HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 285/6/H2437    most recent 00534.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (9) Citing Articles via Google Scholar Google Scholar Articles by Jansen, M. A. Articles by Balschi, J. A. Search for Related Content PubMed PubMed Citation Articles by Jansen, M. A. Articles by Balschi, J. A.

Energy requirements for the Na⁺ gradient in the oxygenated isolated heart: effect of changing the free energy of ATP hydrolysis

²Division of Cardiovascular Disease, Department of Medicine, University of Alabama, Birmingham, Alabama 35294-4470; and ¹NMR Laboratory for Physiological Chemistry, Division of Cardiovascular Medicine, Department of Medicine Brigham Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

Submitted 11 June 2003 ; accepted in final form 19 August 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This study tests the hypothesis that a decrease of the free energy of ATP hydrolysis (G_ATP) below a threshold value will inhibit Na⁺-K⁺-ATPase (Na⁺ pump) activity and result in an increase of intracellular Na⁺ concentration ([Na⁺]_i) in the heart. Conditions were designed in which hearts were solely dependent on ATP derived from oxidative phosphorylation. The only substrate supplied was the fatty acid butyrate (Bu) at either low, 0.1 mM (LowBu), or high, 4 mM (HighBu), concentrations. Escalating work demand reduced the G_ATP of the LowBu hearts. ³¹P, ²³Na, and ⁸⁷Rb NMR spectroscopy measured high-energy phosphate metabolites, [Na⁺]_i, and Rb⁺ uptake. Rb⁺ uptake was used to estimate Na⁺ pump activity. To measure [Na⁺]_i using a shift reagent for cations, extracellular Ca²⁺ was reduced to 0.85 mM, which eliminated work demand G_ATP reductions. Increasing extracellular Na⁺ () to 200 mM restored work demand G_ATP reductions. In response to higher [Na⁺]_e, [Na⁺]_i increased equally in LowBu and HighBu hearts to 8.6 mM, but G_ATP decreased only in LowBu hearts. At lowest work demand the LowBu heart G_ATP was –53 kJ/mol, Rb⁺ uptake was similar to that of HighBu hearts, and [Na⁺]_i was constant. At highest work demand the LowBu heart G_ATP decreased to –48 kJ/mol, the [Na⁺]_i increased to 25 mM, and Rb⁺ uptake was 56% of that in HighBu hearts. At the highest work demand the HighBu heart G_ATP was –54 kJ/mol and [Na⁺]_i increased only 10%. We conclude that a G_ATP below –50 kJ/mol limits the Na⁺ pump and prevents maintenance of [Na⁺]_i homeostasis.

energy metabolism; intracellular sodium

Na⁺ pump activity requires ATP. The energy available for chemical work from the ATP concentration ([ATP]) can be computed by the free energy of ATP hydrolysis (G_ATP). The free energy change for the Na⁺ and K⁺ movements of the Na⁺ pump reaction (G_Na-pump) is positive or endergonic. These ion movements occur because they are coupled to ATP hydrolysis, and the G_ATP is negative or exergonic. To obtain the free energy change for any reaction, or series of reactions, the Gs may be added. The sum (G) of G_Na-pump plus G_ATP must either be negative (meaning that the overall reaction is exergonic and can proceed) or zero (meaning that the reaction is at equilibrium). Tanford (24) concluded that the G = –12 kJ/mol in both the red blood cell and the squid axon and, therefore, that the substrates of the Na⁺ pump reaction are not at equilibrium. Kammermeier (13) estimated that the Na⁺ pump ion transport in the heart requires a minimum G_ATP of –46 kJ/mol of ATP or that G_Na-pump is 46 kJ/mol. Because the typical G_ATP in the heart is –56 to –60 kJ/mol, these estimations by Tanford and Kammermeier are similar. In contrast, Masuda et al. (19) concluded that the G was 0 in the perfused rat heart. Although the study of Masuda et al. (19) found a comparable G_ATP to other studies, it reported an intracellular Na⁺ concentration ([Na⁺]_i) of 1.6 mM in the heart, which results in a G_Na-pump of 59 kJ/mol. A [Na⁺]_i of 1.6 mM is much lower than most measurements of [Na⁺]_i in the rat heart (25), including those of the present study. Despite its potential importance, the relationship between [Na⁺]_i and G_ATP in the well-oxygenated heart has not yet been defined. This is the subject of this report.

The hypothesis tested in this study is that a decrease of G_ATP below a threshold value will inhibit Na⁺ pump activity and result in an increase in [Na⁺]_i in the well-oxygenated heart. To test this hypothesis, we employed hearts that are dependent on ATP derived totally from oxidative phosphorylation. In these hearts, G_ATP can be manipulated in the absence of ischemia or hypoxia.

These hearts, which were dependent entirely on exogenous substrate, were perfused with one of two butyrate (Bu) concentrations: one, with 0.1 mM (LowBu); or, two, with 4 mM (HighBu). Both groups of hearts entered a protocol of increasing work demand, which included increasing extracellular Na⁺ () to 200 mM. With the LowBu hearts, increased work demand reduced phosphocreatine (PCr) and ATP and, thus, G_ATP. In the LowBu hearts an increase in [Na⁺]_i was first noted as G_ATP decreased to approximately –52 kJ/mol; large increases in [Na⁺]_i occurred as G_ATP declined to approximately –48 kJ/mol.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Preparation of isolated perfused rat hearts. Male Sprague-Dawley rats (300–350 g), which had been fasted overnight, were treated with heparin (2,850 U/kg ip) and anesthetized with 200 mg/kg ip pentobarbital sodium. Under deep anesthesia, rat hearts were rapidly excised and placed in ice-cold Krebs-Henseleit buffer (KH). The aortas were cannulated, the left ventricle was vented, and constant 80-mmHg perfusion was initiated with KH at 37°C. The KH contained (in mM) 118 NaCl, 5.9 KCl, 1.2 MgSO₄, 25 NaHCO₃, 1.35 CaCl₂, 0.5 Na₂EDTA, and 10 D-glucose and was equilibrated with 95% O₂-5% CO₂, resulting in a pH of 7.4. During perfusion the KH was filtered with an inline 1.2-µm filter. Specific metabolic inhibitors or exogenous substrates were added to this perfusate as noted.

