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Loss of myotubularin function results in T-tubule disorganization in zebrafish and human myotubular myopathy.

Dowling JJ, Vreede AP, Low SE, Gibbs EM, Kuwada JY, Bonnemann CG, Feldman EL - PLoS Genet. (2009)

Bottom Line: Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle.We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin.Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan, USA. jamedowl@umich.edu

ABSTRACT
Myotubularin is a lipid phosphatase implicated in endosomal trafficking in vitro, but with an unknown function in vivo. Mutations in myotubularin cause myotubular myopathy, a devastating congenital myopathy with unclear pathogenesis and no current therapies. Myotubular myopathy was the first described of a growing list of conditions caused by mutations in proteins implicated in membrane trafficking. To advance the understanding of myotubularin function and disease pathogenesis, we have created a zebrafish model of myotubular myopathy using morpholino antisense technology. Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle. These changes include abnormally shaped and positioned nuclei and myofiber hypotrophy. These findings are consistent with those observed in the human disease. We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin. Finally, we identify abnormalities in the tubulo-reticular network in muscle from myotubularin zebrafish morphants and correlate these changes with abnormalities in T-tubule organization in biopsies from patients with myotubular myopathy. In all, we have generated a new model of myotubular myopathy and employed this model to uncover a novel function for myotubularin and a new pathomechanism for the human disease that may explain the weakness associated with the condition (defective excitation-contraction coupling). In addition, our findings of tubuloreticular abnormalities and defective excitation-contraction coupling mechanistically link myotubular myopathy with several other inherited muscle diseases, most notably those due to ryanodine receptor mutations. Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis.

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Related in: MedlinePlus

Alteration of T-tubule/SR component localization in myotubular myopathy.Muscle from 3 myotubular myopathy patients and an age matched control were immunostained with DHPRa1 to mark T-tubules (A) and RYR1 to mark the adjacent sarcoplasmic reticulum (B). Abnormal distribution of DHPRa1 and RYR1 was observed in numerous fibers in all 3 myotubularin patients but in none of the control fibers (arrows). Scale bar = 20 mm.
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pgen-1000372-g011: Alteration of T-tubule/SR component localization in myotubular myopathy.Muscle from 3 myotubular myopathy patients and an age matched control were immunostained with DHPRa1 to mark T-tubules (A) and RYR1 to mark the adjacent sarcoplasmic reticulum (B). Abnormal distribution of DHPRa1 and RYR1 was observed in numerous fibers in all 3 myotubularin patients but in none of the control fibers (arrows). Scale bar = 20 mm.

Mentions: We were interested to correlate our findings with muscle from myotubular myopathy patient biopsies. T-tubule abnormalities have not been specifically mentioned in previous pathologic analyses from myotubular myopathy patients. We examined T-tubule organization in human biopsy samples using immunohistochemistry and antibodies to DHPRa1, a T-tubule marker, and to RYR1, a marker of the adjacent sarcoplasmic reticulum. A similar technical approach was successfully utilized by Laporte and colleagues to examine T-tubule organization in centronuclear myopathy patients with BIN1 mutations [28]. As a staining control, we used muscle from an unaffected, age matched control sample. DHPRa1 and RYR1 staining in the control muscle were found in the expected pattern along the membrane and throughout the cytoplasm (Figure 11A and Figure 11B, respectively). Conversely, samples from three patients revealed clear abnormalities in both DHPR and RYR1 staining patterns. T-tubules were found concentrated around the abnormally located central nuclei, or in irregular densities in the centers of several fibers. Importantly, other plasma membrane components were not found in this distribution (Figure S5), indicating that this disorganization is relatively specific for T-tubules.


Loss of myotubularin function results in T-tubule disorganization in zebrafish and human myotubular myopathy.

