Membrane stretch as the mechanism of activation of PIEZO1 ion channels in chondrocytes.

Osteoarthritis is a chronic disease that can be initiated by altered joint loading or injury of the cartilage. The mechanically sensitive PIEZO ion channels have been shown to transduce injurious levels of biomechanical strain in articular chondrocytes and mediate cell death. However, the mechanisms of channel gating in response to high cellular deformation and the strain thresholds for activating PIEZO channels remain unclear. We coupled studies of single-cell compression using atomic force microscopy (AFM) with finite element modeling (FEM) to identify the biophysical mechanisms of PIEZO-mediated calcium (Ca2+) signaling in chondrocytes. We showed that PIEZO1 and PIEZO2 are needed for initiating Ca2+ signaling at moderately high levels of cellular deformation, but at the highest strains, PIEZO1 functions independently of PIEZO2. Biophysical factors that increase apparent chondrocyte membrane tension, including hypoosmotic prestrain, high compression magnitudes, and low deformation rates, also increased PIEZO1-driven Ca2+ signaling. Combined AFM/FEM studies showed that 50% of chondrocytes exhibit Ca2+ signaling at 80 to 85% nominal cell compression, corresponding to a threshold of apparent membrane finite principal strain of E = 1.31, which represents a membrane stretch ratio (λ) of 1.9. Both intracellular and extracellular Ca2+ are necessary for the PIEZO1-mediated Ca2+ signaling response to compression. Our results suggest that PIEZO1-induced signaling drives chondrocyte mechanical injury due to high membrane tension, and this threshold can be altered by factors that influence membrane prestress, such as cartilage hypoosmolarity, secondary to proteoglycan loss. These findings suggest that modulating PIEZO1 activation or downstream signaling may offer avenues for the prevention or treatment of osteoarthritis.

Skeletal dysplasia-causing TRPV4 mutations suppress the hypertrophic differentiation of human iPSC-derived chondrocytes.

Mutations in the TRPV4 ion channel can lead to a range of skeletal dysplasias. However, the mechanisms by which TRPV4 mutations lead to distinct disease severity remain unknown. Here, we use CRISPR-Cas9-edited human-induced pluripotent stem cells (hiPSCs) harboring either the mild V620I or lethal T89I mutations to elucidate the differential effects on channel function and chondrogenic differentiation. We found that hiPSC-derived chondrocytes with the V620I mutation exhibited increased basal currents through TRPV4. However, both mutations showed more rapid calcium signaling with a reduced overall magnitude in response to TRPV4 agonist GSK1016790A compared to wildtype (WT). There were no differences in overall cartilaginous matrix production, but the V620I mutation resulted in reduced mechanical properties of cartilage matrix later in chondrogenesis. mRNA sequencing revealed that both mutations up-regulated several anterior HOX genes and down-regulated antioxidant genes CAT and GSTA1 throughout chondrogenesis. BMP4 treatment up-regulated several essential hypertrophic genes in WT chondrocytes; however, this hypertrophic maturation response was inhibited in mutant chondrocytes. These results indicate that the TRPV4 mutations alter BMP signaling in chondrocytes and prevent proper chondrocyte hypertrophy, as a potential mechanism for dysfunctional skeletal development. Our findings provide potential therapeutic targets for developing treatments for TRPV4-mediated skeletal dysplasias.

Single-cell RNA sequencing reveals the induction of novel myeloid and myeloid-associated cell populations in visceral fat with long-term obesity.

Macrophages and other immune cells are important contributors to obesity-associated inflammation; however, the cellular identities of these specific populations remain unknown. In this study, we identified individual populations of myeloid cells found in mouse epididymal/visceral adipose tissue by single-cell RNA sequencing, immunofluorescence, and flow cytometry. Multiple canonical correlation analysis identified 11 unique myeloid and myeloid-associate cell populations. In obese mice, we detected an increased percentage of monocyte-derived pro-inflammatory cells expressing Cd9 and Trem2, as well as significantly decreased percentages of multiple cell populations, including tissue-resident cells expressing Lyve1, Mafb, and Mrc1. We have identified and validated a novel myeloid/macrophage population defined by Ly6a expression, exhibiting both myeloid and mesenchymal characteristics, which increased with obesity and showed high pro-fibrotic characteristics in vitro. Our mouse adipose tissue myeloid cell atlas provides an important resource to investigate obesity-associated inflammation and fibrosis.

