Adverse Effects on beta-Adrenergic Receptor Coupling: Ischemic Postconditioning Failed to Preserve Long-Term Cardiac Function

BACKGROUND: Ischemic preconditioning (IPC) and ischemic postconditioning (IPoC) are currently among the most efficient strategies protecting the heart against ischemia/reperfusion injury. However, the effect of IPC and IPoC on functional recovery following ischemia/reperfusion is less clear, particularly with regard to the specific receptor-mediated signaling of the postischemic heart. The current article examines the effect of IPC or IPoC on the regulation and coupling of beta-adrenergic receptors and their effects on postischemic left ventricular function. METHODS AND RESULTS: The beta-adrenergic signal transduction was analyzed in 3-month-old Wistar rats for each of the intervention strategies (Sham, ischemia/reperfusion, IPC, IPoC) immediately and 7 days after myocardial infarction. Directly after the infarction a cardioprotective potential was demonstrated for both IPC and IPoC: the infarct size was reduced, apoptosis and production of reactive oxygen species were lowered, and the myocardial tissue was preserved. Seven days after myocardial ischemia, only IPC hearts showed significant functional improvement. Along with a deterioration in fractional shortening, IPoC hearts no longer responded adequately to beta-adrenergic stimulation. The stabilization of beta-adrenergic receptor kinase-2 via increased phosphorylation of Mdm2 (an E3-ubiquitin ligase) was responsible for desensitization of beta-adrenergic receptors and identified as a characteristic specific to IPoC hearts. CONCLUSIONS: Immediately after myocardial infarction, rapid and transient activation of beta-adrenergic receptor kinase-2 may be an appropriate means to protect the injured heart from excessive stress. In the long term, however, induction and stabilization of beta-adrenergic receptor kinase-2, with the resultant loss of positive inotropic function, leads to the functional picture of heart failure

Protection by the NO-Donor SNAP and BNP against Hypoxia/Reoxygenation in Rat Engineered Heart Tissue

In vitro assays could replace animal experiments in drug screening and disease modeling, but have shortcomings in terms of functional readout. Force-generating engineered heart tissues (EHT) provide simple automated measurements of contractile function. Here we evaluated the response of EHTs to hypoxia/reoxygenation (H/R) and the effect of known cardiocytoprotective molecules. EHTs from neonatal rat heart cells were incubated for 24 h in EHT medium. Then they were subjected to 180 min hypoxia (93% N2, 7% CO2) and 120 min reoxygenation (40% O2, 53% N2, 7% CO2), change of medium and additional follow-up of 48 h. Time-matched controls (40% O2, 53% N2, 7% CO2) were run for comparison. The following conditions were applied during H/R: fresh EHT medium (positive control), the NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 10-7, 10-6, 10-5 M) or the guanylate cyclase activator brain type natriuretic peptide (BNP, 10-9, 10-8, 10-7 M). Frequency and force of contraction were repeatedly monitored over the entire experiment, pH, troponin I (cTnI), lactate dehydrogenase (LDH) and glucose concentrations measured in EHT medium. Beating activity of EHTs in 24 h-medium ceased during hypoxia, partially recovered during reoxygenation and reached time-control values during follow-up. H/R was accompanied by a small increase in LDH and non-significant increase in cTnI. In fresh medium, some EHTs continued beating during hypoxia and all EHTs recovered faster during reoxygenation. SNAP and BNP showed small but significant protective effects during reoxygenation. EHTs are applicable to test potential cardioprotective compounds in vitro, monitoring functional and biochemical endpoints, which otherwise could be only measured by using in vivo or ex vivo heart preparations. The sensitivity of the model needs improvement

Improved Characterization of EV Preparations Based on Protein to Lipid Ratio and Lipid Properties.

In recent years the study of extracellular vesicles has gathered much scientific and clinical interest. As the field is expanding, it is becoming clear that better methods for characterization and quantification of extracellular vesicles as well as better standards to compare studies are warranted. The goal of the present work was to find improved parameters to characterize extracellular vesicle preparations. Here we introduce a simple 96 well plate-based total lipid assay for determination of lipid content and protein to lipid ratios of extracellular vesicle preparations from various myeloid and lymphoid cell lines as well as blood plasma. These preparations included apoptotic bodies, microvesicles/microparticles, and exosomes isolated by size-based fractionation. We also investigated lipid bilayer order of extracellular vesicle subpopulations using Di-4-ANEPPDHQ lipid probe, and lipid composition using affinity reagents to clustered cholesterol (monoclonal anti-cholesterol antibody) and ganglioside GM1 (cholera toxin subunit B). We have consistently found different protein to lipid ratios characteristic for the investigated extracellular vesicle subpopulations which were substantially altered in the case of vesicular damage or protein contamination. Spectral ratiometric imaging and flow cytometric analysis also revealed marked differences between the various vesicle populations in their lipid order and their clustered membrane cholesterol and GM1 content. Our study introduces for the first time a simple and readily available lipid assay to complement the widely used protein assays in order to better characterize extracellular vesicle preparations. Besides differentiating extracellular vesicle subpopulations, the novel parameters introduced in this work (protein to lipid ratio, lipid bilayer order, and lipid composition), may prove useful for quality control of extracellular vesicle related basic and clinical studies

Protection by the NO-Donor SNAP and BNP against Hypoxia/Reoxygenation in Rat Engineered Heart Tissue.

