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Cardiovascular Research 2002 53(2):392-404; doi:10.1016/S0008-6363(01)00490-4
© 2002 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Catecholamine stimulation is associated with impaired myocardial O2 utilization in heart failure

Lazaros A Nikolaidisa, Teresa Hentosza, Aaron Doverspikea, Rhonda Huerbina, Carol Stolarskia, You-Tang Shenb and Richard P Shannona,*

aDepartment of Medicine, Allegheny General Hospital, MCP-Hahneman University School of Medicine, 320 E. North Avenue, Pittsburgh, PA 15212, USA
bMerck Research Laboratories, West Point, PA, USA

* Corresponding author. Tel.: +1-412-359-3022; fax: +1-412-359-8152 rshannon{at}wpahs.org

Received 9 April 2001; accepted 24 September 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: To investigate the effect of {alpha}, β1 and β2 adrenergic receptor (AR) stimulation on coronary hemodynamics, myocardial oxygen consumption (MvO2) and metabolic substrate preference in advanced dilated cardiomyopathy (DCM). Methods: We studied 19 conscious, instrumented dogs with pacing-induced DCM. We evaluated systemic, coronary hemodynamics and MvO2 in response to norepinephrine (NOR, 0.05–0.4 µg/kg per min), dobutamine (DOB, 1–10 µg/kg per min), phenylephrine (PHE, 1–5 µg/kg per min) and isoproterenol (ISO, 0.05–0.4 µg/kg per min) alone or in the presence of metoprolol (ISO+MET). Experiments were conducted in control state and in advanced DCM, 4–5 weeks after the initiation of pacing. Results: Contractile responses (LV dP/dt) to catecholamines were desensitized and accompanied by a parallel decrease in heart rate-adjusted myocardial O2 consumption (MvO2/beat), when {alpha} (PHE) or β1 (DOB) or both {alpha}/β1 (NOR) AR were stimulated in DCM. This was due to impaired transmyocardial (Ao–Cs) O2 extraction rather than limitations in CBF responses. There was an associated shift in myocardial metabolism, evidenced by an increased preference for glycolytic substrates ({uparrow}Respiratory Quotient) following administration of any of these three adrenergic agonists in DCM. Combined β1/β2 stimulation with ISO or β2-AR stimulation (ISO+MET) in DCM resulted in greater MvO2/beat, [(Ao–Cs) O2] extraction, and decreases in myocardial RQ consistent with a shift toward oxidation of FFA. Conclusions: The impairment in contractile responses to dobutamine and norepinephrine in DCM is associated with impaired myocardial O2 extraction, and a shift toward a preference for glycolysis. A different myocardial metabolic pattern suggestive of increased oxidation of FFA with increased myocardial O2 extraction was observed in the presence of combined β1/β2 stimulation with isoproterenol or β2 stimulation (ISO+MET). These data suggest that β2-AR stimulation in DCM shifts substrate preference toward FFA oxidation associated with greater MvO2 requirements. These findings identify a putative metabolic effect of β2 –AR in DCM that may be deleterious.

KEYWORDS Cardiomyopathy; Adrenergic (ant)agonists; Heart failure; Oxygen consumption; Energy metabolism; Hemodynamics


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Catecholamine stimulation is considered deleterious in congestive heart failure (CHF). It has been suggested that the myocardial O2 requirements associated with catecholamine stimulation in CHF are excessive and that catecholamine administration is associated with altered myocardial energetics in CHF [1–3]. Catecholamines modulate several aspects of cardiac function including inotropy, chronotropy, coronary blood flow and metabolic substrate preference. While the β-adrenergic effects of catecholamines on heart rate and contractility are known to be desensitized [4–6], less is known regarding the effects on coronary blood flow, O2 utilization, and substrate preference. The extent to which different adrenergic receptor subtypes (β1,β2, or {alpha}) mediate these effects is also incompletely understood.

