Jianxun Yi

Activation of Protein Kinase A Modulates Trafficking of the Human Cardiac Sodium Channel in Xenopus Oocytes

Voltage-gated Na(+) channels are critical determinants of electrophysiological properties in the heart. Stimulation of beta-adrenergic receptors, which activate cAMP-dependent protein kinase (protein kinase A [PKA]), can alter impulse conduction in normal tissue and promote development of cardiac arrhythmias in pathological states. Recent studies demonstrate that PKA activation increases cardiac Na(+) currents, although the mechanism of this effect is unknown. To explore the molecular basis of Na(+) channel modulation by beta-adrenergic receptors, we have examined the effects of PKA activation on the recombinant human cardiac Na(+) channel, hH1. Both in the absence and the presence of hbeta(1) subunit coexpression, activation of PKA caused a slow increase in Na(+) current that did not saturate despite kinase stimulation for 1 hour. In addition, there was a small shift in the voltage dependence of channel activation and inactivation to more negative voltages. Chloroquine and monensin, compounds that disrupt plasma membrane recycling, reduced hH1 current, suggesting rapid turnover of channels at the cell surface. Preincubation with these agents also prevented the PKA-mediated rise in Na(+) current, indicating that this effect likely resulted from an increased number of Na(+) channels in the plasma membrane. Experiments using chimeric constructs of hH1 and the skeletal muscle Na(+) channel, hSKM1, identified the I-II interdomain loop of hH1 as the region responsible for the PKA effect. These results demonstrate that activation of PKA modulates both trafficking and function of the hH1 channel, with changes in Na(+) current that could either speed or slow conduction, depending on the physiological circumstances.

Hyperactive Intracellular Calcium Signaling Associated with Localized Mitochondrial Defects in Skeletal Muscle of an Animal Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by degeneration of motor neurons and atrophy of skeletal muscle. Mutations in the superoxide dismutase (SOD1) gene are linked to 20% cases of inherited ALS. Mitochondrial dysfunction has been implicated in the pathogenic process, but how it contributes to muscle degeneration of ALS is not known. Here we identify a specific deficit in the cellular physiology of skeletal muscle derived from an ALS mouse model (G93A) with transgenic overexpression of the human SOD1G93A mutant. The G93A skeletal muscle fibers display localized loss of mitochondrial inner membrane potential in fiber segments near the neuromuscular junction. These defects occur in young G93A mice prior to disease onset. Fiber segments with depolarized mitochondria show greater osmotic stress-induced Ca2+ release activity, which can include propagating Ca2+ waves. These Ca2+ waves are confined to regions of depolarized mitochondria and stop propagating shortly upon entering the regions of normal, polarized mitochondria. Uncoupling of mitochondrial membrane potential with FCCP or inhibition of mitochondrial Ca2+ uptake by Ru360 lead to cell-wide propagation of such Ca2+ release events. Our data reveal that mitochondria regulate Ca2+ signaling in skeletal muscle, and loss of this capacity may contribute to the progression of muscle atrophy in ALS.

Phosphorylation and Putative ER Retention Signals Are Required for Protein Kinase A-Mediated Potentiation of Cardiac Sodium Current

Abstract— Activation of protein kinase A (PKA) increases Na+ current derived from the human cardiac Na+ channel, hH1, in a slow, nonsaturable manner. This effect is prevented by compounds that disrupt plasma membrane recycling, implying enhanced trafficking of channels to the cell membrane as the mechanism responsible for Na+ current potentiation. To investigate the molecular basis of this effect, preferred consensus sites (serines 483, 571, and 593) and alternative sites phosphorylated by PKA in the rat heart isoform (serines 525 and 528) were removed in the I-II interdomain linker, a region in the channel previously implicated in the PKA response. Our results demonstrate that the presence of either serine 525 or 528 is required for Na+ current potentiation. The role of amino acid sequences that can mediate channel-protein interactions was also examined. Removal of a PDZ domain-binding motif at the carboxy terminus of hH1 did not alter the PKA response. The I-II interdomain linker of the channel contains 3 sites (479RKR481, 533RRR535, and 659RQR661) with the sequence RXR, a motif known to mediate retention of proteins in the endoplasmic reticulum (ER). The PKA-mediated increase in Na+ current was abolished when all 3 sites were eliminated, with RRR at position 533 to 535 primarily responsible for this effect. These results demonstrate that both &agr;-subunit phosphorylation and the presence of putative ER retention signals are required for the PKA-mediated increase in cardiac Na+ current, an effect that likely involves interaction of the I-II interdomain linker with other proteins or regions of the channel.

