Periodic acceleration (pGz), sinusoidal motion of the whole body in a head-foot direction in the spinal axis, is a novel noninvasive means for cardiopulmonary support and induction of pulsatile shear stress. pGz increases plasma nitrite levels, in vivo and in vitro. Additionally, pGz confers cardioprotection in models of ischemia reperfusion injury. We hypothesize that pGz may also confer a cardiac phenotypic change by upregulation of the expression of the various NO synthase (NOS) isoforms in vivo. pGz was applied for 1h to awake restrained male rats at 2 frequencies (360 and 600 cpm) and acceleration (Gz) of +/-3.4 m/s(2). pGz did not affect arterial blood gases or electrolytes. pGz significantly increased total nitrosylated protein levels, indicating increased NO production. pGz also increased mRNA and protein levels of eNOS and nNOS, and phosphorylated eNOS in heart. pGz increased Akt phosphorylation (p-AKT), but not total Akt, or phosphorylated ERK1/2. Inducible (i) NOS levels were undetectable with or without pGz. Immunoblotting revealed the localization of nNOS, exclusively in cardiomyocyte, and pGz increased its expression. We have demonstrated that pGz changes myocardial NOS phenotypes. Such upregulation of eNOS and nNOS was still evident 24h after pGz. Further studies are needed to understand the biochemical and biomechanical signal transduction pathway for the observed NOS phenotype changed induced by pGz.

Prostate cancer is a bimodal disease with aggressive and indolent forms. Current prostate-specific-antigen testing and digital rectal examination screening provide ambiguous results leading to both under-and over-treatment. Accurate, consistent diagnosis is crucial to risk-stratify patients and facilitate clinical decision making as to treatment versus active surveillance. Diagnosis is currently achieved by needle biopsy, a painful procedure. Thus, there is a clinical need for a minimally-invasive test to determine prostate cancer aggressiveness. A blood sample to predict Gleason score, which is known to reflect aggressiveness of the cancer, could serve as such a test.

Calsequestrin (CS), the major Ca(2+)-binding protein in the sarcoplasmic reticulum (SR), is thought to play a dual role in excitation-contraction coupling: buffering free Ca(2+) increasing SR capacity, and modulating the activity of the Ca(2+) release channels (RyRs). In this study, we generated and characterized the first murine model lacking the skeletal CS isoform (CS1). CS1-null mice are viable and fertile, even though skeletal muscles appear slightly atrophic compared to the control mice. No compensatory increase of the cardiac isoform CS2 is detectable in any type of skeletal muscle. CS1-null muscle fibres are characterized by structural and functional changes, which are much more evident in fast-twitch muscles (EDL) in which most fibres express only CS1, than in slow-twitch muscles (soleus), where CS2 is expressed in about 50% of the fibres. In isolated EDL muscle, force development is preserved, but characterized by prolonged time-to-peak and half-relaxation time, probably related to impaired calcium release from and re-uptake by the SR. Ca(2+)-imaging studies show that the amount of Ca(2+) released from the SR and the amplitude of the Ca(2+) transient are significantly reduced. The lack of CS1 also causes significant ultrastructural changes, which include: (i) striking proliferation of SR junctional domains; (ii) increased density of Ca(2+)-release channels (confirmed also by (3)H-ryanodine binding); (iii) decreased SR terminal cisternae volume; (iv) higher density of mitochondria. Taken together these results demonstrate that CS1 is essential for the normal development of the SR and its calcium release units and for the storage and release of appropriate amounts of SR Ca(2+).

Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RyR1 in skeletal muscle. Here, we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant malignant hyperthermia when exposed to halothane or moderate heat stress. All three genotypes (WT, HET, and HOM) express similar levels of CaV1.1 by quantitative PCR, Western blot, [3H]PN200-110 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of transient receptor potential canonical (TRPC) 3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant malignant hyperthermia response to halothane and/or heat stress in HET and HOM mice.