Clinical studies have demonstrated the benefits of β-adrenergic antagonists in patients with CHF [7–10], in contrast to the adverse outcomes demonstrated with the chronic use of adrenergic agonists [11–14]. It appears that different β-adrenergic antagonists with varying degrees of selectivity exert similar salutary effects, independent of their ability to restore the downregulated and desensitized β1-adrenergic receptors [15–19]. This raises the possibility that metabolic derangements may contribute to the toxic effects of catecholamines in CHF. It is conceivable that some or all of adrenergic receptor subtypes may be implicated in such mechanisms.

The current study was designed to determine the effects of cardiac stimulation with exogenously administered catecholamines with different affinity for the β1, β2, and {alpha} adrenergic receptors on coronary blood flow, myocardial O2 utilization, and metabolic substrate utilization in conscious dogs with pacing induced dilated cardiomyopathy (DCM), compared to the responses elicited in the same animals studied in the control state.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
We studied 19 conscious, chronically instrumented dogs with pacing-induced DCM. This canine model has been previously described from our laboratory [20], and develops a cardiovascular phenotype similar to that seen in human heart failure including comparable alterations in plasma catecholamines and down-regulation and heterologous desensitization or β-adrenergic receptors [21].

Mongrel dogs of either sex weighing 15–20 kg were sedated with xylazine (2 mg/kg im) and anesthetized with halothane (1% vol). Using a sterile technique, catheters were implanted in the descending thoracic aorta (Ao), left atrium (LA), right atrium (RA) and coronary sinus (Cs) through an incision in the left fifth intercostal space. A solid-state pressure transducer was implanted in the left ventricular (LV) apex to measure LV pressures and LV dP/dt. Ultrasonic Doppler flow probes were implanted in the ascending aorta and the left circumflex coronary artery, for determination of cardiac output (CO) and coronary blood flow (CBF), respectively. Piezoelectric ultrasonic dimension crystals were implanted on the anterior and posterior endocardial surfaces of the LV to measure the internal short axis diameter in end-diastole (LVEDD) and end-systole (LVESD). Similar piezoelectric crystals on the endocardial and epicardial surfaces of the posterior wall were utilized to measure wall thickening (WTh). A sutureless pacing lead was implanted on the epicardial surface of the right ventricle.

Experiments were initiated 2 weeks after recovery from surgical instrumentation. Hemodynamic measurements were made with the dogs fully awake, lying quietly on their right side. All experiments were performed in the ‘control’ state, and repeated after 4 weeks of rapid ventricular pacing at 240 min–1 to induce DCM. A 30-min stabilization period following deactivation of the pacemaker preceded all experiments in DCM.

Animals used in this study were maintained in accordance with the Guide for the Care and Use of Laboratory Animal Resources [DHHS Publication No (NIH) 86-23, Revised 1996] and the guidelines of the Institutional Animal Care and Use Committee at Allegheny General Hospital.

Systemic hemodynamics, LV dP/dt, CO and CBF measurements and aortic (Ao) and coronary sinus (Cs) blood samples were obtained at rest and in response to graded infusions of norepinephrine (0.05–0.4 µg/kg per min), dobutamine (1–10 µg/kg per min), phenylephrine (1–5 µg/kg per min) and isoproterenol (0.05–0.4 µg/kg per min). A 30-min period of stabilization between drug infusions allowed all parameters to return to baseline. The agonists were infused in random order, in each dog. Phenylephrine infusion was always performed on a separate day due to its prolonged effects on hemodynamics. Myocardial oxygen consumption (MvO2) was calculated as the product of the left circumflex coronary artery blood flow multiplied by the myocardial arterio-venous O2 content (ct) difference: MvO2=(CBF)x[(Ao–Cs)O2ct]. Similarly, myocardial O2 delivery was calculated as MdO2=(CBF)x(Ao)O2ct. As coronary blood flow was measured only in the LCX distribution, these parameters represent indices of total myocardial O2 delivery and consumption respectively. We measured MvO2 and MdO2 at rest and in response to catecholamine infusions, both at Control and in advanced DCM. We also calculated heart rate adjusted indices reflecting the CBF ‘per beat’ and MvO2 ‘per beat’, to control for the influence of differing heart rate responses to catecholamines under the two different experimental conditions.

