Post-translational modifications of pyruvate dehydrogenase complex in cardiovascular disease.

Pyruvate dehydrogenase complex (PDC) is a crucial enzyme that connects glycolysis and the tricarboxylic acid (TCA) cycle pathway. It plays an essential role in regulating glucose metabolism for energy production by catalyzing the oxidative decarboxylation of pyruvate to acetyl coenzyme A. Importantly, the activity of PDC is regulated through post-translational modifications (PTMs), phosphorylation, acetylation, and O-GlcNAcylation. These PTMs have significant effects on PDC activity under both physiological and pathophysiological conditions, making them potential targets for metabolism-related diseases. This review specifically focuses on the PTMs of PDC in cardiovascular diseases (CVDs) such as myocardial ischemia/reperfusion injury, diabetic cardiomyopathy, obesity-related cardiomyopathy, heart failure (HF), and vascular diseases. The findings from this review offer theoretical references for the diagnosis, treatment, and prognosis of CVD.

Acacetin protects against acute lung injury by upregulating SIRT1/ NF-κB pathway.

Acacetin is one of the natural flavone components found in many plants and possesses diverse pharmacological activities. The anti-inflammatory properties and definite mechanism of acacetin remains incompletely illuminated. Here, we evaluated the efficacy of acacetin on lipopolysaccharide (LPS)-induced acute lung injury in vivo and TNF-α-stimulated cellular injury in vitro. As indicated by survival experiments, acacetin reduced mortality and improved survival time of LPS-induced acute lung injury in mice. 50 mg/kg of acacetin obtained higher survival (about 60 %), and 20 mg/kg of acacetin was about 46.7 %. In addition, 20 mg/kg of acacetin rescued lung histopathologic damage in LPS treated mice, lowered lung-to-body weight and lung wet-to-dry ratios, suppressed myeloperoxidase activity in lung tissue, the contents of protein, the numbers of total cells and neutrophils in bronchoalveolar lavage fluid (BALF), and the contents of inflammatory cytokines such as TNF-α, IL-6, IL-17 and IL-1ß in BALF. Acacetin also increased the activity and expression of SIRT1, thereby suppressing acetylation-dependent activation NF-κB. Similarly, in vitro, acacetin increased cell viability, reduced levels of TNF-α, IL-6, IL-17, and IL-1ß, increased NAD+ levels as well as NAD/NADH ratio, and then up-regulated the activity and expression of SIRT1, and restrained acetylation-dependent activation NF-κB in TNF-α-stimulated A549 cells, which could be abolished by SIRT1 siRNA. Collectively, the current study showed that acacetin exerts a protective effiect on acute lung injury by improving the activity and expression SIRT1, thereby suppressing the acetylation-dependent activation of NF-κB-p65 and the release of downstream inflammatory cytokines.

Biomimetic Nanoparticle-Mediated Target Delivery of Hypoxia-Responsive Plasmid of Angiotensin-Converting Enzyme 2 to Reverse Hypoxic Pulmonary Hypertension.

Hypoxic pulmonary hypertension (HPH) is characterized by pulmonary vascular sustained constriction and progressive remodeling, which are initiated by hypoxia then with hypoxia-induced additive factors including pulmonary vascular endothelium injury, intrapulmonary angiotension system imbalance, and inflammation. Now HPH is still an intractable disease lacking effective treatments. Gene therapy has a massive potential for HPH but is hindered by a lack of efficient targeted delivery and hypoxia-responsive regulation systems for transgenes. Herein, we constructed the hypoxia-responsive plasmid of angiotensin-converting enzyme 2 (ACE2) with endothelial-specific promoter Tie2 and a hypoxia response element and next prepared its biomimetic nanoparticle delivery system, named ACE2-CS-PRT@PM, by encapsulating the plasmid of ACE2 with protamine and chondroitin sulfate as the core then coated it with a platelet membrane as a shell for targeting the injured pulmonary vascular endothelium. ACE2-CS-PRT@PM has a 194.3 nm diameter with a platelet membrane-coating core-shell structure and a negatively charged surface, and it exhibits higher delivery efficiency targeting to pulmonary vascular endothelium and hypoxia-responsive overexpression of ACE2 in endothelial cells in a hypoxia environment. In vitro, ACE2-CS-PRT@PM significantly inhibited the hypoxia-induced proliferation of pulmonary smooth muscle cells. In vivo, ACE2-CS-PRT@PM potently ameliorated the hemodynamic dysfunction and morphological abnormality and largely reversed HPH via inhibiting the hypoxic proliferation of pulmonary artery smooth muscle cells, reducing pulmonary vascular remodeling, restoring balance to the intrapulmonary angiotension system, and improving the inflammatory microenvironment without any detectable toxicity. Therefore, ACE2-CS-PRT@PM is promising for the targeted gene therapy of HPH.

