Cardiomyocyte-specific deletion of endothelin receptor A (ETA) obliterates cardiac aging through regulation of mitophagy and ferroptosis.

Advanced aging evokes unfavorable changes in the heart including cardiac remodeling and contractile dysfunction although the underlying mechanism remains elusive. This study was conducted to evaluate the role of endothelin-1 (ET-1) in the pathogenesis of cardiac aging and mechanism involved. Echocardiographic and cardiomyocyte mechanical properties were determined in young (5-6 mo) and aged (26-28 mo) wild-type (WT) and cardiomyocyte-specific ETA receptor knockout (ETAKO) mice. GSEA enrichment identified differentially expressed genes associated with mitochondrial respiration, mitochondrial protein processing and mitochondrial depolarization in cardiac aging. Aging elevated plasma levels of ET-1, Ang II and suppressed serum Fe2+, evoked cardiac remodeling (hypertrophy and interstitial fibrosis), contractile defects (fractional shortening, ejection fraction, cardiomyocyte peak shortening, maximal velocity of shortening/relengthening and prolonged relengthening) and intracellular Ca2+ mishandling (dampened intracellular Ca2+ release and prolonged decay), the effects with the exception of plasma AngII, ET-1 and Fe2+ were mitigated by ETAKO. Advanced age facilitated O2- production, carbonyl protein damage, cardiac hypertrophy (GATA4, ANP, NFATc3), ER stress, ferroptosis, compromised autophagy (LC3B, Beclin-1, Atg7, Atg5 and p62) and mitophagy (parkin and FUNDC1), and deranged intracellular Ca2+ proteins (SERCA2a and phospholamban), the effects of which were reversed by ETA ablation. ET-1 provoked ferroptosis in vitro, the response was nullified by the ETA receptor antagonist BQ123 and mitophagy inducer CsA. ETA but not ETB receptor antagonism reconciled cardiac aging, which was abrogated by inhibition of mitophagy and ferroptosis. These findings collectively denote promises of targeting ETA, mitophagy and ferroptosis in the management of aging-associated cardiac remodeling and contractile defect.

Cardiac-specific overexpression of catalase attenuates lipopolysaccharide-induced cardiac anomalies through reconciliation of autophagy and ferroptosis.

Lipopolysaccharide (LPS) from Gram-negative bacteria is a major contributor to cardiovascular failure, but the signaling mechanisms underlying its stress response are not fully understood. This study aimed to investigate the effect of the antioxidant enzyme catalase on LPS-induced cardiac abnormalities and the mechanisms involved, with particular focus on the interplay between autophagy, ferroptosis, and apoptosis. Cardiac-specific catalase (CAT) overexpression and wild-type (WT) mice were stimulated with LPS (6 mg/kg, intravenous injection), and cardiac morphology and function were evaluated. Oxidative stress, ferroptosis, apoptosis, and mitochondrial status were monitored, and survival curves were plotted based on the results of LPS stimulation. The results showed that, compared with WT mice, mice overexpressing catalase had a higher survival rate under LPS stimulation. Ultrasound echocardiography, cardiomyocyte characteristics, and Masson's trichrome staining showed that LPS inhibited cardiac function and caused cardiac fibrosis, while catalase alleviated these adverse effects. LPS increased apoptosis (TUNEL, caspase-3 activation, cleaved caspase-3), increased O2·- production, induced inflammation (TNF-α), autophagy, iron toxicity, and carbonyl damage, and significantly damaged mitochondria (mitochondrial membrane potential, mitochondrial proteins, and ultrastructure). These effects were significantly alleviated by catalase. Interestingly, the antioxidant N-acetylcysteine, autophagy inhibitor 3-methyladenine, and ferroptosis inhibitor lipostatin-1 all eliminated the LPS-induced contraction dysfunction and ferroptosis (using lipid peroxidation). Induction of ferroptosis could eliminate the cardioprotective effect of NAC. In conclusion, catalase rescues LPS-induced cardiac dysfunction by regulating oxidative stress, autophagy, ferroptosis, apoptosis, and mitochondrial damage in cardiomyocytes.

