Cardiac Akt Research Articles

Aberrant activation of p53-TRIB3 axis contributes to diabetic myocardial insulin resistance and sulforaphane protection

IntroductionInsulin resistance (IR) is associated with multiple pathological features. Although p53- or TRIB3-orchestrated IR is extensively studied in adipose tissue and liver, the role of p53-TRIB3 axis in myocardial IR remains unknown, and more importantly target-directed therapies of myocardial IR are missing. ObjectivesConsidering the beneficial effects of sulforaphane (SFN) on cardiovascular health, it is of particular interest to explore whether SFN protects against myocardial IR with a focus on the regulatory role of p53-TRIB3 axis. MethodsMouse models including cardiac specific p53-overexpressing transgenic (p53-cTg) mice and Trib3 knockout (Trib3-KO) mice, combined with primary cardiomyocytes treated with p53 activator (nutlin-3a) and inhibitor (pifithrin-α, PFT-α), or transfected with p53-shRNA and Trib3-shRNA, followed by multiple molecular biological methodologies, were used to investigate the role of p53-TRIB3 axis in SFN actions on myocardial IR. ResultsHere, we report that knockdown of p53 rescued cardiac insulin-stimulated AKT phosphorylation, while up-regulation of p53 by nutlin-3a or p53-cTg mice blunted insulin sensitivity in cardiomyocytes under diabetic conditions. Diabetic attenuation of AKT-mediated cardiac insulin signaling was markedly reversed by SFN in p53-Tgfl/fl mice, but not in p53-cTg mice. Importantly, we identified TRIB3 was elevated in p53-cTg diabetic mice, and confirmed the physical interaction between p53 and TRIB3. Trib3-KO diabetic mice displayed improved insulin sensitivity in the heart. More specifically, the AMPKα-triggered CHOP phosphorylation and degradation were essential for p53 on the transcriptional regulation of Trib3. ConclusionOverall, these results indicate that inhibiting the p53-TRIB3 pathway by SFN plays an unsuspected key role in the improvement of myocardial IR, which may be a promising strategy for attenuating diabetic cardiomyopathy (DCM) in diabetic patients.

The effect of electrical stimulation of skeletal muscle on cardioprotection and on muscle-derived myokine levels in rats: A pilot study.

Electrical muscle stimulation (EMS) is a widely used method in sports and rehabilitation therapies to simulate physical exercise. EMS treatment via skeletal muscle activity improves the cardiovascular functions and the overall physical condition of the patients. However, the cardioprotective effect of EMS has not been proven so far, therefore, the aim of this study was to investigate the potential cardiac conditioning effect of EMS in an animal model. Low-frequency 35-min EMS was applied to the gastrocnemius muscle of male Wistar rats for three consecutive days. Their isolated hearts were then subjected to 30 min global ischemia and 120 min reperfusion. At the end of reperfusion cardiac specific creatine kinase (CK-MB) and lactate dehydrogenase (LDH) enzyme release and myocardial infarct size were determined. Additionally, skeletal muscle-driven myokine expression and release were also assessed. Phosphorylation of cardioprotective signaling pathway members AKT, ERK1/2, and STAT3 proteins were also measured. EMS significantly attenuated cardiac LDH and CK-MB enzyme activities in the coronary effluents at the end of the ex vivo reperfusion. EMS treatment considerably altered the myokine content of the stimulated gastrocnemius muscle without altering circulating myokine levels in the serum. Additionally, phosphorylation of cardiac AKT, ERK1/2, and STAT3 was not significantly different in the two groups. Despite the lack of significant infarct size reduction, the EMS treatment seems to influence the course of cellular damage due to ischemia/reperfusion and favorably modifies skeletal muscle myokine expressions. Our results suggest that EMS may have a protective effect on the myocardium, however, further optimization is required.

Sodium butyrate and voluntary exercise through activating VEGF-A downstream signaling pathway improve heart angiogenesis in type 2 diabetes.

Inadequate angiogenesis in patients with type 2 diabetic heart could result in deprived collateral formation. Herein, we aimed to investigate the effects of sodium butyrate (NaB) along with voluntary exercise simultaneously on the mechanisms acting on cardiac angiogenesis. Animals were divided into the following five groups: control (Con), diabetic rats (Dia), diabetic rats treated with NaB (200mg/kg, i.p.) (Dia-NaB), diabetic rats receiving voluntary exercise (Dia-Exe), and diabetic rats treated with NaB and exercise simultaneously (Dia-NaB-Exe). After an eight-week duration, NO metabolites levels were measured using Griess method, the VEGF-A and VEGFR2 expressions was examined by PCR, the expressions of VEGF-A and VEGFR2 proteins was investigated by western blot, and ELISA method was used for Akt, ERK1/2 expression. Cardiac VEGF-A and VEGFR2 expressions were higher in the Dia-Exe and Dia-NaB-Exe groups compared to the Dia group. However, a combination of exercise and NaB enhanced the VEGF-A expression in cardiac tissue compared to the Dia-NaB and Dai-Exe groups. Heart NOx concentration was higher in the treated groups compared to the Dia group. The expression of cardiac Akt levels increased in both the Dia-Exe and Dia-NaB-Exe groups compared to the Dia groups. In addition, cardiac ERK1/2 expression was found to be higher in the Dia-NaB-Exe group compared to the Dia group. The findings of this study showed the therapeutic potential of a novel combination therapy of sodium butyrate and voluntary exercise in improving cardiac angiogenesis with the enhanced involvement mechanism in high fat/STZ-induced type 2 diabetic rats.

