Cardioprotective actions of nitroxyl donor Angeli's salt are preserved in the diabetic heart and vasculature in the face of nitric oxide resistance

Background and Purpose The risk of fatal cardiovascular events is increased in patients with type 2 diabetes mellitus (T2DM). A major contributor to poor prognosis is impaired nitric oxide (NO•) signalling at the level of tissue responsiveness, termed NO• resistance. This study aimed to determine if T2DM promotes NO• resistance in the heart and vasculature and whether tissue responsiveness to nitroxyl (HNO) is affected. Experimental Approach At 8 weeks of age, male Sprague–Dawley rats commenced a high‐fat diet. After 2 weeks, the rats received low‐dose streptozotocin (two intraperitoneal injections, 35 mg·kg−1, over two consecutive days) and continued on the same diet. Twelve weeks later, isolated hearts were Langendorff‐perfused to assess responses to the NO• donor diethylamine NONOate (DEA/NO) and the HNO donor Angeli's salt. Isolated mesenteric arteries were utilised to measure vascular responsiveness to the NO• donors sodium nitroprusside (SNP) and DEA/NO, and the HNO donor Angeli's salt. Key Results Inotropic, lusitropic and coronary vasodilator responses to DEA/NO were impaired in T2DM hearts, whereas responses to Angeli's salt were preserved or enhanced. Vasorelaxation to Angeli's salt was augmented in T2DM mesenteric arteries, which were hyporesponsive to the relaxant effects of SNP and DEA/NO. Conclusion and Implications This is the first evidence that inotropic and lusitropic responses are preserved, and NO• resistance in the coronary and mesenteric vasculature is circumvented, by the HNO donor Angeli's salt in T2DM. These findings highlight the cardiovascular therapeutic potential of HNO donors, especially in emergencies such as acute ischaemia or heart failure.


| INTRODUCTION
Type 2 diabetes mellitus (T2DM) is a growing epidemic with over 463 million individuals affected worldwide, and this number is predicted to increase to 700 million by the year 2045 (International Diabetes Federation, 2019). This rise in prevalence will not only place a greater burden on health care expenditure (Williams et al., 2020) but also will increase the number of patient hospitalisations due to cardiovascular complications, which remain the leading cause of morbidity and mortality in this population (Al-Salameh et al., 2019). Individuals with T2DM have over a two-fold increased risk of developing symptomatic heart failure (Ohkuma et al., 2019), a condition for which long-term prognosis is poor with a 5-year mortality rate of approximately 75%, irrespective of ejection fraction (Shah et al., 2017). Understanding whether, and if so how, T2DM alters cardiovascular responses to pharmacotherapies employed for the management of cardiovascular pathologies, including in the context of acute heart failure, is essential for improving patient prognosis.
Nitric oxide (NO•) plays an important role in maintaining cardiovascular homeostasis due to its cardio-and vaso-protective effects, which include its ability to inhibit platelet aggregation and inflammation, while promoting vasodilation (Kemp-Harper & Schmidt, 2009) as well as cardiac relaxation (Paulus & Bronzwaer, 2004). Impaired responsiveness to NO•, termed 'NO• resistance', has been identified in the vasculature and platelets of patients with heart failure (Anderson et al., 2004;Maguire et al., 1998) and T2DM (Anderson et al., 2005;Williams et al., 1996). This impairment occurs largely due to elevations in reactive oxygen species (ROS), such as superoxide, which 'scavenge' NO• and oxidise its intracellular receptor soluble guanylate cyclase (sGC) (Pacher et al., 2007). Indeed, the degree of impairment in platelet responsiveness to NO• appears to be proportionate to blood glucose levels in patients with diabetes and acute coronary syndromes, which positively correlated with superoxide levels (Worthley et al., 2007). The impact of NO• resistance on long-term prognosis in patients with diabetes presenting with acute coronary syndromes was highlighted in a study by Schachinger et al., where impaired coronary vasodilation in response to NO• was identified as an independent predictor of adverse cardiovascular events such as myocardial infarction and ischaemic stroke (Schachinger et al., 2000). As such, the therapeutic efficacy of NO• donors in T2DM is likely to be impaired (R.A. Anderson et al., 2005) and thus the need for another therapy that circumvents this problem is paramount.
Nitroxyl (HNO) is the one-electron reduced product of NO• (Miranda et al., 2003). Similar to its redox sibling, HNO also has antiaggregatory (Dautov et al., 2013) and vasodilator (Tare et al., 2017) effects. However, unlike NO•, HNO lacks reactivity with the superoxide anion and thus remains effective during oxidative stress (Leo et al., 2012). Another feature of HNO that distinguishes it from NO• is its ability to induce positive inotropic responses via direct interaction with cysteine residues on thiol-containing proteins such as ryanodine receptors (Tocchetti et al., 2007) and phospholamban (Keceli et al., 2019) on the sarcoplasmic reticulum Ca 2+ -ATPase. We have previously demonstrated that inotropic and lusitropic responses to the NO• donor diethylamine-NONOate (DEA/NO) are impaired, whereas responses to the HNO donor iso-propylamine-NONOate (IPA-NO) are enhanced, in hearts from a rat model of type 1 diabetes mellitus (T1DM) . However, whether this outcome extends to other HNO donors and the more prevalent subtype of diabetes, that is, T2DM (International Diabetes Federation, 2019), is unclear. Although both subtypes of diabetes are characterised by hyperglycaemia, the pathogenesis and clinical presentation of T2DM is distinct to that of T1DM (Zaccardi et al., 2016). In patients with T2DM, low insulin sensitivity is negatively correlated with left ventricular ejection fraction (LVEF) (Sasso et al., 2000). The main objective of this study was to determine if T2DM promotes NO• resistance in the heart and vasculature, and whether tissue responsiveness to HNO is affected.

