Improved Myocardial ;-Adrenergic Responsiveness and Signaling With Exercise Training in Hypertension
http://www.100md.com
《循环学杂志》
the Departments of Kinesiology (S.M.M., B.F.R., P.O.R., J.R.L.) and Physiology (S.R.H., J.R.L.) and the Cardiovascular Research Center (H.K., D.L.C., J.M., S.R.H., J.R.L.)
Temple University, and the Center for Translational Medicine, Thomas Jefferson University (A.S., W.J.K.), Philadelphia, Pa.
Abstract
Background— Cardiac responses to ;-adrenergic receptor stimulation are depressed with pressure overload–induced cardiac hypertrophy. We investigated whether exercise training could modify ;-adrenergic receptor responsiveness in a model of spontaneous hypertension by modifying the ;-adrenergic receptor desensitizing kinase GRK2 and the abundance and phosphorylation of some key Ca2+ cycling proteins.
Methods and Results— Female spontaneously hypertensive rats (SHR; age, 4 months) were placed into a treadmill running (SHR-TRD; 20 m/min, 1 h/d, 5 d/wk, 12 weeks) or sedentary group (SHR-SED). Age-matched Wistar Kyoto (WKY) rats were controls. Mean blood pressure was higher in SHR versus WKY (P<0.01) and unaltered with exercise. Left ventricular (LV) diastolic anterior and posterior wall thicknesses were greater in SHR than WKY (P<0.001) and augmented with training (P<0.01). Langendorff LV performance was examined during isoproterenol (ISO) infusions (1x10–10 to 1x10–7 mol/L) and pacing stress (8.5 Hz). The peak LV developed pressure/ISO dose response was shifted rightward 100-fold in SHR relative to WKY. The peak ISO LV developed pressure response was similar between WKY and SHR-SED and increased in SHR-TRD (P<0.05). SHR-TRD showed the greatest lusitropic response to ISO (P<0.05) and offset the pacing-induced increase in LV end-diastolic pressure and the time constant of isovolumic relaxation () observed in WKY and SHR-SED. Improved cardiac responses to ISO in SHR-TRD were associated with normalized myocardial levels of GRK2 (P<0.05). SHR displayed increased L-type Ca2+ channel and sodium calcium exchanger abundance compared with WKY (P<0.001). Training increased ryanodine receptor phosphorylation and phospholamban phosphorylation at both the Ser16 and Thr17 residues (P<0.05).
Conclusions— Exercise training in hypertension improves the inotropic and lusitropic responsiveness to ;-adrenergic receptor stimulation despite augmenting LV wall thickness. A lower GRK2 abundance and an increased phosphorylation of key Ca2+ cycling proteins may be responsible for the above putative effects.
Key Words: diastole ; exercise ; hypertension ; hypertrophy ; proteins
Introduction
Pressure overload–induced concentric hypertrophy is associated with a reduction in ;-adrenergic receptor responsiveness and subsequent altered myocardial contractile function.1,2 Concentric hypertrophy is regarded as an adaptational process to normalize wall stress with hypertension.3 Increased systolic performance with diastolic dysfunction has been documented during this compensatory phase of concentric hypertrophy in spontaneous hypertensive rats (SHR).4 Although some studies have shown a decreased ;-adrenergic receptor density in hypertension,5 it appears likely that abnormalities "downstream" from the ;-adrenergic receptor are mechanistically culpable for the impaired adrenergic responsiveness of the hypertrophied heart.1,6–10 For example, ;-adrenergic receptor kinase (;ARK1 or GRK2) has been shown to be centrally involved in blunting ;-adrenergic receptor signaling in pressure-overload hypertrophy.11
Given that exercise training has been shown to enhance ;-adrenergic responsiveness12–18 without increasing ;-receptor density,18–20 we hypothesized that exercise training could rectify defective myocardial ;-adrenergic receptor responsiveness in hypertrophy. Thus, the purposes of the present study were to use an SHR model to (1) to establish whether exercise training could mitigate hypertrophy associated with hypertension, (2) to examine whether exercise training could increase both inotropic and lusitropic responses to ;-adrenergic stimulation and pacing, (3) to examine how exercise training affects GRK2, and (4) to examine whether exercise training alters the abundance and phosphorylation of Ca2+ cycling proteins.
Methods
Animals and Exercise Training
Thirty-six 16-week-old female Wistar-Kyoto rats (WKY; n=12) and SHR (n=24) (weight, 185 g) were obtained from Charles River Laboratories (St Constant, Quebec, Canada). Animals in the SHR group were randomly assigned into either a sedentary (SHR-SED) group (n=12) or an exercise-trained (SHR-TRD) group (n=12). All rats were housed 3 per cage, maintained on a 12-hour light/dark cycle, and fed ad libitum (Harlan Teklad Global Diets, 18% Protein Diet). Training consisted of low-intensity endurance training at speeds of 20 to 25 m/min, 0% grade, 60 continuous minutes, 5 d/wk for 12 weeks. SHR-SED and WKY were handled each day. Resting heart rates (HRs; mean of 25 cardiac cycles) and blood pressures were collected biweekly with a tail cuff apparatus (XBP1000, Kent Scientific). At 28 weeks of age, all animals were killed, and the functional studies were performed. All animals received humane care in compliance with 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 85-23, revised 1985).
