Scn1b expression in the adult mouse heart modulates Na⁺ influx in myocytes and reveals a mechanistic link between Na⁺ entry and diastolic function

Figures

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  Figure 1.. Cardiac specific, adult Scn1b deletion enhances Na+ influx. A: quantitative data for expression of Scn1b in cMCM (n = 6 cells preparations; 6 male and female mice) and cKO cardiomyocytes (n = 6 cells preparations; 6 male and female mice). Data are shown as median and interquartile ranges. *P < 0.001 vs. cMCM, using unpaired t test. Animals at more than 4 wk after induction of Cre recombinase expression were employed. B: whole cell voltage-gated INa recorded in voltage-clamp in myocytes isolated from cMCM and cKO mouse hearts. The voltage-clamp protocol consisted of a family of depolarizing steps in 5-mV increments. C: current-voltage (I-V) relations, voltage dependence of conductance, and steady-state activation for INa in myocytes from cMCM (n = 21 cells from 5 animals, 2–4 cells/mouse) and cKO mice (n = 18 cells from 6 animals, 3–6 cells/mouse). Male and female mice were employed. *P < 0.05 vs. cMCM, using unpaired t test. C and D: quantitative data for properties of INa decay for cells reported in C. *P < 0.05 vs. cMCM, using Mann–Whitney test (D) or unpaired t test (E). F: steady state inactivation for INa in myocytes from cMCM (n = 21 cells from 5 animals, 3–6 cells/mouse)…

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  Figure 2.. Cardiac specific, adult Scn1b deletion prolongs cardiac electrical recovery and affects electrical stability. A: electrocardiograms (ECG) obtained in anesthetized male cMCM and cKO mice 4 wk after tamoxifen administration. B: quantitative data for electrocardiographic parameters obtained, serially, in cMCM (n = 6) and cKO (n = 10) male mice before (Base) and 2 wk (2w) and 4 wk (4w) after tamoxifen treatment. Data are shown as median and interquartile ranges. *P < 0.01 vs. Base for the same genotype, using one-way repeated measures ANOVA followed by Tukey’s multiple comparison test. †P < 0.05 vs. cMCM at the same time point, using unpaired t test. C: ECGs obtained in anesthetized male cKO mouse 4 wk after gene deletion, before (control, Ctrl, left trace) and after administration of the INaL inhibitor GS967 (0.5 mg/kg body wt ip, right trace). D: quantitative data for electrocardiographic parameters obtained in male cMCM (n = 9) and cKO (n = 8) mice 4 wk after gene deletion before (Ctrl) and after administration of GS967. Data are shown as median and interquartile ranges. *P < 0.01 vs. Ctrl, using Wilcoxon test. †P < 0.01 vs. cMCM in the same experimental condition, using…

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  Figure 3.. Cardiac specific, adult Scn1b deletion alters diastolic function. A: quantitative data for two-dimensional echocardiographic parameters obtained in cMCM (n = 8) and cKO (n = 13) male mice before (Base) and 2 wk (2w) and 4 wk (4w) after tamoxifen treatment. Data are shown as median and interquartile ranges. B: transmitral flow Doppler echocardiograms for a male cMCM mouse before (Base, left) and 4 wk (right) after tamoxifen treatment. C: transmitral flow Doppler echocardiograms for a male cKO mouse before (Base, left) and 4 wk (right) after Scn1b deletion. D: quantitative data for transmitral flow Doppler echocardiographic parameters obtained in cMCM and cKO male mice shown in A are reported as median and interquartile ranges. *P < 0.01 vs. Base for the same genotype, using one-way repeated measures ANOVA followed by Tukey’s multiple comparison test. †P < 0.05 vs. cMCM at the same time point, using unpaired t test. E: transmitral flow Doppler echocardiograms for a male cKO mouse at 4 wk after gene deletion, before (Ctrl, left) and after administration of the INaL inhibitor GS967 (0.5 mg/kg body wt ip, right). F: quantitative data for two-dimensional and transmitral flow Doppler…

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  Figure 4.. Cardiac specific, adult Scn1b deletion alters rate of pressure development and decay and LV compliance. A: traces of ECG, LV pressure, LV volume, and first derivative of LV pressure (dP/dt) for male cMCM and cKO mice. Traces are superimposed in the inbox. B: quantitative data for LV hemodynamic and volumetric parameters obtained in cMCM (n = 17) and cKO (n = 13) male mice are shown as median and interquartile ranges. *P < 0.05 vs. cMCM, using unpaired t test or Mann–Whitney test. C: PV loops obtained during occlusion of the inferior vena cava to unload the LV in a male cMCM mouse and a male cKO mouse. Gray lines reflect the slope of the LV end diastolic PV (LVEDPV) relationship. D: Quantitative data for LVEDPV slope obtained in cMCM (n = 13) and cKO (n = 12) male mice are shown as median and interquartile ranges. *P < 0.05 vs. cMCM, using unpaired t test. cKO, conditional knockout; cMCM, conditional MerCreMer; ECG, electrocardiogram; LV, left ventricle.

