JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY VOL. 75, NO. 12, 2020 ª 2020 THE AUTHORS. PUBLISHED BY ELSEVIER ON BEHALF OF THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER THE CC BY-NC-ND LICENSE (http://creativecommons.org/licenses/by-nc-nd/4.0/). Intravenous Statin Administration During Myocardial Infarction Compared With Oral Post-Infarct Administration Guiomar Mendieta, MSC, MD,a,b Soumaya Ben-Aicha, MSC,a Manuel Gutiérrez, MD, PHD,c Laura Casani, PHD,a,c _ MD,a Francesc Carreras, MD, PHD,d Manel Sabate, MD, PHD,b Lina Badimon, PHD,a,e,* anauskaite, Monika Arz Gemma Vilahur, PHDa,e,* ABSTRACT BACKGROUND Beyond lipid-lowering, statins exert cardioprotective effects. High-dose statin treatment seems to reduce cardiovascular complications in high-risk patients. The ideal timing and administration regime remain unknown. OBJECTIVES This study compared the cardioprotective effects of intravenous statin administration during myocardial infarction (MI) with oral administration immediately post-MI. METHODS Hypercholesterolemic pigs underwent MI induction (90 min of ischemia) and were kept for 42 days. Animals were distributed in 3 arms (A): A1 received an intravenous bolus of atorvastatin during MI; A2 received an intravenous bolus of vehicle during MI; and A3 received oral atorvastatin within 2 h post-MI. A1 and A3 remained on daily oral atorvastatin for the following 42 days. Cardiac magnetic resonance analysis (days 3 and 42 post-MI) and molecular/ histological studies were performed. RESULTS At day 3, A1 showed a 10% reduction in infarct size compared with A3 and A2 and a 50% increase in myocardial salvage. At day 42, both A1 and A3 showed a significant decrease in scar size versus A2; however, A1 showed a further 24% reduction versus A3. Functional analyses revealed improved systolic performance in A1 compared with A2 and less wall motion abnormalities in the jeopardized myocardium versus both groups at day 42. A1 showed enhanced collagen content and AMP-activated protein kinase activation in the scar, increased vessel density in the penumbra, higher tumor necrosis factor a plasma levels and lower peripheral blood mononuclear cell activation versus both groups. CONCLUSIONS Intravenous administration of atorvastatin during MI limits cardiac damage, improves cardiac function, and mitigates remodeling to a larger extent than when administered orally shortly after reperfusion. This therapeutic approach deserves to be investigated in ST-segment elevation MI patients. (J Am Coll Cardiol 2020;75:1386–402) © 2020 The Authors. Published by Elsevier on behalf of the American College of Cardiology Foundation. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). S everal trials have suggested that high-dose statin treatment may reduce cardiovascular complications in patients undergoing invasive management. Through the inhibition of 3-hydroxy- 3-methylglutaryl-coenzyme A reduce low-density lipoprotein (LDL) cholesterol levels and decrease cardiovascular morbidity and reductase, mortality (1). Moreover, intensive statin therapy statins From the aCardiovascular Research Center-ICCC, Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain; bDepartment of Cardiology, Clinic Hospital, Barcelona, Spain; cRadiology, Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain; Listen to this manuscript’s d Cardiology, Hospital de la Santa Creu i Sant Pau, IIB-Sant Pau, Barcelona, Spain; and the eCiberCV, Institute Carlos III, Barcelona, audio summary by Spain. *Drs. Badimon and Vilahur contributed equally to this work. This work was supported by the Fundació Investigació Marato Editor-in-Chief TV3 #20154310 (to Dr. Vilahur), Plan Nacional de Salud (PGC2018-094025-B-I00 to Dr. Vilahur and SAF2016-76819-R to Dr. Bad- Dr. Valentin Fuster on imon) from the Spanish Ministry of Science and Innovation, and funds FEDER “Una Manera de Hacer Europa”; a grant from the JACC.org. Spanish Society of Cardiology (Beca FEC Investigación Básica/2016 to Dr. Vilahur); and CIBERCV (to Dr. Badimon). The Generalitat of Catalunya (Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat, 2017 SGR 1480) and the Fundación Investigación Cardiovascular-Fundación Jesus Serra also provided continuous support. Drs. Badimon and Vilahur are the authors of the patent that includes the use of statins for intravenous administration. All other authors have re- ported that they have no relationships relevant to the contents of this paper to disclose. Manuscript received June 22, 2019; revised manuscript received January 3, 2020, accepted January 23, 2020. ISSN 0735-1097 https://doi.org/10.1016/j.jacc.2020.01.042 JACC VOL. 75, NO. 12, 2020 Mendieta et al. 1387 MARCH 31, 2020:1386–402 Timing and Mode of Statin Administration Matters has demonstrated clinical benefit in non–ST- intravenous statin treatment during ongoing ABBREVIATIONS segment elevation myocardial infarction (STEMI) myocardial ischemia optimizes cardiac AND ACRONYMS patients, an effect also observed in patients sub- protection compared with oral post- ACS = acute coronary jected to elective coronary intervention procedures interventional statin therapy remains to be syndrome (2,3). In addition, statins seem to exert cardiopro- elucidated. AMPK = AMP-activated protein tective effects irrespective of their lipid-lowering In this study, we have hypothesized that kinase properties. However, the ideal timing and adminis- intravenous administration of atorvastatin CMR = cardiac magnetic tration regime are unknown. Post-interventional during MI is superior to an oral dose of ator- resonance oral statin administration is a common practice in vastatin initiated shortly after revasculariza- IL = interleukin cardiology departments in accordance with current tion. To adequately measure the impact of LV = left ventricle/ventricular practice guidelines (4,5). Yet, the value of a loading these interventions, we have used a preclin- MI = myocardial infarction dose of statin before intervention in reducing car- ical pig model of MI with serial cardiac mag- STEMI = ST-segment