Echocardiography
Rats were studied with echocardiography before organ harvest. In brief, animals were lightly sedated with an intraperitoneal injection of xylazine (10 mg/kg) and ketamine (50 mg/kg) to achieve semiconscious sedation. After 5 minutes, images were acquired with the animals in the left lateral decubitus position. A Hewlett-Packard 5500 Sonos machine was used to obtain both 2D and M-mode images with a 12-MHz transducer. Imaging was performed with a depth of 4 cm. Doppler imaging was performed with a sample volume of 6 mm and a paper speed of 100 mm/s. In accordance with the American Society of Echocardiography conventions, M-mode imaging of the parasternal short-axis view allowed measurement of left ventricular (LV) end-systolic and end-diastolic internal dimensions (LVEDD) and anterior and posterior wall thicknesses (AW and PW, respectively). Values were determined by averaging the measurements of 3 consecutive beats. The above measurements were used in conjunction with histological interventricular septal thickness (IVS) for offline calculations of LV mass (LVM) with the following equation:
LVM=0.8[1.04(IVS+LVEDD+PW)3–(LVEDD)3]+0.6 g.21
Noninvasive Hemodynamics
Pulsed-wave Doppler was used to assess isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT), and LV ejection time (ET). The Doppler sample volume was placed at the entrance of the LV outflow tract and the mitral tip, allowing acquisition of both the mitral inflow and LV outflow envelopes. IVRT was defined as the time between aortic valve closure and mitral valve opening. IVCT was defined as the time between mitral value closure and aortic valve opening. LV ET was defined as the duration of the LV outflow envelope. The parasternal short-axis view at the base of the heart was used to obtain the right ventricular outflow tract peak flow velocity and diameter. Cardiac output was determined from the right ventricular outflow tract because it represents an accurate assessment of the cardiac output in the absence of pulmonary insufficiency.22 Stroke volume was calculated as the echocardiography-derived cardiac output (mL/min) divided by the ECG-derived HR.
Isolated Rat Heart Preparation/Experimental Design
Rats were anesthetized with sodium pentobarbital (60 mg/kg IP) and heparinized intravenously (500 U). The heart was excised, trimmed of excess tissue, and rapidly immersed in cold (4°C) Ca2+-free Krebs-Henseleit buffer.23 Hearts were placed on a Langendorff perfusion apparatus (ML785B2, ADInstruments); perfused at 16 mL/min (STH pump controller ML175, ADInstruments) with a modified Krebs-Henseleit solution containing 2.0 mmol/L Ca2+Cl2, 130 mmol/L NaCl, 5.4 mmol/L KCl, 11 mmol/L dextrose, 2 mmol/L pyruvate, 0.5 mmol/L MgCl2, 0.5 mmol/L NaH2PO4, and 25 mmol/L NaHCO3; and aerated with 95% oxygen and 5% carbon dioxide, pH 7.35 to 7.4. Coronary flow rate was selected to mimic the in situ perfusion pressure. 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 12 mm Hg of LV end-diastolic pressure (LVEDP). No further alterations in balloon volume were made. All hearts were immersed in a water-jacketed organ chamber to maintain a temperature of 37°C.
After equilibration, preagonist baseline data were recorded. After baseline, 5-minute infusions of isoproterenol were begun at doses ranging from 1x10–10 to 1x10–7 mol/L. During baseline and the fifth minute of each infusion, hearts were instantly paced at 8.5 Hz for 20 seconds. Isoproterenol infusion was stopped after 5 minutes, and hearts were allowed to return to baseline before the next dose was initiated. LV pressure, LVEDP, the maximum rate of positive and negative change in LV pressure (±LV dP/dt), and coronary perfusion pressure were continuously recorded by means of a data acquisition system (Powerlab/8SP, ADInstruments). LV developed pressure (LV Dev P) was calculated by subtracting the LVEDP from the LV systolic pressure. The peak response to isoproterenol was recorded throughout the dose-response relationship. The time constant of isovolumic pressure relaxation (, ms) was calculated using LV pressure and dP/dt, from peak –dP/dt to 5 mm Hg above LVEDP, allowing a shift of the exponential baseline.24,25
Protein Assay
After 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 buffer containing 25 mmol/L Tris (pH 7.4), 0.5 mmol/L EGTA, 5 mmol/L EDTA, 20 mmol/L NaF, 1.0 mmol/L Na3VO4, 0.5% Triton X-100, and proteinase inhibitors (10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mmol/L aminoethyl benzylsufonyl fluoride, 5 μg/mL pepstatin A, and 8 and 200 μg/mL benzamidine). The supernatant was assayed for total protein concentration, mixed with equal volume of SDS sample buffer (4% SDS, 0.1 mol/L Tris-Cl, pH 6.8, 20% glycerol, 200 mmol/L dithiothreitol, and 0.02% bromphenol blue), and stored at –80°C.
Western Blot Analysis
Tissue protein abundance and phosphorylation levels in isolated protein were analyzed with Western blot analysis as described previously.26 Target antigens were probed with the following non–phosphorylation-specific monoclonal antibodies: SERCA (Sigma), phospholamban total (PLBt; Upstate Biotechnology), ryanodine receptor C3-33 (RyRt; Research Diagnostics), L-type calcium channel 1c subunit (LTCC; Chemicon), sodium-calcium exchanger (NCX; Swant), and GAPDH (Biogenesis). Phosphorylation-specific polyclonal antibodies Ser 2809-RyR (RyRp), Ser16-PLB (Ser16), and Thr17-PLB (Thr17) (gift from Dr J. Colyer, University of Leeds, Leeds, UK) also were probed. In addition, we used antibodies for GRK2 (Upstate) and actin (Sigma).
Films were scanned (UMAX PowerLook 1100) and band intensities were quantified with densitometric analysis using the Scion Image program. To normalize blot-to-blot difference in protein loading or transfer efficiency, a common sample was included. Target bands were normalized to GAPDH measured in the same sample. GRK2 expression was examined by Western blot analysis and detected with an Odyssey Infrared Imaging System (Li-Cor Biosciences). Densitometric analysis was performed with the Odyssey version 1.2 application software with GRK2 normalized to actin expression.
Data Analysis and Interpretation
Hemodynamics, animal characteristics, and pacing data were compared by use of 1-way ANOVA, followed by a Tukey post hoc analysis. The temporal responses of HR, blood pressure, and rate-pressure product (RPP) to exercise training were analyzed with ANOVA for repeated measures and Tukey post hoc analysis. Isoproterenol dose-response relationships were compared by ANOVA for repeated measures, followed by 1-way ANOVA and Tukey post hoc analysis at each isoproterenol concentration. Myocardial protein abundance was compared with 1-way ANOVA, followed by a Tukey post hoc analysis. All analyses were performed on SPSS (SPSS Inc, release 12.0.). Significance was set at an level of P<0.05. Data are reported as mean±SEM.