SIRT6 Protects Against Myocardial Ischemia-Reperfusion Injury by Attenuating Aging-Related CHMP2B Accumulation.

Impaired autophagic flux induces aging-related ischemia vulnerability, which is the hallmark pathology in cardiac aging. Our previous work has confirmed that the accumulation of charged multivesicular body protein 2B (CHMP2B), a subunit of the ESCRT-III complex, in the heart can impair autophagy flux. However, whether CHMP2B accumulation contributes to aging-related intolerance to ischemia/reperfusion (I/R) injury and the regulatory mechanism for CHMP2B in aged heart remain elusive. The cardiac CHMP2B level was significantly higher in aged human myocardium than that in young myocardium. Increased CHMP2B were shown to inhibit autophagic flux leading to the deterioration of MI/R injury in aged mice hearts. Interestingly, a negative correlation was observed between SIRT6 and CHMP2B expression in human heart samples. Specific activation of SIRT6 suppressed CHMP2B accumulation and ameliorated autophagy flux in aged hearts. Using myocardial-specific SIRT6 heterozygous knockout mice and recovery experiments confirmed that SIRT6 regulated myocardial CHMP2B levels. Finally, activation of SIRT6 decreased acetylation of FoxO1 to promote its transcriptional function on Atrogin-1, a muscle-specific ubiquitin ligase, which subsequently enhanced the degradation of CHMP2B by Atrogin-1. This is a novel mechanism for SIRT6 against aging-related myocardial ischemia vulnerability, particularly by preventing excessive accumulation of autophagy key factors.

Association of Metformin with the Mortality and Incidence of Cardiovascular Events in Patients with Pre-existing Cardiovascular Diseases.

INTRODUCTION: Whether metformin reduces all-cause cardiovascular mortality and the incidence of cardiovascular events in patients with pre-existing cardiovascular diseases (CVD) remains inconclusive. Some randomised controlled trials (RCTs) and cohort studies have shown that metformin is associated with an increased risk of mortality and cardiovascular events. METHODS: We conducted a pooling synthesis to assess the effects of metformin in all-cause cardiovascular mortality and incidence of cardiovascular events in patients with CVD. Studies published up to October 2021 in PubMed or Embase with a registration in PROSPERO (CRD42020189905) were collected. Both RCT and cohort studies were included. Hazard ratios (HR) with 95% CI were pooled across various trials using the random-effects model. RESULTS: This study enrolled 35 published studies (in 14 publications) for qualitative synthesis and identified 33 studies (published in 26 publications) for quantitative analysis. We analysed a total of 61,704 patients, among them 58,271 patients were used to calculate all-cause mortality while 12,814 patients were used to calculate cardiovascular mortality. Compared with non-metformin control, metformin usage is associated with a reduction in all-cause mortality (HR: 0.90; 95% CI 0.83, 0.98; p = 0.01), cardiovascular mortality (HR: 0.89; 95% CI 0.85, 0.94; p < 0.0001), incidence of coronary revascularisation (HR: 0.79; 95% CI 0.64, 0.98; p = 0.03), and heart failure (HR: 0.90; 95% CI 0.87, 0.94; p < 0.0001) in patients with pre-existing cardiovascular diseases. CONCLUSION: Metformin use is associated with a reduction in all-cause mortality, cardiovascular mortality, incidence of coronary revascularisation, and heart failure in patients with CVD; however, metformin usage was not associated with reduction in the incidence of myocardial infarction, angina, or stroke.

