Temporal dynamics of inotropic, chronotropic, and metabolic responses during {beta}1- and {beta}2-AR stimulation in the isolated, perfused rat heart

P. McConville,1,2 R. G. Spencer,1 and E. G. Lakatta2

1Nuclear Magnetic Resonance Unit, Laboratory of Clinical Investigation and 2Laboratory for Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland

Submitted 2 February 2004 ; accepted in final form 24 March 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
During the {beta}-adrenergic receptor ({beta}-AR)-mediated stress response in the heart, the relations between functional responses and metabolism are ill defined, with the distinction between {beta}1- and {beta}2-AR subtypes creating further complexity. Specific outstanding questions include the temporal relation between inotropic and chronotropic responses and their metabolic correlates. We sought to elucidate the relative magnitudes and temporal dynamics of the response to {beta}1- and {beta}2-AR stimulation and the energy expenditure and bioenergetic state related to these responses in the isolated perfused rat heart. Inotropic [left ventricular developed pressure (LVDP) and dP/dt], chronotropic [heart rate (HR)], and metabolic responses were measured during {beta}1- (n = 9; agonist: norepinephrine) and {beta}2- (n = 9; agonist: zinterol) AR stimulation. Myocardial oxygen consumption (MO2) was measured using fiber-optic oximetry, and high-energy phosphate levels and intracellular pH were measured using 31P NMR spectroscopy. A multiple-dose protocol was used, with near-maximal {beta}-AR stimulation at the highest doses. In both {beta}1 and {beta}2 groups, there were dose-dependent increases in LVDP, dP/dt, HR, and MO2. The inotropic response showed more rapid onset, washout, and variation during dose than did the chronotropic response and was closely correlated with MO2. This suggests that the myocardial bioenergetic state is more closely related to the inotropic response than to the chronotropic response. In addition, {beta}1-AR stimulation resulted in a greater magnitude and rate of onset of inotropic and MO2 responses than did {beta}2-AR stimulation during maximal stimulation. However, a similar decrease in intracellular energy charge was seen in the two groups, consistent with a greater rate of oxidative phosphorylation during {beta}1- than during {beta}2-AR stimulation.

receptors; adrenergic; metabolism; myocardial oxygen consumption; inotropy; chronotropy; phosphorus-31 nuclear magnetic resonance; rat heart; adrenergic receptor


THE MAGNITUDE AND TIME COURSE of inotropic, chronotropic, and metabolic responses to {beta}1- and {beta}2-adrenergic receptor (AR) stimulation in the isolated, perfused rat heart were measured. Inotropic and metabolic responses were closely correlated, with both being significantly greater during {beta}1 stimulation than during {beta}2 stimulation. These findings are consistent with increased protein kinase A (PKA)-dependent phosphorylation of regulatory proteins and greater intracellular Ca2+ concentration ([Ca2+]i)-dependent regulation of the inotropic response and metabolism during {beta}1-, compared with {beta}2-AR, stimulation.

The response to stress in the heart is influenced by catecholamines, which increase contractile force and heart rate (HR) via stimulation of the {beta}-ARs. Although these effects are well known, the specific relations between the inotropic and chronotropic responses during {beta}-AR stimulation, and the concomitant metabolic response, define much of the physiological action of catecholamines at the organ level. However, these relations remain unknown, with further complexity arising due to the differing responses to stimulation of {beta}1- and {beta}2-AR subtypes.

It is known that contractile dysfunction is associated with a decline in the number of {beta}1-AR and an increased responsiveness to {beta}2-AR stimulation (1). Other differences between the {beta}1- and {beta}2-AR subtypes have been demonstrated in the contractile response, [Ca2+]i dynamics, metabolism, apoptotic and arrhythmogenic potential, and susceptibility to muscarinic accentuated antagonism (13, 5, 15, 16, 1921). These differences have been attributed to the respective signaling cascades for {beta}1- and {beta}2-AR activation. Both {beta}1- and {beta}2-ARs couple to the stimulatory G protein (Gs), but only {beta}2-ARs couple to the inhibitory G protein (Gi) and phosphatidylinositol 3-kinase (PI3K). {beta}1-AR stimulation leads to elevation of cAMP levels, followed by PKA-dependent phosphorylation of key regulatory target proteins such as phospholamban (PLB), myofilament proteins, and the metabolic regulator glycogen phosphorylase kinase. Although cAMP levels are also elevated during {beta}2-AR stimulation, this increase is uncoupled from PKA signaling under a wide range of circumstances. Therefore, subtype-dependent regulation of enzymatic activity during {beta}-AR stimulation may play a key role in determining contractile and metabolic responses. [Ca2+]i may also be important in regulating these responses, acting as a dual messenger by promoting both contraction of the myofilaments and oxidative phosphorylation (16).

