Erratum: Author correction to 'Ablation of Akt2 and AMPKα2 rescues high fat diet-induced obesity and hepatic steatosis through Parkin-mediated mitophagy' [Acta Pharmaceutica Sinica B 11 (2021) 3508-3526].

[This corrects the article DOI: 10.1016/j.apsb.2021.07.006.].

Ablation of Akt2 and AMPKα2 rescues high fat diet-induced obesity and hepatic steatosis through Parkin-mediated mitophagy.

Given the opposing effects of Akt and AMP-activated protein kinase (AMPK) on metabolic homeostasis, this study examined the effects of deletion of Akt2 and AMPKα2 on fat diet-induced hepatic steatosis. Akt2-Ampkα2 double knockout (DKO) mice were placed on high fat diet for 5 months. Glucose metabolism, energy homeostasis, cardiac function, lipid accumulation, and hepatic steatosis were examined. DKO mice were lean without anthropometric defects. High fat intake led to adiposity and decreased respiratory exchange ratio (RER) in wild-type (WT) mice, which were ablated in DKO but not Akt2 -/- and Ampkα2 -/- mice. High fat intake increased blood and hepatic triglycerides and cholesterol, promoted hepatic steatosis and injury in WT mice. These effects were eliminated in DKO but not Akt2 -/- and Ampkα2 -/- mice. Fat diet promoted fat accumulation, and enlarged adipocyte size, the effect was negated in DKO mice. Fat intake elevated fatty acid synthase (FAS), carbohydrate-responsive element-binding protein (CHREBP), sterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), peroxisome proliferator-activated receptor-α (PPARα), PPARγ, stearoyl-CoA desaturase 1 (SCD-1), phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6Pase), and diglyceride O-acyltransferase 1 (DGAT1), the effect was absent in DKO but not Akt2 -/- and Ampkα2 -/- mice. Fat diet dampened mitophagy, promoted inflammation and phosphorylation of forkhead box protein O1 (FoxO1) and AMPKα1 (Ser485), the effects were eradicated by DKO. Deletion of Parkin effectively nullified DKO-induced metabolic benefits against high fat intake. Liver samples from obese humans displayed lowered microtubule-associated proteins 1A/1B light chain 3B (LC3B), Pink1, Parkin, as well as enhanced phosphorylation of Akt, AMPK (Ser485), and FoxO1, which were consolidated by RNA sequencing (RNAseq) and mass spectrometry analyses from rodent and human livers. These data suggest that concurrent deletion of Akt2 and AMPKα2 offers resilience to fat diet-induced obesity and hepatic steatosis, possibly through preservation of Parkin-mediated mitophagy and lipid metabolism.

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.

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.

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 (ETA) ablation on obesity-induced changes in cardiac geometry and contractile function, as well as the mechanisms involved with a focus on autophagy. Cardiomyocyte-specific ETA 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 ETA receptor antagonist BQ123 or mTOR inhibitor rapamycin. These results suggest that inhibition of ETA rescues high-fat intake-induced cardiac anomalies possibly through autophagy regulation.

Spectroscopic and biological activity studies of the chromium-binding peptide EEEEGDD.

While trivalent chromium has been shown at high doses to have pharmacological effects improving insulin resistance in rodent models of insulin resistance, the mechanism of action of chromium at a molecular level is not known. The chromium-binding and transport agent low-molecular-weight chromium-binding substance (LMWCr) has been proposed to be the biologically active form of chromium. LMWCr has recently been shown to be comprised of a heptapeptide of the sequence EEEEDGG. The binding of Cr(3+) to this heptapeptide has been examined. Mass spectrometric and a variety of spectroscopic studies have shown that multiple chromic ions bind to the peptide in an octahedral fashion through carboxylate groups and potentially small anionic ligands such as oxide and hydroxide. A complex of Cr and the peptide when administered intravenously to mice is able to decrease area under the curve in intravenous glucose tolerance tests. It can also restore insulin-stimulated glucose uptake in myotubes rendered insulin resistant by treating them with a high-glucose media.

