GRAF1 Acts as a Downstream Mediator of Parkin to Regulate Mitophagy in Cardiomyocytes.

Cardiomyocytes rely on proper mitochondrial homeostasis to maintain contractility and achieve optimal cardiac performance. Mitochondrial homeostasis is controlled by mitochondrial fission, fusion, and mitochondrial autophagy (mitophagy). Mitophagy plays a particularly important role in promoting the degradation of dysfunctional mitochondria in terminally differentiated cells. However, the precise mechanisms by which this is achieved in cardiomyocytes remain opaque. Our study identifies GRAF1 as an important mediator in PINK1-Parkin pathway-dependent mitophagy. Depletion of GRAF1 (Arhgap26) in cardiomyocytes results in actin remodeling defects, suboptimal mitochondria clustering, and clearance. Mechanistically, GRAF1 promotes Parkin-LC3 complex formation and directs autophagosomes to damaged mitochondria. Herein, we found that these functions are regulated, at least in part, by the direct binding of GRAF1 to phosphoinositides (PI(3)P, PI(4)P, and PI(5)P) on autophagosomes. In addition, PINK1-dependent phosphorylation of Parkin promotes Parkin-GRAF1-LC3 complex formation, and PINK1-dependent phosphorylation of GRAF1 (on S668 and S671) facilitates the clustering and clearance of mitochondria. Herein, we developed new phosphor-specific antibodies to these sites and showed that these post-translational modifications are differentially modified in human hypertrophic cardiomyopathy and dilated cardiomyopathy. Furthermore, our metabolic studies using serum collected from isoproterenol-treated WT and GRAF1CKO mice revealed defects in mitophagy-dependent cardiomyocyte fuel flexibility that have widespread impacts on systemic metabolism. In summary, our study reveals that GRAF1 co-regulates actin and membrane dynamics to promote cardiomyocyte mitophagy and that dysregulation of GRAF1 post-translational modifications may underlie cardiac disease pathogenesis.

GRAF1 integrates PINK1-Parkin signaling and actin dynamics to mediate cardiac mitochondrial homeostasis.

The serine/threonine kinase, PINK1, and the E3 ubiquitin ligase, Parkin, are known to facilitate LC3-dependent autophagosomal encasement and lysosomal clearance of dysfunctional mitochondria, and defects in this process contribute to a variety of cardiometabolic and neurological diseases. Although recent evidence indicates that dynamic actin remodeling plays an important role in PINK1/Parkin-mediated mitochondrial autophagy (mitophagy), the underlying signaling mechanisms remain unknown. Here, we identify the RhoGAP GRAF1 (Arhgap26) as a PINK1 substrate that regulates mitophagy. GRAF1 promotes the release of damaged mitochondria from F-actin anchors, regulates mitochondrial-associated Arp2/3-mediated actin remodeling and facilitates Parkin-LC3 interactions to enhance mitochondria capture by autophagosomes. Graf1 phosphorylation on PINK1-dependent sites is dysregulated in human heart failure, and cardiomyocyte-restricted Graf1 depletion in mice blunts mitochondrial clearance and attenuates compensatory metabolic adaptations to stress. Overall, we identify GRAF1 as an enzyme that coordinates cytoskeletal and metabolic remodeling to promote cardioprotection.

Prdm6 drives ductus arteriosus closure by promoting ductus arteriosus smooth muscle cell identity and contractility.

