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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 59:1089-1098 (2004)
© 2004 The Gerontological Society of America

Chronic Exercise Improves Myocardial Inotropic Reserve Capacity Through {alpha}1-Adrenergic and Protein Kinase C-Dependent Effects in Senescent Rats

Donna H. Korzick1,2,, James C. Hunter1, Mark K. McDowell1, Michael D. Delp3, Marlena M. Tickerhoof1,2 and LaToya D. Carson1,2

1 The Noll Physiological Research Center
2 The Department of Kinesiology, The Pennsylvania State University, University Park.
3 Department of Kinesiology, Texas A&M University, College Station.

Address correspondence to Donna H. Korzick, PhD, 106 Noll Physiological Research Center, The Pennsylvania State University, University Park, PA 16802. E-mail: dhk102{at}psu.edu


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
We have previously demonstrated that {alpha}1-adrenergic (AR)-mediated contraction is diminished in the senescent rat heart, in part due to alterations in protein kinase C (PKC) signaling. Since chronic exercise training (EX) can exert independent effects on increasing {alpha}1-AR contraction in the adult rat heart, we sought to determine whether age-related defects in {alpha}1-AR contraction could be reversed by chronic EX. We further hypothesized that improved {alpha}1-AR contraction by EX may be PKC dependent. Adult (4 months; Y) and aged (24 months; O) male F344 rats were treadmill-trained (n = 12–13/group; TR) at ~70% of VO2max for 12 weeks or remained sedentary (YSED, YTR, OSED, OTR). Training status was verified by plantaris citrate synthase activity and left ventricular (LV) contractile responses (dP/dt) to {alpha}1-AR stimulation were assessed in Langendorff-perfused hearts using the {alpha}1-AR agonist phenylephrine (PE; 10–5 M) with and without the PKC inhibitor chelerythrine (CE; 10–6 M). {alpha}1-AR stimulation elicited greater increases in LV dP/dt in hearts isolated from OTR (4525.4 ± 224.1 mmHg/s) versus OSED (3658.9 ± 291.0 mmHg/s), while CE abolished PE-induced effects (OTR, 4069.2 ± 341.2) versus (OSED, 3608.9 ± 321.2) (p <.01). Upon western blotting, phosphospecific antibodies directed at PKC{varepsilon} (pSer729) revealed greater levels in LV isolated from YTR versus YSED, and EX ameliorated aged-related reductions in OSED (p <.001). Basal PKC{varepsilon} mRNA levels were also greater in YTR and OTR versus YSED (p <.01). PE-induced increases in phosphor-PKC{delta} (pThr507) levels observed in OSED were attenuated in OTR (p <.03). Chronic EX was also associated with significant reductions in PKC{alpha} (pSer657) levels following PE in OTR (p <.002). The results indicate that age-related reductions in {alpha}1-AR contraction can be partially reversed by EX in the rat heart. These results further suggest that alterations in PKC levels underlie, at least in part, EX-induced improvements in {alpha}1-AR contraction.

REDUCTIONS in functional reserve capacity contribute to the diminished ability of the aged heart to respond and adapt to mechanical and ischemic stresses [for review see (1)]. In addition to the well-characterized dysregulation of the cardiac ß1-adrenergic receptor (AR) signaling cascade, we have demonstrated that alterations in the cardiac {alpha}1-AR system also contribute to age-related reductions in functional reserve capacity, in part due to diminished isoform-specific protein kinase C (PKC) translocation and associated membrane-binding proteins (receptors for activated C kinase; RACKs) (2). In contrast, exercise training leads to functionally beneficial adaptations and reverses several hallmark features of pathological hypertrophy and left ventricular (LV) dysfunction [for review see (3)]. Moreover, exercise training improves inotropic responsiveness following {alpha}1-AR stimulation in both the adult and hypertensive heart in male rats (4), as well as in the aged human (5). However, a complete understanding of the cellular mechanisms by which exercise preserves and/or improves cardiac function in the aged heart is lacking. Taffet and colleagues (6) have reported exercise-induced improvements in ß1-AR-mediated cardiac relaxation in senescent rats, but studies on the adrenergic regulation of systolic function with regard to signal transduction in the aged heart are sparse and inconclusive (7).

A likely cellular target of exercise-induced functional adaptation under conditions of {alpha}1-AR stimulation includes alterations in PKC, one candidate signal transducer of {alpha}1-AR inotropic actions (8–10). Interestingly, PKC has also been implicated in cardioprotective responses in a variety of animal species and experimental paradigms, including acute exercise (11–13). Cellular PKC exerts its effects by translocating to multiple cellular locations whereby protein phosphorylation is likely to be achieved (14). Current experimental evidence suggests that multiple isoforms of PKC exist in the heart and subserve a variety of cellular functions depending on the nature of stimulation. For example, low-level activation of PKC{varepsilon} has consistently been found to reduce hypoxic injury in the myocardium (11,15); however, overexpression of PKC{alpha} has been associated with pathological hypertrophic growth and cardiac contractile dysfunction in response to {alpha}1-AR stimulation (13,16).

