Analysis of population structure and genetic diversity of Camellia tachangensis in Guizhou based on SNP markers.

BACKGROUND: Camellia tachangensis F. C. Zhang is a five-compartment species in the ovary of tea group plants, which represents the original germline of early differentiation of some tea group plants. METHODS AND RESULTS: In this study, we analyzed single-nucleotide polymorphisms (SNPs) at the genome level, constructed a phylogenetic tree, analyzed the genetic diversity, and further investigated the population structure of 100 C. tachangensis accessions using the genotyping-by-sequencing (GBS) method. A total of 91,959 high-quality SNPs were obtained. Population structure analysis showed that the 100 C. tachangensis accessions clustered into three groups: YQ-1 (Village Group), YQ-2 (Forest Group) and YQ-3 (Transition Group), which was further consistent with the results of phylogenetic analysis and principal component analyses (PCA). In addition, a comparative analysis of the genetic diversity among the three populations (Forest, Village, and Transition Groups) detected the highest genetic diversity in the Transition Group and the highest differentiation between Forest and Village Groups. CONCLUSIONS: C. tachangensis plants growing in the forest had different genetic backgrounds from those growing in villages. This study provides a basis for the effective protection and utilization of C. tachangensis populations and lays a foundation for future C. tachangensis breeding.

Genome-wide association study, population structure, and genetic diversity of the tea plant in Guizhou Plateau.

BACKGROUND: Guizhou Plateau, as one of the original centers of tea plant, has a profound multi-ethnic cultural heritage and abundant tea germplasm resources. However, the impact of indigenous community factors on the genetic diversity, population structure and geographical distribution of tea plant is still unclear. RESULTS: Using the genotyping-by-sequencing (GBS) approach, we collected 415 tea plant accessions from the study sites, estimated genetic diversity, developed a core collection, and conducted a genome-wide association study (GWAS) based on 99,363 high-quality single-nucleotide polymorphisms (SNPs). A total of 415 tea accessions were clustered into six populations (GP01, GP02, GP03, GP04, GP05 and GP06), and the results showed that GP04 and GP05 had the highest and lowest genetic diversity (Pi = 0.214 and Pi = 0.145, respectively). Moreover, 136 tea accessions (33%) were selected to construct the core set that can represent the genetic diversity of the whole collection. By analyzing seven significant SNP markers associated with the traits such as the germination period of one bud and two leaves (OTL) and the germination period of one bud and three leaves (OtL), four candidate genes possibly related to OTL and OtL were identified. CONCLUSIONS: This study revealed the impact of indigenous communities on the population structure of 415 tea accessions, indicating the importance of cultural practices for protection and utilization of tea plant genetic resources. Four potential candidate genes associated with the OTL and OtL of tea plant were also identified, which will facilitate genetic research, germplasm conservation, and breeding.

Nogo-B mediates endothelial oxidative stress and inflammation to promote coronary atherosclerosis in pressure-overloaded mouse hearts.

AIMS: Endothelial dysfunction plays a pivotal role in atherosclerosis, but the detailed mechanism remains incomplete understood. Nogo-B is an endoplasmic reticulum (ER)-localized protein mediating ER-mitochondrial morphology. We previously showed endothelial Nogo-B as a key regulator of endothelial function in the setting of hypertension. Here, we aim to further assess the role of Nogo-B in coronary atherosclerosis in ApoE-/- mice with pressure overload. METHODS AND RESULTS: We generated double knockout (DKO) mouse models of systemically or endothelium-specifically excising Nogo-A/B gene on an ApoE-/- background. After 7 weeks of transverse aortic constriction (TAC) surgery, compared to ApoE-/- mice DKO mice were resistant to the development of coronary atherosclerotic lesions and plaque rapture. Sustained elevation of Nogo-B and adhesion molecules (VCAM-1/ICAM-1), early markers of atherosclerosis, was identified in heart tissues and endothelial cells (ECs) isolated from TAC ApoE-/- mice, changes that were significantly repressed by Nogo-B deficiency. In cultured human umbilical vein endothelial cells (HUVECs) exposure to inflammatory cytokines (TNF-α, IL-1ß), Nogo-B was upregulated and activated reactive oxide species (ROS)-p38-p65 signaling axis. Mitofusin 2 (Mfn2) is a key protein tethering ER to mitochondria in ECs, and we showed that Nogo-B expression positively correlated with Mfn2 protein level. And Nogo-B deletion in ECs or in ApoE-/- mice reduced Mfn2 protein content and increased ER-mitochondria distance, reduced ER-mitochondrial Ca2+ transport and mitochondrial ROS generation, and prevented VCAM-1/ICAM-1 upregulation and EC dysfunction, eventually restrained atherosclerotic lesions development. CONCLUSION: Our study revealed that Nogo-B is a critical modulator in promoting endothelial dysfunction and consequent pathogenesis of coronary atherosclerosis in pressure overloaded hearts of ApoE-/- mice. Nogo-B may hold the promise to be a common therapeutic target in the setting of hypertension.

