Simultaneous Assessment of Cardiac Inflammation and Extracellular Matrix Remodeling After Myocardial Infarction

Background: Optimal healing of the myocardium after myocardial infarction (MI) requires a suitable degree of inflammation and its timely resolution, together with a well-orchestrated deposition and degradation of ECM (extracellular matrix) proteins. Methods and Results: MI and SHAM-operated animals were imaged at 3, 7, 14, and 21 days with 3T magnetic resonance imaging using a 19F/1H surface coil. Mice were injected with 19F-perfluorocarbon nanoparticles to study inflammatory cell recruitment, and with a gadolinium-based elastin-binding contrast agent to evaluate elastin content. 19F magnetic resonance imaging signal colocalized with infarction areas, as confirmed by late gadolinium enhancement, and was highest 7 days post-MI, correlating with macrophage content (MAC-3 immunohistochemistry; &rgr;=0.89, P<0.0001). 19F quantification with in vivo (magnetic resonance imaging) and ex vivo nuclear magnetic resonance spectroscopy correlated linearly (&rgr;=0.58, P=0.020). T1 mapping after gadolinium-based elastin-binding contrast agent injection showed increased relaxation rate (R1) in the infarcted regions and was significantly higher at 21 days compared with 7 days post-MI (R1 [s−1]: 21 days=2.8 [interquartile range, 2.69–3.30] versus 7 days=2.3 [interquartile range, 2.12–2.5], P<0.05), which agreed with an increased tropoelastin content (&rgr;=0.89, P<0.0001). The predictive value of each contrast agent for beneficial remodeling was evaluated in a longitudinal proof-of-principle study. Neither R1 nor 19F at day 7 were significant predictors for beneficial remodeling (P=0.68; P=0.062). However, the combination of both measurements (R1<2.34 Hz and 0.55⩽19F⩽1.85) resulted in an odds ratio of 30.0 (CI 95%, 1.41–638.15; P=0.029) for favorable post-MI remodeling. Conclusions: Multinuclear 1H/19F magnetic resonance imaging allows the simultaneous assessment of inflammation and elastin remodeling in a murine MI model. The interplay of these biological processes affects cardiac outcome and may have potential for improved diagnosis and personalized treatment.

C ardiac injury activates innate immunity, which initiates an inflammatory response whereby neutrophils and monocytes/macrophages are recruited to the myocardium. 1 Immediately after myocardial infarction (MI), neutrophils are recruited to the site of injury, followed by the recruitment of monocytes/ macrophages that remove dead cells and debris by phagocytosis. 1,2 Studies have shown that an early and aggressive immune response and high levels of neutrophils and monocytes within the infarct may promote adverse remodeling and lead to poor prognosis. 3,4 In addition to their phagocytic properties, inflammatory cells activate reparative pathways, including the formation and deposition of scar tissue, which is mainly composed of collagen and elastin/tropoelastin. 2,5,6 Elastin has been shown to be essential for the stabilization of the scar after MI and improving cardiac function by preserving elasticity. 7,8 While the healthy myocardium contains elastin only to a negligible degree within the interstitium and coronary vasculature, an increase of elastic fibers within the myocardial scar is detected in the first weeks after ischemic injury developing a dense network between the remaining viable myocytes, myofibroblasts, and smooth muscle cells during the maturation of the infarct. 5 Tropoelastin, the soluble precursor of elastin, is synthesized in significant amounts and deposited within the remodeled myocardium, particularly at later stages of the healing process. 9 Magnetic resonance imaging (MRI) has great potential to noninvasively assess both structure and function of the heart. By combining MRI with specific contrast agents, different biological processes can be targeted and tracked over time. In this study, we sought to explore the merits of multinuclear 1 H/ 19 F MRI for the sequential assessment and quantification of cardiac inflammation and elastin remodeling in a murine model of MI using a 3T clinical scanner. Perfluorocarbons (PFCs) were used to assess inflammatory cell recruitment and gadolinium-based elastin-specific magnetic resonance contrast agent (Gd-ESMA) was used for the investigation of elastin deposition and quantification of ECM (extracellular matrix) remodeling of the myocardium. This approach may potentially allow a more accurate characterization of early or persistent inflammation and diffuse myocardial remodeling at the molecular level. It may also serve as a new biomarker for monitoring treatment response and evaluation of novel cardioprotective therapies.

