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Aims: Systemic inflammation occurs commonly during many human disease settings and increases vascular permeability, leading to organ failure, and lethal outcomes. Lipocalin 10 (Lcn10), a poorly characterized member of the lipocalin family, is remarkably altered in the cardiovascular system of human patients with inflammatory conditions. Nonetheless, whether Lcn10 regulates inflammation-induced endothelial permeability remains unknown., Methods and Results: Systemic inflammation models were induced using mice by injection of endotoxin lipopolysaccharide (LPS) or caecal ligation and puncture (CLP) surgery. We observed that the expression of Lcn10 was dynamically altered only in endothelial cells (ECs), but not in either fibroblasts or cardiomyocytes isolated from mouse hearts following the LPS challenge or CLP surgery. Using in vitro gain- and loss-of-function approaches and an in vivo global knockout mouse model, we discovered that Lcn10 negatively regulated endothelial permeability upon inflammatory stimuli. Loss of Lcn10 augmented vascular leakage, leading to severe organ damage and higher mortality following LPS challenge, compared to wild-type controls. By contrast, overexpression of Lcn10 in ECs displayed opposite effects. A mechanistic analysis revealed that both endogenous and exogenous elevation of Lcn10 in ECs could activate slingshot homologue 1 (Ssh1)-Cofilin signalling cascade, a key axis known to control actin filament dynamics. Accordingly, a reduced formation of stress fibre and increased generation of cortical actin band were exhibited in Lcn10-ECs, when compared to controls upon endotoxin insults. Furthermore, we identified that Lcn10 interacted with LDL receptor-related protein 2 (LRP2) in ECs, which acted as an upstream factor of the Ssh1-Confilin signalling. Finally, injection of recombinant Lcn10 protein into endotoxic mice showed therapeutic effects against inflammation-induced vascular leakage., Conclusion: This study identifies Lcn10 as a novel regulator of EC function and illustrates a new link in the Lcn10-LRP2-Ssh1 axis to controlling endothelial barrier integrity. Our findings may provide novel strategies for the treatment of inflammation-related diseases., Competing Interests: Conflict of interest: Dr. Sadayappan provides consulting and collaborative research studies to the Leducq Foundation (CURE-PLAN), Red Saree Inc., Greater Cincinnati Tamil Sangam, Novo Nordisk, Pfizer, AavantioBio, AstraZeneca, MyoKardia, Merck, and Amgen, but such work is unrelated to the content of this article., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aims: Cardiac dysfunction is a prevalent comorbidity of disrupted inflammatory homeostasis observed in conditions such as sepsis (acute) or obesity (chronic). Secreted and transmembrane protein 1a (Sectm1a) has previously been implicated to regulate inflammatory responses, yet its role in inflammation-associated cardiac dysfunction is virtually unknown., Methods and Results: Using the CRISPR/Cas9 system, we generated a global Sectm1a-knockout (KO) mouse model and observed significantly increased mortality and cardiac injury after lipopolysaccharide (LPS) injection, when compared with wild-type (WT) control. Further analysis revealed significantly increased accumulation of inflammatory macrophages in hearts of LPS-treated KO mice. Accordingly, ablation of Sectm1a remarkably increased inflammatory cytokines levels both in vitro [from bone marrow-derived macrophages (BMDMs)] and in vivo (in serum and myocardium) after LPS challenge. RNA-sequencing results and bioinformatics analyses showed that the most significantly down-regulated genes in KO-BMDMs were modulated by LXRα, a nuclear receptor with robust anti-inflammatory activity in macrophages. Indeed, we identified that the nuclear translocation of LXRα was disrupted in KO-BMDMs when treated with GW3965 (LXR agonist), resulting in higher levels of inflammatory cytokines, compared to GW3965-treated WT-cells. Furthermore, using chronic inflammation model of high-fat diet (HFD) feeding, we observed that infiltration of inflammatory monocytes/macrophages into KO-hearts were greatly increased and accordingly, worsened cardiac function, compared to WT-HFD controls., Conclusion: This study defines Sectm1a as a new regulator of inflammatory-induced cardiac dysfunction through modulation of LXRα signalling in macrophages. Our data suggest that augmenting Sectm1a activity may be a potential therapeutic approach to resolve inflammation and associated cardiac dysfunction., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Aims: Doxorubicin causes damage to the heart, which may present as cardiomyopathy. However, the mechanisms by which doxorubicin induces cardiotoxicity remain not fully understood and no effective prevention for doxorubicin cardiomyopathy is available. Calpains, a family of calcium-dependent thiol-proteases, have been implicated in cardiovascular diseases. Their activities are tightly controlled by calpastatin. This study employed transgenic mice over-expressing calpastatin to investigate the role of calpain in doxorubicin-induced cardiotoxicity., Methods and Results: Doxorubicin treatment decreased calpain activities in cultured neonatal mouse cardiomyocytes and in vivo mouse hearts, which correlated with down-regulation of calpain-1 and calpain-2 proteins. Over-expression of calpastatin or incubation with pharmacological calpain inhibitors enhanced apoptosis in neonatal and adult cardiomyocytes induced by doxorubicin. In contrast, over-expression of calpain-2 but not calpain-1 attenuated doxorubicin-induced apoptosis in cardiomyocytes. The pro-apoptotic effects of calpain inhibition were associated with down-regulation of protein kinase B (AKT) protein and mRNA expression, and a concomitant reduction in glycogen synthase kinase-3beta (GSK-3β) phosphorylation (Ser9) in doxorubicin-treated cardiomyocytes. Blocking AKT further increased doxorubicin-induced cardiac injuries, suggesting the effects of calpain inhibition may be mediated by inactivating the AKT signalling. In an in vivo model of doxorubicin-induced cardiotoxicity, over-expression of calpastatin exacerbated myocardial dysfunction as assessed by echocardiography and haemodynamic measurement in transgenic mice 5 days after doxorubicin injection. The 5-day mortality was higher in transgenic mice (29.16%) compared with their wild-type littermates (8%) after doxorubicin treatment., Conclusion: Over-expression of calpastatin enhances doxorubicin-induced cardiac injuries through calpain inhibition and thus, calpains may protect cardiomyocytes against doxorubicin-induced cardiotoxicity.

