Effects of the Delta Opioid Receptor Agonist DADLE in a Novel Hypoxia-Reoxygenation Model on Human and Rat-Engineered Heart Tissue: A Pilot Study

Abstract

Intermittent hypoxia and various pharmacological compounds protect the heart from ischemia reperfusion injury in experimental approaches, but the translation into clinical trials has largely failed. One reason may lie in species differences and the lack of suitable human in vitro models to test for ischemia/reperfusion. We aimed to develop a novel hypoxia-reoxygenation model based on three-dimensional, spontaneously beating and work performing engineered heart tissue (EHT) from rat and human cardiomyocytes. Contractile force, the most important cardiac performance parameter, served as an integrated outcome measure. EHTs from neonatal rat cardiomyocytes were subjected to 90 min of hypoxia which led to cardiomyocyte apoptosis as revealed by caspase 3-staining, increased troponin I release (time control vs. 24 h after hypoxia: cTnI 2.7 vs. 6.3 ng/mL, ** p = 0.002) and decreased contractile force (64 ± 6% of baseline) in the long-term follow-up. The detrimental effects were attenuated by preceding the long-term hypoxia with three cycles of 10 min hypoxia (i.e., hypoxic preconditioning). Similarly, [d-Ala2, d-Leu5]-enkephalin (DADLE) reduced the effect of hypoxia on force (recovery to 78 ± 5% of baseline with DADLE preconditioning vs. 57 ± 5% without, p = 0.012), apoptosis and cardiomyocyte stress. Human EHTs presented a comparable hypoxia-induced reduction in force (55 ± 5% of baseline), but DADLE failed to precondition them, likely due to the absence of δ-opioid receptors. In summary, this hypoxia-reoxygenation in vitro model displays cellular damage and the decline of contractile function after hypoxia allows the investigation of preconditioning strategies and will therefore help us to understand the discrepancy between successful conditioning in vitro experiments and its failure in clinical trials.

Keywords: 3D tissue model; cardiac hypertrophy; cardioprotection; human induced pluripotent stem cells; opioids; preconditioning; reperfusion injury; tissue engineering; translational medicine.

Figure 1

Study design and different hypoxia-reoxygenation experimental groups. (A) Hypoxia-reoxygenation alone (HYP) or (B) preceded by hypoxic preconditioning (HPC) or (C) preceded by drug-induced preconditioning (with [d-Ala2, d-Leu5]-Enkephalin (DADLE): DA, or Naloxone and DADLE: N+DA).

Figure 2

Model of hypoxia-reoxygenation injury in rat-engineered heart tissues (EHTs). (A) Correlation between duration of hypoxia and decrease in force after 120 min of reoxygenation (each dot represents the mean force of one batch of EHTs (n = 8–12), except for 90 min where hypoxia experiments from 9 independent batches were averaged (mean ± SEM). (B) Release of cardiac troponin I (cTnI) over 24 h after the onset of hypoxia (90 min) compared to the time control (TC) group. Data are presented as scatterplot with mean (bars) ± SEM (whiskers). One-way ANOVA with Dunnett‘s post-hoc test (n = 2–13, each measurement from the pooled cell culture media of 2 EHTs; “19 h” vs. “TC”, * p = 0.029; “24 h” vs. “TC”, ** p = 0.002). The values depicted as circles stem from one experimental series. (C) MLC-2V (myosin light chain 2, ventricular isoform) staining of EHTs undergoing 90 min of hypoxia and reoxygenation (HYP) and control group without hypoxia (time control = TC). In some central areas of the hypoxic EHTs (e.g., at the blue rectangle) only roundish cardiomyocytes were detectable. (D) Reactive oxygen species (ROS) measured in the cell culture media of TC and HYP groups. Student’s unpaired t-test (n = 10–21 per group, *** p < 0.0001).

Figure 3

(A,B) Effect of hypoxic preconditioning (HPC, 3 × 10 min) followed by 90 min hypoxia compared to the hypoxic group without preconditioning (HYP). Data are presented as mean ± SEM. (A) Force (mN) before, during and after hypoxia in early (0–2 h) and late (≥2 days) reoxygenation. Two-way ANOVA and Sidak’s post-test at day 12 (yellow box, n = 10 per group, * p = 0.024). (B) Release of cardiac troponin I (cTnI) in the first 24 h (see arrow in A) after reoxygenation. Unpaired t-test (n = 6–7, * p = 0.031). (C,D) Impact of gas flow rates on the O₂-fraction in the medium and on force. (C) O₂-fraction measured in the medium during hypoxia experiments at high (5 L/min) or low gas flow (0.5 L/min) in the hypoxic chamber. The oxygen concentration inside the incubator (gas phases), as well as in the medium (5 mm below the surface, liquid phase), was determined with an oxygen microprobe based on fluorescent light emission (Microx-TX transmitter, tip diameter < 50 µm; Presens Precision Sensing GmbH, Regensburg, Germany). (D) Corresponding changes of the contractile force over time.

