The role of glucose in cardiac physiology and pathophysiology.

PURPOSE OF REVIEW: Heart failure is one of the major causes of death worldwide and continues to increase despite therapeutics and pharmacology advances. Fatty acids and glucose are used as ATP-producing fuels in heart to meet its energy demands. However, dysregulation of metabolites' use plays a pivotal role in cardiac diseases. How glucose becomes toxic or drives cardiac dysfunction is incompletely understood. In the present review, we summarize the recent findings on cardiac cellular and molecular events that are driven by glucose during pathologic conditions and potential therapeutic strategies to tackle hyperglycemia-mediated cardiac dysfunction. RECENT FINDINGS: Several studies have emerged recently, demonstrating that excessive glucose utilization has been correlated with impairment of cellular metabolic homeostasis primarily driven by mitochondrial dysfunction and damage, oxidative stress, and abnormal redox signaling. This disturbance is associated with cardiac remodeling, hypertrophy, and systolic and diastolic dysfunction. Both human and animal heart failure studies, report that glucose is a preferable fuel at the expense of fatty acid oxidation during ischemia and hypertrophy, but the opposite happens in diabetic hearts, which warrants further investigation. SUMMARY: A better understanding of glucose metabolism and its fate during distinct types of heart disease will contribute to developing novel therapeutic options for the prevention and treatment of heart failure.

Impact of obesity on day-night differences in cardiac metabolism.

An intrinsic property of the heart is an ability to rapidly and coordinately adjust flux through metabolic pathways in response to physiologic stimuli (termed metabolic flexibility). Cardiac metabolism also fluctuates across the 24-hours day, in association with diurnal sleep-wake and fasting-feeding cycles. Although loss of metabolic flexibility has been proposed to play a causal role in the pathogenesis of cardiac disease, it is currently unknown whether day-night variations in cardiac metabolism are altered during disease states. Here, we tested the hypothesis that diet-induced obesity disrupts cardiac "diurnal metabolic flexibility", which is normalized by time-of-day-restricted feeding. Chronic high fat feeding (20-wk)-induced obesity in mice, abolished diurnal rhythms in whole body metabolic flexibility, and increased markers of adverse cardiac remodeling (hypertrophy, fibrosis, and steatosis). RNAseq analysis revealed that 24-hours rhythms in the cardiac transcriptome were dramatically altered during obesity; only 22% of rhythmic transcripts in control hearts were unaffected by obesity. However, day-night differences in cardiac substrate oxidation were essentially identical in control and high fat fed mice. In contrast, day-night differences in both cardiac triglyceride synthesis and lipidome were abolished during obesity. Next, a subset of obese mice (induced by 18-wks ad libitum high fat feeding) were allowed access to the high fat diet only during the 12-hours dark (active) phase, for a 2-wk period. Dark phase restricted feeding partially restored whole body metabolic flexibility, as well as day-night differences in cardiac triglyceride synthesis and lipidome. Moreover, this intervention partially reversed adverse cardiac remodeling in obese mice. Collectively, these studies reveal diurnal metabolic inflexibility of the heart during obesity specifically for nonoxidative lipid metabolism (but not for substrate oxidation), and that restricting food intake to the active period partially reverses obesity-induced cardiac lipid metabolism abnormalities and adverse remodeling of the heart.

Branched chain amino acids selectively promote cardiac growth at the end of the awake period.

Essentially all biological processes fluctuate over the course of the day, manifesting as time-of-day-dependent variations with regards to the way in which organ systems respond to normal behaviors. For example, basic, translational, and epidemiologic studies indicate that temporal partitioning of metabolic processes governs the fate of dietary nutrients, in a manner in which concentrating caloric intake towards the end of the day is detrimental to both cardiometabolic and cardiovascular parameters. Despite appreciation that branched chain amino acids impact risk for obesity, diabetes mellitus, and heart failure, it is currently unknown whether the time-of-day at which dietary BCAAs are consumed influence cardiometabolic/cardiovascular outcomes. Here, we report that feeding mice a BCAA-enriched meal at the end of the active period (i.e., last 4 h of the dark phase) rapidly increases cardiac protein synthesis and mass, as well as cardiomyocyte size; consumption of the same meal at the beginning of the active period (i.e., first 4 h of the dark phase) is without effect. This was associated with a greater BCAA-induced activation of mTOR signaling in the heart at the end of the active period; pharmacological inhibition of mTOR (through rapamycin) blocked BCAA-induced augmentation of cardiac mass and cardiomyocyte size. Moreover, genetic disruption of the cardiomyocyte circadian clock abolished time-of-day-dependent fluctuations in BCAA-responsiveness. Finally, we report that repetitive consumption of BCAA-enriched meals at the end of the active period accelerated adverse cardiac remodeling and contractile dysfunction in mice subjected to transverse aortic constriction. Thus, our data demonstrate that the timing of BCAA consumption has significant implications for cardiac health and disease.