Myocardial stroke work (SW) was calculated using the formula SW={Delta}Px{Delta}Vx0.0136, where {Delta}P=LVP–LVEDP and {Delta}V=LVEDV–LVESV, using sonomicrometry-derived data [22–24]. The MvO2 was then normalized for SW (MvO2x105/SW) as an index of contractile efficiency for each agonist in control and in DCM.

2.1 Myocardial acid–base balance and respiratory quotient calculation
Blood samples from the coronary sinus (Cs) were analyzed and the coronary sinus pH (Cs-pH) was plotted in response to the different catecholamine infusions. This was repeated in control and DCM animals and was utilized as a measure of myocardial acidosis. The myocardial respiratory quotient (RQ) was calculated according to the method described by Recchia et al. [25]. Physiologic values for RQ range between 0.7 and 1.0; values close to 1.0 are considered consistent with predominant glucose utilization as energy source, while lower values are seen with free fatty acids (FFA) metabolism. We performed serial calculations of the RQ in control and in advanced DCM and in response to the different catecholamine infusions. This parameter was used as an indicator of metabolic substrate utilization within the myocardium.

2.2 Metabolic responses to selective β2-adrenergic stimulation
To evaluate the metabolic effects of β2-adrenergic receptor (AR) stimulation in DCM, a subgroup of six dogs received isoproterenol at the same dosing protocol in the presence of the β1-AR selective antagonist metoprolol (5–10 mg i.v.). The metoprolol dose was selected to assure adequate β1-AR blockade [at least 50% blockade of both inotropic (LV dP/dt) and chronotropic β1-AR mediated responses to isoproterenol] in each dog. Measurements of oxygen content, pH and RQ were obtained in a similar fashion, to reflect the effects of β2-AR stimulation. To confirm that the reduction in RQ reflected increased free fatty acid (FFA) oxidation, myocardial FFA utilization was estimated from the difference between aortic (ao) and coronary sinus (cs) FFA concentrations before and after administration of the peak dose of isoproterenol (0.4 µg/kg per min) in the presence of metoprolol. All FFA measurements were conducted using a commercially available test kit (Wako Diagnostics, Richmond, VA) in the fasting state.

2.3 Myocardial β-adrenergic receptors
Myocardial β-adrenergic receptors (β-AR) were measured on crude sarcolemmal membrane preparations from eight animals in advanced DCM and were compared to similar membrane preparations from normal dogs (n=3). We performed receptor antagonist binding studies using 125I-cyanopindolol (125I-CYP) to determine total β-AR density, as well as competitive inhibition agonist binding studies using graded concentrations of isoproterenol to determine receptor affinity, as described by Kiuchi et al. [21]. Furthermore, we characterized the relative proportion and affinity of β1 and β2 receptors in myocardium from DCM and control animals, using selective antagonist binding assays to the β1 (Betaxolol) and β2 (ICI-118,551) receptor selective antagonists [21,26].

2.4 Statistical analysis
Data were expressed as mean±S.E.M. Maximal responses to each drug were also expressed as percentage change from the baseline value. They were analyzed using MS Excel for Win ‘98 and Origin 6.0 for graphics analysis. Statistical significance was determined using repeated measures ANOVA for comparisons between multiple groups or the two-tail Student's t-test (two-sample t-test assuming unequal variances) for comparisons between two groups. A P value of <0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Animal model of dilated cardiomyopathy
Table 1 illustrates the hemodynamic perturbations seen in association with the development of advanced DCM. Advanced DCM was characterized by increased heart rate, left ventricular end-diastolic and end-systolic dimensions (LVEDD, LVESD) and decreased LV systolic (LVP) and aortic mean pressure, regional wall thickening (WTh), fractional shortening (FS), ejection fraction (EF) and stroke work (SW). The sinus tachycardia was responsible for maintaining cardiac output (CO) while stroke volume (SV) was reduced significantly. CBF/beat was lower in DCM compared to controls. Although MvO2 at rest was not significantly different in DCM as compared to controls (control vs. DCM: 2.3±0.1 vs. 2.4±0.1 ml O2/min), the HR-adjusted MvO2 (‘resting O2 consumption per beat’) was actually lower in DCM than in control (30±1 vs. 21±1 µl O2/beat, P<0.0001). This was attributable partially to a lower ‘CBF/beat’ (0.33±0.01 vs. 0.26±0.01 ml/beat, P<0.03) and partially to a lower O2 extraction [(a–v) O2 difference] in DCM (9.8±0.2 vs. 8.7±0.2 ml O2/dl, P<0.01).