What is already known
• Impaired tissue responsiveness to NO•, termed NO• resistance, is documented in diabetic vasculature/ platelets.
• NO• resistance contributes to the development of cardiovascular complications in type 2 diabetes.

What does this study add
• Type 2 diabetes-induced NO• resistance in the coronary and mesenteric vasculature is circumvented by nitroxyl.
• Nitroxyl donors act as positive inotropic vasodilators in type 2 diabetes.

What is the clinical significance
• Nitroxyl may represent an effective and rapid intervention for cardiovascular emergencies in type 2 diabetes.  (Lilley et al., 2020). Group sizes were designed to be equal and account for 15% animal loss in diabetic rats due to failure to develop hyperglycaemia despite streptozotocin (STZ) administration or diabetes-associated mortality.
No formal power calculation was performed regarding the primary outcome of NO• resistance (determined by coronary flow rate to DEA/NO in non-diabetic vs. diabetic hearts). However, sample sizes were greater than those in our previous work . In addition, a post hoc power calculation was performed to ensure adequate power using the following online calculator: https://clincalc. com/stats/Power.aspx. Using the mean coronary flow rate response to DEA/NO (non-diabetic: 7.0 ± 0.7 vs. diabetic: 4.6 ± 0.9), this study had a power of >90% (α = 0.05) to detect a significant difference between two groups with n = 8. For all experiments, a flow diagram based on the CONSAERT template is provided for reporting animal use and analysis in preclinical studies (Drucker, 2016), which details animal fate, and any variations/inequalities in sample sizes ( Figure S1).
The low-dose STZ and high-fat diet rat model is an established model of T2DM, and was utilised for this study as it recapitulates features of disease progression observed in humans (Marsh et al., 2009).
Male Sprague-Dawley rats (RRID:RGD_10395233) at three to five weeks of age were obtained from the Animal Resources Centre (ARC; WA, Australia). Only male rats were used, because female rats are regarded as resistant to the effects of low-dose STZ (Furman, 2015). All rats were housed in individually ventilated cages (in groups of two to three per cage) and maintained on a 12-h light/dark cycle at room temperature (22.0 ± 0.1 C) in the PC2-certified Alfred Research Alliance Precinct Animal Centre. Food (standard laboratory chow) and water were provided ad libitum. Every second day, animal welfare was checked, and paper chip bedding, nesting wool and enrichment devices were replaced. At 6 weeks of age, rats were randomly allocated to non-diabetic (n = 30) or diabetic (n = 43) groups using a random number generator. A subset of these animals was allocated to tissue collection only as part of a separate study (detailed in Figure S1). At 8 weeks of age, rats in the diabetic group were placed on a high-fat diet (SF03-002; total digestible energy: 59% lipids, 15% protein; wt/wt: 34.6% sucrose; Specialty Feeds, WA, Australia). The difference in colour and consistency between the two diets precluded blinding of experimental groups. Following 2 weeks, the diabetic group received two consecutive daily i.p. injections of STZ (35 mgÁkg À1 in 0.1-M citric acid vehicle, pH 4.5; Sigma-Aldrich, St. Louis, MO, USA), whereas the non-diabetic group received an equivalent volume of the citric acid vehicle. All animals were maintained on their respective diets for the remainder of the study.
