Increased nitric oxide availability worsens the cardiac performance during early re-perfusion period in adult rats

Objectives Re-perfusion is the standard therapy for acute myocardial infarction, despite the associated pathologies that may contribute to irreversible myocardial injury. The present study aims to clarify the alterations in cardiac activities in response to experimental cardiac ischemic arrest followed by re-perfusion in isolated hearts perfused with nitric oxide (NO) donor, l-arginine, or NO inhibitor, Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME), to shed light on the possible role of NO in the re-perfusion process. Methods Hearts isolated from adult Wistar rats were studied on Langendorff preparation under basal conditions and during 30 min re-perfusion following 30 min of total global ischemia. Rats were randomly divided into three groups; control and l-arginine or l-NAME infused heart groups. Cardiac tissue content of malondialdhyde, catalase and nitrite was also measured. Results Compared to the control group, both l-arginine and l-NAME infused hearts showed increased basal chronotropy and myocardial flow rate. Following ischemia and during the whole period of re-perfusion, the three groups demonstrated significant deterioration in the inotropic activity and compromised myocardial flow rate. l-arginine infused hearts revealed depressed inotropy and chronotropy, weak systolic and diastolic functions with compromised myocardial flow at early 5 min of re-perfusion, yet with significantly higher myocardial flow rate by the end of re-perfusion. Conclusions Reducing NO availability by l-NAME revealed mild impact on the ischemia re-perfusion induced contractile dysfunction, whereas excess NO worsens cardiac performance at the early re-perfusion period.

Sex differences in progesterone-induced relaxation in the coronary bed from normotensive rats

Progesterone seems to play a role in cardiovascular physiology since its receptors are expressed on endothelial cells from both sexes of mammals. However, little is known about its role on the coronary circulation. Thus, this study aims to evaluate the effect of acute administration of progesterone on the coronary bed and the endothelial pathways involved in this action in normotensive rats of both sexes. A dose–response curve of progesterone (1–50 μmol/L) in isolated hearts using the Langendorff preparation was performed. Baseline coronary perfusion pressure (CPP) was determined, and the vasoactive effect of progesterone was evaluated before and after infusion with Nω-nitro-L-arginine methyl ester (L-NAME), indomethacin, catalase, and Tiron. The analysis of nitric oxide (NO) and superoxide anion (O2 · −) was performed by DAF-2DA and DHE, respectively. Female group showed higher CPP. Nevertheless, progesterone promoted a similar relaxing response in both sexes. The use of L-NAME increased vasodilatory response in both sexes. When indomethacin was used, only the males showed a reduced relaxing response, and in the combined inhibition with L-NAME, indomethacin, and catalase, or with the use of Tiron, only the females presented reduced responses. NO and O2 ·− production has increased in female group, while the male group has increased only NO production. Our results suggest that progesterone is able to modulate vascular reactivity in coronary vascular bed with a vasodilatory response in both sexes. These effects seem to be, at least in part, mediated by different endothelial pathways, involving NO and EDH pathways in females and NO and prostanoids pathways in males.

Cell atlas of the foetal human heart and implications for autoimmune-mediated congenital heart block

Aims Investigating human heart development and applying this to deviations resulting in disease is incomplete without molecular characterization of the cell types required for normal functioning. We investigated foetal human heart single-cell transcriptomes from mid-gestational healthy and anti-SSA/Ro associated congenital heart block (CHB) samples. Methods and results Three healthy foetal human hearts (19th to 22nd week of gestation) and one foetal heart affected by autoimmune-associated CHB (21st week of gestation) were subjected to enzymatic dissociation using the Langendorff preparation to obtain single-cell suspensions followed by 10× Genomics- and Illumina-based single-cell RNA-sequencing (scRNA-seq). In addition to the myocytes, fibroblasts, immune cells, and other minor cell types, previously uncharacterized diverse sub-populations of endothelial cells were identified in the human heart. Differential gene expression analysis revealed increased and heterogeneous interferon responses in varied cell types of the CHB heart compared with the healthy controls. In addition, we also identified matrisome transcripts enriched in CHB stromal cells that potentially contribute to extracellular matrix deposition and subsequent fibrosis. Conclusion These data provide an information-rich resource to further our understanding of human heart development, which, as illustrated by comparison to a heart exposed to a maternal autoimmune environment, can be leveraged to provide insight into the pathogenesis of disease.

