Resolution Agonist 15-epi-Lipoxin A4 Programs Early Activation of Resolving Phase in Post-Myocardial Infarction Healing

Following myocardial infarction (MI), overactive inflammation remodels the left ventricle (LV) leading to heart failure coinciding with reduced levels of 15-epi-Lipoxin A4 (15-epi LXA4). However, the role of 15-epi LXA4 in post-MI acute inflammatory response and resolving phase is unclear. We hypothesize that liposomal fusion of 15-epi-LXA4 (Lipo-15-epi-LXA4) or free 15-epi-LXA4 will expedite the resolving phase in post-MI inflammation. 8 to 12-week-old male C57BL/6 mice were subjected to permanent coronary artery ligation. Lipo-15-epi-LXA4 or 15-epi-LXA4 (1 µg/kg/day) was injected 3 hours post-MI for (d)1 or continued daily till d5. 15-epi-LXA4 activated formyl peptide receptor (FPR2) and GPR120 on alternative macrophages but inhibited GPR40 on classical macrophages in-vitro. The 15-epi-LXA4 injected mice displayed reduced LV and lung mass to body weight ratios and improved ejection fraction at d5 post-MI. In the acute phase of inflammation-(d1), 15-epi-LXA4 primes neutrophil infiltration with a robust increase of Ccl2 and FPR2 expression. During the resolving phase-(d5), 15-epi-LXA4 initiated rapid neutrophils clearance with persistent activation of FPR2 in LV. Compared to MI-control, 15-epi-LXA4 injected mice showed reduced renal inflammation along with decreased levels of ngal and plasma creatinine. In summary, 15-epi-LXA4 initiates the resolving phase early to discontinue inflammation post-MI, thereby reducing LV dysfunction.

Myocardial infarction (MI) is the leading cause of heart failure and is responsible for a high number of mortalities resulting from persistent and unresolved inflammation 1 . In heart failure pathology, there has been displayed a consistent increase in the number of cytokines and chemokines that are the hallmark of chronic inflammation and kidney dysfunction 2 . Traditional inflammation treatment using non-steroidal anti-inflammatory agents (e.g. rofecoxib and celecoxib) was unsuccessful to restore the function of the inflamed infarct area in clinical settings; instead, inhibition of inflammation treatments provoked MI events and kidney pathology [3][4][5] . Thus, inhibition of inflammation or suppression of cytokines has been an ineffective approach in delaying heart failure post-MI 6 , indicating the diversified role of MI-induced cytokines in heart failure pathology has been overemphasized for decades. In response to myocardial injury, the physiological innate response consists of programing the clearance and healing of the left ventricle (LV) 7 . However, the non-resolving innate response programs the LV towards heart failure 8 . Thus, by exploring an alternative strategy to resolve uncontrolled inflammation presents an important unmet medical need to re-examine methods to delay or reduce heart failure. Post-MI resolution of inflammation is an active phase that limits subsequent LV remodeling and heart failure. Following MI, an overactive neutrophil and macrophage response adversely remodels the LV to alters size, shape, and function with progressive heart failure pathology 9 . Post-MI healing occurs in two phases: the early acute inflammatory phase and the resolving phase (also called late or reparative phase) 10 . The acute phase is marked by the entry of neutrophils and monocytes/macrophages, followed by the rapid repression of pro-inflammatory genes, indicating the resolution of inflammation 1 . Post-MI, the splenic supply of leukocytes correlates with pro-inflammatory circulating leukocytes that are associated with the acute inflammatory response of the LV, marked by the proliferation of monocyte progenitor cells and activation of monocytes 11,12 . A persistent supply of overactive immune cells post-MI, such as polymorphonuclear leukocytes (PMNs), contributes to the pathology of congestive heart failure 13 . Thus, to resolve the inflamed infarcted area post-MI, the early activation of the resolving phase is essential [13][14][15] .
Lipoxygenase interaction products (lipoxins; LXs) are endogenous, pro-resolving, lipid mediators generated from membrane arachidonic acid through biochemical synthesis involving the enzymes 5-and 15-lipoxygenase (5-LOX and 15-LOX). The primary function of LXs is to coordinate the functions and recruitment of PMNs to promote clearance of debris 16 . The major pathway for generating LXs is augmented in the presence of aspirin, through cyclooxygenase (COX)-2 and 5-LOX activity during the acute inflammatory response 17 . In order to offer pharmacological action, 15-epimer LXA 4   binds to the G-coupled protein receptor FPR2 18,19 . Endogenous natural lipoxin A 4 also inhibits TGF-β1-dependent collagen secretion and α-SMA expression in human lung myofibroblasts 20 . Importantly, 15-epi-LXA 4 levels were lower than normal values in patients experiencing the progressive development of chronic heart failure due to the defective resolution of inflammation 21 .
