Metabolic remodeling is a fundamental mechanism that underlies cardiomyopathy and heart failure. Preclinical and clinical studies in diverse types of heart disease have provided evidence for the activation of the myocardial Hippo pathway and that cardiomyocyte-specific Hippo pathway activation or YAP-TEAD1 inactivation drives cardiomyocyte mitochondrial damage, metabolic abnormalities and fibrosis. Here we studied whether intervention of myocardial lactate metabolism have therapeutic effect on cardiac fibrosis in cardiomyopathy models. In a Hippo pathway-activated mouse model (Mst1-cTG), single-nucleus RNA-sequencing (snRNA-seq) revealed activated fibroblasts while cardiomyocytes displayed downregulation of gene sets of mitochondrial metabolism and augmented HIF-1 signaling. Seahorse assay in isolated cardiomyocytes from adult TG mice showed impaired oxidative respiratory capacity together with enhanced glycolysis and lactate production. In TG mouse myocardium, fibroblast histone H3 lysine 18 lactylation (H3K18la), regulated by p300, was elevated starting from young age. By CUT&Tag, H3K18la was found to be enriched in promoter regions of numerous fibrotic genes with enhanced transcription. Treatment of TG mice with inhibitors to suppress either glycolysis (PX-478 as HIF-1α inhibitor) or lactate production (FX-11 as LDHA inhibitor) reduced both myocardial lactate content and H3K18la level, and ameliorated cardiac fibrosis and dysfunction. Similar efficacy was achieved in vitro or in vivo by fibroblast p300 knockdown using siRNA or AAV9. Treatment with FX-11 was similarly effective in inhibiting histone lactylation and fibrosis in mice subjected to chronic ischemia-reperfusion. Finally, we integrated our snRNA-seq and CUT&Tag studies to identify RUNX2 as a crucial transcription factor that promotes fibrotic gene expression in concert with H3K18la levels. Treatment with RUNX2 inhibitor CADD522 in TG mice improved heart dysfunction and fibrosis. In the setting of myocardial Hippo pathway activation, inhibiting the lactate-histone lactylation-RUNX2 axis effectively attenuated cardiac fibrosis.

Research in the last two decades has well established galectin-3 (Gal3), a member of the lectin family, as a clinical biomarker and mediator of cardiovascular as well as other diseases. Gal3 contributes to progression of diseases by promoting pathological components, including inflammation, fibrosis, cell death or proliferation, and metabolic remodeling, and hence forms an ideal therapeutic target. Notably, nearly all Gal3 inhibitors that are currently under intensive pre-clinical and clinical testing target carbohydrate recognition/binding domain (CRD) of Gal3 molecules. Whereas the role of Gal3 in cardiovascular disease (CVD) has been well established, research on Gal3 in cancer or immunology has been leading the frontiers in this discipline. Therefore, it is important to have an integrated understanding on the biology and pathophysiology of Gal3 in a spectrum of pathological conditions. This review describes current findings from studies on diverse disease conditions and examines the role of Gal3 in the pathogenesis of diseases focusing on its transcription, post-translational modifications, intracellular dynamics, extracellular exporting, and interactions with a variety of signaling molecules. By bridging findings from different disciplines on the role of Gal3 in diseased settings, we explore the diverse anti-Gal3 strategies in addition to inhibition of CRD binding and highlight the significance of interventions targeting the transcription and post-translational modifications of Gal3, as well as intracellular actions of Gal3. At the end of this review, we provide perspectives for future research and therapeutic implications in CVD.

