Genetic Ablation of G Protein-Gated Inwardly Rectifying K+ Channels Prevents Training-Induced Sinus Bradycardia

Figures

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  FIGURE 1. Heart rate recorded in sedentary (left panel) and trained (right panel) WT (A) and Girk4–/– mice (B) at different days during 5 min sham-training (WT and Girk4–/– sedentary, see methods) and training (WT and Girk4–/– trained) protocol. (C) Histogram of the average values of the slopes of the regression line between time and heart rate. Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. (D) Representative examples of ECG traces and averaged heart rate recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4–/– mice (right panel). Statistics: unpaired Student’s t-test. Tertiapin-Q (Tert, 5 mg/kg) effect in WT (E) and Girk4–/– (F) sedentary (empty bar) and trained (filled bars) mice. Statistics: two-way analysis of variance followed by Sidak multiple comparisons test. (G) Close-up of ECG traces and averaged PR interval recorded at day 28 from sedentary (top) and trained (bottom) WT (left panel) and Girk4–/– mice (right panel). Statistics: unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ###p < 0.001, ####p < 0.0001. Error bars indicate s.e.m. WTS: WT sedentary; WTT: WT trained; Girk4–/– S: Girk4–/–…

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  FIGURE 2. Standard deviation of intervals between two consecutive heart beats (SDNN) calculated in 5-min stable ECG periods at day 0 and at day 28 in WT (A) and Girk4–/– (B) sedentary (open bars) and trained (filled bars) animals. Power spectral density (PSD) of heart rate variability determined by Fast Fourier Transformation analysis of 5-min of stable ECG segments at day 0 and at day 28 in WT (C) and Girk4–/– (D) sedentary (open bars) and trained (filled bars) animals. Heart rate (non-invasive ECG recordings) measured in sedentary (open bars, left panel) and trained (filled bars, right panel) WT mice before (day 0) and after (day 28) training period in control condition (ANS +, E) or following intraperitoneal injection of atropine (0.5 mg/kg) and propranolol (5 mg/kg) to inhibit the input of the autonomic nervous system (ANS-, F). (G,H) same as (E,F) but in Girk4–/– animals. Statistics: unpaired Student’s t-test. **p < 0.01, ****p < 0.0001. Error bars indicate s.e.m.

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  FIGURE 3. Action potential recordings of SAN myocytes isolated from WT sedentary (WT S), WT trained (WT T), Girk4–/– sedentary (Girk4–/– S) and Girk4–/– trained (Girk4–/– T) in Tyrode’s solution at the end of training, or sham-training protocol (day 28) (A). The dotted line indicates the 0 mV. Rate of spontaneous action potentials (B) and slope of the linear part of the diastolic depolarization (SLDD, C) recorded at day 28 in SAN myocytes in Tyrode’s solution (n = 6 WT S, n = 10 WT T, n = 7 Girk4–/– S and n = 8 Girk4–/– T). Statistics: one-way analysis of variance followed by Tukey’s multiple comparisons test. *p < 0.05, ****p < 0.0001. Error bars indicate s.e.m.

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  FIGURE 4. Representative traces of If recordings (A) and averaged current-to-voltage (I-V) curve (B) in sedentary (open black circles, n = 15) and trained (filled black circles, n = 23) WT SAN myocytes. If recordings (C) and I-V curve (D) in SAN myocytes from sedentary (open blue circles, n = 12) and trained (filled blue circles, n = 18) isolated Girk4–/– SAN pacemaker cells. The voltage-clamp protocol used for all the recordings is shown at the bottom of panel (C). Statistical significance was tested at each voltage using the unpaired Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. (E, left panel) Steady state If activation curves in isolated SAN cells from sedentary (dotted line) and trained (continuous line), WT (black) and Girk4–/– (blue) mice. Representative ramp current trace recorded in a WT sedentary SAN cell and corresponding voltage protocol are shown under the curves. (E, right panel) Histograms representing averaged half-activation voltages (V1/2) values for If recorded in SAN cells from WT (black bars) and Girk4–/– (blue bars) sedentary (open bars) and trained (filled bars) animals. Data have been collected at day 28 (end of training, or sham-training, protocol).…

