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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-α) 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-α 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-α mRNA expression and protein expression in the LV myocardium. Pre-treatment of the p38 MAPK inhibitor SB202190 (2 mg/kg, i.p.) decreased TNF-α mRNA and protein by 65 and 36%, respectively (P<0.05). Immunohistochemical staining confirmed that cardiomyocytes were the major source of TNF-α production in the myocardium and blocking p38 MAPK activation inhibited TNF-α expression in response to LPS. Pre-treatment of SB202190 or a TNF-α 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-α production and cardiac dysfunction during acute endotoxemia in mice. Our data suggest that p38 MAPK is a potential therapeutic target of endotoxemia. [Copyright &y& Elsevier]

Background: Sepsis-caused multi-organ failure remains the major cause of morbidity and mortality in intensive care units with limited therapeutics. Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD+), has been recently reported to be protective in sepsis; however, its therapeutic effects remain to be determined. This study sought to investigate the therapeutic effects of NMN in septic organ failure and its underlying mechanisms. Methods: Sepsis was induced by feces-injection-in-peritoneum in mice. NMN was given after an hour of sepsis onset. Cultured neutrophils, macrophages and endothelial cells were incubated with various agents. Results: We demonstrate that administration of NMN elevated NAD+ levels and reduced serum lactate levels, oxidative stress, inflammation, and caspase-3 activity in multiple organs of septic mice, which correlated with the attenuation of heart dysfunction, pulmonary microvascular permeability, liver injury, and kidney dysfunction, leading to lower mortality. The therapeutic effects of NMN were associated with lower bacterial burden in blood, and less ROS production in septic mice. NMN improved bacterial phagocytosis and bactericidal activity of macrophages and neutrophils while reducing the lipopolysaccharides-induced inflammatory response of macrophages. In cultured endothelial cells, NMN mitigated mitochondrial dysfunction, inflammation, apoptosis, and barrier dysfunction induced by septic conditions, all of which were offset by SIRT3 inhibition. Conclusion: NAD+ repletion with NMN prevents mitochondrial dysfunction and restrains bacterial dissemination while limiting inflammatory damage through SIRT3 signaling in sepsis. Thus, NMN may represent a therapeutic option for sepsis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Cardiomyocyte death contributes to cardiac pathology of diabetes. Studies have shown that the RIPK3/MLKL necroptosis signaling is activated in diabetic hearts. Deletion of RIPK3 was reported to attenuate myocardial injury and heart dysfunction in streptozocin (STZ)-induced diabetic mice, suggesting a potential role of necroptosis in diabetic cardiomyopathy. This study characterized cardiomyocyte necroptosis in diabetic hearts and investigated whether MLKL-mediated necroptosis is a target for cardiac protection in diabetes. Methods: Type 1 diabetes was induced in RIPK3 knockout, MLKL knockout and wild-type mice. Akita Type-1 diabetic mice were injected with shRNA for MLKL. Myocardial function was assessed by echocardiography. Immuno-histological analyses determined cardiomyocyte death and fibrosis in the heart. Cultured adult mouse cardiomyocytes were incubated with high glucose in the presence of various drugs. Cell death and phosphorylation of RIPK3 and MLKL were analysed. Results: We showed that the levels of phosphorylated RIPK3 and MLKL were higher in high glucose-stimulated cardiomyocytes and hearts of STZ-induced type-1 diabetic mice, akita mice and type-1 diabetic monkeys when compared to non-diabetic controls. Inhibition of RIPK3 by its pharmacological inhibitor or gene deletion, or MLKL deletion prevented high glucose-induced MLKL phosphorylation and attenuated necroptosis in cardiomyocytes. In STZ-induced type-1 diabetic mice, cardiomyocyte necroptosis was present along with elevated cardiac troponin I in serum and MLKL oligomerization, and co-localized with phosphorylated MLKL. Deletion of RIPK3 or MLKL prevented MLKL phosphorylation and cardiac necroptosis, attenuated serum cardiac troponin I levels, reduced myocardial collagen deposition and improved myocardial function in STZ-injected mice. Additionally, shRNA-mediated down-regulation of MLKL reduced cardiomyocyte necroptosis in akita mice. Interestingly, incubation with anti-diabetic drugs (empagliflozin and metformin) prevented phosphorylation of RIPK3 and MLKL, and reduced cell death in high glucose-induced cardiomyocytes. Conclusions: We have provided evidence that cardiomyocyte necroptosis is present in diabetic hearts and that MLKL-mediated cardiomyocyte necroptosis contributes to diabetic cardiomyopathy. These findings highlight MLKL-mediated necroptosis as a target for cardiac protection in diabetes. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this study was to investigate the role of Rac1 and estrogen in sex difference of cardiac tumor necrosis factor-alpha (TNF-α) expression during endotoxemia. Endotoxemia was induced in male and female mice by peritoneal injection of lipopolysaccharide (LPS, 4 mg/kg). Compared with female mice, male mice produced more TNF-α in the heart 4 h after LPS treatment, which were correlated with higher Rac1 and NADPH oxidase activity, more phosphorylation of ERK1/2 and p38 MAPK, and up-regulation of toll-like receptor-4 (TLR-4) expression in male mice. Cardiac specific Rac1 knockout or administration of 17β-estradiol down-regulated Rac1 expression, attenuated gp91 phox -NADPH oxidase expression and activity, decreased phosphorylation of ERK1/2/p38 MAPK and inhibited cardiac TNF-α expression induced by LPS, suggesting an important role of Rac1 and estrogen in LPS-stimulated TNF-α expression in the heart. More importantly, the sex difference in TNF-α expression was abrogated by Rac1 knockout or gp91 phox knockout and by administration of apocynin or N-acetylcysteine in LPS-stimulated mice. To investigate the functional significance of sex difference in endotoxemia, heart function was measured in isolated hearts with a Langendorff system. Male mice exhibited worse myocardial dysfunction compared with female in endotoxemia. Treatment of male mice with 17β-estradiol attenuated myocardial dysfunction during endotoxemia. In conclusion, LPS induces Rac1 activation, which contributes to NADPH oxidase activity and phosphorylation of ERK1/2/p38 MAPK, leading to TNF-α expression in the heart. The sex difference in TNF-α expression is estrogen-dependent and mediated via Rac1 dependent NADPH oxidase/ERK1/2 and p38 MAPK pathway in LPS-stimulated hearts. [Copyright &y& Elsevier]