AGEs-RAGE-KCa3.1 pathway mediates palmitic acid-induced migration of PBMCs from patients with type 2 diabetes.

Type 2 diabetes mellitus (T2DM) is characterized by chronic low-grade systemic inflammation. Tissue infiltration by monocyte migration contributes to the pathogenesis of vascular complications in T2DM. We studied the role of intermediate-conductance Ca2+-activated K+ (KCa3.1) channels in the palmitic acid (PA)-induced migration of peripheral blood mononuclear cells (PBMCs) from T2DM patients and the influence of advanced glycation endproducts (AGEs). A total of 49 T2DM patients and 33 healthy subjects was recruited into this study. Using flow cytometry and Western blotting analysis as well as cell migration assay, we found that there was a significant decrease in frequency of T lymphocytes and monocytes in CD45+ leukocyte population. PA at 100 µM stimulated migration of PBMCs from T2DM individuals, which was inhibited by the specific KCa3.1 channel blocker TRAM-34 (1 µM). The PBMC migration was positively correlated with glycosylated hemoglobin A1 chain (HbA1c) level of T2DM patients, an indicator of AGEs, and PBMCs with higher level of HbA1c showed upregulated expression of toll-like receptor (TLR) 2/4 and KCa3.1 channels. In THP-1 cells, AGEs at 200 µg/ml increased protein expression of TLR 2/4 and KCa3.1 channels, and were synergistically involved in PA-induced migration through receptors of AGEs (RAGE)-mediated KCa3.1 upregulation. In conclusion, in PBMCs of T2DM patients, AGEs promotes PA-induced migration via upregulation of TLR2/4 and KCa3.1 channels.

Mitochondrial damage in a Takotsubo syndrome-like mouse model mediated by activation of ß-adrenoceptor-Hippo signaling pathway.

Takotsubo syndrome (TTS) is characterized by short-term contractile dysfunction with its mechanism undefined. We showed that activation of cardiac Hippo pathway mediates mitochondrial dysfunction and that stimulation of ß-adrenoceptors (ßAR) activates Hippo pathway. Here, we investigated the role of ßAR-Hippo signaling in mediating mitochondrial dysfunction in isoproterenol (Iso)-induced TTS-like mouse model. Elderly postmenopausal female mice were administered with Iso (1.25 mg/kg/h for 23 h). Cardiac function was determined by serially echocardiography. At days 1 and 7 post-Iso exposure, mitochondrial ultrastructure and function were examined by electron microscopy and various assays. Alterations in cardiac Hippo pathway and effects of genetic inactivation of Hippo kinase (Mst1) on mitochondrial damage and dysfunction in the acute phase of TTS were investigated. Isoproterenol exposure induced acute increase in biomarkers of cardiac damage and ventricular contractile dysfunction and dilation. At day 1 post-Iso, we observed extensive abnormalities in mitochondrial ultrastructure, downregulation of mitochondrial marker proteins, and mitochondrial dysfunction evidenced by lower ATP content, increased lipid droplets, higher contents of lactate, and augmented reactive oxygen species (ROS). All changes were reversed by day 7. ßAR stimulation led to activation of cardiac Hippo pathway with enhanced expression of Hippo kinase Mst1 and inhibitory YAP phosphorylation, as well as reduced nuclear YAP-TEAD1 interaction. In mice with cardiac expression of inactive mutant Mst1 gene, acute mitochondrial damage and dysfunction were mitigated. Stimulation of cardiac ßAR activates Hippo pathway that mediates mitochondrial dysfunction with energy insufficiency and enhanced ROS, promoting acute but short-term ventricular dysfunction.NEW & NOTEWORTHY Takotsubo syndrome (TTS) is featured by activation of sympatho-ß-adrenoceptor (ßAR) system leading to acute loss of ventricular contractile performance. However, the molecular mechanism remains undefined. We demonstrated, in an isoproterenol-induced murine TTS-like model, extensive mitochondrial damage, metabolic dysfunction, and downregulated mitochondrial marker proteins, changes temporarily associated with cardiac dysfunction. Mechanistically, stimulation of ßAR activated Hippo signaling pathway and genetic inactivation of Mst1 kinase ameliorated mitochondrial damage and metabolic dysfunction at the acute phase of TTS.

