Mitochondrial aldehyde dehydrogenase obliterates endoplasmic reticulum stress-induced cardiac contractile dysfunction via correction of autophagy

ER stress triggers myocardial contractile dysfunction while effective therapeutic regimen is still lacking. Mitochondrial aldehyde dehydrogenase (ALDH2), an essential mitochondrial enzyme governing mitochondrial and cardiac function, displays distinct beneficial effect on the heart. This study was designed to evaluate the effect of ALDH2 on ER stress-induced cardiac anomalies and the underlying mechanism involved with a special focus on autophagy. WT and ALDH2 transgenic mice were subjected to the ER stress inducer thapsigargin (1 mg/kg, i.p., 48 h). Echocardiographic, cardiomyocyte contractile and intracellular Ca^2 + properties as well as myocardial histology, autophagy and autophagy regulatory proteins were evaluated. ER stress led to compromised echocardiographic indices (elevated LVESD, reduced fractional shortening and cardiac output), cardiomyocyte contractile and intracellular Ca^2 + properties and cell survival, associated with upregulated autophagy, dampened phosphorylation of Akt and its downstream signal molecules TSC2 and mTOR, the effects of which were alleviated or mitigated by ALDH2. Thapsigargin promoted ER stress proteins Gadd153 and GRP78 without altering cardiomyocyte size and interstitial fibrosis, the effects of which were unaffected by ALDH2. Treatment with thapsigargin in vitro mimicked in vivo ER stress-induced cardiomyocyte contractile anomalies including depressed peak shortening and maximal velocity of shortening/relengthening as well as prolonged relengthening duration, the effect of which was abrogated by the autophagy inhibitor 3-methyladenine and the ALDH2 activator Alda-1. Interestingly, Alda-1-induced beneficial effect against ER stress was obliterated by autophagy inducer rapamycin, Akt inhibitor AktI and mTOR inhibitor RAD001. These data suggest a beneficial role of ALDH2 against ER stress-induced cardiac anomalies possibly through autophagy reduction.     

AMP-dependent kinase and autophagic flux are involved in aldehyde dehydrogenase-2-induced protection against cardiac toxicity of ethanol

Mitochondrial aldehyde dehydrogenase-2 (ALDH2) alleviates ethanol toxicity although the precise mechanism is unclear. This study was designed to evaluate the effect of ALDH2 on ethanol-induced myocardial damage with a focus on autophagy. Wild-type FVB and transgenic mice overexpressing ALDH2 were challenged with ethanol (3 g/kg/day, ip) for 3 days and cardiac mechanical function was assessed using the echocardiographic and IonOptix systems. Western blot analysis was used to evaluate essential autophagy markers, Akt and AMPK, and the downstream signal mTOR. Ethanol challenge altered cardiac geometry and function as evidenced by enlarged ventricular end systolic and diastolic diameters, decreased cell shortening and intracellular Ca²⁺ rise, prolonged relengthening and intracellular Ca²⁺ decay, as well as reduced SERCA Ca²⁺ uptake, which effects were mitigated by ALDH2. Ethanol challenge facilitated myocardial autophagy as evidenced by enhanced expression of Beclin, ATG7, and LC3B II, as well as mTOR dephosphorylation, which was alleviated by ALDH2. Ethanol challenge-induced cardiac defect and apoptosis were reversed by the ALDH2 agonist Alda-1, the autophagy inhibitor 3-MA, and the AMPK inhibitor compound C, whereas the autophagy inducer rapamycin and the AMPK activator AICAR mimicked or exacerbated ethanol-induced cell injury. Ethanol promoted or suppressed phosphorylation of AMPK and Akt, respectively, in FVB but not ALDH2 murine hearts. Moreover, AICAR nullified Alda-1-induced protection against ethanol-triggered autophagic and functional changes. Ethanol increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by Alda-1 and 3-MA. Lysosomal inhibition using bafilomycin A1, E64D, and pepstatin A obliterated Alda-1- but not ethanol-induced responses in GFP-LC3 puncta. Our results suggest that ALDH2 protects against ethanol toxicity through altered Akt and AMPK signaling and regulation of autophagic flux.     

