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Fibroblasts can be reprogrammed into cardiovascular progenitor cells (CPCs) using transgenic approaches, although the underlying mechanism remains unclear. We determined whether activation of endogenous genes such as Gata4, Nkx2.5, and Tbx5 can rapidly establish autoregulatory loops and initiate CPC generation in adult extracardiac fibroblasts using a CRISPR activation system. The induced fibroblasts (>80%) showed phenotypic changes as indicated by an Nkx2.5 cardiac enhancer reporter. The progenitor characteristics were confirmed by colony formation and expression of cardiovascular genes. Cardiac sphere induction segregated the early and late reprogrammed cells that can generate functional cardiomyocytes and vascular cells in vitro. Therefore, they were termed CRISPR-induced CPCs (ciCPCs). Transcriptomic analysis showed that cell cycle and heart development pathways were important to accelerate CPC formation during the early reprogramming stage. The CRISPR system opened the silenced chromatin locus, thereby allowing transcriptional factors to access their own promoters and eventually forming a positive feedback loop. The regenerative potential of ciCPCs was assessed after implantation in mouse myocardial infarction models. The engrafted ciCPCs differentiated into cardiovascular cells in vivo but also significantly improved contractile function and scar formation. In conclusion, multiplex gene activation was sufficient to drive CPC reprogramming, providing a new cell source for regenerative therapeutics.

Abnormal cell proliferation caused by abnormal transcription regulation mechanism seems to be one of the reasons for the progression of breast cancer and also the pathological basis. MicroRNA-142-5p (miR-142-5p) is a low-expressed miRNA in breast cancer. The role of MKL-1s regulation of DNMT1 in breast cancer cell proliferation and migration is still unclear. MKL-1 (myocardin related transcription factor A) can bind to the conserved cis-regulatory element CC (A/T) 6GG (called CarG box) in the promoter to regulate the transcription of miR-142-5p. The expressions of miR-142-5p and MKL-1 are positively correlated. In addition, it has been proved that DNMT1 is the target of miR-142-5p, which inhibits the expression of DNMT1 by targeting the 3-UTR of DNMT1, thereby forming a feedback loop and inhibiting the migration and proliferation of breast cancer. Our data provide important and novel insights into the MKL-1/miR-142-5p/DNMT1/maspin signaling pathway and may become a new idea for breast cancer diagnosis, treatment, and prognosis.

The human cardiovascular system has adapted to function optimally in Earth's 1G gravity, and microgravity conditions cause myocardial abnormalities, including atrophy and dysfunction. However, the underlying mechanisms linking microgravity and cardiac anomalies are incompletely understood. In this study, we investigated whether and how calpain activation promotes myocardial abnormalities under simulated microgravity conditions. Simulated microgravity was induced by tail suspension in mice with cardiomyocyte-specific deletion of Capns1, which disrupts activity and stability of calpain-1 and calpain-2, and their WT littermates. Tail suspension time-dependently reduced cardiomyocyte size, heart weight, and myocardial function in WT mice, and these changes were accompanied by calpain activation, NADPH oxidase activation, and oxidative stress in heart tissues. The effects of tail suspension were attenuated by deletion of Capns1. Notably, the protective effects of Capns1 deletion were associated with the prevention of phosphorylation of Ser-345 on p47phox and attenuation of ERK1/2 and p38 activation in hearts of tail-suspended mice. Using a rotary cell culture system, we simulated microgravity in cultured neonatal mouse cardiomyocytes and observed decreased total protein/DNA ratio and induced calpain activation, phosphorylation of Ser-345 on p47phox, and activation of ERK1/2 and p38, all of which were prevented by calpain inhibitor-III. Furthermore, inhibition of ERK1/2 or p38 attenuated phosphorylation of Ser-345 on p47phox in cardiomyocytes under simulated microgravity. This study demonstrates for the first time that calpain promotes NADPH oxidase activation and myocardial abnormalities under microgravity by facilitating p47phox phosphorylation via ERK1/2 and p38 pathways. Thus, calpain inhibition may be an effective therapeutic approach to reduce microgravity-induced myocardial abnormalities.