We have been concerned with organ-level functional consequences of these mechanistic differences. In recent work, we showed that there were large differences in the bioenergetic sequelae of {beta}1- as opposed to {beta}2-AR stimulation in the perfused rat heart (15). These differences were consistent with {beta}-AR subtype-dependent [Ca2+]i regulation and regulatory protein phosphorylation. However, that study did not examine the attendant temporal relationships and did not distinguish between inotropic and chronotropic components of the overall work load response (which was measured as rate-pressure product). Furthermore, this previous work (15) was limited to examination of mean responses during sustained dosing and did not examine the dynamics (onset, washout, and tachyphylaxis) of responses during dose. The rapid changes in intracellular energy charge (IEC) that occurred immediately after the start or end of a given catecholamine dose in our previous study (15) suggest the hypothesis that metabolic state, and chronotropic and/or inotropic state, are closely correlated, not only in terms of mean response but also in terms of peak response and response temporal dynamics, which are also expected to be related to energy charge and metabolic reserve. The magnitude of metabolic and functional changes observed in Ref. 15 further suggest the hypothesis that this correlation differs for {beta}1- and {beta}2-AR stimulation. Accordingly, in this study, we hypothesized that: 1) the time courses of the inotropic and chronotropic contractile responses to {beta}-AR stimulation differ significantly; 2) the dose dependence and dynamics of these responses are correlated with IEC and net cardiac oxygen utilization; and 3) the correlation of functional and metabolic responses differs for {beta}1- compared with {beta}2-AR stimulation.

To test these hypotheses, we simultaneously measured left ventricular developed pressure (LVDP) and dP/dt, cardiac myocardial oxygen consumption (MO2) and intracellular high-energy phosphate levels, and pH during selective {beta}1- or {beta}2-AR stimulation in the isolated, perfused rat heart. We defined the relationships among these variables during the onset, steady-state, and washout periods of administration of multiple sustained doses of selective {beta}1- and {beta}2-AR stimulatory agents.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isolated Heart Preparation

Male Wistar rats, 3–4 mo of age, were injected with 20 mg/kg 6-hydroxydopamine 24 h before experimentation to attenuate the effect of endogenous catecholamines. Hearts were perfused retrogradely 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. The hearts were not paced, in order to specifically quantify chronotropic responses to the adrenergic agents. The buffer consisted of (in mM) 118 NaCl, 5 KCl, 0.5 Na2EDTA, 1.2 MgSO4, 25 NaHCO3, 1.8 CaCl2, 11 glucose, and 1,100 U/l heparin. A water-filled polyethylene balloon connected to a pressure transducer was inserted in the left ventricle (LV) and used to measure the LV pressure. Balloon inflation was adjusted to achieve a preload of 10–15 mmHg.

31P NMR Spectroscopy

Perfused hearts were placed in a 20-mm NMR tube and then inserted in a 9.4-T Magnex magnet (Magnex Scientific) interfaced to a Bruker DMX spectrometer (Bruker Analytik, Rheinstetten, Germany). After a stabilization period, 3-min 31P spectra were acquired continuously with flip angle = 45°, repetition time = 1.5 s, spectral width = 12 kHz, 50 Hz line broadening, and zero filling to 8,000 points. The ratio of the phosphocreatine (PCr) and Pi resonances was used as an index of the IEC (6). Intracellular pH was calculated from the chemical shift difference between the PCr and Pi resonances (9).

MO2 Measurements

An O2-sensitive fiber-optic fluorometer (Presens, Neuburg, Germany) was used to measure the O2 saturation in the arterial line and in the venous effluent via a cannula sutured in the coronary sinus. MO2 was derived from the arteriovenous O2 difference per unit coronary flow.

