Research Article: SIRT1 deficiency interferes with membrane resealing after cell membrane injury

Date Published: June 26, 2019

Publisher: Public Library of Science

Author(s): Daisuke Fujiwara, Naotoshi Iwahara, Rio Sebori, Ryusuke Hosoda, Shun Shimohama, Atsushi Kuno, Yoshiyuki Horio, Atsushi Asakura.


Activation of SIRT1, an NAD+-dependent protein deacetylase, ameliorates muscular pathophysiology of δ-sarcoglycan-deficient TO-2 hamsters and dystrophin-deficient mdx mice. We found that SIRT1 was highly expressed beneath the cellular membranes of muscle cells. To elucidate functional roles of SIRT1 on muscles, skeletal muscle-specific SIRT1 knockout mice (SIRT1-MKO) were generated. SIRT1-MKO mice showed muscular pathology similar to mild muscular dystrophies with increased numbers of centrally nucleated small myofibers and decreased numbers of middle-sized (2000–3001 μm2) myofibers compared to those of wild-type (WT) mice. Accordingly, SIRT1-MKO mice showed significantly decreased exercise capacity in treadmill and inverted hanging tests with higher levels of serum creatine kinase activities compared with those in WT mice. Evans blue dye uptake after exercise was greater in the muscles of SIRT1-MKO than those of WT mice, suggesting membrane fragility in SIRT1-MKO mice. Because SIRT1 was dominantly localized beneath the membranes of muscular cells, SIRT1 may have a new role in the membranes. We found that levels of fluorescent FM1-43 dye intake after laser-induced membrane disruption in C2C12 cells were significantly increased by SIRT1 inhibitors or Sirt1-siRNA compared with those of control cells. Inhibition of SIRT1 or SIRT1-knockdown severely disturbed the dynamic aggregation of membrane vesicles under the injured site but did not affect expression levels of membrane repair proteins. These data suggested that SIRT1 had a critical role in the resealing of membrane-ruptured muscle cells, which could affect phenotypes of SIRT1-MKO mice. To our knowledge, this report is the first to demonstrate that SIRT1 affected plasma-membrane repair mechanisms.

Partial Text

Cycles of contraction and relaxation in skeletal muscles and cardiac cells induce cellular membrane friction and strain that could cause membrane rupture. Plasma membrane disruption is rapidly resealed by membrane repair mechanisms for cell survival [1]. Membrane resealing is triggered by Ca2+ influx through the injured site, where Ca2+ activates Ca2+ binding proteins including calpain 3, which is involved in the resealing of membrane structures through their Ca2+-dependent protease activity [1]. Accordingly, mutations in the calpain 3 gene cause limb girdle muscular dystrophy type 2A [2]. F-actin is accumulated promptly at the site of membrane disruption and annexins, phospholipid-interacting proteins with Ca2+-binding activity, are also recruited to the injured site and contribute to membrane repair [3]. Dysferlin interacts with negatively charged phospholipids in a Ca2+-binding manner and its genetic defects result in limb girdle muscular dystrophy type 2B [4]. After membrane injury, intracellular small vesicles containing dysferlin are recruited to the injured site and form a large vesicle to reseal membranes [1]. Dysferlin interacts with mitsugumin 53 (MG53) and caveolin 3, which are also essential to repair membrane damage [5]. Mutations of caveolin 3 cause limb girdle muscular dystrophy 1C [6] and MG53 knockout mice show dystrophic phenotype [7].

In the present study, we showed that SIRT1-MKO mice had pathological and physiological characteristics similar to those of mild dystrophies, especially dysferlinopathy (Figs 1 and 2). High serum CK and LDH levels by exercise, indicated membrane fragility of muscles in SIRT1-MKO mice. EB uptake in the muscle after exercise in fact was significantly higher than that of WT mice (Fig 2e–2h). Although SIRT1-MKO mice had a high number of regenerating myofibers and lower number of middle-sized myofibers with high serum CK levels compared with those of WT mice, necrotic fibers, fibrosis and inflammatory changes were barely detected in their muscles (Fig 1e–1i). These phenotypes are common with clinical features of dysferlinopathy including limb girdle dystrophy type 2B and Miyoshi myopathy, which show minimal dystrophic change and decreased exercise performance with very high serum CK levels in their early stages [22, 23]. Interestingly, exercise in the early stage of dysferlinopathy accelerated the progression of disease [23]. Dysferlin is indispensable for membrane repair mechanism in which vesicles containing dysferlin aggregate and fuse beneath injured membranes [24]. We found that SIRT1 inhibition or knockdown disturbed membrane repair and inhibited vesicle aggregation and fusion at the injured site (Fig 3). To our knowledge, this is the first report to demonstrate the role of SIRT1 in membrane repair.




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