Spexin Diminishes Atrial Fibrillation Vulnerability by Acting on Galanin Receptor 2.

BACKGROUND: G protein-coupled receptors play a critical role in atrial fibrillation (AF). Spexin is a novel ligand of galanin receptors (GALRs). In this study, we investigated the regulation of spexin and GALRs on AF and the underlying mechanisms. METHODS: Global spexin knockout (SPX-KO) and cardiomyocyte-specific GALRs knockout (GALR-cKO) mice underwent burst pacing electrical stimulation. Optical mapping was used to determine atrial conduction velocity and action potential duration. Atrial myocyte action potential duration and inward rectifying K+ current (IK1) were recorded using whole-cell patch clamps. Isolated cardiomyocytes were stained with Fluo-3/AM dye, and intracellular Ca2+ handling was examined by CCD camera. A mouse model of AF was established by Ang-II (angiotensin II) infusion. RESULTS: Spexin plasma levels in patients with AF were lower than those in subjects without AF, and knockout of spexin increased AF susceptibility in mice. In the atrium of SPX-KO mice, potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) and sarcolipin (SLN) were upregulated; meanwhile, IK1 current was increased and Ca2+ handling was impaired in isolated atrial myocytes of SPX-KO mice. GALR2-cKO mice, but not GALR1-cKO and GALR3-cKO mice, had a higher incidence of AF, which was associated with higher IK1 current and intracellular Ca2+ overload. The phosphorylation level of CREB (cyclic AMP responsive element binding protein 1) was upregulated in atrial tissues of SPX-KO and GALR2-cKO mice. Chromatin immunoprecipitation confirmed the recruitment of p-CREB to the proximal promoter regions of KCNJ2 and SLN. Finally, spexin treatment suppressed CREB signaling, decreased IK1 current and decreased intracellular Ca2+ overload, which thus reduced the inducibility of AF in Ang-II-infused mice. CONCLUSIONS: Spexin reduces atrial fibrillation susceptibility by inhibiting CREB phosphorylation and thus downregulating KCNJ2 and SLN transcription by GALR2 receptor. The spexin/GALR2/CREB signaling pathway represents a novel therapeutic avenue in the development of agents against atrial fibrillation.

Interdependent Nuclear Co-Trafficking of ASPP1 and p53 Aggravates Cardiac Ischemia/Reperfusion Injury.

OBJECTIVE: ASPP1 (apoptosis stimulating of p53 protein 1) is critical in regulating cell apoptosis as a cofactor of p53 to promote its transcriptional activity in the nucleus. However, whether cytoplasmic ASPP1 affects p53 nuclear trafficking and its role in cardiac diseases remains unknown. This study aims to explore the mechanism by which ASPP1 modulates p53 nuclear trafficking and the subsequent contribution to cardiac ischemia/reperfusion (I/R) injury. METHODS AND RESULTS: The immunofluorescent staining showed that under normal condition ASPP1 and p53 colocalized in the cytoplasm of neonatal mouse ventricular cardiomyocytes, while they were both upregulated and translocated to the nuclei upon hypoxia/reoxygenation treatment. The nuclear translocation of ASPP1 and p53 was interdependent, as knockdown of either ASPP1 or p53 attenuated nuclear translocation of the other one. Inhibition of importin-ß1 resulted in the cytoplasmic sequestration of both p53 and ASPP1 in neonatal mouse ventricular cardiomyocytes with hypoxia/reoxygenation stimulation. Overexpression of ASPP1 potentiated, whereas knockdown of ASPP1 inhibited the expression of Bax (Bcl2-associated X), PUMA (p53 upregulated modulator of apoptosis), and Noxa, direct apoptosis-associated targets of p53. ASPP1 was also increased in the I/R myocardium. Cardiomyocyte-specific transgenic overexpression of ASPP1 aggravated I/R injury as indicated by increased infarct size and impaired cardiac function. Conversely, knockout of ASPP1 mitigated cardiac I/R injury. The same qualitative data were observed in neonatal mouse ventricular cardiomyocytes exposed to hypoxia/reoxygenation injury. Furthermore, inhibition of p53 significantly blunted the proapoptotic activity and detrimental effects of ASPP1 both in vitro and in vivo. CONCLUSIONS: Binding of ASPP1 to p53 triggers their nuclear cotranslocation via importin-ß1 that eventually exacerbates cardiac I/R injury. The findings imply that interfering the expression of ASPP1 or the interaction between ASPP1 and p53 to block their nuclear trafficking represents an important therapeutic strategy for cardiac I/R injury.

IEAtlas: an atlas of HLA-presented immune epitopes derived from non-coding regions.