Glucose levels were measured fortnightly, in blood taken from the tail vein and using an Accu-chek ® Performa glucometer (Roche Diagnostics, Basel, Switzerland). When blood glucose levels reached ≥28 mM, rats were administered low-dose insulin (2 IU every second day, s.c.; Humulin ® N intermediate-acting; Eli Lilly, Indianapolis, IN, USA) to reduce animal welfare burden caused by marked hyperglycaemia. Insulin administration was stopped 1 week prior to the study end-point. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were conducted 1 week prior to end-point.
Rats were fasted for 6 h, after which baseline blood glucose measurement was recorded using a glucometer. At time 0, a single i.p. injection of glucose (D-glucose, 50% wt/vol, 2 μlÁg À1 ; Baxter, Viaflex ® ) or insulin (0.5 IUÁkg À1 , Humalog ® rapid-acting; Eli Lilly) was administered for the GTT or ITT, respectively. Then, glucose measurements were obtained in tail vein blood at 15,30,45,60,90, and 120 min. Animal welfare was monitored during and after (2, 4, 24, and 48 h) STZ or citrate vehicle injections, and the GTT or ITT. At 22 weeks of age, rats were anaesthetised using ketamine/xylazine (100/12 mgÁkg À1 , i.p.). Once anaesthetised, whole blood was collected from the portal vein in heparinised tubes, and animals were euthanised via rapid excision of the heart. Whole blood was centrifuged at 1500 rpm for 15 min at 4 C to obtain plasma, which was immediately stored at À80 C for subsequent analysis.

| Plasma insulin, triglyceride, and cholesterol levels
Insulin was measured by a rat-specific ELISA kit, as per the manufac-

| Perfusion of isolated hearts
Hearts isolated from anaesthetised rats were cannulated via the aorta and Langendorff-perfused with Krebs' physiological salt solution (PSS, mmolÁL À1 : 118 NaCl, 4.7 KCl, 1.18 MgSO 4 .7H 2 O, 1.12 KH 2 PO 4 , 25 NaHCO 3 , 11 D-glucose, 0.5 EDTA, and 1.75 CaCl 2 ) and continuously bubbled with carbogen (95% O 2 , 5% CO 2 ) at 37 C. Coronary flow rate was gradually increased to 10 mL min À1 . Then, a fluid-filled latex balloon attached to a pressure transducer was inserted into the left atrium and positioned in the left ventricle to measure left ventricular pressure. Following an equilibration period of 30 min, perfusion pressure was held constant using a STH Pump Controller (ADInstruments, Bella Vista, NSW, Australia). Once readings were stable, the thromboxane A 2 mimetic U46619 (10 μmolÁL À1 , 0.01 to 0.1 mL min À1 ) was infused continuously through the aorta using a two-syringe infusion pump (model sp210iw; World Precision Instruments, SA, Australia) to achieve a 50% reduction in coronary flow rate (i.e., from $10 to $5 mL min À1 ). Then, a single bolus dose of vehicle (NaOH; 10 mmolÁL À1 ) was administered via an injection port above the aortic cannula, followed by construction of serial concentrationresponse curves to the NO• donor DEA/NO (10 À10 to 10 À5 mol) or the HNO donor Angeli's salt (10 À10 to 10 À5 mol) in the presence or absence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ; 10 μmolÁL À1 , 30 min pre-incubation, sGC inhibitor) with each bolus dose administered $1 min apart. Following a washout period of 10 min, a single bolus of Humalog ® rapid-acting insulin (33.3 IUÁmL À1 ; Eli Lilly, Indianapolis, IN, USA) was administered via a second injection port above the aortic cannula to assess acute cardiac responses to insulin in non-diabetic versus diabetic hearts ( Figure S2). Three minutes post-insulin administration, the left ventricle was snap-frozen for subsequent analysis of protein expression via western blotting.

