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Am J Physiol Heart Circ Physiol 281: H581-H589, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 2, H581-H589, August 2001

Diminished alpha 1-adrenergic-mediated contraction and translocation of PKC in senescent rat heart

D. H. Korzick1,2, D. A. Holiman1, M. O. Boluyt2, M. H. Laughlin1, and E. G. Lakatta2

1 Department of Veterinary Biomedical Sciences, University of Missouri, Columbia, Missouri 65211; and 2 Laboratory of Cardiovascular Science, The National Institute on Aging, Baltimore, Maryland 21224


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Myocardial reserve function declines with aging due in part to reduced alpha - and beta -adrenergic receptor (AR)-mediated contractile augmentation. Whereas specific age-associated deficits in beta -AR signaling have been identified, it is not known which components of the alpha 1-AR signaling cascade, e.g., protein kinase C (PKC) and associated anchoring proteins (receptors for activated C kinase; RACKs), underlie deficits in alpha 1-AR contractile function with aging. We therefore assessed cardiac contraction (dP/dt) in Langendorff perfused hearts isolated from adult (5 mo) and senescent (24 mo) Wistar rats following maximal alpha 1-AR stimulation with phenylephrine (PE), and we measured the subcellular distribution of PKCalpha and PKCepsilon , and their respective anchoring proteins RACK1 and RACK2 by Western blotting. The maximum dP/dt response to PE (10-5 M) was significantly reduced by 41% in 24-mo-old vs. 5-mo-old (P < 0.01). Inhibitory effects of PKC blockade (chelerythrine; 10 µM) on dP/dt following alpha 1-AR stimulation with PE observed in adult hearts were absent in 24-mo-old hearts (P < 0.01). In 5-mo-old hearts, PE elicited reductions in soluble PKCalpha and PKCepsilon levels, while increasing particulate PKCalpha and PKCepsilon levels to a similar extent. In contrast, soluble PKCalpha and PKCepsilon levels in 24-mo-old hearts were increased in response to PE; particulate PKCepsilon and PKCalpha were unchanged or reduced and associated with significant reductions in particulate RACK1 and RACK2. The results indicate, for the first time, that selective translocation of PKCalpha and PKCepsilon in response to alpha 1-AR stimulation is disrupted in the senescent myocardium. That age-related reductions in particulate RACK1 and RACK2 levels were also observed provide evidence that alterations in PKC-anchoring proteins may contribute to impaired PKC translocation and defective alpha 1-AR contraction in the aged rat heart.

myocardium; signal transduction; cell surface receptors; aging; receptors for activated C kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AN IMPORTANT ADVERSE CONSEQUENCE of the aging process in both healthy humans and animals is a diminished myocardial functional reserve (for review see Ref. 28). This limitation, in turn, impairs the ability of the aged myocardium to adequately augment contractile performance during periods of increasing circulatory demand, e.g., during the superimposition of an ischemic stress, performance of activities of daily living, or during physical exercise. Whereas age-associated defects in the beta -adrenergic receptor (beta -AR) signaling cascade clearly contribute to diminished inotropic responses and subsequent reductions in contractile reserve (28), the cardiac alpha 1-AR pathway has also been implicated in this process (19, 26, 35). This finding is intriguing because in disease states, such as heart failure, in which the beta -AR system is compromised, alpha 1-AR function is preserved, suggesting an important "back-up" or supporting role for the alpha 1-AR system in the maintenance of contractile function (32, 51). Unlike the beta -AR system, however, age-associated alterations in intracellular events leading to blunted alpha 1-AR contraction have not been examined.

The mechanisms underlying alpha 1-AR-induced inotropy are related, at least in part, to activation of protein kinase C (PKC) through the phospholipase C-beta (PLC-beta ) pathway. In the myocardium, alpha 1-ARs are known to couple to the Gq/G11 family of heterotrimeric G proteins (14), which when stimulated, lead to the hydrolysis of phosphatidylinositol bisphosphate via PLC-beta (11) giving rise to the second messengers inositol (1,4,5)-trisphosphate (InsP3) and diacylgylcerol (DAG). Modulation of several key intracellular processes by subsequent PKC-dependent phosphorylations is thought to play an important role in cardiac excitation contraction coupling following alpha 1-AR stimulation, and immunohistochemical studies of activated PKC in cardiac myocytes have revealed localization of specific PKC isoforms to cellular structures including myofibrils and the cytoskeleton (25).

