Enhanced β-adrenergic response in mice with dominant-negative expression of the PKD2L1 channel

Abstract

Polycystic kidney disease (PKD) is the most common genetic cause of kidney failure in humans. Among the various PKD-related molecules, PKD2L1 forms cation channels, but its physiological importance is obscure. In the present study, we established a transgenic mouse line by overexpressing the dominant-negative form of the mouse PKD2L1 gene (i.e., lacking the pore-forming domain). The resulting PKD2L1del-Tg mice exhibited supraventricular premature contraction, as well as enhanced sensitivity to β-adrenergic stimulation and unstable R-R intervals in electrocardiography. During spontaneous atrial contraction, PKD2L1del-Tg atria showed enhanced sensitivity to isoproterenol, norepinephrine, and epinephrine. Action potential recording revealed a shortened action potential duration in PKD2L1del-Tg atria in response to isoproterenol. These findings indicated increased adrenergic sensitivity in PKD2L1del-Tg mice, suggesting that PKD2L1 is involved in sympathetic regulation.

Fig 1

A) Molecular characterization of PKD2L1del overexpressed in Tg mice. Schematic of the CMV promoter/PKD2L1del construct used for the generation of PKD2L1del-Tg mice. The deletion site is also shown. The primer sets for each PCR amplification (DNA typing and RT-PCR) are indicated with arrows. The inset shows predicted schematic transmembrane constructs of full-length PKD2L1 (upper panel) and PKD2L1del. Scale bar = 1 kb. B) DNA typing of Tg mice. Tg-A mice contained a single copy of the external gene, whereas mice in Tg-B contained two copies. RT-PCR analysis of cardiac tissue from Tg mice. Tg-B mice showed enhanced PKD2L1 expression in the heart. NC, negative control without cDNA. The names of the Tg lines (A and B) are indicated. C) Western blot analysis of the hearts from the WT and Tg mice. Representative immunoblots of membranes from WT and Tg mice analyzed for the expression of PKD2L1 (left panel) and GAPDH (right panel).

Fig 2

A) Typical RT-PCR data from kidney and heart tissues collected from WT and Tg mice. Amplified sequences are shown. Increased expression levels of PKD2L1 were confirmed in kidney and heart tissues from Tg mice. The expression levels of PKD1, PKD2, and PKD2L2 were assessed. Expression of β-actin was evaluated as a control. The primer sets used for PCR amplification are shown. B) Representative ECG tracings of WT and Tg mice under basal conditions (i). Statistical analysis of the heart rates of WT and Tg mice (ii). There were no significant differences in heart rate between WT (n = 10) and Tg (n = 6) mice under basal conditions. Isoproterenol administration resulted in increased heart rate changes in Tg mice (iii). *P < 0.05, between WT (n = 10) and Tg (n = 6) mice. Error bars indicate the standard error of the mean.

Fig 3. HRV analysis.

A) Representative ECG Poincaré plots of WT (left panel) and Tg mice (right panel). Poincaré plots (RRn vs. RRn+1) in which consecutive pairs of R-R intervals during the control period were plotted with the nth+1 R-R interval against the nth R-R period. Note the marked scattering in Tg mice. B) Representative power spectral analysis of WT (left panel) and Tg mice (right panel). C) Statistical analysis of the power spectra (i–iii). LF (i) and HF (ii) components, LF/HF (iii), and SDNN of WT (open circles) and Tg mice (filled circles) are shown. *P < 0.05 vs. WT. Each group consisted of at least six samples. Error bars indicate the standard error of the mean.

Fig 4. Increased contractility in PKD2L1del-Tg atria in response to isoproterenol.

A) Typical traces of atrial contraction in WT (upper panels) and PKD2L1del-Tg (lower panels) mice. Isoproterenol (100 nM) increased atrial contractility in WT mice. Tg mice showed enhanced atrial contractility to isoproterenol. B) Dose-dependent changes in atrial contractility in response to isoproterenol (10–100 nM) in WT and Tg mice. Error bars indicate the standard error of the mean. *P < 0.05, between atria from WT and Tg mice.

Fig 5. Action potential in atria.

A) Representative action potentials of atrial sinus nodes in WT and PKD2L1del-Tg mice. Basal action potential changes in WT and Tg mice are shown (upper panels). Action potential changes are shown in response to isoproterenol (100 nM, lower panels). B) Statistical analysis of the atrial action potential duration at 90% repolarization in WT and Tg mice. Atria from Tg mice showed a shortened action potential duration (red arrow). n = 8–12. P < 0.05.