INTRODUCTION

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ATP-SENSITIVE K⁺ (K_ATP) channels, which are highly expressed in myocardial sarcolemma, serve as membrane metabolic sensors that translate fluctuations in cellular energetics into regulation of electrical activity (1, 24, 25, 40). Nucleotide-dependent K⁺ permeation through Kir6.2, the inwardly rectifying pore-forming core of the K_ATP channel, is gated by ATPase activity of the regulatory subunit SUR2A integrated with cellular metabolism through phosphotransfer networks (1, 2, 4, 13, 33, 42). This metabolic sensor function is underscored in response to ischemic challenge, where sarcolemmal K_ATP channels have been proposed to respond to changes in cellular energetics that regulate ionic homeostasis (1, 5, 11, 15, 24, 25).

In fact, Kir6.2-knockout (Kir6.2-KO) hearts, which lack functional K_ATP channels, display a compromised ability to regulate electrical activity with loss of characteristic ST-segment elevation on the ECG during ischemia and poor contractile recovery (19, 35). Furthermore, intact sarcolemmal K_ATP channel function contributes to the reduction of infarct size afforded by ischemic preconditioning (IPC) (35), a cardioprotective phenomenon by which brief intermittent periods of ischemia protect the myocardium against a prolonged ischemic insult (21). Essential in the IPC-induced injury-tolerant state is the remodeling of energy transduction and cellular phosphotransfer networks, which results in maintained bioenergetic homeostasis and improved contractile recovery (10, 22, 26, 29). Metabolic flux through creatine kinase, the major phosphotransfer enzyme in the myocardium and integrator of cellular metabolism with K_ATP channels (1, 31), tightly correlates with cardioprotection of preconditioning (26). Although this correlation suggests a relationship between metabolic sensor activity and preservation of energetic homeostasis, it remains unexplored whether cardiac K_ATP channels are required for IPC-mediated protection of cellular bioenergetics.

Here, ¹⁸O-assisted ³¹P-NMR spectroscopy captures bioenergetic dynamics in hearts from wild-type (WT) and Kir6.2-KO mice. Deletion of sarcolemmal K_ATP channels did not perturb baseline energetics, but did negate IPC-induced protection of myocardial energy generation, transfer, utilization, and associated contractile recovery. Thus K_ATP channels are an integral component in IPC-induced preservation of cardiac bioenergetics.

	METHODS

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Experimental protocols. This investigation conformed to the Guidelines for the Care and Use of Laboratory Animals of the National Institutes of Health [DHEW Publication No. (NIH) 85-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20205] and was approved by the Institutional Animal Care and Use Committee at the Mayo Clinic. K_ATP channel-deficient mice were generated by targeted disruption of the Kir6.2 gene (20). Excised hearts from heparinized (60 U ip) and anesthetized (75 mg/kg of pentobarbital sodium ip) homozygous Kir6.2-KO or WT C57BL/6 mice (body wt, 25-30 g) were perfused on a Langendorff apparatus with a 95% O₂-5% CO₂ saturated Krebs-Henseleit (KH) solution (in mmol/l: 118 NaCl, 5.3 KCl, 2.0 CaCl₂, 19 NaHCO₃, 1.2 MgSO₄, 11.0 glucose, and 0.5 EDTA) at 37°C at a perfusion pressure of 80 mmHg. Rate-pressure product (RPP) was derived from continuous monitoring of the left ventricular pressure signal with the use of a fluid-filled, balloon-tipped pressure transducer (Harvard Apparatus). Hearts were paced at 500 beats/min. After 30 min of stabilization, WT and Kir6.2-KO hearts were either continuously perfused for 40 min (control) or were subjected to four IPC cycles of 5 min of ischemia and 5 min of reperfusion (Fig. 1). All hearts were then subjected to 30 min of zero-flow normothermic ischemia followed by a 30-min-long reperfusion and were labeled with ¹⁸O at the end of the respective protocols. Baseline data in the absence of ischemic stress were obtained with ¹⁸O labeling performed after 40 min of perfusion in both WT and Kir6.2-KO hearts.

