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Am J Physiol Heart Circ Physiol 292: H1706-H1713, 2007. First published December 22, 2006; doi:10.1152/ajpheart.01305.2006
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KATP channel knockout worsens myocardial calcium stress load in vivo and impairs recovery in stunned heart

Richard J. Gumina,1 D. Fearghas O'Cochlain,1 Christopher E. Kurtz,1 Peter Bast,1 Darko Pucar,1 Prasanna Mishra,2 Takashi Miki,3 Susumu Seino,3 Slobodan Macura,2 and Andre Terzic1

1Marriott Heart Disease Research Program, Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology & Experimental Therapeutics and 2Biochemistry & Molecular Biology, Mayo Clinic, Rochester, Minnesota; 3Division of Cellular and Molecular Medicine, Kobe University Graduate School of Medicine, Kobe, Japan

Submitted 29 November 2006 ; accepted in final form 18 December 2006


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Gene knockout of the KCNJ11-encoded Kir6.2 ATP-sensitive K+ (KATP) channel implicates this stress-response element in the safeguard of cardiac homeostasis under imposed demand. KATP channels are abundant in ventricular sarcolemma, where subunit expression appears to vary between the sexes. A limitation, however, in establishing the full significance of KATP channels in the intact organism has been the inability to monitor in vivo the contribution of the channel to intracellular calcium handling and the superimposed effect of sex that ultimately defines heart function. Here, in vivo manganese-enhanced cardiac magnetic resonance imaging revealed, under dobutamine stress, a significantly greater accumulation of calcium in both male and female KATP channel knockout (Kir6.2-KO) mice compared with sex- and age-matched wild-type (WT) counterparts, with greatest calcium load in Kir6.2-KO females. This translated, poststress, into a sustained contracture manifested by reduced end-diastolic volumes in KATP channel-deficient mice. In response to ischemia-induced stunning, male and female Kir6.2-KO hearts demonstrated accelerated time to contracture and increased peak contracture compared with WT. The outcome on reperfusion, in both male and female Kir6.2-KO hearts, was a transient reduction in systolic performance, measured as rate-pressure product compared with WT, with protracted increase in left ventricular end-diastolic pressure, exaggerated in female knockout hearts, despite comparable leakage of creatine kinase across groups. Kir6.2-KO hearts were rescued from diastolic dysfunction by agents that target alternative pathways of calcium handling. Thus KATP channel deficit confers a greater susceptibility to calcium overload in vivo, accentuated in female hearts, impairing contractile recovery under various conditions of high metabolic demand.

ATP-sensitive K+ channel; Kir6.2; magnetic resonance imaging; myocardium; sex

THE INWARDLY RECTIFYING K+ channel Kir6.2 is the pore-forming subunit of myocardial ATP-sensitive K+ (KATP) channels (21, 22). In association with the regulatory sulfonylurea SUR2A receptor subunit, Kir6.2 generates functional adenine nucleotide-gated KATP channels expressed in high density in the sarcolemma (34, 38, 39). KATP channels are tightly coupled with intracellular energetic networks (1, 7, 10, 25, 58), and metabolic signals of distress are the primary inductors of channel activity with pore opening associated with regulation of action potential duration (48, 60). The property of signal decoding and translation of cellular energetic fluctuations implicates cardiac KATP channels in the feedback regulation of membrane electrical activity (2, 12). In conjunction with an intrinsic ATPase activity, the tandem function of nucleotide binding domains within the SUR2A regulatory subunit gates Kir6.2 (61), securing pore opening and action potential shortening in response to stress (33, 60). The proposed role of a metabolic rheostat associated with preservation of energy economy (2, 30) has prompted investigation to establish the contribution of KATP channels in matching demands for homeostatic maintenance.

