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

Görbe A, Eder A, Varga ZV, Pálóczi J, Hansen A, Ferdinandy P, Eschenhagen T - PLoS ONE (2015)

Bottom Line: H/R was accompanied by a small increase in LDH and non-significant increase in cTnI.SNAP and BNP showed small but significant protective effects during reoxygenation.The sensitivity of the model needs improvement.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Szeged, Hungary; Pharmahungary Group, Szeged, Hungary.

ABSTRACT
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.

No MeSH data available.


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pone.0132186.g001: Study design and experimental groups.

Mentions: To test EHT responses under ischemic stress, we used hypoxia/reoxygenation to simulate in vivo myocardial ischemia/reperfusion injury. For this purpose, the spontaneously contracting 15–22 days old EHTs were placed into fresh EHT medium 24 h before experiments. As a pilot experiment, 4 EHTs were subjected to hypoxia by culturing them in 93% N2 and 7% CO2. Based on their beating activity the 3 h hypoxic period was chosen for further experiments. EHTs were subjected to 180 min hypoxia (93% N2 and 7% CO2, EHTs were covered by 24 h conditioned medium) followed by 120 min of reoxygenation (40% O2 53% N2 and 7% CO2, EHTs were covered by 24 h conditioned medium). Endpoints were recorded before hypoxia (baseline), at 11 time points during 180 min hypoxia (0, 15, 30, 45, 60, 80, 100, 120, 140, 160, 180 min) and at 8 time points during reperfusion (0, 20, 40, 60, 75, 90, 105, 120 min). At the end of experimental protocol the EHT medium was collected, and pH was determined. EHTs were placed into freshly prepared medium and were incubated further for 48 h. Additional 3 time points were recorded during the follow up period (overnight, 1 day, 2 days). In the end EHT medium was collected and pH was determined as previously (Fig 1).


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

Görbe A, Eder A, Varga ZV, Pálóczi J, Hansen A, Ferdinandy P, Eschenhagen T - PLoS ONE (2015)

Study design and experimental groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492769&req=5

pone.0132186.g001: Study design and experimental groups.
Mentions: To test EHT responses under ischemic stress, we used hypoxia/reoxygenation to simulate in vivo myocardial ischemia/reperfusion injury. For this purpose, the spontaneously contracting 15–22 days old EHTs were placed into fresh EHT medium 24 h before experiments. As a pilot experiment, 4 EHTs were subjected to hypoxia by culturing them in 93% N2 and 7% CO2. Based on their beating activity the 3 h hypoxic period was chosen for further experiments. EHTs were subjected to 180 min hypoxia (93% N2 and 7% CO2, EHTs were covered by 24 h conditioned medium) followed by 120 min of reoxygenation (40% O2 53% N2 and 7% CO2, EHTs were covered by 24 h conditioned medium). Endpoints were recorded before hypoxia (baseline), at 11 time points during 180 min hypoxia (0, 15, 30, 45, 60, 80, 100, 120, 140, 160, 180 min) and at 8 time points during reperfusion (0, 20, 40, 60, 75, 90, 105, 120 min). At the end of experimental protocol the EHT medium was collected, and pH was determined. EHTs were placed into freshly prepared medium and were incubated further for 48 h. Additional 3 time points were recorded during the follow up period (overnight, 1 day, 2 days). In the end EHT medium was collected and pH was determined as previously (Fig 1).

Bottom Line: H/R was accompanied by a small increase in LDH and non-significant increase in cTnI.SNAP and BNP showed small but significant protective effects during reoxygenation.The sensitivity of the model needs improvement.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Group, Department of Biochemistry, University of Szeged, Szeged, Hungary; Pharmahungary Group, Szeged, Hungary.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus