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Human Molecular Genetics Advance Access originally published online on June 16, 2006
Human Molecular Genetics 2006 15(15):2285-2297; doi:10.1093/hmg/ddl154
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© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

KCNJ11 gene knockout of the Kir6.2 KATP channel causes maladaptive remodeling and heart failure in hypertension

Garvan C. Kane1,2, Atta Behfar1,2, Roy B. Dyer1,2, D. Fearghas O'Cochlain1,2, Xiao-Ke Liu1,2, Denice M. Hodgson1,2, Santiago Reyes1,2, Takashi Miki3, Susumu Seino3 and Andre Terzic1,2,*

1 Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Department of Medicine, 2 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA and 3 Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan

* To whom correspondence should be addressed at:, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA. Tel: +1 5072847517; Fax: +1 5072849111; Email: terzic.andre{at}mayo.edu

Received April 11, 2006; Accepted June 13, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Heart failure is a growing epidemic, with systemic hypertension a major risk factor for development of disease. However, the molecular determinants that prevent the transition from a state of hypertensive load to that of overt cardiac failure remain largely unknown. Here in experimental hypertension, knockout of the KCNJ11 gene, encoding the Kir6.2 pore-forming subunit of the sarcolemmal ATP-sensitive potassium (KATP) channel, predisposed to heart failure and death. Defective decoding of hypertension-induced metabolic distress signals in the KATP channel knockout set in motion pathological calcium overload and aggravated cardiac remodeling through a calcium/calcineurin-dependent cyclosporine-sensitive pathway. Rescue of the failing KATP knockout phenotype was achieved by alternative control of myocardial calcium influx, bypassing uncoupled metabolic-electrical integration. The intact KCNJ11-encoded KATP channel is thus a required safety element preventing hypertension-induced heart failure, with channel dysfunction a molecular substrate for stress-associated channelopathy in cardiovascular disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Heart failure has been associated with conditions of pathological stress that compromise myocardial homeostasis (1–3). The nature of risk factors, exemplified by hypertension, is increasingly understood, but the genetic make-up that determines which hearts will transition from a state of risk to that of disease remains largely unknown (4–6). The identity of mechanisms that protect against organ failure is ill defined, warranting elucidation of molecular processes by which signals of distress are read and transduced into adaptive responses.

The syndrome of heart failure is characterized by increased metabolic demand mandating maintenance of ionic equilibrium required to safeguard cardiac performance (7–9). Action potential-controlled calcium entry is a prerequisite for excitation–contraction coupling, whereas mishandled calcium is a major elicitor of maladaptive myocardial remodeling precipitating contractile dysfunction and decompensation (10–14). Molecular effectors of calcium overload and determinants of calcium dynamics or myofilament responsiveness are increasingly identified (15–18), but checkpoints that synchronize changes in metabolic state with cellular ionic balance remain only partially understood.

ATP-sensitive K+ (KATP) channels are membrane sensors of energy metabolism that, by virtue of specialized nucleotide responsiveness, supply a feedback mechanism capable of adjusting cell excitability to match demand (19–22). Expressed at high density in cardiac sarcolemma, KATP channels are formed by multimerization of the Kir6.2 pore-forming subunit and the regulatory sulfonylurea receptor, an ATPase-harboring ATP-binding cassette protein that confers energetic decoding abilities to the channel complex (23–28). Energetic signals of distress, received via integration with metabolic pathways, are processed by the regulatory module to gate the nucleotide responsiveness of the KATP channel pore-controlling cardiac action potential duration and associated cellular functions (29–32). The disruption of metabolic signaling or KATP channel malfunction predisposes to stress intolerance causing deficits in cardiac contractile and electrical activity (33–38). The role of KATP channels as mediators of the response to stress is underscored in mice with genetic deletion of Kir6.2 that underperform on exercise stress test, a natural trigger of the general adaptation syndrome (33), and acquire cardiac deficits under imposed chronic exertion (37), despite an apparent absence of phenotypic differences in the unchallenged state (22). The contribution of cardiac KATP channels is demonstrated by failure of Kir6.2 knockout hearts to shorten action potentials or to sustain augmented contractile performance, suffering calcium overload, injury and increased susceptibility to fatal arrhythmia under stress (33,38), with reduced tolerance to ischemic challenge and aberrant response on the electrocardiogram (34). Underscoring the potential significance of KATP channels for cardiac protection, mutations in the cardiac channel isoform that produce abnormal channel phenotypes with compromised metabolic signal decoding have been demonstrated in patients with inherited cardiomyopathy (39). However, a causal relationship between KATP channel dysfunction and the development of heart failure remains untested (40,41).

This study, using knockout of sarcolemmal KATP channels in the setting of systemic hypertension, establishes that the pathogenesis of the heart failure syndrome is precipitated by disruption of cardiac KATP channel activity. Ablation of the KCNJ11-encoded KATP channel pore unmasked a severe vulnerability of the hypertensive heart, inducing excessive calcium/calcineurin-dependent remodeling and congestive cardiac failure with death. These findings identify genetic deficit in the KATP channel as a molecular substrate for the development of heart failure in hypertension.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Survival disadvantage in KATP channel knockout with hypertension
Systemic hypertension is a common condition of stress faced by the heart (42). Here, hypertension was imposed upon C57BL/6 wild-type and age-matched KATP channel-deficient mice (Kir6.2-KO) generated by targeted disruption of the KCNJ11 gene that encodes the pore-forming Kir6.2 channel subunit. Chronic hyperaldosteronism, the leading secondary cause of hypertension (43), was replicated by unilateral nephrectomy and mineralocorticoid salt challenge through a sustained subcutaneous deoxycorticosterone acetate release with oral salt loading. This 21 day-long hemodynamic stress regimen produced a severe but comparable level of systemic hypertension in wild-type and Kir6.2-KO mice (shown at 2 weeks in Fig. 1A). On average, this regimen produced an increase in systolic blood pressure of 25.5±3.5 (n=16) and 24.6±5 mmHg (n=14), respectively, in wild-type and Kir6.2-KO mice, a degree of hypertension considered to represent severe stress in murine models (44). Despite equivalent loading conditions, as further indicated by similar natriuresis-induced increases in fluid intake (Fig. 1B), renal hypertrophy (Fig. 1C) and kidney collagen deposition (Fig. 1D), hypertensive mice displayed a differential exertional response, depending on the presence or absence of functional KATP channels. Unlike the wild-type, genetic deletion of KATP channels produced discernible impairment in exercise stress capacity, an integrative indicator of physical endurance (Fig. 1E). Furthermore, in comparison to the wild-type, the challenge of systemic hypertension halved survival in the Kir6.2-KO cohort, with significant mortality occurring between day 7 and the end of the 3-week protocol (Fig. 1F). In contrast, over 95% of wild-type exposed to the same hypertensive regimen survived as did all wild-type (n=25) or Kir6.2-KO (n=23) treated with nephrectomy alone, which in the absence of mineralocorticoid/salt loading did not induce hypertension (Fig. 1A). Thus, despite exposure to equivalent chronic hemodynamic stress, lack of Kir6.2-containing KATP channels translates into an impaired tolerated workload and an overall marked survival disadvantage.