Since exercise training can increase the inotropic responsiveness to {alpha}1-AR stimulation in the adult rat heart as well as attenuate the functional consequences of diminished {alpha}1-AR sensitivity in the hypertensive heart (4), it is possible that exercise-induced adaptations in {alpha}1-AR signal transduction may occur with aging, thereby providing a potential mechanism to enhance inotropic reserve in the senescent myocardium. Thus, one purpose of the current study was to determine whether chronic exercise could attenuate age-related aberrations in {alpha}1-AR mediated inotropic function. The involvement of PKC in {alpha}1-AR signaling is well characterized (2,10,17) and we have recently reported alterations in PKC{varepsilon} and PKC{alpha} protein and mRNA levels, respectively, in response to acute exercise in the adult myocardium (12). We therefore hypothesized that exercise-induced improvements in {alpha}1-AR contraction in the aged heart would be PKC dependent.


   METHODS
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 Abstract
 Methods
Results
 Discussion
 References
 
Animal Preparation
Adult (4 months) and aged (21 months) male Fischer-344 rats were obtained from Harlan Sprague Dawley (Indianapolis, IN). Rats were exposed to a 12-hour light/dark cycle and received food and water ad libitum. All experiments were approved by the Institutional Animal Care and Use Committee of The Pennsylvania State University and were in agreement with the "Guidelines for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

Exercise Training
Adult and aged animals were randomly assigned to sedentary or run-training groups (adult sedentary [YSED], n = 14, aged sedentary [OSED], n = 14; adult trained [YTR], n = 14; aged trained [OTR], n = 14). The training program consisted of running 60 minutes per day, 5 days per week on a motor-driven treadmill for 14 weeks. Initially, the duration was gradually increased over 2 weeks followed by a gradual increase in intensity over 5 weeks until the desired relative workload of ~70% of VO2max was achieved (YTR: 28 m/min, 10% grade; OTR 16 m/min, 5% grade). Age-matched sedentary controls were placed on the treadmill for 10 minutes twice weekly and pursued normal exploratory behavior. Five animals were excluded from the study; 3 aged (2 OTR, 1 OSED) animals died (exercise-unrelated causes) before the end of the study, 1 YTR animal was excluded for nonadherence to the training protocol, and 1 YSED was excluded due to failed cardiac function assessment.

Citrate Synthase Assay
Verification of training was assessed by citrate synthase activity of plantaris muscle homogenates as described by Srere (18). Briefly, frozen plantaris muscles were homogenized with a glass–glass grinder in 19 volumes of 100 mM Tris buffer (pH 8.0) and exposed to one cycle of freeze-thawing. Samples were then diluted 10 times, and citrate synthase activity was determined from the change in absorption at 412 nm in a reaction mixture containing (in mM): 100 Tris (pH 8.0), 0.3 acetyl CoA, 0.1 5,5'-dithio-bis(2-nitrobenzoic acid), 0.5 oxaloacetate.

Isolated Heart Perfusion
In vitro assessment of left ventricular (LV) function was performed utilizing a modified Langendorff isovolumic heart preparation as described previously (2,4). Briefly, following administration of heparin (2.5 mg i.p.), rats were anesthetized with pentobarbital (35 mg/kg body wt) and hearts were rapidly excised via midline thoracotomy and placed in cold (4°C) saline. Hearts were then secured to the perfusion apparatus and perfused with a Krebs-Henseleit bicarbonate buffer (pH 7.4) containing (in mM) 1.75 CaCl2, 117.4 NaCl, 4.7 KCl, 1.2 MgSO4, 1.3 KH2PO4, 24.7 NaHCO3, 11.0 glucose, 5.0 pyruvate, and 0.5 EDTA, continuously gassed with 95% O2–5% CO2, and maintained at 37°C. Following a 5-minute equilibration period, a fluid-filled latex balloon, attached to a DTX Plus Transducer (Becton-Dickinson, Franklin Lakes, NJ), was inserted into the left ventricle via the mitral valve. Based on previous studies in our laboratory (unpublished observations), the high-compliance balloon was inflated until a minimum pressure of 5 mmHg was achieved, corresponding to ~90%–95% of maximum on the ascending limb of the Starling curve. Hearts were paced at 240 bpm (with atria crushed) via a platinum electrode in the right ventricle, and all hearts were allowed to equilibrate with pacing for 15 minutes prior to assessment of {alpha}1-AR-inotropic effects. Contractile function was continuously monitored by the Ponemah Physiology Platform (Gould Electronics, Valley View, OH) in YSED, OSED, and OTR using the following variables (per beat): LV developed pressure ({Delta}P, mmHg), LV diastolic pressure (mmHg), LV maximal dP/dt (dP/dtmax, mmHg/s), LV negative dP/dtmax (–dP/dtmax, mmHg/s), and time to peak pressure development (TTPK, ms).

