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Effect of Hypoxia and Aging on PKC ╬┤-Mediated SC-35 Phosphorylation in Rat Myocardial Tissue.

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THE ANATOMICAL RECORD 292:1135–1142 (2009)
Effect of Hypoxia and Aging on PKC
d-Mediated SC-35 Phosphorylation in Rat
Myocardial Tissue
AMELIA CATALDI,1,2* MARIA ZINGARIELLO,2 MONICA RAPINO,3 SUSI ZARA,1,2
FRANCA DANIELE,4 CAMILLO DI GIULIO,5 AND ADRIANO ANTONUCCI2
1
Cattedra di Anatomia Umana, Facoltà di Farmacia, Università G. d’Annunzio,
Chieti-Pescara, Chieti, Italy
2
Dipartimento di Biomorfologia, Università G. d’Annunzio, Chieti-Pescara, Chieti, Italy
3
Istituto di Genetica Molecolare del CNR, Unità di Chieti, Chieti, Italy
4
Dipartimento di Scienze Linguistiche e Letterarie, Università G. d’Annunzio,
Chieti-Pescara, Chieti, Italy
5
Dipartimento di Scienze Mediche di Base e Applicate, Università G. d’Annunzio,
Chieti-Pescara, Chieti, Italy
ABSTRACT
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) d 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 d coimmunoprecipitation occurs, suggesting
that SC-35 phosphorylation could be PKC d-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 d
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,
C 2009 Wiley-Liss, Inc.
292:1135–1142, 2009. V
Key words: PKC d; SC-35; hypoxia; aging; myocardial tissue
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
Grant sponsor: MIUR (60% grant 2007).
*Correspondence to: Amelia Cataldi, Dipartimento di Biomorfologia, Università G d’Annunzio, Via dei Vestini, 6, 66100
Chieti, Italy. Fax: 39-0871-3554568. E-mail: cataldi@unich.its
Received 14 January 2009; Accepted 6 May 2009
DOI 10.1002/ar.20936
Published online in Wiley InterScience (www.interscience.wiley.
com).
C 2009 WILEY-LISS, INC.
V
1136
CATALDI ET AL.
Fig. 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.
TABLE 1. Mean value (6SD) of myocardial
cells diametera
TABLE 2. Densitometric analysis of Trichrome
Mallory staining positive area/field (76,000 lm2),
occupied by myocardial cells and connective
compartment, expressed as percentage (6SD)a
Myocardial cells diameter,
mean value SD (lm)
Normoxic young
Hypoxic young
Normoxic old
Hypoxic old
1.05
0.69
1.19
1.35
0.08*
0.05*
0.07
0.09
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).
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.,
Myocardial area
Normoxic young
Hypoxic young
Normoxic old
Hypoxic old
71.7
66.2
62.6
64.4
6.6*
5.8*
5.5
6.1
Connective area
28.3
33.8
37.4
35.6
2.9*
2.3*
3.1
2.4
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).
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).
PKC d-MEDIATED SC-35 ACTIVATION IN RAT MYOCARDIAL TISSUE
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 SC35, because they are frequently located at the nuclear
periphery, in the perichromatin area, which is a region
1137
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 ¼ 3 for all groups, (P < 0.05).
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,
Fig. 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 lg) have been normalized to levels of b-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.
TABLE 3. Apoptotic myocardial cell percentage
detected by TUNEL analysis
Myocardial cell percentage
Normoxic young
Hypoxic young
Normoxic old
Hypoxic old
2.3
22.1
3.8
15.5
0.19
3.10
0.40
1.15
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CATALDI ET AL.
Fig. 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.
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 d and SC-35, to determine possible links
between physiologically important stimuli and premRNA splicing machinery in vivo in rat myocardial tissue.
EXPERIMENTAL PROCEDURES
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
PKC d-MEDIATED SC-35 ACTIVATION IN RAT MYOCARDIAL TISSUE
1139
pentobarbital sodium salt (Nembutal, 40 mg/kg) (SigmaAldrich, 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) phosphatebuffered 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 d, 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 d 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 d, 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.
Fig. 4. A: Western blotting analysis of PKC d and p-PKC d expression. Samples (20 lg) have been normalized to levels of b 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 d and pPKC d expression, defined by I.O.I. (integrated optical intensity). Results
are the mean of five different samples (SD). *p-PKC d/PKC d hypoxic
young heart versus p-PKC d/PKC d normoxic young heart: P < 0.05.
body, and 500 lL 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 lL of PBS. SC35 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 lg) 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 SC35, PKC d monoclonal antibodies, goat phosphorylated
PKC d (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 b 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.
Protein Analysis
For immunoprecipitation, whole cell lysate (500 lg)
was incubated in the presence of 50 lL 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 lL of suspended IP matrix,
3 lg of mouse phosphorylated SF SC-35 monoclonal anti-
RESULTS
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
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CATALDI ET AL.
Fig. 5. A: Immunohistochemical analysis of PKC d 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 d 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.
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).
PKC d-MEDIATED SC-35 ACTIVATION IN RAT MYOCARDIAL TISSUE
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 d has been performed. An increased PKC d 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 d 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 d and SC35 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 d monoclonal antibody. Coimmunoprecipitation of
PKC d 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
d-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.
DISCUSSION
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 d in
1141
Fig. 6. A: Western blotting analysis of SC-35 expression and coimmunoprecipitation of SC-35 and PKC d. Immunoprecipitated SC-35
was probed against rabbit PKC d polyclonal antibody and reprobed
against mouse SC-35 monoclonal antibody. Note that SC-35/PKC d
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 lg) have
been normalized to levels of b-tubulin expression. Results are the
mean of five different samples (SD).
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 d are high. In the hypoxic young heart, both
PKC d activation increase and SC-35/PKC d coimmunoprecipitation occurs, suggesting that SC-35 phosphorylation could be PKC d-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
1142
CATALDI ET AL.
activation of SC-35 mediated by PKC d 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.
ACKNOWLEDGMENTS
Prof. A. Antonucci: ‘‘Effetto dell’ipossia cronica intermittente nella regolazione degli eventi ipertrofici ed
apoptotici nel cuore e nel cervello di ratto’’.
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effect, phosphorylation, pkc, myocardial, rat, tissue, hypoxia, aging, mediated
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