Figure 1541
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  		Figure 1. Survival disadvantage of hypertensive Kir6.2-KO mice with mineralocorticoid salt loading. Mineralocorticoid/salt (DOCA/salt) challenge induced significant but comparable systemic hypertension (A), fluid intake (B), renal hypertrophy (C) with kidney collagen deposition (D) in wild-type (WT) and KATP channel knockout (Kir6.2-KO) mice. In response to hypertension, Kir6.2-KO (KO) mice displayed a compromised exercise capacity on treadmill testing (E) and severe mortality (F; P<0.001) when compared with WT. Asterisk indicates significant difference when compared with control (P<0.001). Treadmill exercise capacity expressed as change from mean of unchallenged WT or KO controls.

 
Congestive heart failure in hypertensive KATP channel knockout
In the hypertensive Kir6.2-KO mice, a progressive decline in cardiac activity preceded death (Fig. 2A) with development of atrio-ventricular conduction delay and bradycardia on electrocardiography (Fig. 2A inset), consistent with the phenotype of a failing heart and not observed in the wild-type. Following mineralocorticoid hypertension, even in surviving animals, hearts lacking KATP channels developed significant impairment in contractile function captured in vivo by echocardiography and cardiac catheterization (Fig. 2 and Table 1). Although having comparable heart rates, multiple indicators of myocardial dysfunction were present in Kir6.2-KO but not in wild-type, following mineralocorticoid-induced hypertensive challenge (Fig. 2 and Table 1). In contrast to non-hypertensive Kir6.2-KO or hypertensive or non-hypertensive wild-type, all had similar and normal systolic function (Table 1), hypertensive Kir6.2-KO mice developed left ventricular chamber dilation (Fig. 2B) and reduced trans-aortic flow velocity (Fig. 2C) with profound systolic contractile impairment (Fig. 2D and Table 1). Left ventricular systolic diameter was 2.3±0.1 versus 1.5±0.1 mm in hypertensive Kir6.2-KO (n=8) and wild-type (n=8), respectively (P=0.0008; Fig. 2E). Left ventricular systolic dysfunction in the hypertensive Kir6.2-KO was associated with pulmonary congestion (Fig. 2F), indicating that the cardiac contractile impairment led to left ventricular failure. In contrast to hypertensive wild-type with preserved cardiac function, by 3 weeks, Kir6.2-KO counterparts demonstrated defective cardiac inotropy and lusitropy with diminished peak left ventricular developed pressure (Fig. 2G–I and Table 1) and abnormal prolongation in cardiac relaxation (Fig. 2H–I and Table 1). Under inotropic challenge with dobutamine, hypertensive Kir6.2-KO mice decompensated to fulminant cardiac dysfunction, with rapid elevation of end-diastolic pressure and deterioration of developed pressure, eventually leading to cardiac collapse and death in all (n=8, P<0.001; Fig. 2J). In contrast, hypertensive wild-type displayed a normal contractile response to the same challenge with no death (n=8; Fig. 2J). Thus, hypertensive KATP channel-deficient mice are predisposed to develop congestive heart failure precipitating fatal outcome.


Figure 1542
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  		Figure 2. Hypertension precipitated congestive heart failure in the absence of KATP channels. Hypertensive KATP channel KO mice demonstrated progressive bradycardia (A) with atrio-ventricular conduction delay (A inset), not observed in WT. In hypertensive KO (KO HTN) but not WT (WT HTN), echocardiography captured left ventricular (LV) dysfunction (B; open arrows: LV chamber dimension in diastole and closed arrow: LV chamber dimension in systole), impaired trans-aortic valve velocity (C) and fractional shortening (D), associated with LV dilatation (E) and pulmonary edema (F). On cardiac catheterization, impaired inotropy (G, diminished LV developed LVdev pressure) and lusitropy (H, prolonged cardiac relaxation) were demonstrated in hypertensive KO (I). With dobutamine challenge, KO rapidly decompensated into overt cardiac failure (J). Asterisk indicates significant difference when compared with control (P<0.01). Horizontal scale bars in A inset, B, C, I and J: 200 ms. Vertical scale bars in B: 1 mm and in C: 2 mm/s.

 


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  		Table 1. Cardiac functional assessments of control and hypertensive wild-type and Kir6.2 knockout mice in vivo

 
Aggravated remodeling in hypertensive KATP channel knockout heart
Underlying the pathogenesis of the heart failure syndrome is an extensive ventricular remodeling comprised of cardiomyocyte hypertrophy and fibrosis (8–10). Indeed, in hypertension, the magnitude of left ventricular mass increase is a predictor of long-term prognosis and of the rate of decompensation to heart failure (45–47). Wild-type hearts responded to hypertension with increased overall cardiac (Fig. 3A) and, more specifically, left ventricular size (Fig. 3B) accompanied by interstitial fibrosis (Fig. 3D–E). Despite equivalent hypertensive load, Kir6.2-KO hearts had, on average, more than three times the increase in left ventricular mass observed in the wild-type (Fig. 3A and B). The composite of left and right atrial weights with right ventricular free wall was higher in the hypertensive Kir6.2-KO (n=20) compared with hypertensive wild-type (n=23), i.e. 1.84±0.5 versus 1.52±0.4 mg/g (P<0.05). This excessive remodeling correlated with poor outcome, as the normalized left ventricular mass from the Kir6.2-KO that died (5.6±4.2 mg/g, n=8) was significantly higher than from those that survived within the 21 days of imposed hypertension (5.02±0.13 mg/g, n=15; P<0.02). The mean surface area of individual cardiomyocytes isolated from hypertensive Kir6.2-KO hearts was also greater than those from hypertensive wild-type hearts (Fig. 3C; P<0.03). Further, the extent of extracellular matrix deposition was significantly higher in Kir6.2-KO hearts compared with wild-type (Fig. 3D and E), as was the left ventricular collagen content with a 3-fold higher increase over control seen in the Kir6.2-KO versus wild-type (P<0.001). Thus, underlying contractile dysfunction and exaggerated mortality in hypertensive KATP channel-deficient mice are an excessive maladaptive cardiac remodeling response.


Figure 1543
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  		Figure 3. Exaggerated maladaptive ventricular remodeling in hypertensive KATP channel KO. KATP KO challenged with HTN developed aggravated cardiac (A) and LV (B) remodeling. Hypertension-stressed KO, immunostaining negative for Kir6.2, had larger cardiomyocyte surface area compared with Kir6.2-positive WT (C). Scale bar in C: 20 µm. Masson's trichrome stained hypertensive KO hearts were significantly more fibrotic than WT (D and E). Asterisk indicates significant difference compared with respective control or hypertensive WT (P<0.05).