Activation of PKC is primarily regulated by selective translocation from the cytosolic to the membrane cellular compartment in an agonist-specific manner (37). This translocation and activation are, in turn, mediated by PKC isoform-specific binding to membrane anchoring proteins (36) termed receptors for activated C kinase (RACKs). It is well-known that preferential translocation and activation of the Ca2+-dependent PKCalpha and Ca2+-independent or novel PKCepsilon occur in response to alpha 1-AR stimulation in adult rat hearts (41, 48). Therefore, these PKC isoforms with their respective RACKs (RACK1 and RACK2) may play a critical role in regulating alpha 1-AR effects on cardiac contraction. Whereas there is growing consensus with regard to developmental downregulation of PKCalpha and PKCepsilon isoform expression in the fetal, neonatal, and adult rat heart (6, 41, 48), it is unknown whether PKC levels are disrupted in the senescent heart and whether activation of these isoforms by alpha 1-AR stimulation is reduced. Interestingly, deficits in PKC signal transduction and impaired translocation of PKC in the aging rat brain appear to be mediated, at least in part, by diminutions in RACK levels (4, 39). Whether and to what extent RACK levels in the presence and absence of alpha 1-AR stimulation are affected by senescence in the myocardium is also unknown. Therefore, the purposes of this investigation were to determine whether alpha 1-AR contraction is impaired in the aging rat heart and to determine whether alterations in the subcellular distribution of PKCalpha and PKCepsilon in response to alpha 1-AR stimulation underlie a defective alpha 1-AR-mediated contraction in the senescent rat heart. We hypothesized that reductions in selective myocardial RACK anchoring proteins would occur with aging and would be correlated with reduced levels of membrane PKCalpha and PKCepsilon , thereby contributing to diminished alpha 1-AR contractile function in hearts of aged rats.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animal Preparation

Male Wistar rats (ages 5 and 24 mo) obtained from the Gerontology Research Center inbred colony were used in this study. All experimental protocols were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Department of Health and Human Services, National Institutes of Health, Publication No. 86-23) and approved by the Animal Care and Use Committees at the National Institute on Aging and the University of Missouri, respectively. Animals with overt pathological lesions such as atrial tumors were eliminated from study (n = 4).

Isolated Heart Perfusion

In vitro assessment of left ventricular (LV) function was performed utilizing a modified Langendorff isovolumic heart preparation as described previously (27). After administration of heparin (2.5 mg) and pentobarbital (35 mg/kg body wt), hearts were excised via midline thoracotomy, placed in cold saline solution, and cannulated and perfused with a Krebs-Henseleit bicarbonate buffer (95% O2-5% CO2) 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. Hearts were perfused at constant pressure (85 mmHg), temperature (37°C) and heart rate (240 beats/min); following a 5-min equilibration, a fluid-filled latex balloon attached to a transducer-tipped catheter was placed in the LV, and balloon volume was adjusted to yield a minimum pressure of 5 mmHg. Contractile hemodynamics were assessed (per beat) as follows: LV developed pressure, LV minimum pressure, LV maximal dP/dt (mmHg/s), LV negative dP/dt (-dP/dt; mmHg/s), time to 50% relaxation (1/2 relax; ms), and time to peak contraction (Dcon; ms).

Experimental Protocol to Determine alpha 1-AR Contractile Effects

After a 20-min equilibration period, hearts from 5-mo-old (n = 6) and 24-mo-old (n = 6) animals were perfused with a concentration of the alpha 1-AR agonist phenylephrine (PE; 10-5 M) known from preliminary experiments to elicit a maximal and sustained contractile response in both young and old hearts (5 min exposure). To provide insight into the role of PKC on alpha 1-AR contraction, in a separate series of experiments, hearts from 5-mo-old (n = 6) and 24-mo-old (n = 6) animals were first perfused with the specific PKC blocker, chelerythrine (CE; 10 µM) for 10 min, as described previously (42). CE inhibits the catalytic domain of PKC (23) and produces similar reductions in PKC activity in adult cardiac myocytes compared with other PKC inhibitors (10). Hearts were then perfused with PE (10-5 M) plus CE as stated above, and contractile function was assessed. All experiments were performed in the presence of the beta -AR antagonist propranolol (10-6 M) following initial equilibration to minimize PE effects through beta -ARs.

