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J Appl Physiol 101: 1149-1161, 2006. First published June 15, 2006; doi:10.1152/japplphysiol.00194.2006
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Increased DNA fragmentation and altered apoptotic protein levels in skeletal muscle of spontaneously hypertensive rats

Joe Quadrilatero and James W. E. Rush

Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada

Submitted 16 February 2006 ; accepted in final form 9 June 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Apoptosis is a highly conserved process that plays an important role in controlling tissue development, homeostasis, and architecture. Dysregulation of apoptosis is a hallmark of numerous human pathologies including hypertension. In the present work we studied the effect of hypertension on apoptosis and the expression of several apoptotic signaling and/or regulatory proteins in four functionally and metabolically distinct muscles. Specifically, we examined these markers in soleus, red gastrocnemius, white gastrocnemius, and left ventricle (LV) of 20-wk-old normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Compared with WKY rats SHR had a significantly greater heart weight, LV weight, and mean arterial pressure. In general, SHR skeletal muscle had increased Bax protein, procaspase-3 protein, caspase-3 activity, cleaved poly(ADP-ribose) polymerase protein, and DNA fragmentation as well as decreased Bcl-2 protein and a lower Bcl-2-to-Bax ratio. Subcellular distribution studies demonstrated increased levels of apoptosis-inducing factor protein in cytosolic or nuclear extracts as well as elevated nuclear Bax protein in SHR skeletal muscle. Moreover, heat shock protein 70 in red gastrocnemius and soleus was significantly correlated to several apoptotic factors. With the exception of lower heat shock protein 90 levels in SHR no additional differences in any apoptotic markers were observed in LV between groups. Collectively, this report provides the first evidence that apoptotic signaling is altered in skeletal muscle of hypertensive animals, an effect that may be mediated by both caspase-dependent and -independent mechanisms. This proapoptotic state may provide some understanding for the morphological and functional abnormalities observed in skeletal muscle of hypertensive animals.

Bcl-2; caspases; cell death; mitochondria

APOPTOSIS OR PROGRAMMED CELL DEATH is a highly conserved biological process that plays an important role in controlling tissue development, homeostasis, and architecture by eliminating redundant, dysfunctional, or injured cells (53, 66). Mitochondria play an essential role during apoptotic cell death. In general, a variety of proapoptotic stimuli can converge on the mitochondria and promote apoptosis by 1) disrupting electron transport and ATP production, 2) altering cellular redox potential, and 3) causing the release of apoptogenic proteins such as apoptosis-inducing factor (AIF) and cytochrome c (28). Release of cytochrome c into the cytosol initiates a cascade of events leading to the activation of a group of apoptosis-related cysteine proteases (i.e., caspases), DNA fragmentation, and apoptotic cell death (8, 69). In addition, caspases can proteolytically cleave the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) from its active 116-kDa full-length form to an inactive 85-kDa fragment (3, 8, 68). AIF released from the mitochondria into the cytosol can subsequently translocate to the nucleus to induce DNA fragmentation and apoptotic cell death independent of caspase activation (17, 64). However, recent evidence suggests that under some conditions AIF release is caspase dependent (33).

The Bcl-2 family of regulatory proteins play a central role in mediating a variety of mitochondrial events including the release of apoptogenic factors. For example, Bcl-2 prevents mitochondrial release of cytochrome c, smac/DIABLO, and AIF, thus preserving cell survival (1, 64, 69). In contrast, Bax can translocate from the cytosol to the mitochondria causing the release of apoptogenic proteins, thereby promoting apoptotic cell death (3, 6, 43). Current evidence also suggests that cytosolic-nuclear translocation of Bax occurs during apoptosis (31, 39), which may influence apoptotic cell death by interacting with p53 (48). Heat shock proteins (Hsp) act as molecular chaperones by facilitating protein folding and import as well as by assisting in the repair and degradation of damaged proteins. During cellular stress Hsp expression is increased, thereby providing cytoprotection, an effect that may be partially attributed to their ability to regulate caspase-dependent and -independent apoptosis (36, 42, 49).

Dysregulation of apoptosis is a hallmark feature of numerous human pathologies and plays a critical role in several cardiovascular diseases including heart failure, ischemic heart disease, atherosclerosis, and hypertension (21, 30). The spontaneously hypertensive rat (SHR) has been extensively used as a genetic model of essential hypertension. It has been shown that apoptosis, susceptibility to apoptosis, or the expression of various apoptosis-associated proteins and genes is altered in aortic smooth muscle (56), smooth muscle, and endothelial cells of heart arterioles and capillaries (67), heart (19, 21, 22, 50, 51), thymus (65), and kidney mesangial cells (52) of SHR compared with Wistar-Kyoto rats (WKY). Interestingly, several skeletal muscle abnormalities including decreased fatigue resistance (27), development of less contractile force (4, 27), increased interstitial norepinephrine levels (13, 14), altered Na+ pump number and activity (44), elevated intracellular free Ca2+ (2), fiber-type redistribution (5, 7), and decreased capillary density (5, 32) have also been reported in SHR compared with WKY rats. However, to date, apoptosis and the profile of various apoptosis-related proteins have not been systematically examined in skeletal muscle of hypertensive animals.

Therefore, the purpose of the present study was to examine apoptotic signaling and Hsp expression in several functionally and metabolically diverse skeletal muscles of 20-wk-old SHR and WKY rats. Specifically, we have studied 1) soleus (Sol), a slow oxidative muscle with a predominantly myosin heavy chain (MHC) type I fiber composition; 2) red gastrocnemius (RG), a mixed slow oxidative-fast oxidative glycolytic muscle with primarily MHC type I and MHC type IIA fibers; and 3) white gastrocnemius (WG), a fast-twitch muscle with predominantly fast glycolytic MHC type IIB fibers (18). Furthermore, we expanded on previous cardiac literature by concurrently examining several apoptotic markers (including AIF) and Hsp expression in the left ventricle (LV) of hypertensive animals. This age cohort is well suited for studying the effect of hypertension on cardiac and skeletal muscle apoptosis because animals display stable, severe hypertension (37) but are not confounded by aging effects and other pathologies (i.e., heart failure) that have also been associated with increased apoptosis in either heart (46, 47) or skeletal muscle (16, 59). We hypothesized that hypertensive animals would have augmented apoptotic signaling and DNA fragmentation relative to normotensive controls across muscle fiber types.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals.   All procedures were performed in accordance with the guidelines of the University of Waterloo Animal Care Committee. Male normotensive WKY and SHR were obtained from Harlan (Indianapolis, IN) and were 20 wk old at the time of investigation. Rats were group housed on a 12:12-h reverse light-dark cycle in a temperature and humidity controlled environment. Standard rodent lab chow and tap water were provided ad libitum.

Blood pressure measurement.   Body mass (BM) was recorded and rats were anesthetized with a pentobarbital sodium injection (0.65 mg/kg ip; MTC Pharmaceuticals). Mean arterial blood pressure (MAP) was measured for 10 min from a transducer (Harvard) attached to a PE-50 cannula in the left common carotid artery. After MAP measurements, rats were killed by removing the heart. Right ventricle (RV), LV, Sol, WG, and RG muscles were removed, immediately frozen in liquid nitrogen, and stored at –80°C. For histochemical analysis muscles were embedded in freezing medium (Shandon Cryomatrix; Thermo), frozen in liquid nitrogen-cooled isopentane, and stored at –80°C until further analysis.

Preparation of whole tissue lysate.   Tissue (~20 mg wet wt) was homogenized in ice-cold lysis buffer (20 mM HEPES, 10 mM NaCl, 1.5 mM MgCl, 1 mM DTT, 20% glycerol, and 0.1% Triton X-100; pH 7.4) by using a glass pestle and mortar to obtain a total cell lysate. Samples were centrifuged at 1,000 g for 10 min at 4°C, the supernatant was collected, and total protein was determined by the bicinchoninic acid (BCA) protein assay.

Subcellular fractionation.   Subcellular fractions were obtained essentially as previously reported (58) with modifications. Tissue (~50 mg wet wt) was homogenized in ice-cold mitochondrial isolation buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT; pH 7.4) containing protease inhibitors (Complete Cocktail; Roche Diagnostics) by using a glass pestle and mortar. The homogenate was centrifuged at 800 g for 10 min at 4°C. The supernatant (S1) was carefully removed from the pellet (P1). The S1 was further centrifuged at 16,000 g for 20 min at 4°C. The supernatant resulting from this procedure (S2) was carefully removed from the pellet (P2) and centrifuged at 16,000 g for 20 min at 4°C to remove residual mitochondria. The resulting supernatant was designated the mitochondrial-free, nuclear-free, cytosolic-enriched protein fraction. The P1 containing nuclei was washed twice (centrifuged at 800 g for 10 min at 4°C) in 1,000 µl of PBS (pH 7.4). Lysis buffer (360 µl; 20 mM HEPES, 10 mM NaCl, 1.5 mM MgCl, 1 mM DTT, 20% glycerol, and 0.1% Triton X-100; pH 7.4) and 40 µl of 5 M NaCl were added to each sample and rotated for 1 h at 4°C. The samples were centrifuged at 20,000 g for 15 min at 4°C and the supernatant collected and designated the nuclear-enriched protein fraction. The P2 containing mitochondria was washed (16,000 g for 20 min at 4°C) twice in 200-µl mitochondrial isolation buffer. The pellet was resuspended in mitochondrial isolation buffer, lysed by three consecutive freeze-thaw cycles, and sonicated on ice for 20 s (2 s on, 5 s off duty cycle). The suspension was designated the mitochondrial-enriched protein fraction. Subcellular protein concentrations were determined by the BCA assay.