Based upon our demonstration that the smooth muscle cell-selective (SMC-selective) putative methyltransferase, Prdm6, interacts with myocardin-related transcription factor-A, we examined Prdm6's role in SMCs in vivo using cell type-specific knockout mouse models. Although SMC-specific depletion of Prdm6 in adult mice was well tolerated, Prdm6 depletion in Wnt1-expressing cells during development resulted in perinatal lethality and a completely penetrant patent ductus arteriosus (DA) phenotype. Lineage tracing experiments in Wnt1Cre2 Prdm6fl/fl ROSA26LacZ mice revealed normal neural crest-derived SMC investment of the outflow tract. In contrast, myography measurements on DA segments isolated from E18.5 embryos indicated that Prdm6 depletion significantly reduced DA tone and contractility. RNA-Seq analyses on DA and ascending aorta samples at E18.5 identified a DA-enriched gene program that included many SMC-selective contractile associated proteins that was downregulated by Prdm6 depletion. Chromatin immunoprecipitation-sequencing experiments in outflow tract SMCs demonstrated that 50% of the genes Prdm6 depletion altered contained Prdm6 binding sites. Finally, using several genome-wide data sets, we identified an SMC-selective enhancer within the Prdm6 third intron that exhibited allele-specific activity, providing evidence that rs17149944 may be the causal SNP for a cardiovascular disease GWAS locus identified within the human PRDM6 gene.

GRAF1 Regulates Brown and Beige Adipose Differentiation and Function.

Adipose tissue, which is crucial for the regulation of energy within the body, contains both white and brown adipocytes. White adipose tissue (WAT) primarily stores energy, while brown adipose tissue (BAT) plays a critical role in energy dissipation as heat, offering potential for therapies aimed at enhancing metabolic health. Regulation of the RhoA/ROCK pathway is crucial for appropriate specification, differentiation and maturation of both white and brown adipocytes. However, our knowledge of how this pathway is controlled within specific adipose depots remains unclear, and to date a RhoA regulator that selectively controls adipocyte browning has not been identified. Our study shows that expression of GRAF1, a RhoGAP highly expressed in metabolically active tissues, closely correlates with brown adipocyte differentiation in culture and in vivo. Mice with either global or adipocyte-specific GRAF1 deficiency exhibit impaired BAT maturation, reduced capacity for WAT browning, and compromised cold-induced thermogenesis. Moreover, defects in differentiation of mouse or human GRAF1-deficient brown preadipocytes can be rescued by treatment with a Rho kinase inhibitor. Collectively, these studies indicate that GRAF1 can selectively induce brown and beige adipocyte differentiation and suggest that manipulating GRAF1 activity may hold promise for the future treatment of diseases related to metabolic dysfunction.

Stretching muscle cells induces transcriptional and splicing transitions and changes in SR proteins.

Alternative splicing is an RNA processing mechanism involved in skeletal muscle development and pathology. Muscular diseases exhibit splicing alterations and changes in mechanobiology leading us to investigate the interconnection between mechanical forces and RNA processing. We performed deep RNA-sequencing after stretching muscle cells. First, we uncovered transcriptional changes in genes encoding proteins involved in muscle function and transcription. Second, we observed that numerous mechanosensitive genes were part of the MAPK pathway which was activated in response to stretching. Third, we revealed that stretching skeletal muscle cells increased the proportion of alternatively spliced cassette exons and their inclusion. Fourth, we demonstrated that the serine and arginine-rich proteins exhibited stronger transcriptional changes than other RNA-binding proteins and that SRSF4 phosphorylation is mechanosensitive. Identifying SRSF4 as a mechanosensitive RNA-binding protein that might contribute to crosstalk between mechanotransduction, transcription, and splicing could potentially reveal novel insights into muscular diseases, particularly those with unknown etiologies.

Exerkines in health, resilience and disease.

The health benefits of exercise are well-recognized and are observed across multiple organ systems. These beneficial effects enhance overall resilience, healthspan and longevity. The molecular mechanisms that underlie the beneficial effects of exercise, however, remain poorly understood. Since the discovery in 2000 that muscle contraction releases IL-6, the number of exercise-associated signalling molecules that have been identified has multiplied. Exerkines are defined as signalling moieties released in response to acute and/or chronic exercise, which exert their effects through endocrine, paracrine and/or autocrine pathways. A multitude of organs, cells and tissues release these factors, including skeletal muscle (myokines), the heart (cardiokines), liver (hepatokines), white adipose tissue (adipokines), brown adipose tissue (baptokines) and neurons (neurokines). Exerkines have potential roles in improving cardiovascular, metabolic, immune and neurological health. As such, exerkines have potential for the treatment of cardiovascular disease, type 2 diabetes mellitus and obesity, and possibly in the facilitation of healthy ageing. This Review summarizes the importance and current state of exerkine research, prevailing challenges and future directions.

