Temperature instability of a mutation at a multidomain junction in Na,K-ATPase isoform ATP1A3 (p.Arg756His) produces a fever-induced neurological syndrome.

ATP1A3 encodes the α3 isoform of Na,K-ATPase. In the brain, it is expressed only in neurons. Human ATP1A3 mutations produce a wide spectrum of phenotypes, but particular syndromes are associated with unique substitutions. For arginine 756, at the junction of membrane and cytoplasmic domains, mutations produce encephalopathy during febrile infections. Here we tested the pathogenicity of p.Arg756His (R756H) in isogenic mammalian cells. R756H protein had sufficient transport activity to support cells when endogenous ATP1A1 was inhibited. It had half the turnover rate of wildtype, reduced affinity for Na+, and increased affinity for K+. There was modest endoplasmic reticulum retention during biosynthesis at 37 °C but little benefit from the folding drug phenylbutyrate (4-PBA), suggesting a tolerated level of misfolding. When cells were incubated at just 39 °C, however, α3 protein level dropped without loss of ß subunit, paralleled by an increase of endogenous α1. Elevated temperature resulted in internalization of α3 from the surface along with some ß subunit, accompanied by cytoplasmic redistribution of a marker of lysosomes and endosomes, lysosomal-associated membrane protein 1. After return to 37 °C, α3 protein levels recovered with cycloheximide-sensitive new protein synthesis. Heating in vitro showed activity loss at a rate 20- to 30-fold faster than wildtype, indicating a temperature-dependent destabilization of protein structure. Arg756 appears to confer thermal resistance as an anchor, forming hydrogen bonds among four linearly distant parts of the Na,K-ATPase structure. Taken together, our observations are consistent with fever-induced symptoms in patients.

Membrane-delimited signaling and cytosolic action of MG53 preserve hepatocyte integrity during drug-induced liver injury.

BACKGROUND & AIMS: Drug-induced liver injury (DILI) remains challenging to treat and is still a leading cause of acute liver failure. MG53 is a muscle-derived tissue-repair protein that circulates in the bloodstream and whose physiological role in protection against DILI has not been examined. METHODS: Recombinant MG53 protein (rhMG53) was administered exogenously, using mice with deletion of Mg53 or Ripk3. Live-cell imaging, histological, biochemical, and molecular studies were used to investigate the mechanisms that underlie the extracellular and intracellular action of rhMG53 in hepatoprotection. RESULTS: Systemic administration of rhMG53 protein, in mice, can prophylactically and therapeutically treat DILI induced through exposure to acetaminophen, tetracycline, concanavalin A, carbon tetrachloride, or thioacetamide. Circulating MG53 protects hepatocytes from injury through direct interaction with MLKL at the plasma membrane. Extracellular MG53 can enter hepatocytes and act as an E3-ligase to mitigate RIPK3-mediated MLKL phosphorylation and membrane translocation. CONCLUSIONS: Our data show that the membrane-delimited signaling and cytosolic dual action of MG53 effectively preserves hepatocyte integrity during DILI. rhMG53 may be a potential treatment option for patients with DILI. LAY SUMMARY: Interventions to treat drug-induced liver injury and halt its progression into liver failure are of great value to society. The present study reveals that muscle-liver cross talk, with MG53 as a messenger, serves an important role in liver cell protection. Thus, MG53 is a potential treatment option for patients with drug-induced liver injury.

A simple, quick, and efficient CRISPR/Cas9 genome editing method for human induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) have become an essential research platform to study different human diseases once being discovered by Dr. Shinya Yamanaka in 2006. Another breakthrough in biomedical research is the application of CRISPR/Cas9 system for genome editing in mammalian cells. Although numerous studies have been done to develop methods for gene editing in iPSCs, the current approaches suffer from several limitations, including time and labor consuming, low editing efficiency, and potential off-target effects. In the current study, we report an electroporation-mediated plasmid CRISPR/Cas9 delivery approach for genome editing in iPSCs. With this approach, an edited iPSC cell line could be obtained within 2 weeks. In addition, the transit introducing of CRISPR/Cas9 machinery could minimize genomic integration of Cas9 gene, which avoided potential long-term side effects of Cas9 enzyme. We showed that CRISPR/Cas9-mediated genomic editing did not affect pluripotency and differentiation ability of iPSCs. With the quickly evolving of both iPSC and CRISPR/Cas9-mediated genome editing research fields, we believe that our method can significantly facilitate the application of genome editing in iPSCs research.

Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store-operated Ca2+ entry (SOCE) channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1-mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, a specific Zn2+ chelator, whereas nontumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn2+ exerted rapid inhibitory effects on Orai1-mediated SOCE and on intracellular Ca2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1-mediated intracellular Ca2+ oscillations and reveal a possible molecular basis for zinc-induced cancer prevention and Orai1-SOCE signaling pathway in cancer cells.-Choi, S., Cui, C., Luo, Y., Kim, S.-H., Ko, J.-K., Huo, X., Ma, J., Fu, L.-W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Zinc Binding to MG53 Protein Facilitates Repair of Injury to Cell Membranes.

Zinc is an essential trace element that participates in a wide range of biological functions, including wound healing. Although Zn(2+) deficiency has been linked to compromised wound healing and tissue repair in human diseases, the molecular mechanisms underlying Zn(2+)-mediated tissue repair remain unknown. Our previous studies established that MG53, a TRIM (tripartite motif) family protein, is an essential component of the cell membrane repair machinery. Domain homology analysis revealed that MG53 contains two Zn(2+)-binding motifs. Here, we show that Zn(2+) binding to MG53 is indispensable to assembly of the cell membrane repair machinery. Live cell imaging illustrated that Zn(2+) entry from extracellular space is essential for translocation of MG53-containing vesicles to the acute membrane injury sites for formation of a repair patch. The effect of Zn(2+) on membrane repair is abolished in mg53(-/-) muscle fibers, suggesting that MG53 functions as a potential target for Zn(2+) during membrane repair. Mutagenesis studies suggested that both RING and B-box motifs of MG53 constitute Zn(2+)-binding domains that contribute to MG53-mediated membrane repair. Overall, this study establishes a base for Zn(2+) interaction with MG53 in protection against injury to the cell membrane.

Melanophilin regulates dendritogenesis in melanocytes for feather pigmentation.

Limited studies using animal models with a few natural mutations in melanophilin (Mlph) provided partial functions of Mlph in melanosome trafficking. To investigate cellular functions of Mlph, especially ZnF motif of Mlph, we analyzed all three Mlph knockout (KO) quail lines, one and two base pair (bp) deletions as models for total KO, and three bp deletion causing deletion of one Cysteine (C84del) in the ZnF motif. All quail lines had diluted feather pigmentation with impaired dendritogenesis and melanosome transport in melanocytes. In vitro studies revealed capability of binding of the ZnF motif to PIP3, and impairment of PI3P binding and mislocalization of MLPH proteins with ZnF motif mutations. The shortened melanocyte dendrites by the C84del mutation were rescued by introducing WT Mlph in vitro. These results revealed the diluted feather pigmentation by Mlph mutations resulted from congregation of melanosomes in the cell bodies with impairment of the dendritogenesis and the transport of melanosomes to the cell periphery.

Quantification of autophagy flux in isolated mouse skeletal muscle fibers with overexpression of fluorescent protein mCherry-EGFP-LC3.

Evaluation of autophagy flux could be challenging for muscle fibers due to the baseline expression of mCherry-EGFP-LC3 along the Z-line. We established a protocol to overcome this difficulty. We overexpress mChery-EGFP-LC3 in the FDB muscle of an adult mouse via electroporation. Then, we enzymatically digest FDB muscle to yield individual fibers for live cell imaging. Finally, we develop an ImageJ-based program to eliminate the baseline striation pattern and semi-automatically quantify autophagosomes (APs) and autolysosomes (ALs) for autophagy flux analysis.

Distinct GSDMB protein isoforms and protease cleavage processes differentially control pyroptotic cell death and mitochondrial damage in cancer cells.