A latex balloon was inserted into the left ventricle (LV) and sutured in place to make the heart isovolumic. LV enddiastolic pressure was set to 8 mmHg. A Stratham P23dB pressure transducer connected by a catheter to the LV balloon measured the pressure and heart rate. All parameters were recorded with a MacLab system (ADInstruments, Mountain View, CA). Developed pressure was calculated as the difference between peak systolic pressure and end-diastolic pressure. All hearts were electrically paced using agarwick electrodes connected to a Grass stimulator (Grass, Quincy, MA). The hearts were placed in a latex bag to capture effluent, which was aspirated above the heart. This reduced the contributions to NMR signals from KH outside the heart.

The experimental protocol was approved by the Harvard Medical Area Standing Committee on Animals and followed the recommendations of the National Institutes of Health and the American Physiological Society guidelines for the use and care of laboratory animals.

Experimental protocol designed to create conditions in which hearts depend solely on oxidative metabolism and to increase the work demand (Table 1). To create conditions in which hearts depend solely on oxidative metabolism, it is necessary to control the availability of endogenous substrates and to eliminate glycolytic activity. First, all hearts were perfused with KH plus 0.3 mM 2-bromo-octanoate (BrO) for 30 min to inhibit 3-ketothiolase activity and, thus, the -oxidation of long-chain fatty acids (23) derived from endogenous triacylglycerols. Inhibition of 3-ketothiolase restricts only the oxidation of fatty acids with six or more carbons. Fatty acids of four carbons, such as Bu, are able to bypass this inhibition by BrO (2). To deplete glycogen, glycogenolysis was activated in these hearts by perfusion with KH containing 4 mM Bu and 0.2 mg/ml epinephrine but no glucose for 10 min. The KH Bu concentration ([Bu]) was then reduced to 0.1 mM for 16 min while hearts were paced at 300 beats/min (Glyc Dep). At the end of Glyc Dep, the hearts were glycogen depleted and, thus, totally dependent on exogenous oxidative substrates. Hence, we describe these hearts as dependent solely on oxidative phosphorylation for production of ATP.

The hearts were then perfused with KH containing 200 mM Na⁺ and either 0.1 mM Bu (LowBu) or 4 mM Bu (HighBu) and began three periods of increasing work demand during which NMR spectra were acquired (Table 1). First, heart rate was maintained at 300 beats/min for 16 min during work demand state 1 (HR300). Second, the heart rate was increased to 450 beats/min for 10 min during work demand state 2 (HR450). Finally, the heart rate was maintained at 450 beats/min, and the perfusate was switched to KH containing 80 µg/l dobutamine for 16 min during work demand state 3 (HR450DB). At the end of the protocol, the dry weight of each heart was determined after hearts were dried at 60°C for at least 48 h.

For this study, the use of the thulium(III) 1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetra(methylene-phosphonate) shift reagent (TmDOTP^5–) to measure intracellular Na⁺ () required a reduction in KH extracellular Ca²⁺ concentration ([Ca²⁺]_e).The reduction in [Ca²⁺]_e reduced changes in G_ATP normally observed during increased work demand with LowBu hearts observed with normal [Ca²⁺]_e. Increasing the to 200 mM restored the changes of G_ATP with increased work demand, allowing us to define the relationship between G_ATP and [Na⁺]_i.

Glycogen measurements. The glycogen content was measured in a separate series of hearts (15). Measurements were obtained in hearts at four times during the experimental protocol: first, after 10 min of perfusion with glucose KH (n = 2); second, after 30 min of perfusion with glucose KH containing BrO (n = 4); third, after 10 min of perfusion with KH containing 4 mM Bu and epinephrine (n = 4); and, fourth, after 15 min of perfusion with KH containing 0.1 mM Bu, which is the end of Glyc Dep (n = 4).

NMR measurements. Because of a change in equipment, half of the experiments of each group were done using a GE Omega (Bruker Instruments, Mountain View, CA) spectrometer and the other half using a Varian Unity INOVA (Varian Instruments, Palo Alto, CA) spectrometer. The spectrometers were equipped with a 9.4-T magnet and multinuclear 20-mm NMR probes.

³¹P NMR spectroscopy of isolated perfused hearts. ³¹P NMR free induction decays (FIDs) were acquired at 161.94 MHz with a 15-kHz spectral width into 1,024 data points. Typically, 48 FIDs were averaged using a 60° pulse width and a recycle time of 2.5 s for 2 min. The FIDs were multiplied by an exponential function and Fourier transformed to spectra. After phasing and baseline correction, the resonance areas and chemical shifts were quantified using the NMR1 curve fitting analysis program (Tripos, St. Louis, MO). All quantification was performed relative to the peak area of the reference signal. The reference capillary contained 15 µl of a 500 mM methylphosphonic acid (MPA) solution. Saturation factors for all resonances were determined from fully relaxed spectra acquired with a recycle time of 60 s.

For ³¹P NMR experiments of HighBu (n = 6) and LowBu hearts (n = 6), the KH included 5 mM phenylphosphonic acid (PPA) to measure extracellular water volume and 10 mM dimethyl-methylphosphonate (DMMP) to measure total water volume (8). The volume in the NMR tube surrounding the latex bag was filled with KH without PPA and DMMP.

²³Na NMR spectroscopy of isolated perfused hearts. ²³Na NMR FIDs were acquired at 105.5 MHz using a 5-kHz spectral width and 512 data points. Typically, 268 FIDs were accumulated after 90° pulses with a repetition time of 0.21 s for 1 min. FIDs were Fourier transformed after Gaussian multiplication. After polynomial baseline correction, the intracellular and reference ²³Na resonances were fitted to Gaussian lines using NMR1. All ²³Na spectra were fully relaxed. Quantification was performed relative to the peak area of the reference resonance. The reference capillary contained a known amount of 250 mM Na⁺ and 5 mM TmDOTP^5–. NMR visibility of all ²³Na signals was assumed to be 100% (25).