Differential effects of REV-ERBα/ß agonism on cardiac gene expression, metabolism, and contractile function in a mouse model of circadian disruption.

Cell-autonomous circadian clocks have emerged as temporal orchestrators of numerous biological processes. For example, the cardiomyocyte circadian clock modulates transcription, translation, posttranslational modifications, ion homeostasis, signaling cascades, metabolism, and contractility of the heart over the course of the day. Circadian clocks are composed of more than 10 interconnected transcriptional modulators, all of which have the potential to influence the cardiac transcriptome (and ultimately cardiac processes). These transcriptional modulators include BMAL1 and REV-ERBα/ß; BMAL1 induces REV-ERBα/ß, which in turn feeds back to inhibit BMAL1. Previous studies indicate that cardiomyocyte-specific BMAL1-knockout (CBK) mice exhibit a dysfunctional circadian clock (including decreased REV-ERBα/ß expression) in the heart associated with abnormalities in cardiac mitochondrial function, metabolism, signaling, and contractile function. Here, we hypothesized that decreased REV-ERBα/ß activity is responsible for distinct phenotypical alterations observed in CBK hearts. To test this hypothesis, CBK (and littermate control) mice were administered with the selective REV-ERBα/ß agonist SR-9009 (100 mg·kg-1·day-1 for 8 days). SR-9009 administration was sufficient to normalize cardiac glycogen synthesis rates, cardiomyocyte size, interstitial fibrosis, and contractility in CBK hearts (without influencing mitochondrial complex activities, nor normalizing substrate oxidation and Akt/mTOR/GSK3ß signaling). Collectively, these observations highlight a role for REV-ERBα/ß as a mediator of a subset of circadian clock-controlled processes in the heart.

AMP-activated protein kinase α1-sensitive activation of AP-1 in cardiomyocytes.

AMP-activated protein kinase (Ampk) regulates myocardial energy metabolism and plays a crucial role in the response to cell stress. In the failing heart, an isoform shift of the predominant Ampkα2 to the Ampkα1 was observed. The present study explored possible isoform specific effects of Ampkα1 in cardiomyocytes. To this end, experiments were performed in HL-1 cardiomyocytes, as well as in Ampkα1-deficient and corresponding wild-type mice and mice following AAV9-mediated cardiac overexpression of constitutively active Ampkα1. As a result, in HL-1 cardiomyocytes, overexpression of constitutively active Ampkα1 increased the phosphorylation of Pkcζ. Constitutively active Ampkα1 further increased AP-1-dependent transcriptional activity and mRNA expression of the AP-1 target genes c-Fos, Il6 and Ncx1, effects blunted by Pkcζ silencing. In HL-1 cardiomyocytes, angiotensin-II activated AP-1, an effect blunted by silencing of Ampkα1 and Pkcζ, but not of Ampkα2. In wild-type mice, angiotensin-II infusion increased cardiac Ampkα1 and cardiac Pkcζ protein levels, as well as c-Fos, Il6 and Ncx1 mRNA expression, effects blunted in Ampkα1-deficient mice. Pressure overload by transverse aortic constriction (TAC) similarly increased cardiac Ampkα1 and Pkcζ abundance as well as c-Fos, Il6 and Ncx1 mRNA expression, effects again blunted in Ampkα1-deficient mice. AAV9-mediated cardiac overexpression of constitutively active Ampkα1 increased Pkcζ protein abundance and the mRNA expression of c-Fos, Il6 and Ncx1 in cardiac tissue. In conclusion, Ampkα1 promotes myocardial AP-1 activation in a Pkcζ-dependent manner and thus contributes to cardiac stress signaling.

Role of AMP-activated protein kinase α1 in angiotensin-II-induced renal Tgfß-activated kinase 1 activation.