Particulate matter 2.5 triggers airway inflammation and bronchial hyperresponsiveness in mice by activating the SIRT2-p65 pathway.

Exposure to particulate matter 2.5 (PM2.5) potentially triggers airway inflammation by activating nuclear factor-κB (NF-κB). Sirtuin 2 (SIRT2) is a key modulator in inflammation. However, the function and specific mechanisms of SIRT2 in PM2.5-induced airway inflammation are largely understudied. Therefore, this work investigated the mechanisms of SIRT2 in regulating the phosphorylation and acetylation of p65 influenced by PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Results revealed that PM2.5 exposure lowered the expression and activity of SIRT2 in bronchial tissues. Subsequently, SIRT2 impairment promoted the phosphorylation and acetylation of p65 and activated the NF-κB signaling pathway. The activation of p65 triggered airway inflammation, increment of mucus secretion by goblet cells, and acceleration of tracheal stenosis. Meanwhile, p65 phosphorylation and acetylation, airway inflammation, and bronchial hyperresponsiveness were deteriorated in SIRT2 knockout mice exposed to PM2.5. Triptolide (a specific p65 inhibitor) reversed p65 activation and ameliorated PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Our findings provide novel insights into the molecular mechanisms underlying the toxicity of PM2.5 exposure. Triptolide inhibition of p65 phosphorylation and acetylation could be an effective therapeutic approach in averting PM2.5-induced airway inflammation and bronchial hyperresponsiveness.

SIRT6 in Senescence and Aging-Related Cardiovascular Diseases.

SIRT6 belongs to the nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases and has established diverse roles in aging, metabolism and disease. Its function is similar to the Silent Information Regulator 2 (SIR2), which prolongs lifespan and regulates genomic stability, telomere integrity, transcription, and DNA repair. It has been demonstrated that increasing the sirtuin level through genetic manipulation extends the lifespan of yeast, nematodes and flies. Deficiency of SIRT6 induces chronic inflammation, autophagy disorder and telomere instability. Also, these cellular processes can lead to the occurrence and progression of cardiovascular diseases (CVDs), such as atherosclerosis, hypertrophic cardiomyopathy and heart failure. Herein, we discuss the implications of SIRT6 regulates multiple cellular processes in cell senescence and aging-related CVDs, and we summarize clinical application of SIRT6 agonists and possible therapeutic interventions in aging-related CVDs.

Association of metformin monotherapy or combined therapy with cardiovascular risks in patients with type 2 diabetes mellitus.

BACKGROUND: Metformin is a first-line drug in type 2 diabetes mellitus (T2DM) treatment, yet whether metformin may increase all-cause or cardiovascular mortality of T2DM patients remains inconclusive. METHODS: We searched PubMed and Embase for data extracted from inception to July 14, 2020, with a registration in PROSPERO (CRD42020177283). This study included randomized controlled trials (RCT) assessing the cardiovascular effects of metformin for T2DM. This study is followed by PRISMA and Cochrane guideline. Risk ratio (RR) with 95% CI was pooled across trials by a random-effects model. Primary outcomes include all-cause mortality and cardiovascular mortality. RESULTS: We identified 29 studies that randomly assigned patients with 371 all-cause and 227 cardiovascular death events. Compared with untreated T2DM patients, metformin-treated patients was not associated with lower risk of all-cause mortality (RR: 0.98; 95%CI: 0.69-1.38; P = 0.90), cardiovascular mortality (RR: 1.13; 95% CI: 0.60, 2.15; P = 0.70), macrovascular events (RR: 0.87; 95%CI: 0.70-1.07; P = 0.19), heart failure (RR: 1.02; 95% CI:0.61-1.71; P = 0.95), and microvascular events (RR: 0.78; 95% CI:0.54-1.13; P = 0.19). Combination of metformin with another hypoglycemic drug was associated with higher risk of all-cause mortality (RR: 1.49; 95% CI: 1.02, 2.16) and cardiovascular mortality (RR: 2.21; 95% CI: 1.22, 4.00) compared with hypoglycemic drug regimens with no metformin. CONCLUSION: The combination of metformin treatment may impose higher risk in all-cause and cardiovascular mortality. This finding, at least in part, shows no evidence for benefits of metformin in combination in terms of all-cause/cardiovascular mortality and cardiovascular events for T2DM. However, the conclusion shall be explained cautiously considering the limitations from UK Prospective Diabetes Study (UKPDS).

