Recovery Of Left Ventricular Developed Pressure Research Articles

Matrix metalloproteinase-2 proteolyzes mitofusin-2 and impairs mitochondrial function during myocardial ischemia-reperfusion injury.

During myocardial ischemia and reperfusion (IR) injury matrix metalloproteinase-2 (MMP-2) is rapidly activated in response to oxidative stress. MMP-2 is a multifunctional protease that cleaves both extracellular and intracellular proteins. Oxidative stress also impairs mitochondrial function which is regulated by different proteins, including mitofusin-2 (Mfn-2), which is lost in IR injury. Oxidative stress and mitochondrial dysfunction trigger the NLRP3 inflammasome and the innate immune response which invokes the de novo expression of an N-terminal truncated isoform of MMP-2 (NTT-MMP-2) at or near mitochondria. We hypothesized that MMP-2 proteolyzes Mfn-2 during myocardial IR injury, impairing mitochondrial function and enhancing the inflammasome response. Isolated hearts from mice subjected to IR injury (30min ischemia/40min reperfusion) showed a significant reduction in left ventricular developed pressure (LVDP) compared to aerobically perfused hearts. IR injury increased MMP-2 activity as observed by gelatin zymography and increased degradation of troponin I, an intracellular MMP-2 target. MMP-2 preferring inhibitors, ARP-100 or ONO-4817, improved post-ischemic recovery of LVDP compared to vehicle perfused IR hearts. In muscle fibers isolated from IR hearts the rates of mitochondrial oxygen consumption and ATP production were impaired compared to those from aerobic hearts, whereas ARP-100 or ONO-4817 attenuated these reductions. IR hearts showed higher levels of NLRP3, cleaved caspase-1 and interleukin-1β in the cytosolic fraction, while the mitochondria-enriched fraction showed reduced levels of Mfn-2, compared to aerobic hearts. ARP-100 or ONO-4817 attenuated these changes. Co-immunoprecipitation showed that MMP-2 is associated with Mfn-2 in aerobic and IR hearts. ARP-100 or ONO-4817 also reduced infarct size and cell death in hearts subjected to 45min ischemia/120min reperfusion. Following myocardial IR injury, impaired contractile function and mitochondrial respiration and elevated inflammasome response could be attributed, at least in part, to MMP-2 activation, which targets and cleaves mitochondrial Mfn-2. Inhibition of MMP-2 activity protects against cardiac contractile dysfunction in IR injury in part by preserving Mfn-2 and suppressing inflammation.

The effect of dapagliflozin on myocardial ischemia–reperfusion injury in diabetic rats

The incidence of ischemic heart disease is 2-3 times higher in diabetic patients. However, the effect of dapagliflozin on ischemia-reperfusion myocardial injury in diabetic rats has not been studied. We examined the effects of dapagliflozin on myocardial IR injury in streptozotocin-nicotinamide-induced diabetic rats. Rats were divided into four groups (n = 7 in each group): control, control-dapagliflozin, diabetes, and diabetes-dapagliflozin. Dapagliflozin (1.5 mg/kg/day) was administered concomitantly in drinking water for 2 months. The hearts were perfused in a Langendorff's apparatus at 2 months and assessed before (baseline) and after myocardial IR for the following parameters: left ventricular developed pressure (LVDP), minimum and maximum rates of pressure change in the left ventricle (±dP/dt), endothelial nitric oxide (NO) synthase (eNOS) and inducible NO synthase (iNOS) mRNA expressions, creatine kinase MB (CK-MB) and troponin imyocardial enzyme extravasation, and lactate dehydrogenase. The recovery of LVDP and±dP/dt in diabetic rats was lower than that in controls but near normal after dapagliflozin treatment. Diabetic rats had decreased eNOS expression and increased iNOS expression at baseline and after IR, whereas dapagliflozin normalized these parameters after IR. Compared with controls, cardiac NOx levels were initially lower in diabetic patients but higher after IR. Baseline MDA levels were higher in diabetic rats after IR, whereas cardiac NOx levels decreased after treatment with dapagliflozin. Dapagliflozin protects the diabetic rat heart from ischemia-reperfusion myocardial injury by regulating the expression of eNOS and iNOS and inhibiting cardiac lipid peroxidation.

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection via the purinergic P2Y13 receptor and nitric oxide signalling

