Jose R. Lopez

Increased intraneuronal resting [Ca2+] in adult Alzheimer’s disease mice

Neurodegeneration in Alzheimer’s disease (AD) has been linked to intracellular accumulation of misfolded proteins and dysregulation of intracellular Ca2+. In the current work, we determined the contribution of specific Ca2+ pathways to an alteration in Ca2+ homeostasis in primary cortical neurons from an adult triple transgenic (3xTg‐AD) mouse model of AD that exhibits intraneuronal accumulation of β‐amyloid proteins. Resting free Ca2+ concentration ([Ca2+]i), as measured with Ca2+‐selective microelectrodes, was greatly elevated in neurons from 3xTg‐AD and APPSWE mouse strains when compared with their respective non‐transgenic neurons, while there was no alteration in the resting membrane potential. In the absence of the extracellular Ca2+, the [Ca2+]i returned to near normal levels in 3xTg‐AD neurons, demonstrating that extracellular Ca2+contributed to elevated [Ca2+]i. Application of nifedipine, or a non‐L‐type channel blocker, SKF‐96365, partially reduced [Ca2+]i. Blocking the ryanodine receptors, with ryanodine or FLA‐365 had no effect, suggesting that these channels do not contribute to the elevated [Ca2+]i. Conversely, inhibition of inositol trisphosphate receptors with xestospongin C produced a partial reduction in [Ca2+]i. These results demonstrate that an elevation in resting [Ca2+]i, contributed by aberrant Ca2+entry and release pathways, should be considered a major component of the abnormal Ca2+ homeostasis associated with AD.

Enhanced excitation-coupled calcium entry in myotubes expressing malignant hyperthermia mutation R163C is attenuated by dantrolene.

Dantrolene is the drug of choice for the treatment of malignant hyperthermia (MH) and is also useful for treatment of spasticity or muscle spasms associated with several clinical conditions. The current study examines the mechanisms of dantrolenes action on skeletal muscle and shows that one of dantrolenes mechanisms of action is to block excitation-coupled calcium entry (ECCE) in both adult mouse flexor digitorum brevis fibers and primary myotubes. A second important new finding is that myotubes isolated from mice heterozygous and homozygous for the ryanodine receptor type 1 R163C MH susceptibility mutation show significantly enhanced ECCE rates that could be restored to those measured in wild-type cells after exposure to clinical concentrations of dantrolene. We propose that this gain of ECCE function is an important etiological component of MH susceptibility and possibly contributes to the fulminant MH episode. The inhibitory potency of dantrolene on ECCE found in wild-type and MH-susceptible muscle is consistent with the drugs clinical potency for reversing the MH syndrome and is incomplete as predicted by its efficacy as a muscle relaxant.

Heart and skeletal muscle are targets of dengue virus infection.

Background: Dengue fever is one of the most significant re-emerging tropical diseases, despite our expanding knowledge of the disease, viral tropism is still not known to target heart tissues or muscle. Methods: A prospective pediatric clinical cohort of 102 dengue hemorrhagic fever patients from Colombia, South America, was followed for 1 year. Clinical diagnosis of myocarditis was routinely performed. Electrocardiograph and echocardiograph analysis were performed to confirm those cases. Immunohistochemistry for detection of dengue virus and inflammatory markers was performed on autopsied heart tissue. In vitro studies of human striated skeletal fibers (myotubes) infected with dengue virus were used as a model for myocyte infection. Measurements of intracellular Ca2+ concentration as well as immunodetection of dengue virus and inflammation markers in infected myotubes were performed. Results: Eleven children with dengue hemorrhagic fever presented with symptoms of myocarditis. Widespread viral infection of the heart, myocardial endothelium, and cardiomyocytes, accompanied by inflammation was observed in 1 fatal case. Immunofluorescence confocal microscopy showed that myotubes were infected by dengue virus and had increased expression of the inflammatory genes and protein IP-10. The infected myotubes also had increases in intracellular Ca2+ concentration. Conclusions: Vigorous infection of heart tissues in vivo and striated skeletal cells in vitro are demonstrated. Derangements of Ca2+ storage in the infected cells may directly contribute to the presentation of myocarditis in pediatric patients.