Cancer-related epitopes can engage the immune system against tumor cells, thus exploring epitopes derived from non-coding regions is emerging as a fascinating field in cancer immunotherapies. Here, we described a database, IEAtlas (http://bio-bigdata.hrbmu.edu.cn/IEAtlas), which aims to provide and visualize the comprehensive atlas of human leukocyte antigen (HLA)-presented immunogenic epitopes derived from non-coding regions. IEAtlas reanalyzed publicly available mass spectrometry-based HLA immunopeptidome datasets against our integrated benchmarked non-canonical open reading frame information. The current IEAtlas identified 245 870 non-canonical epitopes binding to HLA-I/II allotypes across 15 cancer types and 30 non-cancerous tissues, greatly expanding the cancer immunopeptidome. IEAtlas further evaluates the immunogenicity via several commonly used immunogenic features, including HLA binding affinity, stability and T-cell receptor recognition. In addition, IEAtlas provides the biochemical properties of epitopes as well as the clinical relevance of corresponding genes across major cancer types and normal tissues. Several flexible tools were also developed to aid retrieval and to analyze the epitopes derived from non-coding regions. Overall, IEAtlas will serve as a valuable resource for investigating the immunogenic capacity of non-canonical epitopes and the potential as therapeutic cancer vaccines.

CIB2 Is a Novel Endogenous Repressor of Atrial Remodeling.

BACKGROUND: Atrial fibrillation (AF) is a highly prevalent condition that can cause or exacerbate heart failure, is an important risk factor for stroke, and is associated with pronounced morbidity and death. Genes uniquely expressed in the atria are known to be essential for maintaining atrial structure and function. Atrial tissue remodeling contributes to arrhythmia recurrence and maintenance. However, the mechanism underlying atrial remodeling remains poorly understood. This study was designed to investigate whether other uncharacterized atrial specific genes play important roles in atrial physiology and arrhythmogenesis. METHODS: RNA-sequencing analysis was used to identify atrial myocyte specific and angiotensin II-responsive genes. Genetically modified, cardiomyocyte-specific mouse models (knockout and overexpression) were generated. In vivo and in vitro electrophysiological, histology, and biochemical analyses were performed to determine the consequences of CIB2 (calcium and integrin binding family member 2 protein) gain and loss of function in the atrium. RESULTS: Using RNA-sequencing analysis, we identified CIB2 as an atrial-enriched protein that is significantly downregulated in the left atria of patients with AF and mouse models of AF from angiotensin II infusion or pressure overload. Using cardiomyocyte-specific Cib2 knockout (Cib2-/-) and atrial myocyte-specific Cib2-overexpressing mouse models, we found that loss of Cib2 enhances AF occurrence, prolongs AF duration, and correlates with a significant increase in atrial fibrosis under stress. Conversely, Cib2 overexpression mitigates AF occurrence and atrial fibrosis triggered by angiotensin II stress. Mechanistically, we revealed that CIB2 competes with and inhibits CIB1-mediated calcineurin activation, thereby negating stress-induced structural remodeling and AF. CONCLUSIONS: Our data suggest that CIB2 represents a novel endogenous and atrial-enriched regulator that protects against atrial remodeling and AF under stress conditions. Therefore, CIB2 may represent a new potential target for treating AF.

MetBil as a novel molecular regulator in ischemia-induced cardiac fibrosis via METTL3-mediated m6A modification.

Cardiac fibrosis is a common pathological manifestation in multiple cardiovascular diseases and often results in myocardial stiffness and cardiac dysfunctions. LncRNA (long noncoding RNA) participates in a number of pathophysiological processes. However, its role in cardiac fibrosis remains unclear. The purpose of this study was to investigate the role and molecular mechanism of MetBil in regulating cardiac fibrosis. Our data showed that METTL3 binding lncRNA (MetBil) was significantly increased both in fibrotic tissue following myocardial infarction (MI) in mice and in cardiac fibroblasts (CFs) exposed to TGF-ß1 (20 ng/mL) or 20% FBS. Overexpression of MetBil augmented collagen deposition, CF proliferation and activation while silencing MetBil exhibited the opposite effects. Importantly, heterozygous knockout of MetBil alleviated cardiac fibrosis and improved cardiac function after MI. RNA pull-down and RNA-binding protein immunoprecipitation assay showed that METTL3 is a direct downstream target of MetBil; consistently, MetBil and METTL3 were co-localized in both the nucleus and cytoplasm of CFs. Interestingly, MetBil regulated METTL3 expression at protein level, but not mRNA level, in ubiquitin-proteasome pathway. Enforced expression of METTL3 canceled the antifibrotic effects of silencing MetBil reflected by increased collagen production, CF proliferation and activation. Most notably, the m6A-modified fibrosis-regulated genes mediated by METTL3 are profoundly involved in the regulation of MetBil in the cardiac fibrosis following MI. Our study reveals that MetBil as a novel regulator of fibrosis promotes cardiac fibrosis via interacting with METTL3 and regulating the expression of the methylated fibrosis-associated genes, providing a new intervening target for fibrosis-associated cardiac diseases.

