C57BL/6 mice overexpressing the Slit2 gene (Slit2-Tg) were originally generated by the Geng Laboratory (University of Michigan, United States) (Yang et al., 2010). Briefly, full-length human Slit2 cDNA was cloned into a pCEP4F vector containing the CMV promoter, and then injected into the pronuclei of fertilized C57 × CBA F1 oocytes to construct transgenic mice. The genotypes were confirmed by Southern blotting and PCR analysis. PCR screening of Slit2 heterozygotes was performed using a pair of primers specific for Slit2 cDNA (forward: 5′-CCCTCCGGATC CTTTACCTGTCAAGGTCCT-3′; reverse: 5′-TGGAGAGAGCTCACAGAACAAGCCACTGTA-3′). Slit2-Tg mice and C57BL/6J (background strain) mice were bred and maintained in a specific pathogen-free (SPF), AAALAC-accredited facility of the Guangdong Laboratory Animals Monitoring Institute, Guangzhou, China. The temperature and humidity were 24 ± 2°C and 40–60%, respectively, and the light cycle included 12 h of light and 12 h of dark. Three- to five-month-old male mice were used in this study. All animal experiments were approved by the Institutional Animal Care and Use Committee (No. IACUC2017002).

To determine the effects of Slit2 on myocardial IR injury, a Langendorff heart perfusion system for rodents (Radnoti LLC, United States) equipped with a pressure transducer was used (Yang and Pyle, 2012). Briefly, mice were euthanized with isoflurane, and their hearts were excised. The aortas were then cannulated and the hearts were perfused using a modified Krebs-Henseleit (KH) buffer solution (37°C) containing (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 2.5 CaCl₂, 1.2 KH₂PO₄, 25 NaHCO₃, 0.05 EDTA, 0.5 sodium pyruvate, and 11 glucose. The KH buffer was continuously gassed with 95% O₂/5% CO₂, and the perfusion pressure was maintained at 70 mmHg. A water-filled balloon connected to the pressure transducer was inserted into the left ventricle, and left ventricular pressures were recorded with a PowerLab 16/35 system (AD Instruments Inc., United States). Hemodynamic parameters, including the left ventricular developed pressure (LVDP), heart rate (HR), left ventricular end diastolic pressure (LVEDP), rate of pressure product (RPP), and minimum and maximum rates of left ventricular pressure development [dP/dt(min) and dP/dt(max), respectively] were analyzed with Chart 7 Software (ADInstruments Inc., United States).

For the IR protocol, hearts were subjected to 20 min of stable perfusion, followed by 20 min of no-flow global ischemia and 40 min of reperfusion. Control hearts were continuously perfused for 80 min (Figure 1A). At the end of the experiment, some heart tissues were fixed for hematoxylin–eosin (HE) staining, transmission electron microscopy (TEM), and immunofluorescence staining, and some were flash frozen in liquid nitrogen for subsequent RNA sequencing analysis, RT-qPCR, Western blotting, Pro-Q staining, and actomyosin MgATPase activity assay.

To determine post-IR myocardial infarct size, hearts were subjected to TTC staining. Briefly, the frozen tissues were sliced into 2 mm thick sections and incubated in a 1% TTC (Sigma-Aldrich, United States) solution at 37°C for 15 min and in 10% neutral formalin for 1 h. TTC stained the viable areas red, while the unstained areas (white) were infarcted tissue. The infarct size in the myocardial tissue was measured using ImageJ software (NIH, United States), and the infarct size (%) was calculated as a percentage of the total section area. Different technicians performed the sectioning and observations, and the technicians did not know the individual identities of the samples.

Hearts were fixed in 10% neutral formalin and subjected to HE staining, which was used to detect necrosis of heart tissues. First, the hearts were embedded in paraffin, sliced into 3 μm sections, and dewaxed with dimethylbenzene and dehydrated with gradient ethanol solutions. Next, the sections were stained with hematoxylin for 10 min and soaked in 1% hydrochloric acid–ethanol for 2 s. The sections were then stained with alcohol-soluble eosin for 25 s. Finally, the sections were sealed with neutral gum and observed under an optical microscope (Leica DM 2000, Germany). During evaluation of the histological changes, different technicians performed the sectioning and observations, and the technicians did not know the sample IDs.