Calpains belong to the family of calcium-dependent cysteine proteases expressed ubiquitously in mammals and many other organisms. Activation of calpain is observed in diseased hearts and is implicated in cardiac cell death, hypertrophy, fibrosis, and inflammation. However, the underlying mechanisms remain incompletely understood. Recent studies have revealed that calpains target and impair mitochondria in cardiac disease. The objective of this review is to discuss the role of calpains in mediating mitochondrial damage and the underlying mechanisms, and to evaluate whether targeted inhibition of mitochondrial calpain is a potential strategy in treating cardiac disease. We expect to describe the wealth of new evidence surrounding calpain-mediated mitochondrial damage to facilitate future mechanistic studies and therapy development for cardiac disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aims: Clinical use of cisplatin (Cisp), one of the most widely used, common, and effective chemotherapeutic agents, is limited by its side effects, particularly tubular injury-associated nephrotoxicity. Previous studies suggest that hydrogen sulfide (H2S) alleviates Cisp-induced acute kidney injury (AKI). However, the underlying mechanism remains largely unclear. Results: A single intraperitoneal injection of Cisp is employed to induce AKI, and the mice exhibit severe kidney dysfunction and histological damage at day 4 after Cisp injection. Here, we reported that H2S alleviated Cisp-caused renal toxicity via SIRT3 activation and subsequent improvement of mitochondrial ATP production. Using a biotin-switch assay, we showed that H2S increased S-sulfhydration of SIRT3 and induced deacetylation of its target proteins (OPA1, ATP synthase β, and superoxide dismutase 2). These effects of H2S were associated with a reduction of mitochondrial fragmentation, an increase in ATP generation, and less oxidative injury. Notably, the S-sulfhydration of SIRT3 induced by H2S was abrogated when Cys256, Cys259, Cys280, and Cys283 residues on SIRT3 (two zinc finger domains) were mutated. Innovation and Conclusion: Our data suggest that H2S attenuates Cisp-induced AKI by preventing mitochondrial dysfunction via SIRT3 sulfhydrylation. Antioxid. Redox Signal. 31, 1302–1319. [ABSTRACT FROM AUTHOR]

Cardiovascular dysfunction is a common complication among heatstroke patients, but its underlying mechanism is unclear. This study was designed to investigate the role of calpain‐2 and its downstream signal pathway in heat stress‐induced cardiomyocyte apoptosis and heart dysfunction. In cultured primary mouse neonatal cardiomyocytes (MNCs), heat stress (43°C for 2 hr) induced a heat‐shock response, as indicated by upregulated heat‐shock protein 27 (HSP27) expression and cellular apoptosis, as indicated by increased caspase‐3 activity, DNA fragmentation and decreased cell viability. Meanwhile, heat stress decreased calpain activity, which was accompanied by downregulated calpain‐2 expression and increased phosphorylation of p38, extraceIIuIar signaI‐reguIated protein kinase (ERK1/2) and c‐Jun N‐terminaI kinase (JNK). Calpain‐2 overexpression abrogated heat stress‐induced apoptosis and phosphorylation of p38 and JNK, but not of ERK1/2. Blocking only p38 prevented heat stress‐induced apoptosis in MNCs. In cardiac‐specific calpain‐2 overexpressing transgenic mice, p38 phosphorylation and cardiomyocyte apoptosis were decreased in the heart tissue of heatstroke mice, as revealed by western blot and terminal deoxynucleotidyl transferase dUTP nick end labelling assays, respectively. M‐mode echocardiography also demonstrated that calpain‐2 overexpression significantly improved heatstroke‐induced decreases in ventricular end‐diastolic volume and cardiac output. In conclusion, our study suggests that heat stress reduces calpain‐2 expression, which then activates p38, leading to cardiomyocyte apoptosis and heart dysfunction. Heat stress could induce cardiomyocyte apoptosis and decrease calpain‐2 expression and total calpain activity, and increase phosphorylation of p38, extracellular signaI‐reguIated protein kinase (ERK1/2) and c‐Jun N‐terminaI kinase (JNK). Calpain‐2 overexpression could abrogate heat stress‐induced phosphorylation of p38 but not ERK1/2 and JNK, thereby decrease cardiomyocyte apoptosis and heart dysfunction. Targeting calpain‐2 may be a potential therapeutic strategy for heatstroke‐induced myocardial complications.. [ABSTRACT FROM AUTHOR]

We recently reported that doxorubicin decreased the expression of calpain-1/2, while inhibition of calpain activity promoted doxorubicin-induced cardiac injury in mice. In this study, we investigated whether and how elevation of calpain-2 could affect doxorubicin-triggered cardiac injury. Transgenic mice with inducible cardiomyocyte-specific expression of calpain-2 were generated. An acute cardiotoxicity was induced in both transgenic mice and their relevant wild-type littermates by injection of a single dose of doxorubicin (20 mg/kg) and cardiac injury was analyzed 5 days after doxorubicin injection. Cardiomyocyte-specific up-regulation of calpain-2 did not induce any adverse cardiac phenotypes under physiological conditions by age 3 months, but significantly reduced myocardial injury and improved myocardial function in doxorubicin-treated mice. Cardiac protection of calpain-2 up-regulation was also observed in a mouse model of chronic doxorubicin cardiotoxicity. Up-regulation of calpain-2 increased the protein levels of mitogen activated protein kinase phosphatase-1 (MKP-1) in cultured mouse cardiomyocytes and heart tissues. Over-expression of MKP-1 prevented, whereas knockdown of MKP-1 augmented doxorubicin-induced apoptosis in cultured cardiomyocytes. Moreover, knockdown of MKP-1 offset calpain-2-elicited protective effects against doxorubicin-induced injury in cultured cardiomyocytes. Mechanistically, up-regulation of calpain-2 reduced the protein levels of phosphatase and tensin homolog and consequently promoted Akt activation, leading to increased MKP-1 protein steady-state levels by inhibiting its degradation. Collectively, this study reveals a new role of calpain-2 in promoting MKP-1 expression via phosphatase and tensin homolog/Akt signaling. This study also suggests that calpain-2/MKP-1 signaling may represent new therapeutic targets for doxorubicin-induced cardiac injury. [ABSTRACT FROM AUTHOR]