Myocardial infarction (MI) may produce significant inflammatory changes and adverse ventricular remodeling leading to heart failure and premature death. Pharmacologic, stem cell transplantation, and exercise have not halted the inexorable rise in the prevalence and great economic costs of heart failure despite extensive investigations of such treatments. New therapeutic modalities are needed. Whole Body Periodic Acceleration (pGz) is a non-invasive technology that increases pulsatile shear stress to the endothelium thereby producing several beneficial cardiovascular effects as demonstrated in animal models, normal humans and patients with heart disease. pGz upregulates endothelial derived nitric oxide synthase (eNOS) and its phosphorylation (p-eNOS) to improve myocardial function in models of myocardial stunning and preconditioning. Here we test whether pGz applied chronically after focal myocardial infarction in rats improves functional outcomes from MI. Focal MI was produced by left coronary artery ligation. One day after ligation animals were randomized to receive daily treatments of pGz for four weeks (MI-pGz) or serve as controls (MI-CONT), with an additional group as non-infarction controls (Sham). Echocardiograms and invasive pressure volume loop analysis were carried out. Infarct transmurality, myocardial fibrosis, and markers of inflammatory and anti-inflammatory cytokines were determined along with protein analysis of eNOS, p-eNOS and inducible nitric oxide synthase (iNOS).At four weeks, survival was 80% in MI-pGz vs 50% in MI-CONT (p< 0.01). Ejection fraction and fractional shortening and invasive pressure volume relation indices of afterload and contractility were significantly better in MI-pGz. The latter where associated with decreased infarct transmurality and decreased fibrosis along with increased eNOS, p-eNOS. Additionally, MI-pGz had significantly lower levels of iNOS, inflammatory cytokines (IL-6, TNF-α), and higher level of anti-inflammatory cytokine (IL-10). pGz improved survival and contractile performance, associated with improved myocardial remodeling. pGz may serve as a simple, safe, non-invasive therapeutic modality to improve myocardial function after MI.

The medial nucleus of the trapezoid body (MNTB) in the superior olivary complex (SOC) is an inhibitory hub considered critical for binaural sound localization. We show that genetic ablation of MNTB neurons in mice only subtly affects this ability by prolonging the minimum time required to detect shifts in sound location. Furthermore, glycinergic innervation of the SOC is maintained without an MNTB, consistent with the existence of parallel inhibitory inputs. These findings redefine the role of MNTB in sound localization and suggest that the inhibitory network is more complex than previously thought.

Duchenne Muscular Dystrophy (DMD) is a recessive X-linked genetic disease, caused by mutations in the gene encoding dystrophin. DMD is characterized in humans and in mdx mice by a severe and progressive destruction of muscle fibers, inflammation, oxidative/nitrosative stress, and cell death. In mdx muscle fibers, we have shown that basal ATP release is increased and that extracellular ATP stimulation is pro-apoptotic. In normal fibers, depolarization-induced ATP release is blocked by nifedipine, leading us to study the potential therapeutic effect of nifedipine in mdx muscles and its relation with extracellular ATP signaling. Acute exposure to nifedipine (10 µM) decreased [Ca(2+)]r, NF-κB activity and iNOS expression in mdx myotubes. In addition, 6-week-old mdx mice were treated with daily intraperitoneal injections of nifedipine, 1 mg/Kg for 1 week. This treatment lowered the [Ca(2+)]r measured in vivo in the mdx vastus lateralis. We demonstrated that extracellular ATP levels were higher in adult mdx flexor digitorum brevis (FDB) fibers and can be significantly reduced after 1 week of treatment with nifedipine. Interestingly, acute treatment of mdx FDB fibers with apyrase, an enzyme that completely degrades extracellular ATP to AMP, reduced [Ca(2+)]r to a similar extent as was seen in FDB fibers after 1-week of nifedipine treatment. Moreover, we demonstrated that nifedipine treatment reduced mRNA levels of pro-oxidative/nitrosative (iNOS and gp91(phox)/p47(phox) NOX2 subunits) and pro-apoptotic (Bax) genes in mdx diaphragm muscles and lowered serum creatine kinase (CK) levels. In addition, nifedipine treatment increased muscle strength assessed by the inverted grip-hanging test and exercise tolerance measured with forced swimming test in mdx mice. We hypothesize that nifedipine reduces basal ATP release, thereby decreasing purinergic receptor activation, which in turn reduces [Ca(2+)]r in mdx skeletal muscle cells. The results in this work open new perspectives towards possible targets for pharmacological approaches to treat DMD.