Angiotensin-II is a key factor in renal fibrosis. Obstructive nephropathy induces an isoform shift from catalytic Ampkα2 towards Ampkα1 which contributes to signaling involved in renal tissue injury. The present study explored whether the Ampkα1 isoform contributes to the renal effects of angiotensin-II. To this end, angiotensin-II was infused by subcutaneous implantation of osmotic minipumps in gene-targeted mice lacking functional Ampkα1 (Ampkα1(-/-)) and corresponding wild-type mice (Ampkα1(+/+)). Western blotting and qRT-PCR were employed to determine protein abundance and mRNA levels, respectively, in renal tissue. In Ampkα1(+/+) mice, angiotensin-II increased renal Ampkα1 protein expression without significantly modifying renal Ampkα2 protein expression. The renal phosphorylated Ampkα (Thr(172)) protein abundance was not affected by angiotensin-II in neither genotypes, but was significantly lower in Ampkα1(-/-) mice than Ampkα1(+/+) mice. Angiotensin-II increased the phosphorylation of Tak1 (Ser(412)) in renal tissue of Ampkα1(+/+) mice, an effect virtually absent in the Ampkα1(-/-) mice. Furthermore, angiotensin-II treatment significantly increased renal protein and mRNA expression of α-smooth muscle actin (αSma) as well as Tak1-target gene expression: Cox2, Il6 and Pai1 in Ampkα1(+/+) mice, all effects significantly less pronounced in Ampkα1(-/-) mice. In conclusion, angiotensin-II up-regulates the Ampkα1 isoform in renal tissue. Ampkα1 participates in renal Tak1 activation and Tak1-dependent signaling induced by angiotensin-II.

SGK1-Sensitive Regulation of Cyclin-Dependent Kinase Inhibitor 1B (p27) in Cardiomyocyte Hypertrophy.

BACKGROUND/AIMS: The serum- and glucocorticoid-inducible kinase SGK1 participates in the orchestration of cardiac hypertrophy and remodeling. Signaling linking SGK1 activity to cardiac remodeling is, however, incompletely understood. SGK1 phosphorylation targets include cyclin-dependent kinase inhibitor 1B (p27), a protein which suppresses cardiac hypertrophy. The present study explored how effects of SGK1 on nuclear p27 localization might modulate the hypertrophic response in cardiomyocytes. METHODS: Experiments were performed in HL-1 cardiomyocytes and in SGK1-deficient (sgk1-/-) and corresponding wild-type (sgk1+/+) mice following pressure overload by transverse aortic constriction (TAC). Transcript levels were quantified by RT-PCR, protein abundance by Western blotting and protein localization by confocal microscopy. RESULTS: In HL-1 cardiomyocytes, overexpression of constitutively active SGK1 (SGK1S422D) but not of inactive SGK1 (SGK1K127N) increased significantly the cell size and transcript levels encoding Acta1, a molecular marker of hypertrophy. Those effects were paralleled by almost complete relocation of p27 in the cytoplasm. Treatment of HL-1 cardiomyocytes with isoproterenol was followed by up-regulation of SGK1 expression. Moreover, isoproterenol treatment stimulated the hypertrophic response and was followed by disappearance of p27 from the nuclei, effects prevented by the SGK1 inhibitor EMD638683. The effect of SGK1S422D overexpression on Acta1 mRNA levels was disrupted by overexpression of p27 and of the p27T197A mutant lacking the SGK1 phosphorylation site, but not of the phosphomimetic p27T197D mutant. In sgk1+/+ mice, TAC increased significantly SGK1 and Acta1 mRNA levels and decreased the nuclear to cytoplasmic protein ratio of p27 in cardiac tissue, effects blunted in the sgk1-/- mice. CONCLUSION: SGK1-induced hypertrophy of cardiomyocytes involves p27 phosphorylation at T197, which fosters cytoplasmic p27 localization.

Inhibitory effect of NH4Cl treatment on renal Tgfß1 signaling following unilateral ureteral obstruction.