Experimental Protocol

A high, constant-perfusion flow rate (28.4 ± 0.1 ml/min) was used to maintain adequate substrate and oxygen supply during the highest work loads. Hearts underwent either selective {beta}1-AR stimulation ({beta}1 group, n = 9) or selective {beta}2-AR stimulation ({beta}2 group, n = 9) during three 12-min doses of increasing concentrations of the {beta}1-agonist norepinephrine, or the {beta}2-agonist zinterol (Bristol-Myers Squibb). These agonists have been previously shown to elicit their functional responses almost exclusively via {beta}1- and {beta}2-AR stimulation at the doses used (13, 20, 21). Nevertheless, to increase selectivity of the receptor stimulation, an {alpha}-AR antagonist, prazosin, or a {beta}1-AR antagonist, bisoprolol (Merck), was used for {beta}1- and {beta}2-stimulated hearts, respectively, in combination with the agonists (13, 20, 21). The doses chosen ({beta}1-AR stimulation with norepinephrine: 10–8, 10–7, and 10–6 M; {beta}2-AR stimulation with zinterol: 10–7, 10–6, and 10–5 M) elicited a broad range of inotropic and chronotropic responses in both groups, the magnitudes of which were approximately saturated at the highest doses, as shown in preliminary experiments. The highest doses were therefore considered to represent near-maximal inotropic and chronotropic stimulation for each subtype. This multiple-dose protocol allowed us to examine dose-dependent responses while also allowing a comparison of the responses elicited by the {beta}1- and {beta}2-AR subtypes under conditions of near-maximal stimulation. Examination of washout of the {beta}-AR stimulation was performed during replacement of buffer containing the {beta}-AR agonists with baseline buffer.

Throughout each protocol, LVDP, HR, maximum dP/dt during systole (+dP/dtmax), and maximum dP/dt during diastole (–dP/dtmax) were recorded at 400 Hz, MO2 at 10 Hz, and IEC and pH at 3-min intervals. The time courses of the pressure-derived parameters and MO2 were analyzed over 5-s intervals, which provided sufficiently high time resolution for accurate quantification of the most rapid variations in each response. Half-times were calculated for response onset and washout (time taken to increase or decrease, respectively, by half the maximal response). To examine tachyphylaxis in the inotropic and chronotropic responses during each dose, the time courses were also calculated as a percentage of the maximal increase in each response over the dose duration.

Statistics

Data are reported as means ± SE. Inotropic, chronotropic, and metabolic parameters were analyzed for dose, response-type (inotropic vs. chronotropic vs. metabolic), and, when appropriate, group main effects and interactions using repeated-measures ANOVA. Between-group comparisons for maximal stimulation were performed using ANOVA. Differences were considered to be significant at the P < 0.05 level.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Each group showed a statistically significant dose dependence of the inotropic and chronotropic responses, with a substantially greater inotropic response achieved in the {beta}1 group compared with the {beta}2 group (Table 1). In both groups, abrupt increases in LVDP over a period of ~1–2 min occurred at each dose to a maximum; this was followed by a gradual decrease (Fig. 1). The chronotropic responses were similar in magnitude between the groups (Table 1) and developed gradually toward a steady state by the end of each dose (Fig. 1). In both groups, the single-dose experiments (Fig. 1) confirm that the HR remained at a constant level after the initial increase, whereas the inotropic response varied markedly throughout the dose.


View this table:
[in this window]
[in a new window]
 
Table 1. Baseline and peak inotropic and chronotropic responses for each {beta}-AR subtype

 


View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1. Time course of left ventricular developed pressure (LVDP) and heart rate (HR) responses to {beta}1 (A)- and {beta}2 (B)-adrenergic receptor (AR) stimulation. The data are expressed as %baseline. Left: multiple-dose ({beta}1: 10–8, 10–7, and 10–6 M norepinephrine; {beta}2: 10–7, 10–6, and 10–5 M zinterol) mean time courses; right, examples of individual hearts that received single extended doses ({beta}1: 2 x 10–8 M norepinephrine; {beta}2: 10–5 M zinterol) for 24 min. In both groups, the time course of the HR response shows a steady increase toward a constant value that is approximately maintained for the remainder of the dose. In contrast, the LVDP responses show a relatively sudden increase to a local maximum before decay of the response was observed. The magnitude of the HR response was similar in each group. The LVDP response magnitude was substantially greater in the {beta}1 group than in the {beta}2 group.

 
Inotropic and Chronotropic Response Kinetics

Onset rates. In both groups, the inotropic onset half-times decreased in a dose-dependent manner (Table 2). During maximal stimulation, the inotropic response half-time in the {beta}1 group was significantly shorter than that in the {beta}2 group. In both groups, the chronotropic response half-times were longer and showed less dose dependence compared with the inotropic responses (Table 2).