The Pivotal Role of Mitsugumin 53 in Cardiovascular Diseases.

The MG53 (also known as TRIM72) is a conserved, muscle-specific tripartite motif family protein that is abundantly expressed in cardiac or skeletal muscle and present in circulation. Recently, the MG53 had been hypothesized to serve a dual role in the heart: involving in repairing cell membranes that protect myocardial function while acting as an E3 ligase to trigger insulin resistance and cardiovascular complications. This review discusses the roles of MG53 in cardiac physiological function with emphasis on MG53 protective function in the heart and its negative impact on the myocardium due to the continuous elevation of MG53. Besides, this work reviewed the significance of MG53 as a potential therapeutic in human cardiovascular diseases. Despite the expression of MG53 being rare in the human, thus exogenous MG53 can potentially be a new treatment for human cardiovascular diseases. Notably, the specific mechanism of MG53 in cardiovascular diseases remains elusive.

Lipocalin-2-induced proliferative endoplasmic reticulum stress participates in Kawasaki disease-related pulmonary arterial abnormalities.

Clinical cases have reported pulmonary arterial structural and functional abnormalities in patients with Kawasaki disease (KD); however, the underlying mechanisms are unclear. In this study, a KD rat model was established via the intraperitoneal injection of Lactobacillus casei cell wall extract (LCWE). The results showed that pulmonary arterial functional and structural abnormalities were observed in KD rats. Furthermore, proliferative endoplasmic reticulum stress (ER stress) was observed in the pulmonary arteries of KD rats. Notably, the level of lipocalin-2 (Lcn 2), a trigger factor of inflammation, was remarkably elevated in the plasma and lung tissues of KD rats; increased Lcn 2 levels following LCWE stimulation may result from polymorphonuclear neutrophils (PMNs). Correspondingly, in cultured pulmonary artery smooth muscle cells (PASMCs), Lcn 2 markedly augmented the cleavage and nuclear localization of activating transcription factor-6 (ATF6), upregulated the transcription of glucose regulated protein 78 (GRP78) and neurite outgrowth inhibitor (NOGO), and promoted PASMCs proliferation. However, proapoptotic C/EBP homologous protein (CHOP) and caspase 12 levels were not elevated. Treatment with 4-phenyl butyric acid (4-PBA, a specific inhibitor of ER stress) inhibited PASMCs proliferation induced by Lcn 2 and attenuated pulmonary arterial abnormalities and right ventricular hypertrophy and reduced right ventricular systolic pressure in KD rats. In conclusion, Lcn 2 remarkably facilitates proliferative ER stress in PASMCs, which probably accounts for KD-related pulmonary arterial abnormalities.

Clinical Features of COVID-19 Patients in Xiaogan City.

On February 6, 2020, Xiaogan City became the second most seriously affected city with coronavirus disease 2019 (COVID-19), outside Wuhan district, Hubei Province, China. The objectives are to study the clinical features of COVID-19 patients and assess the relationship between the severity of COVID-19, age, and C-reactive protein (CRP) levels. The retrospective data of 134 COVID-19 patients hospitalized in 3 hospitals of Xiaogan City, between February 1 and March 1, 2020, was collected. This study documented COVID-19 patients. Clinical data in terms of body temperature, history of travel, and direct contact with COVID-19 patients, and incubation period was collected. Out of the 134 patients, only 5 required intensive care. Moreover, 2 patients succumbed during this period. The median age of patients was 45 (33-56) years. The most common symptoms at the onset of disease were fever (66.4%), cough (33, 6%), and sore throat (14.7%). Amongst the medicines used, antiviral agents (92.3%) followed by the traditional Chinese medicine (89.5%) were most commonly used. In both the crude and adjusted (I to III) models, odds ratio and its 95% confidence interval for both age and CRP levels were > 1. Moreover, the smooth curve fitting graph reflected that the severity of COVID-19 was positively correlated with both age and CRP levels (all P value < 0.05). The signs and symptoms of COVID-19 patients were fairly moderate. The health care professionals treating the COVID-19 patients should be aware of the increased likelihood of progression to severe COVID-19 in elderly patients and those with high CRP levels.