METHODS
The DICOM MR images will be made available to other researchers for purposes of reproducing the results. 10

Animal Model
The institutional subcommittee on research animal care approved all animal studies. MI was induced in 10-week-old female C57BL/6J mice (n=71; 21% mortality rate; Charles River, United Kingdom) by permanent occlusion of the left anterior descending (LAD) coronary artery. Mice were anesthetized by intraperitoneal injection of 75 mg/kg ketamine (Vetalar V, Vetmedica) and 1 mg/kg medetomidine hydrochloride (Domitor, Orion Corporation, Finland). The animals underwent endotracheal intubation before surgery using an animal ventilator (Hugo Sacks Elektronic, Germany). A left thoracotomy was performed in the fourth intercostal space, the pericardium removed, and the LAD was permanently ligated with an 8-0 nylon suture (Direct Medical Supplies, Alton, United Kingdom). Successful ligation was confirmed by the regional blanching of the left ventricle (LV), extending to the apex. After thoracotomy, subcutaneous tissue and skin were closed in separate layers and the animal was weaned from the ventilator. After the surgery, mice were monitored and maintained in a heated chamber for at least 6 hours. Sham-operated animals underwent the same surgical procedure, without LAD ligation. Thirty minutes before recovery, 0.15 mg/kg buprenorphine (Vetergesic, Alstoe, United Kingdom) was administered for analgesia by intramuscular injection.

CLINICAL PERSPECTIVE
Here, we developed a new multinuclear magnetic resonance imaging protocol that allows visualizing and quantifying myocardial inflammation and ECM (extracellular matrix) remodeling simultaneously in a murine model of permanent coronary occlusion. The imaging approach is based on 19 F/ 1 H multinuclear magnetic resonance imaging in concert with fluorine nanoparticles and a gadoliniumbased elastin-specific magnetic resonance contrast agent. This is the first study that assessed inflammation and ECM remodeling simultaneously with a single noninvasive imaging modality and could provide important insights in post-infarct remodeling in patients developing heart failure. We also demonstrated the prognostic value of quantifying inflammation and ECM remodeling 7 days post-MI. A well-balanced inflammatory response was beneficial for maintaining left ventricular function, whereas extensive ECM remodeling was detrimental. If translated into the clinic, this work could provide clinicians with a new tool to noninvasively assess inflammation and ECM remodeling (focal and diffuse fibrosis), which is not possible with currently available contrast agents or noncontrast enhanced imaging methods. In summary, we demonstrated the feasibility of measuring myocardial inflammation and ECM remodeling (fibrosis) noninvasively, 2 key processes in tissue injury, and has great potential beyond cardiac imaging such as for the assessment of renal, liver or lung inflammation, and fibrosis.

Magnetic Resonance Imaging
In vivo cardiac scans were performed using a 3T Philips Achieva MR scanner (Philips Healthcare, Best, The Netherlands) equipped with a clinical gradient system (30 mT/m, 200 mT/m per ms). Mice (n=8 per time-point) were imaged at 3, 7, 14, and 21 days post-MI. Sham-operated mice (n=6 per time-point) were imaged at the same timepoints and were used as controls ( Figure 1A). Fifteen mice were imaged longitudinally at 7 and 21 days post-MI ( Figure 1B). Animals were placed in a prone position on a 19 F/ 1 H surface coil (Rapid Biomedical, Würzburg, Germany; diameter=23 and 33 mm). Anesthesia was induced with 5% and maintained with 1.5% to 2% isoflurane in medical oxygen during the MRI scan, and the body temperature was measured with a rectal temperature probe and maintained at 35±1ºC using a water-based heating system (SA Instruments, Stony Brook, NY). ECG was monitored with 2 metallic needles placed subcutaneously in the region of the chest. 1 H and 19 F cardiac ECG-triggered MR images were acquired 48 hours after intravenous injection of 400 μL of 10% perfluoro-15-crown-5 ether emulsion (PFC), as previously described 11 and 0.5 mmol/kg of a gadolinium-based MRI contrast agent that targets elastin and tropoelastin, Gd-ESMA (Lantheus Medical Imaging, North Billerica, MA), administered 1 hour before the MRI scan. At the end of the scans, the mice were culled and the heart was extracted for histological and nuclear magnetic resonance (NMR) studies.

