LRP5 regulates cardiomyocyte proliferation and neonatal heart regeneration by the AKT/P21 pathway.

The neonatal heart can efficiently regenerate within a short period after birth, whereas the adult mammalian heart has extremely limited capacity to regenerate. The molecular mechanisms underlying neonatal heart regeneration remain elusive. Here, we revealed that as a coreceptor of Wnt signalling, low-density lipoprotein receptor-related protein 5 (LRP5) is required for neonatal heart regeneration by regulating cardiomyocyte proliferation. The expression of LRP5 in the mouse heart gradually decreased after birth, consistent with the time window during which cardiomyocytes withdrew from the cell cycle. LRP5 downregulation reduced the proliferation of neonatal cardiomyocytes, while LRP5 overexpression promoted cardiomyocyte proliferation. The cardiac-specific deletion of Lrp5 disrupted myocardial regeneration after injury, exhibiting extensive fibrotic scars and cardiac dysfunction. Mechanistically, the decreased heart regeneration ability induced by LRP5 deficiency was mainly due to reduced cardiomyocyte proliferation. Further study identified AKT/P21 signalling as the key pathway accounting for the regulation of cardiomyocyte proliferation mediated by LRP5. LRP5 downregulation accelerated the degradation of AKT, leading to increased expression of the cyclin-dependent kinase inhibitor P21. Our study revealed that LRP5 is necessary for cardiomyocyte proliferation and neonatal heart regeneration, providing a potential strategy to repair myocardial injury.

Toll-like receptor 3 controls QT interval on the electrocardiogram by targeting the degradation of Kv4.2/4.3 channels in the endoplasmic reticulum.

TLRs have been proven to be essential mediators for the early innate immune response. Overactivation of TLR-mediated immune signaling promotes deterioration of cardiovascular diseases; however, the role of TLRs in the heart under physiologic conditions remains neglected. Here, we show that Tlr3 deficiency induced the endoplasmic reticulum (ER) retention of Kv4.2/4.3 proteins and consequent degradation via the ubiquitin-proteasome pathway. Knockout of Tlr3 resulted in a prolonged QT interval (the space between the start of the Q wave and the end of the T wave) in mice with no significant signs of inflammation and tissue abnormality in cardiac muscles. Prolongation of action potential duration resulted from the depression of transient outward potassium channel (Ito) currents in Tlr3-deficient ventricular myocytes mirrored the change in QT interval. Mechanistically, we found that Tlr3 was exclusively localized in the ER of cardiomyocytes where it interacted with Kv4.2/4.3 subunits of Ito channel. Thus, our data indicated that TLR3 directly regulates Ito channel protein dynamics to maintain cardiac repolarization, which may implicate a new molecular surveillance system for cardiac electrophysiological homeostasis.-Gao, X., Gao, S., Guan, Y., Huang, L., Huang, J., Lin, L., Liu, Y., Zhao, H., Huang, B., Yuan, T., Liu, Y., Liang, D., Zhang, Y., Ma, X., Li, L., Li, J., Zhou, D., Shi, D., Xu, L., Chen, Y.-H. Toll-like receptor 3 controls QT interval on the electrocardiogram by targeting the degradation of Kv4.2/4.3 channels in the endoplasmic reticulum.

Cardiac Nav 1.5 is modulated by ubiquitin protein ligase E3 component n-recognin UBR3 and 6.

The voltage-gated Na(+) channel Nav 1.5 is essential for action potential (AP) formation and electrophysiological homoeostasis in the heart. The ubiquitin-proteasome system (UPS) is a major degradative system for intracellular proteins including ion channels. The ubiquitin protein ligase E3 component N-recognin (UBR) family is a part of the UPS; however, their roles in regulating cardiac Nav 1.5 channels remain elusive. Here, we found that all of the UBR members were expressed in cardiomyocytes. Individual knockdown of UBR3 or UBR6, but not of other UBR members, significantly increased Nav 1.5 protein levels in neonatal rat ventricular myocytes, and this effect was verified in HEK293T cells expressing Nav 1.5 channels. The UBR3/6-dependent regulation of Nav 1.5 channels was not transcriptionally mediated, and pharmacological inhibition of protein biosynthesis failed to counteract the increase in Nav 1.5 protein caused by UBR3/6 reduction, suggesting a degradative modulation of UBR3/6 on Nav 1.5. Furthermore, the effects of UBR3/6 knockdown on Nav 1.5 proteins were abolished under the inhibition of proteasome activity, and UBR3/6 knockdown reduced Nav 1.5 ubiquitylation. The double UBR3-UBR6 knockdown resulted in comparable increases in Nav 1.5 proteins to that observed for single knockdown of either UBR3 or UBR6. Electrophysiological recordings showed that UBR3/6 reduction-mediated increase in Nav 1.5 protein enhanced the opening of Nav 1.5 channels and thereby the amplitude of the AP. Thus, our findings indicate that UBR3/6 regulate cardiomyocyte Nav 1.5 channel protein levels via the ubiquitin-proteasome pathway. It is likely that UBR3/6 have the potential to be a therapeutic target for cardiac arrhythmias.

