CD74 knockout protects against LPS‐induced myocardial contractile dysfunction through AMPK‐Skp2‐SUV39H1‐mediated demethylation of BCLB

Abstract

Background and Purpose

Lipopolysaccharides (LPS), an outer membrane component of Gram‐negative bacteria, triggers myocardial anomalies in sepsis. Recent findings indicated a role for inflammatory cytokine MIF and its receptor, CD74, in septic organ injury, although little is known of the role of MIF‐CD74 in septic cardiomyopathy.

Experimental Approach

This study evaluated the impact of CD74 ablation on endotoxaemia‐induced cardiac anomalies. Echocardiographic, cardiomyocyte contractile and intracellular Ca²⁺ properties were examined.

Key Results

Our data revealed compromised cardiac function (lower fractional shortening, enlarged LV end systolic diameter, decreased peak shortening, maximal velocity of shortening/relengthening, prolonged duration of relengthening and intracellular Ca²⁺ mishandling) and ultrastructural derangement associated with inflammation, O₂ ⁻ production, apoptosis, excess autophagy, phosphorylation of AMPK and JNK and dampened mTOR phosphorylation. These effects were attenuated or mitigated by CD74 knockout. LPS challenge also down‐regulated Skp2, an F‐box component of Skp1/Cullin/F‐box protein‐type ubiquitin ligase, while up‐regulating that of SUV39H1 and H3K9 methylation of the Bcl2 protein BCLB. These effects were reversed by CD74 ablation. In vitro study revealed that LPS facilitated GFP‐LC3B formation and cardiomyocyte defects. These effects were prevented by CD74 ablation. Interestingly, the AMPK activator AICAR, the autophagy inducer rapamycin and the demethylation inhibitor difenoconazole inhibited the effects of CD74 ablation against LPS‐induced cardiac dysfunction, while the SUV39H1 inhibitor chaetocin or methylation inhibitor 5‐AzaC ameliorated LPS‐induced GFP‐LC3B formation and cardiomyocyte contractile dysfunction.

Conclusion and Implications

Our data suggested that CD74 ablation protected against LPS‐induced cardiac anomalies, O₂ ⁻ production, inflammation and apoptosis through suppression of autophagy in a Skp2‐SUV39H1‐mediated mechanism.

Abbreviations

−dL/dt

maximal velocity of relengthening

+dL/dt

maximal velocity of shortening

ΔFFI

fura‐fluorescence intensity change

AICAR

5′‐aminoimidasole‐4‐carboxamide‐1‐β‐d‐ribofuranoside

ALDH2

mitochondrial isoform of aldehyde dehydrogenase

AMPK

5′ AMP‐activated protein kinase

DHE

dihydroethidium

EDD

end diastolic dimension

ESD

end systolic dimension

GFP

green fluorescence Protein

HMGB1

high mobility group 1

LV

left ventricle

MIF

migration inhibitory factor

mTOR

mechanistic target of rapamycin

MyD88

myeloid differentiation factor 88

NCM

neonatal cardiomyocyte

PS

peak shortening

Sirt

sirtuin

Skp2

S‐phase kinase‐associated protein 2

SUV39H1

shttps://www.ncbi.nlm.nih.gov/gene/6839

TLR4

toll‐like receptor 4

TPS

time‐to‐peak shortening

TR₉₀

time‐to‐90% relengthening

TRAF6

TNF receptor associated factor 6

WT

wild type

What is already known

• Endotoxemia triggers cardiomyopathy accompanied with dysregulated autophagy.

• CD74 ablation reduces alcoholic cardiomyopathy through regulation of autophagy.

What this study adds

• CD74 ablation protects against LPS‐induced cardiac dysfunction.

• CD74 ablation rescues endotoxemia‐induced cardiomyopathy through regulation of autophagy.

What is the clinical significance

• CD74 may serve as a therapeutic target against sepsis‐induced cardiac dysfunction.

1. INTRODUCTION

Septic cardiomyopathy represents a severe form of multi‐organ anomalies in sepsis, leading to unfavourable changes in cardiac structure and function including dilated ventricles, impaired cardiac contractility and reduced cardiac output (Arfaras‐Melainis et al., 2019; Ceylan‐Isik et al., 2010; Pang, Zheng et al., 2019; Ren & Wu, 2006; Tan, Chen, Zhong, Ren, & Dong, 2019; Turdi et al., 2012; Zhang et al., 2014). Patients with sepsis commonly develop septic cardiomyopathy enroute to cardiogenic shock, contributing to a high mortality rate of ~70–90% in sepsis (Merx & Weber, 2007; Ren & Wu, 2006; Rudiger & Singer, 2007). Despite the advance in medical technology and clinical management of sepsis (Ren & Wu, 2006; Stanzani, Duchen, & Singer, 2019), targeted therapy is still dismal for septic cardiomyopathy. Evidence from our lab and others has demonstrated a major role for endotoxin lipopolysaccharides (LPS) released from Gram‐negative bacteria in the inflammatory response and cardiovascular homeostasis in sepsis (Pfalzgraff & Weindl, 2019; Ren et al., 2016; Zhang et al., 2014). Although antioxidants (such as metallothionein, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4971 and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2979), mitochondrial protein aldehyde dehydrogenase (https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2595) and ER chaperones have shown some promise in the treatment of septic cardiomyopathy (Ceylan‐Isik et al., 2010; Durand et al., 2017; Pang, Peng et al., 2019; Turdi et al., 2012), clinical validation has not been consolidated for antioxidants and mitochondrial drugs in sepsis and septic hearts. More recent findings from our group and others depicted a rather pivotal role for autophagy dysregulation in the onset and development of septic cardiomyopathy (Pang, Peng, et al., 2019; Pang, Zheng, et al., 2019; Piquereau et al., 2013; Ren et al., 2016; Sun et al., 2018), although the mechanism behind sepsis‐induced autophagy dysregulation is still unclear.

