Renzhi Han

TRPM2 links oxidative stress to NLRP3 inflammasome activation

Exposure to particulate crystals can induce oxidative stress in phagocytes, which triggers NLRP3 inflammasome-mediated interleukin 1β (IL-1β) secretion to initiate undesirable inflammatory responses that are associated with both autoinflammatory and metabolic diseases. Although mitochondrial reactive oxygen species (ROS) play a central role in NLRP3 inflammasome activation, how ROS signal assembly of the NLRP3 inflammasome remains elusive. Here, we identify liposomes as novel activators of NLRP3 inflammasome and further demonstrate that liposome-induced inflammasome activation also requires mitochondrial ROS. Moreover, we found that stimulation with liposomes/crystals induced ROS-dependent calcium influx via the TRPM2 channel and that macrophages deficient in TRPM2 displayed drastically impaired NLRP3 inflammasome activation and IL-1β secretion. Consistently, Trpm2−/− mice were resistant to crystal-/liposome-induced IL-1β-mediated peritonitis in vivo. Together, these results identify TRPM2 as a key player that links oxidative stress to the NLRP3 inflammasome activation. Therefore, targeting TRPM2 may be effective for the treatment of NLRP3 inflamamsome-associated inflammatory disorders.

Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury

Dilated cardiomyopathy is a life-threatening syndrome that can arise from a myriad of causes, but predisposition toward this malady is inherited in many cases. A number of inherited forms of dilated cardiomyopathy arise from mutations in genes that encode proteins involved in linking the cytoskeleton to the extracellular matrix, and disruption of this link renders the cell membrane more susceptible to injury. Membrane repair is an important cellular mechanism that animal cells have developed to survive membrane disruption. We have previously shown that dysferlin deficiency leads to defective membrane resealing in skeletal muscle and muscle necrosis; however, the function of dysferlin in the heart remains to be determined. Here, we demonstrate that dysferlin is also involved in cardiomyocyte membrane repair and that dysferlin deficiency leads to cardiomyopathy. In particular, stress exercise disturbs left ventricular function in dysferlin-null mice and increases Evans blue dye uptake in dysferlin-deficient cardiomyocytes. Furthermore, a combined deficiency of dystrophin and dysferlin leads to early onset cardiomyopathy. Our results suggest that dysferlin-mediated membrane repair is important for maintaining membrane integrity of cardiomyocytes, particularly under conditions of mechanical stress. Thus, our study establishes what we believe is a novel mechanism underlying the cardiomyopathy that results from a defective membrane repair in the absence of dysferlin.

Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of α-dystroglycan

Skeletal muscle basal lamina is linked to the sarcolemma through transmembrane receptors, including integrins and dystroglycan. The function of dystroglycan relies critically on posttranslational glycosylation, a common target shared by a genetically heterogeneous group of muscular dystrophies characterized by α-dystroglycan hypoglycosylation. Here we show that both dystroglycan and integrin α7 contribute to force-production of muscles, but that only disruption of dystroglycan causes detachment of the basal lamina from the sarcolemma and renders muscle prone to contraction-induced injury. These phenotypes of dystroglycan-null muscles are recapitulated by Largemyd muscles, which have an intact dystrophin–glycoprotein complex and lack only the laminin globular domain-binding motif on α-dystroglycan. Compromised sarcolemmal integrity is directly shown in Largemyd muscles and similarly in normal muscles when arenaviruses compete with matrix proteins for binding α-dystroglycan. These data provide direct mechanistic insight into how the dystroglycan-linked basal lamina contributes to the maintenance of sarcolemmal integrity and protects muscles from damage.

CRISPR-mediated Genome Editing Restores Dystrophin Expression and Function in mdx Mice

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by genetic mutations that lead to the disruption of dystrophin in muscle fibers. There is no curative treatment for this devastating disease. Clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) has emerged as a powerful tool for genetic manipulation and potential therapy. Here we demonstrate that CRIPSR-mediated genome editing efficiently excised a 23-kb genomic region on the X-chromosome covering the mutant exon 23 in a mouse model of DMD, and restored dystrophin expression and the dystrophin-glycoprotein complex at the sarcolemma of skeletal muscles in live mdx mice. Electroporation-mediated transfection of the Cas9/gRNA constructs in the skeletal muscles of mdx mice normalized the calcium sparks in response to osmotic shock. Adenovirus-mediated transduction of Cas9/gRNA greatly reduced the Evans blue dye uptake of skeletal muscles at rest and after downhill treadmill running. This study provides proof evidence for permanent gene correction in DMD.