Deletion of protein tyrosine phosphatase 1B rescues against myocardial anomalies in high fat diet-induced obesity: Role of AMPK-dependent autophagy.

Obesity-induced cardiomyopathy may be mediated by alterations in multiple signaling cascades involved in glucose and lipid metabolism. Protein tyrosine phosphatase-1B (PTP1B) is an important negative regulator of insulin signaling. This study was designed to evaluate the role of PTP1B in high fat diet-induced cardiac contractile anomalies. Wild-type and PTP1B knockout mice were fed normal (10%) or high (45%) fat diet for 5months prior to evaluation of cardiac function. Myocardial function was assessed using echocardiography and an Ion-Optix MyoCam system. Western blot analysis was employed to evaluate levels of AMPK, mTOR, raptor, Beclin-1, p62 and LC3-II. RT-PCR technique was employed to assess genes involved in hypertrophy and lipid metabolism. Our data revealed increased LV thickness and LV chamber size as well as decreased fractional shortening following high fat diet intake, the effect was nullified by PTP1B knockout. High fat diet intake compromised cardiomyocyte contractile function as evidenced by decreased peak shortening, maximal velocity of shortening/relengthening, intracellular Ca²âº release as well as prolonged duration of relengthening and intracellular Ca²âº decay, the effects of which were alleviated by PTP1B knockout. High fat diet resulted in enlarged cardiomyocyte area and increased lipid accumulation, which were attenuated by PTP1B knockout. High fat diet intake dampened myocardial autophagy as evidenced by decreased LC3-II conversion and Beclin-1, increased p62 levels as well as decreased phosphorylation of AMPK and raptor, the effects of which were significantly alleviated by PTP1B knockout. Pharmacological inhibition of AMPK using compound C disengaged PTP1B knockout-conferred protection against fatty acid-induced cardiomyocyte contractile anomalies. Taken together, our results suggest that PTP1B knockout offers cardioprotection against high fat diet intake through activation of AMPK. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Targeted deletion of PTEN in cardiomyocytes renders cardiac contractile dysfunction through interruption of Pink1-AMPK signaling and autophagy.

Phosphatase and tensin homolog (PTEN) deleted from chromosome 10 has been implicated in the maintenance of cardiac homeostasis although the underlying mechanism(s) remains elusive. We generated a murine model of cardiomyocyte-specific knockout of PTEN to evaluate cardiac geometry and contractile function, as well as the effect of metformin on PTEN deficiency-induced cardiac anomalies, if any. Cardiac histology, autophagy and related signaling molecules were evaluated. Cardiomyocyte-specific PTEN deletion elicited cardiac hypertrophy and contractile anomalies (echocardiographic and cardiomyocyte contractile dysfunction) associated with compromised intracellular Ca(2+) handling. PTEN deletion-induced cardiac hypertrophy and contractile anomalies were associated with dampened phosphorylation of PTEN-inducible kinase 1 (Pink1) and AMPK. Interestingly, administration of AMPK activator metformin (200mg/kg/d, in drinking H2O for 4weeks) rescued against PTEN deletion-induced geometric and functional defects as well as interrupted autophagy and autophagic flux in the heart. Moreover, metformin administration partially although significantly attenuated PTEN deletion-induced accumulation of superoxide. RNA interference against Pink1 in H9C2 myoblasts overtly increased intracellular ATP levels and suppressed AMPK phosphorylation, confirming the role of AMPK as a downstream target for PTEN-Pink1. Further scrutiny revealed that activation of AMPK and autophagy using metformin and rapamycin, respectively, rescued against PTEN deletion-induced mechanical anomalies with little additive effect. These data demonstrated that cardiomyocyte-specific deletion of PTEN leads to the loss of Pink1-AMPK signaling, development of cardiac hypertrophy and contractile defect. Activation of AMPK rescued against PTEN deletion-induced cardiac anomalies associated with restoration of autophagy and autophagic flux. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Autophagy inhibition rescues against leptin-induced cardiac contractile dysfunction.