Recently, targeted disruption of the KCNJ11 gene, which encodes Kir6.2, has been applied to generate knockout mice, lacking functional KATP channels in cardiomyocytes (36, 49). Knockout of KATP channels mitigates the regulation of cardiac repolarization (33, 52) and impairs the tolerance of the myocardium to sustain injury (30, 50, 59). Moreover, the Kir6.2-knockout heart demonstrates an abnormal vulnerability to physiological and pathological stress, including ischemia (16, 50), sympathetic surge (52, 60), physical exertion (28), and volume and pressure overload (27, 57), indicating a general role for KATP channels in the innate mechanisms of cardiac adaptation. Whereas the KATP channel knockout phenotype is increasingly recognized as a genetic model of compromised cardiac homeostasis, the intimate derangements that develop in stressed KATP channel-deficient hearts are partially understood (3, 15, 30). A major limitation in gaining full insight into the significance of cardiac KATP channels in the safeguard of cellular well being in the intact organism has been the inability to directly monitor, in vivo, the dynamics of intracellular fluctuations under stress challenge. Furthermore, whether the potential for sex-dependent differential expression of KATP channels in the heart, as recently proposed (6, 43, 45), has further impact in the Kir6.2-knockout remains unknown.

In this paper, we demonstrate in vivo, for the first time, the role played in the stressed heart by KATP channels in handling intracellular calcium, the fluxes of which define proper excitation-contraction coupling in the working myocardium. Under dobutamine challenge (56), in vivo manganese-enhanced cardiac magnetic resonance imaging (13, 19, 20, 35) captured excessive calcium accumulation in mice deficient in KATP channels, precipitating poor recovery. Deficit in KATP channels translated into compromised function in the stunned heart, with the knockout phenotype rescued by targeting alternative pathways of calcium handling. Whereas both male and female KATP channel knockout mice were vulnerable to aggravated calcium accumulation and functional compromise, the sequelae were typically accentuated in the female knockout. Thus KATP channels are integral in the maintenance of calcium homeostasis in vivo, and ultimately in the preservation of contractile, including lusitropic function in the stressed myocardium, with an implication of an even higher dependence upon this metabolic sensor within the female heart.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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The investigation conformed to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health and was approved by the Mayo Clinic Institutional Animal Care and Use Committee. Mice deficient in KATP channels were generated by targeted disruption of the KCNJ11 gene encoding the Kir6.2 channel pore and backcrossed for five generations into a C57BL/6 background (36). Age- and sex-matched, 8- to 12-wk-old (25–30 g), KATP channel-knockout (Kir6.2-KO) and C57BL/6 WT mice were used in all protocols.

Dobutamine Stress Under Manganese-Enhanced Cardiac MRI

Animal preparation. Male (n = 4 each) and female (n = 4 each) Kir6.2-KO mice or WT counterparts were anesthetized via intraperitoneal administration of 2,2,2-tribromoethanol (Avertin) at a dose of 250 mg/kg body wt. Under sterile conditions, a heparinized 10-µl total volume polyethylene-10 catheter was introduced into the right internal jugular and sutured in place for infusion of the Mn2+ and dobutamine solutions, used for tracing calcium influx and stress induction, respectively (13, 19, 20, 35, 56). Anesthesia was then maintained with <1% inhaled isoflurane to allow for a constant and controllable level of anesthesia and heart rate during the manganese-enhanced cardiac MRI procedure.

Mn2+ and dobutamine infusion. To limit the variability for the time due to the low infusion rates, a microinfusion pump and catheter were attached to the 10-µl catheter in the internal jugular vein. In this way, the precise time when the Mn2+ solution entered the circulation could be determined. MnCl2 dissolved in saline was infused into the mice via the right internal jugular vein line at 7.0 ± 0.4 nmol·min–1·g body wt–1, for a total of 15 min. For dobutamine stimulation, dobutamine was administered concomitantly at a rate of 30 ng·min–1·g–1 to cause inotropic stress and associated increase in myocyte Ca2+ influx.

Cardiac MRI. Images were acquired on a 7-T, 15-cm horizontal bore Bruker Avance spectrometer (Bruker Instruments, Billerica, MA) equipped with a 4.3-cm microimaging gradient insert. The ECG signals were obtained with an SA Diagnostics ECG system designed specifically for use in MRI (Boston MA). The ECG signals were used to trigger the fast low-angle shot acquisition sequence (46). End diastole, single-slice, mid-left ventricle, short-axis heart images were acquired with the cardiac-gated FLASH imaging gated directly after the R wave of the ECG. The imaging parameters were as follows: matrix = 128 x 128; angle = 90°; echo time = 1.3 ms; repetition time = 300 ms; slice thickness = 1.00 mm; field of view = 2.5 cm and eight averages. Image intensities in the midseptum, midanterior wall, and midlateral wall were averaged and normalized to an external gadolinium-spiked water phantom placed to the side of the mouse (19). Images were taken every 3 min to monitor signal changes during the experiment. For all protocols, control images were taken before any infusion and used to normalize the individual time course.

