Reduced Sarcolemmal Membrane Repair Exacerbates Striated Muscle Pathology in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a common X-linked degenerative muscle disorder that involves mutations in the DMD gene that frequently reduce the expression of the dystrophin protein, compromising the structural integrity of the sarcolemmal membrane and leaving it vulnerable to injury during cycles of muscle contraction and relaxation. This results in an increased frequency of sarcolemma disruptions that can compromise the barrier function of the membrane and lead to death of the myocyte. Sarcolemmal membrane repair processes can potentially compensate for increased membrane disruptions in DMD myocytes. Previous studies demonstrated that TRIM72, a muscle-enriched tripartite motif (TRIM) family protein also known as mitsugumin 53 (MG53), is a component of the cell membrane repair machinery in striated muscle. To test the importance of membrane repair in striated muscle in compensating for the membrane fragility in DMD, we crossed TRIM72/MG53 knockout mice into the mdx mouse model of DMD. These double knockout (DKO) mice showed compromised sarcolemmal membrane integrity compared to mdx mice, as measured by immunoglobulin G staining and ex vivo muscle laser microscopy wounding assays. We also found a significant decrease in muscle ex vivo contractile function as compared to mdx mice at both 6 weeks and 1.5 years of age. As the DKO mice aged, they developed more extensive fibrosis in skeletal muscles compared to mdx. Our findings indicate that TRIM72/MG53-mediated membrane repair can partially compensate for the sarcolemmal fragility associated with DMD and that the loss of membrane repair results in increased pathology in the DKO mice.

Autoantibodies targeting TRIM72 compromise membrane repair and contribute to inflammatory myopathy.

Idiopathic inflammatory myopathies (IIM) involve chronic inflammation of skeletal muscle and subsequent muscle degeneration due to an uncontrolled autoimmune response; however, the mechanisms leading to pathogenesis are not well understood. A compromised sarcolemmal repair process could promote an aberrant exposure of intramuscular antigens with the subsequent initiation of an inflammatory response that contributes to IIM. Using an adoptive transfer mouse model of IIM, we show that sarcolemmal repair is significantly compromised in distal skeletal muscle in the absence of inflammation. We identified autoantibodies against TRIM72 (also known as MG53), a muscle-enriched membrane repair protein, in IIM patient sera and in our mouse model of IIM by ELISA. We found that patient sera with elevated levels of TRIM72 autoantibodies suppress sarcolemmal resealing in healthy skeletal muscle, and depletion of TRIM72 antibodies from these same serum samples rescues sarcolemmal repair capacity. Autoantibodies targeting TRIM72 lead to skeletal muscle fibers with compromised membrane barrier function, providing a continuous source of autoantigens to promote autoimmunity and further amplifying humoral responses. These findings reveal a potential pathogenic mechanism that acts as a feedback loop contributing to the progression of IIM.

A Murine Model of Myocardial Ischemia-Reperfusion Injury.

Ligation of the left anterior descending (LAD) coronary artery in the mouse heart is a widely used model to simulate myocardial infarction and ischemia-reperfusion injury. Here we describe a ligation technique routinely performed in our laboratory to induce myocardial infarction that may be used to study ischemia-reperfusion injury in the myocardium. The methods described enhance location of the LAD coronary artery to allow for accurate ligation, thus increasing reproducibility of infarct size and location.

Non-contrast estimation of diffuse myocardial fibrosis with dual energy CT: A phantom study.

BACKGROUND: Estimation of diffuse myocardial fibrosis, substrate for adverse events such as heart failure and arrhythmias in patients with various cardiac disorders, is presently done by histopathology or cardiac magnetic resonance. We sought to develop a non-contrast method to estimate the amount of diffuse myocardial fibrosis leveraging dual energy computed tomography (DECT) in phantoms and a suitable small animal model. METHODS AND RESULTS: Phantoms consisted of homogenized bovine myocardium with varying amounts of Type 1 collagen. Fifteen mice underwent sham surgery, no procedure, or transverse aortic constriction (TAC) for 5 or 8 weeks to produce moderate or severe fibrosis, respectively. Phantoms and ex vivo mouse hearts were imaged on a single source, DECT scanner equipped with kVp switching. Monochromatic images were reconstructed at 40-140 keV. Linear discriminant analysis (LDA) was performed on mean myocardial CT numbers derived from single energy (70 keV) images as well as images reconstructed across multiple energies. Classification of myocardial fibrosis severity as low, moderate or severe was more often correct using the multi-energy CT/LDA approach vs. single energy CT/LDA in both phantoms (80.0% vs. 70.0%) and mice (93.3% vs. 33.3%). CONCLUSIONS: DECT myocardial imaging with multi-energy analysis better classifies myocardial fibrosis severity compared to a single energy-based approach. Non-contrast DECT can accurately and non-invasively estimate the extent of diffuse myocardial fibrosis in phantom and animal models. These data support further evaluation of this approach for in vivo myocardial fibrosis estimation.