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  		Table 1 Baseline hemodynamics between dogs studied at control and in advanced dilated cardiomyopathy

 
3.2 Hemodynamic and metabolic responses to norepinephrine
Table 2 illustrates the peak responses to IV norepinephrine infusion (0.4 µg/kg per min). Severe DCM was associated with depressed peak contractile responses to norepinephrine (153±5.1 vs. 85±7.5%, P<0.001), and a similar impairment in MvO2 and MdO2 response, which persisted after adjusting for HR (MvO2/beat: 150.7±10 vs. 75.3±7.6%, P<0.001). While the CBF response to norepinephrine was significantly less in DCM than in controls (89.8±9.1 vs. 43.8±10%, P<0.001), the difference was not significant on a ‘per beat’ basis (80±10.5 vs. 64±7.7%). The principal determinant of the impaired peak MvO2 response to norepinephrine (169±9 vs. 62±6.9%, P<0.001) was the failure to augment the myocardial arterio-venous O2 extraction [(a-v)O2] in response to norepinephrine stimulation (33.8±4.5 vs. 8.9±4.2%, P<0.001). Fig. 1 demonstrates the relative changes in contractile and metabolic parameters as a percentage (%) change from the baseline in response to norepinephrine 0.4 µg/kg per min, in control and in advanced DCM.


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  		Table 2 Peak responses to norepinephrine at control and in advanced DCM

 

Figure 1
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  		Fig. 1 The peak response to norepinephrine (0.4 µg/kg/min) depicted as a percentage (%) change from resting values. The ‘MvO2/beat’ responses were depressed, due to limited transmyocardial ‘(a-v)O2’ extraction, while the ‘CBF/beat’ responses remained intact in DCM.

 
3.3 Hemodynamic and metabolic responses to dobutamine
Table 3 summarizes the peak responses to dobutamine (10 µg/kg per min). The peak inotropic and chronotropic responses were desensitized in DCM (LV dP/dt: 105±3.7 vs. 67±10.7%, P<0.001). MvO2 responses were also impaired in DCM (156±8 vs. 58.4±9%, P<0.001), even when adjusted for heart rate (MvO2/beat: 88.8±8 vs. 45±7.8%, P<0.001). On the other hand, the CBF/beat response was not significantly different (42±6.7 vs. 36±10.4%). The failure of the animals with DCM to augment [(a-v)O2] extraction in response to dobutamine was striking in comparison to the control animals (26.2±5.8 vs. 3.2±5.2%, P<0.0001). Fig. 2 depicts the relative changes as (%) of the baseline in control and in severe heart failure in response to dobutamine (10 µg/kg per min).


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  		Table 3 Peak responses to dobutamine at control and in advanced DCM

 

Figure 2
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  		Fig. 2 The peak response to dobutamine (10 µg/kg/min) depicted as a percentage (%) change from resting values. Similar to norepinephrine, the ‘MvO2/beat’ responses were depressed, due to limited transmyocardial ‘(a-v)O2’ extraction, while the ‘CBF/beat’ responses remained intact in DCM.

 
3.4 Hemodynamic and metabolic responses to phenylephrine
Table 4 illustrates the hemodynamic responses to phenylephrine (5 µg/kg per min). Phenylephrine did not exert any significant inotropic or chronotropic effect in either group. The MvO2 and MdO2 responses were markedly impaired in DCM (MvO2/beat: 80.8±8.6 vs. 29.2±6.3%, P<0.001), as was the CBF response, even when adjusted for HR (CBF/beat: 42.8±7 vs. 7±5.2%, P<0.001). This suggests an increased coronary vasoconstriction response to {alpha}-adrenergic stimulation in DCM (CVR response: 8±7 vs. 34±7%, P<0.02). However, myocardial [(a-v)O2] extraction in response to the {alpha}-agonist was also limited in DCM compared to controls (32.3±3.1 vs. 16.5±3.2%, P<0.003), suggesting both limited myocardial O2 delivery and utilization. The peak responses to phenylephrine (5 µg/kg per min) are illustrated in Fig. 3.


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  		Table 4 Peak responses to phenylephrine at control and in advanced DCM

 

Figure 3
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  		Fig. 3 The peak response to phenylephrine (5 µg/kg/min) depicted as a percentage (%) change from resting values. The {alpha}-agonist resulted in a marked reduction of the ‘MvO2/beat’ in DCM, due to limitations in both ‘CBF/beat’ response and transmyocardial ‘(a-v)O2’ extraction.

 
3.5 Hemodynamic and metabolic responses to isoproterenol
We observed different responses with isoproterenol, a combined β1 and β2-adrenergic agonist (Table 5). The peak inotropic (LV dP/dt: 235±4 vs. 132±9.5% P<0.001) and chronotropic (145±4.8 vs. 58±6.5%, P<0.001) responses were significantly attenuated in response to isoproterenol infusion in DCM. When adjusted for HR, there was no significant difference in the MvO2/beat (59.3±10.6 vs. 56.8±10%) or CBF/beat responses (29.3±7 vs. 21.4±12.9%) between the DCM and the control groups (Fig. 4).


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  		Table 5 Peak responses to isoproterenol at control and in advanced DCM

 

Figure 4
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  		Fig. 4 The peak response to isoproterenol (0.4 µg/kg/min) depicted as a percentage (%) change from resting values. Unlike the other catecholamines, the ‘MvO2/beat’, ‘CBF/beat’ and ‘(a-v)O2’ extraction were preserved in DCM. There were, however, reduced MvO2 and CBF responses, which were solely due to impaired chronotropic response.

 
Notably, the myocardial [(a-v)O2] difference in response to exogenous stimulation was preserved with isoproterenol (24.6±5.8 vs. 20±7.2%), in contrast to that observed with other agonists studied.

3.6 Myocardial stroke work – MvO2 relationships
Myocardial stroke work was significantly reduced in DCM compared to controls, both at baseline (15±1 vs. 8±1 g m, P<0.0001), and following adrenergic stimulation. MvO2 normalized for SW (MvO2x105/SW) was significantly greater in DCM compared to controls, both at baseline (17±1 vs. 40±1 mm2/g, P<0.001) or following stimulation with any agonist (Tables 1–5GoGoGoGo). However, the effect of pharmacologic stimulation with peak doses of dobutamine (10 µg/kg per min), norepinephrine (0.04 µg/kg per min) or phenylephrine (5 µg/kg per min) on MvO2/SW was limited in DCM, compared to the significant increases in MvO2/SW in control animals (Fig. 5). In contrast, isoproterenol preserved the ability to augment MvO2/SW in DCM, although to a lesser extent (C: 111±9%, CHF: 74±11%, P<0.05) than in the control state.


Figure 5
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  		Fig. 5 Impact of Dobutamine, Norepinephrine, Phenylephrine and Isoproterenol on MvO2 normalized for myocardial stroke work (MvO2x105/SW) in control and in DCM. White bars represent resting values and are consistently higher in animals with DCM compared to control animals. Shaded bars reflect the effect of pharmacologic stimulation, which was minimal in advanced DCM, with the exception of isoproterenol. Isoproterenol infusion in DCM was still capable of further augmentation of an already high ‘SW-normalized MvO2’ by approximately 70%. While all other agonists significantly augmented the amount of O2 consumed per unit increase in SW in controls, only isoproterenol exhibited a similar (although less pronounced) metabolic effect in animals with dilated cardiomyopathy.

