Mesirca P et al. (2014), Cardiac arrhythmia induced by genetic silencing...

XB-ART-53261

Nat Commun 2014 Aug 21;5:4664. doi: 10.1038/ncomms5664.

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Cardiac arrhythmia induced by genetic silencing of 'funny' (f) channels is rescued by GIRK4 inactivation.

Mesirca P , Alig J , Torrente AG , Müller JC , Marger L , Rollin A , Marquilly C , Vincent A , Dubel S , Bidaud I , Fernandez A , Seniuk A , Engeland B , Singh J , Miquerol L , Ehmke H , Eschenhagen T , Nargeot J , Wickman K , Isbrandt D , Mangoni ME .

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The mechanisms underlying cardiac automaticity are still incompletely understood and controversial. Here we report the complete conditional and time-controlled silencing of the 'funny' current (If) by expression of a dominant-negative, non-conductive HCN4-channel subunit (hHCN4-AYA). Heart-specific If silencing caused altered [Ca(2+)]i release and Ca(2+) handling in the sinoatrial node, impaired pacemaker activity and symptoms reminiscent of severe human disease of pacemaking. The effects of If silencing critically depended on the activity of the autonomic nervous system. We were able to rescue the failure of impulse generation and conduction by additional genetic deletion of cardiac muscarinic G-protein-activated (GIRK4) channels in If-deficient mice without impairing heartbeat regulation. Our study establishes the role of f-channels in cardiac automaticity and indicates that arrhythmia related to HCN loss-of-function may be managed by pharmacological or genetic inhibition of GIRK4 channels, thus offering a new therapeutic strategy for the treatment of heart rhythm diseases.

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Species referenced: Xenopus
Genes referenced: hcn1 hcn4 kcnj5 naa50 nodal

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	Figure 2. Transgene expression in the SAN and AVN regions of heart nodal tissuesRepresentative whole-mount stainings of n=4 mouse heart nodal tissues with close-up views of the mutant SAN (a) and AVN (b) regions stained with anti-HA (green) and anti-HCN1 (red) antibodies. Stainings of nuclei are shown in blue. See Supplementary Fig. 3 for the localization of the close-up views in whole-mount right atrial preparations containing the nodes. Scale bar: 100 µm.
	Figure 3. If in control and mutant pacemaker myocytesRepresentative examples of If recordings (Cs+-sensitive current, a) averaged current-to-voltage (I/V) curve (b) in control (n=11) and mutant (n=14) SAN pacemakers myocytes, (c) and (d) If recordings and I/V curve in AVN myocytes from control (n=16) and mutant (n=10) mice. (e) and (f) If recordings and I/V curve in myocytes from control (n=12) and mutant (n=9) PF myocytes. In (a), (b), and (e), the top panel shows If in control myocytes and the bottom panel shows If in mutant myocytes. In (b), (d), and (f), black circles indicate control and red squares indicate mutant myocytes. Statistical significance was tested at each voltage using the unpaired t-test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars indicate S.E.M. The voltage-clamp protocol used for all the recordings is shown in (e).