MiR-25 protects cardiomyocytes against oxidative damage by targeting the mitochondrial calcium uniporter.

MicroRNAs (miRNAs) are a class of small non-coding RNAs, whose expression levels vary in different cell types and tissues. Emerging evidence indicates that tissue-specific and -enriched miRNAs are closely associated with cellular development and stress responses in their tissues. MiR-25 has been documented to be abundant in cardiomyocytes, but its function in the heart remains unknown. Here, we report that miR-25 can protect cardiomyocytes against oxidative damage by down-regulating mitochondrial calcium uniporter (MCU). MiR-25 was markedly elevated in response to oxidative stimulation in cardiomyocytes. Further overexpression of miR-25 protected cardiomyocytes against oxidative damage by inactivating the mitochondrial apoptosis pathway. MCU was identified as a potential target of miR-25 by bioinformatical analysis. MCU mRNA level was reversely correlated with miR-25 under the exposure of H2O2, and MCU protein level was largely decreased by miR-25 overexpression. The luciferase reporter assay confirmed that miR-25 bound directly to the 3' untranslated region (UTR) of MCU mRNA. MiR-25 significantly decreased H2O2-induced elevation of mitochondrial Ca2+ concentration, which is likely to be the result of decreased activity of MCU. We conclude that miR-25 targets MCU to protect cardiomyocytes against oxidative damages. This finding provides novel insights into the involvement of miRNAs in oxidative stress in cardiomyocytes.

Integrated analysis reveals the dysfunction of intercellular communication and metabolic signals in dilated cardiomyopathy.

Aims: Dilated cardiomyopathy refers to a heart muscle condition characterized by structural and functional irregularities in the myocardium that are not related to ischemia. Due to diverse etiologies such as genetic mutations, infections, and exposure to toxins, dilated cardiomyopathy can lead to substantial morbidity and mortality despite advances in the management of heart failure in dilated cardiomyopathy patients. We sought to analyze the characteristics of cell-cell communication and the metabolic signaling pathways in dilated cardiomyopathy. Methods and results: The single-nucleus sequencing data of left ventricle samples were acquired from two donor datasets and two dilated cardiomyopathy datasets. Three dilated cardiomyopathy bulk-sequencing datasets were included to determine the shared dilated cardiomyopathy-specific alterations in differentially expressed genes and signaling pathways. Using "CellChat," we analyzed intercellular communication to grasp how cell clusters interact and to map out the impaired signaling pathways in both donor and dilated cardiomyopathy conditions. Gene set enrichment analysis was applied to compare the metabolic signaling before and after dilated cardiomyopathy. We showcased how cell clusters exhibited abnormal cell-to-cell signaling transduction and how each cell type displayed dysfunctional metabolic signaling pathways through the integration of various datasets. The crucial ligand-receptor signaling contributing to outgoing or incoming signaling of dilated cardiomyopathy was identified in a cell-type dependent way, and the cell-specific metabolic alterations in glucose, lipid and amino acid were determined. The expression of gene pairs in BMP and NOTCH signal, as well as the gene expression in the arginine metabolism was validated. Conclusions: We reveal the key signals and metabolic pathways for dilated cardiomyopathy adaptation and maintenance, providing potential targets for dilated cardiomyopathy interference.

CDC-like kinase 3 deficiency aggravates hypoxia-induced cardiomyocyte apoptosis through AKT signaling pathway.