 
3.7 Myocardial acid–base balance
Baseline coronary sinus pH (Cs-pH) was lower in DCM compared to controls (7.38±0.01 vs. 7.36±0.01, P<0.05). In control animals, administration of the catecholamines did not result in significant changes in Cs-pH, as illustrated in Fig. 6. In contrast, administration of peak doses of phenylephrine, norepinephrine or dobutamine resulted in significant further decline in Cs-pH. (Cs-pH: PHE: 7.32±0.01, NOR: 7.33±0.01, DOB: 7.34±0.01, all P<0.05 compared to resting values in DCM, Fig. 6) in dogs with DCM. This was not observed with isoproterenol alone (Cs-pH ISO: 7.36±0.007) or in combination with metoprolol (β-2stimulation) in dogs with DCM (Cs-pH: ISO+MET: 7.38±0.007), where the Cs-pH response was not different than that observed in the control state.


Figure 6
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  		Fig. 6 Changes in coronary sinus pH (Cs-pH) in response to IV infusions of different adrenergic agonists in control animals and in animals with DCM. Square dots ({blacksquare}) represent control animals and round dots (bullet) represent animals with DCM. None of the drugs infused resulted in a statistically significant alteration in Cs-pH in control animals. Contrast with the significant decline in Cs-pH when animals with DCM were stimulated with phenylephrine, norepinephrine or dobutamine, but not with isoproterenol.

 
3.8 Metabolic substrate utilization-myocardial respiratory quotient
The myocardial respiratory quotients were higher in DCM than in control (control: 0.65±0.06, DCM: 0.75±0.05, P<0.05), suggesting a trend toward glycolysis as a preferred metabolic substrate in DCM. Administration of phenylephrine, norepinephrine, or dobutamine in animals with DCM resulted in further increases of the RQ (PHE: 0.87±0.04, NOR: 0.84±0.07, DOB: 0.82±0.05, all P<0.05 compared to RQ in DCM), suggestive of a further shift toward glycolysis. In contrast, isoproterenol significantly lowered the RQ in the dogs with DCM to values similar to those seen in the control group (ISO: 0.67±0.06, P<0.02 compared to RQ in DCM). These findings (Fig. 7) suggest that β-2AR stimulation in DCM shifts metabolic preference toward FFA oxidation.


Figure 7
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  		Fig. 7 Myocardial respiratory quotient (RQ). Administration of phenylephrine, norepinephrine or dobutamine to animals with DCM resulted in even further increase in RQ, favoring glycolysis. Administration of isoproterenol (alone or in combination with metoprolol) in these animals resulted in a significant decline in RQ to values similar to those measured in ‘control’ state. This implies that β2-AR stimulation favors FFA oxidation in dilated cardiomyopathy.

 
3.9 Hemodynamic and metabolic responses to isoproterenol+metoprolol
By experimental design, inotropic (LV dP/dt: 1540±125 to 1466±94 mmHg/s) and chronotropic (106±5 to 110±4 min–1) responses to isoproterenol in the presence of metoprolol were blunted in dogs with DCM. However, combined isoproterenol and metoprolol administration (ISO+MET) in dogs with DCM resulted in augmentation in [(a-v)O2] extraction by 25±1% (from 8.3±0.5 to 10.4±0.6 mlO2/dl, P<0.05), similar to what was observed with isoproterenol alone (ISO) in either control or advanced DCM animals. Coronary sinus pH was preserved (7.37±0.01 to 7.38±0.07), as well. Myocardial RQ (Fig. 7) decreased to control levels in the ISO+MET group (0.88±0.05 to 0.66±0.07, P<0.01). This was associated with a trend toward increased myocardial FFA utilization [(ao-cs) FFA: 145±12 to 233±27 µmol/l, P<0.07] following combined ISO+MET administration.

