https://doi.org/10.1007/s00392-024-02526-y
1Universitäres Herz- und Gefäßzentrum Hamburg Klinik für Kardiologie mit Schwerpunkt Elektrophysiologie Hamburg, Deutschland; 2Universitätsklinikum Hamburg-Eppendorf Institut für Experimentelle Pharmakologie und Toxikologie Hamburg, Deutschland; 3Universitätsklinikum Hamburg-Eppendorf Institut für Klinische Pharmakologie und Toxikologie Hamburg, Deutschland
Background
Engineered heart tissue (EHT) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) beat spontaneously. The spontaneous beating rate (SBR) is higher in EHT produced from atrial hiPSC-CM cultured in the presence of retinoic acid (RA) than in the absence of RA for ventricular hiPSC-CM. Many studies report coexistence of pacemaker cells (nodal-like) and non-pacemaker cells (quiescent) in hiPSC-CM cultures. In this study, we analysed the expression of the hyperpolarization-activated cyclic nucleotidegated channel (HCN) and funny currents (If) in hiPSC-CM isolated from ventricular (vEHT) and atrial EHT (aEHT) in order to quantify the percentage of pacemaker cells in aEHT and vEHT.
Methods
Intact EHT was used to measure HCN expression by immunofluorescence. HCN-mRNA expression of EHT was analysed by RNA sequencing. Human iPSC-CM were enzymatically isolated from intact EHT and used for patch clamping at 37 °C. If currents were quantified as CsCl-sensitive currents at the end of a 2 s long test pulse to -130 mV (holding potential -40 mV).
Results
HCN protein was uniformly expressed in both aEHT and vEHT. The mRNA expression (fold change of normalized RNA count over vEHT) of HCN1 was higher in aEHT (10,5±7,4) whereas the expression of HCN2 and HCN4 was not significantly different. We found If currents in every of 56 cells from vEHT and 33 cells from aEHT. Current densities were log normal distributed with slightly larger values of current density in hiPSC-aCM vs, hiPSC-vCM (9.1±1.8 vs. 5.0±2.3 pA/pF, p <0.05). Neither voltage-dependency nor time-dependency of activation differed between atrial hiPSC-CM and ventricular hiPSC-CM (V0.5act 89±2.3 vs. 92.6±2.5 mV and τact 270±80 vs. 215±40 ms, 26/3 atrial hiPSC-CM vs. 12/3 venticular hiPSC-CM).
Conclusions
If currents are uniformly expressed in EHT and hiPSC-CM and show biophysical properties as seen in mammalian sinus node cells. Larger If currents in atrial hiPSC-CM vs. ventricular hiPSC-CM associates to faster spontaneous beating. The uniform expression of If currents argues against the existence of distinct pacemaker cells within EHT.