Double knockout of Akt2 and AMPK accentuates high fat diet-induced cardiac anomalies through a cGAS-STING-mediated mechanism

High fat diet intake contributes to undesired cardiac geometric and functional changes although the underlying mechanism remains elusive. Akt and AMPK govern to cardiac homeostasis. This study examined the impact of deletion of Akt2 (main cardiac isoform of Akt) and AMPKα2 on high fat diet intake-induced cardiac remodeling and contractile anomalies and mechanisms involved. Cardiac geometry, contractile, and intracellular Ca²⁺ properties were evaluated using echocardiography, IonOptix® edge-detection and fura-2 techniques in wild-type (WT) and Akt2-AMPK double knockout (DKO) mice receiving low fat (LF) or high fat (HF) diet for 4 months. Our results revealed that fat diet intake elicit obesity, cardiac remodeling (hypertrophy, LV mass, LVESD, and cross-sectional area), contractile dysfunction (fractional shortening, peak shortening, maximal velocity of shortening/relengthening, time-to-90% relengthening, and intracellular Ca²⁺ handling), ultrastructural disarray, apoptosis, O₂⁻, inflammation, dampened autophagy and mitophagy. Although DKO did not affect these parameters, it accentuated high fat diet-induced cardiac remodeling and contractile anomalies. High fat intake upregulated levels of cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and STING phosphorylation while suppressing phosphorylation of ULK1 (Ser⁷⁵⁷ and Ser⁷⁷⁷), with a more pronounced effect in DKO mice. In vitro data revealed that inhibition of cGAS and STING using PF-06928215 and Astin C negated palmitic acid-induced cardiomyocyte contractile dysfunction. Biological function analysis for all differentially expressed genes (DEGs) depicted that gene ontology terms associated with Akt and AMPK signaling processes were notably changed in high fat-fed hearts. Our data indicate that Akt2-AMPK ablation accentuated high fat diet-induced cardiac anomalies possibly through a cGAS-STING-mechanism.     

Mitochondrial aldehyde dehydrogenase 2 accentuates aging-induced cardiac remodeling and contractile dysfunction: role of AMPK,Sirt1, and mitochondrial function

Cardiac aging is associated with compromised myocardial function and morphology although the underlying mechanism remains elusive. Aldehyde dehydrogenase 2 (ALDH2), an essential mitochondrial enzyme governing cardiac function, displays polymorphism in humans. This study was designed to examine the role of ALDH2 in aging-induced myocardial anomalies. Myocardial mechanical and intracellular Ca²⁺ properties were examined in young (4–5 months) and old (26–28 months) wild-type and ALDH2 transgenic mice. Cardiac histology, mitochondrial integrity, O₂⁻ generation, apoptosis, and signaling cascades, including AMPK activation and Sirt1 level were evaluated. Myocardial function and intracellular Ca²⁺ handling were compromised with advanced aging; the effects were accentuated by ALDH2. Hematoxylin and eosin and Masson trichrome staining revealed cardiac hypertrophy and interstitial fibrosis associated with greater left-ventricular mass and wall thickness in aged mice. ALDH2 accentuated aging-induced cardiac hypertrophy but not fibrosis. Aging promoted O₂⁻ release, apoptosis, and mitochondrial injury (mitochondrial membrane potential, levels of UCP-2 and PGC-1α), and the effects were also exacerbated by ALDH2. Aging dampened AMPK phosphorylation and Sirt1, the effects of which were exaggerated by ALDH2. Treatment with the ALDH2 activator Alda-1 accentuated aging-induced O₂⁻ generation and mechanical dysfunction in cardiomyocytes, the effects of which were mitigated by cotreatment with activators of AMPK and Sirt1, AICAR, resveratrol, and SRT1720. Examination of human longevity revealed a positive correlation between life span and ALDH2 gene mutation. Taken together, our data revealed that ALDH2 enzyme may accentuate myocardial remodeling and contractile dysfunction in aging, possibly through AMPK/Sirt1-mediated mitochondrial injury.     

