HMG-CoA reductase inhibition prior reperfusion improves reparative fibrosis post-myocardial infarction in a preclinical experimental model

International Journal of Cardiology 175 (2014) 528–538

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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard

HMG-CoA reductase inhibition prior reperfusion improves reparative fibrosis post-myocardial infarction in a preclinical experimental model☆ Gemma Vilahur a, Laura Casani a, Esther Peña a, Oriol Juan-Babot a, Guiomar Mendieta a, Javier Crespo a, Lina Badimon a,b,⁎ a b

Cardiovascular Research Center, CSIC-ICCC, Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain Cardiovascular Research Chair, UAB, Barcelona, Spain

a r t i c l e

i n f o

Article history: Received 20 December 2013 Received in revised form 13 May 2014 Accepted 24 June 2014 Available online 2 July 2014 Keywords: HMG-CoA reductase inhibition Myocardial infarction Swine Cardiac remodeling

a b s t r a c t Background: Studies in patients support a beneficial effect of statin treatment early after acute coronary syndrome and/or prior percutaneous coronary intervention. However, statin effect during total occlusion remains unknown. Objectives: To investigate whether infusion of activated simvastatin during ischemia and prior reperfusion and oral administration thereafter confers cardioprotection and improves cardiac healing in a preclinical model of myocardial infarction. Methods: Pigs (n = 24) fed a 10 day Western-type diet underwent a 90 min coronary-balloon occlusion (MI) being randomized to a single intravenous infusion of active β-hydroxy acid derivative of simvastatin (β-OH-S; 0.3 mg/kg) 15 min prior to reperfusion or vehicle. Animals were either sacrificed 2.5 h post-reperfusion or kept under the same regime ± simvastatin (p.o., 20 mg/day) for 3 weeks. Jeopardized and remote myocardium was obtained for molecular/histological studies. Echocardiography was assessed. Results: β-OH-S infusion prior to reperfusion reduced coronary and cardiac oxidative DNA-damage, diminished neutrophil infiltration at the site of ischemia, preserved mitochondrial membrane potential and reduced apoptosis in the ischemic myocardium (lower mRNA levels of Fas, casp8, p53, and casp3 and mitochondrial-p-Bcl2; and reduced TUNEL and active caspase-3; p b 0.05 vs. vehicle/control). This treatment regime attenuated reperfusion-related arrhythmias and stunning leading to a 40% increased myocardial salvage (p b 0.05 vs. vehicle/control). 3 weeks post-MI simvastatin-treated animals showed P-PKCε increase, lower intramyocardial lipotoxicity, TβRII/Smad2/3 signaling restoration and subsequent myofibroblast differentiation and collagen-fibril formation in the evolving scar (p b 0.05 vs. control). Simvastatin suppressed cardiac RhoA mobilization and triggered Akt/eNOS signaling. Conclusions: Acute HMG-CoA-reductase inhibition during total ischemia and prior reperfusion limits reperfusion injury and prolonged oral simvastatin treatment thereafter improves cardiac healing post-MI. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Statins, by inhibiting the HMG-CoA reductase, have become the firstline therapy for lowering lipid levels and reduce cardiovascular (CV) morbidity/mortality across the spectrum of atherosclerosis [1]. In recent years, however, a number of additional effects not associated to reducing plasma LDL-cholesterol levels have been identified in the statin mechanism of action that contributes to their efficacy in CV disease. Clinically, early statin treatment following acute myocardial infarction ☆ Grant support: This work was supported by RD12/0019/0026 — TerCel and RD12/ 0042/0027 — Red de Investigación Cardiovascular from Institute Carlos III (to LB) and SAF 2010-16549 (to LB) and SAF 2012-40208 (to GV), Spain. We thank Fundacion Investigación Cardiovascular-Fundación Jesus Serra, Barcelona, for their continuous support. GV is a recipient of a contract from the Innovation and Science Spanish Ministry (RyC-2009-5495, MICINN, Spain). ⁎ Corresponding author at: Cardiovascular Research Center, c/Sant Antoni Ma. Claret 167, 08025 Barcelona, Spain. Tel.: +34 935565880; fax: +34 935565559. E-mail address: [email protected] (L. Badimon).

http://dx.doi.org/10.1016/j.ijcard.2014.06.040 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.

(MI) has been associated with a better 1-year survival [2]. Moreover, in the acute phase of MI intensive statin therapy has demonstrated clinical benefit in non-ST-elevation myocardial infarction (NSTEMI) patients, an effect also extended to patients subjected to coronary intervention procedures [3,4]. So far, experimental studies in isolated perfused mouse hearts and rats fed a normocholesterolemic diet have demonstrated the capability of statins to exert cardioprotection in the setting of ischemia/ reperfusion (I/R) when administered either prior induction of ischemia (chronic and/or early deliver before induction of infarction) or after MI-induction [5–8]. These reported cardioprotective effects are thought to be largely mediated by the drug's ability to block the synthesis of important isoprenoids as well as increase nitric oxide (NO) availability via PI3K/Akt/eNOS activation. However, the optimization of the time of use and loading dose of statin during ischemia and prior reperfusion remains to be investigated. On the other hand, after MI a tightlyregulated repair process is triggered in the left ventricle (LV; LV remodeling) which is ultimately associated with a fibrogenic response targeted

52 [36–54] 43 [36–45]

Sacrifice MI-induction

37 [31–41] 30 [28–31] 38 [38–42] 25 [21–27]

Baseline Sacrifice

41 [37–46] 35 [30–37] 29 [27–41] 33 [24–42]

Baseline

28 [27–29] 32 [21–42]

GPT (IU/L) GOT (IU/L)

MI-induction C.

