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Am J Physiol Endocrinol Metab 293: E1828-E1835, 2007. First published October 2, 2007; doi:10.1152/ajpendo.00288.2007
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Greater glycogen utilization during β1- than β2-adrenergic receptor stimulation in the isolated perfused rat heart

Patrick McConville,1,2 Edward G. Lakatta,2 and Richard G. Spencer1

1Magnetic Resonance Imaging and Spectroscopy Section, Laboratory of Clinical Investigation, and 2Laboratory for Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Bethesda, Maryland

Submitted 9 May 2007 ; accepted in final form 28 September 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Differences in energy metabolism during β1- and β2-adrenergic receptor (AR) stimulation have been shown to translate to differences in the elicited functional responses. It has been suggested that differential access to glycogen during β1- compared with β2-AR stimulation may influence the peak functional response and modulation of the response during sustained adrenergic stimulation. Interleaved 13C- and 31P-NMR spectroscopy was used during β1- and β2-AR stimulation at matched peak workload (2.5 times baseline) in the isolated perfused rat heart to monitor glycogen levels, phosphorylation potential, and intracellular pH. Simultaneous measurements of left ventricular (LV) function [LV developed pressure (LVDP)], heart rate (HR), and rate-pressure product (RPP = LVDP x HR) were also performed. The heart was perfused under both substrate-free (SF) conditions and with exogenous glucose (G). The greater glycogenolysis was observed during β1- than β2-AR stimulation with G (54% vs. 38% reduction, P = 0.006) and SF (92% vs. 79% reduction, P = 0.04) perfusions. The greater β1-AR-mediated glycogenolysis was correlated with greater ability to sustain the initial contractile response. However, with SF perfusion, the duration of this ability was limited: excessive early glycogen depletion caused an earlier decline in LVDP and phosphorylation potential during β1- than β2-AR stimulation. Therefore, endogenous glycogen stores are depleted earlier and to a greater extent, despite a slightly weaker overall inotropic response, during β1- than β2-AR stimulation. These findings are consistent with β1-AR-specific PKA-dependent glycogen phosphorylase kinase signaling.

receptors; adrenergic; metabolism; magnetic resonance spectroscopy

METABOLISM IN THE HEART is modulated not only by changes in the rate of substrate utilization, but also by the choice of substrate and corresponding bioenergetic pathways. Stimulation of cardiac β-adrenergic receptors (β-ARs) by catecholamines leads to increased myocardial workload, supported by increased metabolic rate. However, workload and metabolic responses are strikingly different during stimulation of β1- compared with β2-ARs (23, 24), which act through distinct signaling pathways (27, 29).

Although free fatty acids (FFAs) are the preferred substrate of the heart under fasted conditions, the heart responds to an acute increase in workload by switching from FFAs to carbohydrates as the main source of fuel for respiration (5). During β-AR stimulation, there is a significantly greater increase in glucose and glycogen utilization than in FFA utilization (4, 7) and preferential oxidation of glycogen (7, 8). Glucose offers an energetic advantage over FFAs, providing more ATP per mole of oxygen consumed. Recent work in isolated, glucose-perfused rat hearts demonstrated a substantially greater maximal contractile response to β1- than β2-AR stimulation, accompanied by a similar decrease in intracellular energy charge (23). This raises a central question: Is the greater β1-AR-mediated contractile response supported by greater access to an additional metabolic substrate, such as glycogen? The hypothesis that preferential glycmetabolic substrate, such as glycogen.ogenolysis occurs during β1- compared with β2-AR stimulation is supported by previous work that demonstrated PKA-dependent phosphorylation of glycogen phosphorylase kinase during β1- but not β2-AR stimulation (19). However, this hypothesis has not been tested directly. In the normal heart, glycogenolysis, an inorganic phosphate (Pi)-dependent process, is upregulated by AMP and Pi and downregulated by ATP. These mechanisms provide a feedback loop between phosphorylation potential (PP) and glycogenolysis. However, although access to endogenous substrates would provide short-term benefits for the heart, the following question remains: Does excessive glycogen depletion have delayed effects on cardiac function (16, 28)?