Leptin hormone plays a vital role in the pathophysiological changes in heart geometry and function. Nonetheless, the precise mechanism(s) triggering leptin-induced cardiomyocyte contractile dysfunction is not well understood. The present study was designed to examine if autophagy plays a role in leptin-induced cardiac contractile anomalies. Cardiomyocyte contractile function was evaluated using an IonOptix edge detection system in cardiomyocytes following treatment with leptin in the presence or absence of the autophagy inhibiting chemical 3-methyladenine (3-MA). Immunoblotting was employed to evaluate expression of AMPK, Beclin1, Atg 5, p62 and LC3-II. GFP-LC3 puncta was used to assess autophagosome formation. Leptin suppressed cardiac contractile function as evidenced by decreased peak shortening, maximal velocity of shortening and relengthening, increased time-to-90% relengthening, all the observed effects were reduced or obliterated by autophagy inhibition. Leptin promoted superoxide generation, AMPK activation and overt autophagy induction. Leptin promoted autophagy as evidenced by enhanced LC3-II, Beclin, Atg 5 and decreased p62 levels. Pharmacological inhibition of reactive oxygen species (ROS) using tempol significantly attenuated leptin-induced autophagosome formation and cardiac contractile anomalies. In addition, genetic deletion of AMPKα2 or pharmacological inhibition of AMPK using compound C abrogated leptin or superoxide induced cardiac contractile dysfunction and autophagosome formation. In summary, our data revealed that leptin impairs cardiac contractile function through a superoxide generation-AMPK activation-and autophagy dependent mechanism.

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.

Apelin administration ameliorates high fat diet-induced cardiac hypertrophy and contractile dysfunction.

Apelin has been recognized as an adipokine that plays an important role in regulating energy metabolism and is credited with antiobesity and antidiabetic properties. This study was designed to examine the effect of exogenous apelin on obesity-associated cardiac dysfunction. Oral glucose tolerance test, echocardiography, cardiomyocyte contractile and intracellular Ca(2+) properties were assessed in adult C57BL/6J mice fed - low or a - high-fat diet for 24weeks followed by apelin treatment (100nmol/kg, i.p. for 2weeks). High-fat diet resulted in increased left ventricular diastolic and systolic diameters, and wall thickness, compromised fractional shortening, impaired cardiomyocyte mechanics (peak-shortening, maximal velocity of shortening/relengthening, and duration of shortening and relengthening) and compromised intracellular Ca(2+) handling, all of which were reconciled by apelin. Apelin treatment also reversed high fat diet-induced changes in intracellular Ca(2+) regulatory proteins, ER stress, and autophagy. In addition, microRNAs (miR) -133a, miR-208 and miR-1 which were elevated following high-fat feeding were attenuated by apelin treatment. In cultured cardiomyocytes apelin reconciled palmitic acid-induced cardiomyocyte contractile anomalies. Collectively, these data depict a pivotal role of apelin in obesity-associated cardiac contractile dysfunction, suggesting a therapeutic potential of apelin in the management of cardiac dysfunction associated with obesity.

ULK1 plays a critical role in AMPK-mediated myocardial autophagy and contractile dysfunction following acute alcohol challenge.

This study was designed to evaluate the role of ULK1 in AMPK-mediated myocardial autophagy and contractile dysfunction following acute alcohol challenge. Wild-type and AMPK knockout mice were challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial function was evaluated using echocardiography and edge-detection. Western blot analysis was employed to evaluate the levels of AMPK, Raptor, mTOR, the AMPK downstream signal ULK1 and autophagy markers Beclin-1 and LC3-II. siRNA was used to knockdown ULK1 in H9C2 myoblasts. GFP-LC3 puncta was used to evaluate autophagosome formation. Alcohol challenge compromised cardiac function as evidenced by decreased fractional shortening, peak shortening and intracellular Ca²âº rise, prolonged relengthening and intracellular Ca²âº decay in WT mice, the effects of which were mitigated by AMPK knockout. Ethanol exposure facilitated myocardial autophagy as evidenced by enhanced LC3-II level, as well as phosphorylation of AMPK, Raptor, and dephosphorylation of mTOR and ULKI in WT hearts, which were alleviated by AMPK knockout. Pharmacological inhibition of AMPK using compound C attenuated ethanol-induced autophagosome formation, AMPK phosphorylation, ULK1 dephosphorylation and apoptosis. Ethanol exposure-induced cardiomyocyte contractile defects and autophagosome accumulation were reversed by the autophagy inhibitor 3-MA. Similarly, knockdown of ULK1 using siRNA in H9C2 cells ablated ethanol-induced autophagosome accumulation, LC3-II expression and cell death. Lysosomal inhibition using bafilomycin, E64-D and pepstatin A potentiated ethanol-induced increase in autophagosome formation. Taken together, our results suggest that ULK1 may play a critical role in AMPK-mediated myocardial autophagy, apoptosis and contractile dysfunction following acute alcohol challenge.

Chronic Akt activation attenuated lipopolysaccharide-induced cardiac dysfunction via Akt/GSK3ß-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 Ca²âº 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-1ß, 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 Ca²âº 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 Ca²âº 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 GSK3ß. In vitro study using the GSK3ß 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 Ca²âº anomalies through inhibition of apoptosis and ER stress, possibly involving an Akt/GSK3ß-dependent mechanism.

Cardiac-specific catalase overexpression rescues anthrax lethal toxin-induced cardiac contractile dysfunction: role of oxidative stress and autophagy.

BACKGROUND: Lethal and edema toxins secreted by Bacillus anthracis during anthrax infection were found to incite serious cardiovascular complications. However, the underlying mechanisms in anthrax lethal toxin-induced cardiac anomalies remain unknown. This study was designed to evaluate the impact of antioxidant enzyme catalase in anthrax lethal toxin-induced cardiomyocyte contractile dysfunction. METHODS: Wild type (WT) and cardiac-specific catalase overexpression mice were challenged with lethal toxin (2 µg/g, intraperotineally (i.p.)). Cardiomyocyte contractile and intracellular Ca(2+) properties were assessed 18 h later using an IonOptix edge-detection system. Proteasome function was assessed using chymotrypsin-like and caspase-like activities. GFP-LC3 puncta and Western blot analysis were used to evaluate autophagy and protein ubiquitination. RESULTS: Lethal toxin exposure suppressed cardiomyocyte contractile function (suppressed peak shortening, maximal velocity of shortening/re-lengthening, prolonged duration of shortening/re-lengthening, and impaired intracellular Ca(2+) handling), the effects of which were alleviated by catalase. In addition, lethal toxin triggered autophagy, mitochondrial and ubiquitin-proteasome defects, the effects of which were mitigated by catalase. Pretreatment of cardiomyocytes from catalase mice with the autophagy inducer rapamycin significantly attenuated or ablated catalase-offered protection against lethal toxin-induced cardiomyocyte dysfunction. On the other hand, the autophagy inhibitor 3-MA ablated or significantly attenuated lethal toxin-induced cardiomyocyte contractile anomalies. CONCLUSIONS: Our results suggest that catalase is protective against anthrax lethal toxin-induced cardiomyocyte contractile and intracellular Ca(2+) anomalies, possibly through regulation of autophagy and mitochondrial function.

Ca+2/calmodulin-dependent protein kinase mediates glucose toxicity-induced cardiomyocyte contractile dysfunction.