elevation diovascular events in patients with acute coronary netic resonance (CMR) follow-up. myocardial infarction syndrome (ACS) remains to be demonstrated. As TNF = tumor necrosis factor such, the SECURE-PCI (Statins Evaluation in Coro- METHODS nary Procedures and Revascularization Trial) (6) showed that routine administration of 2 early doses The animal experimental procedures were reviewed of atorvastatin (80 mg) is not superior to placebo in and approved by the Institutional Animal Care and reducing major adverse cardiovascular events Use Committees and authorized by the Animal (MACE) at 30 days among patients presenting with Experimental Committee of the local government ACS and scheduled to undergo an early invasive (#5601) in accordance to the Spanish law (RD 53/2013) approach. Yet, a subgroup analysis of the STEMI pa- and European Directive 2010/63/EU. In addition, the tients revealed a significant reduction in MACE at investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. Na- SEE PAGE 1403 tional Institutes of Health (NIH Publication No. 85-23, 30 days in those administered the statin regime, revised 1985), has followed the ARRIVE (Animal likely supporting statin-pleotropic effects. Indeed, Research: Reporting of In Vivo Experiments) guide- beyond their lipid-lowering effects, statins have lines and committed to the “3Rs” of laboratory animal direct cardioprotective and vasculoprotective prop- research, and consequently, used the minimal num- erties that may explain their beneficial effects in ber of animals to reach statistical conclusion (17). All the acute phase of MI (7–10) and post-MI healing animals were allowed to acclimate 7 days prior to any (11,12). Thus far, experimental studies have shown intervention. that statin treatment reduces infarct size when EXPERIMENTAL DESIGN, DRUG ADMINISTRATION, administered prior to or post-MI induction. In this AND MI INDUCTION. The experimental design is context, our group demonstrated that administra- shown in Figure 1. Pigs (n ¼ 21; 4-month-old sows; tion of oral rosuvastatin early after reperfusion weight: 38  3 kg) were fed a high cholesterol diet reduced infarct size (36% smaller infarcts) in a (Western-type hypercholesterolemic diet; 20% satu- closed-chest pig model of MI (13). This effect was rated fat, 2% cholesterol, 1% cholic acid) during due to a reduction in apoptotic cell death and in- 10 days. We have already reported that intake of this flammatory cell infiltration, leading to improved fat-rich diet for 10 days raises cholesterol to levels cardiac function. The effects of rosuvastatin were comparable to those found in hypercholesterolemic not related to changes in low-density lipoprotein humans (18). At day 10, all pigs underwent MI in- (LDL) cholesterol levels, but rather to the activation duction, and then reperfusion was established and of cardioprotective kinases; thus, the effect was animals were kept for 42 days. Closed-chest MI was reversed by the addition of the isoprenoid geranyl- experimentally induced by percutaneous total geranyl pyrophosphate (13,14). Of note, we have balloon occlusion of the mid-portion of the left recently evidenced that the intravenous administra- anterior descending artery, as previously described tion of statin during early ischemia (mimicking first (19,20) (expanded in the Supplemental Appendix, medical contact in the setting of STEMI) reduces the Supplemental Methods). Although pigs have a sparse progression of ischemic injury (15). Additionally, we collateral circulation, we routinely monitored have shown that atorvastatin and b-OH–simvastatin myocardial collateral circulation by fluoroscopy (a modified active simvastatin) exert similar guidance in all pigs to ensure a comparable degree of cardioprotective effects (16). However, whether ischemic damage after MI induction. 1388 Mendieta et al. JACC VOL. 75, NO. 12, 2020 Timing and Mode of Statin Administration Matters MARCH 31, 2020:1386–402 F I G U R E 1 Experimental Design Arm 1: Atorvastatin during MI (i.v.; 0.5 mg/kg; n = 7) Arm 2: Vehicle- control (i.v.; n = 7) Reperfusion CMR CMR (day 3) (day 42) 75 min 15 min 10 days MI-induction (90 min) Baseline Sacrifice Arm 1: Atorvastatin post-MI (p.o.; 1 mg/kg day) Arm 2: Vehicle - control Arm 3: Atorvastatin post-MI (p.o.; 1 mg/kg day; n = 7) Diet-induced hypercholesterolemia CMR ¼ cardiac magnetic resonance; i.v. ¼ intravenous; MI ¼ myocardial infarction; p.o. ¼ per os. Animals were randomly and blindly distributed in drawn at 15, 45, and 75 min after the intravenous the following 3 study arms (7 animals/group). Arm 1: infusion of atorvastatin or vehicle. animals were administered an intravenous bolus of At days 3 and 42 post-MI, animals were anes- atorvastatin (a modified intravenous atorvastatin thetized and brought to the 3T-CMR facility for blind preparation, patent PCT/EP2018/058158; 0.5 mg/kg; assessment of global and regional parameters. Ani- n ¼ 7) during ongoing ischemia (after 75 min of MI) mals were euthanized after the last CMR analysis (day (atorva-during-MI); arm 2: animals received an 42), and blood and tissue samples were collected for intravenous bolus of vehicle (dimethylformamide, molecular analysis. diluted in saline) during MI (after 75 min of MI) 3.0-T CMR ACQUISITION. CMR studies were con- (vehicle-control); and arm 3: animals were adminis- ducted serially in all animals at days 3 (early remod- tered per os atorvastatin, initiated within the first 2 h eling phase) and 42 (late remodeling phase) post-MI. post-MI (atorva-post-MI) (1 mg/kg). Animals in arms The studies were performed on a 3.0-T CMR system 1 and 3 remained on per os atorvastatin (1 mg/kg/day) (Achieva, Philips, Best, the Netherlands) and CMR therapy for the following 42 days, whereas animals in image acquisition was carried out by a CMR- arm 2 received placebo pills. To ensure the blind specialized technician blinded to treatment. For CMR administration of the therapies, an animal facility studies, animals were anesthetized with an intramus- technician who was not involved in