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Am J Physiol Heart Circ Physiol 293: H3617-H3626, 2007. First published September 21, 2007; doi:10.1152/ajpheart.00875.2007
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Effect of pravastatin on sympathetic reinnervation in postinfarcted rats

Tsung-Ming Lee,1,2 Mei-Shu Lin,3 and Nen-Chung Chang4

1Cardiology Section, Department of Medicine, Taipei Medical University, Taipei; 2Chi-Mei Medical Center, Tainan; 3Department of Pharmacy, National Taiwan University Hospital, Taipei; and 4Cardiology Section, Department of Medicine, Taipei Medical University and Hospital, Taipei, Taiwan

Submitted 26 July 2007 ; accepted in final form 13 September 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We assessed whether pravastatin attenuates cardiac sympathetic reinnervation after myocardial infarction through the activation of ATP-sensitive K+ (KATP) channels. Epidemiological studies have shown that men treated with statins appear to have a lower incidence of sudden death than men without statins. However, the specific factor for this has remained disappointingly elusive. Twenty-four hours after ligation of the anterior descending artery, male Wistar rats were randomized to groups treated with either vehicle, nicorandil (a specific mitochondrial KATP channel agonist), pinacidil (a nonspecific KATP channel agonist), pravastatin, glibenclamide (a KATP channel blocker), or a combination of nicorandil and glibenclamide, pinacidil and glibenclamide, or pravastatin and glibenclamide for 4 wk. Myocardial norepinephrine levels revealed a significant elevation in vehicle-treated rats at the remote zone compared with sham-operated rats (2.54 ± 0.17 vs. 1.26 ± 0.36 µg/g protein, P < 0.0001), consistent with excessive sympathetic reinnervation after infarction. Immunohistochemical analysis for tyrosine hydroxylase, growth-associated factor 43, and neurofilament also confirmed the change of myocardial norepinephrine. This was paralleled by a significant upregulation of tyrosine hydroxylase protein expression and mRNA in vehicle-treated rats, which was reduced after the administration of either nicorandil, pinacidil, or pravastatin. Arrhythmic scores during programmed stimulation in vehicle-treated rats were significantly higher than those treated with pravastatin. In contrast, the beneficial effects of pravastatin were reversed by the addition of glibenclamide, implicating KATP channels as the relevant target. The sympathetic reinnervation after infarction is modulated by the activation of KATP channels. Chronic use of pravastatin after infarction, resulting in attenuated sympathetic reinnervation by the activation of KATP channels, may modify the arrhythomogenic response to programmed electrical stimulation.

immunohistochemistry; ion channels; myocardial infarction; norepinephrine; polymerase chain reaction

PHARMACOLOGICAL ACTIONS of 3-hydroxy-3-methyglutaryl-CoA reductase inhibitors, commonly referred to as "statins," are not merely due to blockade of cholesterol synthesis. Statins possess important adjunctive properties that may confer additional benefits beyond changes in plasma cholesterol concentrations (25). Epidemiological studies have shown that men treated with statin administration appear to have a lower incidence of sudden death than men without statins (40). Patterson et al. (35) have shown that statins improved baroreflex sensitivity, a predictor of sudden cardiac death. There is evidence that many effects of statins are the result of reduced synthesis of isoprenoid intermediates of the cholesterol biosynthetic pathway, which serve as lipid attachments for intracellular signaling molecules (45). By changing signaling pathways, the beneficial prognostic effect of statins may result from a favorable modulation of the autonomic nervous system (8). However, the specific factors for affecting various signal transduction pathways have remained disappointingly elusive.

There is general agreement that the activation of ATP-sensitive K+ (KATP) channels in neurons has a protective effect during acute settings of cell-damaging conditions (50). In cardiac tissues, KATP channels are observed on cardiomyocytes (31) and pre- and postsynaptic membranes of sympathetic nerves, which can be inhibited by glibenclamide (15). Burgdorf et al. (4) have shown that the release of norepinephrine from sympathetic nerves supplying the heart can be modulated via KATP channels. Although previous studies have demonstrated neuroprotection by the activation of KATP channels in acute settings, whether long-term administration of KATP channel agonists can modulate sympathetic reinnervation in chronic settings remains unknown. Previous studies have demonstrated that statins induce ecto-5'-nucletidase activation and increased interstitial adenosine formation in a setting of acute myocardial infarction in rabbits (46). Statin administration increases ATP breakdown and adenosine formation in human umbilical vein endothelial cells and human microvascular endothelial cells (34). The increased interstitial adenosine is an important trigger of KATP channel opening (46). We have previously demonstrated that adenosine-induced cardioprotection provided by pravastatin can be abolished by an adenosine inhibitor, aminophylline (24). Furthermore, statins have been shown to worsen myocardial mitochondrial respiration in ischemic rat hearts (39). The KATP channel is a high-fidelity metabolic sensor that adjusts membrane potential-dependent cell functions to match metabolic state (30). The effect of statins on inhibition of myocardial mitochondrial respiration renders the remodeled heart more likely to activate KATP channels. Recently, we showed that statin administration can activate KATP channels in hyperlipidemic rabbits (20). Thus, it is of great interest to assess whether pravastatin administration can modulate sympathetic reinnervation by the activation of KATP channels after infarction.

