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Background: Patients with acute myocardial infarction suffer systemic metabolic dysfunction via incompletely understood mechanisms. Adipocytes play critical role in metabolic homeostasis. The impact of acute myocardial infarction upon adipocyte function is unclear. Small extracellular vesicles (sEVs) critically contribute to organ-organ communication. Whether and how small extracellular vesicle mediate post-MI cardiomyocyte/adipocyte communication remain unknown. Methods: Plasma sEVs were isolated from sham control (Pla-sEV Sham ) or 3 hours after myocardial ischemia/reperfusion (Pla-sEV MI/R ) and incubated with adipocytes for 24 hours. Compared with Pla-sEV Sham , Pla-sEV MI/R significantly altered expression of genes known to be important in adipocyte function, including a well-known metabolic regulatory/cardioprotective adipokine, APN (adiponectin). Pla-sEV MI/R activated 2 (PERK-CHOP and ATF6 [transcription factor 6]-EDEM [ER degradation enhancing alpha-mannosidase like protein 1] pathways) of the 3 endoplasmic reticulum (ER) stress pathways in adipocytes. These pathological alterations were also observed in adipocytes treated with sEVs isolated from adult cardiomyocytes subjected to in vivo myocardial ischemia/reperfusion (MI/R) (Myo-sEV MI/R ). Bioinformatic/RT-qPCR analysis demonstrates that the members of miR-23-27-24 cluster are significantly increased in Pla-sEV MI/R , Myo-sEV MI/R , and adipose tissue of MI/R animals. Administration of cardiomyocyte-specific miR-23-27-24 sponges abolished adipocyte miR-23-27-24 elevation in MI/R animals, supporting the cardiomyocyte origin of adipocyte miR-23-27-24 cluster. In similar fashion to Myo-sEV MI/R , a miR-27a mimic activated PERK-CHOP and ATF6-EDEM-mediated ER stress. Conversely, a miR-27a inhibitor significantly attenuated Myo-sEV MI/R -induced ER stress and restored APN production. Results: An unbiased approach identified EDEM3 (ER degradation enhancing alpha-mannosidase like protein 3) as a novel downstream target of miR-27a. Adipocyte EDEM3 deficiency phenocopied multiple pathological alterations caused by Myo-sEV MI/R , whereas EDEM3 overexpression attenuated Myo-sEV MI/R -resulted ER stress. Finally, administration of GW4869 or cardiomyocyte-specific miR-23-27-24 cluster sponges attenuated adipocyte ER stress, improved adipocyte endocrine function, and restored plasma APN levels in MI/R animals. Conclusions: We demonstrate for the first time that MI/R causes significant adipocyte ER stress and endocrine dysfunction by releasing miR-23-27-24 cluster-enriched small extracellular vesicle. Targeting small extracellular vesicle–mediated cardiomyocyte-adipocyte pathological communication may be of therapeutic potential to prevent metabolic dysfunction after MI/R.

Cardiovascular disease is the leading cause of morbidity in patients with diabetes mellitus. In 2019, the American Heart Association and the American Diabetes Association (along with industry leaders) launched the groundbreaking collaborative initiative “Know Diabetes by Heart™” to reduce cardiovascular deaths in type 2 diabetic patients. The molecular basis linking diabetes with cardiovascular complications has not yet been fully defined. Recent clinical and experimental studies strongly suggest that adipocyte dysfunction and subsequent pathological communications between adipocyte and cardiomyocytes play important roles in diabetic cardiac injury. This perspective article will review recent development concerning adipocyte-cardiomyocyte communications, and identify the most critical questions remain to be answered in this filed.

Background: Dilated cardiomyopathy (DCM) is a common cause of heart failure and an indication for heart transplantation worldwide. FUN14 Domain Containing 1 (FUNDC1), a canonical mitophagy receptor and mitochondria-associated ER membranes (MAMs) protein, plays an essential role in ischemic heart disease. However, the role of FUNDC1 in DCM remains unidentified. The current study attempted to clarify whether and how FUNDC1 regulates DCM development. Methods: Cardiomyocyte-specific F UNDC 1 gene knockout (CM-FUNDC1 KO) mice were generated to study the impact and potential mechanisms of FUNDC1 in the heart. Mitochondria were isolated from heart tissues, primary mouse neonatal/adult cardiomyocytes, or H9C2 cell lines to probe the role of FUNDC1 in mitochondrial protein import (MPI). Results: All CM-FUNDC1 KO mice developed severe DCM spontaneously and died before 15 months old. The expression of MPI-associated proteins was significantly upregulated in the hearts of CM-FUNDC1 KO mice. Experiments with purified mitochondria demonstrated that MPI efficiency was enhanced by CM-FUNDC1 KO, whereas reduced by FUNDC1 overexpression in an autophagy-independent fashion. We further identified that FUNDC1 selectively downregulated the TIM23 complex without altering other MPI translocases (e.g., TOM20, TIM22 complexes). Downregulation of subunits of TIM23 complex partly reversed the FUNDC1 KO-induced MPI enhancement. Importantly, we demonstrated that FUNDC1 selectively inhibited the import of nuclear gene-encoded respiratory subunits on complex I, IV but not on complex II, III, V or mitochondrial gene-encoded subunits. Finally, FUNDC1 deleted cells exhibited enlarged cell size, retarded cell growth, impaired oxygen consumption rate, and increased mitochondrial superoxide level. All of these phenomena were partly reversed by silence TIM23 complex or reproduced by TIM23 complex overexpression. Conclusions: We demonstrate for the first time that: 1) MPI system plays an important role in the progress of DCM; 2) FUNDC1 is a novel negative regulator of MPI via TIM23 pathway; and 3) FUNDC1 is a promising therapeutic target for DCM.