Aims Sepsis-caused multiple organ failure remains the major cause of morbidity and mortality in intensive care units. Nicotinamide riboside (NR) is a precursor of nicotinamide adenine dinucleotide (NAD + ), which is important in regulating oxidative stress. This study investigated whether administration of NR prevented oxidative stress and organ injury in sepsis. Methods Mouse sepsis models were induced by injection of lipopolysaccharides (LPS) or feces-injection-in-peritoneum. NR was given before sepsis onset. Cultured macrophages and endothelial cells were incubated with various agents. Results Administration of NR elevated the NAD + levels, and elicited a reduction of oxidative stress, inflammation and caspase-3 activity in lung and heart tissues, which correlated with attenuation of pulmonary microvascular permeability and myocardial dysfunction, leading to less mortality in sepsis models. These protective effects of NR were associated with decreased levels of plasma high mobility group box-1 (HMGB1) in septic mice. Consistently, pre-treatment of macrophages with NR increased NAD + content and reduced HMGB1 release upon LPS stimulation. NR also prevented reactive oxygen species (ROS) production and apoptosis in endothelial cells induced by a conditioned-medium collected from LPS-treated macrophages. Furthermore, inhibition of SIRT1 by EX527 offset the negative effects of NR on HMGB1 release in macrophages, and ROS and apoptosis in endothelial cells. Conclusions Administration of NR prevents lung and heart injury, and improves the survival in sepsis, likely by inhibiting HMGB1 release and oxidative stress via the NAD + /SIRT1 signaling. Given NR has been used as a health supplement, it may be a useful agent to prevent organ injury in sepsis. [ABSTRACT FROM AUTHOR]

Diabetes and obesity are prevalent in westernized countries. In both conditions, excessive fatty acid uptake by cardiomyocytes induces cardiac lipotoxicity, an important mechanism contributing to diabetic cardiomyopathy. This study investigated the effect of calpain disruption on cardiac lipotoxicity. Cardiac-specific capns1 knockout mice and their wild-type littermates (male, age of 4 weeks) were fed a high fat diet (HFD) or normal diet for 20 weeks. HFD increased body weight, altered blood lipid profiles and impaired glucose tolerance comparably in both capns1 knockout mice and their wild-type littermates. Calpain activity, cardiomyocyte cross-sectional areas, collagen deposition and triglyceride were significantly increased in HFD-fed mouse hearts, and these were accompanied by myocardial dysfunction and up-regulation of hypertrophic and fibrotic collagen genes as well as pro-inflammatory cytokines. These effects of HFD were attenuated by disruption of calpain in capns1 knockout mice. Mechanistically, deletion of capns1 in HFD-fed mouse hearts and disruption of calpain with calpain inhibitor-III, silencing of capn1 , or deletion of capns1 in palmitate-stimulated cardiomyocytes prevented endoplasmic reticulum stress, apoptosis, cleavage of caspase-12 and junctophilin-2, and pro-inflammatory cytokine expression. Pharmacological inhibition of endoplasmic reticulum stress diminished palmitate-induced apoptosis and pro-inflammatory cytokine expression in cardiomyocytes. In summary, disruption of calpain prevents lipotoxicity-induced apoptosis in cardiomyocytes and cardiac injury in mice fed a HFD. The role of calpain is mediated, at least partially, through endoplasmic reticulum stress. Thus, calpain/endoplasmic reticulum stress may represent a new mechanism and potential therapeutic targets for cardiac lipotoxicity. [ABSTRACT FROM AUTHOR]

Background: We discovered that lipopolysaccharide (LPS, an initiating factor in sepsis) and hypoxia-reoxygenation (H/R, a confounding factor) reduce electrical coupling between microvascular endothelial cells from wild-type (WT) but not Cx40-/- mice. Because Cx40 knockout could result in nonspecific effects, this discovery may not establish the causal relationship between Cx40 and reduced coupling. Using the same cell culture model, we aimed to address this uncertainty by using the rescue-of-function approach. Methods/Results: Electrical coupling between endothelial cells (hindlimb muscle origin) was determined by electrophysiology. LPS, H/R and concurrent LPS + H/R reduced coupling between WT but not Cx40-/- cells. The defect in Cx40-/- cells was rescued by ectopic expression of Cx40, after infecting the cells with adenovirus encoding Cx40. Cx40-/- cells were also engineered to express mutant Cx40 that lacked the carboxyl terminal domain beginning at residue 236 (Cx40Δ237-358) or 344 (Cx40Δ345-358). No response to inflammatory stimuli was observed in cells expressing either of these 2 mutants. Conclusion: Our data establish the causal relationship between Cx40 and reduced coupling and suggest that the 345-358 amino acid motif of the Cx40 carboxyl terminal is required for reduced coupling. Cx40 may participate in compromised conducted response in the microvasculature during sepsis. [ABSTRACT FROM AUTHOR]

Background: MicroRNAs (miRs) are a class of short RNA molecules, which negatively regulate gene expression. The levels of circulating miR-15 family members are elevated in septic patients and may be associated with septic death. This study investigated whether inhibition of miR-195, a member of the miR-15 family, provided beneficial effects in sepsis.Methods and Results: Sepsis was induced by injection of feces into the peritoneum in mice. miR-195 was upregulated in the lung and liver of septic mice. Silencing of miR-195 increased the protein levels of BCL-2, Sirt1, and Pim-1; prevented apoptosis; reduced liver and lung injury; and improved the survival in septic mice. Silencing of miR-195 provided similar protection in lipopolysaccharide-induced endotoxemic mice. In endothelial cells, upregulation of miR-195 induced apoptosis, and inhibition of miR-195 prevented lipopolysaccharide-induced apoptosis. miR-195 repressed expression of its protein targets, BCL-2, Sirt1, and Pim-1. Furthermore, overexpression of Pim-1 prevented apoptosis induced by lipopolysaccharide and miR-195 mimic. Inhibition of Pim-1 attenuated the protective effects of miR-195 silencing in septic mice.Conclusions: Silencing of miR-195 reduced multiple-organ injury and improved the survival in sepsis, and the protective effects of miR-195 inhibition were associated with upregulation of Bcl-2, Sirt1, and Pim-1. Thus, inhibition of miR-195 may represent a new therapeutic approach for sepsis. [ABSTRACT FROM AUTHOR]