(1) Hyperglycemia leads to cytotoxicity in the heart. Although several theories are postulated for glucose toxicity-induced cardiomyocyte dysfunction, the precise mechanism still remains unclear. (2) This study was designed to evaluate the impact of elevated extracellular Ca(2+) on glucose toxicity-induced cardiac contractile and intracellular Ca(2+) anomalies as well as the mechanism(s) involved with a focus on Ca(2+)/calmodulin (CaM)-dependent kinase. Isolated adult rat cardiomyocytes were maintained in normal (NG, 5.5 mM) or high glucose (HG, 25.5 mM) media for 6-12 hours. Contractile indices were measured including peak shortening (PS), maximal velocity of shortening/relengthening (±dL/dt), time-to-PS (TPS), and time-to-90% relengthening (TR(90)). (3) Cardiomyocytes maintained with HG displayed abnormal mechanical function including reduced PS, ±dL/dt, and prolonged TPS, TR(90) and intracellular Ca(2+) clearance. Expression of intracellular Ca(2+) regulatory proteins including SERCA2a, phospholamban and Na(+)-Ca(2+) exchanger were unaffected whereas SERCA activity was inhibited by HG. Interestingly, the HG-induced mechanical anomalies were abolished by elevated extracellular Ca(2+) (from 1.0 to 2.7 mM). Interestingly, the high extracellular Ca(2+)-induced beneficial effect against HG was abolished by the CaM kinase inhibitor KN93. (4) These data suggest that elevated extracellular Ca(2+) protects against glucose toxicity-induced cardiomyocyte contractile defects through a mechanism associated with CaM kinase.

Toll-like receptor 4 knockout protects against anthrax lethal toxin-induced cardiac contractile dysfunction: role of autophagy.

BACKGROUND AND PURPOSE: Anthrax lethal toxin (LeTx) is known to induce circulatory shock and death, although the underlying mechanisms have not been elucidated. This study was designed to evaluate the role of toll-like receptor 4 (TLR4) in anthrax lethal toxin-induced cardiac contractile dysfunction. EXPERIMENTAL APPROACH: Wild-type (WT) and TLR4 knockout (TLR⁻/⁻) mice were challenged with lethal toxin (2 µg·g⁻¹, i.p.), and cardiac function was assessed 18 h later using echocardiography and edge detection. Small interfering RNA (siRNA) was employed to knockdown TLR4 receptor or class III PI3K in H9C2 myoblasts. GFP-LC3 puncta was used to assess autophagosome formation. Western blot analysis was performed to evaluate autophagy (LC3, Becline-1, Agt5 and Agt7) and endoplasmic reticulum (ER) stress (BiP, eIF2α and calreticulin). KEY RESULTS: In WT mice, lethal toxin exposure induced cardiac contractile dysfunction, as evidenced by reduced fractional shortening, peak shortening, maximal velocity of shortening/re-lengthening, prolonged re-lengthening duration and intracellular Ca²âº derangement. These effects were significantly attenuated or absent in the TLR4 knockout mice. In addition, lethal toxin elicited autophagy in the absence of change in ER stress. Knockdown of TLR4 or class III PI3 kinase using siRNA but not the autophagy inhibitor 3-methyladenine significantly attenuated or inhibited lethal toxin-induced autophagy in H9C2 cells. CONCLUSION AND IMPLICATIONS: Our results suggest that TLR4 may be pivotal in mediating the lethal cardiac toxicity induced by anthrax possibly through induction of autophagy. These findings suggest that compounds that negatively modulate TLR4 signalling and autophagy could be used to treat anthrax infection-induced cardiovascular complications.

Facilitated ethanol metabolism promotes cardiomyocyte contractile dysfunction through autophagy in murine hearts.