View larger version (6K): [in this window] [in a new window]   Fig. 1.   Experimental protocol. Isolated hearts from wild-type (WT) or Kir6.2-knockout (KO) mice were subjected to either 40-min perfusion (control) or ischemic preconditioning (IPC) that consisted of four cycles of 5-min of ischemia followed by 5-min of reperfusion. All hearts were further subjected to 30 min of zero-flow global normothermic ischemia followed by a 30-min reperfusion. At the end of the reperfusion period, all hearts were labeled for 30 s with [18O]H2O perfusion buffer.

Labeling with ¹⁸O. The ¹⁸O-labeling technique allows for measurement of synthesis, transfer, and consumption of high-energy phosphoryl-carrying molecules (7, 41). Labeling with ¹⁸O-phosphoryl permits assessment of net phosphoryl flux through individual pathways, because it detects only newly generated molecules that contain ¹⁸O-labeled phosphoryls (26, 41). To capture the pseudolinear phase of labeling, ¹⁸O was delivered for 30 s in KH buffer supplemented with 30% of ¹⁸O[H₂O] (Isotec) (27). Hearts were freeze-clamped, pulverized under liquid N₂, and extracted in a solution that contained 0.6 M HClO₄ and 1 mM EDTA, and protein content was determined with a DC Protein Assay Kit (Bio-Rad). Extracts were neutralized with 2 M KHCO₃, and after processing, extracts were used for measurements of ¹⁸O-labeling rates and metabolite levels via ³¹P-NMR spectroscopy (27).

NMR spectroscopy. High-resolution ³¹P-NMR spectra were recorded in 5-mm tubes at ambient temperature on a Bruker 11 T spectrometer (Avance) at 202.5 MHz (26). With the use of a pulse width of 10 µs (53° angle), 21,000 scans were acquired without relaxation delay (acquisition time, 1.61 s). During signal acquisition, proton decoupling was applied with a 506-µs pulse width using the WALTZ-16 sequence at a 3-kHz radio frequency. Free induction decays were Fourier-transformed after zero filling to 32 K and filtered with a line-broadening factor of 0.3 Hz. Phase and baseline values were automatically corrected, and peak integrals were determined with a built-in integration routine (XWIN-NMR 2.5 software; Bruker). Signal intensities were corrected for the effects of nuclear Overhauser enhancement based on factors derived from recordings of typical samples with and without decoupling. The T₁ times, determined in typical samples by the inversion-recovery technique, were used to correct for metabolite signal attenuation that was produced by incomplete relaxation (26).

Calculation of ¹⁸O-labeling rates and metabolite levels. Cumulative percentage of ¹⁸O labeling in P_i was calculated as [percent ¹⁸O₁ + 2(percent ¹⁸O₂) + 3(percent ¹⁸O₃) + 4(percent ¹⁸O₄)]/[4(percent ¹⁸O in H₂O)], whereas the ¹⁸O labeling percentage values in -ATP and creatine phosphate (CrP) were calculated as [percent ¹⁸O₁ + 2(percent ¹⁸O₂) + 3(percent ¹⁸O₃)]/[3(percent ¹⁸O in H₂O)] (7). Cellular ATP turnover was estimated from the total number of ¹⁸O atoms that appeared in phosphoryls of P_i, -ATP, and CrP. Creatine kinase phosphotransfer flux was determined using pseudolinear approximation from the rate of appearance of CrP species that contain ¹⁸O-labeled phosphoryls, which reflects net flux through the creatine kinase reaction (9, 26, 27, 41). Tissue levels of P_i, ATP, and CrP were determined by ³¹P-NMR using methylene diphosphonic acid as a standard (26, 27).

