The tumor suppressor RASSF1A modulates inflammation and injury in the reperfused murine myocardium

Inflammation is a central feature of cardiovascular disease, including myocardial infarction and heart failure. Reperfusion of the ischemic myocardium triggers a complex inflammatory response that can exacerbate injury and worsen heart function, as well as prevent myocardial rupture and mediate wound healing. Therefore, a more complete understanding of this process could contribute to interventions that properly balance inflammatory responses for improved outcomes. In this study, we leveraged several approaches, including global and regional ischemia/reperfusion (I/R), genetically modified mice, and primary cell culture, to investigate the cell type–specific function of the tumor suppressor Ras association domain family member 1 isoform A (RASSF1A) in cardiac inflammation. Our results revealed that genetic inhibition of RASSF1A in cardiomyocytes affords cardioprotection, whereas myeloid-specific deletion of RASSF1A exacerbates inflammation and injury caused by I/R in mice. Cell-based studies revealed that RASSF1A negatively regulates NF-κB and thereby attenuates inflammatory cytokine expression. These findings indicate that myeloid RASSF1A antagonizes I/R-induced myocardial inflammation and suggest that RASSF1A may be a promising target in immunomodulatory therapy for the management of acute heart injury.

Aberrant inflammation is thought to underlie a host of pathologies, including those related to cardiovascular disease such as atherosclerosis, injury caused by myocardial infarction (MI), 3 and the progression to heart failure (1). Ischemia, either accompanied by reperfusion (I/R) or not, elicits massive cardiomyocyte loss. Damage-associated molecular patterns released from dead cells trigger robust activation of the innate immune response, which is associated with adverse clinical outcomes (2)(3)(4). Although a balanced inflammatory reaction is necessary for optimal wound healing, prolonged inflammation can exert deleterious effects within the myocardium leading to greater cardiomyocyte loss, increased fibrosis, and worsened heart function (4,5). Moreover, efforts to attenuate this response have shown therapeutic promise in mouse MI models (6,7). To date, results from clinical trials targeting inflammation have been inconclusive (8 -10). Importantly, the molecular mechanisms that modulate cardiac inflammation remain incompletely understood, representing a potential barrier to improved therapeutics for patients suffering MI.
Ras association domain family member 1 isoform A (RASSF1A) is a tumor suppressor and protein scaffold that lacks catalytic activity and regulates signaling through protein interactions (11). Loss of RASSF1A function, either through point mutations or epigenetic silencing, is observed with high frequency in multiple tumor types (12). However, its role in heart disease is far less established. Our previous work demonstrated that cardiomyocyte RASSF1A mediates apoptosis and promotes cardiac dysfunction and pathological remodeling in a pressure overload (PO)-induced model of heart failure (13). Cardiomyocyte-specific deletion of RASSF1A afforded protection against PO-induced failure, whereas cardiomyocyte-specific transgenic overexpression of RASSF1A exacerbated the heart failure phenotype in response to PO. Interestingly, systemic RASSF1A deletion resulted in augmented cardiac fibrosis and did not afford cardioprotection against PO-induced heart failure (13,14). These results indicated cell type specificity of RASSF1A signaling that facilitated robust effects on cardiac remodeling and dysfunction. Our prior studies also indicated a role for RASSF1A in facilitating the activation of Mst1, and subsequent inhibition of Bcl-xL, to promote cardiomyocyte apoptosis (15). However, the function of RASSF1A in the heart during acute injury such as I/R has not been directly examined. Moreover, in vivo genetic manipulation to elucidate cell typespecific functions of RASSF1A in this cardiac injury model has not been reported.
The aim of this study was to investigate RASSF1A function during acute I/R injury in the heart. We utilized both systemic and cell type-specific knockout mice to demonstrate opposing effects of RASSF1A deletion in cardiomyocytes versus myeloid cells on myocardial infarction. We report that RASSF1A negatively regulates the activity of NF-B transcriptional output in macrophages. Our findings indicate that RASSF1A works to repress inflammatory cytokine expression in macrophages and propose RASSF1A to be an important endogenous "brake" to prevent excessive NF-B activation and inflammatory cytokine production during I/R.

