© 1999 American Heart Association, Inc.
Integrative Physiology |
Overexpression of the Cardiac ß2-Adrenergic Receptor and Expression of a ß-Adrenergic Receptor Kinase-1 (ßARK1) Inhibitor Both Increase Myocardial Contractility but Have Differential Effects on Susceptibility to Ischemic Injury
From the National Institute of Environmental Health Sciences (H.R.C., E.M.), Research Triangle Park; Department of Pathology (C.S.), Duke University Medical Center, Durham; Departments of Medicine and Biochemistry and the Howard Hughes Medical Institute (R.J.L.), Duke University Medical Center, Durham; and Department of Surgery (W.J.K.), Duke University Medical Center, Durham, NC.
Correspondence to Heather R. Cross, Mail Drop D2-03, NIEHS, Alexander Dr, Research Triangle Park, NC 27709. E-mail cross{at}niehs.nih.gov
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Abstract |
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Abstract—Cardiac ß2-adrenergic receptor (ß2AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing ß2ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of ß2AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20-minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because ß2ARs, unlike ß1ARs, couple to Gi as well as Gs, we pretreated mice with the Gi inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TG4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either ßARK1 (TGßARK1) or a ßARK1 inhibitor (TGßARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGßARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGßARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, ß2AR overexpression increased ischemic injury, whereas ßARK1 overexpression was protective. Ischemic injury in the ß2AR overexpressors was exacerbated by PTX treatment, implying that it was Gs not Gi activity that enhanced injury. Unlike ß2AR overexpression, basal contractility was increased by ßARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure.
Key Words: adrenergic signaling • energetics • G proteins • ischemia • NMR spectroscopy
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Introduction |
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Cardiac ß-adrenergic receptors (ßARs) mediate the myocardial contractile response to the sympathetic transmitters epinephrine and norepinephrine. ßARs are thought to be coupled primarily to the stimulatory guanine nucleotide binding protein Gs. Binding of agonists to ßARs stimulates dissociation of the G
subunit from the
Gß
subunit (Figure 1
). The stimulatory subunit
Gs binds and activates
adenylate cyclase, causing production of cAMP and
activation of protein kinase A (PKA). In the heart, PKA
phosphorylates and activates L-type
Ca2+ channels, the sarcoplasmic reticular
Ca2+ ATPase inhibitor phospholamban,
and the myofibrillar protein troponin I. The net result of these
phosphorylations is an increase in cardiac
contractility.1 Desensitization and
downregulation of ßARs is mediated via
phosphorylation of the activated receptors by
kinases such as ßAR kinase-1 (ßARK1).2 ßARK1 is
targeted to the ßARs via its affinity for membrane-bound
Gß subunits.3
Gß binding is also necessary for ßARK1
activation.4 ßARK1 has been shown to
phosphorylate and uncouple other receptors such as
angiotensin II in in vivo studies5 and
endothelin, M2 muscarinic cholinergic,
2A-adrenergic, thrombin, dopamine, and 
-
and 
-opioid receptors in in vitro studies.6 7 8
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ß1 and ß2ARs exist in
the myocardium, with ß1AR being the
most abundant subtype.9 ß1ARs are
coupled solely to Gs and operate as described
above. Recent findings have, however, revealed that
ß2ARs are coupled to the inhibitory
G protein Gi, in addition to
Gs.10 Although not shown directly
for the ß2AR, on stimulation of other
Gi-coupled receptors, Gi
dissociates from Gß, and
Gi then binds to adenylate cyclase
and inhibits its activity, thereby opposing the action of
Gs. Consistent with a similar
mechanism for the ß2AR,
Gi activation appears to prevent many of the
Gs-mediated downstream events of
ß-adrenergic signaling. ß2AR stimulation does
not lead to PKA-mediated phosphorylation of
phospholamban11 12 or troponin I.13 There is
evidence in rat11 and dog12 that the
increased cytosolic Ca2+ transients and increased
contractility observed on ß2AR
stimulation are dissociated from an increase in cAMP and instead are
caused by localized activation of the L-type Ca2+
channels. This implies that Gs activation via
ß2AR stimulation is only effective locally, at
the sarcolemma, and any distant effects are attenuated, possibly by
Gi-mediated inhibition of adenylate
cyclase. This hypothesis was supported further by the observation
that pertussis toxin (PTX), a Gi
inhibitor, restores the ability of
ß2AR stimulation to mediate phospholamban
phosphorylation.14
The ß-adrenergic signaling cascade is impaired in human congestive heart failure (CHF). ß1ARs are reduced by 50%, whereas ßARK1 levels and activity are increased.15 These changes contribute to the decreased basal and ß-agonist–stimulated contractility observed in CHF patients.9 ß2ARs, however, are not downregulated during CHF,15 and in fact, ß2 sensitivity may be increased.12 These observations led to the proposition that overexpression of ß2ARs in the myocardium could improve cardiac function and therefore be developed as a potential therapy for CHF.16 To investigate this hypothesis, transgenic mice with a 200-fold overexpression of the ß2AR were created; these mice exhibited increased basal cardiac contractility without evidence of cardiac abnormalities or increased mortality.16 Although this approach successfully increased cardiac function under normal physiological conditions, there may be consequences during pathological conditions such as ischemia.
