Abstract
Calcium homeostasis is critical for several vital functions in excitable and nonexcitable cells and has been shown to be impaired in many pathologies including Duchenne muscular dystrophy (DMD). Various studies using murine models showed the implication of calcium entry in the dystrophic phenotype. However, alteration of store-operated calcium entry (SOCE) and transient receptor potential vanilloid 2 (TRPV2)-dependant cation entry has not been investigated yet in human skeletal muscle cells. We pharmacologically characterized basal and store-operated cation entries in primary cultures of myotubes prepared from muscle of normal and DMD patients and found, for the first time, an increased SOCE in DMD myotubes. Moreover, this increase cannot be explained by an over expression of the well-known SOCE actors: TRPC1/4, Orai1, and stromal interaction molecule 1 (STIM1) mRNA and proteins. Thus we investigated the modes of regulation of this cation entry. We firstly demonstrated the important role of the scaffolding protein α1-syntrophin, which regulates SOCE in primary human myotubes through its PDZ domain. We also studied the implication of phospholipase C (PLC) and protein kinase C (PKC) in SOCE and showed that their inhibition restores normal levels of SOCE in DMD human myotubes. In addition, the involvement of TRPV2 in calcium deregulation in DMD human myotubes was explored. We showed an abnormal elevation of TRPV2-dependant cation entry in dystrophic primary human myotubes compared with normal ones. These findings show that calcium homeostasis mishandling in DMD myotubes depends on SOCE under the influence of Ca(2+)/PLC/PKC pathway and α1-syntrophin regulation as well as on TRPV2-dependant cation influx.
Highlights
CALCIUM HOMEOSTASIS IS BASED on strictly cohesive and regulated communication between internal and plasma membrane calcium channels and pumps and has been shown to be altered in several pathologies and in dystrophinopathies including Duchenne muscular dystrophy (DMD; Ref. 5)
Two different cation influxes were recorded in both types of myotube: first, basal calcium entry, a spontaneous influx that occurs subsequent to the addition of manganese ions in the extracellular medium, and second, the cation entry subsequent to sarcoplasmic reticulum (SR) calcium store depletion (Fig. 1D)
storeoperated calcium entry (SOCE) requires depletion of the internal stores and has been mainly characterized as a major calcium entry pathway in nonexcitable cells [47]. It was described in mouse myotubes [22], mouse fibers [35], and normal human myotubes by stimulating ryanodine receptors (RyR) resulting in the liberation of calcium from the SR to the cytoplasm [63]
Summary
CALCIUM HOMEOSTASIS IS BASED on strictly cohesive and regulated communication between internal and plasma membrane calcium channels and pumps and has been shown to be altered in several pathologies and in dystrophinopathies including Duchenne muscular dystrophy (DMD; Ref. 5). The regulation of the free cytosolic calcium concentration is known to be impaired in DMD human myotubes [17], and studies using contracting myotubes suggested that the calcium elevation resulted from abnormal activation of cation channels or from membrane alteration during contraction (24 –26). Expression of a dominant-negative TRPV2 mutant was shown to reduce calcium influx through inhibition of endogenous TRPV2 in FDB fibers and to ameliorate muscular dystrophy in mdx mouse [29]. Two different calcium influx pathway through SOC or TRPV2 channels could be determinant for DMD physiopathology This prompted us to characterize storeoperated cation entry as well as TRPV2-dependent calcium entry of normal and DMD primary cultured human myotubes. Because of our previous findings on dystrophin- or syntrophindeficient myotubes [49], we studied the implication of ␣1-syntrophin and of phospholipase C (PLC) and protein kinase C (PKC) in the impairment of Ca2ϩ entry in human myotubes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