The IGF-1R Inhibitor NVP-AEW541 Causes Insulin-Independent and Reversible Cardiac Contractile Dysfunction.

The antitumor treatment NVP-AEW541 blocks IGF-1R. IGF-1R signaling is crucial for cardiac function, but the cardiac effects of NVP-AEW541 are ill defined. We assessed NVP-AEW541′s effects on cardiac function and insulin response in vivo and in isolated working hearts. We performed a dose–response analysis of NVP-AEW541 in male, 3-week-old rats and assessed the chronic effects of the clinically relevant dose in adult rats. We performed glucose tolerance tests and echocardiography; assessed the expression and phosphorylation of InsR/IGF-1R and Akt in vivo; and measured substrate oxidation, contractile function, and insulin response in the isolated working hearts. NVP-AEW541 caused dose-dependent growth retardation and impaired glucose tolerance in the juvenile rats. In the adults, NVP-AEW541 caused a continuously worsening depression of cardiac contractility, which recovered within 2 weeks after cessation. Cardiac Akt protein and phosphorylation were unchanged and associated with InsR upregulation. An acute application of NVP-AEW541 in the working hearts did not affect cardiac power but eliminated insulin’s effects on glucose and fatty acid oxidation. The systemic administration of NVP-AEW541 caused dose- and time-dependent impairment of glucose tolerance, growth, and cardiac function. Because cardiac insulin signaling was maintained in vivo but absent in vitro and because contractile function was not affected in vitro, a direct link between insulin resistance and contractile dysfunction appears unlikely.

Elimination of AKT in Cardiomyocytes Causes Complex Reorganization of Signal Transduction leading to AMPK Inhibition and Fatal Cardiac Atrophy

Akt1 and Akt2 are the main protein kinase B (Akt) isoforms expressed in the mammalian heart. Tamoxifen(OHTx) ‐inducible, cardiomyocyte‐specific Akt1/ Akt2 double knockout mice (iCM‐Akt12) show progressive cardiac atrophy and loss of contractile function leading to terminal heart failure 23.9 ± 2 days after first OHTx injection.TUNEL staining of iCM‐Akt12 hearts revealed that cardiomyocyte apoptosis did not contribute substantially to cardiac atrophy. Rather, cellular atrophy caused the loss of cardiac mass. The cellular area (a), width (w), and length (l) declined between day 9 and day 14 [‐18 (a)/‐18 (w)/‐9% (l)] as compared to WT controls. This size reduction was retarded between d14 and d21 (‐20 (a)/‐24 (w)/‐8% (l). In vivo 31P‐imaging identified a progressive energetic deficiency of iCM‐Akt12 hearts on d15 and d20 after OHTx injection.Further analyses showed that in the early phase up to day 14 increased autophagic activity and reduced de novo protein synthesis seemed to cause cellular atrophy. As expected, deletion of Akt led to impaired mTORC1 signaling indicated by reduced phosphorylation of S6K, RPS6, 4E‐BP1, all involved in translation. Concomitantly, we measured reduced protein synthesis. Autophagy was increased in iCM‐Akt12 hearts, as shown by increased LC3‐II/I ratio and higher expression of autophagic genes.From day 14 after KO induction (late phase), protein synthesis appeared to increase and mTORC1 substrates involved in translation (S6K, RPS6 and 4E‐BP1) were higher phosphorylated. Of note, also a higher phosphorylation of the inhibitory mTORC1 target site in the central autophagy kinase Ulk1 (S757) was found. Moreover, autophagy appeared to be disturbed, as p62 accumulated.Despite increasing energetic depletion, the cellular energy sensor AMPK was not activated in iCM‐Akt12 hearts. Rather, AMPK was inhibited by phosphorylation of the α‐subunit on serine 485/491. The S485/491 phosphorylation has been attributed to several kinases (Akt, PKD1, S6K, PKC, PKA). Akt deletion led to a pronounced hyperactivation of Pdk1 signaling upstream of Akt, causing an activation of protein kinases regulated by Pdk1 (S6K, PKA, PKC, PKD). This was demonstrated by increased phosphorylation of specific kinase substrates and consensus motifs in late iCM‐Akt12 hearts and might promote the AMPK inactivation.To test to what extent a non‐inhibitable AMPK might attenuate the fatal phenotype of cardiac Akt loss, AAV‐mediated infection of iCM‐Akt12 mice with a phospho‐defective AMPKα2 S491A mutant prior to KO induction was performed. This led to an increased survival and improved heart function, demonstrating that the AMPK inhibition contributes to the lethal phenotype.In conclusion, loss of Akt signaling leads to a widespread dysregulation of intracellular signal transduction. Whereas enhanced autophagy and inhibition of mTORC1 are direct consequences of Akt deletion, the later reactivation of mTORC1 and inhibition of AMPK causes attenuation of autophagy, increased translation, deceleration of cellular atrophy, worsenes energetic shortage, and aggravates cardiac dysfunction.

