BACKGROUND:Heart failure with preserved ejection fraction (HFpEF) has become the most prevalent type of heart failure, but effective treatments are lacking. Cardiac lymphatics play a crucial role in maintaining heart health by draining fluids and immune cells. However, their involvement in HFpEF remains largely unexplored. METHODS:We examined cardiac lymphatic alterations in mice with HFpEF with comorbid obesity and hypertension, and in heart tissues from patients with HFpEF. Using genetically engineered mouse models and various cellular and molecular techniques, we investigated the role of cardiac lymphatics in HFpEF and the underlying mechanisms. RESULTS:In mice with HFpEF, cardiac lymphatics displayed substantial structural and functional anomalies, including decreased lymphatic endothelial cell (LEC) density, vessel fragmentation, reduced branch connections, and impaired capacity to drain fluids and immune cells. LEC numbers and marker expression levels were also decreased in heart tissues from patients with HFpEF. Stimulating lymphangiogenesis with an adeno-associated virus expressing an engineered variant of vascular endothelial growth factor C (VEGFCC156S) that selectively activates vascular endothelial growth factor receptor 3 (VEGFR3) in LECs restored cardiac lymphatic integrity and substantially alleviated HFpEF. Through discovery-driven approaches, defective branched-chain amino acid (BCAA) catabolism was identified as a predominant metabolic signature in HFpEF cardiac LECs. Overexpression of branched-chain ketoacid dehydrogenase kinase (encoded by the Bckdk gene), which inactivates branched-chain ketoacid dehydrogenase (the rate-limiting enzyme in BCAA catabolism), resulted in spontaneous lymphangiogenic defects in LECs. In mice, inducible Bckdk gene deletion in LECs to enhance their BCAA catabolism preserved cardiac lymphatic integrity and protected against HFpEF. BCAA catabolic defects caused ligand-independent phosphorylation of VEGFR3 in the cytoplasm by Src kinase, leading to lysosomal degradation of VEGFR3 instead of its trafficking to the cell membrane. Reduced VEGFR3 availability on the cell surface impeded downstream Akt (protein kinase B) activation, hindered glucose uptake and utilization, and inhibited lymphangiogenesis in LECs with BCAA catabolic defects. CONCLUSIONS:Our study provides evidence that cardiac lymphatic disruption, driven by impaired BCAA catabolism in LECs, is a key factor contributing to HFpEF. These findings unravel the crucial role of BCAA catabolism in modulating lymphatic biology, and suggest that preserving cardiac lymphatic integrity may present a novel therapeutic strategy for HFpEF.

BACKGROUND:Hypertension is a significant global health issue that contributes to chronic kidney disease (CKD). Mitochondrial dysfunction in renal tubular epithelial cells (TECs) plays a crucial role in the progression of CKD. However, the involvement of mitochondrial DNA (mtDNA) methylation in hypertensive CKD and its potential as a therapeutic target remains largely unexamined. METHODS:A hypertensive CKD mouse model was induced by continuous infusion of AngII (angiotensin II). Renal mtDNA methylation types and levels were evaluated using nanopore sequencing and dot blot assays. The impact of N6-methyladenine (6mA) modification, catalyzed by METTL4 (methyltransferase-like protein 4), on hypertensive CKD was assessed using TEC-specific Mettl4 knockout mice. RESULTS:Utilizing high-confidence Nanopore sequencing, we identified 6mA as the predominant methylation type in renal mtDNA, rather than 5-methylcytosine. In mice with hypertensive CKD, renal mtDNA 6mA levels significantly increased, while 5-methylcytosine levels remained stable. METTL4, the only known mammalian methyltransferase for 6mA, was upregulated in the renal tubules of hypertensive CKD mice and patients with hypertension. AngII stimulation increased METTL4 expression and mtDNA 6mA levels in primary TECs. Activated c-Jun/AP-1 (activator protein-1) directly promoted Mettl4 transcription in AngII-treated primary TECs. METTL4-catalyzed mtDNA 6mA impeded mitochondrial transcription initiation complex assembly, thereby halting mtDNA transcription, disrupting mitochondrial function, and resulting in the transition of TECs into a proinflammatory and profibrotic phenotype. TEC-specific Mettl4 gene deletion in mice exhibited reduced mtDNA 6mA, preserved mtDNA transcription, improved tubular mitochondrial function, and alleviation of hypertensive CKD. CONCLUSIONS:This study reveals that METTL4-catalyzed mtDNA 6mA contributes to renal mitochondrial dysfunction and hypertensive CKD, offering novel therapeutic strategies from the perspective of mitochondrial epigenetics.

