Circulating mitochondria promoted endothelial cGAS-derived neuroinflammation in subfornical organ to aggravate sympathetic overdrive in heart failure mice.

BACKGROUND: Sympathoexcitation contributes to myocardial remodeling in heart failure (HF). Increased circulating pro-inflammatory mediators directly act on the Subfornical organ (SFO), the cardiovascular autonomic center, to increase sympathetic outflow. Circulating mitochondria (C-Mito) are the novel discovered mediators for inter-organ communication. Cyclic GMP-AMP synthase (cGAS) is the pro-inflammatory sensor of damaged mitochondria. OBJECTIVES: This study aimed to assess the sympathoexcitation effect of C-Mito in HF mice via promoting endothelial cGAS-derived neuroinflammation in the SFO. METHODS: C-Mito were isolated from HF mice established by isoprenaline (0.0125 mg/kg) infusion via osmotic mini-pumps for 2 weeks. Structural and functional analyses of C-Mito were conducted. Pre-stained C-Mito were intravenously injected every day for 2 weeks. Specific cGAS knockdown (cGAS KD) in the SFO endothelial cells (ECs) was achieved via the administration of AAV9-TIE-shRNA (cGAS) into the SFO. The activation of cGAS in the SFO ECs was assessed. The expression of the mitochondrial redox regulator Dihydroorotate dehydrogenase (DHODH) and its interaction with cGAS were also explored. Neuroinflammation and neuronal activation in the SFO were evaluated. Sympathetic activity, myocardial remodeling, and cardiac systolic dysfunction were measured. RESULTS: C-Mito were successfully isolated, which showed typical structural characteristics of mitochondria with double-membrane and inner crista. Further analysis showed impaired respiratory complexes activities of C-Mito from HF mice (C-MitoHF) accompanied by oxidative damage. C-Mito entered ECs, instead of glial cells and neurons in the SFO of HF mice. C-MitoHF increased the level of ROS and cytosolic free double-strand DNA (dsDNA), and activated cGAS in cultured brain endothelial cells. Furthermore, C-MitoHF highly expressed DHODH, which interacted with cGAS to facilitate endothelial cGAS activation. C-MitoHF aggravated endothelial inflammation, microglial/astroglial activation, and neuronal sensitization in the SFO of HF mice, which could be ameliorated by cGAS KD in the ECs of the SFO. Further analysis showed C-MitoHF failed to exacerbate sympathoexcitation and myocardial sympathetic hyperinnervation in cGAS KD HF mice. C-MitoHF promoted myocardial fibrosis and hypertrophy, and cardiac systolic dysfunction in HF mice, which could be ameliorated by cGAS KD. CONCLUSION: Collectively, we demonstrated that damaged C-MitoHF highly expressed DHODH, which promoted endothelial cGAS activation in the SFO, hence aggravating the sympathoexcitation and myocardial injury in HF mice, suggesting that C-Mito might be the novel therapeutic target for sympathoexcitation in HF.

Cathodic photoelectrochemical sensor developed for glutathione detection based on carrier transport in a Ti3C2Tx/AgI heterojunction.

Recently, cathodic photoelectrochemical (PEC) sensing has emerged as a convenient and efficient method for molecular detection and analysis. Novel photoactive materials are urgently required for the further development of advanced cathodic PEC sensors. In this study, an original photoactive material, the Ti3C2Tx/AgI heterojunction material (HM), was synthesized by combining the two-dimensional layered material Ti3C2Tx MXenes with the p-type semiconductor AgI. The Ti3C2Tx/AgI HM exhibited excellent PEC performance. The PEC process in the Ti3C2Tx/AgI HM under light irradiation was explored and demonstrated using Vienna abinitio simulation package (VASP) calculations. The Ti3C2Tx/AgI HM exhibited enhanced electron-hole separation and charge transport owing to the good electronic conductivity of Ti3C2Tx and improved interfacial electron transport from Ti3C2Tx to AgI. Therefore, a cathodic PEC sensor was developed for glutathione (GSH) detection. GSH acted as a reductant and consumed the electron acceptor to inhibit electron transfer, resulting in a decrease in photocurrent signals that was linear with the GSH concentration. The linear range was wide (1-10 µM), with a detection limit of 0.31 nM. The PEC sensor also displayed satisfactory selectivity and stability; thus, the findings provide insights into the design and construction of a PEC sensing platform without enzymes.