Both systemic and cardiomyocyte-specific RASSF1A deletion are protective against global I/R injury
Our previous work demonstrated that cardiomyocytespecific deletion of RASSF1A (RASSF1A F/F ;␣MHC-Cre and referred to hereafter as RASSF1A CKO), but not systemic RASSF1A deletion (RASSF1A Ϫ/Ϫ ), attenuated cardiac hyper-trophy, remodeling, and dysfunction in response to PO stress (13). Therefore, we sought to determine whether loss of RASSF1A in these genetic mouse models provided cardioprotection against I/R injury. We first used a Langendorff preparation to subject intact hearts to global I/R ex vivo, and we determined the extent of infarct by TTC staining and functional recovery using invasive hemodynamic measurement (16). We found that RASSF1A Ϫ/Ϫ hearts were significantly protected against global I/R, showing smaller infarcts and greater left ventricular developed pressure (LVDP) and dP/dt max and dP/dt min compared with wildtype (WT) littermate controls (Fig. 1, A-E). Cardiomyocyte-specific RASSF1A CKO mice were also subjected to global I/R and assessed. We found that RASSF1A CKO hearts had reduced infarcts and better heart function (LVDP, dP/dt max, and dP/dt min) compared with RASSF1A F/F Figure 1. RASSF1A ؊/؊ and RASSF1A F/F ;␣MHC-Cre (CKO) hearts are protected against global I/R ex vivo using a Langendorff preparation. A and F, representative images of TTC-stained heart sections (upper panel) and Western blottings (lower panel). Scale bar, 1 mm. B and G, percent infarct was determined using TTC staining. C-E and H-J, LVDP, dP/dt max, and dP/dt min were measured during the reperfusion phase using a pressure transducer and graphed as a percentage of baseline measurements (% recovery). *, p Ͻ 0.05. n ϭ 4 -7 mice/group.

RASSF1A modulates cardiac inflammation
controls, and the extent of cardioprotection was comparable with what was observed in RASSF1A Ϫ/Ϫ hearts ( Fig. 1, F-J). Together, these results indicated that loss of RASSF1A function, either globally or restricted to cardiomyocytes, was sufficient to reduce injury caused by I/R in the isolated heart.

RASSF1A ؊/؊ hearts are not protected against in vivo I/R
To determine the role of RASSF1A in a model of I/R injury that is more relevant to MI patients, we subjected our mice to regional I/R by transiently occluding the left anterior descending artery in vivo (15). Surprisingly, we found that RASSF1A Ϫ/Ϫ mice were no longer afforded protection, as determined by no difference in infarct size between knockout and WT controls (Fig. 2, A-C). In contrast, the RASSF1A CKO mice had significantly smaller infarcts versus RASSF1A F/F controls in response to in vivo I/R (Fig. 2, D-F). Because myocyte loss drives infarct, we determined the extent of TUNEL-positive cardiomyocytes in the infarct border zone and remote regions after I/R. We observed no differences between groups in the remote region; however, there was a significant attenuation of apoptosis in RASSF1A CKO border zone myocytes compared with controls, whereas no difference was noted in RASSF1A systemic knockout mice (Fig. 2G). These results indicated that RASSF1A function in nonmyocytes plays an important role in modulating I/R injury, as well as cardiomyocyte apoptosis surrounding the infarct area. Additionally, based on our findings in isolated hearts, we reasoned the cellular source of this RASSF1A signal was likely extra-cardiac.

RASSF1A ؊/؊ hearts have augmented inflammation after injury
To better understand these phenotypic differences, we began to explore the potential role of RASSF1A in cardiac inflammation during I/R. Using the same mouse models as above, we performed additional in vivo I/R experiments and investigated the extent of inflammatory markers within the myocardium. Our results demonstrate that I/R increases the level of TNF␣ protein in WT myocardium compared with sham control (Fig.  3A). In the systemic RASSF1A Ϫ/Ϫ hearts, TNF␣ protein levels were further increased by I/R. In contrast, we did not observe any difference in TNF␣ protein between RASSF1A CKO and RASSF1A F/F control hearts, either in sham or I/R conditions (Fig. 3B). Staining of heart sections to detect the pan-macrophage marker F4/80 demonstrated similar results (Fig. 3C). We also performed immunostaining for TNF␣ and observed a significant increase in TNF␣-positive nonmyocytes in RASSF1A Ϫ/Ϫ hearts after I/R compared with WT (Fig. 3, D and E), whereas the number of TNF␣-positive nonmyocytes in RASSF1A CKO hearts was significantly attenuated compared with controls (Fig. 3, F and G). Additionally, we coun-