| Western blotting
The immuno-related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al., 2018). The procedures used with respect to protein analyses were performed in a blinded-fashion. Left ventricular protein was extracted by homogenisation in RIPA buffer as previously described (Tate et al., 2019). Protein concentration was determined using the BCA protein assay kit (Sigma-Aldrich). Protein lysates containing an equal amount of protein (30 μg) were loaded into 7.5-12% SDS-PAGE gels and electrophoresed prior to transferring to a polyvinylidene difluoride membrane (Immobilon-FL, Millipore). Membranes were blocked with 5% BSA in TBST for 60 min at room temperature and then probed overnight at 4 C with either a rabbit polyclonal (IgG) antibody to detect p-Akt (when phosphorylated at Ser473; #9271; 1:1000 dilution; Cell Signalling; RRID:AB_329825), which is a mediator of insulin-stimulated glucose uptake (Muniyappa et al., 2007); a rabbit monoclonal (IgG) antibody to detect endothelial NO• synthase

| Statistical analyses
The data and statistical analysis in this study complies with the recommendations on experimental design and analysis in pharmacology.
Data are expressed as mean ± SEM and were analysed using Gra- 3 | RESULTS

| Body weights and metabolic characteristics
Fortnightly body weight and blood glucose measurements are displayed in Figure S4. Diabetic rats had a lower body weight during the final 4 weeks of the study ( Figure S4A; P < 0.05 vs. non-diabetic rats) and elevated blood glucose levels throughout the entire study post-STZ administration ( Figure S4B; P < 0.05 vs. non-diabetic rats). Glucose tolerance was impaired in diabetic rats, indicated by a greater area under the curve (AUC; normalised to baseline blood glucose level), and failure for blood glucose levels to return to baseline at test completion ( Figure S4C; P < 0.05 vs. non-diabetic rats). During the insulin tolerance test, diabetic rats displayed elevated blood glucose levels at baseline, which declined considerably 30 min post-insulin administration, but did not reach those of non-diabetic rats, and remained elevated at test completion ( Figure S4D). At study end-point (12 weeks post-vehicle or STZ), glycated haemoglobin, and plasma insulin and triglycerides were elevated in the diabetic group (Table S1; P < 0.05 vs. non-diabetic rats). Plasma levels of total cholesterol did not differ between groups (Table S1). However, HDL levels were lower, and the total cholesterol to HDL ratio and LDL/VLDL to HDL ratio was higher in diabetic rats (Table S1; P < 0.05 vs. non-diabetic rats).

| Baseline haemodynamic parameters in Langendorff-perfused hearts
There were no significant differences in pressure values at baseline following equilibration, after pre-treatment with ODQ or during U46619 infusion between non-diabetic and diabetic Langendorffperfused hearts (Table S2). Heart rate was lower in diabetic hearts, when compared with non-diabetic hearts (Table S2; P < 0.05 vs. nondiabetic group). absence of ODQ), but no significant change was evident in nondiabetic hearts (Figure 4k). There were no significant differences in the increases in LVEDP in response to DEA/NO in the presence of F I G U R E 1 Change in (a) LVDP, (b) LV + dP/dt, (c) LV À dP/dt, and (d) coronary flow rate, in addition to LV protein expression of (f) phospho-Akt/total-Akt, (g) phospho-Akt/β-actin, (h) total-Akt/β-actin, and (j) p22 phox /β-actin following a bolus dose of insulin in non-diabetic and diabetic Langendorff-perfused hearts. Representative LV immunoblot of (e) phospho-Akt, total-Akt and β-actin, and (i) p22 phox and β-actin. Uncropped blots are provided in Figures S7 and S9. Data are presented as mean ± SEM. Data analysed by Student's unpaired t test. *P < 0.05 compared with the non-diabetic group. Δ, change from baseline; LV, left ventricular; LVDP, LV developed pressure; LV + dP/dt, maximal rate of rise in LV pressure; LV À dP/dt, maximal rate of fall in LV pressure; ND, non-diabetic; D, diabetic ODQ in non-diabetic ( Figure S11E) and diabetic hearts ( Figure S11F). Similarly, increases in LVEDP in response to Angeli's salt were not significantly altered by ODQ in non-diabetic hearts ( Figure S11G). In contrast, ODQ attenuated increases in LVEDP in response to Angeli's salt in diabetic hearts ( Figure S11H; P < 0.05 vs. absence of ODQ).