Retraction notice to "Cardiac-specific overexpression of catalase attenuates lipopolysaccharide-induced myocardial contractile dysfunction: Role of autophagy" [Free Radic. Biol. Med. 53/6 (2012) 1327-1338].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. After an institutional investigation into the work of Dr. Jun Ren, University of Wyoming subsequently conducted an examination of other selected publications of Dr. Ren's under the direction of the HHS Office of Research Integrity. Based on the findings of this examination, the University of Wyoming recommended this article be retracted due to concerns regarding data irregularities inconsistent with published conclusions. Specifically, University of Wyoming found evidence of data irregularities and image reuse in Figure 2 that significantly affect the results and conclusions reported in the manuscript.

Mitochondrial aldehyde dehydrogenase (ALDH2) rescues cardiac contractile dysfunction in an APP/PS1 murine model of Alzheimer's disease via inhibition of ACSL4-dependent ferroptosis.

Alzheimer's disease (AD) is associated with high incidence of cardiovascular events but the mechanism remains elusive. Our previous study reveals a tight correlation between cardiac dysfunction and low mitochondrial aldehyde dehydrogenase (ALDH2) activity in elderly AD patients. In the present study we investigated the effect of ALDH2 overexpression on cardiac function in APP/PS1 mouse model of AD. Global ALDH2 transgenic mice were crossed with APP/PS1 mutant mice to generate the ALDH2-APP/PS1 mutant mice. Cognitive function, cardiac contractile, and morphological properties were assessed. We showed that APP/PS1 mice displayed significant cognitive deficit in Morris water maze test, myocardial ultrastructural, geometric (cardiac atrophy, interstitial fibrosis) and functional (reduced fractional shortening and cardiomyocyte contraction) anomalies along with oxidative stress, apoptosis, and inflammation in myocardium. ALDH2 transgene significantly attenuated or mitigated these anomalies. We also noted the markedly elevated levels of lipid peroxidation, the essential lipid peroxidation enzyme acyl-CoA synthetase long-chain family member 4 (ACSL4), the transcriptional regulator for ACLS4 special protein 1 (SP1) and ferroptosis, evidenced by elevated NCOA4, decreased GPx4, and SLC7A11 in myocardium of APP/PS1 mutant mice; these effects were nullified by ALDH2 transgene. In cardiomyocytes isolated from WT mice and in H9C2 myoblasts in vitro, application of Aß (20 µM) decreased cell survival, compromised cardiomyocyte contractile function, and induced lipid peroxidation; ALDH2 transgene or activator Alda-1 rescued Aß-induced deteriorating effects. ALDH2-induced protection against Aß-induced lipid peroxidation was mimicked by the SP1 inhibitor tolfenamic acid (TA) or the ACSL4 inhibitor triacsin C (TC), and mitigated by the lipid peroxidation inducer 5-hydroxyeicosatetraenoic acid (5-HETE) or the ferroptosis inducer erastin. These results demonstrate an essential role for ALDH2 in AD-induced cardiac anomalies through regulation of lipid peroxidation and ferroptosis.

ALDH2 contributes to melatonin-induced protection against APP/PS1 mutation-prompted cardiac anomalies through cGAS-STING-TBK1-mediated regulation of mitophagy.