Pharmacological inhibition of the lipid phosphatase PTEN ameliorates heart damage and adipose tissue inflammation in stressed rats with metabolic syndrome.

Phosphatidylinositol 3‐kinase (PI3K) signaling promotes the differentiation and proliferation of regulatory B (Breg) cells, and the lipid phosphatase phosphatase and tensin homolog deleted on chromosome 10 (PTEN) antagonizes the PI3K–Akt signaling pathway. We previously demonstrated that cardiac Akt activity is increased and that restraint stress exacerbates hypertension and both heart and adipose tissue (AT) inflammation in DS/obese rats, an animal model of metabolic syndrome (MetS). We here examined the effects of restraint stress and pharmacological inhibition of PTEN on heart and AT pathology in such rats. Nine‐week‐old animals were treated with the PTEN inhibitor bisperoxovanadium‐pic [bpV(pic)] or vehicle in the absence or presence of restraint stress for 4 weeks. BpV(pic) treatment had no effect on body weight or fat mass but attenuated hypertension in DS/obese rats subjected to restraint stress. BpV(pic) ameliorated left ventricular (LV) inflammation, fibrosis, and diastolic dysfunction as well as AT inflammation in the stressed rats. Restraint stress reduced myocardial capillary density, and this effect was prevented by bpV(pic). In addition, bpV(pic) increased the proportions of Breg and B‐1 cells as well as reduced those of CD8+ T and B‐2 cells in AT of stressed rats. Our results indicate that inhibition of PTEN by bpV(pic) alleviated heart and AT inflammation in stressed rats with MetS. These positive effects of bpV(pic) are likely due, at least in part, to a reduction in blood pressure, an increase in myocardial capillary formation, and an altered distribution of immune cells in fat tissue that result from the activation of PI3K–Akt signaling.

Vaspin Mediates the Intraorgan Crosstalk Between Heart and Adipose Tissue in Lipoatrophic Mice.

Lipoatrophy is characterized as selective loss of adipose tissues, leading to the severity of cardiovascular disorders. Therefore, there was close intraorgan crosstalk between adipose tissue and cardiovascular in lipoatrophy. A-ZIP/F-1 mouse, a well-established lipoatrophic model, and primary cardiomyocytes were used for investigating the pathophysiological changes and molecular mechanisms. A-ZIP/F-1 mice had severe fat loss and impaired ventricular function during growth, but closely associated with the reduction of circulating vaspin levels. Administration of recombinant vaspin protein improved cardiac structural disorders, left ventricular dysfunction, and inflammatory response in lipoatrophic mice. In detail, vaspin decreased cardiac lipid deposits, but enhanced mitochondrial biogenesis and activities. Interestingly, A-ZIP/F-1 mice transplanted with normal visceral adipose tissues exhibited improvement in cardiac structural remodeling and mitochondrial function. Mechanistically, vaspin increased cardiac AKT activity, which guaranteed the mitochondrial benefits of vaspin in lipoatrophic mice and primary mouse cardiomyocytes. The present study suggested that vaspin possessed biological benefits in attenuating lipoatrophy-induced cardiomyopathy onset, and targeting vaspin/AKT signaling was a potential strategy to maintain heart metabolism.

N-acetylcysteine protects neonatal mice from ventricular hypertrophy induced by maternal obesity in a sex-specific manner

BackgroundMaternal obesity induces adverse cardiac programming in offspring, and effective interventions are needed to prevent cardiovascular ill-health. Herein we hypothesized that exposure to maternal obesogenic diet-induced obesity in mice results in left ventricular remodelling and hypertrophy in early childhood, and that maternal N-acetylcysteine (NAC) treatment alleviates these effects in a sex-dependent manner. Methods and ResultsThe maternal obesity was induced in mice by the consumption of a Western diet accompanied by a 20 % sucrose solution. To determine the effect of NAC on the cardiac outcomes induced by maternal obesity, obese dams were continuously exposed to the obesogenic diet, with or without the oral NAC treatment during pregnancy. Left ventricular remodelling and hypertrophy occurred as early as 7 days after birth in the male offspring of obese dams (O-OB) compared with controls (O-CO). An over-expression of key genes and markers related to cardiac fibrosis accompanied by more disorganized myofibrils was observed in the hearts of neonatal male O-OB mice. When we next evaluated the level of oxidative stress in the hearts of neonatal mice, the activity of enzymatic antioxidants declined and expression of NOX enzyme complex was up-regulated in O-OB offspring hearts, but was normal in the offspring of NAC treated mice (O-OB/NAC). Maternal obesity also activated cardiac Akt and mammalian target of rapamycin (mTOR) signalling in offspring, and NAC treatment restored offspring cardiac Akt-mTOR signalling to normal irrespective of sex. NAC treatment did not prevent cardiomyocyte hypertrophy but did alleviate increased heart weight, interventricular septal thickness, and collagen content in male O-OB/NAC pups. ConclusionsCollectively, our results indicated that NAC blunted cardiac fibrosis and related ventricular hypertrophy of male neonatal offspring in the setting of maternal obesity, potentially acting by reducing oxidative stress. The present study provides a basis for investigating the role of NAC in nutrition-related cardiac programming.

