Age- and sex-dependent alterations in protein kinase C (PKC) and extracellular regulated kinase 1/2 (ERK1/2) in rat myocardium

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Abstract

Cardiovascular morbidity and mortality increase significantly with advancing age, with proportionally higher rates occurring in aged women when compared to aged men. The signaling alterations responsible for age-related reductions in ischemic stress reserves, particularly in aged women, are poorly understood. Accordingly, we sought to determine whether alterations in the cellular location and formation of specific protein kinase C (PKC)-extracellular regulated1/2 (ERK1/2) signaling modules (SMS) might provide insight into known age- and sex-related differences in cardiovascular disease outcomes. Cytosolic (Cyto), mitochondrial (Mito) and nuclear (Nuc) fractions were isolated from left ventricles of male (M) and female (F) adult (6mo), castrated or aged (23mo) F344 rats by centrifugation. Western blotting was used to assess PKC (α, δ, ɛ), p-ERK1/2 and p-Bad(Ser112) levels, and immunoprecipitation to assess PKC-ERK1/2 SMS. Cyto-PKCα levels increased with age (p < 0.0001), whereas increases in cyto-PKCα-ERK1/2 SMS were only observed in aged F (60%; p < 0.01). Mito-PKCδ and Mito-PKCδ-ERK1/2 SMS increased in M and F with age (p < 0.0001); however increases in Cyto-PKCδ were only observed in aged M (80% p < 0.0001). It is important to note that Nuc- and Mito-PKCδ-ERK1/2 SMS were 3.5- and 4.8-fold greater in males versus females, respectively (p < 0001). Increases in Mito-PKCɛ-ERK1/2 SMS (216%) were also specific to aged M (p < 0.0001), however, Mito-p-Bad(Ser112) levels were decreased with age in both M and F. Differences in sex hormone status could not fully account for observed age-related differences in PKC. Collectively, our results provide novel evidence for age and sex-related differences in the magnitude and distribution of cardiac PKC-ERK1/2 SMS consistent with previously described pathological and protective phenotypes, respectively.

Introduction

Salient features of the aged heart include cardiac myocyte hypertrophy, apoptosis and reductions in intrinsic- and adrenergic-mediated contractile function (for review see (Lakatta and Levy, 2003, Lakatta and Sollott, 2002)). Cardiovascular disease affects nearly 3/4 of the population over age 75, and age-related reductions in ischemic tolerance have been repeatedly demonstrated in both humans and animals (Abete et al., 1999, AHA, 2003, Lesnefsky et al., 1994). Epidemiological data also indicate that aged females are at higher risk for cardiovascular morbidity and mortality when compared to aged men (AHA, 2003, Leinwand, 2003). These findings are intriguing as they suggest a sexual dimorphism in cardiovascular disease outcomes with advancing age, which may or may not be estrogen-dependent. The intracellular signaling pathway alterations which distinguish cardiovascular stress responses in adult from aged hearts, particularly in women, remain poorly understood.

One potential mechanism by which differences in stress responses may occur with advancing age includes aberrant regulation of cell signaling pathways critical to cell survival and/or cell death. Activation of protein kinase C (PKC)-dependent mechanisms represents one such candidate pathway. For instance, constitutive activation and/or enhanced membrane partitioning of PKCɛ in the basal state is sufficient to protect against damage from subsequent ischemia/reperfusion injury (Dorn et al., 1999, Gregory et al., 2004, Pass et al., 2001). Recent studies have also highlighted the formation of mitochondrial PKCɛ-extracellular-regulated kinase 1/2 (ERK1/2) signaling modules and subsequent inactivation of the pro-apoptotic protein Bad (Baines et al., 2002, Sugden, 2003) as a partial basis for PKCɛ-mediated protection. The idea that specific PKC isoforms can form signaling modules with ERK1/2 provides for a cellular strategy to achieve distinct cellular responses through differential subcellular targeting. As such, formation of PKC-ERK1/2 signaling modules at specific cellular loci may represent a potentially important regulatory mechanism in response to a variety of imposed stresses, including those associated with cardiac aging.

It is currently unknown whether the balance and cellular distribution of specific PKC-isoforms and PKC-ERK1/2 signaling modules, including those formed with PKCɛ, are altered by advancing age. Furthermore, whether cardiac PKC responses differ according to male or female sex is also unknown. In this regard, estrogen has been shown to activate myocardial ERK1/2 (Geraldes et al., 2003, Nuedling et al., 1999), the effects of which may impact subsequent cardiac PKC-ERK1/2 signaling module formation to a greater extent in females versus males. That ERK1/2 activation is blunted with advanced age (Aoyagi and Izumo, 2001) may also impact PKC-ERK1/2 signaling module formation in a sex-specific manner. The purpose of the present study was to systematically evaluate age-related changes in specific PKC-ERK1/2 signaling module distribution and to determine whether these alterations were sex-dependent.

Section snippets

Methods

Animal preparation: Adult (6mo) and aged (22mo) male (n = 6 adult, n = 5 aged) and female (n = 6/age) Fischer-344 rats were obtained from Harlan Sprague–Dawley (Indianapolis, IN). Rats were exposed to a 12 h light/dark cycle and received food and water ad libitum. All animal handling and utilization was in accordance with the Penn State University Animal Care and the Guide for the Care and Use of Laboratory Animals. Animals were anesthetized with pentobarbital (40 mg/kg body weight (BW) ip), and hearts

Rat heart characteristics

Table 1 shows the body weights and wet ventricular weights for all experimental groups. Consistent with previous studies, LV weight was significantly increased with advanced age in male and female rats (p < 0.05), however the effect was lost once normalized to body weight. Normalized LV weight/BW ratios were significantly greater in female versus male rats (p < 0.05). Sex-related differences in BW persisted across the lifespan, and contributed to greater absolute values for LV/BW in aged female

Discussion

In the aged myocardium, reductions in cellular reserves contribute to the diminished ability of the aged heart to respond and adapt to mechanical and oxidative stresses, rendering the aged heart more vulnerable to ischemic insult (Abete et al., 1999, AHA, 2003, Lesnefsky et al., 1994). We sought to determine whether the cellular location and formation of specific PKC-ERK signaling modules were subject to age- and sex-specific regulation, and as such provide important clues to understanding the

Acknowledgements

The authors thank Kevin Krick, M.S. for excellent technical assistance with Western blotting studies and data analysis. This project was supported by a Penn State College of Health and Human Development Seed Grant (DHK), NIH KO1 AG00875 (DHK), and NIH T32 GM008619 (JCH).

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