Molecular Pathways of Protein Degradation in Muscular Dystrophies: Targets for Therapeutic Intervention

Dhanarajan, R (2011) Molecular Pathways of Protein Degradation in Muscular Dystrophies: Targets for Therapeutic Intervention. Doctoral thesis, The Tamilnadu Dr. M.G.R. Medical University, Chennai.


Download (1MB) | Preview


Muscular dystrophies, characterized by progressive muscle wasting and weakness, are the most common inherited muscle disorders. Twenty-five different muscular dystrophies have been characterized. Limb girdle muscular dystrophies (LGMD) are a group of heterogeneous disorders, prevalent worldwide and common in India, that predominantly affects upper and lower limb musculature. LGMD 2A, due to mutations in muscle specific cysteine protease calpain-3 gene, and LGMD 2B, due to mutations in the dysferlin gene, are major subtypes of LGMD and the focus of this thesis. Mechanisms of muscle wasting are not completely elucidated for any form of muscular dystrophy, which has prevented the development of specific treatment for these disorders. Compounds that inhibit pathogenic pathways of muscle wasting and induce muscle regeneration would be drugs of choice for treatment of muscular dystrophies. Muscle loss as a result of protein degradation through the ubiquitin-proteasomal pathway occurs in several muscle wasting conditions. Oxidant stress and activation of the transcription factor, NF-κB, initiate and mediate the ubiquitin pathway of protein degradation in cachexia. Their role in wasting of target muscles in LGMD 2A and dysferlinopathy has not been studied but would be helpful to examine in considering development of specific pharmacotherapy. If oxidant stress and activation of NF-κB lead to muscle wasting of LGMD, the use of polyphenolic curcumin, an inhibitor of these paths, could be explored. AIM OF THE STUDY: This study were to elucidate (1) Protein degrading pathways of LGMD 2A and dysferlinopathy, common but poorly studied dystrophies in Indian patients and (2) Target the pathways of muscle wasting with drugs that reduce protein degradation and stimulate muscle regeneration. METHODS: Protein content, oxidative and nitrosative stress, NADPH oxidase, NF-κBp65 and IKKβ activation, protein ubiquitinylation and muscle specific E3 ligases of the protein ubquitinylation system were determined in LGMD 2A and dysferlinopathic muscle biopsies and analyzed in comparison to normal muscle. All muscle biopsies were performed under standard conditions and obtained with informed consent from the patient or legal guardian. Further, rat myoblast cell lines and primary myoblast cultures were subject to H2O2 induced oxidative stress and examined for intracellular Ca2+ by live cell fluorescence imaging and for protein ubiquitinylation on immune blots. Myoblasts silenced for NF-κB or treated with curcumin and subject to oxidative stress were similarly studied. RESULTS: Reduced protein, increased ubiquitinylated proteins, oxidative and nitrosative stress and elevated NADPH oxidase activity occurred in LGMD muscle compared to normal. NF-κB was increased in both LGMD 2A and dysferlinopathic muscle. E3 ligases MAFbx and MURF 1 were activated in LGMD 2A but MAFbx not MURF 1 was activated in dysferlinopathic muscle. Myosin heavy chain, actin and antioxidant defense were reduced in wasted muscle but not in spared muscle compared to normal. Oxidative stress led to activation of NK-ƙB that induced protein ubiquitinylation, raised intracellular Ca2+ and led to cell death of rat myoblasts. NF-ƙB knockdown and curcumin prevented oxidative stress induced raised intracellular Ca2+, nitric oxide generation, protein ubiquitinylation and cell death. Curcumin also increased myoblast number. CCONCLUSIONS: The absence of calpain 3 or dysferlin induces oxidative stress in muscle that activates NF-ƙB signaling to induce protein degradation through the ubiquitin-proteasome pathway. NADPH oxidase contributes to oxidative stress of LGMD muscle. Loss of major myofibrillar proteins and failure of the antioxidant system may underlie selective loss of muscle in LGMD. Curcumin is effective in preventing oxidative stress induced myoblast death and stimulating myoblast division. IMPACT OF THE STUDY: There are no comprehensive studies from the Indian subcontinent on muscular dystrophies, especially LGMD and very few centers diagnose LGMD 2A and dysferlinopathy. This thesis describes Western blots suitable for the diagnosis of LGMD in the Indian population, which were used to determine the frequency of LGMD 2A and dysferlinopathy in a series of dystrophic muscles of patients from several parts of India. This study showed the efficacy of the nutraceutical, curcumin, to protect against muscle wasting of muscular dystrophies. Inclusion of curcumin in the therapeutic regimen of muscular dystrophies should be considered.

Item Type: Thesis (Doctoral)
Uncontrolled Keywords: Molecular Pathways, Protein Degradation, Muscular Dystrophies Targets, Therapeutic Intervention.
Subjects: Respiratory Medicine > Neurology
> Respiratory Medicine > Neurology
Depositing User: Subramani R
Date Deposited: 17 Jun 2017 09:41
Last Modified: 22 Sep 2022 11:46

Actions (login required)

View Item View Item