BACKGROUND: Myocardial mitochondrial dysfunction underpins the pathogenesis of heart failure (HF), yet therapeutic options to restore myocardial mitochondrial function are scarce. Epigenetic modifications of mitochondrial DNA (mtDNA), such as methylation, play a pivotal role in modulating mitochondrial homeostasis. However, their involvement in HF remains unclear. METHODS: Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N 6 -methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF. RESULTS: METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury. CONCLUSIONS: Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.

Mesenchymal stromal cell (MSC) implantation is a promising option for liver repair, but their poor retention in the injured liver milieu critically blunts therapeutic effects. The aim is to clarify the mechanisms underlying massive MSC loss post‐implantation and establish corresponding improvement strategies. MSC loss primarily occurs within the initial hours after implantation into the injured liver milieu or under reactive oxygen species (ROS) stress. Surprisingly, ferroptosis is identified as the culprit for rapid depletion. In ferroptosis‐ or ROS‐provoking MSCs, branched‐chain amino acid transaminase‐1 (BCAT1) is dramatically decreased, and its downregulation renders MSC susceptible to ferroptosis via suppressing the transcription of glutathione peroxidase‐4 (GPX4), a vital ferroptosis defensing enzyme. BCAT1 downregulation impedes GPX4 transcription via a rapid‐responsive metabolism‐epigenetics coordinating mechanism, involving α ‐ketoglutarate accumulation, histone 3 lysine 9 trimethylation loss, and early growth response protein‐1 upregulation. Approaches to suppress ferroptosis (e.g., incorporating ferroptosis inhibitors in injection solvent and overexpressing BCAT1) significantly improve MSC retention and liver‐protective effects post‐implantation. This study provides the first evidence indicating that excessive MSC ferroptosis is the nonnegligible culprit for their rapid depletion and insufficient therapeutic efficacy after implantation into the injured liver milieu. Strategies suppressing MSC ferroptosis are conducive to optimizing MSC‐based therapy.

Background Hypertrophic cardiomyopathy (HCM) is a widely distributed, but clinically heterogeneous genetic heart disease, affects approximately 20 million people worldwide. Nowadays, HCM is treatable with the advancement of medical interventions. However, due to occult clinical presentations and a lack of easy, inexpensive, and widely popularized screening approaches in the general population, 80–90% HCM patients are not clinically identifiable, which brings certain safety hazards could have been prevented. The majority HCM patients showed abnormal and diverse electrocardiogram (ECG) presentations, it is unclear which ECG parameters are the most efficient for HCM screening. Objective We aimed to develop a pragmatic prediction model based on the most common ECG features to screen for HCM. Methods Between April 1st and September 30th, 2020, 423 consecutive subjects from the International Cooperation Center for Hypertrophic Cardiomyopathy of Xijing Hospital [172 HCM patients, 251 participants without left ventricular hypertrophy (non-HCM)] were prospectively included in the training cohort. Between January 4th and February 30th, 2021, 163 participants from the same center were included in the temporal internal validation cohort (62 HCM patients, 101 non-HCM participants). External validation was performed using retrospectively collected ECG data from Xijing Hospital (3,232 HCM ECG samples from January 1st, 2000, to March 31st, 2020; 95,184 non-HCM ECG samples from January 1st to December 31st, 2020). The C-statistic was used to measure the discriminative ability of the model. Results Among 30 ECG features examined, all except abnormal Q wave significantly differed between the HCM patients and non-HCM comparators. After several independent feature selection approaches and model evaluation, we included only two ECG features, T wave inversion (TWI) and the amplitude of S wave in lead V1 (SV1), in the HCM prediction model. The model showed a clearly useful discriminative performance (C-statistic > 0.75) in the training [C-statistic 0.857 (0.818–0.896)], and temporal validation cohorts [C-statistic 0.871 (0.812–0.930)]. In the external validation cohort, the C-statistic of the model was 0.833 [0.825–0.841]. A browser-based calculator was generated accordingly. Conclusion The pragmatic model established using only TWI and SV1 may be helpful for predicting the probability of HCM and shows promise for use in population-based HCM screening.