Ample clinical evidence suggests a high incidence of cardiovascular events in Alzheimer's disease (AD), although neither precise etiology nor effective treatment is available. This study was designed to evaluate cardiac function in AD patients and APP/PS1 mutant mice, along with circulating levels of melatonin, mitochondrial aldehyde dehydrogenase (ALDH2) and autophagy. AD patients and APP/PS1 mice displayed cognitive and myocardial deficits, low levels of circulating melatonin, ALDH2 activity, and autophagy, ultrastructural, geometric (cardiac atrophy and interstitial fibrosis) and functional (reduced fractional shortening and cardiomyocyte contraction) anomalies, mitochondrial injury, cytosolic mtDNA buildup, apoptosis, and suppressed autophagy and mitophagy. APP/PS1 mutation downregulated cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) levels and TBK1 phosphorylation, while promoting Aß accumulation. Treatment with melatonin overtly ameliorated unfavorable APP/PS1-induced changes in cardiac geometry and function, apoptosis, mitochondrial integrity, cytosolic mtDNA accumulation (using both immunocytochemistry and qPCR), mitophagy, and cGAS-STING-TBK1 signaling, although these benefits were absent in APP/PS1/ALDH2-/- mice. In vitro evidence indicated that melatonin attenuated APP/PS1-induced suppression of mitophagy and cardiomyocyte function, and the effect was negated by the nonselective melatonin receptor blocker luzindole, inhibitors or RNA interference of cGAS, STING, TBK1, and autophagy. Our data collectively established a correlation among cardiac dysfunction, low levels of melatonin, ALDH2 activity, and autophagy in AD patients, with compelling support in APP/PS1 mice, in which melatonin rescued myopathic changes by promoting cGAS-STING-TBK1 signaling and mitophagy via an ALDH2-dependent mechanism.

Correction to: ALDH2 protects against high fat diet-induced obesity cardiomyopathy and defective autophagy: role of CaM kinase II, histone H3K9 methyltransferase SUV39H, Sirt1, and PGC-1α deacetylation.

The authors found a critical mistake in the assembly of Fig. 2; in Fig. 2A the right two images were erroneously duplicated. The authors have re-analysed all the data, checked for accuracy and provided the updated Fig. 2 here. Nothing is affected with regards to data summary and conclusion.

ALDH2 protects against high fat diet-induced obesity cardiomyopathy and defective autophagy: role of CaM kinase II, histone H3K9 methyltransferase SUV39H, Sirt1, and PGC-1α deacetylation.

BACKGROUND AND AIMS: Uncorrected obesity contributes to cardiac remodeling and contractile dysfunction although the underlying mechanism remains poorly understood. Mitochondrial aldehyde dehydrogenase (ALDH2) is a mitochondrial enzyme with some promises in a number of cardiovascular diseases. This study was designed to evaluate the impact of ALDH2 on cardiac remodeling and contractile property in high fat diet-induced obesity. METHODS: Wild-type (WT) and ALDH2 transgenic mice were fed low (10% calorie from fat) or high (45% calorie from fat) fat diet for 5 months prior to the assessment of cardiac geometry and function using echocardiography, IonOptix system, Lectin, and Masson Trichrome staining. Western blot analysis was employed to evaluate autophagy, CaM kinase II, PGC-1α, histone H3K9 methyltransferase SUV39H, and Sirt-1. RESULTS: Our data revealed that high fat diet intake promoted weight gain, cardiac remodeling (hypertrophy and interstitial fibrosis, p < 0.0001) and contractile dysfunction (reduced fractional shortening (p < 0.0001), cardiomyocyte function (p < 0.0001), and intracellular Ca2+ handling (p = 0.0346)), mitochondrial injury (elevated O2- levels, suppressed PGC-1α, and enhanced PGC-1α acetylation, p < 0.0001), elevated SUV39H, suppressed Sirt1, autophagy and phosphorylation of AMPK and CaM kinase II, the effects of which were negated by ALDH2 (p ≤ 0.0162). In vitro incubation of the ALDH2 activator Alda-1 rescued against palmitic acid-induced changes in cardiomyocyte function, the effect of which was nullified by the Sirt-1 inhibitor nicotinamide and the CaM kinase II inhibitor KN-93 (p < 0.0001). The SUV39H inhibitor chaetocin mimicked Alda-1-induced protection again palmitic acid (p < 0.0001). Examination in overweight human revealed an inverse correlation between diastolic cardiac function and ALDH2 gene mutation (p < 0.05). CONCLUSIONS: Taken together, these data suggest that ALDH2 serves as an indispensable factor against cardiac anomalies in diet-induced obesity through a mechanism related to autophagy regulation and facilitation of the SUV39H-Sirt1-dependent PGC-1α deacetylation.