 
Loss of KATP channel sensing of metabolic distress precipitates calcium overload
Characteristic of metabolic changes in cardiac remodeling (48,49), hypertensive wild-type and Kir6.2-KO hearts demonstrated a greater than 6-fold increase in intracellular nucleotide diphosphate over triphosphate concentrations (Fig. 4A; P<0.005). Such cytosolic energetic compromise in the absence of functional KATP channels resulted in deficient myocardial repolarization characterized by prolonged QTc interval on the electrocardiogram compared with wild-type (Fig. 4B; P=0.003). Inappropriately prolonged repolarization predisposes to excessive cytosolic calcium loading, and in contrast to hearts of hypertensive wild-type (Fig. 4C) or non-hypertensive controls (not illustrated) that maintained steady intracellular calcium levels, lack of KATP channels in hypertension precipitated cytosolic calcium overload (Fig. 4C; P<0.001). Thus, absence of metabolism-sensing KATP channels produces a bioenergetic-electrophysiological uncoupling in hypertensive hearts, leading to abnormal calcium accumulation.


Figure 1544
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  		Figure 4. Aberrant repolarization, excessive calcium loading and activation of remodeling signals in energetically challenged hypertensive KATP channel KO. Altered bioenergetics probed by the DP to TP index in HTN WT and Kir6.2-KO hearts versus respective controls (A; P<0.005). Prolonged repolarization, expressed as corrected QT interval (QTc) on the electrocardiogram in HTN KO compared with WT (B; P=0.003). Calcium loaded cardiomyocytes from hypertensive KO versus WT captured by the fluorescent probe Fluo-4 on laser confocal microscopy (C; P<0.001). Nuclear translocation of the calcium-dependent cardiac transcription factor (MEF2), highlighted in insets, in {alpha}-actinin-stained LV tissue sections (D) was greater in HTN KO versus WT (P<0.001).

 
Knockout of KATP channels discharges uncontrolled calcium–calcineurin-dependent signaling
At the crossroads of multiple calcium-dependent signal pathways in the heart is the transcription factor, myocyte enhancer factor 2 (MEF2), which when activated translocates to the nucleus where it initiates genetic reprogramming (50). Unlike the hypertensive wild-type that displayed limited nuclear translocation of MEF2, hearts lacking Kir6.2-containing KATP channels exhibited nuclei abnormally loaded with MEF2 (Fig. 4D; P<0.001), an indicator of excessive calcium-dependent remodeling signals in the hypertensive Kir6.2-KO hearts. The key calcium-dependent determinant of pathological cardiac hypertrophy, upstream of calcium-dependent transcription factors, is the serine/threonine protein phosphatase calcineurin. Upon activation by the binding of calcium/calmodulin, calcineurin dephosphorylates the nuclear factor of activated T-cells (NF-AT) facilitating nuclear transport where NF-AT further mediates pro-remodeling gene activation (50,51). Calcineurin phosphatase activity measured in cytosolic left ventricular extracts was abnormally upregulated in hypertensive Kir6.2-KO compared with the hypertensive wild-type (Fig. 5A; P<0.03). In the Kir6.2-KO, the degree of calcineurin activity closely correlated (r=0.82, P<0.02) with left ventricular mass where the highest level of calcineurin activity was found in hypertensive hearts with the greater degree of cardiomegaly (Fig. 5B). This association between cardiac mass and calcineurin activity was not evident in the wild-type (r=0.22, P=0.3). Only hypertensive Kir6.2-KO, but not wild-type hearts (in which only limited increases were noted), demonstrated nuclear localization of NF-AT on immunostaining indicative of marked activation and cytosolic-nuclear shuttling of NF-AT (Fig. 5C). This was confirmed on western blot analysis of myocardial nuclear extracts in which there was a 7-fold increase in NF-AT from hypertensive Kir6.2-KO over that from control (Fig. 5D and 5E). Activated NF-AT that has been transported to the nucleus directly mediates genetic reprogramming (50,51). Here, translocated NF-AT in the nuclei of the hypertensive Kir6.2-KO was demonstrated to bind with high specificity to the B-type natriuretic peptide gene, a gene marker of the pathologically remodeled heart (Fig. 5F). Thus, KATP channel deficiency under hypertension translates into a reduced ability of the heart to gate calcium-triggered maladaptive genetic reprogramming.


Figure 1545
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  		Figure 5. Exaggerated activation of calcium–calcineurin-dependent signaling in HTN hearts lacking KATP channels. Calcineurin phosphatase activity significantly increased in Kir6.2-KO versus WT hearts in hypertension (A), with a tight correlation between calcineurin enzyme activity and resulting increase in cardiac mass (B). Change in calcineurin activity expressed as percentage change from mean of unchallenged WT or KO control groups, respectively. (C) Enhanced cytosol to nucleus shuttling of NF-AT in hypertensive KO confirmed by co-localization with the nuclear marker DAPI, in {alpha}-actinin-stained isolated cardiomyocytes, further highlighted in insets. Scale bars: 20 µm. (D) Immunoblot of nuclear extracts demonstrated excessive nuclear accumulation of NF-AT in hypertensive KO compared with modest levels in hypertensive WT, with protein molecular weight standard indicated in kD. (E) Corresponding quantitation expressed as a fold increase in nuclear protein expression over WT control. (F) HTN KO nuclear extracts demonstrated, on electrophoretic mobility shift assay, specific NF-AT/DNA complex formation through specific binding to an NF-AT DNA probe from the promoter region of the B-type natriuretic peptide (BNP) gene (unlabeled lanes). Specificity was demonstrated by competition of binding with excess normal (N) but not mutated (M) NF-AT probe. The identity of the substrate within the nuclear extract was confirmed to be NF-AT, as immunodepletion (D) with an NF-AT antibody prevented DNA complex formation, which was recovered (R) by replenishing the precipitated NF-AT. The DNA/protein complex (bracket) and unbound DNA probe (arrow) are indicated to the right.

 
Calcineurin inhibition negates maladaptive remodeling in hypertensive KATP channel-deficient hearts
To examine the significance of over-activity of the calcium–calcineurin pathway in hypertensive Kir6.2-KO mice, mineralocorticoid/salt-challenged mice were further treated with the calcineurin inhibitor, cyclosporine A (52). Left ventricular sections taken from hypertensive Kir6.2-KO hearts treated with cyclosporine showed no significant evidence of nuclear NF-AT staining, implicating prevention of NF-AT activation by calcineurin inhibition (Fig. 6A). In fact, treatment with the calcineurin inhibitor negated the aggravated increase in cardiac mass observed in Kir6.2-KO hearts (Fig. 6B). Following cyclosporine therapy, the left ventricular mass of hypertensive Kir6.2-KO hearts was equivalent to that of the hypertensive wild-type, reversing the deficit produced by lack of KATP channels (Fig. 6B). Thus, intact KATP channels are required as proximal modulators of the calcium–calcineurin pathway under hemodynamic stress.