| Role of sGC in coronary vasodilation and heart rate responses to exogenous NO•/HNO
In There were no significant differences in plasma levels of GSSG or GSH between groups (Figure 6a

| Relaxation responses in mesenteric arteries
We first explored the impact of diabetes on endothelium-dependent and endothelium-independent relaxation responses in mesenteric arteries. Relaxation in response to the endothelium-dependent vasodilator ACh was impaired in arteries from diabetic rats (Table 1 and Figure 7a; P < 0.05 vs. non-diabetic arteries). Diabetes was also associated with impaired SNP-induced endothelium-independent vasorelaxation (Table 1 and Figure 7b; P < 0.05 vs. non-diabetic arteries). There was however no significant difference in the maximal relaxation response to ACh or SNP between non-diabetic and diabetic arteries (Table 1)  The sGC inhibitor, ODQ, attenuated vasorelaxation to Angeli's salt in arteries from non-diabetic ( Figure S12C) and diabetic rats ( Figure S12D). In a subset of vessels, we also obtained pilot data suggesting that the rightward shift caused by ODQ in the concentration-response curve to Angeli's salt was greater in non-diabetic arteries, when compared with diabetic arteries ( Figure S12E), although, firm conclusions cannot be drawn from this small subset.

| DISCUSSION
This is the first report that the myocardium is susceptible to T2DM-  Figure S13). The presence of NO• resistance in the coronary vasculature represents an independent risk factor for adverse cardiovascular outcomes (e.g. myocardial infarction) in patients with diabetes presenting with acute coronary syndromes (Schachinger et al., 2000). The findings of this study are therefore of clinical importance, as they highlight the ability of HNO donors to improve cardiac contraction and relaxation, and promote vasodilation, through circumvention of NO• resistance in the diabetic heart and vasculature.
The increase in cardiac contraction and coronary flow rate observed in response to insulin in non-diabetic hearts, and reduced response in diabetic hearts, is consistent with results of previous studies (Jagasia et al., 2001;Sasso et al., 2000). Sasso et al., identified that insulin infusion increases LVEF and peak filling rate in healthy participants compared with sex-, age-, and BMI-matched diabetic subjects (Sasso et al., 2000). The authors also found that LVEF was positively correlated with the insulin sensitivity index in diabetic subjects, suggesting that cardiac insulin resistance was responsible for the reduction in LVEF and peak filling rate following insulin infusion (Sasso et al., 2000). Similarly, in a study by Jagasia et al., intracoronary insulin infusion increased coronary blood flow in non-diabetic subjects, but lacked efficacy in those with diabetes (Jagasia et al., 2001).
Previous studies examining cardiac insulin signalling suggest that insulin exerts positive inotropic effects through several phosphoinositide 3-kinase (PI3K)-dependent mechanisms, some of which include activation of L-type Ca 2+ channels and myofilament Ca 2+ sensitisation (Maier et al., 1999;Von Lewinski et al., 2005). Insulin also induces vasodilation by signalling through the PI3K/Akt pathway, leading to downstream activation of eNOS and subsequent NO• generation (Muniyappa et al., 2007). Given that we identified Akt phosphorylation to be lower in left ventricles from diabetic hearts following a bolus dose of insulin during Langendorff-perfusion, this indicates that the cardiovascular actions of insulin were blunted in diabetic hearts due to impaired insulin signalling and therefore cardiac insulin resistance.
Inotropic, lusitropic and coronary vasodilator responses to NO• were impaired, but those to HNO were preserved or enhanced in the insulin-resistant diabetic rat myocardium. At baseline, heart rate was slower in the diabetic group, however pressure values were not different between groups. These observations are consistent with those observed in T1DM rat hearts . The impairment of DEA/NO-induced inotropic, lusitropic, and coronary vasodilator  (Williams et al., 1996) actions F I G U R E 6 Impact of diabetes on metabolites of the methionine and glutathione pathway, and the glutathione redox cycle. Peak area of (a) GSSG, (b) GSH, (c) GSH:GSSG, (d) methionine, (e) serine, (f) SAM, (g) cysteine, and (i) γ-Glu-Cys in plasma and left ventricles from non-diabetic and diabetic rats. Peak area of (i) homocysteine and (j) cystine in plasma from non-diabetic and diabetic rats. Peak area of (k) Cys-Gly in left ventricles from non-diabetic and diabetic rats. Values are expressed as mean ± SEM (relative to that of the non-diabetic group, which was expressed as 1 in each case). N = 6-9 animals. Data analysed by the Mann-Whitney U test. *P < 0.05 compared with the non-diabetic group. GSSG, oxidised glutathione; GSH, reduced glutathione; SAM, S-adenosyl-methionine; γ-Glu-Cys, γ-glutamylcysteine; Cys-Gly, cysteinylglycine of NO• in patients with T2DM. It should be mentioned that a component of the inotropic actions we observed in response to DEA/NO, which are sGC-dependent, may be attributed to the Gregg effect, whereby an increase in coronary flow rate leads to an increase in contractile parameters (Westerhof et al., 2006). Thus, it is possible that the attenuation of positive inotropic effect of DEA/NO in diabetic hearts is secondary to the reduced induction of coronary vasodilatation, rather than a direct effect on the myocardium.

