β-Adrenergic receptor (β-AR) responsiveness is downregulated in left ventricular (LV) hypertrophy induced by chronic hypertension. While exercise training in hypertension enhances β-AR responsiveness, the role of adenylyl cyclase remains unclear. The purpose of the present study was to test whether treadmill running in the spontaneously hypertensive rat (SHR) model improves LV responsiveness to forskolin (FOR) or the combination of FOR + isoproterenol (FOR+ISO). Female SHR (16-wk) were randomly placed into sedentary (SHR-SED; n = 7) or treadmill-trained (SHR-TRD; n = 8) groups. Wistar-Kyoto (WKY; n = 7) animals acted as normotensive controls. Langendorff, isovolumic LV performance was established at baseline and during incremental FOR infusion (1 and 5 μmol/l) and FOR+ISO (5 μmol/l + 1×10−8 mol/l). Heart rate, systolic blood pressure, and heart-to-body weight ratio were lower in WKY relative to both SHR groups (P < 0.05). LV performance and heart rate significantly increased in all groups to a similar extent with incremental FOR infusion. However, in the presence of 5 μmol/l FOR, ISO increased LV developed pressure, positive change in LV pressure, and negative change in LV pressure to a greater extent in SHR-TRD relative to SHR-SED (P < 0.05). Phospholamban phosphorylation at the Thr17 was greater in SHR-TRD relative to SHR-SED and WKY (P < 0.05). Absolute LV developed pressure was moderately correlated with phospholamban phosphorylation at both the Ser16 (r = 0.64; P < 0.05) and Thr17 (r = 0.52; P < 0.05). Our data suggest that the adenylyl cyclase step in the β-AR cascade is not downregulated in the early course of hypertension and that the enhanced β-AR responsiveness with training is likely mediated at levels other than adenylyl cyclase. Our data also suggest that β-AR inotropic responsiveness in the presence of direct adenylyl cyclase agonism is improved in trained compared with sedentary SHR hearts.
- spontaneously hypertensive rat
the β-adrenergic receptor (β-AR) system is an integral pathway in regulating myocardial inotropy and lusitropy, particularly during the stress of exercise (43, 46, 51). With hypertension, however, the β-AR system is downregulated via receptor uncoupling (10, 27) and is clearly one hallmark of the maladaptive phenotype associated with compensatory hypertrophy (50). Recent data from our laboratory have shown that exercise training improves β-AR responsiveness in the spontaneously hypertensive rat (SHR) model by mitigating the left ventricular (LV) abundance of the β-AR receptor desensitizing kinase, G protein receptor kinase 2 (GRK2) (27). As a result of “restored” β-AR signaling, PKA-mediated phosphorylation of key sarcoplasmic reticulum (SR) Ca2+ handling proteins, such as the ryanodine receptor and phospholamban (PLB), were improved with training (27). However, given that direct adenylyl cyclase/cAMP signaling may also be altered with hypertension (9, 28, 31), it remains unclear from our previous work whether “downstream-associated” β-AR mechanisms are likewise improved by training.
Thus one purpose of the present study was to examine whether exercise training superimposed on hypertension would also elicit a putative physiological effect in response to direct adenylyl cyclase agonism via forskolin infusion. Forskolin is a diterpene derivative synthesized from the root of Coleus forskolii (4). Forskolin activates adenylate cyclase by binding to the cytosolic domain of the membrane-bound enzyme and leads to cAMP generation in a reversible manner, irrespective of cell surface receptors (44). This is of particular interest because, although both direct β-AR agonism with isoproterenol and downstream signaling with forskolin result in a dose-dependent increase in intracellular cAMP, differences in cellular compartmentation of these systems allow for a more prolific rise in intracellular Ca2+ concentration ([Ca2+]i) and inotropy with isoproterenol (18, 23, 25, 45, 48). Moreover, since PKA can phosphorylate and uncouple β-AR signaling, we also examined whether concurrent forskolin and isoproterenol infusion would induce a differential inotropic response in the hypertensive sedentary and trained myocardium. We hypothesized that β-AR responsiveness would be enhanced with exercise training in the presence of elevated forskolin-induced increases in cAMP.