BACKGROUND/AIMS: Consequences of obstructive nephropathy include tissue fibrosis, a major pathophysiological mechanism contributing to development of end-stage renal disease. Transforming growth factor ß 1 (Tgfß1) is involved in the progression of renal fibrosis. According to recent observations, ammonium chloride (NH4Cl) prevented phosphate-induced vascular remodeling, effects involving decrease of Tgfß1 expression and inhibition of Tgfß1-dependent signaling. The present study, thus, explored whether NH4Cl influences renal Tgfß1-induced pro-fibrotic signaling in obstructive nephropathy induced by unilateral ureteral obstruction (UUO). METHODS: UUO was induced for seven days in C57Bl6 mice with or without additional treatment with NH4Cl (0.28 M in drinking water). Transcript levels were determined by RT-PCR as well as protein abundance by Western blotting, blood pH was determined utilizing a blood gas and chemistry analyser. RESULTS: UUO increased renal mRNA expression of Tgfb1, Tgfß-activated kinase 1 (Tak1) protein abundance and Smad2 phosphorylation in the nuclear fraction of the obstructed kidney tissues, effects blunted in NH4Cl treated mice as compared to control treated mice. The mRNA levels of the transcription factors nuclear factor of activated T cells 5 (Nfat5) and SRY (sex determining region Y)-box 9 (Sox9) as well as of tumor necrosis factor α (Tnfα), interleukin 6 (Il6), plasminogen activator inhibitor 1 (Pai1) and Snai1 were up-regulated in the obstructed kidney tissues following UUO, effects again significantly ameliorated following NH4Cl treatment. Furthermore, the increased protein and mRNA expression of α-smooth muscle actin (α-Sma), fibronectin and collagen type I in the obstructed kidney tissues following UUO were significantly attenuated following NH4Cl treatment. CONCLUSION: NH4Cl treatment ameliorates Tgfß1-dependent pro-fibrotic signaling and renal tissue fibrosis markers following obstructive nephropathy.

AMP-activated protein kinase α1 regulates cardiac gap junction protein connexin 43 and electrical remodeling following pressure overload.

BACKGROUND/AIMS: Adenosine 5'-monophosphate (AMP)-activated protein kinase (Ampk) modulates a wide array of cellular functions and regulates various ion channels and transporters. In failing human hearts an increased Ampkα1 activity was observed. The present study aimed to uncover the impact of Ampkα1 on cardiac electrical remodeling. METHODS: Gene-targeted mice lacking functional Ampkα1 (Ampkα1-/-) and corresponding wild-type mice were exposed to pressure overload by "transverse aortic constriction" (TAC). In vivo electrophysiology was performed with a single catheter technique, myocardial conduction velocities and conduction characteristics investigated in isolated hearts, transcript levels quantified by RT-PCR and protein abundance determined by Western blotting. Moreover, connexin 43 (Cx43) was expressed in Xenopus oocytes with or without coexpression of wild-type or mutant AMPK and Cx43 protein abundance quantified utilizing confocal microscopy. RESULTS: TAC treatment increased Ampkα1 protein expression in cardiac tissue from wild-type mice. TAC further increased left ventricular conduction inhomogeneity and triggered conduction blocks, effects blunted in the Ampkα1(-/-) mice. TAC treatment decreased Cx43 protein abundance in cardiac tissue, an effect significantly blunted in the Ampkα1(-/-) mice. TAC treatment did not modify Cx43 mRNA levels but increased ubiquitination of Cx43 protein, an effect mitigated by Ampkα1 deficiency. As shown in Xenopus oocytes, Cx43 cell membrane protein abundance was significantly downregulated by wild-type AMPK(WT) and constitutively active AMPK(γR70Q), but not by catalytically inactive AMPK(αK45R). CONCLUSION: Ampkα1 stimulates ubiquitination of the gap junction protein Cx43, thereby contributing to gap junction remodeling following pressure overload.

Annexin A7 deficiency potentiates cardiac NFAT activity promoting hypertrophic signaling.

Annexin A7 (Anxa7) is a cytoskeletal protein interacting with Ca(2+) signaling which in turn is a crucial factor for cardiac remodeling following cardiac injury. The present study explored whether Anxa7 participates in the regulation of cardiac stress signaling. To this end, mice lacking functional Anxa7 (anxa7(-/-)) and wild-type mice (anxa7(+/+)) were investigated following pressure overload by transverse aortic constriction (TAC). In addition, HL-1 cardiomyocytes were silenced with Anxa7 siRNA and treated with isoproterenol. Transcript levels were determined by quantitative RT-PCR, transcriptional activity by luciferase reporter assay and protein abundance by Western blotting and confocal microscopy. As a result, TAC treatment increased the mRNA and protein levels of Anxa7 in wild-type mice. Moreover, TAC increased heart weight to body weight ratio and the cardiac mRNA levels of αSka, Nppb, Col1a1, Col3a1 and Rcan1, effects more pronounced in anxa7(-/-) mice than in anxa7(+/+) mice. Silencing of Anxa7 in HL-1 cardiomyocytes significantly increased nuclear localization of Nfatc1. Furthermore, Anxa7 silencing increased NFAT-dependent transcriptional activity as well as αSka, Nppb, and Rcan1 mRNA levels both, under control conditions and following ß-adrenergic stimulation by isoproterenol. These observations point to an important role of annexin A7 in the regulation of cardiac NFAT activity and hypertrophic response following cardiac stress conditions.