The actin depolymerizing factor destrin serves as a negative feedback inhibitor of smooth muscle cell differentiation.

We have previously shown that several components of the RhoA signaling pathway control smooth muscle cell (SMC) phenotype by altering serum response factor (SRF)-dependent gene expression. Because our genome-wide analyses of chromatin structure and transcription factor binding suggested that the actin depolymerizing factor, destrin (DSTN), was regulated in a SMC-selective fashion, the goals of the current study were to identify the transcription mechanisms that control DSTN expression in SMC and to test whether it regulates SMC function. Immunohistochemical analyses revealed strong and at least partially SMC-selective expression of DSTN in many mouse tissues, a result consistent with human data from the genotype-tissue expression (GTEx) consortium. We identified several regulatory regions that control DSTN expression including a SMC-selective enhancer that was activated by myocardin-related transcription factor-A (MRTF-A), recombination signal binding protein for immunoglobulin κ-J region (RBPJ), and the SMAD transcription factors. Indeed, enhancer activity and endogenous DSTN expression were upregulated by RhoA and transforming growth factor-ß (TGF-ß) signaling and downregulated by inhibition of Notch cleavage. We also showed that DSTN expression was decreased in vivo by carotid artery injury and in cultured SMC cells by platelet-derived growth factor-BB (PDGF-BB) treatment. siRNA-mediated depletion of DSTN significantly enhanced MRTF-A nuclear localization and SMC differentiation marker gene expression, decreased SMC migration in scratch wound assays, and decreased SMC proliferation, as measured by cell number and cyclin-E expression. Taken together our data indicate that DSTN is a negative feedback inhibitor of RhoA/SRF-dependent gene expression in SMC that coordinately promotes SMC phenotypic modulation. Interventions that target DSTN expression or activity could serve as potential therapies for atherosclerosis and restenosis.NEW & NOTEWORTHY First, DSTN is selectively expressed in SMC in RhoA/SRF-dependent manner. Second, a SMC-selective enhancer just upstream of DSTN TSS harbors functional SRF, SMAD, and Notch/RBPJ binding elements. Third, DSTN depletion increased SRF-dependent SMC marker gene expression while inhibiting SMC migration and proliferation. Taken together, our data suggest that DSTN is a critical negative feedback inhibitor of SMC differentiation.

A homozygous stop-gain variant in ARHGAP42 is associated with childhood interstitial lung disease, systemic hypertension, and immunological findings.

ARHGAP42 encodes Rho GTPase activating protein 42 that belongs to a member of the GTPase Regulator Associated with Focal Adhesion Kinase (GRAF) family. ARHGAP42 is involved in blood pressure control by regulating vascular tone. Despite these findings, disorders of human variants in the coding part of ARHGAP42 have not been reported. Here, we describe an 8-year-old girl with childhood interstitial lung disease (chILD), systemic hypertension, and immunological findings who carries a homozygous stop-gain variant (c.469G>T, p.(Glu157Ter)) in the ARHGAP42 gene. The family history is notable for both parents with hypertension. Histopathological examination of the proband lung biopsy showed increased mural smooth muscle in small airways and alveolar septa, and concentric medial hypertrophy in pulmonary arteries. ARHGAP42 stop-gain variant in the proband leads to exon 5 skipping, and reduced ARHGAP42 levels, which was associated with enhanced RhoA and Cdc42 expression. This is the first report linking a homozygous stop-gain variant in ARHGAP42 with a chILD disorder, systemic hypertension, and immunological findings in human patient. Evidence of smooth muscle hypertrophy on lung biopsy and an increase in RhoA/ROCK signaling in patient cells suggests the potential mechanistic link between ARHGAP42 deficiency and the development of chILD disorder.

Cyanocobalamin prevents cardiomyopathy in type 1 diabetes by modulating oxidative stress and DNMT-SOCS1/3-IGF-1 signaling.