View this table:
[in this window]
[in a new window]
 
Table 2. Half-times for response onset at each dose, and washout

 
Response time course during dose and washout. The inotropic response in the {beta}1 group was more sustained than that in the {beta}2 group during each dose, particularly during the higher doses in the {beta}1 group, where a secondary "increase" in the response occurred in the case of the highest dose, leading to a response that transiently exceeded the initial maxima (Fig. 2A). In the {beta}2 group, the responses exhibited a monotonic decrease, declining by >60% of maximum during the submaximal doses (Fig. 2A). The time courses of the chronotropic response were similar in each group, regardless of dose (Fig. 2B). The half-times for washout of the inotropic responses were substantially shorter than those of the chronotropic responses, in each group (Fig. 3 and Table 2).



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2. Time course of LVDP (A) and HR (B) mean responses ({beta}1: n = 9; {beta}2: n = 9) plotted as a %maximum response for a given dose and averaged over 3-min periods in each dose. The LVDP response was less sustained during each dose in the {beta}2 group than in the {beta}1 group; the time course of the LVDP response during a given dose exhibited significant dose dependence in both groups ({beta}1: P = 0.002, {beta}2: P < 0.0001) and a significant dose x time interaction ({beta}1: P = 0.03, {beta}2: P < 0.0001). During maximal stimulation (dose 3), a significant between-group difference (over the entire dose) was found (P = 0.01). In contrast, the time course of the HR responses were not highly dose dependent and not significantly different between the groups (main effect or interaction).

 


View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3. Mean washout time courses for LVDP ({blacksquare} and {square}) and HR ({blacktriangleup} and {triangleup}) for the {beta}1 ({blacksquare} and {blacktriangleup}) and {beta}2 ({square} and {triangleup}) groups, plotted as %{Delta}response ({Delta}response = prewashout response – baseline). Representative SE bars are shown on the time courses, except for the {beta}2 HR response, for which the error bars were smaller than the size of the symbols. The HR response shows a significantly more rapid washout in the {beta}1 group than in the {beta}2 group, where it had only decreased by ~25% by the end of the protocol. However, the LVDP washout was significantly more rapid in the {beta}2 group (decrease to baseline in <5 min) than the {beta}1 group (decrease to baseline in 12 min).

 
Lusitropic response. Dose-dependent responses in +dP/dtmax, and –dP/dtmax were found in both groups. The ratio of +dP/dtmax to –dP/dtmax has been used previously to indicate a true positive lusitropic effect in the isolated heart (4). This effect was found in the {beta}1 group only (Fig. 4).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4. Mean time courses of the ratio of maximum dP/dt during systole (+dP/dtmax) and maximum dP/dt during diastole (–dP/dtmax) for hearts in the {beta}1 and {beta}2 groups. Under baseline conditions, in both groups, +dP/dtmax is greater than –dP/dtmax (+dP/dtmax/–dP/dtmax {approx} 1.6–1.7). In the {beta}2 group, this ratio was generally maintained, even during the highest dose. In the {beta}1 group, however, dose-dependent decreases in +dP/dtmax/–dP/dtmax were observed ({beta}1 vs. {beta}2: P = 0.02).

 
Metabolic Responses

MO2 response time course and washout. MO2 increased at each dose in both groups, showing a strong correlation with the inotropic responses (Fig. 5). The MO2 onset half-times were not dose dependent or statistically different between the groups and were similar to the inotropic response half-times but significantly less than the chronotropic response half-times (Table 2). The MO2 washout half-times showed a similar trend to the inotropic washout half-times, being significantly shorter for the {beta}2 group compared with the {beta}1 group (Table 2). For each group, the half-time for washout of the MO2 response was similar to that of the inotropic response, but generally shorter than that of the chronotropic response (Table 2).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 5. Mean myocardial oxygen consumption (MO2) time courses for the {beta}1 ({blacksquare}) and {beta}2 ({blacktriangleup}) groups, with representative error bars. Thin solid lines show the corresponding LVDP time courses for comparison. A close relationship was observed between the inotropic response and MO2 in both groups. The half-times for onset of the MO2 responses were similar in magnitude to those measured for LVDP, but were not dose dependent in either group. The MO2 and LVDP half-times during washout were also similar in magnitude.

 
IEC. IEC was shown to decrease in a dose-dependent fashion to a similar degree at the highest dose in each group ({beta}1: 36% of baseline, {beta}2: 39% of baseline; Fig. 6A, left). Generally, there was evidence of a small increase in the IEC near the end of each dose in both groups. There was no statistically significant difference between the {beta}1 and {beta}2 IEC time courses during doses 1–3 or the IEC during maximal stimulation. During the early part of the washout, an overshoot in the IEC was found in the {beta}1 group only (Fig. 6A, right).