NO• resistance occurs as a consequence of oxidative stress
whereby elevations in ROS, such as superoxide, leads to scavenging of NO• and oxidation of the ferrous (Fe 2+ ) haem group on its intracellular receptor, sGC, to its ferric (Fe 3+ ) NO•-insensitive state (Pacher et al., 2007). We have demonstrated in our previous work that superoxide generated by pyrogallol induces NO• resistance by impairing the ability of the NO• donor DEA/NO to stimulate the activity of purified sGC (Irvine et al., 2013). Oxidative stress is a characteristic of diabetes that has been previously identified in the myocardium of T1DM and T2DM rats (Hamblin et al., 2007;Qin et al., 2020;Zhou et al., 2010). We observed elevated protein levels of p22 phox in left ventricles from diabetic Langendorff-perfused hearts. p22 phox plays an important role in oxidative stress by mediating superoxide production by NADPH oxidase (Nox) enzymes 1 and 2 (Drummond & Sobey, 2014). Although we did not measure superoxide levels in Langendorff-perfused hearts, an elevation in p22 phox expression suggests that superoxide generation by Nox1 and 2 may have been up-regulated. Further, our observations at the level of GSH/GSSG suggests an environment in which an imbalance in the relative proportions of reduced versus oxidised sGC may be present in the diabetic rat heart. Therefore, it is possible that scavenging of NO• by superoxide, and/or oxidation of sGC due to concomitant oxidative stress, are contributing factors to the NO• resistance observed in the diabetic myocardium and coronary vasculature.
Consistent with previous reports (Leo et al., 2012;Qin et al., 2020;Tare et al., 2017), the vasodilator response to Angeli's salt was preserved in the coronary vasculature in diabetic hearts. This is attributed to the ability of HNO to remain effective in the presence of oxidative stress, as it fails to react with superoxide (Leo et al., 2012).
Interestingly, the positive inotropic and lusitropic effects of Angeli's salt appear to be enhanced in the diabetic myocardium. A possible explanation for the enhanced response to Angeli's salt could be a reduction in thiol availability in diabetic hearts. HNO has high reactivity with thiols. Previous studies have reported that GSH levels are lower, and GSSG levels higher, in atrial tissue from patients with T2DM (Anderson et al., 2009) and left ventricles from diabetic rats (El-Seweidy et al., 2011;Xu et al., 2002). In isolated rat cardiomyocytes, increasing intracellular thiol content quenches HNO and blunts the contractile response to Angeli's salt (Tocchetti et al., 2007). Based on this evidence, a reduction in intracellular thiol content could lead to an enhanced response to Angeli's salt. GSH is the most abundant intracellular thiol and functions as an antioxidant by scavenging ROS (e.g., superoxide) (Anderson et al., 2009;Aquilano et al., 2014). We measured metabolites of the methionine and GSH pathways, and the GSH redox cycle ( Figure S15) to determine whether levels of GSH, GSSG and other thiols were altered by diabetes in left ventricles from Langendorff-perfused hearts. In the present study, both GSH (albeit modestly) and GSSG were higher in left ventricles from diabetic rats, suggesting that GSH production was increased to counteract elevated ROS generation (indicated by increased oxidation of GSH to GSSG). Although our observation of a modest increase in diabetic left ventricular GSH contrasts to the decrease previously observed in left ventricles from diabetic rats (El-Seweidy et al., 2011;Xu et al., 2002), we measured GSH and GSSG directly via mass spectrometry, rather than indirectly via lesssensitive, colorimetric spectrophotometric approaches (where GSH is calculated as the difference between total glutathione and GSSG).