By comparing the response to ischemia in isolated hearts from mice overexpressing the ß2AR with that of wild-type (WT) hearts, we aimed to determine the effect of ß2AR overexpression on ischemic injury. In addition, as the ß2AR activates both Gi and Gs, we aimed to distinguish between the effects of these 2 pathways by pretreating WT and ß2AR overexpressor mice with PTX to inhibit the Gi protein and studying the ischemic response. To assess further the role of ß-adrenergic signaling in ischemic injury, we studied the ischemic response of mice overexpressing ßARK1 and also of mice expressing a ßARK1 inhibitor.17 Finally, by monitoring basal contractility in mouse hearts from transgenic, WT, and PTX-treated animals, we hoped to determine the contribution of the various components of the ß2-adrenergic signaling cascade to the control of myocardial contractility.
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Materials and Methods |
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Animals
Transgenic mice were developed by Milano et al16 and Koch et al.17 TG4 mice exhibit a 200-fold, cardiac-specific overexpression of the ß2AR.16 TGßARK1 mice exhibit a 3-fold higher cardiac ßARK1 activity than WT mice, and in mice expressing a ßARK1 inhibitor peptide (TGßARKct mice), ßARK1 activity was 50% lower than WT.17
Fifteen male adult heterozygous TG4 mice of body weight 28±2 g; 7 heterozygous TGßARK1 mice of body weight 33±1 g and 6 heterozygous TGßARKct mice of body weight 30±1 g were used. Twenty-four WT mice of body weight 33±2 g were used as controls. All animals were treated in accordance with NIH guidelines.
PTX Pretreatment
To inhibit the Gi protein, 8 WT and 6 TG4
mice were injected with 60 µg/kg PTX
intraperitoneally, 24 hours before experimentation.
To confirm inhibition of Gi, hearts were
isolated, perfused, and stimulated with 10–8
mol/L isoproterenol before addition of 10–6
mol/L adenosine. As outlined in Figure 1
, isoproterenol
activates Gs and subsequently
contractility increases, whereas adenosine
activates Gi and opposes the
isoproterenol-induced stimulation of
contractility.18 In non-PTX–treated WT
hearts, addition of 10–8 mol/L isoproterenol
resulted in a 30% increase in contractility, and this
increase was reversed fully by addition of 10–6
mol/L adenosine. In PTX-treated WT hearts, addition of
10–8 mol/L isoproterenol resulted in a 50%
increase in contractility, and there was no decrease in
contractility on subsequent addition of
10–6 mol/L adenosine, confirming the
lack of Gi activity in PTX-treated hearts.
Ischemia/Reperfusion Protocol
Hearts were isolated and perfused in the Langendorff mode as
described previously.19 All hearts were perfused for 30
minutes before being subjected to 20-minute no-flow ischemia
and 40-minute reperfusion. Left ventricular developed
pressure (LVDP), ±dP/dt, and heart rate were monitored via a
water-filled latex balloon inserted into the left ventricle. Recovery
of contractile function was assessed by measurement of LVDP at the end
of reperfusion and was expressed as a percentage of
preischemic LVDP.