conducting the cular injection of a cocktail composed of ketamine, experimental protocol was responsible for daily pill xylazine, and atropine and maintained by a contin- administration. uous intravenous infusion of propofol to ensure me- The intravenous drug dosage was calculated ac- chanical ventilation. Once the animals were cording to a 40-mg oral dose of atorvastatin used in positioned in a head-first supine position with a flex- humans, and converted to pig dosing according to ible phased-array surface coil placed over the chest, body surface area. We chose atorvastatin due to its electrocardiography gating was used to acquire still lipophilicity and use in our previous ischemia studies images of the heart. The following dedicated CMR se- (15), and because it is recommended in current clin- quences were acquired in all cases: “cine” (balanced ical practice guidelines worldwide. In a subgroup of steady-state free-precession) imaging sequence to animals (n ¼ 7 intravenous atorvastatin and n ¼ 7 assess wall motion (WM) and cardiac function, T2- vehicle), we measured statin plasma levels reached weighted short-tau inversion recovery (T2w-STIR) by the intravenous administration of atorvastatin. sequence to assess myocardial edema, early gadolin- Blood was collected, and K2-ethylenediaminetetra- ium enhancement to analyze microvascular obstruc- acetic acid plasma samples were analyzed by high tion (no-reflow phenomenon), and late gadolinium performance liquid chromatography tandem mass enhancement (LGE) to assess the amount and extent of spectrometry. For this purpose, blood was serially myocardial necrosis. All of the CMR studies followed JACC VOL. 75, NO. 12, 2020 Mendieta et al. 1389 MARCH 31, 2020:1386–402 Timing and Mode of Statin Administration Matters the same scheme. First, scout images (T1-TFE also determined the number of dysfunctional seg- sequence) were obtained to localize the true axes of ments and myocardial viability as previously reported the heart and define a field of view involving the whole (24). Briefly, dysfunctional segments were considered heart. Afterward, balanced steady-state free-preces- those segments with a WM <2 mm whereas myocardial sion cine imaging was performed in both horizontal recovery was assessed as the percentage of segments and vertical long axes (4- and 2-chamber views) and in with a WM at day 3 <2 mm (dysfunctional segments) multiple contiguous short-axis images covering the that presented a WM at day 42 $2 mm (recovered whole left ventricle (LV). In the short-axis cine segments). These measurements and analyses were sequence, we acquired 24 cardiac phases of every slice performed only including the jeopardized segments to guarantee a correct evaluation of the WM and heart (mid and apical antero/septal segments; target seg- function. Once the cine sequences were acquired, a ments 7, 8, 13, and 14) and including those segments T2w-STIR sequence was obtained to assess myocardial contralateral to the infracted region (basal, mid, and edema. Thereafter, a gadolinium-based contrast agent apical inferior/lateral segments; target segments 4, 5, was injected intravenously (Gd-GTPA, Magnevist, 10, 11, 15, and 16). Berlex Laboratories Inc., Wayne, New Jersey) at a dose SCAR SIZE ANALYSIS BY TRIPHENYLTETRAZOLIUM of 0.1 mmol/kg. The early gadolinium enhancement CHLORIDE STAINING. Although the investigation of sequence was acquired 1 min after the administration scar size by triphenyltetrazolium chloride (TTC) of the contrast. The LGE sequences were obtained staining late after reperfusion is not universally 10 min after the administration of contrast. Details of accepted, we performed the analysis with the inten- the technical parameters for CMR sequences have been tion of gathering some additional information. At day previously published (19,20). 42 and after CMR analysis, hearts were arrested with 3T-CMR DATA ANALYSIS. G l o b a l f u n c t i o n a l a n d potassium chloride, rapidly excised, sliced, and a n a t o m i c a l p a r a m e t e r s . CMR data were indepen- stained with TTC to determine the size of the scar by dently analyzed using dedicated software (QMass MR planimetry using the National Institutes of Health version 7.6, Medis, Leiden, the Netherlands) by a software ImageJ. CMR-trained radiologist (M.G.) blinded to the study MYOCARDIAL CHARACTERIZATION OF VESSEL medication. The protocol of analysis for global DENSITY AND FIBROSIS: MOLECULAR AND functional/anatomical parameters was performed as HISTOLOGICAL APPROACHES. To investigate the previously reported (19,21). Briefly, LV epicardial effect of statin treatment on cardiac remodeling and and endocardial borders were traced in each image scar tissue, we examined: 1) neovessel formation in of the cardiac phases representing the end-diastole the penumbra and remote myocardium via the anal- and end-systole to obtain the left ventricular end- ysis of angiogenic markers (CD105, von Willebrand diastolic volume (LVEDV), left ventricular end- factor [vWF], CD31) and lectin and vWF staining; and systolic volume (LVESV), and left ventricular 2) myocardial fibrosis in the scar and remote ejection fraction (LVEF). LGE scar size was assessed myocardium by assessing mRNA levels for collagen by using manual planimetric segmentation on each 3A1, collagen 1A1; protein expression/activation of slice. The same method was used for the assessment transforming growth factor (TGF)-b type II receptor of myocardial edema. The area of myocardial edema (Abcam, Cambridge, United Kingdom), was defined as the extent of the LV demonstrating phosphorylated-Smad2/3 (Ser423/425, Santa Cruz, high signal intensity on T2w-STIR images. The size Dallas, Texas)], AMP-activated protein kinase of infarction (day 3) or scar (day 42) was quantified (AMPK), and AMPK phosphorylated at Thr 172 from the extent of myocardial enhancement in the (P-AMPK); and, collagen deposition by Sirius red LGE CMR sequence. staining. We investigated the effects of statin treat- Cardiac remodeling parameters: WM and ment on AMPK activation given its previously re- m y o c a r d i a l v i a b i l i t y . LV regional analysis for each ported cardioprotective effects (16,25). of the 16 American Heart Association myocardial seg- For histological analyses, images were captured ments was performed (22). The endocardial and with a Nikon eclipse 80i microscope (Nikon, Tokyo, epicardial borders of the LV were defined in all slices Japan) and digitalized by a Retiga-1300i (Teledyne representing the end-diastolic and -systolic phases. QImaging, Surrey, British Columbia, Canada). Thereafter the end-systolic wall thickness (EST), the Staining was calculated by a single blinded observer end-diastolic wall thickness and the WM (WM ¼ EST  (S.B.