Earlier evidence has indicated rapid fatal outcomes in septic patients with high levels of https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=9981 (MIF; Chuang et al., 2014), consistent with the widely accepted notion that of a provoked pro‐inflammatory response in sepsis (Magrone & Jirillo, 2019). MIF, a pluripotent cytokine, found in various cell types including immune, endothelial and epithelial cells, is deemed a biomarker for inflammatory diseases such as sepsis, systemic infection and autoimmune disease (Hertelendy et al., 2018). Nonetheless, results from our lab revealed a somewhat paradoxical role for MIF in disease settings, such as obesity and pressure overload‐induced cardiac anomalies through autophagy regulation (Xu, Hua, Nair, Bucala, & Ren, 2014; Xu & Ren, 2015). Moreover, MIF‐deficient mice were shown to succumb more quickly to bacterial infection, with more prominent organism burden, lung pathology and reduced innate cytokine production (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5074, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4977, and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4975), while MIF‐deficient macrophages display lower cytokine and reactive oxygen production and are a poor defence against bacterial infection (Das et al., 2013). Likewise, mutations in human MIF are also associated greater bacterial burden (Doernberg et al., 2011; Renner et al., 2012). These findings suggest a unique role for MIF in the innate defence against bacterial infection. However, earlier finding suggested that MIF may mediate LPS‐induced cardiac defects, while MIF knockout attenuates endotoxin‐induced cardiac anomalies. These authors suggested that MIF‐induced oxidative stress, through activation of stress signalling including JNK and ER stress contributes to MIF‐induced cardiac anomalies (Zhang, Zhang, Cui, Cui, & Zhao, 2018). Nonetheless, the precise mechanism behind MIF‐mediated cardiac dysfunction in sepsis remains determined. To this end, our present study was designed to examine the impact of the knockout of the MIF membrane receptor https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2840 on endotoxemia‐induced cardiac dysfunction, in an effort to determine the therapeutic potential of targeting MIF‐CD74 in septic cardiomyopathy. CD74 is a non‐polymorphic type II transmembrane glycoprotein, participating in T‐cell and B‐cell development, dendritic cell motility and macrophage inflammation in inflammatory diseases including liver fibrosis, diabetes mellitus, systemic lupus erythematosus and Alzheimer disease (Su, Na, Zhang, & Zhao, 2017). Given the important role of autophagy, oxidative stress and inflammation in septic hearts (Ren et al., 2016; Sun et al., 2018; Sun, Cai, & Zang, 2019), levels of autophagy, O₂ ⁻ production, apoptosis and proinflammatory markers were monitored in WT and Cd74 ^−/− mice challenged with LPS. Levels of key autophagy regulatory molecules including https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1479&familyId=285&familyType=ENZYME, AMP‐dependent protein kinase (https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1540) and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2109 were evaluated in murine hearts. Very recent evidence from our group has revealed a novel role for S‐phase kinase‐associated protein 2 (Skp2), an F‐box content of the Skp1/Cullin/F‐box protein‐type ubiquitin ligase, in CD74 ablation‐induced benefit against alcoholic cardiomyopathy (Yang et al., 2019). Skp2 is believed to function as an AMPK downstream signal molecule for regulation of ubiquitination, proteasomal degradation and autophagy (Shin et al., 2016). Therefore, possible involvements of Skp2 and its downstream signalling were examined in Cd74 ablation‐induced responses against endotoxemia.

2. METHODS

2.1. Experimental animals, LPS challenge and MIF levels

All animal procedures performed were approved by the Animal Care and Use Committee at the Fudan University Zhongshan Hospital (Shanghai, China). Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010) and with the recommendations made by the British Journal of Pharmacology. All procedures were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. CD74 knockout (Cd74 ^−/−) mice were described previously (Shachar & Flavell, 1996). In brief, 4‐ to 5‐month‐old mice (both sexes) were housed in individual cages (six per cage) in a climate‐controlled environment (22.8 ± 2.0°C, 45–50% humidity) with a 12/12–light/dark cycle with access to food and water ad libitum until experimentation. On the day of experimentation, Cd74 ^−/− and WT mice were randomly assigned to receive LPS or saline injection intraperitoneally (4 mg·kg⁻¹ Escherichia coli O55:B5 LPS dissolved in sterile saline or an equivalent volume of pathogen‐free saline for control; Zhang et al., 2014). Six hours later mice were killed. Given that systemic inflammation is among the most prominent responses to trigger a wide range of behaviour and organ damage in sepsis, administration of LPS is used to establish an endotoxemia model to provoke transient systemic inflammation, mimicking septic shock (Schedlowski, Engler, & Grigoleit, 2014). The selection of 6 hr for LPS challenge was based on the established model of endotoxemia‐induced cardiomyopathy from our labatory and others (Dong et al., 2013; Koentges et al., 2019; Pang, Peng, et al., 2019; Ren et al., 2016). Immediately following LPS injection, mice were closely monitored for signs of pain or discomfort including obviously decreased activity, abnormal postures, hunched back, muscle flaccidity or rigidity, decreased food or water consumption, vomiting or diarrhoea, cachexia, dehydration, photophobia, abrupt decrease or increase in pulse or respiratory rate and overt physical response to touch (withdrawal, lameness, abnormal aggression, abdominal splinting and increase in pulse or respiration). Mice were killed using the cervical dislocation method should obvious pain or discomfort develop. Sample size was determined using SigmaStat power analysis according to several pre‐determined parameters, including minimal detectable difference of the mean, expected SD, number groups, power and α. WT and Cd74 ^−/− mice were each injected with saline and LPS, with a four‐group in design. Then we estimated the minimal difference and expected SD of parameters between saline control and LPS groups. The power was preset to be 0.8 and α value was preset to be .05. After calculations using this software, a total of ~30 adult WT mice and ~30 adult Cd74 ^−/− mice (both sexes) were used for both in vivo and in vitro studies. Plasma MIF levels were measured using a commercial R&D human MIF kit per manufacturer's instruction (Cui et al., 2018).

2.2. Echocardiographic assessment

Cardiac function were monitored in anesthetized (ketamine 80 mg/kg and xylazine 12 mg/kg, i.p.) mice (placed on a heating pad) 6 hrs after LPS challenge (4 mg·kg⁻¹, i.p.) using a two‐dimensional (2‐D) guided M‐mode echocardiography (Vevo 2100, FUJIFILM Visualsonics, Toronto, ON, Canada) equipped with a 22–55 MHz linear transducer (MS550D, FUJIFILM VisualSonics, Toronto, ON, Canada). The echocardiographic technician was blind to the groups pretreatments. Hearts were imaged in the 2‐D mode using the parasternal long‐axis view before switching to M‐mode positioned perpendicular to interventricular septum and posterior left ventricular (LV) wall. LV wall thickness, LV end diastolic, and end systolic dimensions (EDD and ESD) were measured. Fractional shortening was calculated as [(EDD − ESD)/EDD] × 100. Heart rate was averaged over 10 cardiac cycles (Wang, Zhu, Xiong, & Ren, 2017).