Endothelial KCa3.1 and KCa2.3 Mediate S1P (Sphingosine-1-Phosphate)-Dependent Vasodilation and Blood Pressure Homeostasis.

BACKGROUND: S1P (sphingosine-1-phosphate) has been reported to possess vasodilatory properties, but the underlying pathways are largely unknown. METHODS: Isolated mouse mesenteric artery and endothelial cell models were used to determine S1P-induced vasodilation, intracellular calcium, membrane potentials, and calcium-activated potassium channels (KCa2.3 and KCa3.1 [endothelial small- and intermediate-conductance calcium-activated potassium channels]). Effect of deletion of endothelial S1PR1 (type 1 S1P receptor) on vasodilation and blood pressure was evaluated. RESULTS: Mesenteric arteries subjected to acute S1P stimulation displayed a dose-dependent vasodilation response, which was attenuated by blocking endothelial KCa2.3 or KCa3.1 channels. In cultured human umbilical vein endothelial cells, S1P stimulated immediate membrane potential hyperpolarization following activation of KCa2.3/KCa3.1 with elevated cytosolic Ca2+. Further, chronic S1P stimulation enhanced expression of KCa2.3 and KCa3.1 in human umbilical vein endothelial cells in dose- and time-dependent manners, which was abolished by disrupting either S1PR1-Ca2+ signaling or downstream Ca2+-activated calcineurin/NFAT (nuclear factor of activated T-cells) signaling. By combination of bioinformatics-based binding site prediction and chromatin immunoprecipitation assay, we revealed in human umbilical vein endothelial cells that chronic activation of S1P/S1PR1 promoted NFATc2 nuclear translocation and binding to promoter regions of KCa2.3 and KCa3.1 genes thus to upregulate transcription of these channels. Deletion of endothelial S1PR1 reduced expression of KCa2.3 and KCa3.1 in mesenteric arteries and exacerbated hypertension in mice with angiotensin II infusion. CONCLUSIONS: This study provides evidence for the mechanistic role of KCa2.3/KCa3.1-activated endothelium-dependent hyperpolarization in vasodilation and blood pressure homeostasis in response to S1P. This mechanistic demonstration would facilitate the development of new therapies for cardiovascular diseases associated with hypertension.

Hippo pathway activation mediates chemotherapy-induced anti-cancer effect and cardiomyopathy through causing mitochondrial damage and dysfunction.

Rationale: Chemotherapy is a common clinical strategy for cancer treatment. However, the accompanied cardiomyopathy renders cancer patients under risk of another life-threatening condition. Whereas Hippo pathway is known to play key roles in both cancerogenesis and heart disease, it remains unclear whether Hippo pathway activation mediates chemotherapy-induced cardiomyopathy. Methods and Results: In human breast cancer cells, doxorubicin (DOX) significantly induced upregulation of Hippo kinase Mst1, inhibitory phosphorylation of YAP, mitochondrial damage, reduced cell viability and increased apoptosis. Hippo pathway inactivation by Mst1-siRNA transfection effectively improved cell survival and mitigated mitochondrial damage and cell apoptosis. Another anti-cancer drug YAP inhibitor verteporfin also induced lower cancer cell viability, apoptosis and mitochondrial injury. Chronic treatment with DOX in vivo (4 mg/kg/week for 6 weeks) caused mitochondrial damage and dysfunction, oxidative stress and cardiac fibrosis, while acute DOX treatment (16 mg/kg single bolus) also induced myocardial oxidative stress and mitochondrial abnormalities. Chronic treatment with verteporfin (2 months) resulted in cardiomyopathy phenotypes comparable to that by chronic DOX regimen. In transgenic mice with cardiac overexpression of kinase-dead mutant Mst1 gene, these adverse cardiac effects of DOX were significantly attenuated relative to wild-type littermates. Conclusions: Anti-cancer action of both DOX and verteporfin is associated with Hippo pathway activation. Such action on cardiac Hippo pathway mediates mitochondrial damage and cardiomyopathy.