Myocardial Stunning

Heart preparation. Excised hearts from heparinized (60 units ip) and anesthetized (75 mg/kg ip pentobarbital) male (n = 8) or female (n = 10) Kir6.2-KO or male (n = 8) or female (n = 10) WT mice were perfused on a Langendorff apparatus with a 95% O2-5% CO2-saturated Krebs-Henseleit solution (in mmol/l: 118 NaCl, 5.3 KCl, 2.0 CaCl2, 19 NaHCO3, 1.2 MgSO4, 11.0 glucose, and 0.5 EDTA; 37°C) at a perfusion pressure of 80 mmHg. Contractile parameters were derived from the left ventricular pressure signal continuously monitored using a fluid-filled balloon-tipped pressure transducer (Harvard Apparatus). The pressure curve was used to calculate the rate of pressure development (+dP/dt) and decline (–dP/dt). Hearts were paced at 500 beats/min. After 30 min of stabilization, hearts were subjected to 10 min of global ischemia followed by a 30-min long reperfusion. N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7, 10 µM; Sigma-Aldrich) or 1-[(5-(p-nitrophenyl)furfurylidene)amino]hydantoin (Dantrolene 50 µM; Sigma-Aldrich) was added to the perfusate solution to evaluate the effects of calcium modulators. Cardiac effluents were collected at baseline and throughout the reperfusion period. Total creatine kinase activity was measured in the effluent using a spectrophotometric assay (Sigma-Aldrich) and reported as total creatine kinase units released during the reperfusion period. After the stunning protocol, hearts were freeze-clamped for measurements of high energy metabolites by 31P NMR spectroscopy as previously described (42).

Statistical Analysis

Multifactorial ANOVA with post hoc comparison of means was used for statistical analysis. Data are expressed as means ± SE, and a difference at P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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KATP Channel Knockout Causes Excessive Cardiac Calcium Stress Load With Poor Recovery

Manganese-induced enhancement of the magnetic resonance cardiac signal is indicative of calcium influx into the heart (13, 19, 20, 35). Accordingly, manganese-enhanced cardiac MRI was employed to probe the consequence of KATP channel deletion on calcium loading under stress in vivo. This imaging approach captures, in response to dobutamine challenge, progressive myocardial accumulation of intracellular calcium based on manganese-induced enhancement. Short-axis scanning revealed, at baseline, comparable relative enhancement with manganese in WT and KATP channel knockout (KIr6.2-KO) mice (Fig. 1, A and B). However, whereas both WT and Kir6.2-KO mice demonstrated a progressive increase in relative enhancement upon initiation of dobutamine infusion, mice lacking functional KATP channels showed aggravated calcium accumulation (Fig. 1, A and B). When compared with WT, Kir6.2 deficiency within 9 min of dobutamine administration significantly worsened myocardial calcium stress load in both male and female mice, with greatest accumulation observed in female hearts (Fig. 1, C and D). At 15 min postdobutamine, excessive calcium loading translated into a sustained contracture manifested on MRI slices by reduced end-diastolic volumes in KATP channel-deficient mice (Fig. 1, E–G). Thus, deficiency in KATP channels compromises the aptitude of the myocardium to regulate calcium entry, an effect exaggerated in female heart, precipitating poor poststress recovery in vivo.