Here, we hypothesize that the stable form of LXA 4 ; 15-epi-LXA 4 with liposomal fusion (Lipo-15-epi-LXA 4 ) or free 15-epi-LXA 4 would initiate the resolving phase at the precise time in post-MI healing to limit cardiac remodeling and subsequent heart failure. To address the instability of the hydrophobic compound, 15-epi-LXA 4 was incorporated into a liposomal fusion 15 . Our in-vitro and in-vivo results indicate that post-MI treatment of pure 15-epi-LXA 4 or liposomal 15-epi-LXA 4 initiates the early resolving phase, suggesting a therapeutic potential of the lipoxin biomolecule to delay MI-induced cardiorenal pathology.

15-epi-LXA 4 limited TGF-β induced myofibroblast formation in cardiac fibroblast in-vitro. The
transition of fibroblast to secretory myofibroblast leads to collagen deposition and is involved in the pathophysiological process of scar formation in post-MI healing. To test 15-epi-LXA 4 , we differentiated cardiac fibroblast into myofibroblast using TGF-β. As shown in Fig. 1E, TGF-β induced fibroblast hypertrophy within 18 hours transitioning fibroblast into myofibroblast. The formation of myofibroblast is indicated by higher expression of α-SMA along with DDR2 and having a typical myofibroblast phenotype. Co-incubation of 15-epi-LXA 4 (100 nM) with TGF-β (15 ng/ml) for 18 hours reduced α-SMA expression. Cultured fibroblast treated with 15-epi-LXA 4 were less stellate and became more spindle shape, displaying a significant decrease in stellate:spindle cell ratio compared with TGF-β treated cells, thereby having relatively similar morphology to fibroblasts ( Fig. 1E and Supplementary Figure 1). Thus, 15-epi-LXA 4 limited TGF-β induced hypertrophy as well as the differentiation of fibroblast into myofibroblast in-vitro.
Quality control outcome for 15-epi-LXA 4 liposomes. EPC/DSPE-PEG (2000) liposomes were investigated here to achieve a stable 15-epi-LXA 4 loaded system by overcoming the natural unstable properties of the liposome. The attached PEG chain can stabilize the drug loaded liposomes for storage up to 300 days at 2 °C to 8 °C 22 . Instead of using passive and active loading methods, 15-epi-LXA 4 was loaded in the liposomes by using thin layer dehydration method to achieve higher encapsulation efficiency. As illustrated (Fig. 1F), the average particle size of blank liposome was 87.80 nm with a polydispersity index (PDI) of 0.096 after extrusion, indicating the monodisperse distributions based on this formulation. For extruded 15-epi-LXA 4 liposomes, the average size slightly increased exhibiting at 93.75 nm with the PDI at 0.086, which also confirms the monodispersity for 15-epi-LXA 4 loaded liposomes. The low PDI of the liposomes resulted from the uniform incorporation of 15-epi-LXA 4 and the extrusions (Fig. 1F) 23, 24 . 15-epi-LXA 4 treated mice attenuated LV dysfunction and reduced pulmonary edema post-MI. Echocardiography measurements were performed to assess the effect of 15-epi-LXA 4 treatment on LV function post-MI. Echocardiography data ( Fig. 2A) showed higher, ejection fraction (19% (Lipo 15-epi-LXA 4 ) and 18% (15-epi-LXA 4 ); p < 0.05) with improved EDV (end-diastolic volume and ESV (end systolic volume) in 15-epi-LXA 4 treated mice compared with MI control mice at d5 post-MI ( Fig. 2B and C). The 15-epi-LXA 4 treated mice showed a trend of improvement in fractional shortening, EDD (end-diastolic dimension), ESD (end-systolic dimension) and IVSd (interventricular septal end diastole) compared with MI-control mice. Both Lipo-and free 15-epi-LXA 4 treated mice showed reduced hypertrophy with a decrease in LV/Body weight ratio (4.0 ± 0.10 vs. 4.8 ± 0.12) compared to MI-control mice ( Table 1).
