Protection conferred by myocardial ATP-sensitive K+ channels in pressure overload-induced congestive heart failure revealed in KCNJ11 Kir6.2-null mutant

Authors

  • Satsuki Yamada,

    1. Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA
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  • Garvan C. Kane,

    1. Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA
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  • Atta Behfar,

    1. Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA
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  • Xiao-Ke Liu,

    1. Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA
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  • Roy B. Dyer,

    1. Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA
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  • Randolph S. Faustino,

    1. Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA
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  • Takashi Miki,

    1. Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan
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  • Susumu Seino,

    1. Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan
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  • Andre Terzic

    1. Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA
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Corresponding A. Terzic: Division of Cardiovascular Diseases, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Email: terzic.andre@mayo.edu

Abstract

Ventricular load can precipitate development of the heart failure syndrome, yet the molecular components that control the cardiac adaptive response to imposed demand remain partly understood. Compromised ATP-sensitive K+ (KATP) channel function renders the heart vulnerable to stress, implicating this metabolic sensor in the homeostatic response that would normally prevent progression of cardiac disease. Here, pressure overload was imposed on the left ventricle by transverse aortic constriction in the wild-type and in mice lacking sarcolemmal KATP channels through Kir6.2 pore knockout (Kir6.2-KO). Despite equivalent haemodynamic loads, within 30 min of aortic constriction, Kir6.2-KO showed an aberrant prolongation of action potentials with intracellular calcium overload and ATP depletion, whereas wild-type maintained ionic and energetic handling. On catheterization, constricted Kir6.2-KO displayed compromised myocardial performance with elevated left ventricular end-diastolic pressure, not seen in the wild-type. Glyburide, a KATP channel inhibitor, reproduced the knockout phenotype in the wild-type, whereas the calcium channel antagonist, verapamil, prevented abnormal outcome in Kir6.2-KO. Within 48 h following aortic constriction, fulminant biventricular congestive heart failure, characterized by exercise intolerance, cardiac contractile dysfunction, hepatopulmonary congestion and ascites, halved the Kir6.2-KO cohort, while no signs of organ failure or mortality were seen in wild-type. Surviving Kir6.2-KO developed premature and exaggerated fibrotic myocardial hypertrophy associated with nuclear up-regulation of calcium-dependent pro-remodelling MEF2 and NF-AT pathways, precipitating chamber dilatation within 3 weeks. Thus, KATP channels appear mandatory in acute and chronic cardiac adaptation to imposed haemodynamic load, protecting against congestive heart failure and death.

Unique among ion channels, ATP-sensitive K+ (KATP) channels operate as molecular rheostats adjusting membrane potential-dependent functions to match cellular energetic demands (Alekseev et al. 2005; Miki & Seino, 2005; Nichols, 2006). Underscoring the critical role for KATP channels in coupling metabolic dynamics with electrical activity is the recognition that disruption of channel function can compromise vital homeostatic functions leading to life-threatening disease (Ashcroft, 2005). In particular, dysfunction in KATP channel gating has been linked with insulin secretory disorders, such as congenital hyperinsulinism and more recently neonatal diabetes (Thomas et al. 1995; Dunne et al. 2004; Gloyn et al. 2004; Ashcroft, 2005; Babenko et al. 2006; Pearson et al. 2006). Beyond an isolated failure of pancreatic β-cells, mutations in KCNJ11, the gene encoding the pore-forming Kir6.2 subunit of KATP channels (Aguilar-Bryan et al. 1995; Inagaki et al. 1995), have been reported to be pathogenic in a discrete syndrome encompassing, with diabetes, developmental delay and epilepsy (Proks et al. 2004; Hattersley & Ashcroft, 2005; Gloyn et al. 2006). Kir6.2 is also integral in the make-up of myocardial KATP channels (Inagaki et al. 1996), and targeted disruption of KCNJ11 generates Kir6.2-deficient mice that lack functional KATP channels in ventricular myocytes (Suzuki et al. 2001). Although intact Kir6.2 is required in cardiac adaptation to physiological stress (Zingman et al. 2002a, 2003), limited information is presently available on the repercussions of KATP channel malfunction in the development and progression of heart disease (Kane et al. 2005).