Increased sympathetic nerve density after myocardial injury has been shown to be responsible for the occurrence of lethal arrhythmias and sudden cardiac death in humans (5). Transmural myocardial infarction interrupts efferent sympathetic nerves and denervates viable muscle distal to the myocardial infarction. During the chronic stage of myocardial infarction, regional and global increases of sympathetic nerves have been commonly observed (51). Increased sympathetic nerve activity plays an important role in the generation of ventricular arrhythmia and sudden cardiac death (5). We assessed whether chronic administration of pravastatin can result in attenuated heart reinnervation after infarction through the activation of KATP channels. Because regional sympathetic reinnervation reflects electrophysiological differences, we explored the downstream functional significance of attenuated heart reinnervation by ventricular pacing in a rat myocardial infarction model.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male normocholesterolemic Wistar rats (300–350 g) were subjected to ligation of the anterior descending artery as previously described (23), resulting in infarction of the left ventricular (LV) free wall. Rats were randomly assigned into the following eight treatment groups so as to have approximately the same number of survivors in each group: 1) vehicle; 2) nicorandil (0.1 mg·kg–1·day–1, Chugai Pharmaceutical), a specific mitochondrial KATP channel agonist; 3) pinacidil (0.1 mg·kg–1·day–1, Sigma), a nonspecific KATP channel agonist; 4) pravastatin (5 mg·kg–1·day–1, Sankyo); 5) glibenclamide (1.4 mg·kg–1·day–1), a KATP channel blocker; 6) a combination of nicorandil and glibenclamide; 7) a combination of pinacidil and glibenclamide; and 8) a combination of pravastatin and glibenclamide. Pinacidil has been shown to activate both sarcolemmal and mitochondrial KATP channels, whereas nicorandil activates only mitochondrial KATP channels (32). The doses of nicorandil (9), pinacidil (2), pravastatin (46), and glibenclamide (1) used in this study have been shown to specifically influence KATP channels without the interference of hemodynamics. Drugs were started 24 h after infarction, a time window during which drugs can exert maximum benefits (49). The study duration was designed to be 4 wk because the majority of the myocardial remodeling process in the rat (70–80%) is complete within 3 wk (3). Sympathetic reinnervation has been shown to be present 6 days after injury (33). Drugs were given orally by gastric gavage once a day. To prevent hypoglycemic attacks during the administration of glibenclamide, glucose was supplied, and frequent glucose examinations were performed by the one-touch method. Sham operation served as a control to exclude the possibility of drugs themselves directly altering sympathetic reinnervation. In each treated group, drugs were withdrawn ~24 h before the end of the experiments to eliminate their pharmacological actions. The animal experiments were approved by the Institutional Animal Care and Use Committee and conducted in accordance with local institutional guidelines for the care and use of laboratory animals in the Chi-Mei Medical Center.

Hemodynamics and infarct size measurements. Hemodynamic parameters were measured in anesthetized rats with ketamine (90 mg/kg) intraperitoneally at the end of the study as previously described (23). A polyethylene Millar catheter was inserted into the right carotid artery and connected to a transducer (model SPR-407, Miller Instruments, Houston, TX) to measure LV systolic and diastolic pressure as the mean of measurements of five consecutive pressure cycles. The maximal rates of LV pressure rise and decrease were measured. After the arterial pressure measurements, the heart was rapidly excised and suspended for retrograde perfusion with a Langendorff apparatus for the electrophysiological tests. At the completion of the electrophysiological tests, the atria, right ventricle, and LV were rinsed in cold physiological saline solution, weighed, and immediately frozen in liquid nitrogen after a coronal section of the LV was obtained for infarct size estimation. A section taken from the equator of the LV was fixed in 10% formalin and embedded in paraffin for the determination of infarct size. The infarct size was determined as previously described (23). It has been shown that ventricular remodeling including hypertrophy of the residual myocardium progresses after myocardial infarction only if the infarct size is >30% of the LV (36). With respect to clinical importance, only rats with large infarctions (>30%) were selected for analysis.

Spontaneous and induced arrhythmias. Each heart was perfused with modified Tyrode solution containing (in mM) 117.0 NaCl, 23.0 NaHCO3, 4.6 KCl, 0.8 NaH2PO4, 1.0 MgCl2, 2.0 CaCl2, and 5.5 glucose equilibrated at 37°C and oxygenated with a 95% O2-5% CO2 gas mixture. The perfusion medium was maintained at a constant temperature of 37°C with a constant flow at 4 ml/min as previously described (23). Atrial and ventricular epicardial electrocardiograms were continuously recorded.