Hypoxia-induced cardiomyocyte apoptosis is one major pathological change of acute myocardial infarction (AMI), but the underlying mechanism remains unexplored. CDC-like kinase 3 (CLK3) plays crucial roles in cell proliferation, migration and invasion, and nucleotide metabolism, however, the role of CLK3 in AMI, especially hypoxia-induced apoptosis, is largely unknown. The expression of CLK3 was elevated in mouse myocardial infarction (MI) models and neonatal rat ventricular myocytes (NRVMs) under hypoxia. Furthermore, CLK3 knockdown significantly promoted apoptosis and inhibited NRVM survival, while CLK3 overexpression promoted NRVM survival and inhibited apoptosis under hypoxic conditions. Mechanistically, CLK3 regulated the phosphorylation status of AKT, a key player in the regulation of apoptosis. Furthermore, overexpression of AKT rescued hypoxia-induced apoptosis in NRVMs caused by CLK3 deficiency. Taken together, CLK3 deficiency promotes hypoxia-induced cardiomyocyte apoptosis through AKT signaling pathway.

CDC-like kinase 4 deficiency contributes to pathological cardiac hypertrophy by modulating NEXN phosphorylation.

Kinase-catalyzed phosphorylation plays a crucial role in pathological cardiac hypertrophy. Here, we show that CDC-like kinase 4 (CLK4) is a critical regulator of cardiomyocyte hypertrophy and heart failure. Knockdown of Clk4 leads to pathological cardiomyocyte hypertrophy, while overexpression of Clk4 confers resistance to phenylephrine-induced cardiomyocyte hypertrophy. Cardiac-specific Clk4-knockout mice manifest pathological myocardial hypertrophy with progressive left ventricular systolic dysfunction and heart dilation. Further investigation identifies nexilin (NEXN) as the direct substrate of CLK4, and overexpression of a phosphorylation-mimic mutant of NEXN is sufficient to reverse the hypertrophic growth of cardiomyocytes induced by Clk4 knockdown. Importantly, restoring phosphorylation of NEXN ameliorates myocardial hypertrophy in mice with cardiac-specific Clk4 deletion. We conclude that CLK4 regulates cardiac function through phosphorylation of NEXN, and its deficiency may lead to pathological cardiac hypertrophy. CLK4 is a potential intervention target for the prevention and treatment of heart failure.

Modulation of Ca2+-induced Ca2+ release by ubiquitin protein ligase E3 component n-recognin UBR3 and 6 in cardiac myocytes.

Ca2+-induced Ca2+ release (CICR) from sarcoplasmic reticulum is a finely tuned process responsible for cardiac excitation and contraction. The ubiquitin-proteasome system (UPS) as a major degradative system plays a crucial role in the maintenance of Ca2+ homeostasis. The E3 component N-recognin (UBR) subfamily is a part of the UPS; however, the role of UBR in regulating cardiac CICR is unknown. In the present study, we found that among the UBR family, single knockdown of UBR3 or UBR6 significantly elevated the amplitude of sarcoplasmic reticulum Ca2+ release without affecting Ca2+ transient decay time in neonatal rat ventricular myocytes. The protein expression of alpha 1 C subunit of L-type voltage-dependent Ca2+ channel (Cav1.2) was increased after UBR3/6 knockdown, whereas the protein levels of RyR2, SERCA2a, and PLB remained unchanged. In line with the increase in Cav1.2 proteins, the UBR3/6 knockdown enhanced the current of Cav1.2 channels. Furthermore, the increase in Cav1.2 proteins caused by UBR3/6 reduction was not counteracted by a protein biosynthesis inhibitor, cycloheximide, suggesting a degradative regulation of UBR3/6 on Cav1.2 channels. Our results indicate that UBR3/6 modulates cardiac CICR via targeting Cav1.2 protein degradation.

Cold-inducible RNA-binding protein modulates atrial fibrillation onset by targeting multiple ion channels.