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

[This corrects the article DOI: 10.1371/journal.pone.0063615.].

FasL expression in cardiomyocytes activates the ERK1/2 pathway, leading to dilated cardiomyopathy and advanced heart failure.

Increase in the apoptotic molecule Fas ligand (FasL) in serum and cardiomyocytes has been shown to be associated with progressive dilated cardiomyopathy (DCM) and congestive heart failure (CHF) in humans. However, the underlying mechanism(s) of FasL-related deterioration of heart function remain obscure. The aim of the present study is to determine roles of myocardial FasL in the activation of alternative pathways such as extracellular-signal-regulated kinase 1/2 (ERK1/2), inflammation or fibrosis and to identify effective treatments of progressive DCM and advanced CHF. Transgenic mice with cardiomyocyte-specific overexpression of FasL were investigated and treated with an ERK1/2 inhibitor (U-0126), losartan (los), prednisolone (pred) or placebo. Morpho-histological and molecular studies were subsequently performed. FasL mice showed significantly higher mortality compared with wild-type (WT) littermates due to DCM and advanced CHF. Prominent perivascular and interstitial fibrosis, increased interleukin secretion and diffuse CD3-positive cell infiltration were evident in FasL hearts. Up-regulation of the short form of Fas-associated death domain (FADD)-like interleukin 1ß-converting enzyme (FLICE) inhibitory protein (s-FLIP), RIP (receptor-interacting protein) and ERK1/2 and down-regulation of transforming growth factor beta 1 (TGFß1) and nuclear factor-κB (NF-κB) was determined in the myocardium, whereas expression of ERK1/2, periostin (Postn) and osteopontin increased in cardiac fibroblasts. U-0126 and los increased CHF survival by 75% compared with pred and placebo groups. U-0126 had both anti-fibrotic and anti-apoptotic effects, whereas los reduced fibrosis only. Myocardial FasL expression in mice activates differential robust fibrotic, apoptotic and inflammatory responses via ERK1/2 in cardiomyocytes and cardiac fibroblasts inducing DCM and CHF. Blocking the ERK1/2 pathway prevented progression of FasL-induced DCM and CHF by reducing fibrosis, inflammation and apoptosis in the myocardium.

Accelerated cardiac remodeling in desmoplakin transgenic mice in response to endurance exercise is associated with perturbed Wnt/ß-catenin signaling.

Arrhythmogenic ventricular cardiomyopathy (AVC) is a frequent underlying cause for arrhythmias and sudden cardiac death especially during intense exercise. The mechanisms involved remain largely unknown. The purpose of this study was to investigate how chronic endurance exercise contributes to desmoplakin (DSP) mutation-induced AVC pathogenesis. Transgenic mice with overexpression of desmoplakin, wild-type (Tg-DSP(WT)), or the R2834H mutant (Tg-DSP(R2834H)) along with control nontransgenic (NTg) littermates were kept sedentary or exposed to a daily running regimen for 12 wk. Cardiac function and morphology were analyzed using echocardiography, electrocardiography, histology, immunohistochemistry, RNA, and protein analysis. At baseline, 4-wk-old mice from all groups displayed normal cardiac function. When subjected to exercise, all mice retained normal cardiac function and left ventricular morphology; however, Tg-DSP(R2834H) mutants displayed right ventricular (RV) dilation and wall thinning, unlike NTg and Tg-DSP(WT). The Tg-DSP(R2834H) hearts demonstrated focal fat infiltrations in RV and cytoplasmic aggregations consisting of desmoplakin, plakoglobin, and connexin 43. These aggregates coincided with disruption of the intercalated disks, intermediate filaments, and microtubules. Although Tg-DSP(R2834H) mice already displayed high levels of p-GSK3-ß(Ser9) and p-AKT1(Ser473) under sedentary conditions, decrease of nuclear GSK3-ß and AKT1 levels with reduced p-GSK3-ß(Ser9), p-AKT1(Ser473), and p-AKT1(Ser308) and loss of nuclear junctional plakoglobin was apparent after exercise. In contrast, Tg-DSP(WT) showed upregulation of p-AKT1(Ser473), p-AKT1(Ser308), and p-GSK3-ß(Ser9) in response to exercise. Our data suggest that endurance exercise accelerates AVC pathogenesis in Tg-DSP(R2834H) mice and this event is associated with perturbed AKT1 and GSK3-ß signaling. Our study suggests a potential mechanism-based approach to exercise management in patients with AVC.

