Loss of Na⁺,HCO₃⁻‐Cotransporter NBCn1 Inhibits Net Acid Extrusion in the Atria and Causes Hypertension‐Associated Cardiac Hypertrophy

Figures

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  FIGURE 1. NBCn1 expression is higher in atrial than ventricular cardiomyocytes. (A) Representative immunoblot for NBCn1 on atrial and ventricular lysates from wild type and NBCn1 knockout mice and quantification of total protein using stain‐free gel. (B + C) Quantified protein expression levels for NBCn1 relative to total protein (B) and apparent molecular weight based on the electrophoretic mobility (C). (D–G) Human single‐cell transcript levels in atrial and ventricular cardiomyocytes for SLC9A1 (NHE1; D), SLC4A3 (AE3; E), SLC4A4 (NBCe1; F), and SLC4A7 (NBCn1; G). Data were compared by two‐way ANOVA followed by Šidák's posttests (panel B) or by paired two‐tailed Student's t‐tests (panel C–G). p‐values refer to comparisons vs. wild type or as indicated. KO, knockout; WT, wild type.

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  FIGURE 2. NBCn1 contributes substantially to net acid extrusion in the atria. (A) Average traces of atrial pHi dynamics during NH4 +‐prepulses using 40 mM NH4Cl to induce intracellular acidification in the presence and absence of CO2/HCO3 −. Atrial strips from wild type and NBCn1 knockout mice (n = 12–14) were investigated at an extracellular pH of 7.4. To not obscure the pHi patterns, individual traces were aligned at each wash event and the duration of each interval adjusted accordingly. (B + C) Steady‐state pHi (B) and cellular net acid extrusion calculated at pHi 6.8 (C) for atrial strips from wild type and NBCn1 knockout mice. The 95% confidence interval for the difference between NBCn1 knockout and wild type mice in steady‐state pHi change upon omission of CO2/HCO3 − is −0.04 to 0.20. Thus, it is possible that a larger sample size would reveal an attenuated decrease in pHi upon omission of CO2/HCO3 − in the atria of NBCn1 knockout relative to wild type mice, although the effect would most likely be of small magnitude. Data were compared by two‐way ANOVA followed by Šidák's posttests. p‐values refer to comparisons vs. wild type or as indicated. KO, knockout; WT, wild type.

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  FIGURE 3. NBCn1 Plays no discernible role for net acid extrusion from ventricular cardiomyocytes. (A + B) Steady‐state pHi (A) and cellular net acid extrusion calculated at pHi 6.9 (B) for ventricular cardiomyocytes from wild type and NBCn1 knockout mice. Experiments were performed at an extracellular pH of 7.4 with intracellular acidification induced by NH4 +‐prepulse technique using 10 mM NH4Cl. (C) Immunoblots and quantified expression levels for NBCn1, NHE1, and NBCe1 relative to the pan‐actin loading controls. As previously reported, NHE1 migrates as two separate bands, which has been explained by differences in glycosylation [33, 38]. The relative pan‐actin levels did not differ (p = 0.31; unpaired two‐tailed Student's t‐test) between the ventricular samples from wild type (1.00 ± 0.08; n = 18) and NBCn1 knockout (1.15 ± 0.12; n = 10) mice. The plotted data were compared by two‐way ANOVA followed by Šidák's posttests. p‐values refer to comparisons vs. wild type or as indicated. KO, knockout; MW, molecular weight marker; WT, wild type.

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  FIGURE 4. The electrocardiogram and electrophysiology of ventricular cardiomyocytes are unaffected by NBCn1 knockout. (A) Exemplar extracts of electrocardiograms from wild type and NBCn1 knockout mice. (B) Durations of the RR interval, QRS complex, PR interval, and QT interval. (C) Duration of the PR interval as function of heart rate. (D) Exemplar action potentials recorded from isolated ventricular cardiomyocytes at an extracellular pH of 7.4. (E‐G) Resting membrane potential (E), action potential amplitude (F), and duration of action potential decay (G) in isolated ventricular cardiomyocytes. APD50, APD75, and APD90, indicate the time for 50%, 75%, and 90% decay of the action potential, respectively. Data in panel B were compared by three‐way ANOVA followed by Šidák's posttests, in panel C and G by two‐way ANOVA followed by Šidák's posttests, and in panel D and E by unpaired two‐tailed Student's t‐tests. p‐values refer to comparisons vs. wild type or as indicated. KO, knockout; WT, wild type.

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  FIGURE 5. NBCn1 knockout causes hypertension and cardiac hypertrophy and prolongs isovolumic relaxation. (A + B) Exemplar extracts of telemetric blood pressure measurements (A) and average values (B) for systolic (SAP), diastolic (DAP), and mean (MAP) arterial pressure during day‐ and night‐time. (C) Telemetric measurements of heart rate during day‐ and night‐time. (D) Physical activity quantified for the periods where telemetric blood pressure measurements were performed. (E + F) Postmortem measure of heart weight to body weight ratio (E) and echocardiographic measure of left ventricular mass normalized to total body surface area (F). (G) Short axis echocardiographic M‐mode images for ventricular structure analyses. LVAW, left ventricular anterior wall; LVPW, left ventricular posterior wall. (H) Echocardiographic measure of end‐diastolic posterior wall thickness normalized to total body surface area. (I‐K) Echocardiographic measures of end‐diastolic volume (I), end‐systolic volume (J), and stroke volume (K) normalized to the total body surface area. (L–N) Echocardiographic measures of left ventricular ejection fraction (L), cardiac index (M), and left ventricular myocardial global…

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  FIGURE 6. Hearts from NBCn1 knockout mice do not show fibrotic changes and cardiomyocytes have normal dimensions. (A) Cardiomyocyte sizes evaluated as projected areas of BCECF‐loaded isolated cells. We analyzed 5–20 cells from each atrium and 30–100 cells from each ventricle. The overall 95% confidence interval for the difference between NBCn1 knockout and wild type mice is −236 to 144 μm2/cell. Thus, it is very unlikely that increased cardiomyocyte size—to a quantitatively meaningful extent—contributes toward the cardiac hypertrophy in NBCn1 knockout mice. (B) Collagen content estimated from picrosirius red staining of collagen. The overall 95% confidence interval for the difference between NBCn1 knockout and wild type mice across atria and ventricles is −0.60% to 0.09%. Thus, it is very unlikely that the cardiac hypertrophy in NBCn1 knockout mice is explained by increased collagen content. The scale bars are 50 μm. Data were compared by two‐way ANOVA followed by Šidák's posttests. p‐values refer to comparisons vs. wild type or as indicated. KO, knockout; WT, wild type.