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  FIGURE 5. Representative Ca2+ current traces of the L-type Ca2+ current (ICaL) measured from a holding potential (HP) of −55 mV (A) and current-to-voltage (I-V) relationships (B) from sedentary (open black circles, n = 14) and trained (filled black circles, n = 13) WT SAN myocytes. Traces of ICaL (C) and I-V curves (D) from sedentary (open blue circles, n = 15) compared with trained (filled blue circles, n = 22) Girk4–/– SAN myocytes. Data, collected at day 28 were fitted with a modified Boltzmann equation. Voltage protocol is shown in panel C (bottom). (E) Sample traces of ICa (ICaT + ICaL) recorded from a HP of −80 mV in WT and Girk4–/–SAN myocytes. (F) Sample ICa traces for same myocytes as in (E), but after switching to HP = −55 to inactivate ICaT. (G) Net peak ICaT I-V curves measured following subtraction of traces recorded from HP = −55 mV from traces obtained at HP = −80 mV in WT (left panel) and in Girk4–/– (right panel) SAN myocytes from sedentary and trained mice. Statistical significance was tested at each voltage using the unpaired Student’s t-test. *p < 0.05, **p < 0.01, ****p < 0.0001. Error bars indicate s.e.m. WT S: WT sedentary; WT T: WT trained; Girk4–/– S: Girk4–/–…

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  FIGURE 6. Expression of HCN4 mRNA (A) normalized to expression of Tbp in SAN biopsies of from WT sedentary (n = 8), WT trained (n = 8), Girk4–/– sedentary (n = 7), and Girk4–/– trained (n = 6) mice. Statistics: two-way analysis of variance with Sidak’s multiple comparisons test. Representative HCN4 western blot (B, left panel) with corresponding stain-free total protein gel used for quantification (lower left panel). (B, right panel) Protein expression determined by western blot in individual SAN biopsies isolated from WT sedentary (n = 4), WT trained (n = 4) sedentary Girk4–/– mice (n = 5) and trained Girk4–/–mice (n = 4). Statistics: Students t-test. SAN Expression of miR-423-5p (C) in WT sedentary (n = 8), WT trained (n = 7) sedentary Girk4–/– mice (n = 8) and trained Girk4–/–mice (n = 7). (D) Fold change in expression of selected miRs in the SAN of WT trained (n = 8) and Girk4–/– trained mice (n = 7) relative to respective WT sedentary (n = 8) and Girk4–/– sedentary mice (n = 7). Statistics: one-way analysis of variance with Tukey’s multiple comparisons test. *p < 0.05. miR expression in (C,D) was normalized to expression of Snord61 and Snord95.

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  FIGURE 7. mRNA expression of L-type calcium channel subunits Cav1.2, Cav1.3, Cav3.1, and Cav3.2 (A) (normalized to expression of Tbp) in SAN biopsies from from WT sedentary (n = 10), WT trained (n = 10), Girk4–/– sedentary (n = 9) and Girk4–/– trained (n = 10) mice. Protein expression determined by western blot using antibodies directed against Cav1.2 (B) and Cav1.3 (C) in individual sinus node biopsies isolated from sedentary WT (n = 6), trained WT (n = 4) sedentary Girk4–/– mice (n = 6) and trained Girk4–/–mice (n = 6). Representative western blots with corresponding stain-free total protein blot used for quantification shown in lower panel. Statistics: Students t-test. *p < 0.05.

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  FIGURE 8. Numerical simulation of current traces for If (A), ICaT (B), and ICaL (C) calculated using current density and activation parameters recorded in WT sedentary (WT S), WT trained (WT T), Girk4–/– sedentary (Girk4–/– S) and Girk4–/– trained (Girk4–/– T) at day 28 of experimental protocol. (D) Corresponding predicted I-V curves of If (left panel), ICaT (central panel) and ICaL (right panel), calculated at the peak of current density from simulations in (A–C). In I-V curves, open circles show predicted current densities of WT sedentary SAN cells and black circles densities of WT trained SAN cells. (E) Comparison between predicted pacemaker activities simulated for control (sedentary) condition (left panel), or including training-dependent changes of If, ICaT and ICaL. Abbreviations: CL, pacemaker activity cycle length; DI, diastolic interval.