Background Myocardial fibrosis is a pivotal pathological component of the failing heart where the sympatho‐β‐adrenergic receptor (β‐AR) signaling is augmented. Intermediate‐conductance Ca2+‐activated K+ channel (KCa3.1) is expressed in fibroblasts and mediates fibrosis, but the regulation of KCa3.1 expression by β‐AR activation remains unclear. Used for treatment of skin diseases, dimethyl fumarate (DMF) is recently found to suppress transcription cofactor yes‐associated protein (YAP). Here, we examined whether DMF ameliorates myocardial fibrosis induced by β‐AR activation through inhibiting YAP‐mediated KCa3.1 expression in fibroblasts. Methods We used 2 mouse models of established cardiac fibrosis induced by administration of isoproterenol (30 mg/kg per d SC) for 1 week with animals studied 4 weeks afterwards, or transgenic overexpression of β2‐AR (β2‐TG) exhibiting age‐related worsening of cardiac fibrosis. In both models, DMF was administered (50 mg/kg per d IP) for 4 weeks. The mechanism of DMF in regulating KCa3.1 expression was studied in cultured adult mouse cardiac fibroblasts exposed to isoproterenol. Results Mice subjected to repeated isoproterenol injections developed myocardial inflammation and fibrosis with irreversible cardiac dysfunction. These phenotypes seen in both models were reversed by DMF treatment in vivo. Mechanistically, in both in vivo models and in cultured fibroblasts, we observed declined YAP phosphorylation and enhanced YAP nuclear localization induced by isoproterenol, changes accompanied by upregulation of KCa3.1 expression at mRNA and protein levels. These changes were largely abolished by DMF treatment. Conclusions DMF reverses the established myocardial fibrosis following β‐AR activation by inhibiting YAP nuclear localization and resultant KCa3.1 expression in fibroblasts.

Metabolic reprogramming occurs in cardiomyopathy and heart failure contributing to progression of the disease. Activation of cardiac Hippo pathway signaling has been implicated in mediating mitochondrial dysfunction and metabolic reprogramming in cardiomyopathy, albeit influence of Hippo pathway on lipid profile is unclear. Using a dual-omics approach, we determined alterations of cardiac lipids in a mouse model of cardiomyopathy due to enhanced Hippo signaling and explored molecular mechanisms. Lipidomic profiling discovered multiple alterations in lipid classes, notably reduction of triacylglycerol, diacylglycerol, phospholipids and ether lipids, and elevation of sphingolipids and lysophosphatidylcholine. Mechanistically, we found downregulated expression of PPARα and PGC-1α at mRNA and protein levels, and downregulated expression of PPARα-target genes, indicating attenuated transcriptional activity of PPARα/PGC-1α. Lipidomics-guided transcriptomic analysis revealed dysregulated expression of gene sets that were responsible for enhanced biosynthesis of ceramides, suppression of TG biosynthesis, storage, hydrolysis and mitochondrial fatty acid oxidation, and reduction of peroxisome-localized biosynthesis of ether lipids. Collectively, Hippo pathway activation with attenuated PPARα/PGC-1α signaling is the underlying mechanism for alterations in cardiac lipids in cardiomyopathy and failing heart.

Endothelial dysfunction of pulmonary arteries is important in the initiation of pulmonary hypertension (PH). Pulmonary vascular tone is regulated by endothelium-dependent hyperpolarization (EDH) that induces vasodilation. Although KCa2.3 channels are involved as a key initiator of EDH response, therapeutic potential of endothelial KCa2.3 channels in PH remains unclear. Bioinformatic and biochemical analyses were used to explore KCa2.3 expression in patients with PH. Two mouse PH models were created by injection of Sugen 5416 plus hypoxia or injection with monocrotaline. Endothelial-specific KCa2.3 adeno-associated virus (AAV-Kcnn3) was constructed, and the efficacy in both PH models was tested using immunohistochemistry, myograph system, and echocardiography. Expression of KCa2.3 was decreased in pulmonary arterial endothelial cells or lung tissues from patients with PH and both experimental PH models. AAV-Kcnn3 treatment increased KCa2.3 expression in pulmonary endothelium and ameliorated KCa2.3-medieated vasodilation of small pulmonary arteries and pulmonary vascular endothelial dysfunction in both PH models. The key PH phenotypes, including elevated right ventricular pressure, Fulton index, pulmonary artery wall thickness, and the free wall thickness of the right ventricle, were remarkably alleviated by AAV-Kcnn3 treatment in both PH models. In conclusion, augmented expression of endothelium-specific KCa2.3 channels markedly inhibits the development of PH by improving endothelium-dependent relaxation. Significance Statement This study demonstrated downregulated expression of KCa2.3 channels in lung tissues, specifically in pulmonary artery endothelial cells from patients or mice with pulmonary hypertension. Upregulation of endothelial KCa2.3 might serve as a therapeutic strategy in the early-stage pulmonary hypertension.