Aims: Doxorubicin causes damage to the heart, often leading to irreversible cardiomyopathy, which is fatal. Reactive oxygen species (ROS) or oxidative stress is involved in cardiomyocyte death, contributing to doxorubicin-induced cardiotoxicity. This study investigated the role of Rac1, an important subunit of NADPH oxidase, in doxorubicin-induced cardiotoxicity and the underlying mechanisms., Methods and Results: In a mouse model of acute doxorubicin-induced cardiotoxicity, cardiomyocyte-specific deletion of Rac1 inhibited NADPH oxidase activation and ROS production, prevented cardiac cell death, and improved myocardial function in Rac1 knockout mice. Therapeutic administration of the specific Rac1 inhibitor NSC23766 achieved similar cardio-protective effects in doxorubicin-stimulated mice. In rat cardiomyoblasts (H9c2 cells) and cultured neonatal mouse cardiomyocytes, Rac1 inhibition attenuated apoptosis as evidenced by decreases in caspase-3 activity and DNA fragmentation in response to doxorubicin, which correlated with a reduction in ROS production and down-regulation of p53 acetylation and histone H2AX phosphorylation. In contrast, overexpression of Rac1 enhanced apoptosis. Doxorubicin also inhibited the activity of classical histone deacetylases (HDAC), which was preserved by Rac1 inhibition and further decreased by Rac1 overexpression. Interestingly, scavenging ROS mitigated apoptosis but did not change HDAC activity and p53 acetylation stimulated by doxorubicin, suggesting both ROS-dependent and -independent pathways are involved in Rac1-mediated cardiotoxicity. Furthermore, the HDAC inhibitor trichostatin A enhanced apoptosis, p53 acetylation and H2AX phosphorylation in doxorubicin-treated cardiomyocytes., Conclusions: Rac1 signalling contributes to doxorubicin-induced cardiotoxicity through both a ROS-dependent mechanism and ROS-independent HDAC/p53 signalling in cardiomyocytes. Thus, inhibition of Rac1 may be a useful therapy for doxorubicin-induced cardiotoxicity.