Gravimetric analyses indicated reduced lung pulmonary edema in 15-epi-LXA 4 treated mice with a decrease in lung mass-to-body weight ratios compared to MI-control at d5 post-MI (Table 2). Thus, reduced pulmonary edema decreased LV dilation and increased ejection fraction leading to improved LV function in 15-epi-LXA 4 treated mice post-MI.

15-epi-LXA 4 activated FPR2 in cardiosplenic and cardiorenal network post-MI. Post-MI
15-epi-LXA 4 action was determined in the acute and resolving phase, at the site of LV injury and distant organs like spleen and kidney. FPR2 and Ccl2 expression were measured in the LV, kidney, and spleen. In the acute phase of post-MI healing at d1 both Lipo (2.4 fold) and free 15-epi-LXA 4 (9.2 fold) injected groups displayed a robust increase of FPR2; p < 0.05 in infarcted LV (LVI) compared with MI-control group (Fig. 2D). There was a minimal increase in FPR2 expression at d1 post-MI in the spleen (Supplementary Figure 2A   Heart rate (bpm) 433 ± 14 450 ± 9 464 ± 9 480 ± 13 440 ± 12 504 ± 20 485 ± 13  MI-control mice decreased GPR120 expression in both the acute and resolving phases in LVI and spleen. In the kidneys, GPR120 expression was increased in the acute phase (d1) and decreased during the resolving phase (d5) in MI-control group. Higher expression of GPR120 is known for anti-inflammatory effect, here in post-MI setting 15-epi-LXA 4 activated GPR120 without affecting macrophages density ( Fig. 5I and Supplementary 4A). The reparative macrophage phenotype was confirmed by co-localization (yellow) of macrophage surface marker F4/80 (green) and resolving marker MRC-1 (red) (Fig. 5J and Supplementary 3B and 4). Thus, 15-epi-LXA 4 injected mice displayed an increase in GPR120 expression in LVI with no change in spleen and decreased expression in the kidney at d5 post-MI (Fig. 5D-F).    (Fig. 6E). Post-MI, collagen deposition occurs in response to the acute inflammatory response in the infarcted LV. Quantitative analyses of infarcted area collagen density stained using picrosirius red revealed that there was increased collagen density in response to MI in controls and lipo-and 15-epi-LXA 4 -injected mice (Fig. 6F,G). Of note, 15-epi-LXA 4 injected mice displayed a decrease in α-SMA expression compared to MI-control group at d5 post-MI ( Fig. 6H and I) indicated non-canonical pathway regulation in 15-epi-LXA 4 treated mice compared with MI-control mice. Thus, post-MI treatment of 15-epi-LXA 4 limited acute inflammation in the kidney with no effect on LV collagen density post-MI.

Discussion
Complications in post-MI acute healing lead to recurrent ischemic, mechanical, arrhythmic, and inflammatory disturbances, which together account for a majority of fatalities that occur in heart failure patients 10 .
Anti-inflammatory agents, including tumor-necrosis factor inhibitors, impair the resolution of inflammation and promote heart failure 13 . Therefore, novel alternative treatments for delaying heart failure could be attempted to resolve post-MI inflammation by using proresolving mediators like 15-epi-LXA 4 . Endogenous bioactive 15-epi-LXA 4 is a stable analog of LXA 4 produced by acetylation of COX-2 16 . The presented results show that 15-epi-LXA 4 improved LV function by early activation of the resolving phase post-MI. The key evidence of early activation of the resolving phase with unaltered innate response are: 1) increased FPR2 and Ccl2 thereby leading to rapid neutrophil clearance by d5 post-MI; 2) reduced proinflammatory response at LV and remote organs, i.e. spleen and kidney; and 3) activated GPR120 and inhibited GPR40, along with increased proresolving markers during the resolving phase post-MI. Thus, the present study revealed that bioactive 15-epi-LXA 4 exerts proresolving properties and thus has potential that demands further long-term exploration in the chronic heart failure setting. Resolution of inflammation is an active process regulated by leukocytes (neutrophils, monocytes/macrophages) and some other proresolving lipid mediators 13,14 . Fatty acid receptors are known to play a role in the resolution of inflammation and FPR2 is a receptor for 15-epi-LXA 4 . GPR120, GPR40, and FPR2 are highly expressed on macrophages, thus based on expression pattern, GPR40, GPR120, and FPR2 emerged as a receptor of particular interest in achieving the resolution of inflammation post-MI. Lipoxins and their 15-epimers are both known to exert proresolving responses through activation of GPCR 26 . Many reports suggest that FPR2 is present on neutrophils, monocytes, and macrophages