Experimental Protocol to Determine Time Course of PKC Subcellular Distribution

LV myocardium from the 5-min PE-only experimental condition was quick frozen in liquid N2. To better assess the time course of PKC isoform-specific translocation in response to alpha 1-AR stimulation, an additional group of experiments was performed on hearts from 5-mo-old (n = 6) and 24-mo-old (n = 6) animals following Langendorff perfusion with PE (10-5 M) plus propranolol (10-6 M) for 1 min; additional hearts were also perfused in the absence of any pharmacological intervention to serve as baseline controls (5 mo, n = 5; 24 mo, n = 5).

Western Immunoblotting Analysis for PKC Subcellular Distribution and RACKs

Tissue sample preparation. Preparation of protein extracts from frozen adult and senescent intact LV myocardium from baseline, 1-min, and 5-min PE conditions were prepared with minor modifications of the protocol described by Rybin and Steinburg (48). Briefly, LV tissue was homogenized in lysis buffer (20 mmol/l Tris · HCl, pH 7.5; 2 mmol/l each EDTA and EGTA, pH 7.0-8.0; 6 mmol/l beta -mercaptoethanol; 50 mg/ml each aprotinin and leupeptin; 5 mmol/l pepstatin A; 1 mmol/l phenylmethylsulfonyl fluoride; 0.1 mmol/l orthovanadate) and subjected to centrifugation at 100,000 g for 1 h (4°C). The supernatant (soluble fraction) was removed, and the pellet (particulate fraction) was resuspended in lysis buffer containing 1% sodium dodecyl sulfate (SDS). Protein content was determined according to the method of Bradford (8).

Western immunoblotting analysis for PKCs. Cytosolic and particulate protein fractions were subjected to protein immunoblotting as previously described (7). Briefly, equal amounts of sample per lane for soluble (75 µg) and particulate (50 µg) fractions were electrophoresed on 7.5% SDS-polyacrylamide gels and transferred to polyvinylidine difluoride membranes overnight (4°C). After membrane incubation with blocking solution (PBS-Tween 20 and 1% bovine serum albumen) for 2 h and appropriate washing, blots were probed with rabbit polyclonal antibodies against PKCalpha (80 kDa) PKCbeta I (78 kDa), PKCbeta II (78 kDa), PKCdelta (73 kDa), PKCepsilon (97 kDa), or PKCzeta (67 kDa) at room temperature for 3 h (1:400; Santa Cruz). Subsequent to incubation with secondary antibody (1:10,000 dilution; anti-rabbit IgG conjugated with horseradish peroxidase, Amersham) for 1 h, antibody binding was detected using the enhanced chemiluminescence method (ECL kit; Amersham). Rat brain (25 µg) was used as a positive control for each immunoblot. Western blotting was also performed in the presence and absence of competing immunizing peptide (40-fold excess; Santa Cruz Biochemicals) for each respective PKC isoform to ensure specificity of the antibody response. Densitometric analysis of Western blot films was performed using NIH Image Analysis Software, and all immunoblots were performed in duplicate for each specific PKC isoform.

Western immunoblotting analysis for RACKs. Monoclonal antibodies against RACK1 (mouse IgM; 36 kDa) were purchased from Transduction Laboratories, and immunoblots were prepared as above with the exception that blots were incubated with primary anti-RACK1 antibody for 1 h at room temp (1:750). In addition to PKCbeta , recent findings (46) suggest that RACK1 binds with a high degree of specificity to PKCalpha and thus provide experimental rationale for use in the present study. A rat monoclonal antibody specific for the COOH-terminus of mouse ICP-1alpha (StressGen), a cytosolic hetero-oligomer chaperone known to be involved in the folding of actin and tubulin (13), was utilized to assess RACK2 levels. This antibody recognizes p102beta '-COP (60 kDa), a coat protein I (COPI) coatomer complex protein that binds the V1 region of PKC-epsilon and has been identified as a PKCepsilon -selective RACK (13). Immunoblots were processed as above (1:1,000 dilution).

