Inhibition of AMPK accentuates prolonged caloric restriction-induced change in cardiac contractile function through disruption of compensatory autophagy.

Prolonged caloric restriction often results in alteration in heart geometry and function although the underlying mechanism remains poorly defined. Autophagy, a conserved pathway for bulk degradation of intracellular proteins and organelles, preserves energy and nutrient in the face of caloric insufficiency. This study was designed to examine the role of AMPK in prolonged caloric restriction-induced change in cardiac homeostasis and the underlying mechanism(s) involved with a focus on autophagy. Wild-type (WT) and AMPK kinase dead (KD) mice were caloric restricted (by 40%) for 30 weeks. Echocardiographic, cardiomyocyte contractile and intracellular Ca²âº properties, autophagy and autophagy regulatory proteins were evaluated. Caloric restriction compromised echocardiographic indices (decreased ventricular mass, left ventricular diameters, and cardiac output), cardiomyocyte contractile and intracellular Ca²âº properties associated with upregulated autophagy (Beclin-1, Atg5 and LC3BII-to-LC3BI ratio), increased autophagy adaptor protein p62, elevated phosphorylation of AMPK and TSC1/2, depressed phosphorylation of mTOR and ULK1. Although AMPK inhibition did not affect cardiac mechanical function, autophagy and autophagy signaling proteins, it significantly accentuated caloric restriction-induced changes in myocardial contractile function and intracellular Ca²âº handling. Interestingly, AMPK inhibition reversed caloric restriction-induced changes in autophagy and autophagy signaling. AMPK inhibition led to dampened levels of Beclin-1, Atg 5 and LC3B ratio along with suppressed phosphorylation of AMPK and TSC1/2 as well as elevated phosphorylation of mTOR and ULK1. Taken together, these data suggest an indispensible role for AMPK in the maintenance of cardiac homeostasis under prolonged caloric restriction-induced pathological changes possibly through autophagy regulation. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Akt2 knockout alleviates prolonged caloric restriction-induced change in cardiac contractile function through regulation of autophagy.

Caloric restriction leads to changes in heart geometry and function although the underlying mechanism remains elusive. Autophagy, a conserved pathway for degradation of intracellular proteins and organelles, preserves energy and nutrient in the face of caloric insufficiency. This study was designed to examine the role of Akt2 in prolonged caloric restriction-induced change in cardiac homeostasis and the underlying mechanism(s) involved. Wild-type (WT) and Akt2 knockout mice were calorie restricted (by 40%) for 30weeks. Echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, autophagy and its regulatory proteins were evaluated. Caloric restriction compromised echocardiographic indices (decreased left ventricular mass, left ventricular diameters and cardiac output), cardiomyocyte contractile and intracellular Ca(2+) properties associated with dampened SERCA2a phosphorylation, upregulated phospholamban and autophagy (Beclin-1, Atg7, LC3BII-to-LC3BI ratio), increased autophagy adaptor protein p62, elevated phosphorylation of AMPK, Akt2 and the Akt downstream signal molecule TSC2, the effects of which with the exception of autophagy protein markers (Beclin-1, Atg7, LC3B) and AMPK were mitigated or significantly alleviated by Akt2 knockout. Lysosomal inhibition using bafilomycin A1 negated Akt2 knockout-induced protective effect on p62. Evaluation of downstream signaling molecules of Akt and AMPK including mTOR and ULK1 revealed that caloric restriction suppressed and promoted phosphorylation of mTOR and ULK1, respectively, without affecting total mTOR and ULK1 expression. Akt2 knockout significantly augmented caloric restriction-induced responses on mTOR and ULK1. Taken together, these data suggest a beneficial role of Akt2 knockout in preservation of cardiac homeostasis against prolonged caloric restriction-induced pathological changes possibly through facilitating autophagy. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy."

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.

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.

Aldehyde dehydrogenase-2 transgene ameliorates chronic alcohol ingestion-induced apoptosis in cerebral cortex.

Chronic intake of alcohol results in multiple organ damage including brain. This study was designed to examine the impact of facilitated acetaldehyde breakdown via transgenic overexpression of mitochondrial aldehyde dehydrogenase-2 (ALDH2) on alcohol-induced cerebral cortical injury. ALDH2 transgenic mice were produced using the chicken beta-actin promoter. Wild-type FVB and ALDH2 mice were placed on a 4% alcohol or control diet for 12 weeks. Protein damage and apoptosis were evaluated with carbonyl formation, caspase and TUNEL assays. Western blot was performed to examine expression (or its activation) of ALDH2, the pro- and anti-apoptotic proteins caspase-8, Bax, Bcl-2, Omi/HtrA2, apoptosis repressor with caspase recruitment domain (ARC), FLICE-like inhibitory protein (FLIP), X-linked inhibitor of apoptosis protein (XIAP), Akt, glycogen synthase kinase-3beta (GSK-3beta), p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Chronic alcohol intake led to elevated apoptosis in the absence of overt protein damage, the effect of which was ablated by the overexpression of ALDH2 transgene. Consistently, ALDH2 transgene significantly attenuated alcohol-induced upregulation of Bax, Omi/HtrA2 and XIAP as well as downregulation of Bcl-2 and ARC without affecting alcohol-induced increase of FLIP in cerebral cortex. Phosphorylation of Akt and GSK-3beta was dampened while total/phosphorylated JNK and p38 phosphorylation were elevated following chronic alcohol intake, the effects of which were abrogated by ALDH2 transgene. Expression of total Akt, GSK-3beta, p38 and ERK (total or phosphorylated) was not affected by either chronic alcohol intake or ALDH2 transgene. Our results suggested that transgenic overexpression of ALDH2 rescues chronic alcoholism-elicited cerebral injury possibly via a mechanism associated with Akt, GSK-3beta, p38 and JNK signaling.