The inability to effectively control invading bacteria or other pathogens is a major cause of multiple organ dysfunction and death in sepsis. As the first-line defense of the immune system, macrophages play a crucial role in the removal of pathogens during sepsis. In this study, we define secreted and transmembrane 1A (Sectm1a) as a novel ligand of glucocorticoid-induced TNFR (GITR) that greatly boosts macrophage phagocytosis and bactericidal capacity. Using a global Sectm1a knockout (KO) mouse model, we observed that Sectm1a deficiency significantly suppressed phagocytosis and bactericidal activity in both recruited macrophages and tissue-resident macrophages, which consequently aggravated bacterial burden in the blood and multiple organs and further increased systemic inflammation, leading to multiple organ injury and increased mortality during polymicrobial sepsis. By contrast, treatment of septic mice with recombinant Sectm1a protein (rSectm1a) not only promoted macrophage phagocytosis and bactericidal activity but also significantly improved survival outcome. Mechanistically, we identified that Sectm1a could bind to GITR in the surface of macrophages and thereby activate its downstream PI3K–Akt pathway. Accordingly, rSectm1a-mediated phagocytosis and bacterial killing were abolished in macrophages by either KO of GITR or pharmacological inhibition of the PI3K–Akt pathway. In addition, rSectm1a-induced therapeutic effects on sepsis injury were negated in GITR KO mice. Taken together, these results uncover that Sectm1a may represent a novel target for drug development to control bacterial dissemination during sepsis or other infectious diseases.

Aim 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 LPS injection, when compared to 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 downregulated 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 signaling in macrophages. Our data suggest that augmenting Sectm1a activity may be a potential therapeutic approach to resolve inflammation and associated cardiac dysfunction. Translational perspective Better understanding on the interaction between inflammatory responses and cardiac health is prominent for the development of safer and more efficacious therapies for heart failure patients. The present study, using both acute (LPS) and chronic (high-fat diet) inflammation models, reiterated the adverse effects of abnormal macrophages activation on cardiac function. Our Sectm1a knockout mouse model showed exacerbated cardiac and systemic inflammatory responses, resulting in further aggravation of contractile dysfunction on the heart after endotoxin challenge. We also demonstrated Sectm1a as a new regulator of macrophage function through LXRα pathway. These data suggest a novel approach to regulate macrophage-elicited inflammation.

N6-Methyladenosine (m6A), a unique and common mRNA modification method in eukaryotes, is involved in the occurrence and development of many diseases. Liver fibrosis (LF) is a common response to chronic liver injury and may lead to cirrhosis and even liver cancer. However, the involvement of m6A methylation in the development of LF is still unknown. In this study, we performed a systematic evaluation of hepatic genome-wide m6A modification and mRNA expression by m6A-seq and RNA-seq using LF mice. There were 3,315 genes with significant differential m6A levels, of which 2,498 were hypermethylated and 817 hypomethylated. GO and KEGG analyses illustrated that differentially expressed m6A genes were closely correlated with processes such as the endoplasmic reticulum stress response, PPAR signaling pathway and TGF-β signaling pathway. Moreover, a total of 90 genes had both a significant change in the m6A level and mRNA expression shown by joint analysis of m6A-seq and RNA-seq. Hence, the critical elements of m6A modification, including methyltransferase WTAP, demethylases ALKBH5 and binding proteins YTHDF1 were confirmed by RT-qPCR and Western blot. In an additional cell experiment, we also observed that the decreased expression of WTAP induced the development of LF as a result of promoting hepatic stellate cell (HSC) activation. Therefore, this study revealed unique differential m6A methylation patterns in LF mice and suggested that m6A methylation was associated with the occurrence and course of LF to some extent.

A fluorescence-enhanced and lysosome-targeted Al3+ probe (1) based on a pyrazole-containing hydrazone was developed in this work. The fluorescent probe 1 displays an OFF–ON fluorescence response to Al3+ in the lysosome pH environment. In addition, the resultant 1–Al3+ complex could act as an ON–OFF fluorescence sensor for F−. Fluorescence imaging shows that 1 is suitable for visualization of Al3+ in lysosomes of HeLa cells.

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 p47 phox , p67 phox and Rac1. Heart tissues were also assayed for oxidative stress, p47 phox 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 p47 phox 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 p47 phox phosphorylation and NADPH oxidase activation, and by inhibiting MuRF1 expression. Thus, losartan may be a useful drug to prevent microgravity-induced myocardial abnormalities.