BACKGROUND:Coronary microvascular hyperpermeability and the subsequent inflammation infiltration are the key early characteristics of pressure overload-induced myocardial injury. However, how changes in the coronary endothelial barrier function in response to cardiac pressure overload are less explored. Here, we investigated the specific role of S1PR1 (sphingosine-1-phosphate receptor type 1) on coronary endothelial permeability and the signaling pathways involved during pressure overload. METHODS:Mice with endothelial deletion of S1PR1 or MYPT1 (myosin phosphatase target subunit 1) were subjected to transverse aortic constriction. We also studied cultured human umbilical vein endothelial cells (ECs) in vitro. RESULTS:We found upregulated S1PR1 in cardiac ECs at 24 hours and 3 days after transverse aortic constriction, and EC-specific deletion of S1PR1 (S1pr1ΔEC) led to coronary endothelial hyperpermeability, myocardial edema, and inflammatory infiltration in mice subjected to transverse aortic constriction. In cultured human umbilical vein ECs, silencing S1PR1 reduced total MYPT1 but increased phosphorylated MYPT1, and under TNF-α (tumor necrosis factor-α) stimulation led to MLC (myosin light chain) phosphorylation and actin cytoskeletal contraction. Although S1PR1-NFATc2 (Nuclear Factor of Activated T Cells 2) signaling was essential for maintaining MYPT1 expression, S1PR1 deficiency increased TRPV4 (transient receptor potential vanilloid 4) expression, enhancing extracellular Ca2+ entry and MYPT1 phosphorylation. Mice with EC-specifically MYPT1-deficient (Mypt1ΔEC) also showed coronary endothelial hyperpermeability, and treatment with the S1PR1 agonist FTY720 failed in alleviating the pathological effects. At 1 month post-transverse aortic constriction, both Mypt1ΔEC and S1pr1ΔEC mice displayed aggravated pathological cardiac remodeling. CONCLUSIONS:These findings suggest that the S1PR1-MYPT1 signaling is crucial for coronary endothelial permeability and myocardial microenvironmental homeostasis under pressure overload, targeting which may offer therapeutic potential for related heart diseases.

Heart failure is associated with myocardial fibrosis, a pivotal histopathological feature arising from β-adrenergic receptor (β-AR) stimulation through sympathetic nervous system activation. Augmented glutaminolysis with increased bioavailability of α-ketoglutarate (α-KG) is suggested to contribute to fibrogenesis and changes in cellular gene expression. KCa3.1 is a calcium-activated potassium channel expressed in fibroblasts and has been implicated in mediating fibrosis, yet the putative interactions between glutaminolysis and KCa3.1 in β-AR-mediated cardiac fibrosis remain poorly understood. Here, we performed a series of in vitro and in vivo experiments to investigate how α-KG might influence the expression of KCa3.1 in the context of experimental myocardial fibrosis driven by β-AR activation. In cultured adult mouse cardiac fibroblasts, α-KG exposure resulted in the upregulation of KCa3.1 mRNA and protein levels that were commensurate with the dose and duration of exposure, and also led to increased KCa3.1 channel currents. Exposure to α-KG led to a significant decrease in levels of histone methylation (H3K27me3) within the KCa3.1 promoter, a decrease in the association of the transcription repressor REST from this site, as well as an enrichment of transcription activator AP-1 binding. The exacerbated fibrotic signaling induced by α-KG in cultured fibroblasts was suppressed by functional inhibition of KCa3.1 or by genetic knockdown of Kcnn4. Moreover, β-AR activation by isoproterenol significantly augmented glutaminolysis mediated by glutaminase 1 (GLS1) and significantly increased α-KG levels detected in the supernatant of cultured fibroblasts and cardiomyocytes. In addition, isoproterenol-induced KCa3.1 expression in fibroblasts was curtailed by treatment with the GLS1 inhibitor CB-839, or by GLS1 gene knockdown, or by treatment with the selective β2-AR antagonist, ICI118551. In mouse models of established cardiac fibrosis evoked by isoproterenol-stimulation or β2-AR overexpression, treatment with CB-839 for 4 weeks suppressed the phenotypic features of fibrosis, and this was associated with a decline in α-KG tissue content, a lack of histone demethylation at the KCa3.1 promoter, as well as suppression of KCa3.1 expression. Taken together, our study demonstrates for the first time that glutaminolysis contributes to β-AR activation-induced myocardial fibrosis via α-KG-stimulated KCa3.1 expression. We anticipate that treatments which target the β-AR/GLS1/α-KG/KCa3.1 signaling pathway might be effective for cardiac fibrosis.