Control Simvastatin

48 [25–103] 37 [17–60]

MI-induction Sacrifice Baseline

7.8 [7.6–8.9]* 42 [39–77] 23 [11–31] 8.1 [7.6–8.2]* 35 [28–40] 58 [51–68]

MI-induction Sacrifice Baseline

68 [47–74]* 2.3 [2.3–2.6] 7.9 [5.7–8.8]* 66 [60–70]* 1.9 [1.4–2.4] 6.3 [5.6–6.7]*

MI-induction Sacrifice Baseline MI-induction

Sacrifice Baseline

MI-induction

Sacrifice

Baseline

Triglycerides (mg/dL) Total-cholesterol/HDL

32.1 [29.3–45.3] 36.9 [32.1–45.9]

HDL-cholesterol (mg/dL)

91 [79–95] 434 [403–504]* 536 [440–601] 36 [32–48] 278 [273–290]* 337 [311–347]* 37 [33–38] 55 [53–76] 85 [71–102] 375 [337–400]* 583 [535–603]* 13 [11–31] 234 [221–245]* 324 [193–474]* 50 [38–56] 63 [55–66] Control Simvastatin

2.5 h post-reperfusion Evan's Blue dye was injected in anesthetized animals through the left atrium to outline the AAR after which the animals' hearts were arrested with potassium chloride and rapidly excised. Hearts were sectioned into 6 transverse slices parallel to the atrioventricular ring. Consecutive slices were alternatively collected for infarct size analysis (TTC) and molecular studies of the ischemic myocardium (IM) and non-ischemic myocardium (NIM).

LDL-cholesterol (mg/dL)

2.3. Morphometric determination of infarct size and sample collection

Cholesterol (mg/dL)

Transthoracic echocardiography assessment was performed in all animals before inducing ischemia (baseline), after 75 min of ischemia (prior to β-OH-S infusion), 2.5 h post-reperfusion and 3 weeks post-MI using an echocardiographic system, Phillips iE33, equipped with a S5-1 sector array transducer. Left ventricle ejection fraction (LVEF) and shortening fraction (SF) were measured in the short-axis M-mode right parasternal projection in a plane below the mitral valves and perpendicular to the LV [16].

B.

2.2. Echocardiography

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17.3 [16.9–17.9] 17.7 [16.7–22.4]

Twenty-four cross-bred commercial female swine (3 month-old; average of 40 kg in weight) were fed a Western-type hypercholesterolemic diet [20% — saturated fat (beef tallow), 2% — cholesterol, 1% — cholic acid]. This hyperlipidemic diet contains 24.82% proteins, 64.20% of carbohydrates, 10.98% of fats and 16,834.7 kJ. We have already demonstrated that intake of this fat-rich diet for 10 days raises cholesterol to levels comparable to that found on dyslipidemic humans and induces endothelial dysfunction [11]. After this 10 day diet period animals underwent a closed-chest 90 min mid-left anterior descending (LAD) coronary balloon occlusion [12]. After 75 min of ischemia (15 min before reperfusion) animals were randomized to intravenous infusion (femoral vein) of an active β-hydroxy acid derivative of simvastatin (β-OH-S; 0.3 mg/kg; n = 12) or vehicle (0.9% NaCl; n = 12). At minute 90, the balloon was deflated to achieve coronary artery reperfusion. Animals were either sacrificed 2.5 h post-reperfusion [n = 6 animals/group; to evaluate myocardial injury] or kept under their starting high-cholesterol diet regime plus oral simvastatin treatment (20 mg/day; simvastatin group; n = 6) or placebo (control group; n = 6) for the following 3 weeks and then sacrificed (to evaluate cardiac reparative fibrosis). The incidence of reperfusion-related arrhythmias during the first 2.5 h upon balloon deflation was documented in all animals. Animals had continuous electrocardiogram (ECG) and hemodynamic monitoring throughout all procedures. The β-hydroxy acid form (active form) was prepared from simvastatin (Sigma) by conventional hydrolysis with sodium hydroxide and the concomitant exposure to heat. Simvastatin is an inactive lactone (closed form) which must be metabolized to its hydroxy-acid form (open form) in order to gain activity. Drug dosages were determined on the basis of the loading dose (80 mg) in the setting of acute coronary syndrome and/or previous acute percutaneous coronary intervention (PCI) [13, 14] and were converted to the pig dose according to body surface area [15]. At the beginning of the experimental procedure animal weight was evenly matched between the two groups.

