Myocardial Dysfunction In Mice Research Articles

Gossypol improves myocardial dysfunction caused by sepsis by regulating histone acetylation.

Gossypol is a polyphenol from the cotton plant with anti-inflammatory and anti-oxidation activities and can also function as a histone deacetylase (HDAC) inhibitor. Sepsis is an inflammatory disease with high mortality. Inflammation, oxidative stress, and epigenetic factors are involved in sepsis and its complications. The biological activities of gossypol strongly suggest the potential effects of gossypol on sepsis. In the present study, the beneficial effects of gossypol on sepsis were evaluated. We established a cecal ligation and puncture (CLP) mouse model of sepsis and treated CLP mice with gossypol. The survival rate, serum level of myocardial injury markers, and myocardial level of oxidation markers were measured. We also administered gossypol to lipopolysaccharide (LPS)-treated primary cardiomyocytes. The production of pro-inflammatory cytokines, activation of protein kinase B (AKT) and IκB kinase (IKK), acetylation of histone, and expression of HDACs were measured. Gossypol prevented the death of CLP mice and ameliorated myocardial damage in CLP mice. Moreover, gossypol decreased oxidative factors, while promoting antioxidant production in CLP mice. Gossypol prevented LPS and cytosine-phosphate-guanosine-induced expression of pro-inflammatory cytokines, suppressed LPS-induced activation of AKT and IKK, inhibited histone acetylation, and decreased the expression of HDACs. In conclusion, gossypol ameliorates myocardial dysfunction in mice with sepsis.

Integration of transcriptomics, metabolomics, and lipidomics reveals the mechanisms of doxorubicin-induced inflammatory responses and myocardial dysfunction in mice

Doxorubicin (DOX) is an anthracycline antineoplastic agent that has limited clinical utility due to its dose-dependent cardiotoxicity. Although the exact mechanism remains unknown, inflammatory responses have been implicated in DOX-induced cardiotoxicity (DIC). In this study, we analyzed the transcriptomic, metabolomic as well as lipidomic changes in the DOX-treated mice to explore the underlying mechanisms of DIC. We found that continuous intraperitoneal DOX injections (3 mg/kg/d) for a period of five days significantly induced cardiac dysfunction and cardiac injury in male C57BL/6 J mice (8 weeks old). This corresponded to a significant increase in the myocardial levels of IL-4, IL-6, IL-10, IL-17 and IL-12p70. Furthermore, inflammation-related genes such as Ptgs2, Il1b, Cxcl5, Cxcl1, Cxcl2, Mmp3, Ccl2, Ccl12, Nfkbia, Fos, Mapk11 and Tnf were differentially expressed in the DOX-treated group, and enriched in the IL-17 and TNF signaling pathways. Besides, amino acids, peptides, imidazoles, toluenes, hybrid peptides, fatty acids and lipids such as Hex1Cer, Cer, SM, PG and ACCa were significantly associated with the expression pattern of inflammation-related genes. In conclusion, the integration of transcriptomic, metabolomic and lipidomic data identified potential new targets and biomarkers of DIC.

N6-methyladenosine of Spi2a attenuates inflammation and sepsis-associated myocardial dysfunction in mice

Bacteria-triggered sepsis is characterized by systemic, uncontrolled inflammation in affected individuals. Controlling the excessive production of pro-inflammatory cytokines and subsequent organ dysfunction in sepsis remains challenging. Here, we demonstrate that Spi2a upregulation in lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages reduces the production of pro-inflammatory cytokines and myocardial impairment. In addition, exposure to LPS upregulates the lysine acetyltransferase, KAT2B, to promote METTL14 protein stability through acetylation at K398, leading to the increased m6A methylation of Spi2a in macrophages. m6A-methylated Spi2a directly binds to IKKβ to impair IKK complex formation and inactivate the NF-κB pathway. The loss of m6A methylation in macrophages aggravates cytokine production and myocardial damage in mice under septic conditions, whereas forced expression of Spi2a reverses this phenotype. In septic patients, the mRNA expression levels of the human orthologue SERPINA3 negatively correlates with those of the cytokines, TNF, IL-6, IL-1β and IFNγ. Altogether, these findings suggest that m6A methylation of Spi2a negatively regulates macrophage activation in the context of sepsis.