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 6. Mean time courses for intracellular energy charge (IEC; A) and pH (B) in the {beta}1 ({bullet}, solid line) and {beta}2 ({circ} and {triangleup}, broken line) groups during baseline and doses (left) and washout (right). *P < 0.05 ({beta}1 vs. {beta}2, dose 3). There was no statistical difference in the IEC time courses or IEC during maximal stimulation (dose 3) between the {beta}1 and {beta}2 groups. In each group, a dose-dependent decrease in the IEC was measured to a similar extent at the highest doses (~35% of baseline). Statistical analysis of the washout time courses showed a significant group main effect (P = 0.02) and group x time interaction (P = 0.05). A baseline overshoot was observed in IEC after the start of the washout period in the {beta}1 group only. During maximal stimulation, significant acidosis was measured in the {beta}1 group (decrease = 0.06, P = 0.01) but not in the {beta}2 group (decrease = 0.01, P = 0.9). A pH overshoot occurred after washout in the {beta}1 group only; however, the small size of the Pi peak at this time led to considerable uncertainty in the pH determination.

 
pH. pH was found to decrease in both groups (Fig. 6B, left), but to a significant extent in the {beta}1 group only (0.06 pH unit decrease). During maximal stimulation (dose 3), there was a significant between-group difference. No systematic variation in pH was found during each dose. After the washout, the pH showed an overshoot in the {beta}1 group only (Fig. 6B, right).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The {beta}-AR-mediated Inotropic Response Shows More Dynamic Behavior Than the Chronotripic Response and Is Predictive of the Metabolic Response

Our findings indicate a substantially more rapid and extensive inotropic than chronotropic response to {beta}-AR stimulation in the isolated rat heart. MO2 was shown to parallel these responses in both the {beta}1 and {beta}2 groups and showed a remarkably similar profile to the inotropic response (Fig. 5). Although the direct contribution to MO2 of the chronotropic response and its own influence on the inotropic response cannot be ignored, our data show that, under normal physiological conditions, the inotropic response magnitude alone is predictive of the relative MO2 response during both {beta}1- and {beta}2-AR stimulation. In both groups, dose-dependent decreases in the IEC occurred in each group (Fig. 6A), consistent with our previous findings (15). The secondary increase in the IEC observed toward the end of most doses in both groups can be correlated with the decline in the inotropic response, whereas the chronotropic response was relatively steady in all cases (Figs. 2 and 6A). These findings extend our previous work by showing that, under physiological conditions, changes in the inotropic response alone correlate with the MO2 and IEC metabolic responses.

Greater Inotropic Response and Lusitropic Response During {beta}1- Than {beta}2-AR Stimulation

Substantially greater inotropic responses were observed in the {beta}1 group (3.7x increase, highest dose) than in the {beta}2 group (1.6x increase, highest dose). In contrast, the chronotropic responses in the two groups were similar ({beta}1: 1.4x; {beta}2: 1.3x increases, highest doses). In addition, a positive lusitropic effect was found in the {beta}1 group only. These results demonstrate that, under conditions in which both inotropic and chronotropic responses occur, the measured inotropic response distinguishes the {beta}1 and {beta}2 groups, whereas the measured chronotropic response does not.

The larger inotropic effect in the {beta}1 group is consistent with increased [Ca2+]i levels and increased [Ca2+]i cycling in and out of the cytoplasm, promoting activation of the myofilaments during {beta}1- compared with {beta}2-AR stimulation. {beta}1-AR-mediated [Ca2+]i modulation occurs via phosphorylation of L-type Ca2+ channels, troponin I, PLB, and ryanodine receptors. Phosphorylation of PLB promotes more rapid reuptake of [Ca2+]i by the sarcoplasmic reticulum (SR), phosphorylation of troponin I, and increased dissociation of [Ca2+]i from troponin C, consistent with the positive lusitropic response observed in the {beta}1-AR-stimulated hearts only. A similar lusitropic effect has been found during {beta}1-AR stimulation in cardiomyocytes (20).