Weight gain at MI induction (kg)

2.1. Animal model and study design

Control Simvastatin

All procedures fulfilled the criteria established by the “Guide for the Care and Use of Laboratory Animals” published by the United States National Institutes of Health (NIH Publication No. 85-23, revised 1996).

A.

2. Methods

Table 1 Follow-up data of A. Weight progression; B. Lipid profile; and C. Liver parameters. *p b 0.05 vs. baseline. All animals are included in the measurements. MI: acute myocardial infarction.

to replace the cardiomyocyte loss and restore cardiac structures. Regardless of the initiating stimulus (MI, aortic banding or a variety of transgenic animals) studies in rodents have reported the capability of statins to confer beneficial effects on adverse myocardial remodeling under normocholesterolemic conditions [9,10]. However, the mechanisms underlying such protective effects remain poorly understood as well as whether dyslipidemia may abrogate statin beneficial effects. In this regard, we have recently reported that short-term diet-induced dyslipidemia favors myocardial lipotoxicity and impairs the activation of the reparative myofibroblasts in the jeopardized myocardium resulting in defective scar formation and worse cardiac performance, key myocardial features for heart failure [11]. With all this in mind, we hypothesized that acute fast acting intravenous administration of a loading dose of simvastatin during ischemia prior reperfusion would limit reperfusion-related myocardial damage and that maintenance of treatment with oral simvastatin thereafter, as used clinically, would favor cardiac healing post-MI despite the presence of co-morbid conditions. The mechanisms behind such potential beneficial effects have been investigated in this study conducted in a diet-induced hyperlipidemic pig model of closed-chest coronary balloon occlusion/reperfusion.

Weight gain at day 21 post-MI induction (kg)

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Scar tissue and NIM were obtained from animals 3 weeks post-MI for molecular/ histological studies.

and 3) fibrous tissue formation (collagen, Smad2 and Smad3). The threshold cycle values were determined and normalized to the housekeeping gene 18SrRNA.

2.4. Molecular studies

2.4.2. Western Blot analysis We assessed protein levels of: 1) apoptosis markers (truncated-caspase-3); 2) cardioprotective kinases [PKC-ε and PKC-ε phosphorylated at Ser729 (Santa Cruz) and Akt/PKB (Santa Cruz) and Akt phosphorylated at Ser473 (Cell Signaling)]; 3) total eNOS and eNOS phosphorylated at Ser1177 (Cell Signaling); and 4) fibrosis [TGFβ type II receptor (TβRII; Abcam) and phosphorylated-Smad2/3 (Ser423/425; Santa Cruz)]; isolated mitochondrial extracts were incubated against Bcl-2 phosphorylated at Ser87. RhoA (Santa Cruz) was determined in total and cytosolic-membrane fractions of jeopardized cardiac tissue of all animals as previously described [6]. In addition, 3 weeks post-MI, the coronary arteries of all animals were isolated, cleaned of surrounding tissue and snap frozen for further analysis of coronary phosphorylated-eNOS

Tissue samples obtained from the IM (2.5 h post-reperfusion)/scar (3 weeks post-MI) and NIM of all animals were pulverized and homogenized in TriPure® or lysis buffer for RNA and protein isolation, respectively. 2.4.1. Transcriptomic analysis We analyzed by real-time PCR (Applied Biosystems) mRNA levels for: 1) apoptosisrelated markers [extrinsic-(Fas receptor/CD95 and caspase-8), intrinsic-(Bax, Bcl2, and P53) pathways and caspase-3]; 2) intracellular calcium handling receptors [sarco– endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) and the ryanodine receptor (RyR2)];

Fig. 1. Protective effect of intravenous β-OH-S administration against reperfusion-injury. Infarct size (A), neutrophil myocardial recruitment (B) and oxidative DNA-damage in the coronary artery (C) and ischemic myocardium (D) 2.5 h post-reperfusion. *p b 0.05 vs. vehicle/control animals. Black arrows depict neutrophils or positive staining. N = 6 animals/group.

G. Vilahur et al. / International Journal of Cardiology 175 (2014) 528–538 protein expression. Where appropriate the intensity ratio of the target band to β-actin was applied to provide the relative amounts of the target protein.

2.5. Systemic oxidative stress and vascular/cardiac DNA-oxidative damage Blood samples were collected at 2.5 h post-reperfusion and 3 weeks post-MI to evaluate lipoprotein resistance to oxidation. We firstly assessed HDL-antioxidant activity by a method based on the ability of HDL to reverse oxidation of LDL [17]. Results were expressed as the percentage of total oxidized LDL considering that control oxLDL is 100%. In addition, resistance of LDL against copper induced in vitro oxidation was also determined in EDTA-blood samples as previously described [18]. We also determined the lipid peroxide content of oxidized-LDL by assessing thiobarbituric-acid-reactive substances (TBARS) [19]. Paraffin-embedded LAD and ischemic myocardial tissue of animals sacrificed 2.5 h post-reperfusion were cut into 5-μm-thick slices for 8-hydroxyguanosine staining (Abcam ab48508), a measurement indicative of oxidative stress-induced DNA-damage. In addition, OCT-embedded ischemic myocardial tissue of animals sacrificed 2.5 h postreperfusion was stained for neutrophil detection (Anti-Neutrophil Elastase; Abcam). Staining was calculated by a single blinded observer from an average of 5-fields/animal as % of stained area. Images were captured by a Nikon Eclipse 80i microscope and digitized by a Retiga 1300i Fast camera.