Figure 1546
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  		Figure 6. Calcineurin inhibition negated excessive remodeling in KATP channel KO. In the HTN KATP channel KO, NF-AT nuclear accumulation (boxed in A) was prevented by calcineurin inhibition with daily cyclosporine A (CsA) therapy (B). In the absence of CsA treatment (HTN-CsA), KO demonstrated excessive cardiac (C) and LV (D) remodeling compared with WT, with differences negated by daily CsA treatment (HTN+CsA). Change in cardiac or LV mass expressed as percentage change from mean of unchallenged WT or KO controls. Asterisk indicates significant difference compared with respective hypertensive WT (P<0.005).

 
Calcium channel antagonism rescues hypertensive Kir6.2-KO phenotype
KATP channels respond to derangement of the intracellular nucleotide balance, characteristic of a stressed state, with homeostatic shortening of the cardiac action potential securing a feedback mechanism that gates calcium influx through voltage-dependent calcium channels (53,54). The importance of calcium in the Kir6.2-KO response to mineralocorticoid hypertension was probed by treatment of mice with the L-type calcium channel antagonist verapamil, which, by directly blocking calcium influx, bypasses the site of KATP channel action in the calcium-handling pathway. Although the modest increase in left ventricular mass in the hypertensive wild-type was not affected by verapamil therapy (P=0.72), verapamil significantly attenuated the increase in left ventricular mass induced by mineralocorticoid/salt loading in the hypertensive Kir6.2-KO (Fig. 7 inset; P<0.03) at a dose of the calcium channel antagonist that did not affect the degree of systemic hypertension (P=0.52). In fact, verapamil therapy abolished the differential hypertrophic response between the wild-type and Kir6.2-KO (Fig. 7 inset; P=0.49). Moreover, verapamil treatment averted the syndrome of heart failure, including lung congestion in hypertensive Kir6.2-KO (Fig. 7 inset). Verapamil-mediated prevention of all mortality in the hypertensive Kir6.2-KO (Fig. 7) underscored the pathological role for calcium loading in the hypertensive KATP channel-deficient failing heart with rescue of the phenotype through restoration of a control in calcium influx.


Figure 1547
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  		Figure 7. Calcium channel antagonism rescued hypertensive KATP channel KO phenotype. Treatment with the calcium channel antagonist verapamil (ver) prevented aggravated remodeling (inset), pulmonary congestion (inset) and death (P=0.03) in HTN Kir6.2-KO. Changes in LV mass and lung weight (wgt) expressed as percentage change from mean of unchallenged KO controls. Asterisk indicates significant difference compared with respective control or HTN WT (P<0.001).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The transition from conditions of imposed hemodynamic load to maladaptive pathological remodeling and heart failure is characterized by myocardial bioenergetic compromise and misbalanced calcium homeostasis (55–57). To date, the molecular checkpoints that gate ionic balance in response to metabolic distress are poorly understood. In the setting of hypertension, the present study uncovers that gene knockout of the metabolic-sensing sarcolemmal KATP channels predisposes the energetically compromised myocardium to calcium overload, precipitating aggravated calcium–calcineurin remodeling and causing congestive heart failure with death. These results identify the KATP channel as a proximal metabolic checkpoint in structural reprogramming and the adaptive response to sustained hemodynamic stress, with channel deficit a molecular substrate for the development of cardiac disease.

KATP channels are well suited to serve a safety role in matching demands of imposed distress with membrane potential-dependent homeostatic functions (58). It is established that cardiac KATP channels assemble in the sarcolemma as complexes of pore-forming Kir6.2 and regulatory SUR2A subunits to provide efficient metabolic sensing and to adjust membrane excitability in accord with the cellular energetic state (19,21,24). While high subsarcolemmal myocardial ATP concentrations maintain the channel closed in the healthy myocardium, stress-induced metabolic alterations are transduced by the regulatory subunit to channel pore opening, permitting potassium efflux and an aptitude towards a protective shortening of the cardiac action potential which in turn effectively gates calcium entry through membrane potential-dependent L-type calcium channels (33,40,54). The remodeled myocardium has a higher energetic turnover with a relative shift towards diphosphate nucleotides (48,49), established KATP channel activators (22). Consistent with promotion of KATP channel activity in the hypertrophied cardiomyocyte (59), knockout of the channel rendered the myocardium insensitive to energetic distress, translating into impaired myocardial repolarization and demonstrable cellular calcium loading. The requirement for intact KATP channel activity in coupling calcium handling with load-induced changes in the cellular energetic state indicates a pivotal role for the KATP channel complex in the myocardium where ionic signaling must properly interface with metabolic homeostasis to secure adaptation without distress.

The calcium-overloaded KATP channel-deficient heart was predisposed to malignant calcium-triggered structural remodeling and contractile dysfunction. Evidence of high calcineurin phosphatase activity and nuclear translocation of the transcription factors MEF2 and NF-AT, underlying the exaggerated remodeling response in the hypertensive KATP channel knockout, was supported by negation of difference between wild-type and the KATP channel knockout by pharmacological calcineurin inhibition. Further rescue of the failing phenotype in the KATP channel knockout by restoring the overall aptitude of the hypertensive heart to limit calcium loading with verapamil therapy, reversing the propensity for maladaptive remodeling, organ failure and survival disadvantage, underscores the requirement for an intact proximal checkpoint in the form of KATP channels to safeguard the stressed myocyte from uncontrolled and potentially deleterious calcium overload.