Patients with long-standing diabetes have a high risk for cardiac complications that is exacerbated by increased reactive oxygen species (ROS) production. We found that feeding cyanocobalamin (B12), a scavenger of superoxide, not only prevented but reversed signs of cardiomyopathy in type 1 diabetic Elmo1H/H Ins2Akita/+ mice. ROS reductions in plasma and hearts were comparable to those in mice treated with other antioxidants, N-acetyl-L-cysteine or tempol, but B12 produced better cardioprotective effects. Diabetes markedly decreased plasma insulin-like growth factor (IGF)-1 levels, while B12, but not N-acetyl-L-cysteine nor tempol, restored them. B12 activated hepatic IGF-1 production via normalization of S-adenosylmethionine levels, DNA methyltransferase (DNMT)-1/3a/3b mRNA, and DNA methylation of promoters for suppressor of cytokine signaling (SOCS)-1/3. Reductions of cardiac IGF-1 mRNA and phosphorylated IGF-1 receptors were also restored. Thus, B12 is a promising option for preventing diabetic cardiomyopathy via ROS reduction and IGF-1 retrieval through DNMT-SOCS1/3 signaling.

Down-regulation of Beclin1 promotes direct cardiac reprogramming.

Direct reprogramming of fibroblasts to alternative cell fates by forced expression of transcription factors offers a platform to explore fundamental molecular events governing cell fate identity. The discovery and study of induced cardiomyocytes (iCMs) not only provides alternative therapeutic strategies for heart disease but also sheds lights on basic biology underlying CM fate determination. The iCM field has primarily focused on early transcriptome and epigenome repatterning, whereas little is known about how reprogramming iCMs remodel, erase, and exit the initial fibroblast lineage to acquire final cell identity. Here, we show that autophagy-related 5 (Atg5)-dependent autophagy, an evolutionarily conserved self-digestion process, was induced and required for iCM reprogramming. Unexpectedly, the autophagic factor Beclin1 (Becn1) was found to suppress iCM induction in an autophagy-independent manner. Depletion of Becn1 resulted in improved iCM induction from both murine and human fibroblasts. In a mouse genetic model, Becn1 haploinsufficiency further enhanced reprogramming factor-mediated heart function recovery and scar size reduction after myocardial infarction. Mechanistically, loss of Becn1 up-regulated Lef1 and down-regulated Wnt inhibitors, leading to activation of the canonical Wnt/ß-catenin signaling pathway. In addition, Becn1 physically interacts with other classical class III phosphatidylinositol 3-kinase (PI3K III) complex components, the knockdown of which phenocopied Becn1 depletion in cardiac reprogramming. Collectively, our study revealed an inductive role of Atg5-dependent autophagy as well as a previously unrecognized autophagy-independent inhibitory function of Becn1 in iCM reprogramming.

Molecular Regulation of the RhoGAP GRAF3 and Its Capacity to Limit Blood Pressure In Vivo.

Anti-hypertensive therapies are usually prescribed empirically and are often ineffective. Given the prevalence and deleterious outcomes of hypertension (HTN), improved strategies are needed. We reported that the Rho-GAP GRAF3 is selectively expressed in smooth muscle cells (SMC) and controls blood pressure (BP) by limiting the RhoA-dependent contractility of resistance arterioles. Importantly, genetic variants at the GRAF3 locus controls BP in patients. The goal of this study was to validate GRAF3 as a druggable candidate for future anti-HTN therapies. Importantly, using a novel mouse model, we found that modest induction of GRAF3 in SMC significantly decreased basal and vasoconstrictor-induced BP. Moreover, we found that GRAF3 protein toggles between inactive and active states by processes controlled by the mechano-sensing kinase, focal adhesion kinase (FAK). Using resonance energy transfer methods, we showed that agonist-induced FAK-dependent phosphorylation at Y376GRAF3 reverses an auto-inhibitory interaction between the GAP and BAR-PH domains. Y376 is located in a linker between the PH and GAP domains and is invariant in GRAF3 homologues and a phosphomimetic E376GRAF3 variant exhibited elevated GAP activity. Collectively, these data provide strong support for the future identification of allosteric activators of GRAF3 for targeted anti-hypertensive therapies.