NMR Spectroscopy
Relative changes in concentrations of phosphorus metabolites
were observed during the ischemia/reperfusion protocol by
acquiring consecutive 31P NMR spectra as
described previously.19
The areas of the spectral peaks were expressed as a percentage of the peak areas of an initial, preischemic control spectrum from each heart. The ratios of the phosphocreatine (PCr)/ATP peaks in the preischemic control spectra were lower in the TG4 and TG4+PTX hearts, compared with the other groups. PCr levels decrease on ß-adrenergic stimulation,20 independent of the increased workload,21 but ATP remains at normal levels. The lower PCr/ATP ratio therefore implies that preischemic PCr levels were lower in the 2 TG4 groups. PCr values were therefore normalized by expressing PCr peak areas as a percentage of the ATP peak area in the initial preischemic control spectrum. Intracellular pH was estimated from the chemical shift of the Pi peak relative to PCr using previously obtained titration curves.
Statistics
Results are expressed as mean±SEM. Significance
(P<0.05) was determined by ANOVA followed by a Fisher post
hoc test.
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Contractile Function
Myocardial functional parameters for the 6 groups of mice are shown in the Table
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Effects of Overexpression of the ß2AR
During the preischemic period, LVDP, +dP/dt, and
–dP/dt were higher in the TG4 hearts, at 134 cm
H2O, 5.1 cm H2O/ms, and
–4.3 cm H2O/ms, respectively, than in the WT
hearts, at 113 cm H2O, 4.2 cm
H2O/ms, and –3.4 cm
H2O/ms, respectively (P<0.05).
Overexpression of the ß2AR, therefore,
increased basal myocardial contractility.
Ischemic contracture began at 14 minutes and reached a maximum pressure at 19 minutes in the WT hearts. Contracture occurred earlier in TG4 hearts, beginning at 11 minutes and ending at 16 minutes (P<0.01).
By the end of the 40-minute reperfusion period, recovery of contractile function was significantly lower in the TG4 hearts, at 8% of initial LVDP, than in the WT hearts, at 31% of initial LVDP (P<0.0001). Overexpression of the ß2AR, therefore, resulted in increased ischemic injury.
Effects of Pretreatment With the Gi Inhibitor PTX
During the preischemic period, LVDP and –dP/dt were
higher in the TG4+PTX hearts, at 157 cm H2O and
–5.6 cm H2O/ms, respectively, than in the
untreated TG4 hearts, at 134 cm H2O and –4.3 cm
H2O/ms, respectively (P<0.05). There
was no effect of PTX pretreatment on contractility in
WT hearts. Pretreatment with PTX, therefore, had no effect on WT
hearts, whereas, in the TG4 hearts, PTX pretreatment resulted in
increased myocardial contractility. These results are
consistent with the observation in guinea pig
myocytes22 that PTX treatment had no effect on
contractility in unstimulated myocytes but increased
myocyte sensitivity to isoproterenol stimulation.
Ischemic contracture began at 11 minutes and reached a maximum pressure at 16 minutes in the untreated TG4 hearts. Contracture occurred earlier in TG4+PTX hearts, beginning at 9 minutes and ending at 13 minutes (P<0.05). There was no effect of PTX pretreatment on the timing of contracture in WT hearts.
By the end of the 40-minute reperfusion period, recovery of contractile function was significantly lower in the TG4+PTX hearts, at 2% of initial LVDP, than in the untreated TG4 hearts, at 8% of initial LVDP (P<0.05). There was no effect of PTX pretreatment on postischemic recovery of contractile function in WT hearts. Therefore, pretreatment with PTX had no effect on WT hearts, whereas, in the TG4 hearts, PTX pretreatment resulted in increased ischemic injury. These results imply that Gi has no significant role in ischemic injury in unstimulated WT hearts, but given the findings in TG4 hearts, Gi may be expected to play a role in WT hearts if ischemia is preceded by ß2AR stimulation by, for example, release of epinephrine or norepinephrine or administration of ß-agonists.