Results
In Vivo Hemodynamics
Animal Characteristics
Langendorff Isovolumic Performance
Baseline
In our model, all hearts were perfused at constant flow (16 mL/min) with a crystalloid perfusate, allowing differences in coronary perfusion pressure to be illustrative of coronary vascular resistance. This coronary flow yielded baseline perfusion pressures that were well correlated with in vivo mean tail cuff blood pressures immediately before death (r=0.74, P<0.01). At baseline in the absence of pacing, perfusion pressure was higher in both SHR groups compared with WKY (WKY, 77±7 mm Hg; SHR-SED, 143±9 mm Hg; SHR-TRD, 112±9 mm Hg; P<0.05). Interestingly, baseline perfusion pressure was significantly lower in SHR-TRD versus SHR-SED (P<0.05). Baseline perfusion pressure with pacing tended to increase and remained higher in both SHR groups (WKY, 87±7 mm Hg; SHR-SED, 143±8 mm Hg; SHR-TRD, 115±8 mm Hg; P<0.05). During pacing, SHR-TRD elicited a lower perfusion pressure than SHR-SED (P<0.05).
Isoproterenol Dose-Response Relationship
Isoproterenol significantly increased LV Dev P in all groups (P<0.05) (Figure 4A). The peak response to isoproterenol was shifted rightward 100-fold in both SHR groups relative to WKY, representing a reduced ;-adrenergic agonist sensitivity in SHR. Despite the rightward shift in peak response, the peak LV Dev P response to ;-adrenergic receptor stimulation was greatest in SHR-TRD but similar between WKY and SHR-SED (WKY, 47±8 mm Hg; SHR-SED, 35±13 mm Hg; SHR-TRD, 84±12 mm Hg; P<0.05). As Figure 4B shows, LVEDP fell among all groups with isoproterenol administration up to 1x10–8 mol/L. Interestingly, exercise training in hypertension (SHR-TRD) increased the lusitropic response to isoproterenol compared with both WKY and SHR-SED (P<0.05). (Figure 4C) was significantly lower at 1x10–8 in SHR-TRD versus both WKY and SHR-SED (WKY, 51.3±5.6 ms; SHR-SED, 39.9±2.3 ms; SHR-TRD, 25.8±3.3 ms; P<0.05). Isoproterenol significantly increased the +dP/dt in all groups (P<0.05) (Figure 4D). The peak +dP/dt response to isoproterenol was observed at 1x10–9 mol/L in WKY; the peak +dP/dt response occurred at 1x10–8 mol/L in both SHR groups. The peak +dP/dt response to ;-adrenergic receptor stimulation was greatest in SHR-TRD versus the peak response in WKY (1x10–9 mol/L) and SHR-SED. Figure 4E documents the –dP/dt response to isoproterenol among groups. A significant increase in –dP/dt occurred in all groups with the administration of isoproterenol (P<0.05). The peak response was shifted rightward 10-fold in SHR-TRD and 100-fold in SHR-SED compared with WKY, with the greatest response observed in SHR-TRD. At baseline and at isoproterenol infusions of 1x10–10 and 1x10–9 mol/L, perfusion pressure of WKY was significantly lower than both SHR-SED and SHR-TRD (P<0.05) (Figure 4F). Additionally, at baseline and all isoproterenol doses except 1x10–9 mol/L, SHR-TRD had a lower perfusion pressure than SHR-SED (P<0.05). At isoproterenol doses of 1x10–8 and 1x10–7 mol/L, SHR-SED had a significantly greater perfusion pressure compared with WKY (P<0.05).
Western Blots
Both hypertension and exercise training independently altered protein abundance and phosphorylation of several key Ca2+ cycling proteins (Figures 5 and 6). As Figure 5 illustrates, total ryanodine tended to be elevated in SHR-TRD versus both WKY and SHR-SED, whereas phosphorylated ryanodine (RyRp) was increased in SHR-TRD versus SHR-SED (P<0.05). Hypertension significantly increased LTCC abundance in SHR compared with the normotensive WKY (P<0.001). No difference in LTCC abundance resulted from chronic exercise training. NCX abundance was significantly increased in both SHR groups compared with WKY (P<0.001), with no difference observed with exercise training. PLBt abundance was elevated with training in hypertension (SHR-TRD) versus WKY (P<0.05), with similar SERCA2A among groups (Figure 6). Phospholamban phosphorylated at Ser16 was significantly lower in SHR-SED than both WKY (P<0.05) and SHR-TRD (P<0.01). Additionally, greater phospholamban phosphorylated at Thr17 was observed in SHR-TRD versus SHR-SED (P<0.01), whereas no difference was observed between WKY and SHR-SED. Myocardial levels of the ;-adrenergic receptor kinase (;ARK1 or GRK2), previously shown to be associated with pressure-overload cardiac hypertrophy,11 were increased in SHR-SED versus WKY (P<0.05) but decreased in SHR-TRD (P<0.05) (Figure 7).
Discussion
A major finding of this study is that exercise training improved ;-adrenergic receptor responsiveness in the SHR model, perhaps by decreasing myocardial levels of the ;-adrenergic receptor desensitizing kinase GRK2. As a result of "restored" or improved ;-adrenergic receptor signaling, protein kinase A–mediated "downstream" phosphorylation of key SR Ca2+ handling proteins such as the ryanodine receptor and phospholamban (Ser16) was enhanced with training. Training also increased calcium/calmodulin-dependent protein kinase phosphorylation of phospholamban (Thr17). Exercise training did not alter the observed increases in protein abundance of the LTCC or NCX in SHR. Additionally, exercise training reduced coronary vascular resistance and attenuated pacing-induced increases in both the time constant of isovolumic pressure relaxation () and LV end-diastolic pressure. The above putative effects of exercise are interesting in light of the fact that training increased LV wall thickness in SHR.