in order to regulate chemotaxis 27,28 . In the current study, we further delineated the macrophage class switching plasticity using 15-epi-LXA 4 and interaction with GPCR not only on FPR2 but also with GPR40 and GPR120. First, in-vitro results indicated classical (M1) macrophages expressed FPR2 and GPR40 while alternative (M2) macrophages expressed FPR2, along with GPR120 and a lower expression of GPR40. GPR40 acted as a functional sensor to promote the acute inflammatory response, while GPR120 promoted the resolution process. Since, 15-epi-LXA 4 acts as a sensor for FPR2 and GPR120, while also being an inhibitor for GPR40 suggests that 15-epi-LXA 4 monitors resolution receptors. Second, in-vitro results are further validated in-vivo post-MI studies where 15-epi-LXA 4 in both liposomal and free form activated FPR2 at d1 post-MI. Several in-vitro, pre-clinical and clinical human studies have shown that LXA 4 acts as an agonist to FPR2 in order to exert a proresolving response [29][30][31] . Our study indicates that 15-epi-LXA 4 also acts as an agonist to GPR120 while being an antagonist for GPR40 to heal the infarcted LV and reduce post-MI LV dysfunction. In response to inflammation, a diverse nature of chemo-attractants regulates immune cell kinetics with sequential modification of acute and healing phases at the inflammatory sites 32 . In this post-MI study, the 15-epi-LXA 4 treated mice activated Ccl2 simultaneously with FPR2 at d1 post-MI, which enables leukocyte trafficking to the inflammatory site, i.e. LV but also at remote organs such as the spleen and kidneys. Thus, 15-epi-LXA 4 mediated the simultaneous early activation of FPR2 and Ccl2, suggesting a robust activation of the innate inflammatory phase, which is essential for the physiological healing of the inflammatory response post-MI.
15-epi-LXA 4 inhibits chemotaxis, adherence, and transmigration of neutrophils along with inhibiting neutrophil-epithelial and endothelial cell interactions 33,34 . Post-MI, 15-epi-LXA 4 promoted LV healing through increased neutrophil infiltration in the acute phase and during the resolution phase promotion of the rapid clearance of neutrophils. In-vitro and in-vivo results provide evidence that 15-epi-LXA 4 promotes the clearance of neutrophils in order to achieve the early initiation of the resolving phase thereby shorten the inflammatory phase. Lipoxins (lipoxygenase interaction products; e.g. LXA 4 ) are endogenous bioactive molecules generated by transcellular metabolism and are actively known to promote resolution pathway like other proresolving lipid mediators, such as resolvins 15 . Like LXA 4 , Resolvin D1 interacts with FPR2 and GPR32 that are responsible to mediate the proresolving signal 35 . Similarly, 15-epi-LXA 4 mediates the proresolving signal through FPR2, GPR40, and GPR120. 15-epi-LXA 4 shortens the proinflammatory window without altering the acute inflammatory response that is required immediately after MI for phagocytic clearance of necrotic or apoptotic myocyte 36 . Since the active resolution of inflammation depends on a successful and coordinated transition from the initial recruitment of neutrophils to the more sustained population of mononuclear cells. Post-MI, increased levels of cytokines IL-1β and IL-6 at d1 modulates the inflammation axis. IL-6 represents a checkpoint regulator of neutrophil trafficking while IL-1β regulates neutrophil migration during the inflammatory response to coordinate resolution of inflammation 37,38 . Post-MI, IL-6 is produced by some different cell types including monocytes, macrophages, and fibroblast while IL-1β is produced by monocytes and macrophages, both contributing to the active inflammatory Early activation of the resolving phase following the innate response determines successful healing to control an overactive and unresolved inflammation post-MI. The capacity of macrophages to sequentially exhibit predominant anti-inflammatory properties than classical phenotypes defines the resolution process. Macrophages are a multifunctional diverse cell type, to simplify the resolution process in healing, for easiness macrophages are defined as M1 when possessing a proinflammatory role, and M2, when possessing tissue repair and proresolving functions. Early activation of the resolution process in 15-epi-LXA 4 treated mice was supported by a consistent increase in Mrc-1, Ym-1, and Arg-1 post-MI. Though the lipo-15-epi-LXA 4 injected mice did display an increase in Ym-1 and Arg-1 at d1; this could be due to a slow release of 15-epi-LXA 4 from liposomes.