C1q/tumor necrosis factor-related protein-3 improves microvascular endothelial function in diabetes through the AMPK/eNOS/NO· signaling pathway.

The repair of vascular endothelial cell dysfunction is an encouraging approach for the treatment of vascular complications associated with diabetes. It has been demonstrated that members of C1q/tumor necrosis factor-related protein (CTRP) family may improve endothelial function. Nevertheless, the protective properties of CTRPs in diabetic microvascular complications continue to be mostly unknown. Here, we demonstrate that the C1q-like globular domain of CTRP3, CTRP5, and CTRP9 (gCTRP3, 5, 9) exerted a vasorelaxant effect on the microvasculature, of which gCTRP3 was the most powerful one. In a murine model of type 2 diabetes mellitus, serum gCTRP3 level and endothelial function decreased markedly compared with controls. Two weeks of gCTRP3 treatment (0.5 µg/g/d) enhanced endothelium-dependent relaxation in microvessels, increased nitric oxide (NO·) production, and reduced retinal vascular leakage. In addition, Western blotting in human retinal microvascular endothelial cells indicated that gCTRP3 triggered AMP-activated protein kinase-α (AMPKα), hence increasing the endothelial NO synthase (eNOS) level and NO· production. In addition, incubation with gCTRP3 in vitro ameliorated the endothelial dysfunction induced by high glucose in the branch of the mesenteric artery. Blockade of either eNOS or AMPKα completely abolished the effects of gCTRP3 described above. Taken together, we demonstrate for the first time that gCTRP3 improves impaired vasodilatation of microvasculature in diabetes by ameliorating endothelial cell function through the AMPK/eNOS/NO· signaling pathway. This finding may suggest an effective intervention against diabetes-associated microvascular complications.

Experimental and numerical investigation of the fatigue behaviour and crack evolution mechanism of granite under ultra-high-frequency loading.

Assisted ultrasonic vibration technology has received great interest in the past few years for petroleum and mining engineering related to hard rock breaking. Understanding the fatigue behaviour and damage characteristics of rock subject to ultra-high-frequency loading is vital for its application. In this research, we conducted ultrasonic vibration breaking rock experiments combined with an ultra-dynamic data receiver and strain gauges to monitor the development of strain in real time. The experimental results show that the strain curve is U-shaped, and it can be divided into three stages: the strain first decreases, then remains steady (with light fluctuations) and finally increases. The sample first underwent compressive deformation, and no rupture occurred. As the vibration continued, the compressive deformation decreased with the initiation and propagation of cracks, and fragmentation occurred. To elucidate the crack evolution mechanism of the granite specimens, numerical simulations were performed using particle flow code in two dimensions (PFC2D), and an improved fatigue damage model based on the flat-joint contact model was proposed. The numerical results indicate that this model can effectively reproduce the fatigue characteristics of hard brittle rocks under ultrasonic vibration. By analysing the stress and strain fields and cracking process, the crack evolution mechanism in the brittle hard rock under ultra-high-frequency loading is revealed. These experimental and numerical results are expected to improve the understanding of the fragmentation mechanism of rock under assisted ultrasonic vibration.

Cardiac-derived CTRP9 protects against myocardial ischemia/reperfusion injury via calreticulin-dependent inhibition of apoptosis.