Adiponectin is required for cardiac MEF2 activation during pressure overload induced hypertrophy.

Cardiomyocyte (CM) hypertrophy and increased heart mass in response to pressure overload are associated with hyper-activation of the myocyte enhancer factor-2 (MEF2) family of transcriptional regulators, and concomitant initiation of the fetal gene program. Adiponectin, an adipokine that is reduced in individuals with obesity and diabetes, has been characterized both as a negative regulator or permissive factor in cardiac hypertrophy. We therefore sought to analyze temporal regulation of MEF2 activity in response to pressure overload (PO) and changes in adiponectin status. To address this we crossed a well characterized transgenic MEF2 "sensor" mouse (MEF2-lacZ) with adiponectin null mice (Ad-KO) to create compound MEF2 lacZ/Ad-KO mice. Initially, we established that transverse aortic banding induced PO in wild-type (WT) mice increased heart mass and CM hypertrophy from 1 to 4weeks following surgery, indicated by increased CM diameter and heart weight/tibia length ratio. This was associated with cardiac dysfunction determined by echocardiography. Hypertrophic changes and dysfunction were observed in Ad-KO mice 4weeks following surgery. MEF2 lacZ activity and endogenous ANF mRNA levels, used as indicators of hypertrophic gene activation, were both robustly increased in WT mice after MTAB but attenuated in the Ad-KO background. Furthermore, activation of the pro-hypertrophic molecule p38 was increased following MTAB surgery in WT mice, but not in Ad-KO animals, and treatment of primary isolated CM with recombinant adiponectin induced p38 phosphorylation in a time dependent manner. Adiponectin also increased MEF2 activation in primary cardiomyocytes, an effect attenuated by p38 MAPK inhibition. In conclusion, our data indicate that robust hypertrophic MEF2 activation in the heart in vivo requires a background of adiponectin signaling and that adiponectin signaling in primary isolated CM directly enhances MEF2 activity through activation of p38 MAPK. We conclude that adiponectin is required for full induction of cardiomyocyte MEF2 activation, thus contributing to the myocardial hypertrophic gene expression program in response to PO.

Pressure Overload-Induced Cardiac Dysfunction in Aged Male Adiponectin Knockout Mice Is Associated With Autophagy Deficiency.

Heart failure is a leading cause of death, especially in the elderly or obese and diabetic populations. Various remodeling events have been characterized, which collectively contribute to the progression of heart failure. Of particular interest, autophagy has recently emerged as an important determinant of cardiac remodeling and function. Here, we used aged, 13-month-old, male adiponectin knockout (Ad-KO) or wild-type (wt) mice subjected to aortic banding to induce pressure overload (PO). Cardiac strain analysis using speckle tracking echocardiography indicated significant dysfunction at an earlier stage in Ad-KO than wt. Analysis of autophagy by Western blotting for Light Chain 3 or microtubule-associated proteins 1B and Sequestosome 1 together with transmission electron microscopy of left ventricular tissue indicated a lack of PO-induced cardiac autophagy in Ad-KO compared with wt mice. Associated with this was mitochondrial degeneration and evidence of enhanced endoplasmic reticulum stress. Western blotting for Light Chain 3 or microtubule-associated proteins 1B, examination of flux using tandem fluoresent tagged-Light Chain 3, and analysis of lysosomal activity in H9c2 cardiac myoblasts treated with adiponectin indicated that adiponectin enhanced autophagy flux. In conclusion, adiponectin directly stimulates autophagic flux and the lack of autophagy in response to PO in aged mice lacking adiponectin may contribute to cellular events which exacerbate the development of cardiac dysfunction.

