Impact of hydroxymethylglutaryl coenzyme a reductase inhibition on left ventricular remodeling after myocardial infarction

Journal of the American College of Cardiology © 2002 by the American College of Cardiology Foundation Published by Elsevier Science Inc.

Vol. 40, No. 9, 2002 ISSN 0735-1097/02/$22.00 PII S0735-1097(02)02375-6

Impact of Hydroxymethylglutaryl Coenzyme A Reductase Inhibition on Left Ventricular Remodeling After Myocardial Infarction An Experimental Serial Cardiac Magnetic Resonance Imaging Study Matthias Nahrendorf, MD,* Kai Hu, MD,* Karl-Heinz Hiller, PHD,† Paolo Galuppo, PHD* Daniela Fraccarollo, PHD,* German Schweizer,* Axel Haase, PHD,† Georg Ertl, MD,* Wolfgang R. Bauer, MD, PHD,* Johann Bauersachs, MD* Wu¨rzburg, Germany We sought to assess the influence of long-term hydroxymethylglutaryl coenzyme A reductase inhibition (statin) therapy on left ventricular (LV) remodeling after myocardial infarction (MI) by use of serial cardiac magnetic resonance imaging (CMRI) studies. BACKGROUND Statin therapy has been shown to reduce cardiac hypertrophy in vitro and in vivo, but the influence on LV post-MI remodeling is largely unknown. METHODS The CMRI measurements were taken four and 12 weeks after left coronary artery ligation in a 7.05-tesla Biospec. The MI size, LV mass and volumes, cardiac output (CO), and ejection fraction were determined. Rats were treated for 12 weeks with either placebo (P), cerivastatin (C; 0.6 mg/kg body weight per day) as a dietary supplement, or cerivastatin plus the nitric oxide synthase (NOS) inhibitor N-methyl-L-arginine methyl ester (L-NAME, 76 mg/100 ml) and hydralazine (8 mg/100 ml) in drinking water (CLH) to assess the contribution of endogenous nitric oxide formation. RESULTS Administration of cerivastatin attenuated hypertrophy after MI, and this effect was completely abolished by NOS inhibition (increase of LV mass from 4 to 12 weeks after MI: 235.3 ⫾ 33.7 mg with P vs. 59.8 ⫾ 20.5 mg with C vs. 239.5 ⫾ 16.0 mg with CLH; p ⬍ 0.05 vs. P and CLH). Left ventricular dilation was not changed (increase of end-diastolic volume from 4 to 12 weeks after MI: 108.7 ⫾ 28.8 with P vs. 126.6 ⫾ 20.5 with C vs. 173.7 ⫾ 25.1 with CLH; p ⫽ NS). The CO was higher in the cerivastatin group (12 weeks: 76.1 ⫾ 2.9 ml/min with P vs. 95.8 ⫾ 4.8 ml/min with C; p ⬍ 0.05). The effects of cerivastatin were abolished by NOS inhibition in the CLH group (CO at 12 weeks: 69.3 ⫾ 2.8 ml/min, p ⬍ 0.05 vs. C). CONCLUSIONS Left ventricular remodeling was profoundly changed by statin treatment. Hypertrophy was attenuated, and global function was improved. These positive effects were abolished by NOS inhibition. (J Am Coll Cardiol 2002;40:1695–700) © 2002 by the American College of Cardiology Foundation OBJECTIVES

Statin therapy has been shown to lower the incidence of myocardial infarction (MI) through its positive effects on atherosclerosis (1). Recently, it has been reported that statins have effects beyond lipid lowering, and treatment after MI may be beneficial not only for the secondary prevention of repeat infarction (2–11). Proposed mechanisms include antioxidant (2) and anti-inflammatory (3) effects, upregulation of endogenous nitric oxide synthase (eNOS) (4,5), reversal of endothelial dysfunction (6,7), and induction of angiogenesis (8). In addition, statins attenuate myocardial hypertrophy in vitro (9,10) and in vivo (11). Furthermore, cerivastatin has been shown to improve blood flow and reduce ischemic brain lesions in mice (12). Little is known about the action of statins in left ventricular (LV) remodeling post MI. From the *Medizinische Universita¨tsklinik Wu¨rzburg, and †Experimentelle Physik 5, Universita¨t Wu¨rzburg, Wu¨rzburg, Germany. This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich “Pathophysiologie der Herzinsuffizienz” SFB 355/B9, B10, and A8 and by Bayer AG, Wuppertal, Germany. Manuscript received January 24, 2002; revised manuscript received June 11, 2002, accepted June 26, 2002.

