Cirrincione, Georgina M. BS*; Boluyt, Marvin O. PhD*; Hwang, Hyun Seok MS*; Bleske, Barry E. PharmD†
HMG-CoA reductase inhibitors (statins) have the therapeutic action of reducing levels of circulating cholesterol. A number of statins are on the market and are approved by the FDA for the treatment of hyperlipidemia.1,2 Statins do not affect cholesterol exclusively, but do appear to act by additional mechanisms on the kidneys, bone, cardiovascular system, and glucose metabolism. Specific nonlipid lowering effects of statins include antioxidant properties,3 atherosclerotic plaque stabilization,4 anti-inflammatory action,4,5 modulation of endothelial function,3 improvement of left ventricular (LV) remodeling and function,6–8 beneficial vascular effects,9 and effects on thrombosis and vasculogenesis.5,10 Potential effects of statin therapy on LV remodeling and function are of particular importance because of the prevalence of congestive heart failure in older individuals.11
Interstitial fibrosis is characteristic of the failing heart.12,13 Alterations in extracellular matrix (ECM) components contribute to left ventricular hypertrophy and ventricular dysfunction. A considerable body of research has focused on preventing or reversing pathological fibrosis in the treatment of heart failure.13 Several lines of evidence indicate that statins have benefits for cardiac remodeling and LV function. Administration of cerivastatin after myocardial infarction in rats attenuated declines in LV function and increases in collagen gene expression.6 Fluvastatin therapy in mice with myocardial infarction improved LV function, reduced myocyte hypertrophy, and attenuated increases in collagen volume fraction.7 Cerivastatin treatment attenuated hypertension, cardiac hypertrophy, and cardiac fibrosis in transgenic rats that overexpressed renin and angiotensinogen.8 These data suggest that statin therapy influences cardiac remodeling positively in hemodynamic-overload models of heart failure; the mechanisms by which statins exert beneficial effects on remodeling require clarification.
The influence of statins on fibrosis may relate to their effects on transforming growth factor-β1 (TGF-β1) and angiotensin II. TGF-β1 regulates expression of collagen and other ECM components in the heart; expression of TGF-β1 peaks at 1 day and expression of fibronectin and procollagen genes peaks at 3 days in the heart after aortic constriction.14 Angiotensin II promotes the synthesis of collagen I and depresses the collagenase-mediated extracellular degradation of collagen fibers.13 Statins decrease heart angiotensin II content after aortic stenosis and inhibit TGF-β1 expression and increases in ECM-degrading protease activity in the glomeruli and renal mesangial cells.15,16 These findings suggest that statins may alter pathological remodeling in heart failure by influencing the expression of TGF-β1 and multiple ECM components.
Rosuvastatin (RSV) is a relatively new hydrophilic 3-hydroxy-3-methylglutaryl CoA reductase inhibitor with unique pharmacological and pharmacokinetic properties including but not limited to enhanced potency and longer plasma half-life.1 The benefits of RSV cannot be fully explained on the basis of cholesterol reduction alone. Hydophilic statins may be less likely to exert pleiotropic effects because the uptake into nonhepatic cells is reduced compared with its lipophilic relatives, but several studies have reported pleiotropic effects.5,9 RSV has beneficial cardiovascular effects,9 but the specific mechanisms are unclear.
Based on the aforementioned studies, it was hypothesized that HMG-CoA reductase inhibition would modulate myocardial remodeling in maladaptive cardiac hypertrophy by attenuating the induction of TGF-β1 after 1 day and downstream ECM target genes after 3 days of pressure overload. To evaluate this possibility, the effects of RSV on the expression of TGF-β1, fibronectin, and collagen were studied in a model of pressure overload by aortic constriction, which induces a marked increase in the expression of TGF-β1, fibronectin, and collagen genes.14
MATERIALS AND METHODS
RSV was a generous gift from AstraZeneca (London). Reagents were from Sigma (St Louis, MO), unless otherwise stated. Radioactively labeled 32P was from Amersham (Piscataway, NJ).