Cardiokines play an essential role in maintaining normal cardiac functions and responding to acute myocardial injury. Studies have demonstrated the heart itself is a significant source of C1q/TNF-related protein 9 (CTRP9). However, the biological role of cardiac-derived CTRP9 remains unclear. We hypothesize cardiac-derived CTRP9 responds to acute myocardial ischemia/reperfusion (MI/R) injury as a cardiokine. We explored the role of cardiac-derived CTRP9 in MI/R injury via genetic manipulation and a CTRP9-knockout (CTRP9-KO) animal model. Inhibition of cardiac CTRP9 exacerbated, whereas its overexpression ameliorated, left ventricular dysfunction and myocardial apoptosis. Endothelial CTRP9 expression was unchanged while cardiomyocyte CTRP9 levels decreased after simulated ischemia/`reperfusion (SI/R) in vitro. Cardiomyocyte CTRP9 overexpression inhibited SI/R-induced apoptosis, an effect abrogated by CTRP9 antibody. Mechanistically, cardiac-derived CTRP9 activated anti-apoptotic signaling pathways and inhibited endoplasmic reticulum (ER) stress-related apoptosis in MI/R injury. Notably, CTRP9 interacted with the ER molecular chaperone calreticulin (CRT) located on the cell surface and in the cytoplasm of cardiomyocytes. The CTRP9-CRT interaction activated the protein kinase A-cAMP response element binding protein (PKA-CREB) signaling pathway, blocked by functional neutralization of the autocrine CTRP9. Inhibition of either CRT or PKA blunted cardiac-derived CTRP9's anti-apoptotic actions against MI/R injury. We further confirmed these findings in CTRP9-KO rats. Together, these results demonstrate that autocrine CTRP9 of cardiomyocyte origin protects against MI/R injury via CRT association, activation of the PKA-CREB pathway, ultimately inhibiting cardiomyocyte apoptosis.

Short-Duration Swimming Exercise after Myocardial Infarction Attenuates Cardiac Dysfunction and Regulates Mitochondrial Quality Control in Aged Mice.

BACKGROUND: Exercise benefits to cardiac rehabilitation (CR) following stable myocardial infarction (MI). The suitable exercise duration for aged patients with coronary heart disease (CHD) remains controversial, and the underlying molecular mechanism is still unclear. METHODS AND RESULTS: 18-Month-old mice after stable MI were randomly submitted to different durations of exercise, including 15 and 60 min swimming training (ST) once per day, five times a week for 8 weeks. Compared to sedentary mice, 15 min ST, rather than 60 min ST, significantly augmented left ventricular function, increased survival rate, and suppressed myocardial fibrosis and apoptosis. 15 min ST improved mitochondrial morphology via regulating mitochondrial fission-fusion signaling. 15 min ST regulated mitophagy signaling via inhibiting LC3-II and P62 levels and increasing PINK/Parkin expression. 15 min ST also inhibited ROS production and enhanced antioxidant SOD2 activity. Notably, 15 min ST significantly increased sirtuin (SIRT) 3 level (2.7-fold) in vivo while the inhibition of SIRT3 exacerbated senescent H9c2 cellular LDH release and ROS production under hypoxia. In addition, SIRT3 silencing impairs mitochondrial dynamics and mitophagy in senescent cardiomyocytes against simulated ischemia (SI) injury. CONCLUSION: Collectively, our study demonstrated for the first time that sustained short-duration exercise, rather than long-duration exercise, attenuates cardiac dysfunction after MI in aged mice. It is likely that the positive regulation induced by a short-duration ST regimen on the elevated SIRT3 protein level improved mitochondrial quality control and decreased apoptosis and fibrosis contributed to the observed more resistant phenotype.

Insights for Oxidative Stress and mTOR Signaling in Myocardial Ischemia/Reperfusion Injury under Diabetes.

Diabetes mellitus (DM) displays a high morbidity. The diabetic heart is susceptible to myocardial ischemia/reperfusion (MI/R) injury. Impaired activation of prosurvival pathways, endoplasmic reticulum (ER) stress, increased basal oxidative state, and decreased antioxidant defense and autophagy may render diabetic hearts more vulnerable to MI/R injury. Oxidative stress and mTOR signaling crucially regulate cardiometabolism, affecting MI/R injury under diabetes. Producing reactive oxygen species (ROS) and reactive nitrogen species (RNS), uncoupling nitric oxide synthase (NOS), and disturbing the mitochondrial quality control may be three major mechanisms of oxidative stress. mTOR signaling presents both cardioprotective and cardiotoxic effects on the diabetic heart, which interplays with oxidative stress directly or indirectly. Antihyperglycemic agent metformin and newly found free radicals scavengers, Sirt1 and CTRP9, may serve as promising pharmacological therapeutic targets. In this review, we will focus on the role of oxidative stress and mTOR signaling in the pathophysiology of MI/R injury in diabetes and discuss potential mechanisms and their interactions in an effort to provide some evidence for cardiometabolic targeted therapies for ischemic heart disease (IHD).