Assessment of {alpha}1-AR Inotropic Effects
Two distinct pharmacological protocols were utilized in this study, and animals were randomly assigned to a given protocol. Following equilibration, hearts from adult (YSED n = 7, YTR n = 6) and aged (OSED n = 7, OTR n = 7) rats were perfused with the specific {alpha}1-AR agonist phenylephrine (PE; 10–5 M) for 5 minutes. To minimize effects of ß-adrenergic receptor stimulation, hearts were perfused with propranolol (ß-AR antagonist; 10–7 M) 5 minutes prior to (and during) PE perfusion. To determine the role of PKC in {alpha}1-AR-mediated contraction, the PKC inhibitor chelerythrine (CE; 10–6 M) was used in a separate set of experiments. Adult (YSED n = 6, YTR = 7) and aged (OSED n = 6, OTR n = 5) hearts were perfused with CE for 20 minutes before perfusion with PE (10–5 M), and contractile function was assessed as described. Given our previous studies utilizing the F344 rodent model (4), contractile function was assessed in a limited number of YTR (n = 2) for the PE-only studies.

Tissue Preparation
At the conclusion of PE-only contractile function studies, the atria, great vessels, and right ventricle were removed, and the left ventricle was blotted dry, weighed, quartered, and frozen in liquid nitrogen. Hearts obtained from animals subjected to a similar exercise training protocol as described above were isolated and served as baseline controls. Samples were stored at –80°C until protein analysis. The frozen septal quarter of LV myocardium from adult and aged rats were prepared as described (2) with modifications. Briefly, LV tissue was minced and homogenized in cold (4°C) lysis buffer (20 mM Tris, pH 7.5; 2 mM each of EDTA and EGTA, pH 7.5–8.0; 5 mM sodium fluoride; 5 µg/ml each of leupeptin and aprotinin; 0.5 µg/ml pepstatin A; 0.3 mM phenylmethylsulphonyl fluoride; 1 µM vanadate; 0.03% 2-mercaptoethanol) using a Polytron PTA-10 (Brinkmann/Kinematica, Westbury, NY) at 60% power. Samples were then subjected to centrifugation at 100,000 g for 1 hour at 4°C. The supernatant was defined as the cytosolic fraction, and the pellet was resuspended in lysis buffer containing 1% Triton X-100 and shaken on ice for 2 hours before centrifugation at 100,000 g for 1 hour at 4°C. The resulting supernatant was defined as the particulate fraction. Protein concentration was determined by the method of Bradford (19).

Western Blotting for PKC Subcellular Distribution
Cytosolic and particulate fractions were subjected to Western blotting as described previously (2). Briefly, equal amounts of protein (6.5–26 µg for 16 cm x 16 cm gels; 10–20 µg for 9 cm x 16 cm gels) were electrophoresed on 7.5% SDS polyacrylamide gels and electrophoretically transferred for 4 hours to polyvinylidine difluoride membranes. Membranes were then blocked with 6% nonfat dry milk in TBS Tween-20 for 2 hours. Following a 3-hour incubation at room temperature with rabbit polyclonal antibodies against PKC{alpha} (1:1500), PKC{delta} (1:1200), PKC{varepsilon} (1:1500), and appropriate washes, membranes were incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-linked antirabbit IgG (1:25,000). Following appropriate washes, antibodies were detected using enhanced chemiluminescence (ECL).

As an index of activation, the phosphorylation state of PKC isoforms was assessed using phosphospecific antibodies. Increases in the phosphorylation state of PKC isoforms have been shown to occur with PKC stimulation (20,21). Western blotting was performed as described with the following antibody concentrations (rabbit antiphospho-PKC; HRP-linked antirabbit): p-PKC{alpha} (Ser657) (1:800; 1:15000); p-PKC{delta} (Thr507) (1:800; 1:12,000); p-PKC{varepsilon} (Ser729) (1:1200; 1:20,000). Densitometry of Western blot films was performed using Scion Image software (Scion Corporation, Frederick, MD).

Western Blotting for Extracellular-Regulated Kinase (ERK1/2) Activation
Tissue homogenates were electrophoresed on 10% SDS-polyacrylamide gels, transferred, and blocked as described above. Following incubation with mouse monoclonal antibody against p-p44/p42 (p-ERK1/2) (1:2000) and appropriate washes, membranes were incubated with HRP-linked antimouse IgG (1:2500), and visualized as described. To control for total ERK1/2 protein levels, membranes were then stripped by 30-minute incubation in stripping buffer (62.5 mM Tris pH 6.80, 7% 2-mercaptoethanol, 2% SDS) at 70°C and reprobed with rabbit polyclonal antibody against ERK2 (1:1000). Following appropriate washing, membranes were incubated with HRP-linked antirabbit IgG, visualized by ECL, and densitometry was performed as described.

Ponceau Correction
The Ponceau staining method, as described by Ping and colleagues (30) was used to correct errors introduced during sample loading or gel transfer. Briefly, membranes were incubated in Ponceau S solution for 5 minutes and washed appropriately. The nonspecific band with the largest molecular weight was then subjected to densitometric analysis, and the average density of the bands was calculated across the lanes. For value correction, the density of the PKC band in each lane was divided by the ratio of the density of the Ponceau stain in that lane to the average Ponceau stain density.