Down-regulation of renal klotho expression by Shiga toxin 2.

BACKGROUND/AIMS: Shiga toxin 2 may trigger classical hemolytic uremic syndrome (HUS) eventually leading to renal failure. Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney is involved in the regulation of renal phosphate excretion and also retains renal protective effects. Renal failure is associated with renal depletion of klotho. The present study explored the influence of Shiga toxin 2 on renal klotho expression. METHODS: Mice were injected with either solvent or Shiga toxin 2 and urinary flow rate and phosphate excretion were determined in metabolic cages. Renal transcript levels were measured by quantitative RT-PCR and renal protein abundance by Western blotting. Plasma concentrations of 1,25(OH)2D3 and FGF23 were determined by ELISA and plasma phosphate and urea concentrations by photometry. RESULTS: Shiga toxin 2 treatment was followed by increase of plasma urea concentration, urinary flow rate and renal phosphate excretion but not of plasma phosphate concentration. Shiga toxin 2 treatment strongly decreased klotho mRNA expression and klotho protein abundance in renal tissue. Shiga toxin 2 treatment further increased tumor necrosis factor (Tnfα) mRNA levels, as well as protein abundance of phosphorylated p38 MAPK in renal tissue. The treatment significantly increased renal Cyp27b1 and decreased renal Cyp24a1 mRNA levels without significantly altering plasma 1,25(OH)2D3 levels. Shiga toxin 2 treatment was further followed by increase of plasma FGF23 concentrations. CONCLUSION: Shiga toxin 2 treatment stimulated Tnfα transcription, down-regulated renal klotho expression and increased FGF23 formation, effects presumably contributing to renal tissue injury.

Annexin 7 in the regulation of gastric acid secretion.

BACKGROUND/AIMS: Glucocorticoids enhance gastric acid secretion and inhibit gastric cyclooxygenase, thus downregulating formation of PGE2, an inhibitor of gastric acid secretion. In erythrocytes, PGE2 formation is inhibited by annexin 7. The present study thus explored whether annexin 7 participates in the regulation of gastric acid secretion. METHODS: Annexin 7 protein expression was determined by Western blotting, cytosolic pH (pHi) of parietal cells utilizing BCECF-fluorescence, and gastric acid secretion by determination of Na(+)-independent pHi recovery from an ammonium pulse (∆pHi/min). Experiments were performed in isolated glands from gene targeted mice lacking annexin 7 (anx7(-/-)) and in respective wild type animals (anx7(+/+)). RESULTS: Prior to treatment pHi and ∆pHi/min were similar in isolated gastric glands from anx7(-/-) and from anx7(+/+) mice. Aspirin (100 µM added to the glands 1 hr prior to the experiment) significantly increased ∆pHi/min to similar values in both genotypes. The administration of dexamethasone (10 µg/g BW subcutaneously for 4 consecutive days prior to the experiments) significantly increased ∆pH/min in anx7(+/+) mice but not in anx7(-/-) mice. Following dexamethasone treatment, the luminal pH was significantly lower and the acid content significantly higher in anx7(+/+) mice than in anx7(-/-) mice. An increase of extracellular K(+) concentration to 35 mM (replacing Na(+)/NMDG(+)) significantly increased ∆pHi/min in both genotypes. In neither genotype dexamethasone increased ∆pH/min further in the presence of 35 mM K(+) or presence of aspirin. CONCLUSIONS: Annexin 7 is required for the stimulation of gastric acid secretion by glucocorticoids.

25-Hydroxyvitamin D3 1-α-hydroxylase-dependent stimulation of renal klotho expression by spironolactone.