Effects of Overexpression of ßARK1 and the ßARK1
Inhibitor
During the preischemic period, LVDP, +dP/dt and
–dP/dt were lower in the TGßARK1 hearts, at 90 cm
H2O, 3.3 cm H2O/ms, and
–2.4 cm H2O/ms, respectively, than the WT
hearts, at 113 cm H2O, 4.2 cm
H2O/ms, and –3.4 cm
H2O/ms, respectively (P<0.05). Heart
rate was also lower in the TGßARK1 hearts, at 352 bpm, than in the WT
hearts, at 400 bpm (P<0.01). Overexpression of ßARK1,
therefore, decreased basal myocardial contractility.
LVDP and +dP/dt were significantly higher in the TGßARKct hearts than
in the WT hearts, at 132 cm H2O and 5.1 cm
H2O/ms, respectively (P<0.05).
By the end of the 40-minute reperfusion period, recovery of contractile function was significantly higher in the TGßARK1 hearts, at 48% of initial LVDP, than in the WT hearts, at 31% of initial LVDP (P<0.01). Recovery of function was similar in the TGßARKct hearts, at 27% of initial LVDP, as in WT. Therefore, overexpression of the ßARK1 decreased ischemic injury, whereas expression of the ßARK1 inhibitor had no significant effect on ischemic injury. It should be noted that use of shorter ischemic periods, at least in a working heart model, has been shown to give different results. Chen et al23 demonstrated that after 6 minutes of ischemia, cardiac output in the TGßARK1 hearts was depressed during reperfusion relative to WT.
Isoproterenol Stimulation of Contractile Function
In WT hearts there was an increase in contraction in response to
isoproterenol with an EC50 of
10–6 mol/L. In TG4 hearts, however, there was
no contractile response to isoproterenol doses in the range
10–8 to 10–4 mol/L,
confirming original observations.16 In PTX-treated TG4
hearts, there was a 20% increase in contractility in
response to 10–8 mol/L isoproterenol. Therefore,
the contractile resistance to isoproterenol observed in TG4 hearts was
relieved by PTX pretreatment, supporting recent findings in
myocytes.24
Phosphate Metabolite Levels and pHi
Phosphate metabolite levels and pHi were
measured in all hearts to determine whether the manipulations of
ß2AR signaling altered myocardial energetics or
pH regulation.
Effects of Overexpression of the ß2AR
During the preischemic period, the PCr/ATP ratio was
lower in the TG4 hearts, at 1.17±0.09, than in the WT hearts, at
1.51±0.08 (P<0.01), consistent with increased
ß-adrenergic signaling.
During ischemia, ATP levels fell lower in the TG4 hearts,
reaching 16% of initial ATP, than in the WT hearts, in which ATP
levels fell to 40% of initial ATP (Figure 2A; P<0.0001). During
reperfusion, ATP levels increased in all hearts, however, ATP levels
remained lower in the TG4 hearts, reaching 29% of initial ATP by the
end of reperfusion, compared with 47% of initial ATP in the WT hearts
(P<0.01).
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PCr decreased rapidly in all hearts at the onset of ischemia
(Figure 2B). At the end of ischemia, there was no
significant difference in PCr levels between the TG4 and WT hearts. On
reperfusion, PCr levels increased in all hearts. At the end of
reperfusion, the PCr level was lower in the TG4 hearts, at 53% of
initial ATP, than in WT hearts, at 89% of initial ATP
(P<0.01).
Intracellular pH decreased during ischemia in all hearts
(Figure 3). At the end of
ischemia, pH was lower in the TG4 hearts, at pH 5.52, than in
WT hearts, at pH 5.85 (P<0.0001). There were no significant
differences in pH between the WT and TG4 hearts during reperfusion.
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In summary, hearts from mice overexpressing the ß2AR, in addition to an increase in basal contractility, had a lower PCr/ATP ratio during basal perfusion than WT hearts. During ischemia, these hearts exhibited a greater loss of ATP and a lower pHi than WT hearts. During reperfusion, recoveries of ATP and PCr were lower in ß2AR overexpressor hearts than in WT hearts, therefore correlating with the lower recovery of contractile function observed and indicating greater injury.
