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Background: Tetraspanin CD151 is highly expressed in endothelia and reinforces cell adhesion, but its role in vascular inflammation remains largely unknown., Methods: In vitro molecular and cellular biological analyses on genetically modified endothelial cells, in vivo vascular biological analyses on genetically engineered mouse models, and in silico systems biology and bioinformatics analyses on CD151-related events., Results: Endothelial ablation of Cd151 leads to pulmonary and cardiac inflammation, severe sepsis, and perilous COVID-19, and endothelial CD151 becomes downregulated in inflammation. Mechanistically, CD151 restrains endothelial release of proinflammatory molecules for less leukocyte infiltration. At the subcellular level, CD151 determines the integrity of multivesicular bodies/lysosomes and confines the production of exosomes that carry cytokines such as ANGPT2 (angiopoietin-2) and proteases such as cathepsin-D. At the molecular level, CD151 docks VCP (valosin-containing protein)/p97, which controls protein quality via mediating deubiquitination for proteolytic degradation, onto endolysosomes to facilitate VCP/p97 function. At the endolysosome membrane, CD151 links VCP/p97 to (1) IFITM3 (interferon-induced transmembrane protein 3), which regulates multivesicular body functions, to restrain IFITM3-mediated exosomal sorting, and (2) V-ATPase, which dictates endolysosome pH, to support functional assembly of V-ATPase., Conclusions: Distinct from its canonical function in strengthening cell adhesion at cell surface, CD151 maintains endolysosome function by sustaining VCP/p97-mediated protein unfolding and turnover. By supporting protein quality control and protein degradation, CD151 prevents proteins from (1) buildup in endolysosomes and (2) discharge through exosomes, to limit vascular inflammation. Also, our study conceptualizes that balance between degradation and discharge of proteins in endothelial cells determines vascular information. Thus, the IFITM3/V-ATPase-tetraspanin-VCP/p97 complexes on endolysosome, as a protein quality control and inflammation-inhibitory machinery, could be beneficial for therapeutic intervention against vascular inflammation., Competing Interests: Disclosures None.

Background: Human cardiac long noncoding RNA (lncRNA) profiles in patients with dilated cardiomyopathy (DCM) were previously analyzed, and the long noncoding RNA CHKB (choline kinase beta) divergent transcript (CHKB-DT) levels were found to be mostly downregulated in the heart. In this study, the function of CHKB-DT in DCM was determined., Methods: Long noncoding RNA expression levels in the human heart tissues were measured via quantitative reverse transcription-polymerase chain reaction and in situ hybridization assays. A CHKB-DT heterozygous or homozygous knockout mouse model was generated using the clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, and the adeno-associated virus with a cardiac-specific promoter was used to deliver the RNA in vivo. Sarcomere shortening was performed to assess the primary cardiomyocyte contractility. The Seahorse XF cell mitochondrial stress test was performed to determine the energy metabolism and ATP production. Furthermore, the underlying mechanisms were explored using quantitative proteomics, ribosome profiling, RNA antisense purification assays, mass spectrometry, RNA pull-down, luciferase assay, RNA-fluorescence in situ hybridization, and Western blotting., Results: CHKB-DT levels were remarkably decreased in patients with DCM and mice with transverse aortic constriction-induced heart failure. Heterozygous knockout of CHKB-DT in cardiomyocytes caused cardiac dilation and dysfunction and reduced the contractility of primary cardiomyocytes. Moreover, CHKB-DT heterozygous knockout impaired mitochondrial function and decreased ATP production as well as cardiac energy metabolism. Mechanistically, ALDH2 (aldehyde dehydrogenase 2) was a direct target of CHKB-DT. CHKB-DT physically interacted with the mRNA of ALDH2 and fused in sarcoma (FUS) through the GGUG motif. CHKB-DT knockdown aggravated ALDH2 mRNA degradation and 4-HNE (4-hydroxy-2-nonenal) production, whereas overexpression of CHKB-DT reversed these molecular changes. Furthermore, restoring ALDH2 expression in CHKB-DT +/ - mice alleviated cardiac dilation and dysfunction., Conclusions: CHKB-DT is significantly downregulated in DCM. CHKB-DT acts as an energy metabolism-associated long noncoding RNA and represents a promising therapeutic target against DCM., Competing Interests: Disclosures None.

