W F Denetclaw

University of California, Berkeley

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Richard A. Steinhardt

University of California System

F. Woodward Hopf

Indiana University – Purdue University Indianapolis

Paul R. Turner

University of Otago

Praveen Reddy

Florida College

RA Steinhardt

University of California, Berkeley

Don Pham

University of California, Berkeley

P Y Fong

University of Hong Kong

Jeffrey S. Chamberlain

University of Washington

GC McCarter

University of California, San Francisco

Guo‐Qiang Bi

Shenzhen Institutes of Advanced Technology

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University of California, Berkeley

University of California, San Francisco

Johns Hopkins Medicine

University of California, Davis

Oak Ridge National Laboratory

Indiana University – Purdue University Indianapolis

Indiana University School of Medicine

Neurosciences Institute

Indiana University

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Heterokaryon myotubes with normal mouse and Duchenne nuclei exhibit sarcolemmal dystrophin staining and efficient intracellular free calcium control.

Molecular Biology of the Cell (1993)

W F Denetclaw Guo‐Qiang Bi Don Pham Richard A. Steinhardt

Duchenne and mdx muscle tissues lack dystrophin where it normally interacts with glycoproteins in the sarcolemma. Intracellular free calcium ([Ca2+]i) is elevated in Duchenne and mdx myotubes and is correlated with abnormally active calcium-specific leak channels in dystrophic myotubes. We fused Duchenne human and normal mouse myoblasts and identified heterokaryon myotubes by Hoechst 33342 staining to measure the degree to which dystrophin introduced by normal nuclei could incorporate throughout the myotube at the sarcolemma and restore normal calcium homeostasis. Dystrophin expression in myotubes was determined by immunofluorescence and confocal laser scanning microscopy. Dystrophin was expressed at the sarcolemma in normal mouse and heterokaryon myotubes, but not in Duchenne myotubes. In heterokaryons, extensive dystrophin localization occurred at the sarcolemma even where only Duchenne nuclei were present, indicating that dystrophin does not exhibit nuclear domains. Heterokaryon, normal mouse and Duchenne myotube [Ca2+]i was measured using fura-2 and fluorescence ratio imaging. Heterokaryon and normal mouse myotubes were found to maintain similar levels of [Ca2+]i. In contrast, Duchenne myotubes had significantly higher [Ca2+]i (p < 0.001). Furthermore, the ability of heterokaryons to maintain normal [Ca2+]i did not depend on greater numbers of normal nuclei than Duchenne being present in the myotube. These results support the view that dystrophin expression in heterokaryons allows for efficient control of [Ca2+]i.

Sarcolemma

Heterokaryon

10.1091/mbc.4.9.963

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Citations (8)

Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

Molecular Biology of the Cell (1994)

W F Denetclaw F. Woodward Hopf Gregory A. Cox Jeffrey S. Chamberlain Richard A. Steinhardt

A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.

mdx mouse

Calcium in biology

Utrophin

10.1091/mbc.5.10.1159

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Lipofection of a cDNA plasmid containing the dystrophin gene lowers intracellular free calcium and calcium leak channel activity in mdx myotubes

Gene Therapy (1997)

GC McCarter W F Denetclaw Praveen Reddy RA Steinhardt

Calcium in biology

mdx mouse

Utrophin

10.1038/sj.gt.3300405

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Increased calcium influx in dystrophic muscle.

The Journal of Cell Biology (1991)

Paul R. Turner P Y Fong W F Denetclaw Richard A. Steinhardt

We examined pathways which might result in the elevated resting free calcium [( Ca2+]i) levels observed in dystrophic mouse (mdx) skeletal muscle fibers and myotubes and human Duchenne muscular dystrophy myotubes. We found that mdx fibers, loaded with the calcium indicator fura-2, were less able to regulate [Ca2+]i levels in the region near the sarcolemma. Increased calcium influx or decreased efflux could lead to elevated [Ca2+]i levels. Calcium transient decay times were identical in normal and mdx fibers if resting [Ca2+]i levels were similar, suggesting that calcium-sequestering mechanisms are not altered in dystrophic muscle, but are slowed by the higher resting [Ca2+]i. The defect appears to be specific for calcium since resting free sodium levels and sodium influx rates in the absence of Na+/K(+)-ATPase activity were identical in normal and dystrophic cells when measured with sodium-binding benzofuran isophthalate. Calcium leak channels, whose opening probabilities (Po) were voltage independent, could be the major calcium influx pathway at rest. We have shown previously that calcium leak channel Po is significantly higher in dystrophic myotubes. These leak channels were selective for calcium over sodium under physiological conditions. Agents that increased leak channel activity also increased [Ca2+]i in fibers and myotubes. These results suggest that increased calcium influx, as a result of increased leak channel activity, could result in the elevated [Ca2+]i in dystrophic muscle.

Sarcolemma

10.1083/jcb.115.6.1701

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Citations (271)

A critical evaluation of resting intracellular free calcium regulation in dystrophic mdx muscle

AJP Cell Physiology (1996)

F. Woodward Hopf Paul R. Turner W F Denetclaw Praveen Reddy Richard A. Steinhardt

There are conflicting reports regarding whether resting free calcium levels ([Ca2+]i) are elevated in dystrophic mouse (mdx) myotubes and adult myofibers. We reinvestigated this question and found several lines of evidence supporting the hypothesis that increased calcium influx via leak channels leads to increases in resting [Ca2+]i. 1) Step calibration of fura 2/free acid in myofibers with use of microinjected Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffers revealed greater [Ca2+]i in dystrophic cells. Careful calibration of fura PE3-AM, a compartmentalization-resistant derivative of fura 2, also showed elevated [Ca2+]i in mdx myotubes. 2) Chronic, but not acute, application of tetrodotoxin reduced resting [Ca2+]i in dystrophic myotubes, suggesting that elevated resting [Ca2+]i is a consequence of previous long-term contractile activity. 3) Rates of manganese quenching of fura 2 fluorescence, an indirect indicator of calcium influx, were significantly higher in mdx myotubes and were increased by nifedipine, a calcium leak channel agonist. 4) Calcium leak channel activity, measured using patch clamping, was greater in the sarcolemma of adult non-enzyme-treated mdx myofibers.

Fura-2

Calcium in biology

Sarcolemma

10.1152/ajpcell.1996.271.4.c1325

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Citations (127)