Double knockout of Akt2 and AMPK predisposes cardiac aging without affecting lifespan: Role of autophagy and mitophagy

Increased age often leads to a gradual deterioration in cardiac geometry and contractile function although the precise mechanism remains elusive. Both Akt and AMPK play an essential role in the maintenance of cardiac homeostasis. This study examined the impact of ablation of Akt2 (the main cardiac isoform of Akt) and AMPKα2 on development of cardiac aging and the potential mechanisms involved with a focus on autophagy. Cardiac geometry, contractile, and intracellular Ca²⁺ properties were evaluated in young (4-month-old) and old (12-month-old) wild-type (WT) and Akt2-AMPK double knockout mice using echocardiography, IonOptix® edge-detection and fura-2 techniques. Levels of autophagy and mitophagy were evaluated using western blot. Our results revealed that increased age (12 months) did not elicit any notable effects on cardiac geometry, contractile function, morphology, ultrastructure, autophagy and mitophagy, although Akt2-AMPK double knockout predisposed aging-related unfavorable changes in geometry (heart weight, LVESD, LVEDD, cross-sectional area and interstitial fibrosis), TEM ultrastructure, and function (fractional shortening, peak shortening, maximal velocity of shortening/relengthening, time-to-90% relengthening, intracellular Ca²⁺ release and clearance rate). Double knockout of Akt2 and AMPK unmasked age-induced cardiac autophagy loss including decreased Atg5, Atg7, Beclin1, LC3BII-to-LC3BI ratio and increased p62. Double knockout of Akt2 and AMPK also unmasked age-related loss in mitophagy markers PTEN-induced putative kinase 1 (Pink1), Parkin, Bnip3, and FundC1, the mitochondrial biogenesis cofactor PGC-1α, and lysosomal biogenesis factor TFEB. In conclusion, our data indicate that Akt2-AMPK double ablation predisposes cardiac aging possibly related to compromised autophagy and mitophagy. This article is part of a Special Issue entitled: Genetic and epigenetic regulation of aging and longevity edited by Jun Ren & Megan Yingmei Zhang.     

Deficiency of insulin‐like growth factor 1 reduces vulnerability to chronic alcohol intake‐induced cardiomyocyte mechanical dysfunction: role of AMPK

Circulating insulin‐like growth factor I (IGF‐1) levels are closely associated with cardiac performance although the role of IGF‐1 in alcoholic cardiac dysfunction is unknown. This study was designed to evaluate the impact of severe liver IGF‐1 deficiency (LID) on chronic alcohol‐induced cardiomyocyte contractile and intracellular Ca²⁺ dysfunction. Adult male C57 and LID mice were placed on a 4% alcohol diet for 15 weeks. Cardiomyocyte contractile and intracellular Ca²⁺ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time‐to‐relengthening (TR₉₀), change in fura‐fluorescence intensity (ΔFFI) and intracellular Ca²⁺ decay. Levels of apoptotic regulators caspase‐3, Bcl‐2 and c‐Jun NH2‐terminal kinase (JNK), the ethanol metabolizing enzyme mitochondrial aldehyde dehydrogenase (ALDH2), as well as the cellular fuel gauge AMP‐activated protein kinase (AMPK) were evaluated. Chronic alcohol intake enlarged myocyte cross‐sectional area, reduced PS, ± dL/dt and ΔFFI as well as prolonged TR₉₀ and intracellular Ca²⁺ decay, the effect of which was greatly attenuated by IGF‐1 deficiency. The beneficial effect of LID against alcoholic cardiac mechanical defect was ablated by IGF‐1 replenishment. Alcohol intake increased caspase‐3 activity/expression although it down‐regulated Bcl‐2, ALDH2 and pAMPK without affecting JNK and AMPK. IGF‐1 deficiency attenuated alcoholism‐induced responses in all these proteins with the exception of Bcl‐2. In addition, the AMPK agonist 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside abrogated short‐term ethanol incubation‐elicited cardiac mechanical dysfunction. Taken together, these data suggested that IGF‐1 deficiency may reduce the sensitivity to ethanol‐induced myocardial mechanical dysfunction. Our data further depicted a likely role of Caspase‐3, ALDH2 and AMPK activation in IGF‐1 deficiency induced ‘desensitization’ of alcoholic cardiomyopathy.     