Myocarditis is a challenging inflammatory disease of the heart, and better understanding of its pathogenesis is needed to develop specific drug therapies. Epoxyeicosatrienoic acids (EETs), active molecules synthesized by CYP (cytochrome P450) enzymes from arachidonic acids and hydrolyzed to less active dihydroxyeicosatrienoic acids by sEH (soluble epoxide hydrolase), have been attributed anti-inflammatory activity. Here, we investigated whether EETs have immunomodulatory activity and exert protective effects on coxsackie B3 virus-induced myocarditis. Viral infection altered eicosanoid epoxide and diol levels in both patients with myocarditis and in the murine heart and correlated with the increased expression and activity of sEH after coxsackie B3 virus infection. Administration of a sEH inhibitor prevented coxsackie B3 virus-induced cardiac dysfunction and inflammatory infiltration. Importantly, EET/sEH inhibitor treatment attenuated viral infection or improved viral resistance by activating type I IFN (interferon) signaling. At the molecular level, EETs enhanced the interaction between GSK3β (glycogen synthase kinase-3 beta) and TBK1 (TANK-binding kinase 1) to promote IFN-β production. Our findings revealed that EETs and sEH inhibitors prevent the progress of coxsackie B3 virus-induced myocarditis, particularly by promoting viral resistance by increasing IFN production., Competing Interests: Disclosures None.

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Rationale: Diabetes mellitus is often associated with cardiovascular complications, which is the leading cause of morbidity and mortality among patients with diabetes mellitus, but little is known about the mechanism that connects diabetes mellitus to the development of cardiovascular dysfunction., Objective: We aim to elucidate the mechanism underlying hyperglycemia-induced cardiac dysfunction on a well-established db/db mouse model for diabetes mellitus and diabetic complications that lead to heart failure., Methods and Results: We first profiled the expression of microRNAs (miRNAs) by microarray and quantitative reverse transcription polymerase chain reaction on db/db mice and identified miR-320 as a key miRNA associated with the disease phenotype. We next established the clinical relevance of this finding by showing the upregulation of the same miRNA in the failing heart of patients with diabetes mellitus. We demonstrated the causal role of miR-320 in inducing diabetic cardiomyopathy, showing that miR-320 overexpression exacerbated while its inhibition improved the cardiac phenotype in db/db mice. Unexpectedly, we found that miR-320 acts as a small activating RNA in the nucleus at the level of transcription. By chromatin immunoprecipitation sequencing and chromatin immunoprecipitation quantitive polymerase chain reaction analysis of Ago2 (argonaute RISC catalytic component 2) and RNA polymerase II in response to miR-320 induction, we identified CD36 (fatty acid translocase) as a key target gene for this miRNA and showed that the induced expression of CD36 is responsible for increased fatty acid uptake, thereby causing lipotoxicity in the heart., Conclusions: These findings uncover a novel mechanism for diabetes mellitus-triggered cardiac dysfunction, provide an endogenous case for small activating RNA that has been demonstrated to date only with synthetic RNAs in transfected cells, and suggest a potential strategy to develop a miRNA-based therapy to treat diabetes mellitus-associated cardiovascular complications.

Rationale: Mitochondrial dysfunction plays an important role in heart failure (HF). However, the molecular mechanisms regulating mitochondrial functions via selective mitochondrial autophagy (mitophagy) are poorly understood., Objective: We sought to determine the role of AMPK (AMP-activated protein kinase) in selective mitophagy during HF., Methods and Results: An isoform shift from AMPKα2 to AMPKα1 was observed in failing heart samples from HF patients and transverse aortic constriction-induced mice, accompanied by decreased mitophagy and mitochondrial function. The recombinant adeno-associated virus Serotype 9-mediated overexpression of AMPKα2 in mouse hearts prevented the development of transverse aortic constriction-induced chronic HF by increasing mitophagy and improving mitochondrial function. In contrast, AMPKα2 -/- mutant mice exhibited an exacerbation of the early progression of transverse aortic constriction-induced HF via decreases in cardiac mitophagy. In isolated adult mouse cardiomyocytes, AMPKα2 overexpression mechanistically rescued the impairment of mitophagy after phenylephrine stimulation for 24 hours. Genetic knockdown of AMPKα2, but not AMPKα1, by short interfering RNA suppressed the early phase (6 hours) of phenylephrine-induced compensatory increases in mitophagy. Furthermore, AMPKα2 specifically interacted with phosphorylated PINK1 (PTEN-induced putative kinase 1) at Ser495 after phenylephrine stimulation. Subsequently, phosphorylated PINK1 recruited the E3 ubiquitin ligase, Parkin, to depolarized mitochondria, and then enhanced the role of the PINK1-Parkin-SQSTM1 (sequestosome-1) pathway involved in cardiac mitophagy. This increase in cardiac mitophagy was accompanied by the elimination of damaged mitochondria, improvement in mitochondrial function, decrease in reactive oxygen species production, and apoptosis of cardiomyocytes. Finally, Ala mutation of PINK1 at Ser495 partially suppressed AMPKα2 overexpression-induced mitophagy and improvement of mitochondrial function in phenylephrine-stimulated cardiomyocytes, whereas Asp (phosphorylation mimic) mutation promoted mitophagy after phenylephrine stimulation., Conclusions: In failing hearts, the dominant AMPKα isoform switched from AMPKα2 to AMPKα1, which accelerated HF. The results show that phosphorylation of Ser495 in PINK1 by AMPKα2 was essential for efficient mitophagy to prevent the progression of HF., (© 2017 American Heart Association, Inc.)