Statistical Analysis

All variables of interest are reported as means ± SE. Group comparisons of functional and PKC/RACK immunoreactivity data were made using a 2 × 2 ANOVA (age × drug) using the Statistical Analysis System (SAS). Higher-order terms were employed to reveal significant interaction effects with respect to varying drug treatment on myocardial contractile function during the isovolumic heart experiments across groups. Additional analyses were performed to ensure aptness of the model and normality of the data. The Tukey-Kramer method was used for all pairwise comparisons on significant interaction terms. An alpha  level of P <=  0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Baseline Contractile Function and Morphology

Selected parameters of contractile function and morphology from Langendorff perfused hearts (pooled from two sets of experiments) in the absence of pharmacological manipulation are presented in Table 1. LV weight was ~55% greater (P < 0.01) in hearts isolated from 24-mo-old animals compared with their 5-mo-old adult counterparts. Whereas LV developed pressure, diastolic pressure, LV -dP/dt, Dcon, and 1/2 relax were not significantly different between groups, a small but statistically significant reduction (~7%) in LV dP/dt was observed in the aged animals. Our results are consistent with previous findings suggesting increased LV mass and minimal differences in LV contractile function occur under baseline conditions in old versus young animals.

                              
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  		Table 1.   Selected baseline parameters of contractile function and morphology in the absence of phenylephrine and/or chelerythrine

Effect of alpha 1-AR Stimulation and PKC Blockade on Cardiac Function

Experiments were performed to determine whether 24-mo-old male Wistar rats exhibit blunted alpha 1-AR inotropic effects. Hearts from 5-mo-old (n = 6) and 24-mo-old animals (n = 6) were Langendorff perfused with a single concentration of the alpha 1-AR agonist PE (10-5 M) known to elicit a maximal contractile response (5 min exposure). In response to PE, a positive inotropic effect was observed in hearts isolated from both 5-mo-old and 24-mo-old animals (Figs. 1 and 2); however, the response was significantly greater in hearts isolated from 5-mo-old versus 24-mo-old animals (P < 0.001; Figs. 1 and 2) whether expressed as LV developed pressure (50.8% in 5-mo-old vs. 24.2% in 24-mo-old) or LV dP/dt (77.9% in 5-mo-old vs. 36.6% in 24-mo-old). Similarly, age-associated reductions occurred when LV developed pressure (144.9 ± 5.3 mmHg in 5-mo-old vs. 114.5 ± 7.2 mmHg in 24-mo-old) and dP/dt (4,560.4 ± 282.3 mmHg/s in 5-mo-old vs. 3,338.6 ± 252.9 mmHg/s in 24-mo-old) were expressed in absolute terms (P < 0.0001). Reductions in -dP/dt in response to PE were also observed and were significantly less in hearts of aged rats (-2,122.7 ± 222.5 mmHg/s vs. -1,783.5 ± 218.0 mmHg/s) compared with young adult hearts (P < 0.001). PE also elicited significant reductions in LV diastolic pressure, Dcon, and 1/2 relax; however, differences between groups were not observed (data not shown). These results are consistent with a previous study (26) suggesting that alpha 1-AR mediated contraction is diminished in the aged rat heart.


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  		Fig. 1.   Continuous chart recording of left ventricular (LV) pressure during steady-state stimulation from a representative 5-mo-old heart (top) and 24-mo-old heart (bottom) in the absence and presence of the alpha 1-adrenergic receptor (AR) agonist phenylephrine (PE; 10-5 M) + the beta 1-AR inhibitor propranolol (10-6 M). Positive inotropic effect elicited by PE was significantly greater in hearts isolated from 5-mo-old vs. 24-mo-old animals.



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  		Fig. 2.   Effect of maximal alpha 1-AR stimulation by PE (10-5 M; 5 min) on LV cardiac contraction (dP/dt) in the presence (n = 6/age group) and absence (n = 6/age group) of the protein kinase C (PKC) blocker chelerythrine (CE; 10 µM; 10 min) in 5- and 24-mo-old isolated rat hearts. All experiments were performed in the presence of 10-6 M propranolol. Independent effects of the CE or propranolol on dP/dt were not observed. Data are expressed as means ± SE; *P < 0.01.