GRAF3 serves as a blood volume-sensitive rheostat to control smooth muscle contractility and blood pressure.

Vascular resistance is a major determinant of BP and is controlled, in large part, by RhoA-dependent smooth muscle cell (SMC) contraction within small peripheral arterioles and previous studies from our lab indicate that GRAF3 is a critical regulator of RhoA in vascular SMC. The elevated contractile responses we observed in GRAF3 deficient vessels coupled with the hypertensive phenotype provided a mechanistic link for the hypertensive locus recently identified within the GRAF3 gene. On the basis of our previous findings that the RhoA signaling axis also controls SMC contractile gene expression and that GRAF3 expression was itself controlled by this pathway, we postulated that GRAF3 serves as an important counter-regulator of SMC phenotype. Indeed, our new findings presented herein indicate that GRAF3 expression acts as a pressure-sensitive rheostat to control vessel tone by both reducing calcium sensitivity and restraining expression of the SMC-specific contractile proteins that support this function. Collectively, these studies highlight the potential therapeutic value of GRAF3 in the control of human hypertension.

Transcriptional and posttranscriptional regulation of the SMC-selective blood pressure-associated gene, ARHGAP42.

We previously showed that ARHGAP42 is a smooth muscle cell (SMC)-selective, RhoA-specific GTPase activating protein that regulates blood pressure and that a minor allele single nucleotide variation within a DNAse hypersensitive regulatory element in intron1 (Int1DHS) increased ARHGAP42 expression by promoting serum response factor binding. The goal of the current study was to identify additional transcriptional and posttranscriptional mechanisms that control ARHGAP42 expression. Using deletion/mutation, gel shift, and chromatin immunoprecipitation experiments, we showed that recombination signal binding protein for immunoglobulin κ-J region (RBPJ) and TEA domain family member 1 (TEAD1) binding to a conserved core region was required for full IntDHS transcriptional activity. Importantly, overexpression of the notch intracellular domain (NICD) or plating SMCs on recombinant jagged-1 increased IntDHS activity and endogenous ARHGAP42 expression while siRNA-mediated knockdown of TEAD1 inhibited ARHGAP42 mRNA levels. Re-chromatin immunoprecipitation experiments indicated that RBPJ and TEAD1 were bound to the Int1DHS enhancer at the same time, and coimmunoprecipitation assays indicated that these factors interacted physically. Our results also suggest TEAD1 and RBPJ bound cooperatively to the Int1DHS and that the presence of TEAD1 promoted the recruitment of NICD by RBPJ. Finally, we showed that ARHGAP42 expression was inhibited by micro-RNA 505 (miR505) which interacted with the ARHGAP42 3'-untranslated region (UTR) to facilitate its degradation and by AK124326, a long noncoding RNA that overlaps with the ARHGAP42 transcription start site on the opposite DNA strand. Since siRNA-mediated depletion of AK124326 was associated with increased H3K9 acetylation and RNA Pol-II binding at the ARHGAP42 gene, it is likely that AK124326 inhibits ARHGAP42 transcription.NEW & NOTEWORTHY First, RBPJ and TEAD1 converge at an intronic enhancer to regulate ARHGAP42 expression in SMCs. Second, TEAD1 and RBPJ interact physically and bind cooperatively to the ARHGAP42 enhancer. Third, miR505 interacts with the ARHGAP42 3'-UTR to facilitate its degradation. Finally, LncRNA, AK124326, inhibits ARHGAP42 transcription.

Engulfment and cell motility protein 1 potentiates diabetic cardiomyopathy via Rac-dependent and Rac-independent ROS production.