Rationale: Long-term exercise provides reliable cardioprotection via mechanisms still incompletely understood. Although traditionally considered a thermogenic tissue, brown adipose tissue (BAT) communicates with remote organs (eg, the heart) through its endocrine function. BAT expands in response to exercise, but its involvement in exercise cardioprotection remains undefined. Objective: This study investigated whether small extracellular vesicles (sEVs) secreted by BAT and their contained microRNAs (miRNAs) regulate cardiomyocyte survival and participate in exercise cardioprotection in the context of myocardial ischemia/reperfusion (MI/R) injury. Methods and Results: Four weeks of exercise resulted in a significant BAT expansion in mice. Surgical BAT ablation before MI/R weakened the salutary effects of exercise. Adeno-associated virus 9 vectors carrying short hairpin RNA targeting Rab27a (a GTPase required for sEV secretion) or control viruses were injected in situ into the interscapular BAT. Exercise-mediated protection against MI/R injury was greatly attenuated in mice whose BAT sEV secretion was suppressed by Rab27a silencing. Intramyocardial injection of the BAT sEVs ameliorated MI/R injury, revealing the cardioprotective potential of BAT sEVs. Discovery-driven experiments identified miR-125b-5p, miR-128-3p, and miR-30d-5p (referred to as the BAT miRNAs) as essential BAT sEV components for mediating cardioprotection. BAT-specific inhibition of the BAT miRNAs prevented their upregulation in plasma sEVs and hearts of exercised mice and attenuated exercise cardioprotection. Mechanistically, the BAT miRNAs cooperatively suppressed the proapoptotic MAPK (mitogen-associated protein kinase) pathway by targeting a series of molecules (eg, Map3k5 , Map2k7 , and Map2k4 ) in the signaling cascade. Delivery of BAT sEVs into hearts or cardiomyocytes suppressed MI/R-related MAPK pathway activation, an effect that disappeared with the combined use of the BAT miRNA inhibitors. Conclusions: The sEVs secreted by BAT participate in exercise cardioprotection via delivering the cardioprotective miRNAs into the heart. These results provide novel insights into the mechanisms underlying the BAT-cardiomyocyte interaction and highlight BAT sEVs and their contained miRNAs as alternative candidates for exercise cardioprotection.

Mesenchymal stem cells (MSCs) delivered into the post-ischemic heart milieu have a low survival and retention rate, thus restricting the cardioreparative efficacy of MSC-based therapy. Chronic ischemia results in metabolic reprogramming in the heart, but little is known about how these metabolic changes influence implanted MSCs. Here, we found that excessive branched-chain amino acid (BCAA) accumulation, a metabolic signature seen in the post-ischemic heart, was disadvantageous to the retention and cardioprotection of intramyocardially injected MSCs. Discovery-driven experiments revealed that BCAA at pathological levels sensitized MSCs to stress-induced cell death and premature senescence via accelerating the loss of histone 3 lysine 9 trimethylation (H3K9me3). A novel mTORC1/DUX4/KDM4E axis was identified as the cause of BCAA-induced H3K9me3 loss and adverse phenotype acquisition. Enhancing BCAA catabolic capability in MSCs via genetic/pharmacological approaches greatly improved their adaptation to the high BCAA milieu and strengthened their cardioprotective efficacy. We conclude that aberrant BCAA accumulation is detrimental to implanted MSCs via a previously unknown metabolite-signaling-epigenetic mechanism, emphasizing that the metabolic changes of the post-ischemic heart crucially influence the fate of implanted MSCs and their therapeutic benefits.