Aims The mitochondria are important sources of reactive oxygen species (ROS) in the heart. Mitochondrial ROS production has been implicated in the pathogenesis of diabetic cardiomyopathy, suggesting that therapeutic strategies specifically targeting mitochondrial ROS may have benefit in this disease. We investigated the therapeutic effects of mitochondria-targeted antioxidant mito-TEMPO on diabetic cardiomyopathy. Methods The mitochondria-targeted antioxidant mito-TEMPO was administrated after diabetes onset in a mouse model of streptozotocin-induced type-1 diabetes and type-2 diabetic db/db mice. Cardiac adverse changes were analyzed and myocardial function assessed. Cultured adult cardiomyocytes were stimulated with high glucose, and mitochondrial superoxide generation and cell death were measured. Results Incubation with high glucose increased mitochondria superoxide generation in cultured cardiomyocytes, which was prevented by mito-TEMPO. Co-incubation with mito-TEMPO abrogated high glucose-induced cell death. Mitochondrial ROS generation, and intracellular oxidative stress levels were induced in both type-1 and type-2 diabetic mouse hearts. Daily injection of mito-TEMPO for 30 days inhibited mitochondrial ROS generation, prevented intracellular oxidative stress levels, decreased apoptosis and reduced myocardial hypertrophy in diabetic hearts, leading to improvement of myocardial function in both type-1 and type-2 diabetic mice. Incubation with mito-TEMPO or inhibition of Nox2-containing NADPH oxidase prevented oxidative stress levels and cell death in high glucose-stimulated cardiomyocytes. Mechanistic study revealed that the protective effects of mito-TEMPO were associated with down-regulation of ERK1/2 phosphorylation. Conclusions Therapeutic inhibition of mitochondrial ROS by mito-TEMPO reduced adverse cardiac changes and mitigated myocardial dysfunction in diabetic mice. Thus, mitochondria-targeted antioxidants may be an effective therapy for diabetic cardiac complications. [ABSTRACT FROM AUTHOR]

Calpain plays a critical role in cardiomyopathic changes in type 1 diabetes (T1D). This study investigated how calpain regulates mitochondrial reactive oxygen species (ROS) generation in the development of diabetic cardiomyopathy. T1D was induced in transgenic mice overexpressing calpastatin, in mice with cardiomyocyte-specific capn4 deletion, or in their wild-type littermates by injection of streptozotocin. Calpain-1 protein and activity in mitochondria were elevated in diabetic mouse hearts. The increased mitochondrial calpain-1 was associated with an increase in mitochondrial ROS generation and oxidative damage and a reduction in ATP synthase-α (ATP5A1) protein and ATP synthase activity. Genetic inhibition of calpain or upregulation of ATP5A1 increased ATP5A1 and ATP synthase activity, prevented mitochondrial ROS generation and oxidative damage, and reduced cardiomyopathic changes in diabetic mice. High glucose concentration induced ATP synthase disruption, mitochondrial superoxide generation, and cell death in cardiomyocytes, all of which were prevented by overexpression of mitochondria-targeted calpastatin or ATP5A1. Moreover, upregulation of calpain-1 specifically in mitochondria induced the cleavage of ATP5A1, superoxide generation, and apoptosis in cardiomyocytes. In summary, calpain-1 accumulation in mitochondria disrupts ATP synthase and induces ROS generation, which promotes diabetic cardiomyopathy. These findings suggest a novel mechanism for and may have significant implications in diabetic cardiac complications. [ABSTRACT FROM AUTHOR]

Sepsis is an infection-induced severe inflammatory disorder that leads to multiple organ failure. Amongst organs affected, myocardial depression is believed to be a major contributor to septic death. While it has been identified that large amounts of circulating pro-inflammatory cytokines are culprit for triggering cardiac dysfunction in sepsis, the underlying mechanisms remain obscure. Additionally, recent studies have shown that exosomes released from bacteria-infected macrophages are pro-inflammatory. Hence, we examined in this study whether blocking the generation of exosomes would be protective against sepsis-induced inflammatory response and cardiac dysfunction. To this end, we pre-treated RAW264.7 macrophages with GW4869, an inhibitor of exosome biogenesis/release, followed by endotoxin (LPS) challenge. In vivo , we injected wild-type (WT) mice with GW4869 for 1 h prior to endotoxin treatment or cecal ligation/puncture (CLP) surgery. We observed that pre-treatment with GW4869 significantly impaired release of both exosomes and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in RAW264.7 macrophages. At 12 h after LPS treatment or CLP surgery, WT mice pre-treated with GW4869 displayed lower amounts of exosomes and pro-inflammatory cytokines in the serum than control PBS-injected mice. Accordingly, GW4869 treatment diminished the sepsis-induced cardiac inflammation, attenuated myocardial depression and prolonged survival. Together, our findings indicate that blockade of exosome generation in sepsis dampens the sepsis-triggered inflammatory response and thereby, improves cardiac function and survival. [ABSTRACT FROM AUTHOR]

Both calpain activation and endoplasmic reticulum (ER) stress are implicated in ischemic heart injury. However, the role of calpain in ER stress remains largely elusive. This study investigated whether calpain activation causes ER stress, thereby mediating cardiomyocyte apoptosis in an in vitro model of hypoxia/re-oxygenation (H/R). In neonatal mouse cardiomyocytes and rat cardiomyocyte-like H9c2 cells, up-regulation of calpain-1 sufficiently induced ER stress, c-Jun N-terminal protein kinase1/2 (JNK1/2) activation and apoptosis. Inhibition of ER stress or JNK1/2 prevented apoptosis induced by calpain-1. In an in vitro model of H/R-induced injury in cardiomyocytes, H/R was induced by a 24-hour hypoxia followed by a 24-hour re-oxygenation. H/R activated calpain-1, induced ER stress and JNK1/2 activation, and triggered apoptosis. Inhibition of calpain and ER stress blocked JNK1/2 activation and prevented H/R-induced apoptosis. Furthermore, blockade of JNK1/2 signaling inhibited apoptosis following H/R. The role of calpain in ER stress was also demonstrated in an in vivo model of ischemia/reperfusion using transgenic mice over-expressing calpastatin. In summary, calpain-1 induces ER stress and JNK1/2 activation, thereby mediating apoptosis in cardiomyocytes. Accordingly, inhibition of calpain prevents ER stress, JNK1/2 activation and apoptosis in H/R-induced cardiomyocytes. Thus, ER stress/JNK1/2 activation may represent an important mechanism linking calpain-1 to ischemic injury. [ABSTRACT FROM AUTHOR]