MATERIALS AND METHODS
Animal care and exercise training.
Twenty-two 16-wk-old female Wistar Kyoto (WKY, n = 7) and SHR (n = 15) (weighing ∼200 g) were obtained from Charles River Laboratories (St. Constant, Quebec, Canada). Animals within the SHR group were randomly assigned into either a sedentary (SHR-SED) (N = 7) or an exercise-trained (SHR-TRD) (N = 8) group. All rats were housed three to five per cage. Animals were maintained on a 12:12-h light-dark cycle and fed ad libitum (Harlan Teklad Global Diets, 18% protein diet, Madison, WI). Exercise training consisted of low-intensity endurance training at a speed of 22 m/min, 0% grade, 60 continuous minutes, 5 days/wk, for a period of 12 wk. Resting heart rates (HRs) (mean of 25 cardiac cycles) and blood pressures were collected before death, utilizing a tail cuff apparatus (Kent Scientific, Torrington, CT). At 28 wk of age, animals were killed, and functional studies were performed. All animals received humane care in compliance with Temple University Institutional Animal Care and Use Committee Standards and the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication no. 85–23, revised 1985).
Langendorff isovolumic preparation.
Rats were anesthetized with pentobarbital sodium (60 mg/kg ip) and heparinized intravenously (500 units iv). The heart was excised, trimmed of excess tissue, and rapidly immersed in cold (4°C) Ca2+-free Krebs-Henseleit buffer. As previously described (27, 40, 41), hearts were placed on a Langendorff perfusion apparatus (ML785B2, ADInstruments, Colorado Springs, CO) and perfused at 16 ml/min (STH pump controller ML175, ADInstruments, Colorado Springs, CO) with a modified Krebs-Henseleit solution containing (in mM) 2.0 Ca2+ Cl2, 130 NaCl, 5.4 KCl, 11 dextrose, 2 pyruvate, 0.5 MgCl2, 0.5 NaH2PO4, and 25 NaHCO3, and aerated with 95% oxygen and 5% carbon dioxide, pH 7.35–7.4. A drainage cannula was inserted into the apex of the LV cavity through a left atrial incision. A balloon was inserted into the LV cavity, and the balloon volume was adjusted to yield 9–10 mmHg of LV end-diastolic pressure (LVEDP), and no further alterations in balloon volume were made. All hearts were immersed in a water-jacketed organ chamber to maintain a constant temperature of 37°C and allowed to equilibrate for 20 min.
LV pressure, LVEDP, the maximum rate of positive and negative change in LV pressure (±dP/dt), and coronary flow rate were continuously recorded by a data-acquisition system (Powerlab/8SP, ADI Instruments, Colorado Springs, CO). LV developed pressure (LVDP) was calculated by subtracting the LVEDP from the LV systolic pressure. The mean of five cardiac cycles was used to quantify LV performance.
Following equilibration, preagonist baseline data were recorded. Following baseline, isolated hearts underwent incremental forskolin infusion at 5-min intervals at the following concentrations: 1 and 5 μmol/l. The peak LV response was analyzed at both concentrations. Isoproterenol infusion (1×10−8 mol/l) was then superimposed on 5 μmol/l forskolin to examine whether direct adenylyl cyclase activation would lead to β-AR uncoupling.
Western blot analysis.
Following the isolated heart experiments, an apical section of the LV was frozen in liquid nitrogen. Frozen tissue was weighed and homogenized on ice in a PBS lysis buffer containing 2% SDS, 1% Igepal CA-630, 0.5% deoxycholate, 5 mM EDTA (pH 7.4), and proteinase inhibitors [10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 5 μg/ml pepstatin A, and 200 μg/ml benzamidine]. The supernatant was assayed for total protein concentration, mixed with equal volume of SDS sample buffer [2% SDS, 0.1 M Tris·HCl (pH 6.8), 20% glycerol, 100 mM dithiothreitol, and 0.02% bromphenol blue], and stored at −80°C.