BACKGROUND: Klotho, a transmembrane protein, protease and hormone mainly expressed in kidney, is required for the suppression of 1,25(OH)2D3-generating 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1) by FGF23. Conversely, 1,25(OH)2D3 stimulates, by activating the vitamin D3 receptor (Vdr), the expression of klotho, thus establishing a negative feedback loop. Klotho protects against renal and vascular injury. Klotho deficiency accelerates aging and early death, effects at least partially due to excessive formation of 1,25(OH)2D3 and subsequent hyperphosphatemia. Klotho expression is inhibited by aldosterone. The present study explored the interaction of aldosterone and DOCA as well as the moderately selective mineralocorticoid receptor antagonist spironolactone on klotho expression. METHODS: mRNA levels were determined utilizing quantitative RT-PCR in human embryonic kidney cells (HEK293) or in renal tissues from mice without or with prior mineralocorticoid (aldosterone or DOCA) and/or spironolactone treatment. In HEK293 cells, protein levels were determined by western blotting. The experiments in HEK293 cells were performed without or with silencing of CYP27B1, of vitamin D3 receptor (VDR) or of mineralocorticoid receptor (NR3C2). RESULTS: In HEK293 cells aldosterone and in mice DOCA significantly decreased KLOTHO gene expression, effects opposed by spironolactone treatment. Spironolactone treatment alone significantly increased KLOTHO and CYP27B1 transcript levels in HEK293 cells (24 hours) and mice (8 hours or 5 days). Moreover, spironolactone significantly increased klotho and CYP27B1 protein levels in HEK293 cells (48 hours). Reduced NR3C2 expression following silencing did not significantly affect KLOTHO and CYP27B1 transcript levels in presence or absence of spironolactone. Silencing of CYP27B1 and VDR significantly blunted the stimulating effect of spironolactone on KLOTHO mRNA levels in HEK293 cells. CONCLUSION: Besides blocking the effects of aldosterone, spironolactone upregulates KLOTHO gene expression by upregulation of 25-hydroxyvitamin D3 1-alpha-hydroxylase with subsequent activation of the vitamin D3 receptor by 1,25(OH)2D3, an effect possibly independent from the mineralocorticoid receptor.

PKB/SGK-resistant GSK-3 signaling following unilateral ureteral obstruction.

BACKGROUND/AIMS: Renal tissue fibrosis contributes to the development of end-stage renal disease. Causes for renal tissue fibrosis include obstructive nephropathy. The development of renal fibrosis following unilateral ureteral obstruction (UUO) is blunted in gene-targeted mice lacking functional serum- and glucocorticoid-inducible kinase SGK1. Similar to Akt isoforms, SGK1 phosphorylates and thus inactivates glycogen synthase kinase GSK-3. The present study explored whether PKB/SGK-dependent phoshorylation of GSK-3α/ß impacts on pro-fibrotic signaling following UUO. METHODS: UUO was induced in mice carrying a PKB/SGK-resistant GSK-3α/ß (gsk-3(KI)) and corresponding wild-type mice (gsk-3(WT)). Three days after the obstructive injury, expression of fibrosis markers in kidney tissues was analyzed by quantitative RT-PCR and western blotting. RESULTS: GSK-3α and GSK-3ß phosphorylation was absent in both, the non-obstructed and the obstructed kidney tissues from gsk-3(KI) mice but was increased by UUO in kidney tissues from gsk-3(WT) mice. Expression of α-smooth muscle actin, type I collagen and type III collagen in the non-obstructed kidney tissues was not significantly different between gsk-3(KI) mice and gsk-3(WT) mice but was significantly less increased in the obstructed kidney tissues from gsk-3(KI) mice than from gsk-3(WT) mice. After UUO treatment, renal ß-catenin protein abundance and renal expression of the ß-catenin sensitive genes: c-Myc, Dkk1, Twist and Lef1 were again significantly less increased in kidney tissues from gsk-3(KI) mice than from gsk-3(WT) mice. CONCLUSIONS: PKB/SGK-dependent phosphorylation of glycogen synthase kinase GSK-3 contributes to the pro-fibrotic signaling leading to renal tissue fibrosis in obstructive nephropathy.

Novel Roles for the Transcriptional Repressor E4BP4 in Both Cardiac Physiology and Pathophysiology.

Circadian clocks temporally orchestrate biological processes critical for cellular/organ function. For example, the cardiomyocyte circadian clock modulates cardiac metabolism, signaling, and electrophysiology over the course of the day, such that, disruption of the clock leads to age-onset cardiomyopathy (through unknown mechanisms). Here, we report that genetic disruption of the cardiomyocyte clock results in chronic induction of the transcriptional repressor E4BP4. Importantly, E4BP4 deletion prevents age-onset cardiomyopathy following clock disruption. These studies also indicate that E4BP4 regulates both cardiac metabolism (eg, fatty acid oxidation) and electrophysiology (eg, QT interval). Collectively, these studies reveal that E4BP4 is a novel regulator of both cardiac physiology and pathophysiology.