By quantifying temporal responses, we found that the inotropic response was more sustained in the {beta}1 group compared with the {beta}2 group (Fig. 2). This finding is consistent with the fact that PLB acts to retain increased [Ca2+]i levels in the SR. During {beta}2-AR stimulation, the role of PI3K signaling in the Gi-dependent pathway has been established and is now emerging as a mechanism in a {beta}2-AR-mediated antiapoptotic effect (3, 19). There is evidence that this signaling also acts to negate the {beta}2-AR-Gs-dependent pathway, possibly involving activation of protein phosphatases, offsetting {beta}2-AR-Gs-dependent activation of protein kinases and inhibiting {beta}2-AR-mediated PKA signaling and subsequent phosphorylation of PLB and other regulatory proteins (12). Therefore, {beta}2-AR-mediated PI3K signaling may limit [Ca2+]i levels, [Ca2+]i cycling and retainment, and, hence, the inotropic and lusitropic responses to {beta}2-AR stimulation. This is consistent with our observation of a smaller and less sustained inotropic response, and lack of lusitropic response, in the {beta}2 group compared with the {beta}1 group (Fig. 2).

Greater Metabolic Reserve During {beta}1- Than {beta}2-AR Stimulation

In a dual messenger role, increased [Ca2+]i levels not only directly stimulate the inotropic state but can also promote oxidative phosphorylation both indirectly via increased ADP levels, due to ATP hydrolysis supporting the increased contractile response of the myofilaments, and directly, due to activation of mitochondrial dehydrogenases (16). This is consistent with the close correlation between MO2 and the inotropic responses in both groups (Fig. 5).

The IEC and intracellular pH overshoots after washout, which occurred in the {beta}1 group only (Fig. 6), have been observed during reperfusion after ischemia in isolated, perfused hearts and have been attributed to a temporary mismatch between energy supply and demand (7, 10). This implies a substantially greater flux through the creatine kinase and ATP hydrolysis reactions and a greater rate of oxidative phosphorylation during maximal {beta}1- compared with maximal {beta}2-AR stimulation. The more rapid termination of the inotropic responses compared with the chronotropic responses (Fig. 3 and Table 2) suggest that it is the former that is associated with this energy mismatch and the metabolic overshoot. We would therefore expect this to occur more prominently in the {beta}1 group compared with the {beta}2 group (Fig. 6).

The greater contractile and metabolic responses observed during {beta}1-AR stimulation may additionally indicate a relative bioenergetic deficit during {beta}2- compared with {beta}1-AR stimulation. This deficiency is evidenced by the similar IEC time courses observed for each group (Fig. 6A) despite the substantially greater work load in the {beta}1 group. This suggests that the {beta}1 group was able to develop high work load in a more efficient manner, or else had increased access to a metabolic substrate, compared with the {beta}2 group. This is consistent with increased endogenous glucose delivery via {beta}1-AR-mediated activation of glycogen phosphorylase kinase (13) and consequent increased glycogen breakdown. In addition, {beta}2-AR-PI3K-dependent inhibition of PKA signaling may inhibit the phosphorylation of glycogen phosphorylase kinase during {beta}2-AR stimulation. This would limit the glycogen availability during {beta}2-AR stimulation, consistent with the significantly greater inotropic response in the {beta}1 group for a given IEC compared with the {beta}2 group (Figs. 1 and 6). The lower MO2 response in the {beta}2 group also indicates a smaller capacity for oxidative metabolism during maximal stimulation in this group (Fig. 5).

Although the signaling mechanisms discussed above are consistent with our data, we note that additional mechanisms also contribute explanations for our central observations. Saito et al. (18) found that the density of the {beta}2-AR is substantially greater in the SA node than in surrounding atrial myocardium, with the opposite being true for the {beta}1-AR. For rat ventricular myocardium, Kuznetsov et al. (14) and Hilal-Dandan et al. (11) report an approximately fourfold greater density of {beta}1-AR than {beta}2-AR. These findings support a relatively more pronounced effect of {beta}2-AR stimulation on HR than on contractile function and a relatively greater inotropic response to {beta}1-AR stimulation than to {beta}2-AR stimulation, as found in the functional studies reported here. Of course, receptor density by itself does not account for potential differential degrees of signal amplification secondary to agonist binding, as discussed in detail above. In addition, the more sustained inotropic response to {beta}1-AR compared with {beta}2-AR stimulation may be due, at least in part, to myocyte membrane subdomains, resulting in differences in compartmentation and turnover of these receptor subtypes. Rybin et al. (17) found that {beta}2-AR, although more highly concentrated in caveolae than are {beta}1-AR, are preferentially depleted from them in response to stimulation compared with {beta}1-AR.

Limitations and Future Directions

In this work, we have reported effects due to selective {beta}1- or selective {beta}2-AR stimulation. However, the complexity of adrenergic pharmacology dictates that further studies examining simultaneous {beta}1-, {beta}2-, and {alpha}-AR stimulation may provide further insight into the basis for cardiac functional responses. In addition, further experiments with inhibitors of PI3K, PKA, or Gi would provide whole-heart analogs to mechanistic studies previously performed in myocytes. We also note that our experiments incorporated perfusion under constant flow; further studies are required to directly examine vasodilatory and vasoconstrictory responses during {beta}1- and {beta}2-AR stimulation.