YAP-galectin-3 signaling mediates endothelial dysfunction in angiotensin II-induced hypertension in mice.

BACKGROUND: Vascular endothelial dysfunction is regarded as an early event of hypertension. Galectin-3 (Gal-3) is known to participate in various pathological processes. Whilst previous studies showed that inhibition of Gal-3 effectively ameliorates angiotensin II (Ang II)-induced atherosclerosis or hypertension, it remains unclear whether Ang II regulates Gal-3 expression and actions in vascular endothelium. METHODS: Using techniques of molecular biology and myograph, we investigated Ang II-mediated changes in Gal-3 expression and activity in thoracic aortas and mesenteric arteries from wild-type and Gal-3 gene deleted (Gal-3-/-) mice and cultured endothelial cells. RESULTS: The serum level of Gal-3 was significantly higher in hypertensive patients or in mice with chronic Ang II-infusion. Ang II infusion to wild-type mice enhanced Gal-3 expression in the aortic and mesenteric arteries, elevated systolic blood pressure and impaired endothelium-dependent relaxation of the thoracic aortas and mesenteric arteries, changes that were abolished in Gal-3-/- mice. In human umbilical vein endothelial cells, Ang II significantly upregulated Gal-3 expression by promoting nuclear localization of Yes-associated protein (YAP) and its interaction with transcription factor Tead1 with enhanced YAP/Tead1 binding to Gal-3 gene promoter region. Furthermore, Gal-3 deletion augmented the bioavailability of nitric oxide, suppressed oxidative stress, and alleviated inflammation in the thoracic aorta of Ang II-infused mice or endothelial cells exposed to Ang II. CONCLUSIONS: Our results demonstrate for the first time that Ang II upregulates Gal-3 expression via increment in YAP nuclear localization in vascular endothelium, and that Gal-3 mediates endothelial dysfunction contributing to the development of hypertension.

Evaluation of Coenzyme Q10 (CoQ10) Deficiency and Therapy in Mouse Models of Cardiomyopathy.

ABSTRACT: Mitochondrial dysfunction plays a key role in the development of heart failure, but targeted therapeutic interventions remain elusive. Previous studies have shown coenzyme Q10 (CoQ10) insufficiency in patients with heart disease with undefined mechanism and modest effectiveness of CoQ10 supplement therapy. Using 2 transgenic mouse models of cardiomyopathy owing to cardiac overexpression of Mst1 (Mst1-TG) or ß 2 -adrenoceptor (ß 2 AR-TG), we studied changes in cardiac CoQ10 content and alterations in CoQ10 biosynthesis genes. We also studied in Mst1-TG mice effects of CoQ10, delivered by oral or injection regimens, on both cardiac CoQ10 content and cardiomyopathy phenotypes. High performance liquid chromatography and RNA sequencing revealed in both models significant reduction in cardiac content of CoQ10 and downregulation of most genes encoding CoQ10 biosynthesis enzymes. Mst1-TG mice with 70% reduction in cardiac CoQ10 were treated with CoQ10 either by oral gavage or i.p. injection for 4-8 weeks. Oral regimens failed in increasing cardiac CoQ10 content, whereas injection regimen effectively restored the cardiac CoQ10 level in a time-dependent manner. However, CoQ10 restoration in Mst1-TG mice did not correct mitochondrial dysfunction measured by energy metabolism, downregulated expression of marker proteins, and oxidative stress nor to preserve cardiac contractile function. In conclusion, mouse models of cardiomyopathy exhibited myocardial CoQ10 deficiency likely due to suppressed endogenous synthesis of CoQ10. In contrast to ineffectiveness of oral administration, CoQ10 administration by injection regimen in cardiomyopathy mice restored cardiac CoQ10 content, which, however, failed in achieving detectable efficacy at molecular and global functional levels.

Galectin-3-centered paracrine network mediates cardiac inflammation and fibrosis upon ß-adrenergic insult.

