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Keywords:

  • PKC δ;
  • SC-35;
  • hypoxia;
  • aging;
  • myocardial tissue

Abstract

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Nuclear speckles, which are sites of pre-mRNA splicing and/or assembly components, are diffusely distributed throughout the nucleoplasm. They are composed of splicing factors (SFs), including SC-35, which are nuclear proteins that remove introns (noncoding sequences in the genes) from precursor mRNA molecules, to form mature RNA, which will be transported to the cytoplasm, site of protein synthesis and activation. In light of such evidences, here we report that hypoxia modulates in vivo SC-35 SF phosphorylation via protein kinase C (PKC) δ in young rat heart. Trichrome Mallory staining and TUNEL analysis along with immunohistochemistry and Western blotting have been performed on left ventricles excised from young and old rats exposed to intermittent hypoxia. Although young hypoxic myocardial cells appear smaller than normoxic cells, connective and endothelial components increase, SC-35 phosphorylation is particularly evident in the endothelium and paralleled by an increased expression of vascular endothelial growth factor (VEGF). In addition, SC-35 and PKC δ coimmunoprecipitation occurs, suggesting that SC-35 phosphorylation could be PKC δ-mediated and that hypoxic young heart needs to counteract the damage through a process of neoangiogenesis involving such SF. Even though the levels of SC-35 and PKC δ are high, the similar response disclosed by normoxic and hypoxic old rat hearts (both showing a fibrotic organization, similar endothelial components and VEGF levels) could be due to the existence of an impaired oxygen sensing mechanism and thus to a low rate of angiogenesis. Anat Rec, 292:1135–1142, 2009. © 2009 Wiley-Liss, Inc.

The cell nucleus contains several domains with specialized functions that have been reported as subnuclear organelles, among which nucleoplasm, nuclear lamina filaments, nucleoli, Cajal bodies, and speckles are included, which nuclear matrix has been considered as being the substratum of all of them (Wilson,2005). Nuclear speckles are formed by 25–50 nm particles (Handwerger and Gall,2006) and are sites of pre-mRNA splicing and/or assembly components diffusely distributed throughout the nucleoplasm. Many of the larger speckles correspond to interchromatin granule clusters (IGCs). Speckles are dynamic structures, composed of splicing factors (SFs), responding specifically to activation of nearby genes (Misteli et al.,1997,2001; Spector,2001) and phosphorylation and dephosphorylation modulate their organization (Eils et al.,2000; Misteli,2000; Pederson,2000; Phair and Misteli,2000; Carrero et al.,2006). SFs are nuclear proteins that remove introns (noncoding sequences in the genes) from precursor mRNA molecules, to form mature RNA, which will be transported to the cytoplasm. Thus, the traffic of RNA and ribonuclear proteins from the nucleus to the cytoplasm plays a crucial role in the control of cell processes, such as proliferation, differentiation, senescence, neoplastic transformation, and DNA damage response (Misteli,2001; Zimber et al.,2004).

SC-35 is a SF associated with multiple active genes (70%–100%) (Moen et al.,2004), whereas inactive genes show only low (10%–20%) apparent association with SC-35, because they are frequently located at the nuclear periphery, in the perichromatin area, which is a region devoid of SC-35 domains and rich in heterochromatin (Smith et al.,1999). Moreover, it is now well accepted that “cycles” of phosphorylation and dephosphorylation control the neat assembly and functioning of major nuclear macromolecular complexes. These reactions control DNA transcription and pre-mRNA splicing, regulate mitosis-interphase, and M-G1 and G1-S cell cycle transitions (Bollen and Beullens,2002).

In addition, expression and activation of these subnuclear structure proteins seem to undergo modifications during development and aging in different experimental models (Dell'Orco and Whittle,1994; Gruenbaum et al.,2005), suggesting their involvement in the functional changes that characterize these phases of life. Indeed, molecules crucial to nuclear architecture and assembly are required to achieve a normal lifespan, and compromised nuclear architecture could be a central cause of aging in non-neuronal tissue (Wilson,2005). Moreover, members of the nuclear inositol lipid signaling system, to which protein kinases C (PKCs) belong, are located and/or translocated to the subnuclear structures (i.e., nuclear lamina, nuclear speckles, nuclear matrix) (Tabellini et al.,2002; Martelli et al.,2003,2006), inducing the phosphorylation of specific inner proteins. This phenomenon determines cardiovascular modifications occurring upon hypertrophic, ischemic, or atherosclerotic events (Arnaud et al.,2004; Vijayan et al.,2004; Dorn and Force,2005; Nordlie et al.,2005; Salamanca and Khalil,2005), and subcellular distribution of PKC isozymes in myocardial tissue is strongly related to development, aging (Centurione et al.,2003; Hunter and Korzick,2005), and hypoxic challenge response (Cataldi et al.,2004; Ikeda,2005). Thus, the aims of this work were to investigate whether hypoxic injury and aging could influence the phosphorylation of SC-35 and to assess the possible interactions occurring between PKC δ and SC-35, to determine possible links between physiologically important stimuli and pre-mRNA splicing machinery in vivo in rat myocardial tissue.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Animals