MR Image Analysis
Ejection fraction (%), LV end-diastolic volume (LVEDV, μL) and LV mass (mg) were calculated from the cine images, using an automated segmentation software (ClinicalVolumes, King's College London, www.clinicalvolumes.com). 13 Total infarct size was calculated by adding the LGE measured on consecutive slices after the administration of Gd-ESMA (LGE-ESMA). The sum of the total infarcted area was multiplied by the slice thickness to generate a volume (mm 3 ) and was then divided by the total LV myocardium and expressed as percentage. T 1 relaxation times (R 1 ) were calculated by manually segmenting T 1 map regions corresponding to the LGE-ESMA using OsiriX (Osirix Foundation, Geneva, Switzerland). For 19 F measurements, regions of interest were defined as areas of enhancement seen on the LGE-ESMA images. For these areas, 19F

Histology
After the MRI scans, anesthetized mice were culled by neck dislocation and hearts were collected for ex vivo analysis (n=4 MI mice per time-point, and n=3 SHAM-operated animals per time-point). Hearts were harvested, the atriums were removed and the ventricles were washed in saline solution followed by immersion in 10% formaldehyde solution for 24 hours at room temperature. Hearts were then dehydrated, paraffin-embedded, and transversely sectioned (5 μm thick). Immunohistochemistry (IHC) was used to quantify the amount of tropoelastin and macrophages in the myocardium. Tropoelastin was detected with anti-mouse rabbit polyclonal antibody (21600, Abcam; dilution 1:100) using an avidin-biotin-peroxidase method (Vector SG Peroxidase substrate; Vector Laboratories, Burlingame, CA). A monoclonal rat anti-mouse antibody (550292, BD Pharmingen; CD107b; MAC-3; dilution 1:100) was used for macrophage detection. The antibody was revealed with streptavidin-peroxidase (Dako, Ely, United Kingdom; ABC kit, 1:100). Digital images were analyzed using ImageJ (National Institute of Health, Bethesda, MD). Tropoelastin and MAC-3 were quantified and expressed as percentage of the infarcted myocardium using ImageJ, and were manually segmented and normalized with the total area of infarction for each histology slice calculated from Masson's trichrome staining.

Statistical Analysis
GraphPad Prism 5.00 (GraphPad Software, Inc, La Jolla, CA) was used for statistical analysis. Nonparametric exact tests were used for analysis. Differences between different time-point measurements were analyzed using Kruskal-Wallis test for multiple group comparisons, and if this test was significant, it was followed by Dunn's post hoc test. Differences between different time-points and different groups: SHAM/scar/remote were analyzed using a 2-way ANOVA. Correlations were assessed using Spearman rank test. To study the nonlinear behavior of 19 F in the longitudinal study, the linear correlation model was compared with second-order polynomial model regression; both variables (EDV at day 21 and 19 F SNR) were first tested for normal distribution using the D'Agostino-Pearson omnibus normality test. Receiver operating characteristic curve analysis was performed to identify the cutoff point of imaging biomarkers to predict the evolution of functional cardiac parameters; in this case, the increase in the LVEDV between day 7 and day 21 was considered as dysfunctional remodeling, and the decrease of EDV between day 7 and day 21 was considered as a beneficial remodeling. P<0.05 was considered statistically significant. Data are presented as median, and interquartile range (IQR).