Myocardial ;-adrenergic receptors are a member of the superfamily of G-protein–coupled receptors (GPCRs), which control cardiac function by modulating inotropy, lusitropy, and chronotropy.7 Altered contractile function and reduced myocardial responsiveness to adrenergic stimulation reflect some of the earliest changes associated with hypertension-induced, compensatory hypertrophy.1 Although some studies have shown a decreased ;-adrenergic receptor density in hypertension,5 it appears likely that abnormalities "downstream" from the ;-adrenergic receptor are mechanistically culpable in the hypertrophied hearts impaired responsiveness to adrenergic stimulation.1,6–9 In ventricular hypertrophy11 and heart failure,29 ;-adrenergic receptors are overly desensitized and downregulated, which has been attributed to increased myocardial levels of the GPCR kinase, GRK2 (also known as ;ARK1), seen in these conditions. GRKs, such as GRK2, phosphorylate GPCRs, leading to functional uncoupling between the receptor and downstream effector systems.30 GRK2 is the most abundant GRK in the heart, and in conditions of ventricular hypertrophy and dysfunction, increased expression and activity of myocardial GRK2 uncouple ;-adrenergic receptors, causing dysregulation of myocyte excitation-contraction coupling.7,29 As expected, our data show that GRK2 abundance was increased in SHR sedentary animals, an effect consistent with the observed blunted ;-adrenergic receptor responsiveness in SHR sedentary hearts. However, a novel finding in the present study is that exercise training attenuated the increase in GRK2 associated with hypertension and may explain part of the enhanced functional and molecular performance in SHR-TRD.
Although exercise training blunted the increase in GRK2 associated with hypertension, it did not fully restore the impaired ;-adrenergic sensitivity in SHR. The present data show a 100-fold rightward shift in the isoproterenol–LV Dev P and the isoproterenol–±dP/dt dose-response curves in both SHR groups relative to WKY. These data are similar to those reported by Atkins et al,1 who showed a rightward shift in the isoproterenol and forskolin dose-response relationships in compensatory-hypertrophied SHR hearts. Atkins et al also showed that ;-adrenergic receptor density was unchanged in nonfailing SHR and that receptor affinity did not differ between WKY or nonfailing SHR hearts.1 These data suggest that impaired inotropic responsiveness of the LV myocardium to ;-adrenergic stimulation is not associated with alterations in the density or affinity of ;-adrenergic receptors but rather downstream cellular events.
Of particular interest is that exercise training in SHR improved lusitropic responsiveness to adrenergic stimulation and offset the pacing-induced increases in and LV diastolic chamber stiffness, even beyond that of WKY. Although in the present study SERCA abundance was similar among all groups, phospholamban abundance was greater in SHR-TRD animals compared with WKY. Our results show that in hypertension, exercise training increased phospholamban phosphorylation at both Ser16 and Thr17 sites relative to sedentary animals. The increased phospholamban phosphorylation likely facilitates sarcoplasmic reticulum Ca2+ loading and subsequent release.31 Along these lines, our results also show that exercise training increased ryanodine receptor phosphorylation without altering ryanodine receptor density among groups. Our data support results presented by Shorofsky et al,31 who showed similar SERCA and ryanodine receptor abundance in WKY and SHR hearts. However, Shorofsky et al did not report alterations in phospholamban abundance or phosphorylation in SHR hearts. The differences may reflect our methodological approach, which included the administration of a ;- agonist (isoproterenol) before the heart was arrested.
SBP was markedly elevated throughout the protocol in SHR; at the time of death, both SHR groups exhibited a 25% greater SBP relative to normotensive controls. The antihypertensive effect of exercise in the present study was minimal and smaller than reports from longer training paradigms.32 In our study, exercise training superimposed on hypertension augmented the extent of myocardial hypertrophy. The hypertensive animals displayed greater LV mass and ratio of LV mass to BW than the normotensive controls. Additionally, echocardiography-derived AW and PW thicknesses during both the systolic and diastolic phases of the cardiac cycle were greater in SHR than WKY. The increased wall thickness translated to a 10% increase in LV mass and a 7% increase in the ratio of LV mass to tibial length after training. The increased LV wall thickness observed with exercise training is interesting in light of the lower resting RPP in trained animals relative to hypertensive sedentary animals. Seminal work also has shown that exercise training potentiates LV hypertrophy in hypertension.33,34 The present results show that baseline inotropy and abundance of both LTCC and NCX were increased in SHR relative to WKY. The present data support experiments showing that SHR hearts with compensatory hypertrophy have enhanced contractility4,31 amplitude of [Ca2+]i and a mean amplitude of Ca2+ sparks.31
Study Limitations
We selected the SHR model for our studies because it mimics the clinical course of untreated essential hypertension in humans. It is well documented that concentric hypertrophy occurs in SHR within 6 to 12 months of age. The SHR model gradually decompensates into heart failure sometime after 15 months of age.35,36 We chose to study the animals at 7 months of age, a time in which fibrosis is not accelerated in SHR.36 The interpretability of our study is limited in several ways. First, the causes for hypertension in SHR are polygenic and do not necessarily reflect the genetic anomalies associated with hypertension in humans.31,35 Second, the absence of an exercise-trained WKY group limits our understanding of the exercise adaptation independent of hypertension. Third, we did not examine ;AR density or subtype on the basis of previous data suggesting that impaired responsiveness to ;-adrenergic stimulation is regulated distal to the receptor primarily by GRK2 expression.1,6–9,30,37,38 Fourth, we did not examine myocardial basal phosphorylation state because previous studies reported no differences between SHR and WKY.31,39 Finally, the relative isoproterenol doses were likely extreme for WKY and may have internalized the receptors as evidenced by the negative LV Dev P at 10–7 mol/L.
In summary, exercise training in hypertension increased the inotropic and lusitropic responsiveness to ;-adrenergic receptor stimulation and increased the phosphorylation of key Ca2+ cycling proteins while attenuating GRK2 abundance with hypertension. Future experiments are required to elucidate the putative interaction between the above molecular observations and cardiac function.
Acknowledgments
This study was supported by a beginning-grant-in-aide from the American Heart Association, Mid-Atlantic Affiliate (Dr Libonati), and the National Institute of Health (HL-33921 to Dr Houser, HL-61690 and HL-56205 to Dr Koch). We thank Leigh Ann Hewsten for technical support.
