https://doi.org/10.1007/s00392-024-02526-y
1Universitätsklinikum Hamburg-Eppendorf Institut für Experimentelle Pharmakologie und Toxikologie Hamburg, Deutschland; 2Universitätsklinikum Hamburg-Eppendorf Hamburg, Deutschland; 3Universitätsklinikum Hamburg-Eppendorf Institut für Klinische Pharmakologie und Toxikologie Hamburg, Deutschland
Introduction
Atrial fibrillation (AF) is the most prevalent type of cardiac arrhythmia and is often characterized by rapid asynchronous electrical activation of the atria. The resulting uncoordinated contraction of the atria and ventricles, in addition to high-beating frequencies, can lead to reduced ventricular blood ejection but can also cause a certain degree of fibrosis and inflammation. We aim to establish a multi-cell-type engineered heart tissue (EHT) model to investigate the impact of arrhythmic excitation on contractile function and fibroinflammatory cross-talk.
Methods
To generate multi-cell-type EHTs, atrial cardiomyocytes (aCM), quiescent cardiac fibroblasts (CF) and macrophages (Mφs) were differentiated from the same isogenic hiPSC line. Full characterization of quiescent cardiac fibroblasts was achieved by analyzing collagen secretion and activation of fibroblast associated genes by RT-qPCR. ACMs and CFs were cast (aCMs:CFs-ratio=90:10) into a fibrin matrix which over time led to spontaneously beating EHT. Viral transduction with the light-sensitive cation channel AAV6 expressing CheRiff2.0 under the control of the cardiac specific promoter cTnT allowed for optical pacing. After 19 days of culture, the EHTs were subjected to rhythmic pacing at 4 Hz for 18 days. Measurements of contractile function (force, frequency, rhythmicity) were performed repetitively. Further structural analyses were done by confocal microscopy.
Results
The characterization of fibroblasts showed a higher concentration of collagen secreted into the medium after activation (with 10% serum containing medium) of the cells (26 µg/mL) compared to the quiescent group (14 µg/mL). After activation of fibroblasts, genes encoding for extracellular matrix remodeling and fibrotic processes were upregulated. For example, COL1A1 was three times more highly expressed in the activated group than in the quiescent group. Two-cell-type EHTs remodeled quickly and when subjected to rhythmic pacing could be stimulated with a frequency of up to 4 Hz. The force increased under continuous rhythmic pacing. Confocal microscopy showed a distinct expression of CheRiff2.0 in the cardiomyocyte membrane only. Fibroblasts showed no sign of CheRiff2.0 expression and were mainly situated at the structural edge or interspersed between the cardiomyocytes.
Conclusion
The establishment of a multi-cell-type model will provide further insights into the direct harmful effects of irregular excitation on cardiomyocytes and highlight cellular cross-talk between non-myocytes that aggravates AF-related remodelling. Furthermore, it highlights the significance of multi-cell-type interactions in in vitro modelling. Future experiments will establish a three-cell-type model by adding macrophages and by submitting the EHTs to arrhythmic pacing strategies as well as afterload enhancement to potentially show a more enhanced effect of arrhythmic excitation and to simulate heart failure situations. More knowledge will be expected by performing Western blotting and qPCR.