Left Ventricular Systolic Dysfunction in NBCe1-B/C-Knockout Mice

Abstract

Congenital proximal renal tubular acidosis (pRTA) is a rare systemic disease caused by mutations in the SLC4A4 gene that encodes the electrogenic sodium bicarbonate cotransporter, NBCe1. The major NBCe1 protein variants are designated NBCe1-A, NBCe1-B, and NBCe1-C. NBCe1-A expression is kidney-specific, NBCe1-B is broadly expressed and is the only NBCe1 variant expressed in the heart, and NBCe1-C is a splice variant of NBCe1-B that is expressed in the brain. No cardiac manifestations have been reported from patients with pRTA, but studies in adult rats with virally induced reduction in cardiac NBCe1-B expression indicate that NBCe1-B loss leads to cardiac hypertrophy and prolonged QT intervals in rodents. NBCe1-null mice die shortly after weaning, so the consequence of congenital, global NBCe1 loss on the heart is unknown. To circumvent this issue, we characterized the cardiac function of NBCe1-B/C-null (KO_b/c) mice that survive up to 2 months of age and which, due to the uninterrupted expression of NBCe1-A, do not exhibit the confounding acidemia of the globally null mice. In contrast to the viral knockdown model, cardiac hypertrophy was not present in KO_b/c mice as assessed by heart-weight-to-body-weight ratios and cardiomyocyte cross-sectional area. However, echocardiographic analysis revealed reduced left ventricular ejection fraction, and intraventricular pressure-volume measurements demonstrated reduced load-independent contractility. We also observed increased QT length variation in KO_b/c mice. Finally, using the calcium indicator Fura-2 AM, we observed a significant reduction in the amplitude of Ca²⁺ transients in paced KO_b/c cardiomyocytes. These data indicate that congenital, global absence of NBCe1-B/C leads to impaired cardiac contractility and increased QT length variation in juvenile mice. It remains to be determined whether the cardiac phenotype in KO_b/c mice is influenced by the absence of NBCe1-B/C from neuronal and endocrine tissues.

Keywords: NBCe1; acid–base; calcium; contractility; heart failure; pRTA.

Figure 1

Structure and expression of NBCe1 major isoforms. (A) An illustration of NBCe1-B protein topology. All NBCe1 isoforms have 14 transmembrane spans (TM1–14), with soluble N-terminal and C-terminal (Nt and Ct) domains located within the cytoplasm. A glycosylated extracellular loop joins TMs 5 and 6. (B) An illustration of sequence differences between NBCe1 isoforms. Due to an alternative upstream promoter that controls NBCe1-B translation, there is a different 85-amino acid (aa) Nt sequence in NBCe1-B (shown in blue) that replaces the first 41 aa residues of NBCe1-A (shown in red). NBCe1-C is identical to NBCe1-B except that the last 46 aa residues of the Ct sequence in NBCe1-B (shown in yellow) are replaced by a different 61 aa sequence (shown in grey) as a consequence of alternative splicing. (C) An illustration of the expression pattern of NBCe1 protein isoforms. The figure was created using BioRender.com.

Figure 2

Echocardiography demonstrates impaired left ventricular function in KO_b/c mice. (A) Representative cross-sectional M-mode images of the left ventricle of WT and KO_b/c mice between 4–5 weeks of age. (B) Heart rates were titrated to between ~400–500 BPM via isoflurane anesthesia. (C) KO_b/c mice were found to have significantly greater left ventricle internal diameters during diastole (LVIDd) and systole (LVIDs). (D) KO_b/c mice also had significantly greater end-diastolic volume (EDV) and end-systolic volume (ESV) than WT mice as calculated from LVID measurements. (E) There was no significant difference in stroke volume between WT and KO_b/c mice. (F) The fractional shortening of KO_b/c mice was significantly less than that of WT mice. (G) The ejection fraction of KO_b/c mice was significantly less than that of WT mice. Data presented as mean ± SEM, n = 11–14 per group. Outliers were defined a priori as any point >2 standard deviations from the mean and were excluded from analysis. WT outliers (n) were excluded from LVIDd (1), LVIDs (1), EDV (1), ESV (1), SV (1), FS (1), and EF (2) data sets. KO_b/c outliers (n) were excluded from heart rate (1), LVIDd (1), LVIDs (1), EDV (1), ESV (1), SV (2), FS (2), and EF (2) data sets. A significant difference between WT and KO_b/c groups is indicated in the figure by * p < 0.05, ** p < 0.01, and *** p < 0.001 calculated using Student’s unpaired 2-tailed T-test; ns (non-significant).

Figure 3

Impaired left ventricular function in KO_b/c mice is not attributable to differences in left ventricle wall thickness or systemic vascular resistance. During diastole there was no significant difference between the width of the WT and KO_b/c left ventricle anterior (A) or posterior (B) wall. Similarly, during systole, there was no significant difference between the width of the WT and KO_b/c left ventricle anterior (C) or posterior (D) wall. (E) There was no significant difference in systolic, diastolic, or mean arterial pressures of awake WT and KO_b/c. Data presented as mean ± SEM, n = 11–14 per group (panels A–D) or 7–11 per group (panel E). Outliers were defined a priori as any point >2 standard deviations from the mean and were excluded from analysis. WT outliers (n) were excluded from LVAWd (1), LVPWd (1), and LVPWs (1) data sets. KO_b/c outliers (n) were excluded from LVAWd (1) and LVAWs (1) data sets. Statistical significance calculated using Student’s unpaired 2-tailed T-test; ns (non-significant).