Assessment of Cardiac Function by MRI at 3T
Cardiac function was assessed using cine MRI and results are summarized in Figure 2 (detailed results in Table I 19 F signal was negligible and significantly lower compared with 3 days (P<0.05) and 7 days (P<0.001), consistent with the resolution of inflammation. 19 F signal was also detected at the site of thoracotomy, in the liver and lymph nodes, as these are major sites of macrophage clearance. Although the spleen was outside our imaging volume, PFC uptake would be also expected in this organ (as seen by other studies). 16 To verify the in vivo results and to quantify the evolution of the 19 F signal, infarcted and remote areas were dissected and separated for ex vivo NMR spectroscopy acquired on whole tissue samples. NMR spectra were in good agreement with the in vivo MRI findings, where infarcted regions showed high PFC signal that was absent in the remote myocardium (Figure 3C). NMR analysis showed a maximum 19 Figure 3E).

Assessment of Elastin Remodeling Post-MI Using Gd-ESMA
ECM remodeling post-MI was evaluated using Gd-ESMA, a contrast agent that binds to both cross-linked  elastin and immature tropoelastin. 17,18 One hour post-Gd-ESMA injection, infarcted areas were enhanced at all time-points post-MI, allowing quantification of infarct size. Trichrome staining was performed and used to quantify infarct size ex vivo. There was a strong linear correlation between infarct size measured by in vivo MRI and histology (ρ=0.85; P<0.0001; Figure II in the Data Supplement).
To understand the contribution of fluid accumulation in the interstitial space to the MR signal early after MI, myocardial edema was assessed using a T 2 -weighted sequence. [19][20][21] The high signal was observed at day 3 but not at day 7 post-MI. The signal intensity seen on T 2 -weighted images at day 3 was associated with edema and increased extracellular volume, but did not reflect deposition of elastin/tropoelastin as confirmed by histology (Figure 4).
In vivo quantification of Gd-ESMA uptake was performed using a Modified Look-Locker inversion T 1 -mapping sequence. Relaxation rate (R 1 ) maps showed uptake of Gd-ESMA in the infarcted area (increased R 1 ) at 3, 7, 14, and 21 days post-MI, whereas no enhancement was observed in the remote myocardium (infarct versus remote, P<0.01, Figure 5B) nor in the SHAM-operated animals (infarct versus SHAM, P<0.001, Figure 5B). R 1 values were also significantly higher within the infarct area at 21 days (R 1  To analyze the deposition of elastin fibers in the heart after MI, Elastica van Gieson staining was performed. Mature fibers could be visualized, however, quantification was challenging ( Figure III in the Data Supplement). For that reason, tropoelastin IHC was performed ( Figure 5A and 5C) revealing a dense fiber network within the infarcted myocardium at 14 and 21 days post-MI but not in the remote myocardium.  P<0.01). For the reason abovementioned, the 3 days' time-point was excluded from the correlation analysis between R 1 and tropoelastin IHC analyses. There was a statistically significant correlation between R 1 values from 7, 14, and 21 days measured in vivo and ex vivo IHC analysis (ρ=0.89; P<0.0001; Figure 5D).

F Versus R 1 Can Be Used to Predict Cardiac Remodeling: Longitudinal Study
Remodeling post-MI is a dynamic and complex process. To understand the potential prognostic value of in vivo 19 F and Gd-ESMA MRI, a longitudinal proof-of-principle study was performed. Fifteen animals were scanned twice at days 7 and 21 post-MI. 19 F MRI was performed at 7 days post-MI to assess the peak in the inflammatory response and MRI with Gd-ESMA was performed at days 7 and 21 post-MI. No correlation was found between 19 F at day 7 post-MI and Gd-ESMA uptake at day 7 ( Figure 6A) and at day 21 post-MI ( Figure 6B), suggesting that these biological processes are independent/decoupled from each other.
The presence of elastin at day 7 (measured as R 1 ) showed a linear correlation with the EDV measured at day 21, suggesting that early accumulation of elastin/ tropoelastin (larger Gd-ESMA uptake at day 7) might not be beneficial for the healing of the myocardium at day 21 ( Figure 7A). In contrast, the inflammatory process measured at day 7 showed a more complex behavior (second-order polynomial model regression showed significant correlation than the linear model (P=0.030). PFCs data suggest that an optimal inflammatory response was observed for a 19 F signal range between 0.55 and 1.85. Both an increased ( 19 F>>1.85) or weak ( 19 F<<0.55) inflammatory response at early stages post-MI resulted in large EDV (EDV>100 μL) at day 21 suggesting adverse cardiac outcome ( Figure 7B).
The prognostic value of the 19 F signal ( 19 F SNR) and quantitative assessment of elastin/tropoelastin deposition (R 1 ) was investigated with receiver