Abstract Background Red blood cells (RBC) are suggested to act as important mediators in the regulation of cardiovascular function by exporting nitric oxide (NO) bioactivity and ATP under hypoxic/ischemic conditions. In addition, RBCs are known to protect from ischemia-reperfusion injury via the export of NO bioactivity in experimental settings. However, it remains unknown if such beneficial effects of RBCs are protective in patients with acute myocardial infarction. Purpose To investigate whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia-reperfusion injury and whether such effect involves activation of purinergic and NO signalling in the RBCs. Methods RBCs were collected from patients with STEMI undergoing primary percutaneous coronary intervention and age- and gender-matched healthy controls. The RBCs were administered into the coronary circulation of isolated Langendorff-perfused rat hearts at the onset of global ischemia for 25 min followed by reperfusion of 60 min. Recovery of left ventricular developed pressure (LVDP) during reperfusion and infarct size were determined. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996). The present study was performed following The Code of Ethics of the World Medical Association outlined in the Declaration of Helsinki of 1975 and revised in 1983 for experiments that involve human subjects. Results Administration of RBCs from STEMI patients improved recovery of LVDP and reduced infarct size in hearts subjected to ischemia-reperfusion in comparison with RBCs from healthy controls (Figure 1A, B). Pre-incubation of the RBCs with the NO synthase (NOS) inhibitor L-NAME (Figure 1C, D) and the inhibitor of the NO receptor soluble guanylyl cyclase (sGC) ODQ abolished the cardioprotective effect of RBCs from STEMI patients. The cardioprotective effect was also attenuated by inhibition of cardiac cGMP-dependent protein kinase (PKG). Further, the purinergic P2Y13 receptor antagonist MRS2211 (Figure 1E, F), but not the P1 receptor antagonist 8PT applied to RBCs, attenuated the cardioprotection induced by RBCs from STEMI patients. Moreover, administration of RBCs from healthy subjects pre-incubated with a cell permeable ATP analogue improved post-ischemic recovery of LVDP and reduced infarct size. This cardioprotective effect was abolished by co-incubation of the RBCs with ODQ (Figure 2) and MRS2211. Conclusion Our findings demonstrate a novel function of RBCs in patients with STEMI that provides protection against myocardial ischemia-reperfusion injury via the activation of P2Y13 receptor and the NO-sGC pathway in RBCs and cardiac PKG. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation; Swedish Research Council

Arginase 1 in erythrocytes is a critical modulator of cardioprotective signalling by nitric oxide and soluble guanylyl cyclase

Abstract Background Arginase is involved in the development of ischemia-reperfusion injury by regulating nitric oxide (NO) bioactivity via competition with NO synthase (NOS) for their common substrate L-arginine. Erythrocytes are known to contain high levels of arginase that may reduce export of cardioprotective NO bioactivity. Aim To determine the role of arginase 1 in erythrocytes for cardiac protection against myocardial ischemia-reperfusion injury. Methods A tie2 cre-flox mouse model, in which arginase 1 was deleted (Arg 1-KO) in hematopoietic and endothelial cells, was developed. In vivo ischemia-reperfusion was performed in anaesthetized mice by left anterior descending coronary artery ligation (30 min ischemia and 120 min reperfusion). To determine the specific role of erythrocytes, an isolated perfused mouse heart model was applied. Erythrocytes from Arg 1-KO and wild type (WT) mice were given to isolated hearts from WT mice at the onset of global ischemia. After 40 min ischemia, the recovery of left ventricular developed pressure (LVDP) during 60 min reperfusion was recorded as an indicator of cardiac functional recovery. All animal experiments and procedures were performed according to the guidelines by the U.S National Institutes of Health (NIH publication no 85–23, revised 1996) Results Following in vivo ischemia-reperfusion, infarct size was smaller in Arg 1-KO mice than in WT mice (Fig. A). A similar degree of infarct size reduction was obtained by i.v. administration of an arginase inhibitor in WT mice. The cardioprotective effect observed in Arg 1-KO mice was abolished by the NOS inhibitor L-NMMA (Fig. A). In isolated buffer-perfused hearts, there was no difference in the post-ischemic recovery of LVDP between Arg 1-KO and WT hearts. When erythrocytes from Arg 1-KO mice were administrated to isolated WT mouse hearts, the post-ischemic recovery of LVDP was significantly improved compared to hearts given erythrocytes from WT mice. Also, the cardioprotective effect of erythrocytes from Arg 1-KO mice was abolished by pre-incubation of the erythrocytes with the NOS inhibitor L-NAME (Fig. B) or the inhibitor of soluble guanylyl cyclase ODQ (Fig. C). Conclusion Arginase 1 in erythrocyte plays an important role in regulating cardioprotection mediated via the NO-soluble guanylyl cyclase pathway. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation (20190266),The Swedish Research Council (2020-01372)

Erythrocytes from patients with ST-elevation myocardial infarction induce cardioprotection through the purinergic P2Y13 receptor and nitric oxide signaling

Red blood cells (RBCs) are suggested to play a role in cardiovascular regulation by exporting nitric oxide (NO) bioactivity and ATP under hypoxia. It remains unknown whether such beneficial effects of RBCs are protective in patients with acute myocardial infarction. We investigated whether RBCs from patients with ST-elevation myocardial infarction (STEMI) protect against myocardial ischemia–reperfusion injury and whether such effect involves NO and purinergic signaling in the RBCs. RBCs from patients with STEMI undergoing primary coronary intervention and healthy controls were administered to isolated rat hearts subjected to global ischemia and reperfusion. Compared to RBCs from healthy controls, RBCs from STEMI patients reduced myocardial infarct size (30 ± 12% RBC healthy vs. 11 ± 5% RBC STEMI patients, P < 0.001), improved recovery of left-ventricular developed pressure and dP/dt and reduced left-ventricular end-diastolic pressure in hearts subjected to ischemia–reperfusion. Inhibition of RBC NO synthase with L-NAME or soluble guanylyl cyclase (sGC) with ODQ, and inhibition of cardiac protein kinase G (PKG) abolished the cardioprotective effect. Furthermore, the non-selective purinergic P2 receptor antagonist PPADS but not the P1 receptor antagonist 8PT attenuated the cardioprotection induced by RBCs from STEMI patients. The P2Y13 receptor was expressed in RBCs and the cardioprotection was abolished by the P2Y13 receptor antagonist MRS2211. By contrast, perfusion with PPADS, L-NAME, or ODQ prior to RBCs administration failed to block the cardioprotection induced by RBCs from STEMI patients. Administration of RBCs from healthy subjects following pre-incubation with an ATP analog reduced infarct size from 20 ± 6 to 7 ± 2% (P < 0.001), and this effect was abolished by ODQ and MRS2211. This study demonstrates a novel function of RBCs in STEMI patients providing protection against myocardial ischemia–reperfusion injury through the P2Y13 receptor and the NO–sGC–PKG pathway.

