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

PLoS One. 2015 Jul 6;10(7):e0132186. doi: 10.1371/journal.pone.0132186. eCollection 2015.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Glucose / metabolism
  • L-Lactate Dehydrogenase / metabolism
  • Myocardial Contraction / drug effects*
  • Myocardial Ischemia / metabolism
  • Myocardial Ischemia / pathology
  • Myocardial Ischemia / physiopathology
  • Myocardial Ischemia / prevention & control*
  • Myocardium / metabolism*
  • Natriuretic Peptide, Brain / pharmacology*
  • Rats
  • Rats, Inbred Lew
  • Rats, Wistar
  • S-Nitroso-N-Acetylpenicillamine / pharmacology*
  • Tissue Engineering
  • Troponin I / metabolism

Substances

  • Troponin I
  • Natriuretic Peptide, Brain
  • S-Nitroso-N-Acetylpenicillamine
  • L-Lactate Dehydrogenase
  • Glucose

Grant support

This study was supported by grants from the German Research Foundation (DFG Es/88-12), the European Commission (FP7 Angioscaff, FP7 Biodesign) (EA, HA, TE), NKFP 07 1-ES2HEART-HU (OM-00202/2007), National Development Agency – New Hungary Development Plan (TÁMOP-4.2.2-08/1/2008-0013, TÁMOP-4.2.1/B-09/1); OTKA PD 106001 (GA, PJ, VZV, FP). GA holds a "János Bolyai Fellowship” from the Hungarian Academy of Sciences. PJ holds a "Apáczai-Csere János Fellowship” from TÁMOP-4.2.4.A/2-11/1-2012-0001 (National Excellence Program). PF was a Szentágothai Fellow of the National Program of Excellence (TAMOP 4.2.4.A/2-11-1-2012-0001). Pharmahungary Group provided support in the form of salaries for authors AG, JP, and PF, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the "author contributions" section.