CIRKIL Exacerbates Cardiac Ischemia/Reperfusion Injury by Interacting With Ku70.

BACKGROUND: Ku70 participates in several pathological processes through mediating repair of DNA double-strand breaks. Our previous study has identified a highly conserved long noncoding RNA cardiac ischemia reperfusion associated Ku70 interacting lncRNA (CIRKIL) that was upregulated in myocardial infarction. The study aims to investigate whether CIRKIL regulates myocardial ischemia/reperfusion (I/R) through binding to Ku70. METHODS: CIRKIL transgenic and knockout mice were subjected to 45-minute ischemia and 24-hour reperfusion to establish myocardial I/R model. RNA pull-down and RNA immunoprecipitation assay were used to detect the interaction between CIRKIL and Ku70. RESULTS: The expression of CIRKIL was increased in I/R myocardium and H2O2-treated cardiomyocytes. Overexpression of CIRKIL increased the expression of γH2A.X, a specific marker of DNA double-strand breaks and aggravated cardiomyocyte apoptosis, whereas knockdown of CIRKIL produced the opposite changes. Transgenic overexpression of CIRKIL aggravated cardiac dysfunction, enlarged infarct area, and worsened cardiomyocyte damage in I/R mice. Knockout of CIRKIL alleviated myocardial I/R injury. Mechanistically, CIRKIL directly bound to Ku70 to subsequently decrease nuclear translocation of Ku70 and impair DNA double-strand breaks repair. Concurrent overexpression of Ku70 mitigated CIRKIL overexpression-induced myocardial I/R injury. Furthermore, knockdown of human CIRKIL significantly suppressed cell damage induced by H2O2 in adult human ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: CIRKIL is a detrimental factor in I/R injury acting via regulating nuclear translocation of Ku70 and DNA double-strand breaks repair. Thus, CIRKIL might be considered as a novel molecular target for the treatment of cardiac conditions associated with I/R injury.

Long Noncoding RNA Gpr137b-ps Promotes Advanced Atherosclerosis via the Regulation of Autophagy in Macrophages.

BACKGROUND: Current therapies cannot completely reverse advanced atherosclerosis. High levels of amino acids, induced by Western diet, stimulate mTORC1 (mammalian target of rapamycin complex 1)-autophagy defects in macrophages, accelerating atherosclerotic plaque progression. In addition, autophagy-lysosomal dysfunction contributes to plaque necrotic core enlargement and lipid accumulation. Therefore, it is essential to investigate the novel mechanism and molecules to reverse amino acid-mTORC1-autophagy signaling dysfunction in macrophages of patients with advanced atherosclerosis. METHODS: We observed that Gpr137b-ps (G-protein-coupled receptor 137B, pseudogene) was upregulated in advanced atherosclerotic plaques. The effect of Gpr137b-ps on the progression of atherosclerosis was studied by generating advanced plaques in ApoE-/- mice with cardiac-specific knockout of Gpr137b-ps. Bone marrow-derived macrophages and mouse mononuclear macrophage cell line RAW264.7 cells were subjected to starvation or amino acid stimulation to study amino acid-mTORC1-autophagy signaling. Using both gain- and loss-of-function approaches, we explored the mechanism of Gpr137b-ps-regulated autophagy. RESULTS: Our results demonstrated that Gpr137b-ps deficiency led to enhanced autophagy in macrophages and reduced atherosclerotic lesions, characterized by fewer necrotic cores and less lipid accumulation. Knockdown of Gpr137b-ps increased autophagy and prevented amino acid-induced mTORC1 signaling activation. As the downstream binding protein of Gpr137b-ps, HSC70 (heat shock cognate 70) rescued the impaired autophagy induced by Gpr137b-ps. Furthermore, Gpr137b-ps interfered with the HSC70 binding to G3BP (Ras GTPase-activating protein-binding protein), which tethers the TSC (tuberous sclerosis complex) complex to lysosomes and suppresses mTORC1 signaling. In addition to verifying that the NTF2 (nuclear transport factor 2) domain of G3BP binds to HSC70 by in vitro protein synthesis, we further demonstrated that HSC70 binds to the NTF2 domain of G3BP through its W90-F92 motif by using computational modeling. CONCLUSIONS: These findings reveal that Gpr137b-ps plays an essential role in the regulation of macrophage autophagy, which is crucial for the progression of advanced atherosclerosis. Gpr137b-ps impairs the interaction of HSC70 with G3BP to regulate amino acid-mTORC1-autophagy signaling, and these results provide a new potential therapeutic direction for the treatment of advanced atherosclerosis.

