Figure 1
Measuring protein kinase activity in RNCMs and hiPSC-CMs. (A) Depiction of the experimental pipeline used to compare signaling signatures in two cardiomyocyte model systems. The experimental pipeline begins with the differentiation of hiPSCs into cardiomyocytes or the isolation of rat neonatal cardiomyocytes. Cardiomyocyte cultures are then transduced with adeno-associated virus serotype 6 (AAV6) to introduce the biosensor of interest. Next, RNCM and hiPSC-CM cultures are imaged using high content microscopy and further analyzed using a single cell analytical approach. Representative fluorescent microscopy images illustrating the expression of ExRai-AKAR2-NLS biosensor in (B) hiPSC-CMs and (C) RNCMs. (D) Diagram depicting the drugs used in this study. After a baseline reading was taken, RNCMs and hiPSC-CMs were stimulated with saturating doses of a panel of ligands that targeted cardiac relevant GPCRs. Norepinephrine, epinephrine, isoproterenol, phenylephrine, SI and SII were used at 10 μM. Ang II and PMA were used at 1 μM. Forskolin was prepared as 5 μM and ET-1 at 100 nM. (E) Basal, ligand independent single fluorophore intensities of hiPSC-CM and RNCM ExRai-AKAR2-NLS datasets. Biosensors were expressed at higher levels in hiPSC-CMs compared to RNCMs despite being transduced for the same length of time.