Figure 1
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  		Fig. 1. In vivo manganese enhanced cardiac MRI. A: magnetic resonance imaging (MRI) acquired sections over 15 min of concomitant dobutamine stimulation and Mn2+ infusion, initiated at baseline (B), in wild-type (WT) and Kir6.2-KO (KO) mice. A, anterior wall; L, lateral wall; S, septum. B: relative enhancement over 15 min of concomitant dobutamine stimulation and Mn2+ infusion in WT (open) and Kir6.2-KO (filled) mice. C: representative images at baseline and at 15 min of concomitant dobutamine stimulation and Mn2+ infusion in male or female WT and Kir6.2-KO mice. D: relative enhancement at 15 min of concomitant dobutamine stimulation and Mn2+ infusion in male or female WT (open) and Kir6.2-KO (filled) mice. Values normalized to gadolinium-spiked water. *P < 0.05 compared with WT values. **P < 0.05 compared with male Kir6.2-KO mice values. Relaxation abnormalities following myocardial stress. E: end-diastolic slice volumes at baseline and at 15 min after dobutamine stress MRI in WT and Kir6.2-KO mice. F: end-diastolic slice volumes at baseline and at 15 min after dobutamine stress MRI in male WT and Kir6.2-KO mice. G: end-diastolic slice volumes at baseline and at 15 min after dobutamine stress MRI in female WT and Kir6.2-KO mice.

 
Deficit in KATP Channels Translates Into Compromised Function in Stunned Heart

With the demonstration of the consequences of KATP channel deficit on improper calcium handling and functional recovery in vivo, the implications for the vulnerable Kir6.2-KO heart were validated in a model of myocardial stunning established to be mediated in part by calcium overload (3, 5, 17). In response to brief 10-min ischemia, both male and female Kir6.2-KO hearts demonstrated a significantly accelerated time to contracture and an increased peak contracture when compared with WT (Fig. 2, A–D). After ischemia-induced stunning, WT and KATP channel knockout hearts displayed reduced performance, measured by the rate-pressure product, more pronounced in Kir6.2-deficient hearts, both male and females, during the first 10-min of reperfusion (Fig. 3, A–C). Concomitantly, diastolic dysfunction, as measured by increase in left ventricular end-diastolic pressure (LVEDP), was markedly exaggerated following 10 min ischemia in the Kir6.2-KO hearts with LVEDP remaining elevated during the entire reperfusion period compared with WT hearts (Fig. 4A). When stratified by sex, the degree of LVEDP elevation in the KATP channel knockout was significantly higher in female hearts (Fig. 4, B and C). In addition, KATP channel knockout hearts demonstrated a reduction in the maximum –dP/dt, a sensitive lusitropic index, with the difference from WT predominantly associated with impaired relaxation of the female hearts (Fig. 4, D and E). The impaired tolerance of KATP channel knockout hearts to stunning developed in the setting of similar ischemic indexes across groups (Table 1). Thus KATP channel deficit in a stunned myocardium aggravates ischemic contracture, reduces systolic performance, and culminates into diastolic dysfunction with compromised relaxation on reperfusion exaggerated in female hearts.


Figure 2
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  		Fig. 2. Ischemic contracture with brief ischemia. A: time to contracture during 10 min of global ischemia in WT (open) and Kir6.2-KO (filled) hearts. B: time to contracture during 10 min of global ischemia in male and female WT (open) and Kir6.2-KO (filled) hearts. C: peak ischemic contracture (mmHg) during 10 min of global ischemia in WT (open) and Kir6.2-KO (filled) hearts. D: peak ischemic contracture (mmHg) during 10 min of global ischemia in male and female WT (open) and Kir6.2-KO (filled) hearts. *P < 0.05 compared with WT values.

 

Figure 3
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  		Fig. 3. Systolic dysfunction following brief ischemia. A: rate-pressure product (heart rate x maximal left ventricular systolic pressure; bpm/mmHg) at baseline and at designated time points of reperfusion after 10 min of global ischemia in WT (open) and Kir6.2-KO (filled) hearts. B: rate-pressure product at baseline and at designated time points of reperfusion after 10 min of global ischemia in male WT (open) and Kir6.2-KO (filled) hearts. C: rate-pressure product at baseline and at designated time points of reperfusion after 10 min of global ischemia in female WT (open) and Kir6.2-KO (filled) hearts. *P < 0.05 compared with WT values.