After the perfusion of the isolated hearts was completed, hearts were observed for 20 min to allow stabilization of contraction and rhythm. Because the residual neural integrity at the infarcted site is one of the determinants of the response to electrical induction of ventricular arrhythmias (11), only rats with the infarcted area of the LV totally replaced by scar tissue were included. The protocol for pacing and the arrhythmia scoring system used were as previously described (23). When multiple forms of arrhythmias occurred in one heart, the highest score was used. Experimental protocols were typically completed within 10 min.

Real-time RT-PCR of tyrosine hydroxylase mRNA. Real-time quantitative RT-PCR was performed from samples obtained from the remote zone with the TaqMan system (Prism 7700 Sequence Detection System, PE Biosystems) as previously described (23). Tyrosine hydroxylase, a neuron-specific enzyme found in sympathetic efferent nerves, is a commonly used anatomic marker of regional reinnervation (41). For tyrosine hydroxylase, the primers were 5'-TCGCCACAGCCCAAGGGCTTCAGAA-3' (sense) and 5'-CCTCGAAGCGCACAAAATAC-3' (antisense). Coamplification of GAPDH was used as an internal control as previously described (19). For GAPDH, the primers were 5'-CTTCACCACCATGGAGAAGGC-3' (sense) and 5'-GGCATGGACTGTGGTCATGAG-3' (antisense). Standard curves were plotted with the threshold cycle versus log template quantities. For quantification, tyrosine hydroxylase expression was normalized to the expression of the housekeeping gene GAPDH. Reaction conditions were programmed on a computer linked to the detector for 33 cycles of the amplification step.

Western blot analysis of tyrosine hydroxylase. Samples obtained from the remote zone were homogenized with a kinametic Polytron blender in 100 mM Tris HCl (pH 7.4) supplemented with 20 mmol/l EDTA, 1 mg/ml pepstatin A, 1 mg/ml antipain, and 1 mmol/l benzamidin. Homogenates were centrifuged at 10,000 g for 30 min to pellet the particulate fraction. The supernatant protein concentration was determined with the BCA protein assay reagent kit (Pierce). Protein (20 µg) was separated by 10% SDS-PAGE and electrotransferred onto a nitrocellulose membrane. After an incubation with rabbit polyclonal anti-tyrosine hydroxylase antibodies (Chemicon) at 1:500 dilution, the nitrocellulose membrane was then rinsed with a blocking solution and incubated for 2 h at room temperature. Antigen-antibody complexes were detected with 5-bromo-4-chloro-3-indolyl-phosphate and nitroblue tetrazolium chloride (Sigma). Films were volume integrated within the linear range of the exposure using a scanning densitometer. Experiments were replicated three times, and results are expressed as the mean value.

Immunohistochemical experiments of tyrosine hydroxylase, growth-associated factor 43, and neurofilament. To investigate the spatial distribution and quantification of sympathetic nerve fibers, analysis of immunohistochemical staining for tyrosine hydroxylase, growth-associated factor 43 (GAP43; a marker peptide for neuronal regeneration and outgrowth), and neurofilament (a marker for sympathetic nerves) (27) was performed on LV muscles from remote regions (>2 mm outside the infarct). Papillary muscles were excluded from the experiment because variable sympathetic innervation has been reported (6). Paraffin-embedded sections were sliced at a thickness of 5 µm. Tissues were incubated with anti-tyrosine hydroxylase (1:200, Chemicon), anti-GAP43 (1:400, Chemicon), and anti-neurofilament (1:1,000, Chemicon) antibodies in 0.5% BSA in PBS overnight at 37°C. Immunostaining was performed using standard immunoperoxidase techniques (N-Histofine Simple Stain MAX PO kit, Nichirei, Tokyo, Japan). Isotype-identical directly conjugated antibodies served as negative controls.

The density of tyrosine hydroxylase-labeled nerve fibers was qualitatively estimated from 10 randomly selected fields at a magnification of x400. The nerve density was measured on tracings by computerized planimetry (Image Pro Plus) as previously described (21). The value was expressed as the ratio of tyrosine hydroxylase-labeled nerve fiber area to total area. Slides were coded so that the investigator was blinded to rat identification.

Morphometric determination of myocyte size and fibrosis. Because ventricular remodeling after infarction is a combination of reactive fibrosis and myocyte hypertrophy, we measured cardiomyocyte sizes in addition to myocardial weight to avoid the confounding influence of nonmyocytes on cardiac hypertrophy. LV sections from the remote zone were stained with hematoxylin and eosin. For consistency of results, myocytes positioned perpendicularly to the plane of the section with a visible nucleus and cell membrane clearly outlined and unbroken were then selected for the cross-sectional area measurements (22). This area was determined by manually tracing the cell contour on a digitized image acquired on the image-analysis system at a magnification of x400 using computerized planimetry (Image Pro Plus) as previously described (21). A total of 100 myocytes were selected in the LVof each heart and analyzed by an observer blinded to the experimental treatment.