Abstract Background: Hypertrophic cardiomyopathy (HCM) is an underdiagnosed genetic heart disease worldwide. The management and prognosis of obstructive HCM (HOCM) and non-obstructive HCM (HNCM) are quite different, but it also remains challenging to discriminate these two subtypes. HCM is characterized by dysmetabolism, and myocardial amino acid (AA) metabolism is robustly changed. The present study aimed to delineate plasma AA and derivatives profiles, and identify potential biomarkers for HCM. Methods: Plasma samples from 166 participants, including 57 cases of HOCM, 52 cases of HNCM, and 57 normal controls (NCs), who first visited the International Cooperation Center for HCM, Xijing Hospital between December 2019 and September 2020, were collected and analyzed by high-performance liquid chromatography–mass spectrometry based on targeted AA metabolomics. Three separate classification algorithms, including random forest, support vector machine, and logistic regression, were applied for the identification of specific AA and derivatives compositions for HCM and the development of screening models to discriminate HCM from NC as well as HOCM from HNCM. Results: The univariate analysis showed that the serine, glycine, proline, citrulline, glutamine, cystine, creatinine, cysteine, choline, and aminoadipic acid levels in the HCM group were significantly different from those in the NC group. Four AAs and derivatives (Panel A; proline, glycine, cysteine, and choline) were screened out by multiple feature selection algorithms for discriminating HCM patients from NCs. The receiver operating characteristic (ROC) analysis in Panel A yielded an area under the ROC curve (AUC) of 0.83 (0.75–0.91) in the training set and 0.79 (0.65–0.94) in the validation set. Moreover, among 10 AAs and derivatives (arginine, phenylalanine, tyrosine, proline, alanine, asparagine, creatine, tryptophan, ornithine, and choline) with statistical significance between HOCM and HNCM, 3 AAs (Panel B; arginine, proline, and ornithine) were selected to differentiate the two subgroups. The AUC values in the training and validation sets for Panel B were 0.83 (0.74–0.93) and 0.82 (0.66–0.98), respectively. Conclusions: The plasma AA and derivatives profiles were distinct between the HCM and NC groups. Based on the differential profiles, the two established screening models have potential value in assisting HCM screening and identifying whether it is obstructive.

目的 研究Fas激活的丝氨酸/苏氨酸激酶(Fas-activated serine/threonine kinase,FASTK)在缺氧/复氧(hypoxia/reoxygenation,H/R)损伤导致的心肌细胞线粒体呼吸功能障碍中的作用.方法 分离(1-2)d的野生C57BL/6小鼠原代心肌细胞并进行体外培养.转染对照或FASTK过表达的腺病毒后,心肌细胞接受H/R损伤.分别采用Western blot和RT-PCR检测FASTK蛋白和mRNA表达水平,采用Seahorse能量分析仪检测心肌细胞线粒体呼吸功能,采用caspase-3活性检测试剂盒、乳酸脱氢酶(lactate dehydrogenase,LDH)活性检测试剂盒、MTT细胞活力检测试剂盒评估心肌细胞存活情况.结果 较常氧组,H/R损伤导致心肌细胞FASTK蛋白和mRNA表达显著降低(均P＜ 0.01).转染FASTK过表达腺病毒可显著上调心肌细胞FASTK蛋白和mRNA水平(均P＜ 0.01).H/R损伤抑制心肌细胞基础和最大线粒体氧耗率并降低三磷酸腺苷(adenosine triphosphate,ATP)生成速率(均P＜ 0.01),而FASTK过表达可显著减轻H/R导致的线粒体呼吸功能障碍(均P＜ 0.001).FASTK过表达抑制了caspase-3活化,也显著减轻了H/R损伤导致的心肌细胞死亡和LDH释放(均P＜ 0.001).结论 过表达FASTK可显著减轻H/R损伤导致的心肌细胞线粒体呼吸功能障碍.