Immunoblot analysis.   Equal amounts of protein were electrophoresed on a 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene difluoride membrane (Roche Diagnostics), and blocked with 10% milk/Tris-buffered saline/Tween 20 at 4°C. Membranes were incubated at room temperature for 1 h with primary antibodies against AIF (1:200), Bcl-2 (1:200), Bax (1:200), PARP (1:200), procaspase-3 (1:200) (Santa Cruz Biotechnology), heat shock protein 70 (Hsp70; 1:1,000), and Hsp90 (1:1,000) (Stressgen Bioreagents). Membranes were then washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. Bands were visualized by use of Enhanced Chemiluminescence Western blotting detection reagents (Amersham Biosciences) and the ChemiGenius 2 Bio-Imaging system (Syngene). For whole tissue studies all samples within a given tissue (i.e., Sol) were run on a single gel. For subcellular fraction studies all samples within a given fraction (e.g., all mitochondrial fractions) were run on a single gel to examine differences between groups. In separate studies all three fractions for a given animal were run on a single gel to examine relative subcellular protein distribution within a sample. The purity of the subcellular fractions was verified by probing membranes for cytosolic, mitochondrial, and nuclear protein markers using primary antibodies for CuZn-superoxide dismutase (1:2,500) (Stressgen Bioreagents), adenine nucleotide translator (1:200) (Santa Cruz Biotechnology), and histone H2B (1:2,500) (Upstate Cell Signaling), respectively. Membranes were stained with Ponceau S (Sigma Chemical) to verify quality of transfer and equal loading and further confirmed by actin (1:1,000 Sigma) staining. The molecular weight of the immunoblotted protein was identified by using a biotinylated protein ladder and anti-biotin horseradish peroxidase-linked secondary antibody (Cell Signaling Technology).

Caspase-3 activity.   Caspase-3 activity was determined in tissue homogenate using the EnzChek caspase-3 assay kit (Molecular Probes) according to the manufacturer's instructions. In this assay, the 7-amino-4-methylcoumarin-derived substrate Z-DEVD-AMC is weakly fluorescent but yields a highly fluorescent product after proteolytic cleavage. Briefly, 50 µl of tissue homogenate was incubated with 50 µl of Z-DEVD-AMC substrate at 37°C for 2 h. Fluorescence was measured with a SPECTRAmax Gemini XS microplate spectrofluorometer (Molecular Devices) with excitation and emission wavelengths of 360 and 465 nm, respectively. In control experiments, incubation of tissue homogenate with the caspase-3 inhibitor Ac-DEVD-CHO completely inhibited the fluorescent signal (data not shown). Caspase-3 activity was expressed as arbitrary units per milligram protein.

Detection of apoptotic nuclei.   Apoptotic nuclei were identified by terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) using the TdT-FragEL DNA fragmentation detection kit (Calbiochem). Briefly, 10-µm cardiac and skeletal muscle sections were fixed in 4% paraformaldehyde for 15 min, permeabilized with 20 µg/ml proteinase K for 10 min, and blocked in 3% H2O2 for 5 min all at room temperature. Samples were incubated in equilibration buffer (1 M sodium cacodylate, 0.15 M Tris, 1.5 mg/ml BSA, 3.75 mM CoCl2; pH 6.6) for 20 min at room temperature and then incubated with 1x TdT labeling reaction mixture for 90 min at 37°C. The reaction was terminated with 0.5 M EDTA (pH 8.0) for 5 min at room temperature and blocked with 4% BSA in PBS for 10 min at room temperature. Sections were then incubated with a peroxidase streptavidin conjugate for 30 min at room temperature, reacted with a 3,3' diaminobenzidine-H2O2-urea substrate for 15 min at room temperature, and visualized by light microscopy.