Additionally, heart sections were stained with Sirius red stain to distinguish areas of connective tissue as previously described (22). The percentage of red staining, indicative of fibrosis, was measured (10 fields randomly selected on each section). The value was expressed as the ratio of Sirius red-stained fibrosis area to total infarct area. All sections were evaluated under blinded conditions without prior knowledge as to which section belonged to which rat.

Laboratory measurements. To determine the confounding roles of glucose, insulin, and cholesterol in ventricular remodeling, blood samples from the aorta were assayed at the end of the study. The plasma insulin concentration was measured by collecting 4 ml of blood in test tubes containing 2% EDTA (80 µl/ml blood). Blood samples were immediately centrifuged at 3,000 g for 10 min, and the plasma was stored at –70°C until further analysis. Insulin was measured by an ultrasensitive rat enzyme immunoassay (Mercodia, Uppsala, Sweden).

Because of local release of norepinephrine after sympathetic reinnervation, the tissue from the border zone and remote interventricular zone were obtained for measurements of local norepinephrine levels at the end of the study. Because the samples were collected after perfusion with modified Tyrode solution during electrophysiological experiments, catecholamine in the blood was eliminated. Myocardiums were minced and suspended in 0.4 N perchloric acid with 5 mmol/l reduced glutathione (pH 7.4) and homogenized with a Polytron homogenizer for 60 s in 10 volumes. Samples were immediately centrifuged at 3,000 g for 10 min, and the supernatant was stored at –70°C until further analysis. The supernatant protein concentration was determined with the BCA protein assay reagent kit (Pierce). Total norepinephrine was measured using a commercial ELISA kit (Noradrenalin ELISA, IBL Immuno-Biological Laboratories, Hamburg, Germany).

Statistical analysis. Results are presented as means ± SD. Differences among groups of rats were tested by two-way ANOVA. Subsequently analysis for significant differences between the two groups was performed with a multiple-comparison test (Schèffe's method). The correlation between continuously distributed variables was tested by univariate regression analysis. Electrophysiological data (scoring of programmed electrical stimulation-induced arrhythmias) were compared by a Kruskal-Wallis test followed by a Mann-Whitney test. The significant level was assumed at a value of P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Differences in mortality among the infarcted groups were not found throughout the study. Nicorandil, pinacidil, pravastatin, or glibenclamide had little effect on cardiac gross morphology in sham-operated rats (Table 1). Four weeks after infarction, the infarcted area of the LV was very thin and was totally replaced by fully differentiated scar tissue. The ratio of LV weight to body weight inclusive of the septum remained essentially constant 4 wk after coronary artery occlusion among the infarcted groups. Hemodynamics and infarct size did not differ among the infarcted groups.


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  		Table 1. Cardiac morphology and hemodynamics at the end of the study

 
Pravastatin did not lower serum cholesterol in rats (Table 2), consistent with the notion that compensatory increases in hepatic enzyme production were observed in rats treated with statins. Insulin concentrations were significantly increased in rats administered glibenclamide.


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  		Table 2. Cholesterol, glucose, and insulin levels and plasma and tissue NE concentrations

 
Ventricular remodeling. To characterize the cardiac hypertrophy on a cellular level, morphometric analyses of LV sections from the remote zone were performed on different treatment groups (Fig. 1). Myocytes were significantly hypertrophied in the vehicle-treated infarcted group compared with those in the sham-operated group. Nicorandil, pinacidil, and pravastatin reduced cell areas by 29%, 35%, and 24% compared with the vehicle-treated infarcted group (all P < 0.05). Conversely, rats administered glibenclamide developed cardiomyocyte hypertrophy compared with pravastatin-treated group.


Figure 1
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  		Fig. 1. Morphometric analyses of left ventricular (LV) sections from the remote zone at 4 wk after infarction (magnification: x400). LV cardiomyocyte cross-sectional areas in rats treated with the indicated agents were examined by hematoxylin-eosin staining. The relative myocyte cross-sectional area was normalized to the mean value of sham-operated rats at the end of the study. Top: representative stained images. A, sham; B, vehicle; C, nicorandil (Nic); D, pinacidil (Pin); E, pravastatin (Pra); F, glibenclamide (Glib); G, Nic + Glib; H, Pin + Glib; I, Prav + Glib. Bar = 50 µm. Bottom: quantitative analysis of cardiomyocyte sizes in different treatment groups. *P < 0.05 compared with vehicle-, Glib-, Nic + Glib-, Pin + Glib-, and Pra + Glib-treated groups.