Originally discovered in cardiomyocytes (Noma, 1983), KATP channels are abundant within the sarcolemma where they assemble as heteromultimers of Kir6.2, the inwardly rectifying K+ channel pore, and SUR2A, the regulatory sulphonylurea receptor subunit (Inagaki et al. 1996; Lorenz & Terzic, 1999; Nichols, 2006). Integrated with cellular metabolic pathways (Dzeja & Terzic 1998; Carrasco et al. 2001; Abraham et al. 2002; Selivanov et al. 2004; Dhar-Chowdhury et al. 2005; Jovanovic et al. 2005), SUR2A contains nucleotide binding domains and intrinsic ATPase activity, endowing this regulatory KATP channel subunit with the ability to process energetic signals of distress under conditions of increased cardiac workload (Bienengraeber et al. 2000; Zingman et al. 2001; Alekseev et al. 2005). The tandem function of nucleotide binding domains confers Kir6.2-gating competence to SUR2A (Zingman et al. 2002b), leading to pore opening and action potential shortening under stress (Zingman et al. 2002a; Liu et al. 2004; Nichols, 2006). Within the working myocardium, this homeostatic role for cardiac KATP channels translates into preventing intracellular calcium loading and preserving energy supplies (Terzic et al. 1995; Zingman et al. 2002a; Hodgson et al. 2003; Kane et al. 2005). A deficit in cardiac KATP channels impairs tolerance to systemic stressors imposed by a sympathetic surge (Zingman et al. 2002a), endurance challenge (Kane et al. 2004) or hypertension (Kane et al. 2006a). Genetic disruption of KATP channels results in poor recovery following coronary hypoperfusion, and compromises the protective benefits of ischaemic preconditioning (Suzuki et al. 2002; Gumina et al. 2003), while overexpression of channel subunits generates a phenotype resistant to ischaemia (Du et al. 2006). Moreover, missense and frameshift mutations in the cardiac KATP channel isoform identified in dilated cardiomyopathy suggest, in the setting of compromised channel function, a genetic susceptibility that renders the myocardium vulnerable to failure (Bienengraeber et al. 2004; Kane et al. 2005). Despite these advances, the role for KATP channels in adequate myocardial adaptation under imposed left ventricular load, a recognized risk factor of heart failure and cardiac death, remains only partially understood.

Experimentally, transverse aortic constriction directly imposes a pressure overload on the left ventricle (Rockman et al. 1991). Mechanisms of myocardial response result in concentric ventricular hypertrophy, and preservation of cardiac contractile function with maintenance of ionic and energetic homeostasis within a normal heart (Hunter & Chien, 1999; Barki-Harrington & Rockman, 2003). Here, the acute and chronic consequences of myocardial KATP channel deficit, through knockout of the Kir6.2 pore, were longitudinally tested in this established model of haemodynamic ventricular load. In the absence of functional KATP channels, physical constriction of the aorta precipitated cardiac dysfunction through dysregulation of action potential control of ionic balance thereby translating into fulminant congestive heart failure and death.

Methods

Protocols were carried out in accordance with the National Institutes of Health guidelines, and with approval of the Mayo Clinic Institutional Animal Care and Use Committee.

Kir6.2-knockout mice

Mice deficient in KATP channels were generated by targeted disruption of the KCNJ11 gene and backcrossed for five generations into a C57BL/6 background (Miki et al. 1998). Due to the proximity of the mutated KCNJ11 gene with the gene encoding albino hair colour in the SV129 embryonic stem cells used to create the knockout, the Kir6.2-knockout (Kir6.2-KO) mice remain white upon backbreeding into the black C57BL/6 line (Kane et al. 2004). Mice were kept under a 12 h light–dark cycle and allowed free access to tap water and standard chow.

Transverse aortic constriction

Isoflurane-anaesthetized (2–3%), self-ventilating, 8- to 12-week-old, male, C57BL/6 wild-type and Kir6.2-KO mice underwent aortic constriction at the level of the thoracic aorta, between the origin of the right innominate and left common carotid arteries (Fig. 1A). Heart and respiration rates, as well as animal reflexes were monitored throughout. The aortic constriction was imposed using a 27-gauge needle to standardize the extent of stenosis (Fig. 1B; Rockman et al. 1991). A subgroup of wild-type and Kir6.2-KO mice underwent sham surgery consisting of aortic exposure without ligation. All mice were followed for up to 3 weeks.

Figure 1.

Transverse aortic constriction
A, aortic constriction (x) was secured by placement of a suture (s) at the level of the thoracic aorta (Ao), between the origin of the right innominate (ria; brachiocephalic) and left common carotid (Icca) arteries. LV, left ventricle. B, transverse aortic constriction (TAC) was imposed using a 27-gauge needle to standardize the extent of aortic stenosis compared to preconstriction (Pre).

Treadmill exercise stress test and glycaemic levels

Mice were evaluated on a two-track treadmill fitted with a shock grid which delivered a mild electrical stimulus to encourage running (Columbus Instruments, Columbus, OH, USA), as previously described (Zingman et al. 2002a). The exercise-stress protocol consisted of stepwise increases in either incline or velocity at 3 min intervals. Workload (J), a composite parameter incorporating time, speed and incline, was calculated as the sum of kinetic (Ek=mv2/2) and potential (Ep=mgvtsinθ) energy, where m represents animal mass, v treadmill velocity, g acceleration due to gravity, t elapsed time at a protocol level, and θ angle of incline (Zingman et al. 2002a). Separately, blood glucose levels were measured by tail sampling (OneTouch Ultra, Lifescan, Milpitas, CA, USA) after a 16 h overnight fast (‘fasting’) as previously described (Kane et al. 2004).

Echocardiography

Serial transthoracic echocardiography was performed prior to and at predetermined time points after transverse aortic constriction under light sedation (1.5% isoflurane). Images were digitally acquired and stored for off-line blinded analysis. Measurements of left ventricular (LV) dimensions were recorded at end-diastole (LVDd) and end-systole (LVDs) from three consecutive cardiac cycles using the leading edge convention of the American Society of Echocardiography (Kane et al. 2004). Left ventricular fractional shortening (%FS) was defined as percentage FS =[(LVDd − LVDs)/LVDd]× 100. Ejection time (Et) was determined from the actual pulsed-wave Doppler tracings on the parasternal long-axis view of transaortic flow by measuring the interval from the beginning of the acceleration to the end of the deceleration. Myocardial velocity of left ventricular circumferential shortening (vcf expressed in circumferences per second) was calculated as vcf=[(LVDd − LVDs)/LVDd]×Et (Kane et al. 2006b).