TEM was further used to determine the subcellular changes associated with Slit2 in the post-IR myocardium. Left ventricular tissues were cut into small blocks (about 1 mm³), fixed with 2.5% glutaraldehyde, fixed with 1% OsO₄, dehydrated in ethanol, and embedded in Araldite. The tissue blocks were cut into slices at a thickness of 60 nm using a Leica cryostat system (EM UC7/FC7, Germany) and collected on copper grids. The ultrathin sections were double stained with 3% uranyl acetate and lead citrate. The subcellular structure was observed with a Tecnai G2 Spirit transmission electron microscope (FEI Company, United States). During evaluation of the subcellular differences between the groups, different technicians performed the slice preparation and observations, and the technicians did not know the sample IDs.

Total RNA was isolated using TRIzol^® Reagent (Thermo Fisher Scientific, United States), and RNA concentrations were determined using a Qubit^® 2.0 Fluorometer and an assay kit (Life Technologies, United States). RNA integrity was assessed using an RNA Nano 6000 Assay Kit with a Bioanalyzer 2100 system (Agilent Technologies, United States). Sequencing libraries were generated using an NEBNext^® Ultra^TM RNA Library Prep Kit for Illumina^® (NEB, United States). After adding index codes, the samples were clustered with a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina, United States). An Illumina HiSeq platform (Illumina, United States) was employed to sequence the libraries, generating 125 bp/150 bp paired-end reads. Raw data with proper paired-end clean reads were mapped for further analysis. The original reads of the genes for the samples in the four groups (the C57-non-IR, C57-IR, Slit2-non-IR, and Slit2-IR groups) were compared in pairs, and the fold changes in the expressed genes between the groups were log2 transformed. A P value < 0.05 was the cutoff for identification of differentially expressed genes (DEGs). Furthermore, DEGs with log2 (fold change) values > 0 were considered upregulated, while those with log2 (fold change) values < 0 were considered downregulated. Thus, we defined eight datasets of DEGs: an upregulated dataset and a downregulated dataset each for the C57-IR vs. C57-non-IR, Slit2-IR vs. Slit2-non-IR, Slit2-non-IR vs. C57-non-IR, and Slit2-IR vs. C57-IR comparisons. The gene expression profiles are presented using volcano plots (Supplementary Figure S3). The raw RNA-seq data have been deposited in NCBI’s Gene Expression Omnibus (GEO) (#GSE133796)¹.

First, to identify genes regulated by Slit2 in post-IR hearts, five datasets of DEGs (the downregulated DEGs in the C57-IR vs. C57-non-IR comparison, the upregulated DEGs in the C57-IR vs. C57-non-IR upregulated, the downregulated DEGs in the Slit2-IR vs. C57-IR comparison, the upregulated DEGs in the Slit2-IR vs. C57-IR comparison, and the up-/downregulated DEGs in the Slit2-non-IR vs. C57-non-IR comparison) were input into the TBtools program² to generate a Venn diagram. By analyzing this diagram, two final intersections were obtained: (1) the intersection of the upregulated DEGs in the C57-non-IR vs. C57-IR comparison and the downregulated DEGs in the Slit2-IR vs. C57-IR comparison, and (2) the intersection of the downregulated DEGs in the C57-IR vs. C57-non-IR comparison and the upregulated DEGs in the Slit2-IR vs. C57-IR comparison. These two final intersections further excluded the up-/downregulated DEGs in theSlit2-non-IR vs. C57-non-IR comparison. We therefore identified two new sets of DEGs: one set was upregulated by IR in C57BL/6J hearts but downregulated by Slit2 overexpression in post-IR hearts, and another was downregulated by IR in C57BL/6J hearts but upregulated by Slit2 overexpression in post-IR hearts. The Venn diagram results are presented in Figures 4B,C.

Second, to predict the effects of Slit2 on cardiac function, the GSEA tool Metascape (Zhou et al., 2019) was used to construct protein interaction networks of gene sets. Briefly, the enrichment gene sets (Biological Processes, KEGG pathways, or Reactome pathways) with similar functions were clustered, and the identification algorithm “MCODE” revealed that these enrichment clusters formed interaction networks. The interaction network of the Slit2-non-IR vs. C57-non-IR suggested the effects of Slit2 on the molecular function of the heart (Figures 3A,B), and the interaction network of the Slit2-IR vs. C57-IR suggested the effects of Slit2 on molecular function in post-IR hearts (Figures 3C,D).