The initiation and development of diabetes are mainly ascribed to the loss of functional β-cells. Therapies designed to regenerate β-cells provide great potential for controlling glucose levels and thereby preventing the devastating complications associated with diabetes. This requires detailed knowledge of the molecular events and underlying mechanisms in this disorder. Here, we report that expression of microRNA-223 (miR-223) is up-regulated in islets from diabetic mice and humans, as well as in murine Min6 β-cells exposed to tumor necrosis factor α (TNFα) or high glucose. Interestingly, miR-223 knockout (KO) mice exhibit impaired glucose tolerance and insulin resistance. Further analysis reveals that miR-223 deficiency dramatically suppresses β-cell proliferation and insulin secretion. Mechanistically, using luciferase reporter gene assays, histological analysis, and immunoblotting, we demonstrate that miR-223 inhibits both forkhead box O1 (FOXO1) and SRY-box 6 (SOX6) signaling, a unique bipartite mechanism that modulates expression of several β-cell markers (pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), and urocortin 3 (UCN3)) and cell cycle-related genes (cyclin D1, cyclin E1, and cyclin-dependent kinase inhibitor P27 (P27)). Importantly, miR-223 overexpression in β-cells could promote β-cell proliferation and improve β-cell function. Taken together, our results suggest that miR-223 is a critical factor for maintaining functional β-cell mass and adaptation during metabolic stress.

Doxorubicin (DOX) is widely used as a first-line chemotherapeutic drug for various malignancies. However, DOX causes severe cardiotoxicity, which limits its clinical uses. Oxidative stress is one of major contributors to DOX-induced cardiotoxicity. While autophagic flux serves as an important defense mechanism against oxidative stress in cardiomyocytes, recent studies have demonstrated that DOX induces the blockage of autophagic flux, which contributes to DOX cardiotoxicity. The present study investigated whether nicotinamide riboside (NR), a precursor of nicotinamide adenine dinucleotide (NAD)+, prevents DOX cardiotoxicity by improving autophagic flux. We report that administration of NR elevated NAD+ levels, and reduced cardiac injury and myocardial dysfunction in DOX-injected mice. These protective effects of NR were recapitulated in cultured cardiomyocytes upon DOX treatment. Mechanistically, NR prevented the blockage of autophagic flux, accumulation of autolysosomes, and oxidative stress in DOX-treated cardiomyocytes, the effects of which were associated with restoration of lysosomal acidification. Furthermore, inhibition of lysosomal acidification or SIRT1 abrogated these protective effects of NR during DOX-induced cardiotoxicity. Collectively, our study shows that NR enhances autolysosome clearance via the NAD+/SIRT1 signaling, thereby preventing DOX-triggered cardiotoxicity.

Visual Abstract, Highlights • PKA-phosphorylation of Hsp20 is elevated in human failing hearts. • Increases in phosphorylated Hsp20 in vivo are associated with fibrotic remodeling and reduced left ventricular function. • The phosphorylated Hsp20 in cardiomyocyte promotes upregulation of IL-6 and its subsequent paracrine actions on the cardiac fibroblast. • Blockade of IL-6 effects ex vivo and in vivo reduces the pro-fibrotic effects of phosphorylated Hsp20. • Targeting phosphorylated Hsp20 in the cardiomyocyte may represent a potential therapeutic strategy to mitigate fibrotic remodeling and preserve function in the failing heart., Summary Cardiomyocyte-specific increases in phosphorylated Hsp20 (S16D-Hsp20) to levels similar to those observed in human failing hearts are associated with early fibrotic remodeling and depressed left ventricular function, symptoms which progress to heart failure and early death. The underlying mechanisms appear to involve translocation of phosphorylated Hsp20 to the nucleus and upregulation of interleukin (IL)-6, which subsequently activates cardiac fibroblasts in a paracrine fashion through transcription factor STAT3 signaling. Accordingly, treatment of S16D-Hsp20 mice with a rat anti-mouse IL-6 receptor monoclonal antibody (MR16-1) attenuated interstitial fibrosis and preserved cardiac function. These findings suggest that phosphorylated Hsp20 may be a potential therapeutic target in heart failure.