Adipose tissue is a critical regulator of osteoarthritis.

Osteoarthritis (OA), the leading cause of pain and disability worldwide, disproportionally affects individuals with obesity. The mechanisms by which obesity leads to the onset and progression of OA are unclear due to the complex interactions among the metabolic, biomechanical, and inflammatory factors that accompany increased adiposity. We used a murine preclinical model of lipodystrophy (LD) to examine the direct contribution of adipose tissue to OA. Knee joints of LD mice were protected from spontaneous or posttraumatic OA, on either a chow or high-fat diet, despite similar body weight and the presence of systemic inflammation. These findings indicate that adipose tissue itself plays a critical role in the pathophysiology of OA. Susceptibility to posttraumatic OA was reintroduced into LD mice using implantation of a small adipose tissue depot derived from wild-type animals or mouse embryonic fibroblasts that undergo spontaneous adipogenesis, implicating paracrine signaling from fat, rather than body weight, as a mediator of joint degeneration.

Single cell transcriptomic analysis of human pluripotent stem cell chondrogenesis.

The therapeutic application of human induced pluripotent stem cells (hiPSCs) for cartilage regeneration is largely hindered by the low yield of chondrocytes accompanied by unpredictable and heterogeneous off-target differentiation of cells during chondrogenesis. Here, we combine bulk RNA sequencing, single cell RNA sequencing, and bioinformatic analyses, including weighted gene co-expression analysis (WGCNA), to investigate the gene regulatory networks regulating hiPSC differentiation under chondrogenic conditions. We identify specific WNTs and MITF as hub genes governing the generation of off-target differentiation into neural cells and melanocytes during hiPSC chondrogenesis. With heterocellular signaling models, we further show that WNT signaling produced by off-target cells is responsible for inducing chondrocyte hypertrophy. By targeting WNTs and MITF, we eliminate these cell lineages, significantly enhancing the yield and homogeneity of hiPSC-derived chondrocytes. Collectively, our findings identify the trajectories and molecular mechanisms governing cell fate decision in hiPSC chondrogenesis, as well as dynamic transcriptome profiles orchestrating chondrocyte proliferation and differentiation.

Gene therapy for follistatin mitigates systemic metabolic inflammation and post-traumatic arthritis in high-fat diet-induced obesity.

Obesity-associated inflammation and loss of muscle function play critical roles in the development of osteoarthritis (OA); thus, therapies that target muscle tissue may provide novel approaches to restoring metabolic and biomechanical dysfunction associated with obesity. Follistatin (FST), a protein that binds myostatin and activin, may have the potential to enhance muscle formation while inhibiting inflammation. Here, we hypothesized that adeno-associated virus 9 (AAV9) delivery of FST enhances muscle formation and mitigates metabolic inflammation and knee OA caused by a high-fat diet in mice. AAV-mediated FST delivery exhibited decreased obesity-induced inflammatory adipokines and cytokines systemically and in the joint synovial fluid. Regardless of diet, mice receiving FST gene therapy were protected from post-traumatic OA and bone remodeling induced by joint injury. Together, these findings suggest that FST gene therapy may provide a multifactorial therapeutic approach for injury-induced OA and metabolic inflammation in obesity.

Long non-coding RNA GRASLND enhances chondrogenesis via suppression of the interferon type II signaling pathway.