Background: Despite significantly reduced acute myocardial infarction (MI) mortality in recent years, ischemic heart failure continues to escalate. Therapeutic interventions effectively reversing pathological remodeling are an urgent unmet medical need. We recently demonstrated that AdipoR1 (APN [adiponectin] receptor 1) phosphorylation by GRK2 (G-protein–coupled receptor kinase 2) contributes to maladaptive remodeling in the ischemic heart. The current study clarified the underlying mechanisms leading to AdipoR1 phosphorylative desensitization and investigated whether blocking AdipoR1 phosphorylation may restore its protective signaling, reversing post-MI remodeling. Methods: Specific sites and underlying molecular mechanisms responsible for AdipoR1 phosphorylative desensitization were investigated in vitro (neonatal and adult cardiomyocytes). The effects of AdipoR1 phosphorylation inhibition upon APN post-MI remodeling and heart failure progression were investigated in vivo. Results: Among 4 previously identified sites sensitive to GRK2 phosphorylation, alanine substitution of Ser 205 (AdipoR1 S205A ), but not other 3 sites, rescued GRK2-suppressed AdipoR1 functions, restoring APN-induced cell salvage kinase activation and reducing oxidative cell death. The molecular investigation followed by functional determination demonstrated that AdipoR1 phosphorylation promoted clathrin-dependent (not caveolae) endocytosis and lysosomal-mediated (not proteasome) degradation, reducing AdipoR1 protein level and suppressing AdipoR1-mediated cytoprotective action. GRK2-induced AdipoR1 endocytosis and degradation were blocked by AdipoR1 S205A overexpression. Moreover, AdipoR1 S205E (pseudophosphorylation) phenocopied GRK2 effects, promoted AdipoR1 endocytosis and degradation, and inhibited AdipoR1 biological function. Most importantly, AdipoR1 function was preserved during heart failure development in AdipoR1-KO (AdipoR1 knockout) mice reexpressing hAdipoR1 S205A . APN administration in the failing heart reversed post-MI remodeling and improved cardiac function. However, reexpressing hAdipoR1 WT in AdipoR1-KO mice failed to restore APN cardioprotection. Conclusions: Ser 205 is responsible for AdipoR1 phosphorylative desensitization in the failing heart. Blockade of AdipoR1 phosphorylation followed by pharmacological APN administration is a novel therapy effective in reversing post-MI remodeling and mitigating heart failure progression.

Chronic exposure to hexavalent chromium compounds [Cr(VI)] is associated with an increased risk of cancers, but the molecular mechanisms remain to be elucidated. In this study, we found that CXCL5 levels in peripheral blood monocytes (PBMCs) and plasma from workers with occupational exposure to Cr(VI) were dramatically upregulated compared to non-exposure healthy subjects, and plasma C-X-C Motif Chemokine Ligand 5 (CXCL5) CXCL5 levels were positively correlated with Cr concentrations in subjects’ toenails. Zinc chromate exposed mice showed higher levels of CXCL5 and its receptor CXCR2 in lung tissues, and in PBMCs. Similar CXCL5 upregulation was evident in Cr(VI)-induced transformed (Cr-T) cells with long-term Cr(VI) treatment. Mechanistic studies showed that elevated CXCL5 expression levels were regulated by Cr(VI)-induced histone modifications and DNA hypomethylation, and that the c-Myc/p300 complex was a key upstream regulator of histone H3 acetylation. CXCL5 overexpression promoted Cr (VI)-induced the epithelial to mesenchyme transition (EMT) by upregulating zinc finger E-box binding homeobox 1 (ZEB1) to promote tumor development. Our findings identify a novel mechanism by which CXCL5 is upregulated and promotes EMT and carcinogenesis upon chronic Cr(VI) exposure. Our work also implies that CXCL5 mRNA and protein levels will elevate in PBMCs and serum after occupational Cr(VI) exposure, which may be a potential target and biomarker for cancer prevention and health surveillance among populations exposed to Cr(VI).

Hypertensive patients have impaired sodium excretion. However, the mechanisms are incompletely understood. Despite the established association between obesity/excess adiposity and hypertension, whether and how adiponectin, one of the adipokines, contributes to impaired sodium excretion in hypertension has not been previously investigated. The current study tested the hypothesis that adiponectin promotes natriuresis and diuresis in the normotensive state. However, impaired adiponectin-mediated natriuresis and diuresis are involved in pathogenesis of hypertension. We found that sodium excretion was reduced in adiponectin knockout (Adipo−/−) mice; intrarenal arterial infusion of adiponectin-induced natriuresis and diuresis in Wistar–Kyoto (WKY) rats. However, the natriuretic and diuretic effects of adiponectin were impaired in spontaneously hypertensive rats (SHRs), which were ascribed to the hyperphosphorylation of adiponectin receptor and subsequent uncoupling from Gαi. Inhibition of adiponectin receptor phosphorylation by a specific point mutation restored its coupling with Gαi and the adiponectin-mediated inhibition of Na+-K+-ATPase activity in renal proximal tubule (RPT) cells from SHRs. Finally, we identified G protein-coupled receptor kinase 4 (GRK4) as a mediator of adiponectin receptor hyperphosphorylation; mice transgenic for a hyperphosphorylating variant of GRK4 replicated the abnormal adiponectin function observed in SHRs, whereas down-regulation of GRK4 by renal ultrasound-directed small interfering RNA (siRNA) restored the adiponectin-mediated sodium excretion and reduced the blood pressure in SHRs. We conclude that the stimulatory effect of adiponectin on sodium excretion is impaired in hypertension, which is ascribed to the increased renal GRK4 expression and activity. Targeting GRK4 restores impaired adiponectin-mediated sodium excretion in hypertension, thus representing a novel strategy against hypertension.

Rationale: Obstructive sleep apnea-hypopnea syndrome, a sleep breathing disorder in which chronic intermittent hypoxia (CIH) is the primary pathology, is associated with multiple cardiovascular diseases. However, whether and how CIH may affect cardiac remodeling following myocardial infarction (MI) remains unknown. Objective: To determine whether CIH exposure at different periods of MI may exacerbate post-MI heart failure and to identify the mechanisms underlying CIH-exacerbated post-MI remodeling. Methods and Results: Adult male mice were subjected to MI (4 weeks) with and without CIH (4 or 8 weeks). CIH before MI (CIH+MI) had no significant effect on post-MI remodeling. However, double CIH exposure (CIH+MI+CIH) or CIH only during the MI period (MI+CIH) significantly exacerbated pathological remodeling and reduced survival rate. Mechanistically, CIH activated TGF-β (tumor growth factor-β)/Smad (homologs of both the Drosophila protein MAD and the C. elegans protein SMA) signaling and enhanced cardiac epithelial to mesenchymal transition, markedly increasing post-MI cardiac fibrosis. Transcriptome analysis revealed that, among 15 genes significantly downregulated (MI+CIH versus MI), Ctrp9 (a novel cardioprotective cardiokine) was one of the most significantly inhibited genes. Real-time polymerase chain reaction/Western analysis confirmed that cardiomyocyte CTRP9 expression was significantly reduced in MI+CIH mice. RNA-sequencing, real-time polymerase chain reaction, and dual-luciferase reporter assays identified that microRNA-214-3p is a novel Ctrp9 targeting miRNA. Its upregulation is responsible for Ctrp9 gene suppression in MI+CIH. Finally, AAV9 (adeno-associated virus 9)-mediated cardiac-specific CTRP9 overexpression or rCTRP9 (recombinated CTRP9) administration inhibited TGF-β/Smad and Wnt/β-catenin pathways, attenuated interstitial fibrosis, improved cardiac function, and enhanced survival rate in MI+CIH animals. Conclusions: This study provides the first evidence that MI+CIH upregulates miR-214-3p, suppresses cardiac CTRP9 (C1q tumor necrosis factor-related protein-9) expression, and exacerbates cardiac remodeling, suggesting that CTRP9 may be a novel therapeutic target against pathological remodeling in MI patients with obstructive sleep apnea-hypopnea syndrome.

