Institute of Experimental Cardiovascular Research, University Medical Center Hamburg -Eppendorf (UKE), Hamburg, Germany.
Germany DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.
Methods Mol Biol. 2022;2483:195-204. doi: 10.1007/978-1-0716-2245-2_12.
The ubiquitous second messengers' 3',5'-cyclic adenosine monophosphate (cAMP ) and 3',5'-cyclic guanosine monophosphate (cGMP) are crucial in regulating cardiomyocyte function, as well as pathological processes, by acting in distinct subcellular microdomains and thus controlling excitation-contraction coupling. Spatio-temporal intracellular dynamics of cyclic nucleotides can be measured in living cells using fluorescence resonance energy transfer (FRET ) by transducing isolated cells with genetically encoded biosensors. While FRET experiments have been regularly performed in cardiomyocytes from different animal models, human-based translational experiments are very challenging due to the difficulty to culture and transduce adult human cardiomyocytes. Here, we describe a technique for obtaining human atrial and ventricular myocytes which allows to keep them alive in culture long enough to transduce them and visualize cAMP and cGMP in physiological and pathological human settings.
无处不在的第二信使 3'，5'-环腺苷酸（cAMP）和 3'，5'-环鸟苷酸（cGMP）通过在不同的亚细胞微域中发挥作用，从而控制兴奋-收缩偶联，在调节心肌细胞功能以及病理过程中起着至关重要的作用。通过用遗传编码的生物传感器转导分离的细胞，可以使用荧光共振能量转移（FRET）在活细胞中测量环核苷酸的时空细胞内动力学。虽然 FRET 实验已在来自不同动物模型的心肌细胞中定期进行，但由于培养和转导成人人类心肌细胞非常具有挑战性，基于人类的转化实验非常具有挑战性。在这里，我们描述了一种获取人心房和心室肌细胞的技术，该技术可使它们在培养中保持足够长的时间以进行转导，并在生理和病理的人类环境中可视化 cAMP 和 cGMP。