A reduction in the ratio between the reduced and oxidised form of glutathione (GSH/GSSG) is an indicator of redox imbalance. We observed that the GSH/GSSG ratio tended to be lower in left ventricles from diabetic (compared with non-diabetic) rats, although this did not reach statistical significance. The thiols γ-Glu-Cys and Cys-Gly were elevated in left ventricles from diabetic hearts, and levels of cysteine remained unchanged across groups. This outcome suggests that T A B L E 1 Relaxation to endotheliumdependent and endotheliumindependent vasodilators in mesenteric arteries from non-diabetic and diabetic rats shown that insulin normalises glutathione levels in cardiomyocytes from T1DM rats (Li et al., 2007). Although it is unclear why responses to Angeli's salt were enhanced in the diabetic myocardium, this observation is consistent with our previous findings in the T1DM myocardium . Further studies are required to elucidate the mechanism underlying this observation.
The contribution of sGC to inotropic, lusitropic, and coronary vasodilator effects of DEA/NO and Angeli's salt was examined using ODQ, which irreversibly inhibits sGC by oxidising its ferrous (Fe 2+ ) heme group to its ferric (Fe 3+ ) state (Zhao et al., 2000). Such findings may indicate that HNO is able to activate sGC in its oxidised form; however, this remains a matter of contention due to conflicting evidence that either supports this hypothesis (Dautov et al., 2013;Miranda et al., 2003;Qin et al., 2020) or refutes it (Miller et al., 2009;Zeller et al., 2009). We also demonstrated that ODQ attenuated coronary vasodilation in response to Angeli's salt in non-diabetic and diabetic hearts. In accordance with previous studies (Chin et al., 2014;Favaloro & Kemp-Harper, 2007;Qin et al., 2020), these results suggest that the vasodilator effects of HNO in the coronary vasculature are mediated predominantly via sGC-dependent signalling.
Consistent with previous reports, we demonstrated that sensitivity to the endothelium-dependent vasodilator, ACh, was decreased in mesenteric arteries from diabetic rats (Leo et al., 2011;Tare et al., 2017;Wigg et al., 2001). Impaired vascular responsiveness to the endothelium-independent NO• donor, SNP, has been identified in the brachial (van Etten et al., 2002;Williams et al., 1996) (Irvine et al., 2003). We performed additional wire myography experiments in mesenteric arteries from naïve rats (aged 8-10 weeks) to evaluate whether inclusion or exclusion of EDTA in Krebs' buffer influences relaxation responses to Angeli's salt in the presence or absence of L-cysteine (Methods S1 and Results S2). In the presence of EDTA, L-cysteine caused a rightward shift in the concentration-response curve to Angeli's salt. This inhibitory effect of L-cysteine on relaxation to Angeli's salt was not evident when EDTA was excluded from the Krebs' buffer ( Figure S14). It appears, therefore, that inclusion of EDTA in the Krebs' buffer prevents conversion of HNO to NO•. The reason for the observed lack of a detectable NO• component of effect in the presence of HXC in the EDTA exclusion experiments is unclear, but it suggests that any potential incremental release of NO• from Angeli's salt may be small.