Reversal of Hypoxic Pulmonary Hypertension by Hypoxia-Inducible Overexpression of Angiotensin-(1-7) in Pulmonary Endothelial Cells.

Hypoxia-induced pulmonary vascular constriction and structure remodeling are the main causes of hypoxic pulmonary hypertension. In the present study, an adeno-associated virus vector, containing Tie2 promoter and hypoxia response elements, was designed and named HTSFcAng(1-7). Its targeting, hypoxic inducibility, and vascular relaxation were examined in vitro, and its therapeutic effects on hypobaric hypoxia-induced pulmonary hypertension were examined in rats. Transfection of HTSFcAng(1-7) specifically increased the expression of angiotensin-(1-7) in endothelial cells in normoxia. Hypoxia increased the expression of angiotensin-(1-7) in HTSFcAng(1-7)-transfected endothelial cells. The condition medium from HTSFcAng(1-7)-transfected endothelial cells inhibited the hypoxia-induced proliferation of pulmonary artery smooth muscle cells, relaxed the pulmonary artery rings, totally inhibited hypoxia-induced early contraction, enhanced maximum relaxation, and reversed phase II constriction to sustained relaxation. In hypoxic pulmonary hypertension rats, treatment with HTSFcAng(1-7) by nasal drip adeno-associated virus significantly reversed hypoxia-induced hemodynamic changes and pulmonary artery-wall remodeling, accompanied by the concomitant overexpression of angiotensin-(1-7), mainly in the endothelial cells in the lung. Therefore, hypoxia-inducible overexpression of angiotensin-(1-7) in pulmonary endothelial cells may be a potential strategy for the gene therapy of hypoxic pulmonary hypertension.

Conversion between porcine naïve-like and primed ESCs and specific pluripotency marker identification.

Researchers currently lack standardized porcine-specific markers that would aid in distinguishing the naïve and primed states of porcine embryonic stem cells (ESCs). Here, we converted naïve-like porcine ESCs (nESCs, established in our lab) into primed-state cells, and we proposed a set of molecular criteria for evaluating the naïve porcine ESCs by comparing the two cell states. The reverse-primed porcine ESCs (rpESCs) are phenotypically stable and karyotypically intact. Alkaline phosphatase positivity and the ability to form embryonic bodies suggest that rpESCs still retain the capacity for self-renewal. Lineage-associated genes, such as Cdx2, Sox17, Eomes, Foxa, Fgf5, and Pitx2, exhibited significant expression in rpESCs. Nonetheless, LIF/3i-grown porcine ESCs treated with the small molecular weight inhibitors CHIR99021, PD0325901, and SB431542 expressed the greatest number of pluripotency marker genes, including Oct4, Sox2, Nog, Dppa5, Nr0b1, and Klf4, and at higher levels than were observed in rpESCs. Despite their general trend toward higher expression of critical pluripotency factors, the nESCs showed downregulation of Tbx3, Nanog, and c-Myc, which are considered typical naïve factors in other species. Entry of the nESCs into the developmentally primed state was also associated with a marked reduction in Lin28 expression. These findings extend the knowledge of porcine pluripotency markers and provide a backdrop for future analysis of naïve porcine pluripotency.

Bioinformatics analysis reveals novel core genes associated with nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.