1824-P: Activation of Cardiac Mitochondrial Akt1 Improved Diabetic Cardiomyopathy through Inhibition of Melatonin Degradation and Remotely Improved Liver Metabolism

Insulin resistance impaired Akt1 translocation to mitochondria in myocardium and contributed to diabetic cardiomyopathy (DCM). It is unknown whether activation of mitochondrial Akt in cardiac muscle can protect against DCM. To this end, we generated an inducible heart-specific transgenic mice with a mitochondria-targeting constitutively active Akt (CAMCAKT). A high fat-high fructose (HFF) diet induced myocardial fibrosis and heart failure, but cardiomyopathy was reversed in the CAMCAKT mice, indicating a mechanistic role of mitochondrial Akt1 signaling in DCM. RNAseq analysis revealed myocardial transcriptome changes, including suppressed Col1a1 and Col3a1 expression (fibrosis markers) recapitulating the mechanism of fibrosis. Accompanying lowered GP6 expression suggested a reduction in collagen-induced platelet activation. Pathway analysis revealed significant inhibition in multiple steps of melatonin degradation. Enhancing melatonin signaling, an emerging myocardial protective factor that reduces oxidative stress/apoptosis, is a novel mechanism through which mitochondrial Akt1 could protect against DCM. Under the HFF diet, cardiac mitochondrial Akt1 signaling also caused whole body changes; CAMCAKT mice had 15% less body fat, 30% lower total cholesterol and 42% lower LDL (p<0.05), and showed higher energy expenditure during night cycles and an elevated RER, an indicator for increased carbohydrate metabolism. The diet-induced fatty liver in the CAMCAKT mice was significantly lowered, suggesting that cardiac mitochondrial Akt signaling could remotely modulate hepatic fat metabolism. Liver RNAseq revealed that cholesterol synthesis in the liver was modulated by cardiac mitochondria. In summary, activating cardiac mitochondrial Akt1 protected against DCM by inhibiting melatonin degradation, and attenuated the development of fatty liver by regulating cholesterol synthesis. Disclosure A. Ta: None. Y. Chen: None. Y. Chen: None. H.Y.H. Lin: None. P.H. Wang: Board Member; Self; Dianavi. Funding National Institutes of Health (R01HL096987)

Hypercholesterolemia Interferes with Induction of miR-125b-1-3p in Preconditioned Hearts.

Ischemic preconditioning (IPre) reduces ischemia/reperfusion (I/R) injury in the heart. The non-coding microRNA miR-125b-1-3p has been demonstrated to play a role in the mechanism of IPre. Hypercholesterolemia is known to attenuate the cardioprotective effect of preconditioning; nevertheless, the exact underlying mechanisms are not clear. Here we investigated, whether hypercholesterolemia influences the induction of miR-125b-1-3p by IPre. Male Wistar rats were fed with a rodent chow supplemented with 2% cholesterol and 0.25% sodium-cholate hydrate for 8 weeks to induce high blood cholesterol levels. The hearts of normo- and hypercholesterolemic animals were then isolated and perfused according to Langendorff, and were subjected to 35 min global ischemia and 120 min reperfusion with or without IPre (3 × 5 min I/R cycles applied before index ischemia). IPre significantly reduced infarct size in the hearts of normocholesterolemic rats; however, IPre was ineffective in the hearts of hypercholesterolemic animals. Similarly, miR-125b-1-3p was upregulated by IPre in hearts of normocholesterolemic rats, while in the hearts of hypercholesterolemic animals IPre failed to increase miR-125b-1-3p significantly. Phosphorylation of cardiac Akt, ERK, and STAT3 was not significantly different in any of the groups at the end of reperfusion. Based on these results we propose here that hypercholesterolemia attenuates the upregulation of miR-125b-1-3p by IPre, which seems to be associated with the loss of cardioprotection.