The roles of long noncoding RNAs (lncRNAs) in musculoskeletal development, disease, and regeneration remain poorly understood. Here, we identified the novel lncRNA GRASLND (originally named RNF144A-AS1) as a regulator of mesenchymal stem cell (MSC) chondrogenesis. GRASLND, a primate-specific lncRNA, is upregulated during MSC chondrogenesis and appears to act directly downstream of SOX9, but not TGF-ß3. We showed that the silencing of GRASLND resulted in lower accumulation of cartilage-like extracellular matrix in a pellet assay, while GRASLND overexpression - either via transgene ectopic expression or by endogenous activation via CRISPR-dCas9-VP64 - significantly enhanced cartilage matrix production. GRASLND acts to inhibit IFN-γ by binding to EIF2AK2, and we further demonstrated that GRASLND exhibits a protective effect in engineered cartilage against interferon type II. Our results indicate an important role of GRASLND in regulating stem cell chondrogenesis, as well as its therapeutic potential in the treatment of cartilage-related diseases, such as osteoarthritis.

Intergenerational Transmission of Diet-Induced Obesity, Metabolic Imbalance, and Osteoarthritis in Mice.

OBJECTIVE: Obesity and osteoarthritis (OA) are 2 major public health issues affecting millions of people worldwide. Whereas parental obesity affects the predisposition to diseases such as cancer or diabetes in children, transgenerational influences on musculoskeletal conditions such as OA are poorly understood. This study was undertaken to assess the intergenerational effects of a parental/grandparental high-fat diet on the metabolic and skeletal phenotype, systemic inflammation, and predisposition to OA in 2 generations of offspring in mice. METHODS: Metabolic phenotype and predisposition to OA were investigated in the first and second (F1 and F2) generations of offspring (n = 10-16 mice per sex per diet) bred from mice fed a high-fat diet (HFD) or a low-fat control diet. OA was induced by destabilizing the medial meniscus. OA, synovitis, and adipose tissue inflammation were determined histologically, while bone changes were measured using micro-computed tomography. Serum and synovial cytokines were measured by multiplex assay. RESULTS: Parental high-fat feeding showed an intergenerational effect, with inheritance of increased weight gain (up to 19% in the F1 generation and 9% in F2), metabolic imbalance, and injury-induced OA in at least 2 generations of mice, despite the fact that the offspring were fed the low-fat diet. Strikingly, both F1 and F2 female mice showed an increased predisposition to injury-induced OA (48% higher predisposition in F1 and 19% in F2 female mice fed the HFD) and developed bone microarchitectural changes that were attributable to parental and grandparental high-fat feeding. CONCLUSION: The results of this study reveal a detrimental effect of parental HFD and obesity on the musculoskeletal integrity of 2 generations of offspring, indicating the importance of further investigation of these effects. An improved understanding of the mechanisms involved in the transmissibility of diet-induced changes through multiple generations may help in the development of future therapies that would target the effects of obesity on OA and related conditions.

Differentiation of human induced pluripotent stem cells into nucleus pulposus-like cells.

BACKGROUND: Intervertebral disc (IVD) degeneration is characterized by an early decrease in cellularity of the nucleus pulposus (NP) region, and associated extracellular matrix changes, reduced hydration, and progressive degeneration. Cell-based IVD therapy has emerged as an area of great interest, with studies reporting regenerative potential for many cell sources, including autologous or allogeneic chondrocytes, primary IVD cells, and stem cells. Few approaches, however, have clear strategies to promote the NP phenotype, in part due to a limited knowledge of the defined markers and differentiation protocols for this lineage. Here, we developed a new protocol for the efficient differentiation of human induced pluripotent stem cells (hiPSCs) into NP-like cells in vitro. This differentiation strategy derives from our knowledge of the embryonic notochordal lineage of NP cells as well as strategies used to support healthy NP cell phenotypes for primary cells in vitro. METHODS: An NP-genic phenotype of hiPSCs was promoted in undifferentiated hiPSCs using a stepwise, directed differentiation toward mesodermal, and subsequently notochordal, lineages via chemically defined medium and growth factor supplementation. Fluorescent cell imaging was used to test for pluripotency markers in undifferentiated cells. RT-PCR was used to test for potential cell lineages at the early stage of differentiation. Cells were checked for NP differentiation using immunohistochemistry and histological staining at the end of differentiation. To enrich notochordal progenitor cells, hiPSCs were transduced using lentivirus containing reporter constructs for transcription factor brachyury (T) promoter and green fluorescent protein (GFP) fluorescence, and then sorted on T expression based on GFP intensity by flow cytometry. RESULTS: Periods of pellet culture following initial induction were shown to promote the vacuolated NP cell morphology and NP surface marker expression, including CD24, LMα5, and Basp1. Enrichment of brachyury (T) positive cells using fluorescence-activated cell sorting was shown to further enhance the differentiation efficiency of NP-like cells. CONCLUSIONS: The ability to efficiently differentiate human iPSCs toward NP-like cells may provide insights into the processes of NP cell differentiation and provide a cell source for the development of new therapies for IVD diseases.