Two groups, each composed of 10 male Wistar rats, 3 (250–300 g) and 24 (400–450 g) months old, were used according to the guidelines of Helsinki Declaration. Only animals free of acute and chronic illness were used. Five animals from each group were kept under physiological conditions (21% O2); five young and five old were exposed to intermittent hypoxic challenge (12 hr 10% O2 followed by 12 hr 21% O2) for 8 days in a large plexiglass chamber (80 cm × 40 cm × 65 cm). Chamber air was recirculated with a pump, CO2 was removed from the chamber air with baralyme, and was continuously monitored with a capnograph. During all the hypoxic exposure, the CO2 remained in physiological ranges under 0.01%. Boric acid was mixed with the litter to minimize emission of urinary ammonia. The temperature was maintained at 25°C. Rats were euthanized with pentobarbital sodium salt (Nembutal, 40 mg/kg) (Sigma-Aldrich, St. Louis, MI) and left ventricles were excised from each rat and processed for experiments.

Light Microscopy and Immunohistochemistry

TUNEL (terminal-deoxinucleotidyl-transferase-mediated dUTP nick end-labeling) analysis, which allows to identify DNA strand breaks, yielded during apoptosis, was performed according to the manufacturer's explanations (Boheringer Mannheim, Germany) (Cataldi et al.,2004).

Heart samples were fixed in 10% (vol/vol) phosphate-buffered formalin and then paraffin embedded. The samples were then dewaxed (xylene and alcohol progressively lower concentrations) and processed for Trichrome Mallory staining (Tricromica kit) (Bio Optica, Milano, Italy), as suggested by the data sheet, to distinguish connective and endothelial compartment from myocardial cells.

To detect phosphorylated SC-35, PKC δ, and vascular endothelial growth factor (VEGF) proteins, slides were first blocked in 5% normal goat serum (NGS). Immunohistochemical analysis was performed with an immunoperoxidase two-step staining Autoprobe II kit (Biomeda, CA). Slides were incubated in the presence of mouse phosphorylated SF SC-35 monoclonal antibody (Sigma, St. Louis, MO), mouse PKC δ monoclonal antibody, and rabbit VEGF polyclonal antibody (Santa Cruz Biotechnology, CA). Sections were incubated in the presence of HRP-conjugated secondary antibodies. Peroxidase was developed using diaminobenzidin chromogen (DAB) (Biomeda, CA) and nuclei were hematoxylin counterstained. Negative controls were performed by omitting the primary antibody.

Samples were then observed with a light microscope (Leica) equipped with a Coolsnap video camera for computerized images (RS Photometrics, Tucson, AZ).

Computerized Morphometry Measurements and Image Analysis

After digitizing the images deriving from Trichrome Mallory-stained sections, MetaMorph Software System (Universal Imaging Corporation, Molecular Device Corporation, PA) (Crysel Instruments, Rome, Italy) overlay tools were used to measure interstitial area and myocardial fiber diameters or to evaluate SC-35, PKC δ, and VEGF expression.

Morphometric computerized analysis of interstitial area and myocardial diameters was performed after calibrating the program for the magnification used (40×).

Image analysis of protein expression was performed through the quantification of thresholded area for immunohistochemical brown colors per field of light microscope observation.

MetaMorph assessments were logged to Microsoft Excel and processed for standard deviation and histograms.

Protein Analysis

For immunoprecipitation, whole cell lysate (500 μg) was incubated in the presence of 50 μL of the suspended IP matrix (Exacta CRUZ, Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at 4°C. Matrix was pelleted for 30 min at 4°C and 50 μL of suspended IP matrix, 3 μg of mouse phosphorylated SF SC-35 monoclonal antibody, and 500 μL of PBS were added to the supernatant and incubated at 4°C on a rotator for 1 hr. Matrix was then pelleted and washed twice with 500 μL of PBS. SC-35 antibody-IP matrix complex was incubated with the lysate at 4°C on a rotator overnight. Matrix containing the immunoprecipitated sample was then pelleted and washed three times with RIPA buffer. Samples were boiled and stored at −80°C.

