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Background: Excitation-contraction (E-C) coupling processes become disrupted in heart failure (HF), resulting in abnormal Ca 2+ homeostasis, maladaptive structural and transcriptional remodeling, and cardiac dysfunction. Junctophilin-2 (JP2) is an essential component of the E-C coupling apparatus but becomes site-specifically cleaved by calpain, leading to disruption of E-C coupling, plasmalemmal transverse tubule degeneration, abnormal Ca 2+ homeostasis, and HF. However, it is not clear whether preventing site-specific calpain cleavage of JP2 is sufficient to protect the heart against stress-induced pathological cardiac remodeling in vivo., Methods: Calpain-resistant JP2 knock-in mice (JP2 CR ) were generated by deleting the primary JP2 calpain cleavage site. Stress-dependent JP2 cleavage was assessed through in vitro cleavage assays and in isolated cardiomyocytes treated with 1 μmol/L isoproterenol by immunofluorescence. Cardiac outcomes were assessed in wild-type and JP2 CR mice 5 weeks after transverse aortic constriction compared with sham surgery using echocardiography, histology, and RNA-sequencing methods. E-C coupling efficiency was measured by in situ confocal microscopy. E-C coupling proteins were evaluated by calpain assays and Western blotting. The effectiveness of adeno-associated virus gene therapy with JP2 CR , JP2, or green fluorescent protein to slow HF progression was evaluated in mice with established cardiac dysfunction., Results: JP2 proteolysis by calpain and in response to transverse aortic constriction and isoproterenol was blocked in JP2 CR cardiomyocytes. JP2 CR hearts are more resistant to pressure-overload stress, having significantly improved Ca 2+ homeostasis and transverse tubule organization with significantly attenuated cardiac dysfunction, hypertrophy, lung edema, fibrosis, and gene expression changes relative to wild-type mice. JP2 CR preserves the integrity of calpain-sensitive E-C coupling-related proteins, including ryanodine receptor 2, Ca V 1.2, and sarcoplasmic reticulum calcium ATPase 2a, by attenuating transverse aortic constriction-induced increases in calpain activity. Furthermore, JP2 CR gene therapy after the onset of cardiac dysfunction was found to be effective at slowing the progression of HF and superior to wild-type JP2., Conclusions: The data presented here demonstrate that preserving JP2-dependent E-C coupling by prohibiting the site-specific calpain cleavage of JP2 offers multifaceted beneficial effects, conferring cardiac protection against stress-induced proteolysis, hypertrophy, and HF. Our data also indicate that specifically targeting the primary calpain cleavage site of JP2 by gene therapy approaches holds great therapeutic potential as a novel precision medicine for treating HF.

Background: Preeclampsia is a serious disease of pregnancy that lacks early diagnosis methods or effective treatment, except delivery. Dysregulated uterine immune cells and spiral arteries are implicated in preeclampsia, but the mechanistic link remains unclear., Methods: Single-cell RNA sequencing and spatial transcriptomics were used to identify immune cell subsets associated with preeclampsia. Cell-based studies and animal models including conditional knockout mice and a new preeclampsia mouse model induced by recombinant mouse galectin-9 were applied to validate the pathogenic role of a CD11c high subpopulation of decidual macrophages (dMφ) and to determine its underlying regulatory mechanisms in preeclampsia. A retrospective preeclampsia cohort study was performed to determine the value of circulating galectin-9 in predicting preeclampsia., Results: We discovered a distinct CD11c high dMφ subset that inhibits spiral artery remodeling in preeclampsia. The proinflammatory CD11c high dMφ exhibits perivascular enrichment in the decidua from patients with preeclampsia. We also showed that trophoblast-derived galectin-9 activates CD11c high dMφ by means of CD44 binding to suppress spiral artery remodeling. In 3 independent preeclampsia mouse models, placental and plasma galectin-9 levels were elevated. Galectin-9 administration in mice induces preeclampsia-like phenotypes with increased CD11c high dMφ and defective spiral arteries, whereas galectin-9 blockade or macrophage-specific CD44 deletion prevents such phenotypes. In pregnant women, increased circulating galectin-9 levels in the first trimester and at 16 to 20 gestational weeks can predict subsequent preeclampsia onset., Conclusions: These findings highlight a key role of a distinct perivascular inflammatory CD11c high dMφ subpopulation in the pathogenesis of preeclampsia. CD11c high dMφ activated by increased galectin-9 from trophoblasts suppresses uterine spiral artery remodeling, contributing to preeclampsia. Increased circulating galectin-9 may be a biomarker for preeclampsia prediction and intervention., Competing Interests: Disclosures None.