2.6. Mitochondrial membrane potential Mitochondria were isolated from the ischemic myocardium of pigs subjected to 2.5 h of reperfusion as previously reported [20]. Mitochondrial membrane potential (ΔΨm) was measured by flow cytometry using the ratiometric dye 5,5′,6,6′-tetrachloro1,1′,3,3′-tetraethylbenzimidazol carbocyanine iodide (JC-1; Molecular Probes). Values were expressed as red fluorescence activity. Isolated mitochondria treated with valinomycin (Sigma), which decreases ΔΨm, were used as control (data not shown). 2.7. Characterization of the forming scar Histological analysis was performed on the myocardium (scar and NIM) of all animals kept 3 weeks post-MI in order to analyze the effect of simvastatin treatment on the reparative fibrosis on the evolving scar. To that end, serially cut 5 μm sections from OCT-embedded samples were stained for lipids (Oil Red-O, ORO) and interstitial collagen (Sirius Red). Images were captured with a Nikon eclipse 80i microscope and digitalized by a Retiga-1300i. Staining was calculated by a single blinded observer from an average of 5-fields/sample as content (%) = [positive stained area / (total tissue area − vascular luminal areas)] × 100 using ImageJ®. Lipid characterization (cholesteryl ester, triglycerides and free cholesterol content) was also performed in the same cardiac regions by thin layer chromatography (TLC) and also in the liver (statin target) following lipid extraction as previously reported [21]. In addition, myofibroblast detection in the scar tissue was performed by double staining with mouse monoclonal vimentin (Dako) and rabbit polyclonal alpha-SMA 1:50 (Abcam). Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 546 goat anti-rabbit were used as secondary antibodies, respectively and Hoechst for nuclear staining. Images were captured by confocal microscopy (20×).

2.8. Hematological and biochemical follow-up Blood samples were collected at baseline (prior diet administration), 2.5 h post-MI induction, and 3 weeks post-MI for lipid and liver parameter assessment and hematological counts.

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2.9. Statistical analysis Because data were not normally distributed as observed by applying the Shapiro– Wilk test, a non-parametric statistical analysis was applied and results are reported as medians and interquartile range [IQR]. For independent factors (comparisons between groups) we performed Mann–Whitney analysis; for repeated measurements Wilcoxon and Friedman analysis were appropriate. χ2-analyses of contingency tables were used to evaluate associations. All statistical tests conducted were two-sided and p b 0.05 was considered significant. Statistical analysis was performed with StatView.

3. Results 3.1. Animal follow-up Weight gain throughout the study was comparable among the simvastatin and placebo/control animals (Table 1A). The lipid profile is shown in Table 1B. Baseline values were all within the pig physiological range. The high-cholesterol feeding model used led to a totalcholesterol/HDL ratio similar to human hypercholesterolemia at the moment of MI induction. Treatment with simvastatin did not induce significant changes in the plasma lipid profile. No alterations were observed in liver function-related parameters (Table 1C) and no variations were detected in hematological counts over time and across both animal groups (data not shown). After 3 weeks, characterization of liver lipid extracts revealed a significant reduction in liver neutral lipids [cholesteryl esters (CEs) and triglycerides (TGs)] in the simvastatin-treated animals with respect to controls (CE: 2.6 [2.4–2.7] vs. 3.0 [2.8–3.3] μg/mg protein; TG: 19.4 [16.9–20.5] vs. 24.2 [21.7–28.9] μg/mg protein, respectively; p b 0.05). No differences were observed as to free cholesterol content (1.4 [1.3– 1.5] vs. 1.2 [1.1–1.5] μg/mg protein, respectively). 3.2. Intravenous infusion of β-OH-S prior reperfusion protects against reperfusion injury 3.2.1. Limits infarct size AAR was similar between both groups reaching 37% [32–38] % of the LV (Fig. 1A). Yet, infarct size was 39.6% [38.4–41.5] % AAR in placebo/ control animals and 23.5% [21.5–37.9] % AAR in β-OH-S-treated animals indicating that a single loading dose of active simvastatin prior reperfusion resulted in a 40% salvage of the LV-at-risk as compared to reperfusion alone (p b 0.05). 3.2.2. Reduces neutrophil recruitment β-OH-S infusion exerted a powerful anti-inflammatory affect in the ischemic damaged tissue resulting in a marked attenuation of neutrophil infiltration by almost 70% as compared to vehicle/control (p b 0.05; Fig. 1B).