Atrial fibrillation (AF) is a prevalent cardiac arrhythmia characterized by atrial fibrosis which involves excessed proliferation and increased activity of fibroblast and myofibroblast, as well as alterations in the extracellular matrix (ECM). The specific mechanism driving fibrosis in atrial fibroblasts and myofibroblsats remains incompletely understood. This study investigates the role of the intermediate-conductance Ca2+-activated K+ channel (KCa3.1) in Angiotensin II (Ang II)-induced atrial fibrosis and elucidates the underlying mechanisms. Primary rat atrial fibroblasts/myofibroblasts were treated with Ang II to evaluate KCa3.1 expression, cells proliferation and ECM production. The involvement of ERK/NF-κB signaling pathway was assessed using specific inhibitors. Ang II treatment increased KCa3.1 expression, stimulated the proliferation of fibroblasts/myofibroblasts, and enhanced ECM production, effects that were attenuated by the Ang II receptor antagonist Losartan and the KCa3.1 inhibitor TRAM-34. Knockdown of KCa3.1 using siRNA significantly reduced Ang II-induced collagen synthesis, confirming its critical role in fibrosis. The ERK/NF-κB pathway was found to mediate Ang II-induced upregulation of KCa3.1, as evidenced by inhibition with specific inhibitors. In vivo, Ang II infusion in rats increased KCa3.1 expression and atrial fibrosis, with atria showing greater susceptibility to fibrosis compared to ventricle. These effects were mitigated by losartan and TRAM-34. In conclusion, our findings demonstrate that Ang II-induced upregulation of KCa3.1 through ERK/NF-κB pathway activation in atrial fibroblasts/myofibroblasts promotes cellular proliferation and collagen deposition, ultimately contributing to atrial fibrosis. KCa3.1 represents a promising therapeutic target for the treatment of atrial fibrosis in AF.

The endothelial small-conductance calcium-activated potassium channels (KCa2.3) are indispensable for endothelium-dependent hyperpolarization (EDH) response, mainly in resistance arteries. We recently demonstrated in diet-induced obese mice that adenosine monophosphate-activated protein kinase (AMPK) upregulates endothelial KCa2.3 expression and improves endothelial function. However, the molecular mechanism of regulation of KCa2.3 by AMPK remains less explored. Using techniques of bioinformatics, molecular biology and wire myograph system, we examined KCa2.3 phosphorylation by AMPK in human umbilical vein endothelial cells (HUVECs), human embryonic kidney 293 (HEK-293T) cells and second-order mesenteric resistance arteries from angiotensin II-induced hypertensive mice. In HUVECs, treatment with activators of AMPK (AICAR, metformin, and MK-8722) significantly increased phosphorylation of KCa2.3 Thr106 (human), which was antagonized by AMPK inhibitor compound C. In HEK-293T cells, KCa2.3 current was enhanced by AMPK activation or phosphomimetic mutant KCa2.3 (T106D), which was abolished after de-phosphomimetic mutant (T106A) or deletion of KCa2.3 of Thr106 site (T106Del). In mice with angiotensin II infusion, 2-week treatment with AICAR or overexpressing phosphomimetic mutant KCa2.3 Thr107D (mouse) restored KCa2.3-mediated EDH-dependent relaxation in mesenteric resistance arteries together with reversal of early phase hypertension. Our study demonstrates for the first time that AMPK activation mediates KCa2.3 phosphorylation in endothelial cells with enhanced channel activity. This effect ameliorates endothelial dysfunction of mesenteric resistance arteries and alleviates angiotensin II-induced early phase hypertension in mice.