Rapid over-activation of ß-adrenergic receptors (ß-AR) following acute stress initiates cardiac inflammation and injury by activating interleukin-18 (IL-18), however, the process of inflammation cascades has not been fully illustrated. The present study aimed to determine the mechanisms of cardiac inflammatory amplification following acute sympathetic activation. With bioinformatics analysis, galectin-3 was identified as a potential key downstream effector of ß-AR and IL-18 activation. The serum level of galectin-3 was positively correlated with norepinephrine or IL-18 in patients with chest pain. In the heart of mice treated with ß-AR agonist isoproterenol (ISO, 5 mg kg-1), galectin-3 expression was upregulated markedly later than IL-18 activation, and Nlrp3-/- and Il18-/- mice did not show ISO-induced galectin-3 upregulation. It was further revealed that cardiomyocyte-derived IL-18 induced galectin-3 expression in macrophages following ISO treatment. Moreover, galectin-3 deficiency suppressed ISO-induced cardiac inflammation and fibrosis without blocking ISO-induced IL-18 increase. Treatment with a galectin-3 inhibitor, but not a ß-blocker, one day after ISO treatment effectively attenuated cardiac inflammation and injury. In conclusion, galectin-3 is upregulated to exaggerate cardiac inflammation and injury following acute ß-AR activation, a galectin-3 inhibitor effectively blocks cardiac injury one day after ß-AR insult.

Case Report: A Novel Truncating Variant of NR0B1 Presented With X-Linked Late-Onset Adrenal Hypoplasia Congenita With Hypogonadotropic Hypogonadism.

Nuclear receptor subfamily 0 group B member 1 gene (NR0B1) encodes an orphan nuclear receptor that plays a critical role in the development and regulation of the adrenal gland and hypothalamic-pituitary-gonadal axis. In this study, we report a novel mutation in NR0B1 that led to adult-onset adrenal hypoplasia congenita (AHC) and pubertal development failure in a male adult. Clinical examinations revealed hyponatremia, elevated adrenocorticotropic hormone levels, reduced testosterone and gonadotropin levels, and hyper-responses to gonadotropin-releasing hormone and human chorionic gonadotropin stimulation tests. Whole-exome sequencing and Sanger sequencing were performed to identify the potential causes of AHC. Candidate variants were shortlisted based on the X-linked recessive models. Sequence analyses identified a novel hemizygous variant of c.1034delC in exon 1 of NR0B1 at Xp21.2, resulting in a frameshift mutation and premature stop codon formation. The c.1034delC/p.Pro345Argfs*27 in the NR0B1 gene was detected in the hemizygous state in affected males and in the heterozygous state in healthy female family carriers. These results expand the clinical features of AHC as well as the mutation profile of the causative gene NR0B1. Further studies are needed to elucidate the biological effects of the mutation on the development and function of the adrenal gland and the hypothalamic-pituitary-gonadal axis.

Transcriptomic Analysis of Dysregulated Genes of the nDNA-mtDNA Axis in a Mouse Model of Dilated Cardiomyopathy.

Background: Mitochondrial dysfunction is implicated in the development of cardiomyopathy and heart failure. Transcription of mitochondrial DNA (mtDNA) encoded genes and subsequent protein synthesis are tightly regulated by nuclear DNA (nDNA) encoded proteins forming the nDNA-mtDNA axis. The scale of abnormalities in this axis in dilated cardiomyopathy (DCM) is unclear. We previously demonstrated, in a mouse DCM model with cardiac Mst1 overexpression, extensive downregulation of mitochondrial genes and mitochondrial dysfunction. Using the pre-acquired transcriptome sequencing database, we studied expression of gene sets of the nDNA-mtDNA axis. Methods: Using RNA-sequencing data from DCM hearts of mice at early and severe disease stages, transcriptome was performed for dysregulated nDNA-encoded gene sets that govern mtDNA transcription and in situ protein synthesis. To validate gene data, expression of a panel of proteins was determined by immunoblotting. Results: Relative to littermate controls, DCM hearts showed significant downregulation of all mtDNA encoded mRNAs, as well as mtDNA transcriptional activators. Downregulation was also evident for gene sets of mt-rRNA processing, aminoacyl-tRNA synthases, and mitoribosome subunits for in situ protein synthesis. Multiple downregulated genes belong to mitochondrial protein-importing machinery indicating compromised importing of proteins for mtDNA transcription and translation. Diverse changes were genes of mtRNA-binding proteins that govern maturation and stability of mtDNA-derived RNAs. Expression of mtDNA replicome genes was largely unchanged. These changes were similarly observed in mouse hearts at early and severe stages of DCM. Conclusion: Transcriptome revealed in our DCM model dysregulation of multiple gene sets of the nDNA-mtDNA axis, that is, expected to interfere with mtDNA transcription and in situ protein synthesis. Dysfunction of the nDNA-mtDNA axis might contribute to mitochondrial dysfunction and ultimately development of DCM.