Table 2 A. HDL-antioxidant potential. oxLDL = 100% B. Oxidation of LDL particles. *p b 0.05 vs. 2.5 h post-AMI induction and † p b 0.05 vs. control. All the animals were included in the data. βOH-S: active β-hydroxy acid derivative of simvastatin. A. HDL antioxidant potential (expressed as % of oxLDL) 2.5 h post-reperfusion 3 weeks post-MI

oxLDL oxLDL oxLDL oxLDL

+ + + +

HDL from control plasma HDL from β-OH-S plasma HDL from control plasma HDL from simvastatin plasma

76 [72–81] 56 [55–58]† 107 [102–112] 66 [57–68]†

B. 2.5 h post-reperfusion

Max CD, nmol DC/mg prot LDL lag time, min V max nmol ∗ min(−1) ∗ mg prot LDL(−1) TBARS, nmol MDA/mg prot

3 weeks post-MI

Control

β-OH-S

Control

Simvastatin

237 [230–257] 27 [22–28] 8 [6–14] 87 [75–93]

210 [193–233] 25 [23–27] 9 [7–11] 95 [91–105]

204 [186–227] 8 [7–15]* 5 [4–48] 110 [93–126]*

216 [180–230] 49 [47–53]*† 8 [6–9] 65 [63–92]*†

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3.2.3. Diminishes oxidative damage β-OH-S infusion exerted a powerful protective effect against reperfusion-related oxidative damage to the coronary artery (Fig. 1C) and the ischemic myocardial tissue (Fig. 1D) as observed by the absence of DNA oxidative damage. In contrast, high 8-OHdG staining was observed in vehicle-treated animals. Systemically, intravenous administration of β-OH-S enhanced HDL antioxidant potential as compared to vehicle-administered animals (Table 2A). As such, HDL isolated 2.5 h post-reperfusion displayed a 25% higher antioxidant activity against LDL oxidation relative to controls (p b 0.05). As to LDL oxidative parameters, no differences were detected between both animal groups at 2.5 h post-reperfusion (Table 2B).

3.2.4. Reduces apoptosis execution β-OH-S infusion prevented the upregulation of several genes encoding for molecules downstream of both the death receptor- and mitochondrial-related apoptotic pathways (Fig. 2A) in the jeopardized myocardium. In addition, β-OH-S diminished mitochondrial Bcl-2 activation (Fig. 2B) and reduced truncated caspase-3 expression (execution of apoptosis; Fig. 2C). Accordingly, we detected low apoptotic cell counts (identified by TUNEL staining) within the ischemic myocardium of βOH-S-treated animals as compared to vehicle/control (Fig. 2C). No differences in any of the apoptosis-related parameters were detected in the non-ischemic myocardium showing values similar to those detected in the ischemic myocardium of β-OH-S-treated animals (data not shown).

Fig. 2. Protective effect of β-OH-S on myocardial cell preservation. A. Gene expression of apoptosis-related markers; B. Mitochondrial P-Bcl2 expression; C. Caspase-3 activation and apoptosis execution (TUNEL staining); D. Myocardial mitochondrial membrane potential; E. Contingency tables to assess the potential association between oxidative damage, apoptosis and membrane potential. *p b 0.05 vs. vehicle/control animals. N = 6 animals/group.

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3.2.5. Preserves myocardial mitochondrial membrane potential (ΔΨm) ΔΨm was 2-times higher in mitochondria isolated from the ischemic myocardium of β-OH-S-treated animals relative to vehicle/control (p b 0.05) (Fig. 2D). Interestingly, a two-by-two contingency table revealed a significant inverse association between mitochondrial membrane potential and myocardial apoptosis and/or oxidative stress and a direct correlation between TUNEL and myocardial 8-OHdG staining in the ischemic myocardium (Fig. 2E). 3.2.6. Improves cardiac performance As detailed in Fig. 3A, the incidence of reperfusion-related ventricular arrhythmias was higher in the control group (83% animals; 10 out of 12) than in the β-OH-S-treated group (25%; 3 out of 12). Of note, throughout the 2.5 h reperfusion period we only detected monomorphic ventricular tachycardia without hemodynamic instability and therefore no electrical cardioversion was required. As shown in Fig. 3B ischemia induced a comparable deterioration in both LVFE (≈24% decrease) and SF (≈16% decrease) in all animals. Yet, both LVEF and SF were improved by 9.4% and 6.0%, respectively, in β-OH-S-treated animals 2.5 h after reperfusion, an effect that persisted up to 3 weeks post-MI in simvastatin-treated animals. In contrast, placebo animals displayed a further 5% decrease in both LVEF and SF. All animals displayed similar hemodynamic parameters (heart rate and mean blood pressure) throughout the study (average: 55 [54–55] mm Hg and 74 [73–75] bpm) and they were not affected by β-OH-S treatment. Calcium regulatory proteins SERCA2 and RyR2 mRNA transcripts were not affected by β-OH-S intravenous administration (ischemic