Development of physiologic cardiac hypertrophy has primarily been ascribed to the IGF-1 and its receptor, IGF-1 receptor (IGF-1R), and subsequent activation of the protein kinase B (Akt) pathway. However, regulation of endosome-mediated recycling and degradation of IGF-1R during physiologic hypertrophy has not been investigated. In a physiologic hypertrophy model of treadmill-exercised mice, we observed that levels of tumor susceptibility gene 101 (Tsg101), a key member of the endosomal sorting complex required for transport, were dramatically elevated in the heart compared with sedentary controls. To determine the role of Tsg101 on physiologic hypertrophy, we generated a transgenic (TG) mouse model with cardiac-specific overexpression of Tsg101. These TG mice exhibited a physiologic-like cardiac hypertrophy phenotype at 8 wk evidenced by: 1) the absence of cardiac fibrosis, 2) significant improvement of cardiac function, and 3) increased total and plasma membrane levels of IGF-1R and increased phosphorylation of Akt. Mechanistically, we identified that Tsg101 interacted with family-interacting protein 3 (FIP3) and IGF-1R, thereby stabilizing FIP3 and enhancing recycling of IGF-1R. In vitro, adenovirus-mediated overexpression of Tsg101 in neonatal rat cardiomyocytes resulted in cell hypertrophy, which was blocked by addition of monensin, an inhibitor of endosome-mediated recycling, and by small interfering RNA–mediated knockdown (KD) of FIP3. Furthermore, cardiac-specific KD of Tsg101 showed a significant reduction in levels of endosomal recycling compartment members (Rab11a and FIP3), IGF-1R, and Akt phosphorylation. Most interestingly, Tsg101-KD mice failed to develop cardiac hypertrophy after intense treadmill training. Taken together, our data identify Tsg101 as a novel positive regulator of physiologic cardiac hypertrophy through facilitating the FIP3-mediated endosomal recycling of IGF-1R.—Essandoh, K., Deng, S., Wang, X., Jiang, M., Mu, X., Peng, J., Li, Y., Peng, T., Wagner, K.-U., Rubinstein, J., Fan, G.-C. Tsg101 positively regulates physiologic-like cardiac hypertrophy through FIP3-mediated endosomal recycling of IGF-1R.

Timely reperfusion is still the most effective approach to limit infarct size in humans. Yet, despite advances in care and reduction in door-to-balloon times, nearly 25% of patients develop heart failure postmyocardial infarction, with its attendant morbidity and mortality. We previously showed that cardioprotection results from a skin incision through the umbilicus in a murine model of myocardial infarction. In the present study, we show that an electrical stimulus or topical capsaicin applied to the skin in the same region induces significantly reduced infarct size in a murine model. We define this class of phenomena as nociceptor-induced conditioning (NIC) based on the peripheral nerve mechanism of initiation. We show that NIC is effective both as a preconditioning and postconditioning remote stimulus, reducing infarct size by 86% and 80%, respectively. NIC is induced via activation of skin C-fiber nerves. Interestingly, the skin region that activates NIC is limited to the anterior of the T9−T10 vertebral region of the abdomen. Cardioprotection after NIC requires the integrity of the spinal cord from the region of stimulation to the thoracic vertebral region of the origin of the cardiac nerves but does not require that the cord be intact in the cervical region. Thus, we show that NIC is a reflex and not a central nervous system-mediated effect. The mechanism involves bradykinin 2 receptor activity and activation of PKC, specifically, PKC-α. The similarity of the neuroanatomy and conservation of the effectors of cardioprotection supports that NIC may be translatable to humans as a nontraumatic and practical adjunct therapy against ischemic disease. NEW & NOTEWORTHY This study shows that an electrical stimulus to skin sensory nerves elicits a very powerful cardioprotection against myocardial infarction. This stimulus works by a neurogenic mechanism similar to that previously elucidated for remote cardioprotection of trauma. Nociceptor-induced conditioning is equally potent when applied before ischemia or at reperfusion and has great potential clinically.