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McConnell BK, Moravec CS, Morano I, Bond M. Troponin I phosphorylation in spontaneously hypertensive rat heart: effect of ;-adrenergic stimulation. Am J Physiol. 1997; 273 (pt 2): H1440–H1451.(Scott M. MacDonnell, MS; )
Temple University, and the Center for Translational Medicine, Thomas Jefferson University (A.S., W.J.K.), Philadelphia, Pa.
Abstract
Background— Cardiac responses to ;-adrenergic receptor stimulation are depressed with pressure overload–induced cardiac hypertrophy. We investigated whether exercise training could modify ;-adrenergic receptor responsiveness in a model of spontaneous hypertension by modifying the ;-adrenergic receptor desensitizing kinase GRK2 and the abundance and phosphorylation of some key Ca2+ cycling proteins.
Methods and Results— Female spontaneously hypertensive rats (SHR; age, 4 months) were placed into a treadmill running (SHR-TRD; 20 m/min, 1 h/d, 5 d/wk, 12 weeks) or sedentary group (SHR-SED). Age-matched Wistar Kyoto (WKY) rats were controls. Mean blood pressure was higher in SHR versus WKY (P<0.01) and unaltered with exercise. Left ventricular (LV) diastolic anterior and posterior wall thicknesses were greater in SHR than WKY (P<0.001) and augmented with training (P<0.01). Langendorff LV performance was examined during isoproterenol (ISO) infusions (1x10–10 to 1x10–7 mol/L) and pacing stress (8.5 Hz). The peak LV developed pressure/ISO dose response was shifted rightward 100-fold in SHR relative to WKY. The peak ISO LV developed pressure response was similar between WKY and SHR-SED and increased in SHR-TRD (P<0.05). SHR-TRD showed the greatest lusitropic response to ISO (P<0.05) and offset the pacing-induced increase in LV end-diastolic pressure and the time constant of isovolumic relaxation () observed in WKY and SHR-SED. Improved cardiac responses to ISO in SHR-TRD were associated with normalized myocardial levels of GRK2 (P<0.05). SHR displayed increased L-type Ca2+ channel and sodium calcium exchanger abundance compared with WKY (P<0.001). Training increased ryanodine receptor phosphorylation and phospholamban phosphorylation at both the Ser16 and Thr17 residues (P<0.05).
Conclusions— Exercise training in hypertension improves the inotropic and lusitropic responsiveness to ;-adrenergic receptor stimulation despite augmenting LV wall thickness. A lower GRK2 abundance and an increased phosphorylation of key Ca2+ cycling proteins may be responsible for the above putative effects.
Key Words: diastole ; exercise ; hypertension ; hypertrophy ; proteins
Introduction
Pressure overload–induced concentric hypertrophy is associated with a reduction in ;-adrenergic receptor responsiveness and subsequent altered myocardial contractile function.1,2 Concentric hypertrophy is regarded as an adaptational process to normalize wall stress with hypertension.3 Increased systolic performance with diastolic dysfunction has been documented during this compensatory phase of concentric hypertrophy in spontaneous hypertensive rats (SHR).4 Although some studies have shown a decreased ;-adrenergic receptor density in hypertension,5 it appears likely that abnormalities "downstream" from the ;-adrenergic receptor are mechanistically culpable for the impaired adrenergic responsiveness of the hypertrophied heart.1,6–10 For example, ;-adrenergic receptor kinase (;ARK1 or GRK2) has been shown to be centrally involved in blunting ;-adrenergic receptor signaling in pressure-overload hypertrophy.11
Given that exercise training has been shown to enhance ;-adrenergic responsiveness12–18 without increasing ;-receptor density,18–20 we hypothesized that exercise training could rectify defective myocardial ;-adrenergic receptor responsiveness in hypertrophy. Thus, the purposes of the present study were to use an SHR model to (1) to establish whether exercise training could mitigate hypertrophy associated with hypertension, (2) to examine whether exercise training could increase both inotropic and lusitropic responses to ;-adrenergic stimulation and pacing, (3) to examine how exercise training affects GRK2, and (4) to examine whether exercise training alters the abundance and phosphorylation of Ca2+ cycling proteins.
Methods
Animals and Exercise Training
Thirty-six 16-week-old female Wistar-Kyoto rats (WKY; n=12) and SHR (n=24) (weight, 185 g) were obtained from Charles River Laboratories (St Constant, Quebec, Canada). Animals in the SHR group were randomly assigned into either a sedentary (SHR-SED) group (n=12) or an exercise-trained (SHR-TRD) group (n=12). All rats were housed 3 per cage, maintained on a 12-hour light/dark cycle, and fed ad libitum (Harlan Teklad Global Diets, 18% Protein Diet). Training consisted of low-intensity endurance training at speeds of 20 to 25 m/min, 0% grade, 60 continuous minutes, 5 d/wk for 12 weeks. SHR-SED and WKY were handled each day. Resting heart rates (HRs; mean of 25 cardiac cycles) and blood pressures were collected biweekly with a tail cuff apparatus (XBP1000, Kent Scientific). At 28 weeks of age, all animals were killed, and the functional studies were performed. All animals received humane care in compliance with 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 85-23, revised 1985).