Sphingomyelinase-induced adhesion of eryptotic erythrocytes to endothelial cells.

Eryptosis, the suicidal erythrocyte death, leads to cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to the vascular wall by binding of phosphatidylserine to the CXC chemokine ligand 16 (CXCL16). Stimulators of eryptosis include increased cytosolic Ca(2+) activity, energy depletion, and activation of ceramide-producing sphingomyelinase. The present study explored whether sphingomyelinase triggers erythrocyte adhesion to endothelial cells. To this end, human erythrocytes were exposed for 6 h to bacterial sphingomyelinase (1-10 mU/ml) and phosphatidylserine exposure was estimated from fluorescent annexin-V-binding, cell volume from forward scatter in FACS-analysis, erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber under flow conditions at arterial shear rates, and CXCL16 protein abundance utilizing Western blotting and FACS analysis of fluorescent antibody binding. As a result, sphingomyelinase (≥1 mU/ml) triggered cell shrinkage, phosphatidylserine exposure and erythrocyte adhesion to HUVEC, effects blunted by Ca(2+) removal. Adhesion was significantly blunted by phosphatidylserine-coating annexin-V (5 µl/ml), following addition of neutralizing antibodies against endothelial CXCL16 (4 µg/ml) and following silencing of the CXCL16 gene with small interfering RNA. Pretreatment of HUVEC with sphingomyelinase upregulated CXCL16 protein abundance. Six hours pretreatment of HUVEC with sphingomyelinase (10 mU/ml) or C6-ceramide (50 µM) augmented erythrocyte adhesion following a 30-min treatment with Ca(2+) ionophore ionomycin (1 µM) or following energy depletion by 48-h glucose removal. Thus exposure to sphingomyelinase or C6-ceramide triggers eryptosis followed by phosphatidylserine- and CXCL16-sensitive adhesion of eryptotic erythrocytes to HUVEC.

Downregulation of Kv1.5 K channels by the AMP-activated protein kinase.

BACKGROUND: The voltage gated K(+) channel Kv1.5 participates in the repolarization of a wide variety of cell types. Kv1.5 is downregulated during hypoxia, which is known to stimulate the energy-sensing AMP-activated serine/threonine protein kinase (AMPK). AMPK is a powerful regulator of nutrient transport and metabolism. Moreover, AMPK is known to downregulate several ion channels, an effect at least in part due to stimulation of the ubiquitin ligase Nedd4- 2. The present study explored whether AMPK regulates Kv1.5. METHODS: cRNA encoding Kv1.5 was injected into Xenopus oocytes with and without additional injection of wild-type AMPK (α1 ß 1γ1), of constitutively active (γR70Q)AMPK (α1 ß 1γ1(R70Q)), of inactive mutant (αK45R)AMPK (α1(K45R)ß1γ1), or of Nedd4-2. Kv1.5 activity was determined by two-electrode voltage-clamp. Moreover, Kv1.5 protein abundance in the cell membrane was determined by chemiluminescence and immunostaining with subsequent confocal microscopy. RESULTS: Coexpression of wild-type AMPK(WT) and constitutively active AMPK(γR70Q), but not of inactive AMPK(αK45R) significantly reduced Kv1.5-mediated currents. Coexpression of constitutively active AMPKγR70Q further reduced Kv1.5 K(+) channel protein abundance in the cell membrane. Co-expression of Nedd4-2 similarly downregulated Kv1.5-mediated currents. CONCLUSION: AMPK is a potent regulator of Kv1.5. AMPK inhibits Kv1.5 presumably in part by activation of Nedd4- 2 with subsequent clearance of channel protein from the cell membrane.

Sgk1 sensitivity of Na(+)/H(+) exchanger activity and cardiac remodeling following pressure overload.