In vitro assays could replace animal experiments in drug screening and disease modeling, but have shortcomings in terms of functional readout. Force-generating engineered heart tissues (EHT) provide simple automated measurements of contractile function. Here we evaluated the response of EHTs to hypoxia/reoxygenation (H/R) and the effect of known cardiocytoprotective molecules. EHTs from neonatal rat heart cells were incubated for 24 h in EHT medium. Then they were subjected to 180 min hypoxia (93% N2, 7% CO2) and 120 min reoxygenation (40% O2, 53% N2, 7% CO2), change of medium and additional follow-up of 48 h. Time-matched controls (40% O2, 53% N2, 7% CO2) were run for comparison. The following conditions were applied during H/R: fresh EHT medium (positive control), the NO-donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 10(-7), 10(-6), 10(-5) M) or the guanylate cyclase activator brain type natriuretic peptide (BNP, 10(-9), 10(-8), 10(-7) M). Frequency and force of contraction were repeatedly monitored over the entire experiment, pH, troponin I (cTnI), lactate dehydrogenase (LDH) and glucose concentrations measured in EHT medium. Beating activity of EHTs in 24 h-medium ceased during hypoxia, partially recovered during reoxygenation and reached time-control values during follow-up. H/R was accompanied by a small increase in LDH and non-significant increase in cTnI. In fresh medium, some EHTs continued beating during hypoxia and all EHTs recovered faster during reoxygenation. SNAP and BNP showed small but significant protective effects during reoxygenation. EHTs are applicable to test potential cardioprotective compounds in vitro, monitoring functional and biochemical endpoints, which otherwise could be only measured by using in vivo or ex vivo heart preparations. The sensitivity of the model needs improvement

Comparison of EHT contractile behavior in 24 h medium control (24 h MC) and SNAP treated groups (n = 6).

<p>(A) Beating pattern of EHTs. Grey boxes represent beating periods of EHTs whereas black boxes indicate non-beating phases. (B) Total number of beats is expressed as the percentage of baseline. (C) Rate force product of EHTs during reoxygenation. (D) Histological parameters of EHT sections. (E-F) cTnI, LDH release and glucose consumption are shown respectively. Data are expressed as mean ± SEM; **p<0.001 one-way ANOVA, followed by Dunett’s post hoc test, n = 6.</p

Study design and experimental groups.

<p>Study design and experimental groups.</p

Cardioprotection by Farnesol: Role of the Mevalonate Pathway

Purpose: Farnesol is a key metabolite of the mevalonate pathway and known as an antioxidant. We examined whether farnesol treatment protects the ischemic heart. Methods: Male Wistar rats were treated orally with 0.2, 1, 5, and 50 mg/kg/day farnesol/vehicle for 12 days, respectively. On day 13, the effect of farnesol treatment on cardiac ischemic tolerance and biochemical changes was tested. Therefore, hearts were isolated and subjected either to 30 min coronary occlusion followed by 120 min reperfusion to measure infarct size or to 10 min aerobic perfusion to measure cardiac mevalonate pathway end-products (protein prenylation, cholesterol, coenzyme Q9, coenzyme Q10, dolichol), and 3-nitrotyrosine (oxidative/nitrosative stress marker), respectively. The cytoprotective effect of farnesol was also tested in cardiomyocytes subjected to simulated ischemia/reperfusion. Results: Farnesol pretreatment decreased infarct size in a U-shaped dose-response manner where 1 mg/kg/day dose reached a statistically significant reduction (22.3 ± 3.9 % vs. 40.9 ± 6.1 % of the area at risk, p < 0.05). Farnesol showed a similar cytoprotection in cardiomyocytes. The cardioprotective dose of farnesol (1 mg/kg/day) significantly increased the marker of protein geranylgeranylation, but did not influence protein farnesylation, cardiac tissue cholesterol, coenzyme Q9, coenzyme Q10, and dolichol. While the cardioprotective dose of farnesol did not influence 3-nitrotyrosine, the highest dose of farnesol (50 mg/kg/day) tested did not show cardioprotection, however, it significantly decreased cardiac 3-nitrotyrosine. Conclusions: This is the first demonstration that oral farnesol treatment reduces infarct size. The cardioprotective effect of farnesol likely involves increased protein geranylgeranylation and seems to be independent of the antioxidant effect of farnesol. © 2013 Springer Science+Business Media New York

Cytoprotection by the NO-donor SNAP against ischemia/reoxygenation injury in mouse embryonic stem cell-derived cardiomyocytes

Embryonic stem cell (ESC)-derived cardiomyocytes are a promising cell source for the screening for potential cytoprotective molecules against ischemia/reperfusion injury, however, little is known on their behavior in hypoxia/reoxygenation conditions. Here we tested the cytoprotective effect of the NO-donor SNAP and its downstream cellular pathway. Mouse ESC-derived cardiomyocytes were subjected to 150-min simulated ischemia (SI) followed by 120-min reoxygenation or corresponding non-ischemic conditions. The following treatments were applied during SI or normoxia: the NO-donor S-Nitroso-N-acetyl-D,L-penicillamine (SNAP), the protein kinase G (PKG) inhibitor, the KATP channel blocker glibenclamide, the particulate guanylate cyclase activator brain type natriuretic peptide (BNP), and a non- specific NO synthase inhibitor (N-Nitro-L-arginine, L-NNA) alone or in different combinations. Viability of cells was assayed by propidium iodide staining. SNAP attenuated SI-induced cell death in a concentration-dependent manner, and this protection was attenuated by inhibition of either PKG or KATP channels. However, SI-induced cell death was not affected by BNP or by L- NNA. We conclude that SNAP protects mESC-derived cardiomyocytes against SI/R injury and that soluble guanylate-cyclase, PKG, and KATP channels play a role in the downstream pathway of SNAP- induced cytoprotection. The present mESC-derived cardiomyocyte- based screening platform is a useful tool for discovery of cytoprotective molecules