Tissue protein abundance and phosphorylation levels in isolated protein were analyzed using Western blot analysis, as described previously (27). Target antigens were probed with the nonphosphorylation-specific monoclonal antibodies: PLB total (Upstate Biotechnology, Waltham, MA) and sarcomeric actin (Sigma, St. Louis, MO). Phosphorylation-specific polyclonal antibodies Ser16-PLB (Ser16) and Thr17-PLB (Thr17) (gift from Dr. J Colyer, University of Leeds, UK) also were probed.
Films were scanned (UMAX PowerLook 1100), and band intensities were quantified with densitometric analysis using the Scion Image program (version 1.62). To normalize blot-to-blot difference in protein loading or transfer efficiency, a common sample was included. Target bands were normalized to actin measured in the same sample. All of the PLB phosphorylation data reported in this paper are following joint agonism with both forskolin and isoproterenol stimulation.
Soleus citrate synthase activity.
Immediately following death, the right soleus from a subset of animals was dissected from each hindlimb, frozen in liquid nitrogen, and stored at −80°C until analysis. In brief, samples were homogenized on ice in a cell lysis reagent (Sigma) and centrifuged at 4°C for 15 min at 12,000 rpm. The supernatant was collected, and a standardized concentration of protein was assayed for citrate synthase activity using a commercial reagent kit (Sigma) by methods previously described by Srere (47).
Data analysis and interpretation.
In vivo hemodynamics, animal characteristics, Western blots, and citrate synthase activity were compared with one-way analysis of variance followed by a least significant difference post hoc analysis. The LV response to forskolin was compared with ANOVA for repeated measures followed by one-way analysis of variance and Fisher's least significant difference post hoc comparisons at each forskolin concentration. Pearson product correlations were performed between LVDP and phosphamban phosphorylation. All analyses were performed on SPSS (SPSS, Chicago, IL, release 13.0). Significance was set at an α-level of P < 0.05. All data are reported as the means ± SE.
In vivo hemodynamics and physical characteristics.
Resting HR and systolic blood pressure (SBP) immediately before the time of death are presented in Table 1. HR and SBP were significantly lower in WKY vs. both SHR groups (P < 0.01), while SBP was unaffected by training. The physical characteristics of the animals are also presented in Table 1. Before death, body weight was significantly lower in SHR-SED relative to SHR-TRD. Absolute heart weight (HW) was greater in SHR-TRD relative to WKY (P < 0.05). HW-to-body weight ratio was significantly lower in WKY vs. both SHR groups (P < 0.01). Absolute tibial length and HW-to-tibial length ratio were similar between groups. Table 2 shows that soleus citrate synthase activity was increased in SHR-TRD relative to both WKY and SHR-SED (P < 0.01).
Langendorff LV performance.
Table 3 shows that the LV baseline performance was similar between groups when the LVEDP was experimentally fixed at 9–10 mmHg. Coronary perfusion pressure was greater in SHR-SED relative to WKY (P < 0.05), and SHR-TRD was similar to WKY. Figure 1 illustrates the LV responsiveness to incremental forskolin infusion and forskolin + isoproterenol. LVDP, +dP/dt, −dP/dt, and HR increased similarly from baseline in all groups with incremental forskolin infusion (Fig. 1, A, C, D, and E). In the presence of 5 μmol/l forskolin, isoproterenol increased LVDP, +dP/dt, and −dP/dt to a significantly greater extent in SHR-TRD relative to SHR-SED. Incremental forskolin infusion did not impact coronary perfusion pressure differently between groups (Table 4), with the caveat that differences in baseline coronary perfusion pressure between WKY and SHR persisted throughout the protocol.
Western blot analysis.
