Jianxun Yi

Zunyi Medical University, Tsun-i-ch’eng, Guizhou Sheng, China

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Publications (20)93.83 Total impact

  • Biophysical Journal 01/2013; 104(2):289-. · 3.67 Impact Factor
  • Biophysical Journal 01/2013; 104(2):659-. · 3.67 Impact Factor
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    ABSTRACT: 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 (SOD1(G93A)). 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 SOD1(G93A) in skeletal muscle of normal mice, indicating the SOD1 mutation drives ALS-like muscle pathology in the absence of motor neuron degeneration. Mutant SOD1(G93A) 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 SOD1(G93A) model muscle. A specific mitochondrial fission inhibitor (Mdivi-1) reversed the SOD1(G93A) action on mitochondrial dynamics, indicating SOD1(G93A) likely promotes mitochondrial fission process. Our results suggest that accumulation of mutant SOD1(G93A) 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.
    PLoS ONE 01/2013; 8(12):e82112. · 3.53 Impact Factor
  • Biophysical Journal 01/2013; 104(2):659-. · 3.67 Impact Factor
  • Biophysical Journal 01/2012; 102(3):166-. · 3.67 Impact Factor
  • Biophysical Journal 01/2012; 102(3):362-. · 3.67 Impact Factor
  • Biophysical Journal 01/2012; 102(3):362-. · 3.67 Impact Factor
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    ABSTRACT: Current fluorescent monitors of free [Ca(2+)] 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 Ca(2+). "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 K(D) of 222 µM and a dynamic range [(R(max) - R(min))/R(min)] of 2.5, which are improvements over comparable sensors. Both the maximal ratio, R(max), 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 [Ca(2+)](SR) in 74 viable cells at rest was 416 µM. The distribution of individual ratio values was Gaussian, but that of the calculated [Ca(2+)](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 Ca(2+) concentration.
    The Journal of General Physiology 08/2011; 138(2):211-29. · 4.73 Impact Factor
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    ABSTRACT: The mechanisms that terminate Ca(2+) 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 Ca(2+) concentration inside the sarcoplasmic reticulum (SR), [Ca(2+)](SR), simultaneously with that in the cytosol, [Ca(2+)](c), during the response to long-lasting depolarization of the plasma membrane. The ratio of Ca(2+) release flux (derived from [Ca(2+)](c)(t)) over the gradient that drives it (essentially equal to [Ca(2+)](SR)) provided directly, for the first time, a dynamic measure of the permeability to Ca(2+) of the releasing SR membrane. During maximal depolarization, flux rapidly rises to a peak and then decays. Before 0.5 s, [Ca(2+)](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 Ca(2+) 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 [Ca(2+)](SR), sensed by Casq and transmitted to the channels presumably via connecting proteins, is determinant to cause the closure that terminates Ca(2+) release.
    The Journal of General Physiology 08/2011; 138(2):231-47. · 4.73 Impact Factor
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    ABSTRACT: Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca(2+) 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 Ca(2+) release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca(2+) removal during excitation-contraction coupling by comparing Ca(2+) transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca(2+) transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ~10% increased amplitude. These regional differences in Ca(2+) transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a fast Ca(2+) chelator that reduces mitochondrial Ca(2+) uptake. Using a mitochondria-targeted Ca(2+) biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca(2+) levels during voltage-induced Ca(2+) release and detected a reduced Ca(2+) uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca(2+) transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca(2+) signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.
    Journal of Biological Chemistry 07/2011; 286(37):32436-43. · 4.65 Impact Factor
  • Biophysical Journal 02/2011; 100(3). · 3.67 Impact Factor
  • Biophysical Journal 01/2011; 100(3). · 3.67 Impact Factor
  • Biophysical Journal 01/2010; 98(3). · 3.67 Impact Factor
  • Biophysical Journal 01/2010; 98(3). · 3.67 Impact Factor
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    ABSTRACT: 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 SOD1(G93A) 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 Ca(2+) release activity, which can include propagating Ca(2+) waves. These Ca(2+) 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 Ca(2+) uptake by Ru360 lead to cell-wide propagation of such Ca(2+) release events. Our data reveal that mitochondria regulate Ca(2+) signaling in skeletal muscle, and loss of this capacity may contribute to the progression of muscle atrophy in ALS.
    Journal of Biological Chemistry 11/2009; 285(1):705-12. · 4.65 Impact Factor
  • Biophysical Journal 01/2009; 96(3). · 3.67 Impact Factor
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    ABSTRACT: 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 Ca(2+) 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 Ca(2+) 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 Ca(2+)-induced Ca(2+) 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 Ca(2+), secondarily to Ca(2+) release through junctional RyR1. Physiologically voltage-controlled Ca(2+) sparks thus require a voltage sensor, a master junctional RyR1 channel that provides trigger Ca(2+), and a slave parajunctional RyR3 cohort.
    Proceedings of the National Academy of Sciences 04/2007; 104(12):5235-40. · 9.81 Impact Factor
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    ABSTRACT: Na(+) current derived from expression of the principal cardiac Na(+) channel, Na(v)1.5, is increased by activation of protein kinase A (PKA). This effect is blocked by inhibitors of cell membrane recycling, or removal of a cytoplasmic endoplasmic reticulum (ER) retention motif, suggesting that PKA stimulation increases trafficking of cardiac Na(+) channels to the plasma membrane. To test this hypothesis, green fluorescent protein (GFP) was fused to Na(v)1.5 (Na(v)1.5-GFP), and the effects of PKA activation were investigated in intact, living cells that stably expressed the fusion protein. Using confocal microscopy, the spatial relationship of GFP-tagged channels relative to the plasma membrane was quantitated using a measurement that could control for variables present during live-cell imaging, and permit an unbiased analysis for all cells in a given field. In the absence of kinase stimulation, intracellular fluorescence representing Na(v)1.5-GFP channels was greatest in the perinuclear area, with additional concentration of channels beneath the cell surface. Activation of PKA promoted trafficking of Na(+) channels from both regions to the plasma membrane. Experimental results using a chemiluminescence-based assay further confirmed that PKA stimulation increased expression of Na(v)1.5 channels at the cell membrane. Our results provide direct evidence for PKA-mediated trafficking of cardiac Na(+) channels into the plasma membrane in living, mammalian cells, and they support the existence of multiple intracellular storage pools of channel protein that can be mobilized following a physiologic stimulus.
    Cardiovascular Research 12/2006; 72(2):250-61. · 5.81 Impact Factor
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    ABSTRACT: To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca(2+) release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca(2+) release. Murine primary cultures were confocally imaged for Ca(2+) detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca(2+) sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice (mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca(2+) saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 microM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca(2+) saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca(2+)] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca(2+)] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca(2+) release channels and/or their susceptibility to Ca(2+)-induced activation, thereby suppressing the production of Ca(2+) sparks.
    AJP Cell Physiology 03/2006; 290(2):C539-53. · 3.71 Impact Factor
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    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 alpha-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.
    Circulation Research 10/2002; 91(6):540-6. · 11.86 Impact Factor