BACKGROUND: Atrial fibrillation (AF), the most common sustained arrhythmia, significantly increases cardiovascular and cerebrovascular morbidity and mortality. The pathogenesis and treatment of AF remain a major challenge in the field of cardiology. We previously found that cold-inducible RNA-binding protein (CIRP) regulated ventricular repolarization by posttranscriptionally regulating Kv4.2/4.3 ion channels in rats, but the role of CIRP in AF is not clear. OBJECTIVE: The purpose of this study was to confirm that CIRP participates in atrial electrophysiological remodeling and AF occurrence by regulating atrial channels posttranscriptionally. METHODS: Programmed intra-atrial stimulation was used to induce AF in wild-type or transcription activator-like effector nucleases-based CIRP knockout (KO) rats. Atrial optical mapping, patch clamp, Western blotting, RNA immunoprecipitation, and luciferase reporter assays were performed to evaluate the underlying mechanism of atrial electrical remodeling. RESULTS: First, we observed a shortened atrial effective refractory period and increased susceptibility to AF in CIRP KO rats. Second, atria-specific CIRP delivery through an adeno-associated viral vector serotype 9 prolonged the atrial effective refractory period and attenuated AF development in CIRP KO rats. Third, we observed the shortened action potential duration and enhanced expression of Kv1.5 and Kv4.2/4.3 in KO rats. The transient outward current blocker 4-Aminopyridine and ultrarapid component of the delayed rectifier current blocker Diphenyl phosphine oxide-1 restored the shortened action potential duration in KO atria. Finally, we demonstrated that CIRP suppressed Kv1.5 and Kv4.2/4.3 expression by directly targeting their 3'-untranslated regions. CONCLUSION: CIRP plays a protective role in preventing AF onset through the posttranscriptional regulation of Kv1.5 and Kv4.2/4.3.

Characterization of circRNA­associated ceRNA networks in patients with nonvalvular persistent atrial fibrillation.

Circular RNAs (circRNAs) are non-coding RNAs forming closed-loop structures, and their aberrant expression may lead to disease. However, the potential network of circRNA­associated competing endogenous RNA (ceRNA) involved in nonvalvular persistent atrial fibrillation (NPAF) has not been previously reported. In the present study, four left atrial appendages (LAA) of patients with NPAF and four normal LAAs were examined via RNA sequencing, and their potential functions were investigated via bioinformatics analysis. The circRNA­enriched genes were analyzed using Gene Ontology (GO) categories, while the enrichment of circRNAs was detected via the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A total of 296 significantly dysregulated circRNA transcripts were obtained, with 238 upregulated and 58 downregulated. A number of circRNAs were further confirmed using reverse transcription­quantitative polymerase chain reaction analysis. Furthermore, the more comprehensive circRNA­associated ceRNA networks were examined in patients with NPAF. GO categories and KEGG annotation analysis of circRNAs revealed that the circRNA­associated ceRNA networks were likely to influence AF though alterations in calcium and cardiac muscle contraction. The circRNA­associated ceRNA networks revealed that dysregulated circRNAs in NPAF may be involved in regulating hsa­microRNA (miR)­208b and hsa­miR­21. To the best of our knowledge, this study presents the circRNA­associated ceRNA networks in NPAF for the first time, which may have potential implications for the pathogenesis of AF. This study reveals a potential perspective from which to investigate circRNAs in circRNA­associated ceRNA networks (hsa_circRNA002085, hsa_circRNA001321) in NPAF, and provides a potential biomarker for AF.

Validation of SinoSCORE for isolated CABG operation in East China.

From January 2010 to December 2016, 1616 consecutive patients who underwent isolated coronary artery bypass grafting (CABG) were evaluated for their predicted mortality according to the online Sino System for Coronary Operative Risk Evaluation (SinoSCORE), European System for Cardiac Operative Risk Evaluation II (EuroSCORE II) and Society of Thoracic Surgeons (STS) risk evaluation system. The calibration and discrimination in the total and in the subsets were assessed by the Hosmer-Lemeshow (H-L) statistics and by the C statistics respectively, to evaluate the efficiency of the three risk evaluation systems. The realized mortality was 1.92% (31/1616). The predictive mortality of SinoSCORE, EuroSCORE II and STS risk evaluation system were 1.35%, 1.74% and 1.05%, respectively. SinoSCORE achieved best discrimination. When grouping by risk, SinoSCORE also achieved the best discrimination in high-risk group, followed by STS risk evaluation system and EuroSCORE II while SinoSCORE and EuroSCORE II had excellent performance in low-risk group. In terms of calibration, SinoSCORE, EuroSCORE II and STS risk evaluation system all achieved positive calibrations (H-L: P > 0.05) in the overall population and grouped subsets. SinoSCORE achieved good predictive efficiency in East China patients undergoing isolated CABG and showed no compromise when compared with EuroSCORE II and STS risk evaluation system.

