CaMKIIδ, Stabilized by RNA N6-Methyladenosine Reader IGF2BP2, Boosts Coxsackievirus B3-Induced Myocardial Inflammation via Interacting with TIRAP.

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to be aberrantly activated in viral myocarditis (VMC), but the role of its subtype CaMKIIδ in VMC remains unclear.VMC mice and cardiomyocytes models were induced by Coxsackievirus B3 (CVB3) treatment. Mice that underwent sham surgery and saline-treated cardiomyocytes served as controls. Body weight, survival, left ventricular ejection fraction (LVEF), and fractional shortening (LVFS) were measured, and HE staining was performed to evaluate heart function in VMC mice model and sham control. Inflammation factors in serum or cell supernatant were detected by ELISA. Expressions of CaMKIIδ, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), nuclear factor NF-kappaB (NF-κB) signals, and inflammation factors were examined by quantitative real time polymerase chain reaction (qRT-PCR) or western blot. CCK-8, EdU, and flow cytometry were used to evaluate cell behaviors. Co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down were utilized to validate molecule interaction. Methylated RNA immunoprecipitation (MeRIP) was performed to measure N6-methyladenosine (m6A) level of specific molecule.CaMKIIδ was upregulated in VMC mice and CVB3-treated primary cardiomyocytes, of which knockdown improved cell viability, proliferation, and suppressed cell apoptosis in vitro, thereby alleviating myocarditis in vivo. The stability of CaMKIIδ was attributed to the presence of IGF2BP2 through m6A modification. Loss of CaMKIIδ repressed NF-κB pathway via negatively and directly regulating TIRAP to be involved in inflammatory damage.CaMKIIδ, stabilized by m6A reader IGF2BP2, modulated NF-κB pathway via interacting with TIRAP to alter cell viability, proliferation, and apoptosis, thereby affecting VMC outcome.

Interfering with Dusp2 alleviates high glucose-induced vascular endothelial cell dysfunction by promoting p38 MAPK pathway activation.

BACKGROUND: Hyperglycemia-induced vascular endothelial cell dysfunction is a major factor contributing to diabetic lower extremity ischemia. We intend to investigate the role of Dusp2 in hyperglycemia-induced vascular endothelial cell dysfunction and related mechanisms. METHODS: The human umbilical vein endothelial cells (HUVECs) were treated with high glucose (HG) as the cell model. Streptozotocin injection was performed to induce diabetes and femoral artery ligation was to induce hind limb ischemia in mice. The levels of Dusp2, p-p38 MAPK, E2F4, and p38 MAPK were evaluated by Western blot or quantitative real-time PCR. The laser Doppler perfusion imaging was conducted to measure blood flow recovery. The cell counting kit-8, transwell, and tube formation assay were performed to evaluate cell proliferation, migration, and angiogenesis, respectively. CD31 immunohistochemical staining was carried out to detect the capillary density of gastrocnemius. The dual-luciferase reporter gene assay and Chromatin immunoprecipitation assay were executed to explore the interaction between E2F4 and Dusp2. RESULTS: Dusp2 was highly expressed in HG-induced HUVECs and diabetic lower extremity ischemia model mice. Interference with Dusp2 promoted cell proliferation, migration, and angiogenesis, as well as alleviated mouse diabetic hindlimb ischemia. Dusp2 knockdown up-regulated p-p38 MAPK levels. We verified the binding between E2F4 and Dusp2. Overexpressing E2F4 suppressed Dusp2 levels and promoted cell proliferation, migration, and angiogenesis, co-overexpression of Dusp2 reversed the results. CONCLUSIONS: Overexpressing E2F4 promotes endothelial cell proliferation, migration, and angiogenesis by inhibiting Dusp2 expression and activating p38 MAPK to alleviate vascular endothelial cell dysfunction under HG stimulation.

MiR-100-5p regulates cardiac hypertrophy through activation of autophagy by targeting mTOR.