Temporal and Molecular Analyses of Cardiac Extracellular Matrix Remodeling following Pressure Overload in Adiponectin Deficient Mice.

Adiponectin, circulating levels of which are reduced in obesity and diabetes, mediates cardiac extracellular matrix (ECM) remodeling in response to pressure overload (PO). Here, we performed a detailed temporal analysis of progressive cardiac ECM remodelling in adiponectin knockout (AdKO) and wild-type (WT) mice at 3 days and 1, 2, 3 and 4 weeks following the induction of mild PO via minimally invasive transverse aortic banding. We first observed that myocardial adiponectin gene expression was reduced after 4 weeks of PO, whereas increased adiponectin levels were detected in cardiac homogenates at this time despite decreased circulating levels of adiponectin. Scanning electron microscopy and Masson's trichrome staining showed collagen accumulation increased in response to 2 and 4 weeks of PO in WT mice, while fibrosis in AdKO mice was notably absent after 2 weeks but highly apparent after 4 weeks of PO. Time and intensity of fibroblast appearance after PO was not significantly different between AdKO and WT animals. Gene array analysis indicated that MMP2, TIMP2, collagen 1α1 and collagen 1α3 were induced after 2 weeks of PO in WT but not AdKO mice. After 4 weeks MMP8 was induced in both genotypes, MMP9 only in WT mice and MMP1α only in AdKO mice. Direct stimulation of primary cardiac fibroblasts with adiponectin induced a transient increase in total collagen detected by picrosirius red staining and collagen III levels synthesis, as well as enhanced MMP2 activity detected via gelatin zymography. Adiponectin also enhanced fibroblast migration and attenuated angiotensin-II induced differentiation to a myofibroblast phenotype. In conclusion, these data indicate that increased myocardial bioavailability of adiponectin mediates ECM remodeling following PO and that adiponectin deficiency delays these effects.

17-ß estradiol attenuates ovariectomy-induced changes in cardiomyocyte contractile function via activation of AMP-activated protein kinase.

Menopause increases the risk of cardiometabolic diseases in women. This circumstance is usually attributed to a deficiency in circulating estrogen levels although the underlying mechanism remains elusive. Given the pivotal role of AMP-activated protein kinase (AMPK) in the regulation of energy metabolism and cardiac function, this study was designed to examine the role of AMPK in estrogen deficiency and replacement-exerted cardiomyocyte responses. Adult female WT and AMPK kinase dead (KD) mice were subjected to bilateral ovariectomy (OVX) or sham operation. A cohort of ovariectomized mice received 17ß-estradiol (E2) (40µg/kg/day, i.p.) for 6 weeks. Mechanical and intracellular Ca(2+) properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-90%-relengthening (TR90), and maximal velocity of shortening/relengthening (±dL/dt). Levels of AMPK, Akt JNK, ACC, SERCA, membrane Glut4, AS160 and PGC-1α were assessed using Western blot. OVX significantly decreased PS, ±dL/dt and intracellular Ca(2+) rise in responsible to electric stimulus, prolonged TR90 and intracellular Ca(2+) decay without affecting TPS and resting intracellular Ca(2+), the effects of which were reconciled by E2 replacement. Western blot analysis depicted that OVX suppressed phosphorylation of Akt AMPK and ACC although it promoted JNK phosphorylation, the effects of which were mitigated or significantly attenuated by E2 treatment in WT but not KD mice. Moreover, OVX procedure downregulated SERCA2a and membrane Glut4 while inhibiting AS160 phosphorylation without affecting PGC-1α levels. In vitro study revealed that E2 corrected cardiomyocyte contractile dysfunction elicited by OVX in cardiomyocytes from WT but not the AMPK kinase dead mice. Taken together, these data suggest that E2 treatment ameliorates estrogen deficiency-induced changes in cardiac contractile function possibly through an AMPK-dependent mechanism.

