Neonatal Scn1b-null mice have sinoatrial node dysfunction, altered atrial structure, and atrial fibrillation

Figures

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  Figure 1. Differential gene expression in P16 Scn1b-null versus WT atria. (A) Volcano plot showing upregulated (red) and downregulated (blue) gene expression in Scn1b-null atrium compared with WT. (B) GO groups overrepresented in analysis of Scn1b-null atrium compared with WT. (C) Heat maps depicting altered genes related to calcium ion binding, actin binding, and cytoskeleton structure.

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  Figure 2. Fibrosis in P16–17 WT and Scn1b-null mouse hearts. (A) Representative images of histological coronal sections of whole heart fibrosis with picrosirius red staining. Insets show left and right atria. (B) Percent fibrosis area in WT and Scn1b-null mouse atria (n = 5). (C) Transcript levels of Col1a1, Col1a2, Postn, CTGF, Vim, and Acta2 in the left versus right atria from WT and Scn1b-null mice (n = 3 per group). Data are represented as the mean ± SEM. **P < 0.01, using Students t test. ****P < 0.0001 using 2-way ANOVA with multiple comparisons.

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  Figure 3. Neonatal Scn1b-null mice have increased susceptibility to AF in vivo. (A) Representative recordings of surface ECGs (top) and atrial electrograms (bottom) from P16 WT or Scn1b-null mice. Atrial burst pacing was delivered at 50 Hz for 2 seconds. A period of AF in the null mouse is indicated in red. (B) AF incidence under basal and vagotonic stimulation. AF testing was performed before and after i.p. administration of carbachol (Cch) (0.025 mg/kg). (C) Durations of basal and induced AF episodes in the mice shown in B. Each dot represents a single episode. Mean values are indicated by the bar. (D) Incidence of AF in the null mice before (red) and after i.p. administration of atropine (blue, 1 mg/kg). (E) Incidence of AF in the null mice before and after i.p. administration of atropine (1 mg/kg). Numbers of animals tested are shown in the bar graphs. *P < 0.05 using Fisher’s exact test.

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  Figure 4. Neonatal Scn1b-null mice exhibit SAN dysregulation. (A) Comparison of heart rates between anesthetized WT (black) and null (red) mice at P16 (bpm). (B) Example of a sinus pause (arrow) during the monitoring of surface ECG in a null mouse. (C) Representative ECG recordings showing the time required to resume sinus rhythm following electrical stimulation, or SNRT. “P” indicates a P wave in each trace. (D) Comparisons of SNRT between genotypes assessed at cycle lengths of 100 or 80 ms. Each dot represents the value from 1 animal. Data are presented as mean ± SEM. *P < 0.05 using Student’s t test.

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  Figure 5. Immunofluorescence staining of murine atrial intrinsic ganglia. Merged, zoomed images from whole-mount atrial preparations shown in Supplemental Figure 9. (A) P16 Scn1b-null heart. (B) P16 WT heart. ChAT+ neurons labeled in gray; TH+ neurons labeled in red. Scale bars: 100 μm. (C) The mean and range of ChAT+ and biphenotypic intrinsic cardiac neurons are presented for all ganglia (LGC + RGC) versus the right (RGC) or left (LGC) ganglionic clusters from WT and null mouse whole mount atrial preparations. Data are presented as mean ± SEM. *P < 0.05 versus WT using Student’s t test.

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  Figure 6. Neonatal Scn1b-null atrial myocytes show AP prolongation. (A) Representative recordings of APs from acutely isolated P15–18 WT (black) or Scn1b-null (red) atria myocytes. Scale bar: 30 ms. (B) Resting membrane potential (RMP) and AP amplitude values for WT and null atrial myocytes. (C) AP upstroke velocity (dV/dt) values for WT and null atrial myocytes. (D) APDs at 30%, 50%, and 90 % of membrane repolarization for WT and null atrial myocytes. Each dot represents the value from 1 cell. The number of myocytes (n) and mice (N) used are indicated in the inset. Data are presented as mean ± SEM. *P < 0.01 versus WT using Student’s t test.

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  Figure 7. Neonatal Scn1b-null atrial myocytes have increased INa,L. (A) Membrane capacitance (Cm) of P15–18 WT (black) or Scn1b-null (red) acutely isolated atrial myocytes. Plot shows combined Cm values obtained from the INa, ICa, and IK recordings. (B) I-V relationships for INa,T are similar between genotypes. (C) Superimposed representative INa,L recordings from a WT and null atrial myocyte. (D) Current densities for WT and null atrial myocyte INa,L (pA/pF). (E) INa,L expressed as percentage of INa,T. Each dot represents the value from 1 cell. Data are presented as mean ± SEM. *P < 0.01 versus WT using Student’s t test.

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  Figure 8. Neonatal Scn1b-null atrial myocytes have reduced ICa,L. (A) Representative recordings of ICa,L in P15–18 WT (top) or Scn1b-null (bottom) acutely isolated atrial myocytes. A short prepulse of –30 mV was delivered in the presence of 10 μM TTX to inactivate INa. The membrane voltage was then set to –50 mV to ensure inactivation of INa before delivery of voltage pulses to record ICa,L. (B) I-V relationships for ICa,L for WT (black) and Scn1b-null (red) atrial myocytes. The number of myocytes (n) and mice (N) used are indicated in the inset. Data are presented as mean ± SEM. *P < 0.01 versus WT using Student’s t test.