Renzhi Han, PhD - US grants

DESCRIPTION (provided by applicant): Inflammatory response is associated with various muscular dystrophies and exacerbates the disease progression. In particular, dysferlin-deficient muscular dystrophy has been found to exhibit extensive muscle inflammation. Previous studies established a role of dysferlin in plasma membrane repair. We and other groups recently demonstrated that the innate immune system including the complement pathway and the NLRP3 (Nod-like receptor family, pyrin domain containing 3) inflammasome are activated in dysferlin-deficient muscle. However, the molecular mechanisms that initiate and perpetuate the immune activation are not well understood. The long-term goal of this research proposal is to understand the molecular mechanisms of muscle inflammation in dysferlin-deficient muscular dystrophy and explore the therapeutic potential of targeting the inflammatory signaling pathways in the treatment of this disease. Our pilot studies found that vesicles (in particular, the vesicle composed of charged lipids) strongly induce IL-1? secretion from macrophages through activation of the NLRP3 inflammasome. This observation led us to hypothesize that intracellular vesicles leaked out from dysferlin-deficient muscle activates the NLRP3 inflammasome, causing muscle inflammation. Our planned experiments will significantly advance understanding of the interplay between skeletal muscle with membrane repair defect and the immune system by measuring responses of macrophages to skeletal muscle-derived vesicles, manipulating expression of the NLRP3 inflammasome components and ex vivo and in vivo animal model studies. These data will begin to define potential therapeutic targets for regulation of inflammatory responses, thereby treating the diseases associated with defective membrane repair.

DESCRIPTION (provided by applicant): The plasma membrane integrity is of critical importance for cell homeostasis and function. Physical, chemical or metabolic disruption of the plasma membrane leads to a repair-or-die emergency in the cell. Thus, an efficient plasma membrane repair mechanism is essential for life since disruption of this process due to genetic mutations can result in a number of diseases including muscular dystrophy and associated cardiomyopathy. Previous studies from others and us demonstrated that the membrane repair response in cardiomyocytes is mediated by several proteins including dysferlin and MG53. However, the molecular mechanisms underlying this important physiological process have not been fully defined. Our preliminary data found that anoctamin 5 (Ano5) plays an essential role in membrane repair in myocytes. Ano5 belongs to the anoctamin protein family that includes at least ten proteins all possessing eight transmembrane domains with proved or putative calcium-activated chloride channel (CaCC) functions. Mutations in the ANO5 gene (encoding Ano5) lead to muscular dystrophies in human patients. However, there is little known about the molecular and cellular functions of Ano5 in cardiomyocytes and the molecular mechanisms underlying Ano5-mediated membrane repair remain poorly understood. The long-term goal of this research proposal is to understand the molecular and cellular mechanisms for Ano5 in heart physiology and disease. In pilot studies, we found that Ano5 is primarily localized on the endoplasmic/sarcoplasmic reticulum (ER/SR) and RNAi-silencing of Ano5 shows defective membrane repair in myocytes. Thus, our data present a new biological function for Ano5 in the cellular physiology of muscle cells. In this project, we will focus on testing the hypothesis that Ano5 is involved in the calcium-activated chloride channel (CaCC) activity and plays an essential role in plasma membrane repair of cardiomyocytes. Through manipulating expression of Ano5 and the use of live cell imaging, biochemical markers, ex vivo and in vivo animal model studies, our planned experiments will significantly advance understanding of the mechanisms underlying membrane repair of cardiomyocytes, and begin to define potential therapeutic targets for the regulation of membrane repair capacity to treat the diseases associated with abnormal membrane stability. Disrupted plasma membrane integrity underlies a number of diseases including cardiomyopathy. Our project is designed to understand the molecular and cellular functions of Ano5 in muscle physiology and disease. These studies will aid in defining therapeutic target for the treatment of treatment of heart diseases associated with compromised plasma membrane integrity through the regulation of Ano5-mediated membrane repair capacity.

PROJECT SUMMARY Mutations in ANO5 have been linked to several human diseases including muscular dystrophy. Ano5 is an intracellular membrane protein, belonging to the anoctamin protein family. Many of the proteins in this family have been found to possess the Ca2+-activated phospholipid scrambling activity. Despite the clear genetic linkage between ANO5 and muscular dystrophy in patients, we found that complete KO of Ano5 in mice showed no overt muscle pathology during our last funding period. This was independently confirmed by other investigators using a different line of complete Ano5-KO mice. These findings indicate that a potential compensatory mechanism, likely through other anoctamin proteins, is involved in minimizing the impact of complete Ano5 deficiency. Intriguingly, an Ano5-KO mouse expressing putatively a truncated Ano5 peptide developed clinical signs of muscular dystrophy with intracellular aggregates and defective membrane repair. Many of the ANO5 mutations associated with human muscular dystrophy are premature termination mutations. These findings raise an interesting question about how ANO5 mutations cause muscle degeneration in human patients: does the expression of mutant amino-terminal Ano5 peptide lead to muscular dystrophy by promoting the formation of intracellular aggregates and compromising membrane repair machinery? Our continuing research in this proposal is centered on determining the fundamental role of the amino-terminus of Ano5 in regulating the intrinsic lipid scrambling function of anoctamins proteins, membrane repair and its contribution to the pathogenesis of muscular dystrophy caused by ANO5 mutations. Moreover, our studies will reveal the compensatory mechanism underlying the lack of muscular dystrophy phenotype in complete Ano5-KO mice. Through the use of in vivo CRISPR gene editing, biochemical, histopathological, and living cell imaging studies with animal models, our planned experiments shall advance our understanding of the physiological and pathological roles of amino-terminal Ano5 peptides in muscle and also shed critical insights into the development of novel therapeutic strategies for the treatment of Ano5-related muscular dystrophy.