Figure 2 illustrates total PLB abundance normalized to actin (A) and PLB phosphorylation at the Ser16 (B) and Thr17 (C) residues. Although PLB phosphorylation at Ser16 was similar between groups, PLB phosphorylation at Thr17 was significantly greater in SHR-TRD relative to SHR-SED and WKY. Furthermore, absolute LVDP was moderately correlated with PLB phosphorylation at both the Ser16 (r = 0.64; P < 0.05) and Thr17 (r = 0.52; P < 0.05) residues (see Fig. 3).
The major findings of this study are that, in myocardium harvested from the female SHR model: 1) the adenylyl cyclase step in the β-AR cascade is not downregulated in the early course of hypertension; 2) exercise training-induced improvements in β-AR responsiveness are not adenylyl cyclase dependent; and 3) in the presence of direct adenylyl cyclase agonism, β-AR inotropic responsiveness is improved in SHR-TRD relative to SHR-SED hearts.
Our laboratory recently reported that exercise training improved β-AR receptor responsiveness in the SHR model by attenuating myocardial levels of the β-AR desensitizing kinase GRK2 (27). Improved β-AR signaling with exercise training provided one mechanism for increased PKA-mediated phosphorylation of key SR Ca2+ handling proteins, such as the ryanodine receptor and PLB (Ser16 and Thr17). However, given that several studies have shown that G protein to adenylyl cyclase signaling is impaired in hypertension (1, 2, 5, 9, 14, 26, 28, 31, 32), we further examined whether improved β-AR signaling with exercise training in hypertension was also, in part, mediated at the level of adenylyl cyclase. To our knowledge, the present study is the first to explore this topic.
In the present study, LV inotropic and lusitropic function was increased to a similar extent between groups with infusion of low doses of forskolin (1 and 5 μmol/l), suggesting that the adenylyl cyclase step in the β-AR cascade is not downregulated in early compensated hypertrophy. These data are consistent with the paradigm that the training-induced improvement in β-AR responsiveness in hypertension occurs at steps preceding the generation of cAMP and are consistent with previous studies that reported no differences in forskolin-stimulated inotropic responsiveness between SHR and WKY (2, 3).
Increased [Ca2+]i with β-AR agonism activates Ca2+ calmodulin kinase, which, in turn, phosphorylates PLB, resulting in enhanced SR Ca2+ loading and subsequent increases in LV inotropic performance. LV inotropic performance is also regulated via PKA activation and subsequent PLB phosphorylation at the Ser16 residue. Our previous study showed that both Ser16 and Thr17 phosphorylation were attenuated in isoproterenol-treated SHR-SED hearts relative to WKY and SHR-TRD hearts (27). In the present study, however, PLB phosphorylation at both the Ser16 and Thr17 residues was normalized in SHR-SED after the infusion of concomitant forskolin and isoproterenol. These data reiterate the significance of steps preceding the generation of cAMP in dictating β-AR downregulation in hypertension.
Protein kinase A exerts negative feedback control on the β-AR by phosphorylating and uncoupling the receptor (21, 24). One aim of the present study was to determine whether adenylyl cyclase agonism with forskolin can exhibit inhibition on β-AR differently in hypertensive sedentary and trained myocardium. These studies were performed on the basis that the β-AR can, in seconds, become acutely desensitized via phosphorylation of its serine residue on the carboxy terminus, either by the GRK2 or by PKA directly. In the phosphorylated state, β-arrestin can then induce internalization of the β-AR. Our data show that β-AR inotropic responsiveness with forskolin + isoproterenol was improved in trained vs. sedentary hypertensive hearts. Furthermore, Ca2+ calmodulin kinase-mediated PLB phosphorylation at the Thr17 residue was augmented with training. Training-induced alterations in β-AR phosphorylation status and/or a reduction GRK2 abundance (27) are likely mechanistic candidates in explaining the enhanced inotropic responsiveness to forskolin and isoproterenol in exercise-trained hearts. However, differences in the cellular compartmentation of the cAMP system are also an important consideration. Although both isoproterenol and forskolin are known to increase LV inotropic and lusitropic function via increasing cAMP (8, 18, 22), it is hypothesized that cellular compartmentation allows for a more profuse increase in [Ca2+]i and contractility with isoproterenol (18, 23, 48). Patch-clamp experiments in LV cardiomyocytes show L-type Ca2+ current is more localized to the β-AR with isoproterenol, whereas L-type Ca2+ current is more widely dispersed throughout the entire sarcolemma with forskolin (25). Thus, although speculative, another potential mechanism is that training may alter cellular compartmentation of the β-AR system, such that the trained myocardium elicits a more localized and prolific response to β-AR agonism compared with SHR-SED hearts.