To provide insight into the role of PKC on the diminished alpha 1-AR contractile response, a separate set of experiments was conducted. Hearts from 5-mo-old (n = 6) and 24-mo-old (n = 6) animals were first perfused with the PKC blocker CE (10 µM) followed by perfusion with PE (10-5 M) plus CE. Significant effects of CE on baseline contraction were not observed in young or old hearts (data not shown). Figure 2 shows that the increase in alpha 1-AR-mediated dP/dt induced by PE was significantly diminished by ~40% in hearts isolated from young animals; however, effects of CE on alpha 1-AR-mediated LV dP/dt in aged hearts averaged ~7% (age × drug interaction, P < 0.01; Fig. 2). These findings suggest that while the PKC arm of the alpha 1-AR signal transduction pathway is operative in generating the alpha 1-AR contractile response in hearts isolated from young rats, PKC plays a less significant role in alpha 1-AR-mediated inotropic effects in senescent animals, and defective PKC signaling may underlie diminished alpha 1-AR contraction in aged rat hearts.

Effect of alpha 1-AR Stimulation on Subcellular Distribution of PKCalpha and PKCepsilon

We then determined whether age-associated alterations occurred in the subcellular distribution of selective PKC isoforms known to undergo translocation and activation by alpha 1-AR stimulation. Subcellular distributions of PKCalpha and PKCepsilon immunoreactivity in 5- and 24-mo-old rat hearts in the presence and absence (CTL; n = 5/group) of the alpha 1-AR agonist PE following 1-min (PE-1; n = 6/group) and 5-min (PE-5; n = 6/group) exposures are presented in Figs. 3 and 4. In the soluble fraction, PKCalpha immunoreactivity at baseline was not significantly different between 5-mo-old and 24-mo-old hearts. In 5-mo-old adult hearts, stimulation by PE was associated with an approximately twofold reduction in soluble PKCalpha levels (P < 0.01; Fig. 3) while concomitantly increasing particulate PKCalpha levels by a similar magnitude, consistent with previous observations (41). In contrast, soluble PKCalpha levels increased in response to PE in aged hearts (P < 0.01; Fig. 3). Whereas particulate-associated PKCalpha was significantly greater in 24- versus 5-mo-old hearts under baseline conditions, an age-related reduction (~40%) in particulate PKCalpha in response to PE was observed (P < 0.01; Fig. 3).


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  		Fig. 3.   Subcellular distribution of PKCalpha immunoreactivity in 5- and 24-mo-old rat hearts in the presence and absence (CTL; n = 5/group) of the alpha 1-AR agonist PE (10-5 M) after 1-min (PE-1; n = 6/group) and 5-min (PE-5; n = 6/group) exposures (bottom). Representative immunoblots from soluble and particulate fractions are presented for PKCalpha at PE-5 (top). Equal amounts of protein per gel were loaded for the soluble (75 µg) and particulate (50 µg) fractions, respectively. Data are expressed as means ± SE.



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  		Fig. 4.   Subcellular distribution of PKCepsilon immunoreactivity in 5- and 24-mo-old rat hearts in the presence and absence (CTL; n = 5/group) of the alpha 1-AR agonist PE (10-5 M) after PE-1 (n = 6/group) and PE-5 (n = 6/group) exposures (bottom). Representative immunoblots from soluble and particulate fractions are presented for PKCepsilon at PE-5 (top). Equal amounts of protein per gel were loaded for the soluble (75 µg) and particulate (50 µg) fractions, respectively. Data are expressed as means ± SE.