Exercise training suppresses Mst1 activation and attenuates myocardial dysfunction in mice with type 1 diabetes.

Our study was to test the effects of aerobic exercise on myocardial function in mice with type 1 diabetes and investigate the underlying mechanism associated with mammalian sterile 20-like kinase 1 (Mst1). Wild-type mice and Mst1(-/-) mice were injected with streptozotocin to induce diabetes and given moderate-intensity exercise for 12 weeks. Phosphorylation of Mst1 was significantly enhanced in the left ventricles of diabetic mice, which was reversed by exercise training. Exercise training or Mst1 deficiency improved myocardial function and reduced myocardial fibrosis in diabetic mice. Exercise training or Mst1 deficiency reduced TUNEL-positive cells and caspase-3 activity in the myocardium of diabetic mice. Exercise training or Mst1 deficiency abated oxidative stress and reduced mitochondrial reactive oxygen species formation, attenuated mitochondrial swelling, and enhanced mitochondrial adenosine triphosphate formation and mitochondrial membrane potential in the myocardium of diabetic mice. Exercise training or Mst1 deficiency suppressed inflammation in the myocardium of diabetic mice. Furthermore, exercise training did not provide further protection in Mst1 knockout mice in diabetes. In conclusion, chronic exercise training attenuated myocardial dysfunction in mice with type 1 diabetes, at least in part, through suppressing Mst1 activation.

MiR-214-3p Attenuates Sepsis-Induced Myocardial Dysfunction in Mice by Inhibiting Autophagy through PTEN/AKT/mTOR Pathway

Aims. More than half of the patients with sepsis would develop cardiac dysfunction, which is termed as sepsis-induced myocardial dysfunction (SIMD). Previous studies suggest that autophagy may play an important role in SIMD. The present study investigated whether miR-214-3p could attenuate SIMD by inhibiting autophagy. Main Methods. In this article, we investigated the role of autophagy in a mouse model of cecal ligation and puncture (CLP). The structure and function of hearts harvested from the mice were evaluated. Myocardial autophagy levels were detected with immunohistochemical, immunofluorescent, and Western blot. Key Findings. miR-214-3p can alleviate SIMD in septic mice by inhibiting the level of cardiac autophagy to attenuate myocardial dysfunction. Moreover, this study showed that miR-214-3p inhibited autophagy by silencing PTEN expression in the myocardial tissues of septic mice. Significance. This study showed that miR-214-3p attenuated SIMD through myocardial autophagy inhibition by silencing PTEN expression and activating the AKT/mTOR pathway. The present findings supported that miR-214-3p may be a potential therapeutic target for SIMD.

Late‐In‐Life Treadmill‐Training Improves Autophagy, Protein Clearance, and Contractile Function in Mouse Hearts

Evidence exists that a progressive decline of the cellular quality control mechanism called macroautophagy occurs during the process of primary aging. This leads to the accumulation of protein aggregates (i.e., proteotoxicity) that would otherwise be transported to and degraded in the lysosome. Post‐mitotic cells like cardiomyocytes rely heavily on macroautophagy because of their inability to clear defective proteins via cell division. While voluntary wheel running improves cardiac autophagy, lessens cardiac proteotoxicity, and attenuates myocardial dysfunction in mice with desmin‐related cardiomyopathy (Bhuiyan et al., JCI, 2013), the ability for a physiological intervention to improve these indexes in aged mice is unknown. We hypothesized that late‐in‐life exercise training improves autophagy, protein aggregate clearance, and function that is otherwise dysregulated in hearts from old vs adult mice. As expected, 24‐month old male C57Bl6 mice (old) exhibited repressed autophagosome formation in the heart, myocardial protein aggregate accumulation, systolic and diastolic dysfunction, and reduced exercise capacity vs. 6‐month old (adult) mice (all p< 0.05; n=10 per group) Separate cohorts of 21 month old mice completed a 10‐week progressive resistance / duration treadmill‐running program (old‐ETR) that improved (all p<0.05): (i) body composition; (ii) maximal workload capacity; and (iii) soleus muscle citrate synthase activity vs. age‐matched sedentary mice (old‐SED). Of note, (iv) mRNA and protein expression of autophagy markers indicated trafficking of the autophagosome to the lysosome increased, (v) protein aggregate clearance improved, and (vi) contractile function was enhanced (all p<0.05), in hearts from old‐ETR vs. old‐SED mice. Dietary maneuvers (e.g., caloric restriction, nutraceutical supplementation) and pharmacological interventions (e.g., rapamycin) are reported to elevate myocardial autophagy and mitigate / reverse age‐associated cardiac dysfunction. Here we show the first evidence that a physiological intervention i.e., late‐in‐life exercise training, improves autophagic flux, protein aggregate clearance, and contractile function in hearts from aged mice.Support or Funding InformationKL [APS STRIDE, UU Undergraduate Research Program (UROP)], JMC (UU Research Fellowship), SKP (AHA 17POST33670663), CR (APS UGRF, UROP), LT (UROP), SB (NIDDK R01‐DK‐098646‐01A1, R01‐DK‐099110, AHA 16GRNT30990018), JDS (AHA16GRNT31050004, NIH RO3AGO52848, NIHRO1HL141540).