Magnetic resonance imaging

Magnetic resonance imaging with a 7 T scanner (Bruker, Billerica, MA, USA) was performed on 2% isofluorane-anaesthetized mice with an electro-cardiogram-triggered fast-gradient echo cine sequence, through a short-axis slice of 1.0-mm thickness at the midpapillary muscle level (Zingman et al. 2002a). The cardio-thoracic ratio (CTR) was measured in the transverse plane.

Left ventricular catheterization

In vivo haemodynamics were recorded, in 2% isofluorane-anaesthetized mechanically ventilated mice, directly by a 1.4-Fr micropressure catheter (SPR-671, Millar Instruments, Houston, TX, USA; Zingman et al. 2002a; Kane et al. 2006a) following carotid arterial cannulation and advancement across the aortic valve, before or after transverse aortic constriction. Transaortic gradient was defined as the difference between peak systemic pressure and the peak left ventricular pressure.

Telemetry and electrocardiography

Heart rate and rhythm were measured in the conscious state at rest and following aortic constriction with implantable telemetry devices (Data Sciences International, St Paul, MN, USA). Electrocardiogram signals were acquired at 2 kHz (Zingman et al. 2002a; Kane et al. 2006b).

Epicardial electrophysiology

In vivo electrophysiological measurements were obtained by a stably placed epicardial probe (EP Technologies, San Jose, CA, USA) to record monophasic action potentials (MAP) in 2% isofluorane-anaesthetized, mechanically ventilated mice before or after transverse aortic constriction. Animals were right ventricularly paced to maintain the heart rate of 500 bpm (catheter; NuMed, Hopkinton, NY, USA, stimulator; A310 Accupulser, World Precision Instruments, Sarasota, FL, USA). Action potential duration at 90% repolarization (APD90) was compared between groups.

Calcium imaging

Thirty minutes after transverse aortic constriction, cardiectomy was performed under 5% isoflurane terminal anaesthesia and hearts in a subgroup of wild-type and Kir6.2-KO animals rapidly excised for isolation of cardiomyocytes (Hodgson et al. 2003; Liu et al. 2004). Rod-shaped single cardiomyocytes were loaded with the calcium-fluorescent probe Fluo-4-acetoxymethyl ester (2 μm; Molecular Probes), and scanned using the 488 nm line of an argon/krypton laser in an oxygenated chamber at 36 ± 1°C. Two-dimensional confocal images (Zeiss LSM 510 Axiovert, Thornwood, NY, USA) were deconvoluted, and analysed using Metamorph (Visitron Universal Imaging, Downingtown, PA, USA) normalized to the degree of background fluorescence (O'Cochlain et al. 2004; Hodgson et al. 2004).

Metabolic probing

Whole heart ATP levels were determined in 0.6 m perchloric acid–1 mm EDTA extracts from liquid N2 freeze-clamped myocardium. Extracts were neutralized with 2 m K2HCO3, and adenine nucleotides eluted with a linear gradient of triethylammonium bicarbonate buffer and profiled by high-performance liquid chromatography (HP1100, Hewlett-Packard, Ajax, Ontario, Canada) using a Mono Q HR5/5 column (Amersham Pharmacia Biotech Inc, Piscataway, NJ, USA) as described (Perez-Terzic et al. 2001; Dzeja et al. 2002).

Pathology and immunochemistry

The whole heart, left ventricle, liver and lungs were removed under 5% isoflurane terminal anaesthesia, rinsed, blotted dry, and weighed. Lung and liver samples were dried at 65°C for 48 h, and reweighed. Interstitial fibrosis was quantified by computer analysis (MetaMorph, Visitron Universal Imaging, Downingtown, PA, USA) of 0.5 μm thick, paraffin-embedded, Masson's trichrome-stained sections as described (Kane et al. 2006a). To probe the expression of the pro-hypertrophic cardiac transcription factor Myocyte Enhancing Factor 2 (MEF2), isolated cardiomyocytes were fixed in 3% paraformaldehyde, and primary antibodies to the cardiac sarcomeric protein α-actinin (mouse polyclonal, 1 : 500; Sigma, St Louis, MO, USA) and MEF2 (rabbit polyclonal, 1 : 300; Cell Signalling Technologies, Danvers, MA, USA; Behfar et al. 2005) applied at 4°C overnight. Respective secondary antibodies (Molecular Probes) were incubated with the sample for 60 min, along with nuclear counter-staining achieved by a 3 min application of 300 nm 4′,6′-diamidino-2-phenylindole hydrochloride (DAPI; Molecular Probes). Images were acquired by laser confocal microscopy (Zeiss LSM 510 Axiovert, Thornwood, NY, USA) as described (Behfar & Terzic, 2006). For downstream immunoblotting analysis, left ventricular tissue was homogenized and the lysate assessed for protein content using a DC Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). A final amount of 60 μg of total protein was loaded onto a 10% SDS-PAGE gel and subsequently transferred to nitrocellulose. To investigate levels of the stress-activated kinase, p38, the membrane was incubated with anti-p38 primary antibodies (rabbit polyclonal, 1 : 1000; Cell Signalling Technologies, Danvers, MA, USA) overnight at 4°C after blocking with 5% skim milk. Goat anti-rabbit secondary antibodies (Chemicon International Inc., Temecula, CA, USA) were applied the next day for 60 min and then developed using the Pierce SuperSignal chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA). Bands were visualized using the UVP imager (UVP Inc, Upland, CA, USA) and analysis performed using NIH ImageJ software (http://rsb.info.nih.gov/ij/).

