Blockade of Hemichannels Normalizes the Differentiation Fate of Myoblasts and Features of Skeletal Muscles from Dysferlin-Deficient Mice

Abstract

Dysferlinopathies are muscle dystrophies caused by mutations in the gene encoding dysferlin, a relevant protein for membrane repair and trafficking. These diseases are untreatable, possibly due to the poor knowledge of relevant molecular targets. Previously, we have shown that human myofibers from patient biopsies as well as myotubes derived from immortalized human myoblasts carrying a mutated form of dysferlin express connexin proteins, but their relevance in myoblasts fate and function remained unknown. In the present work, we found that numerous myoblasts bearing a mutated dysferlin when induced to acquire myogenic commitment express PPARγ, revealing adipogenic instead of myogenic commitment. These cell cultures presented many mononucleated cells with fat accumulation and within 48 h of differentiation formed fewer multinucleated cells. In contrast, dysferlin deficient myoblasts treated with boldine, a connexin hemichannels blocker, neither expressed PPARγ, nor accumulated fat and formed similar amount of multinucleated cells as wild type precursor cells. We recently demonstrated that myofibers of skeletal muscles from blAJ mice (an animal model of dysferlinopathies) express three connexins (Cx39, Cx43, and Cx45) that form functional hemichannels (HCs) in the sarcolemma. In symptomatic blAJ mice, we now show that eight-week treatment with a daily dose of boldine showed a progressive recovery of motor activity reaching normality. At the end of this treatment, skeletal muscles were comparable to those of wild type mice and presented normal CK activity in serum. Myofibers of boldine-treated blAJ mice also showed strong dysferlin-like immunoreactivity. These findings reveal that muscle dysfunction results from a pathophysiologic mechanism triggered by mutated dysferlin and downstream connexin hemichannels expressed de novo lead to a drastic reduction of myogenesis and favor muscle damage. Thus, boldine could represent a therapeutic opportunity to treat dysfernilopathies.

Oxidative Stress, Inflammation and Connexin Hemichannels in Muscular Dystrophies

“…In dysferlinopathy, a de novo expression of Cxs 39, 43 and 45 has also been reported [ 151 , 198 , 199 ]. As discussed above, it is accompanied with inflammation [ 35 ] and intracellular Ca 2+ deregulation [ 151 , 152 ].…”

“…In human myoblasts lacking dysferlin, the de novo expression of Cx40.1, an ortholog of the rodent Cx39, has been reported in addition to Cxs 43 and 45 [ 152 ]. Similarly, skeletal muscles of blAJ mice exhibit an elevated expression of Cx39, 43 and 45 associated to elevated basal intracellular Ca 2+ in myofibers, and muscle atrophy and lipid accumulation [ 198 , 199 ]. Remarkably, the downregulation of Cx43 and Cx45 prevents increases in intracellular Ca 2+ and normalizes aberrant lipogenic/muscular commitment, eliminating lipid accumulation and recovering the muscular performance in blAJ mice [ 199 ].…”

“…These findings suggest that Cx and Panx hemichannels constitute a potential therapeutic target for the treatment of dysferlinopathies. In this regard, the use of boldine, a Cx/Panx-hemichannel blocker that does not affect gap junction channels [ 200 ] prevents muscle alterations induced by mutations in the DYSF gene [ 198 ].…”

Assessing the impact of boldine on the gastrocnemius using multiomic profiling at 7 and 28 days post-complete spinal cord injury in young male mice

“…The copyright holder for this preprint this version posted August 17, 2022. ; https://doi.org/10.1101/2022.08.17.503230 doi: bioRxiv preprint have been well-described (23)(24)(25) but there remain some intriguing targets that have not been extensively studied. Connexin hemichannels (CxHC) are a potential target known to drive muscle atrophy (26)(27)(28)(29)(30)(31).…”

“…Boldine can inhibit CxHC activity without blocking GJ function (37), and has been shown to prevent sepsis-induced increases in cytosolic calcium concentrations and cytokine-induced small molecule dye uptake in isolated muscle fibers (35). Furthermore, 8 wks of boldine treatment in mice with dysferlinopathy improved motor function, reduced skeletal muscle fat accumulation and rescued aberrant dysferlin protein expression (29).…”