Therefore, the purpose of the present study was to investigate the influence of statin therapy on cardiac remodeling in the rat model of coronary ligation and the role of nitric oxide synthase (NOS) in its actions. The rat model of MI has proved its usefulness in the introduction of angiotensin-converting enzyme (ACE) inhibition therapy after MI (13–15). It has an additional advantage for testing the non–lipid-lowering effects of statin therapy, because atherosclerosis is absent in this model. The remodeling process was followed by serial cardiac magnetic resonance imaging (CMRI) measurements, a noninvasive reference standard for in vivo volumetry, which has previously been validated in the rat MI model (16).

METHODS Animals, experimental MI, and pharmacologic interventions. Adult male Wistar rats weighing 269 ⫾ 3 g were used when the study was started. Coronary artery ligation was performed as described previously (17–19). In brief, rats were anesthetized by isoflurane, intubated, and ventilated by

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Abbreviations and Acronyms ACE ⫽ angiotensin-converting enzyme CMRI ⫽ cardiac magnetic resonance imaging CO ⫽ cardiac output EF ⫽ ejection fraction eNOS ⫽ endogenous nitric oxide synthase L-NAME ⫽ N-methyl-L-arginine methyl ester LV ⫽ left ventricle or ventricular MI ⫽ myocardial infarction SV ⫽ stroke volume

a volume-constant rodent ventilator (UB 7025 rodent ventilator, Hugo Sachs Elektronik, March, Germany), and a left thoracotomy was performed. The heart was exteriorized from the thorax, and the left coronary artery was ligated using a 5.0 suture between the pulmonary artery outflow tract and left atrium. The heart was then returned to its normal position, and the thorax was closed. Animals were housed in polyethylene cages in climatized rooms with a 12-h light/dark cycle and fed with standard laboratory food and tap water. Rats were treated with either placebo, cerivastatin (0.6 mg/kg body weight) starting on the seventh postoperative day as a dietary supplement, or cerivastatin plus N-methyl-L-arginine methyl ester (L-NAME) (76 mg/100 ml) and hydralazine (8 mg/100 ml) in drinking water daily for 12 weeks. This dosage of hydralazine was shown to counteract the increase of blood pressure induced by L-NAME (20,21). All procedures conformed to the guiding principles of the American Physiological Society and were approved by the institutional Animal Research Committee. Cardiac magnetic resonance imaging. Non-invasive CMRI was used to monitor the typical features of post-MI remodeling consisting of hypertrophy and dilation of the LV. In serial investigations, the influence of statin therapy was assessed. Experiments were performed at 4 and 12 weeks after MI on a 7.05-tesla Biospec 70/21 (Bruker, Germany) under inhalation anesthesia applied by a nose cone (1.5% isoflurane supplemented with 0.5 l oxygen per minute) using a rat-size whole-body coil. An electrocardiographically triggered fast gradient echo sequence (22) was used with the following parameters: flip angle 30° to 40°, echo time 1.1 ms, repetition time (TR) 3.2 ms, and 12 frames per heart cycle. The total acquisition time for one cine sequence was 40 to 50 s, depending on the heart rate, and the acquisition window per frame was 6.2 ms (2 ⫻ TR). Data acquisition per slice was averaged four times to increase the signal to noise ratio. For quantitative determination of the morphology and function, 18 to 22 contiguous ventricular short-axis slices of 1-mm thickness were acquired to cover the entire heart. With a field of view of 50 mm and an image matrix of 128 ⫻ 128, in-plane resolution was 390 ␮m. Data analysis was performed using an operator-

