Increasing T-type calcium channel activity by β-adrenergic stimulation contributes to β-adrenergic regulation of heart rates

Abstract

Cav3.1 T-type Ca²⁺ channel current (I_Ca-T ) contributes to heart rate genesis but is not known to contribute to heart rate regulation by the sympathetic/β-adrenergic system (SAS). We show that the loss of Cav3.1 makes the beating rates of the heart in vivo and perfused hearts ex vivo, as well as sinoatrial node cells, less sensitive to β-adrenergic stimulation; it also renders less conduction acceleration through the atrioventricular node by β-adrenergic stimulation. Increasing Cav3.1 in cardiomyocytes has the opposite effects. I_Ca-T in sinoatrial nodal cells can be upregulated by β-adrenergic stimulation. The results of the present study add a new contribution to heart rate regulation by the SAS system and provide potential new mechanisms for the dysregulation of heart rate and conduction by the SAS in the heart. T-type Ca²⁺ channel can be a target for heart disease treatments that aim to slow down the heart rate ABSTRACT: Cav3.1 (α_1G ) T-type Ca²⁺ channel (TTCC) is expressed in mouse sinoatrial node cells (SANCs) and atrioventricular (AV) nodal cells and contributes to heart rate (HR) genesis and AV conduction. However, its role in HR regulation and AV conduction acceleration by the β-adrenergic system (SAS) is unclear. In the present study, L- (I_Ca-L ) and T-type (I_Ca-T ) Ca²⁺ currents were recorded in SANCs from Cav3.1 transgenic (TG) and knockout (KO), and control mice. I_Ca-T was absent in KO SANCs but enhanced in TG SANCs. In anaesthetized animals, different doses of isoproterenol (ISO) were infused via the jugular vein and the HR was recorded. The EC₅₀ of the HR response to ISO was lower in TG mice but higher in KO mice, and the maximal percentage of HR increase by ISO was greater in TG mice but less in KO mice. In Langendorff-perfused hearts, ISO increased HR and shortened PR intervals to a greater extent in TG but to a less extent in KO hearts. KO SANCs had significantly slower spontaneous beating rates than control SANCs before and after ISO; TG SANCs had similar basal beating rates as control SANCs probably as a result of decreased I_Ca-L but a greater response to ISO than control SANCs. I_Ca-T in SANCs was significantly increased by ISO. I_Ca-T upregulation by β-adrenergic stimulation contributes to HR and conduction regulation by the SAS. TTCC can be a target for slowing the HR.

Keywords: Cav3.1/α1G T-type calcium channel; atrioventricular node; heart rate; sinoatrial node cells; β-adrenergic.

Figure 1. Total, L‐ and T‐ type Ca²⁺ currents in SAN cells of control (WT), Cav3.1 KO and Cav3.1 TG mice

A and B, α‐MHC promoter‐driven expression of GFP in SANCs. A SANC from α‐MHC‐driven GFP transgenic mice in bright field (A) and GFP fluorescence (B). L‐ and T‐type Ca²⁺ currents were separated by holding the cells at −90 and −50 mV. At the V _h of −50 mV, TTCC was fully inactivated and only I _Ca‐L remained. The T‐type Ca²⁺ current was obtained by subtracting the raw L‐type Ca²⁺ current from the raw total Ca²⁺ current. Examples of recordings of total I _Ca (V _h = −90 mV), I _Ca‐L (V _h = −50 mV) and I _Ca‐T (difference between total I _Ca and I _Ca‐L) in one WT (C), one Cav3.1 KO (D) and one Cav3.1 TG (E) SAN cell are shown. The insert in (A) was the voltage clamp protocol. I–V curves of total (F), L‐type (I _Ca‐L) (G) and T‐type Ca²⁺ (I _Ca‐T) (H) currents in SANCs of control (WT), Cav3.1 KO and Cav3.1 TG mice. I–V curves were constructed by examining the peak of total, L‐type and derived T‐type Ca²⁺ currents at different test potentials. All currents were normalized to the cell capacitance. ^@ P < 0.05, TG versus control; ^# P < 0.05, KO versus control; $P < 0.05, TG versus KO at the same voltage determined by two‐way repeated ANOVA and post hoc tests with Bonferroni adjustment.