Hydrogen sulfide potentiates the protective effects of nitrite against myocardial ischemia-reperfusion injury in type 2 diabetic rats

Decreased heart levels of nitric oxide (NO) and hydrogen sulfide (H2S) in type 2 diabetes (T2D) are associated with a higher risk of mortality following ischemia-reperfusion (IR) injury. This study aimed to determine the effects of co-administration of sodium nitrite and sodium hydrosulfide (NaSH) on IR injury in the isolated heart from rats with T2D. Two-month-old male rats were divided into 5 groups (n = 7/group): Control, T2D, T2D + nitrite, T2D + NaSH, and T2D + nitrite + NaSH. T2D was induced using a high-fat diet and a single low dose streptozotocin (30 mg/kg) in intraperitoneal injection. Nitrite (50 mg/L in drinking water) and NaSH (0.28 mg/kg, daily intraperitoneal injection) were administrated for 9 weeks. At the end of the study, hemodynamic parameters were recorded, and infarct size and mRNA expression of H2S- and NO-producing enzymes were measured in the isolated hearts. Nitrite administration to rats with T2D improved recovery of left ventricular developed pressure (LVDP) and the peak rates of positive and negative changes in LV pressure (±dp/dt) by 30%, 17%, and 7.9%, respectively, and decreased infarct size by 18.4%. Co-administration of nitrite and NaSH resulted in further improve in recovery of LVDP, +dp/dt, and –dp/dt by 8.3% (P = 0.0478), 8.4% (P = 0.0085), and 9.0% (P = 0.0004), respectively, and also further decrease in infarct size by 24% (P = 0.0473). Nitrite treatment decreased inducible and neuronal NO synthases (iNOS, 0.4-fold; nNOS, 0.4-fold) and cystathionine β-synthase (CBS, 0.1-fold) expression in the isolated heart from rats with T2D. Co-administration of nitrite and NaSH further increased cystathionine γ-lyase (CSE, 2.8-fold) and endothelial NOS (eNOS, 2.0-fold) expression and further decreased iNOS (0.4-fold) expression. In conclusion, NaSH at a low dose potentiates the favorable effects of inorganic nitrite against myocardial IR injury in a rat model of T2D. These anti-ischemic effects, following co-administration of nitrite and NaSH, were associated with higher CSE-derived H2S and eNOS-derived NO as well as lower iNOS-derived NO in the diabetic hearts.

Stress-induced body weight loss and improvements in cardiometabolic risk factors do not translate to improved myocardial ischemic tolerance in western diet-fed mice.

Although both diet‐induced obesity and psychological stress are recognized as significant independent contributors to cardiometabolic and behavioral disorders, our understanding of how these two disorders interact and influence cardiometabolic risk and myocardial ischemic tolerance is limited. The aim of this study was to assess the combined effects of an obesogenic diet and psychological stress on cardiometabolic risk factors (body weight, dyslipidemia, insulin sensitivity) and postischemic cardiovascular outcomes. C57Bl/6J mice (n = 48) were subject to a combination of 22 weeks of western diet (WD) feeding and chronic restraint stress (CRS) for the last 4 weeks. Metabolic and behavioral changes were assessed using glucose tolerance tests and open field tests (OFTs), respectively. After 22 weeks, cardiac function and ischemic tolerance were assessed in Langendorff perfused hearts. WD feeding increased body weight and worsened blood lipids and insulin sensitivity. WD‐fed mice also exhibited reduced exploratory behavior within the OFT. CRS reduced body weight and increased locomotion in both dietary groups and had differential effects on fasting glucose metabolism in the two dietary groups while not impacting non‐fasting insulin. Although the WD only marginally reduced reperfusion left ventricular developed pressure recovery, CRS worsened reperfusion diastolic dysfunction in both dietary groups. Interestingly, despite WD+CRS animals exhibiting improved cardiometabolic parameters compared to the WD group, these changes did not translate to marked improvements to postischemic cardiac outcomes. In conclusion, in this study, combined WD feeding and CRS did not act synergistically to worsen cardiometabolic risk factors but instead improved them. Despite these cardiometabolic improvements, WD+CRS increased reperfusion end diastolic pressure which may be indicative of worsened ischemia/reperfusion injury.

Abstract 12104: Treatment With Exogenous Atrial Natriuretic Peptide Ameliorates Myocardial Insulin Resistance and Protects Against Ischemia-Reperfusion Injury in Diet-Induced Obesity