Follistatin N terminus differentially regulates muscle size and fat in vivo.

Delivery of follistatin (FST) represents a promising strategy for both muscular dystrophies and diabetes, as FST is a robust antagonist of myostatin and activin, which are critical regulators of skeletal muscle and adipose tissues. FST is a multi-domain protein, and deciphering the function of different domains will facilitate novel designs for FST-based therapy. Our study aims to investigate the role of the N-terminal domain (ND) of FST in regulating muscle and fat mass in vivo. Different FST constructs were created and packaged into the adeno-associated viral vector (AAV). Overexpression of wild-type FST in normal mice greatly increased muscle mass while decreasing fat accumulation, whereas overexpression of an N terminus mutant or N terminus-deleted FST had no effect on muscle mass but moderately decreased fat mass. In contrast, FST-I-I containing the complete N terminus and double domain I without domain II and III had no effect on fat but increased skeletal muscle mass. The effects of different constructs on differentiated C2C12 myotubes were consistent with the in vivo finding. We hypothesized that ND was critical for myostatin blockade, mediating the increase in muscle mass, and was less pivotal for activin binding, which accounts for the decrease in the fat tissue. An in vitro TGF-beta1-responsive reporter assay revealed that FST-I-I and N terminus-mutated or -deleted FST showed differential responses to blockade of activin and myostatin. Our study provided direct in vivo evidence for a role of the ND of FST, shedding light on future potential molecular designs for FST-based gene therapy.

N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions.

Nucleus pulposus (NP) cells of the intervertebral disc are essential for synthesizing extracellular matrix that contributes to disc health and mechanical function. NP cells have a unique morphology and molecular expression pattern derived from their notochordal origin, and reside in N-cadherin (CDH2) positive cell clusters in vivo. With disc degeneration, NP cells undergo morphologic and phenotypic changes including loss of CDH2 expression and ability to form cell clusters. Here, we investigate the role of CDH2 positive cell clusters in preserving healthy, biosynthetically active NP cells. Using a laminin-functionalized hydrogel system designed to mimic features of the native NP microenvironment, we demonstrate NP cell phenotype and morphology is preserved only when NP cells form CDH2 positive cell clusters. Knockdown (CRISPRi) or blocking CDH2 expression in vitro and in vivo results in loss of a healthy NP cell. Findings also reveal that degenerate human NP cells that are CDH2 negative can be promoted to re-express CDH2 and healthy, juvenile NP matrix synthesis patterns by promoting cell clustering for controlled microenvironment conditions. This work also identifies CDH2 interactions with ß-catenin-regulated signaling as one mechanism by which CDH2-mediated cell interactions can control NP cell phenotype and biosynthesis towards maintenance of healthy intervertebral disc tissues.

Overcoming Insulin Insufficiency by Forced Follistatin Expression in ß-cells of db/db Mice.

Diabetes poses a substantial burden to society as it can lead to serious complications and premature death. The number of cases continues to increase worldwide. Two major causes of diabetes are insulin resistance and insulin insufficiency. Currently, there are few antidiabetic drugs available that can preserve or protect ß-cell function to overcome insulin insufficiency in diabetes. We describe a therapeutic strategy to preserve ß-cell function by overexpression of follistatin (FST) using an AAV vector (AAV8-Ins-FST) in diabetic mouse model. Overexpression of FST in the pancreas of db/db mouse increased ß-cell islet mass, decreased fasting glucose level, alleviated diabetic symptoms, and essentially doubled lifespan of the treated mice. The observed islet enlargement was attributed to ß-cell proliferation as a result of bioneutralization of myostatin and activin by FST. Overall, our study indicates overexpression of FST in the diabetic pancreas preserves ß-cell function by promoting ß-cell proliferation, opening up a new therapeutic avenue for the treatment of diabetes.