| Limitations
Angeli's salt was utilised as a source of HNO in this study, as it is the only commercially-available HNO donor. As noted earlier, the corelease of HNO and nitrite by Angeli's salt raises the possibility that the cardio-and vaso-protective effects of Angeli's salt could be mediated, at least partially, by nitrite. The use of next-generation HNO donors (e.g., BMS-986231, cimlanod) that do not release active by-products would eliminate this confounding factor . However, these next-generation HNO donors are currently undergoing clinical trials and are not yet commercially available (Kemp-Harper et al., 2016). Other limitations are that we did not measure superoxide levels in hearts, as they were Langendorff-perfused.
To overcome this, future experiments could measure superoxide levels in the perfusate prior to construction of serial dose-response curves in Langendorff-perfused hearts (Paolocci et al., 2001). The very acute nature of the exposure to HNO and NO• precluded determining their impact on sGC expression and activity. However, the mechanisms of action were explored via the well-known pharmacological inhibitor, ODQ. Further, we did not incorporate assessment of basal coronary blood flow in vivo into our study design, hence whether there were any differences in basal coronary blood flow as a result of diabetes prior to the ex vivo interrogation of the dilator responses to Angeli's salt or DEA/NO was not determined. We also acknowledge that the rat model of T2DM used in this study did not exhibit obesity, which is a risk factor for T2DM and present in a large proportion of the human population with the disease (Almourani et al., 2019).
Despite this, our animal model displayed other features of T2DM such as hyperglycaemia, hyperinsulinaemia, impaired glucose tolerance, decreased insulin sensitivity and cardiac insulin resistance. Diabetic rats also exhibited dyslipidaemia evident by higher plasma levels of triglycerides, lower HDL, and a higher total cholesterol to HDL ratio and LDL/VLDL to HDL ratio, relative to non-diabetic rats. HDL levels are decreased in patients with T2DM and are an independent predictor of adverse cardiovascular events (Drexel et al., 2005;Grant & Meigs, 2007). Similarly, an increase in the total/HDL and LDL/HDL cholesterol ratios is predictive of greater cardiovascular risk (Millán et al., 2009). It should also be noted that performing a hyperinsulinaemic-euglycaemic clamp would have improved the experimental design of this study, as this method is considered 'gold standard' in assessing insulin sensitivity in pre-clinical models and humans with T2DM (Kim, 2009). A final limitation is that the precise contribution of hyperglycaemia, as distinct from diabetes per se, was not ascertained. In a previously reported clinical study, NO• resistance at the platelet level was directly proportional to extent of hyperglycaemia, as was superoxide production, and correction of hyperglycaemia ameliorated NO• resistance (Worthley et al., 2007).
Similarly, Malmberg et al., showed that correction of hyperglycaemia improved long-term prognosis in diabetic patients presenting with acute myocardial infarction: however, the contribution of amelioration of NO• resistance to improve prognosis was not evaluated (Malmberg et al., 1995).

| CLINICAL IMPLICATIONS AND CONCLUSION
HNO donors have been clinically developed for heart failure-related scenarios including heart failure with reduced ejection fraction (HFrEF)  This is an arena where nitrates, which are the current standard therapy for acute heart failure, have not been able to enter, due both to their susceptibility to rapid development of nitrate tolerance (Irvine et al., 2011) and diminished efficacy in the presence of NO• resistance (Anderson et al., 2005). In comparison, repeated exposure to HNO donors does not lead to tolerance development (Irvine et al., 2011), and the efficacy of this drug class is maintained when responsiveness to NO• is impaired, creating a potential competitive advantage over nitrate therapy. This study reveals that HNO