Background: Increased cardiac apoptosis is a hallmark of the elderly, which in turn increases the risk for developing cardiac disease. The overexpression of Omi/HtrA2 mRNA and protein contributes to apoptosis in the aged heart. Heat shock factor 1 (HSF1) is a transcription factor that binds to the promoter of Omi/HtrA2 in the aging myocardium. However, whether HSF1 participates in cardiomyocyte apoptosis via transcriptional regulation of Omi/HtrA2 remains unclear. The present study was designed to investigate whether HSF1 plays a role in Omi/HtrA2 transcriptional regulation and myocardial apoptosis. Methods and Results: Assessment of the hearts of mice of different ages was performed, which indicated a decrease in cardiac function reserve and an increase in mitochondrial apoptosis. Omi/HtrA2 overexpression in the elderly was negatively correlated with left ventricular function after exercise overload and positively correlated with myocardial Caspase-9 apoptosis. Chromatin immunoprecipitation (ChIP) of aging hearts and plasmid transfection/RNA interference of H9C2 cells revealed that enhancement of HSF1 expression promotes Omi/HtrA2 expression by inducing the promoter activity of Omi/HtrA2 while also increasing mitochondrial apoptosis by upregulating Omi/HtrA2 expression. Conclusions: HSF1 acts as a transcriptional factor that induces Omi/HtrA2 expression and Caspase-9 apoptosis in aged cardiomyocytes, while also decreasing cardiac function reserve.

Background: Myocardial ischemia-reperfusion (MI/R) injury is a significant clinical problem without effective therapy. Unbiased omics approaches may reveal key MI/R mediators to initiate MI/R injury. Methods: We used a dynamic transcriptome analysis of mouse heart exposed to various MI/R periods to identify S100a8/a9 as an early mediator. Using loss/gain-of-function approaches to understand the role of S100a8/a9 in MI/R injury, we explored the mechanisms through transcriptome and functional experiment. Dynamic serum S100a8/a9 levels were measured in patients with acute myocardial infarction before and after percutaneous coronary intervention. Patients were prospectively followed for the occurrence of major adverse cardiovascular events. Results: S100a8/a9 was identified as the most significantly upregulated gene during the early reperfusion stage. Knockout of S100a9 markedly decreased cardiomyocyte death and improved heart function, whereas hematopoietic overexpression of S100a9 exacerbated MI/R injury. Transcriptome/functional studies revealed that S100a8/a9 caused mitochondrial respiratory dysfunction in cardiomyocytes. Mechanistically, S100a8/a9 downregulated NDUF gene expression with subsequent mitochondrial complex I inhibition via Toll-like receptor 4/Erk–mediated Pparg coactivator 1 alpha/nuclear respiratory factor 1 signaling suppression. Administration of S100a9 neutralizing antibody significantly reduced MI/R injury and improved cardiac function. Finally, we demonstrated that serum S100a8/a9 levels were significantly increased 1 day after percutaneous coronary intervention in patients with acute myocardial infarction, and elevated S100a8/a9 levels were associated with the incidence of major adverse cardiovascular events. Conclusions: Our study identified S100a8/a9 as a master regulator causing cardiomyocyte death in the early stage of MI/R injury via the suppression of mitochondrial function. Targeting S100a8/a9-intiated signaling may represent a novel therapeutic intervention against MI/R injury. Clinical Trial Registration: URL: https://www.clinicaltrials.gov . Unique identifier: NCT03752515

Background Withaferin A (WFA), an anticancer constituent of the plant Withania somnifera, inhibits tumor growth in association with apoptosis induction. However, the potential role of WFA in the cardiovascular system is little-studied and controversial.Methods and Results:Two different doses of WFA were tested to determine their cardioprotective effects in myocardial ischemia/reperfusion (MI/R) injury through evaluation of cardiofunction in wild-type and AMP-activated protein kinase domain negative (AMPK-DN) gentransgenic mice. Surprisingly, cardioprotective effects (improved cardiac function and reduced infarct size) were observed with low-dose WFA (1 mg/kg) delivery but not high-dose (5 mg/kg). Mechanistically, low-dose WFA attenuated myocardial apoptosis. It decreased MI/R-induced activation of caspase 9, the indicator of the intrinsic mitochondrial pathway, but not caspase 8. It also upregulated the level of AMP-activated protein kinase (AMPK) phosphorylation and increased the MI/R inhibited ratio of Bcl2/Bax. In AMPK-deficient mice, WFA did not ameliorate MI/R-induced cardiac dysfunction, attenuate infarct size, or restore the Bcl2/Bax (B-cell lymphoma2/Mcl-2-like protein 4) ratio. Conclusions These results demonstrated for the first time that low-dose WFA is cardioprotective via upregulation of the anti-apoptotic mitochondrial pathway in an AMPK-dependent manner.

