Aims: Sodium-glucose-cotransporter-2 inhibitors showed favourable cardiovascular outcomes, but the underlying mechanisms are still elusive. This study investigated the mechanisms of empagliflozin in human and murine heart failure with preserved ejection fraction (HFpEF). Methods and results: The acute mechanisms of empagliflozin were investigated in human myocardium from patients with HFpEF and murine ZDF obese rats, which were treated in vivo. As shown with immunoblots and ELISA, empagliflozin significantly suppressed increased levels of ICAM-1, VCAM-1, TNF-α, and IL-6 in human and murine HFpEF myocardium and attenuated pathological oxidative parameters (H2O2, 3-nitrotyrosine, GSH, lipid peroxide) in both cardiomyocyte cytosol and mitochondria in addition to improved endothelial vasorelaxation. In HFpEF, we found higher oxidative stress-dependent activation of eNOS leading to PKGIα oxidation. Interestingly, immunofluorescence imaging and electron microscopy revealed that oxidized PKG1α in HFpEF appeared as dimers/polymers localized to the outer-membrane of the cardiomyocyte. Empagliflozin reduced oxidative stress/eNOS-dependent PKGIα oxidation and polymerization resulting in a higher fraction of PKGIα monomers, which translocated back to the cytosol. Consequently, diminished NO levels, sGC activity, cGMP concentration, and PKGIα activity in HFpEF increased upon empagliflozin leading to improved phosphorylation of myofilament proteins. In skinned HFpEF cardiomyocytes, empagliflozin improved cardiomyocyte stiffness in an anti-oxidative/PKGIα-dependent manner. Monovariate linear regression analysis confirmed the correlation of oxidative stress and PKGIα polymerization with increased cardiomyocyte stiffness and diastolic dysfunction of the HFpEF patients. Conclusion: Empagliflozin reduces inflammatory and oxidative stress in HFpEF and thereby improves the NO–sGC–cGMP–cascade and PKGIα activity via reduced PKGIα oxidation and polymerization leading to less pathological cardiomyocyte stiffness.

Peroxynitrite (ONOO⁻) contributes to coronary microvascular dysfunction in diabetes mellitus (DM). We hypothesized that in DM, ONOO⁻ interferes with the function of coronary endothelial caveolae, which plays an important role in nitric oxide (NO)-dependent vasomotor regulation. Flow-mediated dilation (FMD) of coronary arterioles was investigated in DM (n = 41) and non-DM (n = 37) patients undergoing heart surgery. NO-mediated coronary FMD was significantly reduced in DM patients, which was restored by ONOO⁻ scavenger, iron-(III)-tetrakis(N-methyl-49pyridyl)porphyrin-pentachloride, or uric acid, whereas exogenous ONOO⁻ reduced FMD in non-DM subjects. Immunoelectron microscopy demonstrated an increased 3-nitrotyrosine formation (ONOO⁻-specific protein nitration) in endothelial plasma membrane in DM, which colocalized with caveolin-1 (Cav-1), the key structural protein of caveolae. The membrane-localized Cav-1 was significantly reduced in DM and also in high glucose–exposed coronary endothelial cells. We also found that DM patients exhibited a decreased number of endothelial caveolae, whereas exogenous ONOO⁻ reduced caveolae number. Correspondingly, pharmacological (methyl-β-cyclodextrin) or genetic disruption of caveolae (Cav-1 knockout mice) abolished coronary FMD, which was rescued by sepiapterin, the stable precursor of NO synthase (NOS) cofactor, tetrahydrobiopterin. Sepiapterin also restored coronary FMD in DM patients. Thus, we propose that ONOO⁻ selectively targets and disrupts endothelial caveolae, which contributes to NOS uncoupling, and, hence, reduced NO-mediated coronary vasodilation in DM patients.

Objective—: A disintegrin and metalloproteinase ADAM17 (tumor necrosis factor-α [TNF]–converting enzyme) regulates soluble TNF levels. We tested the hypothesis that aging-induced activation in adipose tissue (AT)-expressed ADAM17 contributes to the development of remote coronary microvascular dysfunction in obesity. Approach and Results—: Coronary arterioles (CAs, ≈90 µm) from right atrial appendages and mediastinal AT were examined in patients (aged: 69±11 years, BMI: 30.2±5.6 kg/m ² ) who underwent open heart surgery. CA and AT were also studied in 6-month and 24-month lean and obese mice fed a normal or high-fat diet. We found that obesity elicited impaired endothelium-dependent CA dilations only in older patients and in aged high-fat diet mice. Transplantation of AT from aged obese, but not from young or aged, mice increased serum cytokine levels, including TNF, and impaired CA dilation in the young recipient mice. In patients and mice, obesity was accompanied by age-related activation of ADAM17, which was attributed to vascular endothelium–expressed ADAM17. Excess, ADAM17-shed TNF from AT arteries in older obese patients was sufficient to impair CA dilation in a bioassay in which the AT artery was serially connected to a CA. Moreover, we found that the increased activity of endothelial ADAM17 is mediated by a diminished inhibitory interaction with caveolin-1, owing to age-related decline in caveolin-1 expression in obese patients and mice or to genetic deletion of caveolin-1. Conclusions—: The present study indicates that aging and obesity cooperatively reduce caveolin-1 expression and increase vascular endothelial ADAM17 activity and soluble TNF release in AT, which may contribute to the development of remote coronary microvascular dysfunction in older obese patients.

It is believed that obesity has detrimental effects on the coronary circulation. These include immediate changes in coronary arterial vasomotor responsiveness and the development of occlusive large coronary artery disease. Despite its critical role in regulating myocardial perfusion, the altered behavior of coronary resistance arteries, which gives rise to coronary microvascular disease (CMD) is poorly understood in obesity. A chronic, low-grade vascular inflammation has been long considered as one of the main underlying pathology behind CMD. The expanded adipose tissue and the infiltrating macrophages are the major sources of pro-inflammatory mediators that have been implicated in causing inadequate myocardial perfusion and, in a long term, development of heart failure in obese patients. Much less is known the mechanisms regulating the release of these cytokines into the circulation that enable them to exert their remote effects in the coronary microcirculation. This mini review aims to examine recent studies describing alterations in the vasomotor function of coronary resistance arteries and the role of adipose tissue-derived pro-inflammatory cytokines and adipokines in contributing to CMD in obesity. We provide examples of regulatory mechanisms by which adipokines are released from adipose tissue to exert their remote inflammatory effects on coronary microvessels. We identify some of the important challenges and opportunities going forward.

Rationale. Diabetic nephropathy is a major cause of end-stage renal disease, associated with endothelial dysfunction. Chronic supplementation of L-arginine (L-arg), the substrate for endothelial nitric oxide synthase (eNOS), failed to improve vascular function. L-citrulline (L-cit) supplementation not only increases L-arg synthesis, but also inhibits cytosolic arginase I (Arg I), a competitor of eNOS for the use of L-arg, in the vasculature. Aims. To investigate whether L-cit treatment reduces diabetic nephropathy in streptozotocin (STZ)-induced type 1 diabetes in mice and rats and to study its effects on arginase II (ArgII) function, the main renal isoform. Methods. STZ-C57BL6 mice received L-cit or vehicle supplemented in the drinking water. For comparative analysis, diabetic ArgII knock out mice and L-cit-treated STZ-rats were evaluated. Results. L-cit exerted protective effects in kidneys of STZ-rats, and markedly reduced urinary albumin excretion, tubulo-interstitial fibrosis and kidney hypertrophy, observed in untreated diabetic mice. Intriguingly, L-cit treatment was accompanied by a sustained elevation of tubular ArgII at 16 wks and significantly enhanced plasma levels of the anti-inflammatory cytokine IL-10. Diabetic ArgII knock out mice showed greater BUN levels, hypertrophy, and dilated tubules than diabetic wild type mice. Despite a marked reduction in collagen deposition in ArgII knock out mice, their albuminuria was not significantly different from diabetic wild type animals. L-cit also restored NO/ROS balance and barrier function in high glucose-treated monolayers of human glomerular endothelial cells. Moreover, L-cit also has the ability to establish an anti-inflammatory profile, characterized by increased IL-10 and reduced IL-1beta and IL-12(p70) generation in the human proximal tubular cells. Conclusions. L-cit supplementation established an anti-inflammatory profile and significantly preserved the nephron function during type 1 diabetes.

Conducted vasodilation is essential to coordinate vascular resistance along distances to ensure adequate tissue perfusion. We hypothesized that conducted vasodilation of coronary resistance arteries declines with age. Coronary arterioles were dissected from right atrial appendage of patients ( n = 27) undergoing cardiac surgery. Arterioles (∼100 μm) were cannulated and pressurized (80 mmHg), and developed spontaneous myogenic tone. Conducted vasodilation was initiated by locally administering the endothelium-dependent agonist bradykinin (BK; 100 μM) ejected from a glass micropipette (∼3 μm tip opening, positioned in close proximity to the vessel wall). Diameter changes were measured at local and upstream sites (500 and 1,000 μm from the stimulus) with videomicroscopy. Local administration of BK elicited vasodilation, the magnitude of which increased with the duration of stimulus (69 ± 6, 81 ± 6, 90 ± 2%, after 1, 3, and 5 × 100 ms, respectively). BK-induced dilation remained substantial at upstream sites (500 μm: 53 ± 7%; 1,000 μm: 46 ± 9%). The gap junction uncoupler carbenoxolone or 18-α-glycyrrhetinic acid did not affect local responses, but diminished conducted vasodilation. Inhibitors of small/intermediate conductance calcium-activated potassium channels (SK_Ca/IK_Ca), apamin and TRAM34, reduced dilations both at local and remote sites. We found that conducted dilation, but not the local response, was significantly reduced in older (≥64 yr) patients. The nitric oxide (NO) synthesis inhibitor N^ω-nitro-l-arginine methyl ester did not affect local responses, but markedly reduced conducted dilation in younger (<64 yr) individuals. Collectively, we show that human coronary arterioles exhibit SK_Ca/IK_Ca-mediated hyperpolarization spread through gap junctions, which contributes to conducted vasodilation initiated by focal application of BK. We demonstrate that conducted dilation declines with age, likely due to reduced NO availability, which plays a permissive role in propagating longitudinal vasomotor signaling.