Palmitate induces ER stress and autophagy in H9c2 cells: implications for apoptosis and adiponectin resistance.

The association between obesity and heart failure is well documented and recent studies have indicated that understanding the physiological role of autophagy will be of great significance. Cardiomyocyte apoptosis is one component of cardiac remodeling which leads to heart failure and in this study we used palmitate-treated H9c2 cells as an in vitro model of lipotoxicity to investigate the role of autophagy in cell death. Temporal analysis revealed that palmitate (100 µM) treatment induced a gradual increase of intracellular lipid accumulation as well as apoptotic cell death. Palmitate induced autophagic flux, determined via increased LC3-II formation and p62 degradation as well as by detecting reduced colocalization of GFP with RFP in cells overexpressing tandem fluorescent GFP/RFP-LC3. The increased level of autophagy indicated by these measures were confirmed using transmission electron microscopy (TEM). Upon inhibiting autophagy using bafilomycin we observed an increased level of palmitate-induced cell death assessed by Annexin V/PI staining, detection of active caspase-3 and MTT cell viability assay. Interestingly, using TEM and p-PERK or p-eIF2α detection we observed increased endoplasmic reticulum (ER) stress in response to palmitate. Autophagy was induced as an adaptive response against ER stress since it was sensitive to ER stress inhibition. Palmitate-induced ER stress also induced adiponectin resistance, assessed via AMPK phosphorylation, via reducing APPL1 expression. This effect was independent of palmitate-induced autophagy. In summary, our data indicate that palmitate induces autophagy subsequent to ER stress and that this confers a prosurvival effect against lipotoxicity-induced cell death. Palmitate-induced ER stress also led to adiponecin resistance.

Adiponectin stimulates Rho-mediated actin cytoskeleton remodeling and glucose uptake via APPL1 in primary cardiomyocytes.

OBJECTIVE: Adiponectin is known to confer its cardioprotective effects in obesity and type 2 diabetes, mainly by regulating glucose and fatty acid metabolism in cardiomyocytes. Dynamic actin cytoskeleton remodeling is involved in regulation of multiple biological functions, including glucose uptake. Here we investigated in neonatal cardiomyocytes whether adiponectin induced actin cytoskeleton remodeling and if this played a role in adiponectin-stimulated glucose uptake. MATERIALS/METHODS: Primary cardiomyocytes were treated with full-length and globular adiponectin (fAd and gAd, respectively). RESULTS: Both fAd and gAd increased RhoA activity, phosphorylation of the Rho/ROCK signaling target cofilin and actin polymerization to form filamentous actin as determined by rhodamine-phallodin immunofluorescence and quantitative analysis of filamentous to globular actin ratio. Scanning electron microscopy also demonstrated structural remodeling. Adiponectin stimulated glucose uptake, was significantly abrogated in the presence of inhibitors of actin cytoskeleton remodeling (cytochalasin D) and Rho/ROCK signaling (C3 transferase, Y27632). We showed that adiponectin increased colocalization of actin and APPL1 and that actin remodeling, phosphorylation of AMPK, p38MAPK and cofilin, glucose uptake and oxidation were all attenuated after siRNA-mediated knockdown of APPL1. CONCLUSION: We show that adiponectin mediates Rho/ROCK-dependent actin cytoskeleton remodeling to increase glucose uptake and metabolism via APPL1 signaling.