RT-qPCR was used to verify the expression of genes identified by the Venn diagram analysis and to determine the expression of Robo receptors. Total RNA extracted from the left ventricle was subjected to reverse transcription and quantitative PCR. The primers are listed in Supplementary Table S1). TB Green^® Premix Ex Taq^TM II (Takara Bio Inc., Japan) was used. The program was as follows: 95°C for 30 s (1 cycle); 95°C for 5 s, 60°C for 34 s (40 cycles); and 72°C for 10 min (1 cycle). Gene expression levels were normalized to GAPDH levels, and the results were expressed as the ratio of the expression in each experimental group to that in the C57-non-IR group.

Immunofluorescence detection of Slamf7 and nuclear translocation of NFκB were used to assess inflammatory responses. Heart tissues were fixed in 10% neutral formalin, cryoprotected in 30% sucrose for 24 h, embedded in optimal cutting temperature (OCT) compound, and sectioned with a freezing microtome at a thickness of 5 μm (Leica CM1950, Germany). The heart sections were processed using a standard immunostaining protocol. After routine hydration, the sections were subjected to permeabilization with PBS containing 0.05% Triton X-100 followed by incubation with a primary antibody against Slamf7 (A5782, ABclonal, China) or NFκB p65 (8242S, CST, United States) at 4°C overnight. The primary antibodies were visualized using Alexa Fluor 594- and Alexa Fluor 488-conjugated secondary antibodies (Invitrogen, CA). The sections were photographed under a confocal microscope (Leica TCS SP5, Germany). The technicians performing immunofluorescent staining and observations were different and did not know the sample ID.

Heart perfusates were collected at the perfusion baseline and 0, 5, 10, and 40 min after reperfusion. ELISA kits (Cusabio Biotech Co., China) were used to test the levels of the proinflammatory cytokines IL-1β and IL-18 in the perfusates according to the manufacturer’s instructions. Briefly, 100 μl of the standard or sample was added to each well, and the plates were incubated for 2 h at 37°C. After removing the liquid, 100 μl/well of anti-biotin antibody was added, and the plates were incubated for 1 h at 37°C before being incubated with 100 μl of HRP-avidin and 90 μl of TMB substrate for 25 min at 37°C in the dark. The optical density was measured within 5 min using a microplate reader at 450 nm.

Myofilaments were extracted according to a modified protocol from Yang et al. (2008) and Yang and Pyle (2011, 2012). Briefly, hearts were homogenized in ice-cold lysis buffer composed of (in mM) 60 KCl, 30 imidazole (pH 7.0), 2 MgCl₂, 0.01 leupeptin, 0.1 phenylmethylsulfonyl fluoride (PMSF), and 0.2 benzamidine and then centrifuged at 12,000 × g and 4°C for 15 min. The pellets were extracted with ice-cold lysis buffer containing 1% Triton X-100 and centrifuged at 1,100 × g to obtain myofilaments. The protein concentrations of the myofilaments were determined with a bicinchoninic acid (BCA) protein assay kit (Pierce, United States).

First, cytosolic fractions extracted from cardiac tissues were used to detect the expression of protein-encoding genes selected from RNA-seq analyses, Slit2 and the Slit2 fragment. Briefly, hearts were homogenized with a cell lysis buffer (#9803, CST, United States) containing protease inhibitors and 1% Triton X-100. The samples were centrifuged at 12,000 × g and 4°C for 15 min, and the supernatants were retained for use. Primary antibodies against Slamf7 (A5782, ABclonal, China), RasGRP1 (sc-365358, Santa Cruz, United States), Slit2 (ab7665, Abcam, United States), and Slit2-C (BM5312, Bostern, China) were used. Second, myofilament fractions extracted from cardiac tissues were used to determine myofilament-associated PKCs, PKA, protein phosphatases (PPs), and phosphorylation sites of cardiac myofilaments. Primary antibodies against PKCδ (2058S, CST, United States), PKCε (2683S, CST, United States), PKA (4782S, CST, United States), PP1α (2582S, CST, United States), PP type 2A (PP2A; 2038S, CST, United States), p-cTnISer43 (PA5-35412, Invitrogen, CA), p-cTnISer23/24 (4004S, CST, United States), Slit2 (ab7665, Abcam, United States), Slit2-C (BM5312, Bostern, China), GAPDH (2118S, CST, United States), and actin (MAB1501R, Millipore, United States) were used.