Critical in orchestrating membrane-dependent cellular functions, ion channels have the potential, when defective, to form the basis for human disease, so called channelopathies (60). Aberrant KATP channel regulation and/or function may result in significant human pathology as demonstrated in the pancreas, where these channels modulate insulin secretion in response to metabolic needs (61). The disease states of persistent hyperinsulinemic hypoglycemia of infancy and neonatal diabetes mellitus have been linked to mutations that impair trafficking and/or function of KATP channels in pancreatic ß-cells (61–63). In the heart, where KATP channels were first described and are present in highest abundance (19), normal cardiac function is particularly dependent on the proper movement of ions across the sarcolemma, with channelopathies composing an increasing number of heart disease entities (60,64). However, mutations in cardiac KATP channel subunits have until very recently not been reported, and hence an association between channel mutations and cardiac disease not established. Our recent findings of mutations in the gene encoding the cardiac-specific metabolism-sensing component of the KATP channel in patients with cardiomyopathy (39) and the development of cardiac structural and functional impairment in Kir6.2-knockout mice stressed with chronic repetitive exertion (37) or transverse aortic constriction (65), when placed in the context of this current analysis by which loss of channel function renders the heart vulnerable to heart failure, present KATP channel dysfunction as a novel channelopathy in heart failure. The recent identification of genetic variants in KCNJ11 in the population (66), in fact, raises the possibility of a broader role for KATP channels in disease susceptibility. In light of the use of KATP channel modulators in clinical medicine, and their apparent effect on cardiovascular disease (67–69), defects in KATP channel proteins and/or modulators of channel function thus warrant further investigation to advance molecular diagnostics and targeted therapeutics of myocardial adaptation to hemodynamic load.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
KATP channel knockout and experimental hypertension
KATP channel-deficient mice (Kir6.2-KO) were generated by targeted disruption of the KCNJ11 gene (70), which encodes the pore-forming Kir6.2 channel subunit of myocardial but not renal or vascular KATP channels (71,72), and backcrossed for five generations to a C57BL/6 background. With approval of the Mayo Clinic Institutional Animal Care and Use Committee, Kir6.2-KO or 8- to 12-week-old, age-matched, C57BL/6 male wild-type mice had left nephrectomy through a retroperitoneal flank excision under isoflurane-anesthesia to reduce clearance, and mineralocorticoid-hypertension was induced for 21 days by subcutaneous implantation of a 50 mg 21 day-release deoxycorticosterone-acetate pellet (Innovative Research of America) and drinking water supplementation with 1%NaCl/0.2% KCl. Control wild-type or Kir6.2-KO mice underwent nephrectomy alone. Water or salt water intake was measured weekly. All mice were given standard rodent chow, had housed individually with a 12 h day/night cycle and were observed daily until termination of the study at day 21. Mice had similar awake non-fasting blood glucose levels measured by tail sampling (OneTouch Ultra, Lifescan). Values in normotensive and hypertensive mice were 159±6 (n=8) and 141±7 mg/dl (n=10), respectively, for the wild-type, and 139±4 (n=8) and 135±7 mg/dl (n=10), respectively, for the Kir6.2-KO (P=0.3 across all groups). Following 1 week of acclimatization, blood pressure was measured by automated tail-cuff recording (Columbus Instruments) in awake restrained wild-type and Kir6.2-KO mice, 2 weeks post-nephrectomy with or without mineralocorticoid/salt loading. Systolic blood pressure was digitally derived from 10 sequential recordings.

Gross pathology and tissue fibrosis
At 21 days, whole heart, left ventricle including the septum, kidney and lungs were removed, rinsed, blotted dry and weighed ex vivo. Lung samples were dried at 65°C for 48 h and reweighed with pulmonary congestion assessed by comparing wet-to-dry lung weight ratios. Interstitial fibrosis was quantified by computer analysis (MetaMorph, Visitron Universal Imaging) of 0.5 µM thick, paraffin-embedded, Masson's trichrome-stained sections. Kidney or left ventricular collagen content was determined by assaying hydroxyproline content after overnight hydrolysis by 12N HCl with samples run in duplicate against standard hydroxyproline (Sigma). All quantification was performed blinded to the sample origin.

Treadmill exertion
To assess the impact of hypertension on exercise capacity, a comparison of performance was made on a graded treadmill exercise test at day 21 between hypertensive and control wild-type and Kir6.2-KO mice. Workload (J) was calculated as the sum of kinetic energy (Ek=mv2/2) and potential energy (Ep=mgvt[sin {theta}]), where m is animal mass, v treadmill velocity, g acceleration due to gravity, t elapsed time at a protocol level and {theta} the angle of incline.

Electrocardiography and telemetry
To continuously monitor heart rate and rhythm in the conscious state, telemetry devices (Data Sciences International) were implanted in the peritoneum and leads tunneled subcutaneously in a lead II configuration under isoflurane anesthesia in wild-type and Kir6.2-KO mice. Following 2 week recovery from surgery, serial signals were acquired at 2 kHz before and during mineralocorticoid/salt challenge. Surface electrocardiogram recordings were obtained under light anesthesia (1.25% isoflurane) via limb lead electrodes. The QT interval was defined as the time from start of the Q wave to the end of the T wave (time when repolarization returned to the isoelectric point) of the electrogram. Corrected for heart rate, the QT interval (QTc) was calculated as Formula, where RR (in milliseconds) is the interval between two consecutive R waves (33).

Cardiac ultrasound
Echocardiography (c256 and 15L8, Acuson) was performed in lightly sedated (1.25% isoflurane) mice at the end of the 21 day protocol. Images were digitally acquired and stored for offline blinded analysis. Echocardiographic measurements of left ventricular dimensions were recorded at end-diastole (EDD) and end-systole (ESD) from three consecutive cardiac cycles, using the leading edge method. LV fractional shortening (%FS) was calculated as: %FS=[(EDD–ESD)/EDD]/100. Ejection time (Et) was determined from the actual pulsed-wave Doppler tracings on the parasternal long-axis view of trans-aortic flow by measuring the interval from the beginning of the acceleration to the end of the deceleration. The myocardial velocity of left ventricular circumferential shortening (Vcf, expressed in circumferences per second) was calculated as: Vcf=[(EDD–ESD)/EDD]/Et. Stroke volume was determined by the sum of aortic root cross-sectional area and the velocity time integral taken from peak trans-aortic Doppler tracings. The product of stroke volume and heart rate, expressed as ml/min, was used to calculate cardiac output.

Left ventricular catheterization
Invasive left ventricular pressure recordings were measured in vivo by a 1.4 Fr micropressure catheter (SPR-671, Millar Instruments), following carotid arterial cannulation and advancement across the aortic valve under 2,2,2-tri-bromoethanol (375 µg/kg i.p.; Sigma) anesthesia before and after dobutamine (15 mg/kg i.p.) challenge. Left ventricular developed pressure was defined as the absolute difference between the maximum and minimum left ventricular pressures (mmHg) and the relaxation time as the time from maximal rate of pressure decay to minimum left ventricular pressure (ms).

Isolated cardiomyocytes, immunohistochemistry and calcium imaging
The aorta was cannulated in situ, heart rapidly excised, retrogradely perfused and cardiomyocytes were enzymatically dissociated (36). Surface area measurements of rod-shaped striated calcium-tolerant ventricular cardiomyocytes were performed by digital planimetry (MetaMorph; Visitron Universal Imaging). For immunohistochemistry, formalin-fixed, paraffin-embedded left ventricular sections were deparaffinized with xylene and rehydrated in serial alcohol washes. To optimize antigen retrieval, sections were incubated in 0.5 M NH4Cl with 0.25% Triton X-100 for 30 min, and then for an additional 30 min in 1 mM EDTA in a pressure cooker. Left ventricular sections or 3% paraformaldehyde-fixed isolated cardiomyocytes were probed with primary antibodies applied at 4°C overnight to the cardiac sarcomeric protein {alpha}-actinin (mouse polyclonal, 1:500; Sigma), the KATP channel pore Kir6.2 (goat polyclonal, 1:300; Santa Cruz) and the cardiac transcription factors MEF2C (rabbit polyclonal, 1:300; Cell Signaling Technologies) and NF-ATc4 (rabbit polyclonal, 1:300; Santa Cruz). Accordingly, Alexa 568-labeled anti-mouse (1:200), Alexa 488-labeled anti-goat (1:200) and Alexa 488-labeled anti-rabbit (1:200) secondary antibodies (Molecular Probes) were applied 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) as described (73,74). For calcium measurements, freshly isolated rod-shaped striated ventricular cardiomyocytes loaded with the calcium-fluorescent probe Fluo-4-acetoxymethyl ester (2 µM; Molecular Probes) were 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) of cells (n≥50 from each heart) from matched hypertensive wild-type and Kir6.2-KO hearts (n≥3 from each group) were deconvoluted, and analyzed using Metamorph, normalized to the degree of background fluorescence (33).