Angiotensin II type 1 receptor autoantibodies (AT1-AA) cause endothelial-dependent smooth muscle relaxation disorder. It is well understood that impairment of the NO–cGMP signaling pathway is one of the mechanisms of endothelial-dependent smooth muscle relaxation disorder. However, it is still unclear whether AT1-AA induces endothelial-dependent smooth muscle relaxation disorder via the impairment of the NO–cGMP signaling pathway. In addition, adiponectin exerts vascular endothelial protection through the NO–cGMP signaling pathway. Therefore, the purpose of this investigation was to assess the mechanism of vascular endothelial-dependent smooth muscle relaxation disorder induced by AT1-AA and the role of adiponectin in attenuating this dysregulation. Serum endothelin-1 (ET-1), adiponectin and AT1-AA were detected by enzyme-linked immunosorbent assay. In preeclamptic patients, there was an increased level of AT1-AA, which was positively correlated with ET-1 and negatively correlated with adiponectin, as elevated levels of ET-1 suggested endothelial injury. AT1-AA-positive animal models were actively immunized with the second extracellular loop of the angiotensin II type 1 receptor (AT1R-ECII) for eight weeks. In thoracic aortas of AT1-AA positive rats, ET-1 was elevated, endothelium-dependent vasodilation was decreased. Paradoxically, as the upstream element of the NO-cGMP signaling pathway, NO production was not decreased but increased, and the ratio of p-VASP/VASP, an established biochemical endpoint of NO-cGMP signaling pathway, was reduced. Moreover, the levels of nitrotyrosine and gp91phox which indicate the presence of peroxynitrite (ONOO-) and superoxide anion (O2·−) were increased. Pretreatment with the ONOO- scavenger FeTMPyP or O2·−scavenger Tempol normalized vasorelaxation. Key enzymes responsible for NO synthesis were also assessed. iNOS protein expression was increased, but p-eNOS(Ser1177)/eNOS was reduced. Preincubation with the iNOS inhibitor 1400 W or eNOS agonist nebivolol restored vasorelaxation. Further experiments showed levels of p-AMPKα (Thr172)/AMPKα, which controls iNOS expression and eNOS activity, to have been reduced. Furthermore, levels of the upstream component of AMPK, adiponectin, was reduced in sera of AT1-AA positive rats and supplementation of adiponectin significantly decreased ET-1 contents, improved endothelial-dependent vasodilation, reduced NO production, elevated p-VASP/VASP, inhibited protein expression of nitrotyrosine and gp91phox, reduced iNOS overexpression, and increased eNOS phosphorylation at Ser1177 in the thoracic aorta of AT1-AA positive rats. These results established that impairment NO–cGMP pathway may aggravate the endothelial-dependent smooth muscle relaxation disorder in AT1-AA positive rats and adiponectin improved endothelial-dependent smooth muscle relaxation disorder by enhancing NO–cGMP pathway. This discovery may shed a novel light on clinical treatment of vascular diseases associated with AT1-AA.

Numerous studies have reported significantly elevated titers of serum autoantibody against the second extracellular loop of β1-adrenoceptor (β1-AA), a catecholamine-like substance with β1-adrenergic activity, in patients with heart failure. Although evidence demonstrates that this autoantibody may alter T cell proliferation and secretion, the role of T lymphocytes in heart failure induced by β1-AA remains unclear. The current study was designed to determine whether T cell disorder contributes to heart failure induced by β1-AA. β1-AA monoclonal antibodies (β1-AAmAb) produced using the hybridoma technique were administered in wild-type mice or T lymphocyte deficiency nudes for 12 weeks. T lymphocytes from heart failure patients and neonatal cardiomyocytes were utilized in vitro. Mouse protein antibody array analysis was employed to detect the cytokines responsible for β1-AAmAb-induced heart failure. Compared to wild-type mice, T lymphocyte deficiency mice prevented cardiac function from getting worse, attenuated adverse remodeling, and ameliorated cardiomyocyte apoptosis and fibrosis. As shown by protein array, the serum level of interleukin (IL)-6 was significantly lower in the nude group as compared to wild-type after β1-AAmAb treatment. Mechanistic studies in vitro demonstrated that T lymphocyte culture supernatants stimulated by β1-AAmAb caused direct damage in the cardiomyocytes, and β1-AAmAb promoted proliferation of T lymphocytes isolated from patients with heart failure and increased IL-6 release. IL-6-specific siRNA virtually abolished cardiomyocyte apoptosis, suggesting that IL-6 may be a key cytokine released by T lymphocytes and responsible for β1-AAmAb-induced cardiac remodeling. Collectively, we demonstrate that β1-AAmAb-induced cardiac remodeling via mediating T lymphocyte disorder and releasing a variety of IL-6.

Setting up an animal model by using active immunization methods is a common means of studying immune-related diseases or producing antibodies with high titer and high activities. However, the security during the process of pathogen emulsification remains unclear. In a physical examination, we unexpectedly noticed high levels of angiotensin II type 1 receptor autoantibody (AT1-AA) specific to the immunizing antigen in the sera of some researchers who had participated in setting up active immunization animal models, and we were puzzled about the cause of AT1-AA production. In this study, we intended to investigate whether the emulsified antigen was the source of infection in these researchers, and if so, how to prevent it from occurring. AT1-AA was detected by advanced ELISA method. The participants presented higher levels of AT1-AA compared with non-participants of the same laboratory. This phenomenon remained that some factors during the process of rat model establishment may contribute to AT1-AA production. Animal and glove penetration studies indicated the emulsified antigen infection was attributed to neither aerosol or fur touch nor penetrating through gloves. However, AT1-AA level was largely decreased in the participants after they used an automatic emulsification device. Because of the strong permeability of the adjuvant, we speculated that emulsified antigen might get access to the unprotected skin of the participants accidentally during the immunization process. These results demonstrated that accidental contacts of emulsified antigens may infect researchers during the process of traditional hand-push emulsification, resulting in high specific autoantibody levels, which can be prevented by using appropriate tools.

There is limited and discordant evidence on the role of nicotine in diabetic vascular disease. Exacerbated endothelial cell dysregulation in smokers with diabetes is associated with the disrupted adipose function. Adipokines possess vascular protective, anti-inflammatory, and anti-diabetic properties. However, whether and how nicotine primes and aggravates diabetic vascular disorders remain uncertain. In this study, we evaluated the alteration of adiponectin (APN) level in high-fat diet (HFD) mice with nicotine (NIC) administration. The vascular pathophysiological response was evaluated with vascular ring assay. Confocal and co-immunoprecipitation analysis were applied to identify the signal interaction and transduction. These results indicated that the circulating APN level in nicotine-administrated diabetic Apolipoprotein E-deficient (ApoE−/−) mice was elevated in advance of 2 weeks of diabetic ApoE−/− mice. NIC and NIC addition in HFD groups (NIC + HFD) reduced the vascular relaxation and signaling response to APN at 6 weeks. Mechanistically, APN receptor 1 (AdipoR1) level was decreased in NIC and further significantly reduced in NIC + HFD group at 6 weeks, while elevated suppressor of cytokine signaling 3 (SOCS3) expression was induced by NIC and further augmented in NIC + HFD group. Additionally, nicotine provoked SOCS3, degraded AdipoR1, and attenuated APN-activated ERK1/2 in the presence of high glucose and high lipid (HG/HL) in human umbilical vein endothelial cells (HUVECs). MG132 (proteasome inhibitor) administration manifested that AdipoR1 was ubiquitinated, while inhibited SOCS3 rescued the reduced AdipoR1. In summary, this study demonstrated for the first time that nicotine primed vascular APN resistance via SOCS3-mediated degradation of ubiquitinated AdipoR1, accelerating diabetic endothelial dysfunction. This discovery provides a potential therapeutic target for preventing nicotine-accelerated diabetic vascular dysfunction.