Cytosolic (40 μg) or myofilament (60 μg) proteins were separated using 10% SDS-PAGE and transferred to PVDF blotting membranes (Millipore, United States). After blocking with 5% skim milk, the membranes were incubated with primary antibodies (as listed above) overnight at 4°C before being incubated with species-appropriate secondary antibodies (CST, United States). The bands were detected with Immobilon Western chemiluminescent HRP substrate (Millipore, United States). The densities of the bands were analyzed using ImageJ software (NIH, United States).

The phosphorylation levels of myofilament proteins (MyBP-C, desmin, cTnT, and cTnI) were measured using the Pro-Q Diamond phosphorylation gel staining method, as previously described (Yang and Pyle, 2011, 2012). Briefly, myofilaments (60 μg) were separated using 12% SDS-PAGE. After fixation with 50% methanol/10% acetic acid for 30 min, the gels were incubated with Pro-Q Diamond phosphorylation gel staining solution (Molecular Probes, United States) for 90 min. The phosphorylated bands were visualized using a Gel Doc^TM XR + gel documentation system (Bio-Rad Laboratories Ltd., United States), and the band densities were quantified using ImageJ (NIH, United States). Total protein was stained with Coomassie solution, and actin bands were used as the loading controls.

An actomyosin MgATPase activity assay was performed as described previously (Yang et al., 2008; Yang and Pyle, 2011, 2012) to assess myofilament function. Briefly, myofilaments (50 μg) were incubated in reaction solutions containing various concentrations of free calcium. The free calcium concentration in each solution was calculated using a Patton assay program (Patton et al., 2004). The myofilaments were incubated in the reaction solutions for 5 min at 32°C. After the reactions were stopped with 10% trichloroacetic acid, the amount of inorganic phosphate produced was measured after adding 0.5% FeSO₄ and 0.5% ammonium molybdate in 0.5 M H₂SO₄ at 630 nm using a spectrophotometer (Biochrom, United Kingdom).

All experimental data are presented as the mean ± SEM. Statistical differences between two groups were analyzed with unpaired Student’s t-tests, and differences among multiple groups of data were analyzed by two-way ANOVA followed by Tukey’s multiple comparisons test (GraphPad Prism 7, United States). P < 0.05 were considered to indicate statistical significance.

First, we examined cardiac function in all groups. There were no significant differences in cardiac function (HR, LVDP, and RPP) between C57BL/6J and Slit2-Tg mice at baseline (0–20 min, Figures 1C–E). However, following 20 min of ischemia, HR, LVDP, RPP, and dP/dt(max) were significantly higher and dP/dt(min) was significantly lower in Slit2-Tg mice than in C57BL/6J mice at the time points of 60, 65, 70, 75, and 80 min (Figures 1C–H). These improvements in contractility were associated with lower LVEDP values at the above time points in post-IR Slit2-Tg hearts compared to control hearts (Figure 1H, Table 1, and Supplementary Figure S2) as well as with markedly reduced infarct size in post-IR Slit2-Tg hearts (Figures 1I,J). These results establish that Slit2 protein overexpression significantly improves cardiac systolic and diastolic function following IR and reduces infarct area. The functional parameters are presented in Table 1. Next, we explored the effects of Slit2 on cardiac cellular damage post IR. Under a microscope, we found focal necrosis, some vacuoles, and swollen and dissolved myofibers in cardiac tissues of post-IR C57BL/6J mice (Figure 2A). However, Slit2 overexpression reduced the IR-induced myofiber swelling and vacuole formation between fibers seen in C57BL/6J hearts (Figure 2A). We further employed TEM technology to examine the subcellular structure in post-IR hearts. We found that reperfused C57BL/6J hearts displayed edematous mitochondria, fractured internal crests of mitochondria, ruptured myofibrils, and ruptured Z-discs in cardiomyocytes (Figure 2B). Compared with C57BL/6J hearts, Slit2-Tg hearts had less mitochondrial edema and better myofilament maintenance and Z-line morphology (Figure 2B) after reperfusion. The results from HE staining and TEM examination demonstrate that Slit2 reduces tissue and cellular damage in post-IR hearts.