Previous studies have confirmed that astragaloside (AST) exerts a positive effect on alleviating synovial and joint injury in rheumatoid arthritis (RA). However, the precise mechanisms through which AST acts in the treatment of RA remain unclear. Long non-coding RNA (lncRNA) LOC100912373 was identified as a key gene related to RA and has been proven to interact with miR-17-5p, in order to regulate the pyruvate dehydrogenase kinase 1 and protein kinase B axis (PDK1/AKT axis). The present study aimed to determine whether AST may treat RA through the interaction between lncRNA LOC100912373 and the miR-17-5p/PDK1 axis. MTT assays and flow cytometry were used to detect the proliferation and cell cycle progression of AST-treated fibroblast-like synoviocytes (FLSs). The expression of lncRNA LOC100912373 and miR-17-5p, as well as relative the mRNA expression of the PDK1 and AKT genes following AST intervention was detected by reverse transcription-quantitative PCR (RT-qPCR), immunofluorescence and western blot analysis. The results revealed that AST inhibited FLS proliferation, reduced lncRNA LOC100912373 expression levels, increased miR-17-5p expression levels, and decreased the PDK1 and p-AKT expression levels. Additionally, consecutive rescue experiments revealed that AST counteracted the effects of lncRNA LOC100912373 overexpression on FLS proliferation and cell cycle progression. On the whole, the present study demonstrates that AST inhibits FLS proliferation by regulating the expression of lncRNA LOC100912373 and the miR-17-5p/PDK1 axis.

Hypochlorite plays a significant role in physiological processes, particularly regulation of lysosomal functions, and is involved in various diseases. Thus, it is crucial to develop highly sensitive and selective molecule tools to detect HClO in lysosomes. Herein, a novel 2H-benzo[h]chromene-pyridine derivative (1) was synthesized through condensation reaction, which exhibited a notable deep-red emission at 640 nm in pure water. This deep-red emission was specifically quenched by adding ClO−. The response of probe 1 toward ClO− was rapid (within 10 s), sensitive (detection limit of 0.012 μM), and effective over a wide range of pH (1.0–12.0). Due to the existence of morpholine as the lysosome-targeting unit, the probe was successfully utilized to monitor lysosomal ClO−. Moreover, the probe 1 was also applied to detecting ClO− in zebrafish.

The defective eradication of invading pathogens is a major cause of death in sepsis. As professional phagocytic cells, macrophages actively engulf/kill microorganisms and play essential roles in innate immune response against pathogens. Growth differentiation factor 3 (GDF3) was previously implicated as an important modulator of inflammatory response upon acute sterile injury. In this study, administration of recombinant GDF3 protein (rGDF3) either before or after CLP surgery remarkably improved mouse survival, along with significant reductions in bacterial load, plasma pro-inflammatory cytokine levels, and organ damage. Notably, our in vitro experiments revealed that rGDF3 treatment substantially promoted macrophage phagocytosis and intracellular killing of bacteria in a dose-dependent manner. Mechanistically, RNA-seq analysis results showed that CD5L, known to be regulated by liver X receptor α (LXRα), was the most significantly upregulated gene in rGDF3-treated macrophages. Furthermore, we observed that rGDF3 could promote LXRα nuclear translocation and thereby, augmented phagocytosis activity in macrophages, which was similar as LXRα agonist GW3965 did. By contrast, pre-treating macrophages with LXRα antagonist GSK2033 abolished beneficial effects of rGDF3 in macrophages. In addition, rGDF3 treatment failed to enhance bacteria uptake and killing in LXRα-knockout (KO) macrophages. Taken together, these results uncover that GDF3 may represent a novel mediator for controlling bacterial infection.

Tissue-resident macrophages (TRMs) are sentinel cells for maintaining tissue homeostasis and organ function. In this study, we discovered that lipopolysaccharide (LPS) administration dramatically reduced TRM populations and suppressed their self-renewal capacities in multiple organs. Using loss- and gain-of-function approaches, we define Sectm1a as a novel regulator of TRM self-renewal. Specifically, at the earlier stage of endotoxemia, Sectm1a deficiency exaggerated acute inflammation-induced reduction of TRM numbers in multiple organs by suppressing their proliferation, which was associated with more infiltrations of inflammatory monocytes/neutrophils and more serious organ damage. By contrast, administration of recombinant Sectm1a enhanced TRM populations and improved animal survival upon endotoxin challenge. Mechanistically, we identified that Sectm1a-induced upregulation in the self-renewal capacity of TRM is dependent on GITR-activated T helper cell expansion and cytokine production. Meanwhile, we found that TRMs may play an important role in protecting local vascular integrity during endotoxemia. Our study demonstrates that Sectm1a contributes to stabling TRM populations through maintaining their self-renewal capacities, which benefits the host immune response to acute inflammation. Therefore, Sectm1a may serve as a new therapeutic agent for the treatment of inflammatory diseases.