2.3. Isolation of cardiomyocytes and in vitro drug treatment

Under anaesthesia, hearts were removed from mice and were perfused with a thermostat (37°C) Langendorff system with a modified Tyrode solution prior to digestion with Liberase Blendzyme 4. After complete digestion, left ventricles were isolated and minced into small pieces before being resuspended (Li, Gilbert, Li, & Ren, 2009). Cardiomyocyte yield was approximately 65–75% which was unaffected by CD74 KO or LPS challenge. To assess the role of AMPK, autophagy, SUV39H1 and autophagy protein methylation in LPS‐induced cardiomyocyte dysfunction, cardiomyocytes from WT and Cd74 ^−/− mice were exposed to LPS (4 μg·ml⁻¹; Ceylan‐Isik et al., 2010) for 6 hr in the presence or absence of the AMPK activator AICAR (500 μM; Nyblom, Sargsyan, & Bergsten, 2008), the autophagy inducer rapamycin (5 μM; Yuan et al., 2009), the methylation inhibitor 5‐aza‐20‐deoxycytidine (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6796,) 10 μM; Liu et al., 2017; Onay, Yapici Eser, Ulasoglu Yildiz, Aslan, & Tali, 2017), the demethylation inhibitor difenoconazole (DIF, 0.5 μg·ml⁻¹; Ali & Amiri, 2018), or the SUV39H1 (suppressor of variegation 3‐9 homolog 1) inhibitor https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=8388 (50 nM; Yang et al., 2017) prior to mechanical assessment.

2.4. Cell shortening/relengthening

Mechanical properties of cardiomyocytes were assessed using a SoftEdge Myocam (IonOptix, Milton, MA). Cardiomyocytes were placed in a chamber mounted on the stage of an inverted microscope (Olympus IX‐70) and superfused (~2 ml·min⁻¹ at 25°C) with a Krebs–Henseleit bicarbonate buffer containing 1‐mM CaCl₂. Myocytes were field stimulated at 0.5 Hz. Cell shortening was assessed including peak shortening (PS), indicating peak contractility; time‐to‐PS (TPS), indicating contraction duration; time‐to‐90% relengthening (TR₉₀), indicating relaxation duration; and maximal velocities of shortening/relengthening (±dL/dt), indicating maximal pressure development and decline (Li et al., 2009).

2.5. Intracellular Ca²⁺ transient

Cardiomyocytes were loaded with fura‐2/AM (0.5 μM) for 10 min, and fluorescence measurements were recorded with dual‐excitation fluorescence photo multiplier tube (PMT, Ionoptix). Cardiomyocytes were placed on an Olympus IX‐70 inverted microscope and imaged through a Fluor 40× oil objective. Cells were exposed to light emitted by a 75‐W lamp and passed through a 360‐nm or 380 nm filter, while being stimulated to contract at 0.5 Hz. Fluorescence emissions were detected between 480 and 520 nm by PMT after first illuminating cells at a 360 nm for 500 msec, then at 380 nm for the duration of the recording protocol (333‐Hz sampling rate). The 360‐nm excitation scan was repeated at the end of the protocol, and qualitative changes in intracellular Ca²⁺ levels were inferred from the ratio of fura‐2 fluorescence intensity at two wavelengths (360/380). Fluorescence decay time was assessed as an indication of intracellular Ca²⁺ clearing. Single exponential curve fit was applied to calculate intracellular Ca²⁺ decay rate (Xu, Hua, Nair, Zhang, & Ren, 2013).

2.6. Histological staining and electron microscopy

Heart samples were fixed with 10% formalin for 24 hr, dehydrated with alcohol gradient, and embedded in paraffin. Subsequently, samples (5‐μm sections) were stained with Lectin from Triticum vulgaris FITC‐conjugated wheat germ agglutinin (Sigma catalog # L4895, St. Louis, MO) to assess cardiomyocyte cross‐sectional areas. For electron microscopy study, myocardial samples were taken from the midventricular region and were trimmed to 1‐mm³ blocks. The blocks were fixed using a 10:1 fluid/tissue ratio overnight at 4°C. RMC‐MTXL ultramicrotome and a Diatome diamond knife were used to obtain thin sections. Sections were labelled with lead citrate and uranyl acetate (in absolute ethanol). Micrographic pictures were taken using a Hitachi 7500 transmission electron microscope (Wang et al., 2018).

2.7. Fluorescence measurement of superoxide (O₂⁻)

Intracellular O₂ ⁻ was monitored by changes in fluorescence intensity from intracellular probe oxidation. In brief, cardiomyocytes were loaded with 5‐μM dihydroethidium (DHE; Molecular Probes, Eugene, OR) for 30 min at 37°C. Cells were evaluated using an Olympus BX‐51 microscope with Olympus MagnaFire™ SP digital camera. Fluorescence was calibrated using InSpeck microspheres (Molecular Probes) and was quantitated using an ImagePro analysis software (Media Cybernetics, Silver Spring, MD; Wang et al., 2017). To determine the cause–effect role of O₂ ⁻ production in various cell signalling pathways‐mediated cardiac contractile responses, cardiomyocytes from WT and Cd74 ^−/− mice were exposed to LPS (4 μg·ml⁻¹; Ceylan‐Isik et al., 2010) for 6 hr in the presence or absence of the AMPK activator AICAR (500 μM; Nyblom et al., 2008), the autophagy inducer rapamycin (5 μM; Yuan et al., 2009), the methylation inhibitor 5‐aza‐20‐deoxycytidine (5‐AzaC, 10 μM; Liu et al., 2017; Onay et al., 2017), the demethylation inhibitor difenoconazole (DIF, 0.5 μg·ml⁻¹; Ali & Amiri, 2018), or the SUV39H1 inhibitor chaetocin (50 nM; Yang et al., 2017) prior to the loading with DHE (5 μM) at 37°C for 30 min. Cardiomyocytes were then rinsed, and DHE fluorescence intensity was measured using a fluorescent microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 530 nm. The final fluorescent intensity was normalized to the protein content in each group (Wang et al., 2013; Zhang, Xia, La Cour, & Ren, 2011).