Pathogenetic Link of Cardiac Rupture and Left Ventricular Thrombus Following Acute Myocardial Infarction: A Joint Preclinical and Clinical Study.

Background: Cardiac rupture (CR) and left ventricular thrombus (LVT) remain important complications of acute myocardial infarction (MI), and they are currently regarded as independent events. We explored the pathogenetic link between CR and LVT by investigating a murine model of MI with a high frequency of CR and in patients with acute MI. Methods: MI was induced in mice, the onset of CR was monitored, and the hearts of mice with or without fatal CR were histologically examined. Between 2015 and 2022, from patients admitted due to acute MI, the data of patients with CR or LVT were retrospectively collected and compared to uncomplicated patients (control). Results: A total of 75% of mice (n = 65) with MI developed CR 2-4 days after MI. A histological examination of CR hearts revealed the existence of platelet-rich intramural thrombi in the rupture tunnel, which was connected at the endocardial site to platelet-fibrin thrombi within an LVT. In CR or non-CR mouse hearts, LV blood clots often contained a portion of platelet-fibrin thrombi that adhered to the infarct wall. In non-CR hearts, sites of incomplete CR or erosion of the infarct wall were typically coated with platelet thrombi and dense inflammatory cells. Of 8,936 patients with acute MI, CR and LVT occurred in 102 (1.14%) and 130 (1.45%) patients, respectively, with three cases having both complications. CR accounted for 32.8% of in-hospital deaths. The majority of CR (95%) or LVT (63%, early LVT) occurred within 7 days. In comparison to the control or LVT-late groups, patients with CR or early LVT reported increased levels of cellular and biochemical markers for inflammation or cardiac injury. Conclusion: CR and LVT after MI are potentially linked in their pathogenesis. LVT occurring early after MI may be triggered by a thrombo-inflammatory response following wall rupture or endocardial erosion.

Activation of Hippo signaling pathway mediates mitochondria dysfunction and dilated cardiomyopathy in mice.

Rationale: Mitochondrial dysfunction facilitates heart failure development forming a therapeutic target, but the mechanism involved remains unclear. We studied whether the Hippo signaling pathway mediates mitochondrial abnormalities that results in onset of dilated cardiomyopathy (DCM). Methods: Mice with DCM due to overexpression of Hippo pathway kinase Mst1 were studied. DCM phenotype was evident in adult animals but contractile dysfunction was identified as an early sign of DCM at 3 weeks postnatal. Electron microscopy, multi-omics and biochemical assays were employed. Results: In 3-week and adult DCM mouse hearts, cardiomyocyte mitochondria exhibited overt structural abnormalities, smaller size and greater number. RNA sequencing revealed comprehensive suppression of nuclear-DNA (nDNA) encoded gene-sets involved in mitochondria turnover and all aspects of metabolism. Changes in cardiotranscriptome were confirmed by lower protein levels of multiple mitochondrial proteins in DCM heart of both ages. Mitochondrial DNA-encoded genes were also downregulated; due apparently to repression of nDNA-encoded transcriptional factors. Lipidomics identified remodeling in cardiolipin acyl-chains, increased acylcarnitine content but lower coenzyme Q10 level. Mitochondrial dysfunction was featured by lower ATP content and elevated levels of lactate, branched-chain amino acids and reactive oxidative species. Mechanistically, inhibitory YAP-phosphorylation was enhanced, which was associated with attenuated binding of transcription factor TEAD1. Numerous suppressed mitochondrial genes were identified as YAP-targets. Conclusion: Hippo signaling activation mediates mitochondrial damage by repressing mitochondrial genes, which causally promotes the development of DCM. The Hippo pathway therefore represents a therapeutic target against mitochondrial dysfunction in cardiomyopathy.