Increasing evidence suggests natriuretic peptide (NP) coordinate inter-organ metabolic crosstalk in addition to its classical actions on hemodynamic regulation. We recently reported exogenous atrial NP (ANP) treatment ameliorated systemic insulin resistance by inducing adipose tissue browning and by attenuating hepatic steatosis in diet-induced obesity. We herein hypothesized that exogenous ANP treatment also ameliorates insulin resistance of the myocardium in which endogenous NP is produced, leading to the cardioprotection during ischemia-reperfusion injury (IRI) in diet-induced obesity. The mice fed a high fat diet (HFD) or normal fat diet (NFD) for 13 weeks were treated with or without ANP infusion subcutaneously via osmotic pump for another 3 weeks (0.5 μg/kg/min). Intraperitoneal administration of insulin induced Akt phosphorylation in NFD hearts, which was reduced in HFD. ANP treatment restored the impaired insulin-induced Akt phosphorylation in HFD hearts, suggesting that exogenous ANP ameliorated myocardial insulin resistance. To test the functional significance of this, hearts were exposed to IRI using Langendorff model. After IRI, HFD resulted in an impaired left ventricular developed pressure (LVDP) recovery with corresponding increase in infarct size. However, ANP treatment improved functional recovery and reduced injury along with restoration of the impaired IRI-induced Akt phosphorylation in HFD hearts. (% LVDP recovery of baseline: NFD 62.9%±3.6 &lt;n=8&gt;, NFD+ANP 64.5%±4.2% &lt;n=8&gt;, HFD 20.8%±3.3 &lt;n=9&gt;, HFD+ANP 31.4%±2.8% &lt;n=9&gt;, p&lt;0.05) Myocardial ultrastructural analyses by electron microscopy showed number of peri-mitochondrial lipid droplets was increased in HFD treated with ANP, suggesting that ANP protects mitochondria from lipid overload. Of note, ANP treatment substantially attenuated mitochondria cristae disruption after IRI in HFD hearts. In conclusion, exogenous ANP treatment ameliorates myocardial insulin resistance and protects against IRI associated with restoration of disrupted mitochondrial structure in diet-induced obesity. The findings that exogenous NP has significant impact on the NP-rich failing myocardium provide new insight into the biological properties and dynamics of NP.

Attenuated cardiac post-ischemic injury and endothelial dysfunction by stimulation of soluble guanylyl cyclase in red blood cells from patients with type 2 diabetes

Abstract Background Reduced bioavailability of nitric oxide (NO) contributes to ischemic heart disease in type 2 diabetes (T2D). Red blood cells (RBCs) are known to produce NO bioactivity and to contain a functional soluble guanylyl cyclase (sGC) that is activated by NO. Recent studies revealed that RBCs from patients with T2D exacerbates ischemia-reperfusion (I/R) injury and induces endothelial dysfunction via a mechanism that is dependent on reduced export of NO bioactivity from RBCs. It remains unknown whether stimulation of sGC in RBCs from patients with T2D protects against myocardial I/R injury and endothelial dysfunction. Purpose To test the hypothesis that stimulation of sGC in RBCs from T2D patients protects against myocardial I/R injury and improves endothelial function. Methods RBCs collected from T2D patients and healthy subjects were incubated with vehicle or the sGC stimulator CYR715 before being administered to isolated Langendorff-perfused rat hearts subjected to 25 min global ischemia and 60 min reperfusion. Left ventricular developed pressure (LVDP) and infarct size were determined. In addition, isolated rat aortic rings were incubated with RBCs subsequent determination of endothelium-dependent relaxation (EDR). Results Administration of RBCs from T2D patients impaired post-ischemic recovery of LVDP and induced endothelial dysfunction in comparison with RBCs from healthy subjects (P&amp;lt;0.001). Pre-incubation of RBCs from patients with T2D with CYR715 prior to administration to the isolated heart enhanced the recovery of LVDP (Fig.1A), reduced infarct size (Fig.1B), and attenuated endothelial dysfunction (Fig.1C). CYR715 did not induce cardioprotection in the absence of RBCs. The sGC inhibitor ODQ did not significantly affect cardiac recovery per se but totally abolished the protective effect of CYR715 (Fig.1A). Conclusions Stimulation of sGC in RBCs from patients with T2D protects against cardiac I/R injury and prevents development of endothelial dysfunction. Stimulation of the NO-sGC pathway in RBCs appears to be an attractive therapeutic strategy to prevent cardiovascular injury in T2D. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Swedish Heart and Lung Foundation, the Swedish Research Council

Cardioprotective Effects of PPARβ/δ Activation against Ischemia/Reperfusion Injury in Rat Heart Are Associated with ALDH2 Upregulation, Amelioration of Oxidative Stress and Preservation of Mitochondrial Energy Production.

Accumulating evidence support the cardioprotective properties of the nuclear receptor peroxisome proliferator activated receptor β/δ (PPARβ/δ); however, the underlying mechanisms are not yet fully elucidated. The aim of the study was to further investigate the mechanisms underlying PPARβ/δ-mediated cardioprotection in the setting of myocardial ischemia/reperfusion (I/R). For this purpose, rats were treated with PPARβ/δ agonist GW0742 and/or antagonist GSK0660 in vivo and hearts were subjected to ex vivo global ischemia followed by reperfusion. PPARβ/δ activation improved left ventricular developed pressure recovery, reduced infarct size (IS) and incidence of reperfusion-induced ventricular arrhythmias while it also up-regulated superoxide dismutase 2, catalase and uncoupling protein 3 resulting in attenuation of oxidative stress as evidenced by the reduction in 4-hydroxy-2-nonenal protein adducts and protein carbonyl formation. PPARβ/δ activation also increased both mRNA expression and enzymatic activity of aldehyde dehydrogenase 2 (ALDH2); inhibition of ALDH2 abrogated the IS limiting effect of PPARβ/δ activation. Furthermore, upregulation of PGC-1α and isocitrate dehydrogenase 2 mRNA expression, increased citrate synthase activity as well as mitochondrial ATP content indicated improvement in mitochondrial content and energy production. These data provide new mechanistic insight into the cardioprotective properties of PPARβ/δ in I/R pointing to ALDH2 as a direct downstream target and suggesting that PPARβ/δ activation alleviates myocardial I/R injury through coordinated stimulation of the antioxidant defense of the heart and preservation of mitochondrial function.