 
Fibrosis of the LV from the remote zone was examined in tissue sections after Sirius red staining, as shown in Fig. 2. Infarcted rats treated with vehicle had significantly larger areas of intense focal fibrosis compared with sham-operated rats (0.11 ± 0.03% vs. 0.03 ± 0.02%, P < 0.05). Compared with vehicle, treatment with either nicorandil, pinacidil, or pravastatin in infarcted rats attenuated fibrosis, as observed by reduced collagen staining. Quantitative analysis showed that collagen formation in the LV was significantly increased in infarcted rats treated with a combination of pravastatin and glibenclamide compared with pravastatin alone-treated rats.


Figure 2
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  		Fig. 2. Top: representative sections from the remote zone with Sirius red staining (red; magnification: x400) at 4 wk after infarction. Collagen deposition within the LV was reduced after the administration of either Nic, Pin, or Pra. A, sham; B, vehicle; C, Nic; D, Pin; E, Pra; F, Glib; G, Nic + Glib; H, Pin + Glib; I, Pra + Glib. Bar = 50 µm. Bottom: LV collagen area fraction (in %). Values are means ± SD. *P < 0.05 compared with vehicle-, Glib-, Nic + Glib-, Pin + Glib-, and Pra + Glib-treated groups.

 
Circulating and myocardial norepinephrine levels. Circulating plasma norepinephrine levels remained similar among the infarcted groups (Table 2). Although cardiac reinnervation has been shown in immunohistochemical staining of tyrosine hydroxylase, GAP43, and neurofilament, it does not imply that the nerves are functional. Thus, to investigate cardiac sympathetic function, we determined ventricular norepinephrine levels. Nicorandil, pinacidil, pravastatin, or glibenclamide administration did not affect basal tissue norepinephrine concentrations in the sham-operated group (data not shown). LV norepinephrine levels were significantly upregulated by 2.02-fold at the remote zone in vehicle-treated rats compared with sham-operated rats (2.54 ± 0.17 vs. 1.26 ± 0.36 µg/g protein, P < 0.0001). Expression was region dependent with a significant increase at the remote zone (2.54 ± 0.17 µg/g protein) compared with the border zone (1.74 ± 0.25 µg/g protein, P = 0.002) after infarction in the vehicle-treated group. In vehicle-treated rats compared with nicorandil-, pinacidil-, or pravastatin-treated rats, LV norepinephrine levels were significantly lower at remote regions. The beneficial effect of nicorandil, pinacidil, and pravastatin on LV norepinephrine levels was reversed by the administration of glibenclamide.

Immunohistochemical analysis, Western blot analysis, and real-time PCR. Tyrosine hydroxylase-immunostained nerve fibers appeared to be oriented in the longitudinal axis of adjacent myofibers (Fig. 3, top). Tyrosine hydroxylase-positive nerve density was significantly increased in vehicle-treated infarcted rats compared with sham-operated rats (Fig. 3, bottom). Rats in the nicorandil-, pinacidil-, and pravastatin-treated groups showed less nerve density at remote regions than rats in the vehicle-treated group (0.03 ± 0.02%, 0.07 ± 0.02%, and 0.06 ± 0.01% vs. 0.92 ± 0.23% in the vehicle group, all P < 0.001). Rats administered glibenclamide developed stronger signals of the immunostained profile than rats treated with pravastatin alone (0.85 ± 0.21% vs. 0.06 ± 0.01%, P < 0.001). Similar to the tyrosine hydroxylase results, GAP43-positive (Fig. 4) and neurofilament-positive (Fig. 5) nerve densities were significantly increased in vehicle-treated infarcted rats than in sham-operated rats. These morphometric results mirrored those of norepinephrine contents.


Figure 3
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  		Fig. 3. Top: immunohistochemical staining for tyrosine hydroxylase from the remote region (magnification: x400). Tyrosine hydroxylase-positive nerve fibers (brown color) were located between myofibrils and oriented in a longitudinal direction. Myocytes were not stained and appear pale in this view. A, sham; B, vehicle; C, Nic; D, Pin; E, Pra; F, Glib; G, Nic + Glib; H, Pin + Glib; I, Pra + Glib. Bar = 50 µm. Bottom: nerve density area fraction (in %) at the remote zone. *P < 0.05 compared with vehicle-, Glib-, Nic + Glib-, Pin + Glib-, and Pra + Glib-treated groups.

 

Figure 4
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  		Fig. 4. Top: immunohistochemical staining for growth-associated factor 43 (GAP43) from the remote region (magnification: x400). GAP43-positive staining was markedly increased in groups treated with vehicle, Glib, Nic + Glib, Pin + Glib, and Pra + Glib. A, sham; B, vehicle; C, Nic; D, Pin; E, Pra; F, Glib; G, Nic + Glib; H, Pin + Glib; I, Pra + Glib. Bar = 50 µm. Bottom: nerve density area fraction (in %) at the remote zone. *P < 0.05 compared with vehicle-, Glib-, Nic + Glib-, Pin + Glib-, and Pra + Glib-treated groups.