Empagliflozin improves chronic hypercortisolism-induced abnormal myocardial structure and cardiac function in mice.

Background:Chronic exposure to excess glucocorticoids is frequently associated with a specific cardiomyopathy. Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, has beneficial effects as it aids in the reduction of heart failure and cardiovascular mortality in hospitalized patients. The aim of this study was to investigate the effects of empagliflozin on chronic hypercortisolism-induced myocardial fibrosis and myocardial dysfunction in mice.Methods:Male C57BL/6J mice (6 weeks old) were randomized to control, corticosterone (CORT), and empagliflozin + CORT groups. After 4 weeks of administration, heart structure and function were evaluated by echocardiography, and peripheral blood and tissue samples were collected. Expressions of Ccl2, Itgax, Mrc1, and Adgre1 mRNA in heart tissue were evaluated by RT-PCR, and signal transducer and activator of transcription 3 (STAT3) and Toll-like receptor 4 (TLR4) protein expression were analyzed by Western blotting.Results:Empagliflozin effectively reduced body weight, liver triglyceride, visceral adipose volume, and uric acid in CORT-treated mice. Left ventricular hypertrophy and cardiac dysfunction were improved significantly, phosphorylated STAT3 and TLR4 were alleviated, and macrophage infiltration in the myocardium was inhibited after administration of empagliflozin in CORT-treated mice.Conclusion:Empagliflozin has beneficial effects on specific cardiomyopathy associated with CORT, and the results provide new evidence that empagliflozin might be a potential drug for the prevention of this disease.

Blueberry anthocyanin‑enriched extract ameliorates transverse aortic constriction‑induced myocardial dysfunction via the DDAH1/ADMA/NO signaling pathway in mice.

Blueberry anthocyanin‑enriched extract (BAE) has been demonstrated to protect against cardiovascular diseases by activating multiple target genes. The present study investigated the effects of BAE on transverse aortic constriction (TAC)‑induced myocardial dysfunction in mice and explored its possible molecular mechanisms. A total of 30 male mice were divided randomly into control, TAC and TAC + BAE groups. Mice in the TAC + BAE groups were administered BAE by oral gavage for 6 consecutive weeks. Myocardial dysfunction was assessed using echocardiogram, histopathology, TUNEL assay, immunofluorescence staining, reverse transcription‑quantitative PCR and western blot analysis. The results demonstrated that BAE treatment significantly ameliorated heart weight, left ventricular weight, myocardial dysfunction, left ventricular hypertrophy and fibrosis. In addition, BAE treatment alleviated TAC‑induced inflammation, oxidative stress and apoptosis. Notably, BAE treatment markedly reduced asymmetric dimethylarginine (ADMA) concentration and significantly increased dimethylarginine dimethylaminohydrolase 1 (DDAH1) expression and nitric oxide (NO) production. The present data indicated that BAE treatment ameliorated TAC‑induced myocardial dysfunction, oxidative stress, inflammatory response and apoptosis via the DDAH1/ADMA/NO signaling pathway.