2.8. Western blot analysis

Ventricular tissues or isolated cardiomyocytes were homogenized and sonicated in RIPA buffer containing 20‐mM Tris (pH 7.4), 150‐mM NaCl, 1‐mM EDTA, 1‐mM EGTA, 1% Triton, 0.1% sodium dodecyl sulfate (SDS), and a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Tissue or cell homogenates (50 μg per lane) were resolved in SDS‐PAGE in a mini‐gel apparatus (Mini‐PROTEAN II, Bio‐Rad, Hercules, CA), and proteins were transferred onto nitrocellulose membranes. The membranes were blocked in 5% (w/v) non‐fat milk in TBS‐T buffer and were then incubated overnight with various primary antibodies at 4°C. The dilutions of primary antibodies (in 5% BSA) were maintained at 4°C and were re‐used no more than three times. Membranes were incubated with an anti‐mouse or anti‐rabbit IgG HRP (HRP)‐coupled secondary antibody (1:5,000 in 5% milk) for 1 hr at room temperature. Signal was detected quantified with a Bio‐Rad Calibrated Densitometer, and the intensity of immunoblot bands was normalized to that of GAPDH. For reprobing, membranes were tripped with 50‐mM Tris–HCl, 2% SDS, and 0.1‐M β‐mercaptoethanol. Antibodies against CD74 (1:1,000, Santa Cruz, sc‐5438, RRID:AB_638241), TLR4 (1:1,000, Cell Signaling, 2219S, RRID:AB_2256152), TNF‐α (1:1,000, Cell Signaling, 3707S, RRID:AB_2240625), HMGB1 (1:1,000, Cell Signaling, 6893S, RRID:AB_10827882), MyD88 (1:1,000, Cell Signaling, 4283S, RRID:AB_10547882), IL‐1β (1:1,000, Santa Cruz, CA, sc‐7884, RRID:AB_2124476), TRAF6 (1:1,000, Cell Signaling, 2219S, RRID:AB_2256152), Bax (1:500, Cell Signaling, 2772, RRID:AB_10695870), Bcl‐2 (1:500, Cell Signaling, 2876S, RRID:AB_2064177), cleaved caspase‐3 (1:500, SCBT, sc‐7272, RRID:AB_626803), Beclin1 (1:1,000, Cell Signaling, 3738S, RRID:AB_490837), Atg5 (1:1,000, Cell Signaling, 2630S, RRID:AB_2062340), LC3B (1:1,000, Cell Signaling, 3868S, RRID:AB_2137707), p62 (1:1,000, Cell Signaling, 5114, RRID:AB_10624872), AMPK (1:1,000, Cell Signaling, 2532, RRID:AB_330331), phosphorylated AMPK (p‐AMPK, Thr¹⁷², 1:1,000, Cell Signaling, 2535, RRID:AB_331250), Akt (1:1,000, Cell Signaling, 9272, RRID:AB_329827), phosphorylated Akt (p‐Akt, Ser⁴⁷³, 1:1,000, Cell Signaling, 4060S, RRID:AB_2315049), mTOR (1:1,000, Cell Signaling, 2972, RRID:AB_330978), phosphorylated mTOR (p‐mTOR, 1:1,000, Ser²⁴⁴⁸, Cell Signaling, 2971, RRID:AB_330970), JNK (1:1,000, Cell Signaling, 9252S, RRID:AB_2250373), phosphorylated JNK (Thr¹⁸³/Tyr¹⁸⁵, 1:1,000, Cell Signaling, 9251S, RRID:AB_331659), Skp2 (1:1,000, Santa Cruz, sc‐74477, RRID:AB_2187653), the Bcl‐2 protein BCLB (1:1,000, Cell Signaling, 3869, RRID:AB_2274786), H3K9 Di‐methyl‐histone (1:1,000, Cell Signaling, 4658, RRID:AB_10544405), SUV39H1 (1:1,000, Abcam, ab12405, RRID:AB_299072), SUV39H2 (1:1,000, Abcam, ab126895, RRID:AB_11130120), and GAPDH (1:1,000, Cell Signaling, 2118 L, RRID:AB_561053) were employed for immunoblotting (Ren et al., 2016). The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology (Alexander et al., 2018).

2.9. Neonatal cardiomyocyte (NCM) isolation

Neonatal (1–2 days old) WT and Cd74 ^−/− (homozygous, no genotyping needed) mice were sterilized with ethanol. Hearts were rinsed with PBS three times and were cut into small pieces prior to four to five rounds of digestion using 0.25% trypsin (Carolina, Burlington, NC, USA). For each round, 2–4 ml of trypsin was added and heart tissues were incubated at 37°C for 10 min. The supernatant was filtered with a 200‐μm cell strainer (Biologix, Lenexa, KS, USA) and neutralized in DMEM medium containing FBS (20%) and 1% penicillin and streptomycin (Gibco, Grand Island, NY, USA) to stop digestion. Tissue fragments were subjected to another round of digestion until they defragmented. Cells were centrifuged at 800× g for 10 min at room temperature, and cell pellets were resuspended in DMEM medium containing FBS (20%) with 1% penicillin and streptomycin before being plated in an uncoated dish for 1 hr at 37°C. The suspending cardiomyocytes were plated in a confocal plate pre‐coated with 1% gelatin, and cultured for 48 hr at 37°C in the presence of 95% O₂ and 5% CO₂ (Wang, Ge, et al., 2018). A total of ~60 neonatal WT pups and ~60 neonatal Cd74 ^−/− pups were used here. Sample size was determined using power analysis.

2.10. LC3B‐GFP‐adenoviral transfection and drug treatment

NCM were transfected with GFP‐LC3 adenovirus for 24 hr (Wang, Ge, et al., 2018) and were treated with LPS (4 μg·ml⁻¹) at 37°C for 24 hr in the absence or presence of the AMPK activator AICAR (500 μM; Nyblom et al., 2008), the autophagy inducer rapamycin (5 μM; Yuan et al., 2009), the methylation inhibitor 5‐AzaC (10 μM; Liu et al., 2017; Onay et al., 2017), the demethylation inhibitor DIF (0.5 μg·ml⁻¹; Ali & Amiri, 2018) or the SUV39H1 inhibitor chaetocin (50 nM; Yang et al., 2017). After treatment, cells were gently rinsed with PBS three times before being fixed in 4% paraformaldehyde (20 min at room temperature). Cells were rinsed with PBS after fixation. For autophagy visualization, cells were imaged using the EVOS® FL Imaging System at 20× magnification (Life Technologies, Carlsbad, CA, USA), and a number of GFP‐LC3 puncta per cell were counted.

2.11. Data analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). Studies were designed to generate groups of equal size, using randomization and blinded animal grouping or data analysis. Statistical analysis was undertaken only for studies where same size was at least five for each group. In certain cases (such as Figure S1), additional data were included but were not subject to statistical analysis owing to small sample size. Sample size was determined using power analysis with SEM expected to be less than 10–15% of the mean value for a reliable sample size. Group size refers to the number of independent values, and statistical analysis was done using these independent values. Data were expressed as mean ± SEM. Statistical significance (P < .05) was deemed by the authors to constitute the threshold for statistical significance among groups and were estimated by multi‐ANOVA followed by a Tukey's test for post hoc analysis (only if in the absence of significant variance inhomogeneity). Outliers were excluded if there was clear evidence for problematic data (signs of arrhythmia or high levels of noise in cardiac function recording, air bubbles in gel blots). All statistics was performed with GraphPad Prism 4.0 software (GraphPad, San Diego, CA).

2.12. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2016) and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander, Fabbro, et al., 2019).