Background: Progressive remodeling of cardiac gene expression underlies decline in cardiac function, eventually leading to heart failure. However, the major determinants of transcriptional network switching from normal to failed hearts remain to be determined., Methods: In this study, we integrated human samples, genetic mouse models, and genomic approaches, including bulk RNA sequencing, single-cell RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, to identify the role of chromatin remodeling complex INO80 in heart homeostasis and dysfunction., Results: The INO80 chromatin remodeling complex was abundantly expressed in mature cardiomyocytes, and its expression further increased in mouse and human heart failure. Cardiomyocyte-specific overexpression of Ino80 , its core catalytic subunit, induced heart failure within 4 days. Combining RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, we revealed INO80 overexpression-dependent reshaping of the nucleosomal landscape that remodeled a core set of transcription factors, most notably the MEF2 (Myocyte Enhancer Factor 2) family, whose target genes were closely associated with cardiac function. Conditional cardiomyocyte-specific deletion of Ino80 in an established mouse model of heart failure demonstrated remarkable preservation of cardiac function., Conclusions: In summary, our findings shed light on the INO80-dependent remodeling of the chromatin landscape and transcriptional networks as a major mechanism underlying cardiac dysfunction in heart failure, and suggest INO80 as a potential preventative or interventional target., Competing Interests: Disclosures None.

Background: Vein graft occlusion is deemed a major challenge in coronary artery bypass grafting. Previous studies implied that the no-touch technique for vein graft harvesting could reduce occlusion rate compared with the conventional approach; however, evidence on the clinical benefit and generalizability of the no-touch technique is scare., Methods: From April 2017 to June 2019, we randomly assigned 2655 patients undergoing coronary artery bypass grafting at 7 hospitals in a 1:1 ratio to receive no-touch technique or conventional approach for vein harvesting. The primary outcome was vein graft occlusion on computed tomography angiography at 3 months and the secondary outcomes included 12-month vein graft occlusion, recurrence of angina, and major adverse cardiac and cerebrovascular events. The generalized estimate equation model was used to account for the cluster effect of grafts from the same patient., Results: During the follow-up, 2533 (96.0%) participants received computed tomography angiography at 3 months after coronary artery bypass grafting and 2434 (92.2%) received it at 12 months. The no-touch group had significantly lower rates of vein graft occlusion than the conventional group both at 3 months (2.8% versus 4.8%; odds ratio, 0.57 [95% CI, 0.41-0.80]; P <0.001) and 12 months (3.7% versus 6.5%; odds ratio, 0.56 [95% CI, 0.41-0.76]; P <0.001). Recurrence of angina was also less common in the no-touch group at 12 months (2.3% versus 4.1%; odds ratio, 0.55 [95% CI, 0.35-0.85]; P <0.01). Rates of major adverse cardiac and cerebrovascular events were of no significant difference between the 2 groups. The no-touch technique was associated with higher rates of leg wound surgical interventions at 3-month follow-up (10.3% versus 4.3%; odds ratio, 2.55 [95% CI, 1.85-3.52]; P <0.001)., Conclusions: Compared with the conventional vein harvesting approach in coronary artery bypass grafting, the no-touch technique significantly reduced the risk of vein graft occlusion and improved patient prognosis. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03126409.

