Figures legends:
Figure 1. Lebanese family with atrial fibrillation, heart block and non-ischemic cardiomyopathy. All patients underwent exome sequencing and the mutations were confirmed by sanger sequencing, III-2 is the index case. E1: evaluation for S13F in DES and E2: evaluation for A123T in PDE4DIP .
Figure 2. Pedigrees with early onset atrial fibrillation and slow ventricular response and their corresponding PDE4DIP mutations. The index cases are indicated by arrows. Individuals with slow AF are indicated by black symbols; unaffected individuals are shown as unfilled symbols. Circles represent females; squares represent males. Symbols with a slash through them indicate deceased subjects.
Figure 3. Segments of the protein sequences flanking the substituted amino acids in the four kindreds with slow AF are shown from diverse vertebrate species. As demonstrated, all substitutions in the familial AF kindreds occurred at evolutionarily conserved sites.
Figure 4. Colocalization of PDE4DIP with PDE4D in C2C12 cells expressing wildtype and mutant (p.A123T) PDE4DIP before and 8 minutes after isoproterenol exposure. The arrows show colocalization of PDE4DIP with PDE4D in wildtype and the absent thereof in the mutant C2C12 cells.
Figure 5. Fluorescence resonance energy transfer (FRET) imaging of Isoproterenol (ISO 1uM) and Forskolin (FOR 25uM) activation of cAMP. Images were obtained under live confocal microscopy at baseline and at 4 minutes after stimulation with 1uM ISO. Further imaging was done 4 minutes after stimulation with 25 uM of Forskolin. The upper and lower limits of the FRET ranges are shown on the right in the colored keys. The quantification of 18-20 fields of two independent experiments per groups are shown in bar graph. FRET imaging shows a significant increase of cAMP activity (decreased FRET) in cells transduced by the mutant (p.A123T) compared to wild type PDE4DIP after stimulation with Isoproterenol (ISO 1uM).
Figure 6. Western blot analysis of β2AR phosphorylation. A, shows increased β2AR phosphorylation at the PKA but not GRK sites in PDE4DIP mutant cells compared to wildtype 8 minutes after stimulation with isoproterenol (1uM). B, the ratio of β2AR phosphorylation at PKA site (S346) to the total β2AR shows increased baseline phosphorylation in the wild type but much higher levels of phosphorylation in the mutant following isoproterenol stimulation. C, the ratio of β2AR phosphorylation at GRK sites (S355, S356) to the total β2AR shows increased baseline phosphorylation in the wild type but no significant increase in the phosphorylation levels in the mutant after isoproterenol stimulation compared to wild type or control. The “*” indicates significant changes when comparing unstimulated to post-isoproterenol signal.
Figure 7. Reduction of total phospho-Desmin in C2C12 cells transduced with the mutant (p.A123T) versus wildtype PDE4DIP. Red: PDE4DIP mCherry, green: anti phospho-Desmin antibody staining.
Figure 8. Graphical abstract.