For ²³Na NMR measurements of HighBu (n = 5) and LowBu hearts (n = 6), the KH was modified by removal of EDTA and addition of the shift reagent for cations, TmDOTP^5–. Solid Na₄HTmDOTP (Magnetic Resonance Solutions, Dallas, TX) was added to the KH to attain a concentration of 3.5 mM. For KH with a [Na⁺] of 144 mM, NaCl was removed; for KH with a [Na⁺] of 200 mM, NaCl was added. To correct for the Ca²⁺ binding by the shift reagent, the total Ca²⁺ added to the standard perfusate was increased to 3.42 mM, which resulted in a free [Ca²⁺] of 0.85 mM measured by Ca²⁺-sensitive electrode. After each experiment, TmDOTP^5– was recovered and reused (21). The volume in the NMR tube surrounding the latex bag was filled with a solution of 148 mM LiCl, 1.2 mM MgSO₄, 3.4 mM CaCl₂, 10 mM HEPES, and 3.5 mM TmDOTP.

⁸⁷Rb NMR spectroscopy of isolated perfused hearts. ⁸⁷Rb NMR FIDs were acquired at 130.9 MHz into 128 data points with an 18-kHz spectral width. For each fully relaxed ⁸⁷Rb spectrum, 4,400 FIDs were accumulated after 90° pulses with a repetition time of 0.013 s for 1 min. FIDs were Fourier transformed after Lorentzian multiplication. After baseline correction, the ⁸⁷Rb resonances from the heart and reference capillary were fitted to Lorentzian lines using NMR1. Quantification was performed relative to the peak area of the reference signal. The reference capillary contained 10 µl of a 600 mM RbCl plus 5 M KI. NMR visibility of all ⁸⁷Rb signals was assumed to be 100% (9).

For ⁸⁷Rb NMR experiments of HighBu (n = 9) and LowBu hearts (n = 10), 20% of the perfusate KCl was replaced by 1.18 mM RbCl during part of HR300 and HR450DB; HR450 was not studied. The volume in the NMR tube surrounding the latex bag was filled with KH without RbCl. The ⁸⁷Rb NMR spectra consist of two resonances, one from the reference capillary and the resonance from the heart (). The resonance includes intracellular, extracellular, and KH perfusate surrounding the heart within the latex bag. The change in reports the accumulation of intracellular Rb⁺ () by the hearts after the extracellular Rb⁺ reaches steady state, typically 4 min. Using our estimates of extracellular and total volume from ³¹P NMR experiments, we estimated concentration ([Rb⁺]_i) (see below).

Heart ³¹P metabolite content. The concentration of the ³¹P NMR-visible metabolites was derived as follows

(1)

NMR-derived intracellular volume (V_i) was determined using the method of Clarke et al. (8).

The G_ATP was calculated from

(2)

where is –30.5 kJ/mol ATP (14), the gas constant (R) is 8.314 J/mol K, and temperature (T) is 310 K. The creatine kinase equilibrium expression was used to calculate free ADP (26)

(3)

where K_eq is 1.66 x 10⁹ M^–1. The creatine (Cr) content was estimated as the difference between heart PCr content, measured by ³¹P NMR, and the total Cr content in the heart (4).

Intracellular pH was calculated from the chemical shift of intracellular P_i (d) relative to that of PCr, by (6)

(4)

G_Na-pump was calculated from

(5)

where G_Na = RT ln [Na⁺]_e/[Na⁺]_i + zFV_m, G_K = RT ln [K⁺]_i/[K⁺]_e – zFV_m; extracellular K concentration ([K⁺]_e) = 5.9 mM, intracellular K⁺ concentration ([K⁺]_i) = 142 mM; extracellular Na⁺ concentration ([Na⁺]_e) = 144 mM in Glyc Dep or 200 mM in HR300-HR450DB; F is Faraday's constant (96,485 J/V mol); V_m is the membrane potential (0.085 V), and z is the valence of the ion.

Heart ²³Na⁺ content. The concentration of the ²³Na NMR-visible [Na⁺]_i was derived as follows

(6)

V_i was as determined from the ³¹P NMR experiments.

Heart ⁸⁷Rb⁺ uptake. The concentration of the heart ⁸⁷Rb NMR-visible Rb⁺ was derived as follows

(7)

(8)

where 1.18 mM = [Rb⁺] in the KH; extracellular volume (V_e) and V_i were determined in ³¹P NMR experiments.

Statistical analysis. The data are summarized as means ± SE for each measurement. A repeated-measures ANOVA compared measurements within each group. The test between any two means used Fisher's least protected significant difference test. Differences between the groups were analyzed using one-way ANOVA; the test between any two means used Fisher's least-protected significant difference test. Differences were declared statistically significant if P < 0.05. Statistical computations were performed with GBSTAT version 6.0 (Silver Spring, MD).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Glycogen content during the protocol to create conditions in which hearts are dependent solely on oxidative phosphorylation for the production of ATP. The glycogen content of an isolated rat heart perfused for 10 min with glucose KH was 194 ± 25 µmol glucose/g dry wt. Elimination of fatty acid oxidation by the addition of BrO to the perfusate (Table 1) further reduced glycogen to 62 ± 7 µmol glucose/g dry wt. Perfusion of the hearts with glucose-free KH containing 4.0 mM Bu plus epinephrine for 10 min reduced glycogen content to 32 ± 9 µmol glucose/g dry wt. Finally, at the end of Glyc Dep the glycogen content of the hearts was 8 ± 5 µmol glucose/g dry wt.

LV pressures during the protocol. All groups of hearts displayed similar LV pressures before and during Glyc Dep. With the start of the HR300 period and the creation of the LowBu (0.1 mM Bu) and HighBu (4.0 mM Bu) groups, the pressures began to differ. The function of hearts from both groups stabilized within 5 min after a change in the work demand state and remained stable in each state, except that during the latter part of HR450DB about one-half of the LowBu hearts stopped beating regularly.

³¹P NMR-measured metabolite content of the hearts during the protocol. ³¹P NMR measured the concentration of high-energy phosphate metabolites of the two experimental groups (Fig. 1). During HR300 when the perfusion with different KH [Bu] for the two groups began and the KH [Na⁺]_e increased, the PCr of LowBu hearts decreased, whereas that of HighBu increased. During the increased work demand of HR450 and HR450DB, PCr decreased and P_i increased in both groups.