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.

As an integral component of cardiac tissue, macrophages are critical for cardiac development, adult heart homeostasis, as well as cardiac healing. One fundamental function of macrophages involves the clearance of dying cells or debris, a process termed efferocytosis. Current literature primarily pays attention to the impact of efferocytosis on apoptotic cells. However, emerging evidence suggests that necrotic cells and their released cellular debris can also be removed by cardiac macrophages through efferocytosis. Importantly, recent studies have demonstrated that macrophage efferocytosis plays an essential role in cardiac pathophysiology and repair. Therefore, understanding macrophage efferocytosis would provide valuable insights on cardiac health, and may offer new therapeutic strategies for the treatment of patients with heart failure. In this review, we first summarize the molecular signals that are associated with macrophage efferocytosis of apoptotic and necrotic cells, and then discuss how the linkage of efferocytosis to the resolution of inflammation affects cardiac function and recovery under normal and diseased conditions. Lastly, we highlight new discoveries related to the effects of macrophage efferocytosis on cardiac injury and repair.

Macrophage, as an integral component of the immune system and the first responder to local damage, is on the front line of defense against infection. Over the past century, the prevailing view of macrophage origin states that all macrophage populations resided in tissues are terminally differentiated and replenished by monocytes from bone-marrow progenitors. Nonetheless, this theory has been reformed by ground-breaking discoveries from the past decades. It is now believed that tissue-resident macrophages (TRMs) are originated from the embryonic precursors and seeded in tissue prenatally. They can replenish via self-renewal throughout the lifespan. Indeed, recent studies have demonstrated that tissue-resident macrophages should not be classified by the over-simplified macrophage polarization (M1/M2) dogma during inflammation. Moreover, multiple lines of evidence have indicated that tissue-resident macrophages play critical roles in maintaining tissue homeostasis and facilitating tissue repair through controlling infection and resolving inflammation. In this review, we summarize the properties of resident macrophages in the lung, spleen, and heart, and further highlight the impact of TRM populations on inflammation control and tissue repair. We also discuss the potential role of local proliferation in maintaining a physiologically stable TRM pool in response to acute inflammation.

Macrophages are critical for regulation of inflammatory response during endotoxemia and septic shock. However, the mediators underlying their regulatory function remain obscure. Growth differentiation factor 3 (GDF3), a member of transforming growth factor beta (TGF-&beta, ) superfamily, has been implicated in inflammatory response. Nonetheless, the role of GDF3 in macrophage-regulated endotoxemia/sepsis is unknown. Here, we show that serum GDF3 levels in septic patients are elevated and strongly correlate with severity of sepsis and 28-day mortality. Interestingly, macrophages treated with recombinant GDF3 protein (rGDF3) exhibit greatly reduced production of pro-inflammatory cytokines, comparing to controls upon endotoxin challenge. Moreover, acute administration of rGDF3 to endotoxin-treated mice suppresses macrophage infiltration to the heart, attenuates systemic and cardiac inflammation with less pro-inflammatory macrophages (M1) and more anti-inflammatory macrophages (M2), as well as prolongs mouse survival. Mechanistically, GDF3 is able to activate Smad2/Smad3 phosphorylation, and consequently inhibits the expression of nod-like receptor protein-3 (NLRP3) in macrophages. Accordingly, blockade of Smad2/Smad3 phosphorylation with SB431542 significantly offsets rGDF3-mediated anti-inflammatory effects. Taken together, this study uncovers that GDF3, as a novel sepsis-associated factor, may have a dual role in the pathophysiology of sepsis. Acute administration of rGDF3 into endotoxic shock mice could increase survival outcome and improve cardiac function through anti-inflammatory response by suppression of M1 macrophage phenotype. However, constitutive high levels of GDF3 in human sepsis patients are associated with lethality, suggesting that GDF3 may promote macrophage polarization toward M2 phenotype which could lead to immunosuppression.