 

Figure 4
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  		Fig. 4. Diastolic dysfunction after brief ischemia. A: left ventricular end-diastolic pressure (mmHg) at baseline and at designated time points of reperfusion after 10 min of global ischemia in WT and Kir6.2-KO hearts. B: left ventricular end-diastolic pressure (mmHg) at baseline and at designated time points of reperfusion after 10 min of global ischemia in male WT (open) and Kir6.2-KO (filled) hearts. C: left ventricular end-diastolic pressure (mmHg) at baseline and at designated time points of reperfusion after 10 min of global ischemia in female WT (open) and Kir6.2-KO (filled) hearts. *P < 0.05 compared with WT values; {dagger}P < 0.05 compared with male Kir6.2-KO. Relaxation abnormalities following myocardial ischemia. D: relaxation index –dP/dt (mmHg/s) at baseline and after 20 min of reperfusion (poststunning) in WT (open) and Kir6.2-KO (filled) isolated hearts. E: relaxation index –dP/dt (mmHg/s) following 20 min of reperfusion (poststunning) in male and female WT (open) and Kir6.2-KO (filled) isolated hearts. *P < 0.05 compared with baseline WT values; **P < 0.05 compared with poststunning WT control, ***P < 0.05 compared with poststunning female WT.

 

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  		Table 1. Metabolite levels and creatine kinase leakage: effect of stunning

 
Rescue of KATP Channel Knockout Phenotype

With the underscore of calcium mishandling in the KATP channel knockout phenotype, the poor contractile recovery in Kir6.2-KO hearts was prevented by treatment with the calcium/calmodulin cytosolic antagonist W7 or the regulator of intracellular calcium stores dantrolene, agents that share the aptitude to limit calcium accumulation and reduce ischemia-reperfusion injury (17, 37, 51). Specifically, pretreatment with W7 negated the elevation of LVEDP throughout the reperfusion period to levels similar to WT (Fig. 5). Similar rescue was observed with dantrolene (not illustrated). Thus restoring the ability of the KATP channel knockout heart to regulate calcium under ischemic stress prevented the functional consequences of their propensity to calcium overload.


Figure 5
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  		Fig. 5. Pharmacological rescue of Kir6.2-KO hearts from calcium mishandling abrogates diastolic dysfunction. Left ventricular end-diastolic pressure (mmHg) at baseline and at designated time points of reperfusion after 10 min of global ischemia in female WT (open) and Kir6.2-KO (filled) hearts untreated (light-shaded bars) or treated (dark-shaded bars) with 10 µM of the calcium/calmodulin antagonist W7. *P < 0.05 compared with WT ± W7 and Kir6.2-KO + W7.

 

   DISCUSSION
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 METHODS
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GRANTS
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In response to stress, maintenance of cardiac performance critically depends on efficient metabolic signaling and rapid adjustment of cardiomyocyte metabolism to preserve the intracellular milieu essential for contractile performance (8, 16, 18, 29, 47, 54). Sarcolemmal Kir6.2-containing KATP channels, tightly coupled with cellular metabolic pathways, have been implicated in the execution of the cardiac protective program in both health and disease (14, 30, 38). Genetic disruption of KATP channels has been associated with poor recovery following coronary hypoperfusion and compromised protective benefits of ischemic preconditioning (16, 50), whereas overexpression of channel subunits has been associated with increased resistance to ischemia (11). In fact, mutations in cardiac KATP channels have been identified in cardiomyopathy and associated with genetic predisposition to failure and/or arrhythmia (4, 40). It has been proposed that stress-induced KATP channel activity provides a metabolism-sensing feedback, shortening cardiac action potential duration that, in principle, would limit calcium influx and associated energy expenditure (2, 33, 38, 57, 60). Studies, to date, in isolated cardiomyocytes in vitro have indicated that deficiency in KATP channel function under stress may precipitate intracellular calcium loading (57, 60). Moreover, in cells transfected with functional KATP channels, calcium accumulation can be minimized in response to metabolic challenge (24). However, the inability to accurately replicate the metabolic demands of the intact working heart has limited our understanding of the role of KATP channels in securing ionic homeostasis in vivo.