Effects of PTX Pretreatment
During the preischemic period, the PCr/ATP ratio was
lower in the TG4+PTX hearts, at 1.11±0.07, than in the WT+PTX hearts,
at 1.56±0.10 (P<0.01), again consistent with
increased ß-adrenergic signaling. There was no significant difference
in PCr/ATP ratio between TG4 and TG4+PTX hearts.
During ischemia, ATP levels fell lower in the TG4+PTX hearts,
reaching 6% of initial ATP, than in the TG4 hearts, in which ATP
levels fell to 16% of initial ATP (Figure 2A;
P<0.05). During reperfusion, ATP levels remained lower in
the TG4+PTX hearts, reaching 18% of initial ATP, compared with 29% of
initial ATP in the TG4 hearts (P<0.05). At no time during
the protocol were there any differences in ATP levels between WT+PTX
and WT hearts.
PCr decreased rapidly in all hearts at the onset of ischemia
and increased in all hearts during reperfusion (Figure 2B). At
no time during the protocol were there any significant differences in
PCr levels between TG4+PTX and TG4 hearts or between WT+PTX and WT
hearts.
Intracellular pH decreased during ischemia in all hearts
(Figure 3). Although pH was slightly lower at the end of
ischemia in the TG4+PTX hearts, at pH 5.39, than in TG4 hearts,
at pH 5.52, there were no significant differences in pH between any of
the groups of hearts during the protocol.
To summarize, hearts from PTX-treated mice overexpressing the ß2AR exhibited a greater loss of ATP during ischemia than did untreated ß2AR overexpressor hearts. During reperfusion, recovery of ATP was also lower in PTX-treated ß2AR overexpressor hearts than in untreated ß2AR overexpressor hearts, therefore correlating with the lower recovery of contractile function observed in the PTX-treated hearts and indicating greater injury. There was no effect of PTX pretreatment, with respect to ischemic energetics or pH, on WT mice, consistent with the lack of functional effects of PTX pretreatment observed in these hearts.
Effects of Overexpression of ßARK1 and the ßARK1
Inhibitor
During the preischemic period, PCr/ATP ratios were the
same in the TGßARK1 hearts, at 1.51±0.09, as in WT hearts, at
1.51±0.08. Despite the increase in contractility and
the presumed increase in ß-adrenergic signaling in TGßARKct hearts,
PCr/ATP ratios were the same as in WT hearts, at 1.49±0.07.
During ischemia, there were no significant differences in ATP
levels between the groups of hearts (Figure 4A). Although ATP levels fluctuated in
the TGßARK1 hearts during reperfusion, at the end of reperfusion ATP
levels were higher in the TGßARK1 hearts, at 61% of initial ATP,
compared with 47% of initial ATP in the WT hearts
(P<0.05). ATP levels were the same in the TGßARKct as in
WT hearts.
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PCr decreased during ischemia and increased during reperfusion
in all hearts (Figure 4B). At the end of reperfusion, PCr levels
were higher in the TGßARK1 hearts, at 122% of initial ATP, than in
the WT hearts, at 89% of initial ATP (P<0.05). There were
no differences in PCr levels between TGßARKct and WT hearts.
Intracellular pH in the TGßARK1 and TGßARKct hearts is shown in
Figure 5. At no time during the protocol
were there any significant differences in pH between the WT and
TGßARK1 or TGßARKct hearts.
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In summary, recoveries of energy metabolites during reperfusion were higher in ßARK1 overexpressor hearts than in WT hearts, therefore correlating with the higher recovery of contractile function observed in these hearts and indicating less injury. Despite the increase in contractility and the presumed increase in ß-adrenergic signaling observed in hearts overexpressing the ßARK1 inhibitor, PCr/ATP ratios during basal perfusion were the same as in WT hearts, and there were no significant differences in energy metabolites or pHi during ischemia and reperfusion when compared with WT hearts.
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Effects of ß2AR Signaling on Basal Contractility
In the present study, perfused hearts from ß2AR overexpressor mice exhibited increased peak contraction (LVDP), as well as increased rate of contraction (+dP/dt) and rate of relaxation (–dP/dt), compared with WT hearts. This is consistent with in vivo findings16 and supports the hypothesis that overexpression of ß2ARs can improve cardiac function.