Aims: Free fatty acids induce apoptosis in cardiomyocytes, which is implicated in lipotoxic cardiomyopathy. However, the underlying mechanisms remain not fully understood. MicroRNAs (miRNAs) are non-coding small RNAs that control gene expression at the post-transcriptional level. Dysregulated miRNAs have been shown to be involved in heart diseases. This study was to examine whether miR-195 regulates palmitate-induced cardiomyocyte apoptosis by targeting Sirt1, a known anti-apoptotic protein., Methods and Results: In cultured neonatal mouse cardiomyocytes, palmitate up-regulated miR-195 expression, increased reactive oxygen species (ROS) production, and induced apoptosis as determined by up-regulation of caspase-3 activity and DNA fragmentation. Inhibition of miR-195 decreased ROS production and apoptosis in palmitate-stimulated cardiomyocytes. In contrast, a miR-195 mimic enhanced palmitate-induced ROS production and apoptosis. The induction of miR-195 correlated with a reduction in Sirt1 and Bcl-2. We further showed that miR-195 targeted and inhibited Sirt1 expression through two target sites located in the 3' un-translational region of Sirt1 mRNA. In concordance, inhibition of miR-195 increased Sirt1 protein in cardiomyocytes whereas the miR-195 mimic reduced it. Activation of Sirt1 or overexpression of Bcl-2 inhibited palmitate-induced apoptosis. On the other hand, inhibition of Sirt1 enhanced apoptosis. The inhibitory effect of Sirt1 on apoptosis was associated with a reduction in ROS., Conclusions: This study demonstrates a pro-apoptotic role of miR-195 in cardiomyocytes and identifies Sirt1 as a direct target of miR-195. The effect of miR-195 on apoptosis is mediated through down-regulation of Sirt1 and Bcl-2 and ROS production. Thus, miR-195 may be a new therapeutic target for lipotoxic cardiomyopathy.

Aims: Cardiomyocyte apoptosis contributes to cardiac complications of diabetes. The aim of this study was to investigate the role of calpain in cardiomyocyte apoptosis induced by hyperglycaemia., Methods and Results: In cultured adult rat ventricular cardiomyocytes, high glucose (33 mM) increased calpain activity and induced apoptosis, concomitant with the impairment of Na+/K+ ATPase activity. These effects of high glucose on cardiomyocytes were abolished by various pharmacological calpain inhibitors, knockdown of calpain-1 but not calpain-2 using siRNA, or over-expression of calpastatin, a specific endogenous calpain inhibitor. The effect of calpain inhibition on cardiomyocyte apoptosis was abrogated by ouabain, a selective inhibitor of Na+/K+ ATPase. Furthermore, blocking gp91(phox)-NADPH oxidase activation, L-type calcium channels, or ryanodine receptors prevented calpain activation and apoptosis in high glucose-stimulated cardiomyocytes. In a mouse model of streptozotocin-induced diabetes, administration of different calpain inhibitors blocked calpain activation, increased the Na+/K+ ATPase activity, and decreased apoptosis in the heart., Conclusion: Calpain-1 activation induces apoptosis through down-regulation of the Na+/K+ ATPase activity in high glucose-stimulated cardiomyocytes and in vivo hyperglycaemic hearts. High glucose-induced calpain-1 activation is mediated through the NADPH oxidase-dependent pathway and associated with activation of L-type calcium channels and ryanodine receptors. Our data suggest that calpain activation may be important in the development of diabetic cardiomyopathy and thus may represent a potential therapeutic target for diabetic heart diseases.

Aims: Lipopolysaccharide (LPS) induces cardiomyocyte caspase-3 activation and proinflammatory factors, in particular tumour necrosis factor-alpha (TNF-alpha) production, both of which contribute to myocardial dysfunction during sepsis. The present study was to investigate the roles of calpain/calpastatin system in cardiomyocyte caspase-3 activation, TNF-alpha expression, and myocardial dysfunction during LPS stimulation., Methods and Results: In cultured adult rat cardiomyocytes, LPS (1 microg/mL) induced calpain and caspase-3 activity, and up-regulated TNF-alpha expression. These effects of LPS were abrogated by over-expression of calpastatin, an endogenous calpain inhibitor, transfection of calpain-1 siRNA, or various pharmacological calpain inhibitors. Furthermore, blocking gp91(phox)-NADPH oxidase prevented calpain and caspase-3 activation and decreased TNF-alpha expression in LPS-stimulated cardiomyocytes. To investigate the role of calpastatin in endotoxaemia, transgenic mice with calpastatin over-expression (CAST-Tg) and wild-type mice were treated with LPS (4 mg/kg, i.p.) or saline in the presence of calpain inhibitor-III (10 mg/kg, i.p.) for 4 h, and their heart function was measured with a Langendorff system. Over-expression of calpastatin significantly attenuated myocardial dysfunction (P < 0.05). Consistently, calpain activity, caspase-3 activity, and TNF-alpha expression were also reduced in CAST-Tg and calpain inhibitor-III compared with wild-type and vehicle-treated hearts, respectively., Conclusion: gp91(phox)-NADPH oxidase-mediated calpain-1 activation induces caspase-3 activation and TNF-alpha expression in cardiomyocytes during LPS stimulation. Over-expression of calpastatin inhibits calpain activation and improves myocardial function in endotoxaemia. The present study suggests that targeting calpain/calpastatin system may be a potential therapeutic intervention for septic hearts.