Bidirectional communication between the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor (RYR1) in the sarcoplasmic reticulum (SR) is responsible for both skeletal-type excitation-contraction coupling (voltage-gated Ca(2+) release from the SR) and increased amplitude of L-type Ca(2+) current via the DHPR. Because the DHPR and RYR1 are functionally coupled, mutations in RYR1 that are linked to malignant hyperthermia (MH) may affect DHPR activity. For this reason, we investigated whether cultured myotubes originating from mice carrying an MH-linked mutation in RYR1 (R163C) had altered voltage-gated Ca(2+) release from the SR, membrane-bound charge movement, and/or L-type Ca(2+) current. In myotubes homozygous (Hom) for the R163C mutation, voltage-gated Ca(2+) release from the SR was substantially reduced and shifted ( approximately 10 mV) to more hyperpolarizing potentials compared with wild-type (WT) myotubes. Intramembrane charge movements of both Hom and heterozygous (Het) myotubes displayed hyperpolarizing shifts similar to that observed in voltage-gated SR Ca(2+) release. The current-voltage relationships for L-type currents in both Hom and Het myotubes were also shifted to more hyperpolarizing potentials ( approximately 7 and 5 mV, respectively). Compared with WT myotubes, Het and Hom myotubes both displayed a greater sensitivity to the L-type channel agonist +/-Bay K 8644 (10 microM). In general, L-type currents in WT, Het, and Hom myotubes inactivated modestly after 30-s prepulses to -50, -10, 0, 10, 20, and 30 mV. However, L-type currents in Hom myotubes displayed a hyperpolarizing shift in inactivation relative to L-type currents in either WT or Het myotubes. Our present results indicate that mutations in RYR1 can alter DHPR activity and raise the possibility that this altered DHPR function may contribute to MH episodes.

Developing skeletal myofibers in vertebrates are intrinsically 'pre-patterned' for motor nerve innervation. However, the intrinsic factors that regulate muscle pre-patterning remain unknown. We found that a functional skeletal muscle dihydropyridine receptor (DHPR, the L-type Ca(2+) channel in muscle) was required for muscle pre-patterning during the development of the neuromuscular junction (NMJ). Targeted deletion of the β1 subunit of DHPR (Cacnb1) in mice led to muscle pre-patterning defects, aberrant innervation and precocious maturation of the NMJ. Reintroducing Cacnb1 into Cacnb1(-/-) muscles reversed the pre-patterning defects and restored normal development of the NMJ. The mechanism by which DHPRs govern muscle pre-patterning is independent of their role in excitation-contraction coupling, but requires Ca(2+) influx through the L-type Ca(2+) channel. Our findings indicate that the skeletal muscle DHPR retrogradely regulates the patterning and formation of the NMJ.

Sepsis is a life threatening condition which produces multi-organ dysfunction with profound circulatory and cellular derangements. Administration of E.Coli endotoxin (LPS) produces systemic inflammatory effects of sepsis including disruption of endothelial barrier, and if severe enough death. Whole body periodic acceleration (pGz) is the headward-footward motion of the body. pGz has been shown to induce pulsatile shear stress to the endothelium, thereby releasing vascular and cardio protective mediators. The purpose of this study was to determine whether or not pGz performed as a pre-treatment or post-treatment strategy improves survival in a lethal murine endotoxin model.This study was designed as a prospective randomized controlled study in mice. pGz was performed in mice as pre-treatment (pGz-LPS, 3 days prior to LPS), post-treatment (LPS- pGz, 30 min after LPS) strategies or Control (LPS-CONT), in a lethal murine model of endotoxemia. Endotoxemia was induced with intraperitoneal injection of E.Coli LPS (40mg/kg). In a separate group of mice, a nonspecific nitric oxide synthase inhibitor (L-NAME) was provided in their drinking water and pGz-LPS and LPS-pGz performed to determine the effect of nitric oxide (NO) inhibition on survival. In another subset of mice, micro vascular leakage was determined. Behavioral scoring around the clock was performed in all mice at 30 min intervals after LPS administration, until 48 hrs. survival or death. LPS induced 100% mortality in LPS-CONT animals by 30 hrs. In contrast, survival to 48 hrs. occurred in 60% of pGz-LPS and 80% of LPS-pGz. L-NAME abolished the survival effects of pGz. Microvascular leakage was markedly reduced in both pre and post pGz treated animals and was associated with increased tyrosine kinase endothelial-enriched tunica interna endothelial cell kinase 2 (TIE2) receptor and its phosphorylation (p-TIE2). In a murine model of lethal endotoxemia, pGz performed as a pre or post treatment strategy significantly improved survival, and markedly reduced microvascular leakage. The effect was modulated, in part, by NO since a non-selective inhibitor of NO abolished the pGz survival effect.