Augmentation of Ouabain-Induced Increase in Heart Muscle Contractility by Akt Inhibitor MK-2206

Cardiac steroids (CSs), such as ouabain and digoxin, increase the force of contraction of heart muscle and are used for the treatment of congestive heart failure (CHF). However, their small therapeutic window limits their use. It is well established that Na+, K+-ATPase inhibition mediates CS-induced increase in heart contractility. Recently, the involvement of intracellular signal transduction was implicated in this effect. The aim of the present study was to test the hypothesis that combined treatment with ouabain and Akt inhibitor (MK-2206) augments ouabain-induced inotropy in mammalian models. We demonstrate that the combined treatment led to an ouabain-induced increase in contractility at concentrations at which ouabain alone was ineffective. This was shown in 3 experimental systems: neonatal primary rat cardiomyocytes, a Langendorff preparation, and an in vivo myocardial infarction induced by left anterior descending coronary artery (LAD) ligation. Furthermore, cell viability experiments revealed that this treatment protected primary cardiomyocytes from MK-2206 toxicity and in vivo reduced the size of scar tissue 10 days post-LAD ligation. We propose that Akt activity imposes a constant inhibitory force on muscle contraction, which is attenuated by low concentrations of MK-2206, resulting in potentiation of the ouabain effect. This demonstration of the increase in the CS effect advocates the development of the combined treatment in CHF.

Abstract 33: Prostaglandin E2 Reduces Cardiac Contractility via EP3 Receptor

Prostaglandin E2 (PGE2) EP receptors EP3 and EP4 are present in the heart and signal via decreased and increased cAMP production, respectively. Previously we reported that cardiomyocyte-specific EP4 KO mice develop a phenotype of dilated cardiomyopathy with reduced ejection fraction. We thus hypothesized that PGE2 decreases contractility via EP3. To test this hypothesis, the effects of PGE2 and the EP1/EP3 agonist sulprostone (sulp) were examined in the mouse langendorff preparation and in adult mouse cardiomyocytes (AVM) using the IonOptix cell contractility system. Isolated hearts of 18-20 wk old male C57Bl/6 mice were mounted and equilibrated for 10 min, then perfused with PGE2 (10 -6 mol/l) or sulp (10 -6 mol/l) for 30 min. Values at the end of equilibration were set to 100%. Compared to vehicle, PGE2 decreased +dp/dt (77.8±3% vs 96.7±3%, p<0.01) and left ventricular developed pressure, LVDP (77.2±2% vs 96.8±3%, p<0.001). Sulp decreased +dp/dt (75.9±2% vs 96.7±3%, p<0.001), -dp/dt (72.2±1% vs 85.7±1%, p<0.01) and LVDP (70.9±1% vs 96.8±3%, p<0.001). The effects of both PGE2 and sulp were reversed by the EP3 antagonist, L789,106 (10 -6 mol/l). Myocyte contractility was evaluated on the IonOptix system with pacing at 1Hz. Treatment with PGE2 (10 -9 M) for 10 min reduced contractility as measured by peak height (3.69 ± 0.48% for vehicle vs 2.00 ± 0.22% for PGE2, p < 0.05 ), departure velocity (-171.9 ± 22.9 um/sec for vehicle vs -106.3± 12.5 um/sec for PGE2, p < 0.05) and return velocity (87.7 ± 16.3 um/sec for vehicle vs 36.7 ± 6.6 um/sec for PGE2, p < 0.05) with similar effects noted for sulp. Sulp reduced change in peak height (4.79 ± 1.15% for vehicle vs 1.81 ± 0.37% for sulp, p < 0.05), departure velocity (-169.1 ± 35.8 um/sec for vehicle vs -59.4 ± 10.3 um/sec for sulp, p < 0.05) and return velocity (86.5 ± 23.8 um/sec for vehicle vs 16.9 ± 14.7 um/sec for sulp, p < 0.05). We then examined the acute effects of PGE2 and sulp on expression of phosphorylated phospholamban (PLN) and SERCA using Western blot. Treatment of AVM for 15min with either PGE2 or sulp decreased expression of phosphorylated PLN corrected to total PLN, by 67% and 43%. SERCA2a expression was unaffected. In conclusion, PGE2 and sulp reduce contractility via the EP3 receptor through effects on PLN.

