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
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ABSTRACT |
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receptors; adrenergic; metabolism; myocardial oxygen consumption; inotropy; chronotropy; phosphorus-31 nuclear magnetic resonance; rat heart; adrenergic receptor
The response to stress in the heart is influenced by catecholamines, which increase contractile force and heart rate (HR) via stimulation of the -ARs. Although these effects are well known, the specific relations between the inotropic and chronotropic responses during
-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
1- and
2-AR subtypes.
It is known that contractile dysfunction is associated with a decline in the number of 1-AR and an increased responsiveness to
2-AR stimulation (1). Other differences between the
1- and
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
1- and
2-AR activation. Both
1- and
2-ARs couple to the stimulatory G protein (Gs), but only
2-ARs couple to the inhibitory G protein (Gi) and phosphatidylinositol 3-kinase (PI3K).
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
2-AR stimulation, this increase is uncoupled from PKA signaling under a wide range of circumstances. Therefore, subtype-dependent regulation of enzymatic activity during
-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 1- as opposed to
2-AR stimulation in the perfused rat heart (15). These differences were consistent with
-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
1- and
2-AR stimulation. Accordingly, in this study, we hypothesized that: 1) the time courses of the inotropic and chronotropic contractile responses to
-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
1- compared with
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
1- or
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
1- and
2-AR stimulatory agents.
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MATERIALS AND METHODS |
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Male Wistar rats, 34 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 1015 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 1-AR stimulation (
1 group, n = 9) or selective
2-AR stimulation (
2 group, n = 9) during three 12-min doses of increasing concentrations of the
1-agonist norepinephrine, or the
2-agonist zinterol (Bristol-Myers Squibb). These agonists have been previously shown to elicit their functional responses almost exclusively via
1- and
2-AR stimulation at the doses used (13, 20, 21). Nevertheless, to increase selectivity of the receptor stimulation, an
-AR antagonist, prazosin, or a
1-AR antagonist, bisoprolol (Merck), was used for
1- and
2-stimulated hearts, respectively, in combination with the agonists (13, 20, 21). The doses chosen (
1-AR stimulation with norepinephrine: 108, 107, and 106 M;
2-AR stimulation with zinterol: 107, 106, and 105 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
1- and
2-AR subtypes under conditions of near-maximal stimulation. Examination of washout of the
-AR stimulation was performed during replacement of buffer containing the
-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 M
O2 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.
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RESULTS |
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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 1 group was significantly shorter than that in the
2 group. In both groups, the chronotropic response half-times were longer and showed less dose dependence compared with the inotropic responses (Table 2).
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MO2 response time course and washout.
M
O2 increased at each dose in both groups, showing a strong correlation with the inotropic responses (Fig. 5). The M
O2 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 M
O2 washout half-times showed a similar trend to the inotropic washout half-times, being significantly shorter for the
2 group compared with the
1 group (Table 2). For each group, the half-time for washout of the M
O2 response was similar to that of the inotropic response, but generally shorter than that of the chronotropic response (Table 2).
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DISCUSSION |
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Our findings indicate a substantially more rapid and extensive inotropic than chronotropic response to -AR stimulation in the isolated rat heart. M
O2 was shown to parallel these responses in both the
1 and
2 groups and showed a remarkably similar profile to the inotropic response (Fig. 5). Although the direct contribution to M
O2 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 M
O2 response during both
1- and
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 M
O2 and IEC metabolic responses.
Greater Inotropic Response and Lusitropic Response During 1- Than
2-AR Stimulation
Substantially greater inotropic responses were observed in the 1 group (3.7x increase, highest dose) than in the
2 group (1.6x increase, highest dose). In contrast, the chronotropic responses in the two groups were similar (
1: 1.4x;
2: 1.3x increases, highest doses). In addition, a positive lusitropic effect was found in the
1 group only. These results demonstrate that, under conditions in which both inotropic and chronotropic responses occur, the measured inotropic response distinguishes the
1 and
2 groups, whereas the measured chronotropic response does not.
The larger inotropic effect in the 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
1- compared with
2-AR stimulation.
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
1-AR-stimulated hearts only. A similar lusitropic effect has been found during
1-AR stimulation in cardiomyocytes (20).
By quantifying temporal responses, we found that the inotropic response was more sustained in the 1 group compared with the
2 group (Fig. 2). This finding is consistent with the fact that PLB acts to retain increased [Ca2+]i levels in the SR. During
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
2-AR-mediated antiapoptotic effect (3, 19). There is evidence that this signaling also acts to negate the
2-AR-Gs-dependent pathway, possibly involving activation of protein phosphatases, offsetting
2-AR-Gs-dependent activation of protein kinases and inhibiting
2-AR-mediated PKA signaling and subsequent phosphorylation of PLB and other regulatory proteins (12). Therefore,
2-AR-mediated PI3K signaling may limit [Ca2+]i levels, [Ca2+]i cycling and retainment, and, hence, the inotropic and lusitropic responses to
2-AR stimulation. This is consistent with our observation of a smaller and less sustained inotropic response, and lack of lusitropic response, in the
2 group compared with the
1 group (Fig. 2).
Greater Metabolic Reserve During 1- Than
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 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
1- compared with maximal
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
1 group compared with the
2 group (Fig. 6).
The greater contractile and metabolic responses observed during 1-AR stimulation may additionally indicate a relative bioenergetic deficit during
2- compared with
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
1 group. This suggests that the
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
2 group. This is consistent with increased endogenous glucose delivery via
1-AR-mediated activation of glycogen phosphorylase kinase (13) and consequent increased glycogen breakdown. In addition,
2-AR-PI3K-dependent inhibition of PKA signaling may inhibit the phosphorylation of glycogen phosphorylase kinase during
2-AR stimulation. This would limit the glycogen availability during
2-AR stimulation, consistent with the significantly greater inotropic response in the
1 group for a given IEC compared with the
2 group (Figs. 1 and 6). The lower M
O2 response in the
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 2-AR is substantially greater in the SA node than in surrounding atrial myocardium, with the opposite being true for the
1-AR. For rat ventricular myocardium, Kuznetsov et al. (14) and Hilal-Dandan et al. (11) report an approximately fourfold greater density of
1-AR than
2-AR. These findings support a relatively more pronounced effect of
2-AR stimulation on HR than on contractile function and a relatively greater inotropic response to
1-AR stimulation than to
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
1-AR compared with
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
2-AR, although more highly concentrated in caveolae than are
1-AR, are preferentially depleted from them in response to stimulation compared with
1-AR.
Limitations and Future Directions
In this work, we have reported effects due to selective 1- or selective
2-AR stimulation. However, the complexity of adrenergic pharmacology dictates that further studies examining simultaneous
1-,
2-, and
-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
1- and
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 1- and
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 M
O2 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
1 and
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 -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 -adrenergic stimulation of the heart. Our findings demonstrate substantial differences between the inotropic and chronotropic responses to
1- compared with
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
-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
1-AR stimulation may potentiate this larger inotropic response. These findings are consistent with greater Gs-PKA-dependent phosphorylation of regulatory proteins during
1-AR stimulation, and inhibition of this pathway through Gi-PI3K-dependent signaling during
2-AR stimulation.
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ACKNOWLEDGMENTS |
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Present address of P. McConville: MIR Preclinical Services, Ann Arbor, MI 48104.
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FOOTNOTES |
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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.
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REFERENCES |
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