DNA fragmentation assay.   Quantification of cytoplasmic histone associated mono- and oligonucleosomes (i.e., DNA fragmentation) was determined using the Cell Death Detection ELISAPLUS kit (Roche Diagnostics) according to the manufacturer's protocol. Briefly, tissue was homogenized in lysis buffer followed by centrifugation at 200 g for 10 min. Subsequently, 20 µl of supernatant and 80 µl of anti-histone-biotin/anti-DNA-POD reagent were incubated in a streptavidin-coated microplate for 2 h at room temperature under gently shaking. 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate (6)] substrate solution (100 µl) was added to each well, incubated for 20 min, and measured at 405 and 490 nm via a SPECTRAmax Plus spectrophotometer (Molecular Devices). A control DNA-histone-complex sample was included to confirm a positive signal. DNA fragmentation was expressed as arbitrary units per milligram protein.

Statistical analysis.   Group data were analyzed by a two-tailed independent sample t-test. Relationships between variables were determined by calculating the Pearson correlation coefficient. For all analyses P < 0.05 was considered statistically significant. All results are given as means ± SE.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Anatomical data and blood pressure.   As shown in Table 1, body weight, kidney weight, and right ventricle weight were not significantly different between groups. Heart weight, LV weight, and MAP were significantly (P < 0.001) higher in SHR compared with age-matched WKY rats.


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  		Table 1. Group characteristics

 
Whole tissue protein levels.   No significant group differences were found in apoptotic-associated proteins (Bcl-2, Bax, Bcl-2-to-Bax ratio, procaspase-3, and AIF) or Hsp70 in LV whole tissue extracts between groups. Hsp90 protein was significantly (P < 0.05) reduced in LV of SHR compared with WKY rats (Fig. 1A).


Figure 1
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  		Fig. 1. Whole tissue apoptotic and heat shock protein (Hsp) expression. Representative immunoblots and quantitative analysis of apoptotic and stress protein expression in whole tissue extracts of left ventricle (LV; A), soleus (Sol; B), red gastrocnemius (RG; C), and white gastrocnemius (WG; D). AIF, apoptosis-inducing factor. Bars are means ± SE of Wistar-Kyoto rats (WKY; solid bars; n = 6) and spontaneously hypertensive rats (SHR; open bars; n = 6). *P < 0.05 vs. WKY; {ddagger}P < 0.005 vs. WKY; ¶P < 0.001 vs. WKY.

 
Relative to WKY rats, Sol whole tissue extracts from SHR had significantly higher protein levels of Bax (P < 0.05), AIF (P < 0.05), and procaspase-3 (P < 0.001) as well as lower levels of Bcl-2 (P < 0.05) and a decreased Bcl-2-to-Bax ratio (P < 0.05) (Fig. 1B). Hsp70 protein levels in Sol were elevated by ~32% in SHR compared with WKY; however, this change did not reach statistical significance (P < 0.10). Hsp90 protein levels in Sol were not different between groups (Fig. 1B).

The relative protein levels of Bax, Bcl-2, and AIF in RG whole tissue extracts were not significantly different between groups (Fig. 1C). There was a significant elevation in procaspase-3 (P < 0.005) and Hsp90 (P < 0.05) protein levels in SHR compared with WKY rats. A lower Bcl-2-to-Bax ratio (~37%; P < 0.10) and higher Hsp70 (~66%; P < 0.07) protein level were also observed in RG muscle of SHR compared with WKY rats; however, these changes were not statistical significant (Fig. 1C).

In WG whole tissue extracts there was a significant reduction in Bcl-2 protein (P < 0.005) and in the Bcl-2-to-Bax ratio (P < 0.005) in SHR compared with WKY rats (Fig. 1D). In addition, there were nonsignificant increases in Bax (~19%; P < 0.08) and procaspase-3 (~61%; P < 0.07) protein levels in SHR WG tissue relative to WKY rats. No significant differences in AIF, Hsp70, and Hsp90 protein expression were found in WG muscle between groups (Fig. 1D).

Subcellular apoptotic protein expression.   As shown in Figs. 2A, 3A, 4A, and 5A, the majority of AIF was localized to the mitochondria-enriched fraction with lower amounts in the nuclear-enriched and cytosolic-enriched fractions. In addition, Bax was primarily localized to the cytosol with lesser amounts in the mitochondrial and nuclear fraction. No significant differences in the subcellular distribution of AIF and Bax were found between groups in LV tissue (Fig. 2, B and C).