Background: G protein-coupled receptors are important regulators of contractility and differentiation in vascular smooth muscle cells (SMCs), but the specific function of SMC-expressed orphan G protein-coupled receptor class C group 5 member B (GPRC5B) is unclear., Methods: We studied the role of GPRC5B in the regulation of contractility and dedifferentiation in human and murine SMCs in vitro and in iSM- Gprc5b -KO (tamoxifen-inducible, SMC-specific knockout) mice under conditions of arterial hypertension and atherosclerosis in vivo., Results: Mesenteric arteries from SMC-specific Gprc5b -KOs showed ex vivo significantly enhanced prostacyclin receptor (IP)-dependent relaxation, whereas responses to other relaxant or contractile factors were normal. In vitro, knockdown of GPRC5B in human aortic SMCs resulted in increased IP-dependent cAMP production and consecutive facilitation of SMC relaxation. In line with this facilitation of IP-mediated relaxation, iSM- Gprc5b -KO mice were protected from arterial hypertension, and this protective effect was abrogated by IP antagonists. Mechanistically, we show that knockdown of GPRC5B increased the membrane localization of IP both in vitro and in vivo and that GPRC5B, but not other G protein-coupled receptors, physically interacts with IP. Last, we show that enhanced IP signaling in GPRC5B-deficient SMCs not only facilitates relaxation but also prevents dedifferentiation during atherosclerosis development, resulting in reduced plaque load and increased differentiation of SMCs in the fibrous cap., Conclusions: Taken together, our data show that GPRC5B regulates vascular SMC tone and differentiation by negatively regulating IP signaling.

Background: Ceramides have been implicated in the pathophysiology of HIV infection and cardiovascular disease. However, no study, to our knowledge, has evaluated circulating ceramide levels in association with subclinical cardiovascular disease risk among HIV-infected individuals., Methods: Plasma levels of 4 ceramide species (C16:0, C22:0, C24:0, and C24:1) were measured among 398 women (73% HIV+) and 339 men (68% HIV+) without carotid artery plaques at baseline from the Women's Interagency HIV Study and the Multicenter AIDS Cohort Study. We examined associations between baseline plasma ceramides and risk of carotid artery plaque formation, assessed by repeated B-mode carotid artery ultrasound imaging over a median 7-year follow-up., Results: Plasma levels of C16:0, C22:0, and C24:1 ceramides were significantly higher in HIV-infected individuals compared with those without HIV infection (all P<0.001), and further analysis indicated that elevated ceramide levels were associated with antiretroviral therapy use, particularly protease inhibitor use, in HIV-infected individuals (all P<0.001). All 4 ceramides were highly correlated with each other ( r=0.70-0.94; all P<0.001) and significantly correlated with total-cholesterol ( r=0.42-0.58; all P<0.001) and low-density lipoprotein cholesterol ( r=0.24-0.42; all P<0.001) levels. Of note, C16:0 and C24:1 ceramides, rather than C22:0 and C24:0 ceramides, were more closely correlated with specific monocyte activation and inflammation markers (eg, r=0.30 between C16:0 ceramide and soluble CD14; P<0.001) and surface markers of CD4 + T-cell activation. A total of 112 participants developed carotid artery plaques over 7 years, and higher levels of C16:0 and C24:1 ceramides were significantly associated with increased risk of carotid artery plaques (relative risk [95% CI]=1.55 [1.29, 1.86] and 1.51 [1.26, 1.82] per standard deviation increment, respectively; both P<0.001), after adjusting for demographic and behavioral factors. After further adjustment for cardiovascular disease risk factors and immune activation markers, these associations were attenuated but remained significant. The results were consistent between men and women and between HIV-infected and HIV-uninfected participants., Conclusions: In 2 HIV cohorts, elevated plasma levels of C16:0 and C24:1 ceramides, correlating with immune activation and inflammation, were associated with antiretroviral therapy use and progression of carotid artery atherosclerosis.

Background: Common diseases such as coronary heart disease (CHD) are complex in etiology. The interaction of genetic susceptibility with lifestyle factors may play a prominent role. However, gene-lifestyle interactions for CHD have been difficult to identify. Here, we investigate interaction of smoking behavior, a potent lifestyle factor, with genotypes that have been shown to associate with CHD risk., Methods: We analyzed data on 60 919 CHD cases and 80 243 controls from 29 studies for gene-smoking interactions for genetic variants at 45 loci previously reported to be associated with CHD risk. We also studied 5 loci associated with smoking behavior. Study-specific gene-smoking interaction effects were calculated and pooled using fixed-effects meta-analyses. Interaction analyses were declared to be significant at a P value of <1.0×10 -3 (Bonferroni correction for 50 tests)., Results: We identified novel gene-smoking interaction for a variant upstream of the ADAMTS7 gene. Every T allele of rs7178051 was associated with lower CHD risk by 12% in never-smokers ( P =1.3×10 -16 ) in comparison with 5% in ever-smokers ( P =2.5×10 -4 ), translating to a 60% loss of CHD protection conferred by this allelic variation in people who smoked tobacco (interaction P value=8.7×10 -5 ). The protective T allele at rs7178051 was also associated with reduced ADAMTS7 expression in human aortic endothelial cells and lymphoblastoid cell lines. Exposure of human coronary artery smooth muscle cells to cigarette smoke extract led to induction of ADAMTS7. CONCLUSIONS: Allelic variation at rs7178051 that associates with reduced ADAMTS7 expression confers stronger CHD protection in never-smokers than in ever-smokers. Increased vascular ADAMTS7 expression may contribute to the loss of CHD protection in smokers., (© 2017 American Heart Association, Inc.)

Background: Remote ischemic preconditioning (RIPC) can inhibit recurrent ischemic events effectively in patients with acute or chronic cerebral ischemia. However, it is still unclear whether RIPC can impede ischemic injury after carotid artery stenting (CAS) in patients with severe carotid artery stenosis., Methods: Subjects with severe carotid artery stenosis were recruited in this randomized controlled study, and assigned to RIPC, sham, and no intervention (control) groups. All subjects received standard medical therapy. Subjects in the RIPC and sham groups underwent RIPC and sham RIPC twice daily, respectively, for 2 weeks before CAS. Plasma neuron-specific enolase and S-100B were used to evaluate safety, hypersensitive C-reactive protein, and new ischemic diffusion-weighted imaging lesions were used to determine treatment efficacy. The primary outcomes were the presence of ≥1 newly ischemic brain lesions on diffusion-weighted imaging within 48 hours after stenting and clinical events within 6 months after stenting., Results: We randomly assigned 189 subjects in this study (63 subjects in each group). Both RIPC and sham RIPC procedures were well tolerated and completed with high compliance (98.41% and 95.24%, respectively). Neither plasma neuron-specific enolase levels nor S-100B levels changed significantly before and after treatment. No severe adverse event was attributed to RIPC and sham RIPC procedures. The incidence of new diffusion-weighted imaging lesions in the RIPC group (15.87%) was significantly lower than in the sham group (36.51%; relative risk, 0.44; 96% confidence interval, 0.20-0.91; P <0.01) and the control group (41.27%; relative risk, 0.39; 96% confidence interval, 0.21-0.82; P <0.01). The volumes of lesions were smaller in the RIPC group than in the control and sham groups ( P <0.01 each). Ischemic events that occurred after CAS were 1 transient ischemic attack in the RIPC group, 2 strokes in the control group, and 2 strokes and 1 transient ischemic attack in the sham group, but these results were not significantly different among the 3 groups ( P =0.597)., Conclusions: RIPC is safe in patients undergoing CAS, which may be able to decrease ischemic brain injury secondary to CAS. However, the mechanisms and effects of RIPC on clinical outcomes in this cohort of patients need further investigation., Clinical Trial Registration: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01654666., (© 2017 American Heart Association, Inc.)