RNA Isolation and Real-Time Polymerase Chain Reaction
Quantitative real-time polymerase chain reaction (PCR) was used to assess PKC{varepsilon} mRNA levels in LV myocardium. Total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction and reverse-transcribed into cDNA as previously described in our laboratory (12). Forward and reverse primers for PKC{varepsilon} were designed as follows: PKC{epsilon}, 5'-TCA ATG GCC TCC TTA AGA TCA AA-3' and 5'-ATG GCG CAG CGA CCA G-3'. Carboxyfluorescein-labeled oligonucleotide probe was synthesized by Biosearch Technologies, Inc. (Novato, CA) with the following sequence for PKC{epsilon}: 5'-TCT GCG AGG CCG TGA GCT TGA AG-3'. RT-PCR using the ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) was used to assess gene expression levels relative to expression of an internal control (glyceraldehyde-3-phosphate dehydrogenase, GAPDH).

Materials
Tris and NaCl were obtained from Fisher Scientific (Pittsburgh, PA); SDS and 2-mercaptoethanol were obtained from BioRad Laboratories (Hercules, CA); horseradish peroxidase-linked IgG, polyvinylidine difluoride membranes and enhanced chemiluminescence kits were obtained from Amersham-Pharmacia Biotech (Piscataway, NJ). All other chemicals and pharmacological agents were obtained from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal antibodies against PKC{alpha}, PKC{delta}, PKC{varepsilon}, p-PKC{alpha} (Ser657), p-PKC{delta} (Thr507), and p-PKC{epsilon} (Ser729) and ERK2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and mouse monoclonal antibody against p-p44/p42 (p-ERK1/2) was obtained from Cell Signaling Technology (Beverly, MA).

Statistical Analysis
Group comparisons of contractile function in response to PE or PE + CE were analyzed with analysis of variance (ANOVA) using the PROC Mixed General Linear Models program for an unbalanced and mixed design (2 x 2 x 2 ANOVA) utilizing Statistical Analysis Software (SAS Institute, Inc., Cary, NC). For immunoblotting, two-way ANOVA was used to determine group differences between mean values of protein levels. Significance of differences between mean values for the physical and physiological characteristics of animals was determined by one-way ANOVA. The Least Significant Difference method was employed for all post hoc comparisons. All variables are reported as mean ± standard error (SE). Significance was defined as p <.05.


   RESULTS
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 Abstract
 Methods
 Results
Discussion
 References
 
Efficacy of the Endurance Training Program
Animal characteristics at necropsy are presented in Table 1. Chronic training resulted in significant body weight reductions when compared with age-matched sedentary controls. The OTR animals consistently lost weight during the training program and weighed ~13% less at the time of necropsy versus OSED. The YTR animals weighed on average 7% less each week compared with the sedentary control group, and LV weight/body weight ratios increased by 10% and 22% in the YTR and OTR, respectively (p <.01). There was an insignificant increase in the LV weight/tibial length ratio in YTR (2%) and OTR (6%) compared with sedentary controls, consistent with similar studies of chronic training in male rats. Analysis of excised plantaris muscle for citrate synthase activity revealed that both groups of trained animals had significantly higher activities compared with controls (Table 1). Citrate synthase activity was 61% and 63% greater in the YTR and OTR, respectively, when compared with their sedentary counterparts (p <.01). Furthermore, citrate synthase activity was significantly less in muscles isolated from aged compared with adult rats, independent of exercise training status (p <.01).


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  		Table 1. Selected Rat Characteristics at Necropsy.

 
Baseline Cardiac Function
Left ventricular contractile responses were assessed in Landendorff-perfused hearts, and baseline values are presented in Table 2. During the equilibration period, +dP/dt was ~10% less in OSED when compared with YSED, consistent with our previous findings of minor reductions in basal contractile function with senescence (2). As expected, significant increases were observed in TTPK with advanced age; differences in TTPK due to chronic training were not observed (Table 2). Importantly, no significant group differences were observed in indices of contractile function following administration of propranolol or CE prior to administration of PE (data not shown).


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  		Table 2. Selected Baseline Parameters of Contractile Function and Morphology in the Absence of Phenylephrine and/or Chelrythrine.