Renal dysfunction is important in post-MI comorbidities for heart failure with diminished kidney function. The interaction between heart failure and kidney dysfunction is bidirectional to acute or chronic dysfunction of the heart and vice versa. NGAL is a potent acute kidney injury biomarker, which is immediately elevated within 24 hr post-MI as a result of the acute inflammatory and cardiorenal axis 40 . Similarly, Kim-1 is another biomarker that validates renal injury 41  the possible mechanism of 15-epi-LXA 4 is helping to build less inflamed stable extracellular matrix due to early turn-off inflammation with immediate turn-on resolution post-MI 43 . The current study monitored the proresolving capacity of 15-epi-LXA 4 in response to acute post-MI healing, the long-term studies are warranted to validate the role of 15-epi-LXA 4 in chronic heart failure.
Our study has highlighted the significant advantages for liposomal drug delivery, achieved from the liposomes' controllable size ranging 10-200 nm, which enhances penetration into blood vessels and helps to reduce systemic degradation of biomolecules compared to a non-liposomal or free drug 44,45 . More importantly, liposomal drug delivery for small bioactive molecules, such as 15-epi-LXA 4 provides the protection in the blood stream during circulation that avoids the fast clearance from the reticulo-endothelial system and depresses the immune response before reaching the targeting tissues 46,47 . Liposomal delivery of 15-epi-LXA 4 showed similar protective function as free 15-epi-LXA 4 , improving ventricular function to a similar extent. Furthermore, liposomal delivery of 15-epi-LXA 4 limited changes in the proinflammatory markers IL-6 and IL-1β in the remote organs spleen and kidneys ( Fig. 3D and G) compared to free 15-epi-LXA 4 delivery.
In conclusion, we demonstrated that 15-epi-LXA 4 initiates early activation of the resolving phase ( Fig. 7) thereby improving LV function post-MI. 15-epi-LXA 4 activated neutrophil clearance in the LV during the resolving phase with an increase in FPR2 and Ccl2 during the acute inflammatory phase. Additionally, 15-epi-LXA 4 promotes the resolution of inflammation by activating GPR120 and inhibiting GPR40. 15-epi-LXA 4 potentially acts on the cardiorenal axis by inhibiting kidney ngal and Kim-1 expression post-MI. In summary, our findings indicate that 15-epi-LXA 4 possesses the high therapeutic potential to modulate neutrophil biology and resolution physiology to limit cardiac remodeling, thus regulating renal pathology and thereby delaying cardiorenal failure post-MI. Macrophages isolation and 15-epi-LXA 4 treatment. Peritoneal macrophages (PM) were isolated from C57BL/6 J mice as previously described 48 with slight modifications. Briefly, the mice were anesthetized with 2% isoflurane using a tabletop anesthesia machine (SurgiVet TM ), and the peritoneal cavity was lavaged twice with 10 ml of ice-cold RPMI 1640 media with 10% FBS) and 1% antibiotics. The recovered media was centrifuged at 250 x g for 10 min. The cell pellet was re-suspended in 6 ml of RPMI 1640 media. The cells were plated in a 6-well plate (1 × 10 6 cells/ well), incubated at 37 °C overnight to allow the cells to adhere, and subsequently washed with fresh media to remove any unattached cells. To differentiate PM into M1 or M2 phenotype, the cells were stimulated with LPS (1 µg/ml) + IFNγ (20 ng/ml) and IL-4 (20 ng/ml) + IL-13 (20 ng/ml) respectively for 4 hrs. Both M1 and M2 macrophages were treated with 15-epi-LXA 4 (100 nM) for 2 hrs.