Tauroursodeoxycholic Acid Mitigates High Fat Diet-Induced Cardiomyocyte Contractile and Intracellular Ca2+ Anomalies

ObjectivesThe endoplasmic reticulum (ER) chaperone tauroursodeoxycholic acid (TUDCA) has exhibited promises in the treatment of obesity, although its impact on obesity-induced cardiac dysfunction is unknown. This study examined the effect of TUDCA on cardiomyocyte function in high-fat diet-induced obesity.MethodsAdult mice were fed low or high fat diet for 5 months prior to treatment of TUDCA (300 mg/kg. i.p., for 15d). Intraperitoneal glucose tolerance test (IPGTT), cardiomyocyte mechanical and intracellular Ca²⁺ property, insulin signaling molecules including IRS-1, Akt, AMPK, ACC, GSK-3β, c-Jun, ERK and c-Jun N terminal kinase (JNK) as well as ER stress and intracellular Ca²⁺ regulatory proteins were examined. Myocardial ultrastructure was evaluated using transmission electron microscopy (TEM).ResultsHigh-fat diet depressed peak shortening (PS) and maximal velocity of shortening/relengthenin as well as prolonged relengthening duration. TUDCA reversed or overtly ameliorated high fat diet-induced cardiomyocyte dysfunction including prolongation in relengthening. TUDCA alleviated high-fat diet-induced decrease in SERCA2a and phosphorylation of phospholamban, increase in ER stress (GRP78/BiP, CHOP, phosphorylation of PERK, IRE1α and eIF2α), ultrastructural changes and mitochondrial permeation pore opening. High-fat diet feeding inhibited phosphorylation of AMPK and promoted phosphorylation of GSK-3β. TUDCA prevented high fat-induced dephosphorylation of AMPK but not GSK-3β. High fat diet promoted phosphorylation of IRS-1 (Ser³⁰⁷), JNK, and ERK without affecting c-Jun phosphorylation, the effect of which with the exception of ERK phosphorylation was attenuated by TUDCA.ConclusionsThese data depict that TUDCA may ameliorate high fat diet feeding-induced cardiomyocyte contractile and intracellular Ca²⁺ defects through mechanisms associated with mitochondrial integrity, AMPK, JNK and IRS-1 serine phosphorylation.     

Cardiomyocyte-specific knockout of endothelin receptor a attenuates obesity cardiomyopathy

Endothelin (ET)-1 is implicated in the pathophysiology of cardiovascular diseases although its role in obesity anomalies has not been fully elucidated. This study was designed to examine the impact of ET-1 receptor A (ET_A) ablation on obesity-induced changes in cardiac geometry and contractile function, as well as the mechanisms involved with a focus on autophagy. Cardiomyocyte-specific ET_A receptor knockout (ETAKO) and WT mice were fed either low-fat (10% calorie from fat) or high-fat (45% calorie from fat) diet for 24?weeks. Glucose tolerance test was examined to confirm insulin resistance. High-fat diet intake compromised myocardial geometry (enlarged left ventricular diameters in systole and diastole), morphology (cardiac hypertrophy, increased wall thickness and interstitial fibrosis), contractile function (reduced fractional shortening, ejection fraction and cardiomyocyte shortening) and intracellular Ca²⁺ handling, the effect of which was significantly attenuated by ETAKO. TUNEL staining revealed overt apoptosis in high-fat-fed group, the effect was reverted by ETAKO. Western blot analysis noted that high-fat intake downregulated leptin receptor and PPARγ, insulin signaling (elevated basal/dampened insulin-stimulated phosphorylation of Akt and IRS1), phosphorylation of AMPK, ACC, upregulated GATA-4, ANP, NFATc3, PPARα, m-TOR/p70s6k signaling, which were attenuated by ETAKO with the exception of AMPK/ACC. Furthermore, high-fat intake suppressed cardiac autophagy, which was abrogated by ETAKO. In cultured murine cardiomyocytes, palmitic acid challenged mimicked high-fat diet-induced hypertrophic and autophagic responses, the effect of which were abolished by the ET_A receptor antagonist BQ123 or mTOR inhibitor rapamycin. These results suggest that inhibition of ET_A rescues high-fat intake-induced cardiac anomalies possibly through autophagy regulation.     

Alpha1 catalytic subunit of AMPK modulates contractile function of cardiomyocytes through phosphorylation of troponin I

Aims

The specific role of AMPKα1 or AMPKα2 in mediating cardiomyocyte contractile function remains elusive. The present study investigated how AMPK activation modulates the contractility of isolated cardiomyocytes.