Treatment with Recombinant Human MG53 Protein Increases Membrane Integrity in a Mouse Model of Limb Girdle Muscular Dystrophy 2B.

Limb girdle muscular dystrophy type 2B (LGMD2B) and other dysferlinopathies are degenerative muscle diseases that result from mutations in the dysferlin gene and have limited treatment options. The dysferlin protein has been linked to multiple cellular functions including a Ca2+-dependent membrane repair process that reseals disruptions in the sarcolemmal membrane. Recombinant human MG53 protein (rhMG53) can increase the membrane repair process in multiple cell types both in vitro and in vivo. Here, we tested whether rhMG53 protein can improve membrane repair in a dysferlin-deficient mouse model of LGMD2B (B6.129-Dysftm1Kcam/J). We found that rhMG53 can increase the integrity of the sarcolemmal membrane of isolated muscle fibers and whole muscles in a Ca2+-independent fashion when assayed by a multi-photon laser wounding assay. Intraperitoneal injection of rhMG53 into mice before acute eccentric treadmill exercise can decrease the release of intracellular enzymes from skeletal muscle and decrease the entry of immunoglobulin G and Evans blue dye into muscle fibers in vivo. These results indicate that short-term rhMG53 treatment can ameliorate one of the underlying defects in dysferlin-deficient muscle by increasing sarcolemmal membrane integrity. We also provide evidence that rhMG53 protein increases membrane integrity independently of the canonical dysferlin-mediated, Ca2+-dependent pathway known to be important for sarcolemmal membrane repair.

Expression levels of sarcolemmal membrane repair proteins following prolonged exercise training in mice.

Membrane repair is a conserved cellular process, where intracellular vesicles translocate to sites of plasma membrane injury to actively reseal membrane disruptions. Such membrane disruptions commonly occur in the course of normal physiology, particularly in skeletal muscles due to repeated contraction producing small tears in the sarcolemmal membrane. Here, we investigated whether prolonged exercise could produce adaptive changes in expression levels of proteins associated with the membrane repair process, including mitsugumin 53/tripartite motif-containing protein 72 (MG53/TRIM72), dysferlin and caveolin-3 (cav3). Mice were exercised using a treadmill running protocol and protein levels were measured by immunoblotting. The specificity of the antibodies used was established by immunoblot testing of various tissue lysates from both mice and rats. We found that MG53/TRIM72 immunostaining on isolated mouse skeletal muscle fibers showed protein localization at sites of membrane disruption created by the isolation of these muscle fibers. However, no significant changes in the expression levels of the tested membrane repair proteins were observed following prolonged treadmill running for eight weeks (30 to 80 min/day). These findings suggest that any compensation occurring in the membrane repair process in skeletal muscle following prolonged exercise does not affect the expression levels of these three key membrane repair proteins.

R-spondin1, a novel intestinotrophic mitogen, ameliorates experimental colitis in mice.

BACKGROUND & AIMS: R-spondin 1 (Rspo1) is a novel epithelial mitogen that stimulates the growth of mucosa in both the small and large intestine. METHODS: We investigated the therapeutic potential of Rspo1 in ameliorating experimental colitis induced by dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) as well as nonsteroidal anti-inflammatory drug-induced colitis in interleukin (IL)-10-deficient mice. RESULTS: Therapeutic administration of recombinant Rspo1 protein reduced the loss of body weight, diarrhea, and rectal bleeding in a mouse model of acute or chronic DSS-induced colitis. Histologic evaluation revealed that Rspo1 improved mucosal integrity in both villus and/or crypt compartments in the small intestine and colon by stimulating crypt cell growth and mucosal regeneration in DSS-treated mice. Moreover, Rspo1 significantly reduced DSS-induced myeloperoxidase activity and inhibited the overproduction of proinflammatory cytokines, including tumor necrosis factor-alpha, IL-1alpha, IL-6, interferon-gamma, and granulocyte-macrophage colony-stimulating factor, in mouse intestinal tissue, indicating that Rspo1 may reduce DSS-induced inflammation by preserving the mucosal barrier function. Likewise, Rspo1 therapy also alleviated TNBS-induced interstitial inflammation and mucosal erosion in the mouse colon. Furthermore, Rspo1 substantially decreased the histopathologic severity of chronic enterocolitis by repairing crypt epithelium and simultaneously suppressing inflammatory infiltration in piroxicam-exposed IL-10(-/-) mice. Endogenous Rspo1 protein was localized to villus epithelium and crypt Paneth cells in mouse small intestine. CONCLUSIONS: Our results show that Rspo1 may be clinically useful in the therapeutic treatment of inflammatory bowel disease by stimulating crypt cell growth, accelerating mucosal regeneration, and restoring intestinal architecture.