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. Linear correlation was performed, and the fidelity of fit was assessed by the least-square method.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Deletion of K_ATP channels negates IPC-induced protection of ATP turnover required for functional recovery. Myocardial ATP turnover is a global parameter of cellular energetic dynamics (37). ATP turnover values at baseline in WT (n = 3) and Kir6.2-KO (n = 3) hearts were comparable at 560 ± 55 and 624 ± 55 nmol · mg protein¹ · min¹, respectively, with no significant difference in total ATP or CrP levels in the presence or absence of K_ATP channels (Table 1). After ischemia-reperfusion, a significant but comparable decrease in total ATP turnover was observed in WT (n = 7) and Kir6.2-KO (n = 7) hearts of 161 ± 32 and 151 ± 28 nmol · mg protein¹ · min¹, respectively. In ischemia-preconditioned WT hearts (n = 6), ATP turnover reached 236 ± 22 nmol · mg protein¹ · min¹, a value that is significantly higher than in the nonconditioned controls (P = 0.04; Fig. 2A). However, in Kir6.2-KO hearts (n = 7), IPC failed to improve ATP turnover, which remained at 159 ± 16 nmol · mg protein¹ · min¹, a value similar to control KO hearts (P = 0.81) and significantly lower than in preconditioned WT hearts (P = 0.04; Fig. 2A). IPC-induced protection of ATP turnover in WT hearts was associated with improved postischemic performance. On average, there was no difference in the RPP at baseline between WT and Kir6.2-KO hearts (21,933 ± 3,943 and 25,577 ± 608 mmHg/min, respectively). However, in WT control and preconditioned hearts, after 30 min of reperfusion, RPP values were 3,500 ± 1,363 (n = 7) vs. 13,645 ± 2,939 mmHg/min (n = 6), respectively (P = 0.001; Fig. 2B). In contrast with WT hearts, IPC failed to preserve contractile recovery in Kir6.2-KO hearts. Specifically, RPP values after 30 min of reperfusion were 1,821 ± 540 in Kir6.2-KO control (n = 7) vs. 1,772 ± 397 mmHg/min in Kir6.2-KO preconditioned (n = 6) hearts (P = 0.78). Thus hearts that lack functional sarcolemmal K_ATP channels fail to improve global cellular energetics that are required for contractile recovery induced by preconditioning.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   IPC-mediated protection of the rate of ATP turnover and functional recovery negated in KATP channel-KO hearts. A: total ATP turnover in WT control (WT-C) or ischemic preconditioned (WT-IPC) hearts and in Kir6.2-KO control (KO-C) or ischemic preconditioned (KO-IPC) hearts. B: rate-pressure product (RPP) in WT-C, WT-IPC, KO-C, and KO-IPC hearts.