Aims: Myocardial tumour necrosis factor-alpha (TNF-alpha) production plays an important role in cardiac dysfunction during sepsis. The aim of this study was to investigate the role of c-Jun NH2-terminal kinases (JNK) signalling in cardiomyocyte TNF-alpha expression during lipopolysaccharide (LPS) stimulation and myocardial function in endotoxaemic mice., Methods and Results: In cultured neonatal mouse cardiomyocytes, deficiency of JNK1 or selective inhibition of JNK1 signalling by over-expression of a dominant negative mutant of JNK1 enhanced LPS-induced TNF-alpha expression, which was associated with elevations in phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK). At the organ level, LPS-induced TNF-alpha expression was significantly increased in JNK1(-/-) compared with wild-type hearts. JNK1 activation by LPS also induced immediate c-fos expression in cardiomyocytes, which was blocked by inhibition of JNK1 signalling. The role of c-fos expression in LPS-induced TNF-alpha expression was investigated in both cultured c-fos(-/-) cardiomyocytes and isolated c-fos(-/-) hearts. Deficiency of c-fos significantly enhanced LPS-induced TNF-alpha expression in cardiomyocytes and isolated hearts. Over-expression of c-fos decreased TNF-alpha expression in LPS-stimulated cardiomyocytes, which was associated with a decrease in phosphorylation of ERK1/2 and p38. In mice with endotoxaemia, deficiency of either JNK1 or c-fos further decreased cardiac function compared with corresponding wild-type controls., Conclusion: JNK1/c-fos inhibits ERK1/2 and p38 MAPK signalling, leading to decreased cardiomyocyte TNF-alpha expression and improvements in cardiac function during endotoxaemia.

Aims: Lipopolysaccharide (LPS) induces tumor necrosis factor-alpha (TNF-alpha) expression in cardiomyocytes, which contributes to myocardial dysfunction during sepsis. The purpose of this study was to investigate the role of phosphatidylinositol (PI) phospholipase Cgamma1 (PLCgamma1) in cardiac TNF-alpha expression, and myocardial dysfunction during endotoxemia., Methods and Results: In cultured mouse neonatal cardiomyocytes, LPS increased PLCgamma1 phosphorylation. Knockdown of PLCgamma1 with specific siRNA or inhibition of PLCgamma1 with U73122 attenuated TNF-alpha expression induced by LPS. This action of PLCgamma1 was mediated through inositol-1,4,5-trisphosphate (IP3)/IP3 receptor (IP3R) pathways since blocking either IP3 or IP3R decreased LPS-induced TNF-alpha expression. In contrast, neither diacylglycerol agonist nor antagonist had any evident effect on LPS-induced TNF-alpha expression in cardiomyocytes. To investigate the role of PLCgamma1 in endotoxemia in vivo, wild-type and heterozygous PLCgamma1 knockout (PLCgamma1(+/-)) mice were pre-treated with either U73122, or its inactive analog U73343, or vehicle for 15 min, followed by LPS for 4 h. Inhibition of PLCgamma1 by U73122 or by heterozygous deletion of the PLCgamma1 gene decreased cardiac TNF-alpha expression. More importantly, LPS-induced myocardial dysfunction was also attenuated in PLCgamma1(+/-) mice or by U73122 treatment., Conclusion: PLCgamma1 signalling induces cardiac TNF-alpha expression and myocardial dysfunction during LPS stimulation. The action of PLCgamma1 on TNF-alpha expression is mediated through IP3/IP3R pathways. The present results suggest that PLCgamma1 may be a potential therapeutic target for myocardial dysfunction in sepsis.

