Differences in analytical approach during conduction velocity analysis of optically mapped hearts affect reported data and conclusions

Figures

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  Figure 1:. Scn1b+/− mice have reduced Scn1b mRNA and β1 protein, with no change in canonical conduction protein expression. A. Quantification from RT-PCR demonstrating a significant, ~50% reduction in Scn1b mRNA between WT and Scn1b+/− mice. *, P < 0.05, one-sample t-test. B. Representative Western Blot image from left ventricular tissue. C. Quantification of β1 expression from two technical replicate Western Blots (n=2) with three biological replicates (N=3) each, demonstrating a significant reduction in β1 protein in Scn1b+/− hearts relative to WT, normalized to GAPDH. D. Quantification of NaV1.5 expression from Western Blot with three biological replicates (N=3) showing no difference between WT and Scn1b+/−, normalized to GAPDH. E. Quantification of Total Cx43 expression from Western Blot (N=3) showing no difference between WT and Scn1b+/−, normalized to GAPDH. F. Quantification of serine-368-phosphorylated Cx43 from Western Blot (N=3) showing no difference between WT and Scn1b+/−, relative to total Cx43. *, P < 0.05, unpaired t-test.

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  Figure 2:. Ventricular INa is preserved between WT and Scn1b+/− cardiomyocytes. A. Representative INa traces from a WT cardiomyocyte. B. Representative INa traces from a Scn1b+/− cardiomyocyte. C. Average I-V curves from WT (N=3 hearts, n=21 cells) and Scn1b+/− (N=4 hearts, n=18 cells) cardiomyocytes. Data is presented as mean and standard error of the mean. D. Groupwise quantification of INa peak current, i.e. the average value of the mean peak INa in each heart, demonstrating no difference between WT and Scn1b+/− cardiomyocytes. Unpaired t-test.

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  Figure 3:. WP is wider in Scn1b+/− hearts, and more sensitive to osmotic stress. A. Representative perinexal tracing (yellow) from WT (top) and Scn1b+/− (bottom) hearts under the three interventions: baseline (left), mannitol (center), and dextran (right). B. Quantification of perinexal width (WP) from N=4 hearts per intervention, with n=15–20 perinexal images per heart. Scn1b+/− hearts have wider perinexi at baseline relative to WT. Mannitol significantly increases WP in both genotypes. Dextran 2MDa selectively narrows perinexi in Scn1b+/− hearts. *, P < 0.05, Ordinary two-way ANOVA.

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  Figure 4:. Scn1b+/− hearts have preserved conduction at baseline but are more sensitive to osmotic stress. A. Representative isochrone activation maps of WT (top, N=8) and Scn1b+/− (bottom, N=4) hearts at baseline (left) and after 15-minutes continued perfusion for time control (right). B. Quantification of CV in the transverse direction for WT and Scn1b+/− hearts at baseline and time control demonstrating no significant differences. C. Delta CV (time control – baseline) for WT and Scn1b+/− hearts. D. Representative isochrone activation maps of WT (N=8) and Scn1b+/− (N=8) hearts at baseline and after 15-minutes perfusion of 26.1g/L mannitol. E. Quantification of CV in the transverse direction for WT and Scn1b+/− hearts at baseline and mannitol demonstrating mannitol significantly slows CV in both genotypes. F. Delta CV (mannitol – baseline) for WT and Scn1b+/− hearts demonstrating mannitol slows CV to a significantly greater degree in Scn1b+/− hearts. G. Representative isochrone activation maps of WT (N=5) and Scn1b+/− (N=6) hearts at baseline and after 15-minutes perfusion of 10g/L dextran 2MDa. H. Quantification of CV in the transverse direction for WT and Scn1b+/− hearts at baseline and…

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  Figure 5:. Concealed phenotype and conduction sensitivity to osmotic challenge are preserved in vivo. A. Representative trace of 5s ECG recording of sinus rhythm in Scn1b+/− mouse. B. Exemplar trace of 5s ECG recording of arrhythmia in Scn1b+/− mouse after mannitol injection. C. Average QRS traces (black) from n=10 traces (color) for WT (left) and Scn1b+/− (right) mice after each intervention: baseline (top), 0.9% saline injection (center), and 26.1g/L mannitol injection (bottom). D. Quantification of average QRS durations from entire 3-minute recording for WT before and after saline (N=5) or mannitol (N=5) injection and for Scn1b+/− before and after saline (N=5) or mannitol (N=5) injection demonstrating significant prolongation of QRS duration in Scn1b+/− mice after mannitol injection. *, P < 0.05, paired t-test.