Diabetic patients are sensitive to myocardial ischemia-reperfusion (MI/R) injury. During diabetes, branched-chain amino acid (BCAA) catabolism is defective and mitochondrial phosphatase 2C (PP2Cm) expression is reduced. This study aims to elucidate the relationship between PP2Cm downregulation and BCAA catabolism defect in diabetic mice against MI/R injury. PP2Cm was significantly downregulated in hearts of diabetic mice. The cardiac function was improved and the myocardial infarct size and apoptosis were decreased in diabetic mice overexpressing PP2Cm after MI/R. In diabetic mice, the cardiac BCAA and its metabolites branched-chain keto-acids (BCKA) levels, and p-BCKDE1α (E1 subunit of BCKA dehydrogenase)/BCKDE1α ratio were increased while the BCKD activity was decreased. Treatment of diabetic mice subjected to MI/R injury with BT2, a BCKD kinase (BDK) inhibitor, alleviated the BCAA catabolism defect, and improved the cardiac function alongside reduced apoptosis. PP2Cm overexpression alleviated the BCAA catabolism defect and MI/R injury. Similarly, MnTBAP ameliorated the oxidative stress and MI/R injury. BCKA treatment of H9C2 cells under simulated ischemia/reperfusion (SI/R) injury significantly decreased cell viability and increased LDH release and apoptosis. These effects were alleviated by BT2 and MnTBAP treatments. These results suggested that PP2Cm directly mediates the BCAA catabolism defect and oxidative stress observed after MI/R in diabetes. Overexpression of PP2Cm alleviates MI/R injury by reducing the catabolism of BCAA and oxidative stress.

Objective To investigate the regulatory effect of branched chain amino acids (BCAA) on the expression of apoptosis related proteins after cerebral ischemia reperfusion injury and the protective effects of BCAA on ischemic brain injury in rats.Methods 40 male SD rats were randomly divided into normal diet group (n =20) and branched chain amino acid (BCAA) group (n=20) according to the random number table,and each group was randomly divided into control group (n=6),sham operation group (n=6) and model group (n =8) which used suture method to make ischemia reperfusion model.After modeling,modified Neurological Severity Scores (mNSS) was used to access the neurological impairment degree of 2,6,24,48 and 72 h in each group.The expression of apoptosis related proteins (Cleaved,Bax/Bcl-2) after 72 h was detected by the method of immune protein imprinting (Caspase3) and compared between normal diet group and BCAA group.Results Compared with the normal diet rats,the mNSS of BCAA diet rats after modeling at 2,6,24,48,72 h decreased (11.35±2.78 vs.7.15±2.41,P=0.019;9.35±1.75 vs.5.82±1.17,P=0.002;6.11±1.16vs.4.39±1.46,P=0.048;5.87±1.32vs.3.55±1.94,P=0.036;4.98±2.24vs.2.09±1.33,P=0.022).The expression of cleaved caspase3 protein and the ratio of Bax/Bcl-2 decreased in BCAA group.Conclusion BCAA can alleviate the apoptosis of rats after ischemia and reperfusion,reduce the damage of nerve function,and has a positive protective effect on ischemic brain injury.

Smooth muscle cells (SMCs), which form the walls of blood vessels, play an important role in vascular development and the pathogenic process of vascular remodeling. However, the molecular mechanisms governing SMC differentiation remain poorly understood. Glycoprotein M6B (GPM6B) is a four-transmembrane protein that belongs to the proteolipid protein family and is widely expressed in neurons, oligodendrocytes, and astrocytes. Previous studies have revealed that GPM6B plays a role in neuronal differentiation, myelination, and osteoblast differentiation. In the present study, we found that the GPM6B gene and protein expression levels were significantly upregulated during transforming growth factor-β1 (TGF-β1)-induced SMC differentiation. The knockdown of GPM6B resulted in the downregulation of SMC-specific marker expression and repressed the activation of Smad2/3 signaling. Moreover, GPM6B regulates SMC Differentiation by Controlling TGF-β-Smad2/3 Signaling. Furthermore, we demonstrated that similar to p-Smad2/3, GPM6B was profoundly expressed and coexpressed with SMC differentiation markers in embryonic SMCs. Moreover, GPM6B can regulate the tightness between TβRI, TβRII, or Smad2/3 by directly binding to TβRI to activate Smad2/3 signaling during SMC differentiation, and activation of TGF-β-Smad2/3 signaling also facilitate the expression of GPM6B. Taken together, these findings demonstrate that GPM6B plays a crucial role in SMC differentiation and regulates SMC differentiation through the activation of TGF-β-Smad2/3 signaling via direct interactions with TβRI. This finding indicates that GPM6B is a potential target for deriving SMCs from stem cells in cardiovascular regenerative medicine. Stem Cells 2018 Stem Cells 2019;37:190–201