Engulfment and cell motility protein 1 (ELMO1) is part of a guanine nucleotide exchange factor for Ras-related C3 botulinum toxin substrate (Rac), and ELMO1 polymorphisms were identified to be associated with diabetic nephropathy in genome-wide association studies. We generated a set of Akita Ins2C96Y diabetic mice having 5 graded cardiac mRNA levels of ELMO1 from 30% to 200% of normal and found that severe dilated cardiomyopathy develops in ELMO1-hypermorphic mice independent of renal function at age 16 weeks, whereas ELMO1-hypomorphic mice were completely protected. As ELMO1 expression increased, reactive oxygen species indicators, dissociation of the intercalated disc, mitochondrial fragmentation/dysfunction, cleaved caspase-3 levels, and actin polymerization increased in hearts from Akita mice. Cardiomyocyte-specific overexpression in otherwise ELMO1-hypomorphic Akita mice was sufficient to promote cardiomyopathy. Cardiac Rac1 activity was positively correlated with the ELMO1 levels, and oral administration of a pan-Rac inhibitor, EHT1864, partially mitigated cardiomyopathy of the ELMO1 hypermorphs. Disrupting Nox4, a Rac-independent NADPH oxidase, also partially mitigated it. In contrast, a pan-NADPH oxidase inhibitor, VAS3947, markedly prevented cardiomyopathy. Our data demonstrate that in diabetes mellitus ELMO1 is the "rate-limiting" factor of reactive oxygen species production via both Rac-dependent and Rac-independent NADPH oxidases, which in turn trigger cellular signaling cascades toward cardiomyopathy.

Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-Mediated Cyclin D1 Transcription.

RATIONALE: Neointimal hyperplasia is characterized by excessive accumulation of vascular smooth muscle cells (SMCs) leading to occlusive disorders, such as atherosclerosis and stenosis. Blood vessel injury increases growth factor secretion and matrix synthesis, which promotes SMC proliferation and neointimal hyperplasia via FAK (focal adhesion kinase). OBJECTIVE: To understand the mechanism of FAK action in SMC proliferation and neointimal hyperplasia. METHODS AND RESULTS: Using combined pharmacological FAK catalytic inhibition (VS-4718) and SMC-specific FAK kinase-dead (Myh11-Cre-ERT2) mouse models, we report that FAK regulates SMC proliferation and neointimal hyperplasia in part by governing GATA4- (GATA-binding protein 4) cyclin D1 signaling. Inhibition of FAK catalytic activity facilitates FAK nuclear localization, which is required for proteasome-mediated GATA4 degradation in the cytoplasm. Chromatin immunoprecipitation identified GATA4 binding to the mouse cyclin D1 promoter, and loss of GATA4-mediated cyclin D1 transcription diminished SMC proliferation. Stimulation with platelet-derived growth factor or serum activated FAK and redistributed FAK from the nucleus to cytoplasm, leading to concomitant increase in GATA4 protein and cyclin D1 expression. In a femoral artery wire injury model, increased neointimal hyperplasia was observed in parallel with elevated FAK activity, GATA4 and cyclin D1 expression following injury in control mice, but not in VS-4718-treated and SMC-specific FAK kinase-dead mice. Finally, lentiviral shGATA4 knockdown in the wire injury significantly reduced cyclin D1 expression, SMC proliferation, and neointimal hyperplasia compared with control mice. CONCLUSIONS: Nuclear enrichment of FAK by inhibition of FAK catalytic activity during vessel injury blocks SMC proliferation and neointimal hyperplasia through regulation of GATA4-mediated cyclin D1 transcription.

Lin28a Regulates Pathological Cardiac Hypertrophic Growth Through Pck2-Mediated Enhancement of Anabolic Synthesis.