Gasdermin (GSDM)-mediated pyroptosis is functionally involved in multiple diseases, but Gasdermin-B (GSDMB) exhibit cell death-dependent and independent activities in several pathologies including cancer. When the GSDMB pore-forming N-terminal domain is released by Granzyme-A cleavage, it provokes cancer cell death, but uncleaved GSDMB promotes multiple pro-tumoral effects (invasion, metastasis, and drug resistance). To uncover the mechanisms of GSDMB pyroptosis, here we determined the GSDMB regions essential for cell death and described for the first time a differential role of the four translated GSDMB isoforms (GSDMB1-4, that differ in the alternative usage of exons 6-7) in this process. Accordingly, we here prove that exon 6 translation is essential for GSDMB mediated pyroptosis, and therefore, GSDMB isoforms lacking this exon (GSDMB1-2) cannot provoke cancer cell death. Consistently, in breast carcinomas the expression of GSDMB2, and not exon 6-containing variants (GSDMB3-4), associates with unfavourable clinical-pathological parameters. Mechanistically, we show that GSDMB N-terminal constructs containing exon-6 provoke cell membrane lysis and a concomitant mitochondrial damage. Moreover, we have identified specific residues within exon 6 and other regions of the N-terminal domain that are important for GSDMB-triggered cell death as well as for mitochondrial impairment. Additionally, we demonstrated that GSDMB cleavage by specific proteases (Granzyme-A, Neutrophil Elastase and caspases) have different effects on pyroptosis regulation. Thus, immunocyte-derived Granzyme-A can cleave all GSDMB isoforms, but in only those containing exon 6, this processing results in pyroptosis induction. By contrast, the cleavage of GSDMB isoforms by Neutrophil Elastase or caspases produces short N-terminal fragments with no cytotoxic activity, thus suggesting that these proteases act as inhibitory mechanisms of pyroptosis. Summarizing, our results have important implications for understanding the complex roles of GSDMB isoforms in cancer or other pathologies and for the future design of GSDMB-targeted therapies.

Amphipathic tail-anchoring peptide and Bcl-2 homology domain-3 (BH3) peptides from Bcl-2 family proteins induce apoptosis through different mechanisms.

Bcl-2 homology domain-3 (BH3) peptides are potent cancer therapeutic reagents that target regulators of apoptotic cell death in cancer cells. However, their cytotoxic effects are affected by different expression levels of Bcl-2 family proteins. We recently found that the amphipathic tail-anchoring peptide (ATAP) from Bfl-1, a bifunctional Bcl-2 family member, produced strong pro-apoptotic activity by permeabilizing the mitochondrial outer membrane. Here, we test whether the activity of ATAP requires other cellular factors and whether ATAP has an advantage over the BH3 peptides in targeting cancer cells. Confocal microscopic imaging illustrates specific targeting of ATAP to mitochondria, whereas BH3 peptides show diffuse patterns of cytosolic distribution. Although the pro-apoptotic activities of BH3 peptides are largely inhibited by either overexpression of anti-apoptotic Bcl-2 or Bcl-xL or nullification of pro-apoptotic Bax and Bak in cells, the pro-apoptotic function of ATAP is not affected by these cellular factors. Reconstitution of synthetic ATAP into liposomal membranes results in release of fluorescent molecules of the size of cytochrome c from the liposomes, suggesting that the membrane permeabilizing activity of ATAP does not require additional protein factors. Because ATAP can target to the mitochondrial membrane and its pro-apoptotic activity does not depend on the content of Bcl-2 family proteins, it represents a promising candidate for anti-cancer drugs that can potentially overcome the intrinsic apoptosis-resistant nature of cancer cells.

A versatile single-plasmid system for tissue-specific and inducible control of gene expression in transgenic mice.

We describe a novel transgenic system for tissue-specific and inducible control of gene expression in mice. The system employs a tetracycline-responsive CMV promoter that controls transcription of a short-hairpin RNA (shRNA) that remains nonfunctional until an interrupting reporter cassette is excised by Cre recombinase. Insertion of Dicer and Drosha RNase processing sites within the shRNA allows generation of siRNA to knock down a target gene efficiently. Tissue-specific shRNA expression is achieved through the use of appropriate inducer mice with tissue-specific expression of Cre. We applied this system to regulate expression of junctophilins (JPs), genes essential for maintenance of membrane ultrastructure and Ca(2+) signaling in muscle. Transgenic mice with skeletal muscle-specific expression of shRNA against JP mRNAs displayed no basal change of JP expression before treatment with doxycycline (Dox), while inducible and reversible knockdown of JPs was achieved by feeding mice with Dox-containing water. Dox-induced knockdown of JPs led to abnormal junctional membrane structure and Ca(2+) signaling in adult muscle fibers, consistent with essential roles of JPs in muscle development and function. This transgenic approach can be applied for inducible and reversible gene knockdown or gene overexpression in many different tissues, thus providing a versatile system for elucidating the physiological gene function in viable animal models.