Nucleotide content
Nucleotide levels were determined in 0.6 M perchloric acid/1 mM EDTA extracts from liquid N2 freeze-clamped hearts. Extracts were neutralized with 2 M K2HCO3, and nucleotides, eluted with a linear gradient of triethylammonium bicarbonate buffer, were profiled by high-performance liquid chromatography (HP 1100, Hewlett-Packard) with a MonoQ HR5/5 column (Amersham Pharmacia) (75). As an integrated indicator of the overall myocardial bioenergetic state, the DP/TP nucleotide index was defined as the product of the ratios of nucleotide diphosphates (DP) to their respective triphosphates (TP), i.e. [ADP/ATP]·[GDP/GTP]·[UDP/UTP].

Calcineurin activity
Calcineurin enzyme activity was obtained by quantification of phosphatase activity in left ventricular desalted cytosolic extracts, normalized to protein content, as determined by assay (Bio-Rad). Calcineurin phosphatase activity was measured in duplicate using the RII phosphopeptide as substrate in okadaic acid by the average difference in free-phosphate released in the presence and absence of EGTA as detected by the Malachite green assay at OD620 nm (CN Biosciences Corporation).

Nuclear extracts and electrophoretic mobility shift assay
Nuclear extracts were prepared from left ventricular tissue in the presence of complete mini protease inhibitor (Roche), phosphatase inhibitors, 10 nM staurosporine, 1 mM dithiothreitol and 1 mM phenylmethylsulfonyl fluoride at 4°C. Following tissue resuspension in 10 ml/g of hypotonic buffer (10 mM Tris, pH 7.5; 1 mM MgCl2; 10 mM NaCl; 5 mM CaCl2) and mechanical dissociation, cellular debris was removed and nuclei recovered by centrifugation. Nuclei were washed in 1 ml of nuclear resuspension buffer (10 mM Tris, pH 7.5; 250 mM sucrose; 1 mM MgCl2; 0.1 mM EDTA) and collected by centrifugation. Nuclei were resuspended in low salt buffer (20 mM HEPES, pH 7.9 at 4°C; 25% glycerol; 1.5 mM MgCl2; 20 mM KCl; 0.2 mM EDTA), and one packed nuclear volume of high salt buffer (20 mM HEPES pH 7.9 at 4°C; 25% glycerol; 1.5 mM MgCl2; 800 mM KCl; 0.2 mM EDTA) added. Samples were incubated on ice for 30 min with intermittent mixing. Salt concentration was brought to 833 mM by addition of 2.2 µl of 3 M KCl per 10 µl of sample volume and samples were incubated on ice for 30 min. The nuclear extract was diluted with 1.5 volumes of nuclear diluent (25 mM HEPES, pH 7.9 at 4°C; 0.1 mM EDTA) and clarified by centrifugation at 16 000g for 15 min. Electrophoretic mobility shift assays were performed with 6 µg of nuclear extract and 20 fmole of end-labeled probe (BNP-927 sequence; 14) in binding buffer (10 mM HEPES, pH 7.9 at 4°C; 50 mM NaCl; 1 mM MgCl2; 1 mM CaCl2; 10% glycerol; 100 µg/ml bovine serum albumin; 0.167 mg/ml polydI-dC). For competition reactions, a 50-fold molar excess (i.e. 1 pmol) of unlabeled wild-type or mutated NF-AT consensus sequence (Santa Cruz Biotechnology) was added prior to probe addition. For immunodepletion experiments, nuclear extract was incubated overnight with 1 µg of NFATc4 (NFAT3) antibody (Santa Cruz Biotechnology). Protein A precipitates were washed three times with PBS containing 0.1% nonidet P40 and once in PBS. NF-AT was recovered from the precipitate by elution for 10 min on ice in citrate buffer, pH 3, then neutralized by 0.05 volumes of 1 M Tris, pH 8.9.

Immunoblotting
Proteins were resolved from myocardial nuclear extracts, and the presence of NF-AT was probed by incubation with 2 µg/ml of NF-AT antibody (Santa Cruz Biotechnology) counterstained by anti-rabbit IgG horse raddish peroxidase conjugate diluted 1:24 000 in TBS. The membranes were incubated in Super Signal West Pico Substrate (Pierce) and the signals captured on the AutoChemi System (UVP, Inc.) and quantified (LabWorks software, UVP, Inc.).

Pharmacological intervention in vivo
Murine subgroups were treated with either 25 mg/kg i.p. every 12 h of the calcineurin inhibitor cyclosporine A (Sandimmune, Novartis) or with 250 µg of the L-type calcium channel antagonist verapamil (Sigma) orally daily, starting the day of nephrectomy with or without deoxycorticosterone-acetate/salt loading.

Statistical analysis
Comparisons between groups were performed by log-rank, analysis of variance, Student's t-tests or non-parametric tests as appropriate (JMP; SAS). Data are presented as mean±SEM; n refers to sample size. P<0.05 was predetermined.


   ACKNOWLEDGEMENTS
 
The authors wish to thank Jonathan Nesbitt for excellent technical assistance and the Translational Ultrasound Research Core for the 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 and Japanese Ministry of Education, Science, Sports, Culture and Technology. Funding to pay the Open Access publication charges for this article was provided by Marriott Foundation.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Braunwald E. (2000) Simon Dack lecture–Cardiology: the past, the present, and the future. J. Am. Coll. Cardiol. 42:2031–2041.