-A.) from an average of 5-fields/sample and end-diastolic wall thickness) were automatically expressed as: (%) ¼ (positive stained area/[total tissue calculated as previously reported (23). In addition, we area  vascular luminal areas])  100 using ImageJ. 1390 Mendieta et al. JACC VOL. 75, NO. 12, 2020 Timing and Mode of Statin Administration Matters MARCH 31, 2020:1386–402 cyclooxygenase-2, and Toll-like receptor-4 T A B L E 1 Serial Cardiac Magnetic Resonance Analyses were performed. Treatment 3 Days Post-MI 42 Days Post-MI C i r c u l a t i n g c y t o k i n e s a n d C R P m e a s u r e m e n t . We Anatomical parameters determined circulating levels of cytokines (inter- LV mass, g Vehicle 70.1  11.7 79.6  13.8 leukin [IL]-6 and tumor necrosis factor [TNF]-a ) and Atorva post-MI (p.o.) 60.3  6.2 72.8  7.4 CRP in plasma and serum, respectively, at baseline, Atorva during MI (i.v.) 62.6  8.9 71.4  12.1 Edema, g LV Vehicle 24.4  2.8 acutely after MI induction (ischemia), and at 3 days Atorva post-MI (p.o.) 21.5  2.3 post-MI by using commercially available enzyme- Atorva during MI (i.v.) 20.7  3.3 linked immunosorbent assay kits (porcine IL-6 and Edema, % LV Vehicle 35.5  5.0 porcine TNF-a from R&D Systems, Minneapolis, Atorva post-MI (p.o.) 35.8  4.2 Minnesota; porcine CRP from Abcam). According to Atorva during MI (i.v.) 33.6  7.0 the manufacturers, the minimum detection limits for Infarct mass, g LV Vehicle 20.2  2.3 18.0  2.1 IL-6, TNF- a , and CRP are 10 pg/ml, 3.7 pg/ml, and Atorva post-MI (p.o.) 17.4  2.5 13.7  2.3† 2.15 ng/ml, respectively. Atorva during MI (i.v.) 12.2  4.9* 10.3  2.4* Necrosis, % LV Vehicle 29.3  5.2 22.9  2.5 HEMATOLOGICAL AND BIOCHEMICAL FOLLOW-UP. Atorva post-MI (p.o.) 29.0  4.4 18.8  2.5† Blood samples were collected at baseline, prior to MI, Atorva during-MI (i.v.) 19.1  6.6* 14.4  .4* post-MI, and 3 and 42 days post-MI for lipid levels Myocardial salvage, g LV Vehicle 4.3  2.1 and liver and renal parameters assessment. Atorva post-MI (p.o.) 4.1  2.3 Atorva during MI (i.v.) 8.6  2.4* STATISTICAL ANALYSIS. After testing for normal Myocardial salvage index Vehicle 17.2  6.3 distribution (Shapiro-Wilk test) repeated measures Atorva post-MI (p.o.) 19.0  8.5 analysis of variance and paired Student’s t-test was Atorva during MI (i.v.) 42.1  18.3* used to analyze variables within each group, whereas No-reflow Vehicle 1.9  0.8 unpaired Student’s t-test was used for all single time- Atorva post-MI (p.o.) 2.2  1.3 Atorva during MI (i.v.) 1.4  0.7 point measurements to compare the 3 animal groups Functional parameters in the absence of multiple testing correction. A value LVEF Vehicle 49.1  5.2 49.5  5.8 of p < 0.05 was considered significant. Continuous Atorva post-MI (p.o.) 52.4  8.0 46.7  9.9 variables are expressed as mean  SD. The statistical Atorva during MI (i.v.) 52.5  6.6 54.0  5.0 analyses were performed with the statistical LVEDV Vehicle 80.8  11.1 110.8  18.2‡ software package StatView (SAS Institute, Cary, Atorva post-MI (p.o.) 67.4  10.5 97.0  16.7‡ North Carolina). Atorva during MI (i.v.) 69.5  4.5 90.9  15.1‡ LVESV Vehicle 41.3  8.1 55.2  5.3‡ RESULTS Atorva post-MI (p.o.) 32.3  8.0 52.1  15.1‡ Atorva during MI (i.v.) 33.2  6.7 42.2  10.2† GLOBAL ANATOMICAL AND FUNCTIONAL PARAMETERS. Values are mean  SD. *p < 0.05 versus vehicle and atorvastatin post-MI. †p < 0.05 versus vehicle. ‡p < 0.05 C a r d i a c d a m a g e . The extent of area-at-risk (edema) versus day 3. i.v. ¼ intravenous. LV ¼ left ventricle; LVEDV ¼ left ventricular end-diastolic volume; LVEF ¼ left ventricular and the no-reflow were similar between the 3 ejection fraction; LVESV ¼ left ventricular end-systolic volume.; MI ¼ myocardial infarction; p.o. ¼ per os. treatment arms at day 3 post-MI. Administration of intravenous atorvastatin during MI resulted in a significant and marked reduction on infarct size INFLAMMATORY RESPONSE. P e r i p h e r a l blood (either expressed as grams or % of LV) in comparison m o n o n u c l e a r c e l l s i s o l a t i o n . Blood (20 ml) was to both oral administration of atorvastatin post-MI collected into ethylenediaminetetraacetic acid tubes and vehicle (p < 0.05 vs. both). Accordingly, at baseline and 3 days post-MI to evaluate any administering atorvastatin during MI resulted in 50% potential effect of the treatment on MI-induced increase in myocardial salvage (p < 0.05 vs. both). peripheral blood mononuclear cell (PBMC) activation The 42-day CMR analysis revealed that orally treated (26). All blood samples were immediately subjected atorvastatin animals after MI developed smaller scars to Ficoll-paque Plus (Amersham Biosciences, Little as compared to vehicle-administered animals Chalfont, United Kingdom) density gradient (p < 0.05) (Table 1, Figure 2A). Interestingly, atorva- centrifugation to isolate PBMCs. Samples were during-MI animals consistently showed a more counted and then directly frozen in liquid nitrogen pronounced reduction in the size of the scar, with a and stored at 80o C until mRNA and protein further 24% reduction, compared with atorva-post- analysis of monocyte-chemoattractant protein-1, MI pigs (p < 0.05 vs. both groups). Histopathological JACC VOL. 75, NO. 12, 2020 Mendieta et al. 1391 MARCH 31, 2020:1386–402 Timing and Mode of Statin Administration Matters F I G U R E 2 Cardiac Damage Assessed by CMR at Days 3 and 42 Post-MI Induction (A) Cardiac magnetic resonance imaging (CMR) representative images of edema, infarct size (day 3), and scar size (day 42). (B) Gross pathological images. MI ¼ myocardial infarction. 