Protective effect of intermediate doses of hydrogen sulfide against myocardial ischemia-reperfusion injury in obese type 2 diabetic rats

ObjectiveSubjects with type 2 diabetes (T2D) have lower circulating hydrogen sulfide (H2S) levels following myocardial ischemia and a higher risk of mortality. The aim of this study was to determine the dose-dependent favorable effects of sodium hydrosulfide (NaSH) on myocardial ischemia-reperfusion (IR) injury in rats with T2D. MethodsT2D was induced using a high-fat diet (HFD) and low-dose of streptozotocin. Rats were divided into control, T2D, and T2D + NaSH groups. NaSH (0.28, 0.56, 1.6, 2.8, and 5.6 mg/kg) was administered intraperitoneally for 9 weeks. At the end of the study, heart from all rats were isolated and left ventricular developed pressure (LVDP) and the peak rates of positive and negative changes in LV pressure (±dp/dt) were recorded during baseline and following myocardial IR injury. In addition, infarct size as well as mRNA expression of H2S- and nitric oxide (NO)-producing enzymes were measured. ResultsIn diabetic rats, NaSH only at doses of 0.56 and 1.6 mg/kg increased recovery of LVDP (16% and 42%), +dp/dt (25% and 35%) and –dp/dt (23% and 32%) as well as decreased infarct size (44% and 35%). At these doses, NaSH increased expressions of cystathionine γ-lyase (CSE) (440% and 271%) and endothelial NO synthase (eNOS) (232% and 148%) but it decreased the expressions of inducible NOS (iNOS) (55% and 71%). NaSH at 0.28, 2.8 and 5.6 mg/kg had no significant effects on these parameters. ConclusionNaSH had a bell-shaped cardioprotective effect against myocardial IR injury in rats with T2D. Higher tolerance to IR injury in heart isolated from type 2 diabetic rats treated with intermediate doses of NaSH is associated with higher CSE-derived H2S and eNOS-derived NO as well as lower iNOS-derived NO.

P1592A cardioprotective effect of dietary nitrate mediated by red blood cells

Abstract Background Reduced bioavailability of nitric oxide (NO) is a key factor behind coronary artery disease and ischemic heart disease. Besides being produced from L-arginine by NO synthase, reduction of nitrate/nitrite to NO is also an important pathway for NO generation, especially during hypoxic/ischemic conditions. Existing evidences suggest that dietary nitrate improves endothelial function and ischemic tolerance via a NO-dependent mechanism. Recent data indicate that red blood cells (RBCs) are an important source of NO bioactivity that protects the heart from ischemia-reperfusion injury. It is unknown whether dietary nitrate exerts cardioprotection by increasing export of RBC NO bioactivity. Purpose To investigate whether dietary nitrate protects the heart against ischemia-reperfusion injury via a mechanism mediated by RBCs. Methods Patients with mild hypertension (systolic blood pressure &gt;130, ≤159 mmHg) were randomly assigned to three groups after a 2-week run-in period on a diet low in nitrate: group 1 received nitrate-rich vegetables (green leafy, ∼150 g/day) plus capsules with placebo salt (KCl), group 2 received low nitrate vegetables (cherry tomato, sweet corn, capsicum, carrot, ∼150 g/day) and capsules with nitrate salt (KNO3, 300mg) and group 3 received low nitrate vegetables and placebo capsules for 5 weeks. The nitrate content of the pills and nitrate-rich vegetables were precisely matched. As a pre-specified substudy, RBCs were collected blindly from 48 subjects before (baseline) and after the 5-week treatment (follow-up). The RBCs were given to isolated Langendorff-perfused rat hearts at the onset of ischemia with and without the soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ). The hearts were subjected to 25 min global ischemia following 60 min reperfusion. Left ventricular developed pressure (LVDP) was recorded as an indicator of cardiac function. Results The recovery in LVDP during reperfusion following global ischemia in hearts given RBCs collected at baseline was similar in the three groups (Fig. A). Of note, post-ischemic recovery of LVDP was significantly improved by administration of RBCs from patients randomized to high nitrate vegetables or nitrate capsule compared to the group randomized to low nitrate and placebo (Fig. B). There was no difference in LVDP recovery between the groups receiving high nitrate vegetables or nitrate capsule (Fig. B). The nitrate-induced improvement in post-ischemic cardiac recovery was totally abolished by the sGC inhibitor ODQ (Fig. C), indicating that the protective effect induced by RBCs from subjects given nitrate is NO-sGC dependent. Conclusion Dietary nitrate in the form of leafy vegetables induces protection against myocardial ischemia-reperfusion injury via a mechanism involving the NO-sGC signaling pathway in RBCs.