Acetylcytidine modification of Amotl1 by N-acetyltransferase 10 contributes to cardiac fibrotic expansion in mice after myocardial infarction.

Cardiac fibrosis is a pathological scarring process that impairs cardiac function. N-acetyltransferase 10 (Nat10) is recently identified as the key enzyme for the N4-acetylcytidine (ac4C) modification of mRNAs. In this study, we investigated the role of Nat10 in cardiac fibrosis following myocardial infarction (MI) and the related mechanisms. MI was induced in mice by ligation of the left anterior descending coronary artery; cardiac function was assessed with echocardiography. We showed that both the mRNA and protein expression levels of Nat10 were significantly increased in the infarct zone and border zone 4 weeks post-MI, and the expression of Nat10 in cardiac fibroblasts was significantly higher compared with that in cardiomyocytes after MI. Fibroblast-specific overexpression of Nat10 promoted collagen deposition and induced cardiac systolic dysfunction post-MI in mice. Conversely, fibroblast-specific knockout of Nat10 markedly relieved cardiac function impairment and extracellular matrix remodeling following MI. We then conducted ac4C-RNA binding protein immunoprecipitation-sequencing (RIP-seq) in cardiac fibroblasts transfected with Nat10 siRNA, and revealed that angiomotin-like 1 (Amotl1), an upstream regulator of the Hippo signaling pathway, was the target gene of Nat10. We demonstrated that Nat10-mediated ac4C modification of Amotl1 increased its mRNA stability and translation in neonatal cardiac fibroblasts, thereby increasing the interaction of Amotl1 with yes-associated protein 1 (Yap) and facilitating Yap translocation into the nucleus. Intriguingly, silencing of Amotl1 or Yap, as well as treatment with verteporfin, a selective and potent Yap inhibitor, attenuated the Nat10 overexpression-induced proliferation of cardiac fibroblasts and prevented their differentiation into myofibroblasts in vitro. In conclusion, this study highlights Nat10 as a crucial regulator of myocardial fibrosis following MI injury through ac4C modification of upstream activators within the Hippo/Yap signaling pathway.

Microprotein Dysregulation in the Serum of Patients with Atrial Fibrillation.

The incidence rate of atrial fibrillation (AF) has stayed at a high level in recent years. Despite the intensive efforts to study the pathologic changes of AF, the molecular mechanism of disease development remains unclarified. Microproteins are ribosomally translated gene products from small open reading frames (sORFs) and are found to play crucial biological functions, while remain rare attention and indistinct in AF study. In this work, we recruited 65 AF patients and 65 healthy subjects for microproteomic profiling. By differential analysis and cross-validation between independent datasets, a total of 4 microproteins were identified as significantly different, including 3 annotated ones and 1 novel one. Additionally, we established a diagnostic model with either microproteins or global proteins by machine learning methods and found the model with microproteins achieved comparable and excellent performance as that with global proteins. Our results confirmed the abnormal expression of microproteins in AF and may provide new perspectives on the mechanism study of AF.

Forced activation of dystrophin transcription by CRISPR/dCas9 reduced arrhythmia susceptibility via restoring membrane Nav1.5 distribution.

Dystrophin deficiency due to genetic mutations causes cardiac abnormalities in Duchenne's muscular dystrophy. Dystrophin is also shown to be downregulated in conventional failing hearts. Whether restoration of dystrophin expression possesses any therapeutic potential for conventional heart failure (HF) remains to be examined. HF mouse model was generated by transverse aortic constriction (TAC). In vivo activation of dystrophin transcription was achieved by tail-vein injection of adeno-associated virus 9 carrying CRISPR/dCas system for dystrophin. We found that activation of dystrophin expression in TAC mice significantly reduced the susceptibility to arrhythmia of TAC mice and the mortality rate. We further demonstrated that over-expression of dystrophin increased cardiac conduction of hearts in TAC mice by optical mapping evaluation. Activation of dystrophin expression also increased peak sodium current in isolated ventricular myocytes from hearts of TAC mice as recorded by the patch-clamp technique. Immunoblotting and immunofluorescence showed that increased dystrophin transcription restored the membrane distribution of Nav1.5 in the hearts of TAC mice. In summary, correction of dystrophin downregulation by the CRISPR-dCas9 system reduced the susceptibility to arrhythmia of conventional HF mice through restoring Nav1.5 membrane distribution. This study paved the way to develop a new therapeutic strategy for HF-related ventricular arrhythmia.

