Genetic Disruption of Kir6.2, the Pore-Forming Subunit of ATP-Sensitive K⁺ Channel, Predisposes to Catecholamine-Induced Ventricular Dysrhythmia

Mice deficient in K_ATP channels were generated by targeted disruption of the KCNJ11 gene, which encodes the pore-forming Kir6.2 subunit of the channel complex (12). Kir6.2-knockout mice were backcrossed for five generations into a C57BL/6 background. This investigation was approved by the Mayo Clinic Institutional Animal Care and Use Committee.

Mice were anesthetized with intraperitoneal injection of 2,2,2-tribromoethanol (0.375 mg/g body wt; Sigma), intubated, and ventilated, and the aortic root was cannulated in situ (10). Perfusion was sustained ex vivo on a Langendorff system, at 90 cm H₂O with 37°C-prewarmed and 100% O₂-bubbled Tyrode solution (in mmol/l: NaCl 137, KCl 5.4, CaCl₂ 2, MgCl₂ 1, HEPES 10, and glucose 10, pH 7.4 with NaOH). After a 10-min equilibration, KCl was reduced to 2.7 mmol/l and MgCl₂ to 0.5 mmol/l, with the atrioventricular node cauterized to allow ventricular pacing (13). Coronary flow was monitored with a T106 blood flow meter (Transonic Systems).

Orthogonal electrogram signals were simultaneously recorded using four silver-silver chloride electrodes surrounding the perfused heart in a simulated “Einthoven” configuration, and signals were amplified by an electrocardiographic amplifier (Gould Electronics). A catheter (NuMed) was placed in the left ventricular endocardium to pace the heart at twice diastolic threshold intensity with 2-ms pulse width and 100-ms cycle length using a pulse generator (A310 Accupulser; World Precision Instruments). Monophasic action potentials were continuously recorded from the left ventricle by a probe (EP Technologies) positioned on the epicardial surface, and amplified signals (IsoDam; World Precision Instruments) were acquired at 11.8 kHz and stored for off-line digital analysis (9).

Cardiomyocytes were enzymatically dissociated from the ventricular myocardium (10). Action potentials were recorded at 30 ± 1°C from current-clamped isolated cells paced at 1 Hz, and were superfused with Tyrode solution (pH 7.2 adjusted with KOH) using the whole-cell patch clamp technique with 5–10 mol/lΩ pipettes containing (in mmol/l) KCl 120, MgCl₂ 1, Na₂ATP 5, HEPES 10, EGTA 0.5, and CaCl₂ 0.01 (14).

Comparisons were made using the Student’s t test. A significance level of 0.05 was preselected. Data are reported as means ± SE.

Whereas at baseline the action potentials were similar, the metabolic challenge of adrenergic stimulation induced distinct outcomes depending on the presence of functional K_ATP channels, with significant shortening of the action potential duration observed in wild-type hearts but not in age- and sex-matched counterparts lacking the Kir6.2 pore-forming channel subunit (Kir6.2-KO) (Fig. 1A and B). After a 10-min perfusion with the sympathomimetic isoproterenol (1 μmol/l), monophasic action potential duration at 90% repolarization (APD₉₀) shortened from 82 ± 2 to 74 ± 2 ms in wild-type hearts (P < 0.01, n = 6; Fig. 1A). In contrast, APD₉₀ remained at 79 ± 3 and 80 ± 3 ms before and following isoproterenol treatment, respectively, in Kir6.2-KO hearts (n = 6) (Fig. 1B). This deficit in repolarization led to distorted action potential profiles with characteristic phase 3 early afterdepolarizations manifested as distinct humps in hearts lacking functional K_ATP channels (Fig. 1B and C). In all Kir6.2-KO hearts (n = 8), adrenergic challenge induced early afterdepolarizations, which occurred in 97 ± 2% of the action potentials examined (Fig. 1D). This is in contrast to the action potential profile of the wild type (n = 6) that maintained a smooth repolarization contour following isoproterenol challenge (Fig. 1A) without significant afterdepolarizations (1 ± 1%; P < 0.01 vs. Kir6.2-KO) (Fig. 1D).

Abnormal electrical response of Kir6.2-KO hearts under adrenergic challenge was not associated with an isoproterenol-induced deficit in coronary perfusion (Fig. 2A). In fact, abnormal electrical activity during repolarization observed at the whole-heart level was reproduced at the single-cell level using action potential recording in isoproterenol-stressed current-clamped Kir6.2-KO cardiomyocytes (Fig. 2B).

Afterdepolarizations in isoproterenol-challenged Kir6.2-KO hearts translated into increased electrical vulnerability (Fig. 3). In the absence of functional K_ATP channels, afterdepolarizations induced triggered activity, disrupting regular rhythm and manifesting as premature ventricular complexes on the electrogram (Fig. 3A). On average, isoproterenol-induced afterdepolarizations complicated by triggered activity were observed in six of eight Kir6.2-KO (75%) compared with one of six wild-type (16%) hearts, translating into a 16-fold higher risk (P < 0.05) of developing premature ventricular complexes (Fig. 3B). Hence, absence of K_ATP channels produces a deficit in the repolarization reserve leading to a pronounced susceptibility of Kir6.2-KO hearts to isoproterenol-induced ventricular dysrhythmia. Thus, sarcolemmal K_ATP channels provide for membrane electrical stability that reduces the risk for arrhythmia under hyperadrenergic conditions.

Imposed catecholamine stress is a well-established precipitator of triggered activity and arrhythmia (15,16). Analogous to hearts with genetic and/or environmental compromise of repolarizing currents, as observed in congenital or acquired long QT syndrome (17), here isoproterenol challenge was proarrhythmic in the K_ATP channel–deficient myocardium provoking afterdepolarizations and triggered activity. This is in line with pharmacological studies that demonstrate that K_ATP channel activation with potassium channel openers prevents—whereas channel blockade with sulfonylurea drugs enhances—afterdepolarizations and triggered activity (18–20). In fact, in a recent randomized clinical trial in patients with type 2 diabetes, the sulfonylurea glyburide, but not the alternative oral hypoglycemic agent metformin, caused an increase in QT prolongation with QT dispersion on the electrocardiogram (21). Moreover, mutations in the cardiac sulfonylurea receptor inducing deficits in K_ATP channel function have been identified in patients with cardiomyopathy and ventricular arrhythmia (8).

Suppression of K_ATP channel activity, whether by genetic deletion of channel subunits or through use of channel antagonists, predisposes to inadequate calcium handling and intracellular calcium overload (5,9). In turn, excessive cytosolic calcium acts as a trigger for early depolarizations (22,23). Conversely, in the intact heart, where K_ATP channel opening is promoted by β-adrenergic–mediated phosphorylation of channel proteins (24,25) or subsarcolemmal ATP depletion (26), shortening of cardiac action potentials would balance catecholamine-induced increase in calcium influx protecting against triggered arrhythmia.

In summary, the present study underscores the homeostatic requirement for functional K_ATP channels in the adaptation of cardiac repolarization under adrenergic stress. In this regard, a deficit in K_ATP channel function is here identified as a previously unrecognized risk factor for the development of catecholamine-induced afterdepolarizations and triggered arrhythmia.

Supported by the National Institutes of Health (HL64822, GM08685, HL07111, and AG21201), American Heart Association, Miami Heart Research Institute, Marriott Foundation, Mayo Foundation Clinician-Investigator Program, Japan Heart Foundation, Mayo-Dubai Healthcare City Research Project, and Japanese Ministry of Education, Science, Sports, Culture and Technology. A.T. is an Established Investigator of the American Heart Association.