Dowling JJ, Vreede AP, Low SE, Gibbs EM, Kuwada JY, Bonnemann CG, Feldman EL - PLoS Genet. (2009)

Alteration of T-tubule/SR component localization in myotubular myopathy.Muscle from 3 myotubular myopathy patients and an age matched control were immunostained with DHPRa1 to mark T-tubules (A) and RYR1 to mark the adjacent sarcoplasmic reticulum (B). Abnormal distribution of DHPRa1 and RYR1 was observed in numerous fibers in all 3 myotubularin patients but in none of the control fibers (arrows). Scale bar = 20 mm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2631153&req=5

pgen-1000372-g011: Alteration of T-tubule/SR component localization in myotubular myopathy.Muscle from 3 myotubular myopathy patients and an age matched control were immunostained with DHPRa1 to mark T-tubules (A) and RYR1 to mark the adjacent sarcoplasmic reticulum (B). Abnormal distribution of DHPRa1 and RYR1 was observed in numerous fibers in all 3 myotubularin patients but in none of the control fibers (arrows). Scale bar = 20 mm.
Mentions: We were interested to correlate our findings with muscle from myotubular myopathy patient biopsies. T-tubule abnormalities have not been specifically mentioned in previous pathologic analyses from myotubular myopathy patients. We examined T-tubule organization in human biopsy samples using immunohistochemistry and antibodies to DHPRa1, a T-tubule marker, and to RYR1, a marker of the adjacent sarcoplasmic reticulum. A similar technical approach was successfully utilized by Laporte and colleagues to examine T-tubule organization in centronuclear myopathy patients with BIN1 mutations [28]. As a staining control, we used muscle from an unaffected, age matched control sample. DHPRa1 and RYR1 staining in the control muscle were found in the expected pattern along the membrane and throughout the cytoplasm (Figure 11A and Figure 11B, respectively). Conversely, samples from three patients revealed clear abnormalities in both DHPR and RYR1 staining patterns. T-tubules were found concentrated around the abnormally located central nuclei, or in irregular densities in the centers of several fibers. Importantly, other plasma membrane components were not found in this distribution (Figure S5), indicating that this disorganization is relatively specific for T-tubules.

Bottom Line: Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle.We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin.Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan, USA. jamedowl@umich.edu

ABSTRACT
Myotubularin is a lipid phosphatase implicated in endosomal trafficking in vitro, but with an unknown function in vivo. Mutations in myotubularin cause myotubular myopathy, a devastating congenital myopathy with unclear pathogenesis and no current therapies. Myotubular myopathy was the first described of a growing list of conditions caused by mutations in proteins implicated in membrane trafficking. To advance the understanding of myotubularin function and disease pathogenesis, we have created a zebrafish model of myotubular myopathy using morpholino antisense technology. Zebrafish with reduced levels of myotubularin have significantly impaired motor function and obvious histopathologic changes in their muscle. These changes include abnormally shaped and positioned nuclei and myofiber hypotrophy. These findings are consistent with those observed in the human disease. We demonstrate for the first time that myotubularin functions to regulate PI3P levels in a vertebrate in vivo, and that homologous myotubularin-related proteins can functionally compensate for the loss of myotubularin. Finally, we identify abnormalities in the tubulo-reticular network in muscle from myotubularin zebrafish morphants and correlate these changes with abnormalities in T-tubule organization in biopsies from patients with myotubular myopathy. In all, we have generated a new model of myotubular myopathy and employed this model to uncover a novel function for myotubularin and a new pathomechanism for the human disease that may explain the weakness associated with the condition (defective excitation-contraction coupling). In addition, our findings of tubuloreticular abnormalities and defective excitation-contraction coupling mechanistically link myotubular myopathy with several other inherited muscle diseases, most notably those due to ryanodine receptor mutations. Based on our findings, we speculate that congenital myopathies, usually considered entities with similar clinical features but very disparate pathomechanisms, may at their root be disorders of calcium homeostasis.

Show MeSH
Related in: MedlinePlus