GDF11 promotes wound healing in diabetic mice via stimulating HIF-1ɑ-VEGF/SDF-1ɑ-mediated endothelial progenitor cell mobilization and neovascularization.

Non-healing diabetic wounds (DW) are a serious clinical problem that remained poorly understood. We recently found that topical application of growth differentiation factor 11 (GDF11) accelerated skin wound healing in both Type 1 DM (T1DM) and genetically engineered Type 2 diabetic db/db (T2DM) mice. In the present study, we elucidated the cellular and molecular mechanisms underlying the action of GDF11 on healing of small skin wound. Single round-shape full-thickness wound of 5-mm diameter with muscle and bone exposed was made on mouse dorsum using a sterile punch biopsy 7 days following the onset of DM. Recombinant human GDF11 (rGDF11, 50 ng/mL, 10 µL) was topically applied onto the wound area twice a day until epidermal closure (maximum 14 days). Digital images of wound were obtained once a day from D0 to D14 post-wounding. We showed that topical application of GDF11 accelerated the healing of full-thickness skin wounds in both type 1 and type 2 diabetic mice, even after GDF8 (a muscle growth factor) had been silenced. At the cellular level, GDF11 significantly facilitated neovascularization to enhance regeneration of skin tissues by stimulating mobilization, migration and homing of endothelial progenitor cells (EPCs) to the wounded area. At the molecular level, GDF11 greatly increased HIF-1ɑ expression to enhance the activities of VEGF and SDF-1ɑ, thereby neovascularization. We found that endogenous GDF11 level was robustly decreased in skin tissue of diabetic wounds. The specific antibody against GDF11 or silence of GDF11 by siRNA in healthy mice mimicked the non-healing property of diabetic wound. Thus, we demonstrate that GDF11 promotes diabetic wound healing via stimulating endothelial progenitor cells mobilization and neovascularization mediated by HIF-1ɑ-VEGF/SDF-1ɑ pathway. Our results support the potential of GDF11 as a therapeutic agent for non-healing DW.

Silencing of METTL3 attenuates cardiac fibrosis induced by myocardial infarction via inhibiting the activation of cardiac fibroblasts.

Cardiac fibrosis is characterized by the activation of cardiac fibroblasts and accumulation of extracellular matrix. METTL3, a component of methyltransferase complex, participates in multiple biological processes associated with mammalian development and disease progression. However, the role of METTL3 in cardiac fibrosis is still unknown. We performed fibroblasts activation with TGF-ß1 (20 ng/mL) in vitro and established in vivo mouse models with lentivirus to assess the effects of METTL3 on cardiac fibroblasts proliferation and collagen formation. Methylated RNA immunoprecipitation (MeRIP) was used to define the potential fibrosis-regulated gene. The expression level of METTL3 was increased in cardiac fibrotic tissue of mice with chronic myocardial infarction and cultured cardiac fibroblats (CFs) treated with TGF-ß1. Enforced expression of METTL3 promoted proliferation and fibroblast-to-myofibroblast transition and collagens accumulation, while silence of METTL3 did the opposite. Silence of METTL3 by lentivirus carrying METTL3 siRNA markedly alleviated cardiac fibrosis in MI mice. Transcriptome and N6-methyladenosine (m6 A) profiling analyses revealed that the expression and m6 A level of collagen-related genes were altered after silence of METTL3. METTL3-mediated m6 A modification is critical for the development of cardiac fibrosis, providing a molecular target for manipulating fibrosis and the associated cardiac diseases.

Cullin-associated and neddylation-dissociated 1 protein (CAND1) governs cardiac hypertrophy and heart failure partially through regulating calcineurin degradation.

Pathological cardiac hypertrophy is a process characterized by significant disturbance of protein turnover. Cullin-associated and Neddylation-dissociated 1 (CAND1) acts as a coordinator to modulate substrate protein degradation by promoting the formation of specific cullin-based ubiquitin ligase 3 complex in response to substrate accumulation, which thereby facilitate the maintaining of normal protein homeostasis. Accumulation of calcineurin is critical in the pathogenesis of cardiac hypertrophy and heart failure. However, whether CAND1 titrates the degradation of hypertrophy related protein eg. calcineurin and regulates cardiac hypertrophy remains unknown. Therefore, we aim to explore the role of CAND1 in cardiac hypertrophy and heart failure and the underlying molecular mechanism. Here, we found that the protein level of CAND1 was increased in cardiac tissues from heart failure (HF) patients and TAC mice, whereas the mRNA level did not change. CAND1-KO+ /- aggravated TAC-induced cardiac hypertrophic phenotypes; in contrast, CAND1-Tg attenuated the maladaptive cardiac remodeling. At the molecular level, CAND1 overexpression downregulated, whereas CAND1-KO+ /- or knockdown upregulated calcineurin expression at both in vivo and in vitro conditions. Mechanistically, CAND1 overexpression favored the assembly of Cul1/atrogin1/calcineurin complex and rendered the ubiquitination and degradation of calcineurin. Notably, CAND1 deficiency-induced hypertrophic phenotypes were partially rescued by knockdown of calcineurin, and application of exogenous CAND1 prevented TAC-induced cardiac hypertrophy. Taken together, our findings demonstrate that CAND1 exerts a protective effect against cardiac hypertrophy and heart failure partially by inducing the degradation of calcineurin.