Electrophoretic mobility shift assay

To determine the DNA binding activity of the nuclear factor of activated T cells (NF-AT), nuclear extracts were prepared by hypotonic lysis of left ventricles followed by enrichment using density centrifugation. Non-specific binding was prevented by addition of the mild detergent sodium deoxycholate, which was added to the purified nuclear extract to a final concentration of 0.4%, incubated on ice 1 h, and clarified by centrifugation through 1 m sucrose at 200 000 g for 1 h at 4°C. A labelled DNA element from the B-type natriuretic peptide promoter served as a probe for electrophoretic mobility shift assays performed as described (Kane et al. 2006a). Specificity of binding was assessed by competition with an excess of unlabelled probe, further validated by two-point mutations of the NF-AT binding region.

Statistical analysis

Data are presented as the mean ±s.e.m. Comparison of parameters was performed using Student's t test. Kaplan-Meier analysis with log-rank testing was employed for survival analysis. A P-value < 0.05 was considered significant.

Results

KATP channel deletion disrupts electrical and ionic homeostasis under acute pressure overload predisposing to myocardial dysfunction

Transverse aortic constriction (Fig. 1A and B), an established in vivo model of haemodynamic overload (Rockman et al. 1991), induced significant and equivalent left ventricle pressure load on both wild-type and KATP channel knockout (Kir6.2-KO) hearts (Fig. 2A and B). Following transverse aortic constriction, the transaortic pressure gradient acutely increased from 6 ± 6 to 59 ± 6 mmHg in wild-type (n= 5), and from 8 ± 5 to 64 ± 10 mmHg in Kir6.2-KO (n= 5; Fig. 2B), in the absence of electrocardiographic evidence of myocardial injury (not illustrated). Despite this equivalent haemodynamic stress (P > 0.05, wild-type versus Kir6.2-KO), there was a divergent electrophysiological response between wild-type and KATP channel knockout mice within 5 min of transverse aortic constriction (Fig. 2C). While monophasic action potential duration, measured at 90% repolarization (APD90), shortened in the wild-type by −4.8 ± 1.8% (n= 6, P= 0.03), it was prolonged in Kir6.2-KO by +34.2 ± 11.8% (n= 8, P= 0.004; Fig. 2D). The aberrant prolongation in repolarization seen in the Kir6.2-KO was also observed in wild-type mice pretreated with the KATP channel inhibitor glyburide (20 μg g−1, oral, n= 5; Fig. 2D). Treatment with the calcium channel antagonist verapamil (5 μg g−1, i.p.), prior to transverse aortic constriction, prevented the action potential prolongation in the Kir6.2-KO (n= 7; Fig. 2D), indicating that in the absence of functional KATP channels prolongation of action potential is mediated by exaggerated calcium influx. The reduced repolarization reserve in KATP channel knockout mice precipitated myocyte calcium overload, as evidenced by the elevated intensity of the calcium-sensitive fluorescent probe Fluo-4 in cardiomyocytes acutely isolated from constricted Kir6.2-KO versus equivalently stressed wild-type hearts, i.e. 185 ± 17 AU (n= 8) versus 59 ± 9 AU (n= 6), respectively (P < 0.001; Fig. 2E).

Figure 2.

KATP channel knockout demonstrated disrupted electrical, ionic and metabolic balance precipitating myocardial dysfunction
A, representative serial tracings from wild-type (WT) and KATP channel knockout (KO) illustrate acute elevation of left ventricular pressure upon transverse aortic constriction (TAC) under direct recording compared to preconstriction (Pre). Vertical bar corresponds to 25 mmHg. B, transaortic gradient measured by aortic and left ventricular catheterization demonstrated similar values in WT and KO, under both Pre and TAC conditions. C and D, divergent response to TAC in action potential duration, measured at 90% repolarization (APD90), with shortening in WT and prolongation in KO. Glyburide (Gly; 20 μg g−1, oral), a KATP channel inhibitor, replicated the KO phenotype when administered to WT. Treatment of KO with the calcium channel antagonist, verapamil (Ver; 5 μg g−1, i.p.), prior to TAC prevented abnormal action potential prolongation. E, evidence of calcium overload indicated by increased fluorescence intensity in KO cardiomyocytes, but not WT, loaded with the calcium-sensitive probe Fluo-4. F, myocardial energetic deficit in KO versus WT, measured by ATP levels in extracted hearts post-TAC. G and H, left ventricular pressure (LVP) recordings demonstrate myocardial dysfunction with elevation in left ventricular end-diastolic pressure (LVEDP) seen in KO, but not WT or KO pretreated with verapamil. In B, D and H, †P < 0.05 TAC versus pre; in D, E, F and H, *P < 0.05 KO TAC versus WT TAC.

