Increased GIRK channel activity prevents arrhythmia in mice with heart failure by enhancing ventricular repolarization

Abstract

Ventricular arrhythmia causing sudden cardiac death is the leading mode of death in patients with heart failure. Yet, the mechanisms that prevent ventricular arrhythmias in heart failure are not well characterized. Using a mouse model of heart failure created by transverse aorta constriction, we show that GIRK channel, an important regulator of cardiac action potentials, is constitutively active in failing ventricles in contrast to normal cells. Evidence is presented indicating that the tonic activation of M₂ muscarinic acetylcholine receptors by endogenously released acetylcholine contributes to the constitutive GIRK activity. This constitutive GIRK activity prevents the action potential prolongation in heart failure ventricles. Consistently, GIRK channel blockade with tertiapin-Q induces QT interval prolongation and increases the incidence of arrhythmia in heart failure, but not in control mice. These results suggest that constitutive GIRK channels comprise a key mechanism to protect against arrhythmia by providing repolarizing currents in heart failure ventricles.

Figure 1

Basal and acetylcholine (ACh)-induced inward rectifier currents are increased in left ventricular myocytes from transverse aorta constriction (TAC) mice. Mice were subjected to the TAC (n = 57) or sham (n = 50) operation, as described in the Methods. (a) The temporal changes in ejection fraction (EF) over 11 weeks after surgery in sham and TAC mice. In TAC mice, the EF decreased gradually. (b) Representative M-mode echocardiographic images of the left ventricle (LV), time stamp: 100 ms, vertical bar: 2 mm. (c,d) Average values for the LV mass (c) and the indicated echocardiographic parameter (d) from sham and TAC mice at 11 weeks after surgery. TAC mice showed clear LV structural abnormalities compared with age-matched sham-operated mice. LVID;d or LVID;s the internal dimension of LV; diastolic or systolic, LVPW;d or LVPW;s the postwall thickness of LV; diastolic or systolic, IVS;d or IVS;s the interventricular septum; diastolic or systolic. (e) Capacitance of myocytes from the left atrium (LA), right atrium (RA), LV, and right ventricle (RV) of sham and TAC mice. (f) Representative current trace of membrane currents in LV myocytes of sham and TAC mice at a holding potential of − 40 mV. ACh (10 µM) was applied during the periods indicated. The dotted line indicates zero current level. (g) Current–voltage relationship under basal conditions (basal current) and after ACh treatment in response to a ramp pulse from − 120 to + 60 mV (at a speed of ± 0.6Vs^-1) from the holding potential of − 40 mV. (h,i) Summary data for basal current and ACh-activated GIRK current density (h) and resting membrane potential (RMP) (i) in sham and TAC. Data represent means ± SEM. The numbers indicate number of myocytes/mice. NS indicates not significantly different. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t-test.

Figure 2

Constitutive GIRK current is increased in left ventricular myocytes from transverse aorta constriction (TAC) mice. (a) Tertiapin-Q (300 nM) was applied before acetylcholine (ACh; 10 μM) treatment in left ventricular (LV) myocytes from sham (upper panel) and TAC (lower panel) mice. Currents were recorded at a holding potential of − 40 mV. (b–d) Summarized data for the effects of 300 nM tertiapin-Q on basal current density (b), ACh-activated GIRK current density (c), and resting membrane potential (RMP) (d) in sham and TAC LV myocytes. The numbers indicate number of myocytes/mice. NS indicates not significantly different. *P < 0.05, **P < 0.01, Student’s t-test.

Figure 3

Transverse aorta constriction (TAC) ventricular myocytes exhibit enhanced susceptibility of tertiapin-Q and acetylcholine (ACh)-induced action potential (AP) modulation. (a,b) Representative AP traces in left ventricular (LV) myocytes stimulated at 1 Hz from sham (upper panel) and TAC (lower panel) before (black) and after 300 nM tertiapin-Q (a, blue) or 10 μM ACh treatment (b, red). Right, superimposed AP traces measured before and after exposure of tertiapin-Q (a) or ACh (b). Tertiapin-Q or ACh altered action potential duration (APD) only in TAC myocytes. (c,d) Summary data of APD at 90% repolarization (APD₉₀) in LV myocytes from sham and TAC stimulated at 0.5 Hz, 1 Hz, 2 Hz, and 4 Hz in response to tertiapin-Q or ACh treatment. Data represent means ± SEM. The numbers indicate number of myocytes/mice. NS indicates not significantly different. *P < 0.05, Student’s paired or unpaired t-test was used as indicated.