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myocardium: 1.9 ± 0.5 vs. 1.8 ± 0.2 SERCA/18SrRNA and 0.14 ± 0.03 vs. 0.08 ± 0.03 RyR2/18SrRNA and NIM: 1.5 ± 0.3 vs. 1.6 ± 0.2 SERCA/18SrRNA 0.2 ± 0.07 vs. 0.11 ± 0.03 RyR2/18SrRNA; β-OH-S vs. control, respectively). 3.3. Simvastatin favors reparative scar formation 3.3.1. Attenuates myocardial lipotoxicity ORO staining evidenced significantly lower lipid infiltration in the evolving scar of simvastatin-treated animals 3 weeks post-MI induction as compared to control animals (Fig. 4A). In fact, only 2 out of 6 simvastatin-treated animals showed ORO staining whereas 6 out of 6 animals in the control group showed lipid infiltration in the forming scar. Lipids were undetectable in the non-ischemic myocardium of all animals. Lipid characterization revealed 50% less cholesteryl-ester accumulation in the forming scar of simvastatin-treated animals as compared to placebo–controls (Fig. 4B). Triglycerides were found in similar levels in the scar tissue of simvastatin and control animals and were significantly higher as compared to the NIM-area. No differences were detected in free-cholesterol content among the different groups of animals across the different cardiac zones (Fig. 4B). 3.3.2. Preserves myofibroblast transdifferentiation Simvastatin treatment was associated with a marked and significant increase in TβRII protein expression and subsequent activation of its downstream effector Smad2/3 in the scar area (Fig. 5A). Gene expression analysis of the transcription factors Smad2 and Smad3 revealed that Smad2 was significantly upregulated in the scar tissue of simvastatin animals whereas no changes were detected as to Smad3 mRNA levels throughout the different cardiac regions (Fig. 5B). Simvastatin-related activation of the TβRII/SMAD2/3 pathway in the healing scar was accordingly associated with a higher detection of α-SMA/vimentin positive myofibroblasts (Fig. 5C) and subsequent higher collagen 1A1/ collagen 1A3 mRNA ratio (Fig. 5D) and collagen fibril deposition as compared to control animals and NIM (Fig. 5E). 3.3.3. Inhibits RhoA mobilization and activates Akt/eNOS and PKCε Total myocardial RhoA protein expression as well as both the cytosolic (inactive) and membrane (active) fractions were determined by immunoblotting in all animals (2.5 h post-reperfusion and 3 weeks post-MI). As depicted in Fig. 6A the total amount of RhoA was unchanged. However, intravenous infusion of β-OH-S markedly decreased RhoA membrane translocation (activation), measured as a prominent decrease in the membrane-to-cytosol ratio, an effect that persisted after the 3-week oral treatment (p b 0.001 vs. control; Fig. 6A). In contrast to vehicle/control animals, acute β-OH-S administration was also capable of activating the P-Akt/P-eNOS pathway in the ischemic myocardium an effect that persisted in the evolving scar up to 3 weeks post-AMI (Fig. 6B). Interestingly, P-eNOS was also found to be enhanced in the coronary arteries of simvastatin-fed animals at 3-weeks post-MI (Fig. 6C). Finally we assessed PKCε in the forming scar, an isozyme known to participate in physiological remodeling. As shown in Fig. 6D simvastatin treatment enhanced both the expression and activation of PKCε in the scar tissue as compared to placebo–control animals (Fig. 6D).

Fig. 3. Effect of β-OH-S on cardiac performance. Reperfusion-related arrhythmias (A), left ventricle ejection fraction (LVEF; B), and shortening fraction (SF; C). *p b 0.05 vs. vehicle/ control. N = 6 animals/group.

3.3.4. Diminishes oxidative stress HDL antioxidant potential, already observed upon reperfusion, was maintained by daily oral simvastatin administration up to 3-weeks post-MI (Table 2A). Conversely, control animals only subjected to the Western-type diet decreased their HDL antioxidant capacity by 30% over time (76 [72–81] % vs. 107 [102–112] %; Table 2A). A 3 week oral simvastatin treatment also led to a significant increase in LDL resistance to in vitro oxidation determined as a prolongation of the lag time (almost 2 times longer vs. acute-MI induction and 4-times longer as compared to control/placebo animals; p b 0.05; Table 2B). In

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Fig. 4. Protective effect of simvastatin on myocardial lipotoxicity assessed 3-weeks post-MI. Oil Red-O staining of the scar and remote/non-ischemic myocardium (NIM; A) and myocardial lipid-characterization (B). CE: cholesterol ester; TG: triglycerides; and FC: free-cholesterol. *p b 0.05 vs. control; †p b 0.05 vs. NIM. N = 6 animals/group.

contrast, non-treated animals showed a higher susceptibility to LDL oxidation (lag time was shortened by 50% compared to the experimental day). TBARS levels were also found elevated in control/placebo animals after a 3 week high fat/high cholesterol diet. In contrast, oral simvastatin treatment was associated with a 32% decrease in TBARS levels as compared to the experimental day (p b 0.05) indicating a marked diminishment in the degree of lipid peroxidation. No differences were detected in the capacity of LDL to reach oxidation (maximal conjugated dienes and maximal velocity of conjugated diene formation) among both animal groups (Table 2B). 4. Discussion Here we have used a swine model of myocardial infarction and short-term diet-induced hypercholesterolemia that has previously been well-characterized [11]. We have recently reported in this model that hypercholesterolemia is associated with diminished coronary vasodilation, intramyocardial lipotoxicity and the impairment of the reparative fibrotic response (TGF-β/TβRII/Smad2/3 signaling pathway) adversely affecting collagen deposition in the forming scar leading to enlarged myocardial infarcts and cardiac dysfunction [11]. The present study demonstrates that intravenous infusion of a loading dose of activated simvastatin (β-OH-S) 15 min prior the restoration of the coronary flow limits myocardial injury by counteracting the inflammatory response and oxidative damage, diminishing apoptosis execution, preserving mitochondrial membrane potential and attenuating both reperfusion-related arrhythmias and cardiac stunning. Moreover, we can evidence, as soon as 3 weeks post-MI, that continuing with simvastatin oral treatment leads to a reduction in cardiac lipotoxicity (mainly

reduction in intracellular cholesteryl-ester lipid accumulation) and stimulation of the TβRII/Smad2 signaling pathway and PKCε activation favoring the subsequent reparative fibrotic response in the evolving scar. Such beneficial effects are due to the direct inhibitory effect of the statin on RhoA membrane translocation allowing Akt/eNOS activation. Firstly, we demonstrate that a single infusion of β-OH-S markedly attenuates reperfusion-related inflammatory response by modulating myocardial infiltration of neutrophils, a key component of reperfusion injury, at the site of ischemia [22]. Neutrophils are not only an important source of reactive oxygen species but also largely contribute to the nonreflow phenomena thereby exacerbating tissue damage and subsequently amplifying inflammation-derived deleterious effects [23]. In line with these observations we also observe a powerful β-OH-S-related antioxidant effect in both the coronary artery and myocardium. Statin vascular and myocardial preservation against the overall injurious burst of oxygen-free-radicals upon reperfusion may have limited apoptotic cell death and preserved cardiac mitochondrial membrane potential. Moreover, taking into consideration that reactive oxygen species generation has shown to collapse mitochondrial ΔΨ inducing electrophysiological alterations in healthy hearts [24], the detected maintenance of mitochondrial ΔΨ in β-OH-S-treated animals may have largely contributed to prevent the appearance of reperfusionrelated arrhythmias. Altogether, these beneficial effects may have contributed to decrease apoptosis execution in β-OH-S-treated animals. Indeed, we observe an increase in anti-apoptotic Bcl2 expression in control animals. Yet, anti-apoptotic Bcl2, which mostly localizes in the cytoplasm under healthy conditions, translocates to the mitochondrial membrane

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Fig. 5. Simvastatin favors cardiac reparative fibrosis. A. Sirius-Red staining in control and simvastatin treated animals in the different cardiac regions. Simvastatin preserves TBRII/Smad2/3 signaling pathway activation (A), up-regulates Smad2 gene expression without altering Smad3 mRNA (B), stimulates fibroblast transdifferentiation into myofibroblasts (C; depicted in yellow in the representative image), subsequently increasing collagen mRNA expression (D) and fibril deposition (E; Sirius Red staining) in the forming scar. *p b 0.05 vs. control animals. N = 6 animals/group.

in order to bind and inactivate Bax, as a mechanism to counteract myocardial injury [25]. In line with this observation, we have previously reported in this animal model that upon reperfusion mitochondrial Bcl-2 activation remains high for up to 3 days post-reperfusion of the ischemic heart [26]. A recent study in swine has demonstrated that a single oral dose of simvastatin administered 1 h before ischemia attenuates myocardial no-reflow through increasing myocardial eNOS activation [27]. Here we provide evidence that intravenous β-OH-S is capable of inducing eNOS activation during ischemia even under dyslipidemic conditions. Such eNOS preservation may have favored myocardial perfusion and microvascular permeability partly attenuating cardiac dysfunction (i.e., stunning) and limiting infarct size. As such, we detect a rapid recovery in heart contractibility post-MI which persists up to 3-weeks.

The cardioprotective effects of simvastatin can be attributed to the inhibition of isoprenylation of small G-proteins rather than to a reduction in serum cholesterol. We observe a pronounced myocardial inhibition of RhoA translocation to the membrane (i.e., inactivation state) after a single intravenous dose of β-OH-S. RhoA inhibition by statins has shown to induce the activation and translocation of Akt to the cell plasma membrane in concurrence with our findings [28]. Moreover, such Akt activation has been found to be inhibited upon loading the cells with cholesterol [29]. Interestingly, in the forming scar control animals showed a significant myocardial lipotoxicity in concurrence with Akt inactivation whereas simvastatin-treated animals displayed lower lipid infiltration (cholesteryl ester accumulation) and enhanced Akt activation. We have recently evidenced cardiomyocyte capacity to internalize and accumulate cholesteryl-ester via LRP-1 receptor

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Fig. 6. Effect of β-OH-S and oral simvastatin on myocardial RhoA and cardioprotective kinases. RhoA expression in the ischemic myocardium (IM) and scar, respectively (A). Activation of the Akt/eNOS signaling pathway (B). Coronary eNOS activation 3-weeks post-MI (C). Epsilon-PKC isozyme myocardial expression/activation (D). *p b 0.05 vs. vehicle/control animals and †p b 0.05 vs. non-ischemic cardiac tissue. N = 6 animals/group.