Figure 2
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  		Fig. 2. Subcellular distribution of AIF and Bax in left ventricle. A: representative immunoblots of subcellular AIF and Bax distribution in LV tissue. M, mitochondrial-enriched fraction; C, cytosolic-enriched fraction; N, nuclear-enriched fraction. Purity of fractions was confirmed by positive and negative staining of adenine nucleotide translator (ANT), CuZn-superoxide dismutase (CuZnSOD), and histone H2B as described inMETHODS. B: representative immunoblots of mitochondrial, cytosolic, and nuclear AIF and Bax of WKY and SHR. C: quantitative analysis of mitochondrial, cytosolic, and nuclear AIF and Bax from WKY (solid bars; n = 6) and SHR (open bars; n = 6). Bars are means ± SE.

 

Figure 3
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  		Fig. 3. Subcellular distribution of AIF and Bax in soleus. A: representative immunoblots of subcellular AIF and Bax distribution in Sol tissue. Purity of fractions was confirmed by positive and negative staining of ANT, CuZnSOD, and histone H2B as described in METHODS. B: representative immunoblots of mitochondrial, cytosolic, and nuclear AIF and Bax of WKY and SHR. C: quantitative analysis of mitochondrial, cytosolic, and nuclear AIF and Bax from WKY (solid bars; n = 6) and SHR (open bars; n = 6). Bars are means ± SE. *P < 0.05 vs. WKY.

 

Figure 4
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  		Fig. 4. Subcellular distribution of AIF and Bax in red gastrocnemius. A: representative immunoblots of subcellular AIF and Bax distribution in RG tissue. Purity of fractions was confirmed by positive and negative staining of ANT, CuZnSOD, and histone H2B as described in METHODS. B: representative immunoblots of mitochondrial, cytosolic, and nuclear AIF and Bax of WKY and SHR. C: quantitative analysis of mitochondrial, cytosolic, and nuclear AIF and Bax from WKY (solid bars; n = 6) and SHR (open bars; n = 6). Bars are means ± SE. *P < 0.05 vs. WKY; {dagger}P < 0.01 vs. WKY; ¶P < 0.001 vs. WKY.

 

Figure 5
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  		Fig. 5. Subcellular distribution of AIF and Bax in white gastrocnemius. A: representative immunoblots of subcellular AIF and Bax distribution in WG tissue. Purity of fractions was confirmed by positive and negative staining of ANT, CuZnSOD, and histone H2B as described in METHODS. B: representative immunoblots of mitochondrial, cytosolic, and nuclear AIF and Bax of WKY and SHR. C: quantitative analysis of mitochondrial, cytosolic, and nuclear AIF and Bax from WKY (solid bars; n = 6) and SHR (open bars; n = 6). Bars are means ± SE. *P < 0.05 vs. WKY; {ddagger}P < 0.005 vs. WKY; ¶P < 0.001 vs. WKY.

 
The protein levels of mitochondrial AIF, cytosolic AIF, and cytosolic Bax in Sol tissue were not different between groups (Fig. 3, B and C). There was a nonsignificant increase in mitochondrial Bax protein (~43%; P < 0.09) along with significant increases in nuclear AIF (P < 0.05) and nuclear Bax (P < 0.05) protein in Sol of SHR relative to WKY rats (Fig. 3, B and C).

Mitochondrial AIF, mitochondrial Bax, and cytosolic Bax protein levels in RG were not different between groups (Fig. 4, B and C). However, RG muscle from SHR had significantly higher protein levels of cytosolic AIF (P < 0.01), nuclear AIF (P < 0.001), and nuclear Bax (P < 0.05) relative to WKY animals (Fig. 4, B and C).

No significant group differences were found in mitochondrial AIF, mitochondrial Bax, and nuclear AIF protein content in WG muscle (Fig. 5, B and C). In contrast, WG from SHR had significantly higher levels of cytosolic AIF (P < 0.001), cytosolic Bax (P < 0.05), and nuclear Bax (P < 0.005) protein compared with WKY controls (Fig. 5, B and C).

Caspase-3 activity and PARP cleavage.   Caspase-3 activity, as measured by Z-DEVD-AMC cleavage, was not different between groups in LV but was significantly increased in Sol (P < 0.005) and RG (P < 0.01) tissue of SHR compared with WKY rats. A small nonsignificant increase (~13%; P < 0.07) in WG caspase-3 activity was also observed in SHR compared with WKY rats. An immunoreactive band corresponding to the 85-kDa cleaved PARP fragment was detected in cardiac and skeletal muscle. There was no difference in cleaved PARP protein in LV between groups. Cleaved PARP levels were significantly elevated in SHR compared with WKY rats in Sol (P < 0.005) and RG (P < 0.05) muscle but did not reach statistical significance (~73% P < 0.08) in WG muscle (Fig. 6, A and B).