BACKGROUND: Hypertrophic response to pathological stimuli is a complex biological process that involves transcriptional and epigenetic regulation of the cardiac transcriptome. Although previous studies have implicated transcriptional factors and signaling molecules in pathological hypertrophy, the role of RNA-binding protein in this process has received little attention. METHODS: Here we used transverse aortic constriction and in vitro cardiac hypertrophy models to characterize the role of an evolutionary conserved RNA-binding protein Lin28a in pathological cardiac hypertrophy. Next-generation sequencing, RNA immunoprecipitation, and gene expression analyses were applied to identify the downstream targets of Lin28a. Epistatic analysis, metabolic assays, and flux analysis were further used to characterize the effects of Lin28a and its downstream mediator in cardiomyocyte hypertrophic growth and metabolic remodeling. RESULTS: Cardiac-specific deletion of Lin28a attenuated pressure overload-induced hypertrophic growth, cardiac dysfunction, and alterations in cardiac transcriptome. Mechanistically, Lin28a directly bound to mitochondrial phosphoenolpyruvate carboxykinase 2 ( Pck2) mRNA and increased its transcript level. Increasing Pck2 was sufficient to promote hypertrophic growth similar to that caused by increasing Lin28a, whereas knocking down Pck2 attenuated norepinephrine-induced cardiac hypertrophy. Epistatic analysis demonstrated that Pck2 mediated, at least in part, the role of Lin28a in cardiac hypertrophic growth. Furthermore, metabolomic analyses highlighted the role for Lin28a and Pck2 in promoting cardiac biosynthesis required for cell growth. CONCLUSIONS: Our study demonstrates that Lin28a promotes pathological cardiac hypertrophy and glycolytic reprograming, at least in part, by binding to and stabilizing Pck2 mRNA.

Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis.

Regulation of cellular death is central to nearly all physiological routines and is dysregulated in virtually all diseases. Cell death occurs by two major processes, necrosis which culminates in a pervasive inflammatory response and apoptosis which is largely immunologically inert. As necrosis has long been considered an accidental, unregulated form of cellular death that occurred in response to a harsh environmental stimulus, it was largely ignored as a clinical target. However, recent elegant studies suggest that certain forms of necrosis can be reprogrammed. However, scant little is known about the molecules and pathways that orchestrate calcium-overload-induced necrosis, a main mediator of ischemia/reperfusion (IR)-induced cardiomyocyte cell death. To rectify this critical gap in our knowledge, we performed a novel genome-wide siRNA screen to identify modulators of calcium-induced necrosis in human muscle cells. Our screen identified multiple molecular circuitries that either enhance or inhibit this process, including lysosomal calcium channel TPCN1, mitophagy mediatorTOMM7, Ran-binding protein RanBP9, Histone deacetylase HDAC2, chemokine CCL11, and the Arp2/3 complex regulator glia maturation factor-γ (GMFG). Notably, a number of druggable enzymes were identified, including the proteasome ß5 subunit (encoded by PSMB5 gene), which controls the proteasomal chymotrypsin-like peptidase activity. Such findings open up the possibility for the discovery of pharmacological interventions that could provide therapeutic benefits to patients affected by myriad disorders characterized by excessive (or too little) necrotic cell loss, including but not limited to IR injury in the heart and kidney, chronic neurodegenerative disorders, muscular dystrophies, sepsis, and cancers.

Druggable targets in the Rho pathway and their promise for therapeutic control of blood pressure.

The prevalence of high blood pressure (also known as hypertension) has steadily increased over the last few decades. Known as a silent killer, hypertension increases the risk for cardiovascular disease and can lead to stroke, heart attack, kidney failure and associated sequela. While numerous hypertensive therapies are currently available, it is estimated that only half of medicated patients exhibit blood pressure control. This signifies the need for a better understanding of the underlying cause of disease and for more effective therapies. While blood pressure homeostasis is very complex and involves the integrated control of multiple body systems, smooth muscle contractility and arterial resistance are important contributors. Strong evidence from pre-clinical animal models and genome-wide association studies indicate that smooth muscle contraction and BP homeostasis are governed by the small GTPase RhoA and its downstream target, Rho kinase. In this review, we summarize the signaling pathways and regulators that impart tight spatial-temporal control of RhoA activity in smooth muscle cells and discuss current therapeutic strategies to target these RhoA pathway components. We also discuss known allelic variations in the RhoA pathway and consider how these polymorphisms may affect genetic risk for hypertension and its clinical manifestations.