Chronic drinking leads to myocardial contractile dysfunction where ethanol metabolism plays an essential role. Acetaldehyde, the main ethanol metabolite, mediates alcohol-induced cell injury although the underlying mechanism is still elusive. This study was designed to examine the mechanism involved in accelerated ethanol metabolism-induced cardiac defect with a focus on autophagy. Wild-type FVB and cardiac-specific overexpression of alcohol dehydrogenase mice were placed on a 4% nutrition-balanced alcohol diet for 8 weeks. Myocardial histology, immunohistochemistry, autophagy markers and signal molecules were examined. Expression of micro RNA miR-30a, a potential target of Beclin 1, was evaluated by real-time PCR. Chronic alcohol intake led to cardiac acetaldehyde accumulation, hypertrophy and overt autophagosome accumulation (LC3-II and Atg7), the effect of which was accentuated by ADH. Signaling molecules governing autophagy initiation including class III PtdIns3K, phosphorylation of mTOR and p70S6K were enhanced and dampened, respectively, following alcohol intake. These alcohol-induced signaling responses were augmented by ADH. ADH accentuated or unmasked alcohol-induced downregulation of Bcl-2, Bcl-xL and MiR-30a. Interestingly, ADH aggravated alcohol-induced p62 accumulation. Autophagy inhibition using 3-MA abolished alcohol-induced cardiomyocyte contractile anomalies. Moreover, acetaldehyde led to cardiomyocyte contractile dysfunction and autophagy induction, which was ablated by 3-MA. Ethanol or acetaldehyde increased GFP-LC3 puncta in H9c2 cells, the effect of which was ablated by 3-MA but unaffected by lysosomal inhibition using bafilomycin A(1), E64D and pepstatin A. In summary, these data suggested that facilitated acetaldehyde production via ADH following alcohol intake triggered cardiac autophagosome formation along with impaired lysosomal degradation, en route to myocardial defect.

Deficiency in AMP-activated protein kinase exaggerates high fat diet-induced cardiac hypertrophy and contractile dysfunction.

AMPK, a metabolic sensor, protects against ischemic injury and cardiac hypertrophy although its role in obesity is unclear. This study was designed to examine the impact of AMPK deficiency on cardiac dysfunction following high fat feeding. Adult WT and transgenic mice overexpressing a kinase dead (KD) α2 isoform (K45R mutation) of AMPK were fed a low or high fat diet for 20 weeks. DEXA was used to confirm adiposity. Wheat germ agglutinin immunostaining was used to evaluate myocardial histology. Myocardial function was evaluated using echocardiography and edge-detection. AMPK activity was analyzed using fluorescence polarization assays. [1-(14)C] oleate was used to determine fatty acid oxidation. Expression of AMPK, α1, α2, ACC, Akt, the Glut-4 translocation mediator Akt substrate of 160KD (AS160), mTOR, total and membrane Glut-4 was evaluated using Western blot. AMPK activity was decreased in KD mice regardless of diet regimen. High fat diet led to obesity, glucose intolerance and cardiac hypertrophy with accentuated glucose intolerance, dampened fatty acid oxidation and cardiac hypertrophy in KD mice. High fat feeding triggered lower fractional shortening, increased LV mass, left ventricular end diastolic/systolic diameter, decreased PS, ± dL/dt, prolonged TR(90) and intracellular Ca(2+) mishandling with a more pronounced effect in KD mice. High fat diet and AMPK KD lessened AMPKα2 isoform activity and ACC phosphorylation. AMPK deficiency unveiled or accentuated high fat diet-induced decrease in phosphorylation of Akt and AS160, membrane fraction of Glut-4 and mTOR expression (a greater mTOR phosphorylation). Taken together, these data suggest that AMPK deficiency exacerbates obesity-induced cardiac hypertrophy and contractile dysfunction, possibly associated with AS160 and mTOR signaling.

Anthrax lethal toxin suppresses murine cardiomyocyte contractile function and intracellular Ca2+ handling via a NADPH oxidase-dependent mechanism.

OBJECTIVES: Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca(2+) properties. METHODS: Murine cardiomyocyte contractile function and intracellular Ca(2+) handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR(90)), intracellular Ca(2+) rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca(2+) decay rate. Stress signaling and Ca(2+) regulatory proteins were assessed using Western blot analysis. RESULTS: In vitro exposure to a lethal toxin (0.05-50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca(2+) properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca(2+) decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca(2+) responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca(2+) regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure. CONCLUSIONS: Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca(2+) through a NADPH oxidase-dependent mechanism.