Figure 6
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  		Fig. 6. Caspase-3 activation in cardiac and skeletal muscle. A: caspase-3 activity in LV, Sol, RG, and WG. B: representative immunoblots and quantitative analysis of the 85-kDa cleaved poly(ADP-ribose) polymerase (PARP) fragment in LV, Sol, RG, and WG tissue. Bars are means ± SE of WKY (solid bars; n = 6) and SHR (open bars; n = 6). *P < 0.05 vs. WKY; {dagger}P < 0.01 vs. WKY; {ddagger}P < 0.005 vs. WKY.

 
DNA fragmentation.   Histochemical analysis was performed on cardiac and skeletal muscle cryosections to assess muscle structure and to determine the location of nuclei with fragmented DNA (i.e., apoptotic nuclei). The cytoplasm and nuclei of LV and Sol muscle are easily distinguished after hematoxylin and eosin staining (Fig. 7, A–D). Photographs showing representative TUNEL staining of apoptotic myonuclei in LV and Sol muscle of WKY and SHR are shown in Fig. 7, E–H. Similar results were observed in RG and WG muscle (data not shown). DNA fragmentation was quantified in whole muscle homogenates by determining cytoplasmic histone associated mono- and oligonucleosomes. Consistent with the qualitative TUNEL staining results, DNA fragmentation as determined by ELISA was not different in LV between groups but was significantly increased in Sol (P < 0.01), RG (P < 0.05), and WG (P < 0.05) of SHR compared with WKY rats (Fig. 7I).


Figure 7
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  		Fig. 7. DNA fragmentation. Representative photographs of hematoxylin and eosin (A–D) and terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL; E–H) staining of WKY LV (A and E), SHR LV (B and F), WKY Sol (C and G), and SHR Sol (D and H) tissue sections. Arrows indicate TUNEL-positive nuclei. Bar in H represents 50 µm for all pictures. I: differences between WKY and SHR cardiac and skeletal muscle DNA fragmentation were quantified by measuring mono- and oligonucleosomes in whole muscle homogenates. Bars are means ± SE of WKY (solid bars; n = 6–9) and SHR (open bars; n = 6–9). *P < 0.05 vs. WKY; {dagger}P < 0.01 vs. WKY.

 
Relationship between apoptotic markers and stress protein expression.   Correlation analysis was preformed with all data collapsed across groups. The expression of Hsp70 was significantly correlated to several apoptotic markers in Sol and RG (Table 2 and Fig. 8) but not in WG and LV (data not shown). Hsp90 expression was also significantly correlated to procaspase-3 protein (r = 0.828; P < 0.001), cytosolic AIF protein (r = 0.662; P < 0.05), caspase-3 activity (r = 0.846; P < 0.001), and DNA fragmentation (r = 0.624; P < 0.05) in RG muscle. Hsp90 was not significantly correlated to any apoptotic markers in LV, Sol, or WG (data not shown).


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  		Table 2. Relationship between Hsp70 and apoptotic markers

 

Figure 8
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  		Fig. 8. Relationship between Hsp70 and apoptotic markers. Correlation of Hsp70 expression and total Bax or caspase 3 activity in Sol (A and B) and RG (C and D) of WKY ({blacksquare}; n = 6) and SHR ({square}; n = 6). Data were collapsed across groups for analysis.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this paper we observed apoptotic myonuclei (i.e., TUNEL-positive staining) as well as increased DNA fragmentation and altered apoptotic protein levels in skeletal muscle of 20-wk-old hypertensive rats (SHR) compared with age-matched normotensive controls (WKY). Although some differences were observed between specific skeletal muscles, in general, a proapoptotic state was evident in slow oxidative muscle (i.e., Sol), mixed slow oxidative-fast oxidative glycolytic muscle (i.e., RG) and fast glycolytic muscle (i.e., WG) of SHR compared with WKY animals. The increased procaspase-3 levels, caspase-3 activity, and PARP cleavage suggest that increased skeletal muscle apoptotic signaling in SHR may be mediated by a caspase-dependent pathway. Furthermore, higher cytosolic and/or nuclear AIF in SHR muscle may provide additional support for caspase-dependent and possibly caspase-independent signaling. Together, these data support the hypothesis that skeletal muscle apoptotic signaling is augmented in hypertensive animals. In contrast, we failed to find any signs of increased apoptotic signaling in cardiac muscle of hypertensive animals.