Total cell lysates (20 μg) or immunoprecipitates were electrophoresed and transferred to nitrocellulose membrane. Nitrocellulose membranes, blocked in 5% nonfat milk, 10 mmol/L Tris pH 7.5, 100 mmol/L NaCl, 0.1% Tween-20, were probed with mouse phosphorylated SC-35, PKC δ monoclonal antibodies, goat phosphorylated PKC δ (Ser-643) polyclonal antibody (Santa Cruz, Santa Cruz Biotechnology, CA), and then incubated in the presence of specific enzyme-conjugated IgG horseradish peroxidase. Samples were normalized by incubation in the presence of mouse β tubulin monoclonal antibody. Immunoreactive bands were detected by ECL detection system (Amersham, Buckinghamshire, UK) and analyzed by densitometry.

Densitometric Analysis

Densitometric values of Western blotting, expressed as integrated optical intensity, were estimated by a CHEMIDOC XRS System using QuantiOne 1D analysis software (BIORAD).

Statistical analysis was performed using the analysis of variance (ANOVA). Probability of null hypothesis of 5% (P <0.05) was considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

After hypoxia exposure, rat weights undergo a physiological decline. In fact young animal weight, which ranges between 250 and 300 g, lowers to 200 and 250 g, after hypoxia exposure, whereas old animal weight drops from 400–450 g to 320–360 g. This effect is due to lower intake of food and to a loss of muscular proteins, which leads to sarcopenia. Similarly, the heart weight of the young ranges between 700 and 850 mg, whereas the old one ranges between 1.1 and 1.4 g.

To check the effect of hypoxia at tissue level, Trichrome Mallory staining, which detects connective and endothelial compartments from myocardial cells, has been performed. As evidenced in Fig. 1 and in Table 1, myocardial cells become smaller in hypoxic young heart, when compared with normoxic one, while the connective compartment increases as well as the endothelial component (Table 2). The old heart discloses an increased connective compartment, rich in collagen fibers along with cell enlargement, already in normoxic conditions, when compared with the young heart, not significantly affected by hypoxia exposure. Obviously, these morphological modifications are associated with the functional state of the cells. In fact, as previously described by our group (Cataldi et al.,2004), the hypoxic young heart seems to be the most stressed in our experimental protocol, because it discloses the most apoptosis, when compared with the normoxic young one and with the old heart, which in normoxic conditions, shows a low rate of apoptosis, increasing after hypoxia exposure (Table 3). Low oxygen tension, moreover, determines also an increased expression of VEGF (belonging to a family of proteins that are central to angiogenesis and lymphangiogenesis) (McColl et al.,2004) in the young heart, when compared with the normoxic one, whereas no significant difference occurs between the two old. (Fig. 2). In parallel, SC-35 monoclonal antibody, which recognizes a phosphoepitope on the non-SnRNP protein (a small nuclear ribonucleoprotein component factor), marks consistently the endothelial compartment in the hypoxic young heart, when compared with the normoxic one, whereas the old shows the same distribution in the two experimental conditions, although SC-35 expression does not significantly modify (Fig. 3). To elucidate the signaling pathway mediating the effect of hypoxia and aging on SC-35 phosphorylation, a Western blotting analysis of PKC δ has been performed. An increased PKC δ phosphorylation, which means protein activation, in the hypoxic young heart is shown, when compared with the normoxic one, although no important difference in protein expression is evidenced. On the other hand, in the old, no statistically significant difference occurs in the two experimental conditions, even though both expression and phosphorylation levels are high (Fig. 4). By immunohistochemistry, PKC δ is largely showed in the endothelial compartment of hypoxic young and old hearts, when compared with normoxic ones (Fig. 5). As already evidenced by immunohistochemistry, Western blotting analysis of SC-35 expression reveals no significant difference in the various experimental conditions (Fig. 6). Because the interaction between PKC δ and SC-35 suggests a possible function for these structures in the transcription and processing of pre-mRNAs, SC-35 has been immunoprecipitated and probed against mouse PKC δ monoclonal antibody. Coimmunoprecipitation of PKC δ and SC-35 is evidenced in the young heart after hypoxic stress, and to a lesser extent in the normoxic old one. No immune complex is revealed in the normoxic young heart and in the hypoxic old heart (Fig. 6). PKC δ-SC-35 coimmunoprecipitation and increased expression of VEGF in the hypoxic young heart could suggest the need for the hypoxic young to counteract the damage occurring during hypoxia exposure through a process of neoangiogenesis.