Main methods

Mechanical properties and intracellular Ca^2 + properties were measured in isolated cardiomyocytes. The stress signaling was evaluated using western blot and immunoprecipitation analysis.

Key findings

AMPK activator, A-769662 induced maximal velocity of shortening (+ dL/dt) and relengthening (− dL/dt), peak height and peak shortening (PS) amplitude in both WT and AMPKα2 KO cardiomyocytes, but did not affect time-to-90% relengthening (TR90). AMPK KD cardiomyocytes demonstrated contractile dysfunction compared with cardiomyocytes from WT and AMPKα2 KO hearts. However, the rise of intracellular Ca^2 + levels as well as intracellular ATP levels has no significant difference among WT, AMPKα2 KO and AMPK KD groups with and without the presence of A-769662. Besides, WT, AMPKα2 KO and AMPK KD group displayed a phosphorylated AMPK and downstream acetyl-CoA carboxylase (ACC) phosphorylation. Interestingly, A-769662 also triggered troponin I (cTnI) phosphorylation at Ser¹⁴⁹ site which is related to contractility of cardiomyocytes. Furthermore, the immunoprecipitation analysis revealed that AMPKα1 of cardiomyocytes was phosphorylated by A-769662.

Significance

This is the first study illustrating that activation of AMPK plays a significant role in mediating the contractile function of cardiomyocytes using transgenic animal models. AMPK activator facilitates the contractility of cardiomyocytes via activating AMPKα1 catalytic subunit. The phosphorylation of cTnI by AMPK could be a factor attributing to the regulation of contractility of cardiomyocytes.     

Live-cell imaging of Aspergillus nidulans autophagy

We exploited the amenability of the fungus Aspergillus nidulans to genetics and live-cell microscopy to investigate autophagy. Upon nitrogen starvation, GFP-Atg8-containing pre-autophagosomal puncta give rise to cup-shaped phagophores and circular (0.9-μm diameter) autophagosomes that disappear in the vicinity of the vacuoles after their shape becomes irregular and their GFP-Atg8 fluorescence decays. This ‘autophagosome cycle’ gives rise to characteristic cone-shaped traces in kymographs. Autophagy does not require endosome maturation or ESCRTs, as autophagosomes fuse with vacuoles directly in a RabS (homolog of Saccharomyces cerevisiae Ypt7 and mammalian RAB7; written hereafter as RabS^RAB7)-HOPS-(homotypic fusion and vacuole protein sorting complex)-dependent manner. However, by removing RabS^RAB7 or Vps41 (a component of the HOPS complex), we show that autophagosomes may still fuse, albeit inefficiently, with the endovacuolar system in a process almost certainly mediated by RabA^RAB5/RabB^RAB5 (yeast Vps21 homologs)-CORVET (class C core vacuole/endosome tethering complex), because acute inactivation of HbrA/Vps33, a key component of HOPS and CORVET, completely precludes access of GFP-Atg8 to vacuoles without affecting autophagosome biogenesis. Using a FYVE₂-GFP probe and endosomal PtdIns3P-depleted cells, we imaged PtdIns3P on autophagic membranes. PtdIns3P present on autophagosomes decays at late stages of the cycle, preceding fusion with the vacuole. Autophagy does not require Golgi traffic, but it is crucially dependent on RabO^RAB1. TRAPPIII-specific factor AN7311 (yeast Trs85) localizes to the phagophore assembly site (PAS) and RabO^RAB1 localizes to phagophores and autophagosomes. The Golgi and autophagy roles of RabO^RAB1 are dissociable by mutation: rabO^A136D hyphae show relatively normal secretion at 28°C but are completely blocked in autophagy. This finding and the lack of Golgi traffic involvement pointed to the ER as one potential source of membranes for autophagy. In agreement, autophagosomes form in close association with ring-shaped omegasome-like ER structures resembling those described in mammalian cells.     

Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3β-dependent inhibition of apoptosis and ER stress

Sepsis is characterized by systematic inflammation and contributes to cardiac dysfunction. This study was designed to examine the effect of protein kinase B (Akt) activation on lipopolysaccharide-induced cardiac anomalies and underlying mechanism(s) involved. Mechanical and intracellular Ca^2 + properties were examined in myocardium from wild-type and transgenic mice with cardiac-specific chronic Akt overexpression following LPS (4 mg/kg, i.p.) challenge. Akt signaling cascade (Akt, phosphatase and tensin homologue deleted on chromosome ten, glycogen synthase kinase 3 beta), stress signal (extracellular-signal-regulated kinases, c-Jun N-terminal kinases, p38), apoptotic markers (Bcl-2 associated X protein, caspase-3/-9), endoplasmic reticulum (ER) stress markers (glucose-regulated protein 78, growth arrest and DNA damage induced gene-153, eukaryotic initiation factor 2α), inflammatory markers (tumor necrosis factor α, interleukin-1β, interleukin-6) and autophagic markers (Beclin-1, light chain 3B, autophagy-related gene 7 and sequestosome 1) were evaluated. Our results revealed that LPS induced marked decrease in ejection fraction, fractional shortening, cardiomyocyte contractile capacity with dampened intracellular Ca^2 + release and clearance, elevated reactive oxygen species (ROS) generation and decreased glutathione and glutathione disulfide (GSH/GSSG) ratio, increased ERK, JNK, p38, GRP78, Gadd153, eIF2α, BAX, caspase-3 and -9, downregulated B cell lymphoma 2 (Bcl-2), the effects of which were significantly attenuated or obliterated by Akt activation. Akt activation itself did not affect cardiac contractile and intracellular Ca^2 + properties, ROS production, oxidative stress, apoptosis and ER stress. In addition, LPS upregulated levels of Beclin-1, LC3B and Atg7, while suppressing p62 accumulation. Akt activation did not affect Beclin-1, LC3B, Atg7 and p62 in the presence or absence of LPS. Akt overexpression promoted phosphorylation of Akt and GSK3β. In vitro study using the GSK3β inhibitor SB216763 mimicked the response elicited by chronic Akt activation. Taken together, these data showed that Akt activation ameliorated LPS-induced cardiac contractile and intracellular Ca^2 + anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3β-dependent mechanism.     

高糖对线粒体乙醛脱氢酶2在大鼠心肌成纤维细胞中表达的影响

目的：观察心肌成纤维细胞是否存在线粒体乙醛脱氢酶2（ALDH2）的表达,探讨ALDH2在高糖诱导的心肌成纤维细胞引起纤维化发生中的作用。方法：原代培养心肌成纤维细胞,分为正常对照组（5.5 mmol/L）、正常+ALDH2激动剂Alda-1（20μmol/L）组、高糖组（30 mmol/L）、高糖+ Alda-1组。免疫荧光鉴定心肌成纤维细胞。各组细胞分别培养48 h后应用MTT法检测成纤维细胞增殖活力,RT-PCR和Western blot检测ALDH2 mRNA及蛋白的表达。结果：RT-PCR和Western blot结果显示心肌成纤维细胞ALDH2 mRNA和蛋白均有表达。与正常对照组相比,高糖组心肌成纤维细胞增殖能力提高（P < 0.01）,ALDH2蛋白表达下降（P < 0.05）;与高糖组相比,高糖+ Alda-1组心肌成纤维细胞增殖能力降低（P < 0.01）,ALDH2的蛋白表达增加（P < 0.05）。结论：心肌成纤维细胞存在ALDH2的表达,ALDH2激动剂Alda-1提高ALDH2的表达后可以抑制高糖引起的心肌成纤维细胞的增殖。     

PAR2 promotes high-fat diet-induced hepatic steatosis by inhibiting AMPK-mediated autophagy