LRP6 acts as a scaffold protein in cardiac gap junction assembly.

Low-density lipoprotein receptor-related protein 6 (LRP6) is a Wnt co-receptor in the canonical Wnt/ß-catenin signalling. Here, we report the scaffold function of LRP6 in gap junction formation of cardiomyocytes. Cardiac LRP6 is spatially restricted to intercalated discs and binds to gap junction protein connexin 43 (Cx43). A deficiency in LRP6 disrupts Cx43 gap junction formation and thereby impairs the cell-to-cell coupling, which is independent of Wnt/ß-catenin signalling. The defect in Cx43 gap junction resulting from LRP6 reduction is attributable to the defective traffic of de novo Cx43 proteins from the endoplasmic reticulum to the Golgi apparatus, leading to the lysosomal degradation of Cx43 proteins. Accordingly, the hearts of conditional cardiac-specific Lrp6-knockout mice consistently exhibit overt reduction of Cx43 gap junction plaques without any abnormality in Wnt signalling and are predisposed to lethal arrhythmias. These findings uncover a distinct role of LRP6 as a platform for intracellular protein trafficking.

miRNA-204 drives cardiomyocyte proliferation via targeting Jarid2.

OBJECTIVES: In mammals, the heart grows by hypertrophy but not proliferation of cardiomyocytes after birth. The paucity of cardiomyocyte proliferation limits cardiac regeneration in a variety of heart diseases. To explore the efficient strategies that drive cardiomyocyte proliferation, we employed in vitro and in vivo models to investigate the function of miRNA-204, which was demonstrated to regulate the proliferation and differentiation of human cardiac progenitor cells in our previous study. METHODS AND RESULTS: miRNA-204 overexpression markedly promoted cardiomyocyte proliferation in both neonatal and adult rat cardiomyocytes in vitro. Transgenic mice with the cardiac-specific overexpression of miRNA-204 exhibited excessive cardiomyocyte proliferation throughout the embryonic and adult stages, leading to a pronounced increase in ventricular mass. Accordingly, the cell cycle regulators, including Cyclin A, Cyclin B, Cyclin D2, Cyclin E, CDC2 and PCNA, were upregulated in miRNA-204 transgenic embryonic hearts. Furthermore, we demonstrated that miRNA-204 directly targeted Jarid2. Knockdown of Jarid2 mimicked the pro-proliferative effect of miRNA-204 overexpression on cultured rat cardiomyocytes, whereas enhanced expression of Jarid2 conferred the myocytes with substantial resistance to proliferation by miRNA-204 overexpression. CONCLUSION: Our findings identify a conserved role for miRNA-204 in regulating cardiomyocyte proliferation by targeting the Jarid2 signaling pathway.

Low expression of lncRNA-GAS5 is implicated in human primary varicose great saphenous veins.

The cellular mechanisms of primary varicose great saphenous veins (GSVs) involve inflammation, apoptosis, and proliferation of local cells and extracellular matrix degradation. Long non-coding RNAs (lncRNAs) play important roles in these cellular processes; however, which and how lncRNAs related to these mechanisms take effect on GSVs remain unclear. By screening lncRNAs that might experience changes in GSV varicosities, we selected the lower expressed lncRNA-GAS5 (growth arrest specific transcript 5) for functional assessments. Silencing of lncRNA-GAS5 promoted cell proliferation and migration, and cell cycle of the human saphenous vein smooth muscle cells (HSVSMCs), whereas overexpressing it inhibited these cellular behaviors and reduced apoptosis of HSVSMCs. RNA pull-down experiment revealed a direct bind of lncRNA-GAS5 to a Ca2+-dependent RNA-binding protein, Annexin A2. Further experiments showed that silencing of Annexin A2 reduced the HSVSMCs proliferation and vice versa. In the context of lncRNA-GAS5 knockdown, silencing of Annexin A2 reduced the proliferation of HSVSMCs while overexpression of Annexin A2 increased the proliferation. Thus, the low expression of lncRNA-GAS5 may facilitate HSVSMCs proliferation and migration through Annexin A2 and thereby the pathogenesis of GSV varicosities.