Overexpression of CTRP3 protects against sepsis-induced myocardial dysfunction in mice.

C1q/tumor necrosis factor-related protein-3 (CTRP3) shows striking homologies of genomic structure to the adiponectin. In this study, we aimed to investigate the protective role of CTRP3 against sepsis-induced cardiomyopathy. Here, we overexpressed CTRP3 in myocardium by direct intramyocardial injection and constructed a model of lipopolysaccharide (LPS)-induced sepsis in mice. Our results demonstrated that cardiac-specific overexpression of CTRP3 remarkably attenuated myocardial dysfunction and increased the phosphorylation level of AMPKα during LPS-induced sepsis. The anti-inflammatory effects of CTRP3, as determined by decreased mRNA levels of TNF-α, IL-6 and a lower protein expression of phosphorylated NF-κB p65 and IκBα, was detected in mice following LPS treatment. Additionally, CTRP3 suppressed cardiac apoptosis induced by LPS in mice as indicated by terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) staining and western blot for Cleaved-caspase3, Bax and Bcl-2. In conclusion, CTRP3 could protect against sepsis-induced myocardial dysfunction in mice. The cardioprotective effects of CTRP3 might be mediated by activating AMPKα signaling pathway and blunting inflammatory response and apoptosis.

Irisin inhibits high glucose-induced endothelial-to-mesenchymal transition and exerts a dose-dependent bidirectional effect on diabetic cardiomyopathy.

Emerging evidence indicates that irisin provides beneficial effects in diabetes. However, whether irisin influences the development of diabetic cardiomyopathy (DCM) remains unclear. Therefore, we investigated the potential role and mechanism of action of irisin in diabetes‐induced myocardial dysfunction in mice. Type 1 diabetes was induced in mice by injecting streptozotocin, and the diabetic mice were administered recombinant r‐irisin (low or high dose: 0.5 or 1.5 μg/g body weight/day, I.P.) or PBS for 16 weeks. Irisin treatment did not alter blood glucose levels in the diabetic mice. However, the results of echocardiographical and histopathological assays indicated that low‐dose irisin treatment alleviated cardiac fibrosis and left ventricular function in the diabetic mice, whereas high‐dose irisin failed to mitigate the ventricular function impairment and increased collagen deposition. The potential mechanism underlying the effect of low‐dose irisin involved irisin‐mediated inhibition of high glucose‐induced endothelial‐to‐mesenchymal transition (EndMT); conversely, high‐dose irisin treatment enhanced high glucose‐induced MMP expression by stimulating MAPK (p38 and ERK) signalling and cardiac fibroblast proliferation and migration. Low‐dose irisin alleviated DCM development by inhibiting high glucose‐induced EndMT. By contrast, high‐dose irisin disrupted normal MMP expression and induced cardiac fibroblast proliferation and migration, which results in excess collagen deposition. Thus, irisin can inhibit high glucose‐induced EndMT and exert a dose‐dependent bidirectional effect on DCM.

Delta opioid receptor agonist attenuates lipopolysaccharide-induced myocardial injury by regulating autophagy

BackgroundPrevious studies have described the protective effects of DADLE on myocardial injury in sepsis. Recently, autophagy has been shown to be an innate defense mechanism in sepsis-related myocardial injury. However, whether DADLE has an pro-autophagic effect is yet to be elucidated. The present study aimed to investigate the effect of DADLE on the regulation of autophagy during sepsis. MethodsMale mice were subjected to LPS or vehicle intraperitoneal injection. After LPS injection, mice received either DADLE, Naltrindole or vehicle. ELISA and JC-1 were used to evaluate the level cTnI and Mitochondrial membrane potential. Cardiac ultrastructural and autophagosomes were visualized by transmission electron microscopy. The relative protein levels were analyzed by Western blot. ResultsThe results showed that treatment with DADLE both immediately or 4 h after LPS intraperitoneal injection could improve the survival rate of mice with endotoxemic. DADLE could ease myocardium ultrastructure injury induced by LPS, this cardioprotective effect was also seen in increased MMP levels, and decreased cTnI levels. Through observation of transmission electron microscopy and Western blot we have discovered that the amount of autophagosome and the expression of autophagy related protein LC3II, Beclin1 were significantly increased with DADLE treatment. DADLE promoted LPS-induced autophagosome maturation as indicated by the increased LAMP-1 protein level and decreased SQSTM1/p62 protein level. The selective δ-opioid receptor antagonist Naltrindole play an opposite effects. ConclusionsDADLE could improve the survival and protect myocardial dysfunction in mice with LPS-induced endotoxemia. This effect was related to the increase of autophagy.