Abstract: Sepsis is the leading cause of death in critically ill patients. While myocardial dysfunction has been recognized as a major manifestation in severe sepsis, the underlying molecular mechanisms associated with septic cardiomyopathy remain unclear. In this study, we performed a miRNA array analysis in hearts collected from a severe septic mouse model induced by cecal ligation and puncture (CLP). Among the 19 miRNAs that were dys-regulated in CLP-mouse hearts, miR-223(3p) and miR-223*(5p) were most significantly downregulated, compared with sham-operated mouse hearts. To test whether a drop of miR-223 duplex plays any roles in sepsis-induced cardiac dysfunction and inflammation, a knockout (KO) mouse model with a deletion of the miR-223 gene locus and wild-type (WT) mice were subjected to CLP or sham surgery. We observed that sepsis-induced cardiac dysfunction, inflammatory response and mortality were remarkably aggravated in CLP-treated KO mice, compared with control WTs. Using Western-blotting and luciferase reporter assays, we identified Sema3A, an activator of cytokine storm and a neural chemorepellent for sympathetic axons, as an authentic target of miR-223* in the myocardium. In addition, we validated that miR-223 negatively regulated the expression of STAT-3 and IL-6 in mouse hearts. Furthermore, injection of Sema3A protein into WT mice revealed an exacerbation of sepsis-triggered inflammatory response and myocardial depression, compared with control IgG1 protein-treated WT mice following CLP surgery. Taken together, these data indicate that loss of miR-223/-223* causes an aggravation of sepsis-induced inflammation, myocardial dysfunction and mortality. Our study uncovers a previously unrecognized mechanism underlying septic cardiomyopathy and thereby, may provide a new strategy to treat sepsis. [Copyright &y& Elsevier]

Highlights: [•] Establishing an in vitro model of Coxsackievirus B3 (CVB3) infection in H9c2 cells. [•] CVB3 infection triggers intracellular calpain activity. [•] Calpain activity facilitates CVB3 replication via enhancing autophagy. [•] Both calpain and autophagy hinder the early phase host cell apoptosis. [Copyright &y& Elsevier]

Lipid overload contributes to cardiac complications of diabetes and obesity. However, the underlying mechanisms remain obscure. This study investigates the role of gamma-aminobutyrate transaminase (ABAT), the key enzyme involved in the catabolism of γ-aminobutyric acid (GABA), in lipid overload-induced cardiac injury. Microarray revealed a down-regulation of ABAT mRNA expression in high fat diet (HFD)-fed mouse hearts, which correlated with a reduction in ABAT protein level and its GABA catabolic activity. Transgenic mice with cardiomyocyte-specific ABAT over-expression (Tg-ABAT/tTA) were generated to determine the role of ABAT in lipid overload-induced cardiac injury. Feeding with a HFD to control mice for 4 months reduced ATP production and the mitochondrial DNA copy number, and induced myocardial oxidative stress, hypertrophy, fibrosis and dysfunction. Such pathological effects of HFD were mitigated by ABAT over-expression in Tg-ABAT/tTA mice. In cultured cardiomyocytes, palmitate increased mitochondrial ROS production, depleted ATP production and promoted apoptosis, all of which were attenuated by ABAT over-expression. With the inhibition of ABAT's GABA catabolic activity, the protective effects of ABAT remained unchanged in palmitate-induced cardiomyocytes. Thus, ABAT protects the mitochondrial function in defending the heart against lipid overload-induced injury through mechanisms independent of its GABA catabolic activity, and may represent a new therapeutic target for lipid overload-induced cardiac injury. [ABSTRACT FROM AUTHOR]

