Expression of Functional Transgenic Alpha1s-DHPR Channels in Adult Mammalian Skeletal Muscle Fibers

Abstract

Central Core Disease (CCD) and Malignant Hyperthermia (MH) are linked to single amino acid substitutions in the skeletal muscle Ca 2þ release channel, the type 1 ryanodine receptor (RyR1). We focus on two autosomal dominant (AD) 712a

Pathogenity of some limb girdle muscular dystrophy mutations can result from reduced anchorage to myofibrils and altered stability of calpain 3

“…In those studies, CAPN3 was shown to exist in numerous sites including at the N2-line, Z disc, M-line, costameres, nuclei and myotendinous junctions. As an alternative approach and to localize CAPN3 in live muscle, the FDB muscle was electroporated with the GFP-plasmids, and after 3 days, the muscles were dissected and visualized by two-photon laser-scanning microscopy (TPLSM) as previously carried out for other proteins (32,36,37). For these studies, we used two additional indicators as landmarks for the sarcomeric structure: (i) the second harmonic generation (SHG) imaging, which demarcates the A band (myosin thick filaments) and (ii) the staining of a fluorescent non-penetrating potentiometric dye called di-8-ANEPPS, which labels the surface and T-tubule membranes.…”

A shortcut to a skeletal muscle DHPR knock‐in?

“…The Vergara laboratory has had good success expressing non‐fused EGFP and an EYFP‐β 1a fusion construct in adult fibres (239 and 789 residues, respectively) but expression of the considerably larger EGFP‐α 1S construct (∼2100 residues) was by no means a trivial matter. However, much of the uncertainty concerning EGFP‐α 1S expression was relieved when successfully transfected flexor digitorum brevis (FDB) or interosseus (IO) fibres were identified by the fluorescence in the transverse tubules generated by the EGFP tag (DiFranco et al 2011, Fig. 2).…”

“…In conclusion, the work of DiFranco et al (2011) represents a step forward in the development of an adult model for the study of conformational coupling between the DHPR and RyR1, but the current approach requires refinement to be truly useful. Perhaps species‐specific sequence differences between the transfected rabbit α 1S clone and the endogenous mouse α 1S subunit could be exploited.…”

“…The attempt to create effectively dysgenic adult fibres was not entirely successful because of the inability of the siRNA to quash α 1S expression completely (an inherent weakness of siRNA-based strategies), but a key role for DHPR expression in maintaining healthy muscle mass was revealed. In the study discussed below, the Vergara laboratory (DiFranco et al 2011) has now taken the in vivo electroporation strategy to the next level; they attempt to create pharmacologically inducible α 1S 'knock-in' fibres by silencing the endogenous protein with a dihydropyridine (DHP) antagonist and replacing it with a DHP-insensitive counterpart. Their success would provide EC coupling investigators with a suitable system in which to investigate DHPR-RyR1 conformational coupling in fully differentiated muscle fibres.…”