3. RESULTS

3.1. CD74 knockout attenuates echocardiographic dysfunction in LPS challenge

With LPS injection (4 mg·kg⁻¹, i.p., 6 hr), mice from both groups displayed typical but moderate signs of endotoximic shock including lessened motor activity, ruffled fur, diarrhoea and ocular exudates. LPS challenge did not overtly affect body or organ (heart, liver and kidney) weights (or size) in WT or Cd74 ^−/− mice. Neither systolic nor diastolic BP was affected by LPS challenge or CD74 ablation. Echocardiographic assessment indicated that LPS challenge significantly increased LV end systolic diameter (LVESD) and decreased fractional shortening, without affecting LV wall thickness, LV end diastolic diameter (LVEDD) and LV mass, consistent with previous findings (Dong et al., 2013; Turdi et al., 2012). Although CD74 ablation itself did not affect any of echocardiographic indices, it block LPS‐induced echocardiographic anomalies. Neither LPS challenge nor CD74 ablation or both, altered LV wall thickness, left ventricular end diastolic diameter (LVEDD) and LV mass and heart rate. LPS challenge raised plasma MIF levels in a comparable manner in both WT and Cd74 ^−/− mice while CD74 ablation itself did not affect plasma MIF levels (Table 1).

Table 1.

Biometric and echocardiographic properties features of WT and Cd74 ^−/− mice treated with or without LPS (4 mg·kg⁻¹, i.p. for 6 hr)

Mouse group	WT	WT‐LPS	CD74−/−	CD74−/−‐LPS
Body weight (g)	26.0 ± 0.5	27.2 ± 2.5	26.6 ± 1.1	25.6 ± 1.3
Heart weight (mg)	143 ± 4	141 ± 9	139 ± 3	144 ± 5
Heart/weight (mg·g−1)	5.51 ± 0.14	5.32 ± 0.25	5.30 ± 0.19	5.67 ± 0.14
Liver weight (mg)	1.39 ± 0.03	1.40 ± 0.10	1.37 ± 0.03	1.37 ± 0.08
Liver/body weight (mg·g−1)	53.7 ± 1.0	52.4 ± 1.8	52.1 ± 1.8	53.6 ± 1.3
Kidney weight (mg)	354 ± 9	358 ± 20	352 ± 8	353 ± 18
Kidney/body weight (mg·g−1)	13.7 ± 0.3	13.6 ± 0.7	13.3 ± 0.4	13.9 ± 0.4
Diastolic BP (mmHg)	78.7 ± 2.2	79.5 ± 3.1	76.8 ± 1.8	79.2 ± 2.1
Systolic BP (mmHg)	109.8 ± 2.1	112.8 ± 2.7	110.7 ± 2.2	111.3 ± 2.6
Heart rate (bpm)	465 ± 23	473 ± 44	490 ± 22	466 ± 15
LV Wall thickness (mm)	1.08 ± 0.06	1.03 ± 0.08	0.97 ± 0.03	1.00 ± 0.07
LV EDD (mm)	2.43 ± 0.08	2.35 ± 0.16	2.45 ± 0.06	2.48 ± 0.13
LV ESD (mm)	1.26 ± 0.06	1.53 ± 0.13*	1.16 ± 0.05	1.20 ± 0.15#
Fractional shortening (%)	47.9 ± 2.7	35.1 ± 2.1*	52.7 ± 1.9	52.0 ± 4.4#
Calculated LV mass (mg)	93.7 ± 4.1	84.0 ± 9.4	87.9 ± 3.4	86.4 ± 11.1
Plasma MIF levels (ng·ml−1)	3.20 ± 0.47	12.07 ± 0.97*	2.99 ± 0.41	13.83 ± 1.49*

Note. Mean ± SEM, n = 9–10 mice per group.

Abbreviations: EDD, end diastolic diameter; ESD, end systolic diameter; LV, left ventricular.

^* P < .05 versus WT group

^# P < .05 versus WT‐LPS group.

3.2. CD74 ablation nullified LPS‐induced cardiomyocyte contractile and intracellular Ca²⁺ defects

In line with echocardiographic findings, LPS significantly suppressed cardiomyocyte contractile function, as manifested by decreased peaking shortening (PS) and maximal velocity of shortening/relengthening (±dL/dt), as well as prolonged TR₉₀ with little changes in resting cell length and TPS. CD74 ablation nullified LPS‐induced cardiomyocyte anomalies without notable effect itself (Figure 1a–f). To further determine the possible mechanisms behind CD74 ablation protection against LPS‐induced cardiac defects, intracellular Ca²⁺ handling was evaluated using Fura‐2. Our data revealed that LPS challenge overtly decreased intracellular Ca²⁺ release in response to electrical stimuli (ΔFFI) and prolonged intracellular Ca²⁺ decay with unchanged resting intracellular Ca²⁺ (resting FFI). CD74 ablation blocked LPS‐induced changes in intracellular Ca²⁺ handling properties without notable effect on its own (Figure 1g–i).

Figure 1.

Effect of LPS challenge (4 mg·kg⁻¹, i.p., for 6 hr) on cardiomyocyte contractile and intracellular Ca²⁺ properties in WT and Cd74 ^−/− mice. (a) Resting cell length; (b) peak shortening (PS); (c) maximal velocity of shortening (+dL/dt); (d) maximal velocity of relengthening (−dL/dt); (e) time‐to‐PS (TPS); (f) time‐to‐90% relengthening (TR₉₀); (g) resting Fura‐2 fluorescence intensity (FFI); (h) electrically stimulated rise in FFI (ΔFFI); and (i) intracellular Ca²⁺ decay rate. Mean ± SEM, multiple cardiomyocytes obtained from individual mouse hearts were considered technical replicates, and the number of hearts studies is given in the boxes, *P < .05 between denoted groups

3.3. CD74 ameliorated LPS‐induced mitochondrial O₂⁻ production and ultrastructural changes

Neither LPS challenge nor CD74 ablation (or both) overtly affected cardiomyocyte cross‐sectional area (Figure 2a). LPS increased mitochondrial O₂ ⁻ production as evidenced by DHE staining and the effect was blocked by CD74 knockout, with little effect from CD74 ablation itself (Figure 2b,c). Transmission electronic microscopy was employed to evaluate the ultrastructure of sarcomere and mitochondria. Our data presented in Figure 2d depicted notable cytoarchitectural aberrations such as disrupted mitochondria, distortion of sarcomeres and myofilaments in LPS‐challenged myocardium. These responses were much less severe in LPS‐challenged Cd74 ^−/− mice, which also had little ultrastructural change from the CD74 knockout itself.

Figure 2.

