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.

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.

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.

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.

Ablation of interleukin-17 alleviated cardiac interstitial fibrosis and improved cardiac function via inhibiting long non-coding RNA-AK081284 in diabetic mice.

Interleukin 17 (IL-17) plays an important role in the pathogenesis of cardiac interstitial fibrosis. In this study, we explored the role of interleukin-17 in the development of diabetic cardiomyopathy and the underlying mechanisms. The level of IL-17 increased in both the serum and cardiac tissue of diabetic mice. Knockout of IL-17 improved cardiac function of diabetic mice induced by streptozotocin (STZ), and significantly alleviated interstitial fibrosis as manifested by reduced collagen mRNA expression and collagen deposition evaluated by Masson's staining. High glucose treatment induced collagen production were abolished in cultured IL-17 knockout cardiac fibroblasts (CFs). The levels of long noncoding RNA-AK081284 were increased in the CFs treated with high glucose or IL-17. Knockout of IL-17 abrogated high glucose induced upregulation of AK081284. Overexpression of AK081284 in cultured CFs promoted the production of collagen and TGFß1. Both high glucose and IL-17 induced collagen and TGFß1 production were mitigated by the application of the siRNA for AK081284. In summary, deletion of IL-17 is able to mitigate myocardial fibrosis and improve cardiac function of diabetic mice. The IL-17/AK081284/TGFß1 signaling pathway mediates high glucose induced collagen production. This study indicates the therapeutic potential of IL-17 inhibition on diabetic cardiomyopathy disease associated with fibrosis.

Valsartan reduced the vulnerability to atrial fibrillation by preventing action potential prolongation and conduction slowing in castrated male mice.

INTRODUCTION: Deficiency of testosterone was associated with the susceptibility of atrial fibrillation (AF). Angiotensin-II (AngII) receptor antagonists were shown to reduce AF by improving atrial electrical remodeling. This study investigated the effects and mechanism of valsartan, an AngII receptor antagonist, on the susceptibility to AF with testosterone deficiency. METHODS AND RESULTS: Five-week-old male ICR mice were castrated and valsartan was administered orally (50 mg/kg/d). High-frequency electrical stimulation method was used to induce atrial arrhythmia. Patch-clamp technique was used for recording action potential duration (APD), transient outward potassium current ( I to ), sustained outward potassium current ( I ksus ), and late sodium current ( I Na-L ). Optical mapping technique was used to examine atrial conduction velocity (CV). The expression of connexin40 (Cx40) and Cx43 were detected by Western blot analysis. The occurrence rate of AF was significantly increased in castrated mice and APDs measured at 50% and 90% repolarization were markedly prolonged in castrated mice than controls, which were alleviated by the administration of valsartan. Valsartan suppressed the increase of INa-L and rescued the reduction of Ito and Iksus in castrated mice. The left atrial CV in castrated mice was decreased and the expression of Cx43 reduced than controls, which were restored after valsartan treatment. CONCLUSIONS: Valsartan reduced the susceptibility of AF in castrated mice, which may be related to the inhibition of action potential prolongation and improvement of atrial conduction impairment. This study indicates that valsartan may represent a useful agent for the prevention of AF pathogenesis in elderly male patients.

Anti-CTLA-4 m2a Antibody Exacerbates Cardiac Injury in Experimental Autoimmune Myocarditis Mice By Promoting Ccl5-Neutrophil Infiltration.

The risk for suffering immune checkpoint inhibitors (ICIs)-associated myocarditis increases in patients with pre-existing conditions and the mechanisms remain to be clarified. Spatial transcriptomics, single-cell RNA sequencing, and flow cytometry are used to decipher how anti-cytotoxic T lymphocyte antigen-4 m2a antibody (anti-CTLA-4 m2a antibody) aggravated cardiac injury in experimental autoimmune myocarditis (EAM) mice. It is found that anti-CTLA-4 m2a antibody increases cardiac fibroblast-derived C-X-C motif chemokine ligand 1 (Cxcl1), which promots neutrophil infiltration to the myocarditic zones (MZs) of EAM mice via enhanced Cxcl1-Cxcr2 chemotaxis. It is identified that the C-C motif chemokine ligand 5 (Ccl5)-neutrophil subpopulation is responsible for high activity of cytokine production, adaptive immune response, NF-κB signaling, and cellular response to interferon-gamma and that the Ccl5-neutrophil subpopulation and its-associated proinflammatory cytokines/chemokines promoted macrophage (Mφ) polarization to M1 Mφ. These altered infiltrating landscape and phenotypic switch of immune cells, and proinflammatory factors synergistically aggravated anti-CTLA-4 m2a antibody-induced cardiac injury in EAM mice. Neutralizing neutrophils, Cxcl1, and applying Cxcr2 antagonist dramatically alleviates anti-CTLA-4 m2a antibody-induced leukocyte infiltration, cardiac fibrosis, and dysfunction. It is suggested that Ccl5-neutrophil subpopulation plays a critical role in aggravating anti-CTLA-4 m2a antibody-induced cardiac injury in EAM mice. This data may provide a strategic rational for preventing/curing ICIs-associated myocarditis.