  2. Jessup M. and Brozena S. (2003) Heart failure. N. Engl. J. Med. 348:2007–2018.[Free Full Text]

  3. Redfield M.M., Jacobsen S., Burnett J.C., Mahoney D.W., Bailey K., Rodeheffer R. (2003) Burden of systolic and diastolic ventricular dysfunction in the community: appreciating the scope of the heart failure epidemic. JAMA 289:194–202.[Abstract/Free Full Text]

  4. Seidman J.G. and Seidman C.E. (2001) The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms. Cell 104:557–567.[CrossRef][ISI][Medline]

  5. Kostis J.B. (2003) From hypertension to heart failure. Am. J. Hyper. 16:18S–22S.[ISI][Medline]

  6. Watkins H. and Farrall M. (2006) Genetic susceptibility to coronary artery disease: from promise to progress. Nat. Rev. Genet. 7:163–173.[CrossRef][ISI][Medline]

  7. Bers D.M. (2002) Cardiac excitation–contraction coupling. Nature 415:180–205.[CrossRef][Medline]

  8. Chien K.R. (1999) Stress pathways and heart failure. Cell 98:555–558.[CrossRef][ISI][Medline]

  9. Towbin J.A. and Bowles N.E. (2002) The failing heart. Nature 415:227–233.[CrossRef][Medline]

  10. Chien K.R. and Olson E.N. (2002) Converging pathways and principles in heart development and disease. Cell 110:153–162.[CrossRef][ISI][Medline]

  11. Chien K.R., Ross J., Hoshijima M. (2003) Calcium and heart failure. Nat. Med. 9:508–509.[CrossRef][ISI][Medline]

  12. Ahmad F., Seidman J.G., Seidman C.E. (2005) The genetic basis for cardiac remodeling. Annu. Rev. Genom. Hum. Genet. 6:185–216.[CrossRef][ISI][Medline]

  13. Wehrens X.H., Lehnart S.E., Marks A.R. (2005) Intracellular calcium release and cardiac disease. Annu. Rev. Physiol. 67:69–98.[CrossRef][ISI][Medline]

  14. Molkentin J.D. (2006) Dichotomy of Ca2+ in the heart: contraction versus intracellular signaling. J. Clin. Invest. 116:623–626.[CrossRef][ISI][Medline]

  15. Molkentin J.D., Lu J.R., Antos C.L., Markham B., Richardson J., Robbins J., Grant S.R., Olson E.N. (1998) A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93:215–228.[CrossRef][ISI][Medline]

  16. Zhang C.L., McKinsey T.A., Chang S., Antos C.L., Hill J.A., Olson E.N. (2002) Class II histone deacetylases act as signal-responsive repressors of cardiac hypertrophy. Cell 110:479–488.[CrossRef][ISI][Medline]

  17. Schmitt J.P., Kamisago M., Asahi M., Li G.H., Ahmad F., Mende U., Kranias E.G., MacLennan D.H., Seidman J.G., Seidman C.E. (2003) Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban. Science 299:1410–1413.[Abstract/Free Full Text]

  18. Zhang R., Khoo M.S., Wu Y., Yang Y., Grueter C.E., Ni G., Price E.E., Thiel W., Guatimosim S., Song L.S., et al. (2005) Calmodulin kinase II inhibition protects against structural heart disease. Nat. Med. 11:409–417.[CrossRef][ISI][Medline]

  19. Noma A. (1983) ATP-regulated K+ channels in cardiac muscle. Nature 305:147–148.[CrossRef][Medline]

  20. Ashcroft F.M. (1988) Adenosine 5'-triphosphate-sensitive potassium channels. Annu. Rev. Neurosci. 11:7–118.

  21. Alekseev A.E., Hodgson D.M., Karger A.B., Park S., Zingman L.V., Terzic A. (2005) ATP-sensitive K+ channel channel/enzyme multimer: metabolic gating in the heart. J. Mol. Cell. Cardiol. 38:895–905.[CrossRef][ISI][Medline]

  22. Nichols C.G. (2006) KATP channels as molecular sensors of cellular metabolism. Nature 440:470–476.[CrossRef][Medline]

  23. Inagaki N., Gonoi T., Clement J.P., Namba N., Inazawa J., Gonzalez G., Aguilar-Bryan L., Seino S., Bryan J. (1995) Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270:1166–1170.[Abstract/Free Full Text]

  24. Inagaki N., Gonoi T., Clement J.P., Wang C.Z., Aguilar-Bryan L., Bryan J., Seino S. (1996) A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron 16:1011–1017.[CrossRef][ISI][Medline]

  25. Tucker S.J., Gribble F.M., Zhao C., Trapp S., Ashcroft F.M. (1997) Truncation of Kir6.2 produces ATP-sensitive K+ channels in the absence of the sulphonylurea receptor. Nature 387:179–183.[CrossRef][Medline]

  26. Lorenz E. and Terzic A. (1999) Physical association between recombinant cardiac ATP-sensitive K+ channel subunits Kir6.2 and SUR2A. J. Mol. Cell. Cardiol. 31:425–434.[CrossRef][ISI][Medline]

  27. Bienengraeber M., Alekseev A.E., Abraham M.R., Carrasco A.J., Moreau C., Vivaudou M., Dzeja P.P., Terzic A. (2000) ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex. FASEB J. 14:1943–1952.[Abstract/Free Full Text]

  28. Zingman L.V., Alekseev A.E., Bienengraeber M., Hodgson D., Karger A.B., Dzeja P.P., Terzic A. (2001) Signaling in channel/enzyme multimers: ATPase transitions in SUR module gate ATP-sensitive K+ conductance. Neuron. 31:233–245.[CrossRef][ISI][Medline]

  29. Carrasco A.J., Dzeja P.P., Alekseev A.E., Pucar D., Zingman L.V., Abraham M.R., Hodgson D., Bienengraeber M., Puceat M., Janssen E., Wieringa B., Terzic A. (2001) Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels. Proc. Natl Acad. Sci. USA 98:7623–7628.[Abstract/Free Full Text]

  30. Abraham M.R., Selivanov V.A., Hodgson D.M., Pucar D., Zingman L.V., Wieringa B., Dzeja P.P., Alekseev A.E., Terzic A. (2002) Coupling of cell energetics with membrane metabolic sensing. Integrative signaling through creatine kinase phosphotransfer disrupted by M-CK gene knock-out. J. Biol. Chem. 277:24427–24434.[Abstract/Free Full Text]

  31. Jovanovic S., Du Q., Crawford R.M., Budas G.R., Stagljar I., Jovanovic A. (2005) Glyceraldehyde 3-phosphate dehydrogenase serves as an accessory protein of the cardiac sarcolemmal KATP channel. EMBO Rep. 6:848–852.[CrossRef][ISI][Medline]

  32. Dhar-Chowdhury P., Harrell M.D., Han S.Y., Jankowska D., Parachuru L., Morrissey A., Srivastava S., Liu W., Malester B., Yoshida H., Coetzee W.A. (2005) The glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, and pyruvate kinase are components of the KATP channel macromolecular complex and regulate its function. J. Biol. Chem. 280:38464–38470.[Abstract/Free Full Text]

  33. Zingman L.V., Hodgson D.M., Bast P.H., Kane G.C., Perez-Terzic C., Gumina R.J., Pucar D., Bienengraeber M., Dzeja P.P., Miki T., et al. (2002) Kir6.2 is required for adaptation to stress. Proc. Natl Acad. Sci. USA 99:13278–13283.[Abstract/Free Full Text]

  34. Suzuki M., Sasaki N., Miki T., Sakamoto N., Ohmoto-Sekine Y., Tamagawa M., Seino S., Marban E., Nakaya H. (2002) Role of sarcolemmal KATP channels in cardioprotection against ischemia/reperfusion injury in mice. J. Clin. Invest. 109:509–516.[CrossRef][ISI][Medline]

  35. Gumina R.J., Pucar D., Bast P., Hodgson D.M., Kurtz C.E., Dzeja P.P., Miki T., Seino S., Terzic A. (2003) Knockout of Kir6.2 negates ischemic preconditioning-induced protection of myocardial energetics. Am. J. Physiol. 284:H2106–H2113.