DISCUSSION
In this cross-sectional study we showed that (1) PFCs 19 F MRI can be used to assess and monitor inflammatory cell recruitment in vivo in the injured myocardium at a clinical field strength, as confirmed by ex vivo NMR and histological studies; (2) Gd-ESMA MRI allows quantification and visualization of scar size and elastin/ tropoelastin deposition in the myocardium during the scar maturation phase; and (3) in a longitudinal proofof-concept study, we further investigated the merits of  A, Linear correlation was found between EDV and R 1 (gadolinium-based elastin/tropoelastin specific MR contrast agent [Gd-ESMA] uptake). B, Quadratic regression was found between EDV and 19 F signal-to-noise ratio (SNR). both biomarkers for the prediction of the amount of cardiac remodeling (measured as EDV). We found that at the early stages post-MI a weak or strong inflammatory response results in dysfunctional MI healing and that increased elastin/tropoelastin deposition within the scar tissue at the 7 day's time-point is detrimental for cardiac remodeling. Our results suggest that multinuclear 19 F/ 1 H MRI may provide a better understanding of the biological processes underlying post-MI remodeling in vivo and PFCs and Gd-ESMA may serve as new imaging biomarkers for monitoring the progression of cardiac disease and allow predicting outcome.
Within the first week after MI inflammatory cells are recruited to the site of injury. Phagocytes avidly take up PFCs, which can be imaged by 19 F MRI with excellent contrast and without unwanted background signal, as shown previously. 11,14,22,23 However, no study has described the temporal evolution of 19 F signal in vivo at clinical field strength. Here we investigated the time course of inflammation using PFCs in vivo in a model of MI and related with the presence of immune cells. We successfully demonstrated the noninvasive visualization and quantification of inflammatory cells with PFCs in the infarcted region in a murine model of MI by in vivo MRI, validated ex vivo by NMR. MRI signal intensity measurements demonstrated that 19 F is detectable within the first-week post-MI, with a peak at 7 days, and importantly, PFC accumulation was restricted only to the infarcted region. Consistent with these findings, histological analysis showed that monocyte/ macrophage populations are significantly increased up to 7 days after LAD occlusion as detected by immune positive MAC-3 staining and, furthermore, we found a strong correlation between MAC-3 and 19 F MRI signal. Our results are also in good agreement with the resolution of inflammatory response at days 14 and 21 as described in animal models of MI. 2, 24,25 Noninvasive imaging of inflammatory cells recruitment to the injured and remote myocardium has been shown by positron emission tomography in vivo 26 but requires special patient preparation to suppress myocardial glucose metabolism. Alternatively, magnetic nanoparticles have been used for macrophage imaging post-MI in vivo 27 ; however, despite their excellent sensitivity, they generate negative contrast because of shortened T 2 /T 2 * relaxation times of nearby water protons, creating hypointense regions and consequently making quantification challenging. Moreover, magnetic nanoparticles cannot be used in combination with other Gd-based contrast agents. In contrast, PFCs are detected directly and, therefore, generate positive signal contrast, and more importantly can be imaged simultaneously with, for example, Gd-based contrast agents affecting only the extracellular 1 H signal while 19 F PFCs will be located intracellularly after phagocytosis. The potential of 19 F particles has been extensively explored in preclinical models; they are chemically stable and can be further functionalized by adding fluorochromes, thus allowing multimodal imaging (eg, MRI and fluorescence imaging). In this work, we did not investigate if PFCs can differentiate between the monocyte/macrophage subpopulations (Ly6C high versus Ly6C low ; M1 versus M2) during post-MI remodeling in vivo, which would be of great interest. Previous publications have suggested the recruitment of macrophages to the remote myocardium 26,27 ; here, because of sensitivity we could not detect myocardial leukocyte enrichment. Nevertheless, 19 F MRI may provide an in vivo readout for monitoring treatment-related changes in total inflammatory cell infiltration.