Ca2+ sparks operated by membrane depolarization require isoform 3 ryanodine receptor channels in skeletal muscle

Stimuli are translated to intracellular calcium signals via opening of inositol trisphosphate receptor and ryanodine receptor (RyR) channels of the sarcoplasmic reticulum or endoplasmic reticulum. In cardiac and skeletal muscle of amphibians the stimulus is depolarization of the transverse tubular membrane, transduced by voltage sensors at tubular–sarcoplasmic reticulum junctions, and the unit signal is the Ca2+ spark, caused by concerted opening of multiple RyR channels. Mammalian muscles instead lose postnatally the ability to produce sparks, and they also lose RyR3, an isoform abundant in spark-producing skeletal muscles. What does it take for cells to respond to membrane depolarization with Ca2+ sparks? To answer this question we made skeletal muscles of adult mice expressing exogenous RyR3, demonstrated as immunoreactivity at triad junctions. These muscles showed abundant sparks upon depolarization. Sparks produced thusly were found to amplify the response to depolarization in a manner characteristic of Ca2+-induced Ca2+ release processes. The amplification was particularly effective in responses to brief depolarizations, as in action potentials. We also induced expression of exogenous RyR1 or yellow fluorescent protein-tagged RyR1 in muscles of adult mice. In these, tag fluorescence was present at triad junctions. RyR1-transfected muscle lacked voltage-operated sparks. Therefore, the voltage-operated sparks phenotype is specific to the RyR3 isoform. Because RyR3 does not contact voltage sensors, their opening was probably activated by Ca2+, secondarily to Ca2+ release through junctional RyR1. Physiologically voltage-controlled Ca2+ sparks thus require a voltage sensor, a master junctional RyR1 channel that provides trigger Ca2+, and a slave parajunctional RyR3 cohort.

Defective mitochondrial dynamics is an early event in skeletal muscle of an amyotrophic lateral sclerosis mouse model.

Mitochondria are dynamic organelles that constantly undergo fusion and fission to maintain their normal functionality. Impairment of mitochondrial dynamics is implicated in various neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is an adult-onset neuromuscular degenerative disorder characterized by motor neuron death and muscle atrophy. ALS onset and progression clearly involve motor neuron degeneration but accumulating evidence suggests primary muscle pathology may also be involved. Here, we examined mitochondrial dynamics in live skeletal muscle of an ALS mouse model (G93A) harboring a superoxide dismutase mutation (SOD1G93A). Using confocal microscopy combined with overexpression of mitochondria-targeted photoactivatable fluorescent proteins, we discovered abnormal mitochondrial dynamics in skeletal muscle of young G93A mice before disease onset. We further demonstrated that similar abnormalities in mitochondrial dynamics were induced by overexpression of mutant SOD1G93A in skeletal muscle of normal mice, indicating the SOD1 mutation drives ALS-like muscle pathology in the absence of motor neuron degeneration. Mutant SOD1G93A forms aggregates inside muscle mitochondria and leads to fragmentation of the mitochondrial network as well as mitochondrial depolarization. Partial depolarization of mitochondrial membrane potential in normal muscle by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) caused abnormalities in mitochondrial dynamics similar to that in the SOD1G93A model muscle. A specific mitochondrial fission inhibitor (Mdivi-1) reversed the SOD1G93A action on mitochondrial dynamics, indicating SOD1G93A likely promotes mitochondrial fission process. Our results suggest that accumulation of mutant SOD1G93A inside mitochondria, depolarization of mitochondrial membrane potential and abnormal mitochondrial dynamics are causally linked and cause intrinsic muscle pathology, which occurs early in the course of ALS and may actively promote ALS progression.