Figure 4

Both constitutive and exogenous acetylcholine (ACh)-induced activation of GIRK channels of transverse aorta constriction (TAC) mice is mediated by M₂ muscarinic ACh receptors (mAChRs). (a) Representative current trance in LV myocytes from sham (left panel) and TAC (right panel) mice exposed to a nonselective muscarinic antagonist, atropine (1 μM) before ACh treatment (10 μM) at a holding potential of − 40 mV for 5 min. (b,c) Summarized data for the effects of 1 μM atropine on ACh-activated GIRK channels (b) and basal current density (c) in sham and TAC left ventricular (LV) myocytes. d Same as in (a), but for the M₂ mAChR selective antagonist methoctramine. (e,f) Same as in (b,c), but for the M₂ mAChR selective antagonist methoctramine. (g) Representative AP traces in LV myocytes stimulated at 1 Hz from sham (upper panel) and TAC (lower panel) before (black) and after (blue) 1 μM methoctramine treatment. Right, superimposed AP traces measured before and after exposure of methoctramine. Methoctramine prolonged action potential duration (APD) only in TAC. (h) Summary data of APD at 90% repolarization (APD₉₀) in LV myocytes from sham (upper panel) and TAC (lower panel) stimulated at 0.5 Hz, 1 Hz, 2 Hz, and 4 Hz in response to methoctramine. Data represent means ± SEM. The numbers indicate number of myocytes/mice. NS indicates not significantly different. *P < 0.05, **P < 0.01, Student’s paired or unpaired t-test was used as indicated.

Figure 5

Endogenous acetylcholine (ACh) tonically stimulates GIRK activities in left ventricular (LV) myocytes from transverse aorta constriction (TAC) mice. (a,b) Representative current trance in LV myocytes from sham (a) and TAC (b) mice exposed to HC-3 (10 μM) for 24 min at a holding potential of − 40 mV. (c) Summarized data for the effects of 10 μM HC-3 on the tertiapinQ-sensitive current density in sham and TAC LV myocytes. Tertiapin Q-sensitive currents were obtained by subtraction. (d) Representative action potential (AP) traces in LV myocytes stimulated at 1 Hz from sham (upper panel) and TAC (lower panel) before (black) and after (blue) 10 μM HC-3 treatment. Right, superimposed AP traces measured before and after exposure of HC-3. HC-3 prolonged action potential duration (APD) only in TAC. (e) Summary data of APD at 90% repolarization (APD₉₀) in LV myocytes from sham (upper panel) and TAC (lower panel) stimulated at 0.5 Hz, 1 Hz, 2 Hz, and 4 Hz in response to HC-3. Data represent means ± SEM. The numbers indicate number of myocytes/mice. NS indicates not significantly different. *P < 0.05, Student’s paired or unpaired t-test was used as indicated.

Figure 6

Inhibition of GIRK activity with tertiapin-Q induces QT prolongation and arrhythmia in transverse aorta constriction (TAC) mice. (a,b) Representative lead-II electrocardiogram (ECG) traces from sham (a) and TAC (b) mice. Mice were anesthetized using isoflurane, and the basal ECG was monitored for 10–15 min. The ECG was further monitored for 15 min following an intraperitoneal injection of tertiapin-Q (5 mg/kg). The trace from sham mice indicates sinus rhythm. ECGs from TAC mice show premature ventricular event (arrowhead, 1st and 2nd traces) and a episode of ventricular tachycardia (3rd trace) after tertiapin-Q treatment. (c–h) Summarized data for heart rate (HR) (c), QRS (d), QT (e), QTc (f), premature ventricular event (g), and incidence of the enlarged QRS complex (h). n = 6 in the sham group and n = 7 in the TAC group. NS indicates not significantly different. *P < 0.05, **P < 0.01, Student’s t-test.