	Figure 4. Pacemaker activity and DADs in control and mutant myocytes(a) Action potential recordings of control (left) or mutant (right) SAN myocytes in Tyrode’s solution (black line) and ISO (100 nM, gray line). (b) Averaged spontaneous beating rates of SAN myocytes in Tyrode’s solution (0; n=11 control, n=11 mutant), ISO 2 nM (n=10 control, n=11 mutant), or 100 nM (n=7 control, n=10 mutant) expressed in beats min−1 (bpm). (c) Frequency of DADs (defined as a variation of at least 5 mV of the membrane potential) in control and mutant SAN myocytes in Tyrode’s solution (n=10 control, n=14 mutant) or ISO 100 nM (n=10 control, n=12 mutant). (d) Averaged time interval between a DAD and the following action potential (DAD) in comparison to the cycle length (CL) of the two consecutive action potentials preceding the DAD in n=8 mutant SAN myocytes. (e) Action potentials of control (left) or mutant (right) AVN myocytes in Tyrode’s solution (black line) and after ISO (100 nM) perfusion (gray line). (f) Spontaneous rates of AVN myocytes in Tyrode’s and ISO (0: n=12 control, n=9 mutant cells; 2: n=9 control, n=7 mutant; 100; n=6 control, n=7 mutant). (g) Frequency of DADs in control and mutant AVN myocytes in Tyrode’s and ISO (0; n=11 control, n=11 mutant; 100; n=11 control, n=13 mutant). (h) Same data as in (d) for AVN mutant myocytes (0, n=9) and in ISO (100, n=6). (i) Spontaneous action potentials recorded from PF myocytes from control (left) or mutant (right) mice in Tyrode’s solution (black line) and after perfusion of ISO (100 nM, gray line). (j) Spontaneous beating rates of PF myocytes (0: n=10 control, n=7 mutant; 2: n=9 control, n=6 mutant; 100: n=7 control, n=5 mutant). (k) Frequency of DADs in control and mutant PF myocytes (0: n=10 control and n=8 mutant;100: n=5 control, n=7 mutant). (l) Same data as in (d and h) for PF mutant myocytes in Tyrode’s solution (n=6) and ISO (n=3) in mutant PF myocytes. Statistics: 2-way ANOVA followed by Sidak multiple comparisons test and unpaired or paired t-test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars indicate S.E.M. Dotted line indicates the 0 mV. Open bars represent data from control, filled bars from mutant mice.
	Figure 5. [Ca2+]i handling in If –deficient SAN myocytes(a) Averaged frequency of spontaneous [Ca2+]i transients of SAN myocytes in Tyrode’s solution (n=20 control, n=29 mutant cells), ISO 2 nM (n=16 control, n=29 mutant cells), and in ISO 100 nM (n=14 control, n=12 mutant cells). (b) Frequency of LCRs in Tyrode-perfused SAN myocytes (n=19 control, n=24 mutant cells), in ISO 2 nM (n=18 control and n=26 mutant cells), and ISO 100 nM (n=14 control, n=13 mutant cells). (c) Averaged amplitude of spontaneous [Ca2+]i transients of SAN myocytes in Tyrode’s solution (n=19 control, n=19 mutant cells), in ISO 2 nM (n=18, n=28 mutant cells), and ISO 100 nM (n=14 control, n=13 mutant cells). Perfusion with ISO reduced transient amplitude in mutant but not in control SAN cells (d). Statistics: 2-way ANOVA, followed by Sidak multiple comparisons test. (e) Histograms of caffeine-evoked (10 mM) Ca2+ release (n=12 control, n=14 mutant cells, e-left) and decay (n=9 control, n=11 mutant cells, e-middle) in SAN myocytes. e-right. Examples of line scan in control (top) and mutant (bottom) cell during caffeine application. Statistics: unpaired t-test, (f) and (g) confocal images of spontaneously active control (f) and mutant (g) SAN myocytes loaded with Fluo-4 perfused with Tyrode’s solution (left) or 100 nM ISO (right). Red circle indicates [Ca2+]i wave, (h) and (i) averaged frequency of [Ca2+]i waves (defined as a [Ca2+]i release larger than 4 µm and/or with an intrinsic light intensity of more than 10% of the intensity measured during the following spontaneous [Ca2+]i transient) recorded in control and mutant SAN myocytes under Tyrode’s solution (n=12 control, n=11 mutant cells, h) and ISO 100 nM perfusion (n=12 control, n=11 mutant cells i), (j) and (k) histograms comparing the averaged interval of the cycle length (CL) between two consecutive spontaneous [Ca2+]i transients preceding the wave and those between the wave and the following spontaneous [Ca2+]i transient (DAD) in Tyrode’s solution (n=11, j) and in ISO 100 nM (n=6, k) in mutant SAN myocytes. Statistics: t-test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars indicate S.E.M.