Mitochondrial Calcium Uptake Regulates Rapid Calcium Transients in Skeletal Muscle during Excitation-Contraction (E-C) Coupling

Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca2+ signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca2+ release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca2+ removal during excitation-contraction coupling by comparing Ca2+ transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca2+ transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ∼10% increased amplitude. These regional differences in Ca2+ transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, a fast Ca2+ chelator that reduces mitochondrial Ca2+ uptake. Using a mitochondria-targeted Ca2+ biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca2+ levels during voltage-induced Ca2+ release and detected a reduced Ca2+ uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca2+ transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca2+ signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.

Measurement of RyR permeability reveals a role of calsequestrin in termination of SR Ca 2+ release in skeletal muscle

The mechanisms that terminate Ca2+ release from the sarcoplasmic reticulum are not fully understood. D4cpv-Casq1 (Sztretye et al. 2011. J. Gen. Physiol. doi:10.1085/jgp.201010591) was used in mouse skeletal muscle cells under voltage clamp to measure free Ca2+ concentration inside the sarcoplasmic reticulum (SR), [Ca2+]SR, simultaneously with that in the cytosol, [Ca2+]c, during the response to long-lasting depolarization of the plasma membrane. The ratio of Ca2+ release flux (derived from [Ca2+]c(t)) over the gradient that drives it (essentially equal to [Ca2+]SR) provided directly, for the first time, a dynamic measure of the permeability to Ca2+ of the releasing SR membrane. During maximal depolarization, flux rapidly rises to a peak and then decays. Before 0.5 s, [Ca2+]SR stabilized at ∼35% of its resting level; depletion was therefore incomplete. By 0.4 s of depolarization, the measured permeability decayed to ∼10% of maximum, indicating ryanodine receptor channel closure. Inactivation of the t tubule voltage sensor was immeasurably small by this time and thus not a significant factor in channel closure. In cells of mice null for Casq1, permeability did not decrease in the same way, indicating that calsequestrin (Casq) is essential in the mechanism of channel closure and termination of Ca2+ release. The absence of this mechanism explains why the total amount of calcium releasable by depolarization is not greatly reduced in Casq-null muscle (Royer et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.201010454). When the fast buffer BAPTA was introduced in the cytosol, release flux became more intense, and the SR emptied earlier. The consequent reduction in permeability accelerated as well, reaching comparable decay at earlier times but comparable levels of depletion. This observation indicates that [Ca2+]SR, sensed by Casq and transmitted to the channels presumably via connecting proteins, is determinant to cause the closure that terminates Ca2+ release.

D4cpv-calsequestrin: a sensitive ratiometric biosensor accurately targeted to the calcium store of skeletal muscle.

Current fluorescent monitors of free [Ca2+] in the sarcoplasmic reticulum (SR) of skeletal muscle cells are of limited quantitative value. They provide either a nonratio signal that is difficult to calibrate and is not specific or, in the case of Forster resonant energy transfer (FRET) biosensors, a signal of small dynamic range, which may be degraded further by imperfect targeting and interference from endogenous ligands of calsequestrin. We describe a novel tool that uses the cameleon D4cpv, which has a greater dynamic range and lower susceptibility to endogenous ligands than earlier cameleons. D4cpv was targeted to the SR by fusion with the cDNA of calsequestrin 1 or a variant that binds less Ca2+. “D4cpv-Casq1,” expressed in adult mouse at concentrations up to 22 µmole/liter of muscle cell, displayed the accurate targeting of calsequestrin and stayed inside cells after permeabilization of surface and t system membranes, which confirmed its strict targeting. FRET ratio changes of D4cpv-Casq1 were calibrated inside cells, with an effective KD of 222 µM and a dynamic range [(Rmax − Rmin)/Rmin] of 2.5, which are improvements over comparable sensors. Both the maximal ratio, Rmax, and its resting value were slightly lower in areas of high expression, a variation that was inversely correlated to distance from the sites of protein synthesis. The average [Ca2+]SR in 74 viable cells at rest was 416 µM. The distribution of individual ratio values was Gaussian, but that of the calculated [Ca2+]SR was skewed, with a tail of very large values, up to 6 mM. Model calculations reproduce this skewness as the consequence of quantifiably small variations in biosensor performance. Local variability, a perceived weakness of biosensors, thus becomes quantifiable. It is demonstrably small in D4cpv. D4cpv-Casq1 therefore provides substantial improvements in sensitivity, specificity, and reproducibility over existing monitors of SR free Ca2+ concentration.