Figure 4

Left intraventricular pressure–volume (PV) assessment reveals no significant difference between WT and KO_b/c mice in load-dependent measures of contractility or relaxation. There was no significant difference between WT and KO_b/c left ventricular end-systolic pressure (A) or end-diastolic pressure (B). There was no significant difference between WT and KO_b/c mice in their left ventricular maximum rate of pressure change (dP/dt max, representing load-dependent contractility) (C) or in their minimum rate of pressure change (dP/dt min, representing load-dependent relaxation) (D). Heart rates were titrated to between ~300–500 BPM via isoflurane anesthesia (E). Data presented as mean ± SEM, n = 13–14 per group. Outliers were defined a priori as any point >2 standard deviations from the mean and were excluded from analysis. WT outliers (n) were excluded from end-systolic pressure (1), dP/dt max (1), and heart rate (1) data sets. KO_b/c outliers (n) were excluded from end-systolic pressure (1), end-diastolic pressure (1), dP/dt max (1), dP/dt min (1), and heart rate (1) data sets. A significant difference between WT and KO_b/c groups is indicated in the figure by *** p < 0.001 calculated using Student’s unpaired 2-tailed T-test; ns (non-significant).

Figure 5

Left intraventricular pressure–volume (PV) assessment during IVC occlusion reveals diminished load-independent contractility in KO_b/c mice. Representative PV loops obtained in WT (A) and KO_b/c (B) mice during IVC occlusion used as a preload reduction maneuver to assess load-independent contractility (slope of the end-systolic pressure volume relationship [ESPVR]) and relaxation (slope of the end-diastolic pressure volume relationship [EDPVR]). (C) The slope of the ESPVR was significantly reduced in KO_b/c mice. (D) The slope of the EDPVR was not significantly different between WT and KO_b/c mice. (E) Plotting ESPVR against heart rate for individual mice illustrates that ESPVR is independent of heart rate, supporting that although KO_b/c mice have a slower heart rate than WT during this experiment, this does not account for the observed reduction in their ESPVR. Data presented as mean ± SEM, n = 12–15 per group. Outliers were defined a priori as any point >2 standard deviations from the mean and were excluded from analysis. A single WT outlier was excluded from the EDPVR data set. A single KO_b/c outlier was excluded from the ESPVR data set. A significant difference between WT and KO_b/c groups is indicated in the figure by ** p < 0.01 calculated using Student’s unpaired 2-tailed T-test; ns (non-significant).

Figure 6

Absence of cardiac hypertrophy in KO_b/c hearts. (A) Representative low-magnification tiled images, with higher magnified regions of interest (black boxes in low-magnification images), taken of WT and KO_b/c heart sections stained with H&E. (B) The HW/BW ratio, an index of heart size, was not significantly different between WT and KO_b/c mice. (C) There was also no significant difference in cross-sectional area between genotypes. Data presented as mean ± SEM, n = 16–18 per group (panel B) or 13–10 per group (panel C). Outliers were defined a priori as any point >2 standard deviations from the mean and were excluded from analysis. A single WT outlier was excluded from the HW/BW ratio data set. A single KO_b/c outlier was excluded from the HW/BW ratio data set. For panel (B), the statistical significance was calculated using Student’s unpaired 2-tailed T-test. For panel (C), the cross-sectional area of 25–29 cardiomyocytes was measured across 5 images taken around the left ventricle and averaged for each individual mouse, with the statistical significance calculated using hierarchal statistical analysis (nested T-test). ns (non-significant).

Figure 7

Increased QT length variation in KO_b/c mice. Representative average ECG cycles of WT (A) and KO_b/c (B) mice were created from 5 s segments of Lead-I recordings. The black line represents the average trace, with underlying grey lines representing each individual cycle. This method was applied to 30 s Lead-I recordings of WT and KO_b/c mice from which QT length and QT length variation were assessed. QT length variation was calculated as the coefficient of variation (SD/mean) across 5 s intervals from a continuous 30 s ECG trace (i.e., 6 × 5 s intervals). (C) Heart rates were titrated to between ~350–500 BPM via isoflurane anesthesia. (D) There was no significant difference between the length of the QT interval in WT and KO_b/c mice. (E) The QT length variation in KO_b/c was significantly greater than in WT mice. Data presented as mean ± SEM, n = 11–13 per group. A significant difference between WT and KO_b/c groups is indicated in the figure by *** p < 0.001 calculated using Student’s unpaired 2-tailed T-test; ns (non-significant).

Figure 8

KO_b/c cardiomyocytes have reduced Ca²⁺-transient amplitude. (A) Representative Ca²⁺ transients recorded in individual cardiomyocytes isolated from WT and KO_b/c mice loaded with the intracellular Ca²⁺ indicator Fura-2 AM. Traces represent the average of ~100 consecutive transients recorded in a single cardiomyocyte while paced at 5 Hz. (B) There was no significant difference between WT and KO_b/c ‘baseline’ F_340/380 ratio. (C) The ‘peak amplitude’ was significantly decreased in KO_b/c cardiomyocytes. (D) The ‘peak amplitude as % baseline’ (describing the % change from baseline of the Ca²⁺ transient) was also significantly decreased in KO_b/c cardiomyocytes. (E) There was no significant difference between WT and KO_b/c in ‘time to peak’. (F) There was no significant difference between WT and KO_b/c in ‘time to 90% baseline’. (G) There was no significant difference between WT and KO_b/c the Ca²⁺ exponential ‘decay constant (tau)’. Data presented as mean ± SEM, n = 8–9 per group with each point representing the mean of 9–12 cells. Outliers were defined a priori as any point >2 standard deviations from the mean and were excluded from analysis. A single WT outlier was excluded from the ‘time to 90% baseline’ data set. A significant difference between WT and KO_b/c groups is indicated in the figure by * p < 0.05 calculated using hierarchal statistical analysis (nested T-test); ns (non-significant).