Deficits in repolarization response and calcium handling were associated with energetic and haemodynamic consequences in Kir6.2-KO mice. Within 30 min of transverse aortic constriction, Kir6.2-KO had evidence of energetic compromise with significantly lower myocardial ATP concentrations compared to constricted wild-type counterparts, i.e. 17.0 ± 0.6 nmol (mg protein)−1 (n= 7) versus 23.0 ± 1.6 nmol (mg protein)−1 (n= 7), respectively (P= 0.003; Fig. 2F). Energetically impaired Kir6.2-KO displayed evidence of diastolic dysfunction with significantly elevated left ventricular end-diastolic pressures (18.9 ± 4.4 mmHg, n= 5) compared to similarily constricted wild-type mice (3.2 ± 1.2 mmHg, n= 5, P= 0.02; Fig. 2G and H). As with the electrophysiological response, elevations in the end-diastolic pressure in constricted Kir6.2-KO could be prevented with verapamil (2.7 ± 3.3 mmHg, n= 5, P= 0.03; Fig. 2H) to levels similar to those in constricted wild-type (P > 0.05; Fig. 2H), demonstrating the requirement of intact KATP channels in preserving electrical and ionic homeostasis, and thereby safeguarding myocardial performance under imposed pressure overload.

Absence of KATP channels induces fulminant heart failure and high mortality under pressure overload

These early signs of myocardial dysfunction rapidly progressed to overt heart failure (Fig. 3). Within hours of aortic constriction, Kir6.2-KO mice displayed typical physical signs of the biventricular congestive heart failure syndrome, including severely reduced cardiac function (Fig. 3BD), hepatopulmonary congestion (Fig. 3EH) and ascites, along with systemic fluid retention and anasarca (Fig. 3A and G). Transthoracic echocardiograms within 24 h of aortic constriction demonstrated severely impaired ventricular systolic function in Kir6.2-KO, but not wild-type mice, as indicated by reduced fractional shortening, i.e. 35 ± 4% (n= 6) versus 49 ± 2% (n= 4), respectively (P= 0.03; Fig. 3B and C), as well as reduced circumferential shortening velocity, a preload independent measure of ventricular function (wild-type: 8.5 ± 0.7 circumferences s−1, n= 4; Kir6.2-KO: 5.7 ± 0.9 circumferences s−1, n= 6, P= 0.04; Fig. 3D).

Figure 3.

Pressure overload induces fulminant heart failure in KATP channel knockout
Within 1 day of transverse aortic constriction (TAC), unlike the wild-type (WT), Kir6.2-knockout (KO) mice were inactive and oedematous (A). Echocardiograms within 24 h of TAC showed left ventricular systolic dysfunction in KO, but not WT mice (B). Arrows indicate diastolic (dotted) and systolic (continuous) dimensions. C and D, echocardiographic measures of systolic dysfunction were abnormal in KO versus WT only after TAC. E, evidence in KO, but not WT, following TAC for left ventricular failure with pulmonary congestion (E and F) and right ventricular failure with fluid accumulation in the extravascular space (G) and hepatic congestion (H). H, heart; L, lung; BW, body weight; Flu, subcutaneous fluid (arrows in G). In C, D, F and H, †P < 0.05 TAC versus pre; *P < 0.05 KO TAC versus WT TAC.

Consistent with the rapid decline in cardiac function leading to fulminant heart failure, telemetry readings detected progressive sinus bradycardia followed by gradual prolongation of atrio-ventricular conduction, ultimately developing into atrio-ventricular conduction block preceding death in the Kir6.2-KO (Fig. 4A). Consistent with KATP channel knockout mice dying of progressive pump failure, no episodes of ventricular tachyarrhythmia were detected (Fig. 4A). Half of the Kir6.2-KO (n= 20 out of 40) died within 48 h of aortic constriction, compared to no mortality in similarly challenged wild-type mice (n= 31), sham-operated wild-type (n= 16) or KATP channel knockout (n= 15) controls (P < 0.001; Fig. 4B). Thus, following the imposition of pressure loading on the myocardium, the absence of functional KATP channels results in the rapid development of severe congestive heart failure and massive death, not experienced in equivalently challenged wild-type mice.

Figure 4.

Absence of KATP channels imparts high mortality under pressure overload
A, compatible with the failing heart phenotype, Kir6.2-knockout (KO) mice, but not wild-type (WT), displayed progressive decline in heart function, i.e. bradycardia, following transverse aortic constriction (TAC) detected by telemetry. Scale bar: 200 ms. B, within 48 h, TAC reduced the survival by half in the KO cohort. No mortality was seen in either WT or KO undergoing sham surgery or in WT after TAC. Number in parentheses indicates respective numbers of animals in each group.