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Figure 1. Trichrome Mallory staining of rat myocardial tissue. Connective compartment is indicated by purple stain (arrows), endothelial cells by light blue stain (arrowheads). A: normoxic young heart; B: hypoxic young heart; C: normoxic old heart; D: hypoxic old heart.

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Figure 2. A: Immunohistochemical analysis of VEGF expression. Arrow indicates endothelial structure. (a) normoxic young heart, (b) hypoxic young heart, (c) normoxic old heart, and (d) hypoxic old heart. B: Densitometric analysis of VEGF positive area, expressed as % ± SD, assessed by direct visual counting of three fields for each of five slides per each of five samples at ×40 magnification by MetaMorph Software System. C: Western blotting analysis of VEGF expression. Samples (20 μg) have been normalized to levels of β-tubulin expression. A representative of three separate experiments is shown. Densitometric analysis is defined by I.O.I. (integrated optical intensity). Results are the mean of five different samples ± SD *VEGF hypoxic young heart versus VEGF normoxic young heart: P < 0.05.

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Figure 3. A: Immunohistochemical analysis of SC-35 expression. Endothelial structure is indicated by arrows. (a) normoxic young heart, (b) hypoxic young heart, (c) normoxic old heart, and (d) hypoxic old heart. B: Densitometric analysis of SC-35 positive area, expressed as % ± SD, assessed by direct visual counting of three fields for each of five slides per each of five samples at ×40 magnification by MetaMorph Software System. Inset indicates nuclear localization of SC-35.

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Figure 4. A: Western blotting analysis of PKC δ and p-PKC δ expression. Samples (20 μg) have been normalized to levels of β tubulin expression. A representative of three separate experiments is shown. ny, normoxic young heart; hy, hypoxic young heart; no, normoxic old heart; ho, hypoxic old heart. B: Densitometric analysis of PKC δ and p-PKC δ expression, defined by I.O.I. (integrated optical intensity). Results are the mean of five different samples (±SD). *p-PKC δ/PKC δ hypoxic young heart versus p-PKC δ/PKC δ normoxic young heart: P < 0.05.

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Figure 5. A: Immunohistochemical analysis of PKC δ expression. Magnification: ×40. Arrow indicates endothelial structure. (a) normoxic young heart, (b) hypoxic young heart, (c) normoxic old heart, and (d) hypoxic old heart. B: Densitometric analysis of PKC δ positive area, expressed as % ± SD, determined by direct visual counting of three fields for each of five slides per each of five samples at ×40 magnification, by MetaMorph Software System.

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Figure 6. A: Western blotting analysis of SC-35 expression and coimmunoprecipitation of SC-35 and PKC δ. Immunoprecipitated SC-35 was probed against rabbit PKC δ polyclonal antibody and reprobed against mouse SC-35 monoclonal antibody. Note that SC-35/PKC δ immune complex is present in hypoxic young and normoxic old samples. A representative of three separate experiments is shown. ny, normoxic young heart; hy, hypoxic young heart; no, normoxic old heart; ho, hypoxic old heart. B: Densitometric analysis of SC-35 expression, defined by I.O.I. (integrated optical intensity). Samples (20 μg) have been normalized to levels of β-tubulin expression. Results are the mean of five different samples (±SD).

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Table 1. Mean value (±SD) of myocardial cells diametera
 Myocardial cells diameter, mean value ± SD (μm)
  • a

    Acquired at 40× magnification on 10 fields for each of the five different longitudinal Trichrome Mallory-stained sections per five samples using the MetaMorph Software System.

  • *

    Hypoxic young vs normoxic young (P < 0.05).

Normoxic young1.05 ± 0.08*
Hypoxic young0.69 ± 0.05*
Normoxic old1.19 ± 0.07
Hypoxic old1.35 ± 0.09
Table 2. Densitometric analysis of Trichrome Mallory staining positive area/field (76,000 μm2), occupied by myocardial cells and connective compartment, expressed as percentage (±SD)a
 Myocardial areaConnective area
  • a, *

    Acquired at 40× magnification on 10 fields for each of the five different longitudinal sections per five samples using the MetaMorph Software System.