Objective Atherosclerotic vascular disease remains the principal cause of death and disability among patients with type 2 diabetes. Unfortunately, the problem is not adequately resolved by therapeutic strategies with currently available drugs or approaches that solely focus on optimal glycemic control. To identify the key contributors and better understand the mechanism of diabetic atherosclerotic vascular disease, we aimed to elucidate the key genetic characteristics and pathological pathways in atherosclerotic vascular disease through nonbiased bioinformatics analysis and subsequent experimental demonstration and exploration in diabetic atherosclerotic vascular disease. Methods and results Sixty-eight upregulated and 23 downregulated genes were identified from the analysis of gene expression profiles (GSE30169 and GSE6584). A comprehensive bioinformatic assay further identified that ferroptosis, a new type of programmed cell death and HMOX1 (a gene that encodes heme oxygenase), were vital factors in atherosclerotic vascular disease. We further demonstrated that diabetes significantly increased ferroptosis and HMOX1 levels compared to normal controls. Importantly, the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively attenuated diabetic atherosclerosis, suggesting the causative role of ferroptosis in diabetic atherosclerosis development. At the cellular level, Fer-1 ameliorated high glucose high lipid-induced lipid peroxidation and downregulated ROS production. More importantly, HMOX1 knockdown attenuated Fe2+ overload, reduced iron content and ROS, and alleviated lipid peroxidation, which led to a reduction in ferroptosis in diabetic human endothelial cells. Conclusions We demonstrated that HMOX1 upregulation is responsible for the increased ferroptosis in diabetic atherosclerosis development, suggesting that HMOX1 may serve as a potential therapeutic or drug development target for diabetic atherosclerosis.

Background To develop a sensitive risk score predicting the risk of mortality in patients with coronavirus disease 2019 (COVID-19) using complete blood count (CBC). Methods We performed a retrospective cohort study from a total of 13,138 inpatients with COVID-19 in Hubei, China, and Milan, Italy. Among them, 9,810 patients with ≥2 CBC records from Hubei were assigned to the training cohort. CBC parameters were analyzed as potential predictors for all-cause mortality and were selected by the generalized linear mixed model (GLMM). Findings Five risk factors were derived to construct a composite score (PAWNN score) using the Cox regression model, including platelet counts, age, white blood cell counts, neutrophil counts, and neutrophil:lymphocyte ratio. The PAWNN score showed good accuracy for predicting mortality in 10-fold cross-validation (AUROCs 0.92–0.93) and subsets with different quartile intervals of follow-up and preexisting diseases. The performance of the score was further validated in 2,949 patients with only 1 CBC record from the Hubei cohort (AUROC 0.97) and 227 patients from the Italian cohort (AUROC 0.80). The latent Markov model (LMM) demonstrated that the PAWNN score has good prediction power for transition probabilities between different latent conditions. Conclusions The PAWNN score is a simple and accurate risk assessment tool that can predict the mortality for COVID-19 patients during their entire hospitalization. This tool can assist clinicians in prioritizing medical treatment of COVID-19 patients. Funding This work was supported by National Key R&D Program of China (2016YFF0101504, 2016YFF0101505, 2020YFC2004702, 2020YFC0845500), the Key R&D Program of Guangdong Province (2020B1111330003), and the medical flight plan of Wuhan University (TFJH2018006)., Context and significance Researchers from Wuhan, China developed a complete blood count-based risk score (PAWNN score) that can predict mortality during the entire course of hospitalization in a large cohort with 13,138 COVID-19 patients. This risk score was validated in two independent cohorts from China and Italy and in a longitudinal model. The model can serve as a valuable tool for physicians with very limited medical resources to properly monitor and stratify COVID-19 patients and to reduce mortality., Graphical Abstract, Highlights The blood count-based PAWNN score can accurately predict mortality risk of COVID-19 PAWNN can properly stratify COVID-19 patients with limited medical resources, Liu et al. developed and validated a complete blood count-based score (PAWNN) that can predict mortality during the entire course of hospitalization in a cohort of 13,138 COVID-19 patients. The model can serve as a valuable tool to properly monitor and stratify COVID-19 patients with very limited medical resources.

Background. Obstructive sleep apnea (OSA) is closely related to the incidence and progression of coronary artery disease (CAD), and the mechanisms linking OSA and CAD are multifactorial. C1q/TNF-related protein-9 (CTRP9) is a novel adipokine that protects the heart against ischemic injury and ameliorates cardiac remodeling. We aimed to ascertain the clinical relevance of CTRP9 with OSA prevalence in patients with CAD. Methods. From August 2016 to March 2019, consecutive eligible patients with CAD (n=154; angina pectoris, n=88; acute myocardial infarction [AMI], n=66) underwent cardiorespiratory polygraphy. OSA was defined as an apnea-hypopnea index (AHI) ≥15 events·h−1. Plasma CTRP9 concentrations were measured by ELISA method. Results. Moderate/severe OSA was present in 89 patients (57.8%). CTRP9 levels were significantly decreased in the moderate/severe OSA group than in the no/mild OSA group (4.7 [4.1-5.2] ng/mL vs. 4.9 [4.4-6.0] ng/mL, P=0.003). The difference between groups was only observed in patients with AMI (3.0 [2.3-4.9] vs. 4.5 [3.2-7.9], P=0.009). Correlation analysis showed that CTRP9 levels were negatively correlated with AHI (r=−0.238, P=0.003) and oxygen desaturation index (r=−0.234, P=0.004) and positively correlated with left ventricular ejection fraction (r=0.251, P=0.004) in all subjects. Multivariate analysis showed that male gender (OR 3.099, 95% CI 1.029-9.330, P=0.044), BMI (OR 1.148, 95% CI 1.040-1.268, P=0.006), and CTRP9 levels (OR 0.726, 95% CI 0.592-0.890, P=0.002) were independently associated with the prevalence of moderate/severe OSA. Conclusions. Plasma CTRP9 levels were independently related to the prevalence of moderate/severe OSA in patients with CAD, suggesting that CTRP9 might play a role in the pathogenesis of CAD exacerbated by OSA.