Neuronal intracellular Ca2+ homeostasis is critical to the normal physiological functions of neurons and neuronal Ca2+ dyshomeostasis has been associated with the age-related decline of cognitive functions. Accumulated evidence indicates that the underlying mechanism for this is that abnormal intracellular Ca2+ levels stimulate the dysregulation of intracellular signaling, which subsequently induces neuronal cell death. We examined intracellular Ca2+ homeostasis in cortical (in vivo) and hippocampal (in vitro) neurons from young (3-months), middle-age (12-months), and aged (24-months) wild type C57BL6J mice. We found a progressive age-related elevation of intracellular resting calcium ([Ca2+]r) in cortical (in vivo) and hippocampal (in vitro) neurons associated with increased hippocampal neuronal calpain activity and reduced cell viability. In vitro, removal of extracellular Ca2+ or treatment with SAR7334 or dantrolene reduced [Ca2+]r in all age groups and dantrolene treatment lowered calpain activity and increased cell viability. In vivo, both middle-aged and aged mice showed cognitive deficits compared to young mice, which improved after dantrolene treatment. These findings support the hypothesis that intracellular Ca2+ dyshomeostasis is a major mechanism underlying the cognitive deficits seen in both normal aging and degenerative neurologic diseases.

In skeletal muscle, the release of calcium (Ca(2+)) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca(2+) release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca(2+) buffering as well as its potential for modulating RyR1, the L-type Ca(2+) channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca(2+)]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca(2+) content and SR Ca(2+) release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca(2+) release with single action potentials and a collapse of the Ca(2+) release with repetitive trains. Under voltage clamp, SR Ca(2+) release flux and total SR Ca(2+) release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca(2+) release flux appears to be solely due to elimination of the slowly decaying component of SR Ca(2+) release, whereas the rapidly decaying component of SR Ca(2+) release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca(2+)] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca(2+)](free) in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca(2+) buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca(2+) release.

Triadin (Tdn) and Junctin (Jct) are structurally related transmembrane proteins thought to be key mediators of structural and functional interactions between calsequestrin (CASQ) and ryanodine receptor (RyRs) at the junctional sarcoplasmic reticulum (jSR). However, the specific contribution of each protein to the jSR architecture and to excitation-contraction (e-c) coupling has not been fully established. Here, using mouse models lacking either Tdn (Tdn-null), Jct (Jct-null) or both (Tdn/Jct-null), we identify Tdn as the main component of periodically located anchors connecting CASQ to the RyR-bearing jSR membrane. Both proteins proved to be important for the structural organization of jSR cisternae and retention of CASQ within them, but with different degrees of impact. Our results also suggest that the presence of CASQ is responsible for the wide lumen of the jSR cisternae. Using Ca(2+) imaging and Ca(2+) selective microelectrodes we found that changes in e-c coupling, SR Ca(2+)content and resting [Ca(2+)] in Jct, Tdn and Tdn/Jct-null muscles are directly correlated to the effect of each deletion on CASQ content and its organization within the jSR. These data suggest that in skeletal muscle the disruption of Tdn/CASQ link has a more profound effect on jSR architecture and myoplasmic Ca(2+) regulation than Jct/CASQ association.

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca2+, inhibits halothane- and isoflurane-induced Ca2+ release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.