 
{alpha}1-AR Inotropic Effects With and Without PKC Inhibition
The inotropic response following stimulation of the {alpha}1-adrenergic signaling pathway with PE (10–5 M) in the presence of the ß-AR blocker propranolol is illustrated in Figure 1 (left panel). A separate series of experiments were conducted and hearts were first perfused with the PKC inhibitor CE (10–6 M) followed by PE (10–5 M; Figure 1, right panel). As we have previously demonstrated (2), PE resulted in a significant increase in +dP/dt from baseline across all groups studied, while attenuated {alpha}1-AR inotropism was observed in the senescent myocardium (p <.01; Figure 1). Importantly, {alpha}1-AR stimulation elicited greater increases in LV dP/dt in hearts isolated from OTR versus OSED (4525.4 ± 274.1 mmHg/s vs 3658.9 ± 291.0 mmHg/s). These data are consistent with an exercise-induced augmentation in {alpha}1-AR-mediated contractile responses in the aged heart, and extend our previous findings of {alpha}1-AR adaptations following chronic training in the adult rat heart (4). Significant inhibitory effects of CE on contractile responses following PE were observed in YSED, YTR, and OTR but not OSED (p <.01). Specifically, we observed a ~66% inhibition of {alpha}1-AR-mediated inotropic effects in YTR versus ~47% inhibition in YSED (3462.5 mmHg/s ± 336.9 vs 3784.7 mmHg/s ± 100.8). In the aged heart, greater inhibitory effects of CE on PE-mediated contraction were observed in OTR versus OSED (4069.2 mmHg/s ± 341.2 vs 3608.9 mmHg/s ± 321.2; Figure 1B). We believe these results suggest an important role for PKC in attenuating age-related reductions in {alpha}1-AR contraction. Reductions in –dP/dt were observed following {alpha}1-AR stimulation in all groups studied (YSED = 1460.9 ± 174.4, YTR = 1360.5 ± 112.6, OSED = 1218.37 ± 91.2, OTR = 1334.08 ± 261.2), as were decreases in TTPK (NSD = 74.4 ± 0.7; NTR = 76.3 ± 0.3; OSED = 81.4 ± 2.0, and OTR = 81.4 ± 0.9; p <.01).



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  		Figure 1. Effects of advanced age and chronic exercise training on left ventricular (LV) dP/dt following {alpha}1-AR (adrenergic) stimulation in the presence and absence of protein kinase C (PKC) inhibition in the rat heart. Hearts were stimulated with 10–5 M phenylephrine (PE) for 5 minutes (left). In a separate series of experiments, hearts were perfused with the PKC inhibitor chelerythrine (CE) (10–6 M, 20 min) followed by PE (10–5 M) (right). All experiments were performed in the presence of 10–6 M propranolol, and independent effects of the CE or propranolol on dP/dt were not observed. A significant main effect of exercise was present for {alpha}1-AR inotropic effects in aged hearts for PE-only studies, while exercise was associated with greater inhibitory effects of CE on PE-mediated contractile responses. YSED = young sedentary (n = 7 for PE, n = 6 for PE + CE); YTR = young trained (n = 2 for PE, n = 7 for PE + CE); OSED = old sedentary (n = 7 for PE, n = 6 for PE + CE); OTR = old trained (n = 7 for PE, n = 5 for PE + CE). Data are expressed as mean ± SE (standard error). *p <.01 versus SED; {dagger}p <.01 versus young; {ddagger}p <.01 versus PE

 
Western Blotting for PKC Isoforms and ERK1/2
Left ventricular myocardium was fractionated using differential centrifugation for PKC localization in soluble and particulate cellular compartments, respectively, and subjected to Western blotting to assess isoform-specific PKC levels. Consistent with our previous findings utilizing a Wistar model of rodent aging (2), a significant main effect of age was observed revealing greater PKC{alpha} levels in the soluble compartment following stimulation with PE in OSED and OTR as compared with YSED and YTR (Figure 2A). Similar group differences for PKC{alpha} were also observed under basal conditions in the F344 model of aging. Advanced aged was also associated with significant increases in particulate PKC{delta} levels (Figure 2C). Interestingly, we observed no group differences for soluble or particulate PKC{varepsilon} levels in response to advanced age or chronic training with PE stimulation (Figure 2B). However, significantly lower PKC{varepsilon} levels prior to PE stimulation in LV isolated from YTR and OTR relative to control suggested greater PKC{varepsilon} translocation as a result of exercise training.



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  		Figure 2. Effects of advanced age on the subcellular distribution of protein kinase C alpha (PKC{alpha}), PKC{varepsilon}, and PKC{delta} immunoreactivity following {alpha}1-AR (adrenergic) stimulation in the rat heart. Hearts were stimulated with 10–5 M phenylephrine (PE) for 5 minutes. Equal amounts of protein were loaded per lane (6.5–26 µg/lane), and densities were indexed to Ponceau corrected. YSED = young sedentary (n = 5 for baseline, n = 5 for PE); YTR = young trained (n = 5 for baseline, n = 6 for PE); OSED = old sedentary (n = 5 for baseline, n = 7 for PE); OTR = old trained (n = 5 for baseline, n = 7 for PE). Data are expressed as mean ± SE (standard error). *p <.01 versus SED; {dagger}p <.01 versus young

 
Because the phosphorylation state of PKC is regulated by the phosphoinositide-dependent kinase PDK-1 and requisite for subsequent PKC catalytic activity (14,22,23), we also sought to determine whether PKC phosphorylation was altered by chronic exercise and/or senescence. Phosphospecific antibodies directed toward the activation loop of PKC{alpha} (pSer657), PKC{delta} (pThr507), and PKC{varepsilon} (pSer729) were utilized in the current investigation. Upon Western blotting, antiphospho-PKC{varepsilon} (pSer729) levels were significantly greater in LV isolated from YTR versus YSED in response to PE stimulation (Figure 3). We also observed a 40% reduction in PE-stimulated phospho-PKC{varepsilon} (pSer729) levels in OSED relative to YSED, which was partially reversed by chronic exercise in OTR (p <.001; Figure 3). These findings suggest that the upstream activation of PKC{varepsilon} may represent a critical event underlying exercise-induced adaptation to {alpha}1-AR stimulation.