3.10 Myocardial β-adrenergic receptor studies
Fig. 8 reveals the change in receptor density and affinity as well as the β-1/β-2distribution of receptors in DCM compared to controls. Decreased total receptor density (137±24 vs. 82±10 fmol/mg protein) as well as a decreased proportion of β-1 receptors in a ‘high affinity’-state (68±10 vs. 38±11%) was observed in DCM animals compared to controls. This was accompanied by a relative increase in both density and affinity, of β-2ARs, that constituted a higher proportion of the total β-ARs in the failing as opposed to the normal myocardium (control vs. DCM: 40±8 vs. 62±4%).


Figure 8
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  		Fig. 8 Myocardial adrenergic receptor density and affinity in tissue obtained from control (dark bars) and DCM animals (light bars). There is selective down-regulation and desensitization of β1-ARs, with relative increase in β2-ARs density and affinity in DCM compared to controls.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
We observed a significantly desensitized inotropic response (LV dP/dt) to norepinephrine, dobutamine and isoproterenol, consistent with previously published reports [4,5,27–30]. However, it was also apparent that the desensitized contractile response (LV dP/dt) to exogenously administered β-1 or {alpha}- (dobutamine, norepinephrine) adrenergic agonists was accompanied by a parallel decrease in both MvO2 and MvO2 per beat in DCM. The latter was due to failure to increase transmyocardial [(a-v)O2] difference in response to catecholamine administration. Heart rate –adjusted CBF response was only impaired (and CVR response was enhanced) in response to phenylephrine, signifying a potentially enhanced {alpha}-vasoconstrictor effect in DCM. There was impaired O2 utilization ({downarrow}MvO2/beat) in conscious dogs with pacing induced DCM compared to the same animals in control state. This was accompanied by an increased preference for glycolytic substrates ({uparrow}RQ) and a decrease in Cs-pH, compared to controls.

Conventionally, FFA are the preferred metabolic substrate for normal myocardium. However, in states of stress such as heart failure, glycolysis has been demonstrated to assume a greater role in ATP synthesis [25,31]. The reason involves the greater efficiency of glycolysis in terms of ATP generated per mole O2 consumed. We observed an even greater shift of the myocardial metabolism toward glycolytic substrates ({uparrow}RQ) and further declines in Cs-pH with either {alpha} or β-1adrenergic agonists in DCM.

In contrast, combined β1/β2 stimulation with isoproterenol caused a similar impairment in contractile and chronotropic response, but a different metabolic pattern, with a shift toward FFA oxidation ({downarrow}RQ), preserved O2 utilization (MvO2/beat) and (a-v)O2 extraction compared to control responses. These data suggest that β-2adrenergic stimulation in CHF favors FFA oxidation and requires greater O2 extraction.

As it has been described in both human myocardial tissue biopsies [32,33] and pacing-induced DCM in dogs, there is down-regulation of β1-AR with preservation or up-regulation β2-AR in CHF (Fig. 8), suggesting that β2-ARs may be more important in heart failure. Taken together, these data define a putative role for β2-AR stimulation in myocardial substrate preference in heart failure.

The physiological role of β2-AR in larger mammals in heart failure is poorly understood. The functions include improvement in diastolic relaxation, and reduced phospholamban levels [34], as well as anti-apoptotic effects [35]. The pathophysiologic role of β2-AR in heart failure is even less well understood. Transgenic murine models of cardiac specific overexpression of β2-AR have yielded conflicting results [36,37]. Notably, over-expression has been at many fold higher levels than observed in human or mammalian models of CHF. Data from these transgenic murine models suggest that while β1-AR over-expression may lead to deleterious myocardial sequelae, this may not be true for β2-AR, where an ‘optimal range’ of β2-AR over-expression may be salutary. Our study is the first to implicate β2-AR stimulation in dictating metabolic substrate preference in the heart. Moreover, preference for FFA oxidation during DCM contributes to further impairment in myocardial efficiency.

It has been hypothesized that the increased energy demands of the failing myocardium lead to a state of relative energy depletion, through an initial compensatory phase of increased O2 extraction [1,38]. This paradigm [39] suggests that further inotropic stimulation would impose further energy demands and ultimately accelerate myocardial cell death. Investigators have attempted to confirm or refute it, utilizing a variety of in-vitro and in-vivo models. The heterogeneity of those models and methods to investigate MvO2 has led to conflicting results. Our study attempted to minimize these variables by investigating a well-validated model of end-stage dilated cardiomyopathy, with no evidence of hypertrophy or myocardial ischemia, in the conscious state. To normalize for differences in heart rate that accompany the progression of dilated cardiomyopathy, we expressed O2 indices on a ‘per-beat’ basis.