Protease-activated receptor 2 (PAR2) is a member of G protein-coupled receptors. There are two types of PAR2 signaling pathways: Canonical G-protein signaling and β-arrestin signaling. Although PAR2 signaling has been reported to aggravate hepatic steatosis, the exact mechanism is still unclear, and the role of PAR2 in autophagy remains unknown. In this study, we investigated the regulatory role of PAR2 in autophagy during high-fat diet (HFD)-induced hepatic steatosis in mice. Increased protein levels of PAR2 and β-arrestin-2 and their interactions were detected after four months of HFD. To further investigate the role of PAR2, male and female wild-type (WT) and PAR2-knockout (PAR2 KO) mice were fed HFD. PAR2 deficiency protected HFD-induced hepatic steatosis in male mice, but not in female mice. Interestingly, PAR2-deficient liver showed increased AMP-activated protein kinase (AMPK) activation with decreased interaction between Ca²⁺/calmodulin-dependent protein kinase kinase β (CAMKKβ) and β-arrestin-2. In addition, PAR2 deficiency up-regulated autophagy in the liver. To elucidate whether PAR2 plays a role in the regulation of autophagy and lipid accumulation in vitro, PAR2 was overexpressed in HepG2 cells. Overexpression of PAR2 decreased AMPK activation with increased interaction of CAMKKβ with β-arrestin-2 and significantly inhibited autophagic responses in HepG2 cells. Inhibition of autophagy by PAR2 overexpression further exacerbated palmitate-induced lipid accumulation in HepG2 cells. Collectively, these findings suggest that the increase in the PAR2-β-arrestin-2-CAMKKβ complex by HFD inhibits AMPK-mediated autophagy, leading to the alleviation of hepatic steatosis.     

线粒体乙醛脱氢酶2(ALDH2)通过AMPK/FOXO3a通路改善高糖导致的心肌细胞凋亡

目的:观察乙醛脱氢酶2(ALDH2)对高糖诱导的H9C2心肌细胞存活及凋亡的影响,并探讨腺苷酸活化蛋白激酶(AMPK)/FOXO3a信号通路在高糖导致的心肌细胞凋亡中的调控作用。方法:以30 mmol/L葡萄糖诱导培养H9C2心肌细胞48 h,经ALDH2激动剂Alda-1及AMPK抑制剂Compound C干预后,用MTT法检测细胞的存活情况,TUNEL试剂盒检测细胞凋亡情况,Western blot检测ALDH2、磷酸化AMPK和FOXO3a蛋白的表达水平。结果:与对照组相比,高浓度葡萄糖培养H9C2心肌细胞后,细胞的存活率显著降低、凋亡指数明显升高,磷酸化AMPK的表达水平明显上调,ALDH2和磷酸化FOXO3a的蛋白表达显著降低(P0.05)。ALDH2的激动剂Alda-1处理可显著提高高糖诱导的H9C2心肌细胞的存活率、降低其凋亡率,减少磷酸化AMPK的蛋白表达,增加ALDH2的表达和FOXO3a蛋白的磷酸化;而进一步采用AMPK的抑制剂Compound C处理,可显著抑制Alda-1对高糖诱导的H9C2心肌细胞的这些影响。结论:ALDH2的激动剂Alda-1对高糖诱导的心肌细胞凋亡具有保护作用,可能与其激活AMPK,进而抑制心肌细胞FOXO3a的活性有关。     

Influence of long-term caloric restriction on myocardial and cardiomyocyte contractile function and autophagy in mice

Both clinical and experimental evidence has revealed that calorie restriction (CR) is capable of improving heart function. However, most the reports are focused on the effect of CR on the pathological states such as obesity, while the effect of CR on heart function in otherwise healthy subjects is not well understood. This study examined the long-term CR effect on cardiac contractile function and possible underlying mechanisms involved. C57BL/6 mice were subjected to a 40% CR or ad libitum feeding for 20 weeks. Echocardiographic and cardiomyocyte contractile properties were evaluated. Intracellular signaling pathways were examined using Western blot analysis. Our results showed that CR overtly lessened glucose intolerance, lessened body and heart weights (although not heart size), lowered fat tissue density, decreased left ventricular (LV) wall thickness (septum and posterior wall) in both systole and diastole, and reduced LV mass (not normalized LV mass) without affecting fractional shortening. Cardiomyocyte cell length and cross-sectional area were reduced, while peak shortening amplitude was increased following CR. CR failed to affect maximal velocity of shortening/relengthening and duration of shortening and relengthening. Immunoblotting data depicted decreased and increased phosphorylation of Akt/glycogen synthase kinase-3β and AMP-dependent protein kinase/acetyl-CoA carboxylase, respectively, following CR. CR also dampened the phosphorylation of mammalian target of rapamycin, extracellular-signal-regulated protein kinase 1/2 and c-Jun, while it increased the phosphorylation of c-Jun NH2-terminal kinase. Last but not least, CR significantly promoted cardiac autophagy as evidenced by increased expression of LC3B-II (and LC3B-II to LC3B-I ratio) and Beclin-1. In summary, our data suggested that long-term CR may preserve cardiac contractile function with improved cardiomyocyte function, lessen cardiac remodeling and promote autophagy.     