P6299Dietary nitrate restores cardiac ischemic tolerance in type 2 diabetic mice and protects against ischemia-reperfusion injury via soluble guanylate cyclase in red blood cells

Abstract Background Inorganic nitrate has been shown to exert beneficial cardiovascular effects, which are thought to be mediated via sequential reduction of nitrate to nitrite and nitric oxide (NO). We have previously reported that hearts from type 2 diabetic db/db mice have impaired cardiac ischemic tolerance and that this effect involves reduced export of NO-like bioactivity from red blood cells (RBCs). It remains unknown whether nitrate supplementation may affect cardiac ischemic tolerance in diabetes through interference with RBC function. Purpose To test the hypothesis that dietary nitrate supplementation improves cardiac ischemic tolerance of hearts via an effect mediated through RBCs in type 2 diabetes. Methods Type 2 diabetic (db/db) and wild type (WT) mice on nitrate-free chow were treated with vehicle or nitrate (1 mM) in the drinking water for 4 weeks. Hearts were isolated and perfused using the Langendorff technique. After 30 min stabilization, the hearts were subjected to 40 min global ischemia followed by 60 min reperfusion. In protocol 1, isolated hearts from db/db and WT mice given vehicle or nitrate were perfused with buffer. In protocol 2, only hearts from untreated WT mice were used. Washed RBCs from WT or db/db mice treated with vehicle or nitrate were administered to WT hearts at the onset of ischemia with and without the soluble guanylyl cyclase (sGC) inhibitor (1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one, ODQ). In both protocols post-ischemic recovery of cardiac function was evaluated by determination of left ventricular developed pressure (LVDP). Results In Protocol 1, post-ischemic recovery of LVDP was impaired in hearts from db/db mice in comparison with hearts from WT mice (Fig. A). Dietary nitrate restored the ischemic tolerance of hearts from db/db mice but did not affect post-ischemic recovery of hearts from WT mice (Fig. A). In Protocol 2, administration of RBCs collected from vehicle-treated db/db mice significantly impaired post-ischemic recovery of hearts from WT mice (Fig. B). Notably, administration of RBCs from nitrate-treated db/db mice completely reversed the impairment of post-ischemic cardiac function induced by diabetic RBCs (Fig. B). Interestingly, post-ischemic cardiac function did not differ between hearts given RBCs from nitrate-treated db/db and WT mice (Fig. B). The protective effect of RBCs from nitrate-treated mice was abolished by pre-incubation of the RBCs with ODQ, an inhibitor of soluble guanylate cyclase (sGC) (Fig. C). By contrast, pretreatment of isolated WT hearts with ODQ failed to block the protective effect of RBCs from nitrate-treated mice (Fig C) indicating that sGC in the RBC but not in the heart is critical for nitrate-induced cardiac protection. Conclusion Dietary nitrate restores cardiac ischemic tolerance in db/db mice and protects the heart against ischemia–reperfusion injury via an RBC NO-sGC pathway.

Cardiovascular effects of diesel exhaust inhalation: photochemically altered versus freshly emitted in mice

ABSTRACTThis study was designed to compare the cardiovascular effects of inhaled photochemically altered diesel exhaust (aged DE) to freshly emitted DE (fresh DE) in female C57Bl/6 mice. Mice were exposed to either fresh DE, aged DE, or filtered air (FA) for 4 hr using an environmental irradiation chamber. Cardiac responses were assessed 8 hr after exposure utilizing Langendorff preparation with a protocol consisting of 20 min of perfusion and 20 min of ischemia followed by 2 hr of reperfusion. Cardiac function was measured by indices of left-ventricular-developed pressure (LVDP) and contractility (dP/dt) prior to ischemia. Recovery of post-ischemic LVDP was examined on reperfusion following ischemia. Fresh DE contained 460 µg/m3 of particulate matter (PM), 0.29 ppm of nitrogen dioxide (NO2) and no ozone (O3), while aged DE consisted of 330 µg/m3 of PM, 0.23 ppm O3 and no NO2. Fresh DE significantly decreased LVDP, dP/dtmax, and dP/dtmin compared to FA. Aged DE also significantly reduced LVDP and dP/dtmax. Data demonstrated that acute inhalation to either fresh or aged DE lowered LVDP and dP/dt, with a greater fall noted with fresh DE, suggesting that the composition of DE may play a key role in DE-induced adverse cardiovascular effects in female C57Bl/6 mice.

Abstract 300: Ischemia and Reperfusion Injury Following Cardioplegic Arrest is Attenuated by Age and Testosterone Deficiency in Male but Not Female Mice

Cardiovascular disease increases with age in both sexes and treatment can require cardiac surgery, where the heart is pre-treated with a protective cardioplegic solution before exposure to ischemia and reperfusion (I/R). While endogenous estrogen is thought to be beneficial in I/R, the role of testosterone in cardioprotection is not well understood and whether age modifies responses to I/R is unclear. We investigated sex- and age-specific differences in I/R injury in hearts pre-treated with a clinically relevant cardioplegic solution. Hearts were isolated from adult (6-9 mos) and old (22-28 mos) C57BL/6 mice of both sexes (n=27). They were Langendorff-perfused with Krebs-Henseleit buffer (15 min; 37°C), followed by St. Thomas’2 cardioplegia (6 min; 6-7°C). This was followed by 90 min of global ischemia (room temperature) and 30 min of reperfusion (37°C). Hearts were then perfused with triphenyltetrazolium chloride to quantify infarct area. The role of testosterone was investigated in gonadectomized (GDX; 6-9 mos) male mice and serum testosterone was measured with an ELISA. Functional recovery in reperfusion was significantly better in old males than in adult males. Left ventricular developed pressure (LVDP) recovered to 67.3 ± 7.4% in old compared to 31.6 ± 12.3% in adult male hearts (p&lt;0.05). Similar results were seen for rates of pressure development (+dP/dt; p&lt;0.05) and decay (-dP/dt; p&lt;0.05). Also, infarct areas were markedly smaller in old male hearts (15.5 ± 1.8%) than in younger hearts (48.1 ± 7.7%; p&lt;0.05). By contrast, hearts from adult and old females exhibited a similar degree of post-ischemic functional recovery and showed no age-dependent difference in infarct size. Serum testosterone levels declined with age in males but were low regardless of age in females, which suggested a potential benefit of low testosterone. In support of this, hearts from GDX males exhibited much better recovery of LVDP in reperfusion when compared to hearts from intact males (values were 64.8 ± 7.7% vs. 29.9 ± 12.3%; p&lt;0.05). GDX hearts also had smaller infarcts than hearts from intact males (p&lt;0.05). These results suggest that low testosterone levels may be protective against I/R injury following cardioplegic arrest in the setting of aging, especially in old males.