Th17 cells have been implicated in the pathogenesis of myocarditis. Interleukin (IL)-17A produced by Th17 cells is dispensable for viral myocarditis but essential for the progression to dilated cardiomyopathy (DCM). This study investigated whether the adenoviral transfer of the IL-17 receptor A reduces myocardial remodeling and dysfunction in viral myocarditis leading to DCM. In a mouse model of Coxsackievirus B3 (CVB3)-induced chronic myocarditis, the delivery of the adenovirus-containing IL-17 receptor A (Ad-IL17RA:Fc) reduced IL-17A production and decreased the number of Th17 cells in the spleen and heart, leading to the down-regulation of systemic TNF-α and IL-6 production. Cardiac function improved significantly in the Ad-IL17R:Fc- compared with the Ad-null-treated mice 3 months after the first CVB3 infection. Ad-IL17R:Fc reduced the left ventricle dilation and decreased the mortality in viral myocarditis, leading to DCM (56% in the Ad-IL17R:Fc versus 76% in the Ad-null group). The protective effects of Ad-IL17R-Fc on remodeling correlated with the attenuation of myocardial collagen deposition and the reduction of fibroblasts in CVB3-infected hearts, which was accompanied by the down-regulation of A distintegrin and metalloprotease with thrombospondin type 1 motifs (ADAMTS-1), Matrix metalloproteinase-2(MMP-2), and collagen subtypes I and III in the heart. Moreover, in cultured cardiac fibroblasts, IL-17A induced the expression of ADAMTS-1, MMP-2, and collagen subtypes I and III and increased the proliferation of fibroblasts. We determined that the delivery of IL-17-RA:Fc reduces cardiac remodeling, improves function, and decreases mortality in viral myocarditis leading to DCM, possibly by suppressing fibrosis. Therefore, the adenoviral transfer of the IL-17 receptor A may represent an alternative therapy for chronic viral myocarditis and its progression to DCM. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The outer membrane protein RagB is one of the major virulence factors of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis). In order to induce protective immune response against P. gingivalis infection, an mGITRL gene-linked ragB DNA vaccine (pIRES-ragB-mGITRL ) was constructed. Six-week-old female BALB/c mice were immunized with pIRES-ragB-mGITRL through intramuscular injection and then challenged by subcutaneous injection in the abdomen with P. gingivalis. RagB-specific antibody-forming cells were evaluated by an Enzyme-linked immunosorbent spot, and specific antibody was determined by enzyme-linked immunosorbent assay. In addition, the frequencies of Tfh and IFN-γ+ T cells in spleen were measured using flow cytometer, and the levels of IL-21 and IFN-γ mRNA or proteins were detected by real time RT-PCR or ELISA. The data showed that the mGITRL-linked ragB DNA vaccine induced higher levels of RagB-specific IgG in serum and RagB-specific antibody-forming cells in spleen. The frequencies of Tfh and IFN-γ+ T cells were obviously expanded in mice immunized by pIRES-ragB-mGITRL compared with other groups (pIRES or pIRES-ragB ). The levels of Tfh and IFN-γ+ T cells associated cytokines were also significantly increased in pIRES-ragB-mGITRL group. Therefore, the mice immunized with ragB plus mGITRL showed the stronger resistant to P. gingivalis infection and a significant reduction of the lesion size caused by P. gingivalis infection comparing with other groups. Taken together, our findings demonstrated that intramuscular injection of DNA vaccine ragB together with mGITRL induced protective immune response dramatically by increasing Tfh and IFN-γ+ T cells and antibody production to P. gingivalis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Calpain has been implicated in acute myocardial injury after myocardial infarction (MI). However, the causal relationship between calpain and post-MI myocardial remodeling has not been fully understood. This study examined whether deletion of Capn4, essential for calpain-1 and calpain-2 activities, reduces myocardial remodeling and dysfunction following MI, and if yes, whether these effects of Capn4 deletion are associated with NF-κB signaling and inflammatory responses in the MI heart. A novel mouse model with cardiomyocyte-specific deletion of Capn4 (Capn4-ko) was employed. MI was induced by left coronary artery ligation. Deficiency of Capn4 dramatically reduced the protein levels and activities of calpain-1 and calpain-2 in the Capn4-ko heart. In vivo cardiac function was relatively improved in Capn4-ko mice at 7 and 30 days after MI when compared with their wild-type littermates. Deletion of Capn4 reduced apoptosis, limited infarct expansion, prevented left ventricle dilation, and reduced mortality in Capn4-ko mice. Furthermore, cardiomyocyte cross-sectional areas and myocardial collagen deposition were significantly attenuated in Capn4-ko mice, which were accompanied by down-regulation of hypertrophic genes and profibrotic genes. These effects of Capn4 knock-out correlated with restoration of IκB protein and inhibition of NF-κB activation, leading to suppression of proinflammatory cytokine expression and inflammatory cell infiltration in the Capn4-ko heart after MI. In conclusion, deficiency of Capn4 reduces adverse myocardial remodeling and myocardial dysfunction after MI. These effects of Capn4 deletion may be mediated through prevention of IκB degradation and NF-κB activation, resulting in inhibition of inflammatory responses. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Recently we have shown that calpain-1 activation contributes to cardiomyocyte apoptosis induced by hyperglycemia. This study was undertaken to investigate whether targeted disruption of calpain would reduce myocardial hypertrophy and fibrosis in mouse models of type 1 diabetes. RESEARCH DESIGN AND METHODS--Diabetes in mice was induced by injection of streptozotocin (STZ), and OVE26 mice were also used as a type 1 diabetic model. The function of calpain was genetically manipulated by cardiomyocyte-specific knockout Capn4 in mice and the use of calpastatin transgenic mice. Myocardial hypertrophy and fibrosis were investigated 2 and 5 months after STZ injection or in OVE26 diabetic mice at the age of 5 months. Cultured isolated adult mouse cardiac fibroblast cells were also investigated under high glucose conditions. RESULTS--Calpain activity, cardiomyocyte cross-sectional areas, and myocardial collagen deposition were significantly increased in both STZ-induced and OVE26 diabetic hearts, and these were accompanied by elevated expression of hypertrophic and fibrotic collagen genes. Deficiency of Capn4 or overexpression of calpastatin reduced myocardial hypertrophy and fibrosis in both diabetic models, leading to the improvement of myocardial function. These effects were associated with a normalization of the nuclear factor of activated T-cell nuclear factor-κB and matrix metalloproteinase (MMP) activities in diabetic hearts. In cultured cardiac fibroblasts, high glucose--induced proliferation and MMP activities were prevented by calpain inhibition. CONCLUSIONS--Myocardial hypertrophy and fibrosis in diabetic mice are attenuated by reduction of calpain function. Thus targeted inhibition of calpain represents a potential novel therapeutic strategy for reversing diabetic cardiomyopathy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Abstract: Diabetes increases myocardial ischemia/reperfusion (I/R) injury. However, the underlying mechanisms remain incompletely understood. This study investigated the role of Rac1 signaling and calpain in exacerbated I/R injury in diabetic hearts. Mice with cardiac-specific deletion of Rac1 (Rac1-ko) and transgenic mice with cardiac-specific superoxide dismutase-2 (SOD2) or calpastatin overexpression were rendered diabetic with streptozotocin. Isolated perfused hearts were subjected to global I/R. After I/R, Rac1 activity was significantly enhanced in diabetic compared with nondiabetic hearts. Diabetic hearts displayed more severe I/R injury than nondiabetic hearts, as evidenced by more lactate dehydrogenase release and apoptosis and decreased cardiac function. These adverse impacts of diabetes were abrogated in Rac1-ko hearts or by perfusion with the Rac1 inhibitor NSC23766. In an in vivo I/R mouse model, infarct size was much smaller in diabetic Rac1-ko compared with wild-type mice. Inhibition of Rac1 signaling prevented NADPH oxidase activation, reactive oxygen species production, and protein carbonyl accumulation, leading to inhibition of calpain activation. Furthermore, SOD2 or calpastatin overexpression significantly reduced I/R injury in diabetic hearts and improved cardiac function after I/R. In summary, Rac1 activation increases I/R injury in diabetic hearts and the role of Rac1 signaling is mediated, at least in part, through calpain activation. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Our recent study demonstrated that Rac1 and NADPH oxidase activation contributes to cardiomyocyte apoptosis in short-term diabetes. This study was undertaken to investigate if disruption of Rac1 and inhibition of NADPH oxidase would prevent myocardial remodeling in chronic diabetes. RESEARCH DESIGN AND METHODS--Diabetes was induced by injection of streptozotocin in mice with cardiomyocyte-specific Rac1 knockout and their wild-type littermates. In a separate experiment, wild-type diabetic mice were treated with vehicle or apocynin in drinking water. Myocardial hypertrophy, fibrosis, endoplasmic reticulum (ER) stress, inflammatory response, and myocardial function were investigated after 2 months of diabetes. Isolated adult rat cardiomyocytes were cultured and stimulated with high glucose. RESULTS--In diabetic hearts, NADPH oxidase activation, its subunits' expression, and reactive oxygen species production were inhibited by Rac1 knockout or apocynin treatment. Myocardial collagen deposition and cardiomyocyte cross-sectional areas were significantly increased in diabetic mice, which were accompanied by elevated expression of pro-fibrotic genes and hypertrophic genes. Deficiency of Rac1 or apocynin administration reduced myocardial fibrosis and hypertrophy, resulting in improved myocardial function. These effects were associated with a normalization of ER stress markers' expression and inflammatory response in diabetic hearts. In cultured cardiomyocytes, high glucose-induced ER stress was inhibited by blocking Rac1 or NADPH oxidase. CONCLUSIONS--Rac1 via NADPH oxidase activation induces myocardial remodeling and dysfunction in diabetic mice. The role of Rac1 signaling may be associated with ER stress and inflammation. Thus, targeting inhibition of Rac1 and NADPH oxidase may be a therapeutic approach for diabetic cardiomyopathy. Diabetes 59:2033-2042, 2010 [ABSTRACT FROM AUTHOR]