A similar pattern of response was observed for PKCepsilon levels in 5- and 24-mo-old hearts in response to alpha 1-AR stimulation (Fig. 4). Despite a lower level of soluble PKCepsilon under baseline conditions in 24-mo-old versus 5-mo-old hearts (P < 0.01), PE stimulation resulted in significant increases in soluble PKCepsilon in 24-mo-old hearts, and reductions in soluble PKCepsilon levels in 5-mo-old hearts. PE increased particulate PKCepsilon levels in young hearts but did not alter particulate PKCepsilon immunoreactivity in hearts isolated from aged animals. Age-related differences in PKCbeta I, PKCbeta II, PKCdelta , or PKCzeta in response to PE were not observed (data not shown). Taken together, the apparent reductions in particulate-associated PKCalpha and PKCepsilon in response to alpha 1-AR stimulation in hearts isolated from senescent animals and the lack of an apparent effect of CE on the contractile response to PE provide support for the hypothesis that alterations in PKC translocation following alpha 1-AR stimulation contribute to the diminished responses to PE in the aged heart.

Effect of alpha 1-AR Stimulation on RACK1 and RACK2 Levels

Activation of membrane-bound PKC is thought to occur by specific protein-to-protein interactions via a class of proteins known as RACKs. To investigate the interaction between PKCalpha and PKCepsilon and their specific RACKs (RACK1 and RACK2) particulate fractions from young and old rat hearts -/+ PE were subjected to Western blotting. Under baseline conditions, differences in RACK1 and RACK2 levels were not observed in young versus old rat hearts (Fig. 5). After stimulation with PE, RACK levels increased in particulate homogenates isolated from hearts of both age groups, but the increase was ~45% less for RACK1 and ~60% less for RACK2 in 24-mo-old versus 5-mo-old hearts. These data suggest that a lower level of particulate bound RACKs (following PE stimulation) may contribute to impaired PKC translocation in the senescent rat heart.


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  		Fig. 5.   Particulate-associated receptors for activated C kinase (RACK)1 and RACK2 immunoreactivity (bottom) in LV homogenates isolated from 5- and 24-mo-old rat hearts before (CTL; n = 5/age group) and after stimulation with alpha 1-AR agonist PE (10-5 M, 5 min; n = 6/age group). Representative immunoblots are presented (top). Equal amounts of protein were loaded per lane (50 µg). Data are expressed as means ± SE; *P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

An important compensatory role for the alpha 1-AR system in the regulation of cardiac contractile function in a variety of cardiac disease states has recently been proposed (5, 32). In a setting such as chronic heart failure when cardiac beta 1-AR responsiveness is impaired (50), alpha 1-AR regulation is preserved and/or enhanced, which suggests a potentially important therapeutic role for the alpha 1-AR in the maintenance of optimal levels of cardiac performance in the face of decreasing beta 1-AR inotropic support (12, 50). A principal finding of the present study is that the inotropic responsiveness of the senescent heart to alpha 1-AR stimulation is blunted, thereby diminishing the functional reserve capacity of the aged rat myocardium. Regulation of contractile function by alpha 1-AR stimulation is thought to involve modulation of several key intracellular processes by a series of PKC-dependent protein phosphorylations (16-18). In this regard, localization of activated PKCepsilon to myofibrillar and cytoskeletal structures in cardiac myocytes by immunocytochemistry (25) provides a physical link between PKC and potential phosphorylation targets. Aberrant signaling via one or all of these PKC-mediated effects may account, at least in part, for the decline in alpha 1-AR-mediated contraction observed in the senescent rat heart. Accordingly, our results also indicate that translocation of both PKCalpha and PKCepsilon in response to alpha 1-AR stimulation is impaired in the senescent rat heart and suggests that this impairment may be mediated, in part, by reductions in RACK anchoring proteins. To our knowledge, these are the first studies to characterize alterations in PKC signal transduction in senescent myocardium.