Differences have also been described in the correlations between bioenergetic response and inotropic and chronotropic components of the response to altered workload. It was found that oxygen consumption and intracellular energy charge were not closely correlated with the chronotropic response, in contrast to the inotropic response (24). Whether the relative uncoupling of the chronotropic response extends to glycogenolysis is a further unanswered question.

Our laboratory has studied organ-level functional consequences of previously demonstrated mechanistic differences (23, 24). The isolated perfused heart model allows a convenient characterization of whole organ inotropic [left ventricular (LV) developed pressure (LVDP)] and chronotropic [heart rate (HR)] function. 31P-NMR permits real-time characterization of bioenergetic status via analysis of creatine kinase equilibria and intracellular pH (6, 12) and has been used to examine the metabolic effects of β-AR stimulation in isolated (15, 18) and in situ (2) rat hearts, isolated guinea pig hearts (13), in situ rabbit hearts (14), and human hearts (25). 13C-NMR can yield direct measurements of certain metabolic substrate concentrations, including glucose and glycogen, which provide distinct NMR signals (20, 21).

In the present study, we used 13C-NMR to test the hypothesis that glycogenolysis occurs to a greater extent during β1- than β2-AR stimulation under conditions of matched peak workload with substrate-free (SF) and glucose (G) perfusions. Additionally, we used 31P-NMR spectroscopy to determine phosphocreatine (PCr) and Pi content, as well as intracellular pH, under matched workload during β1- and β2-AR stimulation and SF and G perfusion to examine the bioenergetic consequences of β-AR-induced glycogen utilization.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolated heart preparation. Male Wistar rats (3–4 mo of age) were injected with 6-hydroxydopamine (20 mg/kg ip) 24 h before experimentation to attenuate the effect of endogenous catecholamines, as previously described (3). Hearts were rapidly excised under pentobarbital sodium anesthesia, perfused retrograde through the aorta with a filtered (0.45 µm), warmed (37°C), and gassed (95% O2-5% CO2) buffer in a nonrecirculating system, and allowed to beat spontaneously to allow examination of the inotropic effects of β-AR stimulation, as well as the stimulatory effects on the atrioventricular and sinoatrial nodes (chronotropic effects). The basic buffer consisted of 118 mM NaCl, 5 mM KCl, 0.5 mM Na2EDTA, 1.2 mM MgSO4, 25 mM NaHCO3, 1.8 mM CaCl2, and 1,100 U/l heparin. Glucose, lactate, insulin, or catecholamines were included in the perfusate as indicated. A water-filled polyethylene balloon connected to a pressure transducer was inserted into the LV and used to measure LV pressure. Balloon inflation was adjusted to achieve a preload of 10–15 mmHg.

13C- and 31P-NMR spectroscopy. Perfused hearts were placed in a 20-mm NMR tube and then inserted into a 9.4-T magnet (Magnex Scientific) interfaced to a spectrometer (model DMX; Bruker Analytik, Rheinstetten, Germany). Shimming was performed to achieve a water-proton line width of ≤40 Hz. After a stabilization period, 13C and 31P spectra were acquired continuously. By interleaving the excitation pulses and signal acquisitions for 13C and 31P, individual spectra for both nuclei were obtained every 3 min. Pulse flip angles of 30° and 45°, respectively, and repetition times of 0.2 and 1.5 s, respectively, were used for 13C and 31P acquisitions. In both cases, the spectral width was 12 kHz. Quantitation was performed with Lorentzian deconvolution. Small tubes containing 400 mM methylenediphosphonate and β-dioxane were placed external to the heart and used as intensity and chemical shift references for 31P and 13C spectra, respectively. For 31P spectra, resonance peaks corresponding to PCr and Pi were located at chemical shifts of +20 and +16 ppm relative to methylenediphosphonate, respectively. The ratio of the PCr peak magnitude to the sum of the PCr and Pi magnitudes was used as an index of PP (6), and intracellular pH was calculated from the chemical shift difference between the PCr and Pi resonances (12). For 13C spectra, a doublet resonance corresponding to {alpha}- and β-glucose isomers (93.0 and 96.9 ppm, respectively) was present during the [13C]glucose loading period (see below), with a [13C]glycogen peak (100.6 ppm) observed after 30–40 min of loading. Changes in glycogen concentration were followed throughout the adrenergic stimulation by changes in the [13C]glycogen peak area.