Dietary Fat and Sugar Differentially Affect β-Adrenergic Stimulation of Cardiac ERK and AKT Pathways in C57BL/6 Male Mice Subjected to High-Calorie Feeding

High dietary fat and sugar promote cardiac hypertrophy independently from an increase in blood pressure. The respective contribution that each macronutrient exerts on cardiac growth signaling pathways remains unclear. The goal of this study was to investigate the mechanisms by which high amounts of dietary fat and sugar affect cardiac growth regulatory pathways. Male C57BL/6 mice (9 wk old; n=20/group) were fed a standard rodent diet (STD; kcal% protein-fat-carbohydrate, 29-17-54), a high-fat diet (HFD; 20-60-20), a high-fat and high-sugar Western diet (WD; 20-45-35), a high-sugar diet with mixed carbohydrates (HCD; 20-10-70), or a high-sucrose diet (HSD; 20-10-70). Body composition was assessed weekly by EchoMRI. Whole-body glucose utilization was assessed with an intraperitoneal glucose tolerance test. After 6 wk on diets, mice were treated with saline or 20 mg/kg isoproterenol (ISO), and the activity of cardiac growth regulatory pathways was analyzed by immunoblotting. Data were analyzed by ANOVA with data from the STD group included for references only. Compared with HCD and HSD, WD and HFD increased body fat mass 2.7- to 3.8-fold (P<0.001), induced glucose intolerance (P<0.001), and increased insulin concentrations >1.5-fold (P<0.05), thereby enhancing basal and ISO-stimulated AKT phosphorylation at both threonine 308 and serine 473 residues (+25-63%; P<0.05). Compared with HFD, the high-sugar diets potentiated ISO-mediated stimulation of the glucose-sensitive kinases PYK2 (>47%; P<0.05 for HCD and HSD) and ERK (>34%; P<0.05 for WD, HCD, and HSD), thereby leading to increased phosphorylation of protein synthesis regulator S6K1 at threonine 389 residue (>64%; P<0.05 for WD, HCD, and HSD). Dietary fat and sugar affect cardiac growth signaling pathways in C57BL/6 mice through distinct and additive mechanisms. The findings may provide new insights into the role of overnutrition in pathological cardiac remodeling.

Dietary Fat and Sugar Differentially Affect Beta‐Adrenergic Stimulation of Cardiac ERK and AKT Pathways in Mice Subjected to High‐Calorie Feeding

Introduction. High dietary fat and sugar promote cardiac hypertrophy independently from an increase in blood pressure. The respective contribution that each macronutrient exerts on cardiac growth signaling pathways remains unclear. The goal of this study was to investigate the mechanisms by which high dietary fat and sugar levels affect cardiac growth regulatory pathways in the mouse heart.Methods. Nine‐week‐old male C57BL/6 mice (n = 20/group) were fed a standard rodent diet (STD; 3.0 kcal/g), a high‐fat diet (HFD; 5.24 kcal/g), a high‐fat and high‐sugar Western diet (WD; 4.73 kcal/g), a high‐sugar diet with mixed carbohydrates (HCD; 3.85 kcal/g), or a high‐sucrose diet (HSD; 3.85 kcal/g). Body composition was assessed weekly by EchoMRI. Whole‐body glucose utilization was assessed with an intraperitoneal glucose tolerance test. After six weeks on diets, mice were treated with saline or 20 mg/kg isoproterenol (ISO) and the activity of cardiac growth regulatory pathways was analyzed by immunoblotting. One‐ or two‐way ANOVA was used for statistical comparisons.Results. Compared with STD, HFD increased body fat mass 4.5‐fold (P &lt; 0.0001), induced glucose intolerance (P &lt; 0.0001), and increased insulin levels 3.6‐fold (P &lt; 0.0001), thereby enhancing basal and ISO‐stimulated AKT phosphorylation at both threonine 308 and serine 473 residues (+37–100%; P &lt; 0.05). Both HCD and HSD potentiated ISO‐mediated stimulation of the glucose‐sensitive kinases PYK2 (&gt;50%; P &lt; 0.05) and ERK (&gt;30%; P &lt; 0.05), thereby leading to increased phosphorylation of protein synthesis regulator S6K1 at threonine 389 residue (≥70%; P &lt; 0.05). All these anthropometric, metabolic and cardiac growth signaling alterations occurred simultaneously in WD‐fed mice.Conclusions. The results indicate that dietary fat and sugar affect cardiac cell growth signaling pathways in C57BL/6 mice through distinct and additive mechanisms. The findings may provide new insights into the role of overnutrition in pathological remodeling of the heart.Support or Funding InformationThis work was supported by grants R01HL136438, P20GM104357 and P01HL051971 from the National Institutes of Health.