PROJECT SUMMARY/ABSTRACT Recent therapeutic advances considerably reduced the incidence of cardiovascular disease (CVD). The contribution of low-density (LDL) and very low density (VLDL) lipoproteins is critical in atherogenesis. Regardless the progress of clinical treatments in the past 30 years, for a significant proportion of statin-treated patients, with or without combination therapy, insufficient LDL-C reduction and relatively high residual risk still remain, likely associated with persistent relatively high triglyceride (TG) levels, thus limiting the benefits of these therapies. This underscores the need for additional new therapies targeting lipid metabolism in CVD prevention and treatment. In this proposal, we propose to develop genetic deficiency of ApoC3 through an adeno-associated virus (AAV) mediated in vivo silencing of ApoC3 by Cas9 base editor (Cas9-BE) strategy (AAV-Cas9-BE-ApoC3) to render protection against high cholesterol diet-induced hypercholesterolemia and atherosclerosis in rabbits. Specifically, we will 1) develop AAV-Cas9-BE-ApoC3 in a preclinical model species, the New Zealand White rabbits. We will determine the optimal targeting strategies using in vitro cultured rabbit cells, followed by experiments to determine the optimal delivery parameters to achieve effective ApoC3 gene knockout in rabbit hepatocytes; 2) evaluate the efficacy of AAV-Cas9-BE-ApoC3 using optimal conditions determined in Aim 1 to knockout ApoC3 in rabbits; 3) conduct multiple-year evaluation of the safety of AAV- Cas9-BE-ApoC3 in rabbits. Completion of these aims by leveraging new CRISPR/Cas9 technology to target ApoC3 will provide compelling evidence to establish ApoC3 as a novel feasible target for gene editing-based therapy for hyperlipidemia and CVD.

SUMMARY Skeletal muscle injury-repair and regeneration is a multi-cellular process that involves repair of acute injury to the sarcolemma, mobilization of satellite cells to replace the lost-muscle fibers, and control of fibrotic remodeling for maintenance of muscle integrity. In muscular dystrophy, compromised sarcolemma integrity or membrane repair triggers the cascade of muscle degeneration that incurs progressive, severe morbidity and ultimately mortality. Developing therapeutic approaches to improve sarcolemma integrity while facilitating regeneration of injured muscle fibers remain a major challenge in muscle physiology research. This project builds on the discovery of MG53, a member of the TRIM-family protein, as an essential component of the cell membrane repair machinery. MG53 functions in vesicle trafficking and facilitates the nucleation of intracellular vesicles to sites of membrane disruption for repair patch formation. Native MG53 is present in blood circulation, at levels directly correlating with injury or secretory activity of the muscle. Administration of recombinant human MG53 (rhMG53) protein protects muscle fibers and stem cells from injury, and reduces muscle fibrosis in the mdx mouse model. Our research with MG53 over the past few years has established a potential tri-functional role for MG53 in muscle injury-regeneration, as a facilitator to repair acute sarcolemma injury, a contributor to activate satellite cells during the early phase of muscle injury, and a modulator of fibrosis by controlling fibroblast differentiation associated with chronic muscle injury. We envision that targeting the tri-functional role of MG53 will have advantage over the current paradigms for treating muscular dystrophy. In Aim 1, we will determine the pathways that transduce the newly identified myokine function of MG53 into activation of satellite cells in response to acute muscle injury; define the mechanisms that underlie MG53?s function in regulating fibrosis during chronic muscle injury; and test if non-invasive interventions can modulate circulating MG53 levels toward muscle injury-regeneration. If circulating MG53 plays a role in satellite cell activation, we predict that ischemia-preconditioning that releases MG53 without muscle injury, or inducible secretion of MG53 from a transgenic mouse model, will effectively activate satellite cells and muscle regeneration following injury. In Aim 2, we will evaluate the safety and efficacy for sustained elevation of MG53 in circulation to preserve muscle integrity/satellite cell activation/fibrosis control in animal models of muscular dystrophy. Fulfillment of the studies in this project will advance the biology of MG53 in muscle injury-repair and regeneration, and lay the foundation for our translational approach for targeting MG53 function for treatment of muscular dystrophy.