The role of FK506-binding proteins 12 and 12.6 in regulating cardiac function.

Specifically, FK506-binding proteins 12 (FKBP12) and 12.6 (FKBP12.6) are cis-trans peptidyl prolyl isomerases that are expressed in the heart. Both FKBP12 and FKBP12.6 were previously known to interact with ryanodine receptors in striated muscles. Although FKBP12 is abundantly present in the heart, its function in the heart is largely uncertain. Recently, by generating FKBP12 transgenic overexpression and cardiac-restricted knockout mice, we showed that FKBP12 is critically important in regulating trans-sarcolemmal ionic currents, predominately the voltage-gated Na+ current, I(Na), but it appears to be less important for regulating cardiac ryanodine receptor function. Similar genetic approaches also confirm the role of FKBP12.6 in regulating cardiac ryanodine receptors. The current study demonstrated that FKBP12 and FKBP12.6 have very different physiologic functions in the heart.

Calcium-sensing receptor activation contributed to apoptosis stimulates TRPC6 channel in rat neonatal ventricular myocytes.

Capacitative calcium entry (CCE) refers to the influx of calcium through plasma membrane channels activated on depletion of endoplasmic sarcoplasmic/reticulum (ER/SR) Ca(2+) stores, which is performed mainly by the transient receptor potential (TRP) channels. TRP channels are expressed in cardiomyocytes. Calcium-sensing receptor (CaR) is also expressed in rat cardiac tissue and plays an important role in mediating cardiomyocyte apoptosis. However, there are no data regarding the link between CaR and TRP channels in rat heart. In this study, in rat neonatal myocytes, by Ca(2+) imaging, we found that the depletion of ER/SR Ca(2+) stores by thapsigargin (TG) elicited a transient rise in cytoplasmic Ca(2+) ([Ca(2+)](i)), followed by sustained increase depending on extracellular Ca(2+). But, TRP channels inhibitor (SKF96365), not L-type channels or the Na(+)/Ca(2+) exchanger inhibitors, inhibited [Ca(2+)](i) relatively high. Then, we found that the stimulation of CaR with its activator gadolinium chloride (GdCl(3)) or by an increased extracellular Ca(2+)([Ca(2+)](o)) increased the concentration of intracelluar Ca(2+), whereas, the sustained elevation of [Ca(2+)](i) was reduced in the presence of SKF96365. Similarly, the duration of [Ca(2+)](i) increase was also shortened in the absence of extracellular Ca(2+). Western blot analysis showed that GdCl(3) increased the expression of TRPC6, which was reversed by SKF96365. Additionally, SKF96365 reduced cardiomyocyte apoptosis induced by GdCl(3). Our results suggested that CCE exhibited in rat neonatal myocytes and CaR activation induced Ca(2+)-permeable cationic channels TRPCs to gate the CCE, for which TRPC6 was one of the most likely candidates. TRPC6 channel was functionally coupled with CaR to enhance the cardiomyocyte apoptosis.

Post-conditioning protects cardiomyocytes from apoptosis via PKC(epsilon)-interacting with calcium-sensing receptors to inhibit endo(sarco)plasmic reticulum-mitochondria crosstalk.