Previous reports examining cardiomyocyte apoptosis in SHR and WKY rats have been conflicting. Diez and colleagues (19) reported that LV cardiomyocyte apoptosis was elevated in 30- but not 16-wk-old SHR compared with age-matched WKY rats. Along with increased apoptosis this group also reported elevated Bax protein levels and a lower Bcl-2-to-Bax ratio in 30-wk-old SHR compared with WKY rats; however, no group differences were found in Bcl-2 protein, p53 protein, and Bax mRNA (22, 23). Interestingly, cultured LV cardiomyocytes from 30-wk SHR have higher basal apoptosis but show no differences in Bax, Bcl-2, and p53 protein, caspase 3 activation, and Bax mRNA compared with WKY rats (50). In 18- to 20-mo-old rats DNA fragmentation was significantly increased in SHR with failing hearts compared with WKY rats but not in SHR with nonfailing hearts; Bcl-2 protein was not different between the three groups (35). In contrast, Liu et al. (37) found that cardiomyocyte DNA fragmentation was increased as early as 4 wk in SHR compared with WKY rats. This effect reached a plateau at 16 wk, was maintained for up to 64 wk, and was associated with significantly lower Bcl-2 and higher Bax protein levels. Paradoxically, Rodriguez-Feo et al. (51) reported increased apoptosis, PARP cleavage, and Bax-Bcl-2 complex formation concurrently with decreased Bax and increase Bcl-2 protein levels in LV of 19-wk-old SHR compared with WKY rats. To date, the examination of caspase-independent apoptotic markers such as AIF have not been defined in LV of SHR relative to WKY rats. In the present study we have provided a comprehensive analysis of cardiac muscle apoptosis using several approaches but failed to find alterations in apoptotic regulatory proteins (i.e., Bcl-2, Bax, Bcl-2-to-Bax ratio, Bax subcellular distribution), caspase activation (i.e., procaspase-3 protein content, caspase-3 activity and PARP cleavage), mitochondrial apoptotic protein release (i.e., AIF subcellular distribution), anti-apoptotic stress protein expression (i.e., Hsp70) or DNA fragmentation in LV of 20-wk-old hypertensive animals not experiencing heart failure. The present findings are consistent with previous reports showing no increased LV apoptosis in younger (i.e., 16-wk-old) SHR (19) and SHR with nonfailing hearts (35). Therefore, increased LV apoptosis in SHR may be an age-dependent and/or cardiac failure-related event.

In the present study we observed apoptotic myonuclei (i.e., TUNEL-positive staining) and found increased levels of DNA fragmentation in skeletal muscle of hypertensive animals. Bcl-2 and Bax are important apoptotic regulatory proteins acting to inhibit and promote mitochondrial apoptogenic protein (e.g., AIF, cytochrome c, smac/DIABLO) release, respectively (1, 3, 6, 43, 64, 69). In general, we found that skeletal muscle of hypertensive animals display increased Bax and decreased Bcl-2 protein levels as well as a lower Bcl-2-to-Bax ratio. Bcl-2 can also modulate apoptosis by reducing endoplasmic reticulum (ER) Ca2+ stores and release and by decreasing mitochondrial Ca2+ accumulation (24, 45). In contrast, Bax can mediate apoptosis by promoting ER Ca2+ release and mitochondrial Ca2+ uptake (41, 55). Interestingly, cytosolic Ca2+ concentrations are significantly elevated in skeletal muscle of SHR compared with WKY rats (2). It is not known whether increased skeletal muscle cytosolic Ca2+ in SHR is causally related to altered Bcl-2 and/or Bax levels and whether this contributed to the observed apoptotic alterations.

Caspase-3 activation can occur after mitochondria- (8, 20, 36), ER- (57, 60) and death receptor-mediated (9, 54) apoptotic signaling. Although we did not identify the principal pathways involved we observed increased procaspase-3 levels, elevated caspase-3 activity, and PARP cleavage in skeletal muscle of SHR. It has been demonstrated that AIF can be released into the cytosol from the mitochondria and translocate to the nucleus to induce apoptosis either dependent (33) or independent of caspase activation (17, 64). We found significantly increased protein levels of AIF in cytosolic (RG and WG) and nuclear (Sol and RG) fractions of SHR animals, providing some support for mitochondrial involvement in the proapoptotic state observed in skeletal muscle of hypertensive animals. It remains to be determined whether AIF release in this model represents a caspase-dependent or -independent event. Collectively, these data suggest that caspase-dependent and possibly caspase-independent signaling (via AIF) may be involved in the increased DNA fragmentation observed in skeletal muscle of hypertensive animals.