Nonalcoholic fatty liver disease (NAFLD) is the most frequent liver disease and associated with a wide spectrum of hepatic disorders ranging from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). NASH is projected to become the most common indication for liver transplantation, and the annual incidence rate of NASH-related HCC is 5.29 cases per 1000 person-years. Owing to the epidemics of NAFLD and the unclear mechanism of NAFLD progression, it is important to elucidate the underlying NAFLD mechanisms in detail. NASH is mainly caused by the development of NAFL Therefore, it is also of great significance to understand the mechanism of progression from NAFL to NASH. Gene expression chip data for NAFLD and NASH were downloaded from the Gene Expression Omnibus database to identify differentially expressed genes (DEGs) between NAFLD and normal controls (called DEGs for NAFLD), as well as between NASH and normal tissue (called DEGs for NASH-Normal), and between NASH and NAFL tissue (called DEGs for NASH-NAFL). For DEGs for the NAFLD group, key genes were identified by studying the form of intersection. Potential functions of DEGs for NASH were then analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. A protein-protein interaction network (PPI) was constructed using the STRING database. A total of 249 DEGs and one key gene for NAFLD were identified. For NASH-Normal, 514 DEGs and 11 hub genes were identified, three of which were closely related to the survival analysis of HCC, and potentially closely related to progression from NASH to HCC. One key gene for NASH-NAFL (AKR1B10) was identified. These genes appear to mediate the molecular mechanism underlying NAFLD and may be promising biomarkers for the presence of NASH.

The effect of activated κ-opioid receptor (κ-OR) on the role of calcium sensing receptor (CaSR) in preventing hypoxic pulmonary hypertension development.

κ-opioid receptor (κ-OR) plays a key role in preventing hypoxic pulmonary hypertension (HPH) development after activated by exogenous agonist U50,488H. Calcium sensing receptor (CaSR) activation induces HPH by promoting vasoconstriction and vascular remodeling. The activated κ-OR is reported to inhibit the expression of CaSR in pulmonary artery smooth muscle cells (PASMCs). Thus, in this study, we aimed to explore the effect of activated κ-OR on the role of CaSR in preventing HPH development. An HPH rat model was constructed using Sprague-Dawley rats. Changes in mean pulmonary arterial pressure (mPAP) and right ventricular pressure (RVP) mediated by κ-OR agonist U50,488H and CaSR inhibitor NPS2143 were observed. The effects of CaSR agonist spermine and inhibitor NPS2143 on pulmonary artery tension were tested. The expression and localization of κ-OR and CaSR were measured in isolated PASMCs. A cell-counting kit-8 assay was performed to evaluate the effect of spermine in PASMC proliferation. Expression of proliferating cell nuclear antigen (PCNA), Erk, and p-Erk was evaluated by western blot analysis. Results showed that κ-OR and CaSR were co-expressed and colocalized in PASMCs under normoxic and hypoxic conditions. Interactions between κ-OR and CaSR were also observed. Spermine improved vasoconstriction in the pulmonary artery in HPH rats, which was abolished by U50,488H. RVP and mPAP were significantly increased in HPH rats under CaSR stimulation, but were significantly reduced when the rats were pretreated with U50,488H and NPS2143 (P < 0.01). Spermine treatment significantly promoted PASMC proliferation, which was significantly inhibited by U50,488H, p38 inhibitor SB203580, JNK inhibitor SP600125, Erk inhibitor SCH772984, and MEK inhibitor U0126, especially Erk inhibitor (P < 0.01). Spermine significantly increased PCNA and P-Erk expression in hypoxic conditions, which was inhibited by U50,488H and NPS2143. κ-OR stimulation prevented HPH development via the CaSR/MAPK signaling pathway.

Optimized PCR-Based Enrichment Improves Coverage Uniformity and Mutation Detection in Mitochondrial DNA Next-Generation Sequencing.