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  		Figure 3. Effects of advanced age on phosphorylated protein kinase C epsilon (PKC{varepsilon}) and PKC{delta} immunoreactivity following {alpha}1-AR (adrenergic) stimulation in the rat heart. Hearts were stimulated with 10–5 M phenylephrine (PE) for 5 minutes. Equal amounts of protein were loaded per lane (6.5–26 µg/lane). Antibodies were directed at the activation loop of PKC{varepsilon} (pSer729) and PKC{delta} (pThr507), respectively. Prior to PE, group differences in baseline levels of PKC{varepsilon} (pSer729) were not observed, while a significant effect of age was evident for PKC{delta} (pThr507). YSED = young sedentary (n = 5 for baseline, n = 5 for PE); YTR = young trained (n = 5 for baseline, n = 6 for PE); OSED = old sedentary (n = 5 for baseline, n = 7 for PE); OTR = old trained (n = 5 for baseline, n = 7 for PE). Data are expressed as mean ± SE (standard error). *p <.01 versus SED; {dagger}p <.01 versus young

 
Interestingly, directional changes in phosphorylated PKC{delta} (pThr506) and PKC{alpha} (pSer657) levels were also observed with chronic training in the aged heart, PKC isoforms known to be involved in pathological hypertrophic growth, and contractile dysfunction (13,16,24–27). Specifically, we observed age-related increases in membrane-associated phospho-PKC{delta} (pThr507) levels following {alpha}1-AR stimulation, which were significantly attenuated in OTR (p <.03; Figure 3). Chronic exercise also was associated with significant reductions in PKC{alpha} (pSer657) levels in the soluble cellular compartment following PE in 24-month and 5-month hearts, respectively (Figure 4). Age-related increases in phospho-PKC{alpha} (pSer657) levels were attenuated by chronic exercise (Figure 4; p <.002), and we observed similar directional changes in p-ERK1/2 levels (Figure 5; p <.01) across experimental groups. These patterns suggest that, in addition to training-induced adaptations in LV contractile performance in response to {alpha}1-AR stimulation, beneficial adaptations in myocardial growth also may contribute to the protective phenotype associated with chronic exercise training in the aged heart.



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  		Figure 4. Effects of advanced age and exercise on soluble phosphorylated protein kinase C alpha (PKC{alpha}) levels following {alpha}1-AR (adrenergic) stimulation in the rat heart. Hearts were stimulated with 10–5 M phenylephrine (PE) for 5 minutes, and equal amounts of protein were loaded per lane (7.5 µg). Antibodies were directed at the activation loop of PKC{alpha} (pSer657). Prior to PE, PKC{alpha} (pSer657) levels were significantly less in hearts isolated from trained animals. YSED = young sedentary (n = 5 for baseline, n = 5 for PE); YTR = young trained (n = 5 for baseline, n = 6 for PE); OSED = old sedentary (n = 5 for baseline, n = 7 for PE); OTR = old trained (n = 5 for baseline, n = 7 for PE). Representative immunoblot reflects PKC levels following PE stimulation: lanes 1–2, YSED; lanes 3–4, YTR; lanes 5–6, OSED; lanes 7–8, OTR. Data are expressed as mean ± SE (standard error). *p <.01 versus SED; {dagger}p <.01 versus young

 


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  		Figure 5. Effects of advanced age and exercise on phosphorylated extracellular-regulated kinase (ERK) levels following {alpha}1-AR (adrenergic) stimulation in the rat heart. Hearts were stimulated with 10–5 M phenylephrine (PE) for 5 minutes, and equal amounts of protein were loaded per lane (12–20 µg). Antibodies were directed at the activation loop of PKC{alpha} (pSer657). YSED = young sedentary (n = 4); YTR = young trained (n = 5); OSED = old sedentary (n = 5); OTR = old trained (n = 6). Representative immunoblot reflects PKC levels following PE stimulation: lanes 1–2, YSED; lanes 3–4, YTR; lanes 5–6, OSED; lanes 7–8, OTR. Data are expressed as mean ± SE (standard error); *p <.01 versus SED; {dagger}p <.01 versus young

 
PKC{varepsilon} mRNA Levels Are Increased With Chronic Exercise and Senescence
Because of the known causal relationship between PKC{varepsilon} and cardioprotection (11,28–31), we further interrogated the role of PKC{varepsilon} in our experimental paradigm by determining basal PKC{varepsilon} mRNA levels (i.e., in the absence of PE) using real-time PCR. Accordingly, we observed a significant main effect of age and exercise on increasing PKC{varepsilon} mRNA levels (p <.01; Figure 6).