  • *

    Hypoxic young vs normoxic young (P < 0.05).

Normoxic young71.7 ± 6.6*28.3 ± 2.9*
Hypoxic young66.2 ± 5.8*33.8 ± 2.3*
Normoxic old62.6 ± 5.537.4 ± 3.1
Hypoxic old64.4 ± 6.135.6 ± 2.4
Table 3. Apoptotic myocardial cell percentage detected by TUNEL analysis
 Myocardial cell percentage
  1. Five paraffin sections were examined for sample. Numbers represent the mean percentage of positive myocardial cells observed per 10 fields for each of the five different longitudinal sections per five samples by direct visual counting of fluorescent labelled nuclei at 40× magnification. Values are means ± SD. N = 3 for all groups, (P < 0.05).

Normoxic young2.3 ± 0.19
Hypoxic young22.1 ± 3.10
Normoxic old3.8 ± 0.40
Hypoxic old15.5 ± 1.15

DISCUSSION

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Oxidative stress is involved both in the pathogenesis of various degenerative diseases, including cancer, and in aging. Moreover in the heart, low oxygen tension (10%, hypoxia) followed by reoxygenation, increasing generation of reactive oxygen species, is involved in the progression of hypertrophy and failure (Sugden and Clerk,1998; Kang et al.,2000; Dhalla et al.,2000). Hypertrophy is not only hypoxia- but also age-dependent, and it implies increased cell size, protein synthesis, and enhanced sarcomeric organization (Zhang et al.,2005). Because hypoxia induces new blood vessels formation, inside the cell a number of metabolic alterations occurs, including synthesis of proangiogenic growth factors such as VEGF, which is targeted to the vascular endothelium (Shima et al.,2004). The regulation of the angiogenic activities of VEGF via common pathways that include proteolysis, transcription, and RNA SFs may allow coordinated development of lymphatic and blood vasculature, which is necessary for fluid homeostasis in physiological conditions. During disease progression, VEGF may allow remodeling of vasculature, such as angiogenesis, and loss of tissue-specific vascular structure (Rapino et al.,2005). To this aim, here we report that hypoxia modulates in vivo SC-35 SF phosphorylation via PKC δ in young rat heart. Even though hypoxic young myocardial cells are smaller, when compared with the normoxic ones, the connective and the endothelial components increase. Furthermore, SC-35 phosphorylation is particularly evident in the endothelium and is paralleled by an increased expression of VEGF. On the other hand, in the old heart no differences are evidenced in the two experimental conditions, even though the levels of SC-35 and PKC δ are high. In the hypoxic young heart, both PKC δ activation increase and SC-35/PKC δ coimmunoprecipitation occurs, suggesting that SC-35 phosphorylation could be PKC δ-mediated, as already reported in other experimental models (Zhu et al.,2003). Finally, this evidence lets us suppose that the hypoxic young heart needs to counteract the damage through a process of neoangiogenesis. The similar response disclosed by old normoxic and hypoxic rat hearts could be due either to an impaired oxygen sensing mechanism and thus to a low rate of angiogenesis or to an adaptation of the cells to hypoxia (Bianchi et al.,2006). This effect seems to be justified by the fibrotic organization, the endothelial component and VEGF expression similar in the two experimental conditions, as evidenced also in rat brain (Rapino et al.,2005). Thus, such results could indicate that adaptation to low oxygen tension needs the activation of SC-35 mediated by PKC δ and that cardiac function is reduced when this mechanism is partially or total exhausted, as already reported in a human cardiac model (Hein et al.,2003). SC-35 domains may have direct roles in the transcription, processing, and transport of many pre-mRNAs, rather than being reservoirs of factors, and involved in the synthesis of specific signaling proteins (Shopland et al.,2002).

Therefore, the knowledge of the intranuclear specific signaling mediating the cell adaptive response to hypoxic stress, which is similar to that occurring in hypertrophic, ischemic, and atherosclerotic events, allows us to set up targeted molecular therapeutic strategies against cardiovascular and neoplastic diseases.

Acknowledgements

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Prof. A. Antonucci: “Effetto dell'ipossia cronica intermittente nella regolazione degli eventi ipertrofici ed apoptotici nel cuore e nel cervello di ratto”.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. EXPERIMENTAL PROCEDURES
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED