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Purpose Vascular inflammation and disturbed metabolism are observed in heart failure and type 2 diabetes mellitus. Glycolytic enzyme hexokinase II (HKII) is upregulated by inflammation. We hypothesized that SGLT2 inhibitors Canagliflozin (Cana), Empagliflozin (Empa) or Dapagliflozin (Dapa) reduces inflammation via HKII in endothelial cells, and that HKII-dependent inflammation is determined by ERK1/2, NF-κB. and/or AMPK activity in lipopolysaccharide (LPS)-stimulated human coronary artery endothelial cells (HCAECs). Methods HCAECs were pre-incubated with 3 μM or 10 μM Cana, 1 μM, 3 μM or 10 μM Empa or 0.5 μM, 3 μM or 10 μM Dapa (16 h) and subjected to 3 h LPS (1 μg/mL). HKII was silenced via siRNA transfection. Interleukin-6 (IL-6) release was measured by ELISA. Protein levels of HK I and II, ERK1/2, AMPK and NF-κB were detected using infra-red western blot. Results LPS increased IL-6 release and ERK1/2 phosphorylation; Cana prevented these pro-inflammatory responses (IL-6: pg/ml, control 46 ± 2, LPS 280 ± 154 p < 0.01 vs. control, LPS + Cana 96 ± 40, p < 0.05 vs. LPS). Cana reduced HKII expression (HKII/GAPDH, control 0.91 ± 0.16, Cana 0.71 ± 0.13 p < 0.05 vs. control, LPS 1.02 ± 0.25, LPS + Cana 0.82 ± 0.24 p < 0.05 vs. LPS). Empa and Dapa were without effect on IL-6 release and HKII expression in the model used. Knockdown of HKII by 37% resulted caused partial loss of Cana-mediated IL-6 reduction (pg/ml, control 35 ± 5, LPS 188 ± 115 p < 0.05 vs. control, LPS + Cana 124 ± 75) and ERK1/2 activation by LPS. In LPS-stimulated HCAECs, Cana, but not Empa or Dapa, activated AMPK. AMPK activator A769662 reduced IL-6 release. Conclusion Cana conveys anti-inflammatory actions in LPS-treated HCAECs through 1) reductions in HKII and ERK1/2 phosphorylation and 2) AMPK activation. These data suggest a novel anti-inflammatory mechanism of Cana through HKII.

AimsSodium glucose cotransporter 2 (SGLT2) inhibitors have sodium–hydrogen exchanger (NHE) inhibition properties in isolated cardiomyocytes, but it is unknown whether these properties extend to the intact heart during ischaemia–reperfusion (IR) conditions. NHE inhibitors as Cariporide delay time to onset of contracture (TOC) during ischaemia and reduce IR injury. We hypothesized that, in the ex vivo heart, Empagliflozin (Empa) mimics Cariporide during IR by delaying TOC and reducing IR injury. To facilitate translation to in vivo conditions with insulin present, effects were examined in the absence and presence of insulin.Methods and resultsIsolated C57Bl/6NCrl mouse hearts were subjected to 25 min I and 120 min R without and with 50 mU/L insulin. Without insulin, Empa and Cari delayed TOC by 100 and 129 s, respectively, yet only Cariporide reduced IR injury [infarct size (mean ± SEM in %) from 51 ± 6 to 34 ± 5]. Empa did not delay TOC in the presence of the NHE1 inhibitor Eniporide. Insulin perfusion increased tissue glycogen content at baseline (from 2 ± 2 µmol to 42 ± 1 µmol glycosyl units/g heart dry weight), amplified G6P and lactate accumulation at end-ischaemia, thereby decreased mtHKII and exacerbated IR injury. Under these conditions, Empa (1 µM) and Cariporide (10 µM) were without effect on TOC and IR injury. Empa and Cariporide both inhibited NHE activity, in isolated cardiomyocytes, independent of insulin.ConclusionsIn the absence of insulin, Empa and Cariporide strongly delayed the time to onset of contracture during ischaemia. In the presence of insulin, both Empa and Cari were without effect on IR, possibly because of severe ischaemic acidification. Insulin exacerbates IR injury through increased glycogen depletion during ischaemia and consequently mtHKII dissociation. The data suggest that also in the ex vivo intact heart Empa exerts direct cardiac effects by inhibiting NHE during ischaemia, but not during reperfusion.

Contains fulltext : 239064.pdf (Publisher’s version ) (Open Access) BACKGROUND: Proton magnetic resonance spectroscopy ((1) H-MRS) of the human heart is deemed to be a quantitative method to investigate myocardial metabolite content, but thorough validations of in vivo measurements against invasive techniques are lacking. PURPOSE: To determine measurement precision and accuracy for quantifications of myocardial total creatine and triglyceride content with localized (1) H-MRS. STUDY TYPE: Test-retest repeatability and measurement validation study. SUBJECTS: Sixteen volunteers and 22 patients scheduled for open-heart aortic valve replacement or septal myectomy. FIELD STRENGTH/SEQUENCE: Prospectively ECG-triggered respiratory-gated free-breathing single-voxel point-resolved spectroscopy (PRESS) sequence at 3 T. ASSESSMENT: Myocardial total creatine and triglyceride content were quantified relative to the total water content by fitting the (1) H-MR spectra. Precision was assessed with measurement repeatability. Accuracy was assessed by validating in vivo (1) H-MRS measurements against biochemical assays in myocardial tissue from the same subjects. STATISTICAL TESTS: Intrasession and intersession repeatability was assessed using Bland-Altman analyses. Agreement between (1) H-MRS measurements and biochemical assay was tested with regression analyses. RESULTS: The intersession repeatability coefficient for myocardial total creatine content was 41.8% with a mean value of 0.083% ± 0.020% of the total water signal, and 36.7% for myocardial triglyceride content with a mean value of 0.35% ± 0.13% of the total water signal. Ex vivo myocardial total creatine concentrations in tissue samples correlated with the in vivo myocardial total creatine content measured with (1) H-MRS: n = 22, r = 0.44; P

Aims/hypothesis: Changes in cardiac metabolism and ion homeostasis precede and drive cardiac remodeling and heart failure development. We previously demonstrated that sodium/glucose cotransporter 2 inhibitors (SGLT2i's) have direct cardiac effects on ion homeostasis, possibly through inhibition of the cardiac sodium/hydrogen exchanger (NHE-1). Here, we hypothesize that Empagliflozin (EMPA) also possesses direct and acute cardiac effects on glucose and fatty acid metabolism of isolated type II diabetes mellitus (db/db) mouse hearts. In addition, we explore whether direct effects on glucose metabolism are nullified in the presence of an NHE-1 inhibitor.Methods: Langendorff-perfused type II diabetic db/db mouse hearts were examined in three different series: 1: 13C glucose perfusions (n = 32); 2: 13C palmitate perfusions (n = 13); and 3: 13C glucose + 10 μM Cariporide (specific NHE-1 inhibitor) perfusions (n = 17). Within each series, EMPA treated hearts (1 μM EMPA) were compared with vehicle-perfused hearts (0.02% DMSO). Afterwards, hearts were snap frozen and lysed for stable isotope analysis and metabolomics using LC-MS techniques. Hearts from series 1 were also analyzed for phosphorylation status of AKT, STAT3, AMPK, ERK, and eNOS (n = 8 per group).Results: Cardiac mechanical performance, oxygen consumption and protein phosphorylation were not altered by 35 min EMPA treatment. EMPA was without an overall acute and direct effect on glucose or fatty acid metabolism. However, EMPA did specifically decrease cardiac lactate labeling in the 13C glucose perfusions (13C labeling of lactate: 58 ± 2% vs. 50 ± 3%, for vehicle and EMPA, respectively; P = 0.02), without changes in other glucose metabolic pathways. In contrast, EMPA increased cardiac labeling in α-ketoglutarate derived from 13C palmitate perfusions (13C labeling of α-KG: 79 ± 1% vs. 86 ± 1% for vehicle and EMPA, respectively; P = 0.01). Inhibition of the NHE by Cariporide abolished EMPA effects on lactate labeling from 13C glucose.Conclusions: The present study shows for the first time that the SGLT2 inhibitor Empagliflozin has acute specific metabolic effects in isolated diabetic hearts, i.e., decreased lactate generation from labeled glucose and increased α-ketoglutarate synthesis from labeled palmitate. The decreased lactate generation by EMPA seems to be mediated through NHE-1 inhibition.

BACKGROUND/AIMS: Heart failure is characterized by chronic low-grade vascular inflammation, which in itself can lead to endothelial dysfunction. Clinical trials showed reductions in heart failure-related hospitalizations of type 2 diabetic patients using sodium glucose co-transporter 2 inhibitors (SGLT2i's). Whether and how SGLT2i's directly affect the endothelium under inflammatory conditions is not completely understood. The aim of the study was to investigate whether the SGLT2i Empagliflozin (EMPA) and Dapagliflozin (DAPA) reduce tumor necrosis factor α (TNFα) induced endothelial inflammation in vitro. METHODS: Human coronary arterial endothelial cells (HCAECs) and human umbilical vein endothelial cells (HUVECs) were (pre-)incubated with 1 µM EMPA or DAPA and subsequently exposed to 10 ng/ml TNFα. ROS and NO were measured using live cell imaging. Target proteins were either determined by infrared western blotting or fluorescence activated cell sorting (FACS). The connection between Cav-1 and eNOS was determined by co-immunoprecipitation. RESULTS: Nitric oxide (NO) bioavailability was reduced by TNFα and both EMPA and DAPA restored NO levels in TNFα-stimulated HCAECs. Intracellular ROS was increased by TNFα, and this increase was completely abolished by EMPA and DAPA in HCAECs by means of live cell imaging. eNOS signaling was significantly disturbed after 24 h when cells were exposed to TNFα for 24h, yet the presence of both SGLT2is did not prevent this disruption. TNFα-induced enhanced permeability at t=24h was unaffected in HUVECs by EMPA. Similarly, adhesion molecule expression (VCAM-1 and ICAM-1) was elevated after 4h TNFα (1.5-5.5 fold increase of VCAM-1 and 4-12 fold increase of ICAM-1) but were unaffected by EMPA and DAPA in both cell types. Although we detected expression of SGLT2 protein levels, the fact that we could not silence this expression by means of siRNA and the mRNA levels of SGLT2 were not detectable in HCAECs, suggests aspecificity or our SGLT2 antibody and absence of SGLT2 in our cells. CONCLUSION: These data suggest that EMPA and DAPA rather restore NO bioavailability by inhibiting ROS generation than by affecting eNOS expression or signaling, barrier function and adhesion molecules expression in TNFα-induced endothelial cells. Furthermore, the observed effects cannot be ascribed to the inhibition of SGLT2 in endothelial cells.

Sodium glucose cotransporter 2 inhibitors (SGLT2i) are the first antidiabetic compounds that effectively reduce heart failure hospitalization and cardiovascular death in type 2 diabetics. Being explicitly designed to inhibit SGLT2 in the kidney, SGLT2i have lately been investigated for their off-target cardiac actions. Here, we review the direct effects of SGLT2i Empagliflozin (Empa), Dapagliflozin (Dapa), and Canagliflozin (Cana) on various cardiac cell types and cardiac function, and how these may contribute to the cardiovascular benefits observed in large clinical trials. SGLT2i impaired the Na+/H+ exchanger 1 (NHE-1), reduced cytosolic [Ca2+] and [Na+] and increased mitochondrial [Ca2+] in healthy cardiomyocytes. Empa, one of the best studied SGLT2i, maintained cell viability and ATP content following hypoxia/reoxygenation in cardiomyocytes and endothelial cells. SGLT2i recovered vasoreactivity of hyperglycemic and TNF-α-stimulated aortic rings and of hyperglycemic endothelial cells. Anti-inflammatory actions of Cana in IL-1β-treated HUVEC and of Dapa in LPS-treated cardiofibroblast were mediated by AMPK activation. In isolated mouse hearts, Empa and Cana, but not Dapa, induced vasodilation. In ischemia-reperfusion studies of the isolated heart, Empa delayed contracture development during ischemia and increased mitochondrial respiration post-ischemia. Direct cardiac effects of SGLT2i target well-known drivers of diabetes and heart failure (elevated cardiac cytosolic [Ca2+] and [Na+], activated NHE-1, elevated inflammation, impaired vasorelaxation, and reduced AMPK activity). These cardiac effects may contribute to the large beneficial clinical effects of these antidiabetic drugs.

Both the absence of cyclophilin D (CypD) and the presence of mitochondrial bound hexokinase II (mtHKII) protect the heart against ischemia/reperfusion (I/R) injury. It is unknown whether CypD determines the amount of mtHKII in the heart. We examined whether CypD affects mtHK in normoxic, ischemic and preconditioned isolated mouse hearts. Wild type (WT) and CypD−/− mouse hearts were perfused with glucose only and subjected to 25 min ischemia and reperfusion. At baseline, cytosolic and mtHK was similar between hearts. CypD ablation protected against I/R injury and increased ischemic preconditioning (IPC) effects, without affecting end-ischemic mtHK. When hearts were perfused with glucose, glutamine, pyruvate and lactate, the preparation was more stable and CypD ablation−resulted in more protection that was associated with increased mtHK activity, leaving little room for additional protection by IPC. In conclusion, in glucose only-perfused hearts, deletion of CypD is not associated with end-ischemic mitochondrial-HK binding. In contrast, in the physiologically more relevant multiple-substrate perfusion model, deletion of CypD is associated with an increased mtHK activity, possibly explaining the increased protection against I/R injury.