Genetic ablation of complement C3 attenuates muscle pathology in dysferlin-deficient mice

Mutations in the dysferlin gene underlie a group of autosomal recessive muscle-wasting disorders denoted as dysferlinopathies. Dysferlin has been shown to play roles in muscle membrane repair and muscle regeneration, both of which require vesicle-membrane fusion. However, the mechanism by which muscle becomes dystrophic in these disorders remains poorly understood. Although muscle inflammation is widely recognized in dysferlinopathy and dysferlin is expressed in immune cells, the contribution of the immune system to the pathology of dysferlinopathy remains to be fully explored. Here, we show that the complement system plays an important role in muscle pathology in dysferlinopathy. Dysferlin deficiency led to increased expression of complement factors in muscle, while muscle-specific transgenic expression of dysferlin normalized the expression of complement factors and eliminated the dystrophic phenotype present in dysferlin-null mice. Furthermore, genetic disruption of the central component (C3) of the complement system ameliorated muscle pathology in dysferlin-deficient mice but had no significant beneficial effect in a genetically distinct model of muscular dystrophy, mdx mice. These results demonstrate that complement-mediated muscle injury is central to the pathogenesis of dysferlinopathy and suggest that targeting the complement system might serve as a therapeutic approach for this disease.

New emerging recombinant HIV-1 strains and close transmission linkage of HIV-1 strains in the Chinese MSM population indicate a new epidemic risk.

In recent years, the population of men who have sex with men (MSM) have become the most significant increasing group of HIV-1 transmission in China. To identify new recombinant strains and transmission patterns of HIV-1 in Chinese MSM population, a cross-sectional investigation of MSM in Anhui Province (in south-eastern China) was performed in 2011. The diagnosed AIDS case rate, CD4 T-cell counts, HIV subtypes, and origin of the recombinant strains were investigated in 138 collected samples. The phylogenetic and bootscan analyses demonstrated that, apart from three previously reported circulating strains (CRF07_BC, CRF01_AE, subtype B), various recombinant strains among subtype B, subtype C, CRF01_AE, and CRF07_BC were simultaneously identified in Chinese MSM for the first time. The introducing time of B subtype in Chinese MSM populations was estimated in 1985, CRF01_AE in 2000, and CRF07_BC in 2003; the latter two account for more than 85% of MSM infections. Notably, in comparison with B subtype infections in Anhui MSM, CRF01_AE, with the highest prevalence rate, may accelerate AIDS progression. Over half of patients (56%) infected with new recombinant strains infection are diagnosed as progression into AIDS. Both Bayes and phylogenetic analyses indicated that there was active HIV transmission among MSM nationwide, which may facilitate the transmission of the new 01B recombinant strains in MSM. In conclusion, new recombinant strains and active transmission were identified in the Chinese MSM population, which may lead to a new alarming HIV pandemic in this population due to the increased pathogenesis of the newly emerging strains.

Thapsigargin Modulates Osteoclastogenesis Through the Regulation of RANKL‐Induced Signaling Pathways and Reactive Oxygen Species Production

The mechanism by which TG modulates osteoclast formation and apoptosis is not clear. In this study, we showed a biphasic effect of TG on osteoclast formation and apoptosis through the regulation of ROS production, caspase‐3 activity, cytosolic Ca2+, and RANKL‐induced activation of NF‐κB and AP‐1 activities.