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interactive threshold technique by one trained observer. Myocardial and ventricular slice volumes were determined from end-diastolic and end-systolic images by multiplication of the compartment area and slice thickness (1 mm). Total volumes were calculated as the sum of all slice volumes. Left ventricular mass was calculated as LV enddiastolic myocardial volume multiplied by the myocardial specific gravity (1.05 g/cm3). The stroke volume (SV) and ejection fraction (EF) were calculated using the enddiastolic and end-systolic volumes (SV ⫽ EDV ⫺ ESV; EF ⫽ SV/EDV). For cardiac output (CO), SV was multiplied by the heart rate. Myocardial infarct size was determined for every slice as the myocardial portion of the LV with significant thinning and akinesia or dyskinesia during systole (16). The relative MI size was calculated by taking the sum of the endocardial and epicardial circumferences of end-systolic frames occupied by the MI and dividing it by the sum of the total epicardial and endocardial circumferences. This method has been previously validated by histologic determination of infarct size (16). Absolute MI size, as the area covered by MI, was obtained as follows: the average length of the infarct-related section (sum of the endocardial and epicardial circumferential lengths of the infarct area divided by 2) of a single slice was multiplied by slice thickness (1 mm). The infarct areas of all slices were totaled, to calculate the area covered by scar tissue. Polymerase chain reaction. Total ribonucleic acid (RNA) was isolated from the surviving LV myocardium (septum) using TRIzol reagent (Life Technologies), and the RNA concentration was determined spectrophotometrically at 260 nm. After reverse transcription (SuperScript II, Life Technologies), the alpha-actin iso-messenger RNAs (mRNAs) were amplified by polymerase chain reaction (PCR), as previously described (23), using digoxigeninlabeled forward primers. After digestion with the restriction enzyme SacI (Roche-Diagnostics GmbH, Mannheim, Germany), the fragments of the PCR amplification product were separated on 6% polyacrylamide gel (lengths: 202 base pair [bp] for skeletal and 161 bp for cardiac alpha-actin). The deoxyribonucleic acid fragments were transferred onto a nylon membrane positively charged (Roche) and exposed to film suitable for detection of chemiluminescence (Kodak BioMax Light, Eastman Kodak, Rochester, New York). The resultant bands on the autoradiograms were then quantified with NIH Image (version 1.61, National Institute of Health, Bethesda, Maryland), and the results were expressed as the ratio of skeletal alpha-actin mRNA to cardiac alpha-actin mRNA. Data analysis. The results are expressed as the mean value ⫾ SEM. Statistical comparisons among various groups over time were evaluated by analysis of variance, followed by the Duncan test to isolate the significance of differences between individual mean values. A p value ⬍0.05 was considered to indicate statistical significance.

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Figure 1. Typical mid-ventricular diastolic and systolic short-axis slices 12 weeks after myocardial infarction. Heart from a rat treated with cerivastatin (myocardial infarction size 40%).

RESULTS Typical examples of CMRIs are shown in Figure 1. Shortaxis slices were used for quantification. Infarcts were similar in all groups and, on average, ⬎30% (Table 1). As shown in Figure 2, the LV mass increased after MI in the placebo group, associated with a marked increase in skeletal actin expression (Fig. 3). Statin treatment almost completely inhibited the increase in LV mass (Fig. 2) and significantly reduced skeletal actin expression (Fig. 3). Cotreatment with the NOS inhibitor L-NAME and hydralazine reversed the effect of cerivastatin on LV mass (Fig. 2) and significantly reduced skeletal actin expression (Fig. 3). Dilation of the LV after MI was not prevented by cerivastatin (Fig. 4A), and LVEF was not significantly improved (Fig. 4B). Cardiac output was higher in the groups treated with cerivastatin (Fig 5A); this was due to a higher heart rate (Table 1) and larger SVs (12 weeks after MI: 240.7 ⫾ 11.0 ␮l with placebo vs. 273.7 ⫾ 8.9 ␮l with cerivastatin; p ⫽ NS). Wall motion analysis revealed a decline of systolic wall thickening of the remote region during the remodeling process. This was found to be significant in all MI groups, with exception of rats treated with cerivastatin (Fig. 5B). Therefore, treatment with cerivastatin was able to preserve regional function of the remote region to a certain degree. Again, this effect was reversed by co-treatment with L-NAME and hydralazine (Fig. 5B). In the rats given placebo, the area covered by scar tissue increased by 7 ⫾ 5.3 mm2, and in rats treated with

Figure 2. Hypertrophy after myocardial infarction (MI). (A) Increase in left ventricular (LV) mass (mg) from 4 to 12 weeks after MI. (B) Change in wall thickness (mm) in the remote region. Data are presented as the mean value ⫾ SEM. *p ⬍ 0.05 vs. Sham Plac, Sham Cer, and MI Cer. Cer ⫽ cerivastatin; Plac ⫽ placebo.

cerivastatin, by 5.1 ⫾ 5.9 mm2. In the group co-treated with L-NAME, the increase in infarct size was significantly higher (33.8 ⫾ 5.1 mm2; p ⬍ 0.05 vs. placebo and cerivastatin).