Carbon monoxide releasing molecule-3 improves myocardial function in mice with sepsis by inhibiting NLRP3 inflammasome activation in cardiac fibroblasts.

The NLRP3 inflammasome is an intracellular multiple-protein complex that controls the maturation and release of interleukin (IL)-1β and IL-18. Endogenous carbon monoxide (CO) is anti-inflammatory. The aim of this study was to assess the effects/mechanisms of CO-releasing molecule-3 (CORM-3)-dependent modulation of the NLRP3 inflammasome in cardiac fibroblasts (CF) and its effect on myocardial function in sepsis. CF were treated with CORM-3 or inactive CORM-3 (iCORM-3) before NLRP3 inflammasome priming with lipopolysaccharides (LPS) or following activation with adenosine triphosphate (ATP). In parallel, cardiomyocytes (CM) were challenged with supernatants of LPS/ATP-stimulated CF or a cytokine mixture (Cyto-mix) containing IL-1β, IL-18, and HMGB1. In vivo, mice were treated with CORM-3 before or after LPS to induce sepsis (endotoxemia). Pretreatment of CF with CORM-3 prevented an LPS-induced increase in NLRP3 and pro-IL-1β expression. Treatment of CF with CORM-3 before ATP prevented ATP-induced activation of the NLRP3 inflammasome. Challenging CF with LPS/ATP promoted NLRP3 interactions with adaptor ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain), which was prevented by CORM-3. Challenging CM with supernatants of CF with LPS/ATP or Cyto-mix (IL-1β, IL-18, and HMGB1) resulted in CM apoptosis, which was attenuated with either a CORM-3 or IL-1 receptor antagonist. Finally, myocardial NLRP3 inflammasome activation and myocardial dysfunction in septic mice were abolished by CORM-3. In NLRP3-deficient mice with sepsis, CORM-3 did not show additional benefits in improving myocardial function. Our results indicate that CORM-3 suppresses NLRP3 inflammasome activation by blocking NLRP3 interactions with the adaptor protein ASC and attenuates myocardial dysfunction in mice with sepsis.

Protection against doxorubicin-induced myocardial dysfunction in mice by cardiac-specific expression of carboxyl terminus of hsp70-interacting protein.

Carboxyl terminus of Hsp70-interacting protein (CHIP) is a critical ubiquitin ligase/cochaperone to reduce cardiac oxidative stress, inflammation, cardiomyocyte apoptosis and autophage etc. However, it is unclear whether overexpression of CHIP in the heart would exert protective effects against DOX-induced cardiomyopathy. Cardiac-specific CHIP transgenic (CHIP-TG) mice and the wild-type (WT) littermates were treated with DOX or saline. DOX-induced cardiac atrophy, dysfunction, inflammation, oxidative stress and cardiomyocyte apoptosis were significantly attenuated in CHIP-TG mice. CHIP-TG mice also showed higher survival rate than that of WT mice (40% versus 10%) after 10-day administration of DOX. In contrast, knockdown of CHIP by siRNA in vitro further enhanced DOX-induced cardiotoxic effects. Global gene microarray assay revealed that after DOX-treatment, differentially expressed genes between WT and CHIP-TG mice were mainly involved in apoptosis, atrophy, immune/inflammation and oxidative stress. Mechanistically, CHIP directly promotes ubiquitin-mediated degradation of p53 and SHP-1, which results in activation of ERK1/2 and STAT3 pathways thereby ameliorating DOX-induced cardiac toxicity.

The role of speckle tracking echocardiography in assessment of lipopolysaccharide-induced myocardial dysfunction in mice.