Myocardial morphology, ultrastructure, and mitochondrial O₂ ⁻ production in from WT and Cd74 ^−/− mice with or without LPS challenge (4 mg·kg⁻¹, i.p., for 6 hr). (a) Lectin staining (400×); (b) mitochondrial O₂ ⁻ production using DHE staining; (c) pooled data of mitochondrial O₂ ⁻ levels; and (d) mitochondria and sarcomere ultrastructure using transmission electron microscopy. Mean ± SEM, multiple cardiomyocyte images obtained from individual mouse hearts were considered technical replicates, and the number of hearts studied is given in the boxes, *P < .05 between denoted groups

3.4. CD74 ablation ameliorated LPS‐induced inflammation and apoptosis

To examine the potential mechanism of action responsible for CD74 ablation blocking LPS‐induced myocardial injury, we evaluated levels of inflammatory and apoptotic markers. Our data suggested that LPS significantly up‐regulated levels of the proinflammatory https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1754, TNF‐α, TRAF6, HMGB1, MyD88, and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4974, as well as the pro‐apoptotic markers Bax and cleaved caspase‐3 (total caspase‐3 levels were not affected, data not shown), while down‐regulation of Bcl‐2 and all these effects were blocked in CD74 knockout mice with the exception of TLR4 (Figure 3). CD74 ablation overtly decreased CD74 receptor levels in both whole hearts and isolated cardiomyocytes (Figure S1), without affecting other proinflammatory and apoptotic protein makers. Levels of CD74 receptor were not affected by LPS challenge (Figures 3 and S1).

Figure 3.

Effect of LPS on CD74, proinflammatory and pro‐apoptotic protein markers in hearts from WT and Cd74 ^−/− mice challenged with or without LPS (4 mg·kg⁻¹, i.p., for 6 hr). (a and g) Representative gel blots depicting levels of CD74, TLR4, TNF‐α, TRAF6 and HMGB1 (GAPDH as loading control); (b) CD74 levels; (c) TLR4 levels; (d) TNF‐α levels; (e) TRAF6 levels; (f) HMGB1 levels. Mean ± SEM, mouse sample sizes are denoted in boxed numbers, *P < .05 between denoted groups

3.5. Effect of CD74 ablation on LPS‐induced myocardial autophagy response

As shown in Figure 4, our western blot analysis revealed that LPS treatment triggered a robust rise in autophagy response as evidenced by rises in autophagy protein markers including Beclin1, Atg5 and LC3BII‐to‐LC3BI ratio along with decreased p62 and the effect was attenuated by CD74 ablation. CD74 knockout itself did not affect autophagy.

Figure 4.

Autophagy markers in hearts from WT and Cd74 ^−/− mice treated with or without LPS (4 mg·kg⁻¹, i.p., for 6 hr). (a) Beclin1 levels; (b) Atg5 levels; (c) LC3B1 levels; (d) LC3BII levels; (e) LC3BII‐to‐LC3BI ratio; and (f) p62 levels. Insets: Representative gel blots depicting levels of the autophagy markers (GAPDH used as loading control). Mean ± SEM, mouse sample sizes are denoted in boxed numbers, *P < .05 between denoted groups

3.6. Effect of CD74 ablation on LPS‐induced autophagy regulatory molecules

To understand the autophagy regulatory machinery, levels of autophagy regulatory molecules including Akt, AMPK, mTOR and JNK were examined. Our results showed that LPS triggered an overt increase in the phosphorylation of Akt, AMPK and JNK, along with decreased phosphorylation of mTOR without affecting pan protein expression of these signalling molecules. Although CD74 ablation itself did not affect pan and phosphorylated levels of Akt, AMPK, mTOR and JNK, it prevented LPS‐induced hyperphosphorylation of AMPK and JNK, as well as dephosphorylation of mTOR, without affecting LPS‐induced Akt phosphorylation (Figure 5a–d). We further examined the levels of Skp2, SUV39H1/2 and H3K9 methylation of the Bcl2 protein BCLB, which are possible downstream regulators for AMPK. Our data is shown in Figure 5e–h revealed that LPS overtly down‐regulated the levels of Skp2 and up‐regulated SUV39H1 and BCLB H3K9 methylation without affecting that of https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=871#2716 and these effects were not observed after CD74 ablation.

Figure 5.

Autophagy regulatory and stress signalling molecules in hearts from WT and Cd74 ^−/− mice treated with or without LPS (4 mg·kg⁻¹, i.p., for 6 hr). (a) pAkt‐to‐Akt ratio; (b) p‐AMPK‐to‐AMPK ratio; (c) p‐mTOR‐to‐mTOR ratio; (d) pJNK‐to‐JNK ratio; (e) Skp2 levels; (f) SUV39H1 levels; (g) SUV39H2 levels; and (h) BCLB H3K9 levels. Insets: Representative blots depicting levels of the autophagy and stress signalling molecules (GAPDH used as loading control). Mean ± SEM, mouse sample sizes are denoted in boxed numbers, *P < .05 between denoted groups

3.7. Role of AMPK, autophagy, methylation and demethylation in CD74 ablation effects on LPS‐induced autophagy and mechanical dysregulation

In vitro study was then performed to examine the cell signalling mechanism in cardiomyocyte autophagy and contractile response upon LPS challenge in CD74 knockout mice. Neonatal murine cardiomyocytes from WT and Cd74 ^−/− mice were transfected with an adenovirus expressing GFP‐LC3 fusion protein for 24 hr prior to exposure of LPS (4 μg·ml⁻¹) for 6 hr in the presence or absence of the AMPK activator AICAR (500 μM; Nyblom et al., 2008), the autophagy inducer rapamycin (5 μM; Yuan et al., 2009), the methylation inhibitor 5‐AzaC (10 μM; Liu et al., 2017), the demethylation inhibitor DIF (0.5 μg·ml⁻¹; Ali & Amiri, 2018) or the SUV39H1 inhibitor chaetocin (50 nM; Yang et al., 2017). Data shown in Figure 6 that LPS stimulated autophagosome formation was abolished by CD74 ablation. Interestingly, stimulation of AMPK, autophagy and methylation using AICAR, rapamycin and DIF, respectively prevented CD74 ablation‐conferred protection against LPS, without eliciting any effect themselves. In addition, the SUV39H1 inhibitor chaetocin and the methylation inhibitor 5‐AzaC effectively blocked LPS‐induced autophagosome formation (in the presence or absence of CD74 ablation) without generating any effect themselves. These data indicate a likely key role for autophagosome formation in CD74 ablation‐induced protection against LPS‐mediated changes in autophagy. Our data shown in Figure 7 shows that LPS significantly increased O₂ ⁻ production, decreased peak shortening, ±dL/dt and prolonged TR₉₀ without affecting TPS in cardiomyocytes and these effects were prevented by CD74 ablation. However, the beneficial effect of CD74 ablation was prevented by activators of AMPK, autophagy and methylation, namely, AICAR, rapamycin and DIF. Our data further suggested that treatment of the SUV39H1 inhibitor chaetocin or the methylation inhibitor 5‐AzaC blocks LPS‐induced cardiomyocyte O₂ ⁻ production and contractile dysfunction (in the presence or absence of CD74 ablation). None of the pharmacological modulators alone exerted any notable effect.