 

Figure 5
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  		Fig. 5. Quantitative analysis of neurofilament-stained nerve density area fraction (in %) at the remote zone. *P < 0.05 compared with vehicle-, Glib-, Nic + Glib-, Pin + Glib-, and Pra + Glib-treated groups.

 
Western blot analysis showed that tyrosine hydroxylase levels were significantly upregulated by 8.0-fold at the remote zone in vehicle-treated rats compared with sham-operated rats (P < 0.0001; Fig. 6). In vehicle-treated rats compared with nicorandil-, pinacidil-, and pravastatin-treated rats, tyrosine hydroxylase levels were significantly lower at the remote zone. The attenuated expression of pravastatin-related tyrosine hydroxylase could be reversed to levels similar to those of vehicle-treated infarcted rats after the addition of glibenclamide.


Figure 6
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  		Fig. 6. Top: Western blot analysis of tyrosine hydroxylase in homogenates of the LV from the remote zone. Bottom: quantitative analysis results for tyrosine hydroxylase obtained by densitometry from the same blots. In vehicle-treated rats compared with Nic-, Pin-, and Pra-treated rats, tyrosine hydroxylase levels were significantly lower at the remote zone. The attenuated expression of Pra-related tyrosine hydroxylase could be reversed to levels similar to those of vehicle-treated infarcted rats after the addition of Glib. Relative abundance was obtained by normalizing the density of tyrosine hydroxylase protein against that of β-actin. Results are means ± SD of 3 independent experiments. *P < 0.05 compared with vehicle-, Glib-, Nic + Glib-, Pin + Glib-, and Pra + Glib-treated groups.

 
PCR amplification of cDNA revealed that mRNA levels of tyrosine hydroxylase showed a 6.3-fold upregulation at the remote zone in vehicle-treated rats compared with sham-operated rats (P < 0.0001; Fig. 7). Thus, mRNA levels of tyrosine hydroxylase changed in parallel to the tissue immunohistochemical analyses, implying that the production of tyrosine hydroxylase is a critical regulation step for its local activation. In either nicorandil-, pinacidil-, or pravastatin-treated rats, levels of tyrosine hydroxylase mRNA were significantly decreased compared with those in vehicle-treated rats. Conversely, glibenclamide-treated rats showed a marked increase of tyrosine hydroxylase mRNA compared with the pravastatin alone-treated group.


Figure 7
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  		Fig. 7. LV tyrosine hydroxylase mRNA levels. Each mRNA level was normalized to the mRNA level of GAPDH. Values are means ± SD. *P < 0.05 compared with vehicle-, Glib-, Nic + Glib-, Pin + Glib-, and Pra + Glib-treated groups.

 
Electrophysiological stimulation. To further elucidate the physiological effect of attenuated sympathetic reinnervation, ventricular pacing was performed. Arrhythmia scores in sham-operated rats were very low (0) (Fig. 8). In contrast, ventricular tachyarrhythmias consisting of ventricular tachycardia and ventricular fibrillation were inducible by programmed stimulation in vehicle-treated infarcted rats. Nicorandil, pinacidil, and pravastatin treatment significantly decreased the inducibility of ventricular tachyarrhythmias compared with vehicle- and glibenclamide-treated groups. Glibenclamide administration significantly increased arrhythmia scores in pravastatin-treated rats compared with pravastatin alone-treated rats.


Figure 8
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  		Fig. 8. Inducibility quotient of ventricular arrhythmias by programmed electrical stimulation 4 wk after myocardial infarction. *P < 0.05 compared with vehicle-, Glib-, Nic + Glib-, Pin + Glib-, and Pra + Glib-treated groups.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Our present study is the first one to investigate the effect of pravastatin on sympathetic reinnervation after infarction. The major novel findings were twofold. First, KATP channel agonist administration by either nicorandil, pinacidil, or pravastatin attenuated sympathetic reinnervation, which resulted in an attenuated incidence of pacing-induced fatal arrhythmia. Second, the large attenuation of arrhythmia was reversed when the activation of KATP channels produced by pravastatin administration was prevented with glibenclamide. From these observations, we conclude that pravastatin-induced activation of KATP channels is a major mechanism underlying attenuated sympathetic reinnervation after infarction. Our results were compatible with the recent findings of Pliquett et al. (37), who showed that statins normalize autonomic neural control in heart failure induced by ventricular pacing.