Sepsis-induced myocardial dysfunction is a common and severe complication of septic shock. Conventional echocardiography often fails to reveal myocardial depression in severe sepsis due to hemodynamic changes; in contrast, decline of strain measurements by speckle tracking echocardiography (STE) may indicate impaired cardiac function. This study investigates the role of STE in detecting lipopolysaccharide (LPS)-induced cardiac dysfunction with mouse models. We evaluated cardiac function in 20 mice at baseline, 6 h (n=10) and 20 h (n=10) after LPS injection to monitor the development of heart failure induced by severe sepsis using 2-D and M-mode echocardiography. Ejection fraction (EF) and fractional shortening (FS) were measured with standard M-mode tracings, whereas circumferential and radial strain was derived from STE. Serum biochemical and cardiac histopathological examinations were performed to determine sepsis-induced myocardial injury. Left ventricular (LV) myocardial function was significantly reduced at 6 h after LPS treatment assessed by circumferential strain (-14.65%±3.00% to -8.48%±1.72%, P=0.006), whereas there were no significant differences between 6 and 20 h group. Conversely, EF and FS were significantly increased at 20 h when comparing to 6 h (P<0.05) accompanied with marked decreases in EF and FS 6 h following LPS administration. Consistent with strain echocardiographic results, we showed that LPS injection leaded to elevated serum level of cardiac Troponin-T (cTnT), CK-MB and rising leucocytes infiltration into myocardium within 20 h. Altogether, these results demonstrate that, circumferential strain by STE is a specific and reliable value for evaluating LPS-induced cardiac dysfunction in mice.

Lin28a protects against diabetic cardiomyopathy via the PKA/ROCK2 pathway

BackgroundLin28a enhances glucose uptake and insulin-sensitivity. However, the role of Lin28a on experimental diabetic cardiomyopathy (DCM) is not well understood. We investigated the potential role and mechanism ofLin28a in diabetes-induced myocardial dysfunction in mice. MethodsDiabetes was induced by intraperitoneal (i.p.) injections of Streptozocin (STZ) in mice. Animals were randomized to be treated with lentivirus carrying Lin28a siRNA or Lin28a cDNA. Cardiac function, cardiomyocyte autophagy, apoptosis and mitochondria morphology in diabetic mice were compared between groups. The target proteins of Lin28a were examined by western blot analysis. ResultsLin28a levels were markedly reduced in the cardiac tissue compared to the control mice. Lin28a overexpression significantly improved left ventricular ejection fraction (LVEF), promoted autophagy, decreased myocardial apoptotic index and alleviated mitochondria cristae destruction in diabetic mice. Lin28a knockdown exacerbated diabetic injury as evidenced by decreased LVEF, increased apoptotic index and aggravated mitochondria cristae destruction. Interestingly, pretreatment with a PKA inhibitor, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt (H89) abolished the beneficial effects of Lin28a overexpression. RhoA-expression and ROCK2-expression were decreased in vivo after Lin28a overexpression, while Lin28a knockdown increased the expression of RhoA and ROCK2 in diabetic mice. ConclusionsLin28a protects against DCM through PKA/ROCK2 dependent pathway. Lin28a might serve as a potential therapeutic target for the treatment of the patients with DCM.

Cardiac fibroblasts contribute to myocardial dysfunction in mice with sepsis: the role of NLRP3 inflammasome activation.

Myocardial contractile dysfunction in sepsis is associated with the increased morbidity and mortality. Although the underlying mechanisms of the cardiac depression have not been fully elucidated, an exaggerated inflammatory response is believed to be responsible. Nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome is an intracellular platform that is involved in the maturation and release of interleukin (IL)-1β. The aim of the present study is to evaluate whether sepsis activates NLRP3 inflammasome/caspase-1/IL-1β pathway in cardiac fibroblasts (CFs) and whether this cytokine can subsequently impact the function of cardiomyocytes (cardiac fibroblast-myocyte cross-talk). We show that treatment of CFs with lipopolysaccharide (LPS) induces upregulation of NLRP3, activation of caspase-1, as well as the maturation (activation) and release of IL-1β. In addition, the genetic (small interfering ribonucleic acid [siRNA]) and pharmacological (glyburide) inhibition of the NLRP3 inflammasome in CFs can block this signaling pathway. Furthermore, the inhibition of the NLRP3 inflammasome in cardiac fibroblasts ameliorated the ability of LPS-chalenged CFs to impact cardiomyocyte function as assessed by intracellular cyclic adenosine monophosphate (cAMP) responses in cardiomyocytes. Salient features of this the NLP3 inflammasome/ caspase-1 pathway were confirmed in in vivo models of endotoxemia/sepsis. We found that inhibition of the NLRP3 inflammasome attenuated myocardial dysfunction in mice with LPS and increased the survival rate in mice with feces-induced peritonitis. Our results indicate that the activation of the NLRP3 inflammasome in cardiac fibroblasts is pivotal in the induction of myocardial dysfunction in sepsis.