Excess sarcoplasmic reticulum-mitochondria calcium transport induced by Sphingosine-1-phosphate contributes to cardiomyocyte hypertrophy.

Sphingosine-1-phosphate (S1P) has been shown to possess pro-hypertrophic properties in the heart, but the detailed molecular mechanism that underlies the pathological process is rarely explored. In the present study, we aim to explore the role of S1P-mediated intracellular Ca2+ signaling, with a focus on sarcoplasmic reticulum (SR)-mitochondria communication, in cardiomyocyte hypertrophy. Cultured neonatal rat ventricular myocytes (NRVMs) displayed significantly hypertrophic growth after treatment with 1 µmol/L S1P for 48 h, as indicated by the cell surface area or mRNA expressions of hypertrophic marker genes (ANP, BNP and ß-MHC). Importantly, mitochondrial Ca2+ and reactive oxygen species (ROS) levels were dramatically elevated upon S1P stimulation, and pharmacological blockage of which abolished NRVM hypertrophy. 0.5 Hz electrical pacing induced similar cytosolic Ca2+ kinetics to S1P stimulation, but unaffected the peak of mitochondrial [Ca2+]. With interference of the expression of type 2 inositol 1,4,5-trisphosphate receptors (IP3R2), which are unemployed in electrical paced Ca2+ activity but may be activated by S1P, alteration in mitochondrial Ca2+ as well as the hypertrophic effect in NRVMs under S1P stimulation were attenuated. The hypertrophic effect of S1P can also be abolished by pharmacological block of S1PR1 or Gi signaling. Collectively, our study highlights the mechanistic role of IP3R2-mediated excess SR-mitochondria Ca2+ transport in S1P-induced cardiomyocyte hypertrophy.

Stretch-induced sarcoplasmic reticulum calcium leak is causatively associated with atrial fibrillation in pressure-overloaded hearts.

AIMS: Despite numerous reports documenting an important role of hypertension in the development of atrial fibrillation (AF), the detailed mechanism underlying the pathological process remains incompletely understood. Here, we aim to test the hypothesis that diastolic sarcoplasmic reticulum (SR) Ca2+ leak in atrial myocytes, induced by mechanical stretch due to elevated pressure in the left atrium (LA), plays an essential role in the AF development in pressure-overloaded hearts. METHODS AND RESULTS: Isolated mouse atrial myocytes subjected to acute axial stretch displayed an immediate elevation of SR Ca2+ leak. Using a mouse model of transverse aortic constriction (TAC), the relation between stretch, SR Ca2+ leak, and AF susceptibility was further tested. At 36 h post-TAC, SR Ca2+ leak in cardiomyocytes from the LA (with haemodynamic stress), but not right atrium (without haemodynamic stress), significantly increased, which was further elevated at 4 weeks post-TAC. Accordingly, AF susceptibility to atrial burst pacing in the 4-week TAC mice were also significantly increased, which was unaffected by inhibition of atrial fibrosis or inflammation via deletion of galectin-3. Western blotting revealed that type 2 ryanodine receptor (RyR2) in left atrial myocytes of TAC mice was oxidized due to activation and up-regulation of Nox2 and Nox4. Direct rescue of dysfunctional RyR2 with dantrolene or rycal S107 reduced diastolic SR Ca2+ leak in left atrial myocytes and prevented atrial burst pacing stimulated AF. CONCLUSION: Our study demonstrated for the first time the increased SR Ca2+ leak mediated by enhanced oxidative stress in left atrial myocytes that is causatively associated with higher AF susceptibility in pressure-overloaded hearts.

AMPK upregulates KCa2.3 channels and ameliorates endothelial dysfunction in diet-induced obese mice.

