Background                                                                   

There is ongoing interest in generating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to study human cardiac physiology and pathophysiology or to repair tissue. Recently we found calcium currents (I_Ca) stimulated by maximum concentrations of norepinephrine (NE) smaller in hiPSC-CM from conventional monolayers (ML) compared to integrated contracting engineered heart tissue (EHT). In order to elucidate culture specific regulation of β₁-adrenoceptor (β₁-AR) responses we investigated whether PDE-activity may limit NE effects on I_Ca and on cAMP in hiPSC-CM. Results were compared to adult human atrial myocytes (HAMs).

Methods

HAMs were isolated from tissue samples obtained during open heart surgery. All patients were on sinus rhythm. HiPSC-CM were dissociated from ML and EHT. Förster-resonance energy transfer (FRET) was used to monitor cytosolic cAMP (transduced by adenovirus, Epac1-camps sensor). I_Ca was recorded by whole-cell patch clamp technique. Cilostamide (300 nM) and rolipram (10 µM) were used to inhibit PDE3 and PDE4 respectively. β₁-AR were stimulated with the natural agonist NE (100 µM). Results are presented as mean ± SEM. N/N indicates number of cells vs. number of batches for hiPSC-CM and number of cells/number patients for HAMs.

Results

In HAMs NE increased cAMP by 13.7±1.5% (n=10/9) and I_Ca by 10.4±1.5 pA/pF (n=15/10). Effects on both, cAMP and I_Ca, were not larger in the concomitant presence of rolipram, cilostamide and NE (FRET 15.5±1.8% n=12/10 and I_Ca 12.4±1.4 pA/pF n=17/6).

In ML NE-induced increases in cAMP and I_Ca were significantly smaller than in HAMs (FRET 9.6±0.5% n=52/21 and I_Ca 1.4±0.2 pA/pF n=34/7, p<0.05 each). Notably, effects on cAMP as well as on I_Ca were larger in the presence of rolipram and NE (FRET 16.7±0.8% n=94/26 and I_Ca 5.6±1.4 pA/pF n=11/5, p<0.05 each). Cilostamide and NE increased FRET to a larger extent than NE alone (12.7±0.5% n=91/19, p<0.05). However, I_Ca responses were not increased (1.7±0.4 pA/pF n=9/5).

In EHT NE responses on both, FRET and I_Ca, were larger than in ML (FRET 12.1±0.3% n=87/32 and I_Ca 3.3±0.2 pA/pF n=13/5, p<0.05 each). Effects of rolipram and NE on cAMP were not larger than with NE alone (FRET 11.2±0.7% n=87/26), while I_Ca responses were significantly increased to 6.2±1.6 pA/pF (p<0.05 vs. NE alone, n=10/4).

Conclusion

Our results show culture-dependent differences in hiPSC-CM. In conventional ML, but not in EHT maximum NE effects on cytosolic cAMP depend on PDE3 and PDE4, suggesting immaturity when compared to the situation in adult HAMs. The smaller I_Ca responses to NE in ML and EHT vs. adult HAMs depend at least partially on a non-physiological large impact of PDE4 in hiPSC-CM.