Our measurements of functional and metabolic parameters were conducted under conditions in which both inotropic and chronotropic responses were examined simultaneously. Therefore, the specific influences of each response type on the metabolic parameters measured cannot be determined. However, these conditions allowed us to examine our defined set of hypotheses, which involved comparisons of the inotropic and chronotropic responses to {beta}1- and {beta}2-AR stimulation. Indeed, substantive differences in the manner in which the relative inotropic and chronotropic responses evolve were observed, and the inotropic response was shown to be highly predictive of the MO2 changes during sustained dosing. On the basis of these findings, we have discussed possible mechanisms concerning [Ca2+]i cycling and PI3K activation and receptor localization that could potentiate the dominant inotropic response and may explain the observed differences between the {beta}1 and {beta}2 groups.

Finally, we chose to use glucose as the sole substrate in our perfusion protocol since glucose becomes the preferred substrate in the rat heart during {beta}-AR stimulation (8). Although the effect of free fatty acids, the preferred fuel of the heart under resting conditions, is therefore expected to be small under these conditions, further examination of these effects may also be warranted.

In summary, this study has specifically examined the temporal dynamics of the functional and metabolic responses elicited by {beta}-adrenergic stimulation of the heart. Our findings demonstrate substantial differences between the inotropic and chronotropic responses to {beta}1- compared with {beta}2-AR stimulation, in terms of rate of onset, maintenance of the effect during a sustained dose, and the rate of washout of the response. We found a close correlation between the metabolic and inotropic responses, indicating that the {beta}-AR-induced inotropic response may be dominant, as opposed to the chronotropic response, in regulating or responding to cellular processes such as energy generation and [Ca2+]i handling. A greater energy reserve during {beta}1-AR stimulation may potentiate this larger inotropic response. These findings are consistent with greater Gs-PKA-dependent phosphorylation of regulatory proteins during {beta}1-AR stimulation, and inhibition of this pathway through Gi-PI3K-dependent signaling during {beta}2-AR stimulation.


    ACKNOWLEDGMENTS
 
We thank Dr. Kenneth Fishbein for technical support.

Present address of P. McConville: MIR Preclinical Services, Ann Arbor, MI 48104.