CORM‐3 derived CO suppresses NLRP3 inflammasome in cardiac fibroblasts and protects cardiomyocytes from apoptosis in mice with sepsis (1096.6)

The study is to examine the role of CO derived from CORM‐3 on NLRP3 inflammasome activation in cardiac fibroblast (CF) and its effect on cardiomyocyte apoptosis and myocardial dysfunction in mice with sepsis. Methods: CFs were primed with LPS and followed by ATP treatment with or without CORM‐3. CF NLRP3 expression and NLRP3 inflammasome activation assessed. Cardiomyocyte apoptosis and myocardial function were measured in mice with LPS. Results: LPS priming and ATP treatment to CFs resulted in NLRP3 inflammasome activation as indicated by increase in cleaved caspase‐1 p20 and matured IL‐1β. CFs with NLRP3 siRNA abolished caspase‐1 cleavage and IL‐1β maturation. CORM‐3 attenuated the increase in NLRP3 and pro‐IL‐1β expression in LPS primed CFs and prevented NLRP3 inflammasome activation. CORM‐3 inhibited NLRP3‐mediated ASC pyroptosome formation. Myocardial NLRP3, pro‐capase‐1, p20, pro‐IL‐1β and IL‐1β were increased, cardiomyocyte apoptosis was induced and myocardial contractility was decreased in mice with LPS. CORM3 prevented the myocardial NLPR3 inflammasome activation, attenuated cardiomyocytes apoptosis, and improved cardiac function in mice with LPS. Conclusions: CORM‐3‐derived CO suppresses NLRP3 inflammasome priming and activation in CFs and protects myocardium from apoptosis and dysfunction in mice with endotoxemia. (HSFO 2012‐000212)Grant Funding Source: Heart and Stroke Foundation of Ontario

Role of cardiac fibroblasts in myocardial dysfunction in mice with sepsis: effect of NLRP3 inflammasome activation (1096.7)

Background: Inflammasomes are intracellular platforms which promote maturation and releasing of IL‐1β and IL‐18. Cardiac fibroblast is the most abundant non‐cardiomyocytes within the heart. The aim of the present study was to evaluate whether sepsis activates NLRP3 inflammasome in cardiac fibroblasts and subsequently affects function of cardiomyocytes through IL‐1β thereby results in myocardial dysfunction. Materials and Methods: NLPR3, procaspase‐1, caspase‐1 p10, pro‐IL‐1β and IL‐1β in cardiac fibroblasts were assessed with Western blot or ELISA. Cardiomyocyte intracellular cAMP was assessed with direct immunoassay kit. Mouse model of sepsis was induced by intraperitoneal injection of LPS. Mouse myocardial function was evaluated with a mouse pressure‐volume loop analysis system. Results: Treatment of cardiac fibroblasts with LPS resulted in activation of capase‐1 (increased p10) and induced maturation and releasing of IL‐1β. Genetically (siRNA) and pharmacologically (Glyburide) inhibition of NLRP3 inflammasome in cardiac fibroblasts blocked the LPS‐induced activation of the caspase‐1, as well as the maturation and releasing of the IL‐1β. Inhibition of NLRP3 inflammasome in cardiac fibroblasts prevented the decrease in intracellular cAMP in cardiomyocyte conditioned with supernatants of cardiac fibroblasts with LPS. Inhibition of the NLRP3 inflammasome attenuated the myocardial dysfunction in mice with sepsis. Conclusions: Activation of NLRP3 inflammasome in cardiac fibroblasts results in increased IL‐1β production which mediates the cardiac fibroblast‐cardiomyocyte interaction and promotes the induction of myocardial dysfunction in mice with sepsis. (HSFO GIA 2012‐000212 to TR).Grant Funding Source: HSFO