Knockout of interleukin-17A diminishes ventricular arrhythmia susceptibility in diabetic mice via inhibiting NF-κB-mediated electrical remodeling.

Interleukin-17A (IL-17), a potent proinflammatory cytokine, has been shown to participate in cardiac electrical disorders. Diabetes mellitus is an independent risk factor for ventricular arrhythmia. In this study, we investigated the role of IL-17 in ventricular arrhythmia of diabetic mice. Diabetes was induced in both wild-type and IL-17 knockout mice by intraperitoneal injection of streptozotocin (STZ). High-frequency electrical stimuli were delivered into the right ventricle to induce ventricular arrhythmias. We showed that the occurrence rate of ventricular tachycardia was significantly increased in diabetic mice, which was attenuated by IL-17 knockout. We conducted optical mapping on perfused mouse hearts and found that cardiac conduction velocity (CV) was significantly decreased, and action potential duration (APD) was prolonged in diabetic mice, which were mitigated by IL-17 knockout. We performed whole-cell patch clamp recordings from isolated ventricular myocytes, and found that the densities of Ito, INa and ICa,L were reduced, the APDs at 50% and 90% repolarization were increased, and early afterdepolarization (EAD) was markedly increased in diabetic mice. These alterations were alleviated by the knockout of IL-17. Moreover, knockout of IL-17 alleviated the downregulation of Nav1.5 (the pore forming subunit of INa), Cav1.2 (the main component subunit of ICa,L) and KChIP2 (potassium voltage-gated channel interacting protein 2, the regulatory subunit of Ito) in the hearts of diabetic mice. The expression of NF-κB was significantly upregulated in the hearts of diabetic mice, which was suppressed by IL-17 knockout. In neonatal mouse ventricular myocytes, knockdown of NF-κB significantly increased the expression of Nav1.5, Cav1.2 and KChIP2. These results imply that IL-17 may represent a potential target for the development of agents against diabetes-related ventricular arrhythmias.

LncRNA-6395 promotes myocardial ischemia-reperfusion injury in mice through increasing p53 pathway.

Myocardial ischemia-reperfusion (I/R) injury is a pathological process characterized by cardiomyocyte apoptosis, which leads to cardiac dysfunction. Increasing evidence shows that abnormal expression of long noncoding RNAs (lncRNAs) plays a crucial role in cardiovascular diseases. In this study we investigated the role of lncRNAs in myocardial I/R injury. Myocardial I/R injury was induced in mice by ligating left anterior descending coronary artery for 45 min followed by reperfusion for 24 h. We showed that lncRNA KnowTID_00006395, termed lncRNA-6395 was significantly upregulated in the infarct area of mouse hearts following I/R injury as well as in H2O2-treated neonatal mouse ventricular cardiomyocytes (NMVCs). Overexpression of lncRNA-6395 led to cell apoptosis and the expression change of apoptosis-related proteins in NMVCs, whereas knockdown of lncRNA-6395 attenuated H2O2-induced cell apoptosis. LncRNA-6395 knockout mice (lncRNA-6395+/-) displayed improved cardiac function, decreased plasma LDH activity and infarct size following I/R injury. We demonstrated that lncRNA-6395 directly bound to p53, and increased the abundance of p53 protein through inhibiting ubiquitination-mediated p53 degradation and thereby facilitated p53 translocation to the nucleus. More importantly, overexpression of p53 canceled the inhibitory effects of lncRNA-6395 knockdown on cardiomyocyte apoptosis, whereas knockdown of p53 counteracted the apoptotic effects of lncRNA-6395 in cardiomyocytes. Taken together, lncRNA-6395 as an endogenous pro-apoptotic factor, regulates cardiomyocyte apoptosis and myocardial I/R injury by inhibiting degradation and promoting sub-cellular translocation of p53.

Mettl3 deficiency leads to the upregulation of Cav1.2 and increases arrhythmia susceptibility in mice.