	Figure 6. Time course of heart rate slowing in mutant mice following DOX withdrawal(a) Time course of the heart rate (HR, in bpm) slowing in mutant mice expressed as the difference in heart rate between n=3 control and n=5 mutant mice. DOX was removed from the diet at week 0 (W0). The maximum heart rate difference was observed at week 6 (from 525 ± 16 bpm at week 0 to 429 ± 6 bpm at week 6; t test, p<0.001). (b) Relationship between the average increase in the number of atrioventricular blocks (AVB) and the time elapsed after DOX withdrawal in n=5 mutant mice (0.2 ± 0.2 AVB/60s at week 0 vs 15 ± 6 AVB/60s at week 6; t test, p<0.05). (c) Representative examples of ECG traces recorded from mutant mice before (left) and after (right) withdrawal of doxycycline. (d) Mean values of ECG intervals recorded from control and mutant mice. Statistics: t-test. (e) Averaged number of Mobitz type I and type II 2:1 (two P waves for one QRS complex) and 3:1 (three P waves for one QRS complex) AVBs during 30 s of ECG recording (n=9 mutant mice). (f-left) Averaged maximum and minimum heart rates in control (open bars) and mutant (filled bars) mice. Statistics: 2-way ANOVA followed by Sidak multiple comparisons test. (f-right) Range of heart rate regulation calculated as differences between maximum and minimum heart rates during continuous 24-h ECG recordings of n=7 control and n=9 mutant mice (t test, p>0.05). p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars indicate S.E.M.
	Figure 7. Loss of ivabradine-mediated heart rate reduction in mutant miceDose-response relationship of heart rate (RR) to intraperitoneal administration of ivabradine (IVA) in n=13 control (a) and n=7 mutant (b) mice (ANOVA followed by Tukey’s multiple comparisons test). (c). Averaged SAN rate (PP interval) in mutant mice after IVA administration. (d). Comparison between the SAN rate of control (white bar) and mutant (black bar) mice as compared to the ventricular rate of mutant mice (gray bar) after injection of the highest dose of IVA (6 mg/kg, ANOVA followed by Tukey’s multiple comparisons test). (e-left) Averaged spontaneous beating rates of SAN myocytes, isolated from n=9 control (white bars) and n=8 mutant mice (black bars), in Tyrode’s solution (empty bars) and after perfusion with ivabradine (1 µM, dotted bars). Statistics: 2-way ANOVA followed by Sidak multiple comparisons test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars define the S.E.M. (e-right) Sample action potentials recorded from control (top) and mutant (bottom) SAN myocytes before (black line) and after (gray line) application of 1 µM ivabradine.
	Figure 8. Autonomic regulation of heart rate and impulse conduction in mutant miceAveraged heart rate recorded from n=11 control (open bars) and n=11 mutant (filled bars) mice before (pre) and after (post) intraperitoneal injection of propranolol (a), or atropine and propranolol (b). Statistics in (a) and (b): 2-way ANOVA followed by Sidak multiple comparisons test. (c). Number of atrioventricular blocks (AVB) measured in a 30-s window every 5 minutes for a 30-min total recording period before (pre) and after (post) injection. AVB counting started 30 minutes after injection to allow the effect to stabilize. Statistics: t-test. (d). Sample dot plot of beat-to-beat variability (bpm) of heart rate of mutant mice before and after (dotted line) injection of atropine and propranolol. Note slow ventricular beats in the presence of AVB before injection. Arrows indicate AVBs (two P waves for one QRS complex, 2:1; three P waves for one QRS complex, 3:1). (e). Time course of heart rate and the standard deviation of heart rate before and after injection of ISO. (f–h). Heart rate in control and mutant mice before and after injection of atropine (f) or isoproterenol (g) in n=8 control and n=12 mutant mice. (h) Heart rate before and after 5-min swimming test, in n=5 control (open bars) and n=7 mutant (filled bars) mice. (i) Delta of heart rate following different β-adrenergic stimulation. Values represent the mean difference between pre- and post-adrenergic pathway activation (pharmacological or physiological). Statistics: 2-way ANOVA followed by Sidak multiple comparisons test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars indicate S.E.M.