Abstract 18928: Cardiac Soluble Guanylate Cyclase Protects Against Cardiac Dysfunction Induced by Chronic Doxorubicin Treatment in Mice

Background - The chemotherapeutic use of doxorubicin (DOX) is limited by cardiotoxicity. Growing evidence suggests a protective role for cyclic guanosine monophosphate (cGMP) in pathological LV remodeling. We therefore tested the hypothesis that reduced cGMP bioavailability in cardiac myocytes worsens myocardial dysfunction in mice after chronic DOX treatment. Methods and Results - We generated mice with an inducible (Tet-Off) cardiac myocyte-specific expression of a dominant-negative soluble guanylate cyclase (dnsGC) mutant, which inhibits sGC signaling selectively in the heart. Cardiac cGMP levels, measured using an enzyme immunoassay, were lower in dnsGC mice (n=10) than in WT littermates (n=20) 5 minutes after administration of the NO-donor GSNO (1mg/kg) (0.99±0.34 vs 1.85±0.17 nmol/mg protein, respectively, P&lt;0.05). At baseline, transthoracic echocardiography (TTE) demonstrated similar LV dimensions and function in dnsGC and WT (data not shown). Heart to body weight ratio was also comparable (HW/BW; 4.3±0.2 mg/g in dnsGC, n=13, vs 3.9±0.1 mg/g in WT, n=21). After 12 weeks DOX (2 mg/kg weekly), LV function was significantly reduced and LV diameters significantly enlarged as compared to saline-treated WT (Table). The impact of DOX on cardiac function and dimensions was more marked in dnsGC than in WT (P&lt;0.05 for both, Table). Survival was similarly impaired in DOX-treated dnsGC (67%, n=30) and WT (59%, n=24) compared with saline-treated WT (100%, n=25). HW/BW was similar in DOX-treated dnsGC and WT (4.3±0.1 vs 4.2±0.1 mg/g), as was the % bromodeoxyuridine (BrdU)-positive cardiac myocytes or cardiac progenitor cells (0.8±0.4%, n=7 vs 1.6±0.7%, n=9, respectively, P=NS). Conclusions - Reduced myocardial cGMP results in increased LV dysfunction and dilatation in doxorubicin-treated mice. Stimulation of sGC activity in cardiac myocytes may represent a promising therapeutic approach for doxorubicin cardiotoxicity.

Rhynchophylline prevents cardiac dysfunction and improves survival in lipopolysaccharide-challenged mice via suppressing macrophage I-κBα phosphorylation

Myocardial dysfunction is a common complication during sepsis and significantly contributes to the mortality of patients with septic shock. However, none of the available therapeutic strategies proven to be effective in patients with severe sepsis are designed specifically to target myocardial dysfunction. The purpose of the present study is to investigate the effect of rhynchophylline (Rhy) on LPS-induced myocardial dysfunction in mice. We found that pretreatment with Rhy significantly improved cardiac systolic dysfunction, increased stroke volume and cardiac output in mice challenged with LPS. LPS induced cardiac inhibitor-κBα (I-κBα) phosphorylation, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) mRNA expression, and in turn increased cardiac TNF-α and IL-1β protein production, all of which were attenuated by pretreatment with Rhy. Immunohistochemistry revealed that TNF-α was found in infiltrated macrophages (F4/80+) and myocardium, and Rhy reduced TNF-α immunostaining in cardiac infiltrated macrophages in LPS-challenged mice. Furthermore, Rhy inhibited LPS-induced I-κBα phosphorylation and TNF-α production in cultured mouse peritoneal macrophages, but not in neonatal mouse cardiomyocytes. Pretreatment with Rhy significantly decreased the mortality of LPS-challenged mice. These results indicate that Rhy reduces cardiac dysfunction and improves survival via suppression of macrophage I-κBα phosphorylation in LPS-challenged mice, and suggest that Rhy may be a potential agent for the treatment of septic cardiac dysfunction.