Early studies show that one bout of acute exercise increases cAMP levels (19, 35, 36), an effect that is dependent upon the intensity and duration of exercise (20). However, data in chronic exercise training models are less clear. Exercise training studies in nonpathological models have elicited variable results with respect to myocardial β-AR responsiveness, with some studies showing increased (29, 49, 52), decreased (11, 17), or no change (34, 37) with training. The unpredictable adaptive pattern of the β-AR with training in normotensive myocardium is likewise reflected in variable observations reported in the plasticity of the adenylyl cyclase step in the cascade. In the basal state, adenylyl cyclase has been shown to be increased (6), decreased (16, 33), or unaltered (12, 30, 42) with training. Likewise, agonist stimulated adenylyl cyclase has also been reported to be increased (30, 39, 52), decreased (12, 30), or unaltered with training (6, 12, 42). Thus, in normotensive rodent myocardium, it is difficult to generate any categorical conclusions with respect to how exercise training impacts β-AR responsiveness and adenylyl cyclase activity. Furthermore, there is a paucity of data with respect to how training impacts forskolin-stimulated adenylyl cyclase activity in compensatory hypertrophy, illustrating the novelty of the present study.
The SHR model was selected for our study because it mimics the slowly progressing clinical course of untreated essential hypertension in humans. It has been documented that concentric hypertrophy occurs in SHR between 6 and 12 mo of age, decompensating to heart failure near 15 mo (15, 38). We chose to study these animals at 7 mo of age because cardiac function is well maintained and fibrosis is minimal (7). There are several design considerations that may affect the interpretability of the present study. First, inclusion of a normotensive exercise group would have expanded our understanding of the exercise-induced adaptation. However, this was not the direct purpose of the present study, as we instead focused our efforts toward establishing the phenotype of the hypertensive myocardium in response to training. Second, although we utilized forskolin-stimulated Langendorff LV performance as an adenylyl cyclase activity surrogate, we did not directly measure adenylyl cyclase activity, phosphodiesterase activity, or G protein isoforms. Third, we did not uncouple the influence of Gs on adenylate cyclase such that the binding affinity for forskolin is relatively high in the presence of Gsα (EC50 = 0.1 μmol/l) and relatively low in the absence of Gsα (EC50 ∼15 μmol/l) (13). Fourth, we only assessed PLB phosphorylation following agonism with forskolin and isoproterenol, thus not allowing for interpretation of basal differences.
In summary, our data suggest that the adenylyl cyclase step in the β-AR cascade is not downregulated in the early course of hypertension and that the enhanced β-AR responsiveness with training previously established in hypertension (27) is likely mediated at levels other than adenylyl cyclase. Our data also suggest that exercise training in hypertension shifts PKA-mediated phosphorylation and desensitization of the β-AR rightward. The clinical significance of this paper is that, during acute stress and exercise, β-AR responsiveness is more likely to be maintained in patients with hypertension-induced LV hypertrophy who are exercise trained.
This study was supported by a beginning-grant-in-aide from the American Heart Association, Mid-Atlantic Affiliate (J. R. Libonati), and National Heart, Lung, and Blood Institute Grant HL-33921 (S. R. Houser).
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.
- Copyright © 2007 the American Physiological Society