Supplemental Digital Content is available in the text., The prognostic power of circulating cardiac biomarkers, their utility, and pattern of release in coronavirus disease 2019 (COVID-19) patients have not been clearly defined. In this multicentered retrospective study, we enrolled 3219 patients with diagnosed COVID-19 admitted to 9 hospitals from December 31, 2019, to March 4, 2020, to estimate the associations and prognostic power of circulating cardiac injury markers with the poor outcomes of COVID-19. In the mixed-effects Cox model, after adjusting for age, sex, and comorbidities, the adjusted hazard ratio of 28-day mortality for high-sensitivity cardiac troponin I was 7.12 ([95% CI, 4.60–11.03] P

Summary Statins are lipid-lowering therapeutics with favorable anti-inflammatory profiles and have been proposed as an adjunct therapy for COVID-19. However, statins may increase the risk of SARS-CoV-2 viral entry by inducing ACE2 expression. Here, we performed a retrospective study on 13,981 patients with COVID-19 in Hubei Province, China, among which 1,219 received statins. Based on a mixed-effect Cox model after propensity score-matching, we found that the risk for 28-day all-cause mortality was 5.2% and 9.4% in the matched statin and non-statin groups, respectively, with an adjusted hazard ratio of 0.58. The statin use-associated lower risk of mortality was also observed in the Cox time-varying model and marginal structural model analysis. These results give support for the completion of ongoing prospective studies and randomized controlled trials involving statin treatment for COVID-19, which are needed to further validate the utility of this class of drugs to combat the mortality of this pandemic., Graphical Abstract, Highlights • Statin treatment among 13,981 patients with COVID-19 was retrospectively studied • Statin use in this cohort was associated with a lower risk of all-cause mortality • Adding an ACE inhibitor or an ARB did not affect statin-associated outcome in the cohort • The benefit of statins among this cohort may be due to immunomodulatory benefits, Statins have anti-inflammatory benefits and were suggested as an adjunct therapy for COVID-19. But statins may increase the expression of ACE2, the receptor for SARS-CoV-2. Here, Zhang et al. retrospectively analyzed 13,981 COVID-19 cases and found that in-hospital statin use is associated with a lower risk of all-cause mortality.

Background Laboratory studies demonstrate glucose‐insulin‐potassium ( GIK ) as a potent cardioprotective intervention, but clinical trials have yielded mixed results, likely because of varying formulas and timing of GIK treatment and different clinical settings. This study sought to evaluate the effects of modified GIK regimen given perioperatively with an insulin‐glucose ratio of 1:3 in patients undergoing cardiopulmonary bypass surgery. Methods and Results In this prospective, randomized, double‐blinded trial with 930 patients referred for cardiac surgery with cardiopulmonary bypass , GIK (200 g/L glucose, 66.7 U/L insulin, and 80 mmol/L KC l) or placebo treatment was administered intravenously at 1 mL /kg per hour 10 minutes before anesthesia and continuously for 12.5 hours. The primary outcome was the incidence of in‐hospital major adverse cardiac events including all‐cause death, low cardiac output syndrome, acute myocardial infarction, cardiac arrest with successful resuscitation, congestive heart failure, and arrhythmia. GIK therapy reduced the incidence of major adverse cardiac events and enhanced cardiac function recovery without increasing perioperative blood glucose compared with the control group. Mechanistically, this treatment resulted in increased glucose uptake and less lactate excretion calculated by the differences between arterial and coronary sinus, and increased phosphorylation of insulin receptor substrate‐1 and protein kinase B in the hearts of GIK ‐treated patients. Systemic blood lactate was also reduced in GIK ‐treated patients during cardiopulmonary bypass surgery. Conclusions A modified GIK regimen administered perioperatively reduces the incidence of in‐hospital major adverse cardiac events in patients undergoing cardiopulmonary bypass surgery. These benefits are likely a result of enhanced systemic tissue perfusion and improved myocardial metabolism via activation of insulin signaling by GIK . Clinical Trial Registration URL: clinicaltrials.gov. Identifier: NCT 01516138.

Rationale: Mesenchymal stromal cell–based therapy is promising against ischemic heart failure. However, its efficacy is limited due to low cell retention and poor paracrine function. A transmembrane protein capable of enhancing cell-cell adhesion, N-cadherin garnered attention in the field of stem cell biology only recently. Objective: The current study investigates whether and how N-cadherin may regulate mesenchymal stromal cells retention and cardioprotective capability against ischemic heart failure. Methods and Results: Adult mice–derived adipose tissue–derived mesenchymal stromal cells (ADSC) were transfected with adenovirus harboring N-cadherin, T-cadherin, or control adenovirus. CM-DiI-labeled ADSC were intramyocardially injected into the infarct border zone at 3 sites immediately after myocardial infarction (MI) or myocardial ischemia/reperfusion. ADSC retention/survival, cardiomyocyte apoptosis/proliferation, capillary density, cardiac fibrosis, and cardiac function were determined. Discovery-driven/cause-effect analysis was used to determine the molecular mechanisms. Compared with ADSC transfected with adenovirus-control, N-cadherin overexpression (but not T-cadherin) markedly increased engrafted ADSC survival/retention up to 7 days post-MI. Histological analysis revealed that ADSC transfected with adenovirus-N-cadherin significantly preserved capillary density and increased cardiomyocyte proliferation and moderately reduced cardiomyocyte apoptosis 3 days post-MI. More importantly, ADSC transfected with adenovirus-N-cadherin (but not ADSC transfected with adenovirus-T-cadherin) significantly increased left ventricular ejection fraction and reduced fibrosis in both MI and myocardial ischemia/reperfusion mice. In vitro experiments demonstrated that N-cadherin overexpression promoted ADSC-cardiomyocyte adhesion and ADSC migration, enhancing their capability to increase angiogenesis and cardiomyocyte proliferation. MMP (matrix metallopeptidases)-10/13 and HGF (hepatocyte growth factor) upregulation is responsible for N-cadherin’s effect upon ADSC migration and paracrine angiogenesis. N-cadherin overexpression promotes cardiomyocyte proliferation by HGF release. Mechanistically, N-cadherin overexpression significantly increased N-cadherin/β-catenin complex formation and active β-catenin levels in the nucleus. β-catenin knockdown abolished N-cadherin overexpression–induced MMP-10, MMP-13, and HGF expression and blocked the cellular actions and cardioprotective effects of ADSC overexpressing N-cadherin. Conclusions: We demonstrate for the first time that N-cadherin overexpression enhances mesenchymal stromal cells–protective effects against ischemic heart failure via β-catenin-mediated MMP-10/MMP-13/HGF expression and production, promoting ADSC/cardiomyocyte adhesion and ADSC retention.