Muscle membrane repair and inflammatory attack in dysferlinopathy

Repair of plasma membrane tears is an important normal physiological process that enables the cells to survive a variety of physiological and pathological membrane lesions. Dysferlin was the first protein reported to play a crucial role in this repair process in muscle, and recently, several other proteins including Mitsugumin 53 (MG53), annexin and calpain were also found to participate. These findings have now established the framework of the membrane repair mechanism. Defective membrane repair in dysferlin-deficient muscle leads to the development of muscular dystrophy associated with remarkable muscle inflammation. Recent studies have demonstrated a crosstalk between defective membrane repair and immunological attack, thus unveiling a new pathophysiological mechanism of dysferlinopathy. Here I summarize and discuss the latest progress in the molecular mechanisms of membrane repair and the pathogenesis of dysferlinopathy. Discussion about potential therapeutic applications of these findings is also provided.

Dystrophin deficiency exacerbates skeletal muscle pathology in dysferlin-null mice.

BackgroundMutations in the genes coding for either dystrophin or dysferlin cause distinct forms of muscular dystrophy. Dystrophin links the cytoskeleton to the sarcolemma through direct interaction with β-dystroglycan. This link extends to the extracellular matrix by β-dystroglycans interaction with α-dystroglycan, which binds extracellular matrix proteins, including laminin α2, agrin and perlecan, that possess laminin globular domains. The absence of dystrophin disrupts this link, leading to compromised muscle sarcolemmal integrity. Dysferlin, on the other hand, plays an important role in the Ca2+-dependent membrane repair of damaged sarcolemma in skeletal muscle. Because dysferlin and dystrophin play different roles in maintaining muscle cell integrity, we hypothesized that disrupting sarcolemmal integrity with dystrophin deficiency would exacerbate the pathology in dysferlin-null mice and allow further characterization of the role of dysferlin in skeletal muscle.MethodsTo test our hypothesis, we generated dystrophin/dysferlin double-knockout (DKO) mice by breeding mdx mice with dysferlin-null mice and analyzed the effects of a combined deficiency of dysferlin and dystrophin on muscle pathology and sarcolemmal integrity.ResultsThe DKO mice exhibited more severe muscle pathology than either mdx mice or dysferlin-null mice, and, importantly, the onset of the muscle pathology occurred much earlier than it did in dysferlin-deficient mice. The DKO mice showed muscle pathology of various skeletal muscles, including the mandible muscles, as well as a greater number of regenerating muscle fibers, higher serum creatine kinase levels and elevated Evans blue dye uptake into skeletal muscles. Lengthening contractions caused similar force deficits, regardless of dysferlin expression. However, the rate of force recovery within 45 minutes following lengthening contractions was hampered in DKO muscles compared to mdx muscles or dysferlin-null muscles, suggesting that dysferlin is required for the initial recovery from lengthening contraction-induced muscle injury of the dystrophin-glycoprotein complex-compromised muscles.ConclusionsThe results of our study suggest that dysferlin-mediated membrane repair helps to limit the dystrophic changes in dystrophin-deficient skeletal muscle. Dystrophin deficiency unmasks the function of dysferlin in membrane repair during lengthening contractions. Dystrophin/dysferlin-deficient mice provide a very useful model with which to evaluate the effectiveness of therapies designed to treat dysferlin deficiency.

Targeted Myostatin Gene Editing in Multiple Mammalian Species Directed by a Single Pair of TALE Nucleases

Myostatin (MSTN) is a negative regulator of skeletal muscle mass. Strategies to block myostatin signaling pathway have been extensively pursued to increase muscle mass in various disease settings including muscular dystrophy. Here, we report a new class of reagents based on transcription activator-like effector nucleases (TALENs) to disrupt myostatin expression at the genome level. We designed a pair of MSTN TALENs to target a highly conserved sequence in the coding region of the myostatin gene. We demonstrate that codelivery of these MSTN TALENs induce highly specific and efficient gene disruption in a variety of human, cattle, and mouse cells. Based upon sequence analysis, this pair of TALENs is expected to be functional in many other mammalian species. Moreover, we demonstrate that these MSTN TALENs can facilitate targeted integration of a mCherry expression cassette or a larger muscular dystrophy gene (dysferlin) expression cassette into the MSTN locus in mouse or human cells. Therefore, targeted editing of the myostatin gene using our highly specific and efficient TALEN pair would facilitate cell engineering, allowing potential use in translational research for cell-based therapy.