Increased length-dependent activation of human engineered heart tissue after chronic α1A-adrenergic agonist treatment: testing a novel heart failure therapy

Abstract

Chronic stimulation of cardiac α_1A-adrenergic receptors (α_1A-ARs) improves symptoms in multiple preclinical models of heart failure. However, the translational significance remains unclear. Human engineered heart tissues (EHTs) provide a means of quantifying the effects of chronic α_1A-AR stimulation on human cardiomyocyte physiology. EHTs were created from thin slices of decellularized pig myocardium seeded with human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and fibroblasts. With a paired experimental design, EHTs were cultured for 3 wk, mechanically tested, cultured again for 2 wk with α_1A-AR agonist A61603 (10 nM) or vehicle control, and retested after drug washout for 24 h. Separate control experiments determined the effects of EHT age (3-5 wk) or repeat mechanical testing. We found that chronic A61603 treatment caused a 25% increase of length-dependent activation (LDA) of contraction compared with vehicle treatment (n = 7/group, P = 0.035). EHT force was not increased after chronic A61603 treatment. However, after vehicle treatment, EHT force was increased by 35% relative to baseline testing (n = 7/group, P = 0.022), suggesting EHT maturation. Control experiments suggested that increased EHT force resulted from repeat mechanical testing, not from EHT aging. RNA-seq analysis confirmed that the α_1A-AR is expressed in human EHTs and found chronic A61603 treatment affected gene expression in biological pathways known to be activated by α_1A-ARs, including the MAP kinase signaling pathway. In conclusion, increased LDA in human EHT after chronic A61603 treatment raises the possibility that chronic stimulation of the α_1A-AR might be beneficial for increasing LDA in human myocardium and might be beneficial for treating human heart failure by restoring LDA.NEW & NOTEWORTHY Chronic stimulation of α_1A-adrenergic receptors (α_1A-ARs) is known to mediate therapeutic effects in animal heart failure models. To investigate the effects of chronic α_1A-AR stimulation in human cardiomyocytes, we tested engineered heart tissue (EHT) created with iPSC-derived cardiomyocytes. RNA-seq analysis confirmed human EHT expressed α_1A-ARs. Chronic (2 wk) α_1A-AR stimulation with A61603 (10 nM) increased length-dependent activation (LDA) of contraction. Chronic α_1A-AR stimulation might be beneficial for treating human heart failure by restoring LDA.

Keywords: engineered heart tissue; heart failure; human; induced pluripotent stem cell; α1A-adrenergic receptor.

Figure 1.

A: schematic of engineered heart tissue (EHT) mounted in MyoPod cassette. EHT length was varied using an adjustable force transducer claw. B: schematic of the experimental protocol.

Figure 2.

Representative records and pooled data for contractions of engineered heart tissue (EHT) after 3 wk culture, before drug/vehicle treatment. A: superimposed twitch contractions for EHT at various lengths above and below culture length (L_o). B: relationship between force vs. EHT length: same experiment as in A (means ± SE, n = 3 contractions, errors are smaller than symbols). C: baseline parameters before drug/vehicle treatment for all EHTs in cohort (individual and mean values, n = 14, and coefficients of variation).

Figure 3.

Chronic A61603 treatment did not change engineered heart tissue (EHT) intrinsic contraction rate. A: intrinsic rate of spontaneous EHT contractions measured before and after chronic (2 wk) treatment with vehicle or A61603 (means ± SE, n = 5–7/group, P values for mixed effects analysis). B: no change in intrinsic rate after treatment relative to before treatment (P value for unpaired t test).

Figure 4.

Chronic A61603 treatment increased length-dependent activation (LDA). A: LDA measured before and after chronic (2 wk) treatment with vehicle or A61603 (means ± SE, n = 7/group, P values for paired t tests). B: LDA (%initial) was higher after A61603 treatment vs. after vehicle treatment (P value for unpaired t test).

Figure 5.

Chronic A61603 treatment did not increase engineered heart tissue (EHT) force. A: twitch contraction force measured before and after chronic (2 wk) treatment with vehicle or A61603 (means ± SE, n = 7/group, P values for paired t tests). B: EHT force (%initial) was increased after vehicle treatment but not after A61603 treatment (P value for unpaired t test). C: EHT width before and after treatment with vehicle or A61603 (means ± SE, n = 7/group, P values for paired t tests).

Figure 6.

Effect of engineered heart tissue (EHT) age on contraction parameters measured on a single occasion (n = 3–6 per age group; P values for linear regressions). A: length-dependent activation (LDA). B: force. C: time to peak force (TTP). D: time to 50% relaxation (RT₅₀).

Figure 7.

Repeat mechanical testing after 3 wk culture increased engineered heart tissue (EHT) force. A: relationship of EHT force (normalized) to EHT age. First, EHT mechanical testing was at 3 wk (n = 6, same EHT as Fig. 6B), with retesting after 1 day or 1 wk (P value for linear regression). B: effect of both EHT age and repeat testing on EHT force (data from Fig. 6B and Fig. 7A) (P values for unpaired t tests).

Figure 8.

Effect of chronic A61603 treatment on the timing of contraction and relaxation. A: twitch time to peak (TTP) measured before and after chronic (2 wk) treatment with vehicle or A61603 (means ± SE, n = 7/group, P values for paired t tests). B: TTP after treatment relative to before treatment (P value for unpaired t test). C: time to 50% relaxation (RT₅₀) measured before and after chronic (2 wk) treatment with vehicle or A61603 (means ± SE, n = 7/group, P values for paired t tests). D: RT₅₀ after treatment relative to before treatment (P value for unpaired t test).

Figure 9.

Effect of chronic A61603 treatment on the response to rapid pacing. A: maximum capture rate (MCR) measured before and after chronic (2 wk) treatment with vehicle or A61603 (means ± SE, n = 7/group, P values for paired t tests). B: MCR after treatment relative to before treatment (P value for unpaired t test). C: relationship between engineered heart tissue (EHT) force (normalized) and stimulation frequency before treatment with vehicle or A61603 (n = 14) and after vehicle treatment (n = 5) (means ± SE, ****P < 0.0001, **P < 0.01, paired t tests). D: force-frequency relation after A61603 treatment (n = 6) (means ± SE, ns, not significant, paired t test).

Figure 10.

Effects on chronic A61603 treatment on adrenergic receptors and contraction-related gene expression. A: expression of adrenergic receptors in engineered heart tissue (EHT) treated with A61603 (n = 6) or vehicle (n = 7). ****P < 0.0001. B: heat maps for selected contraction-related genes; values scaled from 0.4× (deep blue) to 1.6× (deep red) the average for the vehicle group. C: significantly changed biological pathways associated with prioritized genes in EHT treated with A61603 (n = 6) vs. vehicle (n = 7). Boldface indicates pathways with known links to α₁-AR signaling. Origin value on the abscissa corresponds to P = 0.05.