Male Sprague–Dawley rats were obtained from Charles River Laboratories. Animal protocols were approved by the University Committee on Use and Care of Animals at the University of Michigan. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
Six-week-old male Sprague–Dawley rats weighing 250 to 275 g were maintained on a 12 h:12 h light:dark cycle with food and water ad libitum. When rats weighed 300±10 g they were randomized to 1 of 4 groups: sham-vehicle (SH-V), aortic constriction-vehicle (AC-V), aortic constriction-RSV 2 mg/kg/day (AC-LO), or aortic constriction-RSV 10 mg/kg/day (AC-HI). RSV was dissolved fresh daily in sterile normal NaCl and did not stand more than 30 min before injection. One day before aortic constriction, rats received RSV or vehicle by injection into the intraperitoneal (IP) cavity. IP administration continued once daily throughout the study period. One day after the first injection, animals underwent aortic constriction or sham operation. Rats were killed 1 or 3 days after surgery.
Aortic constriction was conducted as described previously.17
At designated times, the rats were sedated and ventilated with 1.5% isoflurane, balance oxygen, and the chest was opened. A needle-tipped catheter linked to a MLT844 pressure transducer (AD Instruments, Mountain View, CA) was inserted into the apex of the LV for a period not exceeding 3 min, during which LV function was measured. LV function data were collected, stored, and analyzed with a PowerLab 8SP system (AD Instruments). Data from rats with a heart rate <200 bpm were excluded from the analyses of LV function to avoid excessive effects of anesthesia. Blood was collected and LVs were subsequently dissected quickly on iced saline, weighed, and rapidly frozen with liquid nitrogen. The heart was dissected into right ventricle, septum, LV free wall, left atrium, and right atrium. The LV free wall was further subdivided into three pieces of unequal size. One portion of the LV free wall was used for the isolation of total RNA, another for protein, and another for dry weight. The LV dry:wet weight ratio obtained from the small piece of LV was then used to calculate the dry weight of the entire LV including the septum. Tissues were stored at −80°C for subsequent isolation of RNA and protein.
Total RNA was isolated from LVs as described previously.17,18 All mRNA values are expressed in relative amount per gram tissue dry weight, using the 18S-normalized value and adjusting for RNA yield, as described previously.17
Complementary DNA probes were synthesized and radiolabeled from a template by the random prime method. The probes for fibronectin, pro-α1(I) collagen, atrial natriuretic factor, TGF-β1, glyceraldehyde-3-phosphate dehydrogenase, and 18S were described previously.17,18 Oligonucleotides were radiolabeled by terminal deoxynucleotide transferase with [α-32P]D-adenosine triphosphate.
Western blotting procedures were described previously.17 Frozen tissue samples were homogenized with homogenization buffer (tris 62.5 mmol/L, aprotinin 10 mg/L, leupeptin 10 mg/L, phenylmethylsulfonylfluoride 1 mmol/L, orthovanadate 1 mmol/L, Triton X-100 1%), then centrifuged at 4°C at 5000g for 5 min. Protein concentrations were determined by the BCA protein assay (Pierce). Protein samples (20 μg) were heated at 85°C for 3 min and then separated on four 15% Tris-HCl polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride membranes. The membranes were blocked with blocking buffer (0.01 mmol/L Tris pH 6.8, 0.15 mmol/L NaCl, 5% nonfat milk, and 0.1% BSA) for 1 h at room temperature. Then the blots were incubated with monoclonal anti-collagen type III antibody (Sigma) at 1:10,000 diluted with blocking buffer for 2 h. Next, the membranes were washed and incubated with anti-mouse secondary antibody conjugated to horseradish peroxidase at 1:2500 diluted with blocking buffer. The protein bands for collagen type III were visualized with ECL reagents (Amersham, Arlington Heights, IL). We estimated the expression of collagen type III by staining the membrane with Ponceau S and normalizing the collagen type III signal to that of multiple Ponceau S-stained protein bands.
RSV Plasma Concentration
RSV (ZD4522) levels were determined in plasma by reverse-phase liquid chromatography with electrospray ionization tandem mass spectrometry. Plasma standards, quality controls, and study samples were transferred robotically to a 96-well filter plate. Plasma proteins were precipitated by addition of ethanol solution containing the internal standard D6ZD4522. Vacuum was applied to the filter plate and the filtrate was moved to a 96-well collection plate. The filtrate was evaporated to dryness and reconstituted with an acetonitrile:water solution. The extract was then injected onto a Polar RP analytical column connected to the mass spectrometer, where the retained analytes were detected by multiple reaction monitoring via positive electrospray ionization tandem mass spectrometry. The upper and lower limits of quantitation were 100 ng/mL and 0.0500 ng/mL from a 200-μL aliquot of undiluted plasma.
Values are mean±SE. Differences between groups were determined with ANOVA and the LSD post hoc test. The α level was 0.05.
RSV Levels in Plasma
To document that RSV was circulating, plasma concentrations of RSV were measured at harvest, 14 to 24 h after the last injection. Plasma RSV averaged 2.3 ng/mL and 1.3 ng/mL in the AC-LO groups and 8.8 ng/mL and 4.8 ng/mL in the AC-HI groups 1 and 3 days after AC, respectively (Table 1). It is not clear why the levels of RSV were lower in the 3-day groups compared with the 1-day groups. Nevertheless, plasma RSV was appropriately elevated to efficacious levels in the RSV-treated rats at the time of tissue harvest.
Consistent with previous studies,17 robust hypertrophic growth of the LV was observed (Fig. 1, Tables 2 and 3). Aortic constriction induced a 25% increase in LV weight after 3 days; AC-LO and AC-HI-treated rats exhibited similar increases (all P<0.01 vs SH-V). RSV had no effect on the magnitude of LV hypertrophy induced by aortic constriction.
Left Ventricular Function In Situ
LV peak systolic pressure and developed pressure tended to be higher in all AC groups compared to sham-operated rats at 1 day, but not at 3 days post-AC (Fig. 1, Tables 2 and 3). The maximum rates of positive and negative change in LV pressure normalized to developed pressure (normalized +dP/dt and normalized −dP/dt) were not different after 1 day, but were decreased after 3 days of aortic constriction (Fig. 1). The normalized dP/dt values were significantly depressed in each of the AC groups but were unaffected by RSV treatment.
Expression of mRNAs and Proteins Associated With the Extracellular Matrix
Fibronectin mRNA expression is a marker for fibrosis because pressure-overload hypertrophy is associated with a rapid increase in fibronectin expression and a subsequent accumulation of excessive ECM.14,19 LV expression of fibronectin was 4- and 5-fold greater in hearts of AC-V compared to SH-V rats 1 and 3 days post-operation (Fig. 2). The aortic constriction-induced increase in levels of fibronectin mRNA was not significantly attenuated by either dose of RSV (power >0.90). Rather, there was a weak dose-dependent trend for the levels of fibronectin mRNA to be augmented in RSV-treated rats 1 day after aortic constriction. These data strongly refute the hypothesis that HMG-CoA reductase inhibition would attenuate the aortic constriction-induced increase in fibronectin expression.
Expression of pro-α1(I) collagen mRNA is an indication of the acute fibrosis response that peaks after 3 days of pressure-overload hypertrophy.14 Accordingly, LV expression of type I collagen mRNA was 2.5-fold greater in hearts of AC-V compared to SH-V rats 3 days post-operation (Fig. 2). The aortic constriction–induced increase in levels of pro-α1(I) collagen mRNA was not significantly attenuated by either dose of RSV (power >0.80), although there was a significant difference between the high- and low-dose groups. These data refute the hypothesis that HMG-CoA reductase inhibition would attenuate the aortic constriction-induced increase in pro-α1(I) collagen expression.