Adiponectin exerts cardioprotection against ischemia/reperfusion injury partially via calreticulin mediated anti-apoptotic and anti-oxidative actions.

The underlying mechanisms of cardioprotection of adiponectin (APN) against ischemia/reperfusion (I/R) injury remain largely unknown. The present study aimed to investigate whether calreticulin (CRT) mediated APN's cardioprotection against I/R injury. We inhibited mice cardiac CRT expression via intra-myocardial injection of CRT SiRNA, performed transient LAD ligation, measured the cardiac function, apoptosis and oxidative stress to identify CRT's effects on cardioprotective actions of APN against I/R injury in vivo. LDH release and expression of CRT were measured in neonatal cardiomyocytes (NCM) subjected to simulated I/R (SI/R) and APN. CRT specific SiRNA was also utilized in vitro. CRT inhibition partially blunted cardioprotection of APN against I/R injury (evidenced by left ventricular ejection fraction and myocardial infarct size). It also blunted APN's function against I/R induced apoptosis and oxidative stress (evidenced by TUNEL positive staining and reactive oxygen species production). In addition, SI/R increased LDH release, and administration of APN attenuated SI/R-induced cell death significantly. However, neither SI/R nor APN altered CRT expression in NCM. Inhibition of CRT expression blunted cardioprotective action of APN against SI/R induced apoptotic events (evidenced by TUNEL positive staining, LDH release and Caspase 3 activity). Furthermore, CRT inhibition significantly blunted APN's anti-oxidative action (evidenced by gp91phox expression and superoxide generation). However, CRT inhibition did not attenuate AMPK phosphorylation by APN administration in NCM. Therefore, these novel findings strongly indicate that APN exerts cardioprotective effects against I/R injury partially via CRT mediated anti-apoptotic and anti-oxidative actions.

C1q/TNF-Related Protein 9 Protects Diabetic Rat Heart against Ischemia Reperfusion Injury: Role of Endoplasmic Reticulum Stress.

As a newly identified adiponectin paralog, C1q/TNF-related protein 9 (CTRP9) reduces myocardial ischemia reperfusion (IR) injury through partially understood mechanisms. In the present study, we sought to identify the role of endoplasmic reticulum stress (ERS) in CTRP9 induced cardioprotection in diabetic heart. Isolated hearts from high-fat-diet (HFD) induced type 2 diabetic Sprague-Dawley rats were subjected to ex vivo IR protocol via a Langendorff apparatus at the presence of globular CTRP9. CTRP9 significantly improved post-IR heart function and reduced cardiac infarction, cardiomyocytes apoptosis, Caspase-3, Caspase-9, Caspase-12, TNF-α expression, and lactate dehydrogenase activity. The cardioprotective effect of CTRP9 was associated with reduced ERS and increased expression of disulfide-bond A oxidoreductase-like protein (DsbA-L) in diabetic heart. CTRP9 reduced ERS in thapsigargin (TG) treated cardiomyocytes and protected endoplasmic reticulum (ER) stressed H9c2 cells against simulated ischemia reperfusion (SIR) injury, concurrent with increased expression of DsbA-L. Knockdown of DsbA-L increased ERS and attenuated CTRP9 induced protection against SIR injury in H9c2 cells. Our findings demonstrated for the first time that CTRP9 exerts cardioprotection by reducing ERS in diabetic heart through increasing DsbA-L.

cIAP1 attenuates shear stress-induced hBMSC apoptosis for tissue-engineered blood vessels through the inhibition of the mitochondrial apoptosis pathway.