The intracellular Ca(2+) concentration ([Ca(2+)](i)) is increased during cardiac ischemia/reperfusion injury (IRI), leading to endo(sarco)plasmic reticulum (ER) stress. Persistent ER stress, such as with the accumulation of [Ca(2+)](i), results in apoptosis. Ischemic post-conditioning (PC) can protect cardiomyocytes from IRI by reducing the [Ca(2+)](i) via protein kinase C (PKC). The calcium-sensing receptor (CaR), a G protein-coupled receptor, causes the production of inositol phosphate (IP(3)) to increase the release of intracellular Ca(2+) from the ER. This process can be negatively regulated by PKC through the phosphorylation of Thr-888 of the CaR. This study tested the hypothesis that PC prevents cardiomyocyte apoptosis by reducing the [Ca(2+)](i) through an interaction of PKC with CaR to alleviate [Ca(2+)](ER) depletion and [Ca(2+)](m) elevation by the ER-mitochondrial associated membrane (MAM). Cardiomyocytes were post-conditioned after 3 h of ischemia by three cycles of 5 min of reperfusion and 5 min of re-ischemia before 6 h of reperfusion. During PC, PKC(epsilon) translocated to the cell membrane and interacted with CaR. While PC led to a significant decrease in [Ca(2+)](i), the [Ca(2+)](ER) was not reduced and [Ca(2+)](m) was not increased in the PC and GdCl(3)-PC groups. Furthermore, there was no evident psi(m) collapse during PC compared with ischemia/reperfusion (I/R) or PKC inhibitor groups, as evaluated by laser confocal scanning microscopy. The apoptotic rates detected by TUNEL and Hoechst33342 were lower in PC and GdCl(3)-PC groups than those in I/R and PKC inhibitor groups. Apoptotic proteins, including m-calpain, BAP31, and caspase-12, were significantly increased in the I/R and PKC inhibitor groups. These results suggested that PKC(epsilon) interacting with CaR protected post-conditioned cardiomyocytes from programmed cell death by inhibiting disruption of the mitochondria by the ER as well as preventing calcium-induced signaling of the apoptotic pathway.

MicroRNAs: a novel class of potential therapeutic targets for cardiovascular diseases.

Currently, cardiovascular diseases remain one of the leading causes of morbidity and mortality in the world, indicating the need for innovative therapies and diagnosis for heart disease. MicroRNAs (miRNAs) have recently emerged as one of the central players in regulating gene expression. Numerous studies have documented the implications of miRNAs in nearly every pathological process of the cardiovascular system, including cardiac arrhythmia, cardiac hypertrophy, heart failure, cardiac fibrosis, cardiac ischemia and vascular atherosclerosis. More surprisingly, forced expression or suppression of a single miRNA is enough to cause or alleviate the pathological alteration, underscoring the therapeutic potential of miRNAs in cardiovascular diseases. In this review we summarize the key miRNAs that can solely modulate the cardiovascular pathological process and discuss the mechanisms by which they exert their function and the perspective of these miRNAs as novel therapeutic targets and/or diagnostic markers. In addition, current approaches for manipulating the action of miRNAs will be introduced.

M3 muscarinic acetylcholine receptor is associated with beta-catenin in ventricular myocytes during myocardial infarction in the rat.

1. The present study was designed to investigate whether the M(3) muscarinic acetylcholine receptors (mAChR) is associated with beta-catenin in the ventricular myocardium during ischaemic myocardial injury and to determine the possible mechanism/s involved. 2. Rat hearts were subjected to coronary artery ligation for 1 and 6 h or 1 month to establish a myocardial ischaemia (MI) model. In the acute MI model, 16 rats were randomized into four groups: (i) control; (ii) ischaemia (rats were subjected to 20 min coronary occlusion); (iii) choline (10 mg/kg, i.v., choline chloride, an M(3) receptor agonist, was administered 15 min before occlusion); and (iv) 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; 0.12 mg/kg 4-DAMP, an M(3) receptor antagonist, was administered 20 min before occlusion, followed 5 min later by 10 mg/kg, i.v., choline chloride). Immunochemistry, western blot analysis and immunoprecipitation were used to determine the expression and localization of beta-catenin and the M(3) mAChR. 3. Myocardial ischaemia caused a time-dependent increase in the expression of beta-catenin. Moreover, a physical association was found between beta-catenin and the M(3) mAChR in intercalated discs. This association was enhanced by prolonged ischaemia. Administration of choline before ischaemia not only increased beta-catenin expression, but also strengthened the association between beta-catenin and the M(3) mAChR. However, blockade of M(3) mAChR by 4-DAMP completely inhibited the effect of choline on the expression of beta-catenin. In addition, MI increased phosphorylation of the M(3) mAChR. 4. The results indicate that increased beta-catenin activity is associated with M(3) mAChR during MI. This association is likely to play a role in heart signal transduction during ischaemia between neighbouring ventricular myocardiocum.