Next-generation sequencing-based methods have been commonly used for detecting mutations of mitochondrial genome (mtDNA). PCR amplification is a highly effective method of mtDNA enrichment before sequencing. However, it has been observed that highly variable sequencing depth within PCR amplicons severely reduces the coverage uniformity and accuracy of mutation calling. Therefore, it is necessary to develop an optimized PCR-based strategy for mtDNA sequencing. Herein, the effect of DNA quality on the efficiency of PCR amplification was analyzed and the effects of different primer-design methods, including the number of primer pairs, overlap length of amplicons, and modification of primers, on coverage uniformity and mutation calling in mtDNA sequencing were assessed. Results showed that DNA quality significantly affected the efficiency of PCR amplification. Importantly, over- and under-representation of coverage depth at overlap regions of amplicons were observed when amplicons were not modified and overlap was shorter than two sequencing fragment sizes (800 bp). Then, under-representation was overcome by increasing the overlap length of the amplicons, and over-representation was effectively reduced by 5'-block modification of primers and sticky-end ligation of amplicons. Moreover, findings indicated that these two optimized PCR-based sequencing strategies effectively improved mutation calling in primer-binding regions. Optimized PCR-based mtDNA enrichment and sequencing approaches have been established, which laid a foundation for accurate mutation detection of mtDNA in diseases.

Metformin mediates cardioprotection against aging-induced ischemic necroptosis.

Necroptosis is crucially involved in severe cardiac pathological conditions. However, whether necroptosis contributes to age-related intolerance to ischemia/reperfusion (I/R) injury remains elusive. In addition, metformin as a potential anti-aging related injury drug, how it interacts with myocardial necroptosis is not yet clear. Male C57BL/6 mice at 3-4- (young) and 22-24 months of age (aged) and RIPK3-deficient (Ripk3-/- ) mice were used to investigate aging-related I/R injury in vivo. Metformin (125 µg/kg, i.p.), necrostatin-1 (3.5 mg/kg), and adenovirus vector encoding p62-shRNAs (Ad-sh-p62) were used to treat aging mice. I/R-induced myocardial necroptosis was exaggerated in aged mice, which correlated with autophagy defects characterized by p62 accumulation in aged hearts or aged human myocardium. Functionally, blocking autophagic flux promoted H/R-evoked cardiomyocyte necroptosis in vitro. We further revealed that p62 forms a complex with RIP1-RIP3 (necrosome) and promotes the binding of RIP1 and RIP3. In mice, necrostatin-1 treatment (a RIP1 inhibitor), RIP3 deficiency, and cardiac p62 knockdown in vivo demonstrated that p62-RIP1-RIP3-dependent myocardial necroptosis contributes to aging-related myocardial vulnerability to I/R injury. Notably, metformin treatment disrupted p62-RIP1-RIP3 complexes and effectively repressed I/R-induced necroptosis in aged hearts, ultimately reducing mortality in this model. These findings highlight previously unknown mechanisms of aging-related myocardial ischemic vulnerability: p62-necrosome-dependent necroptosis. Metformin acts as a cardioprotective agent that inhibits this unfavorable chain mechanism of aging-related I/R susceptibility.

Metformin-Enhanced Cardiac AMP-Activated Protein Kinase/Atrogin-1 Pathways Inhibit Charged Multivesicular Body Protein 2B Accumulation in Ischemia-Reperfusion Injury.