Cardiomyocyte-specific p65 NF-κB deletion protects the injured heart by preservation of calcium handling.

NF-κB is a well-known transcription factor that is intimately involved with inflammation and immunity. We have previously shown that NF-κB promotes inflammatory events and mediates adverse cardiac remodeling following ischemia reperfusion (I/R). Conversely, others have pointed to the beneficial influence of NF-κB in I/R injury related to its anti-apoptotic effects. Understanding the seemingly disparate influence of manipulating NF-κB is hindered, in part, by current approaches that only indirectly interfere with the function of its most transcriptionally active unit, p65 NF-κB. Mice were generated with cardiomyocyte-specific deletion of p65 NF-κB. Phenotypically, these mice and their hearts appeared normal. Basal and stimulated p65 expression were significantly reduced in whole hearts and completely ablated in isolated cardiomyocytes. When compared with wild-type mice, transgenic animals were protected from both global I/R by Langendorff as well as regional I/R by coronary ligation and release. The protected, transgenic hearts had less cytokine activity and decreased apoptosis. Furthermore, p65 ablation was associated with enhanced calcium reuptake by the sarcoplasmic reticulum. This influence on calcium handling was related to increased expression of phosphorylated phospholamban in conditional p65 null mice. In conclusion, cardiomyocyte-specific deletion of the most active, canonical NF-κB subunit affords cardioprotection to both global and regional I/R injury. The beneficial effects of NF-κB inhibition are related, in part, to modulation of intracellular calcium homeostasis.

Acetylation of myocardin is required for the activation of cardiac and smooth muscle genes.

Myocardin belongs to the SAF-A/B, Acinus, PIAS (SAP) domain family of transcription factors and is specifically expressed in cardiac and smooth muscle. Myocardin functions as a transcriptional coactivator of SRF and is sufficient and necessary for smooth muscle gene expression. We have previously found that myocardin induces the acetylation of nucleosomal histones surrounding SRF-binding sites in the control regions of cardiac and smooth muscle genes through recruiting chromatin-modifying enzyme p300, yet no studies have determined whether myocardin itself is similarly modified. In this study, we show that myocardin is a direct target for p300-mediated acetylation. p300 acetylates lysine residues at the N terminus of the myocardin protein. Interestingly, a direct interaction between p300 and myocardin, which is mediated by the C terminus of myocardin, is required for the acetylation event. Acetylation of myocardin by p300 enhances the association of myocardin and SRF as well as the formation of the myocardin-SRF-CArG box ternary complex. Conversely, acetylation of myocardin decreases the binding of histone deacetylase 5 (HDAC5) to myocardin. Acetylation of myocardin is required for myocardin to activate smooth muscle genes. Our study demonstrates that acetylation plays a key role in modulating myocardin function in controlling cardiac and smooth muscle gene expression.

Recovery from decompensated heart failure is associated with a distinct, phase-dependent gene expression profile.

BACKGROUND: Clinical and experimental studies have traditionally focused on understanding the mechanisms for why a heart fails. We hypothesize that the pathways involved with myocardial recovery are not simply the reverse of those that cause heart failure. However, determining when and how a decompensated heart can recover remains unknown. METHODS: Male C57BL/6 mice underwent minimally invasive aortic banding for 3, 4, or 6 wk with or without subsequent band removal for 1 wk (debanding). Physiologic and genomic characterization was performed with intracardiac pressure-volume recordings, rt-PCR, and microarray analysis. RESULTS: Heart weight/body weight ratios and PV loops demonstrated a transition from compensated left ventricular hypertrophy to decompensated heart failure between 3 and 4 wk. Pressure-relief afforded by debanding allowed functional recovery and normalization of LVH after both 3 and 4, but not 6 wk of banding. Whole genome microarrays demonstrated 397 genes differentially expressed in recovered hearts, 250 genes differentially expressed in the nonrecoverable (6 wk) hearts, and only 10 genes shared by both processes. In particular, altered expression patterns of apoptotic and metalloproteinase genes correlated with the heart's ability to functionally recover. CONCLUSIONS: This clinically-relevant model (1) allows us to temporally and mechanistically characterize the failing heart, (2) demonstrates a unique genomic signature that may predict when a failing heart can recover following pressure relief, and (3) will prove useful as a template for testing therapeutic strategies aimed at recovery of the failing heart.

