Popeye domain containing proteins are essential for stress-mediated modulation of cardiac pacemaking in mice

Abstract

Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention.

Figure 1. Popdc2^–/– mice develop a stress-induced bradycardia.

(A) Consecutive sections of a Popdc2^+/– mouse heart were stained for β-galactosidase (LacZ) and acetylcholine esterase (AChE). (B) A section through the vena cava region was LacZ stained for 10 or 80 minutes. (C) Whole-mount LacZ staining and (D) in situ hybridization for Popdc2. Arrowheads and arrows in A, C, and D denote sinus and AV nodes, respectively. RA, right atrium; SAN, sinus node. Scale bars: 1 mm (A); 200 μm (B); 500 μm (C and D). (E) Heart rate distribution (y axis) of 8-month-old Popdc2^–/– and WT mice before (green), during (blue), and after (red) swim stress, depicted on a beat per beat basis (x axis). (F) Age-dependent mean heart rates of WT and Popdc2^–/– mice during and after swimming. *P < 0.05 between genotypes (n = 5–12). (G) ECG recordings of 8-month-old Popdc2^–/– and WT mice during swim stress test. Asterisks indicate P waves. Horizontal bar: 100 ms. (H) Number of pauses in WT and Popdc2^–/– mice during a 30-minute period after swim stress (log scale on y axis) as a function of age (x axis). *P < 0.05 between genotypes (n = 5–12). (I) PQ intervals measured at 600, 450, and 150 bpm (n = 5 per genotype and heart rate). ne, nonexistent in WT. (J) Number of isolated hearts with sinus pauses after β-adrenergic stimulation. *P < 0.05 between genotypes; ^#P < 0.05, baseline vs. catecholamine.

Figure 2. Functional deficits in Popdc1^–/– mice.

(A–C) Whole-mount LacZ staining of (A) sinus node, (B and C) His bundle (His), and (B) left and (C) right bundle branches (BB). Ao, aorta; SVC, superior vena cava. (D) Consecutive sections through the His bundle were stained for LacZ, acetylcholine esterase, and Popdc1. (E) Immunofluorescent detection of Popdc1 and HCN4 and merged images in the sinus node. Shown are composites of multiple images. endo, endocardial; epi, epicardial. Scale bars: 100 μm. (F) Heart rate (y axis) of Popdc1^–/– and WT mice before (green), during (blue), and after (red) swim stress, depicted on a beat per beat basis (x axis). (G) Age-dependent mean heart rates of WT and Popdc1^–/– mice during and directly after swimming. Shown are mean heart rates (y axes) as a function of age (x axes). *P < 0.05 between genotypes (n = 5–12). (H) ECG recordings of Popdc1^–/– and WT mice during swim stress test. Asterisks indicate P waves. Horizontal bar: 100 ms. (I) Number of pauses in WT and Popdc1^–/– mice during a 30-minute period after swim stress (log scale on y axis) as a function of age (x axis). *P < 0.05 between genotypes (n = 5–12). (J) PQ intervals measured at 600, 450, and 150 bpm (n = 5 per genotype and heart rate).

Figure 3. Structural alterations of the sinus node in Popdc2^–/– mice.

(A–D) Whole-mount immunohistochemistry of HCN4 expression in the sinus node. Boxed regions in A and B are enlarged in C and D, respectively. Arrowheads in C and D denote the network of filopodia-like extensions of nodal myocytes that were structurally abnormal in Popdc2^–/– mice. (E and F) Whole-mount immunofluorescence staining of HCN4 in the sinus node illustrating an overall reduction of pacemaking cells in Popdc2^–/– mice. (G and H) 3D reconstruction of the sinus node based on HCN4 expression. Arrowheads in E–H indicate reduction of HCN4 immunoreactivity in the inferior part of the sinus node in mutant mice. Bars at right denote approximate planes of sections shown in I–P. (I–L) Trichrome staining visualizing the histology and fibrotic tissue content in the superior (I and J) and inferior (K and L) region of the sinus node. (M–P) Immunohistochemistry for HCN4 expression in the superior (M and N) and inferior (O and P) part of the sinus node. Scale bars: 200 μm (A and B); 100 μm (C–P).