Our findings suggest a parallel impairment in contractile performance and myocardial oxygen consumption in response to inotropic stimulation in end-stage cardiomyopathy. Catecholamine mediated increases in glycolytic flux subtended by {alpha}1 and β1 receptor subtype leads to reduced MvO2 due to the greater efficiency of glucose oxidation. This is reflected in less O2 extracted. However, β2-AR stimulation shifts the substrate preference to FFA with additional requirements for O2 extraction and O2 utilization. While most prior studies have focused on the effects of dobutamine on energy utilization by the failing myocardium [40–43], limited data exist on the differential contribution of discrete adrenergic receptor subtypes in these phenomena.

Myocardial ischemia was not present in our studies, as evident by the lack of significant decline in CBF during β1-adrenergic stimulation. With the exception of phenylephrine, no potentation of coronary vasoconstrictor response to any other agonist was observed in animals with DCM. The random order of agonist administration also assured the absence of residual pharmacodynamic effects.

Although statistically significant, the magnitude of acidemia observed in our studies was modest and insufficient to account for a significant shift in Hb–O2 dissociation curve, which usually occurs at pH<7.2. The theoretical change anticipated from a rightward shift in the Hb–O2 dissociation curve induced by acidemia (a slight increase in [a-v]O2 tissue extraction) would have been in the opposite direction of what was observed in our experiments [44,45]. Therefore, the observed limitation in O2 utilization in response to β1 and {alpha}-AR stimulation is unlikely to be a shift in hemoglobin affinity.

In our study, we did not take into account the potential effect of isoproterenol on β-3 adrenergic receptors. While β-3AR have been reported to be stimulated by isoproterenol in human adipocytes in-vitro, a systemic lipolytic effect could not be substantiated in-vivo, following combined β-1 and β-2blockade in healthy volunteers [46]. The contribution of β-3AR stimulation to myocardial effects in different species has also been studied by Shen et al. [47]. This study demonstrated a more pronounced effect on both systemic hemodynamics and FFA utilization in dogs, compared to rats or non-human primates. However the β-3AR specific agonists BRL37344 and CL316243 [GenBank] , rather than isoproterenol were used in this study of normal dogs. Since neither of these studies has investigated the function of β-3ARs in the failing heart, a theoretical up-regulation of these receptors or enhanced affinity of isoproterenol for them in dilated cardiomyopathy, contributing to increased FFA oxidation cannot be entirely excluded.

In conclusion, advanced DCM in our model was associated with impaired inotropic responses to catecholamines. The impairment in contractile responses to dobutamine and norepinephrine in DCM was associated with limitations in O2 utilization, and a metabolic shift toward myocardial anaerobic glycolysis and myocardial acidemia.

In the presence of combined β1/β2 stimulation with isoproterenol, however, there was a similar impairment in contractile response, but a different metabolic pattern, with a shift toward FFA oxidation (decreased RQ, increased myocardial FFA extraction), preserved O2 utilization (MvO2/beat, ability to augment MvO2/SW) and (a-v)O2 extraction. These patterns were even more apparent when isoproterenol was administered in the presence of metoprolol, signifying a β2 receptor-specific effect. These data suggest that β2-adrenergic stimulation in advanced DCM favors FFA oxidation and requires greater O2 extraction and utilization. Whether the metabolic shift to the more O2 demanding process of FFA oxidation is deleterious in the long term, remains to be examined.

Time for primary review 36 days.


   Acknowledgements
 
We thank Robert Jacobs, PhD for his assistance with graphics and statistical analysis. This work was supported in part by US Public Health Service Grants HL-59070 and DA-10480, and by an American Heart Association (PA-DE Affiliate) Fellowship grant (0020248U).


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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