Aldosterone augments Na+-induced reduction of cardiac norepinephrine reuptake

Impairment of the cardiac norepinephrine (NE) reuptake by the neuronal NE transporter contributes to enhanced cardiac NE net release in congestive heart failure. Elevated plasma levels of aldosterone (AL) promote sympathetic overstimulation in failing hearts by unclear mechanisms. Our aim was to evaluate if elevated AL and/or alterations in Na+ intake regulate cardiac NE reuptake. To test the effects of AL and Na+ on cardiac NE reuptake, Wistar rats were fed a normal-salt (NS) diet (0.2% NaCl), a low-salt (LS) diet (0.015% NaCl), or a high-salt (HS) diet (8% NaCl). Another group of animals received AL infusion alone (0.75 μg/h) or AL infusion plus HS diet. Specific cardiac [3H]NE uptake via the NE transporter in a Langendorff preparation and AL plasma levels were measured at different time points between 5 and 42 days of treatment. To compare these findings from healthy animals with a disease model, Dahl salt-sensitive rats were investigated as a model of congestive heart failure with endogenously elevated AL. In summary, neither exogenous nor endogenous elevations of AL alone were sufficient to reduce cardiac NE reuptake. Only the HS diet induced a reduction of NE reuptake by 26%; additional infusion of AL augmented this effect to a further reduction of NE reuptake by 36%. In concordance, Dahl salt-sensitive rats treated with a HS diet displayed elevated AL and a marked reduction of NE reuptake. We conclude that exogenous or endogenous AL elevations alone do not reduce cardiac NE reuptake, but AL serves as an additional factor that negatively regulates cardiac NE reuptake in concert with HS intake.

Lipid Emulsion Reverses Bupivacaine-induced Asystole in Isolated Rat Hearts

Background The concentration-response and time-response relationships of lipid emulsions used to reverse bupivacaine-induced asystole are poorly defined. Methods Concentration response across a range of lipid concentrations (0-16%) to reverse bupivacaine-induced asystole were observed using isolated rat heart Langendorff preparation. Cardiac function parameters were recorded during infusion. Concentrations of bupivacaine in myocardial tissue were measured by liquid chromatography and tandem mass spectrometry at the end of the experiment. Results Although all lipid-treated hearts recovered (cardiac recovery was defined as a rate-pressure product more than 10% baseline), no nonlipid-treated hearts (control group) did so. The ratio of the maximum rate pressure product during recovery to baseline value demonstrated a concentration-dependent relationship among lipid groups, with 0.25, 0.5, 1, 2, 4, 8, and 16%. Mean ± SD values for each corresponding group were 22 ± 4, 24 ± 5, 29 ± 6, 52 ± 11, 73 ± 18, 119 ± 22, and 112 ± 10%, respectively (n = 6, P &lt; 0.01). Rate-pressure product in lipid groups with 4-16% concentrations was lower at 15-40 min than at 1 min, showing a decreasing tendency during recovery phase (P &lt; 0.01). The concentration of myocardial bupivacaine in all lipid-treated groups was significantly lower than in the control group (P &lt; 0.01). It was also lower in lipid groups with 2-16% concentrations than in those with concentrations at 0.25-1% (P &lt; 0.05), with the 16% group lower than groups with 2-8% concentrations (P &lt; 0.001). Conclusion Lipid application in bupivacaine-induced asystole displays a concentration-dependent and time-response relationship in isolated rat hearts.