Slit2 mRNA and protein levels were significantly higher in Slit2-Tg hearts than in C57BL/6J (background strain) hearts, but there were no functional or histological differences (Supplementary Figure S1 and Supplementary Table S2). The overall gene expression profiles are displayed in Supplementary Figure S3 and Supplementary Table S4. Furthermore, the RNA sequencing data revealed that multiple molecular functions were altered in Slit2-Tg hearts (Figure 3). Slit2 overexpression boosted regulation of cell adhesion, protein kinase B (Akt) signaling, the response to hypoxia, organic acid catabolic processes, and the response to oxidative stress (Figure 3A and Supplementary Table S3). Slit2 reduced positive regulation of glycogen metabolic processes, the PPAR signaling pathway, regulation of the inflammatory response, and regulation of the release of cytochrome C from mitochondria (Figure 3B and Supplementary Table S3). These results suggest that hearts overexpressing Slit2 are sensitive to external stimuli, while they resist inflammatory insults and might exhibit improved cell survival.

In hearts with post-IR injury, Slit2 enhanced collagen chain trimerization, regulation of ion transport, regulation of system processes, responses to mechanical stimuli, potassium ion transport, and ion homeostasis (Figure 3C and Supplementary Table S3). Moreover, Slit2 specifically protected cardiac contractile function in the post-IR myocardium, as observed from the enrichment of the “regulation of system process” term: a cluster of gene sets upregulated in post-IR Slit2 hearts was enriched for muscle contraction, regulation of HR, regulation of heart contraction, heart contraction, actin-mediated cell contraction, regulation of blood circulation, and adrenergic signaling in cardiomyocytes (Figure 3C and Supplementary Table S3). In addition, Slit2 reduced regulation of immune effector processes, the inflammatory response, the response to interferon-gamma, monocyte chemotactic protein-1 production, and regulation of leukocyte activation in the post-IR myocardium (Figure 3D and Supplementary Table S3). These results suggest that Slit2 maintains cardiac contractile function and extracellular structure and prevents inflammatory responses during IR.

In exploring the intracellular targets of Slit2 in the protected myocardium, we identified 385, 646, 112, 183, and 431 DEGs from among the downregulated DEGs in the C57-IR vs. C57-non-IR comparison, the upregulated DEGs in the C57-IR vs. C57-non-IR comparison, the downregulated DEGs in the Slit2-IR vs. C57-IR comparison, the upregulated DEGs in the Slit2-IR vs. C57-IR comparison, and the up-/downregulated DEGs in the Slit2-non-IR vs. C57-non-IR comparison, respectively (Figures 4A,B and Supplementary Table S4). By using Venn diagram analysis (Figures 4A,B), we identified 18 genes (6 upregulated and 12 downregulated) that might be regulated by Slit2 to protect the post-IR myocardium. The genes are shown in Figure 4C.

The expression of the two genes most affected by Slit2 overexpression post IR (Slamf7 and RasGRP1) was verified using RT-qPCR and Western blotting. The gene expression and protein expression of Slamf7 were both upregulated by IR in C57BL/6J hearts, but Slit2 overexpression blunted this upregulation in post-IR conditions (Figures 5A,F,G). No differences in RasGRP1 were detected between the groups (Figures 5B,F,H). In addition, RT-qPCR and Western blotting were used to determine the gene and/or protein expression of the Slit2-C fragment and Robo receptors. The protein expression of Slit2-C did not differ between the groups (Figure 5I). Robo1 expression did not change in C57BL/6J hearts after IR but was activated in post-IR Slit2-Tg hearts (Figure 5C). Robo2 was not different between any groups (Figure 5D). Robo4 was upregulated by IR in C57BL/6J hearts, but the IR-induced increases were blunted in Slit2-Tg hearts (Figure 5E). Therefore, we identified Robo1, Robo4, and Slamf7 to be regulated by Slit2 in post-IR hearts.

A previous study has shown that Slit-Robo signaling plays anti-inflammatory roles, and our RNA sequencing data indicated that Slit2 overexpression reduced inflammatory responses in post-IR conditions (Figure 3D and Supplementary Table S3). In addition, RNA sequencing and expression verification confirmed that Slamf7 was an effector of Slit2 overexpression. Slamf7 is known to be expressed on immune cells (Malaer and Mathew, 2017). Thus, we further investigated the inflammatory responses within cells. First, we found that Slamf7 was diffusely distributed in cardiomyocytes in C57BL/6J hearts. The expression of Slamf7 increased significantly when the myocardium was subjected to IR, but this activation was suppressed by Slit2 overexpression (Figures 6A,C). These results were consistent with the Western blot and RT-qPCR findings (Figures 5A,F,G). Moreover, we found that Slit2 overexpression inhibited the IR-induced nuclear translocation of NFκB p65 in cells (mainly fibroblasts) (Figures 6B,D).