The magnitude of the age-associated reduction in alpha 1-AR mediated contractile response in the present study was ~50%, consistent with a previous study that indicated similar deficits in alpha 1-AR-mediated contraction in aged right ventricle (26). alpha 1-AR-mediated activation of PLCbeta via the Gq/G11 family of heterotrimeric G proteins results in production of the second messengers InsP3 and DAG (22, 52). Whereas the importance of InsP3 on cardiac excitation-contraction coupling has been debated, regulation of contraction by alpha 1-AR stimulation via DAG-mediated increases in PKC is well established (9, 15, 24, 29). A major goal of this study was to determine whether PKC and associated anchoring proteins play a role in the alpha 1-AR contractile response in senescent myocardium. The fact that alpha 1-AR-mediated contraction was markedly reduced following PKC inhibition with chelerythrine in the adult rat heart but without effect in the senescent heart suggests that the PKC arm of the alpha 1-AR signal transduction pathway apparently plays a less significant role in alpha 1-AR mediated-contraction in the senescent heart. Thus present results provide support for the hypothesis that aberrations in PKC signaling are associated with and responsible for defective alpha 1-AR contraction in the aged myocardium. These results also imply that the InsP3-related component of the alpha 1-AR contractile response may exert important effects on cardiac contraction. It is noteworthy that steady-state levels of mRNA encoding cardiac InsP3 receptors are similar in young versus old rat ventricle (20) and increased in chronic human heart failure and ischemia (30), supporting a potential compensatory role of the InsP3 receptor in different cardiac pathologies. However, the role of InsP3 or its receptor on cardiac contraction has not been critically examined.

Current experimental evidence from several laboratories suggests that multiple PKC isoforms exist in the rat heart and may undergo developmental downregulation (6, 41, 42, 47, 48). Reductions in PKCalpha , PKdelta , PKCepsilon , and PKCzeta have been reported during the neonatal and adult growth period; however, the present study is the first to examine PKC isoform expression and distribution with adult aging into senescence. In response to alpha 1-AR stimulation with PE, as expected from prior studies, we observed a significant decrease in soluble PKCalpha and PKCepsilon levels in adult myocardium compared with control conditions, i.e., in the absence of pharmacological manipulation. Reductions in particulate PKC were associated with approximately one- to twofold increases in particulate PKCalpha and PKCepsilon , consistent with PKC translocation and activation in response to alpha 1-AR stimulation. In contrast, PE increased soluble levels of PKC in senescent myocardium compared with hearts isolated from adult animals, whereas particulate PKCalpha and PKCepsilon were apparently reduced or unchanged from their baseline levels, respectively. These results are qualitatively similar to those in aged rat brain cortex (3, 39), in which an age-associated impairment in membrane translocation of PKCdelta and PKCepsilon , as well as PKCbeta , in response to phorbol ester stimulation was observed.

Association of PKC with the membrane cellular compartment is a necessary step in PKC activation due to cofactor requirements, including phosphatidylserine, DAG, and in the case of Ca2+-dependent PKCs, Ca2+ (37). It is also apparent that stimulus-specific translocation and intracellular localization of individual PKCs are dependent, in part, on the interaction of PKCs with specific membrane anchoring proteins, i.e., RACKs, which appear requisite for the proper functioning of individual PKC isoforms (36). In this regard, Mochly-Rosen and colleagues (45) were able to effectively abolish phorbol ester-mediated inhibition of L-type Ca2+ current, as well as isoproterenol-induced PKC translocation and regulation of contraction rate in cardiac myocytes (25) utilizing specific peptides corresponding to RACK binding sites on PKC.

A novel finding of the present study is that reductions in particulate PKCalpha and PKCepsilon were associated with concomitant reductions in their respective RACKs, RACK1 and RACK2. We observed ~45% and ~60% reductions in RACK1 and RACK2 immunoreactivity, respectively, in response to alpha 1-AR stimulation in hearts isolated from aged rats compared with adult controls. These reductions occurred in the face of increased levels of soluble PKCalpha and PKCepsilon , suggesting that the inability of individual PKC isoforms to effectively bind their selective RACKs, thereby preventing translocation and subsequent activation, may underlie age-related reductions in alpha 1-AR-mediated contraction. Indeed, deficits in RACK1 in the aging rat brain are apparently responsible for impaired translocation of PKCs known to interact with RACK1 (PKCbeta , PKCdelta , and PKCepsilon ) in response to phorbol ester stimulation (4, 39). Furthermore, our data support recent studies from Ron et al. (44) and Rieger et al. (43), suggesting that PKC activation can induce the recruitment of RACKs to different cellular locations. Specifically, these authors provide compelling evidence that RACKs may serve an important "shuttling" role allowing for PKC movement from one intracellular site to another (44). If this is indeed the case, data from the current study suggest that aging may interfere with this process. Whereas it is also possible that age-associated changes in membrane phospholipid content may contribute to reductions in membrane-associated PKC and RACK immunoreactivity, alterations in phosphatidylserine, a cofactor required for PKC activation, appear minimal in sarcolemmal membranes isolated from aged rat hearts (2).