TGF-β1 is a regulator of ECM production; its peak expression after aortic constriction precedes that of fibronectin and collagen.14 Aortic constriction significantly increased the levels of TGF-β1 mRNA in the LV (Fig. 2) at 3 days post-operation. There was, however, no significant effect of RSV on the aortic constriction-induced increase in abundance of TGF-β1 mRNA after 3 days of aortic constriction (β>0.90). These data fail to support the hypothesis that HMG-CoA reductase inhibition would attenuate the aortic constriction–induced increase in LV TGF-β1 mRNA expression.
To examine a protein constituent of the ECM, the levels of collagen type III protein were measured by immunoblotting. Collagen type III protein levels did not increase after 3 days of aortic constriction, nor were collagen type III protein levels modified in the groups treated with RSV (Fig. 3). These data indicate that collagen type III protein levels do not accumulate disproportionately by 3 days after aortic constriction.
Expression of mRNAs Associated With Cardiac Hypertrophy
To aid interpretation, the expression of 3 well-known markers of LV hypertrophy was studied, atrial natriuretic factor (ANF), α-myosin heavy chain (α-MHC), and β-MHC. Ventricular expression of ANF is a reliable marker of cardiac hypertrophy that typically exhibits elevated levels within several days of the onset of pressure overload and other hypertrophic stimuli.20 There were no differences in LV ANF mRNA levels at 1 day post-operation; a marked increase in expression of ANF in the LV was observed in the pressure-overloaded rats 3 days post-operation (Fig. 4). There were no significant effects of RSV treatment on ANF mRNA levels.
Pressure overload typically induces a decrease in the expression of α-MHC and an increase in the expression of β-MHC. In the present study, there were no significant differences among any of the 4 groups in the expression of α-MHC mRNA at either 1 or 3 days after aortic constriction (Fig. 4). The expression of β-MHC mRNA was significantly elevated 3 days after aortic constriction (Fig. 4). Although RSV treatment did not alter the effect of pressure overload at the 3-day time point, it significantly augmented the aortic constriction-induced increase in β-MHC mRNA at the 1-day point in a manner that appeared to be dose dependent.
Aortic constriction produced the expected changes in LV hypertrophy and expression of TGF-β1, fibronectin, pro-α1(I) collagen, and β-MHC.14,17 In the statin-treated groups, the changes induced by aortic constriction were not different from those in the vehicle-treated group. Thus, the results clearly demonstrate that HMG-CoA reductase inhibition does not attenuate the acute changes in ECM gene expression or fetal gene reexpression that are typically observed in the rat model of pressure overload.
Relationship of This Study to Other Research
Based on the pleiotropic effects of statins and on previous cardiac remodeling studies,6–8,15,16 it was hypothesized that HMG-CoA reductase inhibition would preserve cardiac function and inhibit the expression of genes responsible for the development of fibrosis. The time points of 1- and 3-days post-aortic constriction used in this study were chosen specifically because expression of TGF-β1 peaks at 1 day and expression of fibronectin and procollagen genes peaks at 3 days in the heart after aortic constriction.14 Because the elevation of matrix encoding mRNAs is a likely mechanism of fibrosis that precedes the accumulation of matrix protein, we wanted to determine whether RSV would inhibit the rapid, early induction of the TGF-β1, fibronectin, and procollagen mRNA expression typically produced by acute pressure overload. Evidence also suggested that TGF-β1 may be involved in the beneficial effects of statins.15,16 The presence of clinically relevant levels of the HMG-CoA reductase inhibitor RSV had no effect on the acute expression of TGF-β1 or other ECM genes in this acute pressure overload rat model.