Abstract 312: SGLT1 is Essential for Cardioprotection During Ischemia-Reperfusion Injury via Enhanced Glucose Utilization in Diet-induced Obese Mice

Recent large clinical trials have shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors reduced cardiovascular events in diabetic patients. However, the regulation and functional role of cardiac SGLTs (SGLT1 is the dominant isoform) compared with those of other glucose transporters (insulin-dependent GLUT4 is the major isoform) remain incompletely understood. Given that glucose is a preferential substrate for myocardial metabolism during ischemia-reperfusion injury (IRI), we hypothesized that SGLT1 contributes to cardioprotection during IRI via enhanced glucose transport, particularly in insulin-resistant phenotypes. The hearts from mice fed a high-fat diet (HFD) for 12 weeks or a normal-fat diet (NFD) were perfused with either non-selective SGLT-inhibitor phlorizin (to inhibit SGLT1) or selective SGLT2-inhibitors (tofogliflozin, ipragliflozin, canagliflozin) during IRI using Langendorff model. After ischemia-reperfusion, HFD impaired left ventricular developed pressure (LVDP) recovery compared with NFD. Although phlorizin-perfusion impaired LVDP recovery in NFD, a further impaired LVDP recovery and dramatically increased infarct size (indicated by CPK release into perfusate and TTC staining) were observed in HFD with phlorizin-perfusion. Meanwhile, none of the SGLT2-inhibitors significantly affected cardiac function or myocardial injury after ischemia-reperfusion under either diet condition. The plasma membrane expression of GLUT4 was significantly increased after IRI in NFD but was substantially attenuated in HFD, the latter of which was associated with a significant reduction in both myocardial glucose uptake and cardiac tissue ATP content. In contrast, regardless of the diet condition, SGLT1 expression remained constant during IRI, whereas SGLT2 was not detected. Of note, phlorizin considerably reduced myocardial glucose uptake and decreased cardiac tissue ATP content after IRI, particularly in HFD. In conclusion, cardiac SGLT1 but not SGLT2 plays a compensatory protective role and contributes to cardiac energy metabolism during acute phase of IRI via enhanced glucose utilization, particularly under insulin-resistant conditions, in which IRI-induced GLUT4 upregulation is compromised.

Cardiac ischemia\u2013reperfusion injury under insulin-resistant conditions: SGLT1 but not SGLT2 plays a compensatory protective role in diet-induced obesity

BackgroundRecent large-scale clinical trials have shown that SGLT2-inhibitors reduce cardiovascular events in diabetic patients. However, the regulation and functional role of cardiac sodium–glucose cotransporter (SGLT1 is the dominant isoform) compared with those of other glucose transporters (insulin-dependent GLUT4 is the major isoform) remain incompletely understood. Given that glucose is an important preferential substrate for myocardial energy metabolism under conditions of ischemia–reperfusion injury (IRI), we hypothesized that SGLT1 contributes to cardioprotection during the acute phase of IRI via enhanced glucose transport, particularly in insulin-resistant phenotypes.Methods and resultsThe hearts from mice fed a high-fat diet (HFD) for 12 weeks or a normal-fat diet (NFD) were perfused with either the non-selective SGLT-inhibitor phlorizin or selective SGLT2-inhibitors (tofogliflozin, ipragliflozin, canagliflozin) during IRI using Langendorff model. After ischemia–reperfusion, HFD impaired left ventricular developed pressure (LVDP) recovery compared with the findings in NFD. Although phlorizin-perfusion impaired LVDP recovery in NFD, a further impaired LVDP recovery and a dramatically increased infarct size were observed in HFD with phlorizin-perfusion. Meanwhile, none of the SGLT2-inhibitors significantly affected cardiac function or myocardial injury after ischemia–reperfusion under either diet condition. The plasma membrane expression of GLUT4 was significantly increased after IRI in NFD but was substantially attenuated in HFD, the latter of which was associated with a significant reduction in myocardial glucose uptake. In contrast, SGLT1 expression at the plasma membrane remained constant during IRI, regardless of the diet condition, whereas SGLT2 was not detected in the hearts of any mice. Of note, phlorizin considerably reduced myocardial glucose uptake after IRI, particularly in HFD.ConclusionsCardiac SGLT1 but not SGLT2 plays a compensatory protective role during the acute phase of IRI via enhanced glucose uptake, particularly under insulin-resistant conditions, in which IRI-induced GLUT4 upregulation is compromised.

Neutrophil Elastase Inhibitor Sivelestat Attenuates Myocardial Injury after Cardioplegic Arrest in Rat Hearts.

Purpose: Sivelestat, a neutrophil elastase inhibitor, attenuates global ischemia-induced myocardial damage and coronary endothelial dysfunction. Here, we investigated whether sivelestat exerts the cardioprotective effects against cardioplegic arrest in rat hearts.Methods: Isolated Langendorff-perfused rat hearts were randomly allocated to three groups and subjected to 2-min infusions with St. Thomas’ Hospital cardioplegic solution No. 2 (STH2) and 30-min global ischemia followed by 60-min reperfusion as follows: (i) control (STH2 treatment only), (ii) sivelestat (19 μmol/L) infusion for the first 10 min of reperfusion, and (iii) sivelestat (19 μmol/L) infusion for 10 min before ischemia and for the first 10 min of reperfusion. Left ventricular developed pressure (LVDP) recovery and troponin T leakage were measured at the end of reperfusion. Coronary flow response to acetylcholine (ACh) was assessed.Results: Single and multiple doses of sivelestat significantly improved LVDP recovery (69 ± 15 and 69 ± 14 vs 48 ± 15 [control]; p <0.05) and decreased troponin T leakage (0.4 ± 0.3 and 0.7 ± 0.5 vs 1.7 ± 0.6 [control]; p <0.05). Multiple doses of sivelestat significantly improved coronary flow response to ACh (121 ± 9 vs 105 ± 4; p <0.05).Conclusions: Addition of sivelestat to STH2 attenuates myocardial injury after cardioplegic arrest in rat hearts. This cardioprotective effect was achieved even when sivelestat was administered during early reperfusion.

Abstract 427: Inhibition of Myocardial Romk Channels Blocks Ischemic Preconditioning Induced Cardio-protection

Background: Prior evidence has shown that the renal outer medullary potassium channel (Kir 1.1 or ROMK) may be the pore forming subunit of the mitochondrial KATP channel (mitoKATP). However, it is unknown if ROMK inhibition affects myocardial function after ischemia reperfusion injury or cardio-protection from ischemic preconditioning. Methods: C57/BL6J mice were subjected to ex vivo ischemia (45 min) and then reperfusion (60 min) with or without ischemic preconditioning (3 cycles of 3 min). Mice were treated throughout the protocol with either 5 μM of compound A, a potent ROMK inhibitor, or vehicle, in a modified Krebs Henseliet buffer. Results: WT mice hearts subjected to ischemia reperfusion injury and perfused with 5 μM compound A recovered 73% of baseline left ventricular developed pressure (LVDP) while mouse hearts perfused with vehicle recovered 50% of baseline LVDP (P=0.07, N=3). Infarct size was not significantly different between hearts perfused with 5 μM compound A or vehicle (32% ± 7% vs. 46% ± 10%, P =0.18, N=3). In vehicle perfused mice, IPC significantly improved % recovery of LVDP (IPC 87% ± 9% vs. no IPC 50% ± 7%, P =0.014, N=3) and infarct size (IPC 22% ± 4% vs no IPC 46% ± 11%, P =0.02, N=3). However, IPC did not improve % recovery of LVDP (IPC 69% ± 6% vs no IPC 73% ± 4%, P =0.33, N=3) or infarct size (IPC 46% ± 14% vs no IPC 32% ± 7%, P =0.22, N=3) in mouse hearts perfused with compound A. Conclusions: Myocardial ROMK inhibition does not increase IR injury in an ex vivo mouse preparation, but does block cardio-protection from IPC further supporting ROMK as the potential pore forming channel of mitoKATP.

Role of inducible nitric oxide synthase in myocardial ischemia-reperfusion injury in sleep-deprived rats.

REM sleep deprivation (SD) decreases tolerance of the rat heart to ischemia-reperfusion (IR) injury; the underlying mechanisms, however, are unknown. This study aimed at determining whether changes in iNOS, Bax, and Bcl-2 gene expression are involved in this detrimental effect. SD was induced by flowerpot technique for a period of 4days. This method is simple and able to induce sleep fragmentation which occurs as one of the sleep disorder symptoms in clinical conditions. The hearts of control and SD rats were perfused in Langendorff apparatus and subjected to 30min ischemia, followed by 90min reperfusion. The hemodynamic parameters (left ventricular developed pressure (LVDP), and ±dp/dt), NOx (nitrite+nitrate) level, infarct size, and mRNA expression of iNOS, Bax, and Bcl-2 were measured after IR. SD rats had lower recovery of post-ischemic LVDP (32.8±2.5 vs. 51.5±2.1mmHg; P<0.05), +dp/dt (1555±66 vs. 1119.5±87mmHg/s; P<0.05) and -dp/dt (1437±65 vs. 888±162mmHg/s; P<0.05). SD rats also had higher NOx levels (41.4±3.1 vs. 22.4±3.6μmol/L; P<0.05) and infarct size (64.3±2.3 vs. 38.3±1.6%; P<0.05) after IR, which along with LVDP, ±dp/dt restored to near normal status in the presence of aminoguanidine, a selective iNOS inhibitor. Following IR, expression of iNOS and Bax increased and Bcl-2 decreased (502, 372, and 54%, respectively) in SD rats; whereas in the presence of aminoguanidine, expression of iNOS and Bax significantly decreased and Bcl-2 increased (165, 168, and 19%, respectively). Higher expression of iNOS and subsequent increase in apoptosis in the hearts after IR may contribute to less tolerance to myocardial IR injury in SD rats.