Pharmacologic and Functional Characterization of Malignant Hyperthermia in the R163C RyR1 Knock-in Mouse

Background:Malignant hyperthermia is a pharmacogenetic disorder affecting humans, dogs, pigs, and horses. In the majority of human cases and all cases in animals, malignant hyperthermia has been associated with missense mutations in the skeletal ryanodine receptor (RyR1). Methods:The authors used a “knock-in” targeting vector to create mice carrying the RyR1 R163C malignant hyperthermia mutation. Results:Validation of this new mouse model of human malignant hyperthermia susceptibility includes (1) proof of transcription of the R163C allele and expression of ryanodine receptor protein in R163C heterozygous and R163C homozygous animals; (2) fulminant malignant hyperthermia episodes in R163C heterozygous mice after exposure to 1.25–1.75% halothane or an ambient temperature of 42°C characterized by increased rectal temperature, respiratory rate, and inspiratory effort, with significant blood biochemical changes indicating metabolic acidosis, ending in death and hyperacute rigor mortis; (3) intraperitoneal pretreatment with dantrolene provided 100% protection from the halothane-triggered fulminant malignant hyperthermia episode; (4) significantly increased sensitivity (decreased effective concentration causing 50% of the maximal response) of R163C heterozygous and homozygous myotubes to caffeine, 4-chloro-m-cresol, and K+-induced depolarization; (5) R163C heterozygous and homozygous myotubes have a significantly increased resting intracellular Ca2+ concentration compared with wild type; (6) R163C heterozygous sarcoplasmic reticulum membranes have a twofold higher affinity (Kd = 35.4 nm) for [3H]ryanodine binding compared with wild type (Kd = 80.1 nm) and a diminished inhibitory regulation by Mg2+. Conclusions:Heterozygous R163C mice represent a valid model for studying the mechanisms that cause the human malignant hyperthermia syndrome.

Triadins Modulate Intracellular Ca2+ Homeostasis but Are Not Essential for Excitation-Contraction Coupling in Skeletal Muscle

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca2+ imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca2+ transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca2+.[3H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca2+ activation or Ca2+ and Mg2+ inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca2+ homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.

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 Ca2+ 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 CaV1.1, the principal subunit of the L-type Ca2+ channel. All of the mutations that have been characterized previously augment EC coupling and/or increase the rate of L-type Ca2+ entry. The CaV1.1 mutation R174W associated with MH susceptibility occurs at the innermost basic residue of the IS4 voltage-sensing helix, a residue conserved among all CaV 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 (CaV1.1 null) myotubes. Unlike previously described MH-linked mutations in CaV1.1, R174W ablated the L-type current and had no effect on EC coupling. Nonetheless, R174W increased sensitivity of Ca2+ release to caffeine (used for MH diagnostic in vitro testing) and to volatile anesthetics. Moreover, in CaV1.1 R174W-expressing myotubes, resting myoplasmic Ca2+ levels were elevated, and sarcoplasmic reticulum (SR) stores were partially depleted, compared with myotubes expressing wild-type CaV1.1. Our results indicate that CaV1.1 functions not only to activate RyR1 during EC coupling, but also to suppress resting RyR1-mediated Ca2+ leak from the SR, and that perturbation of CaV1.1 negative regulation of RyR1 leak identifies a unique mechanism that can sensitize muscle cells to MH triggers.

Increased Resting Intracellular Calcium Modulates NF-κB-dependent Inducible Nitric-oxide Synthase Gene Expression in Dystrophic mdx Skeletal Myotubes

Background: The mechanisms by which NF-κB signaling is up-regulated in dystrophic muscles are unclear. Results: [Ca2+]rest is elevated in mdx myotubes as a result of both sarcolemmal Ca2+ entry and SR release, resulting in NF-κB-induced iNOS expression. Conclusion: Ca2+ alterations at rest modulate NF-κB transcriptional activity and pro-inflammatory gene expression. Significance: This allows for understanding the mechanism that relates elevated resting calcium and altered gene expression in muscular dystrophy. Duchenne muscular dystrophy (DMD) is a genetic disorder caused by dystrophin mutations, characterized by chronic inflammation and severe muscle wasting. Dystrophic muscles exhibit activated immune cell infiltrates, up-regulated inflammatory gene expression, and increased NF-κB activity, but the contribution of the skeletal muscle cell to this process has been unclear. The aim of this work was to study the pathways that contribute to the increased resting calcium ([Ca2+]rest) observed in mdx myotubes and its possible link with up-regulation of NF-κB and pro-inflammatory gene expression in dystrophic muscle cells. [Ca2+]rest was higher in mdx than in WT myotubes (308 ± 6 versus 113 ± 2 nm, p < 0.001). In mdx myotubes, both the inhibition of Ca2+ entry (low Ca2+ solution, Ca2+-free solution, and Gd3+) and blockade of either ryanodine receptors or inositol 1,4,5-trisphosphate receptors reduced [Ca2+]rest. Basal activity of NF-κB was significantly up-regulated in mdx versus WT myotubes. There was an increased transcriptional activity and p65 nuclear localization, which could be reversed when [Ca2+]rest was reduced. Levels of mRNA for TNFα, IL-1β, and IL-6 were similar in WT and mdx myotubes, whereas inducible nitric-oxide synthase (iNOS) expression was increased 5-fold. Reducing [Ca2+]rest using different strategies reduced iNOS gene expression presumably as a result of decreased activation of NF-κB. We propose that NF-κB, modulated by increased [Ca2+]rest, is constitutively active in mdx myotubes, and this mechanism can account for iNOS overexpression and the increase in reactive nitrogen species that promote damage in dystrophic skeletal muscle cells.