With the use of manganese-enhanced cardiac MRI, the present study provides first evidence in vivo that myocardial stress results in increased calcium accumulation in the hearts of mice deficient in sarcolemmal KATP channels. The divalent manganese ion is a recognized indicator of Ca2+ influx, as Mn2+ enters cells through voltage-gated Ca2+ channels (41). Mn2+ is also paramagnetic, resulting in shortening of the spin-lattice relaxation time constant T1, which yields positive contrast enhancement in T1-weighted MRI, specific to tissues in which the ion has accumulated (19, 55). Manganese-enhanced MRI, applied in the present study, combines these properties to trace in an MRI-detectable manner regions of Ca2+ uptake. The inaptitude of Kir6.2-knockout hearts to regulate calcium influx manifested herein as an increased myocardial enhancement with Mn2+, correlating with prolongation of action potential and deficient myocardial repolarization associated with deficit in KATP channels (33, 52, 60). The propensity for exaggerated calcium loading in the KATP channel knockout lacking the pore-forming subunit Kir6.2 translated into sustained contracture revealed on imaging by reduced end-diastolic volumes. Moreover, in response to ischemia-induced stunning, Kir6.2-deficient hearts demonstrated both accelerated time to contracture and increased peak contracture compared with WT controls. The outcome on reperfusion was a reduction in systolic performance, with protracted increase in LVEDP and impaired cardiac relaxation and the development of overt diastolic dysfunction, an effect preventable by targeting parallel pathways of intracellular calcium handling. Collectively, these data support a vital role for KATP channels in preventing susceptibility to calcium overload in vivo, preserving contractile recovery under various conditions of high metabolic demand. The observed short- and long-term vulnerability of the Kir6.2 KO underscores the requirement for KATP channel checkpoints in preventing calcium accumulation in the setting of not only ischemia and stunning, but also under broader conditions of ventricular load (30). Calcium-overload predisposes to malignant calcium-triggered gene reprogramming and structural remodeling precipitating pump failure (27, 57). That genetic disruption of the Kir6.2 pore precipitates poor outcome is also in line with reports of compromised myocardial tolerance associated with use of KATP channel blocking agents or with mutations in channel subunits, as both acquired and innate cardiac KATP channelopathies have been documented (4, 30, 40).

Several reports suggest that sex differences exist with regard to the level of expression of KATP channels within the myocardium (6, 43). Here, compared with age-matched KATP channel-deficient males, females exhibited a higher propensity for calcium accumulation resulting in exaggerated diastolic dysfunction despite comparable parameters in ischemic indexes. The present findings thus provide further evidence for the suggested gender-specific resistance to metabolic stress associated with higher levels of KATP channels in females compared with males and a reduction in infarct size following ischemia-reperfusion injury (6, 23, 43). The protective action of estrogen on the ischemic myocardium is abolished by KATP channel blockade (32), whereas treatment with 17beta-estradiol appears to stimulate KATP channel expression (44) and prevent stress-induced calcium loading (26). Conversely, male-derived cardiac cells appear to accumulate higher levels of intracellular calcium in response to metabolic stress compared with female-derived cells (9). Moreover, sex differences in cardiac repolarization, illustrated by differences in action potential duration between the sexes (53), may predispose the female heart to a greater dependence upon KATP channel activity to regulate action potential duration and calcium handling in response to stress (31).

In summary, we demonstrate here in vivo, using live manganese-enhanced cardiac magnetic resonance imaging, that mice deficient in functional KATP channels exhibit a propensity for calcium accumulation in response to metabolic stress. The sequelae of a reduced tolerance toward increased calcium load were manifested in both male and female KATP channel KO hearts as impaired contractile recovery under conditions of high metabolic demand, with marked systolic and diastolic dysfunction demonstrated in the stunned myocardium. Exaggerated consequences were more common in females, suggesting a sex-dependent degree of reliance on KATP channel-mediated protection from calcium overload. From the established linkage between abnormal intracellular calcium handling and cardiac disease (54) and the recently reported KATP channel mutations in human heart disease (4, 40), the present demonstration of a mandatory role for intact KATP channels in the maintenance of ionic homeostasis in the working myocardium warrant further investigation of these cardioprotective channels as determinants of individual myocardial stress tolerance.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by 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. R. J. Gumina has received support from the Mayo Clinic Clinician-Investigator Program and is a Hartz Foundation Young Investigator. A. Terzic holds the Mayo Clinic Marriott Family Professorship in Cardiovascular Research.


   ACKNOWLEDGMENTS
 
This study was presented in abstract form at the American Heart Association Scientific Sessions, New Orleans, Louisiana, 2004 (Circulation 110: III-297, 2004).


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Terzic, Div. of Cardiovascular Diseases, Mayo Clinic, 200 First St. SW, Rochester, MN, 55905 (e-mail: terzic.andre{at}mayo.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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