Experimental protocol. A perfusion flow rate of 28 ml/min was used to maintain adequate supply during the β-AR responses. This flow rate was based on the flow rates observed in previous studies in which a constant pressure of 120 mmHg was used. Labeling with [13C]glycogen was accomplished using a well-known technique (7–11). First, the heart was perfused with substrate-free buffer to deplete endogenous substrate reserves, as evidenced by an abrupt decline in developed pressure after 30–35 min. This decline (30–40% decrease in developed pressure) indicated the lack of sufficient endogenous substrates (e.g., glycogen and endogenous triglycerides) to maintain normal heart function. The heart was then reloaded with [13C]glycogen by perfusion with buffer containing 11 mM [1-13C]glucose, 0.5 mM lactate, and 0.1 U/l insulin, until the glycogen resonance in the 13C-NMR spectrum reached a steady level (50–60 min). During the glycogen reloading period, insulin was used to stimulate glycogen loading, and lactate was used to suppress glucose metabolism (Fig. 1), as described previously (9, 17).


Figure 1
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  		Fig. 1. Perfusion protocols: substrate-free (SF) and exogenous glucose (G). Common to both protocols were glycogen depletion and 13C loading perfusions at baseline (12 min), β-adrenergic receptor (AR) stimulation (24 min), and washout (16 min). Depletion was achieved via SF perfusion. 13C loading was achieved using 11 mM [1-13C]glucose, with lactate as additional exogenous substrate (0.5 mM) and insulin (0.1 U/l). Baseline perfusion also served to wash out the insulin and lactate that were used during the loading perfusion. The SF protocol enabled measurement of glycogen utilization when glycogen was the sole available substrate source during β-AR stimulation. The G protocol permitted assessment of the effect of exogenous glucose supply on glycogen utilization. Respective agonist + antagonist combinations were 3 x 10–8 M norepinephrine + 10–6 M prazosin, and 10–5 M zinterol + 1.5 x 10–6 M bisoprolol, for β1- and β2-AR-stimulated hearts, respectively.

 
There has been debate as to whether glycogen turnover-and-resynthesis is described by a "first glucose on-first glucose off" model or a random resynthesis-turnover model. However, our results are independent of this controversy, since the sharp decline in heart performance after 30–35 min of substrate-free perfusion shows that endogenous substrates, including glycogen, were almost fully depleted. Others have shown that 20 min of substrate-free perfusion results in ~50% depletion of glycogen (10, 11). Our 45-min substrate-free perfusion was therefore likely to have almost completely depleted glycogen and other endogenous substrates (9, 17).

A 12-min perfusion without lactate and insulin and with [12C]glucose substituted for [13C]glucose was then performed; this procedure permitted washout of insulin and lactate and established a baseline before the β-AR stimulation (Fig. 1). In experiments designed to examine glycogen utilization after matched peak workload, hearts underwent selective β1- (β1 group) or β2-AR stimulation (β2 group) during a 24-min dose of the β1-AR agonist norepinephrine (3 x 10–8 M) or the β2-AR agonist zinterol (10–5 M; Bristol-Myers Squibb). The doses were based on approximately maximal response to zinterol (23) and a titrated dose to match this response using norepinephrine. To augment receptor selectivity, an {alpha}-AR antagonist, prazosin (10–6 M), or a β1-AR antagonist, bisoprolol (1.5 x 10–6 M; Merck), was used during β1- and β2-AR stimulation, respectively, in combination with the agonists (19, 30, 31); these antagonists were also used during the baseline perfusion that preceded the β-AR stimulation.

Hearts underwent β-AR stimulation using two different buffer preparations. The first protocol (SF) used substrate-free buffer during the β-AR stimulation to examine glycogen as the sole available substrate. The second protocol (G) was designed to provide a controlled comparison of glycogen utilization when exogenous [13C]glucose substrate was also present. Throughout the protocol, LVDP and HR were recorded and averaged over 30-s intervals, and glycogen concentration, PP, and pH were recorded over 3-min intervals.