Modulation of cardiac AKT and STAT3 signalling in preclinical cancer models and their impact on the heart

BackgroundAdvanced cancer induces fundamental cardiac changes and promotes body wasting and heart failure. We evaluated the impact of cancer on major cardiac signalling pathways, and resulting consequences for the heart. Methods and resultsMetastatic melanoma disease was induced in male C57BL/6 N mice by intraperitoneal injection of the melanoma cell line B16F10 and lead to cardiac atrophy and heart failure. Analyses of key cardiac signalling pathways in left ventricular tissue revealed increased activation of STAT3 and reduced activation of AKT, p38 and ERK1/2. Markers of the ubiquitin proteasomal system (UPS: Atrogin-1) and of mitophagy/autophagy (LC3b, BNIP3) were upregulated. Tumour-bearing C57BL/6 N mice with a cardiomyocyte-specific overexpression of a constitutively active AKT transgene (AKTtg) displayed less cardiac atrophy and dysfunction and normalized Atrogin-1, LC3b and BNIP3 expression while the cardiomyocyte-specific knockout of STAT3 (CKO) had no major effect on these parameters compared to WT. ConclusionCancer alters major cardiac signalling pathways and subsequently the UPS, mitophagy and autophagy. The present study suggests that cancer-induced reduction of cardiomyocyte AKT contributes to these alterations as they were attenuated in tumour-bearing AKTtg mice. In turn, increased cardiomyocyte STAT3 activation appears less relevant, as tumour-induced impairment on the heart was largely similar in CKO and WT mice. Since oncologic therapies frequently target AKT and/or STAT3, their impact on the heart might be different in tumour-bearing mice compared to healthy mice, a feature suggesting to test tumour therapies also in tumour disease models and not only under healthy conditions. This article is part of a Special Issue entitled: Cardiomyocyte biology: new pathways of differentiation and regeneration edited by Marijke Brink, Marcus C. Schaub, and Christian Zuppinger.

Normalisation of circulating adiponectin levels in obese pregnant mice prevents cardiac dysfunction in adult offspring

Background/objectivesAdiponectin concentrations are low in obese pregnant women. Restoring normal adiponectin concentrations by infusion in obese pregnant mice prevents placental dysfunction, foetal overgrowth and metabolic syndrome in the offspring. We hypothesised that normalising maternal adiponectin in obese late pregnant dams prevents cardiac dysfunction in the adult offspring.Subjects/methodsPregnant female mice with diet-induced obesity were infused with adiponectin (0.62 μg g−1 day−1, n = 24) or saline (n = 22) over days 14.5–18.5 of pregnancy (term = day 19.5). Control dams ate standard chow and received saline (n = 22). Offspring were studied at 3 and 6 months of age.ResultsMaternal obesity impaired ventricular diastolic function, increased cardiomyocyte cross-sectional area and upregulated cardiac brain natriuretic peptide (Nppb) and α-skeletal actin (Acta1) gene expression in adult male offspring, compared to control offspring. In adult female offspring, maternal obesity increased Nppb expression, decreased end-diastolic volume and caused age-dependent diastolic dysfunction but not cardiomyocyte hypertrophy. Maternal obesity also activated cardiac Akt and mechanistic target of rapamycin (mTOR) signalling in male, but not in female, offspring and inhibited cardiac extracellular signal-regulated kinase 1/2 (ERK1/2) in both sexes. Normalising maternal circulating adiponectin concentrations by infusing obese dams with adiponectin prevented offspring diastolic dysfunction and ventricular dilation and normalised cardiac Akt-mTOR signalling irrespective of sex. Maternal adiponectin infusion also reduced cardiac Nppb expression and increased ERK1/2 signalling in offspring of obese dams. Adiponectin infusion did not prevent cardiomyocyte hypertrophy but reduced ventricular wall thickness in male offspring and increased collagen content in female offspring of obese dams, compared to controls.ConclusionsLow maternal adiponectin levels in obese mice in late pregnancy are mechanistically linked to in utero programming of cardiac dysfunction in their offspring. Interventions enhancing endogenous adiponectin secretion or signalling in obese pregnant women could prevent the development of cardiac dysfunction in their children.

Activation of Cardiac Mitochondrial Akt Signaling Improved Diabetic Cardiomyopathy and Induced Liver Metabolic Remodeling

Insulin stimulates Akt translocation to cardiac mitochondria, and we have recently shown that inhibition of cardiac mitochondrial Akt led to mitochondria dysfunction and cardiomyopathy. However, it is unknown whether activation of mitochondrial Akt in cardiac muscle can protect against the development of diabetic cardiomyopathy. To this end, we have generated an inducible, heart-specific, transgenic mice harboring a mitochondria-targeting constitutively active Akt (CAMAAKT). After 5 months of high fat-high fructose diet (HFF), the control mice developed myocardial fibrosis and cardiomegaly. In contrast, myocardial fibrosis and cardiomegaly were attenuated in the CAMAAKT mice on HFF. Compared to the control mice, the expression of myocardial BNPa, BNPb, and Myh7 (heart failure genes) were significantly suppressed in the CAMAAKT mice by 80%, 90%, and 80%, respectively (all p&amp;lt;0.01). Col1a1 and Col3a1 expression were reduced in the HFF-CAMAAKT mice, corroborating the reduction of myocardial fibrosis. These data indicated that activation of mitochondrial Akt protected against cardiomyopathy in diet-induced diabetes. In OGTT, fasting glucose level was unchanged but postprandial glucose levels were lower in the CAMAAKT mice. In another heart-specific transgenic mice harboring a mitochondria-targeting dominant negative Akt (CAMDAKT), the glucose curve in OGTT was significantly higher than the control mice. Therefore, mitochondrial Akt signaling in the heart could modulate whole body glucose homeostasis. Interestingly, the diet-induced fatty liver in the CAMAAKT mice was significantly less than the controls, suggesting that cardiac mitochondrial Akt signaling may remotely modulate fat metabolism in the liver. In summary, activating cardiac mitochondrial Akt protected against diabetic cardiomyopathy, improved glucose tolerance, and attenuated fatty liver in HFF-induced diabetes. Disclosure A. Ta: None. Y. Chen: None. Y. Chen: None. H. Lee: None. H. Lin: None. P.H. Wang: None.

