| Autor: |
Skryabin EB; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany., De Jong KA; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.; German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany., Subramanian H; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.; German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany., Bork NI; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.; German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany., Froese A; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.; German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany., Skryabin BV; Core Facility Transgenic Animal and Genetic Engineering Models (TRAM), University of Münster, 48149 Münster, Germany., Nikolaev VO; Institute of Experimental Cardiovascular Research, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany.; German Center for Cardiovascular Research (DZHK), Partner Site Hamburg/Kiel/Lübeck, 20246 Hamburg, Germany. |
| Abstrakt: |
Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50-60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes. |