As the inflammatory process dissipates, fibrotic tissue accumulates in the infarcted regions and is then replaced by ECM proteins. Scar tissue formation commences as early as 1-week post-MI and is mainly composed of collagen type I but also elastin. Elastin is an insoluble protein that has been associated with scar formation and stabilization. 8,28,29 Mature elastin is formed by cross-linking of its soluble precursor, tropoelastin. Here, we took advantage of the increasing expression of elastin/tropoelastin during post-MI remodeling and investigated the use of Gd-ESMA as an imaging biomarker for the assessment of ECM remodeling. Elastin/ tropoelastin was quantified using T 1 mapping to measure R 1 after Gd-ESMA injection with 3T MRI. R 1 values significantly increased from 7 to 21 days post-MI, which was in good agreement with the deposition of tropoelastin in the infarcted area measured by histology. At 3 days post-MI, T 1 mapping showed a significant increase in R 1 in the infarcted area, however, histology showed a lack of elastin/tropoelastin at this time point. We hypothesized that Gd-ESMA acts similarly to other gadoliniumbased contrast agents because of its small size, 17,30 and immediately after MI its retention within the infarcted region may be unspecific and attributed to edema, cellular swelling and rupture, and subsequent increase in extracellular volume, as previously shown in dogs and humans. [19][20][21] Consistent with this hypothesis, we observed a high-signal intensity on native T2-weighted images at day 3 (high water content) in the infarcted area, which decreased at day 7 post-MI. Additionally, Gd-ESMA not only binds to tropoelastin and elastin (41%, K D =9.2±0.7 μM; 40%, K D =1.0±0.5 μM, respectively) but also to other proteins, including BSA (15%, K D =ns) and chondroitin sulfates (5%, K D =ns), 31,32 collagen types I and III (22%, K D =7.3±1.3 μM; 13%, K D =6.8±1.2 μM, respectively) 18 that might be present at day 3, thus increasing its retention and tissue relaxation.
Gd-ESMA has been successfully used for molecular imaging of vessel wall elastin in atherosclerosis (30)(31)(32), and myocardial scar. 15 ECM remodeling could also be assessed with alternative contrast agents specific for other matrix proteins such as collagen thereby providing additional biological information in addition to standard LGE-MRI.
The recovery of cardiac function after MI is highly dependent on a balanced inflammatory response and on the deposition of ECM proteins within the heart. In our longitudinal proof-of-principle study the impact of the inflammatory response on LV remodeling was evaluated with PFCs and ECM remodeling with Gd-ESMA MRI. A moderate inflammatory response at day 3 (intermediate 19 F signal) was associated with a better LV remodeling at day 21 (measured as EDV). The healing process is affected by the exposure and duration of acute inflammation. Prolonged and exacerbated inflammation has been related to worse prognosis and similarly, the lack of inflammation has been associated with thinner infarcts, where the myocardium is more likely to rupture. 3,4 A certain degree of inflammation and a controlled recruitment of monocyte/macrophages populations seems to be desired for optimal MI healing. Likewise, Sahul et al 33 have shown that moderate amounts of metalloproteinases in the infarcted region lead to lower LV remodeling in a pig model of MI. Inflammatory cell migration toward the infarct requires the presence of metalloproteinases to facilitate migration. Interestingly, while metalloproteinases are crucial for the recruitment of inflammatory cells, this study has shown that excessive metalloproteinases activation leads to LV expansion.
Here, we also have shown that higher R 1 values at 7 days (high elastin/tropoelastin deposition) were associated with an unfavorable prognosis at 21 days post-MI (high EDV). In previous work, Gd-ESMA has also been used to assess elastin deposition in the heart in a mouse model of MI. In their cross-sectional study, Wildgruber et al 17 found that a higher contrast to noise ratio between scar and remote myocardium at day 21 correlated with a higher ejection fraction at the same time point, whereas the contrast to noise ratio at day 7 did not predict outcome at day 21. In contrast, we performed a longitudinal study, using T 1 mapping and EDV as a readout for LV remodeling. Other studies showed that modifying the composition of myocardial scar by exogenously increasing elastin content, cardiac function was improved after MI in rats. [7][8][9] Also, increased expression of elastin via cell-based gene therapy improved cardiac function and survival of ischemic hearts in a rat model of MI. 8,34 Further studies are needed to pinpoint the long-term role of elastin turnover/metabolism and cross-linking after MI.
Finally, we have also shown that the combination of the R 1 at day 7 and 19 F at day 7 could predict the beneficial or dysfunctional LV remodeling, although the odds ratio is statistically significant, it has a wide CI, which reflex the complex process of inflammation and remodeling, further studies, including bigger sample size and different animal models, have to be used to validate this conclusion.
Overall, multinuclear 19 F/ 1 H MRI may improve our knowledge of cardiac remodeling in vivo by targeting key biological processes that are responsible for post-MI remodeling. This imaging protocol may be useful for risk stratification or to facilitate the in vivo study of the effects of novel therapeutic procedures in disease progression and potentially personalization of therapy.