Objective: Neuronal nitric oxide synthase (nNOS) has been shown to regulate intracellular calcium in cardiomyocytes. Calcium in turn modulates extracellular signal-related kinase (ERK) signaling, which is important in tumor necrosis factor-alpha (TNF-alpha) expression during lipopolysaccharide (LPS) stimulation. However, the role of nNOS in LPS-induced TNF-alpha expression is not known. We hypothesized that nNOS suppresses LPS-induced TNF-alpha expression by inhibiting the calcium/ERK signaling pathway., Methods and Results: Cultured neonatal mouse cardiomyocytes were challenged with LPS for 4 h. While there was no change in the basal Ca(2+) concentration, LPS increased peak Ca(2+) levels. LPS stimulation increased TNF-alpha mRNA and protein levels in wild-type cells however, the responses were enhanced in nNOS(-/-) cardiomyocytes. Treatment with an antisense oligonucleotide against nNOS also significantly enhanced TNF-alpha expression during LPS stimulation. Furthermore, LPS-induced ERK phosphorylation was significantly increased in the nNOS(-/-) compared to wild-type cardiomyocytes. The enhanced TNF-alpha expression in nNOS(-/-) cardiomyocytes was abrogated by an L-type calcium channel blocker verapamil or ERK1 siRNA. Finally, myocardial ERK phosphorylation and TNF-alpha expression were increased while cardiac function was decreased in endotoxemia in nNOS(-/-) compared to wild-type mice., Conclusions: nNOS inhibits LPS-induced TNF-alpha expression in cardiomyocytes and improves cardiac function in endotoxemia. The inhibitory role of nNOS is mediated by a reduction in L-type calcium channel-dependent ERK signaling in cardiomyocytes.

Objective: Erythropoietin (EPO) prevents the myocardial dysfunction induced by ischemia/reperfusion (I/R). Since I/R-induced myocardial dysfunction is associated with an acute inflammatory response, we assessed the anti-inflammatory properties of EPO using in vitro and in vivo models of I/R., Methods: Isolated cardiac myocytes were exposed to anoxia/reoxygenation (A/R; the in vitro counterpart to I/R). Hearts were challenged with I/R in situ., Results: In vitro, A/R increased myocyte oxidant stress and converted the myocytes to a proinflammatory phenotype (these myocytes induced PMN transendothelial migration). Pretreatment of the myocytes with EPO prevented the A/R-induced proinflammatory effects. EPO increased myocyte (1) nuclear translocation of AP-1 (c-fos/c-jun), (2) eNOS, but not iNOS, protein expression, and (3) NO production. An AP-1 "decoy" oligonucleotide prevented the induction of eNOS by EPO and reversed the beneficial effect of EPO. An inhibitor of phosphatidylinostol 3 (PI3)-kinase prevented the nuclear translocation of AP-1 induced by EPO. In vivo, in wild type mice, I/R induced an increase in myocardial MPO activity (indicative of PMN infiltration); an effect prevented by pretreatment of the mice with EPO. This anti-inflammatory effect of EPO was not observed in cardiac specific c-fos(-/-) mice., Conclusions: Collectively, these findings indicate that EPO can ameliorate the myocardial inflammatory response in both in vitro and in vivo models of I/R. This beneficial effect of EPO is mediated by eNOS-derived NO via a PI3-kinase-dependent activation of AP-1.

Objectives: The role of p38 mitogen-activated protein kinase (MAPK) activation in lipopolysaccharide (LPS)-induced myocardial dysfunction has not been clearly defined. Our aim was to investigate the contribution of p38 MAPK in myocardial tumor necrosis factor-alpha (TNF-alpha) expression, cardiac function and survival during acute endotoxemia in mice., Methods: Acute endotoxemia was induced by LPS (10 mg/kg, i.p.) in mice. Two hours after LPS treatment, left ventricular (LV) function was assessed. Phosphorylation of p38 MAPK was measured by Western blotting. TNF-alpha mRNA and protein levels were determined by semi-quantitative reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay, respectively., Results: LPS rapidly increased phosphorylation of p38 MAPK, followed by TNF-alpha mRNA expression and protein expression in the LV myocardium. Pre-treatment of the p38 MAPK inhibitor SB202190 (2 mg/kg, i.p.) decreased TNF-alpha mRNA and protein by 65 and 36%, respectively (P<0.05). Immunohistochemical staining confirmed that cardiomyocytes were the major source of TNF-alpha production in the myocardium and blocking p38 MAPK activation inhibited TNF-alpha expression in response to LPS. Pre-treatment of SB202190 or a TNF-alpha antagonist etanercept (2 mg/kg, i.p) significantly reversed LPS-induced LV depression (P<0.05). LPS (20 mg/kg, i.p.) induced 94% mortality in mice within 72 h and pre-treatment with SB202190 and etanercept decreased LPS-induced mortality to 65 and 40%, respectively (P<0.01)., Conclusion: p38 MAPK activation represents an important mechanism leading to myocardial TNF-alpha production and cardiac dysfunction during acute endotoxemia in mice. Our data suggest that p38 MAPK is a potential therapeutic target of endotoxemia.