Autophagy has been proved to play a vital role in cardiac hypertrophy. The present study was designed to investigate the relationship between miR-100-5p and autophagy in the development of cardiac hypertrophy. Here, miR-100-5p expression was detected in abdominal aortic coarctation (AAC)-induced cardiac hypertrophy rats and Angiotensin II (Ang II)-stimulated cardiomyocytes. In vitro and in vivo experiments were performed to explore the function of miR-100-5p on autophagy and cardiac hypertrophy. We also investigated the mechanism of miR-100-5p on autophagy with dual-luciferase reporter assays, RNA immunoprecipitation (RIP), quantitative real-time PCR (qRT-PCR), western blot, immunofluorescence, and transmission electron microscopy (TEM). The results showed that miR-100-5p was highly expressed in hypertrophic hearts and Ang II-induced cardiomyocytes. Overexpression of miR-100-5p promoted the expression of cardiac hypertrophy markers ANP, BNP and ß-MHC and cell surface area, while those were suppressed by miR-100-5p inhibitor. Knockdown of miR-100-5p by antagomiR significantly improves cardiac function and attenuate cardiac hypertrophy in vivo. Mechanistic investigation has found that miR-100-5p promote autophagy by targeting mTOR. Inhibition of autophagy by 3-methyladenine (3-MA) or mTOR overexpression could reverse the function of miR-100-5p in cardiac hypertrophy. These results elucidate that miR-100-5p promoted the pathogenesis of cardiac hypertrophy through autophagy activation by targeting mTOR.

LncRNA SNHG12 alleviates hypertensive vascular endothelial injury through miR-25-3p/SIRT6 pathway.

The objective of this study was to find the role of LncRNA SNHG12 in the regulation of hypertensive vascular endothelial injury. LncRNA SNHG12 and miR-25-3p expression were detected by quantitative RT-PCR. Protein levels of Sirtuin 6 (SIRT6), endothelial cell (EC) senescence markers p16 and p21, and EC marker CD31 were measured by Western blot. The apoptosis of HUVECs was detected by flow cytometry. The binding between LncRNA SNHG12 and miR-25-3p was verified by dual luciferase reporter gene assay and RNA pull-down assay. As a result, LncRNA SNHG12 was down-regulated in aortic primary ECs isolated from Ang II-induced hypertensive mice and 1 kidney/deoxycorticosterone acetate/salt-induced hypertensive mice. In Ang II-treated HUVECs, the expression level of SNHG12 was reduced and the overexpression of SNHG12 inhibited EC senescence markers p16 and p21 expressions, the apoptosis of HUVECs, and caspase-3 activity. Further investigation confirmed that LncRNA SNHG12 bound to miR-25-3p, and negatively regulated miR-25-3p expression. MiR-25-3p directly targeted SIRT6 and negatively regulated SIRT6 expression. In addition, SNHG12 overexpression inhibited Ang II-induced HUVECs injury through regulating miR-25-3p. Finally, in vivo experiments showed LncRNA SNHG12 overexpression alleviated vascular endothelial injury in Ang II-induced hypertensive mice. In conclusion, LncRNA SNHG12 alleviates vascular endothelial injury induced by hypertension through miR-25-3p/SIRT6 pathway.

Knockdown of dual oxidase 1 suppresses activin A-induced fibrosis in cardiomyocytes via the reactive oxygen species-dependent pyroptotic pathway.