AIM To determine the effect of nicotine on cardiomyocytes and the mechanism underlying it.METHODS Cultured H9C2 cells were randomly divided into 4 groups:control group,nicotine group(10 μmol/L),control + Akt overexpression group and nicotine (10 μmol/L) + Akt overexpression group.Forty-eight hours after culture,cell viability was detected by CCK-8,apoptosis was detected by Caspase-3 colorimetric activity assay kit and the protein level of Akt was detected by Western blot.RESULTS Compared with the control group,the cell viability of the nicotine group was obviously decreased(P ＜0.05),the cell apoptosis was remarkably improved (P ＜ 0.01) and the protein level of Akt was decreased (P ＜ 0.01).These changes were all reversed by Akt overexpression (P ＜ 0.01).CONCLUSION Nicotine down-regulates the protein level of Akt,which can induce the apoptosis of H9C2 cells.

It has been well documented that hypoadiponectinemia is associated with impaired endothelium-dependent vasodilation. However, the exact molecular mechanism which mediates this process has not been fully described. The current study aimed to investigate the role of hypoadiponectinemia-induced NLRP3 inflammasome activation in diabetic vascular endothelial dysfunction and its molecular mechanism. Male adult adiponectin knockout mice and wild type mice were fed with a high fat diet to establish a type 2 diabetic mellitus model. In addition, human umbilical vein endothelial cells (HUVECs) were cultured and subjected to high glucose/high fat (HG/HF). The NLRP3 inflammasome activation was increased in type 2 diabetic mice and treatment of diabetic aortic segments with MCC950, a potent selective inhibitor of NLRP3 inflammasome ex vivo improved endothelial-dependent vasorelaxation. NLRP3 inflammasome activation and vascular endothelial injury were significantly increased in APN-KO mice compared with WT mice in diabetes and MCC950 decreased diabetic vascular endothelial dysfunction to comparable levels in APN-KO mice and WT mice. Adiponectin could decrease NLRP3 inflammasome activation and attenuate endothelial cell injury, which was abolished by NLRP3 inflammasome overexpression. Inhibition of peroxynitrite formation preferentially attenuated NLRP3 inflammasome activation in APN-KO diabetic mice. The current study demonstrated for the first time that hypoadiponectinemia-induced NLRP3 inflammasome activation was a novel mechanism of diabetic vascular endothelial dysfunction.

Smoking is highly associated with cardiovascular diseases. However, the effect of nicotine, a key ingredient in smoking products, on cardiomyocyte apoptosis remains controversial. The present study aims to clarify the role of nicotine on cardiomyocyte cell apoptosis and to investigate the underlying mechanism. In the present study, H9C2 cells were exposed to nicotine at various concentrations (0, 10 and 100 µM) for 48 h. Cell Counting Kit‑8 and TUNEL assays were performed to assess cell viability and apoptosis, respectively, and reverse transcription‑quantitative polymerase chain reaction and western blot analysis were used to investigate the mRNA and protein expression. PYR‑41, a ubiquitin E1 inhibitor, was employed to investigate whether the ubiquitin‑proteasome system was involved in the downregulation of Akt. An Akt1 overexpression plasmid was used to demonstrate the role of Akt in H9C2 cells apoptosis. Tetratricopeptide repeat domain 3 (TTC3) small interfering RNA (siRNA) was used to investigate the effect of TTC3 on Akt protein degradation. The results demonstrated that nicotine induced apoptosis in H9C2 cells compared with control cells (P<0.05). The protein level of Akt was downregulated by nicotine in a concentration‑dependent manner (P<0.05). PYR‑41 treatment restored the protein level of Akt. The cell viability was significantly improved by Akt overexpression when cells were exposed to nicotine at 10 µM, compared with control cells. Nicotine also upregulated the level of TTC3 mRNA (P<0.05) and the protein level of Akt, and cell viability was recovered by TTC3 siRNA. In conclusion, the current study demonstrated that nicotine induced H9C2 cell apoptosis via Akt protein degradation, which may be mediated by TTC3.