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.

RETRACTED: Cardiac-specific overexpression of catalase attenuates lipopolysaccharide-induced myocardial contractile dysfunction: Role of autophagy

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.

AMP-activated protein kinase deficiency exacerbates aging-induced myocardial contractile dysfunction.

Aging is associated with myocardial dysfunction although the underlying mechanism is unclear. AMPK, a key cellular fuel sensor for energy metabolism, is compromised with aging. This study examined the role of AMPK deficiency in aging-associated myocardial dysfunction. Young or old wild-type (WT) and transgenic mice with overexpression of a mutant AMPK alpha(2) subunit (kinase dead, KD) were used. AMPK alpha isoform activity, myocardial function and morphology were examined. DCF and JC-1 fluorescence probes were employed to quantify reactive oxygen species (ROS) and mitochondrial membrane potential (DeltaPsim), respectively. KD mice displayed significantly reduced alpha(2) but not alpha(1) AMPK isoform activity at both ages with a greater effect at old age. Aging itself decreased alpha(1) isoform activity. Cardiomyocyte contractile function, intracellular Ca(2+) handling, and SERCA2a levels were compromised with aging, the effects of which were exacerbated by AMPK deficiency. H&E staining revealed cardiomyocyte hypertrophy with aging, which was more pronounced in KD mice. TEM micrographs displayed severe disruption of mitochondrial ultrastructure characterized by swollen, irregular shape and disrupted cristae in aged KD compared with WT mice. Aging enhanced ROS production and reduced DeltaPsim, the effects of which were accentuated by AMPK deficiency. Immunoblotting data depicted unchanged Akt phosphorylation and a significant decrease in mitochondrial biogenesis cofactor PGC-1alpha in aged groups. AMPK deficiency but not aging decreased the phosphorylation of ACC and eNOS. Expression of membrane Glut4 and HSP90 was decreased in aged KD mice. Moreover, treatment of the AMPK activator metformin attenuated aging-induced cardiomyocyte contractile defects. Collectively, our data suggest a role for AMPK deficiency in aging-induced cardiac dysfunction possibly through disrupted mitochondrial function and ROS production.

Cardiomyocyte contractile dysfunction in the APPswe/PS1dE9 mouse model of Alzheimer's disease.

OBJECTIVES: Ample clinical and experimental evidence indicated that patients with Alzheimer's disease display a high incidence of cardiovascular events. This study was designed to examine myocardial histology, cardiomyocyte shortening, intracellular Ca(2+) homeostasis and regulatory proteins, electrocardiogram, adrenergic response, endoplasmic reticulum (ER) stress and protein carbonyl formation in C57 wild-type (WT) mice and an APPswe/PS1dE9 transgenic (APP/PS1) model for Alzheimer's disease. METHODS: Cardiomyocyte mechanical properties were evaluated including peak shortening (PS), time-to-PS (TPS), time-to-relengthening (TR), maximal velocity of shortening and relengthening (+/-dL/dt), intracellular Ca(2+) transient rise and decay. RESULTS: Little histological changes were observed in APP/PS1 myocardium. Cardiomyocytes from APP/PS1 but not APP or PS1 single mutation mice exhibited depressed PS, reduced+/-dL/dt, normal TPS and TR compared with WT mice(.) Rise in intracellular Ca(2+) was lower accompanied by unchanged resting/peak intracellular Ca(2+) levels and intracellular Ca(2+) decay in APP/PS1 mice. Cardiomyocytes from APP/PS1 mice exhibited a steeper decline in PS at high frequencies. The responsiveness to adrenergic agonists was dampened although beta(1)-adrenergic receptor expression was unchanged in APP/PS1 hearts. Expression of the Ca(2+) regulatory protein phospholamban and protein carbonyl formation were downregulated and elevated, respectively, associated with unchanged SERCA2a, Na(+)-Ca(2+) exchanger and ER stress markers in APP/PS1 hearts. Our further study revealed that antioxidant N-acetylcysteine attenuated the contractile dysfunction in APP/PS1 mice. CONCLUSIONS: Our results depicted overt cardiomyocyte mechanical dysfunction in the APP/PS1 Alzheimer's disease model, possibly due to oxidative stress.