expression [30] which, in turn, is found to be up-regulated by hypercholesterolemia [31]. Moreover, we have shown that simvastatin reduces cholesterol ester accumulation in vascular smooth muscle cells induced by aggregated LDL, an effect reversed by geranyl-geraniol (involved in RhoA activity) [32]. Therefore, we may conclude that simvastatin, via RhoA inhibition, attenuates cardiomyocyte-lipid overload favoring activation/translocation of Akt and subsequent eNOS phosphorylation. Moreover, taking into consideration that we have recently demonstrated that myocardial-lipid infiltration is a reperfusion-related phenomenon [11] simvastatin may have triggered these protective effects upon reperfusion – likely reducing the extent of myocardial injury – and that continued oral statin administration may have further favored and sustained the healing phase.

Cardiac healing involves fibrous tissue formation at the site of cardiomyocyte loss in order to preserve structural integrity and requires a series of coordinated molecular and cellular events in which the TGFβ/TβRII signaling plays a critical role. TGFβ/TβRII governs fibroblast transdifferentiation into myofibroblasts and via its downstream receptor-associated effectors Smad2/3 regulates the transcription of specific extracellular matrix-related genes, primarily collagen, modulating fibrous tissue deposition [33]. We have previously reported that intramyocardial lipid infiltration is associated with impairment in the TGFβ/Smad2/collagen signaling pathway impeding the reparative fibrotic response post-MI [11]. Herein, we evidence that simvastatin treatment preserves TβRII expression, Smad2/3 activation and collagen synthesis in the evolving scar helping to maintain myocardial shape

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and structure. Previous studies in vascular smooth muscle cells and ApoE-transgenic mice have demonstrated the capability of high cholesterol levels to effectively suppress TGFβ/Smad2/3 activation by diminishing TβRII expression, an effect found to be reversed by statin treatment via RhoA inhibition [34,35]. With the present study we extend these previous results on the vasculature and focus on the effects of statins on myocardial repair post-MI. Finally, our results also attribute to statin treatment a marked upregulation and enhanced activation of ε-protein kinase C, known to modulate cell death and cardiac remodeling. Inagaki et al. [36] demonstrated in a swine model of I/R that intracoronary delivery of ΨεRACK, a selective PKC-ε activator, lowered the appearance of I/R-induced ventricular arrhythmias and reduced infarct size. Moreover, chronic administration of ΨεRACK did not induce PKC-ε desensitization or downregulation supporting a sustained/chronic protective effect. We provide evidence that statins enhance and stimulate the PKC-ε isozyme over a three-week period regardless of the presence of hyperlipidemia. In this latter regard, whereas a high degree of PKCε activation (around a 500% increase in activity) has shown to promote cardiac hypertrophy, low-to-moderate PKCε activation (around a 200% increase in activity) as observed in our study has shown to result in physiological cardiac remodeling [37,38]. 5. Study limitations It deserves to be acknowledged that the following study does not account for important clinical situations found in most STEMI patients. Although it considers the presence of hyperlipidemia (a commonly found risk factor among these patients), the study includes juvenescent animals free from cardiovascular disease and co-medications (adenosine, nitroglycerine, beta-blockers, etc.) that could interfere with the extent of cardiac injury and/or remodeling.

6. Conclusion In summary, this study shows that intravenous delivery of an active β-hydroxy acid derivative of simvastatin before the opening of an occluded artery induces heart protection by attenuating tissue and functional determinants of reperfusion injury despite co-morbid conditions. This therapeutic approach could be easily translated to patient treatment just before primary PCI or surgical revascularization. So far, randomized clinical trials have shown evidence in favor of the administration of statins in the setting of ACS when administered early after revascularization [3,14,39]. In fact, current American College of Cardiology/American Heart Association (ACC/AHA) guidelines recommend (Level of Evidence 1A) starting therapy 24–96 h after hospital admission and continuing treatment at discharge [40]. In addition, decreased rates of myocardial infarction and mortality have also been demonstrated in several smallscale studies where high-dose statin treatment was administered before an elective [41,42] or emergent PCI [3,4]. The efficacy of the therapeutic approach investigated in this experimental study with an intravenous peri-procedural loading dose of statin merits clinical testing. Besides, to date, statins have not appeared to improve outcomes in patients with established/advanced heart failure [43,44]. Yet, our results mechanistically explain why statins seem to limit adverse cardiac remodeling when started early in the setting of acute-MI [3,45]. Interestingly a Pk/ PD study in this same animal model showed efficacy in a treatment regime with metoprolol that has later shown benefit in a recently finished human trial [46–48].

Conflict of interest The authors report no relationships that could be construed as a conflict of interest.

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Acknowledgments The authors gratefully and highly recognized P. Catalina, M.A. Canovas, F.J. Rodriguez and M.A. Velasco for their support in animal handling and care and for properly conducting the experimental work.

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