Transdifferentiation of Myoblasts Into Adipocytes by All-Trans-Retinoic Acid in Avian.

Increased adipogenesis in muscle tissues is related to metabolic syndromes and muscle weakness in humans and improvement of meat quality in animal production. With growing evidence for pro-adipogenic functions of all-trans-retinoic acid (atRA), the current study investigated whether atRA can transdifferentiate myoblasts into adipocytes using a quail myogenic cell line (QM7) and avian primary myoblasts. atRA increased cytoplasmic lipid droplet accumulation and mRNA expression for adipogenic genes in these cells. An acute induction of Pparγ expression by atRA under cycloheximide treatment indicated a direct regulation of Pparγ by atRA. In addition, the induction of Pparγ expression was mediated by retinoic acid receptors . At high levels of Pparγ by atRA, BADGE, an antagonist of Pparγ, inhibited, and rosiglitazone, an agonist of Pparγ, further enhanced atRA-induced transdifferentiation. However, at very low levels of Pparγ in the absence of atRA treatment, rosiglitazone could not induce transdifferentiation of avian myoblasts. These data suggest that the induction of Pparγ expression by atRA is an essential molecular event in myoblasts for atRA-induced transdifferentiation into adipocytes. Based on our findings, atRA can be a new transdifferentiation factor of myoblasts to adipocytes, providing a potential nutrient to enhance marbling in poultry.

Redox-dependent oligomerization through a leucine zipper motif is essential for MG53-mediated cell membrane repair.

We recently discovered that MG53, a muscle-specific tripartite motif (TRIM) family protein, functions as a sensor of oxidation to nucleate the assembly of cell membrane repair machinery. Our data showed that disulfide bond formation mediated by Cys242 is critical for MG53-mediated translocation of intracellular vesicles toward the injury sites. Here we test the hypothesis that leucine zipper motifs in the coiled-coil domain of MG53 constitute an additional mechanism that facilitates oligomerization of MG53 during cell membrane repair. Two leucine zipper motifs in the coiled-coil domain of MG53 (LZ1 - L176/L183/L190/V197 and LZ2 - L205/L212/L219/L226) are highly conserved across the different animal species. Chemical cross-linking studies show that LZ1 is critical for MG53 homodimerization, whereas LZ2 is not. Mutations of the conserved leucines into alanines in LZ1, not in LZ2, diminish the redox-dependent oligomerization of MG53. Live cell imaging studies demonstrate that the movement of green fluorescent protein (GFP)-tagged MG53 mutants (GFP-LA1 and GFP-LA2) is partially compromised in response to mechanical damage of the cell membrane, and the GFP-LA1/2 double mutant is completely ineffective in translocation toward the injury sites. In addition to the leucine zipper-mediated intermolecular interaction, redox-dependent cross talk between MG53 appears to be an obligatory step for cell membrane repair, since in vivo modification of cysteine residues with alkylating reagents can prevent the movement of MG53 toward the injury sites. Our data show that oxidation of the thiol group of Cys242 and leucine zipper-mediated interaction among the MG53 molecules both contribute to the nucleation process for MG53-mediated cell membrane repair.

S165F mutation of junctophilin 2 affects Ca2+ signalling in skeletal muscle.

JPs (junctophilins) contribute to the formation of junctional membrane complexes in muscle cells by physically linking the t-tubule (transverse-tubule) and SR (sarcoplasmic reticulum) membranes. In humans with HCM (hypertrophic cardiomyopathy), mutations in JP2 are linked to altered Ca2+ signalling in cardiomyocytes; however, the effects of these mutations on skeletal muscle function have not been examined. In the present study, we investigated the role of the dominant-negative JP2-S165F mutation (which is associated with human HCM) in skeletal muscle. Consistent with the hypertrophy observed in human cardiac muscle, overexpression of JP2-S165F in primary mouse skeletal myotubes led to a significant increase in myotube diameter and resting cytosolic Ca2+ concentration. Single myotube Ca2+ imaging experiments showed reductions in both the excitation-contraction coupling gain and RyR (ryanodine receptor) 1-mediated Ca2+ release from the SR. Immunoprecipitation assays revealed defects in the PKC (protein kinase C)-mediated phosphorylation of the JP2-S165F mutant protein at Ser165 and in binding of JP2-S165F to the Ca2+ channel TRPC3 (transient receptor potential cation canonical-type channel 3) on the t-tubule membrane. Therefore both the hypertrophy and altered intracellular Ca2+ signalling in the JP2-S165F-expressing skeletal myotubes can be linked to altered phosphorylation of JP2 and/or altered cross-talk among Ca2+ channels on the t-tubule and SR membranes.