Absence of KATP channels accelerates and aggravates cardiac remodelling under chronic pressure overload

When followed for a total of 3 weeks after aortic constriction, 74% (n= 23/31) of wild-type survived in contrast to only 25% (n= 10/40) of Kir6.2-KO mice (P= 0.009). When evaluating the survivors, Kir6.2-KO mice (n= 7) had extreme exercise intolerance compared to the wild-type (n= 6), i.e. 72 ± 16%versus 15 ± 9% decline in exercise capacity (P= 0.02; Fig. 5A) in the absence of a difference in serum glucose levels, i.e. 105 ± 3 mg dl−1versus 86 ± 27 mg dl−1 in wild-type (n= 14) and Kir6.2-KO (n= 8), respectively (P > 0.05). Pressure loaded hearts in Kir6.2-KO mice had demonstrable exaggerated levels of cardiac remodelling in comparison to wild-type (Fig. 5BE). Within 7–10 days following aortic constriction, transthoracic echocardiograms showed significant increase in left ventricular wall thickness, measured as the sum of interventricular septal thickness and posterior wall thickness, in constricted Kir6.2-KO (n= 6) when compared to constricted wild-type (n= 5) mice (P= 0.02; Fig. 5B), suggesting an increased propensity towards cardiac hypertrophy. Indeed, left ventricular dimensions were significantly increased, within 3 weeks postconstriction, in Kir6.2-KO (n= 6) compared to wild-type (n= 8) mice (P= 0.047; Fig. 5C). This was confirmed ex vivo (Fig. 5D), with a 31 ± 8% increase in left ventricular mass measured in Kir6.2-KO mice at 7–10 days postaortic constriction (P= 0.01, n= 8), in contrast to a non-significant change observed in constricted wild-type hearts at the equivalent time point (10 ± 5%, n= 14, P > 0.05; Fig. 5E).

Figure 5.

Exaggerated remodelling response in KATP channel knockout
Following transverse aortic constriction (TAC), and compared with wild-type (WT), surviving Kir6.2-knockout (KO) mice had an impaired exercise capacity (A), an exaggerated left ventricular (LV) wall thickness (B), and chamber dilatation (C) detected by echocardiography. IVST, interventricular septal thickness; PWT, left ventricular posterior wall thickness; LVDd, left ventricular end-diastolic dimension. On autopsy, KO hearts were larger (D) and heavier (E) following TAC. LV, left ventricular mass; BW, body weight. In B, C and E, †P < 0.05 TAC versus pre; in A, B, C and E, *P < 0.05 KO TAC versus WT TAC.

This exaggerated hypertrophic response was associated with an increase in calcium-dependent pro-hypertrophic signalling in isolated cardiomyocytes, indicated by a significantly augmented expression of the transcription factor MEF2 in constricted Kir6.2-KO (195 ± 6 AU, n= 40 cardiomyocytes) compared to wild-type nuclei (135 ± 8 AU, n= 60 cardiomyocytes, P < 0.001; Fig. 6A and B). Additionally, comparison of nuclear extracts demonstrated in the Kir6.2-KO up-regulated calcium-dependent translocation of NF-AT, complexing with high specificity to the B-type natriuretic peptide gene promoter element (Fig. 6C), a marker of pathological cardiac remodelling (Molkentin et al. 1998; Crabtree & Olson, 2002). The p38 stress-activated cascade of cardiac hypertrophy was indistinguishably affected by pressure overload in the presence or absence of KATP channels (n= 3 in each group; P > 0.05). Thus, the excessive myocardial hypertrophy seen in the constricted Kir6.2-KO over that in the respective wild-type is associated with a differential up-regulation of canonical calcium-dependent hypertrophic pathways (Molkentin, 2004). The propensity for myocardial maladaptation was further underlined by an increased degree of interstitial fibrosis in constricted Kir6.2-KO compared to wild-type hearts (P= 0.005; Fig. 6D and E).

Figure 6.

Nuclear up-regulation of calcium-dependent pro-remodelling transcription factors in pressure-overloaded hypertrophic and fibrotic KATP channel knockout hearts
A and B, following transverse aortic constriction (TAC), nuclear translocation of the calcium-dependent pro-remodelling factor MEF2 was revealed using a MEF2C antibody in Kir6.2 knockout (KO), but not wild-type (WT) cardiomyocytes counterstained with the sarcomeric protein α-actinin antibody and the nuclear probe DAPI. C, electrophoretic mobility shift assay demonstrates KATP channel-dependent pressure overload-induced increase in specific binding of NF-AT to the B-type natriuretic peptide promoter element. Left ventricle nuclear extracts plus the radiolabelled DNA probe were loaded as follows: wild-type (WT) prior to (Pre, lane 1) and 48 h following TAC (lane 2); Kir6.2-KO (KO) Pre (lane 3) and 48 h following TAC (lane 4); KO 48 h following TAC in the presence of 50-fold excess of unlabelled DNA probe (lane 5) or unlabelled mutated DNA probe (lane 6). Note the up-regulated NF-AT complex formation with the promoter element (arrows) in TAC KO (lane 4) compared to Pre KO (lane 3) or WT counterparts (lane 1 and 2). The specificity of NF-AT binding to the DNA probe was validated by competition with an excess of cold probe used as control (C; lane 5). Point mutations (M) in the NF-AT binding region of the unlabelled probe prevented competition with the labelled consensus sequence (lane 6). The presented electrophoretic mobility shift assay is representative of a total of 6 wild-type and 6 Kir6.2-KO. D and E, the degree of fibrosis developing post-TAC is greater in KO (n= 5) than WT (n= 7) as indicated by blue staining on Masson trichrome exposed myocardial sections. †P < 0.05 TAC versus pre in E; *P < 0.05 KO TAC versus WT TAC in B and E.