DISCUSSION The major novel result of this study is a substantial reduction of post-MI LV hypertrophy by cerivastatin. Co-treatment with a NOS inhibitor fully counteracted the effects of the statin.

Table 1. Infarct Size, Body Weight, and Cardiac Magnetic Resonance Imaging Parameters of Rats With Myocardial Infarction Placebo Group (n ⴝ 8)

MI size (%) Body weight (g) LV mass (mg) Heart rate (beats/min) EDV (␮l) ⌬SWT (%)

Cerivastatin Group (n ⴝ 11)

Cerivastatin ⴙ L-NAME Group (n ⴝ 11)

4 Weeks

12 Weeks

4 Weeks

12 Weeks

4 Weeks

12 Weeks

32 ⫾ 3 286.6 ⫾ 6.1 500.4 ⫾ 14.8 310 ⫾ 10 591.3 ⫾ 40.8

30 ⫾ 3 305.2 ⫾ 5* 735.7 ⫾ 38.4* 318 ⫾ 11 700.1 ⫾ 67.6* ⫺17.3 ⫾ 4.9

32 ⫾ 1 212.6 ⫾ 6.2† 592.9 ⫾ 15.6† 372 ⫾ 8† 549 ⫾ 21.3

31 ⫾ 2 257 ⫾ 4.7*† 652.7 ⫾ 16.3*† 360 ⫾ 9† 675.7 ⫾ 34.6* 11.1 ⫾ 5.3†

32 ⫾ 3 205.4 ⫾ 6.1† 500.6 ⫾ 18.4‡ 327 ⫾ 5‡ 518.3 ⫾ 17.2†

35 ⫾ 2 246.5 ⫾ 6.9*† 740.1 ⫾ 17.7*‡ 317 ⫾ 13‡ 692.0 ⫾ 34.7* ⫺21.1 ⫾ 5.8‡

*p ⬍ 0.05 vs. 4 weeks. †p ⬍ 0.05 vs. placebo group. ‡p ⬍ 0.05 vs. cerivastatin ⫹ L-NAME group. Data are presented as the mean value ⫾ SEM. EDV ⫽ end-diastolic volume; L-NAME ⫽ N-methyl-L-arginine methyl ester; LV ⫽ left ventricular; MI ⫽ myocardial infarction; ⌬SWT ⫽ change in systolic wall thickening from 4 to 12 weeks.

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Figure 3. Representative autoradiograph and statistical analysis showing the effects of long-term therapy with cerivastatin after myocardial infarction (MI) on left ventricular (LV) gene expression (n ⫽ 5 or 6 per group) of the iso-messenger ribonucleic acids for skeletal and cardiac alpha-actin. Two representative lanes are shown for each group, each from a separate rat. *p ⬍ 0.05 vs. Sham Plac. †p ⬍ 0.05 vs. MI Plac. Cer ⫽ cerivastatin; Plac ⫽ placebo.

Post-MI remodeling. In the placebo group, features of LV remodeling, such as an increase in LV mass and progressive dilation of the LV, were detected by serial measurements, which are feasible due to the noninvasive character of CMRI. The distinct effects of the treatment found in this study are partly due to the possibility of imaging the same animal a second time and hence monitoring the induced change. Changes in LV morphology led to impaired function, as characterized by a reduced EF (Fig. 4B) and impaired systolic wall thickening (Fig. 5B). These findings are in accordance with data obtained by invasive diagnostic procedures (17–19) and previous CMRI studies in rats after MI (24,25). Furthermore, these results resemble observations in patients developing heart failure after MI due to chronic LV remodeling (26). Impact of statin treatment. In this study, evidence for prevention of post-MI hypertrophy by statin is provided by prevention of an increase in LV mass, wall thickness, and expression of skeletal actin. Activation of the renin-