Fibrotic diseases account for more than 8 million deaths worldwide annually. Reactive oxygen species (ROS) has been shown to activate pyroptosis and promote the production of interleukin (IL)-1ß and IL-18, leading to fibrosis development. However, the role of dual oxidase 1 (DUOX1)-induced ROS production and pyroptosis in cardiac fibrosis remains largely unknown. Activin A was used to induce ROS and pyroptosis in cardiomyocytes. ROS level, pyroptosis, and cytokine production were detected using Active Oxygen Detection Kit, flow cytometry, and enzyme-linked immunosorbent assay, respectively. Western blotting analysis was used to measure expression changes of proteins. DUOX1 was silenced or overexpressed to investigate its role in fibrosis. We found that activin A induced ROS production and pyroptosis in cardiomyocytes, which was blocked by the ROS scavenger, N-acetyl-L-cysteine (NAC). Knockdown of DUOX1 reversed activin A-induced ROS production, pyroptosis, cytokine release, and the upregulation of proinflammatory proteins. Overexpression of DUOX1 resulted in opposite effects of knockdown DUOX1. Administration of an ROS scavenger blocked the effect of DUOX1 overexpression. Supplementation of IL-1ß and IL-18 caused significant fibrosis in human cardiac fibroblasts (hCFs). The knockdown of DUOX1 protected cardiomyocytes against activin A-induced fibrosis via the inhibition of ROS, cytokine release, and pyroptosis.

Effects of PGC1α on myocardial ischemia reperfusion injury and the underlying mechanisms. / PGC1αå¯¹å¿ƒè‚Œç¼ºè¡€å†çŒæ³¨æŸä¼¤çš„ä½œç”¨åŠå ¶æœºåˆ¶.

OBJECTIVES: Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) controls mitochondrial biogenesis, but its role in cardiovascular diseases is unclear. The purpose of this study is to explore the effect of PGC1α on myocardial ischemia-reperfusion injury and the underlying mechanisms. METHODS: The transverse coronary artery of SD rat was ligated for 30 minutes followed by 2 hours of reperfusion. Triphenyltetrazolium chloride (TTC) staining was performed to measure the area of myocardial infarction. Immunohistochemistry and Western blotting were used to detect the PGC1α expression in myocardium. The rat cardiomyocyte H9C2 was subjected to hypoxia/reoxygenation (H/R) with the knockdown of PGC1α or hypoxia- inducible factor 1α (HIF-1α), or with treatment of metformin. Western blotting was used to detect the expression of PGC1α, HIF-1α, p21, BAX, and caspase-3. CCK-8 was performed to detect cell viability, and flow cytometry was used to detect apoptosis and mitochondrial superoxide (mitoSOX) release. RT-qPCR was used to detect the mRNA expression of PGC1α and HIF-1α. Besides, chromatin immunoprecipitation (ChIP)-qPCR and luciferase reporter gene assay were applied to detect the transcriptional regulation effect of HIF-1α on PGC1α. RESULTS: After I/R, the PGC1α expression was increased in infarcted myocardium. H/R induced H9C2 cell apoptosis (P<0.001). The release of mitoSOX (P<0.001) and protein expression of PGC1α, and apoptosis-related p21, BAX, and caspase-3 were increased. However, knockdown of PGC1α reduced apoptosis (P<0.01) and mitoSOX release (P<0.001), and decreased protein expression of apoptosis-related gene. HIF-1α is bound to the promoter region of PGC1α. Knockdown of HIF-1α inhibited the transcription and protein expression of PGC1α and further to reduce the apoptosis (all P<0.001) and mitoSOX release (P<0.01). While overexpression of PGC1α reversed the effects caused by HIF-1α knockdown (all P<0.001). Metformin effectively reduced H/R-induced apoptosis (P=0.013), mitoSOX release (P<0.001), HIF-1α, PGC1α and apoptosis-related protein expression, recovered the cell viability (P<0.001), and reduced myocardial infarction (P=0.003). CONCLUSIONS: After I/R, HIF-1α up-regulates the expression of PGC1α, leading to an increase in ROS production and aggravation of injury. Metformin can inhibit the accumulation of HIF-1α during hypoxia and effectively protect myocardium from ischemia/reperfusion injury.

Activation of CaMKII via ER-stress mediates coxsackievirus B3-induced cardiomyocyte apoptosis.