Serial assessment of left ventricular structure indicated a difference not only in the extent, but also in the rate of the remodelling response between pressure overloaded Kir6.2-KO and wild-type mice (Fig. 7). Constricted Kir6.2-KO survivors showed a significant increase of left ventricular mass at both 2 and 7 days determined by serial echocardiograms, while the wild-type required 3 weeks to significantly increase left ventricular weight under equivalent pressure overload (Fig. 7A). At each time point measured, constricted Kir6.2-KO mice had a significantly greater degree of change of left ventricular mass (Fig. 7A). Serial echocardiography in Kir6.2-KO mice demonstrated progressive and accelerated left ventricular wall thickening compared to wild-type (Fig. 7B and C). At 3 weeks postaortic constriction Kir6.2-KO, but not wild-type mice, demonstrated chamber dilatation in addition to thickened walls, suggesting a transition to a decompensated remodelling response. Magnetic resonance imaging independently confirmed cardiomegaly, as defined by the cardio-thoracic ratio, in pressure overloaded Kir6.2-KO compared to wild-type (Fig. 7D). Thus, lack of KATP channels, even in animals that survive the acute phase of pressure overload, produces long-term physical intolerance and myocardial maladaptation.

Figure 7.

Accelerated hypertrophy and dilatation under chronic pressure overload in KATP channel knockout
A, following transverse aortic constriction (TAC), Kir6.2-knockout (KO) hearts showed premature and excessive increase of left ventricular (LV) mass compared to wild-type (WT). †P < 0.05 TAC versus pre; *P < 0.05 KO TAC versus WT TAC. Compared with WT (B), serial echocardiogram demonstrated accelerated left ventricular wall thickening in KO with development of chamber dilatation in KO (C). IVST, interventricular septal thickness; LVDd, left ventricular end-diastolic dimension; PWT, left ventricular posterior wall thickness. D, cardiomegaly defined as increased cardio-thoracic ratio (CTR), was demonstrable by magnetic resonance 3 weeks following TAC in KO but not WT. RV, right ventricle. Continuous and dotted arrows in D indicate cardiac (RV plus LV) and thoracic dimensions, respectively.

Discussion

Heart failure is a leading cause of morbidity and mortality, with haemodynamic loading of the myocardium a recognized risk factor in the pathogenesis of disease (Towbin & Bowles, 2002; Thom et al. 2006). A paradigm of cardiac haemodynamic insult that commonly underlies the syndrome of heart failure is a state of pressure overload imposed upon the left ventricle (Chien, 1999). Yet, the genetic determinants that dictate whether a heart will transition from a state of risk load to one of overt disease remain partially understood (Ahmad et al. 2005). Identifying molecular components vital for myocardial adaptation would advance the understanding of cardioprotective pathways in the haemodynamically challenged heart.

Here, we report that under imposed pressure overload cardiac KATP channels are required to maintain electrical, ionic and metabolic balance, preventing myocardial dysfunction and development of organ failure. Genetic ablation of the KCNJ11-encoded Kir6.2 KATP channel pore, in the setting of transverse aortic constriction, precipitated acutely fulminant congestive heart failure producing a dramatic survival disadvantage, with survivors exhibiting chronically exaggerated cardiac remodelling associated with poor outcome. Thus, the present study, using the Kir6.2 knockout model, unmasks a previously unrecognized protective role for KATP channels in aortic constriction-induced congestive heart failure, establishing in vivo KATP channel deficit as a novel susceptibility mechanism for cardiac disease under conditions creating left ventricular pressure overload.

This is of significance since the left ventricle is exposed to pressure overload in diverse pathological conditions, generating a build-up of biomechanical stress, triggering a neurohumoral surge, and imposing increased energy consumption and metabolic distress upon the myocardium (Hunter & Chien, 1999; Barki-Harrington & Rockman, 2003). As myocardial KATP channels harness mechanosensitive gating and energetic decoding capabilities, they are uniquely positioned to serve as stress-responsive elements providing a high-fidelity feedback mechanism capable of adjusting cellular excitability to match demand and protect the myocardium (Noma, 1983; Van Wagoner, 1993; Zingman et al. 2002a; Alekseev et al. 2005). While the exact mechanism responsible for KATP channel opening under acute pressure overload imposed by transverse aortic constriction has not been identified, channel activation could be the consequence of mechanical stretch (Van Wagoner, 1993; Saegusa et al. 2005) and/or imbalance of supply versus demand (Kane et al. 2006a; Nichols, 2006). Indeed, the findings in this study implicate the cardiac KATP channel as the electrophysiological regulator of ionic balance in the myocardium under haemodynamic stress imposed by sustained aortic constriction. This broadens the homeostatic implications for this K+ conductance beyond ischaemia (Suzuki et al. 2002; Gumina et al. 2003), sympathomimetic challenge (Zingman et al. 2002a; Liu et al. 2004), physical exertion (Kane et al. 2004) or mineralocorticoid-induced hypertension (Kane et al. 2006a), recently linked to myocardial KATP channel-mediated protection in the Kir6.2 knockout model.