[Effects of matrine, oxymatrine and resveratrol on HERG channel expression].

Because HERG potassium channel has important effects on both proarrhythmia and antiarrhythmia, we use immunofluorescence and Western blotting methods to detect the expression of HERG channel of HERG-HEK cells in different concentrations of matrine, oxymatrine and resveratrol. The findings showed that both matrine (1 micromol x L(-1) ) and oxymatrine ( 1micromol x L (-1) ) increased HERG channel expression ( n = 5, P < 0. 05 ) , while matrine (100 micromol x L(-1) ) decreased HERG channel expression ( n = 5, P < 0. 05), resveratrol didn't affect HERG channel expression. In conclusion, different concentrations of matrine and oxymatrine affect HERG channel expression, while there is no relationship between resveratrol and HERG channel expression. It provides a theoretical support for the safety and mechanism of anti-arrhythmic drugs.

Increment of late sodium currents in the left atrial myocytes and its potential contribution to increased susceptibility of atrial fibrillation in castrated male mice.

BACKGROUND: The incidence of atrial fibrillation (AF) is correlated with decreased levels of testosterone in elderly men. Late sodium current may exert a role in AF pathogenesis. OBJECTIVE: The purpose of this study was to explore the effect of testosterone deficiency on AF susceptibility and the therapeutic effect of late sodium current inhibitors in mice. METHODS: Male ICR mice (5 weeks old) were castrated to establish a testosterone deficiency model. One month after castration, dihydrotestosterone 5 mg/kg was administered subcutaneously for 2 months. Serum total testosterone level was assessed by enzyme-linked immunosorbent assay. High-frequency electrical stimulation was used to induce atrial arrhythmias. Whole-cell patch-clamp technique was used to for single-cell electrophysiologic study. RESULTS: Serum dihydrotestosterone levels of castration mice declined significantly but recovered with administration of exogenous dihydrotestosterone. In comparison with sham mice, the number of AF episodes significantly increased by 13.5-fold, AF rate increased by 3.75-fold, and AF duration prolonged in castrated mice. Dihydrotestosterone administration alleviated the occurrence of AF. Action potential duration at both 50% and 90% repolarization were markedly increased in castrated mice compared to sham controls. The late sodium current was enhanced in castrated male mice. These alterations were alleviated by treatment with dihydrotestosterone. Systemic application of the INa-L inhibitors ranolazine, eleclazine, and GS967 inhibited the occurrence of AF in castrated mice. CONCLUSION: Testosterone deficiency contributed to the increased late sodium current, prolonged action potential repolarization, and increased susceptibility to AF. Blocking of late sodium current is beneficial against the occurrence of AF in castrated mice.

[The cardioprotective effect and mechanism of lumbrokinase].

AIM: To investigate the protective effect of lumbrokinase against myocardial ischemia and to further explore its underlying mechanisms. METHODS: The effect of lumbrokinase on myocardial ischemia was observed by a model of acute myocardial infarction due to permanent ligation of the left anterior descending coronary artery in rats. Patch-clamp technique and laser scanning confocal microscopy were utilized to study the action of lumbrokinase on L-type calcium current (ICa-L) and intracellular calcium concentration ([Ca2+]i). RESULTS: Lumbrokinase decreased the infarct size of myocardium in a dose-dependent manner. The inhibitory rate of lumbrokinase at the dose of 20, 40 and 80 mg x kg(-1) was 7.7%, 34.6% and 46.2%, respectively. The electrophysiological studies displayed that, at + 10 mV, the ICa-L was markedly reduced from (-14.42 +/- 1.53) pA/pF to (-11.33 +/- 1.40) pA/pF (decreased by 21.4%, P <0.01) and (-9.92 +/- 1.31) pA/pF (decreased by 36.5%, P <0.01) by lumbrokinase (10 and 50 micromol x L(-1)), respectively. Confocal experiments showed that 10 micromol x L(-1) lumbrokinase showed no obvious effects on [Ca2+]i at resting states (P > 0.05). However, the increase of [Ca2+]i induced by 60 mmol x L(-1) KCl was distinctly limited by 10 micromol x L(-1) lumbrokinase (P <0.01). Within 240 s, the no obvious peak value of fluorescent intensity (FI) was shown. CONCLUSION: Lumbrokinase showed protective action against myocardial infarction in rats. The possible mechanisms of anti-ischemia could be attributed to decreasing ICa-L and [Ca2+] of ventricular myocytes in rats.