Lack of Statin Effect on ECM Gene Expression
The findings of a neutral impact of statin therapy on ECM gene expression and other parameters in this study were unexpected and unambiguous. It is unlikely that the findings are caused by problems associated with drug administration technique, type of statin, or an inadequate number of animals. Plasma measurements of RSV demonstrated drug absorption and a dose-response increase in plasma levels. The doses of 2 and 10 mg/kg that we used exceed the highest Food and Drug Administration – approved human dose by approximately 4-fold and 20-fold, respectively. In response to a single oral dose of 12 mg/kg in rats, plasma levels of RSV peak 1 h after administration, declined to approximately 30% of peak levels by 3 h postadministration, and decline at a much slower rate thereafter. Therefore, the measurements of plasma RSV at 14 to 24 h after the last daily injection (Table 1) represented trough levels rather than peak or average levels during the 24-h period.21 Most animal studies have used doses ranging from 0.2 to 10 mg/kg. Two rat studies, in which multiple doses ranging from 0.2 to 20 mg/kg were used, reported that beneficial extrahepatic effects were observed dose dependently between 0.2 and 10 mg/kg, but that effects were markedly attenuated when 20 mg/kg RSV was given.9,22 To address this issue in more detail we examined the relationship between plasma RSV levels and expression of 3 genes. There was no significant relationship between plasma RSV values and levels of TGF-β1, fibronectin, or β-MHC mRNA, even when data from 4 outlier animals with unusually high plasma RSV levels (100–200 ng/mL) were included in the analyses (data not shown). Therefore, it is extremely unlikely that the neutral effect in the present study is caused by lack of RSV availability. The possibility that RSV may have exerted effects on the expression of ECM genes if administration had been initiated >1 day before aortic constriction, however, cannot be excluded. Both lipophilic and hydrophilic statins have similar profiles regarding the pleiotropic effects.23,24 Furthermore, there is ample evidence that RSV has beneficial cardioprotective effects.9,25 Thus, it seems unlikely that the hydrophilic nature of RSV is responsible for the lack of effect on ECM gene expression. Finally, power analyses indicated that β>0.80 for each of the key variables studied.
Effects of RSV on Expression of β-MHC
The increased expression of β-MHC mRNA in response to pressure overload is typically slow, such that it is slightly but not significantly increased by 1 day post-AC, but is significantly elevated at 2 to 3 days post-AC (unpublished observations of the authors). It appears that RSV, in a dose-dependent manner, accelerates the increase in β-MHC mRNA, but that the untreated groups catch up by 3 days. This suggests that the magnitude of increase in β-MHC mRNA is not affected by RSV, but that it reaches its new elevated level earlier in the presence of RSV.
Implications for the Effects of Statins on Remodeling
Although the present data show that HMG-CoA reductase inhibition does not affect acute expression of TGF-β1 or ECM genes after pressure overload, the data do not preclude the possibility that ECM gene expression could be attenuated by longer treatment. To evaluate this possibility, a long-term (≥1 month) study in this pressure overload model would be required. Long-term treatment of pressure-overloaded rats with statins clearly provides significant benefits in terms of cardiac remodeling.26,27 Alternatively, statin therapy may have no effect on TGF-β1, procollagen 1, or fibronectin expression over either the short- or the long-term. Rather, the statin influence may be directed at ECM component turnover by targeting matrix-degrading enzymes such as the matrix metalloproteinases (MMPs). It is postulated that remodeling of the ECM by activated MMPs contributes to the progression of ventricular remodeling in chronic heart failure.28,29 Studies of animal models of heart disease indicate elevated levels of MMP-2, MMP-9, and MMP-13.29,30 Numerous studies have demonstrated that statins, especially in postinjury models, decrease MMP activity.7,31
At therapeutic concentrations, lipophilic statins are believed to inhibit cardiac hypertrophy and apoptosis by blocking prenylation of the small guanosine triphosphate–binding proteins Rac and Rho.16,32–35 The present data indicate that in the presence of the hydrophilic RSV, cardiac hypertrophy in response to pressure overload was not inhibited (Fig. 1, Table 1).
HMG-CoA reductase inhibition by RSV did not alter the early responses of the rat LV to aortic constriction in terms of hypertrophy, ECM gene expression, or expression of other hypertrophy-associated genes. The findings suggest that a delayed or secondary effect on ECM synthesis or antifibrotic effects by other mechanisms may be responsible for the favorable ventricular remodeling profiles that have been reported with statin therapy.
The authors are grateful to Julie Zalikowski and Carmen DiOrio for making the plasma RSV measurements.
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