Sodium dysregulation coupled with calcium entry leads to muscular dystrophy in mice
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| Title: | Sodium dysregulation coupled with calcium entry leads to muscular dystrophy in mice |
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| Authors: | Burr, Adam R. |
| Committee Members: | Molkentin, Jeff |
| Summary: | Duchenne Muscular Dystrophy (DMD) and many of the limb girdle muscular dystrophies form a family of diseases called sarcoglycanopathies. In these diseases, mutation of any of a host of membrane and membrane associated proteins leads to increased stretch induced damage, aberrant signaling, and increased activity of non-specific cation channels, inducing muscle necrosis. Due to ongoing necrosis, DMD follows a progressive clinical course that leads to death in the mid-twenties. This course is slowed only modestly by high dose corticosteroids, which cause a plethora of harsh side effects. Targeted therapies are needed to ameliorate this disease until a more permanent therapy such as replacement of the mutated gene can be routinely performed. Here, we identified sodium calcium exchanger 1 (NCX1) as a potential therapeutic target. We started from the observation that sodium calcium exchanger 1 (NCX1) was upregulated during the necrotic phase of the disease in Sgcd-/- mice, which have similar pathology and mechanism of disease to boys with DMD. To test the causal effect of NCX1 overexpression on disease, we generated mice that overexpress NCX1 specifically in skeletal muscle. By Western blotting and immunofluorescence, we showed that NCX1 transgenic mice express more NCX1 protein in a similar localization pattern as endogenous NCX1. Sodium calcium exchange activity was also shown to be increased in NCX1 TG mice using an in situ sodium calcium exchange assay. When we examined the histology of NCX1 TG mice, we found that overexpression of NCX1 caused pathologic changes in skeletal muscle of the hindlimb. We then crossed the NCX1 trangene into Sgcd-/-, mdx, and Dysf-/- dystrophic backgrounds and found that the disease of these models was increased by the presence of the NCX1 transgene. Interestingly, NCX1 mediated opposing effects in the diaphragm, where NCX1 transgenic overexpression was protective in dystrophic backgrounds. To determine the effect of endogenous NCX1 on dystrophic pathology we deleted Slc8a1 (NCX1) using a muscle specific Cre recombinase and found that deletion of NCX1 was protective to the hindlimb. We next measured resting intracellular sodium and calcium concentrations and found that NCX1 increased resting sodium and calcium levels. This suggested to us that NCX1 was acting in reverse mode to cause pathology. Thus, we exacerbated reverse mode activity by generating NCX1 TG sodium potassium ATPase heterozygous mice, which have decreased pump function and treated NCX1 TG mice with the NKA inhibitor digoxin. In both cases, we saw a dramatic exacerbation of the NCX1 TG phenotype, strongly suggesting that reverse mode is the mechanism of disease. To translate these findings into a potential therapeutic intervention, we then utilized an FDA approved drug, ranolazine, to treat Sgcd-/- mice. Because ranolazine inhibits sodium channels it should enhance forward mode activity of NCX1. Indeed, we found decreased histopathology in mice treated with ranolazine. Together, these data identify a novel mechanism of dystrophic pathology and a promising therapeutic for future study. |
| URL: | http://rave.ohiolink.edu/etdc/view?acc_num=ucin1414750156 |
| Database: | OpenDissertations |
| Abstract: | Duchenne Muscular Dystrophy (DMD) and many of the limb girdle muscular dystrophies form a family of diseases called sarcoglycanopathies. In these diseases, mutation of any of a host of membrane and membrane associated proteins leads to increased stretch induced damage, aberrant signaling, and increased activity of non-specific cation channels, inducing muscle necrosis. Due to ongoing necrosis, DMD follows a progressive clinical course that leads to death in the mid-twenties. This course is slowed only modestly by high dose corticosteroids, which cause a plethora of harsh side effects. Targeted therapies are needed to ameliorate this disease until a more permanent therapy such as replacement of the mutated gene can be routinely performed. Here, we identified sodium calcium exchanger 1 (NCX1) as a potential therapeutic target. We started from the observation that sodium calcium exchanger 1 (NCX1) was upregulated during the necrotic phase of the disease in Sgcd-/- mice, which have similar pathology and mechanism of disease to boys with DMD. To test the causal effect of NCX1 overexpression on disease, we generated mice that overexpress NCX1 specifically in skeletal muscle. By Western blotting and immunofluorescence, we showed that NCX1 transgenic mice express more NCX1 protein in a similar localization pattern as endogenous NCX1. Sodium calcium exchange activity was also shown to be increased in NCX1 TG mice using an in situ sodium calcium exchange assay. When we examined the histology of NCX1 TG mice, we found that overexpression of NCX1 caused pathologic changes in skeletal muscle of the hindlimb. We then crossed the NCX1 trangene into Sgcd-/-, mdx, and Dysf-/- dystrophic backgrounds and found that the disease of these models was increased by the presence of the NCX1 transgene. Interestingly, NCX1 mediated opposing effects in the diaphragm, where NCX1 transgenic overexpression was protective in dystrophic backgrounds. To determine the effect of endogenous NCX1 on dystrophic pathology we deleted Slc8a1 (NCX1) using a muscle specific Cre recombinase and found that deletion of NCX1 was protective to the hindlimb. We next measured resting intracellular sodium and calcium concentrations and found that NCX1 increased resting sodium and calcium levels. This suggested to us that NCX1 was acting in reverse mode to cause pathology. Thus, we exacerbated reverse mode activity by generating NCX1 TG sodium potassium ATPase heterozygous mice, which have decreased pump function and treated NCX1 TG mice with the NKA inhibitor digoxin. In both cases, we saw a dramatic exacerbation of the NCX1 TG phenotype, strongly suggesting that reverse mode is the mechanism of disease. To translate these findings into a potential therapeutic intervention, we then utilized an FDA approved drug, ranolazine, to treat Sgcd-/- mice. Because ranolazine inhibits sodium channels it should enhance forward mode activity of NCX1. Indeed, we found decreased histopathology in mice treated with ranolazine. Together, these data identify a novel mechanism of dystrophic pathology and a promising therapeutic for future study. |
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