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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.


Related in: MedlinePlus

Comparison of EHT contractile behaviour in time-matched control (TMC) and 24 h medium control (24 h MC) groups (n = 6).(A) Beating pattern of EHTs was recorded during the entire experiment. Grey boxes indicate beating periods of EHTs, black boxes non-beating phases. (B) Total number of beats is expressed as the percentage of baseline. (C) Rate force product of EHTs. Data are expressed as mean ± SEM; **p<0.01 multiple t-test, n = 4–6. (D) Histological analysis of EHT sections (nucleus/cytoplasm ratio, nuclear circularity). (E-F) Biochemical markers of tissue necrosis (cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) release after hypoxia or after 2 days follow up respectively) and glucose consumption are presented respectively. Data are expressed as mean ± SEM; *p<0.05; unpaired t-test, n = 4–6.
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pone.0132186.g002: Comparison of EHT contractile behaviour in time-matched control (TMC) and 24 h medium control (24 h MC) groups (n = 6).(A) Beating pattern of EHTs was recorded during the entire experiment. Grey boxes indicate beating periods of EHTs, black boxes non-beating phases. (B) Total number of beats is expressed as the percentage of baseline. (C) Rate force product of EHTs. Data are expressed as mean ± SEM; **p<0.01 multiple t-test, n = 4–6. (D) Histological analysis of EHT sections (nucleus/cytoplasm ratio, nuclear circularity). (E-F) Biochemical markers of tissue necrosis (cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) release after hypoxia or after 2 days follow up respectively) and glucose consumption are presented respectively. Data are expressed as mean ± SEM; *p<0.05; unpaired t-test, n = 4–6.

Mentions: Spontaneously beating 15–22 days old rat EHTs, incubated for 24 h in EHT medium, were exposed to 180 min hypoxia and 120 min reoxygenation (24 h medium control; 24 h MC) or standard 40% oxygen (time-matched control; TMC). The beating map (Fig 2A) shows the contractile activity of EHTs at each recording time point (beating periods are expressed as grey boxes, non-beating periods are expressed as black boxes). TMC group were essentially stable during the whole experiment, while hypoxia induced a fast decrease in beating rate and complete cease after ~100 min. Three out of 6 EHTs recovered during reoxygenation and all of them during the follow up period.


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)

Comparison of EHT contractile behaviour in time-matched control (TMC) and 24 h medium control (24 h MC) groups (n = 6).(A) Beating pattern of EHTs was recorded during the entire experiment. Grey boxes indicate beating periods of EHTs, black boxes non-beating phases. (B) Total number of beats is expressed as the percentage of baseline. (C) Rate force product of EHTs. Data are expressed as mean ± SEM; **p<0.01 multiple t-test, n = 4–6. (D) Histological analysis of EHT sections (nucleus/cytoplasm ratio, nuclear circularity). (E-F) Biochemical markers of tissue necrosis (cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) release after hypoxia or after 2 days follow up respectively) and glucose consumption are presented respectively. Data are expressed as mean ± SEM; *p<0.05; unpaired t-test, n = 4–6.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132186.g002: Comparison of EHT contractile behaviour in time-matched control (TMC) and 24 h medium control (24 h MC) groups (n = 6).(A) Beating pattern of EHTs was recorded during the entire experiment. Grey boxes indicate beating periods of EHTs, black boxes non-beating phases. (B) Total number of beats is expressed as the percentage of baseline. (C) Rate force product of EHTs. Data are expressed as mean ± SEM; **p<0.01 multiple t-test, n = 4–6. (D) Histological analysis of EHT sections (nucleus/cytoplasm ratio, nuclear circularity). (E-F) Biochemical markers of tissue necrosis (cardiac troponin I (cTnI) and lactate dehydrogenase (LDH) release after hypoxia or after 2 days follow up respectively) and glucose consumption are presented respectively. Data are expressed as mean ± SEM; *p<0.05; unpaired t-test, n = 4–6.
Mentions: Spontaneously beating 15–22 days old rat EHTs, incubated for 24 h in EHT medium, were exposed to 180 min hypoxia and 120 min reoxygenation (24 h medium control; 24 h MC) or standard 40% oxygen (time-matched control; TMC). The beating map (Fig 2A) shows the contractile activity of EHTs at each recording time point (beating periods are expressed as grey boxes, non-beating periods are expressed as black boxes). TMC group were essentially stable during the whole experiment, while hypoxia induced a fast decrease in beating rate and complete cease after ~100 min. Three out of 6 EHTs recovered during reoxygenation and all of them during the follow up period.

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