As discussed previously, ß2ARs can couple to
both Gi and Gs. Treatment
of ß2AR overexpressor hearts with PTX, a
Gi inhibitor, will allow us to
determine whether the coupling of the ß2AR to
Gi modulates contractility in
these hearts. PTX-treated ß2AR overexpressor
hearts exhibited increased LVDP and ±dP/dt compared with untreated
overexpressor hearts, implying that coupling of the
ß2AR to Gi resulted in
attenuation of contractility. This is
consistent with the role of Gi in
inhibiting cAMP production by adenylate cyclase and
therefore opposing the action of Gs. As
described earlier, Gi activation by the
ß2AR prevents many of the
Gs-mediated downstream events, which would
otherwise contribute to increased myocardial
contractility, such as phosphorylation
of phospholamban and troponin I.11 A
Gi-mediated decrease in
phosphorylation of phospholamban and troponin I is
consistent with the observations of the present study.
Phosphorylation of both of these proteins results in an
increased rate of relaxation. In the PTX-treated
ß2AR overexpressor hearts, the rate of
relaxation (–dP/dt) was greatly increased compared with untreated
hearts and, unusually, was greater than the rate of contraction. These
observations confirm the functional role of dual
Gs/Gi coupling of cardiac
ß2ARs in a whole-heart model.
An interesting observation in the ß2AR overexpressor hearts was that, unlike WT, there was no myocardial contractile response to isoproterenol. Originally this was attributed to the ß2AR overexpressor hearts being already maximally stimulated.16 However, we found that treatment of the ß2AR overexpressors with PTX restored the isoproterenol sensitivity of contraction. Thus, the level of Gi activation in the ß2AR overexpressor hearts on stimulation by isoproterenol appears to be sufficient to prevent any increase in contraction, supporting recent findings in myocytes.24 Gi activity therefore attenuates contractility in the ß2AR overexpressor hearts under stimulated, as well as basal, conditions.
Hearts with a 3-fold overexpression of ßARK1 exhibited decreased LVDP, ±dP/dt and heart rate compared with WT hearts. This confirms that high levels of ßARK1 can indeed contribute to decreased contractility, as predicted in CHF. As ßARK1 phosphorylates and desensitizes ßARs, the observation of lower contractility in ßARK1 overexpressor hearts also indicates that ß1 and/or ß2ARs may be activated, presumably by endogenous catecholamines, and contribute to basal contractility in the perfused heart.
We also measured contractility in hearts overexpressing
a ßARK1 inhibitor. The ßARK1 inhibitor is a
peptide (ßARKct) corresponding to the Gß
binding region of ßARK1. As Gß binding is
required for activation of ßARK1,4 expression of the
ßARKct results in competition for Gß, and
ßARK1 activation is attenuated.25 Consequently,
Gß-stimulated ßARK1 activity in extracts
from hearts expressing the ßARKct were 50% lower than in WT
hearts.17 We found that expression of this ßARK1
inhibitor peptide increased LVDP and +dP/dt compared with
WT hearts, consistent with findings in vivo.17
This implies that, even at normal levels, ßARK1 may modulate
contractility in the perfused heart. These findings
also implicate expression of a ßARK1 inhibitor as an
alternate gene therapy approach for increasing
contractility in heart failure patients.
Effects of ß2AR Signaling on Ischemic Injury
To determine the consequences of ß2AR
overexpression with respect to ischemic injury, we compared the
response to ischemia in hearts from mice overexpressing the
ß2AR with that of WT hearts. The
postischemic recoveries of both contractile function and
the energy metabolites, ATP and PCr, were less in the
ß2AR overexpressor hearts, indicating greater
injury. During ischemia, ATP levels fell lower in the
ß2AR overexpressor hearts than in WT hearts,
reflecting greater ischemic energy utilization. The faster rate
of ATP utilization in the ß2AR overexpressor
hearts is also consistent with the earlier onset of contracture
observed. As H+ are produced by ATP degradation,
the greater ATP utilization in the ß2AR
overexpressors may contribute to the lower pH also observed in these
hearts. Low ischemic ATP levels and pH often correlate with
increased ischemic injury. ATP is required to maintain function
of proteins such as the
Na+/K+-ATPase, activity of
which is necessary to prevent ischemic
Na+ overload and consequently, via
Na+/Ca2+ exchange,
Ca2+ overload and injury.19 26 27 28
Low pH activates the
Na+/H+ exchanger, which
also contributes to Na+ overload and
injury.29 30 Although the low ischemic ATP and pH
may be sufficient to cause the increased injury observed in the
ß2AR overexpressor hearts, increased
Ca2+ influx through L-type
Ca2+channels, which are activated by
ß2AR signaling, may also contribute. In
summary, ß2AR overexpression resulted in
greater ischemic energy utilization and increased
ischemic injury. These results suggest that a negative
consequence of overexpressing ß2ARs in CHF
patients may be an increase in susceptibility to ischemic
injury. There is some indication, however, that lower levels of
ß2AR expression may prove to be less
deleterious.31
As the ß2AR is coupled to both Gi and Gs, we determined whether the increased ischemic injury observed in the ß2AR overexpressor hearts was mediated through activation of Gi or Gs. WT and ß2AR overexpressor mice were pretreated with PTX to inhibit the Gi protein, and the response to myocardial ischemia was then studied. The postischemic recoveries of both contractile function and ATP were less in the PTX-treated ß2AR overexpressor hearts compared with untreated hearts, indicating greater injury. During ischemia, ATP levels fell lower and the onset of contracture was earlier in the PTX-treated hearts, reflecting greater ischemic energy utilization. Therefore, PTX treatment increased ischemic energy utilization and exacerbated injury in the ß2AR overexpressor hearts. These results indicate that it is the activation of Gs by ß2ARs that leads to greater energy demand and injury and also suggests that Gi activation attenuates the increased energy demand and is protective. The suggestion of a protective effect of Gi activity with respect to ischemic injury is consistent with findings that pretreatment of hearts with adenosine, a Gi activator, results in greater recovery from ischemia.32
To further assess the role of ßAR signaling in ischemic injury, we studied the ischemic response of mice overexpressing ßARK1 and also of mice expressing a ßARK1 inhibitor.17 We found that hearts with a 3-fold overexpression of ßARK1 exhibited higher postischemic recoveries of contractile function and energy metabolites, indicating less injury. The observation of increased ischemic injury in the ß2AR overexpressor hearts indicated that increased ß2AR signaling can be detrimental. As ßARK1 desensitizes ßARs, the observation of less injury in ßARK1 overexpressor hearts also indicates that even normal levels of ß1 and/or ß2AR signaling can enhance ischemic injury. Schomig et al33 demonstrated extracellular accumulation of endogenous myocardial catecholamines after 10-minute ischemia, even in the isolated perfused heart. Our findings indicate that this ischemic catecholamine release may activate ßAR signaling and exacerbate injury.
Interestingly, use of shorter ischemic periods, at least in a working heart model, has been shown to give different results. Chen et al23 demonstrated that after 6 minutes of ischemia, cardiac output in the TGßARK1 hearts was depressed during reperfusion relative to WT. If an increase in extracellular catecholamines is an important factor in myocardial ischemic injury, as proposed above, then the 6-minute ischemic duration used in the Chen et al23 study would not be sufficient for catecholamine accumulation, which may explain why overexpression of ßARK1 was not protective in their study but does appear to be protective after 20-minute ischemia. Regardless of the mechanism, a short period of ischemia has limited clinical relevance; therefore, we believe the protective effect observed in our study, with clinically relevant longer durations of ischemia, is the more important effect. Other factors that may contribute to the differing findings of our study and that of Chen et al,23 in addition to the different ischemia protocols, are the different model systems and different methods of function measurement used.