The beneficial effect of pravastatin on sympathetic reinnervation was demonstrated by the activation of KATP channels. Our conclusions are supported by three lines of evidence. First, excessive sympathetic reinnervation after infarction, a process modulated by the activation of KATP channels, was noted at the remote zone. Our results are consistent with the notion that the nerve sprouting occurs primarily at the remote zone rather than at the border zone (47, 51). KATP channels have been shown to be associated with attenuated sympathetic reinnervation after infarction, as assessed by KATP channel agonist administration. Either nicorandil or pinacidil administration can attenuate sympathetic reinnervation with similar potency. Although we did not use selective mitochondrial and sarcolemmal KATP channel antagonists to assess sympathetic reinnervation, the similar potency to attenuate reinnervation by nicorandil and pinacidil may suggest that mitochondrial KATP channels, but not sarcolemmal KATP cannels, play a role in mediating sympathetic reinnervation. However, the present results have not totally dismissed a role of sarcolemmal KATP channels in sympathetic reinnervation. Although dissimilar structures, nicorandil and pinacidil appear to share a common mediator, tyrosine hydroxylase, in which transcription levels may play a role in the signal transduction pathway. The involvement of KATP channels is further supported by the antagonizing action of glibenclamide. The finding was consistent with a clinical study (16) showing that KATP channel agonists attenuate sympathetic reinnervation. Second, pravastatin administration can attenuate sympathetic reinnervation at the remote zone, as documented by biochemical, immunohistochemical, molecular, and electrophysiological methods. The effects of pravastatin appear to be specific because no effect of drug on sympathetic reinnervation can be demonstrated in groups without intervening infarction (data not shown) and to be local because nerves at the border zone were not affected by the treatment. Finally, the severity of pacing-induced fatal arrhythmias was associated with the degree of sympathetic reinnervation. This model has the advantage of isolated preparations, with the absence of influence from circulating hormones and hemodynamic reflexes. The finding was further supported by Cao et al. (5), who showed that increased postinjury sympathetic nerve density may be responsible for the occurrence of ventricular arrhythmia and sudden cardiac death in animals and patients.

In this study, we demonstrated that pravastatin administration attenuated sympathetic reinnervation in infarcted hearts. The mechanisms by which pravastatin affects sympathetic reinnervation remain undefined. However, several factors can be excluded. First, hemodynamics can be excluded. A previous study (12) has shown a significantly reverse correlation between LV end-diastolic pressure and tyrosine hydroxylase-immunostained profiles. Pravastatin did not exert any hemodynamic effects, nor was it associated with a change in heart rate. Second, differences in insulin concentrations can be excluded. Although insulin secretion significantly increases in rats treated with glibenclamide, as shown in this study, the increased insulin levels cannot be a confounding factor of sympathetic reinnervation. Hyperinsulinemia has been shown to attenuate sympathetic outflow by interfering primarily with {alpha}2-adrenergic activation (26). Thus, if rats treated with glibenclamide had induced sympathetic reinnervation by increasing insulin levels, we should have obtained a reduction rather than augmentation of sympathetic reinnervation. Furthermore, when KATP channels agonists were administered, sympathetic reinnervation was significantly attenuated with similar insulin levels compared with infarcted rats treated with vehicle, suggesting that factors other than insulin may contribute to the pathogenesis of attenuated sympathetic reinnervation. Our results showed a robust phenomenon despite any potentially confounding effect of insulin-mediated sympathetic reinnervation. Third, differences in glucose concentrations can be excluded. Hypoglycemia has been shown to adversely affect subsequent sympathetic activity (7). However, when KATP channel agonists were administered, sympathetic reinnervation was improved without significant changes in blood glucose levels, suggesting that factors other than glucose may contribute to the pathogenesis of attenuated sympathetic reinnervation. Finally, differences in infarct sizes can be excluded. The degree of sympathetic reinnervation has been shown to be related to the infarct size (10). Successful fiber reinnervation appears to be dependent on repopulating sheaths with Schwann cells (10), which would be injured according to the sizes of infarction. The possibility was excluded in this study due to similar infarct sizes among the groups.

Exactly how the activation of KATP channels leads to attenuated sympathetic reinnervation cannot be determined from this study. The activation of KATP channels induced by adenosine after pravastatin administration has been shown to attenuate sympathetic reinnervation by inhibiting the expression of endothelin-1 (48). Activation of endothelin-1 plays an important role in sympathetic innervation of the heart (14). Endothelin-1 blockade attenuated sympathetic innervation of the heart (14). Thus, pravastatin-mediated KATP channel activation may be critical for sympathetic reinnervation after myocardial infarction. This inference was supported by the observation that pravastatin attenuated sympathetic reinnervation, whereas glibenclamide abolished the protection. A previous study (38) has shown that glibenclamide inhibits the activity of endogenous ecto-5'-nucleotidase and decreases adenosine concentrations in the interstitial space of ventricular muscles. In contrast, pravastatin stimulates ecto-5'-nucleotidase (46), which, in turn, results in increased adenosine concentrations.