Rationale: Asymmetrical dimethylarginine (ADMA), an endogenous arginine analogue, inhibits nitric oxide synthases and plays an important role in endothelial dysfunction., Objective: In the present study, we tested whether a novel genetic variant in dimethylarginine dimethylaminohydrolase 1 (DDAH1), an important ADMA hydrolyzing gene, was associated with stroke and coronary heart disease (CHD) susceptibility in the Chinese Han population., Methods and Results: By resequencing, we identified a novel 4-nucleotide deletion/insertion variant in the DDAH1 promoter. The insertion allele disrupted binding of metal-regulatory transcription factor 1, which resulted in significant reduction of in vitro DDAH1 transcriptional activity and in vivo DDAH1 mRNA level, and in turn, increased plasma ADMA level and the ratio of ADMA to L-arginine. We initially genotyped the polymorphism in 1388 stroke patients and 1027 controls as well as 576 CHD patients and 557 controls and then replicated our study in additional independent case-control cohorts comprising 961 stroke patients and 822 controls and 482 CHD patients and 1072 controls. We identified that the -396 4N ins allele was significantly associated with increased risk of thrombosis stroke and CHD after adjusting for environmental factors in both samples for both diseases (thrombosis stroke discovery set: odds ratio [OR]=1.35, P=0.032; replication set: OR=1.51, P=0.006; CHD discovery set: OR=1.45, P=0.035; replication set: OR=1.47, P=0.003)., Conclusions: Our results suggest that the DDAH1 loss-of-function polymorphism is associated with both increased risk of thrombosis stroke and CHD.

Mutations in SCN5A encoding the principal Na+ channel alpha-subunit expressed in human heart (Na(V)1.5) have recently been linked to an inherited form of dilated cardiomyopathy with atrial and ventricular arrhythmia. We compared the biophysical properties of 2 novel Na(V)1.5 mutations associated with this syndrome (D2/S4--R814W; D4/S3--D1595H) with the wild-type (WT) channel using heterologous expression in cultured tsA201 cells and whole-cell patch-clamp recording. Expression levels were similar among WT and mutant channels, and neither mutation affected persistent sodium current. R814W channels exhibited prominent and novel defects in the kinetics and voltage dependence of activation characterized by slower rise times and a hyperpolarized conductance-voltage relationship resulting in an increased "window current." This mutant also displayed enhanced slow inactivation and greater use-dependent reduction in peak current at fast pulsing frequencies. By contrast, D1595H channels exhibited impaired fast inactivation characterized by slower entry into the inactivated state and a hyperpolarized steady-state inactivation curve. Our findings illustrate the divergent biophysical defects caused by 2 different SCN5A mutations associated with familial dilated cardiomyopathy. Retrospective review of the published clinical data suggested that cardiomyopathy was not common in the family with D1595H, but rather sinus bradycardia was the predominant clinical finding. However, for R814W, we speculate that an increased window current coupled with enhanced slow inactivation and rate-dependent loss of channel availability provided a unique substrate predisposing myocytes to disordered Na+ and Ca2+ homeostasis leading to myocardial dysfunction.

Brugada syndrome is an inherited cardiac disease that causes sudden death related to idiopathic ventricular fibrillation in a structurally normal heart. The disease is characterized by ST-segment elevation in the right precordial ECG leads and is frequently accompanied by an apparent right bundle-branch block. The biophysical properties of the SCN5A mutation T1620M associated with Brugada syndrome were examined for defects in intermediate inactivation (I:(M)), a gating process in Na(+) channels with kinetic features intermediate between fast and slow inactivation. Cultured mammalian cells expressing T1620M Na(+) channels in the presence of the human beta(1) subunit exhibit enhanced intermediate inactivation at both 22 degrees C and 32 degrees C compared with wild-type recombinant human heart Na(+) channels (WT-hH1). Our findings support the hypothesis that Brugada syndrome is caused, in part, by functionally reduced Na(+) current in the myocardium due to an increased proportion of Na(+) channels that enter the I:(M) state. This phenomenon may contribute significantly to arrhythmogenesis in patients with Brugada syndrome. The full text of this article is available at http://www.circresaha.org.