Molecular cloning and functional expression of a skeletal muscle dihydropyridine receptor from Rana catesbeiana.

In skeletal muscle the dihydropyridine receptor is the voltage sensor for excitation-contraction coupling and an L-type Ca2+ channel. We cloned a dihydropyridine receptor (named Fgα1S) from frog skeletal muscle, where excitation-contraction coupling has been studied most extensively. Fgα1S contains 5600 base pairs coding for 1688 amino acids. It is highly homologous with, and of the same length as, the C-truncated form predominant in rabbit muscle. The primary sequence has every feature needed to be an L-type Ca2+ channel and a skeletal-type voltage sensor. Currents expressed in tsA201 cells had rapid activation (5–10 ms half-time) and Ca2+-dependent inactivation. Although functional expression of the full Fgα1S was difficult, the chimera consisting of Fgα1S domain I in the rabbit cardiac Ca channel had high expression and a rapidly activating current. The slow native activation is therefore not determined solely by the α1 subunit sequence. Its Ca2+-dependent inactivation strengthens the notion that in rabbit skeletal muscle this capability is inhibited by a C-terminal stretch (Adams, B., and Tanabe, T. (1997)J. Gen. Physiol. 110, 379–389). This molecule constitutes a new tool for studies of excitation-contraction coupling, gating, modulation, and gene expression.

Impaired Bone Homeostasis in Amyotrophic Lateral Sclerosis Mice with Muscle Atrophy

Background: The effects of ALS on bone homeostasis are poorly understood. Results: ALS mice with muscle atrophy have reduced bone mass associated with multiple impairments of osteoblast properties and striking acceleration of osteoclast formation in bone. Conclusion: Abnormal bone remodeling results in dramatic bone loss during the progression of muscle atrophy in ALS mice. Significance: This study may help to define therapeutic targets for ALS patients. There is an intimate relationship between muscle and bone throughout life. However, how alterations in muscle functions in disease impact bone homeostasis is poorly understood. Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by progressive muscle atrophy. In this study we analyzed the effects of ALS on bone using the well established G93A transgenic mouse model, which harbors an ALS-causing mutation in the gene encoding superoxide dismutase 1. We found that 4-month-old G93A mice with severe muscle atrophy had dramatically reduced trabecular and cortical bone mass compared with their sex-matched wild type (WT) control littermates. Mechanically, we found that multiple osteoblast properties, such as the formation of osteoprogenitors, activation of Akt and Erk1/2 pathways, and osteoblast differentiation capacity, were severely impaired in primary cultures and bones from G93A relative to WT mice; this could contribute to reduced bone formation in the mutant mice. Conversely, osteoclast formation and bone resorption were strikingly enhanced in primary bone marrow cultures and bones of G93A mice compared with WT mice. Furthermore, sclerostin and RANKL expression in osteocytes embedded in the bone matrix were greatly up-regulated, and β-catenin was down-regulated in osteoblasts from G93A mice when compared with those of WT mice. Interestingly, calvarial bone that does not load and long bones from 2-month-old G93A mice without muscle atrophy displayed no detectable changes in parameters for osteoblast and osteoclast functions. Thus, for the first time to our knowledge, we have demonstrated that ALS causes abnormal bone remodeling and defined the underlying molecular and cellular mechanisms.