Real-time records of functional human cardiomyocytes

 

Real-time imaging of Z-discs and cytosolic Ca2+ changes during the contraction of human cardiomyocytes isolated from hiPSCs

Video record 1: Real-time imaging of the Z-discs and contraction of human cardiomyocytes isolated from transgenic hiPSCs: To image the cardiomyocytes cytoskeleton in real time, we first generated transgenic a-cardiac actinin (ACTN2)-enhanced green fluorescent protein (eGFP+)-hiPSCs. The transgenic ACTN2-eGFP+-hiPSCs were generated by the CRISPR methodology by replacing the endogenous ACTN2 with Actn2-eGFP. The ACTN2-eGFP+-hiPSCs were differentiated to human ACTN2-eGFP+-cardiomyocytes (ACTN2-eGFP+-hCMs). Under the fluorescence microscope the Z-discs become visible since ACTN2 is enriched into the Z-discs.

Video record 2: 4x magnification of contacting ACTN2-eGFP+-hCMs

Video record 3: Z-discs distances can be quantified by video recording of the contractile activity of ACTN2-eGPF+-CMs during the systolic and diastolic contractile phases using the ImageJ tool. 

Video record 4: Real-time imaging of Ca2+ waves in contractile human cardiomyocytes isolated from transgenic hiPSCs. We generated first transgenic genetically encoded calcium indicator (GECI)-eGFP+-hiPSCs which were differentiated into GECI-eGFP+-cardiomyocytes (GECI-eGFP+-CMs). After 5 sec, 1 µM Isoproterenol was added resulting to a faster contraction of cardiomyocytes correlating with a higher frequency of the changes of intracellular free Ca2+.