View larger version (26K): [in this window] [in a new window]   Fig. 1. 31P NMR spectra of isolated hearts dependent solely on oxidative phosphorylation for production of ATP during the work demand states. 31P NMR resonances were assigned as follows: 1) dimethyl-methylphosphonate (DMMP) marker for total water space; 2) methylphosphonic acid (MPA) reference in capillary; 3) phenylphosphonate (PPA), the external water; 4) Pi; 5) phosphocreatine (PCr); and 6) the 3 resonances of ATP: -, -, and -phosphate resonance. NMR parameters and calculations are described in MATERIALS AND METHODS. A: LowBu heart. Spectra (bottom to top): Glyc Dep, at a heart rate (HR) of 300 beats/min, perfused with Krebs-Henseleit buffer (KH) containing 0.1 mM butyrate (Bu); HR300, at a HR of 300 beats/min, perfused with KH containing 0.1 mM Bu and 200 mM Na+ for remainder of protocol; HR450, paced at a HR of 450 beats/min; and HR450DB, paced at a HR of 450 beats/min plus 80 µg/l dobutamine. The free energy of ATP hydrolysis (GATP) calculated for each spectrum is shown at right. B: HighBu heart. Spectra (bottom to top): Glyc Dep, at a HR of 300 beats/min, perfused with KH containing 0.1 mM Bu; HR300, at HR of 300 beats/min, perfused with KH containing 4 mM Bu and 200 mM Na+ for remainder of protocol; HR450, paced at a HR of 450 beats/min; and HR450DB, paced at a HR of 450 beats/min plus 80 µg/l dobutamine. The GATP calculated for each spectrum is shown at right.

The changes in G_ATP of the hearts during the work demand states demonstrate four points. First, during the HR300 period when the KH was switched to 200 mM , the G_ATP of the LowBu hearts decreased (Fig. 2). This decrease in G_ATP probably results from the increased ATP demand for Na⁺ pumping. Early during HR300, [Na⁺]_i increased in both LowBu and HighBu hearts, suggesting an increased Na⁺ influx. Within 10 min, the LowBu heart G_ATP stabilized, indicating a new energetic balance between ATP supply and demand. Concurrently, the [Na⁺]_i stabilized, signaling a new balance between Na⁺ influx and efflux. Second, after stabilizing in the second half of HR300, the G_ATP of the LowBu hearts remained constant until the HR450DB period. Third, during the high work demand of HR450DB, the G_ATP of the LowBu hearts rapidly decreased to –48 kJ/mol. Finally, the HighBu hearts maintained values of G_ATP of approximately –56 kJ/mol ATP throughout HR300 and HR450. During the latter half of HR450DB, G_ATP decreased modestly to 54 kJ/mol ATP.

View larger version (19K): [in this window] [in a new window]   Fig. 2. GATP of LowBu and HighBu hearts during the work demand protocol. During Glyc Dep both groups were perfused with KH containing 0.1 mM Bu. Beginning with HR300, the KH for the LowBu hearts () contained 0.1 mM Bu with 200 mM Na+ and that for the HighBu () hearts contained 4.0 mM with 200 mM Na+. The workload challenge protocol consisted of 3 work demand states: HR300, pacing at a HR of 300 beats/min, HR450, pacing at a HR of 450 beats/min; and HR450DB, pacing at 450 beats/min with 80 µg/l dobutamine. Means ± SE are shown. *P < 0.05 within same group vs. measurement at time point A (15.5 min); #P < 0.05 within same group vs. measurement at time point B (41.5 min). At time point C (time = 21.5 min) measurements for the 2 groups become different (P < 0.05) and remain so for the rest of the protocol.

²³Na NMR-measured intracellular Na⁺ content of the hearts during the protocol. Stacked plots of ²³Na NMR spectra of LowBu and HighBu hearts are shown in Fig. 3, A and B, respectively. Perfusion with 144 mM Na⁺ KH containing TmDOTP^5– shifted the resonance downfield by 2.0 ppm. Switching to the 200 mM Na⁺ KH containing TmDOTP^5– at the beginning of HR300 decreased the shift to 1.7 ppm due to the lower ratio of [TmDOTP^5–] to [Na⁺]_e (7).

View larger version (54K): [in this window] [in a new window]   Fig. 3. 23Na NMR spectra of isolated hearts dependent solely on oxidative phosphorylation for production of ATP during the work demand states. 23Na NMR spectra obtained during perfusion with KH containing the shift reagent thulium(III) 1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetra(methylene-phosphonate) (TmDOTP5–). The 23Na NMR resonances of extracellular Na+ (), intracellular Na+ (), and the Na+ resonance of the reference sample () are identified. NMR parameters and calculations are described in MATERIALS AND METHODS. A: LowBu heart. The spectra (1 min each) are displayed from the Glyc Dep (front), HR300, HR450, and HR450DB (rear) periods. During Glyc Dep the heart was paced at a HR of 300 beats/min and perfused with KH containing 0.1 mM Bu and TmDOTP5–. During HR300 the hearts were paced at a rate of 300 beats/min. Perfusion was changed to KH containing 0.1 mM Bu, TmDOTP5–, and 200 mM Na+ for remainder of protocol. A slight decrease in the shift is evident, due to the lower ratio of [TmDOTP5–] to [Na+]e. During HR450 the hearts were paced at a HR of 450 beats/min. Finally, during HR450DB, the hearts were paced at a HR of 450 beats/min, and 80 µg/l dobutamine was added to the KH. B: HighBu heart. The spectra (1 min each) are displayed from the Glyc Dep (front), HR300, HR450, and HR450DB (rear) periods. During Glyc Dep the heart was paced at a HR of 300 beats/min and perfused with KH containing 0.1 mM Butyrate (Bu) and TmDOTP5–. During HR300 the hearts were paced at a rate of 300 beats/min. Perfusion was changed to KH containing 4.0 mM Bu, TmDOTP5–, and 200 mM Na+ for remainder of protocol. A slight decrease in the shift is evident, due to the lower ratio of [TmDOTP5–] to [Na+]e. During HR450 the hearts were paced at a HR of 450 beats/min. Finally, during HR450DB, the hearts were paced at a HR of 450 beats/min, and 80 µg/l dobutamine was added to the KH.