Inhibitory kappa-B kinase-ß inhibition prevents adaptive left ventricular hypertrophy.

BACKGROUND: Most cardiovascular studies have implicated the central transcription factor nuclear factor kappa-B (NF-κB) as contributing to the detrimental effects of cardiac injury. This ostensibly negative view of NF-κB competes with its important role in the normal host inflammatory and immune response. Pressure overload, left ventricular hypertrophy (LVH), and heart failure represent a spectrum of disease that has both adaptive and maladaptive components. In contrast to its known effects related to myocardial ischemia-reperfusion, we hypothesized that NF-κB is necessary for the compensatory phase of cardiac remodeling. METHODS: C57BL6 mice underwent minimally invasive transverse aortic constriction with or without inhibition of the proximal NF-κB kinase, inhibitory kappa-B kinase-ß. Isolated cardiomyocytes were cultured. Transthoracic echocardiography was performed on all mice. RESULTS: Inhibitory kappa-B kinase-ß inhibition successfully decreased cardiomyocyte expression of phosphorylated p65 NF-κB and decreased expression of hypertrophic markers with stimulation in vitro. Three weeks after transverse aortic constriction, the mice treated with inhibitory kappa-B kinase-ß inhibition more aggressively developed LVH, as measured by heart weight/body weight ratio, left ventricular mass, and wall thickness. These mice also demonstrated a functional decline, as measured by decreased fractional shortening and ejection fraction. These findings were associated with decreased protein expression of p65 NF-κB. CONCLUSIONS: Although short-term pressure-overload results in compensatory LVH with normal cardiac function, NF-κB inhibition resulted in increased LVH that was associated with functional deterioration. These observations suggest that NF-κB is an important part of the adaptive phase of LVH, and its inhibition detrimentally affects cardiac remodeling.

Single tyrosine mutation in AAV8 and AAV9 capsids is insufficient to enhance gene delivery to skeletal muscle and heart.

Site-directed mutations of tyrosine (Y) to phenylalanine (F) on the surface of adeno-associated viral (AAV) capsids have been reported as a simple method to greatly enhance gene transfer in vitro and in vivo. To determine whether the Y-to-F mutation could also enhance AAV8 and AAV9 gene transfer in skeletal muscle and heart to facilitate muscular dystrophy gene therapy, we investigated four capsid mutants of AAV8 (Y447F or Y733F) and AAV9 (Y446F or Y731F). The mutants and their wild-type control AAV8 and AAV9 capsids were used to package reporter genes (luciferase or ß-galactosidase) resulting in similar vector yields. To evaluate gene delivery efficiencies, especially in muscle and heart, the vectors were compared side by side in a series of experiments in vivo in two different strains of mice, the outbred ICR and the inbred C57BL/6. Because AAV8 and AAV9 are among the most effective in systemic gene delivery, we first examined the mutant and wild-type vectors in neonatal mice by intraperitoneal injection, or in adult mice by intravenous injection. To our surprise, no statistically significant differences in transgene expression were observed between the mutant and wild-type vectors, regardless of the reporter genes, vector doses, and the ages and strains of mice used. In addition, quantitative analyses of vector DNA copy number in various tissues from mice treated with mutant and wild-type vectors also showed similar results. Finally, direct intramuscular injection of the above-described vectors with the luciferase gene into the hind limb muscles revealed the same levels of gene expression between mutant and wild-type vectors. Our results thus demonstrate that a single mutation of Y447F or Y733F on capsids of AAV8, and of Y446F or Y731F on AAV9, is insufficient to enhance gene delivery to the skeletal muscle and heart.