	Figure 9. Improvement of heart rate in mutant mice by GIRK4 gene knockoutAveraged RR (a), PP (b), PR (c), number of atrioventricular blocks (AVB) (d) and QRS (e) in n=7 control (white bar), n=7 mutant (black bar), and n=8 Mut/GIRK4−/− (black bar with white stripes) mice. Statistics (a–e): ANOVA followed by Tukey’s multiple comparisons test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars define the S.E.M. f. Representative example of ECG recordings from control mice. (g–i). Different examples of ECG recordings showing heart rate dysfunction recorded from mutant mice. (g) Second-degree AVB; (h) ventricular extrasystoles; (i) ventricular tachycardia. (j) Relative percentages of SAN or AVN dysfunctions preceding a period of ventricular tachycardia in n=5 mutant mice (SP: sinus pauses, ESV: ventricular extrasystoles). (k) ECG recorded from Mut/GIRK4−/−, mice. Small circles indicate P waves.

References [+] :

Alig, Control of heart rate by cAMP sensitivity of HCN channels. 2009, Pubmed

	Figure 2. Transgene expression in the SAN and AVN regions of heart nodal tissuesRepresentative whole-mount stainings of n=4 mouse heart nodal tissues with close-up views of the mutant SAN (a) and AVN (b) regions stained with anti-HA (green) and anti-HCN1 (red) antibodies. Stainings of nuclei are shown in blue. See Supplementary Fig. 3 for the localization of the close-up views in whole-mount right atrial preparations containing the nodes. Scale bar: 100 µm.
	Figure 3. If in control and mutant pacemaker myocytesRepresentative examples of If recordings (Cs+-sensitive current, a) averaged current-to-voltage (I/V) curve (b) in control (n=11) and mutant (n=14) SAN pacemakers myocytes, (c) and (d) If recordings and I/V curve in AVN myocytes from control (n=16) and mutant (n=10) mice. (e) and (f) If recordings and I/V curve in myocytes from control (n=12) and mutant (n=9) PF myocytes. In (a), (b), and (e), the top panel shows If in control myocytes and the bottom panel shows If in mutant myocytes. In (b), (d), and (f), black circles indicate control and red squares indicate mutant myocytes. Statistical significance was tested at each voltage using the unpaired t-test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars indicate S.E.M. The voltage-clamp protocol used for all the recordings is shown in (e).
	Figure 4. Pacemaker activity and DADs in control and mutant myocytes(a) Action potential recordings of control (left) or mutant (right) SAN myocytes in Tyrode’s solution (black line) and ISO (100 nM, gray line). (b) Averaged spontaneous beating rates of SAN myocytes in Tyrode’s solution (0; n=11 control, n=11 mutant), ISO 2 nM (n=10 control, n=11 mutant), or 100 nM (n=7 control, n=10 mutant) expressed in beats min−1 (bpm). (c) Frequency of DADs (defined as a variation of at least 5 mV of the membrane potential) in control and mutant SAN myocytes in Tyrode’s solution (n=10 control, n=14 mutant) or ISO 100 nM (n=10 control, n=12 mutant). (d) Averaged time interval between a DAD and the following action potential (DAD) in comparison to the cycle length (CL) of the two consecutive action potentials preceding the DAD in n=8 mutant SAN myocytes. (e) Action potentials of control (left) or mutant (right) AVN myocytes in Tyrode’s solution (black line) and after ISO (100 nM) perfusion (gray line). (f) Spontaneous rates of AVN myocytes in Tyrode’s and ISO (0: n=12 control, n=9 mutant cells; 2: n=9 control, n=7 mutant; 100; n=6 control, n=7 mutant). (g) Frequency of DADs in control and mutant AVN myocytes in Tyrode’s and ISO (0; n=11 control, n=11 mutant; 100; n=11 control, n=13 mutant). (h) Same data as in (d) for AVN mutant myocytes (0, n=9) and in ISO (100, n=6). (i) Spontaneous action potentials recorded from PF myocytes from control (left) or mutant (right) mice in Tyrode’s solution (black line) and after perfusion of ISO (100 nM, gray line). (j) Spontaneous beating rates of PF myocytes (0: n=10 control, n=7 mutant; 2: n=9 control, n=6 mutant; 100: n=7 control, n=5 mutant). (k) Frequency of DADs in control and mutant PF myocytes (0: n=10 control and n=8 mutant;100: n=5 control, n=7 mutant). (l) Same data as in (d and h) for PF mutant myocytes in Tyrode’s solution (n=6) and ISO (n=3) in mutant PF myocytes. Statistics: 2-way ANOVA followed by Sidak multiple comparisons test and unpaired or paired t-test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars indicate S.E.M. Dotted line indicates the 0 mV. Open bars represent data from control, filled bars from mutant mice.