During Glyc Dep when the treatment of both groups was identical, [Na⁺]_i of both groups was the same (Fig. 4). During HR300 the two KH [Bu] were established, and [Na⁺]_e was increased. [Na⁺]_i increased comparably, 20%, in both groups, from 6.4 ± 0.3 to 8.6 ± 0.6 mM in LowBu hearts and from 6.7 ± 0.5 to 8.9 ± 0.7 mM in HighBu hearts. This represents an adjustment to the altered Na⁺ gradient: at the end of Glyc Dep the ratio of [Na⁺]_e to [Na⁺]_i for both groups was 22, at the beginning of HR300 it was 31, and at the end of HR300 the ratio was 23. Midway through HR450 the [Na⁺]_i of LowBu hearts increased to 11 mM while the [Na⁺]_i of HighBu hearts held constant. During HR450DB the LowBu heart [Na⁺]_i increased rapidly from 11 to 25 mM while the [Na⁺]_i of the HighBu hearts increased by only 10% to 9.6 mM.

View larger version (21K): [in this window] [in a new window]   Fig. 4. Intracellular Na+ concentration ([Na+]i) from isolated hearts dependent solely on oxidative phosphorylation for production of ATP during the work demand states. [Na+]i was calculated from the determination of content from 23Na NMR spectra and volume determinations in 31P NMR experiments (see MATERIALS AND METHODS). During Glyc Dep both groups were perfused with KH containing 0.1 mM Bu. Beginning with HR300, the KH for the LowBu hearts () contained 0.1 mM Bu with 200 mM Na+ and that for the HighBu () hearts contained 4.0 mM with 200 mM Na+. The workload challenge protocol consisted of 3 work demand states: HR300, pacing at a HR of 300 beats/min; HR450, pacing at a HR of 450 beats/min; and HR450DB, pacing at 450 beats/min with 80 µg/l dobutamine. Means ± SE are shown. *P < 0.05 vs. HR300 measurement (same group) at t = –31.5 min (point A); #P < 0.05 vs. HR450 measurement (same group) at t = –41.5 min (point B); point C, P < 0.05, LowBu group vs. HighBu group

⁸⁷Rb NMR-measured Rb⁺ uptake of the hearts during the protocol. ⁸⁷Rb NMR spectroscopy measurements of the Rb⁺ uptake were used to estimate Na⁺-K⁺-ATPase activity (Fig. 5). For these measurements, RbCl replaced 20% of the KCl in the KH during HR300 (t = 22 min) and at the beginning of HR450DB (t = 42 min). Net Rb⁺ uptake rates were calculated from the slope of a line fit to the [Rb⁺]_i for 6 min after extracellular Rb⁺ reached steady state, which began 4 min after the arrival of KH-containing Rb⁺. During this period Rb⁺ influx greatly exceeds Rb⁺ efflux.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Intracellular Rb+ concentration ([Rb+]i) in isolated hearts dependent solely on oxidative phosphorylation for production of ATP during the work demand states. A and B: [Rb+]i for the LowBu () and HighBu () group hearts after substitution of Rb+ for part of the K+ of the KH perfusate during HR300 (A) and HR450DB (B). Means ± SE [Rb+]i were calculated from the determination of Rb+ content from 87Rb NMR spectra and volume determinations from 31P NMR experiments. The fitting of the [Rb+]i to the equation of a straight line during HR300 (A) yielded the following parameters: first, for LowBu hearts, slope = 0.41 ± 0.21 mM/min, 95% confidence intervals (–0.02 to 0.84), r2 = 0.12; and, second, for HighBu hearts, slope = 0.66 ± 0.16 mM/min, 95% confidence intervals (0.33 to 0.99), r2 = 0.44. The slopes of the lines for the LowBu and HighBu groups were not different (P = 0.37); pooled slope for all data equals 0.52 mM/min. The fitting of the [Rb+]i to the equation of a straight line during HR450DB (B) yielded the following parameters: first, for LowBu hearts, slope = 0.40 ± 0.09 mM/min, 95% confidence intervals (0.20 to 0.59), r2 = 0.45; and, second, for HighBu hearts, slope = 0.71 ± 0.11 mM/min, 95% confidence intervals (0.49 to 0.94), r2 = 0.60. The slopes of the lines for the LowBu and HighBu groups were different (P = 0.04).

During HR300 the [Rb⁺]_i of LowBu and HighBu hearts was equal 4 min after introduction of Rb⁺ in the KH. Rb⁺ uptake rates during HR300 (time = 26.5 to 31.5 min) were as follows: LowBu hearts, 0.41 ± 0.21 mM/min (P = 0.37 vs. HighBu); and HighBu hearts, 0.66 ± 0.16 mM/min. The Rb⁺ uptake of the LowBu and HighBu hearts was similar during HR300.

Rb⁺ uptake rates during HR450DB (t = 46.5 to 51.5 min) were as follows: LowBu hearts, 0.40 ± 0.09 mM/min (P < 0.05 vs. HighBu); and HighBu hearts, 0.71 ± 0.11 mM/min. The Rb⁺ uptake of the LowBu hearts was 56% that of the HighBu hearts during HR450DB.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
The hypothesis tested here is that a decrease of G_ATP below a threshold value will inhibit Na⁺ pump activity and result in an increase in [Na⁺]_i in well-oxygenated intact hearts. To test this hypothesis, perfused rat hearts that were dependent solely on oxidative phosphorylation for production of ATP were used. A reduction in the G_ATP was achieved by limiting the heart's supply of exogenous substrate, increasing [Na⁺]_e, and by increasing the myocardial work demand. In this way G_ATP was manipulated in the absence of ischemia or hypoxia. We found that large increases in [Na⁺]_i occurred only when G_ATP was less than approximately –50 kJ/mol ATP.