The C1q complement/TNF-related protein superfamily (CTRPs) displays differential effects on the regulation of metabolic homeostasis, governing cardiovascular function. However, whether and how they may serve as predictor/pro-diagnosis factors for assessing the risks of coronary artery disease (CAD) remains controversial. Therefore, we performed a clinical study to elaborate on the implication of CTRPs (CTRP1, CTRP5, CTRP7, and CTRP15) in CAD. CTRP1 were significantly increased, whereas CTRP7 and CTRP15 levels were decreased in CAD patients compared to the non-CAD group. Significant differences in CTRP1 levels were discovered between the single- and triple-vascular-vessel lesion groups. ROC analysis revealed that CTRP7 and CTRP15 may serve as CAD markers, while CTRP1 may serve as a marker for the single-vessel lesion of CAD. CTRP1 and CTRP5 can serve as markers for the triple-vessel lesion. CTRP1 may serve as an independent risk predictor for triple-vessel lesion, whereas CTRP15 alteration may serve for a single-vessel lesion of CAD. CTRP1 may serve as a novel superior biomarker for diagnosis of severity of vessel-lesion of CAD patients. CTRP7, CTRP15 may serve as more suitable biomarker for the diagnosis of CAD patients, whereas CTRP5 may serve as an independent predictor for CAD. These findings suggest CTRPs may be the superior predictive factors for the vascular lesion of CAD and represent novel therapeutic targets against CAD.

Withaferin A (WFA), a withanolide derived from medicinal plant Withania somnifera, possesses anti-tumorigenic and immunomodulatory activities against various cancer cells. However, the role of WFA in myocardial ischemia reperfusion (MI/R) injury remains unclear. In the present study, we determined whether WFA may regulate cardiac ischemia reperfusion injury and elucidate the underlying mechanisms. We demonstrated that WFA enhanced H9c2 cells survival ability against simulated ischemia/reperfusion (SI/R) or hydrogen peroxide (H2O2)-induced cell apoptosis. In addition, the enhanced oxidative stress induced by SI/R was inhibited by WFA. Among the multiple antioxidant molecules determined, antioxidants SOD2, SOD3, Prdx-1 was obviously upregulated by WFA. When Akt inhibitor IV was administrated, WFA's suppression effect on oxidative stress was obviously abolished. Additional experiments demonstrated that WFA successfully inhibited H2O2 induced upregulation of SOD2, SOD3, and Prdx-1, ameliorated cardiomyocyte caspase-3 activity via an Akt dependent manner. Collectively, these results support the therapeutic potential of WFA against cardiac ischemia reperfusion injury and highlight the application of WFA in cardiovascular diseases holding great promise for the future.

ObjectiveThe prognostic significance of obstructive sleep apnea (OSA) in patients with acute coronary syndrome (ACS) according to diabetes mellitus (DM) status remains unclear. We aimed to elucidate the association of OSA with subsequent cardiovascular events in patients with ACS with or without DM.Research design and methodsIn this prospective cohort study, consecutive eligible patients with ACS underwent cardiorespiratory polygraphy between June 2015 and May 2017. OSA was defined as an Apnea Hypopnea Index ≥15 events/hour. The primary end point was major adverse cardiovascular and cerebrovascular events (MACCEs), including cardiovascular death, myocardial infarction, stroke, ischemia-driven revascularization, or hospitalization for unstable angina or heart failure.ResultsAmong 804 patients, 248 (30.8%) had DM and 403 (50.1%) had OSA. OSA was associated with 2.5 times the risk of 1 year MACCE in patients with DM (22.3% vs 7.1% in the non-OSA group; adjusted HR (HR)=2.49, 95% CI 1.16 to 5.35, p=0.019), but not in patients without DM (8.5% vs 7.7% in the non-OSA group, adjusted HR=0.94, 95% CI 0.51 to 1.75, p=0.85). Patients with DM without OSA had a similar 1 year MACCE rate as patients without DM. The increased risk of events was predominately isolated to patients with OSA with baseline glucose or hemoglobin A1c levels above the median. Combined OSA and longer hypoxia duration (time with arterial oxygen saturation 22 min) further increased the MACCE rate to 31.0% in patients with DM.ConclusionsOSA was associated with increased risk of 1 year MACCE following ACS in patients with DM, but not in non-DM patients. Further trials exploring the efficacy of OSA treatment in high-risk patients with ACS and DM are warranted.

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.

Previous studies have demonstrated that aging promotes myocardial apoptosis after ischemia/reperfusion, via unknown specific mechanisms. The present study investigates the potential relationship between lncRNAs and aging-related apoptosis by lncRNA/mRNA microarray technology. The results indicate aging increased myocardial lncRNA ENSMUST00000134285 and mMAPK11, confirmed by both bioinformatics analysis and polymerase chain reaction (PCR). Mouse cardiomyocytes were subjected to gene manipulation (ENSMUST00000134285 knockdown and overexpression). Knockdown of ENSMUST00000134285 inhibited MAPK11 activity and increased the myocardial apoptotic ratio (determined by TUNEL staining and caspase activity assays) after hypoxia/reoxygenation. Conversely, overexpression of ENSMUST00000134285 increased MAPK11 activity and decreased the myocardial apoptotic ratio. Furthermore, luciferase reporter assay revealed that miR760 may be a mediator between ENSMUST00000134285 and mMAPK11. We have provided evidence that lncRNAs are the important regulatory molecules in aging-mediated effects upon apoptosis. The apoptosis regulatory effects of aging are complex. Except apoptosis-promoting effects, aging could also inhibit myocardial apoptosis after hypoxia or ischemia. Further studies investigating the mechanisms that aging inhibit myocardial apoptosis after hypoxia/ischemia.

Background: Cell therapy remains the most promising approach against ischemic heart injury. However, the poor survival of engrafted stem cells in the ischemic environment limits their therapeutic efficacy for cardiac repair after myocardial infarction. CTRP9 (C1q/tumor necrosis factor–related protein-9) is a novel prosurvival cardiokine with significantly downregulated expression after myocardial infarction. Here we tested a hypothesis that CTRP9 might be a cardiokine required for a healthy microenvironment promoting implanted stem cell survival and cardioprotection. Methods: Mice were subjected to myocardial infarction and treated with adipose-derived mesenchymal stem cells (ADSCs, intramyocardial transplantation), CTRP9, or their combination. Survival, cardiac remodeling and function, cardiomyocytes apoptosis, and ADSCs engraftment were evaluated. Whether CTRP9 directly regulates ADSCs function was determined in vitro. Discovery-drive approaches followed by cause-effect analysis were used to uncover the molecular mechanisms of CTRP9. Results: Administration of ADSCs alone failed to exert significant cardioprotection. However, administration of ADSCs in addition to CTRP9 further enhanced the cardioprotective effect of CTRP9 ( P P Conclusions: We provide the first evidence that CTRP9 promotes ADSCs proliferation/survival, stimulates ADSCs migration, and attenuates cardiomyocyte cell death by previously unrecognized signaling mechanisms. These include binding with N-cadherin, activation of ERK-matrix metallopeptidase 9 and ERK-nuclear factor erythroid-derived 2—like 2 signaling, and upregulation/secretion of antioxidative proteins. These results suggest that CTRP9 is a cardiokine critical in maintaining a healthy microenvironment facilitating stem cell engraftment in infarcted myocardial tissue, thereby enhancing stem cell therapeutic efficacy.