Cardiomyocyte loss by regulated death modes, like apoptosis and ferroptosis, has been implicated in the development of dilated cardiomyopathy (DCM). It remains unclear whether cardiomyocyte ferroptosis occurs as a consequence of Hippo pathway activation. Using a mouse model of DCM by overexpression of Mst1 transgene (Mst1-TG) leading to Hippo pathway activation, we showed that cardiomyocyte ferroptosis was evident by transcriptomic profiles, elevated mitochondrial Fe2+ content, increased levels of lipid peroxidation and obvious mitochondrial damage. Transcriptome revealed significant alterations of genes participating in iron metabolism and lipid peroxidation. Treatment of Mst1-TG mice with the ferroptosis inhibitor ferrostatin-1 reduced cardiomyocyte ferroptosis and improved cardiac function. Using heart samples from human patients with DCM, we also found significant cardiomyocyte loss and lipid peroxidation. In cultured cardiomyocytes, ferroptosis was induced by treatment with erastin or YAP inhibitor verteporfin, and cell ferroptosis under these conditions was largely prevented by either iron chelation or Mst1 gene knockdown. In a strain of transgenic mice with cardiomyocyte inactivation of Mst1 (dnMst1-TG), erastin-induced ferroptosis and cardiac dysfunction, seen in control mice, were mitigated. Mechanistically, nuclear YAP and YY1 were shown to interact and bind to the Nfs1 promoter, thus mediating downregulation of Nfs1 (encoding cysteine desulfurase). Subsequent inhibition of iron-sulfur cluster (ISC) biosynthesis promoted cardiomyocyte ferroptosis and DCM phenotype. Restoration of Nfs1 expression was achieved by treatment of Mst1-TG mice with AAV9-Nfs1 virus, which alleviated ferroptosis, mitochondrial damage and DCM phenotype. In conclusion, in the DCM model with Hippo pathway activation, our findings unravel that NFS1 downregulation occurs and leads to insufficient ISC biosynthesis and cardiomyocyte ferroptosis. Our findings implicate that restoration of cardiomyocyte NFS1 level may represent a new therapeutic strategy for DCM.

Atrial fibrillation is strongly associated with an increased risk of embolism, stroke, and heart failure. Current therapeutic approaches often have limited efficacy, and controlling atrial fibrosis remains a critical objective for upstream therapies. The specific mechanisms driving atrial fibrosis remain incompletely understood. The intermediate-conductance calcium-activated potassium channel KCa3.1 has been implicated in promoting fibroblast activation in various fibrotic diseases. This study investigates the role of angiotensin II (Ang II) in regulating KCa3.1, as well as its involvement in the pathogenesis of atrial fibrosis and the underlying signaling mechanisms. In a rat model, chronic Ang II infusion for 4 weeks induced atrial fibrosis, which was significantly attenuated by TRAM-34, a specific KCa3.1 channel blocker. In cultured rat atrial fibroblasts, Ang II treatment promoted fibroblast differentiation, proliferation, migration and collagen production, effects that were suppressed by TRAM-34 and KCa3.1 knockdown. Overexpression of KCa3.1 in fibroblasts further confirmed its pro-fibrotic role. Mechanistically, Ang II upregulated KCa3.1 expression and current density by activating the JNK/AP-1 signaling pathway. This involved phosphorylation of JNK, c-Jun, and c-Fos, leading to the formation of c-Jun/c-Fos heterodimers that directly bound to the KCa3.1 promoter to enhance its transcription. Together, these findings demonstrate that KCa3.1 mediates fibroblast activation and atrial fibrosis through the JNK/AP-1 pathway.

Galectin-3 (Gal3) is known to interact with glycans of proteins and lipids. In cardiovascular disease, the elevated expression of Gal3 mediates inflammation, hypertrophy and fibrosis. We explored the effect of Gal3 on the cardiac lipid profile in healthy mice or in mice with dilated cardiomyopathy (DCM). Using lipidomics and gene-targeted mouse models, we studied the influence of Gal3 gene deletion on cardiac lipid profiles in the healthy mice or mice with DCM. Cardiac-restricted transgenic activation of Hippo pathway led to DCM phenotype and Gal3 upregulation. DCM mice were cross-bred with Gal3 gene knockout (KO) mice to obtain genotypes of non-transgenic (nTG), Gal3-KO, DCM and DCM/KO. Alterations in the lipid classes and species due to Gal3-KO were identified by lipidomics in hearts from mice of four genotypes. In the nTG background, Gal3-KO increased ether lipids and lysophospholipids, and induced diverse changes of sphingolipid subclasses. The DCM hearts exhibited profound lipidomic changes including increase in sphingolipids and reduction in ether lipids and triglycerides, which were partially reversed by Gal3 deletion. We demonstrated the nuclear and mitochondrial localization of Gal-3 in DCM hearts. Transcriptomics revealed that Gal3 deletion in the DCM background partially restored the suppressed expression of mitochondrial lipid metabolic genes. In conclusion, we report multiple alterations in the lipid classes and species in the heart by Gal3 deletion in the healthy mice and, more importantly mice with DCM background. Our findings suggest that Gal3 alters cardiac contents of lipids in the DCM model in part through suppression of mitochondrial metabolism.