	Figure 5. [Ca2+]i handling in If –deficient SAN myocytes(a) Averaged frequency of spontaneous [Ca2+]i transients of SAN myocytes in Tyrode’s solution (n=20 control, n=29 mutant cells), ISO 2 nM (n=16 control, n=29 mutant cells), and in ISO 100 nM (n=14 control, n=12 mutant cells). (b) Frequency of LCRs in Tyrode-perfused SAN myocytes (n=19 control, n=24 mutant cells), in ISO 2 nM (n=18 control and n=26 mutant cells), and ISO 100 nM (n=14 control, n=13 mutant cells). (c) Averaged amplitude of spontaneous [Ca2+]i transients of SAN myocytes in Tyrode’s solution (n=19 control, n=19 mutant cells), in ISO 2 nM (n=18, n=28 mutant cells), and ISO 100 nM (n=14 control, n=13 mutant cells). Perfusion with ISO reduced transient amplitude in mutant but not in control SAN cells (d). Statistics: 2-way ANOVA, followed by Sidak multiple comparisons test. (e) Histograms of caffeine-evoked (10 mM) Ca2+ release (n=12 control, n=14 mutant cells, e-left) and decay (n=9 control, n=11 mutant cells, e-middle) in SAN myocytes. e-right. Examples of line scan in control (top) and mutant (bottom) cell during caffeine application. Statistics: unpaired t-test, (f) and (g) confocal images of spontaneously active control (f) and mutant (g) SAN myocytes loaded with Fluo-4 perfused with Tyrode’s solution (left) or 100 nM ISO (right). Red circle indicates [Ca2+]i wave, (h) and (i) averaged frequency of [Ca2+]i waves (defined as a [Ca2+]i release larger than 4 µm and/or with an intrinsic light intensity of more than 10% of the intensity measured during the following spontaneous [Ca2+]i transient) recorded in control and mutant SAN myocytes under Tyrode’s solution (n=12 control, n=11 mutant cells, h) and ISO 100 nM perfusion (n=12 control, n=11 mutant cells i), (j) and (k) histograms comparing the averaged interval of the cycle length (CL) between two consecutive spontaneous [Ca2+]i transients preceding the wave and those between the wave and the following spontaneous [Ca2+]i transient (DAD) in Tyrode’s solution (n=11, j) and in ISO 100 nM (n=6, k) in mutant SAN myocytes. Statistics: t-test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars indicate S.E.M.
	Figure 6. Time course of heart rate slowing in mutant mice following DOX withdrawal(a) Time course of the heart rate (HR, in bpm) slowing in mutant mice expressed as the difference in heart rate between n=3 control and n=5 mutant mice. DOX was removed from the diet at week 0 (W0). The maximum heart rate difference was observed at week 6 (from 525 ± 16 bpm at week 0 to 429 ± 6 bpm at week 6; t test, p<0.001). (b) Relationship between the average increase in the number of atrioventricular blocks (AVB) and the time elapsed after DOX withdrawal in n=5 mutant mice (0.2 ± 0.2 AVB/60s at week 0 vs 15 ± 6 AVB/60s at week 6; t test, p<0.05). (c) Representative examples of ECG traces recorded from mutant mice before (left) and after (right) withdrawal of doxycycline. (d) Mean values of ECG intervals recorded from control and mutant mice. Statistics: t-test. (e) Averaged number of Mobitz type I and type II 2:1 (two P waves for one QRS complex) and 3:1 (three P waves for one QRS complex) AVBs during 30 s of ECG recording (n=9 mutant mice). (f-left) Averaged maximum and minimum heart rates in control (open bars) and mutant (filled bars) mice. Statistics: 2-way ANOVA followed by Sidak multiple comparisons test. (f-right) Range of heart rate regulation calculated as differences between maximum and minimum heart rates during continuous 24-h ECG recordings of n=7 control and n=9 mutant mice (t test, p>0.05). p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars indicate S.E.M.
