Klotho is a potent regulator of 1,25-hydroxyvitamin D3 [1,25(OH) 2 D 3 ] formation and calcium-phosphate metabolism. Klotho-hypomorphic mice (kl/kl mice) suffer from severe growth deficits, rapid aging, hyperphosphatemia, hyperaldosteronism, and extensive vascular and soft tissue calcification. Sequelae of klotho deficiency are similar to those of end-stage renal disease. We show here that the mineralocorticoid receptor antagonist spironolactone reduced vascular and soft tissue calcification and increased the life span of kl/kl mice, without significant effects on 1,25(OH) 2 D 3 , FGF23, calcium, and phosphate plasma concentrations. Spironolactone also reduced the expression of osteoinductive Pit1 and Tnfa mRNA, osteogenic transcription factors, and alkaline phosphatase (Alpl) in calcified tissues of kl/kl mice. In human aortic smooth muscle cells (HAoSMCs), aldosterone dose-dependently increased PIT1 mRNA expression, an effect paralleled by increased expression of osteogenic transcription factors and enhanced ALP activity. The effects of aldosterone were reversed by both spironolactone treatment and PIT1 silencing and were mitigated by FGF23 cotreatment in HAoSMCs. In conclusion, aldosterone contributes to vascular and soft tissue calcification, an effect due, at least in part, to stimulation of spironolactone-sensitive, PIT1-dependent osteoinductive signaling.

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 2ϩ 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 (Ն1 mU/ml) triggered cell shrinkage, phosphatidylserine exposure and erythrocyte adhesion to HUVEC, effects blunted by Ca 2ϩ removal. Adhesion was significantly blunted by phosphatidylserine-coating annexin-V (5 l/ml), following addition of neutralizing antibodies against endothelial CXCL16 (4 g/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 M) augmented erythrocyte adhesion following a 30-min treatment with Ca 2ϩ ionophore ionomycin (1 M) 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.

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 β 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-erb s, 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.

Background Perturbations in myocardial substrate utilization have been proposed to contribute to the pathogenesis of cardiac dysfunction in diabetic subjects. The failing heart in nondiabetics tends to decrease reliance on fatty acid and glucose oxidation, and increases reliance on ketone body oxidation. In contrast, little is known regarding the mechanisms mediating this shift among all 3 substrates in diabetes mellitus. Therefore, we tested the hypothesis that changes in myocardial glucose utilization directly influence ketone body catabolism. Methods and Results We examined ventricular‐cardiac tissue from the following murine models: (1) streptozotocin‐induced type 1 diabetes mellitus; (2) high‐fat‐diet–induced glucose intolerance; and transgenic inducible cardiac‐restricted expression of (3) glucose transporter 4 (transgenic inducible cardiac restricted expression of glucose transporter 4); or (4) dominant negative O ‐GlcNAcase. Elevated blood glucose (type 1 diabetes mellitus and high‐fat diet mice) was associated with reduced cardiac expression of β‐hydroxybutyrate‐dehydrogenase and succinyl‐CoA:3‐oxoacid CoA transferase. Increased myocardial β‐hydroxybutyrate levels were also observed in type 1 diabetes mellitus mice, suggesting a mismatch between ketone body availability and utilization. Increased cellular glucose delivery in transgenic inducible cardiac restricted expression of glucose transporter 4 mice attenuated cardiac expression of both Bdh1 and Oxct1 and reduced rates of myocardial BDH1 activity and β‐hydroxybutyrate oxidation. Moreover, elevated cardiac protein O ‐GlcNAcylation (a glucose‐derived posttranslational modification) by dominant negative O ‐GlcNAcase suppressed β‐hydroxybutyrate dehydrogenase expression. Consistent with the mouse models, transcriptomic analysis confirmed suppression of BDH1 and OXCT1 in patients with type 2 diabetes mellitus and heart failure compared with nondiabetic patients. Conclusions Our results provide evidence that increased glucose leads to suppression of cardiac ketolytic capacity through multiple mechanisms and identifies a potential crosstalk between glucose and ketone body metabolism in the diabetic myocardium.

Vascular calcification resulting from hyperphosphatemia is a major determinant of mortality in chronic kidney disease (CKD). Vascular calcification is driven by aldosterone-sensitive osteogenic transformation of vascular smooth muscle cells (VSMCs). We show that even in absence of exogenous aldosterone, silencing and pharmacological inhibition (spironolactone, eplerenone) of the mineralocorticoid receptor (MR) ameliorated phosphate-induced osteo-/chondrogenic transformation of primary human aortic smooth muscle cells (HAoSMCs). High phosphate concentrations up-regulated aldosterone synthase (CYP11B2) expression in HAoSMCs. Silencing and deficiency of CYP11B2 in VSMCs ameliorated phosphate-induced osteogenic reprogramming and calcification. Phosphate treatment was followed by nuclear export of APEX1, a CYP11B2 transcriptional repressor. APEX1 silencing up-regulated CYP11B2 expression and stimulated osteo-/chondrogenic transformation. APEX1 overexpression blunted the phosphate-induced osteo-/chondrogenic transformation and calcification of HAoSMCs. Cyp11b2 expression was higher in aortic tissue of hyperphosphatemic klotho-hypomorphic (kl/kl) mice than in wild-type mice. In adrenalectomized kl/kl mice, spironolactone treatment still significantly ameliorated aortic osteoinductive reprogramming. Our findings suggest that VSMCs express aldosterone synthase, which is up-regulated by phosphate-induced disruption of APEX1-dependent gene suppression. Vascular CYP11B2 may contribute to stimulation of VSMCs osteo-/chondrogenic transformation during hyperphosphatemia.

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.

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 β 1γ1), of constitutively active ^γR70QAMPK (α1 β 1γ1(R70Q)), of inactive mutant ^αK45RAMPK (α1(K45R)β1γ1), 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.

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