Efficient and modified protocol for zymography to detect muscle specific calpain activity


Calpain, a proteolytic enzyme plays critical role in the skeletal muscle physiology by maintaining the protein metabolism. Up-regulation in the activity of such enzyme under diverse clinical settings (i.e. diabetes, cancer, AIDS, chronic heart failure, immobilization, aging etc) leads to loss in muscle proteins and causes atrophy/cachexia/sarcopenia. Beyond a reduced survival rate, atrophy is also linked to poor functional status and quality of life. Thus, easy detection of calpain at very early stage is highly desired under such settings so that specific therapy or antagonist could be given in time. Multiple methods are available (zymography, qPCR and immunoblotting) and among these zymography is the only approach which actually detect calpain activity. In the present manuscript, we have improvised two zymography protocols which are able to detect calpain in all muscle cells and tissues. Result shows that modified protocols can detect calpain activity even at low proteins concentration (<10 μg) in muscle cells (C2C12 myoblasts, proliferating state; myotubes, differentiating state) and tissues (cardiac and skeletal) in a single gel in comparatively short time span. Overall, purpose of the present study is to provide relatively simple and well described experimental protocol which can be used for muscle-specific calpain study.



Efficacy and Risk profile of Anti-diabetic therapies: Conventional vs traditional drugs-A mechanistic revisit to understand their mode of action


An increasing array of anti-diabetic drugs are available today, yet Type-2 diabetes mellitus (T2DM) − remains a life threatening disease, causing high mortality and morbidity in developing and developed countries. As of now, no effective therapy is available for the complete eradication/cure of diabetes and its associated complications. Therefore, it is time to re-think and revisit molecular pathways and targets of each existing drug in order to identify multiple targets from different signaling pathways that may be manipulated simultaneously to treat or manage T2DM effectively. Bearing this goal in mind, the article reviews the mechanisms of action of available anti-diabetic drugs with in-depth mechanistic analysis of each therapy. The conventional and herbal strategies are analysed and compared for their benefits and the associated possible side effects. This critical information is necessary not only for the development of better, novel and potent anti-diabetic therapy in future but also for best possible combinational therapies and strategies with the available drugs.

Pharmacological Research, 2016, http://dx.doi.org/10.1016/j.phrs.2016.09.029

Skeletal muscle atrophy: Potential therapeutic agents and their mechanisms of action

a b s t r a c t
Over the last two decades, new insights into the etiology of skeletal muscle wasting/atrophy under diverse clinical settings including denervation, AIDS, cancer, diabetes, and chronic heart failure have been reported in the literature. However, the treatment of skeletal muscle wasting remains an unresolved challenge to this day. About nineteen potential drugs that can regulate loss of muscle mass have been reported in the literature. This paper reviews the mechanisms of action of all these drugs by broadly classifying them into six different categories. Mechanistic data of these drugs illustrate that they regulate skeletal muscle loss either by down-regulating myostatin, cyclooxygenase2, pro-inflammatory cytokines mediated catabolic wasting or by up-regulating cyclic AMP, peroxisome proliferator-activated receptor gamma coactivator-1, growth hormone/insulin-like growth factor1, phosphatidylinositide 3-kinases/protein kinase B(Akt) mediated anabolic pathways. So far, five major proteolytic systems that regulate loss of muscle mass have been identified, but the majority of these drugs control only two or three proteolytic systems. In addition to their beneficial effect on restoring the muscle loss, many of these drugs show some level of toxicity and unwanted side effects such as dizziness, hypertension, and constipation. Therefore, further research is needed to understand and develop treatment strategies for muscle wasting. For successful management of skeletal muscle wasting either therapeutic agent which regulates all five known proteolytic systems or new molecular targets/proteolytic systems must be identified.


Pharmacological Research 99 (2015) 86–100