Activation of the sympatho-β-adrenergic receptor (βAR) system is the hallmark of heart disease with adverse consequences that facilitate the onset and progression of heart failure (HF). Use of β-blocking drugs has become the front-line therapy for HF. Last decade has witnessed progress in research demonstrating a pivotal role of Hippo pathway in cardiomyopathy and HF. Clinical studies have revealed myocardial Hippo pathway activation/YAP-TEAD1 inactivation in several types of human cardiomyopathy. Experimental activation of cardiac Hippo signaling or inhibition of YAP-TEAD1 have been shown to leads dilated cardiomyopathy with severe mitochondrial dysfunction and metabolic reprogramming. Studies have also convincingly shown that stimulation of βAR activates cardiac Hippo pathway with inactivation of the down-stream effector molecules YAP/TAZ. There is strong evidence for the adverse consequences of the βAR-Hippo signaling leading to HF. In addition to promoting cardiomyocyte death and fibrosis, recent progress is the demonstration of mitochondrial dysfunction and metabolic reprogramming mediated by βAR-Hippo pathway signaling. Activation of cardiac βAR-Hippo signaling is potent in downregulating a range of mitochondrial and metabolic genes, whereas expression of pro-inflammatory and pro-fibrotic factors are upregulated. Coupling of βAR-Hippo pathway signaling is mediated by several kinases, mechanotransduction and/or Ca2+ signaling, and can be blocked by β-antagonists. Demonstration of the converge of βAR signaling and Hippo pathway bears implications for a better understanding on the role of enhanced sympathetic nervous activity, efficacy of β-antagonists, and metabolic therapy targeting this pathway in HF. In this review we summarize the progress and discuss future research directions in this field.

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 β-adrenoceptors (βAR) activates Hippo pathway. Here, we investigated the role of βAR-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. βAR 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 βAR 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-β-adrenoceptor (βAR) 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 βAR activated Hippo signaling pathway and genetic inactivation of Mst1 kinase ameliorated mitochondrial damage and metabolic dysfunction at the acute phase of TTS.

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 beta 2-adrenoceptor (beta 2AR-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.

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.

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 endotheliumspecifically 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-alpha, IL-1 beta), 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.

BACKGROUND Takotsubo syndrome (TTS) is characterized by transient contractile dysfunction with its mechanism undefined. We showed that activation of cardiac Hippo pathway mediates mitochondrial dysfunction, and that stimulation of β-adrenoceptors (βAR) activates Hippo pathway. Here we investigated the role of βAR-Hippo signaling in mediating mitochondrial dysfunction in isoproterenol-induced TTS-like mouse model. METHODS Elderly post-menopausal female mice were administered with isoproterenol (1.25 mg/kg/h for 23 hours). Cardiac function was determined by serially echocardiography. At day-1 and day-7 post-isoproterenol 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. RESULTS Isoproterenol exposure induced transient increase in biomarkers of cardiac damage, and ventricular contractile dysfunction and dilation. At day-1 post-isoproterenol, 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 ROS. All changes were reversed by day-7. βAR 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. CONCLUSION Stimulation of cardiac βAR activates Hippo pathway that mediates mitochondrial dysfunction with energy insufficiency and enhanced ROS, promoting acute but transient ventricular dysfunction.

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 (K Ca 2.3 and K Ca 3.1 [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 K Ca 2.3 or K Ca 3.1 channels. In cultured human umbilical vein endothelial cells, S1P stimulated immediate membrane potential hyperpolarization following activation of K Ca 2.3/K Ca 3.1 with elevated cytosolic Ca 2+ . Further, chronic S1P stimulation enhanced expression of K Ca 2.3 and K Ca 3.1 in human umbilical vein endothelial cells in dose- and time-dependent manners, which was abolished by disrupting either S1PR1-Ca 2+ signaling or downstream Ca 2+ -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 K Ca 2.3 and K Ca 3.1 genes thus to upregulate transcription of these channels. Deletion of endothelial S1PR1 reduced expression of K Ca 2.3 and K Ca 3.1 in mesenteric arteries and exacerbated hypertension in mice with angiotensin II infusion. Conclusions: This study provides evidence for the mechanistic role of K Ca 2.3/K Ca 3.1-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.