[Integration between M3 muscarinic acetylcholine receptor and connexin 43 as antiarrhythmic targets in rat ventricular myocardium].

AIM: To optimize the method of investigating structural integration between proteins and study the integration between arrhythmia related proteins in molecular level. METHODS: Immunostaining the normal ventricular myocytes was used to observe the distribution of connexin 43 and muscarinic acetylcholine receptor (mAChR). The five mAChR subtypes were precipitated using immunoprecipitation. Then, SDS-PAGE and Western blotting with the anti-connexin 43 antibody were performed to observe whether they were structurally integrated. Further, different concentrations of detergent were used to observe whether this relationship could be broken. RESULTS: The five subtypes of mAChR existed in the cardiac myocyte of the rat, and all the five mAChR subtypes combined with connexin 43. In the normal rat ventricular myocyte membrane, connexin 43 and M3 receptor are co-located. When adding certain concentration of detergent to the membrane protein, the integration between M3 receptor and connexin 43 was broken, and the phosphorylated form of connexin 43 integrated with M3 receptor. CONCLUSION: The results indicated that the structural integration between mAChR and phosphorylation of connexin 43 existed in rat ventricular myocardium, and this integration could be broken by certain concentration of detergent.

FGF21 ameliorates the neurocontrol of blood pressure in the high fructose-drinking rats.

Fibroblast growth factor-21 (FGF21) is closely related to various metabolic and cardiovascular disorders. However, the direct targets and mechanisms linking FGF21 to blood pressure control and hypertension are still elusive. Here we demonstrated a novel regulatory function of FGF21 in the baroreflex afferent pathway (the nucleus tractus solitarii, NTS; nodose ganglion, NG). As the critical co-receptor of FGF21, ß-klotho (klb) significantly expressed on the NTS and NG. Furthermore, we evaluated the beneficial effects of chronic intraperitoneal infusion of recombinant human FGF21 (rhFGF21) on the dysregulated systolic blood pressure, cardiac parameters, baroreflex sensitivity (BRS) and hyperinsulinemia in the high fructose-drinking (HFD) rats. The BRS up-regulation is associated with Akt-eNOS-NO signaling activation in the NTS and NG induced by acute intravenous rhFGF21 administration in HFD and control rats. Moreover, the expressions of FGF21 receptors were aberrantly down-regulated in HFD rats. In addition, the up-regulated peroxisome proliferator-activated receptor-γ and -α (PPAR-γ/-α) in the NTS and NG in HFD rats were markedly reversed by chronic rhFGF21 infusion. Our study extends the work of the FGF21 actions on the neurocontrol of blood pressure regulations through baroreflex afferent pathway in HFD rats.

Deletion of interleukin-6 alleviated interstitial fibrosis in streptozotocin-induced diabetic cardiomyopathy of mice through affecting TGFß1 and miR-29 pathways.

Interleukin 6 (IL-6) has been shown to be an important regulator of cardiac interstitial fibrosis. In this study, we explored the role of interleukin-6 in the development of diabetic cardiomyopathy and the underlying mechanisms. Cardiac function of IL-6 knockout mice was significantly improved and interstitial fibrosis was apparently alleviated in comparison with wildtype (WT) diabetic mice induced by streptozotocin (STZ). Treatment with IL-6 significantly promoted the proliferation and collagen production of cultured cardiac fibroblasts (CFs). High glucose treatment increased collagen production, which were mitigated in CFs from IL-6 KO mice. Moreover, IL-6 knockout alleviated the up-regulation of TGFß1 in diabetic hearts of mice and cultured CFs treated with high glucose or IL-6. Furthermore, the expression of miR-29 reduced upon IL-6 treatment, while increased in IL-6 KO hearts. Overexpression of miR-29 blocked the pro-fibrotic effects of IL-6 on cultured CFs. In summary, deletion of IL-6 is able to mitigate myocardial fibrosis and improve cardiac function of diabetic mice. The mechanism involves the regulation of IL-6 on TGFß1 and miR-29 pathway. This study indicates the therapeutic potential of IL-6 suppression on diabetic cardiomyopathy disease associated with fibrosis.