The levels of the cytokines IL-1β and IL-18 did not differ between C57BL/6J and Slit2-Tg heart perfusates at baseline. At the beginning of reperfusion, the release of IL-1β sharply increased before exhibiting a U-shaped change in C57BL/6J hearts, but Slit2-Tg hearts showed smaller increases, and the levels rapidly returned to baseline levels during reperfusion (Figure 6E). Elevations in IL-18 release were seen in C57BL/6J hearts but not in Slit2-Tg hearts at the beginning of reperfusion (Figure 6F). The above results establish that Slit2 plays an anti-inflammatory role during IR.

In previous experiments, we found that Slit2 improved contractile function, protected cardiac tissues (Figures 1, 2), and shifted gene expression profiles to enhance the regulation of cardiac contraction (Figure 3C). Consequently, we investigated myofilament-associated PKA and PKC and their counterparts PP type 1 (PP1) and PP2A, which are known to be activated during IR and to regulate cardiac contraction (Lochner et al., 1999; Armstrong, 2004; Nicolaou et al., 2009). Here, we found that myofilament-associated PKCδ was significantly upregulated in C57BL/6J hearts after IR, but the increases in PKCδ were blocked by Slit2 post IR (Figure 7B). Slit2 did not fully block the IR-induced upregulation of PKCε in C57BL/6J mice (Figure 7A). The IR-induced increases in myofilament-associated PKA and PP2A were not altered by Slit2 overexpression post IR (Figures 7C,E). The expression of myofilament-associated PP1α was not different between any groups (Figure 7D). The above results establish that Slit2 targets myofilament-associated PKC phosphorylation signaling in the post-IR myocardium.

Several cardiac myofilament proteins, including cTnI, cTnT, MyBP-C, and desmin, can be targeted by protein kinases (Mohamed et al., 1998; Solaro, 2008; Streng et al., 2013). Following identification of myofilament-associated PKC regulation by Slit2, we explored myofilament phosphorylation levels and specific sites affected by Slit2 in the post-IR myocardium. We found that cTnI phosphorylation was increased in both post-IR C57BL/6J and Slit2-Tg hearts, but the increase was relatively suppressed in post-IR Slit2-Tg hearts (Figures 8A,B). MyBP-C phosphorylation was decreased in C57BL/6J and Slit2-Tg hearts after IR, and the decreases did not differ between the groups (Figures 8A,B). Desmin and cTnT did not show any differences between the groups (Figures 8A,B). Moreover, we found that the phosphorylation levels of cTnI at Ser43 were higher than baseline levels in post-IR C57BL/6J hearts, but Slit2 significantly decreased the IR-induced phosphorylation at this site (Figures 8C,E). These changes were consistent with the findings for cTnI phosphorylation. cTnI phosphorylation at Ser23/24 did not differ between any groups (Figures 8C,D). Thus, we identified that cTnI Ser43 was targeted by Slit2 during IR, and this site is known for phosphorylation by PKCs. Given the findings regarding the activation of myofilament-associated PKC, we conclude that Slit2 regulates myofilament activation at least in part by targeting PKC-dependent cTnI phosphorylation.

Myofilament contractile properties (maximum actomyosin MgATPase activity and calcium sensitivity) were examined to further explain the post-IR protective effects of Slit2. We found that while maximum actomyosin MgATPase activity did not differ among non-ischemic hearts (Figures 8F,H), Slit2-Tg hearts had higher calcium sensitivity than C57BL/6J hearts (Figure 8G). Maximum actomyosin MgATPase activity decreased significantly in both C57BL/6J and Slit2-Tg hearts post IR, but Slit2-Tg hearts maintained relatively higher contractility than C57BL/6J hearts (Figures 8F,H). The calcium sensitivity of C57BL/6J myofilaments was significantly decreased after IR, but it was not altered in post-IR Slit2 myofilaments (Figure 8G). In summary, Slit2 protects myofilament contractile function in post-IR hearts, and this effect is associated with Slit2-mediated depression of cTnI phosphorylation.