Mutation of mouse Akap10. (A) Location of the gene-trap insertional mutation between exons 12 and 13 in Ak10. The inserted sequence (gray box) includes a splice acceptor (SA), selectable marker (“βGeo,” a fusion of β galactosidase and neomycin phosphotransferase II), followed by a stop codon and a polyadenylation signal (pA). The insertion disrupts mRNA splicing and eliminates exons 13–15. (B) PCR products obtained by using primers (a, b, and c as indicated in A) and genomic DNA from WT, heterozygous (Het), and homozygous (Hom) Akap10-mutant mice. (C) Expected domain structures of WT and Akap10-mutant proteins. RGS, domains with homology to regulators of G protein signaling; AKB, PKA-binding domain; PDZ, PDZ-binding motif. (D) Immunoblots of cardiac Akap10 protein using an antibody generated against the 27-residue AKB domain. (E) Species alignment of the final 51 aa that are absent from Akap10-mutant protein. Asterisk indicates the human polymorphic I646V position. A full alignment for 16 species is shown in SI Fig. 4.

Effects of the Akap10 mutation on cholinergic/vagus nerve signaling in vitro and in vivo. (A and B) Response of mESC-derived cardiac myocytes to the cholinergic agonist carbachol. (A) Contractions (indicated by downward deflections in brightness) of representative WT and Akap10-mutant myocyte clusters before and after addition of the mAChR cholinergic agonist, carbachol (1 μM). Contractions were detected by phase microscopy as changes in light transmission during cell contraction. (B) Contraction rates of WT and Akap10-mutant myocytes before and after the mAChR cholinergic agonist. Data are mean (±SEM) for 66 WT and 66 Akpa10-mutant myocyte clusters. (C and D) HR regulation in Akap10-mutant mice. (C) A representative experiment showing HRs from WT (black squares), heterozygous (diamonds), and homozygous (circles) littermate mice before and after low-dose mAChR cholinergic agonist, methacholine (agonist better suited for mice) injection (2 mg/kg i.p.). The control mouse (white squares) was injected with saline vehicle. (D) Vagus nerve sensitivity in homozygous Akap10-mutant mice (n = 12) and WT littermates (n = 12) measured by the baroreflex. Baroreceptors in the carotid body sense hypertension induced by the arterial vasoconstrictor, and α-adrenergic agonist, phenylephrine (2 mg/kg i.p.) and stimulate the vagus nerve to slow HR. Values are means from three replicate experiments with four homozygous mutant and four WT mice.

Effects of Akap10 on heart rhythm and survival in mice. (A and B) Spontaneous bradyarrhythmias in homozygous Akap10-mutant mice. ECG recordings demonstrating a sinus pause with a junctional escape (A) and AV block (loss of R waves) (B). Both arrhythmias include slowing of sinus rate (longer P–P intervals) and of AV conduction (a longer P–R interval before the pause), indicative of simultaneous inhibition of the sinoatrial and AV nodes by the vagus nerve. P, atrial depolarizations; R, ventricular depolarizations. (C) HRV in WT (squares) and homozygous Akap10-mutant (circles) littermates, as measured by the root of the mean of squared successive differences in normal R–R intervals (RMSSD). A higher RMSSD indicates more beat-to-beat variability. (D) Kaplan–Meier analysis of survival in WT and mutant littermates. Differences between the three groups were tested by using Wilcoxon (P < 0.01) and log-rank statistical tests (P < 0.02). All deaths were due to natural causes, but daily monitoring of the mice by our veterinary staff did not identify any evidence of illness or distress before death. Death from arrhythmia is possible but could not be demonstrated, because no mice died during ECG recording.