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.

A zinc finger protein SlSZP1 protects SlSTOP1 from SlRAE1-mediated degradation to modulate aluminum resistance.

In acidic soils, aluminum (Al) toxicity is the main factor inhibiting plant root development and reducing crops yield. STOP1 (SENSITIVE TO PROTON RHIZOTOXICITY 1) was a critical factor in detoxifying Al stress. Under Al stress, STOP1 expression was not induced, although STOP1 protein accumulated, even in the presence of RAE1 (STOP1 DEGRADATION E3-LIGASE). How the Al stress triggers and stabilises the accumulation of STOP1 is still unknown. Here, we characterised SlSTOP1-interacting zinc finger protein (SlSZP1) using a yeast-two-hybrid screening, and generated slstop1, slszp1 and slstop1/slszp1 knockout mutants using clustered regularly interspaced short palindromic repeats (CRISPR) in tomato. SlSZP1 is induced by Al stress but it is not regulated by SlSTOP1. The slstop1, slszp1 and slstop1/slszp1 knockout mutants exhibited hypersensitivity to Al stress. The expression of SlSTOP1-targeted genes, such as SlRAE1 and SlASR2 (ALUMINUM SENSITIVE), was inhibited in both slstop1 and slszp1 mutants, but not directly regulated by SlSZP1. Furthermore, the degradation of SlSTOP1 by SlRAE1 was prevented by SlSZP1. Al stress increased the accumulation of SlSTOP1 in wild-type (WT) but not in slszp1 mutants. The overexpression of either SlSTOP1 or SlSZP1 did not enhance plant Al resistance. Altogether, our results show that SlSZP1 is an important factor for protecting SlSTOP1 from SlRAE1-mediated degradation.

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.