Remodeling of Myocardial Metabolism by Cardiac Mitochondrial Akt Signaling through Modulation of Pyruvate Dehydrogenase Complex

Mitochondria play a critical role in the regulation of myocardial metabolism and function. Diminished insulin-activated Akt translocation into mitochondria matrix contributed to the development of diabetic cardiomyopathy. However, its mechanisms of actions are not fully understood. Using an inducible heart specific transgenic mice harboring a mitochondria-targeting dominant Akt (CAMDAKT), we have observed 50% attenuation of pyruvate dehydrogenase activity in mitochondria. Proteomic study revealed insulin injection promoted formation of pyruvate dehydrogenase supercomplex in the control mice, however, insulin-stimulated formation of pyruvate dehydrogenase supercomplex was inhibited in the CAMDAKT mice. We next used a novel small molecular inhibitor (SMI) to disrupt binding of Akt to the E3 subunit of pyruvate dehydrogenase to dissect how active Akt interacted and modulated pyruvate dehydrogenase complex (PDC). Insulin stimulation of PDC activity and complex formation were inhibited by the SMI. Acetyl-CoA, which bridges PDC to the TCA cycle, was significantly reduced by 40% in the cardiac mitochondria of CAMDAKT mice. Interestingly, this was accompanied by reduced NAD+ and NADH levels (40-50% reduction) and NAD+/NADH ratio (30-40% reduction). Therefore the redox state was compromised. In CAMDAKT mice, cardiac mitochondria underwent adaptive changes once mitochondrial Akt became inhibited, with biphasic alteration of mitochondria abundance (up and down). The expression of PPARγ and PGC1α, showed a similar pattern of changes, suggesting compensatory mitochondria biogenesis. In summary, mitochondrial Akt action in the heart is at least partially mediated through its direct interaction with the E3 subunit of PDC, and inhibition of this pathway led to metabolic remodeling and compensatory mitochondria biogenesis. These data shed new light into the mechanism of mitochondrial Akt actions and how it modulated cardiac metabolism. Disclosure Y. Chen: None. Y. Chen: None. A. Ta: None. H. Lee: None. H. Lin: None. P.H. Wang: None.

Insulin supplementation attenuates cancer-induced cardiomyopathy and slows tumor disease progression.

Advanced cancer induces fundamental changes in metabolism and promotes cardiac atrophy and heart failure. We discovered systemic insulin deficiency in cachectic cancer patients. Similarly, mice with advanced B16F10 melanoma (B16F10-TM) or colon 26 carcinoma (C26-TM) displayed decreased systemic insulin associated with marked cardiac atrophy, metabolic impairment, and function. B16F10 and C26 tumors decrease systemic insulin via high glucose consumption, lowering pancreatic insulin production and producing insulin-degrading enzyme. As tumor cells consume glucose in an insulin-independent manner, they shift glucose away from cardiomyocytes. Since cardiomyocytes in both tumor models remained insulin responsive, low-dose insulin supplementation by subcutaneous implantation of insulin-releasing pellets improved cardiac glucose uptake, atrophy, and function, with no adverse side effects. In addition, by redirecting glucose to the heart in addition to other organs, the systemic insulin treatment lowered glucose usage by the tumor and thereby decreased tumor growth and volume. Insulin corrected the cancer-induced reduction in cardiac Akt activation and the subsequent overactivation of the proteasome and autophagy. Thus, cancer-induced systemic insulin depletion contributes to cardiac wasting and failure and may promote tumor growth. Low-dose insulin supplementation attenuates these processes and may be supportive in cardio-oncologic treatment concepts.

Akt signaling as a mediator of cardiac adaptation to low birth weight.