Saikosaponin-d inhibits proliferation by up-regulating autophagy via the CaMKKβ–AMPK–mTOR pathway in ADPKD cells

Autosomal dominant polycystic kidney disease (ADPKD) is a common heritable human disease. Recently, the role of repressed autophagy in ADPKD has drawn increasing attention. Here, we investigate the mechanism underlying the effect of Saikosaponin-d (SSd), a sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump (SERCA) inhibitor. We show that SSd suppresses proliferation in ADPKD cells by up-regulating autophagy. We found that treatment with SSd results in the accumulation of intracellular calcium, which in turn activates the CaMKKβ–AMPK signalling cascade, inhibits mTOR signalling and induces autophagy. Conversely, we also found that treatment with an autophagy inhibitor (3-methyladenine), AMPK inhibitor (Compound C), CaMKKβ inhibitor (STO-609) and intracellular calcium chelator (BAPTA/AM) could reduce autophagy puncta formation mediated by SSd. Our results demonstrated that SSd induces autophagy through the CaMKKβ–AMPK–mTOR signalling pathway in ADPKD cells, indicating that SSd might be a potential therapy for ADPKD and that SERCA might be a new target for ADPKD treatment.

     

Regulation of expression of cardiac sarcoplasmic reticulum proteins under pathophysiological conditions

Congestive heart failure presents a significant medical problem and accumulating evidence indicates that slow relaxation during diastole maybe at least in part be medlated by decreased expression of the gene coding for the Ca²⁺ ATPase of the sarcoplasmic reticulum (SR). In order to determine if increased expression of the SR Ca²⁺ ATPase gene leads to alterations in calcium transients and in contractile behavior we constructed transgenic mice overexpressing the SERCA2 gene. Measuring dP/dt_max and dpPdt_min with a 2 French Milar catheter we found a significant Increase in systolic contraction and diastolic relaxation in transgene positive versus transgene negative mice. In addition we constructed adenoviruses overexpressing the gene coding for the Ca²⁺ ATPase of the sarcoplasmic reticulum. Infacting cardiac myocytes with the adenovirus expressing this transgene led to an accelerated calcium transient. Determining cell shortening and relengthening with a edge detection method indicated that increased expression of the SERCA2 transgene mediated by adenovirus Infection accelerated contractile parameters. In summary increased expression of the SERCA2 transgene leads to an enhancement of cardiac contrectile parameters under in vivo conditions in transgenic mice and in myocytes in cell culture using an adenovirus based approach to increase expression of the SERCAX gene.     

Regulation of SERCA pumps expression in diabetes

Cytosolic calcium concentration ([Ca²⁺]_c) is fundamental for regulation of many cellular processes such metabolism, proliferation, muscle contraction, cell signaling and insulin secretion. In resting conditions, the sarco/endoplasmic reticulum (ER/SR) Ca²⁺ ATPase's (SERCA) transport Ca²⁺ from the cytosol to the ER or SR lumen, maintaining the resting [Ca²⁺]_c about 25–100 nM. A reduced activity and expression of SERCA2 protein have been described in heart failure and diabetic cardiomyopathy, resulting in an altered Ca²⁺ handling and cardiac contractility. In the diabetic pancreas, there has been reported reduction in SERCA2b and SERCA3 expression in β-cells, resulting in diminished insulin secretion. Evidence obtained from different diabetes models has suggested a role for advanced glycation end products formation, oxidative stress and increased O-GlcNAcylation in the lowered SERCA2 expression observed in diabetic cardiomyopathy. However, the role of SERCA2 down-regulation in the pathophysiology of diabetes mellitus and diabetic cardiomyopathy is not yet well described. In this review, we make a comprehensive analysis of the current knowledge of the role of the SERCA pumps in the pathophysiology of insulin-dependent diabetes mellitus type 1 (TIDM) and type 2 (T2DM) in the heart and β-cells in the pancreas.