Influence of gestational overfeeding on myocardial proinflammatory mediators in fetal sheep heart.

Maternal overnutrition is associated with predisposition of offspring to cardiovascular disease in later life. Since maternal overnutrition may promote fetal and placental inflammatory responses, we hypothesized that maternal overnutrition/obesity increases expression of fetal cardiac proinflammatory mediators and alter cardiac morphometry. Multiparous ewes were fed either 150% of National Research Council (NRC) nutrient recommendations (overfed) or 100% of NRC requirement (control) from 60 days prior to mating to gestation Day 75 (D75), when ewes were euthanized. An additional cohort of overfed and control ewes were necropsied on D135. Cardiac morphometry, histology, mRNA and protein expression of toll-like receptor 4, iNOS, IL-1a, IL-1b, IL-6, IL-18, CD-14, CD-68, M-CSF and protein levels of phosphorylated I-κB and nuclear factor κB (NF-κB) were examined. Immunohistochemistry was performed to assess neutrophil and monocyte infiltration. Crown rump length, left and right ventricular free wall weights as well as left and right ventricular wall thickness were significantly increased in D75 fetuses of overfed mothers. Hematoxylin and eosin staining revealed irregular myofiber orientation and increased interstitial space in fetal ventricular tissues born to overfed mothers. Oil red O staining exhibited marked lipid droplet accumulation in the overfed fetuses. Overfeeding significantly enhanced TLR4, IL-1a, IL-1b IL-6 expression, promoted phosphorylation of IκB, decreased cytoplasmic NF-κB levels and increased neutrophil and monocyte infiltration. Collectively, these data suggest that maternal overfeeding prior to and throughout gestation leads to inflammation in the fetal heart and alters fetal cardiac morphometry.

Inhibition of DNA methylation attenuates low-dose cadmium-induced cardiac contractile and intracellular Ca(2+) anomalies.

(1) Cadmium is a human carcinogen with unfavourable health impacts probably associated with its DNA methylation property. Recent data suggest that environmental cadmium exposure is associated with the incidence of myocardial infarction and peripheral arterial disease. Nonetheless, the effect of chronic cadmium exposure on cardiac contractile function remains unknown. (2) The present study was designed to examine the impact of low-dose cadmium exposure on cardiac contractile function and intracellular Ca2+ homeostasis. Adult male mice were exposed to cadmium for 4 weeks (20 nmol/kg, i.p. every other day for 4 weeks) with or without the DNA methylation inhibitor 5-aza-2'-deoxyctidene (5-AZA; 0.25 mg/kg, i.p., twice a week for 6 weeks, starting at the same time as cadmium administration). Cardiac contractile and intracellular Ca2+ properties were analysed, including echocardiographic left ventricular parameters, fractional shortening (FS), peak shortening (PS) amplitude, maximal velocity of shortening/relengthening (±dL/dt), time to PS (TPS), time to 90% relengthening (TR90 ), electrically stimulated increases in intracellular Ca2+ and intracellular Ca2+ decay. (3) Cadmium exposure depressed FS, PS, ±dL/dt and electrically stimulated increases in intracellular Ca2+ without affecting TPS, TR90 , intracellular Ca2+ levels or the decay rate. The effects of cadmium were significantly attenuated (PS) or blocked altogether (all other parameters) by 5-AZA. Cadmium exposure led to overt interstitial fibrosis (collagen deposition), which was mitigated by 5-AZA treatment. Western blot analysis revealed that cadmium exposure and/or 5-AZA treatment had no effect on the expression of intercellular adhesion molecule-1, tumour necrosis factor-α and cleaved caspase 3, suggesting a relatively minor role of proinflammatory cytokines and apoptosis in the cardiac responses to cadmium and 5-AZA. (4) Together, our data demonstrate, for the first time, direct cardiac depressant effects following cadmium exposure, which may be rescued by inhibition of DNA methylation.