RASSF1A modulates cardiac inflammation
terstained WT heart sections to detect TNF␣ and F4/80 and observed prominent signal overlap, suggesting that macrophages are a major source of cardiac TNF␣ in response to I/R (Fig. 3H). These findings indicated that loss of RASSF1A in nonmyocytes exaggerated inflammation caused by I/R in the heart.

Myeloid deletion of RASSF1A increases I/R injury
Previous work has demonstrated that shortly after ischemic insult, bone marrow and splenic monocytes are recruited to and infiltrate the myocardium, where they differentiate to macrophages and participate in the injury response (17,18). Myeloid cells recruited during this initial phase exhibit a pro-inflammatory phenotype and express high levels of cytokines, including TNF␣, IL-1␤, and IL-6. The function of RASSF1A in the myeloid compartment, and its potential contribution to I/R inflammation and injury, is not known. Therefore, we selectively targeted RASSF1A for deletion in these cells using LysM-Cre transgenic mice (19). Baseline analysis did not reveal any obvious abnormalities in the heart, lung, or spleen in RASSF1A F/F ; LysM-Cre mice. Cardiac systolic function and dimensions, as well as immune cell composition, also showed no difference between RASSF1A F/F ;LysM-Cre and RASSF1A F/F control mice (Table 1). However, following I/R, we observed augmented infarct size in RASSF1A F/F ;LysM-Cre hearts compared with controls ( Fig. 4, A-C). Serum levels of cardiac troponin I were also significantly increased in RASSF1A F/F ;LysM-Cre mice compared with control mice after I/R (Fig. 4D). Taken together,

RASSF1A modulates cardiac inflammation
these results indicate enhanced cardiac injury in RASSF1A F/F ; LysM-Cre mice.

Myeloid deletion of RASSF1A increases cardiac inflammation after I/R
To determine whether RASSF1A F/F ;LysM-Cre mice had an augmented cardiac inflammatory response following in vivo I/R, we performed qPCR to detect mRNA expression of proinflammatory genes from infarcted tissue. Our results demonstrated increased cardiac expression of TNF␣, IL-1␤, Nos2, and Cox2 in both RASSF1A F/F control mice and RASSF1A F/F ; LysM-Cre mice after I/R. However, expression levels of each gene were increased to a greater extent in RASSF1A F/F ;LysM-Cre compared with control hearts, indicating an enhanced immune response to injury (Fig. 4, E-H). Additionally, we detected macrophages using immunofluorescence and found that RASSF1A F/F ;LysM-Cre hearts had higher numbers of F4/80-positive cells compared with RASSF1A F/F controls in response to I/R (Fig. 4, I and J). Together, our data demonstrate a more robust inflammatory response to heart injury in RASSF1A F/F ;LysM-Cre mice.

Cardiac remodeling-associated gene expression is altered in RASSF1A mutant mice
Cardiac inflammation and inflammatory cell infiltration are important modulators of fibrosis, scar formation, and remodeling of the heart after injury. To determine whether these processes may also be altered in the RASSF1A mutant mice, we examined gene expression of established regulators of fibrosis and extracellular matrix (ECM) remodeling. Following 24 h of reperfusion, we isolated and compared infarcted tissue with myocardium from sham-operated control mice. We found that in cardiomyocyte-deficient RASSF1A hearts, expression of most of the genes analyzed was significantly lower compared with control mice, both in sham and I/R conditions (Fig. 5, A-F). Conversely, we observed that expression of most of these genes was significantly greater in myeloid-deficient RASSF1A hearts after I/R, but not at baseline (sham) conditions (Fig. 5, G-L). This opposite cell type-specific effect of RASSF1A targeting is in agreement with our injury and inflammation results and suggests that myeloid deletion of RASSF1A may also exacerbate fibrosis and remodeling of the heart following I/R injury.

RASSF1A represses NF-B activity in macrophages
Next, we questioned whether RASSF1A functionally interacts with NF-B, a master regulator of inflammatory gene expression, using RAW264.7 mouse macrophages. To test this hypothesis, we performed both RASSF1A gain-and loss-offunction experiments. We found that RASSF1A overexpression was sufficient to significantly attenuate both baseline and TNF␣-induced NF-B luciferase activity (Fig. 6, A and B). Conversely, we found that siRNA-mediated knockdown of RASSF1A was sufficient to increase NF-B activation (Fig. 6, C and D). In response to RASSF1A knockdown, we also observed increased mRNA expression of IL-1␤, TNF␣, Nos2, and Cox2, established transcriptional targets of NF-B, whereas no changes in genes associated with the resolution of inflammation were altered by RASSF1A depletion (Fig. 6, E and F). To enhance the relevance of our findings, we isolated and cultured bone marrow-derived macrophages (BMDMs) from control and RASSF1A F/F ;LysM-Cre mice. In BMDMs deficient for RASSF1A, we observed significantly higher expression of IL-1␤, TNF␣, Nos2, and Cox2 mRNA following TNF␣ challenge. However, expression of these genes was comparable between RASSF1A F/F ;LysM-Cre BMDMs and control cells at baseline (Fig. 6, G-K). Importantly, we also found enhanced protein expression of IL-1␤ in LPS-treated RASSF1A F/F ;LysM-Cre BMDMs (Fig. 6L). Together, these data indicate that RASSF1A negatively regulates macrophage NF-B activity and target gene expression in a cell-autonomous manner.

RASSF1A negatively regulates YAP to restrain NF-B in macrophages
Our previous work demonstrated that RASSF1A promotes apoptosis through the activation of Mst1 and stimulation of noncanonical Hippo signaling in the cardiomyocyte (13,15). To investigate whether RASSF1A engages Mst1 and the Hippo pathway in the macrophage, we either overexpressed or knocked down RASSF1A in RAW264.7 cells. Increased RASSF1A expression caused Mst1 activation and the downregulation of YAP protein levels (Fig. 7A). Conversely, siRNA-mediated silencing of RASSF1A increased the presence of nuclear YAP and up-regulated the mRNA levels of established YAP target genes, Cyr61 and Ctgf (Fig. 7, B-D). To determine whether YAP mediated the enhanced inflammatory gene expression elicited by RASSF1A silencing, we simultaneously knocked down RASSF1A and YAP in macrophages. Our results indicated that cytokine mRNA levels were normalized following depletion of both RASSF1A and YAP (Fig. 7, E-G), implicating Hippo signaling in this response. To further elucidate how YAP modulates NF-B, we performed co-immunoprecipitation experiments against FLAG-YAP or endogenous RelA, the p65 subunit of NF-B, in RAW264.7 cells. Our results demonstrate association between YAP and RelA (Fig. 7H). We next examined whether YAP localized to B-binding elements within the promoters of two established NF-B target genes. Indeed, we were able to detect exogenously expressed YAP at the promoters of TNF␣ and IL-1␤ as determined by ChIP assay (Fig. 7, I and J). Together, these results indicate that YAP can bind to RelA and is present on DNA at NF-B recognition sites.

Discussion
Many chronic pathologies, including cardiovascular disease and stroke, metabolic disease and diabetes, autoimmune diseases, and cancers, have heightened inflammatory states that are thought to contribute to disease progression. Therefore, the identification of novel regulatory mechanisms to modulate inflammation may lead to more effective therapeutic options for patients. Prior work has implicated RASSF1A as a negative regulator of inflammation using a mouse model of chemically-induced colitis (20). A separate study demonstrated enhanced immune cell infiltration in RAS-driven lung tumors in mice with global RASSF1A deficiency (21). Importantly, however, whether loss of RASSF1A function modulates cardiac inflammation and injury caused by I/R had not been investigated. Moreover, the important cell type(s) in vivo that mediate the effect of RASSF1A on inflammation remained to be elucidated. The results of our study demonstrate that loss of RASSF1A function selectively within myeloid cells caused increased

RASSF1A modulates cardiac inflammation
inflammation and injury of the myocardium resulting from I/R. This is in contrast to cardiomyocyte-specific RASSF1A deletion, which affords reduced infarct triggered by I/R. Using primary BMDMs and RAW264.7 cells, we demonstrated that modulation of RASSF1A causes altered activation status of NF-B and subsequent pro-inflammatory cytokine expression. Additionally, we observed that RASSF1A engages the Hippo pathway to activate Mst1 and inhibit YAP function and that concomitant RASSF1A and YAP knockdown largely normalized the inflammatory gene expression in macrophages. We also report that YAP associates with RelA and is present on B elements in NF-B target genes. These results provide further evidence that RASSF1A acts as a

RASSF1A modulates cardiac inflammation
negative regulator of inflammation, implicates Hippo-YAP signaling as a mediator of this response, and demonstrates the pathological importance of this mechanism for I/R injury.
NF-B is a central regulator of innate immune responses and stimulates expression of numerous pro-inflammatory cytokines to promote inflammation (22). In this study, we identify

RASSF1A modulates cardiac inflammation
RASSF1A as a modulator of NF-B activity in the macrophage. Specifically, we provide evidence that depletion of endogenous RASSF1A, either through siRNA-mediated silencing in RAW264.7 cells or by Cre-mediated gene deletion in the myeloid compartment in vivo, promotes NF-B activation and target gene expression. Interestingly, we observed acute up-regulation of

RASSF1A modulates cardiac inflammation
pro-inflammatory cytokines in response to RASSF1A knockdown, but we saw no change in genes associated with the reparative macrophage phenotype. Whereas a link between RASSF1A and inflammation has been reported previously (20,21), our study is the first to investigate this mechanism through genetic targeting of RASSF1A in myeloid cells in vivo, and it is the first to demonstrate an associated pathophysiological effect in the myocardium during I/R injury.
There is a growing literature describing the different resident cardiac macrophage subtypes, as well as recruited monocyte subtypes, and their relative contribution to heart injury and the progression of heart disease (17,(23)(24)(25)(26)(27)(28)(29). Our approach is limited in that we used the LysM-Cre driver line to deplete RASSF1A globally in the myeloid compartment (19). This alone does not allow for delineating the relative contribution of resident versus recruited macrophage subtypes. However, as our understanding of these specific cell subtypes advances, as well as our ability to manipulate them (29,30), future experiments may allow us to examine RASSF1A function in cardiac macrophage subsets and determine potential varied effects on inflammation and infarct.
Our results indicate that RASSF1A is an important negative regulator of inflammation and functions to suppress cytokine production following cardiac injury in vivo, and suggest that RASSF1A may be a potential therapeutic target for treatment of inflammatory conditions. This RASSF1A-mediated regulatory mechanism could have broad implications not only for MI and heart disease but may also be applicable to aberrant inflammatory pathologies in general.

Animal models
RASSF1A Ϫ/Ϫ and RASSF1A floxed mutant mice were a kind gift from Dr. Louise van der Weyden (31). The cardiomyocyte (␣MHC) and myeloid (LysM) Cre recombinase transgenic mice have been described previously (19,32). Male mice, aged 8 -12 weeks, weighing 20 -25 g were housed in a temperature-controlled environment with 12-h light/dark cycles where they received food and water ad libitum. All protocols concerning the use of animals were approved by the Institutional Animal Care and Use Committee, New Jersey Medical School, Rutgers University.

Myocardial ischemia/reperfusion
Prior to anesthesia, cephazolin (60 mg/kg) was administered (intraperitoneal injection) to prevent infection. Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg). Once anesthetized, mice were intubated and ventilated with a tidal volume of 0.2 ml and a respiratory rate of 110 breaths/min using 65% oxygen (rodent ventilator model 683; Harvard Apparatus Inc.). The animals were kept warm with heat lamps. Rectal temperature was monitored and maintained between 36 and 37°C. The heart was exposed by a thoracotomy through the 4th and 5th ribs. The left coronary artery was located, and a suture was passed under the artery. To occlude the artery, a short length of tubing was threaded through the suture ends, and occlusion was effected by placing tension on the suture such that the tube compressed the artery.
Ischemia was confirmed by ECG change (ST segment elevation). After occlusion for 30 min, the silicon tubing was removed to achieve reperfusion, and the rib space and overlying muscles were closed in layers using 5.0 nylon sutures. For sham operation, the same protocol was followed; however, no ligation of the coronary artery was performed. Postoperatively, mice were administered Buprenex-SR (1.2 mg/kg) subcutaneously for analgesia. Mice were then allowed to recover under close monitoring in an incubator. During this time, mice were observed for signs of post-operative complications, including pain, pneumothorax, and acute heart failure or sudden death (15).

Measurement of infarct size
Twenty four hours after reperfusion, mice were anesthetized and intubated, and the chest was opened. After the heart was arrested at the diastolic phase by KCl injection, the ascending aorta was cannulated and perfused with saline to wash out blood. The left anterior descending coronary artery was occluded with the same suture, which had been left at the site of the ligation. To demarcate the ischemic area at risk (AAR), Alcian blue dye (1%) was perfused into the aorta and coronary arteries. Hearts were excised, and LVs were sliced into 1-mmthick cross-sections and incubated with a 1% TTC solution at 37°C for 15 min. The infarct area (pale), the AAR (not blue), and the total LV area from both sides of each section were measured with the use of Adobe Photoshop (Adobe Systems Inc.), and the values obtained were averaged. The percentages of area of infarction and AAR of each section were multiplied by the weight of the section and then totaled from all sections. AAR/LV and infarct area/AAR were expressed as percentages (33).

Langendorff perfusion model
Mice were anesthetized with pentobarbital (65 mg/kg, i.p.) and treated intraperitoneally with 50 units of heparin. The heart was quickly removed and catheterized with a 22-gauge needle. The hearts were mounted on a Langendorff-type isolated heart perfusion system and subjected to retrograde coronary artery reperfusion with 37°C oxygenated Krebs-Henseleit bicarbonate buffer (NaCl 120 mmol/liter, glucose 17 mmol/ liter, NaHCO 3 25 mmol/liter, KCl 5.9 mmol/liter, MgCl 2 1.2 mmol/liter, CaCl 2 2.5 mmol/liter, EDTA 0.5 mmol/liter) (pH 7.4), at a constant pressure of 80 mm Hg. A balloon filled with water was introduced into the left ventricle (LV) through the mitral valve orifice and connected to a pressure transducer via a plastic tube primed with water. LV pressures and LV dP/dt were recorded with a strip chart recorder (Astro-Med, Inc.). The LV end-diastolic pressure was set at 4 -10 mm Hg at the beginning of perfusion by adjusting the volume of the balloon in the LV, and the volume was kept constant throughout an experiment. After a 30-min equilibration period, the heart was subjected to 30 min of global ischemia (at 37°C) followed by 60 min of reperfusion.

Echocardiography
Mice were anesthetized using 12 l/g body weight of 2.5% tribromoethanol (Avertin, Sigma), and echocardiography was RASSF1A modulates cardiac inflammation performed, as described previously (34), using a 13-MHz linear ultrasound transducer. Two-dimensional guided M-mode measurements of LV internal diameter were obtained from at least three beats and then averaged. LV end-diastolic dimension (LVDd) was measured at the time of the apparent maximal LV diastolic dimension, and LV end-systolic dimension (LVSd) was measured at the time of the most anterior systolic excursion of the posterior wall. LVEF was calculated using the following formula: LVEF (%) ϭ 100 ϫ (LVDd 3 Ϫ LVSd 3 )/LVDd 3 .

Cell culture and reagents
The RAW264.7 cell line was purchased from ATCC (TIB-71) and maintained according to established protocols (35). For BMDM experiments, bone marrow cells were isolated from adult RASSF1A F/F ;LysM-Cre and control RASSF1A F/F mice (36). Cells were cultured in complete RPMI 1640 medium supplemented with 10 ng/ml recombinant M-CSF (PeproTech) for 8 -9 days. Cells were serum-starved 24 h prior to stimulation. Mouse recombinant TNF␣ was purchased from R&D Systems. LPS was purchased from Sigma.