	Figure 7. Loss of ivabradine-mediated heart rate reduction in mutant miceDose-response relationship of heart rate (RR) to intraperitoneal administration of ivabradine (IVA) in n=13 control (a) and n=7 mutant (b) mice (ANOVA followed by Tukey’s multiple comparisons test). (c). Averaged SAN rate (PP interval) in mutant mice after IVA administration. (d). Comparison between the SAN rate of control (white bar) and mutant (black bar) mice as compared to the ventricular rate of mutant mice (gray bar) after injection of the highest dose of IVA (6 mg/kg, ANOVA followed by Tukey’s multiple comparisons test). (e-left) Averaged spontaneous beating rates of SAN myocytes, isolated from n=9 control (white bars) and n=8 mutant mice (black bars), in Tyrode’s solution (empty bars) and after perfusion with ivabradine (1 µM, dotted bars). Statistics: 2-way ANOVA followed by Sidak multiple comparisons test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars define the S.E.M. (e-right) Sample action potentials recorded from control (top) and mutant (bottom) SAN myocytes before (black line) and after (gray line) application of 1 µM ivabradine.
	Figure 8. Autonomic regulation of heart rate and impulse conduction in mutant miceAveraged heart rate recorded from n=11 control (open bars) and n=11 mutant (filled bars) mice before (pre) and after (post) intraperitoneal injection of propranolol (a), or atropine and propranolol (b). Statistics in (a) and (b): 2-way ANOVA followed by Sidak multiple comparisons test. (c). Number of atrioventricular blocks (AVB) measured in a 30-s window every 5 minutes for a 30-min total recording period before (pre) and after (post) injection. AVB counting started 30 minutes after injection to allow the effect to stabilize. Statistics: t-test. (d). Sample dot plot of beat-to-beat variability (bpm) of heart rate of mutant mice before and after (dotted line) injection of atropine and propranolol. Note slow ventricular beats in the presence of AVB before injection. Arrows indicate AVBs (two P waves for one QRS complex, 2:1; three P waves for one QRS complex, 3:1). (e). Time course of heart rate and the standard deviation of heart rate before and after injection of ISO. (f–h). Heart rate in control and mutant mice before and after injection of atropine (f) or isoproterenol (g) in n=8 control and n=12 mutant mice. (h) Heart rate before and after 5-min swimming test, in n=5 control (open bars) and n=7 mutant (filled bars) mice. (i) Delta of heart rate following different β-adrenergic stimulation. Values represent the mean difference between pre- and post-adrenergic pathway activation (pharmacological or physiological). Statistics: 2-way ANOVA followed by Sidak multiple comparisons test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars indicate S.E.M.
	Figure 9. Improvement of heart rate in mutant mice by GIRK4 gene knockoutAveraged RR (a), PP (b), PR (c), number of atrioventricular blocks (AVB) (d) and QRS (e) in n=7 control (white bar), n=7 mutant (black bar), and n=8 Mut/GIRK4−/− (black bar with white stripes) mice. Statistics (a–e): ANOVA followed by Tukey’s multiple comparisons test. p<0.05, p<0.01, p<0.001, **p<0.0001. Error bars define the S.E.M. f. Representative example of ECG recordings from control mice. (g–i). Different examples of ECG recordings showing heart rate dysfunction recorded from mutant mice. (g) Second-degree AVB; (h) ventricular extrasystoles; (i) ventricular tachycardia. (j) Relative percentages of SAN or AVN dysfunctions preceding a period of ventricular tachycardia in n=5 mutant mice (SP: sinus pauses, ESV: ventricular extrasystoles). (k) ECG recorded from Mut/GIRK4−/−, mice. Small circles indicate P waves.