Possible causes for accumulation of [Na⁺]_i. The [Na⁺]_i of LowBu hearts increased during HR450DB. Increasing [Na⁺]_i indicates that Na⁺ influx exceeds Na⁺ efflux. This may arise from enhanced Na⁺ influx and/or reduced Na⁺ efflux. Possible routes for enhanced Na⁺ influx are the Na⁺/H⁺ exchanger, the Na⁺-K⁺-Cl^– cotransporter, and the fast Na⁺ channel. Decreasing pH_i increases the activity of the Na⁺/H⁺ exchanger (3). In the present study, however, significant increases of occur at near normal intracellular pH so Na⁺ influx via the Na⁺/H⁺ exchanger is unlikely to be significant. The driving force, the chemical potential difference, for the Na⁺-K⁺-Cl^– cotransporter is directed into the cell at or near normal [Na⁺]_i (1). Therefore, the cotransporter is a likely pathway for Na⁺ influx that results during HR300 after the increase in [Na⁺]_e. There was no apparent change in the cotransporter driving force during the first half of HR450DB when the major increase in [Na⁺]_i began in LowBu hearts. Therefore, enhanced Na⁺ influx via the Na⁺-K⁺-Cl^– cotransporter is unlikely during that time. The Na⁺ channel may be the most likely route for increased Na⁺ influx. Simulation of the -receptor enhanced Na⁺ channel current in rat papillary muscle (16). The -receptor stimulation increase in work demand during HR450DB results in an immediate increase in ATP utilization, which suggests an effect on the ATP-dependent Na⁺ efflux by the Na⁺ pump. In the oxidative phosphorylation-inhibited heart, a increase comparable to the one observed in the present study was attributed to an inhibition of the Na⁺ pump (18). Thus an attenuated Na⁺ efflux by the Na⁺ pump is likely to be a significant factor in the [Na⁺]_i increase observed in the LowBu hearts during HR450DB.

Rb⁺ uptake and Na⁺ pump activity. Rb⁺, a surrogate for K⁺, can be transported into the myocyte through the Na⁺ pump, the Na⁺-K⁺-Cl^– cotransporter, and K⁺ channels. In the glucose KH-perfused rat heart, Kupriyanov et al. (17) demonstrated that the Na⁺ pump was responsible for 75–87% of the total Rb⁺ uptake, the Na⁺-K⁺-Cl^– cotransporter contributed <22% of the Rb⁺ uptake, and Rb⁺ uptake through ATP-sensitive K⁺ channels was negligible. In those hearts under steady-state conditions, the rate of Rb⁺ uptake was found to depend on Na⁺ uptake. In rat hearts in which oxidative phosphorylation was inhibited, [Na⁺]_i increased to 250% of control while Rb⁺ influx decreased to 82 ± 17% of control (18). An addition of glibenclamide, an inhibitor of ATP-sensitive K⁺ channels, decreased Rb⁺ influx to 47 ± 17% of control. The gain of intracellular Na⁺ was attributed to an inhibition of the Na⁺ pump. These studies indicate that the degree of Rb⁺ transport by the Na⁺ pump, while variable, is a significant portion of total Rb⁺ uptake.

During HR300 the Rb⁺ uptake by the LowBu and HighBu hearts was similar. The Rb⁺ uptake of the LowBu was, however, 44% below that of the HighBu hearts during HR450DB. This large reduction in Rb⁺ uptake is consistent with a decrease in Na⁺ pump activity.

Possible causes for the decrease in Na⁺ pump activity and increase . Depletion of ATP causes Na⁺-K⁺-ATPase inhibition. In our study the [ATP] of the LowBu hearts was the same during HR300 and HR450. Na⁺ accumulation began when [ATP] was 6.4 mM. The dobutamine stimulation of HR450DB results in a reduction of [ATP] to 2.8 mM. These [ATP] are well above the published K_m of 0.21 mM for the Na⁺-K⁺-ATPase for ATP (20). Consequently, decreased [ATP] is unlikely to kinetically limit Na⁺-K⁺-ATPase activity in this study. This conclusion assumes a homogeneous cytosolic ATP pool in the myocyte. In this regard, several reports suggest that the average cytosolic [ATP] does not represent the [ATP] accessible to the Na⁺ pump. Weiss and Hiltbrand (27) employed the perfused rabbit septum to measure extracellular [K⁺] under a variety of interventions, including hypoxia or perfusion with inhibitors of glycolysis or oxidative metabolism. They concluded that ATP from glycolysis was preferentially used to support sarcolemmal function, as assessed by K⁺ loss. While our metabolic conditions minimize or eliminate any potential contributions from glycolytic ATP, which may contribute to a nonhomogeneous [ATP], we cannot exclude the possibility that the [ATP] is nonhomogeneous.

P_i (5 mM) inhibits the Na⁺-K⁺-ATPase activity by 30% at pH 7.0 in isolated membrane preparations (12). Na⁺-K⁺-ATPase activity reductions of 30% resulted from 15 mM P_i in the presence of saturating levels of ATP (2 mM) (22). In LowBu hearts P_i was 18 mM during HR300 when Rb⁺ uptake was not reduced; it was 38 mM in HR450DB when Rb⁺ uptake was reduced. The [P_i] in the HighBu hearts was 13 and 15 mM in HR300 and HR450DB, respectively. Thus it is possible that P_i contributes to the reduction in Na⁺ pump activity found in this study.

P_i and ADP can combine to form a ternary abortive complex with the Na⁺ pump. Kupriyanov et al. (18) hypothesized that the Na⁺ pump was inhibited by a sevenfold increase of [ADP] x [P_i] in hearts with inhibited oxidative phosphorylation where increased to 250% of control. In that study the increase coincided with a [P_i] of 8 to 13 mM and an [ADP] of 0.100 mM, [ADP] x [P_i] = 1mM². The present results may provide some support for this thesis. The LowBu heart [Na⁺]_i 30% increase to 11 mM during HR450 occurred at a [ADP] x [P_i] of 2 mM². During HR450DB the large increase in [Na⁺]_i to 25 mM coincided with a [ADP] x [P_i] of 4 mM², while a modest 10% increase in [Na⁺]_i to 9.5 mM in the HighBu heart occurred when [ADP][P_i] was 1 mM². Thus elevated [ADP] x [P_i] coincided with increased [Na⁺]_i in the present study. The [ADP] x [P_i] values were, however, four times higher than those of Kupriyanov et al.'s hearts when similarly large increases in [Na⁺]_i occurred and only modest increases were observed at [ADP] x [P_i] = 1 mM². Because the value of G_ATP is dependent in part on [ADP] and [P_i] (see Eq. 2), it is, however, not possible to independently distinguish the effects of G_ATP from those of [ADP] and [P_i] in this study.

