrogen). Conversely, the production of nitric oxide was either increased (in atrial myocytes) or remained unchanged (in ventricular myocytes). Protein levels of endothelial and neuronal nitric oxide synthases (eNOS and nNOS) were also investigated. eNOS values did not exhibit significant changes. By contrast, a dual regulation was observed for nNOS, which consisted of inhibition and stimulation, in ventricular and atrial myocytes, respectively. In the latter cells, therefore, an up-regulation of nNOS was sufficient to stimulate the synthesis of NO. These findings improve our knowledge of molecular mechanisms of PFD action and may also help in explaining the corresponding cardioprotective effects.