Lipopolysaccharides (LPS) induce tumor necrosis factor-α (TNF-Ξ) production in cardiomyocytes, which contributes to myocardial depression during sepsis. However, the underlying mechanisms remain not fully understood. This study was undertaken to investigate the contribution of histone deacetylase (HDAC) to TNF-Ξ expression in cardiomyocytes and the signaling mechanism of LPS-induced HDAC activation. Here, we show for the first time that LPS increases HDAC activity and that inhibition of HDAC decreases LPS-stimulated TNF-Ξ expression via the accumulation of NF-κB/p65 at the TNF-α promoter in cardiomyocytes. Using a positive screen, we have further identified HDAC3 as a specific member of the HDAC family able to regulate TNF-α production. Furthermore, our data reveal that LPS-induced HDAC activity is mediated through reactive oxygen species from mitochondria and c-Src signaling. In summary, this study demonstrates a novel signaling mechanism by which LPS via mitochondrial reactive oxygen species/c-Src/HDAC3 pathways mediate TNF-α expression in cardiomyocytes. [ABSTRACT FROM AUTHOR]

Abstract: This study was to investigate the role of calpain in the apoptosis of pulmonary microvascular endothelial cells (PMEC) during septic plasma stimulation. Septic plasma was collected from endotoxemic mice. In cultured PMEC, incubation with septic plasma stimulated calpain activation, increased caspase-3 activity and induced apoptotic cell death. These effects of septic plasma were abrogated by knockdown of calpain-1 but not calpain-2 using specific siRNA. Consistently, treatment with calpain inhibitor-III, or over-expression of calpastatin, an endogenous calpain inhibitor significantly decreased apoptosis induced by septic plasma. Septic plasma also induced NADPH oxidase activation and reactive oxygen species (ROS) production. Inhibiting NADPH oxidase or scavenging ROS attenuated calpain activity and decreased apoptosis in PMEC during septic plasma stimulation. In summary, our study demonstrates that ROS produced from NADPH oxidase stimulates calpain-1 activation, which induces apoptosis under septic conditions. Thus, targeting calpain-1/calpastatin may represent a potential strategy to protect against endothelial injury in sepsis. [Copyright &y& Elsevier]

Abstract: Taurine has been shown to prevent cardiomyocyte apoptosis. This study investigated the effects of taurine on NADPH oxidase and calpain activation in mediating apoptosis in cardiomyocytes. Apoptosis was induced by norepinephrine (NE) in cultured adult rat ventricular cardiomyocytes. NE (5 μM) increased NADPH oxidase activation and reactive oxygen species (ROS) production and induced apoptosis. These effects of NE on cardiomyocytes were diminished by taurine (0.5 mg/kg) but not β-alanine. Inhibition of gp91 phox -NADPH oxidase or ROS production protected cardiomyocytes from apoptosis. NE also induced calpain-1 activation in cardiomyocytes. This effect of NE on calpain was abrogated by gp91 phox -NADPH oxidase inhibition or ROS scavengers and was mimicked by H2O2 (25 μM) in cardiomyocytes. Pharmacological inhibitors of calpain or overexpression of calpastatin, a specific calpain inhibitor, blocked calpain activation and prevented cardiomyocyte apoptosis during NE stimulation. Furthermore, taurine treatment inhibited NE- or H2O2-induced calpain activation in cardiomyocytes. In conclusion, NADPH oxidase induces calpain activation, leading to apoptosis in NE-induced cardiomyocytes. Taurine inhibits NADPH oxidase and calpain activation. Thus, inhibition of NADPH oxidase-mediated calpain activation may be an important mechanism for taurine''s antiapoptotic action in cardiomyocytes. [Copyright &y& Elsevier]

Abstract: Lipopolysaccharide (LPS) induces cyclooxygenase-2 (COX-2) expression in cardiomyocytes, which plays a role in myocardial depression during endotoxemia. The purpose of this study was to investigate the role of phosphatidylinositol (PI)-phospholipase Cγ1 (PLCγ1) in cardiac COX-2 expression in vitro and in vivo. In cultured mouse neonatal cardiomyocytes, LPS increased PLCγ1 phosphorylation and COX-2 expression. Knockdown of PLCγ1 with specific siRNA or inhibition of PI-PLC with U73122 attenuated COX-2 mRNA and protein expression induced by LPS (1 μg/ml). PLCγ1 activation by LPS also increased ERK1/2 MAPK phosphorylation, and inhibition of ERK1/2 MAPK blocked the effect of PLCγ1 on COX-2 expression. Furthermore, activation of PLCγ1 is a consequence of the Src family activation since inhibition of Src abrogated whereas over-expression of Src enhanced PLCγ1 phosphorylation and COX-2 expression in LPS-stimulated cardiomyocytes. To investigate the role of PLCγ1 in endotoxemia, wild-type and PLCγ1+/− adult mice were pre-treated with U73122, or its inactive analog, U73343 (9 mg/kg, i.p.), or vehicle for 15 min followed by LPS (4 mg/kg, i.p.) for 4 h. U73122 or heterozygous deletion of PLCγ1 decreased cardiac COX-2 expression. The phosphorylation of ERK1/2 MAPK induced by LPS was also attenuated in U73122- or PLCγ1+/− compared to U73343-treated or wild-type littermate hearts, respectively. In conclusion, our study suggests that PLCγ1 signalling represents a novel pathway regulating cardiac COX-2 expression during LPS stimulation. The Src family is responsible for PLCγ1 activation, which signals the ERK1/2 MAPK pathway, resulting in COX-2 production in LPS-stimulated cardiomyocytes. [Copyright &y& Elsevier]