AIMS: Shear stress-induced apoptosis is one of the leading problems in seeding cells of tissue-engineered blood vessels (TEBVs). We aim to determine the human bone mesenchymal stem cell (hBMSC) apoptosis under shear stress and its possible mechanism. MAIN METHODS: hBMSCs were subjected to 3-, 10-, and 30-dyn/cm(2) shear stress in vitro. Cell multiplication and apoptosis were analyzed by flow cytometry. Apoptosis-related genes were screened by a microarray and evidenced by real-time polymerase chain reaction (RT-PCR). hBMSCs were treated with the human recombinant cell inhibitor of apoptosis protein 1 (cIAP1) and its inhibitor, direct IAP-binding protein with low pl (DIABLO), and then cell apoptosis was analyzed. KEY FINDINGS: Exposure to shear stress (3dyn/cm(2) for >6h) activated apoptosis progress of hBMSCs. However, the same degree of shear stress (3dyn/cm(2) for 6h) did not induce apoptosis. Microarray screening and RT-PCR revealed that Bcl-2-related ovarian killer (BOK) and apoptotic protease-activating factor 1 (APAF1), key molecules of the mitochondrial apoptosis pathway, were markedly upregulated under 3-dyn/cm(2) shear stress. Then, we observed that cIAP1, a Caspase 9 inhibitor, was elevated under 3dyn/cm(2) at short-time exposure (2 or 6h), and it was reduced at long-time exposure (24h). When treated with human recombinant cIAP1, Caspase 3 activity and LDH release of hBMSCs were decreased, and vice versa when treated with DIABLO. SIGNIFICANCE: cIAP1 attenuates hBMSC apoptosis when cells were exposed to shear stress through the regulation of the BOK-APAF1-Caspase 9-Caspase 3 pathway. It may present a pharmacological target to enhance hBMSC biological function in the application of TEBVs.

Endoplasmic reticulum stress and protein quality control in diabetic cardiomyopathy.

Endoplasmic reticulum (ER) stress, together with the unfolded protein response (UPR), is initially considered an adaptive response aiming at maintenance of ER homeostasis. Nonetheless, ER stress, when in excess, can eventually trigger cell apoptosis and loss of function. UPR is mediated by three major transmembrane proteins, including inositol-requiring enzyme 1 (IRE1), protein kinase RNA-like ER kinase (PERK), and activating transcription factor (ATF) 6. A unique role has been speculated for ER stress in the pathogenesis of diabetes mellitus (DM) and its complications. Recent studies have shown that ER stress is an early event associated with diabetic cardiomyopathy, and may be triggered by hyperglycemia, free fatty acids (FFAs) and inflammation. In this mini-review, we attempted to discuss the activation machinery for ER stress in response to these triggers en route to disrupted ER function and cellular autophagy or apoptosis, ultimately insulin resistance and development of diabetic cardiomyopathy. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Endoplasmic reticulum stress mediates the anti-inflammatory effect of ethyl pyruvate in endothelial cells.

Ethyl pyruvate (EP) is a simple aliphatic ester of the metabolic intermediate pyruvate that has been demonstrated to be a potent anti-inflammatory agent in a variety of in vivo and in vitro model systems. However, the protective effects and mechanisms underlying the actions of EP against endothelial cell (EC) inflammatory injury are not fully understood. Previous studies have confirmed that endoplasmic reticulum stress (ERS) plays an important role in regulating the pathological process of EC inflammation. In this study, our aim was to explore the effects of EP on tumor necrosis factor-α (TNF-α)-induced inflammatory injury in human umbilical vein endothelial cells (HUVECs) and to explore the role of ERS in this process. TNF-α treatment not only significantly increased the adhesion of monocytes to HUVECs and inflammatory cytokine (sICAM1, sE-selectin, MCP-1 and IL-8) production in cell culture supernatants but it also increased ICAM and MMP9 protein expression in HUVECs. TNF-α also effectively increased the ERS-related molecules in HUVECs (GRP78, ATF4, caspase12 and p-PERK). EP treatment effectively reversed the effects of the TNF-α-induced adhesion of monocytes on HUVECs, inflammatory cytokines and ERS-related molecules. Furthermore, thapsigargin (THA, an ERS inducer) attenuated the protective effects of EP against TNF-α-induced inflammatory injury and ERS. The PERK siRNA treatment not only inhibited ERS-related molecules but also mimicked the protective effects of EP to decrease TNF-α-induced inflammatory injury. In summary, we have demonstrated for the first time that EP can effectively reduce vascular endothelial inflammation and that this effect at least in part depends on the attenuation of ERS.