1392 Mendieta et al. JACC VOL. 75, NO. 12, 2020 Timing and Mode of Statin Administration Matters MARCH 31, 2020:1386–402 F I G U R E 3 Regional Analysis of Functional Parameters According to the 16-Segment Model (American Heart Association) A 7 1 6 7 Wall Motion (mm) 2 5 13 6 *† 8 12 4 14 16 * * 3 9 15 11 3 5 2 10 1 4 0 Vehicle Atorva Post- Atorva During- MI (p.o.) MI (i.v.) Dysfunctional Segments (%) 25 10 Myocardial Viability 20 8 15 6 10 4 * * 5 2 0 0 Vehicle Atorva Post- Atorva During- Vehicle Atorva Atorva MI (p.o.) MI (i.v.) Post-MI During-MI (p.o.) (i.v.) 3 Days Post-MI 42 Days Post-MI B 7 1 6 7 Wall Motion (mm) 2 6 5 8 13 12 4 14 16 3 9 15 11 3 5 2 10 1 4 0 Vehicle Atorva Post- Atorva During- MI (p.o.) MI (i.v.) 25 % Dysfunctional Segments 10 Myocardial Viability 20 8 15 6 10 4 5 2 0 0 Vehicle Atorva Post- Atorva During- Vehicle Atorva Atorva MI (p.o.) MI (i.v.) Post-MI During-MI (p.o.) (i.v.) 3 Days Post-MI 42 Days Post-MI Wall motion (WM) analysis, percentage of dysfunctional segments (WM <2 mm), and myocardial viability (percentage of dysfunctional segments at day 3 post-MI [WM <2 mm] that have been recovered [WM $2 mm] at day 42 post-MI) over time in the jeopardized (A) and remote (B) myocardium. Segments included in the analysis are highlighted in blue. The chi-square test was performed for the regional segmental analyses of the number of dysfunctional/ recovered segments. WM ¼ (end-systolic thickness)  (end-diastolic thickness). n ¼ 7 animals/group. Segmental analysis: chi-square *p < 0.05 versus vehicle. †p < 0.05 versus all. atorva ¼ atorvastatin; other abbreviations as in Figure 1. JACC VOL. 75, NO. 12, 2020 Mendieta et al. 1393 MARCH 31, 2020:1386–402 Timing and Mode of Statin Administration Matters analyses by TTC staining correlated (r ¼ 0.843; Smad3 transcript levels throughout the different p < 0.005) with CMR-assessed scar mass (Figure 2B). cardiac regions and among the different animal As observed in Supplemental Figure 1, no significant groups (Figure 5A). The mRNA expression of collagen differences were detected in the collateral circulation 3A1 was enhanced in the scar region of atorvastatin- among the 3 animal groups excluding the potential treated animals versus vehicle; yet, levels were confounding effect of collaterals on the final size significantly higher in atorva-during-MI treated pigs of infarction. compared with atorva-post-MI animals (p < 0.05). C a r d i a c f u n c t i o n . No significant changes in LVEF Likewise, although collagen 1A1 mRNA was enhanced were detected in the 3 animal groups between days 3 in the scar of all animals, atorva-during-MI pigs and 42 post-MI. Yet, there was a trend toward re- showed a 4-fold increase compared with the other 2 covery in atorva-during-MI administered pigs groups (p < 0.05). In line with this transcriptomic (p ¼ 0.1). In fact, although LV volumes (LVEDV and data, atorva-during-MI animals showed a higher LVESV) significantly increased over time (p < 0.05 vs. activation of Smad2/3 protein in the scar region day 3 post-MI), animals administered atorva-during- compared with all animals (Figures 5B and 5C). This MI showed greater improvement in LVESV increase was accordingly associated with a higher compared with control pigs (p < 0.05) (Table 1). detection of collagen fibrils in the forming scar (Figures 5D and 5E). REGIONAL PARAMETERS ASSOCIATED WITH LV REMODELING. We initially evaluated WM abnormal- IMPACT ON AMPK EXPRESSION AND ACTIVATION. ities in the target segments (i.e., jeopardized As per AMPK mRNA levels, these were only enhanced myocardium; 28 segments/group) (Figure 3A). At day in the scar region of atorva-during-MI pigs 3, segmental WM was severely impaired in vehicle (Figure 6A). Furthermore, although AMPK activation animals (akinetic and dyskinetic; 0.2 mm), whereas was found to be enhanced in all atorvastatin-treated they were mildly dysfunctional (hypokinetic) in all animals, the degree of activation was 2-fold higher atorvastatin-treated pigs (1.4 mm). In line with these in atorva-during-MI animals compared with atorva- observations, the proportion of dysfunctional seg- post-MI pigs (p < 0.05) (Figure 6B). ments (WM <2.0 mm) was higher in the vehicle group (67% of the segments vs. z50% in atorvastatin- EFFECTS ON PBMCs ACTIVATION AND INFLAMMATORY treated animals). At day 42 atorva-post-MI pigs CYTOKINES. MCP-1 and Toll-like receptor-4 mRNA persisted with WM at around 1.0 mm, while the levels were significantly down-regulated in PBMCs of atorva-during-MI pigs showed a marked improvement atorva-during-MI pigs 3 days post-MI compared with up to 2.1 mm (p < 0.05) (Figure 3A). We also assessed the other 2 groups, reaching expression levels similar WM in the inferior-lateral myocardium (i.e., remote to those found at baseline (prior MI induction) myocardium; n ¼ 42 segments/group) (Figure 3B). No (Figure 7A). The same pattern was observed in the WM abnormalities were detected in the treatment protein expression of both inflammatory markers arms, and only the vehicle-untreated group showed a (Figure 7B). No changes were detected in nonsignificant impairment (WM ¼ 1.4 mm). cyclooxygenase-2 expression at day 3 (neither at the mRNA nor at the protein level) (Figures 7A and 7B). IMPACT ON ANGIOGENESIS AND TGF b /SMAD2/3/- As shown in Figure 8, IL-6 and TNF-a plasma levels COLLAGEN SIGNALING AND FIBROUS TISSUE were sharply increased post-MI in all 3 groups. Yet, DEPOSITION. As shown in Figure 4A, no changes TNF-a detection was almost 3-fold higher than that of were detected in the gene transcription of diverse vehicle and atorva-post-MI pigs. CRP was similarly markers of angiogenesis among the different animals enhanced at 3 days post-MI and within the different cardiac regions, indicating a steady state in gene transcription. Lectin and vWF ATORVASTATIN PLASMA LEVELS AND BIOCHEMICAL staining, however, revealed