Sustained increase of cardiac workload is known to trigger cardiac remodeling with eventual development of cardiac failure. Compelling evidence points to a critical role of enhanced cardiac Na(+)/H(+) exchanger (NHE1) activity in the underlying pathophysiology. The signaling triggering up-regulation of NHE1 remained, however, ill defined. The present study explored the involvement of the serum- and glucocorticoid-inducible kinase Sgk1 in cardiac remodeling due to transverse aortic constriction (TAC). To this end, experiments were performed in gene targeted mice lacking functional Sgk1 (sgk1 (-/-)) and their wild-type controls (sgk1 (+/+)). Transcript levels have been determined by RT-PCR, cytosolic pH (pH( i )) utilizing 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) fluorescence, Na(+)/H(+) exchanger activity by the Na(+)-dependent realkalinization after an ammonium pulse, ejection fraction (%) utilizing cardiac cine magnetic resonance imaging and cardiac glucose uptake by PET imaging. As a result, TAC increased the mRNA expression of Sgk1 in sgk1 (+/+) mice, paralleled by an increase in Nhe1 transcript levels as well as Na(+)/H(+) exchanger activity, all effects virtually abrogated in sgk1 (-/-) mice. In sgk1 (+/+) mice, TAC induced a decrease in Pgc1a mRNA expression, while Spp1 mRNA expression was increased, both effects diminished in the sgk1 (-/-) mice. TAC was followed by a significant increase of heart and lung weight in sgk1 (+/+) mice, an effect significantly blunted in sgk1 (-/-) mice. TAC increased the transcript levels of Anp and Bnp, effects again significantly blunted in sgk1 (-/-) mice. TAC increased transcript levels of Collagen I and III as well as Ctgf mRNA and CTGF protein abundance, effects significantly blunted in sgk1 (-/-) mice. TAC further decreased the ejection fraction in sgk1 (+/+) mice, an effect again attenuated in sgk1 (-/-) mice. Also, cardiac FDG-glucose uptake was increased to a larger extent in sgk1 (+/+) mice than in sgk1 (-/-) mice after TAC. These observations point to an important role for SGK1 in cardiac remodeling and development of heart failure following an excessive work load.

Circadian REV-ERBs repress E4bp4 to activate NAMPT-dependent NAD+ biosynthesis and sustain cardiac function.

The heart is a highly metabolic organ that uses multiple energy sources to meet its demand for ATP production. Diurnal feeding-fasting cycles result in substrate availability fluctuations which, together with increased energetic demand during the active period, impose a need for rhythmic cardiac metabolism. The nuclear receptors REV-ERBα and ß are essential repressive components of the molecular circadian clock and major regulators of metabolism. To investigate their role in the heart, here we generated mice with cardiomyocyte (CM)-specific deletion of both Rev-erbs, which died prematurely due to dilated cardiomyopathy. Loss of Rev-erbs markedly downregulated fatty acid oxidation genes prior to overt pathology, which was mediated by induction of the transcriptional repressor E4BP4, a direct target of cardiac REV-ERBs. E4BP4 directly controls circadian expression of Nampt and its biosynthetic product NAD+ via distal cis-regulatory elements. Thus, REV-ERB-mediated E4BP4 repression is required for Nampt expression and NAD+ production by the salvage pathway. Together, these results highlight the indispensable role of circadian REV-ERBs in cardiac gene expression, metabolic homeostasis and function.

Nuclear IL-33 restrains the early conversion of fibroblasts to an extracellular matrix-secreting phenotype.

Interleukin (IL)-33 is a cytokine that appears to mediate fibrosis by signaling via its receptor ST2 (IL-33R/IL1RL1). It is also, however, a protein that after synthesis is sorted to the cell nucleus, where it appears to affect chromatin folding. Here we describe a novel role for nuclear IL-33 in regulating the fibroblast phenotype in murine kidney fibrosis driven by unilateral ureteral obstruction. Transcriptional profiling of IL-33-deficient kidneys 24 h after ligation revealed enhanced expression of fibrogenic genes and enrichment of gene sets involved in extracellular matrix formation and remodeling. These changes relied on intracellular effects of IL-33, because they were not reproduced by treatment with a neutralizing antibody to IL-33 that prevents IL-33R/ST2L receptor signaling nor were they observed in IL-33R/ST2-deficient kidneys. To further explore the intracellular function of IL-33, we established transcription profiles of human fibroblasts, observing that knockdown of IL-33 skewed the transcription profile from an inflammatory towards a myofibroblast phenotype, reflected in higher levels of COL3A1, COL5A1 and transgelin protein, as well as lower expression levels of IL6, CXCL8, CLL7 and CCL8. In conclusion, our findings suggest that nuclear IL-33 in fibroblasts dampens the initial profibrotic response until persistent stimuli, as enforced by UUO, can override this protective mechanism.