G_ATP, [Na⁺]_i, and Na⁺ pump activity. As G_ATP of the LowBu hearts decreased below –50 kJ/mol, [Na⁺]_i doubled (Fig. 6). It has been estimated that the minimal value of G_ATP required for the Na⁺ pump reaction is –46 kJ/mol ATP (14, 24). In ischemic skeletal muscle, Blum and co-workers (5) reported that increases in [Na⁺]_i began after G_ATP decreased below –49 kJ/mol. Glitsch and Tappe (10) measured the Na⁺ pump current (I_p) in Purkinje cells while altering G_ATP by perfusing the cell interior with appropriate amounts of ATP, ADP, and P_i (10). It was observed that at a G_ATP of approximately –49 kJ/mol, the I_p was 0 (indicating little or no Na⁺ pump activity) at membrane potentials more negative than –75 mV. Our results studying well-oxygenated hearts are in accord with these observations in Purkinje cells: the increases in [Na⁺]_i began as G_ATP approached –48 kJ/mol (Fig. 6) and Rb⁺ uptake was greatly diminished (Fig. 5).

View larger version (17K): [in this window] [in a new window]   Fig. 6. [Na+]i vs. GATP for the oxidative phosphorylation ATP-dependent hearts. [Na+]i (mM) is shown as a function of the GATP (kJ/mol) for the LowBu heart during the work demand protocol. Means ± SE are shown.

The relationship between G_ATP and G_Na-pump: G. If G_ATP decreases to less than the value required for the Na⁺-K⁺-ATPase reaction, its ion pumping must either cease or the gradient(s) will diminish to allow the reaction to continue. One way to assess this is to estimate G = G_Na-pump + G_ATP. The G for the HighBu hearts was –7 kJ/mol or more except for the end of HR450DB, when it approached –5 kJ/mol and the modest increase in [Na⁺]_i was observed. The G for the LowBu hearts was –4.6 kJ/mol or more except for the first 6 min of HR450DB, when it transiently decreased to –2.6 kJ/mol due to the rapid decrease in G_ATP. The rapid increases in [Na⁺]_i then increased G_Na-pump and restored G to approximately –4 kJ/mol. It is not known if this would have resulted in a new steady-state [Na⁺]_i. There are limitations to this analysis. The calculation of G_Na-pump used the measured [Na⁺]_i, but [K⁺]_i and membrane potential were not measured. Constant values were assumed for these parameters. It is likely that [K⁺]_i decreased as [Na⁺]_i increased (18). The value of G_Na, however, largely determines the value of G_Na-pump. G_K contribution is much less of an influence. The lack of accurate membrane potentials could have a larger influence. Thus the G_Na-pump provides a valid estimation of trends but may not provide absolute values.

In conclusion, the oxidative phosphorylation ATP-dependent hearts in this study achieved values of G_ATP that ranged from –56 to –47 kJ/mol ATP. This modulation of the value of G_ATP allowed us to define the relationship between [Na⁺]_i and G_ATP in the oxygenated heart. At G_ATP values below –50 kJ/mol ATP, the Rb⁺ uptake was greatly reduced and [Na⁺]_i doubled. We conclude that a G_ATP below –50 kJ/mol limits the Na⁺ pump and prevents maintenance of [Na⁺]_i homeostasis.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-46033 (to J. A. Balschi).

	ACKNOWLEDGMENTS

 
We thank Professor J. Ingwall for discussions and reading the manuscript.

Present address of M. A. Jansen: CardioNMR Laboratory, University Medical Center, Heidelberglaan 100, Room G02.523, 3584 CX Utrecht, The Netherlands.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. A. Balschi, 221 Longwood Ave., BLI 247, Boston, MA 02115 (E-mail: jbalschi{at}rics.bwh.harvard.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