Background: Several prospective studies have evaluated the association between body mass index (BMI) and death risk among patients with diabetes mellitus; however, the results have been inconsistent., Methods and Results: We performed a prospective cohort study of 19 478 black and 15 354 white patients with type 2 diabetes mellitus. Cox proportional hazards regression models were used to estimate the association of different levels of BMI stratification with all-cause mortality. During a mean follow-up of 8.7 years, 4042 deaths were identified. The multivariable-adjusted (age, sex, smoking, income, and type of insurance) hazard ratios for all-cause mortality associated with BMI levels (18.5-22.9, 23-24.9, 25-29.9, 30-34.9 [reference group], 35-39.9, and ≥40 kg/m(2)) at baseline were 2.12 (95% confidence interval [CI], 1.80-2.49), 1.74 (95% CI, 1.46-2.07), 1.23 (95% CI, 1.08-1.41), 1.00, 1.19 (95% CI, 1.03-1.39), and 1.23 (95% CI, 1.05-1.43) for blacks and 1.70 (95% CI, 1.42-2.04), 1.51 (95% CI, 1.27-1.80), 1.07 (95% CI, 0.94-1.21), 1.00, 1.07 (95% CI, 0.93-1.23), and 1.20 (95% CI, 1.05-1.38) for whites, respectively. When stratified by age, smoking status, patient type, or the use of antidiabetic drugs, a U-shaped association was still present. When BMI was included in the Cox model as a time-dependent variable, the U-shaped association of BMI with all-cause mortality risk did not change., Conclusions: The present study indicated a U-shaped association of BMI with all-cause mortality risk among black and white patients with type 2 diabetes mellitus. A significantly increased risk of all-cause mortality was observed among blacks with BMI <30 kg/m(2) and ≥35 kg/m(2) and among whites with BMI <25 kg/m(2) and ≥40 kg/m(2) compared with patients with BMI of 30 to 34.9 kg/m(2)., (© 2014 American Heart Association, Inc.)

Background: Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown., Methods and Results: Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44., Conclusions: Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis., (© 2014 American Heart Association, Inc.)

Background: Abnormal sarcoplasmic reticulum calcium (Ca) cycling is increasingly recognized as an important mechanism for increased ventricular automaticity that leads to lethal ventricular arrhythmias. Previous studies have linked lethal familial arrhythmogenic disorders to mutations in the ryanodine receptor and calsequestrin genes, which interact with junctin and triadin to form a macromolecular Ca-signaling complex. The essential physiological effects of junctin and its potential regulatory roles in sarcoplasmic reticulum Ca cycling and Ca-dependent cardiac functions, such as myocyte contractility and automaticity, are unknown., Methods and Results: The junctin gene was targeted in embryonic stem cells, and a junctin-deficient mouse was generated. Ablation of junctin was associated with enhanced cardiac function in vivo, and junctin-deficient cardiomyocytes exhibited increased contractile and Ca-cycling parameters. Short-term isoproterenol stimulation elicited arrhythmias, including premature ventricular contractions, atrioventricular heart block, and ventricular tachycardia. Long-term isoproterenol infusion also induced premature ventricular contractions and atrioventricular heart block in junctin-null mice. Further examination of the electrical activity revealed a significant increase in the occurrence of delayed afterdepolarizations. Consistently, 25% of the junctin-null mice died by 3 months of age with structurally normal hearts., Conclusions: Junctin is an essential regulator of sarcoplasmic reticulum Ca release and contractility in normal hearts. Ablation of junctin is associated with aberrant Ca homeostasis, which leads to fatal arrhythmias. Thus, normal intracellular Ca cycling relies on maintenance of junctin levels and an intricate balance among the components in the sarcoplasmic reticulum quaternary Ca-signaling complex.