Figure 4

(A–D) Effect of preconditioning with [d-Ala2, d-Leu5]-Enkephalin (DADLE) 100 nM, 3 × 10 min, (DA) or DADLE 100 nM plus naloxone 10 µM (N+DA) compared to the hypoxic group without preconditioning (HYP) and the time control (TC) without hypoxia. Data are presented as scatterplot with mean (bars) ± SEM (whiskers). One-way ANOVA with Dunnett‘s multiple comparisons test, in panels A–D compared to HYP group. (A) Force in early reoxygenation (120 min after the onset of reoxygenation) as percent of initial force before hypoxia (n = 7–8, “DA” vs. “HYP” * p = 0.046; “N+DA” vs. “HYP”, p = 0.165). (B) Hypoxia-reoxygenation injury (of experiment depicted in (A)) quantified by glucose-6-phosphate dehydrogenase (G6PDH) release in the first 24 h after onset of hypoxia normalized to the amount of G6PDH in the hypoxia group without preconditioning (n = 5; “DA” vs. “HYP”, *** p = 0.0003). (C) Release of cardiac troponin I (cTnI) in the first 24 h (of experiment depicted in A) after reoxygenation, n = 3 samples per group, each sample pooled from media from 2 EHTs. (D) Force in late (7 days) reoxygenation as percent of initial force before hypoxia (TC n = 9, HYP n = 71, DA n = 37, N+DA n = 15; “TC” vs. “HYP”, ** p = 0.003; “DA” vs. “HYP”, * p = 0.012; “N+DA” vs. “HYP”, p = 0.523). (E) Reduction in force (relative force in % of baseline compared to the time control) after 7 days of afterload enhancement (hatched bars) without (AE) and with preconditioning (DA and N+DA, n = 7–8 per group; “TC” vs. “AE”, * p = 0.044; “DA” vs. “AE”, * p = 0.022; “N+DA” vs. “AE”, p = 0.682; 1-way ANOVA with Dunnett‘s multiple comparisons test compared to group AE).

Figure 5

Effect of preconditioning with [d-Ala2, d-Leu5]-Enkephalin (DADLE, 100 nM, 3 × 10 min, DA), followed by 90 min hypoxia compared to the hypoxic group without preconditioning (HYP) and to the control group without hypoxia (time control, TC). (A–C) Atrial natriuretic peptide (ANP)-stained paraffin section of the EHT in a longitudinal view. (A) 5.0 ANP⁺ cells/mm² in TC, (B) 31.1 ANP⁺ cells/mm² in HYP, (C) 15.0 ANP⁺ cells/mm² in DA. (D–F) Active Caspase 3 (subunit p17)-stained paraffin section of EHTs in a longitudinal view. (D) 37.7 Casp-3⁺ nuclei (dark brown)/mm² in TC, (E) 222.2 Casp-3⁺ nuclei/mm² in HYP, (F) 43.1 Casp-3⁺ nuclei/mm² in DA. Nuclei in (A–F) were lightly counterstained with hematoxylin.

Figure 6

Hypoxia-reoxygenation injury in human EHTs. (A) 90 min of hypoxia (HYP) followed by reoxygenation in human EHTs (vs. time control without hypoxia, TC) (mean ± SEM). (B) Effect of preconditioning by [d-Ala2,d-Leu5]-Enkephalin (DADLE, 100 nM, 3 × 10 min, DA) followed by 90 min hypoxia compared to the hypoxic group without preconditioning (HYP) and TC on the contractile force (shown in % of the force before hypoxia at baseline) in the early (120 min after the onset of reoxygenation) and late (day 1) reoxygenation, compared by 1-way ANOVA with Sidak‘s multiple comparisons test (HYP vs. DA, n = 4, p = 0.812 (early) and p = 0.891 (late)). Data are presented as scatterplot with mean (bars) ± SEM (whiskers). (C,D) PCR analyses of δ-opioid receptor expression. β-glucuronidase served as positive, water as negative control. M = marker, indicates 500 bp and 1000 bp size. (C) Detection of the δ-opioid receptor in rat in contrast to human EHTs. The arrow indicates the lack of a δ-opioid receptor signal in human EHTs. (D) PCR analysis showing absence of δ-opioid receptor mRNA in human atrial (RA = right atrium, LA = left atrium), septal (S) and ventricular (RV = right ventricle, LV = left ventricle) tissue samples.