higher vessel density in FOLLOW-UP. Plasma levels of atorvastatin were found the penumbra of all atorvastatin-treated pigs; yet, to be markedly increased at 15 min post-infusion and vWF staining (marker of mature endothelial cells) was then gradually declined for up to 75 min, always found to be significantly enhanced in the penumbra remaining above detectable levels (Supplemental of atorva-during-MI administered animals compared Figure 2). Atorvastatin was undetectable in vehicle with that of atorva-post-MI pigs (Figure 4B). Gene administered pigs. levels of TGF-b receptor and the transcription factor Blood cholesterol levels (total, LDL, and high- Smad2 were significantly and similarly up-regulated density lipoprotein cholesterol) (Supplemental in the scar region of all animals compared with the Table 1) were raised by the hypercholesterolemic remote myocardium. No changes were detected as to diet, and remained unchanged throughout the entire 1394 Mendieta et al. JACC VOL. 75, NO. 12, 2020 Timing and Mode of Statin Administration Matters MARCH 31, 2020:1386–402 F I G U R E 4 Vessel Density in the Penumbra and Remote Cardiac Tissue A 2.0 1.5 18S/rRNA 1.0 0.5 0.0 CD105 vWF cd31 CD105 vWF cd31 Penumbra Remote Vehicle Atorva Post-MI (p.o.) Atorva During-MI (i.v.) B Atorva Atorva Vehicle Post-MI (p.o.) During-MI (i.v.) Lectin 4 Penumbra 3 * % Stained Area * 2 * * Remote 1 0 Penumbra Remote Atorva Atorva Vehicle Post-MI (p.o.) During MI (i.v.) vWF 1.0 Penumbra * % Stained Area *† 0.5 * Remote 0.0 Penumbra Remote Vehicle Atorva Post-MI (p.o.) Atorva During-MI (i.v.) (A) Transcript levels of markers of new vessel formation (CD105, von Willebrand factor [vWF], and CD31). (B) Immunological staining against lectin and vWF. *p < 0.05 versus vehicle. †p < 0.05 versus vehicle and atorva-post-MI. Abbreviations as in Figure 1. JACC VOL. 75, NO. 12, 2020 Mendieta et al. 1395 MARCH 31, 2020:1386–402 Timing and Mode of Statin Administration Matters F I G U R E 5 Myocardial Fibrosis in the Evolving Scar A 100 80 § § 18SrRNA 60 §§ § † § 40 §§ 20 *† * 0 Scar Remote Scar Remote Scar Remote Scar Remote Scar Remote Scar Remote TGFβ1 TGFβr Smad2 Smad3 COL3A1 COL1A1 Vehicle Atorva Post-MI (p.o.) Atorva During-MI (i.v.) B C D 8 100 P-Smad 2/3/Smad 2/3 (AU) 7 6 P-Smad2/3 % Stained Area 80 * 5 Scar 60 KDa 60 4 Remote 60 KDa 3 40 Atorva Post-MI (p.o.) Vehicle Atorva During-MI (i.v.) 2 † 20 1 0 0 Scar Remote Scar Remote Vehicle Vehicle Atorva Post-MI (p.o.) Atorva Post-MI (p.o.) Atorva During-MI (i.v.) Atorva During-MI (i.v.) E Vehicle Atorvastatin Post-MI (p.o.) Atorvastatin During-MI (i.v.) Scar (A) Transcript levels of fibrotic markers. (B) Activation of the pSmad2/3 signaling pathway. (C) Representative western blot image of Smad2/3 activation. (D) Sirius red staining. (E) Representative image of Sirius red. *p < 0.05 versus vehicle. †p < 0.05 versus all. §p < 0.05 versus remote myocardium. COL ¼ collagen; TGF-b1 ¼ tissue growth factor-b 1; TGF-br ¼ tissue growth factor b receptor; other abbreviations as in Figure 1. 1396 Mendieta et al. JACC VOL. 75, NO. 12, 2020 Timing and Mode of Statin Administration Matters MARCH 31, 2020:1386–402 F I G U R E 6 Myocardial AMPK Expression and Activation A B 60 4 Vehicle 50 Atorva Post-MI (p.o.) 3 Atorva During-MI (i.v.) 40 18SrRNA †§ 30 AU 2 20 * †§ 1 10 0 0 Scar Remote Scar Remote Scar Remote AMPK CD36 p-AMPK/AMPK Vehicle Scar Remote Atorva Post-MI (p.o.) P-AMPK 63 KDa Atorva During-MI (i.v.) AMPK 63 KDa Vehicle Vehicle Atorva During-MI (i.v.) Atorva Post-MI (p.o.) Atorva Post-MI (p.o.) Atorva During-MI (i.v.) (A) Transcript levels of AMPK. (B) AMPK activation. *p < 0.05 versus vehicle. †p < 0.05 versus all. §p < 0.05 versus remote myocardium. AMPK ¼ AMP- activated protein kinase; other abbreviations as in Figure 1. study. No changes were observed in liver and kidney formation to a larger extent than when atorvastatin is enzymes throughout the study (Supplemental administered orally in the first 2 h after coronary Table 2). reperfusion. Such improved myocardial protection was associated with an up-regulation in collagen DISCUSSION expression and enhanced AMPK activation in the evolving scar and an enhanced vessel density in the The improved management of MI patients has penumbra (Central Illustration). reduced morbidity and mortality in the acute phase; Due to its elevated contrast-to-noise ratio, CMR however, this success has resulted in an increased allows for a precise differentiation between viable incidence of heart failure due to adverse post-MI LV and necrotic tissue, facilitating the noninvasive remodeling (27). At present, treatment efforts are quantification of myocardial necrosis and ultimately focused on limiting the damage in the setting of acute serving as the gold standard in cardioprotection ischemic heart disease as well as on stimulating post- research (28). In our study, animals receiving intra- MI LV repair. venous atorvastatin during ongoing MI exhibited In this study, we have demonstrated that intrave- significantly higher myocardial salvage index at nous atorvastatin treatment during MI followed by 3 days post-MI than animals orally treated with oral administration thereafter limits myocardial atorvastatin early post-MI. In fact, the atorva-post-MI damage, improves cardiac function, and reduces scar animals had similar myocardial salvage index than JACC VOL. 75, NO. 12, 2020 Mendieta et al. 1397 MARCH 31, 2020:1386–402 Timing and Mode of Statin Administration Matters F I G U R E 7 Impact of Statin Treatment on Monocyte Expression of Inflammatory Markers A 15 § 10 18SrRNA § § 5 * † 0 BSL 3 days Post-MI BSL 3 days Post-MI BSL 3 days Post-MI MCP1 COX2 TLR4 Vehicle Atorva Post-MI (p.o.) Atorva During-MI (i.v.) B 10 § 9 8 7 6 AU § 5 4 3 2 1 * *† 0 BSL 3 days Post-MI BSL 3 days Post-MI BSL 3 days Post-MI MCP1 COX2 TLR4 Vehicle Atorva Post-MI (p.o.) Atorva During-MI (i.v.) (A) Transcript levels. (B) Protein levels. *p < 0.05 versus vehicle. †p < 0.05 versus all. §p < 0.05 versus BSL. BSL ¼ baseline; COX2 ¼ cyclooxygenase-2; d ¼ days; MPC ¼ monocyte chemoattractant protein; TLR ¼ toll-like receptor; other abbreviations as in Figure 1. those treated with vehicle. In addition, at day 3 post- related markers (16). These benefits in combination MI, atorva-during-MI animals had reduced infarct with the cardioprotective potential associated with size and necrosis compared with all other animal enhanced TNF-a secretion may have contributed to treatment groups, and this effect persisted and was activate the mechanisms that better preserve the enlarged at day 42 post-MI. At day 42, orally only damaged myocardium ultimately reducing scar size. post-MI treated animals showed reduced scar size Of note, our imaging data allows us to exclude the compared with vehicle, but in a significantly lower contribution of no-reflow phenomenon, myocardial degree than atorvastatin-during-MI and, thereafter, collateral network, or edema formation on the dif- orally treated animals. We have demonstrated that ferences observed in cardiac damage. intravenous administration of atorvastatin during Regarding functional parameters, although no sig- ischemia reduces the levels and activation of senes- nificance was detected, there was a clear trend toward cence-, apoptosis-, and cardioprotective/metabolic- improvement in LVEF in the intravenously treated 1398 Mendieta et al. JACC VOL. 75, NO. 12, 2020 Timing and Mode of Statin Administration Matters MARCH 31, 2020:1386–402 F I G U R E 8 Levels of Circulating Inflammatory Markers IL-6 CRP 1,000 100 * * 800 75 * * * * 600 pg/ml pg/ml 50 400 25 200 0 0 Baseline Post-MI 3 Days Post-MI Baseline Post-MI 3 Days Post-MI TNF-α 5,000 *† 4,000 3,000 pg/ml Vehicle Atorva Post-MI (p.o.) 2,000 Atorva During-MI (i.v.) 1,000 * * 0 Baseline Post-MI 3 Days Post-MI Levels of cytokines (interleukin [IL]-6 and tumor necrosis factor [TNF]-a) and CRP in plasma from all pigs. *p < 0.05 versus baseline values. †p < 0.05 versus vehicle and atorva-post-MI. d ¼ days; other abbreviations as in Figure 1. animals compared with the other 2 groups, largely these segments remained below 2 mm, indicative of derived from the significant recovery of systolic func- persistent dysfunctional contractility over time. Of tion (LVESV). Keeping in mind that LVESV is an note, regional WM analysis showed that despite con- important post-MI predictor of survival (29), these tractile function impairment in the setting of ischemia, observations further underline the benefits derived statin administration improved contractile dysfunc- from intravenous treatment during myocardial tion in the early phase post-MI and myocardial infarction. Beyond LVEF and volumes, CMR-detected viability in later phases (42 days). Altogether, the regional function abnormalities have been related to overall functional/remodeling improvements in com- post-MI death and/or major adverse cardiac events bination with lower size of infarction suggest that (30). Here, severe WM abnormalities were detected in there would be a significant improvement in cardiac the jeopardized myocardium of vehicle-administered performance at longer follow-up periods in animals compared with atorvastatin-treated pigs at intravenous-during-MI atorvastatin-treated pigs. 42 days post-MI. WM recovery in the jeopardized Post-MI LV changes entail complex molecular in- myocardium of intravenously treated animals was teractions (31). Pharmacotherapeutic strategies aimed enhanced with respect to the other treatment arms at at mitigating adverse LV remodeling are currently 42 days post-MI. However, the number of segments under investigation. In this study, we have demon- with improved WM in relation to 3 days post-MI was strated that statin treatment regulates molecular higher in vehicle-administered pigs compared with markers of fibrosis. We previously showed, in the atorvastatin-treated animals, likely because they were porcine model of hypercholesterolemia, that MI im- remarkably dysfunctional at day 3; yet, WM within pairs the reparative fibrotic response mediated by JACC VOL. 75, NO. 12, 2020 Mendieta et al. 1399 MARCH 31, 2020:1386–402 Timing and Mode of Statin Administration Matters C ENTR AL I LL U STRA T I O N Cardiovascular Protection Afforded by Intravenous Administration of Atorvastatin During Myocardial Infarction Induction Mendieta, G. et al. J Am Coll Cardiol. 2020;75(12):1386–402. Administration of intravenous atorvastatin during myocardial infarction (MI) induces acute protective effects both in the myocardium (lower infarct size) and sys- temically (diminished peripheral blood mononuclear cell [PBMC] activation and higher tumor necrosis factor [TNF]-a levels) and mitigates adverse left ventricular remodeling (smaller scar and less wall motion abnormalities) to a larger extent than when administered orally shortly after reperfusion. These benefits are associated with higher collagen content and activation of the AMP activated kinase (AMPK) in the evolving scar and enhanced vessel density in the penumbra. CMR ¼ cardiac magnetic resonance. 1400 Mendieta et al. JACC VOL. 75, NO. 12, 2020 Timing and Mode of Statin Administration Matters MARCH 31, 2020:1386–402 TGF- b1–TGF-b receptor and Smad2/3 signaling path- injury, and thus, the role of statins at a systemic level ways (18). The current study shows that intravenous may also contribute to the beneficial effects observed administration of atorvastatin during MI enhances in the cardiac remodeling response. the TGF- b receptor signaling pathway, favoring a In this study, animals were fed a hypercholes- reparative fibrotic response in the developing scar. terolemic diet prior to MI induction to model this The TGF-b1–TGF-b receptor pathway regulates fibro- common adverse cardiovascular comorbidity, often blast differentiation into myofibroblasts, responsible found in ACS patients. Animals were fed the hy- together with downstream associated effectors percholesterolemic diet during the entire duration Smad2/3 of regulating the transcription of extracel- of the study (42 days) given that our objective was lular matrix genes COL1A1-3A1, which