Background: Cardiac autophagic flux is impaired during myocardial ischemia/reperfusion (MI/R). Impaired autophagic flux may exacerbate MI/R injury. Charged multivesicular body protein 2B (CHMP2B) is a subunit of the endosomal sorting complex required for transport (ESCRT-III) complex that is required for autophagy. However, the reverse role of CHMP2B accumulation in autophagy and MI/R injury has not been established. The objective of this article is to elucidate the roles of AMP-activated protein kinase (AMPK)/atrogin-1 pathways in inhibiting CHMP2B accumulation in ischemia-reperfusion injury. Methods: Male C57BL/6 mice (3-4 months) and H9c2 cardiomyocytes were used to evaluate MI/R and hypoxia/reoxygenation (H/R) injury in vivo and in vitro, respectively. MI/R was built by a left lateral thoracotomy and occluded the left anterior descending artery. H9c2 cells were firstly treated in 95% N2 and 5% CO2 for 15 h and reoxygenation for 1 h. Metformin (100 mg/kg/d) and CHMP2B (Ad-CHMP2B) transfected adenoviruses were administered to the mice. The H9c2 cells were treated with metformin (2.5 mM), MG-132 (10 µM), bafilomycin A1 (10 nM), and compound C (20 µM). Results: Autophagic flux was found to be inhibited in H/R-treated cardiomyocytes and MI/R mice, with elevated cardiac CHMP2B accumulation. Upregulated CHMP2B levels in the in vivo and in vitro experiments were shown to inhibit autophagic flux leading to the deterioration of H/R-cardiomyocytes and MI/R injury. This finding implies that CHMP2B accumulation increases the risk of myocardial ischemia. Metformin suppressed CHMP2B accumulation and ameliorated H/R-induced autophagic dysfunction by activating AMPK. Activated AMPK upregulated the messenger RNA expression and protein levels of atrogin-1, a muscle-specific ubiquitin ligase, in the myocardium. Atrogin-1 significantly enhanced the interaction between atrogin-1 and CHMP2B, therefore, promoting CHMP2B degradation in the MI/R myocardium. Finally, this study revealed that metformin-inhibited CHMP2B accumulation induced autophagic impairment and ischemic susceptibility in vivo through the AMPK-regulated CHMP2B degradation by atrogin-1. Conclusion: Impaired CHMP2B clearance in vitro and in vivo inhibits autophagic flux and weakens the myocardial ischemic tolerance. Metformin treatment degrades CHMP2B through the AMPK-atrogin-1-dependent pathway to maintain the homeostasis of autophagic flux. This is a novel mechanism that enriches the understanding of cardioprotection.

[Bioinformatical analysis of differently expressed genes related to mitochondrial function in systemic sclerosis].

Objective To investigate the relationship between mitochondria and systemic sclerosis (SSc) by analyzing the expression of mitochondrial function-related genes in skin biopsy samples from patients with SSc. Methods Gene chip expression profile of SSc skin biopsy in Gene Expression Omnibus (GEO) database was used, and differently expressed genes (DEGs) related to mitochondrial function were identified by t test and fold change (FC). What's more, functional annotation, functional enrichment and protein interaction network analysis were performed. Results A total of 422 significant DEGs were identified between the SSc group and the normal group. Among them, 23 DEGs were mitochondrial function-related genes. Functional annotation and enrichment analysis of 23 DEGs revealed that abnormally expressed mitochondrial function-related genes mainly affected several biological processes, such as mitochondrial energy supply and cell metabolism. Conclusion The dysregulation of mitochondrial function-related genes in SSc patients affects the function of mitochondria, suggesting that the abnormality of mitochondrial function may be associated with the development of SSc.

Derivation of novel naive-like porcine embryonic stem cells by a reprogramming factor-assisted strategy.

The establishment of ungulate embryonic stem cells (ESCs) has been notoriously difficult via a conventional approach. We combined a traditional ESC culture method with reprogramming factors to assist the establishment of porcine naive-like ESCs (nESCs). Pig embryonic fibroblasts were transfected with a tetracycline-inducible vector carrying 4 classic mouse reprogramming factors, followed by somatic cell nuclear transfer and culturing to the blastocyst stage. Then, the inner cell mass was isolated and seeded in culture medium. The naive-like ESCs had characteristic verys similar to those of mouse ESCs and showed no signs of altered morphology or differentiation, even after 130 passages. They depended on leukemia inhibitory factor signals for maintenance of pluripotency, and the female cell lines had low expression of the X-inactive specific transcript gene and no histone H3 lysine 27 trimethylation spot. Notably, the ESCs differentiated into 3 germ layers in vitro and could be induced to undergo directional neural and kidney precursor differentiation under defined conditions, and the ESCs could keep proliferating after doxycycline was removed. nESCs can be established, and the well-characterized ESC lines will be useful for the research of transgenic pig models for human disease.-Zhang, M., Wang, C., Jiang, H., Liu, M., Yang, N., Zhao, L., Hou, D., Jin, Y., Chen, Q., Chen, Y., Wang, J., Dai, Y., Li, R. Derivation of novel naive-like porcine embryonic stem cells by a reprogramming factor-assisted strategy.