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  		Figure 6. Protein kinase C epsilon (PKC{varepsilon}) mRNA levels are increased with chronic exercise and senescence. PKC{varepsilon} mRNA levels (under basal conditions and in the absence of stimulation) were assessed by real-time polymerase chain reaction in left ventricular homogenates isolated from 5-month-old rats and 24-month-old rats. Sedentary controls (YSED [young sedentary], n = 4; OSED [old sedentary], n = 4) were compared with chronically exercised animals (YTR [young trained], n = 4; OTR [old trained], n = 4). Significant main effects of exercise and age were observed by analysis of variance. Data are expressed as mean ± SE (standard error). *p <.01 versus SED; {dagger}p <.01 versus young

 

   DISCUSSION
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 Abstract
 Methods
 Results
 Discussion
References
 
The potential therapeutic role of the {alpha}1-AR in the maintenance of optimal levels of cardiac performance, particularly under conditions of compromised ß1-AR system regulation, has recently been proposed (27,32). In the current study, we examined whether chronic exercise training could reverse age-related reductions in {alpha}1-AR-mediated LV contractile performance. Given the importance of PKC in mediating cardiac {alpha}1-AR inotropic effects (8,10), and our previous findings of a training-induced augmentation in {alpha}1-AR-contractile responsiveness in the adult heart (4), it also was the purpose of this investigation to determine whether exercise-induced functional adaptations in the adult or aged rat myocardium were PKC dependent. Accordingly, the key findings of this study can be summarized as follows: 1) chronic endurance EX partially reversed age-related defects in {alpha}1-AR-mediated contractile function and improved sensitivity to PKC blockade compared with aged sedentary controls; 2) chronic EX training increased phospho-PKC{varepsilon} (pSer729) levels in the adult myocardium while abrogating age-related reductions; and 3) chronic EX offset age-related increases in cardiac phospho-PKC{alpha} (pSer657) and phospho-PKC{delta} (pThr507) levels, respectively, following {alpha}1-AR stimulation. These data suggest, for the first time, that alterations in PKC may underlie, at least in part, EX-induced improvements in {alpha}1-AR-mediated inotropic actions. The data are also consistent with adaptations in PKC{varepsilon} protein and gene levels following exercise training in the adult and aged heart, which are distinct from adaptations in {alpha}1-AR signaling.

A major finding of the current investigation is that chronic endurance exercise produces adaptive responses in {alpha}1-AR-mediated contraction in the aged rat heart. Regulation of inotropic function by {alpha}1-AR signaling is known to involve production of the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG), and the subsequent activation of PKC in conjunction with the Gq/G11 family of heterotrimeric G proteins (8,33). Localization of PKC to specific cellular locations, particularly translocation of specific PKC isoforms to membrane-bound structures, allows for modulation of several key intracellular processes by multiple PKC-dependent phosphorylations (13,14,22). Accordingly, we assessed {alpha}1-AR-mediated inotropism in response to the specific {alpha}1-AR agonist PE. As hypothesized, and consistent with our previous observations of improved {alpha}1-AR–mediated contraction in the adult and hypertensive male heart by chronic training (4), exercise was able to partially restore the {alpha}1-mediated inotropic effect in the aged rat heart. The magnitude of increase in {alpha}1-AR-mediated contraction as assessed by LV dP/dt in the aged heart was significantly attenuated relative to adult sedentary controls (~37% in OSED vs ~52% in YSED), while we observed a ~53% increase in PE-mediated contractile performance in aged hearts following exercise training. Taken together with our previous studies (2,4,34), the current data suggest that the {alpha}1-AR signaling pathway also is a target of exercise-induced adaptation in the aged heart. One potential explanation for this finding includes directional changes in {alpha}1-AR number with exercise and advancing age. However, we have previously demonstrated that cardiac {alpha}1-AR number is unchanged with either sedentary aging (34) or chronic exercise in adult animals (4). It is therefore plausible to conclude that isoform-specific alterations in PKC may provide a cellular basis for the functional responses observed herein. In this regard, additional novel findings associated with the current study include the observation that age-related reductions in PKC sensitivity associated with {alpha}1-AR stimulation were restored by exercise and accompanied by directional changes in PKC levels.