Figure 4. The Popeye domain functions as a cyclic nucleotide-binding domain.

(A) Secondary structure of human cAMP-dependent protein kinase type II-β regulatory subunit (PRKAR2B) and structural prediction of the Popeye domain of human POPDC1. Pink rods, α helices; yellow arrows, β strands; pink underlay, PBC. (B) Western blot detection of chick Popdc1 after affinity precipitation of cardiac tissue extracts incubated with cAMP-agarose. Bound Popdc1 protein was eluted with increasing amounts of cAMP. P, pellet; S, supernatant; T, total protein; C, control incubation using ethanolamine-agarose. (C) Western blot of Cos-7 cells transfected with Popdc1, Popdc2, and Popdc3 cDNAs. Shown are total protein and protein bound to cAMP-agarose or ethanolamine (EA) agarose. Brackets denote differentially glycosylated Popdc proteins; asterisks denote nonglycosylated form of Popdc proteins; arrowhead denotes unspecific immunoreactive protein. (D) 3D model of the Popeye domain of human POPDC1. Invariant amino acids are colored red. The cAMP moiety is shown as CPK model. (E) Enlargement of the predicted cAMP binding site. Predicted hydrogen bonds between cAMP and the DSPE and FQVT sequence motifs are shown as dashed lines. (F) Western blot of cAMP-agarose precipitations of Cos-7 cells transfected with cDNAs encoding Popdc1 and Popdc1^D200A, Popdc1^P202A, Popdc1^E203A, or Popdc1^V217F mutants. (G) Western blot of cAMP-agarose precipitations of Cos-7 cells transfected with cDNAs encoding Popdc2 or Popdc2^D184A mutant. (H) Radioligand binding assay using [³H]-cAMP and recombinant C terminus of Popdc1. Binding was competed with increasing concentrations of free unlabeled cAMP and cGMP, respectively.

Figure 5. Interaction of Popdc proteins with the 2-pore channel TREK-1.

(A) Relative current amplitudes of TASK-1 alone or in the presence of Popdc2 were assayed in Xenopus oocytes 48–72 hours after injection (n = 28–31). Error bars in A and C–E are SEM. (B) Example of the measurement of TREK-1–mediated outward current after injection of TREK-1 cRNA alone (gray curve) or in the presence of Popdc1 (black curve) in Xenopus oocytes. (C) Relative current amplitudes of TREK-1 alone or in the presence of Popdc1, Popdc2, or Popdc3 (n = 14–22). *P < 0.05. (D) Relative TREK-1 current amplitudes in the presence or absence of Popdc2. The oocytes were incubated for 48 hours with or without theophylline (n = 17–32). *P < 0.05. (E) Quantification of the relative surface expression of HA-tagged rat TREK-1b in the presence or absence of mouse Popdc2 (n = 27 and 19, respectively). ni, noninjected control oocytes (n = 12). *P < 0.05. (F and G) Immunostaining of Popdc1 (red) or TREK-1 (green) transfected individually (F) or both together (G) into Cos-7 cells. Nuclei were counterstained with DAPI. Scale bars: 10 μm. (H) Example of a GST pulldown experiment using the C terminus of Popdc1 fused to GST and Flag-tagged human TREK-1. GST-E12 and GST proteins were used as controls.

Figure 6. The interaction of Popdc1 and TREK-1 is modulated by cAMP.

(A) Schematic depiction of the bimolecular FRET sensor to study the interaction of Popdc1 and TREK-1. (B–E) FRET measurements of 293A cells transfected with YFP–TREK-1 together with Popdc1-CFP (B, D, and E) or Popdc1^D200A-CFP (C). Cells were stimulated with isoproterenol (B and C), forskolin (D), or sodium nitroprusside (SNP) followed by the addition of forskolin (E). Values are normalized YFP/CFP ratios. (F) Summary of the observed relative changes of FRET signals (mean ± SEM). ISO, isoproterenol; Mut ISO, Popdc1^D200A-CFP treated with isoproterenol; H89 ISO, addition of PKA inhibitor H89 and isoproterenol. (G and H) Concentration-dependent response curve for isoproterenol-induced FRET signals using Epac1-camps (G) or Popdc1-CFP/YFP–TREK-1 (H).