Luciferase assay
Cells were transfected using Lipofectamine 2000 transfection reagent (Life Technologies, Inc.) according to the manufacturer's instructions. The NF-B luciferase reporter gene p1242-3x-KB-L was purchased from Addgene (plasmid no. 26699) and was used to assess NF-B activity (39). Following transfection, cells were lysed with Passive Lysis Buffer (Promega), and transcriptional activity was measured using the luciferase assay system (Promega) with an OPTOCOMP I luminometer (MGM instruments). All firefly luciferase results were normalized to total protein content as described previously (16,40,41).

RASSF1A modulates cardiac inflammation Immunostaining
Mouse left ventricles were fixed in formalin and sectioned at 6-m thickness. Tissue sections were then subjected to deparaffinization and antigen unmasking using citrate buffer and washed with PBS containing 0.3% Triton X-100. Samples were blocked with 5% BSA and incubated with primary antibody overnight and with Alexa Fluor 488-and Alexa Fluor 594conjugated secondary antibodies (Molecular Probes) at room temperature. Primary antibodies used were anti-TNF␣ rabbit polyclonal antibody (Abcam), anti-F4/80 rat antibody (Bio-Legend), and anti-cardiac troponin-T mouse mAb (Thermo Fisher Scientific). Nuclei were stained with 4Ј,6-diamidino-2phenylindole. Imaging was performed using a Nikon conventional fluorescence microscope.

Chromatin immunoprecipitation (ChIP)
ChIP assays were performed using the SimpleChIP Plus Enzymatic Chromatin IP kit (Cell Signaling) according to the manufacturer's instructions. Briefly, RAW264.7 cells were transduced with FLAG-YAP construct or control LacZ for 24 h, and then cells were cross-linked using 1% formaldehyde for 10 min. Cells were washed with 1ϫ PBS, and glycine was added to stop the cross-linking reaction. Cells were then scraped; nuclei were isolated and lysed; and sheared chromatin was isolated after sonication. Immunoprecipitation reactions were carried out using chromatin extracts and anti-YAP (Cell Signaling Technology, 14074) or control IgG (Cell Signaling Technology, 2729) antibodies overnight at 4°C. Equal amounts of purified input DNA was used to perform qPCR using primers specific to the promoters of the target genes. Primers (5Ј to 3Ј) used for ChIP-qPCR were as follows: Tnf␣, AGTGTTTAGGA-GTGGGAGGGTG and GGAGCCTCTGCCATATCTTG-ACT; IL-1␤, TGTTGTGAAATCAGTTAACCCAAGGGAA and GAGGATCCCAGATGAGCCTATTAG.

Nuclear fractionation
The nuclear and cytosolic-enriched fractions were prepared using NE-PER nuclear and cytoplasmic extraction kit (Pierce) according to the manufacturer's instructions (43). Western blotting was conducted following isolation of fractions.

TUNEL
DNA fragmentation was detected in situ using TUNEL as described previously (44). Heart sections were incubated with proteinase K, and DNA fragments were labeled with fluoresce-in-conjugated dUTP using TdT (Roche Applied Science), and cardiomyocytes were labeled using anti-cardiac troponin-T mouse mAb (Thermo Fisher Scientific, MA5-12960). TUNELpositive cardiomyocyte nuclei (cardiac troponin-T-positive) were determined as a percentage of total cardiomyocyte nuclei.

ELISA
Following I/R and immediately prior to sacrifice, serum was collected, and the level of cardiac troponin-I was determined using the High Sensitivity Mouse Cardiac Troponin I ELISA according to the manufacturer's instructions (Life Diagnostics, Inc.). Ventricular tissue was homogenized, and TNF␣ protein was determined using a TNF␣ mouse ELISA kit according to the manufacturer's instructions (Thermo Fisher Scientific) (13).

Statistical analysis
All data are reported as mean Ϯ S.E. of the mean. Student's t test was used to evaluate the difference in means between the two groups. One-way ANOVA was used to compare three or more group means with one independent variable. Two-way ANOVA was used to compare three or more group means with two independent variables. Post hoc comparisons were performed using Tukey's test. All mouse experiments used agematched male mice and littermate controls and were performed blinded to genotype. Statistical analyses were performed using SPSS version 24 and Graph Pad Prism 6.0. A p value less than 0.05 was considered significant.