Absence of IPC-induced protection of ATP utilization in K_ATP channel-KO hearts. Failure of IPC to improve total ATP turnover in Kir6.2-KO hearts could be due to effects on ATP generation and/or consumption (26). Here the ATP synthesis rate was monitored in the myocardium at baseline and after ischemia-reperfusion with or without preconditioning through incorporation of ¹⁸O into -phosphoryls of ATP. At baseline, both WT and Kir6.2-KO hearts demonstrated comparable rates of ATP synthesis, which was expressed as the percentage of ¹⁸O labeling of -ATP (Fig. 3, A and B); i.e., 90.4 ± 1.9 vs. 86 ± 5.5%, respectively (P = 0.77). The rates of ATP synthesis were essentially halved after ischemia-reperfusion in both WT and Kir6.2-KO hearts (Fig. 4). Although in WT hearts IPC, compared with nonconditioned controls, induced a trend toward an improved rate of ATP synthesis (57.6 ± 3.1 vs. 43.7 ± 5.3%; P = 0.053; Fig. 4), in Kir6.2-KO hearts, no increase in the rate of ATP production was observed with IPC (45.5 ± 3.6 vs. 43.8 ± 8.2%; Fig. 4). To assess ATP utilization rates, the appearance of ¹⁸O-labeled species in P_i was measured (Fig. 5, A and B). Myocardial ATP utilization rate, which was expressed as a percentage of ¹⁸O labeling of P_i during 30 s of labeling, was comparable at baseline in both WT (n = 3) and Kir6.2-KO (n = 3) hearts (58.4 ± 8.2 vs. 69.5 ± 0.5%, respectively; P = 0.27; see Fig. 3, C and D). The ¹⁸O-labeling values of P_i at reperfusion were 33.5 ± 6.5 and 50.6 ± 4.2% in WT nonconditioned (n = 7) vs. WT preconditioned (n = 6) hearts, respectively (P = 0.005; Fig. 5B), which demonstrates improved ATP consumption in preconditioned hearts. In contrast, no differences in myocardial ATP consumption rate were observed in Kir6.2-KO hearts with and without preconditioning (31.1 ± 3.9 vs. 27.1 ± 5.5%; P = 0.54; Fig. 5B), which indicates that IPC-mediated protection of ATP utilization was compromised in hearts deficient in sarcolemmal K_ATP channels. In fact, WT preconditioned hearts demonstrated a significant correlation (R² = 0.91) between ¹⁸O labeling of P_i and contractile performance (Fig. 5C), which indicates tight coupling between ATP consumption and the recovery of cardiac function. This close correlation was compromised in preconditioned Kir6.2-KO hearts (R² = 0.46; Fig. 5C). Additionally, the intracellular metabolite exchange, assessed as the ratio [¹⁸O]P_i/[¹⁸O]-ATP, was lower in preconditioned Kir6.2-KO hearts (0.67 ± 0.04; n = 7) compared with preconditioned WT hearts (0.88 ± 0.04; n = 6; P < 0.02; Fig. 5D), which indicates compromised communication between ATP-utilizing and ATP-generating processes in the K_ATP channel-deficient myocardium.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Preischemic energetic profile. A: baseline 18O/31P-NMR spectra of 18O-labeled -ATP in WT (WT-B) or Kir6.2-KO (KO-B) hearts. Incorporation of 18O induces an isotope shift of 0.0228 ppm in 31P-NMR spectra of -ATP. 16O, 18O1, and 18O2 designate -ATP phosphoryls with 0, 1, and 2 atoms of 18O. B: percent 18O labeling of -ATP, which is a measure of cellular ATP synthesis rate, in WT-B and KO-B hearts. C: baseline 18O/31P-NMR spectra of 18O-labeled Pi in WT-B and KO-B. Incorporation of 18O induces an isotope shift of 0.0210 ppm in 31P-NMR spectra of Pi. 16O, 18O1, 18O2, and 18O3 designate Pi phosphoryls that contain 0, 1, 2, and 3 atoms of 18O. D: percent 18O labeling of Pi, which is a measure of the cellular ATPase rate, in WT-B and KO-B hearts. E: baseline 18O/31P-NMR spectra of 18O-labeled creatine phosphate (CrP) in WT-B and KO-B hearts. Incorporation of 18O induces an isotope shift of 0.0250 ppm in the 31P-NMR spectra of CrP. 16O, 18O1, and 18O2 designate CrP phosphoryls that contain 0, 1, and 2 atoms of 18O. F: baseline creatine kinase flux presented as nanomoles of CrP produced in WT-B and KO-B hearts.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Deficit in IPC-mediated protection of the rate of ATP synthesis in KATP channel-KO hearts. A: 18O/31P-NMR spectra of 18O-labeled -ATP in WT control (WT-C) or ischemic preconditioned (WT-IPC) hearts and in Kir6.2-KO control (KO-C) or ischemic preconditioned (KO-IPC) hearts. Incorporation of 18O induces an isotope shift of 0.0228 ppm in 31P-NMR spectra of -ATP. 16O, 18O1, and 18O2 designate -ATP phosphoryls with 0, 1, and 2 atoms of 18O. B: percent 18O labeling of -ATP, a measure of cellular ATP synthesis rate, in WT-C, WT-IPC, KO-C, and KO-IPC hearts.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Deficit in IPC-mediated protection of the rate of ATP utilization and metabolite exchange in KATP channel-KO hearts. A: 18O/31P-NMR spectra of 18O-labeled Pi in WT-C, WT-IPC, KO-C, and KO-IPC hearts. Incorporation of 18O induces an isotope shift of 0.0210 ppm in 31P-NMR spectra of Pi. 16O, 18O1, 18O2, and 18O3 designate Pi phosphoryls that contain 0, 1, 2, and 3 atoms of 18O. B: percent 18O labeling of Pi, which is a measure of the cellular ATPase rate, in WT-C, WT-IPC, KO-C, and KO-IPC hearts. C: correlation between cellular ATPase rate, expressed as 18O labeling of Pi, and cardiac performance, expressed as the RPP, in WT-IPC and KO-IPC hearts after ischemia-reperfusion. Data were fit by linear regression, and the fidelity of fit was assessed based on R2 values. D: [18O]Pi/[18O]-ATP ratio in WT-C, WT-IPC, KO-C, and KO-IPC hearts.