Statistics. Values are means ± SE. Pressure-derived and metabolic time courses were analyzed for main effects and interactions using repeated-measures ANOVA, with significance assumed at P = 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Heart function during depletion, loading, and baseline periods. Preliminary experiments demonstrated that the glycogen depletion-and-loading protocol (Fig. 1) resulted in consistent 13C labeling of the glycogen pool (Fig. 2) and consistent glycogen concentration during the postloading baseline period. A decline in LVDP (30–40% in all groups) was observed during the depletion period (data not shown), with a full recovery to baseline by the end of the loading period. During the baseline period, LVDP, HR, and RPP were not statistically different among the groups (Table 1). Between the SF and G protocols, only HR showed a statistically significant difference, which was of the same magnitude for the β1 and β2 groups (Table 1).


Figure 2
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  		Fig. 2. [13C]glycogen loading profiles for β1 and β2 groups with SF and G perfusion. Repeated-measures ANOVA showed no main effect of group and no group x time interaction.

 

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  		Table 1. Baseline and peak parameters for LVDP, HR, and RPP for all perfusion protocols

 
HR and contractile function. The peak RPP responses were well matched across both groups and both protocols, with no statistical differences (Fig. 3A). For both perfusion protocols and both agonists, HR reached a peak after ~10 min and was maintained at a relatively constant level throughout most of the catecholamine perfusion (Fig. 3C). Peak LVDP was reached more rapidly, within 2–3 min of the start of the catecholamine dose, and showed a decline throughout the remainder of the dosing period (Fig. 3B).


Figure 3
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  		Fig. 3. Rate-pressure product (RPP, A), left ventricular developed pressure (LVDP, B), and heart rate (HR, C) time courses for β1 and β2 groups during 24-min catecholamine dosing period with SF (n = 5 in β1 and β2) and G (n = 6 in β1, n = 7 in β2) perfusion. Time axis shows time relative to the start of catecholamine perfusion. Peak RPP, LVDP, and HR responses were similar in all cases. LVDP and RPP responses were not sustained throughout 24-min catecholamine dosing period [3 x 10–8 M norepinephrine (β1) and 10–5 M zinterol (β2)]. Onset of LVDP and RPP responses was slightly earlier in β2-AR-stimulated hearts, but these responses were not sustained to as great an extent initially as in β1-AR-stimulated hearts. HR responses showed a similar time course in all cases and were maintained at a relatively constant level throughout the dosing period. HR responses were greater in the β2 than in the β1 groups with G vs. SF protocol.

 
For the β1 and β2 groups, the G perfusion protocol showed somewhat greater peak LVDP responses (Fig. 3B) but somewhat lower peak HR responses (Fig. 3C) than the SF protocol. For the β1 and β2 groups, the peak LVDP decline was significantly greater for the SF than the G protocol (Fig. 3B). In the β1 group, an abrupt increase in the rate of LVDP decline was observed during SF perfusion after ~10 min, in contrast to the more constant rate of LVDP decrease during the G perfusion (Fig. 3B). By the end of the catecholamine dosing period in the SF protocol, LVDP was below baseline in both groups. In the β2 groups, the LVDP decrease was monotonic in the SF and G protocols (Fig. 3B).

After the start of the catecholamine perfusion in the SF and G protocols, the onset of the inotropic response occurred earlier in the β2 than β1 group (Fig. 3B, Table 1). In the SF and G β2 groups, the LVDP response decreased monotonically within 30 s of the initial peak response and had decreased by 5% after 2 min (Fig. 3B). In contrast, in both β1 groups, LVDP was sustained within 5% of the peak response for a longer period (~3.5 min). With SF and G protocols, a greater HR response was observed in the β2 than in the β1 group (Fig. 3C). Coupled with the earlier onset of the β2 LVDP responses, this resulted in a greater RPP response in the β2 than in the β1 group throughout most of the stimulation period (Fig. 3A).

Glycogen utilization. With the SF protocol, significant glycogenolysis was observed in the β1 and β2 groups during stimulation (Fig. 4). However, the onset of the β-AR-mediated glycogen utilization occurred earlier in the β1 group, despite the later onset of the contractile response in this group (Fig. 3A). This led to a significantly greater overall decrease in glycogen concentration over the course of the catecholamine dosing period in the β1 than in the β2 group (P = 0.006), despite the smaller overall RPP profile in the β1 group (Fig. 3A).


Figure 4
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  		Fig. 4. Glycogen concentration in β1- and β2-AR-stimulated hearts during 24-min catecholamine dosing period with SF (n = 5 in β1 and β2) and G (n = 6 in β1, n = 7 in β2) perfusion. Glycogen concentration was calculated as percent baseline and is shown at 3-min intervals. With SF protocol, a more rapid and greater overall decrease (P = 0.006) in glycogen concentration was observed in the β1 vs. the β2 group. With the G protocol, a greater degree of glycogen depletion (P = 0.04) was also observed in the β1 than in the β2 group. *P < 0.05, β1 vs. β2 (repeated-measures ANOVA).

 
With the G protocol, glycogenolysis again occurred in both groups (Fig. 4) during the β-AR stimulation period, although to less than half the extent observed with the SF protocol. The timing of the onset of glycogen depletion was similar in each group relative to the start of the catecholamine dose, as was the initial rate of glycogen depletion as defined by the slope of the curves in Fig. 4. However, the rate of depletion was maintained to a higher degree in the β1 than β2 group, in which the rate of depletion became progressively slower over the dosing period. These trends resulted in significantly greater overall depletion of glycogen with the G protocol in the β1 than β2 group (P = 0.04).

Phosphorylation potential. With the SF protocol, phosphorylation potential (PP) decreased during the first few minutes of the β-AR stimulation period in the β1 and β2 groups. However, this initial decrease was followed by a transient increase in PP in all six cases in the β2 group (Fig. 5) before a subsequent persistent decrease. In contrast, in the β1 group, a monotonic decrease was observed throughout the β1-AR stimulation period. By the end of the β-AR stimulation period, PP had decreased to the same extent with the SF protocol in each group.


Figure 5
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  		Fig. 5. Phosphorylation potential (PP) for β1- and β2-AR-stimulated hearts during 24-min catecholamine dosing period with SF (n = 5 in β1 and β2) and G (n = 6 in β1, n = 7 in β2) perfusion. PP was calculated as percent baseline and is shown at 3-min intervals. With the SF protocol, a more rapid decrease in PP was observed in the β1 than in the β2 group, which showed a transient increase in PP shortly after the start of the catecholamine dosing period. With the G protocol, pattern and extent of PP decrease were similar in β1 and β2 groups.

 
With the G protocol, the profile of the PP decrease was similar in each group. An initial decrease of ~20% occurred soon after the start of the catecholamine perfusion in both groups. PP was relatively well maintained for the remainder of the dosing period in both groups.

pH. Baseline pH was 7.11–7.18. In both groups, with SF and G perfusion, although changes in pH were relatively small and signal-to-noise considerations limited the accuracy of the pH determinations, pH decreased measurably during the first half of the catecholamine dosing period and then recovered toward baseline. Furthermore, trends that differentiated the β1 from β2 groups were observed. The extent of the initial transient pH decrease was, in general, greater in the β1 groups, in which the decrease was similar for the SF and G protocols, than in the β2 groups, in which the SF and G protocol results were also similar. In addition, there was a trend toward lower pH during the later stage of the catecholamine dosing period in the β1 than β2 groups (Fig. 6).


Figure 6
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  		Fig. 6. Change in intracellular pH in β1- and β2-AR-stimulated hearts during the 24-min catecholamine dosing period with SF (n = 5 in β1 and β2) and G (n = 6 in β1, n = 7 in β2) perfusion. β1-AR-stimulated hearts showed a greater and more rapid decrease in pH than β2-AR stimulated hearts (with SF and G perfusions). Only the β1 group with G perfusion did not show full recovery of the pH decrease by the end of the dose.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
During β-AR stimulation, the heart responds to acute workload increase by preferential metabolism of carbohydrates and oxidative metabolism of glycogen (7, 8). Differential glycogen metabolism has been implicated in the marked functional and mechanistic differences between β1- and β2-AR stimulation (23). From a functional perspective, we showed previously that, for a similar contractile response, intracellular energy charge decreases to a greater extent in β2- than β1-AR-stimulated hearts, suggesting that there may be enhanced access to metabolic substrate during β1- compared with β2-AR stimulation. In the present study, a similar peak contractile response and greater overall contractile response in the β2 than β1 group was shown to elicit greater glycogenolysis in β1- than β2-AR-stimulated hearts. Figure 7 summarizes the factors influencing metabolism and workload response during β-AR stimulation.


Figure 7
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  		Fig. 7. Major factors expected to contribute to feedback between workload and metabolism during β-AR stimulation. Arrows and their directions indicate influences of one factor over another. Thicker lines and dotted lines indicate stronger and weaker effects, respectively. Left: metabolic factors; right: contractile factors. Circles labeled SF and G highlight SF and G perfusions and differential effects between these perfusions as found in the present study; triangles highlight findings in the present study that suggest differential influences during β1- compared with β2-AR stimulation. Major findings of the present study, as indicated by numbers (1–4) are as follows. 1) Glycogenolysis occurs to a greater extent during β1- than β2-AR stimulation; 2 and 3) β1-AR stimulation elicited a greater inotropic than chronotropic component of the contractile response than β2-AR stimulation, which was tied to a greater chronotropic component. A similar trend was observed with G compared with SF protocol. 4) Glycogen use was less efficient during β1- than during β2-AR stimulation. These findings may be explained by β1-AR stimulation-dependent Gs-cAMP-PKA signaling, leading to increased intracellular Ca2+ levels and glycogen phosphorylase kinase activation, associating glycogenolysis with the inotropic response and futile Ca2+ turnover.

 
Greater glycogenolysis during β1- compared with β2-AR stimulation after matched peak workload. With SF perfusion, β1- and β2-AR stimulation resulted in substantial depletion of glycogen (Fig. 4). However, despite a slightly greater overall contractile response of the β2 group (earlier RPP onset, greater peak RPP response, and more sustained RPP response over the dose duration than in the β1 group; Fig. 3A), a greater relative decrease in glycogen concentration was observed in the β1 group at all time points after baseline. With glucose included as an exogenous substrate (G perfusion protocol; Fig. 4), glycogen was significantly depleted again during β1- and β2-AR stimulation, with a greater relative decrease in glycogen concentration during β1-AR stimulation (Fig. 4). These data constitute direct evidence for greater glycogenolysis associated with β1- than β2-AR stimulation and are consistent with β1-AR-mediated activation of glycogen phosphorylase kinase.

Bioenergetic status, as measured by PP, can be correlated with the observed glycogen utilization and contractile responses and reflects the benefit of glycogen availability (high PP) and the consequences of glycogen nonavailability (decreased PP). With the SF protocol, the greater initial decrease in PP in the β2 than β1 group (Fig. 5) corresponds to the initially lower rate of glycogenolysis in the β2 group (Fig. 4), despite a similar, although somewhat earlier, contractile response in the β2 group (Fig. 3A). This relationship between glycogenolysis and PP is consistent with Pi-dependent upregulation of glycogenolysis (Fig. 7). Furthermore, the secondary, transient PP increase in the β2 group with SF perfusion (Fig. 5) is consistent with the observation that provision of glucose derived from glycogenolysis is delayed in this group compared with the β1 group with SF perfusion (Fig. 4), which may have led to a transient mismatch between energy supply and demand (which had decreased during the delay; Fig. 3A). The monotonically decreasing PP observed after this transient increase in the β2 group with SF perfusion and throughout the catecholamine dosing period in all other groups (Fig. 5) indicates limited substrate supply.

With the G protocol, PP decreased in a similar manner in the β1 and β2 groups (Fig. 5), reflecting the similar contractile responses. It is noteworthy that the β2 group with G perfusion showed a greater initial decline in PP, again consistent with initially decreased or delayed glycogenolysis in this group compared with the β1 group with G perfusion. The LVDP inotropic response of the β1 group with G perfusion was maintained to a greater extent than that of the β2 group with G perfusion, yet the respective PP time courses were generally similar, suggesting greater bioenergetic supply in the β1 group with G perfusion. This is consistent with greater glycogenolysis during β1- than β2-AR stimulation (Fig. 4).

Greater workload maintenance during β2- compared with β1-AR stimulation with SF perfusion. With the SF protocol, the generally more sustained RPP during β2- than β1-AR stimulation (Fig. 3A), coupled with the greater glycogenolysis during β1-AR stimulation (Fig. 4), suggests that glycogen-derived glucose was better able to maintain workload during β2- than β1-AR stimulation. PP, a relative measure of the energy available to meet the contractile demands, showed a greater decline with β1- than β2-AR stimulation (Fig. 5). Potential explanations for the lower workload during β1-AR stimulation include greater dependence on less efficient anaerobic bioenergetic pathways and increased futile energy cycling during β1- than β2-AR stimulation. For example, spontaneous oscillations of intracellular Ca2+ concentration have been implicated in energy waste during β1-AR, but not β2-AR, stimulation (Fig. 7, arrow 4) (1, 31). Although it has not been tied specifically to β1-AR stimulation, a possible further source of energy waste is the abnormally increased cross-bridge cycling that has been measured during β-AR stimulation (26).

With the G protocol, the availability of exogenous glucose provided an alternative energy substrate; therefore, conclusions about workload maintenance due to glycogenolysis alone cannot be drawn. Although glycogenolysis was still greater during β1- than β2-AR stimulation with the G protocol, RPP and PP responses were similar. This suggests that the availability of exogenous glucose diminished the different degree for which glycogenolysis supported workload between β1- and β2-AR stimulation, as observed with SF perfusion (Fig. 3A).

Although pH changes were relatively small (within 0.1 unit) throughout the perfusion protocols, there were measurable trends that differentiated the β1 and β2 groups in a consistent manner in the SF and G protocols. The greater and earlier relative pH decline in the β1 than β2 groups suggests a greater degree of anaerobic metabolism during β1-AR stimulation (Fig. 6), especially during the initial part of the dose, when the functional demand was greatest. However, it should be noted that the relatively small Pi resonance amplitudes limited the accuracy of the pH determination.

Uncoupling of the chronotropic response from glycogenolysis and the bioenergetic state. In support of previous findings (24), the constancy of the HR responses during catecholamine dosing (Fig. 3C) suggests that HR during sustained β1- or β2-AR stimulation is relatively uncoupled from metabolic status, including the rate of glycogenolysis (Fig. 4) and PP (Fig. 5). Figure 8B, in which HR is plotted directly against relative glycogen concentration, illustrates this uncoupling directly by showing the relative constancy of HR across a wide range of (decreasing) values for glycogen content. In contrast, the parallel decreases in RPP, PP, and glycogen suggest that the coupling of contractility and substrate demand is seen predominantly through the inotropic response.


Figure 8
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  		Fig. 8. A: RPP vs. relative glycogen content. Data are from Figs. 3A and 4, with the time axis factored out to directly illustrate the relationship between RPP and glycogen. Error bars (which are shown in Figs. 3A and 4) are omitted for clarity. RPP declines when glycogen is depleted by ~20%, with all four groups exhibiting a similar relationship between these two variables. B: HR vs. relative glycogen content. Data are from Figs. 3C and 4, with the time axis factored out. Error bars are omitted for clarity. HR remains fairly constant across a wide range of glycogen concentrations, with the four groups each demonstrating a quantitatively different relationship between these two variables.

 
Furthermore, the HR responses were greater with the SF (30% and 40% increase in β1 and β2, respectively) than the G (20% and 30% increase in β1 and β2, respectively) protocol. The greater depletion of glycogen and greater decline in PP with the SF than the G protocol would be expected to result in diminished contractile response with the SF protocol. Indeed, this was seen in RPP, but not in HR. This is again consistent with an uncoupling of the chronotropic component of the RPP from glycogen depletion and the PP decrease.

Correlation of the inotropic responses to β1- and β2-AR stimulation with glycogenolysis and the bioenergetic state. In contrast to the HR responses, during β1- and β2-AR stimulation with the SF and G protocols, the inotropic responses showed a significant decline over the catecholamine dosing period and, therefore, a stronger correlation with decreasing glycogen levels (Figs. 4 and 8) and PP (Fig. 5). The larger degree of inotropic (>100% increase in LVDP) than chronotropic (<50% increase in HR) responses underscores the correlation between inotropy and metabolism and is consistent with previous findings (23, 24). Previous studies have associated high inotropic state with energy wastage due to Ca2+ mishandling (1) through phenomena such as spontaneous Ca2+ oscillations (31). Increased cross-bridge cycling associated with β-AR-induced inotropy (26) may also be a factor in decreased ability to support the inotropic state. This may account for the decline in the inotropic, but not chronotropic, response during supply limitation, (e.g., in the SF protocol), which could reduce energy wastage associated with high inotropy.

The different time courses of the decrease in inotropic response during β1- and β2-AR stimulation (Fig. 3B) correspond to different time courses for glycogen concentration (Fig. 4), further indicating the coupling between inotropy and glycogenolysis. With SF perfusion, the β1 group showed greater initial glycogen depletion (Fig. 4) than the β2 group, consistent with greater substrate utilization to sustain peak LVDP (Fig. 3B). However, there was a greater subsequent rate of decline of the LVDP response in the β1 group with SF perfusion, beginning at approximately the halfway point of the catecholamine dosing period (Fig. 3B). Additionally, the HR response began to decline at the same time and continued to decrease during the later half of the dosing period for this group (Fig. 3C), the only group for which HR declined from its peak. This is consistent with the interpretation that early glycogen depletion initially supported a sustained high workload but then led to a lack of adequate substrate to support LVDP or HR. Indeed, potential negative sequelae of excessive glycogen depletion have been emphasized in recent studies (16, 28).

Exogenous glucose (G protocol) supported less decline in inotropic response in the β1 and β2 groups than in the SF perfusion (Fig. 3B). After the initial increase in RPP during the dosing period, the LVDP response was sustained to a greater degree with G perfusion in the β1 than β2 group. The early maintenance of this response in the β1 G group is consistent with the greater and earlier glycogenolysis exhibited by the β1 G group (Fig. 4), again indicating a correlation between inotropy and glycogenolysis.

Alternative substrate considerations. The present study was performed under conditions that were designed to consider only glycogen metabolism and, with the G perfusion protocol, exogenous glucose as well. In the whole organism, FFAs are the preferred substrate of the heart in the fasted state. Our choice not to consider alternative substrates that exist under physiological conditions was based on a body of previous work that has demonstrated that carbohydrates become the heart's preferred substrate during increases in β-AR-elicited workload, with significantly greater increases in oxidation of glucose and glycogen than exogenous FFAs and endogenous triglycerides (4, 5, 7, 11). Additionally, the workload capacity of heart glycogen metabolism, which is directly regulated by mitochondrial feedback at the level of the pyruvate dehydrogenase complex, is considered to be substantially greater than that of the endogenous lipolytic reserve. We therefore expect that contributions from exogenous FFAs and endogenous triglycerides would not change the findings of the present study.

Although a recent study demonstrated greater reliance on fatty acid oxidation than glucose and glycogen oxidation during β-AR stimulation (10), it was performed under conditions designed to simulate extreme exercise in an untrained individual, with very high systemic levels of lactate (as would be derived from skeletal muscle) and nonesterified fats (as would be derived from lipolysis in adipose tissue and the heart) included in the perfusate. Our study was designed to more generally examine β1- and β2-AR stimulation and was not tied to a specific physiological condition.

Summary. We have shown that after matched peak workloads elicited by β1- and β2-AR stimulation during both SF and G perfusions, that glycogenolysis is associated with significant functional decline and is coupled more strongly to inotropy than chronotropy. Moreover, endogenous glycogen stores are depleted earlier, to a greater extent, and more inefficiently during β1- than β2-AR stimulation. These findings are consistent with the Gs-PKA-dependent glycogen phosphorylase kinase signaling that occurs during β1-AR stimulation (19), compared with the Gi-dependent signaling that occurs during β2-AR stimulation (29). Greater β1-AR-mediated glycogenolysis was correlated with a greater initial ability to sustain the initial contractile response during β1-AR stimulation. However, with SF perfusion, this ability was transient, with excessive early glycogen depletion causing a greater decline in contractile state and PP during the later stages of β1- compared with β2-AR stimulation.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by the Intramural Research Program of the National Institute on Aging.


   ACKNOWLEDGMENTS
 
We thank Kenneth W. Fishbein for technical support.

Present address of P. McConville: MIR Preclinical Services, Ann Arbor, MI 48108.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. G. Spencer, Laboratory of Clinical Investigation, Box 29, Gerontology Research Center 4D-08, 5600 Nathan Shock Dr., Baltimore, MD 21 224 (e-mail: spencerri{at}grc.nia.nih.gov)

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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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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