Increased store-operated Ca2+ entry in skeletal muscle with reduced calsequestrin-1 expression.

Store-operated Ca(2+) entry (SOCE) contributes to Ca(2+) handling in normal skeletal muscle function, as well as the progression of muscular dystrophy and sarcopenia, yet the mechanisms underlying the change in SOCE in these states remain unclear. Previously we showed that calsequestrin-1 (CSQ1) participated in retrograde regulation of SOCE in cultured skeletal myotubes. In this study, we used small-hairpin RNA to determine whether knockdown of CSQ1 in adult mouse skeletal muscle can influence SOCE activity and muscle function. Small-hairpin RNA against CSQ1 was introduced into flexor digitorum brevis muscles using electroporation. Transfected fibers were isolated for SOCE measurements using the Mn(2+) fluorescence-quenching method. At room temperature, the SOCE induced by submaximal depletion of the SR Ca(2+) store was significantly enhanced in CSQ1-knockdown muscle fibers. When temperature of the bathing solution was increased to 39 degrees C, CSQ1-knockdown muscle fibers displayed a significant increase in Ca(2+) permeability across the surface membrane likely via the SOCE pathway, and a corresponding elevation in cytosolic Ca(2+) as compared to control fibers. Preincubation with azumolene, an analog of dantrolene used for the treatment of malignant hyperthermia (MH), suppressed the elevated SOCE in CSQ1-knockdown fibers. Because the CSQ1-knockout mice develop similar MH phenotypes, this inhibitory effect of azumolene on SOCE suggests that elevated extracellular Ca(2+) entry in skeletal muscle may be a key factor for the pathophysiological changes in intracellular Ca(2+) signaling in MH.

Glutamate at position 227 of junctophilin-2 is involved in binding to TRPC3.

Canonical-type transient receptor potential cation channel type 3 (TRPC3) allows the entry of extracellular Ca(2+) and Na(+) into various cells. In mouse skeletal myotubes, functional interaction between TRPC3 and RyR1 (ryanodine receptor type 1/Ca(2+)-release channel on sarcoplasmic reticulum membrane) regulates the gain of excitation-contraction coupling. Junctophilin-2 (JP2) is a TRPC3-interacting protein in mouse skeletal myotubes. Based on these knowledge from bona-fide TRPC3-expressing cells, to identify critical binding region(s) of JP2 that participate in binding to TRPC3, various JP2 portions were subjected to co-immunoprecipitation assay with intact TRPC3 from rabbit skeletal muscle. A region covering 143 to 234 amino acids of JP2 (F1-2) was the most efficient portion binding to TRPC3. Through mutational studies, we found that the binding ability of JP2 to TRPC3 was mainly due to glutamate in the F1-2 region (E227). This substantial binding between JP2 and TRPC3 suggests that JP2 can be a regulatory protein of TRPC3 and/or TRPC3-mediated Ca(2+) homeostasis in skeletal muscle.

Bfl-1S, a novel alternative splice variant of Bfl-1, localizes in the nucleus via its C-terminus and prevents cell death.

Bfl-1 is an antiapoptotic Bcl-2 family member and a mouse A1 homologue. The mouse A1 has been reported to have three isoforms, but little is known about human Bfl-1. By reverse-transcriptase polymerase chain reaction analysis, we have identified Bfl-1S (short form), an alternative splice variant of Bfl-1. The Bfl-1S primary sequence contains four conserved Bcl-2 homology (BH) domains and a positive-charged C-terminus containing KKRK amino acids. The expression of Bfl-1S mRNA was detected predominantly in normal lymph nodes and in B-lymphoid leukemia cells. Confocal microscopic analysis using green fluorescence protein fusion proteins demonstrated that Bfl-1S is localized in the nucleus by its C-terminus as an intrinsic nuclear localization sequence. Bfl-1S acts as an antiapoptotic agent in coexpression experiments with Bax, a proapoptotic molecule. The expression of Bfl-1S provided significant resistance against staurosporine (STS) treatments in Molt-4 human T-leukemia cells. Bfl-1S also significantly inhibited the cleavage of Bid, and of caspases 3 and 8 against STS treatment. These results indicate that Bfl-1S is a novel human Bcl-2 family member that possesses antiapoptotic function.