IPC-induced protection of creatine kinase-mediated phosphotransfer compromised in K_ATP channel-KO hearts. In the myocardium, the creatine kinase network provides the major phosphotransfer pathway that integrates ATP synthesis with ATP consumption processes (6, 9, 32). The creatine kinase phosphotransfer rate was monitored at baseline (see Fig. 3, E and F) as well as after ischemia-reperfusion in nonconditioned and preconditioned myocardium (Fig. 6, A and B) through the appearance of phosphoryl species of CrP that contain ¹⁸O₁, ¹⁸O₂, and ¹⁸O₃. At baseline, the rate of creatine kinase-catalyzed flux was 466 ± 43 in WT (n = 3) vs. 513 ± 82 nmol · mg protein¹ · min¹ in Kir6.2-KO (n = 3) hearts (P = 0.45; see Fig. 3F). In WT ischemia-reperfused hearts, the creatine kinase-catalyzed flux rate was 149 ± 37 in nonconditioned (n = 7) vs. 242 ± 29 nmol · mg protein¹ · min¹ in preconditioned (n = 6) hearts (P = 0.03; Fig. 6B), which indicates that IPC preserved cellular high-energy phosphoryl transfer. However, in Kir6.2-KO hearts, preconditioning failed to preserve creatine kinase phosphotransfer rates, which were 145 ± 30 in nonconditioned (n = 7) vs. 141 ± 21 nmol · mg protein¹ · min¹ in preconditioned (n = 7) KO hearts (P = 0.86; Fig. 6B). In fact, although functional recovery in WT preconditioned hearts showed a strong correlation with creatine kinase flux (R² = 0.84), this was not maintained in Kir6.2-KO preconditioned hearts (R² = 0.59; Fig. 6C). Thus preconditioning-mediated protection of creatine kinase phosphotransfer is compromised in K_ATP channel-deficient hearts.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   IPC-mediated protection of creatine kinase phosphotransfer compromised in KATP channel-KO hearts. A: 18O/31P-NMR spectra of 18O-labeled CrP in WT-C, WT-IPC, KO-C, and KO-IPC hearts. Incorporation of 18O induces an isotope shift of 0.0250 ppm in the 31P-NMR spectra of CrP. 16O, 18O1, and 18O2 designate CrP phosphoryls that contain 0, 1, and 2 atoms of 18O. B: creatine kinase flux expressed as nanomoles of CrP produced in WT-C, WT-IPC, KO-C, and KO-IPC hearts. C: correlation between creatine kinase flux expressed as nanomoles of CrP generated and cardiac performance expressed as the RPP in WT-IPC and KO-IPC hearts after ischemia-reperfusion. Data were fit by linear regression, and the fidelity of fit was assessed based on R2 values.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In response to ischemic insult, survival of heart muscle critically depends on cellular recognition of metabolic deficits and subsequent preservation of bioenergetic and ionic homeostasis, which are necessary for optimal cardiac performance (9, 14, 26, 37, 39). Here the importance of metabolic sensors is highlighted by the failure of preconditioning to protect intracellular bioenergetics from ischemic injury in the absence of K_ATP channels.