Background/Aims: Obstructive sleep apnea hypoxia syndrome (OSAHS) is an independent risk factor for coronary artery disease (CAD). Treatment of OSAHS improves clinical outcome in some CAD patients, but the relationship between OSAHS and CAD is complex. Microparticles (MPs) are shed by the plasma membrane by either physiologic or pathologic stimulation. In the current study, we investigated the role of MPs in the context of OSAHS. Methods and Results: 54 patients with both suspected coronary artery stenosis and OSAHS were recruited and underwent both coronary arteriography and polysomnography. Circulating MPs were isolated and analyzed by flow cytometry. CAD+OSAHS patients exhibited greater levels of total MPs (Annexin V + ), erythrocyte-derived MPs (CD235 + Annexin V + ), platelet-derived MPs (CD41 + Annexin V + ), and leukocyte-derived MPs (CD45 + Annexin V + ) compared to CAD alone patients or control. CAD+OSAHS patients expressed the greatest level of endothelial-derived MPs of all cellular origin types (CD144 + Annexin V + ). Treatment of human aortic endothelial cells (HAECs) with MPs isolated from CAD+OSAHS patients markedly increased HAEC permeability (as detected by FITC-dextran), and significantly upregulated mRNA levels of ICAM-1, VCAM-1, and MCP-1. Conclusion: OSAHS+CAD patients harbor increased levels of MPs, particularly the endothelial cell-derived subtype. When administered to HAECs, OSAHS+CAD patients MPs increase endothelial cell permeability and dysfunction.

Macroautophagy/autophagy is increasingly recognized as an important regulator of myocardial ischemia-reperfusion (MI-R) injury. However, whether and how diabetes may alter autophagy in response to MI-R remains unknown. Deficiency of ADIPOQ, a cardioprotective molecule, markedly increases MI-R injury. However, the role of diabetic hypoadiponectinemia in cardiac autophagy alteration after MI-R is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes, and Adipoq knockout (adipoq−/−) mice, we demonstrated that autophagosome formation was modestly inhibited and autophagosome clearance was markedly impaired in the diabetic heart subjected to MI-R. adipoq−/− largely reproduced the phenotypic alterations observed in the ischemic-reperfused diabetic heart. Treatment of diabetic and adipoq−/− mice with AdipoRon, a novel ADIPOR (adiponectin receptor) agonist, stimulated autophagosome formation, markedly increased autophagosome clearance, reduced infarct size, and improved cardiac function (P < 0.01 vs vehicle). Mechanistically, AdipoRon caused significant phosphorylation of AMPK-BECN1 (Ser93/Thr119)-class III PtdIns3K (Ser164) and enhanced lysosome protein LAMP2 expression both in vivo and in isolated adult cardiomyocytes. Pharmacological AMPK inhibition or genetic Prkaa2 mutation abolished AdipoRon-induced BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) phosphorylation and AdipoRon-stimulated autophagosome formation. However, AdipoRon-induced LAMP2 expression, AdipoRon-stimulated autophagosome clearance, and AdipoRon-suppressed superoxide generation were not affected by AMPK inhibition. Treatment with MnTMPyP (a superoxide scavenger) increased LAMP2 expression and stimulated autophagosome clearance in simulated ischemic-reperfused cardiomyocytes. However, no additive effect between AdipoRon and MnTMPyP was observed. Collectively, these results demonstrate that hypoadiponectinemia impairs autophagic flux, contributing to enhanced MI-R injury in the diabetic state. ADIPOR activation restores AMPK-mediated autophagosome formation and antioxidant-mediated autophagosome clearance, representing a novel intervention effective against MI-R injury in diabetic conditions.

Mitochondrial homeostasis is a key process involved in cellular destiny and organic function. When mitochondrial status is abnormal, it will become a "death motor." Impaired mitochondria lead to the release of cytochrome c, and then trigger mitochondria-induced caspase activation. Omi/HtrA2, a serine protease, locates in mitochondria and involves in mitochondrial homeostasis. Increased Omi/HtrA2 is observed in aging cardiac tissues, and whether this has effects on mitochondrial status has not been reported. In this study, natural Sprague-Dawley rats (22 months) were used. We detected markedly increased proteolytic activity of Omi/HtrA2 and obvious activation of caspase-9 and caspase-3 in their myocardium. Then, we constructed stably transfected mitochondrial Omi/HtrA2 cells, and decreased mitochondrial membrane potential was detected by JC-1 (a probe for mitochondria) and tetramethylrhodamine methyl ester (TMRM) dyeing and significant release of cytochrome c was observed after separation of mitochondrial fraction and cytosolic fraction. Furthermore, ucf-101 (a special inhibitor of Omi/HtrA2) and HAX-1 siRNA could ameliorate those phenomena above. In conclusion, excessive Omi/HtrA2 in mitochondria induced decreased mitochondrial membrane potential by its proteolytic activity, followed by cytochrome c released from mitochondria into cytosol where cytochrome c promoted caspase activation. Also, Omi/HtrA2-HAX-1 chain played a significant role in mitochondrial homeostasis.

Almost no pharmacokinetic compounds to date have been precisely linked with the activity of their herbal or Traditional Chinese Medicine (TCM) formula. This creates challenges for pharmacokinetic significance and application of the TCM. In our study, a sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantitatively or qualitatively determine multiple-components (tanshinol, ferulic acid, protocatechuic acid, rosmarinic acid, salvianolic acid B, baicalin and 9'-methyl lithospermate B) in rat plasma following the oral administration of Denshen-Chuanxiong-Honghua (DCH) extract (20g/kg). Chromatographic separation was carried out on a 300SB-C18 column using a gradient elution with a mobile phase composed of acetonitrile-water (containing 0.1% formic acid) at a flow rate of 1.0mL/min. Determination by mass spectrometry (MS) was conducted in multiple reaction monitoring (MRM) mode with negative electrospray ionization. The validated method exhibited good linearity, with correlation coefficients greater than 0.9949 over a wide concentration range, and the lower limits of quantification were 2.09-12.2ng/mL for the 5 analytes. This assay was successfully applied to investigate the pharmacokinetics of 5 compounds in rat plasma after the oral administration of DCH extracts. In addition, the anti-oxidant capacities of the 5 active ingredients of DCH extract in vitro and the total absorbed DCH extract in vivo were investigated at different concentrations during pharmacokinetic studies.