ISO dose–HR responses in sedated Cav3.1 KO, Cav3.1 TG and control mice

Figure 2

Mice were anaesthetized and a catheter was inserted into the left jugular vein to infuse a series of ISO from 10⁻¹⁶ g g⁻¹ BW to 10⁻⁶ g g⁻¹ BW. A, examples of HR changes in response to different doses of ISO in anaesthetized Cav3.1 KO (blue) and TG (red) and control (black) mice. B, examples of ECG recorded at baseline and at maximal HR after ISO. C, average HR at different concentrations of ISO in Cav3.1 KO, TG, and control mice. HR–ISO dose relationship were fit with a dose–response curve to derive EC₅₀. The inserted table shows the logEC₅₀ and maximal percentage of HR increase in TG, KO and control mice. ^@ P < 0.05, ^@@ P < 0.01 and ^@@@ P < 0.001 TG versus control; ^# P < 0.05, KO versus control; ^$ P < 0.05, ^$$$ P < 0.001 and ^$$$$ P < 0.0001, TG versus KO at the same ISO dose determined by two‐way repeated ANOVA and post hoc tests with Bonferroni adjustment. D, left ventricular systolic pressure at different doses of ISO. E–G, maximum HRs at different i.p. doses of ISO (10⁻¹⁰, 10⁻⁹ and 10⁻⁸ g g⁻¹ BW) without or with ICI 118,551 (2 μg g⁻¹ BW, i.p., 5 min) or with ICI 118,551 + metoprolol (2 μg g⁻¹ BW, i.p., 5 min) in sedated control, KO and TG animals. ^% P < 0.05, without pretreatment versus with ICI pretreatment; ^# P < 0.05, without treatment versus with ICI + metoprolol pretreatment; ^$ P < 0.05, pretreatment with ICI versus pretreatment with ICI + metoprolol. At the same ISO dose, significance was determined by two‐way repeated ANOVA and post hoc tests with Bonferroni adjustment. [Color figure can be viewed at http://wileyonlinelibrary.com]

HR and PR intervals of Langendorff perfused hearts from Cav3.1 KO, TG and control WT mice before and after ISO (10⁻⁷ m)

Figure 3

A, examples of ECG recorded at baseline and at maximal HR after ISO in isolated and perfused Cav3.1 KO (blue) and TG (red) and control (black) mice. B, HR before and after the application of 10⁻⁷  m ISO. C, the percent increase of HR by ISO in Cav3.1 KO and TG, and control mice. D, PR intervals of Langendorff‐perfused control (black), Cav3.1 KO (blue) and TG (red) hearts before and after the application of ISO. E, the percentage of PR interval decrease (100 – PR interval after ISO/baseline PR interval × 100) induced by ISO in c57bl/6 control and Cav3.1 KO mice. F, examples of surface ECG and atrial electrogram before and during atrial pacing at 600 Hz. G, PR intervals during atrial pacing‐induced ventricular beats. H, heart weight or atrial weight to body weight ratios of control, KO and TG animals. ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001. Data in (B) and (D) were analysed by two‐way repeated ANOVA and post hoc tests with Bonferroni adjustment; data in (C) and (E) were analysed by one‐way ANOVA and post hoc tests with Bonferroni adjustment. n, number of animals studied. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 4. ISO dose–response relationships of spontaneous beating rates of SAN cells of Cav3.1 TG, KO and control mice

A, spontaneous beating rates of control, Cav3.1 KO and TG SANs before and after the application ISO (10^{−9 to} ∼10⁻⁷ m). B, percentage of increases of SAN beating rates by 10⁻⁷  m ISO in control, Cav3.1 KO and TG SANCs. ‘n’ is the number of cells from three animals from each group. A: ^@ P < 0.05 and ^@@ P < 0.01, TG versus control; ^# P < 0.05 and ^## P < 0.01, KO versus control; ^$ P < 0.05, ^$$$ P < 0.001 and ^$$$$ P < 0.0001, TG versus KO at the same ISO dose determined by two‐way repeated ANOVA and post hoc tests with Bonferroni adjustment. B: ^* P < 0.05, ^** P < 0.01 and ^*** P < 0.001, analysed by one‐way ANOVA and post hoc tests with Bonferroni adjustment. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 5. I _Ca‐T in SANCs from control and Cav3.1 TG mice is increased by ISO (1 μm)

A and B, I _Ca‐T traces recorded in one control and one TG SANC bathed in nifedipine (10 μm) then nifedipine (10 μm) + ISO (1 μm). The recording was every 20 s but, for clarity, only I _Ca‐T traces every 40 s (A) or 80 s (B) are shown. The insert is the voltage clamp protocol. C and D, the time course of ISO [nifedipine (10 μm) + ISO (1 μm)] effect on I_Ca‐T amplitude recorded at −30 mV depolarized from −90 mV in the presence of nifedipine (10 μm). E and F, the percentage of increases of I _Ca‐T in WT and Cav3.1 TG SAN cells. A paired t test was used for statistics.