Echocardiography
Rats were studied with echocardiography before organ harvest. In brief, animals were lightly sedated with an intraperitoneal injection of xylazine (10 mg/kg) and ketamine (50 mg/kg) to achieve semiconscious sedation. After 5 minutes, images were acquired with the animals in the left lateral decubitus position. A Hewlett-Packard 5500 Sonos machine was used to obtain both 2D and M-mode images with a 12-MHz transducer. Imaging was performed with a depth of 4 cm. Doppler imaging was performed with a sample volume of 6 mm and a paper speed of 100 mm/s. In accordance with the American Society of Echocardiography conventions, M-mode imaging of the parasternal short-axis view allowed measurement of left ventricular (LV) end-systolic and end-diastolic internal dimensions (LVEDD) and anterior and posterior wall thicknesses (AW and PW, respectively). Values were determined by averaging the measurements of 3 consecutive beats. The above measurements were used in conjunction with histological interventricular septal thickness (IVS) for offline calculations of LV mass (LVM) with the following equation:
LVM=0.8[1.04(IVS+LVEDD+PW)3–(LVEDD)3]+0.6 g.21
Noninvasive Hemodynamics
Pulsed-wave Doppler was used to assess isovolumic relaxation time (IVRT), isovolumic contraction time (IVCT), and LV ejection time (ET). The Doppler sample volume was placed at the entrance of the LV outflow tract and the mitral tip, allowing acquisition of both the mitral inflow and LV outflow envelopes. IVRT was defined as the time between aortic valve closure and mitral valve opening. IVCT was defined as the time between mitral value closure and aortic valve opening. LV ET was defined as the duration of the LV outflow envelope. The parasternal short-axis view at the base of the heart was used to obtain the right ventricular outflow tract peak flow velocity and diameter. Cardiac output was determined from the right ventricular outflow tract because it represents an accurate assessment of the cardiac output in the absence of pulmonary insufficiency.22 Stroke volume was calculated as the echocardiography-derived cardiac output (mL/min) divided by the ECG-derived HR.
Isolated Rat Heart Preparation/Experimental Design
Rats were anesthetized with sodium pentobarbital (60 mg/kg IP) and heparinized intravenously (500 U). The heart was excised, trimmed of excess tissue, and rapidly immersed in cold (4°C) Ca2+-free Krebs-Henseleit buffer.23 Hearts were placed on a Langendorff perfusion apparatus (ML785B2, ADInstruments); perfused at 16 mL/min (STH pump controller ML175, ADInstruments) with a modified Krebs-Henseleit solution containing 2.0 mmol/L Ca2+Cl2, 130 mmol/L NaCl, 5.4 mmol/L KCl, 11 mmol/L dextrose, 2 mmol/L pyruvate, 0.5 mmol/L MgCl2, 0.5 mmol/L NaH2PO4, and 25 mmol/L NaHCO3; and aerated with 95% oxygen and 5% carbon dioxide, pH 7.35 to 7.4. Coronary flow rate was selected to mimic the in situ perfusion pressure. 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 12 mm Hg of LV end-diastolic pressure (LVEDP). No further alterations in balloon volume were made. All hearts were immersed in a water-jacketed organ chamber to maintain a temperature of 37°C.
After equilibration, preagonist baseline data were recorded. After baseline, 5-minute infusions of isoproterenol were begun at doses ranging from 1x10–10 to 1x10–7 mol/L. During baseline and the fifth minute of each infusion, hearts were instantly paced at 8.5 Hz for 20 seconds. Isoproterenol infusion was stopped after 5 minutes, and hearts were allowed to return to baseline before the next dose was initiated. LV pressure, LVEDP, the maximum rate of positive and negative change in LV pressure (±LV dP/dt), and coronary perfusion pressure were continuously recorded by means of a data acquisition system (Powerlab/8SP, ADInstruments). LV developed pressure (LV Dev P) was calculated by subtracting the LVEDP from the LV systolic pressure. The peak response to isoproterenol was recorded throughout the dose-response relationship. The time constant of isovolumic pressure relaxation (, ms) was calculated using LV pressure and dP/dt, from peak –dP/dt to 5 mm Hg above LVEDP, allowing a shift of the exponential baseline.24,25
Protein Assay
After 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 buffer containing 25 mmol/L Tris (pH 7.4), 0.5 mmol/L EGTA, 5 mmol/L EDTA, 20 mmol/L NaF, 1.0 mmol/L Na3VO4, 0.5% Triton X-100, and proteinase inhibitors (10 μg/mL aprotinin, 10 μg/mL leupeptin, 1 mmol/L aminoethyl benzylsufonyl fluoride, 5 μg/mL pepstatin A, and 8 and 200 μg/mL benzamidine). The supernatant was assayed for total protein concentration, mixed with equal volume of SDS sample buffer (4% SDS, 0.1 mol/L Tris-Cl, pH 6.8, 20% glycerol, 200 mmol/L dithiothreitol, and 0.02% bromphenol blue), and stored at –80°C.
Western Blot Analysis
Tissue protein abundance and phosphorylation levels in isolated protein were analyzed with Western blot analysis as described previously.26 Target antigens were probed with the following non–phosphorylation-specific monoclonal antibodies: SERCA (Sigma), phospholamban total (PLBt; Upstate Biotechnology), ryanodine receptor C3-33 (RyRt; Research Diagnostics), L-type calcium channel 1c subunit (LTCC; Chemicon), sodium-calcium exchanger (NCX; Swant), and GAPDH (Biogenesis). Phosphorylation-specific polyclonal antibodies Ser 2809-RyR (RyRp), Ser16-PLB (Ser16), and Thr17-PLB (Thr17) (gift from Dr J. Colyer, University of Leeds, Leeds, UK) also were probed. In addition, we used antibodies for GRK2 (Upstate) and actin (Sigma).
Films were scanned (UMAX PowerLook 1100) and band intensities were quantified with densitometric analysis using the Scion Image program. To normalize blot-to-blot difference in protein loading or transfer efficiency, a common sample was included. Target bands were normalized to GAPDH measured in the same sample. GRK2 expression was examined by Western blot analysis and detected with an Odyssey Infrared Imaging System (Li-Cor Biosciences). Densitometric analysis was performed with the Odyssey version 1.2 application software with GRK2 normalized to actin expression.
Data Analysis and Interpretation
Hemodynamics, animal characteristics, and pacing data were compared by use of 1-way ANOVA, followed by a Tukey post hoc analysis. The temporal responses of HR, blood pressure, and rate-pressure product (RPP) to exercise training were analyzed with ANOVA for repeated measures and Tukey post hoc analysis. Isoproterenol dose-response relationships were compared by ANOVA for repeated measures, followed by 1-way ANOVA and Tukey post hoc analysis at each isoproterenol concentration. Myocardial protein abundance was compared with 1-way ANOVA, followed by a Tukey post hoc analysis. All analyses were performed on SPSS (SPSS Inc, release 12.0.). Significance was set at an level of P<0.05. Data are reported as mean±SEM.