Through safeguarding against cardiomyocellular calcium overload, the KATP channel allowed necessary left ventricular function required to combat imposed challenge of aortic constriction, while maintaining energetic stability. KATP channels are recognized regulators of cardiac repolarization reserve under stress (Liu et al. 2004; Tong et al. 2006), and are integral to energetic circuits and myocardial well-being (Dzeja & Terzic, 1998; Gumina et al. 2003; Zingman et al. 2003). Loss of this stress-monitor rapidly unbalances cellular homeostasis resulting in failure of primary heart function. Specifically, within 30 min of pressure overload, hearts lacking KATP channels lost action potential control of calcium influx, creating a susceptibility to a cascade of contractile dysfunction, heart failure and death. The observed sinus bradycardia is consistent with the failing heart phenotype and the generalized deterioration of myocardial function (Kane et al. 2006a), and has been reported to be common in advanced heart failure (Luu et al. 1989). By 48 h of aortic constriction, half of the Kir6.2 knockout cohort had succumbed, and upon 3 weeks of follow-up even the hearts of those knockout mice who survived were driven to an exaggerated pathological remodelling with compromised organ function. This dramatic short- and long-term vulnerability, under conditions of imposed ventricular load, underscores the requirement for KATP channel checkpoints in preventing calcium accumulation and associated deleterious events. Calcium overload predisposes to malignant calcium-triggered gene reprogramming and structural remodelling precipitating pump failure (Wehrens et al. 2005; Molkentin, 2006). As shown here, distal from defective KATP channels, accumulation of calcium triggered an up-regulation of the calcium-dependent transcription factor MEF2. Identified within the nucleus of cardiomyocytes from constricted Kir6.2 knockout, but not wild-type hearts, MEF2 induction has been associated with activation of pro-remodelling and maladaptive myocardial pathways underlying the development of pathological cardiac hypertrophy and failure (Frey & Olson, 2003). Moreover, KATP channel knockouts are prone under chronic stress to up-regulate expression of the serine/threonine protein phosphatase calcineurin, a key calcium-dependent determinant of pathological cardiac remodelling (Kane et al. 2006a). Upon calcium-dependent activation, calcineurin dephosphorylates NF-AT facilitating its import into the nucleus to mediate pro-remodelling gene activation (Crabtree & Olson, 2002). Indeed, compared to constricted wild-type, Kir6.2-KO counterparts exhibited increased complex formation of NF-AT with DNA encoding the B-type natriuretic peptide promoter region, a prototypic step in the remodelling process (Molkentin et al. 1998). Altogether, these findings provide a mechanistic basis for a causal relationship linking KATP channel deficit with induction of maladaptive signalling pathways leading to development of heart failure under pressure overload.

The present findings are further underscored by the identification in humans that defective KATP channels, induced by mutations in ABCC9 encoding the regulatory SUR2A subunit, confer susceptibility to dilated cardiomyopathy (Bienengraeber et al. 2004). The ultimate phenotype in the Kir6.2 knockout, under pressure overload, is development of heart failure with cardiac chamber dilatation, a phenotype similar to that observed in patients with mutations in KATP channel genes. That gene knockout of the KATP channel compromises cardioprotection precipitating heart failure with fatal outcome could further suggest that treatment with KATP channel blocking agents, such as the sulphonylureas, may also compromise myocardial tolerance to injury. In specific cohorts of patients, sulphonylurea use has been associated with potential deleterious outcomes, including increased risk of early mortality (Brady & Terzic, 1998; Garratt et al. 1999) or increase in left ventricular mass, a risk factor for morbidity and mortality (Lee et al. 2006). Typical for the progression of heart failure is an extensive ventricular remodelling initially comprising cardiomyocyte hypertrophy and fibrosis, resulting in increase in ventricular mass (Chien, 1999), as seen in Kir6.2-knockout mice following aortic constriction. This remodelling process is generally recognized to underlie the pathogenesis of the heart failure syndrome of various aetiologies progressing to decompensated, dilated, cardiomyopathy (Towbin & Bowles, 2002; Ahmad et al. 2005). In view of reported genetic polymorphisms in KCNJ11 encoding Kir6.2 (Riedel et al. 2005), the present demonstration that KATP channels protect against the development of congestive heart failure and death, securing both acute and chronic cardiac adaptation to imposed haemodynamic load, provides a foundation for further investigation of the role of these cardioprotective channels in the population at large.

Appendix

Acknowledgements

The authors wish to thank Jonathan Nesbitt and Nadezhda Alekseyeva for excellent technical assistance, Drs Prasanna Mishra and Slobodan Macura at the Mayo Clinic Analytical Nuclear Magnetic Resonance Facility for magnetic resonance imaging, and the Translational Ultrasound Research Core for use of the echocardiographic machine. This work was supported by grants from the National Institutes of Health, Marriott Heart Disease Research Program, Marriott Foundation, Ted Nash Long Life Foundation, Ralph Wilson Medical Research Foundation, Mayo Clinic Clinician-Investigator Program, and Japanese Ministry of Education, Science, Sports, Culture and Technology. S.Y. is the recipient of the American Heart Association Postdoctoral Fellowship (AHA no. 05-25784Z), and has received support from the Japan Magnetic Health Science Foundation, Japan Heart Foundation and Medtronic Japan. A.T. holds the Mayo Clinic Marriott Family Professorship in Cardiovascular Research.

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