Methyltransferase-like 3 (Mettl3) is a component of methyltransferase complex that mediates mA modification of RNAs, and participates in multiple biological processes. However, the role of Mettl3 in cardiac electrophysiology remains unknown. This study aims to explore the ventricular arrhythmia susceptibility of Mettl3 mice and the underlying mechanisms. Mice were anesthetized with 2% avertin (0.1 mL/ body weight) for echocardiography and programmed electrical pacing. Whole-cell patch clamp technique was used to examine the electrophysiological property of cardiomyocytes. The expression of Cav1.2 was determined by qRT-PCR and western blot analysis. The mA medication of mRNA was examined by MeRIP-Seq and MeRIP-qPCR. No differences are found in the morphology and function of the hearts between Mettl3 mice and wild-type (WT) controls. The QT and QTc intervals of Mettl3 mice are significantly longer. High-frequency electrical stimulation showed that heterozygous knockout of Mettl3 increases ventricular arrhythmia susceptibility. The whole-cell patch-clamp recordings showed that the APD is prolonged in Mettl3 ventricular myocytes and more EADs were observed. The density of is substantially increased in ventricular myocytes of Mettl3 mice. The pore-forming subunit of L-type calcium channel Cav1.2 is upregulated in Mettl3 mice, while the mRNA of its coding gene does not change. MeRIP-Seq and MeRIP-qPCR showed that the mA methylation of mRNA is decreased in cultured Mettl3-knockdown cardiomyocytes and Mettl3 hearts. Collectively, deficiency of Mettl3 increases ventricular arrhythmia susceptibility due to the upregulation of Cav1.2 by reducing mA modification onmRNA in mice. This study highlights the role of mA modification in the regulation of cardiac electrophysiology.

Integrin ß1D Deficiency-Mediated RyR2 Dysfunction Contributes to Catecholamine-Sensitive Ventricular Tachycardia in Arrhythmogenic Right Ventricular Cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a hereditary heart disease characterized by fatty infiltration, life-threatening arrhythmias, and increased risk of sudden cardiac death. The guideline for management of ARVC in patients is to improve quality of life by reducing arrhythmic symptoms and to prevent sudden cardiac death. However, the mechanism underlying ARVC-associated cardiac arrhythmias remains poorly understood. METHODS: Using protein mass spectrometry analyses, we identified that integrin ß1 is downregulated in ARVC hearts without changes to Ca2+-handling proteins. As adult cardiomyocytes express only the ß1D isoform, we generated a cardiac specific ß1D knockout mouse model and performed functional imaging and biochemical analyses to determine the consequences of integrin ß1D loss on function in the heart in vivo and in vitro. RESULTS: Integrin ß1D deficiency and RyR2 Ser-2030 hyperphosphorylation were detected by Western blotting in left ventricular tissues from patients with ARVC but not in patients with ischemic or hypertrophic cardiomyopathy. Using lipid bilayer patch clamp single channel recordings, we found that purified integrin ß1D protein could stabilize RyR2 function by decreasing RyR2 open probability, mean open time, and increasing mean close time. Also, ß1D knockout mice exhibited normal cardiac function and morphology but presented with catecholamine-sensitive polymorphic ventricular tachycardia, consistent with increased RyR2 Ser-2030 phosphorylation and aberrant Ca2+ handling in ß1D knockout cardiomyocytes. Mechanistically, we revealed that loss of DSP (desmoplakin) induces integrin ß1D deficiency in ARVC mediated through an ERK1/2 (extracellular signal-regulated kinase 1 and 2)-fibronectin-ubiquitin/lysosome pathway. CONCLUSIONS: Our data suggest that integrin ß1D deficiency represents a novel mechanism underlying the increased risk of ventricular arrhythmias in patients with ARVC.

SAIL: a new conserved anti-fibrotic lncRNA in the heart.

Long non-coding RNAs (lncRNAs) account for a large proportion of genomic transcripts and are critical regulators in various cardiac diseases. Though lncRNAs have been reported to participate in the process of diverse cardiac diseases, the contribution of lncRNAs in cardiac fibrosis remains to be fully elucidated. Here, we identified a novel anti-fibrotic lncRNA, SAIL (scaffold attachment factor B interacting lncRNA). SAIL was reduced in cardiac fibrotic tissue and activated cardiac fibroblasts. Gain- and loss-of-function studies showed that knockdown of SAIL promoted proliferation and collagen production of cardiac fibroblasts with or without TGF-ß1 (transforming growth factor beta1) treatment, while overexpression of SAIL did the opposite. In mouse cardiac fibrosis induced by myocardial infarction, knockdown of SAIL exacerbated, whereas overexpression of SAIL alleviated cardiac fibrosis. Mechanically, SAIL inhibited the fibrotic process by directly binding with SAFB via 23 conserved nucleotide sequences, which in turn blocked the access of SAFB to RNA pol II (RNA polymerase II) and reduced the transcription of fibrosis-related genes. Intriguingly, the human conserved fragment of SAIL (hSAIL) significantly suppressed the proliferation and collagen production of human cardiac fibroblasts. Our findings demonstrate that SAIL regulates cardiac fibrosis by regulating SAFB-mediated transcription of fibrotic related genes. Both SAIL and SAFB hold the potential to become novel therapeutic targets for cardiac fibrosis.