目的探讨支链氨基酸（Branched chain amino acids,BCAA）对高脂诱导胰岛素抵抗的影响。方法将60只野生C57BL/6J小鼠随机分成4组,分别给予正常饮食+普通饮水（ND）组,正常饮食+含50 g/L BCAA饮水（ND/BCAA）组,高脂饮食（HD）组,高脂饮食+含50 g/L BCAA饮水（HD/BCAA）组,喂养18周,仔细观察小鼠生活状态并每周记录小鼠体质量,18周后进行葡萄糖耐量（IPGTT）和胰岛素耐量（ITT）实验,检测小鼠血清BCAA和胰岛素水平,Western blot检测小鼠肝脏胰岛素信号通路关键分子IRS1及其磷酸化水平〔p-IRS1（ser307）〕、AKT及其磷酸化水平〔p-AKT（ser473）〕。结果 1BCAA抑制高脂诱导的小鼠体质量增加,表现为HD/BCAA组小鼠体质量增长比HD组缓慢（P＜0.05,P＜0.01）;ND/BCAA组和ND组体质量增长无明显差异;2与ND组相比,ND/BCAA组小鼠血清BCAA浓度无明显升高,HD组血清BCAA水平升高（P＜0.05）;与HD组相比,HD/BCAA组血清BCAA浓度进一步升高（P＜0.05）;3与ND组相比,ND/BCAA组小鼠血清胰岛素、IPGTT和ITT均无明显改变;与HD组相比,HD/BCAA组血清胰岛素水平更高,IPGTT和ITT均明显受损（P＜0.05）;4Western blot检测显示:ND组和ND/BCAA组小鼠肝脏p-IRS1（ser307）和p-AKT（ser473）水平无明显改变;与ND组相比,HD组小鼠p-IRS1（ser307）上调（P＜0.05）,IRS1、p-AKT（ser473）均下调（P＜0.05,P＜0.01）;与HD组相比,HD/BCAA组小鼠肝脏p-IRS1（ser307）进一步上调（P＜0.05）,p-AKT（ser473）水平进一步下调（P＜0.01）。结论正常饮食单纯补充BCAA不影响小鼠肝脏胰岛素敏感性,BCAA能够促进高脂诱导的小鼠肝脏胰岛素抵抗。

Diabetic mellitus (DM) portends poor prognosis concerning pressure overloaded heart disease. Branched-chain amino acids (BCAAs), elements of essential amino acids, have been found altered in its catabolism in diabetes decades ago. However, the relationship between BCAAs and DM induced deterioration of pressure overloaded heart disease remains controversial. This study is aimed to investigate the particular effect of BCKA, a metabolite of BCAA, on myocardial injury induced by pressure overloaded. Primary cardiomyocytes were incubated with or without BCKA and followed by treatment with isoproterenol (ISO); then cell viability was detected by CCK8 and apoptosis was examined by TUNNEL stain and caspase-3 activity analysis. Compared to non-BCKA incubated group, BCKA incubation decreased cell survival and increased apoptosis concentration dependently. Furthermore, Western blot assay showed that mTORC2-Akt pathway was significantly inactivated by BCKA incubation. Moreover, overexpression of rictor, a vital component of mTORC2, significantly abolished the adverse effects of BCKA on apoptosis susceptibility of cardiomyocytes. These results indicate that BCKA contribute to vulnerability of cardiomyocytes in stimulated stress via inactivation of mTORC2-Akt pathway.