angiotensin system may contribute to hypertrophy after MI (27). Although in the early post-MI phase, hypertrophy may be necessary to reduce regional wall stress and maintain CO and peripheral perfusion, pathologic hypertrophy promotes the progression of the infarct-related ventricle into failure (28 –30). Statins have been shown to reduce angiotensin II-induced hypertrophy in vitro, mainly by attenuation of p21 ras activity (10) and antioxidant properties (2). In a recent study, cerivastatin prevented the LV expression of fetal genes such as beta-myosin heavy chain, and it reduced collagen I gene expression in rats after MI (31). In transgene cardiomyopathic rabbits, simvastatin induced the regression of hypertrophy and fibrosis (32). In the present study, the attenuated LV remodeling resulted in a higher CO (Fig. 5A) and wall thickening of the remote region (Fig. 5B). However, LV dilation and a decline in EF were not reversed by statin treatment (Fig. 4). Role of nitric oxide (NO). Increased expression and activity of eNOS during statin treatment have been reported in several experimental settings, both in vitro and in vivo

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Figure 4. Dilation after myocardial infarction (MI). (A) Increase in end-diastolic volume (␮l) from 4 to 12 weeks after MI. (B) Ejection fraction (%) 12 weeks after MI. Data are presented as the mean value ⫾ SEM. *p ⬍ 0.05 vs. Sham Plac and Sham Cer. †p ⬍ 0.05 vs. MI Cer. Cer ⫽ cerivastatin; Plac ⫽ placebo.

Figure 5. Left ventricular function. (A) Cardiac output (ml/min) 12 weeks after myocardial infarction (MI). *p ⬍ 0.05 vs. MI Plac and MI Cer plus L-NAME. (B) Systolic wall thickening (%) in the remote region 12 weeks after MI. Data are presented as the mean value ⫾ SEM. *p ⬍ 0.05 vs. Sham Plac.

(4,5). Left ventricular eNOS expression was markedly increased in rats after MI, suggesting that the beneficial effects of cerivastatin may be mediated by an improved NO/O2⫺ balance (31). The present study supports this concept, as inhibition of NOS by L-NAME almost completely abolished the effects of statin treatment. Infarct expansion. Despite an identical relative infarct size at 4 weeks after MI (31.5 ⫾ 2.7% with placebo vs. 31.9 ⫾ 1.4% with cerivastatin vs. 31.9 ⫾ 2.8% with cerivastatin plus L-NAME), only the last group’s relative infarct size further increased to 34.5 ⫾ 2.4% at 12 weeks. We therefore calculated infarct size as an absolute measure by multiplying the circumferential length of the noncontractive zone by slice thickness and found a significant increase in the noncontractive area of the LV only in rats treated with L-NAME. It is therefore likely that inhibition of NOS aggravates late scar remodeling. One could speculate that the shift in the NO/O2⫺ balance with consecutively enhanced generation of reactive oxygen species (31) might lead to infarct expansion. Higher distention forces on the scar are not likely to be the cause of increased infarct expansion,

because co-administration of hydralazine minimized preload and after-load (33). Clinical implications. Many patients who have had an MI will be treated with statins for secondary prevention of coronary heart disease. This study suggests that a pleiotropic effect of statins also appear to retard LV hypertrophy in the rat model of chronic MI. Statin therapy may be an additional option for this group of patients, complementary to ACE inhibitor medication, which can prevent LV dilation, heart failure, and death (34). Study limitations. This animal study may not directly represent the patient’s situation. However, the rat model of heart failure after MI closely mimics the clinical syndrome of heart failure in patients and has proven value for research into pharmacologic intervention. For instance, the beneficial effects of ACE inhibitors after MI were first demonstrated in the rat model (13) and provided the basis for large studies of patients (34). Another concern may be that cerivastatin is not representative of all statins. However, the reduction of LV hypertrophy in several models by other statins (2,32)

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supports the concept of a class effect attributable to all statins, rather than a specific effect of cerivastatin. Conclusions. Serial CMRI facilitated the analysis of statin effects after MI. Left ventricular dilation was unchanged, but cerivastatin drastically reduced LV hypertrophy. The effects are likely to be caused by increased NOS, because inhibition of NOS completely abolished the effects of hydroxymethylglutaryl coenzyme A reductase inhibition. Reprint requests and correspondence: Dr. Matthias Nahrendorf, Medizinische Universita¨ tsklinik, Universita¨ t Wu¨ rzburg, Josef Schneider-Strasse 2, 97080 Wu¨ rzburg, Germany. E-mail: [email protected].

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