We also found that skeletal muscle nuclear extracts of hypertensive rats express higher levels of Bax protein relative to their age-matched normotensive counterparts, supporting previous reports documenting Bax redistribution to the nucleus during apoptosis (31, 39). Raffo and colleagues (48) reported an initial rise in nuclear Bax protein followed by nuclear Bax/p53 complex formation and caspase-3 activation in human melanoma cells undergoing apoptosis. It has been suggested that nuclear Bax localization may promote apoptosis by influencing nuclear ion channel regulation; however, this remains to be determined (39). It is currently unknown whether Bax could interact with other nuclear proteins (e.g., Bcl-2) during apoptosis and whether nuclear Bax translocation occurs via caspase-dependent and/or -independent mechanisms. Regardless of the mechanism of action, the present findings in conjunction with previous literature suggest that nuclear Bax translocation may represent a key signaling event during some modes of apoptosis.

Skeletal muscle of hypertensive animals also showed a nonsignificant increase in Hsp70 expression (32% increase in Sol; P < 0.10 and 66% increase in RG; P < 0.07) and a significant increase in Hsp90 protein (RG only) compared with age-matched normotensive controls. Interestingly, in these tissues Hsp content was significantly correlated to several apoptotic factors. Although Hsp have been shown to inhibit apoptosis in vitro (36, 42, 49), our data are consistent with several in vivo studies showing concurrent increases in Hsp70 expression and apoptosis in skeletal muscle during aging (15) and denervation (58). It is currently unclear whether the increased Hsp response in SHR animals represents a compensatory mechanism invoked to inhibit apoptosis, thereby providing some cytoprotection. Regardless, in this model increased Hsp expression does not appear sufficient to fully protect against elevated skeletal muscle apoptosis. Given that Hsp are upregulated during cellular stress, their involvement in various protein regulatory functions (i.e., repair and degradation of damaged proteins) and the present correlations with several apoptotic markers collectively suggest that skeletal muscle of hypertensive rats may be under increased cellular stress.

Several plausible mechanisms could account for the elevated DNA fragmentation and apoptotic protein alterations observed in skeletal muscle of hypertensive rats. Recently, skeletal muscle apoptosis has been shown to be induced after administration of aldosterone (10), angiotensin II (11, 61), catecholamines (12, 26), and glucocorticoids (34). Interestingly, SHR have been shown to have higher levels of circulating aldosterone (62), angiotensin II (38, 63), and corticosterone (29, 62) as well as elevated plasma and skeletal muscle norepinephrine (13, 14). However, these mechanisms have not been systematically studied in WKY and SHR animals in the context of skeletal muscle apoptosis. Alternately, skeletal muscle apoptosis may occur indirectly through a loss of capillary network. Kobayashi et al. (32) found decreased microvessel length density in skeletal muscle of SHR compared with WKY rats. Furthermore, relative to WKY rats capillary density is decreased in SHR soleus (i.e., slow oxidative) but not plantaris (i.e., fast oxidative glycolytic-fast glycolytic) or extensor digitorum longus (i.e., fast glycolytic) muscle (5). Reduced skeletal muscle capillary density has been shown during hindlimb unweighting (25) and experimental heart failure (40); two models that are also associated with skeletal muscle apoptosis (16, 59). Thus it is possible that decreased skeletal muscle capillary density could potentially promote skeletal muscle apoptosis; however, this remains to be determined in hypertensive animals.

In conclusion, the present findings provide the first evidence that DNA fragmentation, a hallmark of apoptosis, and the levels of several apoptotic signaling and/or regulatory proteins are altered in skeletal muscle of hypertensive animals. The data presented also suggest that caspase-dependent signaling plays an important role in the observed DNA fragmentation (i.e., apoptosis). It is currently unclear what mechanisms account for this increased skeletal muscle apoptotic signaling and DNA fragmentation; however, these effects may be directly modulated by several known neuroendocrine alterations associated with hypertensive animals. Alternatively, this effect may occur indirectly as a consequence of decreased capillary network. Increased apoptotic signaling may provide some understanding for the morphological (i.e., fiber-type redistribution) and functional (i.e., development of less contractile force and decreased fatigue resistance) abnormalities observed in skeletal muscle of hypertensive animals.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by the Heart and Stroke Foundation of Ontario (Grant no. T-5599) and the Natural Sciences and Engineering Research Council of Canada (Grant no. RGPIN-23834-03). J. W. E. Rush holds the Canada Research Chair in Integrative Vascular Biology. J. Quadrilatero is supported by a Natural Sciences and Engineering Research Council of Canada Postdoctoral Fellowship.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. W. E. Rush, Dept. of Kinesiology, Univ. of Waterloo, 200 Univ. Ave. W., Waterloo, Ontario, Canada N2L3G1 (e-mail: jwerush{at}uwaterloo.ca)

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.


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 ABSTRACT
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