Diminished bioavailability of nitric oxide (NO), the gaseous signaling molecule involved in the regulation of numerous vital biological functions, contributes to the development and progression of multiple age- and lifestyle-related diseases. While l-arginine is the precursor for the synthesis of NO by endothelial-nitric oxide synthase (eNOS), oral l-arginine supplementation is largely ineffective at increasing NO synthesis and/or bioavailability for a variety of reasons. l-citrulline, found in high concentrations in watermelon, is a neutral alpha-amino acid formed by enzymes in the mitochondria that also serves as a substrate for recycling l-arginine. Unlike l-arginine, l-citrulline is not quantitatively extracted from the gastrointestinal tract (i.e., enterocytes) or liver and its supplementation is therefore more effective at increasing l-arginine levels and NO synthesis. Supplementation with l-citrulline has shown promise as a blood pressure lowering intervention (both resting and stress-induced) in adults with pre-/hypertension, with pre-clinical (animal) evidence for atherogenic-endothelial protection. Preliminary evidence is also available for l-citrulline-induced benefits to muscle and metabolic health (via vascular and non-vascular pathways) in susceptible/older populations. In this review, we examine the impact of supplementing this important urea cycle intermediate on cardiovascular and metabolic health outcomes and identify future directions for investigating its therapeutic impact on cardiometabolic health.

Background: The renal sympathetic nervous system modulates systemic blood pressure, cardiac performance, and renal function. Pathological increases in renal sympathetic nerve activity contribute to the pathogenesis of heart failure with preserved ejection fraction (HFpEF). We investigated the effects of renal sympathetic denervation performed at early or late stages of HFpEF progression. Methods and Results: Male ZSF1 obese rats were subjected to radiofrequency renal denervation (RF‐RDN) or sham procedure at either 8 weeks or 20 weeks of age and assessed for cardiovascular function, exercise capacity, and cardiorenal fibrosis. Renal norepinephrine and renal nerve tyrosine hydroxylase staining were performed to quantify denervation following RF‐RDN. In addition, renal injury, oxidative stress, inflammation, and profibrotic biomarkers were evaluated to determine pathways associated with RDN. RF‐RDN significantly reduced renal norepinephrine and tyrosine hydroxylase content in both study cohorts. RF‐RDN therapy performed at 8 weeks of age attenuated cardiac dysfunction, reduced cardiorenal fibrosis, and improved endothelial‐dependent vascular reactivity. These improvements were associated with reductions in renal injury markers, expression of renal NLR family pyrin domain containing 3/interleukin 1β, and expression of profibrotic mediators. RF‐RDN failed to exert beneficial effects when administered in the 20‐week‐old HFpEF cohort. Conclusions: Our data demonstrate that early RF‐RDN therapy protects against HFpEF disease progression in part due to the attenuation of renal fibrosis and inflammation. In contrast, the renoprotective and left ventricular functional improvements were lost when RF‐RDN was performed in later HFpEF progression. These results suggest that RDN may be a viable treatment option for HFpEF during the early stages of this systemic inflammatory disease.

• Skeletal muscle metabolism is an important factor for exercise capacity in cardiometabolic HFpEF but is not well understood. • Using state-of-the-art metabolic cage technology and a multiomic approach, we define a metabolic signature of skeletal muscle dysfunction and exercise intolerance in a mouse “2-hit” model of cardiometabolic HFpEF. • We then show voluntary wheel running exercise restores skeletal muscle fatty acid and BCAA substrate use and improves exercise capacity without significant improvement in cardiac hemodynamic function. • FAO and BCAA catabolic pathways are potentially important targets to improve exercise intolerance in cardiometabolic HFpEF.

Background and Purpose: Sodium glucose cotransporter 2 inhibitors (SGLT2i) have emerged as a potent therapy for heart failure with preserved ejection fraction (HFpEF). Hydrogen sulphide (H₂S), a well‐studied cardioprotective agent, could be beneficial in HFpEF. SGLT2i monotherapy and combination therapy involving an SGLT2i and H₂S donor in two preclinical models of cardiometabolic HFpEF was investigated.Experimental Approach: Nine‐week‐old C57BL/6N mice received L‐NAME and a 60% high fat diet for five weeks. Mice were then randomized to either control, SGLT2i monotherapy or SGLT2i and H₂S donor, SG1002, for five additional weeks. Ten‐week‐old ZSF1 obese rats were randomized to control, SGLT2i or SGLT2i and SG1002 for 8 weeks. SG1002 monotherapy was investigated in additional animals. Cardiac function (echocardiography and haemodynamics), exercise capacity, glucose handling and multiorgan pathology were monitored during experimental protocols.Key Results: SGLT2i treatment improved E/e′ ratio and treadmill exercise in both models. Combination therapy afforded increases in cardiovascular sulphur bioavailability that coincided with improved left end‐diastolic function (E/e′ ratio), exercise capacity, metabolic state, cardiorenal fibrosis, and hepatic steatosis. Follow‐up studies with SG1002 monotherapy revealed improvements in diastolic function, exercise capacity and multiorgan histopathology.Conclusions and Implications: SGLT2i monotherapy remediated pathological complications exhibited by two well‐established HFpEF models. Adjunctive H₂S therapy resulted in further improvements of cardiometabolic perturbations beyond SGLT2i monotherapy. Follow‐up SG1002 monotherapy studies inferred an improved phenotype with combination therapy beyond either monotherapy. These data demonstrate the differing effects of SGLT2i and H₂S therapy while also revealing the superior efficacy of the combination therapy in cardiometabolic HFpEF.

BACKGROUND: Recent studies suggest aberrant elevation of iNOS (inducible NO synthase) expression and excessive protein s -nitrosylation promote the pathogenesis of heart failure with preserved ejection fraction (HFpEF). However, the interplay between NO bioavailability, enzymatic regulation of protein s -nitrosylation by transnitrosylase and denitrosylase, and HFpEF progression remains poorly defined. We investigated the molecular basis of nitrosative stress in HFpEF, focusing on alterations in NO signaling and regulation of protein s -nitrosylation. METHODS: Circulating nitrite (NO bioavailability) and nitrosothiols were quantified in patients with HFpEF. Parallel studies using rodent models of cardiometabolic HFpEF were performed to evaluate cardiac function, NO signaling, and total nitroso species during disease progression. Single-nucleus RNA sequencing and proteomic analysis were conducted to identify regulatory genes and cellular targets of pathological s -nitrosylation. RESULTS: In patients with HFpEF, circulating nitrosothiols were significantly elevated, indicating heightened nitrosative stress, whereas nitrite levels remained unchanged. In ZSF1 Obese (ob) rats, NO bioavailability declined with age, whereas total nitroso species progressively increased as HFpEF worsened. Transcriptomic analysis revealed marked upregulation of a transnitrosylase HBb (hemoglobin-β subunit), validated in both rat and human HFpEF hearts. Enzymatic assays demonstrated aberrant functions of Trx2 (thioredoxin 2) and GSNOR (S-nitrosoglutathione reductase) in ZSF1 Ob hearts. Cell-based experiments confirmed that altered expression or function of HBb, Trx2, and GSNOR resulted in elevated cellular RxNO. Additionally, similar dysregulation of s -nitrosylation dynamics was observed in the peripheral organs, such as the kidneys and liver, in HFpEF. CONCLUSIONS: These data demonstrate that nitrosative stress, evidenced by dysregulated protein s -nitrosylation occurs in the heart and peripheral organs in cardiometabolic HFpEF. Pathological alterations in NO bioavailability resulting from alterations in NOS expression or function alone do not account for this phenotype. Instead, pathological protein s -nitrosylation results in part from the imbalance between transnitrosylase and denitrosylase function. Restoration of physiological levels of protein s -nitrosylation and NO signaling may represent an effective therapeutic target for HFpEF.

Exercise has beneficial effects on metabolism and health. Although the skeletal muscle has been a primary focus, exercise also mediates robust adaptations in white adipose tissue. To determine if exercise affects in vivo adipocyte formation, fifty-two, sixteen-week-old C57BL/6J mice were allowed access to unlocked running wheels [Exercise (EX) group; n = 13 males, n = 13 females] or to locked wheels [Sedentary (SED) group; n = 13 males, n = 13 females] for 4-weeks. In vivo adipocyte formation was assessed by the incorporation of deuterium (²H) into the DNA of newly formed adipocytes in the inguinal and gonadal adipose depots. A two-way ANOVA revealed that exercise significantly decreased new adipocyte formation in the adipose tissue of mice in the EX group relative to the SED group (activity effect; P = 0.02). This reduction was observed in male and female mice (activity effect; P = 0.03). Independent analysis of the depots showed a significant reduction in adipocyte formation in the inguinal (P = 0.05) but not in the gonadal (P = 0.18) of the EX group. We report for the first time that exercise significantly reduced in vivo adipocyte formation in the adipose tissue of EX mice using a physiologic metabolic ²H₂O-labeling protocol.