Cardiomyocyte apoptosis contributes to the development of coxsackievirus B3 (CVB3)-induced myocarditis, but the mechanism for the apoptosis by CVB3 infection remains unclear. Here, we showed that CVB3-induced endoplasmic reticulum (ER) stress response and apoptosis in cultured H9c2 cardiomyocytes. We found that Ca2+ -calmodulin-dependent kinase II (CaMKII) was activated by ER stress-dependent intracellular Ca2+ overload in the CVB3-infected H9c2 cardiomyocytes. Treatment with an inhibitor of ER stress, 4-phenylbutyric acid (4-PBA), attenuated intracellular Ca2+ accumulation indirectly and reduced CaMKII activity. Inhibition of CaMKII with pharmacological inhibitor (KN-93) or short hairpin RNA reduced CVB3-induced H9c2 apoptosis and repressed cytochrome c release from mitochondria to cytoplasm; whereas overexpression of the activated mutant of CaMKII (CaMKII-T287D) enhanced CVB3-induced H9c2 apoptosis and mitochondrial cytochrome c release, which could be alleviated by blocking of mitochondrial Ca2+ uniporter or mitochondrial permeability transition pore. Further in vivo investigation revealed that blocking of CaMKII with KN-93 prevented cardiomyocytes apoptosis and improved cardiac contractile function in CVB3-infected mouse heart. Collectively, these findings provide a novel evidence that CaMKII plays a vital role in the promotion of CVB3-induced cardiomyocyte apoptosis, which links ER stress and mitochondrial Ca2+ uptake.

Plasma galectin-3 predicts clinical outcomes after catheter ablation in persistent atrial fibrillation patients without structural heart disease.

AIMS: This study sought to explore the relationship between plasma galectin-3 (Gal-3) and persistent atrial fibrillation (PsAF), and investigate whether Gal-3 predicts clinical outcomes in patients with PsAF undergoing catheter ablation. METHODS: Fifty consecutive PsAF patients without coexisting structural heart disease undergoing first-time catheter ablation and 46 healthy controls were included. Blood samples were collected on admission for analysis of plasma Gal-3. Pre-ablation clinical and laboratory data were also recorded. Persistent atrial fibrillations patients were followed after ablation and AF recurrence was defined as episodes of AF or atrial tachycardia lasting >30 s after the blanking period. RESULTS: Plasma Gal-3 concentrations were higher in PsAF patients than in healthy controls (P < 0.001). In PsAF group, those with AF recurrence had higher plasma Gal-3 than did those without recurrence (P = 0.007). Both Gal-3 (hazard ratio 1.28, P = 0.006) and left atrial diameter (LAD) (hazard ratio 1.1, P = 0.025) were independent predictors of AF recurrence after ablation. Moreover, adding Gal-3 to LAD had an incremental predictive value for ablation outcomes (global χ(2) of LAD alone: 8.2; LAD and Gal-3 concentrations: 15.7; P = 0.006). CONCLUSION: Plasma Gal-3 concentrations are elevated in PsAF patients without structural heart disease and independently predict AF recurrence after ablation. Plasma Gal-3 concentration may be helpful in identifying appropriate candidates for AF ablation.

Pouched mitral isthmus is associated with incomplete linear block in atrial fibrillation patients with mechanical mitral valve replacement.

BACKGROUND: Previous studies have described the impact of mitral isthmus (MI) anatomy on the likelihood of achieving MI linear block in patients with native mitral valves (NMV) who underwent atrial fibrillation (AF) ablation. However, none have investigated that issue in AF patients with mechanical mitral valve replacements (MMVR). METHODS AND RESULTS: Twenty-nine consecutive patients who developed symptomatic persistent AF post-MMVR and referred for ablation were enrolled. Twenty-nine patients with NMV who underwent ablation of persistent AF during the same period were matched. With preprocedural cardiac computed tomographic imaging, MI anatomical features of all the participants were analyzed. Pouched MI was observed in 19 (65.5%) MMVR patients versus to 6 (20.7%) controls (P = 0.001). Bidirectional linear block across MI was achieved in 21 (72.4%) MMVR patients and 22 (75.9%) in the controls (P = 0.764). In the multivariable analysis, pouched MI was an independent predictor of incomplete MI block. CONCLUSIONS: Pouched MI accounts for the majority of AF patients with MMVR and may be associated with incomplete bidirectional linear block of MI.