Other mechanisms. Although the present study suggests that the mechanisms of pravastatin-induced neuroprotection may be related to opening of KATP channels, other potential mechanisms need to be studied. First, statins may directly cause neurite loss by blockade of isoprenylation of p21Ras proteins (43), a member of small membrane-associated GTP-binding proteins. p21Ras proteins play a role in the signal transduction of neuronal differentiation induced by nerve growth factor (43). Inhibition of isoprenylation results in the lack of all subsequent posttranslational processing of Ras proteins and function, including the response to nerve growth factor. However, the use of statins to inhibit the isoprenylation of p21Ras proteins is unlikely because a very high dose of statins is needed to block the process (44). Second, tyrosine hydroxylase can be expressed in intracardiac and extracardiac sympathetic targets. The rat heart possesses intrinsic adrenergic cells (18). These specialized nonneuronal cardiomyocytes serve as sources of myocardial tyrosine hydroxylase. Conceivably, they might provide a substitute adrenergic support system following sympathetic denervation in infarcted hearts. Because sympathetic nerve endings do not contain tyrosine hydroxylase mRNA, which is located in neuronal soma in sympathetic ganglia, the increased mRNA of myocardial tyrosine hydroxylase cannot be explained by sympathetic reinnveration. Tyrosine hydroxylase-containing intrinsic cardiac adrenergic cells might explain why tyrosine hydroxylase mRNA is upregulated in infarcted rats. However, the tyrosine hydroxylase in intrinsic adrenergic cells could not be a confounding factor because other nerve-specific markers, such as GAP43 and neurofilament, which only stained sympathetic nerve fibers, confirmed reinnervation in the ventricle.

Clinical implications. Our results confirm and extend our previous observation that pravastatin not only attenuated ventricular remodeling assessed by cardiomyocyte hypertrophy and fibrosis but also ventricular arrhythmias after infarction. Our finding was consistent with a recent observation that increased sympathetic activity is associated with cardiac hypertrophy (18). So far, no studies have directly addressed the question of whether or not treatment with pravastatin may influence the susceptibility to ventricular arrhythmias. Our results show that an association between pravastatin administration and ventricular arrhythmias is anatomically and functionally linked. The major implications of our results are twofold. First, our results may extend previous studies of activated KATP channels from release to synthesis of norepinephrine (4). The attenuated expression of tyrosine hydroxylase immunoreactivity and tissue norepinephrine levels in the myocardium could reflect the reduced production of norepinephrine. Second, based on our experimental findings in rats, we speculated that in a clinical setting, the use of glibenclamide in patients with Type 2 diabetes mellitus may exert detrimental effect on arrhythmias and, in so doing, potentially contribute to the incidence of sudden cardiac death. The finding may explain, at least in part, why diabetic patients have a higher subsequent risk of mortality than nondiabetic patients at the late stage of postinfarction ventricular remodeling after justification of the adverse baseline characteristics (29).

Study limitations. There are some limitations in the present study that have to be acknowledged. First, only one time point after infarction was studied. The immunohistochemical and electrophysiological experiments were not performed until 4 wk after infarction. The interaction between reinnervation and ventricular arrhythmias over the 4-wk period was not assessed. Nori et al. (33) have shown that reinnervation starts as early as 6 days after infarction in rats. Thus, it remains unclear whether pravastatin either inhibits development (late stage of infarction) or causes degeneration (early stage of infarction) of myocardial sympathetic nerve fibers. Activation of KATP channels has been shown to attenuate cardiac sympathetic nerve injury at the acute stage of myocardial ischemia (28), which was not consistent with the pravastatin-induced changes of sympathetic innervation. This implies that in addition to the severity of nerve injury at the acute stage, nerve sprouting may occur at the late stage of myocardial infarction at which pravastatin attenuates sympathetic reinnervation. Future research should identify these early events and trace their progression after the administration of pravastatin after infarction. In addition, because only rats with large infarcts (>30%) were examined, it is possible that the reaction of sympathetic reinnervation may be different with smaller infarcts. Our finding cannot necessarily be extrapolated to animals with small to moderate infarction. Finally, a potential problem with the present study is the use of glibenclamide as an antagonist of KATP channels when there are many potential nonspecific targets of glibenclamide, including inhibition of Na+ channels and opening of Ca2+ channels (17). Glibenclamide can enhance resting and stimulation-evoked releases of norepinephrine, an effect unrelated to KATP channel blockade (42). These alternative effects could confound the interpretation of the present study. However, it seems unlikely because KATP channel agonists markedly reversed the impaired effect of glibenclamide on tissue norepinephrine levels.

Conclusions. These data show excessive myocardial reinnervation after infarction, which is modulated by the activation of KATP channels. Pravastatin administration after infarction can reduce the inducibility of ventricular arrhythmias as a result of attenuated sympathetic reinnervation, probably through a KATP channel-dependent mechanism. This may be a new beneficial role of pravastatin in decreasing cardiovascular mortality.


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 ABSTRACT
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This work was supported Chi-Mei Medical Center Grants CMFHT9501, CMFHR9502, CMFHR9506, and CMTMU9501 and National Science Council, Republic of China, Grant 95-2314-B-384-009.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N.-C. Chang, Cardiology Sect., Dept. of Medicine, Taipei Medical Univ., 252, Wu-Hsing St., Taipei, Taiwan (e-mail: ncchang{at}tmu.edu.tw)

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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