Figure 6.

Role of AMPK, autophagy and methylation in CD74 ablation protection against LPS‐induced autophagosome formation. Neonatal mouse cardiomyocytes from WT and Cd74 ^−/− mice were first transfected with adenovirus for 24 hr to express the GFP‐LC3 fusion protein. Cells were then exposed to LPS (4 μg·ml⁻¹) for 6 hr in the absence or presence of the AMPK activator AICAR (500 μM), the autophagy inducer rapamycin (RAPA, 10 μM), the demethylation inhibitor DIF (0.5 μg·ml⁻¹), the SUV39H inhibitor chaetocin (Chae, 50 nM), or the methylation inhibitor 5‐AzaC (10 μM). (a–p) Representative images depicting GFP‐LC3 puncta in neonatal mouse cardiomyocytes; arrowheads denote GFP‐LC3 autophagosomes. (q) Pooled data depicting GFP‐LC3B punctate (autophagosomes). Mean ± SEM, n = 5 batches of neonatal hearts (each batch composed of 12 neonatal mice) per group, *P < .05 between denoted groups

Figure 7.

Role of AMPK, autophagy, and methylation in CD74 ablation protection against LPS‐induced cardiomyocyte superoxide production and contractile dysfunction. Mouse cardiomyocytes from adult WT and Cd74 ^−/− mice were exposed to LPS (4 μg·ml⁻¹) for 6 hr in the absence or presence of the AMPK activator AICAR (500 μM), the autophagy inducer rapamycin (10 μM), the demethylation inhibitor DIF (0.5 μg·ml⁻¹), the SUV39H inhibitor chaetocin (50 nM) or the methylation inhibitor 5‐AzaC (10 μM). (a) Representative DHE images depicting O₂ ⁻ fluorescence intensity in respective cardiomyocyte groups; (b) determination of DHE fluorescence intensity in cardiomyocytes from WT and Cd74 ^−/− mice with LPS treatment in the presence and absence of various pharmacological inhibitors using a fluorescent microplate reader; (c) peak shortening (PS); (d) maximal velocity of shortening (+dL/dt); (e) maximal velocity of relengthening (−dL/dt); (f) time‐to‐peak shortening (TPS); and (g) time‐to‐90% relengthening (TR₉₀). Mean ± SEM, n = 6 isolations (b) or 30 cells from six mice (c–g) per group, *P < .05 between denoted groups

4. DISCUSSION

The major observation from our study revealed that deficiency in the MIF receptor CD74 protects against endotoxemia‐induced cardiac injury, possibly through inhibition of AMPK‐Skp2‐SUV39H1‐mediated methylation of Bcl2 protein BCLB and excess autophagy, leading to improvements in apoptosis, inflammation, disturbed intracellular Ca²⁺ handling and contractile property. LPS challenge imposed echocardiographic and ultrastructural changes, cardiomyocyte contractile dysfunction and intracellular Ca²⁺ derangement. These effects were prevented by CD74 ablation. In vitro studies revealed that activation of AMPK, autophagyor methylation using AICAR, rapamycin or DIF, respectively, nullified CD74 ablation protection against LPS‐induced pathological effects. On the other hand, inhibition of SUV39H1 and methylation using chaetocin and 5‐AzaC, respectively, blocked LPS‐induced cardiac contractile dysfunction and autophagosome formation. Given the elevated plasma MIF levels upon LPS challenge, our data favoured a unique role for the MIF receptor CD74 in endotoxemia‐induced cardiac injury through AMPK‐SUV39H1‐BCLB methylation‐mediated maladaptive autophagy. Similar to findings from our earlier study (Pang, Peng, et al., 2019), our data demonstrated pronounced inflammatory, apoptotic and pro‐oxidant responses in LPS‐challenged hearts, possibly downstream responses to autophagy. These findings collectively suggested a possible therapeutic potential for CD74 being target in septic heart injury.

Pathological changes in the heart are evident in endotoxemia as manifested by overtly distorted ultrastructure, dampened contractile function and intracellular Ca²⁺ properties (Lew et al., 2013; Lew et al., 2014; Li et al., 2002; Ren, Ren, & Sharma, 2002; Yasuda & Lew, 1997; Zhang et al., 2014). Our data revealed that LPS challenge prompted reduced fractional shortening, enlarged LVESD, decreased cardiomyocyte contractile capacity, intracellular Ca²⁺ derangement, ultrastructural changes in myofilament and mitochondria with unchanged LVEDD and LV wall thickness. These functional anomalies with LPS challenge are in line with apoptosis (Bax, Bcl‐2 and cleaved caspase‐3), superoxide accumulation and inflammation in septic hearts. Prolonged diastolic duration and intracellular Ca²⁺ clearance indicate diastolic dysfunction in sepsis. Intriguingly, CD74 ablation nullified LPS‐induced ultrastructural, mechanical and intracellular Ca²⁺ anomalies. CD74 ablation‐elicited also improved intracellular Ca²⁺ handling and this may be associated with its ability to counter oxidative stress (e.g. intracellular O₂ ⁻, pro‐apoptotic Bax and cleaved caspase‐3 and anti‐apoptotic Bcl‐2). Of note, our earlier findings showed a beneficial role for anti‐inflammation intervention in septic cardiomyopathy (Ren et al., 2016; Zhang et al., 2014) and this is further supported by our current findings of improved proinflammatory profile in LPS‐challenged Cd74 ^−/− mice.