Conversion of Bfl-1, an anti-apoptotic Bcl-2 family protein, to a potent pro-apoptotic protein by fusion with green fluorescent protein (GFP).

Human Bfl-1 is an anti-apoptotic Bcl-2 family member. Here, we found that Bfl-1 was converted into a potent death-promoting protein by green fluorescent protein (GFP) fusion with its N-terminus. The transient expression of GFP-Bfl-1 induced cytochrome c release and triggered apoptosis in 293T cells, which depended on the mitochondrial localization of GFP-Bfl-1. Apoptosis induced by GFP-Bfl-1 was significantly blocked by the pan-caspase inhibitor carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone, but was not blocked by either Bcl-xL or Bfl-1. Our findings provide a useful model for understanding the structural basis of Bcl-2 family proteins that act in an opposite way despite sharing structural similarity between anti-apoptotic and pro-apoptotic proteins.

Amphipathic tail-anchoring peptide is a promising therapeutic agent for prostate cancer treatment.

Amphipathic tail-anchoring peptide (ATAP) derived from the human anti-apoptotic protein Bfl-1 is a potent inducer of apoptosis by targeting mitochondria permeability transition. By linking ATAP to an internalizing RGD peptide (iRGD), selective targeting for ATAP to tumor cell was achieved. Confocal fluorescence microscopy showed that ATAP-iRGD could penetrate into cancer cells and distribute along the mitochondria network. ATAP-iRGD triggered mitochondria-dependent cell death through release of cytochrome c. In an effort to promote ATAP-iRGD physiochemical properties to approach clinic application, amino acid substitution and chemical modification were made with ATAP-iRGD to improve its bioactivity. One of these modified peptides, ATAP-iRGD-M8, was with improved stability and aqueous solubility without compromising in vitro cytotoxicity in cultured cancer cells. In vivo xenograft studies with multiple prostate cancer cell lines showed that intravenous administration of ATAP-iRGD-M8 suppressed tumor growth. Toxicological studies revealed that repetitive intravenous administration of ATAP-iRGD-M8 did not produce significant toxicity in the SV129 mice. Our data suggest that ATAP-iRGD-M8 is a promising agent with high selectivity and limited systemic toxicity for prostate cancer treatment.

Disrupted membrane structure and intracellular Ca²âº signaling in adult skeletal muscle with acute knockdown of Bin1.

Efficient intracellular Ca²âº ([Ca²âº]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca²âº homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short -hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca²âº release. Voltage-induced Ca²âº released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca²âº current and SR Ca²âº transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca²âº sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca²âº sparks' amplitude that was attributed to decreased total Ca²âº stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca²âº]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy.

Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin.

Defective membrane repair can contribute to the progression of muscular dystrophy. Although mutations in caveolin-3 (Cav3) and dysferlin are linked to muscular dystrophy in human patients, the molecular mechanism underlying the functional interplay between Cav3 and dysferlin in membrane repair of muscle physiology and disease has not been fully resolved. We recently discovered that mitsugumin 53 (MG53), a muscle-specific TRIM (Tri-partite motif) family protein (TRIM72), contributes to intracellular vesicle trafficking and is an essential component of the membrane repair machinery in striated muscle. Here we show that MG53 interacts with dysferlin and Cav3 to regulate membrane repair in skeletal muscle. MG53 mediates active trafficking of intracellular vesicles to the sarcolemma and is required for movement of dysferlin to sites of cell injury during repair patch formation. Mutations in Cav3 (P104L, R26Q) that cause retention of Cav3 in Golgi apparatus result in aberrant localization of MG53 and dysferlin in a dominant-negative fashion, leading to defective membrane repair. Our data reveal that a molecular complex formed by MG53, dysferlin, and Cav3 is essential for repair of muscle membrane damage and also provide a therapeutic target for treatment of muscular and cardiovascular diseases that are linked to compromised membrane repair.