Skeletal muscle is the primary site of insulin-mediated glucose uptake through the body and, therefore, an essential contributor to glucose homeostasis maintenance. We have recently provided evidence that chronic elevated intracellular Ca2+ concentration at rest [(Ca2+)i] compromises glucose homeostasis in malignant hyperthermia muscle cells. To further investigate how chronic elevated muscle [Ca2+]i modifies insulin-mediated glucose homeostasis, we measured [Ca2+]i and glucose uptake in vivo and in vitro in intact polarized muscle cells from glucose-intolerant RYR1-p.R163C and db/db mice. Glucose-intolerant RYR1-p.R163C and db/db mice have significantly elevated muscle [Ca2+]i and reduced muscle glucose uptake compared to WT muscle cells. Dantrolene treatment (1.5 mg/kg IP injection for 2 weeks) caused a significant reduction in fasting blood glucose levels and muscle [Ca2+]i and increased muscle glucose uptake compared to untreated RYR1-p.R163C and db/db mice. Furthermore, RYR1-p.R163C and db/db mice had abnormal basal insulin levels and response to glucose-stimulated insulin secretion. In vitro experiments conducted on single muscle fibers, dantrolene improved insulin-mediated glucose uptake in RYR1-p.R163C and db/db muscle fibers without affecting WT muscle fibers. In muscle cells with chronic elevated [Ca2+]i, GLUT4 expression was significantly lower, and the subcellular fraction (plasma membrane/cytoplasmic) was abnormal compared to WT. The results of this study suggest that i) Chronic elevated muscle [Ca2+]i decreases insulin-stimulated glucose uptake and consequently causes hyperglycemia; ii) Reduced muscle [Ca2+]i by dantrolene improves muscle glucose uptake and subsequent hyperglycemia; iii) The mechanism by which chronic high levels of [Ca2+]i interfere with insulin action appears to involve the expression of GLUT4 and its subcellular fractionation.

The recognition that oxidative stress is a major component of several chronic diseases has engendered numerous trials of antioxidant therapies with minimal or no direct benefits. Nanomolar quantities of nitric oxide released into the circulation by pharmacologic stimulation of eNOS have antioxidant properties but physiologic stimulation as through increased pulsatile shear stress of the endothelium has not been assessed. The present study utilized a non-invasive technology, periodic acceleration (pGz) that increases pulsatile shear stress such that upregulation of cardiac eNOS occurs, We assessed its efficacy in normal mice and mouse models with high levels of oxidative stress, e.g. Diabetes type 1 and mdx (Duchene Muscular Dystrophy). pGz increased protein expression and upregulated eNOS in hearts. Application of pGz was associated with significantly increased expression of endogenous antioxidants (Glutathioneperoxidase-1(GPX-1), Catalase (CAT), Superoxide, Superoxide Dismutase 1(SOD1). This led to an increase of total cardiac antioxidant capacity along with an increase in the antioxidant response element transcription factor Nrf2 translocation to the nucleus. pGz decreased reactive oxygen species in both mice models of oxidative stress. Thus, pGz is a novel non-pharmacologic method to harness endogenous antioxidant capacity.

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca(2+) and Na(+) overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca(2+) and Na(+) overload, diminished abnormal sarcolemmal Ca(2+) entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway.

Contractile activation in striated muscles requires a Ca(2+) reservoir of large capacity inside the sarcoplasmic reticulum (SR), presumably the protein calsequestrin. The buffering power of calsequestrin in vitro has a paradoxical dependence on [Ca(2+)] that should be valuable for function. Here, we demonstrate that this dependence is present in living cells. Ca(2+) signals elicited by membrane depolarization under voltage clamp were compared in single skeletal fibers of wild-type (WT) and double (d) Casq-null mice, which lack both calsequestrin isoforms. In nulls, Ca(2+) release started normally, but the store depleted much more rapidly than in the WT. This deficit was reflected in the evolution of SR evacuability, E, which is directly proportional to SR Ca(2+) permeability and inversely to its Ca(2+) buffering power, B. In WT mice E starts low and increases progressively as the SR is depleted. In dCasq-nulls, E started high and decreased upon Ca(2+) depletion. An elevated E in nulls is consistent with the decrease in B expected upon deletion of calsequestrin. The different value and time course of E in cells without calsequestrin indicate that the normal evolution of E reflects loss of B upon SR Ca(2+) depletion. Decrement of B upon SR depletion was supported further. When SR calcium was reduced by exposure to low extracellular [Ca(2+)], release kinetics in the WT became similar to that in the dCasq-null. E became much higher, similar to that of null cells. These results indicate that calsequestrin not only stores Ca(2+), but also varies its affinity in ways that progressively increase the ability of the store to deliver Ca(2+) as it becomes depleted, a novel feedback mechanism of potentially valuable functional implications. The study revealed a surprisingly modest loss of Ca(2+) storage capacity in null cells, which may reflect concurrent changes, rather than detract from the physiological importance of calsequestrin.