After ischemia-reperfusion, IPC-induced energetic and functional improvements occurred in WT but not Kir6.2-KO hearts. Specifically, ¹⁸O/³¹P-NMR analysis of myocardial phosphotransfer dynamics demonstrated that in hearts with functional K_ATP channels, IPC preserved total myocardial ATP turnover rate associated with concomitant preservation of rates of both ATP generation and hydrolysis as indicated from higher ¹⁸O-labeling rates for -ATP and P_i. This is in line with previous reports that preconditioning preserves mitochondrial functions and the activities of cellular ATPases, thereby resulting in improved substrate oxidation, ionic homeostasis, and contractility (5, 10, 11, 17, 22). However, in hearts devoid of Kir6.2 and subjected to preconditioning episodes, total ATP turnover rate as well as ¹⁸O labeling of -ATP and P_i remained comparable to nonpreconditioned hearts; this indicates failure to induce a stress-tolerant bioenergetic state, which is necessary for maintained metabolic homeostasis.

Preservation of energetic homeostasis requires efficient coordination of ATP utilization and generation that is accomplished via phosphotransfer relays that facilitate high-energy phosphoryl delivery and removal of end products from sites of ATP hydrolysis (3, 9, 30, 36, 38). In this regard, intracellular metabolite exchange, which was assessed as the [¹⁸O]P_i/[¹⁸O]-ATP ratio after preconditioning (26), was improved in WT but not Kir6.2-KO hearts. Compromised metabolic communication after ischemia-reperfusion appears to be exacerbated by the lack of improvement upon the creatine kinase phosphotransfer system in the Kir6.2-KO heart subjected to the preconditioning stimulus. Preconditioning protected the creatine kinase phosphotransfer flux that was associated with higher CrP-labeling rates after ischemic injury in WT but not Kir6.2-KO hearts. Defective metabolic communication with impaired phosphotransfer networking contributes to reduction in the rates of ATP synthesis and/or consumption precipitating contractile dysfunction in the heart under stress (18, 26). In fact, loss of K_ATP channel coupling to creatine kinase phosphotransfer disrupts the integrated mechanism of metabolic sensing that is necessary for adjustment of membrane electrical activity with changes in cell metabolism (1, 31).

Action potentials of cardiomyocytes from Kir6.2-deficient hearts do not exhibit the degree of shortening that is normally observed in WT hearts under metabolic challenge (35), which is critical for accelerated cardiac membrane repolarization and reduction of potentially deleterious intracellular calcium accumulation (23, 43). Indeed, cardiomyocytes from Kir6.2-KO hearts display a propensity for calcium accumulation under stress (43). Although the rapid heart rate in mice may contribute to the relative importance of sarcolemmal K_ATP channels in the setting of ischemia-reperfusion (35), calcium overload is known to affect ATP-generating processes in mitochondria (12, 16, 34), thereby compromising the efficiency of energy transfer and ATP consumption (8, 17, 31) and reducing cardioprotective efficacy. In fact, not only the knockout of Kir6.2 (35), but also the expression of K_ATP channels with abnormal gating properties (28) reduce myocardial tolerance to ischemia or metabolic inhibition. Thus aberrance in K_ATP channel activity compromises sensing of the cellular metabolic state and regulation of ionic homeostasis (43) and ultimately interferes with the complex signaling pathways of IPC that are necessary for execution of the cardioprotective program (28, 35).

In summary, this study demonstrates that in response to preconditioning stimuli, the protection of cellular metabolism with maintained high-energy phosphoryl generation, transfer, and utilization that are required for contractile recovery, is compromised in the absence of functional cardiac K_ATP channels. Any adaptive alterations that may have occurred in the Kir6.2-KO mice, although apparently sufficient to maintain the bioenergetic profile under nonstressful conditions, were not capable of compensating for lack of K_ATP channel function in the setting of IPC. Thus intact K_ATP channel signaling is an integral component of the IPC-induced stress-tolerant bioenergetic state.