Results
In Vivo Hemodynamics
Animal Characteristics
Langendorff Isovolumic Performance
Baseline
In our model, all hearts were perfused at constant flow (16 mL/min) with a crystalloid perfusate, allowing differences in coronary perfusion pressure to be illustrative of coronary vascular resistance. This coronary flow yielded baseline perfusion pressures that were well correlated with in vivo mean tail cuff blood pressures immediately before death (r=0.74, P<0.01). At baseline in the absence of pacing, perfusion pressure was higher in both SHR groups compared with WKY (WKY, 77±7 mm Hg; SHR-SED, 143±9 mm Hg; SHR-TRD, 112±9 mm Hg; P<0.05). Interestingly, baseline perfusion pressure was significantly lower in SHR-TRD versus SHR-SED (P<0.05). Baseline perfusion pressure with pacing tended to increase and remained higher in both SHR groups (WKY, 87±7 mm Hg; SHR-SED, 143±8 mm Hg; SHR-TRD, 115±8 mm Hg; P<0.05). During pacing, SHR-TRD elicited a lower perfusion pressure than SHR-SED (P<0.05).
Isoproterenol Dose-Response Relationship
Isoproterenol significantly increased LV Dev P in all groups (P<0.05) (Figure 4A). The peak response to isoproterenol was shifted rightward 100-fold in both SHR groups relative to WKY, representing a reduced ;-adrenergic agonist sensitivity in SHR. Despite the rightward shift in peak response, the peak LV Dev P response to ;-adrenergic receptor stimulation was greatest in SHR-TRD but similar between WKY and SHR-SED (WKY, 47±8 mm Hg; SHR-SED, 35±13 mm Hg; SHR-TRD, 84±12 mm Hg; P<0.05). As Figure 4B shows, LVEDP fell among all groups with isoproterenol administration up to 1x10–8 mol/L. Interestingly, exercise training in hypertension (SHR-TRD) increased the lusitropic response to isoproterenol compared with both WKY and SHR-SED (P<0.05). (Figure 4C) was significantly lower at 1x10–8 in SHR-TRD versus both WKY and SHR-SED (WKY, 51.3±5.6 ms; SHR-SED, 39.9±2.3 ms; SHR-TRD, 25.8±3.3 ms; P<0.05). Isoproterenol significantly increased the +dP/dt in all groups (P<0.05) (Figure 4D). The peak +dP/dt response to isoproterenol was observed at 1x10–9 mol/L in WKY; the peak +dP/dt response occurred at 1x10–8 mol/L in both SHR groups. The peak +dP/dt response to ;-adrenergic receptor stimulation was greatest in SHR-TRD versus the peak response in WKY (1x10–9 mol/L) and SHR-SED. Figure 4E documents the –dP/dt response to isoproterenol among groups. A significant increase in –dP/dt occurred in all groups with the administration of isoproterenol (P<0.05). The peak response was shifted rightward 10-fold in SHR-TRD and 100-fold in SHR-SED compared with WKY, with the greatest response observed in SHR-TRD. At baseline and at isoproterenol infusions of 1x10–10 and 1x10–9 mol/L, perfusion pressure of WKY was significantly lower than both SHR-SED and SHR-TRD (P<0.05) (Figure 4F). Additionally, at baseline and all isoproterenol doses except 1x10–9 mol/L, SHR-TRD had a lower perfusion pressure than SHR-SED (P<0.05). At isoproterenol doses of 1x10–8 and 1x10–7 mol/L, SHR-SED had a significantly greater perfusion pressure compared with WKY (P<0.05).
Western Blots
Both hypertension and exercise training independently altered protein abundance and phosphorylation of several key Ca2+ cycling proteins (Figures 5 and 6). As Figure 5 illustrates, total ryanodine tended to be elevated in SHR-TRD versus both WKY and SHR-SED, whereas phosphorylated ryanodine (RyRp) was increased in SHR-TRD versus SHR-SED (P<0.05). Hypertension significantly increased LTCC abundance in SHR compared with the normotensive WKY (P<0.001). No difference in LTCC abundance resulted from chronic exercise training. NCX abundance was significantly increased in both SHR groups compared with WKY (P<0.001), with no difference observed with exercise training. PLBt abundance was elevated with training in hypertension (SHR-TRD) versus WKY (P<0.05), with similar SERCA2A among groups (Figure 6). Phospholamban phosphorylated at Ser16 was significantly lower in SHR-SED than both WKY (P<0.05) and SHR-TRD (P<0.01). Additionally, greater phospholamban phosphorylated at Thr17 was observed in SHR-TRD versus SHR-SED (P<0.01), whereas no difference was observed between WKY and SHR-SED. Myocardial levels of the ;-adrenergic receptor kinase (;ARK1 or GRK2), previously shown to be associated with pressure-overload cardiac hypertrophy,11 were increased in SHR-SED versus WKY (P<0.05) but decreased in SHR-TRD (P<0.05) (Figure 7).
Discussion
A major finding of this study is that exercise training improved ;-adrenergic receptor responsiveness in the SHR model, perhaps by decreasing myocardial levels of the ;-adrenergic receptor desensitizing kinase GRK2. As a result of "restored" or improved ;-adrenergic receptor signaling, protein kinase A–mediated "downstream" phosphorylation of key SR Ca2+ handling proteins such as the ryanodine receptor and phospholamban (Ser16) was enhanced with training. Training also increased calcium/calmodulin-dependent protein kinase phosphorylation of phospholamban (Thr17). Exercise training did not alter the observed increases in protein abundance of the LTCC or NCX in SHR. Additionally, exercise training reduced coronary vascular resistance and attenuated pacing-induced increases in both the time constant of isovolumic pressure relaxation () and LV end-diastolic pressure. The above putative effects of exercise are interesting in light of the fact that training increased LV wall thickness in SHR.