Duchenne muscular dystrophy (DMD) is an irreversible muscle disease characterized by a progressive loss of muscle function, decreased ambulation, and ultimately death as a result of cardiac or respiratory failure. DMD is caused by the lack of dystrophin, a protein that is important for membrane stability and signaling in excitable cells. Although vascular smooth muscle cells (VSMCs) dysfunction occurs in many pathological conditions, little is known about vascular smooth muscle function in DMD. We have previously shown that striated muscle cells, as well as neurons isolated from dystrophic (mdx) mice have higher intracellular Ca2+ ([Ca2+]i) and Na+ ([Na+]i) concentrations and decreased cell viability in comparison with wild type (Wt). Experiments were carried out in isolated VSMCs from mdx (a murine model of DMD) and congenic C57BL/10SnJ Wt mice. We found elevated [Ca2+]i and [Na+]i in VSMCs from mdx mice compared to Wt. Exposure to 1-oleoyl-2-acetyl-sn-glycerol (OAG), a TRPC3 and TRPC6 channel activator, induced a greater elevation of [Ca2+]i and [Na+]i in mdx than Wt VSMCs. The OAG induced increases in [Ca2+]i could be abolished by either removal of extracellular Ca2+ or by SAR7334, a blocker of TRPC3 and TRPC 6 channels in both genotypes. Mdx and Wt VSMCs were susceptible to muscle cell stretch-induced elevations of [Ca2+]i and [Na+]i which was completely inhibited by GsMTx-4, a mechanosensitive ion channel inhibitor. Western blots showed a significant upregulation of TRPC1 -3, -6 proteins in mdx VSMCs compare to age-matched Wt. The lack of dystrophin in mdx VSMCs produced a profound alteration of [Ca2+]i and [Na+]i homeostasis that appears to be mediated by TRPC channels. Moreover, we have been able to demonstrate pharmacologically that the enhanced stretch-induced elevation of intracellular [Ca2+] and concomitant cell damage in mdx VSMCs also appears to be mediated through TRPC1, -3 and -6 channel activation.

Bidirectional signaling between the sarcolemmal L-type Ca(2+) channel (1,4-dihydropyridine receptor [DHPR]) and the sarcoplasmic reticulum (SR) Ca(2+) release channel (type 1 ryanodine receptor [RYR1]) of skeletal muscle is essential for excitation-contraction coupling (ECC) and is a well-understood prototype of conformational coupling. Mutations in either channel alter coupling fidelity and with an added pharmacologic stimulus or stress can trigger malignant hyperthermia (MH). In this study, we measured the response of wild-type (WT), heterozygous (Het), or homozygous (Hom) RYR1-R163C knock-in mouse myotubes to maintained K(+) depolarization. The new findings are: (a) For all three genotypes, Ca(2+) transients decay during prolonged depolarization, and this decay is not a consequence of SR depletion or RYR1 inactivation. (b) The R163C mutation retards the decay rate with a rank order WT > Het > Hom. (c) The removal of external Ca(2+) or the addition of Ca(2+) entry blockers (nifedipine, SKF96365, and Ni(2+)) enhanced the rate of decay in all genotypes. (d) When Ca(2+) entry is blocked, the decay rates are slower for Hom and Het than WT, indicating that the rate of inactivation of ECC is affected by the R163C mutation and is genotype dependent (WT > Het > Hom). (e) Reduced ECC inactivation in Het and Hom myotubes was shown directly using two identical K(+) depolarizations separated by varying time intervals. These data suggest that conformational changes induced by the R163C MH mutation alter the retrograde signal that is sent from RYR1 to the DHPR, delaying the inactivation of the DHPR voltage sensor.