At this time, it is unclear whether additional components of the alpha 1-AR signaling cascade upstream of PKC are adversely affected by adult aging to senescence. For instance, results from radioligand binding studies performed on senescent rodent myocardium, including those of Kimball et al. (26), have identified significant reductions in alpha 1-AR maximal receptor binding of ~30% while reporting no changes in the dissociation constant (19, 34, 38). However, reductions in receptor number, as well as alpha 1-AR-Gq interactions, are not universal findings in the aged rat ventricle (1, 31, 49) and therefore unlikely in providing a sole explanation for the findings in the present investigation. Thus whereas multiple defects in the alpha 1-AR signal transduction pathway may converge to negatively impact cardiac contractile response in the aged heart, our results implicate PKCalpha , PKCepsilon , and their respective RACKs as primary targets in the genesis of diminished contractile reserve with senescence.

Recent evidence highlighting the obligate role of PKC, particularly PKCepsilon , in conferring cardioprotection in both early and late ischemic preconditioning (21, 33, 40, 42), suggests that stimulus-specific alterations in PKC isoforms may contribute to contractile decompensation in a variety of cardiopathologies. The results of the present investigation extend these previous findings to alpha 1-AR desensitization in the senescent heart and suggest that both PKCalpha and PKCepsilon are important mediators of the alpha 1-AR contractile response.

Limitations of the Study

Measures of total PKC activity were not examined in this study. However, the results of Ping and colleagues (40) suggest that selective increases and/or decreases in the activity of one or more PKC isoforms could obscure changes in the activity of other PKC isoforms due to the limited sensitivity of substrates used in commercially available PKC activity assays (i.e., PKC substrate peptides are PKC isoform nonspecific). Thus by presently available methods it is not possible to relate measurements of total PKC activity to specific isoforms. Another limitation is that PKC/RACKs were determined in the LV myocardium. Future studies will be needed to determine whether directional changes in PKCalpha , PKCepsilon , and RACK expression observed in LV homogenates are quantitatively similar in isolated cardiac myocyte preparations. Dilution effects secondary to myocardial hypertrophy and/or apoptosis in the senescent myocardium may obscure changes in PKC/RACKs that would otherwise be detected on a per myocyte basis. Finally, results from the current study provide little insight into the cellular location and phosphorylation targets of PKC/RACKs following alpha 1-AR stimulation in young versus aged myocardium. Future studies will be needed to address this important issue.

In summary, we provide evidence that the alpha 1-AR pathway is desensitized with senescence in the rat heart. Taken together, our results show that alterations in alpha 1-AR-mediated particulate PKC/RACK immunoreactivity occur in the senescent rat heart relative to adult animals and may contribute to age-associated reductions in alpha 1-AR contractile responses. That PKC isoform expression, including PKCalpha and PKCepsilon , is similarly reduced in experimental models of heart failure, but in the case of PKCepsilon , selectively activated in various models of cardioprotection, provides impetus and rationale for future studies to identify downstream cellular targets of PKCepsilon and PKCalpha , to develop therapeutics designed to preserve the inotropic reserve capacity of the aged myocardium.


    ACKNOWLEDGEMENTS

The authors thank Dr. Daria Mochly-Rosen for insightful discussions on PKC/RACK methodologies and data interpretation. We also extend thanks to Howard Wilson and Don Connor for assistance with graphic presentation of the data.


    FOOTNOTES

This work was supported by the National Institute on Aging Intramural Research Program (to D. H. Korzick, M. O. Boluyt, and E. G. Lakatta); National Heart, Lung, and Blood Institute Grant PO1-HL-52490 (to D. A. Holiman and M. H. Laughlin), National Institutes of Health Grant T32-HD-07460 (to D. H. Korzick), and National Institute on Aging Grant KO1-AG-00875 (to D. H. Korzick).

Address for reprint requests and other correspondence: E. G. Lakatta, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute On Aging, 4940 Eastern Ave., Box 13, Baltimore, MD 21224 (E-mail: LakattaE{at}grc.nia.nih.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 12 November 1999; accepted in final form 23 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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