The involvement of PKC in mediating {alpha}1-AR inotropic effects is incompletely understood (15,31); however, PKC-dependent phosphorylations are thought to play an important role in cardiac excitation contraction coupling following {alpha}1-AR stimulation (9,35). We therefore hypothesized that exercise-induced adaptations in LV performance following {alpha}1-AR stimulation would be due, at least in part, to subtype-specific adaptations in PKC. Importantly, we observed an exercise-induced increase in soluble phospho-PKC{varepsilon} levels, with reversal of age-related reductions following stimulation with PE. Recent evidence suggests that conventional and novel PKC isoforms are activated by two sequential mechanisms, whereby PKC phosphorylation precedes the allosteric regulation of PKC by membrane-bound cofactors (i.e., DAG). The "priming" phosphorylations associated with PKC are thought to be important for the catalytic competence of the enzyme, thus localization of phosphorylated PKC{varepsilon} to the soluble cellular compartment is consistent with release of phosphorylated mature enzyme. One possible interpretation of our results is that exercise may play an important role in fine-tuning the amplitude control of PKC{varepsilon} phosphorylation regulatory events over time, providing additional supportive evidence for the role of PKC{varepsilon} as a basis for improved functional responses in our experimental model.

Our results also indicate that chronic exercise attenuates age-related increases in {alpha}1-AR-stimulated phosphor-PKC{alpha} (pSer657), PKC{delta} (pThr507), and ERK1/2 levels, respectively. These findings may be related to the role of the {alpha}1-AR in regulating growth of the myocardium, as {alpha}1-AR-mediated increases in PKC{alpha} and PKC{delta} have been implicated in hypertrophic growth and apoptosis, respectively, in a variety of pathological settings (36). Specifically, PKC{alpha} is known to activate ERK1/2 signaling pathways in response to {alpha}1-AR stimulation, and recently has been linked to enhanced interstitial fibrosis and cardiac dysfunction (24,37); whereas, PKC{delta} inhibition has been suggested as a possible therapeutic intervention under conditions of myocardial ischemia (38). Transgenic mice lacking cardiac {alpha}1-ARs demonstrate reduced exercise capacity in conjunction with smaller heart size and reduced PE-stimulated p-ERK1/2 levels, as well as expected diminished inotropic responsiveness (39). As such, PKC{alpha} and/or PKC{delta} may provide important sites of cellular regulation in response to a variety of cellular stresses (11,40,41) such as senescence and exercise. It is therefore intriguing to speculate that training-induced reductions in phosphorylated PKC{alpha} and PKC{delta} levels in the aged heart may serve to limit pathological cardiomyocyte signaling such as hypertrophy, apoptosis, and fibrosis. However, it is currently unknown whether advanced age disrupts the targeting of specific PKC isoforms to cellular loci known to transduce these signaling events.

Interestingly, while we did observe a small but significant increase in PKC{varepsilon} translocation relative to baseline values following exercise training in response to PE, changes in total PKC{varepsilon} levels or translocation patterns following {alpha}1-AR stimulation in either the adult or the aged trained myocardium were not observed. These results were surprising, however, but were not without precedence. Strasser and colleagues (36,42) recently proposed that PKC translocation may not be essential for PKC activation in the presence of increased levels of membrane-associated PKC. Accordingly, we observed a significant proportion of PKC{varepsilon} in the particulate cellular fraction in all experimental groups, and it may be that greater localization to this subcellular compartment could provide for enhanced PKC-dependent signaling even in the absence of significant translocation as recently described by Gregory and colleagues (43). Furthermore, the "transient" nature of PKC translocation in response to physiological activators such as PE of short duration also may account for the less-than-robust PKC translocation in our study (22,42). While we cannot completely rule out the possibility of experimental error introduced by subcellular fractionation procedures, our data suggest that phosphorylated PKC levels may be a more sensitive indicator of isoform-specific changes in PKC{varepsilon}-related signal transduction. In this regard, we provide novel evidence that PKC{varepsilon} mRNA levels are increased by chronic exercise in hearts isolated from both adult and aged animals. That aging was also associated with independent effects on PKC{varepsilon} mRNA levels may subserve divergent functional responses associated with PKC{varepsilon} such as cardiac hypertrophic growth (31,40). Future studies are indicated to discern the dynamic role of PKC{varepsilon} in the aging myocardium.

Summary
The beneficial effects of exercise on human health are well documented. However, the basic mechanisms that distinguish physiological from pathological cardiac phenotypes are only partially understood, and are likely to involve changes in the expression of multiple cardiac proteins, posttranslational modification, and protein–protein interactions. The cellular mechanisms by which exercise confers cardiac benefit are also likely to be age dependent. Our data suggest, for the first time, that alterations in {alpha}1-AR signaling may represent a mechanism whereby LV functional reserve capacity is maintained in the aged heart in response to chronic exercise training. Our data also suggest that adaptive responses in PKC underlie, in part, exercise-induced improvements in {alpha}1-AR-mediated contraction. The results of this study should help expand our understanding of the cellular mechanisms that render the aged heart more susceptible to cardiovascular injury, with the ultimate goal of developing therapeutic preventative strategies in aged humans.


   Acknowledgments
 
This work was supported in part by NIH K01-AG00875 (D.H.K.) and an American College of Sports Medicine Foundation Grant (D.H.K.).


   Footnotes
 
Decision Editor: George E. Taffet, MD

Received March 31, 2004

Accepted July 22, 2004


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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