Myocardial ;-adrenergic receptors are a member of the superfamily of G-protein–coupled receptors (GPCRs), which control cardiac function by modulating inotropy, lusitropy, and chronotropy.7 Altered contractile function and reduced myocardial responsiveness to adrenergic stimulation reflect some of the earliest changes associated with hypertension-induced, compensatory hypertrophy.1 Although some studies have shown a decreased ;-adrenergic receptor density in hypertension,5 it appears likely that abnormalities "downstream" from the ;-adrenergic receptor are mechanistically culpable in the hypertrophied hearts impaired responsiveness to adrenergic stimulation.1,6–9 In ventricular hypertrophy11 and heart failure,29 ;-adrenergic receptors are overly desensitized and downregulated, which has been attributed to increased myocardial levels of the GPCR kinase, GRK2 (also known as ;ARK1), seen in these conditions. GRKs, such as GRK2, phosphorylate GPCRs, leading to functional uncoupling between the receptor and downstream effector systems.30 GRK2 is the most abundant GRK in the heart, and in conditions of ventricular hypertrophy and dysfunction, increased expression and activity of myocardial GRK2 uncouple ;-adrenergic receptors, causing dysregulation of myocyte excitation-contraction coupling.7,29 As expected, our data show that GRK2 abundance was increased in SHR sedentary animals, an effect consistent with the observed blunted ;-adrenergic receptor responsiveness in SHR sedentary hearts. However, a novel finding in the present study is that exercise training attenuated the increase in GRK2 associated with hypertension and may explain part of the enhanced functional and molecular performance in SHR-TRD.
Although exercise training blunted the increase in GRK2 associated with hypertension, it did not fully restore the impaired ;-adrenergic sensitivity in SHR. The present data show a 100-fold rightward shift in the isoproterenol–LV Dev P and the isoproterenol–±dP/dt dose-response curves in both SHR groups relative to WKY. These data are similar to those reported by Atkins et al,1 who showed a rightward shift in the isoproterenol and forskolin dose-response relationships in compensatory-hypertrophied SHR hearts. Atkins et al also showed that ;-adrenergic receptor density was unchanged in nonfailing SHR and that receptor affinity did not differ between WKY or nonfailing SHR hearts.1 These data suggest that impaired inotropic responsiveness of the LV myocardium to ;-adrenergic stimulation is not associated with alterations in the density or affinity of ;-adrenergic receptors but rather downstream cellular events.
Of particular interest is that exercise training in SHR improved lusitropic responsiveness to adrenergic stimulation and offset the pacing-induced increases in and LV diastolic chamber stiffness, even beyond that of WKY. Although in the present study SERCA abundance was similar among all groups, phospholamban abundance was greater in SHR-TRD animals compared with WKY. Our results show that in hypertension, exercise training increased phospholamban phosphorylation at both Ser16 and Thr17 sites relative to sedentary animals. The increased phospholamban phosphorylation likely facilitates sarcoplasmic reticulum Ca2+ loading and subsequent release.31 Along these lines, our results also show that exercise training increased ryanodine receptor phosphorylation without altering ryanodine receptor density among groups. Our data support results presented by Shorofsky et al,31 who showed similar SERCA and ryanodine receptor abundance in WKY and SHR hearts. However, Shorofsky et al did not report alterations in phospholamban abundance or phosphorylation in SHR hearts. The differences may reflect our methodological approach, which included the administration of a ;- agonist (isoproterenol) before the heart was arrested.
SBP was markedly elevated throughout the protocol in SHR; at the time of death, both SHR groups exhibited a 25% greater SBP relative to normotensive controls. The antihypertensive effect of exercise in the present study was minimal and smaller than reports from longer training paradigms.32 In our study, exercise training superimposed on hypertension augmented the extent of myocardial hypertrophy. The hypertensive animals displayed greater LV mass and ratio of LV mass to BW than the normotensive controls. Additionally, echocardiography-derived AW and PW thicknesses during both the systolic and diastolic phases of the cardiac cycle were greater in SHR than WKY. The increased wall thickness translated to a 10% increase in LV mass and a 7% increase in the ratio of LV mass to tibial length after training. The increased LV wall thickness observed with exercise training is interesting in light of the lower resting RPP in trained animals relative to hypertensive sedentary animals. Seminal work also has shown that exercise training potentiates LV hypertrophy in hypertension.33,34 The present results show that baseline inotropy and abundance of both LTCC and NCX were increased in SHR relative to WKY. The present data support experiments showing that SHR hearts with compensatory hypertrophy have enhanced contractility4,31 amplitude of [Ca2+]i and a mean amplitude of Ca2+ sparks.31
Study Limitations
We selected the SHR model for our studies because it mimics the clinical course of untreated essential hypertension in humans. It is well documented that concentric hypertrophy occurs in SHR within 6 to 12 months of age. The SHR model gradually decompensates into heart failure sometime after 15 months of age.35,36 We chose to study the animals at 7 months of age, a time in which fibrosis is not accelerated in SHR.36 The interpretability of our study is limited in several ways. First, the causes for hypertension in SHR are polygenic and do not necessarily reflect the genetic anomalies associated with hypertension in humans.31,35 Second, the absence of an exercise-trained WKY group limits our understanding of the exercise adaptation independent of hypertension. Third, we did not examine ;AR density or subtype on the basis of previous data suggesting that impaired responsiveness to ;-adrenergic stimulation is regulated distal to the receptor primarily by GRK2 expression.1,6–9,30,37,38 Fourth, we did not examine myocardial basal phosphorylation state because previous studies reported no differences between SHR and WKY.31,39 Finally, the relative isoproterenol doses were likely extreme for WKY and may have internalized the receptors as evidenced by the negative LV Dev P at 10–7 mol/L.
In summary, exercise training in hypertension increased the inotropic and lusitropic responsiveness to ;-adrenergic receptor stimulation and increased the phosphorylation of key Ca2+ cycling proteins while attenuating GRK2 abundance with hypertension. Future experiments are required to elucidate the putative interaction between the above molecular observations and cardiac function.
Acknowledgments
This study was supported by a beginning-grant-in-aide from the American Heart Association, Mid-Atlantic Affiliate (Dr Libonati), and the National Institute of Health (HL-33921 to Dr Houser, HL-61690 and HL-56205 to Dr Koch). We thank Leigh Ann Hewsten for technical support.
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