Effects of Dantrolene on Myoplasmic Free [Ca2+] Measured In Vivo in Patients Susceptible to Malignant Hyperthermia

Malignant hyperthermia (MH) is a genetic disease characterized by hypermetabolism in skeletal muscle following a triggering stimulus and can be reversed or pretreated with dantrolene sodium. The myoplasmic free [Ca2+] was measured, using Ca2+ selective microelectrodes in vivo in the superficial fibers of the sartorius muscle of eight MH-susceptible and eight control subjects. Both groups received continuous epidural anesthesia with chloroprocaine 3%. In both the control and MH muscle fibers, the myoplasmic free [Ca2+] was measured before and after the intravenous administration of a cumulative dantrolene dose of 0.5, 1.5, and 2.5 mg/kg. The mean resting myoplasmic free [Ca2+] was 0.112 +/- 0.004 microM (mean +/- SEM n = 32) in the control and 0.485 +/- 0.022 microM (n = 33) in the MH subjects. In the MH subjects, dantrolene induced a dose-dependent reduction in myoplasmic free [Ca2+]. The 0.5-mg/kg dose reduced it to 0.326 +/- 0.017 microM (n = 22), the 1.5-mg/kg dose to 0.233 +/- 0.015 microM (n = 25), and the 2.5-mg/kg dose to 0.092 +/- 0.008 microM (n = 26). In controls, dantrolene also reduced resting myoplasmic free [Ca2+] but to a lesser extent. The 0.5-mg/kg dose reduced it to 0.096 +/- 0.004 microM (n = 22), the 1.5-mg/kg dose to 0.077 +/- 0.003 microM (n = 23), and the 2.5-mg/kg dose to 0.068 +/- 0.002 microM (n = 27). The results of the study extend our previous findings in humans and swine and demonstrate that it is possible to measure myoplasmic free [Ca2+] in vivo in humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced Excitation-coupled Calcium Entry in Myotubes Is Associated with Expression of RyR1 Malignant Hyperthermia Mutations

Myotubes expressing wild type RyR1 (WT) or RyR1 with one of three malignant hyperthermia mutations R615C, R2163C, and T4826I (MH) were exposed sequentially to 60 mm KCl in Ca2+-replete and Ca2+-free external buffers (Ca+ and Ca–, respectively) with 3 min of rest between exposures. Although the maximal peak amplitude of the Ca2+ transients during K+ depolarization was similar for WT and MH in both external buffers, the rate of decay of the sustained phase of the transient during K+ depolarization (decay rate) in Ca+ was 50% slower for MH. This difference was eliminated in Ca–, and the relative decay rates were faster for both genotypes than in Ca+. The integrated Ca2+ transient in Ca–compared with Ca+ was reduced by 50–60% for MH and 20% for WT. The decay rate was not affected by [K+] × [Cl–] product or NiCl2 (2 mm) supplementation of Ca–. The addition of La2+ (0.1 mm), or SKF 96365 (20 μm) to Ca+ significantly accelerated decay rates for both WT and MH, but their effect was significantly greater in MH. Nifedipine (1 μm) had no effect, suggesting that the mechanism for this difference was not a reduction in L-type Ca2+ channel Ca2+ current. These data strongly suggest: 1) the decay rate in skeletal myotubes is related in part to Ca2+ entry through the ECCE channel; 2) the MH mutations enhance ECCE compared with wild type; and 3) the increased Ca2+ entry might play a significant role in the pathophysiology of MH.

α2δ1 Dihydropyridine Receptor Subunit Is a Critical Element for Excitation-Coupled Calcium Entry but Not for Formation of Tetrads in Skeletal Myotubes

It has been shown that small interfering RNA (siRNA) partial knockdown of the alpha(2)delta(1) dihydropyridine receptor subunits cause a significant increase in the rate of activation of the L-type Ca(2+) current in myotubes but have little or no effect on skeletal excitation-contraction coupling. This study used permanent siRNA knockdown of alpha(2)delta(1) to address two important unaddressed questions. First, does the alpha(2)delta(1) subunit contribute to the size and/or spacing of tetradic particles? Second, is the alpha(2)delta(1) subunit important for excitation-coupled calcium entry? We found that the size and spacing of tetradic particles is unaffected by siRNA knockdown of alpha(2)delta(1), indicating that the visible particle represents the alpha(1s) subunit. Strikingly, >97% knockdown of alpha(2)delta(1) leads to a complete loss of excitation-coupled calcium entry during KCl depolarization and a more rapid decay of Ca(2+) transients during bouts of repetitive electrical stimulation like those occurring during normal muscle activation in vivo. Thus, we conclude that the alpha(2)delta(1) dihydropyridine receptor subunit is physiologically necessary for sustaining Ca(2+) transients in response to prolonged depolarization or repeated trains of action potentials.