Melatonin promotes cardiomyocyte proliferation and heart repair in mice with myocardial infarction via miR-143-3p/Yap/Ctnnd1 signaling pathway.

The neonatal heart possesses the ability to proliferate and the capacity to regenerate after injury; however, the mechanisms underlying these processes are not fully understood. Melatonin has been shown to protect the heart against myocardial injury through mitigating oxidative stress, reducing apoptosis, inhibiting mitochondrial fission, etc. In this study, we investigated whether melatonin regulated cardiomyocyte proliferation and promoted cardiac repair in mice with myocardial infarction (MI), which was induced by ligation of the left anterior descending coronary artery. We showed that melatonin administration significantly improved the cardiac functions accompanied by markedly enhanced cardiomyocyte proliferation in MI mice. In neonatal mouse cardiomyocytes, treatment with melatonin (1 µM) greatly suppressed miR-143-3p levels. Silencing of miR-143-3p stimulated cardiomyocytes to re-enter the cell cycle. On the contrary, overexpression of miR-143-3p inhibited the mitosis of cardiomyocytes and abrogated cardiomyocyte mitosis induced by exposure to melatonin. Moreover, Yap and Ctnnd1 were identified as the target genes of miR-143-3p. In cardiomyocytes, inhibition of miR-143-3p increased the protein expression of Yap and Ctnnd1. Melatonin treatment also enhanced Yap and Ctnnd1 protein levels. Furthermore, Yap siRNA and Ctnnd1 siRNA attenuated melatonin-induced cell cycle re-entry of cardiomyocytes. We showed that the effect of melatonin on cardiomyocyte proliferation and cardiac regeneration was impeded by the melatonin receptor inhibitor luzindole. Silencing miR-143-3p abrogated the inhibition of luzindole on cardiomyocyte proliferation. In addition, both MT1 and MT2 siRNA could cancel the beneficial effects of melatonin on cardiomyocyte proliferation. Collectively, the results suggest that melatonin induces cardiomyocyte proliferation and heart regeneration after MI by regulating the miR-143-3p/Yap/Ctnnd1 signaling pathway, providing a new therapeutic strategy for cardiac regeneration.

Interleukin-17 upregulation participates in the pathogenesis of heart failure in mice via NF-κB-dependent suppression of SERCA2a and Cav1.2 expression.

Interleukin-17 (IL-17), also called IL-17A, is an important regulator of cardiac diseases, but its role in calcium-related cardiac dysfunction remains to be explored. Thus, we investigated the influence of IL-17 on calcium handling process and its contribution to the development of heart failure. Mice were subjected to transaortic constriction (TAC) to induce heart failure. In these mice, the levels of IL-17 in the plasma and cardiac tissue were significantly increased compared with the sham group. In 77 heart failure patients, the plasma level of IL-17 was significantly higher than 49 non-failing subjects, and was negatively correlated with cardiac ejection fraction and fractional shortening. In IL-17 knockout mice, the shortening of isolated ventricular myocytes was increased compared with that in wild-type mice, which was accompanied by significantly increased amplitude of calcium transient and the upregulation of SERCA2a and Cav1.2. In cultured neonatal cardiac myocytes, treatment of with IL-17 (0.1, 1 ng/mL) concentration-dependently suppressed the amplitude of calcium transient and reduced the expression of SERCA2a and Cav1.2. Furthermore, IL-17 treatment increased the expression of the NF-κB subunits p50 and p65, whereas knockdown of p50 reversed the inhibitory effects of IL-17 on SERCA2a and Cav1.2 expression. In mice with TAC-induced mouse heart, IL-17 knockout restored the expression of SERCA2a and Cav1.2, increased the amplitude of calcium transient and cell shortening, and in turn improved cardiac function. In addition, IL-17 knockout attenuated cardiac hypertrophy with inhibition of calcium-related signaling pathway. In conclusion, upregulation of IL-17 impairs cardiac function through NF-κB-mediated disturbance of calcium handling and cardiac remodeling. Inhibition of IL-17 represents a potential therapeutic strategy for the treatment of heart failure.