Catheter ablation of atrial fibrillation in patients with atrial septal defect: long-term follow-up results.

INTRODUCTION: Atrial fibrillation (AF) is commonly found in patients with structural heart disease (SHD), including atrial septal defect (ASD). The feasibility and safety of ablation for AF in patients with unrepaired ASD is seldom reported. OBJECTIVES: This study aims to evaluate and compare the long-term efficacy of AF ablation in patients with and without ASD. METHODS: From January 2008 to December 2012, 18 consecutive patients were identified with medically refractory AF and an unrepaired ASD under catheter ablation. For each ASD patient, four control subjects were matched from our database. RESULTS: There were no significant differences between groups in terms of age, sex, type of AF, LA diameter, LVIDD, and EF. The mean procedural and fluoroscopy times were not different between the groups (p = NS). After a median follow-up of 20 months, the patients in the ASD group had 44.4 % AF recurrence after a single procedure compared with 34.7 % in the control group (p = 0.11). The mean LA diameter in non-recurrent patients was smaller than in recurrent patients (p = 0.03). In univariate Cox proportional hazards analyses, the factor found to have a significant association with arrhythmia recurrences was left atrial diameter (hazard ratio 1.059, 95 % confidence interval 1.002 to 1.120, p = 0.03). CONCLUSIONS: These results indicate that in patients with AF and an ASD amenable to percutaneous closure, a staged approach with radiofrequency ablation of AF preceding closure is a rational strategy.

Pacing or ablation: which is better for paroxysmal atrial fibrillation-related tachycardia-bradycardia syndrome?

BACKGROUND: Symptomatic prolonged sinus pauses on termination of atrial fibrillation (AF) are an accepted indication for pacemaker implantation. We evaluated the outcome of AF ablation in patients with paroxysmal AF-related tachycardia-bradycardia syndrome and compared the efficacy of catheter ablation with permanent pacing plus antiarrhythmic drugs (AADs). METHODS AND RESULTS: Patients with prolonged symptomatic sinus pauses on termination of AF were retrospectively analyzed. Forty-three consecutive patients who underwent catheter ablation (ABL group) were compared to 57 patients who underwent permanent pacing plus AADs (PM group). All 43 patients in the ABL group fulfilled Class I indication for pacemaker implantation at baseline but they actually underwent AF ablation. Reevaluation after 20.1 ± 9.6 months of follow-up showed that 41 patients (95.3%) did no longer need a pacemaker (Class III indication). Total cardiac-related rehospitalization was not significantly different between the two groups (P = 0.921). Tachycardia-related hospitalization was significantly higher in the PM group than the ABL group (14.0% and 0%, P = 0.029). More patients in the PM group were on AADs (PM 40.4%, ABL 4.7%, P < 0.001) while sinus rhythm maintenance was remarkably higher in the ABL group at the end of follow-up (83.7% vs 21.1% in PM group, P < 0.001). CONCLUSIONS: In patients with paroxysmal AF-related tachycardia-bradycardia syndrome, AF ablation seems to be superior to a strategy of pacing plus AAD. Pacemaker implantation can be waived in the majority of patients after a successful ablation.

Spontaneous coronary artery dissection and pseudoaneurysm: a case report.

Spontaneous coronary artery dissection (SCAD) is a very rare but potentially fatal condition, which often causes acute myocardial infarction and sudden cardiac death. Spontaneous coronary artery dissection associated with pseudoaneurysm has been rarely reported mostly managed with coronary artery bypass grafting. We report a female patient with SCAD and pseudoaneurysm who was treated by successful percutaneous coronary intervention.