Figure 1.
PDE8A expression in cardiac tissue and effect its absence on Ca2+ transient. (A) PDE8A immunohistochemistry detection in mouse ventricles. (Ai) PDE8A−/− ventricle section stained for β-Gal activity. Lower left inset shows a section from a WT animal used as negative control. (Aii) staining on an isolated WT and PDE8A−/− myocyte (see also supplementary Fig.2). Due to the presence of a nuclear localization sequence (NLS) fused at the 3′ of the LacZ gene, β-Gal activity is confined to the nucleus of PDE8A expressing cells. (B) Western Blot gels of PDE8A immunoprecipitates (IP) and their supernatants (SN) from ventricle (V) or cardiomyocytes (CM) from WT and PDE8A−/− animals. Recombinant PDE8A is used as positive control (n=3). (C) PDE activity assay on IP in presence of 10nmol/L cAMP as substrate and absence or presence of 100μmol/L IBMX (n=3). (D) Typical fluo-4 traces of Ca2+ transients at 2 mmol/L Ca2+ and 1.0-Hz field stimulation, in basal state and after 2 min of 100 nmol/L Isoproterenol (ISO) stimulation. (E) Statistics for Ca2+ transient peak amplitude fold increase over basal at increasing concentration of ISO (n=31 to 51 cells from 5 to 6 hearts for each group; * P<0.05 WT vs. 8A−/−).