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  Figure 5.. Cardiac specific, adult Scn1b deletion alters cell mechanics and Ca2+ homeostasis in myocytes. A: cell shortening traces from myocytes obtained from male cMCM and cKO mice. Superimposed traces are reported in the inset. B: quantitative data for cell shortening in cMCM (n = 283 cells, 5 animals, 44–60 cells/mouse) and cKO myocytes (n = 372 cells, 7 animals 40–59 cells/mouse) obtained from male mice are shown as median and interquartile ranges. *P < 0.0001 vs. cMCM, using unpaired t test or Mann–Whitney test. C: traces of Ca2+ transients from myocytes obtained from cMCM and cKO male mice. Superimposed traces are reported in the inset. D: quantitative data for Ca2+ transients in cMCM (n = 284 cells, 5 animals, 52–60 cells/mouse) and cKO myocytes (n = 332 cells, 6 animals, 44–60 cells/mouse) obtained from male mice are shown as median and interquartile ranges. *P < 0.01 vs. cMCM, using Mann–Whitney test. E: traces of Ca2+ transients from cMCM and cKO myocytes stimulated at 1 Hz and 4 Hz. F: quantitative data for Ca2+ transients at 1 Hz and 4 Hz in cMCM (n = 120 cells, 5 animals, 17–34 cells/mouse) and cKO (n = 76 cells, 5 animals, 8–34 cells/mouse) myocytes obtained from male and…

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  Figure 6.. Inhibition of INaL ameliorates kinetics of Ca2+ cycling and cell mechanics in myocytes with Scn1b deletion. A: quantitative data for Ca2+ transients in cMCM and cKO myocytes in Tyrode solution alone (Tyr, n = 102, 119, respectively) or with 300 nmol/L GS967 (n = 105, 123, respectively) are shown as median and interquartile ranges. Cells were obtained from male and female cMCM (n = 5, 8–36 cells/mouse) cKO (n = 5, 15–47 cells/mouse) mice. *P < 0.01 vs. Tyr, using unpaired t test or Mann–Whitney test; †P < 0.01 vs. cMCM in the same experimental condition, using Mann–Whitney test. B: quantitative data for cell mechanics in cMCM and cKO myocytes in Tyrode solution alone (Tyr, n = 76, 157, respectively) or with 300 nmol/L GS967 (n = 79, 128, respectively) are shown as median and interquartile ranges. Cells were obtained from male and female cMCM (n = 4, 13–20 cells/mouse) cKO (n = 4, 10–46 cells/mouse) mice. *P < 0.0001 vs. Tyr, using Mann–Whitney test; †P < 0.0001 vs. cMCM in the same experimental condition, using Mann–Whitney test. cKO, conditional knockout; cMCM, conditional MerCreMer.

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  Figure 7.. Cardiac specific, adult Scn1b deletion prolongs myocardial repolarization. A: monophasic APs obtained from the LV myocardium of perused hearts from cMCM and cKO male mice stimulated at 8 Hz. B: quantitative data for time to 50 (MAP50), 70 (MAP70), and 90% (MAP90) repolarization of the MAP obtained in the LV myocardium of cMCM (n = 11) and cKO (n = 9) male hearts are shown as median and interquartile ranges with dot plots. *P < 0.05 vs. cMCM, using unpaired t test. C: APs elicited at 1 Hz in the presence of buffered [Ca2+]i in myocytes obtained from the LV of cMCM and cKO male hearts. D: quantitative data for AP properties elicited at 1 Hz pacing rate in the presence of buffered [Ca2+]i. Data obtained from LV myocytes from cMCM (n = 14 cells from 4 animals, 2–5 cells/mouse) and cKO (n = 17 cells from 4 animals, 3–6 cells/mouse) male mice are shown as median and interquartile ranges with dot plots. AP, action potential; cKO, conditional knockout; cMCM, conditional MerCreMer; LV, left ventricle; MAP, monophasic action potential.

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  Figure 8.. Cardiac specific, adult Scn1b deletion and screening of Ca2+ handling proteins. A: expression of SERCA2a and NCX1 the LV myocardium of cMCM and cKO male mice, by Western blotting. GAPDH is the loading condition. B: quantitative data for expression of SERCA2a and NCX1 in the LV myocardium of cMCM (n = 8, 5) and cKO (n = 9, 6) male mice are reported as median and interquartile ranges with dot plots. C: expression of phosphorylated (serine 16/threonine 17, pPLN S16/T17) and total phospholamban (PLN) proteins in the LV myocardium of cMCM and cKO male mice, by Western blotting. GAPDH is the loading condition. D: quantitative data for expression of phosphorylated (pPLN) and total (PLN) phospholamban, and pPLN-to-PLN ratio in the LV myocardium of cMCM (n = 9) and cKO (n = 8) male mice are reported as median and interquartile ranges with dot plots. E: expression of phosphorylated (serine 2808, pRYR2 S2808) and total ryanodine (RYR2) proteins in the LV myocardium of cMCM and cKO male mice, by Western blotting. F: quantitative data for pRYR2-to-RYR2 ratio in the LV myocardium of cMCM (n = 5) and cKO (n = 5) male mice are reported as median and interquartile ranges with dot plots. G:…