Abstract: 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-α) expression during lipopolysaccharide (LPS) stimulation. However, the role of nNOS in LPS-induced TNF-α expression is not known. We hypothesized that nNOS suppresses LPS-induced TNF-α 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 Ca2+ concentration, LPS increased peak Ca2+ levels. LPS stimulation increased TNF-α 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-α expression during LPS stimulation. Furthermore, LPS-induced ERK phosphorylation was significantly increased in the nNOS−/− compared to wild-type cardiomyocytes. The enhanced TNF-α expression in nNOS−/− cardiomyocytes was abrogated by an L-type calcium channel blocker verapamil or ERK1 siRNA. Finally, myocardial ERK phosphorylation and TNF-α expression were increased while cardiac function was decreased in endotoxemia in nNOS−/− compared to wild-type mice. Conclusions: nNOS inhibits LPS-induced TNF-α 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Cardiac cells can adapt to pathological stress-induced energy crisis by shifting from fatty acid oxidation to glycolysis. However, the use of glucose-insulin-potassium (GIK) solution in patients undergoing cardiac surgery does not alleviate ischemia/reperfusion (I/R)-induced energy shortage. This indicates that insulin-mediated translocation of glucose transporter-4 (Glut-4) is impaired in ischemic hearts. Indeed, cardiac myocytes contain two intracellular populations of Glut-4: an insulin-dependent non-endosomal pool (also referred to as Glut-4 storage vesicles, GSVs) and an insulin-independent endosomal pool. Tumor susceptibility gene 101 (Tsg101) has been implicated in the endosomal recycling of membrane proteins. In this study, we aimed to examine whether Tsg101 regulated the sorting and re-distribution of Glut-4 to the sarcolemma membrane of cardiomyocytes under basal and ischemic conditions, using gain- and loss-of-function approaches. Forced overexpression of Tsg101 in mouse hearts and isolated cardiomyocytes could promote Glut-4 re-distribution to the sarcolemma, leading to enhanced glucose entry and adenosine triphosphate (ATP) generation in I/R hearts which in turn, attenuation of I/R-induced cardiac dysfunction. Conversely, knockdown of Tsg101 in cardiac myocytes exhibited opposite effects. Mechanistically, we identified that Tsg101 could interact and co-localize with Glut-4 in the sarcolemma membrane of cardiomyocytes. Our findings define Tsg101 as a novel regulator of cardiac Glut-4 trafficking, which may provide a new therapeutic strategy for the treatment of ischemic heart disease. [ABSTRACT FROM AUTHOR]

Background: Spaceflight or microgravity conditions cause myocardial atrophy and dysfunction, contributing to post-flight orthostatic intolerance. However, the underlying mechanisms remain incompletely understood and preventive approaches are limited. This study investigated whether and how losartan, a blocker of angiotensin-II receptor, preserved cardiomyocyte size and prevented myocardial dysfunction during microgravity.Method: Adult male mice were suspended with their tails to simulate microgravity. Echocardiography was performed to assess myocardial function. Heart weight and cardiomyocyte size were measured. NADPH oxidase activation was determined by analyzing membrane translocation of its cytosolic subunits including p47phox, p67phox and Rac1. Heart tissues were also assayed for oxidative stress, p47phox phosphorylation (Ser345), MuRF1 protein levels and angiotensin-II production.Results: Tail-suspension for 28 days increased angiotensin-II production in hearts, decreased cardiomyocyte size and heart weight, and induced myocardial dysfunction. Administration of losartan preserved cardiomyocyte size and heart weight, and prevented myocardial dysfunction in tail-suspended mice. These cardioprotective effects of losartan were associated with inhibition of p47phox phosphorylation (Ser345), NADPH oxidase and oxidative stress in tail-suspended mouse hearts. Additionally, the NADPH oxidase inhibitor, apocynin, also reduced oxidative stress, preserved cardiomyocyte size and heart weight, and improved myocardial function in tail-suspended mice. Furthermore, losartan but not apocynin attenuated tail-suspension-induced up-regulation of MuRF1 protein in mouse hearts.Conclusions: Administration of losartan preserves cardiomyocyte size and prevents myocardial dysfunction under microgravity by blocking p47phox phosphorylation and NADPH oxidase activation, and by inhibiting MuRF1 expression. Thus, losartan may be a useful drug to prevent microgravity-induced myocardial abnormalities. [ABSTRACT FROM AUTHOR]

We and others have reported that calpain-1 was increased in myocardial mitochondria from various animal models of heart disease. This study investigated whether constitutive up-regulation of calpain-1 restricted to mitochondria induced myocardial injury and heart failure and, if so, whether these phenotypes could be rescued by selective inhibition of mitochondrial superoxide production. Transgenic mice with human CAPN1 up-regulation restricted to mitochondria in cardiomyocytes (Tg-mtCapn1/tTA) were generated and characterized with low and high over-expression of transgenic human CAPN1 restricted to mitochondria, respectively. Transgenic up-regulation of mitochondria-targeted CAPN1 dose-dependently induced cardiac cell death, adverse myocardial remodeling, heart failure, and early death in mice, the changes of which were associated with mitochondrial dysfunction and mitochondrial superoxide generation. Importantly, a daily injection of mitochondria-targeted superoxide dismutase mimetics mito-TEMPO for 1 month starting from age 2 months attenuated cardiac cell death, adverse myocardial remodeling and heart failure, and reduced mortality in Tg-mtCapn1/tTA mice. In contrast, administration of TEMPO did not achieve similar cardiac protection in transgenic mice. Furthermore, transgenic up-regulation of mitochondria-targeted CAPN1 induced a reduction of ATP5A1 protein and ATP synthase activity in hearts. In cultured cardiomyocytes, increased calpain-1 in mitochondria promoted mitochondrial permeability transition pore (mPTP) opening and induced cell death, which were prevented by over-expression of ATP5A1, mito-TEMPO or cyclosporin A, an inhibitor of mPTP opening. In conclusion, this study has provided direct evidence demonstrating that increased mitochondrial calpain-1 is an important mechanism contributing to myocardial injury and heart failure by disrupting ATP synthase, and promoting mitochondrial superoxide generation and mPTP opening. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However, the underlying mechanism remains obscure. While recent studies have indicated that miR-223 is highly enriched in MSC-derived exosomes, whether exosomal miR-223 contributes to MSC-mediated cardio-protection in sepsis is unknown. In this study, loss-of-function approach was utilized, and sepsis was induced by cecal ligation and puncture (CLP). We observed that injection of miR-223-KO MSCs at 1 h post-CLP did not confer protection against CLP-triggered cardiac dysfunction, apoptosis and inflammatory response. However, WT-MSCs were able to provide protection which was associated with exosome release. Next, treatment of CLP mice with exosomes released from miR-223-KO MSCs significantly exaggerated sepsis-induced injury. Conversely, WT-MSC-derived-exosomes displayed protective effects. Mechanistically, we identified that miR-223-KO exosomes contained higher levels of Sema3A and Stat3, two known targets of miR-223 (5p &3p), than WT-exosomes. Accordingly, these exosomal proteins were transferred to cardiomyocytes, leading to increased inflammation and cell death. By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3. These data for the first time indicate that exosomal miR-223 plays an essential role for MSC-induced cardio-protection in sepsis. [ABSTRACT FROM AUTHOR]