LIMITATIONS
This study has shown the feasibility of multinuclear imaging in a murine model of post-MI remodeling via LAD ligation. 19 F imaging was performed in a smallanimal model using a clinical 3T MR scanner, where high amounts of PFCs (ie, 3 mmol PFC per kilogram body weight) are required to generate enough signal. The high-dosage enabled visualization of PFCs in small structures such as the murine heart. For early time-points 19 F deposition colocalized with LGE-ESMA enhancement within the scar area; however, for days 14 and 21 LGE-ESMA images were used as guide for the analysis of PFC uptake. Direct quantification of PFCs from the MRI could not be performed, as a surface coil was used. However, with the use of NMR experiments at 9.4T we were able to quantify PFCs content both in the remote and infarcted myocardium ex vivo. As PFCs used in this study were not specific for macrophages, signal accumulation in the infarct area might also be because of other cells with phagocytic capacity. PFC accumulation is not specific for myocardial inflammation but can also occur at other sites of inflammation (thoracotomy) and organs with high amounts of inflammatory and phagocytic cells such as the liver and lymph nodes. The here used PFCs did not allow to differentiate between the monocyte/macrophage subpopulations (Ly6C high versus Ly6C low ; M1 versus M2) during post-MI remodeling in vivo. PFC injection did not show any adverse effects in the animals throughout the period of time animals were monitored. However, PFCs have long-retention time in the body, making it challenging to obtain approval for clinical application. Further, improvement of the PFC nanoparticles biodistribution properties, while maintaining their specificity for macrophages could aid future use in humans. Additionally, in our study, we used a higher the dose of Gd-ESMA (0.5 mmol/kg) compared with that used in other animal models (0.2 mmol/kg) or nontargeted gadolinium-based agents in clinical studies. Future experiments will require dose optimization in MI models or humans; however, no toxic effects have been observed in the animals at any time-point. Finally, we used a permanent LAD occlusion model demonstrating a strong acute inflammatory response for proofof-concept 19 F/ 1 H MRI of myocardial inflammation and remodeling after MI. In future studies, a reperfusion model will be investigated, as it is more clinically relevant. The permanent LAD occlusion model only produces a mild inflammatory response, whereas the reperfusion model typically results in a stronger inflammatory response. Although the inflammatory response was weaker in our model we were able to detect and quantify both inflammation and ECM deposition in the infarct zone, making these contrast agents promising biomarkers for future studies.

CONCLUSIONS
We successfully demonstrated the feasibility of multinuclear 1 H/ 19 F MRI to noninvasively assess and quantify the inflammatory response and evaluate elastin formation after post-MI remodeling in a murine model in vivo. We further studied the interplay between these biological processes and correlated those with LV remodeling. This novel approach has potential for monitoring treatment effects that aim to modulate the inflammatory or elastin responses in vivo and may aid the prognosis of cardiovascular diseases.