Intrauterine insults, such as poor nutrition and placental insufficiency, can alter cardiomyocyte development, and this can have significant long-term implications for heart health. Consequently, epidemiological studies have shown that low-birth-weight babies have an increased risk of death from cardiovascular disease in adult life. In addition, intrauterine growth restriction can result in increased left ventricular hypertrophy, which is the strongest predictor for poor health outcomes in cardiac patients. The mechanisms responsible for these associations are not clear, but a suboptimal intrauterine environment can program alternative expression of genes such as cardiac IGF-2/H19, IGF-2R and AT1R through either an increase or decrease in DNA methylation or histone acetylation at specific loci. Furthermore, hypoxia and other intrauterine insults can also activate the IGF-1 receptor via IGF-1 and IGF-2, and the AT1 receptor via angiotensin signaling pathways; both of which can result in the phosphorylation of Akt and the activation of a range of downstream pathways. In turn, Akt activation can increase cardiac angiogenesis and cardiomyocyte apoptosis and promote a reversion of metabolism in postnatal life to a fetal phenotype, which involves increased reliance on glucose. Cardiac Akt can also be indirectly regulated by microRNAs and conversely can target microRNAs that will eventually affect other specific cardiac genes and proteins. This review aims to discuss our understanding of this complex network of interactions, which may help explain the link between low birth weight and the increased risk of cardiovascular disease in adult life.

Disrupting the key circadian regulator CLOCK leads to age-dependent cardiovascular disease

The circadian mechanism underlies daily rhythms in cardiovascular physiology and rhythm disruption is a major risk factor for heart disease and worse outcomes. However, the role of circadian rhythms is generally clinically unappreciated. Clock is a core component of the circadian mechanism and here we examine the role of Clock as a vital determinant of cardiac physiology and pathophysiology in aging. ClockΔ19/Δ19 mice develop age-dependent increases in heart weight, hypertrophy, dilation, impaired contractility, and reduced myogenic responsiveness. Young ClockΔ19/Δ19 hearts express dysregulated mRNAs and miRNAs in the PTEN-AKT signal pathways important for cardiac hypertrophy. We found a rhythm in the Pten gene and PTEN protein in WT hearts; rhythmic oscillations are lost in ClockΔ19/Δ19 hearts. Changes in PTEN are associated with reduced AKT activation and changes in downstream mediators GSK-3β, PRAS40, and S6K1. Cardiomyocyte cultures confirm that Clock regulates the AKT signalling pathways crucial for cardiac hypertrophy. In old ClockΔ19/Δ19 mice cardiac AKT, GSK3β, S6K1 phosphorylation are increased, consistent with the development of age-dependent cardiac hypertrophy. Lastly, we show that pharmacological modulation of the circadian mechanism with the REV-ERB agonist SR9009 reduces AKT activation and heart weight in old WT mice. Furthermore, SR9009 attenuates cardiac hypertrophy in mice subjected to transverse aortic constriction (TAC), supporting that the circadian mechanism plays an important role in regulating cardiac growth. These findings demonstrate a crucial role for Clock in growth and renewal; disrupting Clock leads to age-dependent cardiomyopathy. Pharmacological targeting of the circadian mechanism provides a new opportunity for treating heart disease.

Selective inhibition of PTEN preserves ischaemic post-conditioning cardioprotection in STZ-induced Type 1 diabetic rats: role of the PI3K/Akt and JAK2/STAT3 pathways.

Patients with diabetes are vulnerable to MI/R (myocardial ischaemia/reperfusion) injury, but are not responsive to IPostC (ischaemic post-conditioning) which activates PI3K (phosphoinositide 3-kinase)/Akt (also known as PKB or protein kinase B) and JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) pathways to confer cardioprotection. We hypothesized that increased cardiac PTEN (phosphatase and tensin homologue deleted on chromosome 10), a major negative regulator of PI3K/Akt, is responsible for the loss of diabetic heart sensitivity to IPostC cardioprotecton. In STZ (streptozotocin)-induced Type 1 diabetic rats subjected to MI/R (30 min coronary occlusion and 120 min reperfusion), the post-ischaemic myocardial infarct size, CK-MB (creatine kinase-MB) and 15-F2t-isoprostane release, as well as cardiac PTEN expression were significantly higher than those in non-diabetic controls, concomitant with more severe cardiac dysfunction and lower cardiac Akt, STAT3 and GSK-3β (glycogen synthase kinase 3β) phosphorylation. IPostC significantly attenuated post-ischaemic infarct size, decreased PTEN expression and further increased Akt, STAT3 and GSK-3β phosphorylation in non-diabetic, but not in diabetic rats. Application of the PTEN inhibitor BpV (bisperoxovanadium) (1.0 mg/kg) restored IPostC cardioprotection in diabetic rats. HPostC (hypoxic post-conditioning) in combination with PTEN gene knockdown, but not HPostC alone, significantly reduced H/R (hypoxia/reoxygenation) injury in cardiac H9c2 cells exposed to high glucose as was evident from reduced apoptotic cell death and JC-1 monomer in cells, accompanied by increased phosphorylation of Akt, STAT3 and GSK-3β. PTEN inhibition/gene knockdown mediated restoration of IPostC/HPostC cardioprotection was completely reversed by the PI3K inhibitor wortmannin, and partially reversed by the JAK2 inhibitor AG490. Increased cardiac PTEN, by impairing PI3K/Akt and JAK2/STAT3 pathways, is a major mechanism that rendered diabetic hearts not responsive to post-conditioning cardioprotection.