Material and Methods
Cardiac fibroblast isolation, TGF-β-induced differentiation, and 15-epi-LXA 4 treatment. The mice were anesthetized using 2% isoflurane, and the chest cavity was opened to remove the heart. After heart isolation, the atria, and right ventricle were removed, and the LV was minced using surgical blades. Cardiac fibroblasts (CF) from the LV were isolated by enzymatic digestion with 600 U/ml collagenase II and 60 U/ml DNase I. Cells at passage 2 were plated in 6-well plates (5 × 10 4 cells/well) and allowed to attach at 37 °C overnight, then washed using DMEM/F-12 media with 10% FBS and 1% antibiotics to remove unattached cells. The cells were plated on Millicell EZ slide-8-well (Millipore). The cells were serum deprived for 24 hrs and then stimulated with TGF-β (15ng/ml) for 18hrs for myofibroblast differentiation and co-incubated with 15-epi-LXA 4 (100 nM). Cells were fixed using 4% PFA (paraformaldehyde), permeated using 0.1% Triton and blocked for 1hr in 10% goat serum. Cells were subsequently incubated with mouse monoclonal anti-α-SMA antibody (Sigma) overnight and Alexa 555-labeled anti-mouse antibody (Molecular Probes), each for 60 min at room temperature. The nucleus was stained using DAPI (Molecular Probes). Cells were mounted using an anti-fade mounting media (Invitrogen) and then visualized and photographed using a Nikon A1 High Speed Laser Confocal microscope. Total 20 cells were counted per field by the morphology i.e., spindle shape vs stellate. Total 5 fields were counted for each group. The extracted solution was analyzed using UV-vis spectroscopy and quantified based on a pre-established calibration curve. For morphometric quality control, liposome size and distribution were analyzed with Dynamic Light Scattering (DLS) using Nano-ZS Zetasizer (Malvern). The average hydrodynamic particle size was determined from three independent runs. Standard deviation (PDI) was obtained to evaluate the size distribution. LV function measurements by echocardiography. LV function was measured using the Vevo 770 imaging system (VisualSonics Inc.), as described previously 49 . Necropsy and infarct area analysis. No-MI control day (d0), d1 or d5 post-MI 15-epi-LXA 4 injected and untreated mice were anesthetized with isoflurane briefly, then mice were under 2% isoflurane anesthesia in 100% oxygen mix, and heparin (4 IU/g) was injected. The blood was collected from the carotid artery and centrifuged for 5 min to separate plasma. The spleen, lungs, LV and right ventricle were separated and weighed individually. The LV was divided into apex (infarcted area), mid-cavity, and base (remote area) under a microscope. The spleen and kidney are collected, weighed and fixed in 10% zinc formalin for immunohistochemistry (IHC) and molecular analysis as described previously 15 .
Quantitative real-time PCR for measurements of gene transcripts. For qPCR, reverse transcription was performed with 2.0 μg of total RNA using SuperScript ® VILO cDNA Synthesis Kit (Invitrogen, CA, USA). Quantitative PCR for FPR2, IL-6, ccl2, IL-1β, Arg-1, Mrc-1, Ym-1, neutrophil gelatinase-associated lipocalin (ngal), GPR40 and GPR120 genes was performed using taqman probes (Applied Biosystems, CA, USA) on master cycler ABI, 7900HT. Gene levels were normalized to Hprt-1 as the housekeeping control gene. The results were reported as 2 −ΔCt (ΔΔCt) values. All the experiments were performed in duplicates with n = 5/group. LV histology and immunohistochemistry and confocal microscopy. For histological measurements, LV transverse section was embedded in paraffin and sectioned. The assessment of neutrophils and macrophages by immunohistochemistry was done as previously described 49 . The confocal microscopy was performed om LV transverse frozan section using F4/80 and MRC-1 (abcam) as previously described 50 .
Picrosirius red staining. Post-MI collagen measurements were done by picro sirius red (PSR) staining as described previously 15 . Image analysis for Immunohistochemistry and PSR staining. For each slide per mouse, a total of 5-7 images were acquired from LV infarcted area including border zone as previously described 15 . Plasma Creatinine measurement. Plasma creatinine levels from No-MI (d0), MI-control and 15-epi-LXA 4 , injected mice at post-MI d1, and d5 were determined using LC-MS/MS. Briefly, 10 µl of sample was deproteinated and diluted with heavy isotope-labeled internal standard (ISTD) in a single step by adding ISTD in 80% acetonitrile. Twenty microliters of diluted sample is subjected to isocratic, HILIC HPLC with 10 mM ammonium acetate in 65% acetonitrile at the rate of 0.15 ml/min. Creatinine and d3-creatinine (ISTD) are detected by electrospray ionization tandem mass spectrometry (Quattro Micro API) MRM transitions 114 > 44 and 117 > 47, respectively. Quantitation is achieved by comparing results to a synthetic standard calibration curve (0, 0.2, 1, 5, 100 µg/ml for serum).
LV protein extraction for immunoblotting. The left ventricle infarct (LVI) tissues were processed for protein extraction as previously described 15 .