Enhancing muscle membrane repair by gene delivery of MG53 ameliorates muscular dystrophy and heart failure in δ-Sarcoglycan-deficient hamsters.

Muscular dystrophies (MDs) are caused by genetic mutations in over 30 different genes, many of which encode for proteins essential for the integrity of muscle cell structure and membrane. Their deficiencies cause the muscle vulnerable to mechanical and biochemical damages, leading to membrane leakage, dystrophic pathology, and eventual loss of muscle cells. Recent studies report that MG53, a muscle-specific TRIM-family protein, plays an essential role in sarcolemmal membrane repair. Here, we show that systemic delivery and muscle-specific overexpression of human MG53 gene by recombinant adeno-associated virus (AAV) vectors enhanced membrane repair, ameliorated pathology, and improved muscle and heart functions in δ-sarcoglycan (δ-SG)-deficient TO-2 hamsters, an animal model of MD and congestive heart failure. In addition, MG53 overexpression increased dysferlin level and facilitated its trafficking to muscle membrane through participation of caveolin-3. MG53 also protected muscle cells by activating cell survival kinases, such as Akt, extracellular signal-regulated kinases (ERK1/2), and glycogen synthase kinase-3ß (GSK-3ß) and inhibiting proapoptotic protein Bax. Our results suggest that enhancing the muscle membrane repair machinery could be a novel therapeutic approach for MD and cardiomyopathy, as demonstrated here in the limb girdle MD (LGMD) 2F model.

The histone methyltransferase Set7/9 promotes myoblast differentiation and myofibril assembly.

The molecular events that modulate chromatin structure during skeletal muscle differentiation are still poorly understood. We report in this paper that expression of the H3-K4 histone methyltransferase Set7 is increased when myoblasts differentiate into myotubes and is required for skeletal muscle development, expression of muscle contractile proteins, and myofibril assembly. Knockdown of Set7 or expression of a dominant-negative Set7 mutant impairs skeletal muscle differentiation, accompanied by a decrease in levels of histone monomethylation (H3-K4me1). Set7 directly interacts with MyoD to enhance expression of muscle differentiation genes. Expression of myocyte enhancer factor 2 and genes encoding contractile proteins is decreased in Set7 knockdown myocytes. Furthermore, we demonstrate that Set7 also activates muscle gene expression by precluding Suv39h1-mediated H3-K9 methylation on the promoters of myogenic differentiation genes. Together, our experiments define a biological function for Set7 in muscle differentiation and provide a molecular mechanism by which Set7 modulates myogenic transcription factors during muscle differentiation.

Post-Natal knockdown of fukutin-related protein expression in muscle by long-termRNA interference induces dystrophic pathology [corrected].

Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by mutations in the fukutin-related protein (FKRP) gene. Unlike its severe allelic forms, LGMD2I usually involves slower onset and milder course without defects in the central nervous system. The lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes led us to use RNA interference technology to knock down FKRP expression via postnatal gene delivery so as to circumvent embryonic lethality. Specifically, an adeno-associated viral vector was used to deliver short hairpin (shRNA) genes to healthy ICR mice. Adeno-associated viral vectors expressing a single shRNA or two different shRNAs were injected one time into the hind limb muscles. We showed that FKRP expression at 10 months postinjection was reduced by about 50% with a single shRNA and by 75% with the dual shRNA cassette. Dual-cassette injection also reduced a-dystroglycan glycosylation and its affinity to laminin by up to 70% and induced α-dystrophic pathology, including fibrosis and central nucleation, in more than 50% of the myofibers at 10 months after injection. These results suggest that the reduction of approximately or more than 75% of the normal level of FKRP expression induces chronic dystrophic phenotypes in skeletal muscles. Furthermore, the restoration of about 25% of the normal FKRP level could be sufficient for LGMD2I therapy to correct the genetic deficiency effectively and prevent dystrophic pathology.