    FOOTNOTES
 

Address for reprint requests and other correspondence: E. Lakatta, Laboratory for Cardiovascular Science, Gerontology Research Center 4D-08, 5600 Nathan Shock Dr., Baltimore MD 21224 (e-mail: lakattae{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
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Altschuld RA and Billman GE. {beta}2-Adrenoceptors and ventricular fibrillation. Pharmacol Ther 88: 1–14, 2000.[CrossRef][ISI][Medline]
  2. Aprigliano O, Rybin VO, Pak E, Robinson RB, and Steinberg SF. {beta}1-and {beta}2-Adrenergic receptors exhibit differing susceptibility to muscarinic accentuated antagonism. Am J Physiol Heart Circ Physiol 272: H2726–H2735, 1997.[Abstract/Free Full Text]
  3. Chesley A, Lundberg MS, Asai T, Xiao RP, Ohtani S, Lakatta EG, and Crow MT. The {beta}2-adrenergic receptor delivers an antiapoptotic signal to cardiac myocytes through Gi-dependent coupling to phosphatidylinositol 3'-kinase. Circ Res 87: 1172–1179, 2000.[Abstract/Free Full Text]
  4. Cingolani HE, Wiedmann RT, Lynch JJ, Wenger HC, Scott AL, Siegl PK, and Stein RB. Negative lusitropic effect of DPI 201–106 and E4031. Possible role of prolonging action potential duration. J Mol Cell Cardiol 22: 1025–1034, 1990.[CrossRef][ISI][Medline]
  5. Communal C, Singh K, Sawyer DB, and Colucci WS. Opposing effects of {beta}1- and {beta}2-adrenergic receptors on cardiac myocyte apoptosis: role of a pertussis toxin-sensitive G protein. Circulation 100: 2210–2212, 1999.[Abstract/Free Full Text]
  6. Gadian DG and Radda GK. NMR studies of tissue metabolism. Annu Rev Biochem 50: 69–83, 1981.[CrossRef][ISI][Medline]
  7. Garnier A, Rossi A, and Lavanchy N. Importance of the early alterations of energy metabolism in the induction and the disappearance of ischemic preconditioning in the isolated rat heart. J Mol Cell Cardiol 28: 1671–1682, 1996.[CrossRef][ISI][Medline]
  8. Goodwin GW, Taylor CS, and Taegtmeyer H. Regulation of energy metabolism of the heart during acute increase in heart work. J Biol Chem 273: 29530–29539, 1998.[Abstract/Free Full Text]
  9. Gupta RK and Wittenberg BA. 31P-NMR studies of isolated adult heart cells: effect of myoglobin inactivation. Am J Physiol Heart Circ Physiol 261: H1155–H1163, 1991.[Abstract/Free Full Text]
  10. Hendrikx M, Mubagwa K, Verdonck F, Overloop K, Van Hecke P, Vanstapel F, Van Lommel A, Verbeken E, Lauweryns J, and Flameng W. New Na+-H+ exchange inhibitor HOE 694 improves postischemic function and high-energy phosphate resynthesis and reduces Ca2+ overload in isolated perfused rabbit heart. Circulation 89: 2787–2798, 1994.[Abstract/Free Full Text]
  11. Hilal-Dandan R, Kanter JR, and Brunton LL. Characterization of G-protein signaling in ventricular myocytes from the adult mouse heart: differences from the rat. J Mol Cell Cardiol 32: 1211–1221, 2000.[CrossRef][ISI][Medline]
  12. Jo SH, Leblais V, Wang PH, Crow MT, and Xiao RP. Phosphatidylinositol 3-kinase functionally compartmentalizes the concurrent Gs signaling during {beta}2-adrenergic stimulation. Circ Res 91: 46–53, 2002.[Abstract/Free Full Text]
  13. Kuschel M, Zhou YY, Spurgeon HA, Bartel S, Karczewski P, Zhang SJ, Krause EG, Lakatta EG, and Xiao RP. {beta}2-Adrenergic cAMP signaling is uncoupled from phosphorylation of cytoplasmic proteins in canine heart. Circulation 99: 2458–2465, 1999.[Abstract/Free Full Text]
  14. Kuznetsov V, Pak E, Robinson RB, and Steinberg SF. {beta}2-Adrenergic receptor actions in neonatal, and adult rat ventricular myocytes. Circ Res 76: 40–52, 1995.[Abstract/Free Full Text]
  15. McConville P, Lakatta EG, and Spencer RGS. Differences in the bioenergetic response of the isolated perfused rat heart to selective {beta}1- and {beta}2-adrenergic receptor stimulation. Circulation 107: 2146–2152, 2003.[Abstract/Free Full Text]
  16. Pepe S. Mitochondrial function in ischaemia and reperfusion of the ageing heart. Clin Exp Pharmacol Physiol 27: 745–750, 2000.[CrossRef][ISI][Medline]
  17. Rybin VO, Xu X, Lisanti MP, and Steinberg SF. Differential targeting of {beta}-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway. J Biol Chem 275: 41447–41457, 2000.[Abstract/Free Full Text]
  18. Saito K, Torda T, Potter WZ, and Saavedra JM. Characterization of {beta}1- and {beta}2-adrenoceptor subtypes in the rat sinoatrial node and stellate ganglia by quantitative autoradiography. Neurosci Lett 96: 35–41, 1989.[CrossRef][ISI][Medline]
  19. Xiao RP, Cheng H, Zhou YY, Kuschel M, and Lakatta EG. Recent advances in cardiac {beta}2-adrenergic signal transduction. Circ Res 85: 1092–1100, 1999.[Abstract/Free Full Text]
  20. Xiao RP, Hohl C, Altschuld R, Jones L, Livingston B, Ziman B, Tantini B, and Lakatta EG. {beta}2-Adrenergic receptor-stimulated increase in cAMP in rat heart cells is not coupled to changes in Ca2+ dynamics, contractility, or phospholamban phosphorylation. J Biol Chem 269: 19151–19156, 1994.[Abstract/Free Full Text]
  21. Xiao RP and Lakatta EG. {beta}1-Adrenoceptor stimulation and {beta}2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca2+, and Ca2+ current in single rat ventricular cells. Circ Res 73: 286–300, 1993.[Abstract]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
289/3/E412    most recent
00049.2004v1
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 ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by McConville, P.
Articles by Lakatta, E. G.
Articles citing this Article
PubMed
PubMed Citation
Articles by McConville, P.
Articles by Lakatta, E. G.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.