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

1. Anderson SE, Dickinson CZ, Liu H, and Cala PM. Effects of Na-K-2Cl cotransport inhibition on myocardial Na and Ca during ischemia and reperfusion. Am J Physiol Cell Physiol 270: C608–C618, 1996.[Abstract/Free Full Text]
2. Balschi JA, Shen H, Madden MC, Hai JO, Bradley EL Jr, and Wolkowicz PE. Model systems for modulating the free energy of ATP hydrolysis in normoxically perfused rat hearts. J Mol Cell Cardiol 29: 3123–3133, 1997.[Web of Science][Medline]
3. Bielen FV, Bosteels S, and Verdonck F. Consequences of CO₂ acidosis for transmembrane Na⁺ transport and membrane current in rabbit cardiac Purkinje fibres. J Physiol 427: 325–345, 1990.[Abstract/Free Full Text]
4. Bittl JA, DeLayre J, and Ingwall JS. Rate equation for creatine kinase predicts the in vivo reaction velocity: ³¹P NMR surface coil studies in brain, heart, and skeletal muscle of the living rat. Biochemistry 26: 6083–6090, 1987.[Medline]
5. Blum H, Schnall MD, Chance B, and Buzby GP. Intracellular sodium flux and high-energy phosphorus metabolites in ischemic skeletal muscle. Am J Physiol Cell Physiol 255: C377–C384, 1988.[Abstract/Free Full Text]
6. Brindle KM, Rajagopalan B, Williams DS, Detre JA, Simplaceanu E, Ho C, and Radda GK. ³¹P NMR measurements of myocardial pH in vivo. Biochem Biophys Res Commun 151: 70–77, 1988.[Web of Science][Medline]
7. Chu SC, Pike MM, Fossel ET, Smith TW, Balschi JA, and Springer CS Jr. Aqueous shift reagents for high-resolution cationic nuclear magnetic resonance. III. Dy(TTHA)^3–, Tm(TTHA)^3–, and Tm(PPP) ^{7–. 2} J Magn Reson 56: 33–47, 1984.
8. Clarke K, Anderson RE, Nedelec JF, Foster DO, and Ally A. Intracellular and extracellular spaces and the direct quantification of molar intracellular concentrations of phosphorus metabolites in the isolated rat heart using ³¹P NMR spectroscopy and phosphonate markers. Magn Reson Med 32: 181–188, 1994.[Web of Science][Medline]
9. Cross HR, Radda GK, and Clarke K. The role of Na⁺-K⁺ ATPase activity during low flow ischemia in preventing myocardial injury: a ³¹P, ²³Na, and ⁸⁷Rb NMR spectroscopic study. Magn Reson Med 34: 673–685, 1995.[Web of Science][Medline]
10. Glitsch HG and Tappe A. Change of Na⁺ pump current reversal potential in sheep cardiac Purkinje cells with varying free energy of ATP hydrolysis. J Physiol 484: 605–616, 1995.[Abstract/Free Full Text]
11. Harold FM. The Vital Force: A Study of Bioenergetics. New York: Freeman, 1986.
12. Huang WH and Askari A. Regulation of Na⁺/K⁺-ATPase by inorganic phosphate: pH dependence and physiological implications. Biochem Biophys Res Commun 123: 438–443, 1984.[Web of Science][Medline]
13. Kammermeier H. High energy phosphate of the myocardium: concentration versus free energy change. Basic Res Cardiol 82: 31–36, 1987.
14. Kammermeier H. High energy phosphate of the myocardium: Concentration versus free energy change. In: Cardiac Energetics, edited by Jacob R, Just H, and Houlbarsch C. New York: Springer-Verlag, 1984, p. 31–6.
15. Keppler D and Decker K. Glycogen. In: Methods of Enzymatic Analysis (3rd ed.), edited by Bergmeyer HU. Weinheim, Germany: Verlag Chemie, 1983, p. 11–18.
16. Kirstein M, Eickhorn R, Kochsiek K, and Langenfeld H. Dose-dependent alteration of rat cardiac sodium current by isoproterenol: results from direct measurements on multicellular preparations. Pflügers Arch 431: 395–401, 1996.[Web of Science][Medline]
17. Kupriyanov VV, Stewart LC, Xiang B, Kwak J, and Deslauriers R. Pathways of Rb⁺ influx and their relation to intracellular [Na⁺] in the perfused rat heart. A ⁸⁷Rb and ²³Na NMR study. Circ Res 76: 839–851, 1995.[Abstract/Free Full Text]
18. Kupriyanov VV, Yang L, and Deslauriers R. Cytoplasmic phosphates in Na⁽⁺⁾-K⁺ balance in KCN-poisoned rat heart: a ⁸⁷Rb-, ²³Na-, and ³¹P-NMR study. Am J Physiol Heart Circ Physiol 270: H1303–H1311, 1996.[Abstract/Free Full Text]
19. Masuda T, Dobson GP, and Veech RL. The Gibbs-Donnan near-equilibrium system of heart. J Biol Chem 265: 20321–20334, 1990.[Abstract/Free Full Text]
20. Philipson KD and Nishimoto AY. ATP-dependent Na⁺ transport in cardiac sarcolemmal vesicles. Biochim Biophys Acta 733: 133–141, 1983.[Medline]
21. Pike MM, Luo CS, Clark MD, Kirk KA, Kitakaze M, Madden MC, Cragoe EJ Jr, and Pohost GM. NMR measurements of Na⁺ and cellular energy in ischemic rat heart: role of Na⁺/H⁺ exchange. Am J Physiol Heart Circ Physiol 265: H2017–H2026, 1993.[Abstract/Free Full Text]
22. Robinson JD, Flashner MS, and Marin GK. Inhibition of the Na⁺/K⁺-dependent ATPase by inorganic phosphate. Biochim Biophys Acta 509: 419–428, 1978.[Medline]
23. Schulz H. Inhibitors of fatty acid oxidation. Life Sci 40: 1443–1449, 1987.[Web of Science][Medline]
24. Tanford C. Equilibrium state of ATP-driven ion pumps in relation to physiological ion concentration gradients. J Gen Physiol 77: 223–229, 1981.[Free Full Text]
25. Van Echteld CJ, Kirkels JH, Eijgelshoven MH, van der Meer P, and Ruigrok TJ. Intracellular sodium during ischemia and calcium-free perfusion: a ²³Na NMR study. J Mol Cell Cardiol 23: 297–307, 1991.[Web of Science][Medline]
26. Veech RL, Lawson JW, Cornell NW, and Krebs HA. Cytosolic phosphorylation potential. J Biol Chem 254: 6538–6547, 1979.[Abstract/Free Full Text]
27. Weiss J and Hiltbrand B. Functional compartmentation of glycolytic versus oxidative metabolism in isolated rabbit heart. J Clin Invest 75: 436–447, 1985.[Web of Science][Medline]

This article has been cited by other articles:

				J. G. Richards, B. A. Sardella, and P. M. Schulte Regulation of pyruvate dehydrogenase in the common killifish, Fundulus heteroclitus, during hypoxia exposure. Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2008; 295(3): R979 - R990. [Abstract] [Full Text] [PDF]

				P. Crotty, T. Sangrey, and W. B Levy Metabolic Energy Cost of Action Potential Velocity J Neurophysiol, September 1, 2006; 96(3): 1237 - 1246. [Abstract] [Full Text] [PDF]

				J. A. Fraser and C. L.-H. Huang A quantitative analysis of cell volume and resting potential determination and regulation in excitable cells J. Physiol., September 1, 2004; 559(2): 459 - 478. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 285/6/H2437    most recent 00534.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Web of Science (9) Citing Articles via Google Scholar Google Scholar Articles by Jansen, M. A. Articles by Balschi, J. A. Search for Related Content PubMed PubMed Citation Articles by Jansen, M. A. Articles by Balschi, J. A.