Our data showed that autophagy serves as a downstream target for LPS‐ and CD74 ablation‐induced contractile responses. This speculation (as depicted in Figure 8) also received support from O₂ ⁻ production and mechanical findings in Figure 7, where activation of AMPK, autophagy and methylation blocked the CD74 knockout benefit against LPS‐induced cardiomyocyte anomalies. It was reported that oxidative stress induces autophagy through JNK‐, Akt‐ or AMPK‐mediated pathways (Ren et al., 2016; Zhang, Sowers, & Ren, 2018). Our findings suggested a likely role for the Akt‐AMPK‐mTOR signalling cascade in bridging oxidative stress and autophagy in endotoxemia. Nonetheless, CD74 ablation reversed AMPK‐mTOR but not the Akt signalling cascade, suggesting that there is not a major role for Akt in CD74 ablation cardioprotection. Our results also showed that there was elevation of autophagy protein markers and GFP‐LC3 autophagosome formation upon LPS challenge, again these effects were inhibited by CD74 ablation. With LPS challenge, levels of p62 were lowered along with an elevation in the LC3BII‐to‐LC3BI ratio and autophagy protein markers, suggesting facilitated autophagosome and autolysosome formation. This is supported by the reversal by the AMPK inducer AICAR or the autophagy inducer rapamycin (via inhibition of mTOR) on CD74 ablation inhibition of LPS‐induced cardiomyocyte anomalies. Our data also suggested that Skp2, SUV39H1 and BCLB methylation may serve as downstream mediators for AMPK signalling. Skp2 was reported to regulate autophagy downstream of AMPK (Shin et al., 2016). SUV39H, a histone lysine methyltransferase and a hallmark for mammalian heterochromatin, was reported to participate in the regulation of autophagy in tumorigenesis, although little is known about this action in the heart (Dorr et al., 2013). More recent evidence from our laboratory demonstrated a role for Skp2 and SUV39H1 in the regulation of autophagy in alcoholic injury and in high fat diet‐induced obesity (Wang et al., 2018; Yang et al., 2019). In particular, our very recent study revealed a rather similar CD74‐AMPK‐Skp2‐mediated regulation of autophagy in alcoholic cardiomyopathy (Yang et al., 2019), which may be a major limitation of the current study. Perhaps the most intriguing data from our current study are the elucidation of how autophagy is dysregulated by sepsis. We noted up‐regulation of SUV39H1 and H3K9 methylation of the Bcl‐2 protein BCLB along with autophagy excess. Inhibition of SUV39H1 and methylation using chaetocin and 5‐AzaC drastically inhibited LPS‐induced autophagy excess, O₂ ⁻ production and contractile defects. These findings support an AMPK‐Skp2‐SUV39H1‐BCLB methylation axis in LPS‐induced dysregulation of autophagy, oxidative stress and contractile function. In addition, given the controversies on the paradoxical roles of the CD74 ligand MIF in disease progression and prevention (Das et al., 2013; Doernberg et al., 2011; Renner et al., 2012; Xu et al., 2016; Xu & Ren, 2015), it would be pertinent to explore the effect of CD74 ablation (and associated mechanisms) in different cardiac pathological settings. In addition to methylation, recent evidence also depicted a role for SUV39H in ischaemia–reperfusion injury by way of Sirt1 deacetylation (Yang et al., 2017).

Figure 8.

Scheme depicting proposed mechanism(s) of action involved in LPS‐ and CD74 ablation‐induced cardiac autophagy and contractile responses. LPS challenge promotes the activation of the proinflammatory factor MIF, which couples with its membrane receptor CD74, leading to activation of AMPK, suppression of mTOR signalling and Skp2 signalling. Down‐regulation of the F‐box content of the Skp1/Cullin/F‐box protein‐type ubiquitin ligase Skp2 disinhibits histone H3K9 methyltransferase SUV39H1, thus promoting BCLB H3K9 methylation and autophagy excess (likely due to disinhibition of Bcl‐2‐Beclin1 association). In consequence, LPS exposure prompts oxidative stress, inflammation, apoptosis, and contractile dysfunction in the heart. CD74 ablation counters oxidative stress (mitochondrial O₂ ⁻ accumulation), inflammation and excessive autophagy to preserve cardiac function. Arrowheads denote stimulatory effect whereas the “T” ending lines represent inhibition

The Bcl‐2 family proteins regulate various forms of cell death including apoptosis and autophagy. The BCLB gene encodes proteins containing conservative Bcl‐2 homology (BH) domain BH1–BH4 and carboxyl terminal domain. Earlier evidence has suggested that BCLB binds Bcl‐2 to promote its association with Beclin1, resulting in inhibition of autophagy (Robert et al., 2012). With LPS challenge, BCLB function is likely to be down‐regulated through hypermethylation thus causing disinhibition of Bcl‐2‐Beclin1‐associated autophagy. Our data has provided convincing evidence that methylation nullified, whereas demethylation mimicked CD74 ablation‐induced protection against LPS‐induced cardiac anomalies. BCLB has been reported previously to be frequently down regulated by promoter CpG methylation (Liu et al., 2017).

Our data suggested that CD74 ablation inhibits LPS‐induced proinflammatory responses without affecting plasma MIF levels and cardiac TLR4 levels. In our hands, LPS challenge increased levels of TNF‐α, TRAF6, HMGB1, MyD88, IL‐1β and JNK activation, and thees effects were prevented by CD74 ablation. LPS is known activate innate immune responses through TLR4 and then a MyD88‐dependent recruitment of IRAK‐TRAF6, which turns on JNK and the release of proinflammatory cytokines (interleukins and TNF‐α; Takeda & Akira, 2004). Our data suggested that CD74 ablation interferes with LPS‐induced injury possibly at a level of MyD88‐TRAF6 (not at the TLR4 level). The fact that AMPK activation, autophagy induction and methylation prevented CD74 ablation‐induced autophagosome formation and mechanical defect favours a role of these signalling pathways in CD74 ablation protection against septic cardiac injury.

In summary, data from our present study revealed that CD74 ablation rescues against endotoxemia‐induced cardiac injury through an AMPK‐Skp2‐SUV39H1‐BCLB methylation‐mediated autophagy regulation. These findings should shed some lights towards a better understanding of the proinflammatory cytokine MIF receptor CD74 in the pathogenesis and long‐term therapy of septic cardiomyopathy. Inhibition of CD74 is expected to protect against LPS‐induced cardiac anomalies through suppression of AMPK‐Skp2‐SUV39H1‐mediated BCLB methylation, autophagy and oxidative stress, ultimately rescuing septic myocardial contractile dysfunction (Figure 8). These data support a role for the CD74 receptor as a therapeutic target for endotoxemia‐induced cardiac anomalies. Targeting CD74 should offer promises in the therapeutics of sepsis complications in particular in those patients with CD74 mutation although further clinical scrutiny is needed.

AUTHOR CONTRIBUTIONS

Y.L., C.F., M.Y., M.D., and Y.Z. performed the study. Y.Z., Z.P., and J.R., conceived and designed the study and drafted and proved the manuscript. R.B., provided Cd74 ^−/− mice and edited the manuscript.

CONFLICT OF INTEREST

Yale University hold intellectual property rights for the therapeutic augmentation of MIF signalling in cardiac tissue protection. R.B. is a co‐inventor on this patent and a co‐founder of MIFCOR, Inc., which seeks to augment MIF pathways for ischaemic tissue injury. R.B. receives research support from MIFCOR, Inc.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14207, https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14208, and https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14206, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Figure S1: Levels of CD74 in isolated cardiomycocytes from WT and Cd74 ^‐/‐ mice treated with or without LPS (4mg/kg, i.p., for 6 hrs).

Luo Y, Fan C, Yang M, et al. CD74 knockout protects against LPS‐induced myocardial contractile dysfunction through AMPK‐Skp2‐SUV39H1‐mediated demethylation of BCLB. Br J Pharmacol. 2020;177:1881–1897. 10.1111/bph.14959