The opening of endothelial small-conductance calcium-activated potassium channels (KCa2.3) is essential for endothelium-dependent hyperpolarization (EDH), which predominantly occurs in small resistance arteries. Adenosine monophosphate-activated protein kinase (AMPK), an important metabolic regulator, has been implicated in regulating endothelial nitric oxide synthase activity. However, it was unclear whether AMPK regulated endothelial KCa2.3-mediated EDH-type vasodilation. Using bioinformatics analysis and myograph system, we investigated the regulation by AMPK of KCa2.3 in human umbilical vein endothelial cells (HUVECs) or mouse second-order mesenteric resistance arteries. In HUVECs, AMPK activation either by activators (AICAR, A769662 and MK-8722) or expression of the constitutively active form of AMPK significantly upregulated KCa2.3 expression. Such effects were abolished by AMPK inhibitor (compound C) or AMPK α1-/α2-siRNA, extracellular-signal-regulated-kinase 5 (ERK5) inhibitor (ERK5-IN-1), and specific siRNA to myocyte-enhancer factor 2 (MEF2) or krüppel-like factor 2/4 (KLF2/4). KCa2.3 expression was significantly reduced in mesenteric resistance arteries in AMPKα2 knockout mice when compared with littermate control mice. Furthermore, in high-fat diet fed mice, 2-week treatment with AICAR restored endothelial KCa2.3 expression in mesenteric resistance arteries with improved endothelial dysfunction. Our results demonstrate that activation of AMPK upregulates KCa2.3 channel expression through the ERK5-MEF2-KLF2/4 signaling pathway in vascular endothelium, which contributes to benefits through KCa2.3-mediated EDH-type vasodilation in mesenteric resistance arteries.

Cardiac ß-adrenergic receptor activation mediates distinct and cell type-dependent changes in the expression and distribution of connexin 43.

Activation of the sympatho-ß-adrenergic receptors (ß-ARs) system is a hallmark of heart failure, leading to fibrosis and arrhythmias. Connexin 43 (Cx43) is the most abundant gap junctional protein in the myocardium. Current knowledge is limited regarding Cx43 remodelling in diverse cell types in the diseased myocardium and the underlying mechanism. We studied cell type-dependent changes in Cx43 remodelling due to ß-AR overactivation and molecular mechanisms involved. Mouse models of isoproterenol stimulation or transgenic cardiomyocyte overexpression of ß2 -AR were used, which exhibited cardiac fibrosis and up-regulated total Cx43 abundance. In both models, whereas Cx43 expression in cardiomyocytes was reduced and more laterally distributed, fibroblasts exhibited elevated Cx43 expression and enhanced gap junction communication. Mechanistically, activation of ß2 -AR in fibroblasts in vitro elevated Cx43 expression, which was abolished by the ß2 -antagonist ICI-118551 or protein kinase A inhibitor H-89, but simulated by the adenylyl cyclase activator forskolin. Our in vitro and in vivo data showed that ß-AR activation-induced production of IL-18 sequentially stimulated Cx43 expression in fibroblasts in a paracrine fashion. In summary, our findings demonstrate a pivotal role of ß-AR in mediating distinct and cell type-dependent changes in the expression and distribution of Cx43, leading to pathological gap junction remodelling in the myocardium.

CAV1-CAVIN1-LC3B-mediated autophagy regulates high glucose-stimulated LDL transcytosis.

Diabetes is a recognized high-risk factor for the development of atherosclerosis, in which macroautophagy/autophagy is emerging to play essential roles. The retention of low-density lipoprotein (LDL) particles in subendothelial space following transcytosis across the endothelium is the initial step of atherosclerosis. Here, we identified that high glucose could promote atherosclerosis by stimulating transcytosis of LDL. By inhibiting AMPK-MTOR-PIK3C3 pathway, high glucose suppresses the CAV-CAVIN-LC3B-mediated autophagic degradation of CAV1; therefore, more CAV1 is accumulated in the cytosol and utilized to form more caveolae in the cell membrane and facilitates the LDL transcytosis across endothelial cells. For a proof of concept, higher levels of lipids were accumulated in the subendothelial space of umbilical venous walls from pregnant women with gestational diabetes mellitus (GDM), compared to those of pregnant women without GDM. Our results reveal that high glucose stimulates LDL transcytosis by a novel CAV1-CAVIN1-LC3B signaling-mediated autophagic degradation pathway. ABBREVIATIONS: 3-MA: 3-methyladenine; ACTB: actin beta; AMPK: AMP-activated protein kinase; Bafi: bafilomycin A1; CAV1: caveolin-1; CAVIN1: caveolae associated protein 1; CSD: the CAV1 scaffolding domain; GDM: gestational diabetes mellitus; IMD: intramembrane domain; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule- associated protein 1 light chain 3; MFI: mean fluorescence intensity; MTOR: mechanistic target of rapamycin kinase; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; SQSTM1/p62: sequestosome 1.