An intriguing observation was that, despite
contractility being elevated to the same extent as in
ß2AR overexpressor hearts, hearts expressing
the ßARK1 inhibitor did not exhibit increased ATP
depletion during ischemia, and ischemic injury was not
increased, as assessed by recovery of postischemic
contractile function and energy metabolites. Even during basal
perfusion, unlike the ß2AR overexpressor
hearts, PCr/ATP ratios were not decreased relative to WT. Therefore it
appears that expression of the ßARK1 inhibitor results in
increased contractility without depletion of energy
metabolites during basal perfusion or increased energy utilization
during ischemia. The reason for this difference between ßARK1
inhibitor and ß2AR overexpressor
hearts is unclear. As the ßARK1 inhibitor relieves
ßARK1-mediated desensitization of the ß2AR,
one may expect ßARK1 inhibitor and
ß2AR overexpressor hearts to have similar
responses to ischemia as well as similar alterations in
contractility. As ßARK1 desensitizes receptors other
than the ßARs,5 6 7 8 the increased
contractility observed in the ßARK1
inhibitor hearts may be due to increased activity of a
receptor that does not concomitantly increase energy utilization.
Interestingly, agonist activation of the endothelin-B receptor,
a ßARK1 substrate, increases contractility without
decreasing the PCr/ATP ratio.34 Furthermore,
ß-adrenergic stimulation has been shown to increase ATP utilization
via the actomyosin ATPase, an event not observed on stimulation with
endothelin.35 Alternatively, the difference may be related
to the ability of the ßARK1 inhibitor (ßARKct) to also
inhibit Gß-mediated effects.36
Interestingly, differences between the effects of
ß2AR overexpression and ßARK1
inhibitor expression have also been observed in an in vivo
model. When ß2AR overexpressor mice were
crossed with a mouse model of cardiomyopathy and
failure, deterioration of myocardial function and mortality were
increased.37 However, when ßARK1 inhibitor
mice were crossed with the same model, contractility
was increased and myocardial degeneration and failure were prevented.
Regardless of the mechanism, our observation that basal
contractility can be increased by expression of the
ßARK1 inhibitor without a concomitant increase in
ischemic injury suggests a novel and safe therapeutic strategy
for heart failure.
In summary, by comparing basal contractility in mice overexpressing the ß2AR to that of WT mice, we confirmed that overexpression of the cardiac ß2AR increases contractility. Pretreatment of ß2AR overexpressors with the Gi inhibitor, PTX, increased contractility and abolished the contractile resistance to isoproterenol, indicating that coupling of the ß2AR to Gi attenuates contractility in the ß2AR overexpressor hearts under both basal and stimulated conditions. These observations confirm the functional role of dual Gs/Gi coupling of cardiac ß2ARs in a whole-heart model. We also demonstrated that overexpression of ßARK1 resulted in decreased contractility and expression of an inhibitor of ßARK1 increased contractility, suggesting that, at normal levels or when overexpressed, ßARK1 modulates contractility in the perfused heart. These findings also implicate expression of the ßARK1 inhibitor as an alternate gene therapy approach for increasing contractility in heart failure patients.
By comparing the ischemic response of hearts overexpressing the ß2AR with that of WT hearts, we also demonstrated that ß2AR overexpression resulted in greater ischemic energy utilization and increased ischemic injury. These results suggest that a negative consequence of overexpressing ß2ARs in CHF patients, at least at high levels, may be an increase in susceptibility to ischemic injury. The increased energy utilization and injury observed in the ß2AR overexpressors was exacerbated by PTX treatment, which indicates that it is the activation of Gs by ß2ARs that leads to injury and also suggests that Gi activation is partially protective. ßARK1 activity was also shown to be protective, as ßARK1 overexpression decreased ischemic injury. A major finding of this study, however, was that, despite increasing basal contractility, expression of the ßARK1 inhibitor had no effect on ischemic injury. This finding implicates expression of the ßARK1 inhibitor as a novel and safe potential therapy for heart failure.
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Acknowledgments |
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W.J.K., R.J.L., and C.S. thank the NIH for grant support. R.J.L. is an investigator of the Howard Hughes Medical Institute. We thank Sandy J. Duncan for mouse phenotyping, Guido Iaccarino for performing PTX treatments, and Robert E. London for use of NMR facilities.
Received June 7, 1999; accepted September 16, 1999.
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