  36. Hodgson D.M., Zingman L.V., Kane G.C., Perez-Terzic C., Bienengraeber M., Ozcan C., Gumina R.J., Pucar D., O'Coclain F., Mann D.L., Alekseev A.E., Terzic A. (2003) Cellular remodeling in heart failure disrupts KATP channel-dependent stress tolerance. EMBO J 22:1732–1742.[CrossRef][ISI][Medline]

  37. Kane G.C., Behfar A., Yamada S., Perez-Terzic C., O'Cochlain F., Reyes S., Dzeja P.P., Miki T., Seino S., Terzic A. (2004) ATP-sensitive K+ channel knockout compromises the metabolic benefit of exercise training, resulting in cardiac deficits. Diabetes 53:S169–S175.[Abstract/Free Full Text]

  38. Liu X.K., Yamada S., Kane G.C., Alekseev A.E., Hodgson D.M., O'Cochlain F., Jahangir A., Miki T., Seino S., Terzic A. (2004) Genetic disruption of Kir6.2, the pore-forming subunit of ATP-sensitive K+ channel, predisposes to catecholamine-induced ventricular dysrhythmia. Diabetes 53:S165–S168.[Abstract/Free Full Text]

  39. Bienengraeber M., Olson T.M., Selivanov V.A., Kathmann E.C., O'Cochlain F., Gao F., Karger A.B., Ballew J.D., Hodgson D.M., Zingman L.V., et al. (2004) ABCC9 mutations identified in human dilated cardiomyopathy disrupt catalytic KATP channel gating. Nat. Genet. 36:382–387.[CrossRef][ISI][Medline]

  40. Kane G.C., Liu X.K., Yamada S., Olson T.M., Terzic A. (2005) Cardiac KATP channels in health and disease. J. Mol. Cell. Cardiol. 38:937–943.[CrossRef][ISI][Medline]

  41. Morita H., Seidman J., Seidman C.E. (2005) Genetic causes of human heart failure. J. Clin. Invest. 115:518–526.[CrossRef][ISI][Medline]

  42. Lifton R.P., Gharavi A.G., Geller D.S. (2001) Molecular mechanisms of human hypertension. Cell 104:545–556.[CrossRef][ISI][Medline]

  43. Weber K.T. (2001) Aldosterone in congestive heart failure. N. Engl. J. Med. 345:1689–1697.[Free Full Text]

  44. Johns C., Gavras I., Handy D.E., Salomao A., Gavras H. (1996) Models of experimental hypertension in mice. Hypertension 28:1064–1069.[Abstract/Free Full Text]

  45. Levy D., Garrison R.J., Savage D.D., Kannel W.B., Castelli W.P. (1990) Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N. Engl. J. Med. 322:1561–1566.[Abstract]

  46. de Simona G., Verdecchia P., Pede S., Gorini M., Maggioni A.P. (2002) Prognosis of inappropriate left ventricular mass in hypertension. The MAVI study. Hypertension 40:470–476.[Abstract/Free Full Text]

  47. Drazner M.H., Rame J.E., Marino E.K., Gottdiener J.S., Kitzman D.W., Gardin J.M., Manolio T.A., Dries D.L., Siscovick D.S. (2004) Increased left ventricular mass is a risk factor for the development of a depressed left ventricular ejection fraction within 5 years. J. Am. Coll. Cardiol. 43:2207–2215.[Abstract/Free Full Text]

  48. Zhang J., Merkle H., Hendrich K., Garwood M., From A.H.L., Ugurbil K., Bache R.J. (1993) Bioenergetic abnormalities associated with severe left ventricular hypertrophy. J. Clin. Invest. 92:993–1003.[ISI][Medline]

  49. Perez-Terzic C., Gacy A.M., Bortolon R., Dzeja P.P., Puceat M., Jaconi M., Prendergast F.G., Terzic A. (2001) Directed inhibition of nuclear import in cellular hypertrophy. J. Biol. Chem. 276:20566–20571.[Abstract/Free Full Text]

  50. Frey N. and Olson E.N. (2003) Cardiac hypertrophy: the good, the bad, and the ugly. Annu. Rev. Physiol. 65:45–79.[CrossRef][ISI][Medline]

  51. Crabtree G.R. and Olson E.N. (2002) NFAT signaling: choreographing the social lives of cells. Cell 109:S67–S79.[CrossRef][ISI][Medline]

  52. Sussman M.A., Lim H.W., Gude N., Taigen T., Olson E.N., Robbins J., Colbert M.C., Gualberto A., Wieczorek D.F., Molkentin J.D. (1998) Prevention of cardiac hypertrophy in mice by calcineurin inhibition. Science 281:1690–1693.[Abstract/Free Full Text]

  53. Terzic A., Jahangir A., Kurachi Y. (1995) Cardiac ATP-sensitive K+ channels: regulation by intracellular nucleotides and K+ channel-opening drugs. Am. J. Physiol. 269:C525–C545.[ISI][Medline]

  54. Selivanov V.A., Alekseev A.E., Hodgson D.M., Dzeja P.P., Terzic A. (2004) Nucleotide-gated KATP channels integrated with creatine and adenylate kinases: amplification, tuning and sensing of energetic signals in the compartmentalized cellular environment. Mol. Cell. Biochem. 256/257:243–256.[CrossRef]

  55. Braunwald E. and Bristow M.R. (2000) Congestive heart failure: fifty years of progress. Circulation 102:IV14–IV23.[Medline]

  56. Marks A.R. (2003) Calcium and the heart: a question of life and death. J. Clin. Invest. 111:597–600.[CrossRef][ISI][Medline]

  57. Saks V., Dzeja P., Schlattner U., Vendelin M., Terzic A., Wallimann T. (2006) Cardiac system bioenergetics: metabolic basis of the Frank-Starling law. J. Physiol. 571:253–273.[Abstract/Free Full Text]

  58. Zingman L.V., Hodgson D.M., Alekseev A.E., Terzic A. (2003) Stress without distress: homeostatic role for KATP channels. Mol. Psychiat. 8:253–254.