in our study to interfere with the isoprenoid pathway (that is were significantly increased in the evolving scar of also regulated by 3-hidroxi-3-metilglutaril-coenzima intravenously treated animals. However, intravenous A reductase), rather than to lower LDL cholesterol. atorvastatin-administered animals also show higher Either reducing dietary cholesterol or a longer vWF staining in the penumbra zone, further indi- treatment period with atorvastatin would have cating beneficial effects in the remodeling process. reduced lipid levels; in fact, current guidelines Altogether, our data indicates that the intravenous advise lipid level control 3 months after treatment administration of statin during MI mitigates adverse initiation and dietary modifications. Our interven- cardiac remodeling, an unobserved benefit when tion in the study, however, was free of adverse liver administering oral statin early post-MI. and/or renal effects. We have previously demonstrated that myocardial AMPK activation is associated with antiplatelet STUDY LIMITATIONS. This study has several short- agent–related cardioprotective effects (23). In this comings worth discussing. First, the atorvastatin study, we have observed enhanced AMPK activation formulation used herein was readily prepared in the in the developing scar of animals treated with statins laboratory because a good manufacturing practice versus vehicle. Yet, the intravenously statin-treated product was not required in this study; our efforts are animals present a significantly higher degree of currently centered on attaining a good manufacturing AMPK activation compared with the only orally practice product. Second, our closed-chest total treated statin group, which further supports the balloon occlusion MI model includes mechanical oc- concept of intravenous treatment during MI as a clusion but not atherothrombosis as the triggering strategy to enhance myocardial preservation. factor of ischemia; however, the porcine model is the Intravenous statin treatment during MI has also best preclinical experimental model to investigate demonstrated to modulate the inflammatory cardiac events due to the similarity in organ size, cor- response by acutely and sharply enhancing TNF-a . onary anatomy, immunology, and physiology to TNF- a is a cytokine released by multiple cells ranging humans. Third, induction of larger and more from inflammatory cells to cardiomyocytes and fi- severe infarcts would have further evidenced the broblasts. Transient increase in TNF- a has shown to efficacy of intravenous atorvastatin-related car- protect against ischemia–reperfusion injury by inter- dioprotective effects; however, in this first statin long- acting with myocardial TNF receptor type 2 and term study, we adopted a conservative approach to consequent downstream activation of the car- reduce peri-intervention animal mortality. Fourth, we dioprotective SAFE pathway (32–34). Whereas acute did not apply multiple testing corrections because of ischemia has shown to induce the secretion of pre- the low N (according to the ARRIVE guidelines in ani- formed TNF- a from macrophages and mast cells, mal testing) and the many time points and groups persistent ischemia (as in our study) has been analyzed. Finally, hypercholesterolemic patients are demonstrated to induce cardiomyocyte release of chronically treated with oral lipid-lowering doses of TNF- a , which, in turn, induces SAFE pathway acti- statins; however, in this study, our objective was to vation (35). Further studies are needed to evaluate compare the effects of intravenous administration of whether such acute TNF-a induction leads to early statins during MI (not the “classic” lipid-lowering myocardial STAT-3 activation. However, in the properties but rather assessing the effect of intravenously atorvastatin administered animals, we isoprenoid-blockage) versus oral post-MI therapy, in also detected a reduction in systemic PBMCs activa- accordance with current clinical practice guidelines. tion through the down-regulation of inflammatory Notwithstanding, the impact of being on statin treat- mediators at 3 days post-MI. Indeed, excessive in- ment for lipid lowering prior to MI is presently be- flammatory cell activation worsens myocardial ing investigated. JACC VOL. 75, NO. 12, 2020 Mendieta et al. 1401 MARCH 31, 2020:1386–402 Timing and Mode of Statin Administration Matters CONCLUSIONS ADDRESS FOR CORRESPONDENCE: Dr. Gemma Vila- In this study, we have demonstrated that intravenous hur, Cardiovascular ICCC Program, Research Institute– administration of statin during MI followed by oral Hospital de la Santa Creu i Sant Pau, IIB Sant Pau, Avda. therapy thereafter is superior to the administration of S. Antoni María Claret 167, 08025 Barcelona, Spain. E-mail: atorvastatin orally shortly after reperfusion, in that it gvilahur@santpau.cat. Twitter: @HospitalSantPau. better protects the myocardium, significantly reduces scar size, and mitigates LV adverse remodeling. The PERSPECTIVES potential therapeutic implications of this novel administration regime should be explored in the management of ACS patients. COMPETENCY IN PATIENT CARE AND PROCEDURAL SKILLS: Intravenous administration of statin medication during ACKNOWLEDGMENTS The authors are grateful for MI is associated with less adverse cardiac remodeling than oral the valuable help and support of M.A. Canovas, administration following myocardial reperfusion. P. Catalina, O. García, J. Moreno, F.J. Rodriguez, and M. Pescador with animal handling and for the proper TRANSLATIONAL OUTLOOK: Clinical studies should seek to conduct of all the experimental and molecular work. establish the optimum timing and mode of statin administration This paper is part of the PhD studies of G. Mendieta at in patients with acute MI. the Medical School of the University of Barcelona and at the ICCC. REFERENCES 1. Baigent C, Blackwell L, Emberson J, et al. Effi- 8. de Lemos JA, Blazing MA, Wiviott SD, et al. cardioprotective effects. 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