Endotoxin-triggered systemic inflammation and multiple organ dysfunction are major culprits to sepsis death. Here, we observed that the plasma levels of SECTM1 in sepsis patients were significantly lower than that in healthy donors. Similarly, the mouse homologous SECTM1A was dramatically down-regulated in multiple organs following LPS injection. Using a global SECTM1A-KO mouse model, we found that SECTM1A depletion exaggerated LPS-caused reduction of tissue-resident macrophage numbers in multi-organs by suppressing their proliferation at earlier stage, leading to more serious organ damage. By contrast, administration of recombinant SECTM1A significantly attenuated macrophage reduction and improved animal survival upon LPS challenge. Mechanistically, we discovered that SECTM1A, as a GITR ligand, contributed to maintaining self-renewal capacity of tissue-resident macrophages during endotoxemia through promoting expansions of T helper cells and releases of macrophage self-renewal cytokines. Our study suggests that SECTM1A is essential for macrophage self-renewal and may serve as a new therapeutic agent for sepsis.

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.

Background Cancer susceptibility candidate 2 (CASC2), a recently discovered long non-coding RNA (lncRNA), was confirmed to play numerous roles in several human cancers. However, the involvement and concrete mechanism of CASC2 in hepatocellular carcinoma (HCC) still need to be further elucidated. Methods The relative expressions of CASC2 and miR-24-3p in HCC tissue and cell lines were determined by quantitative real-time PCR (qRT-PCR). The effects of CASC2 and miR-24-3p on HCC cells were further assessed via cell viability and apoptosis. In vivo tumorigenesis assay was performed to verify the inhibition effect of CASC2 on the tumor growth and further clarify the important role of miR-24-3p in this mechanism. Results Compared with the paired normal tissues, the relative expression of CASC2 significantly reduced in the HCC tissues, while miR-24-3p as determined by qRT-PCR obviously increased in the HCC tissues. This observation was also found in HCC cell lines. Meanwhile, the expression of CASC2 was negatively related to miR-24-3p expression in the HCC tissues (r = -0.804, p

HSPB6/Hsp20 (heat shock protein family B [small] member 6) has emerged as a novel cardioprotector against stress-induced injury. We identified a human mutant of HSPB6 (HSPB6S10F) exclusively present in dilated cardiomyopathy (DCM) patients. Cardiac expression of this mutant in mouse hearts resulted in remodeling and dysfunction, which progressed to heart failure and early death. These detrimental effects were associated with reduced interaction of mutant HSPB6S10F with BECN1/Beclin 1, leading to BECN1 ubiquitination and its proteosomal degradation. As a result, autophagy flux was substantially inhibited and apoptosis was increased in HSPB6S10F-mutant hearts. In contrast, overexpression of wild-type HSPB6 (HSPB6 WT) not only increased BECN1 levels, but also competitively suppressed binding of BECN1 to BCL2, resulting in stimulated autophagy. Indeed, preinhibition of autophagy attenuated the cardioprotective effects of HSPB6 WT. Taken together, these findings reveal a new regulatory mechanism of HSPB6 in cell survival through its interaction with BECN1. Furthermore, Ser10 appears to be crucial for the protective effects of HSPB6 and transversion of this amino acid to Phe contributes to cardiomyopathy.

A morpholine-functionalized pyrrole-cyanine probe was synthesized via a simple condensation reaction in high yield. This probe exhibits high selectivity toward ClO− on fluorescence and UV–vis spectra in neat aqueous solution. The strong green emission of the probe solution was quenched and the yellow color faded immediately upon the addition of ClO−. The detection limit of the probe for ClO− was 0.165 μM. The mechanism of hypochlorite-induced C C breakage was supposed on the basis of EIS-MS, NMR, and density functional theory (DFT) calculation. Finally, the probe was utilized to image ClO− in lysosomes of living cells.

Cardiac mitochondrial damage and subsequent inflammation are hallmarks of endotoxin-induced myocardial depression. Activation of the Parkin/PTEN-induced kinase 1 (PINK1) pathway has been shown to promote autophagy of damaged mitochondria (mitophagy) and to protect from endotoxin-induced cardiac dysfunction. Tumor susceptibility gene 101 (TSG101) is a key member of the endosomal recycling complexes required for transport, which may affect autophagic flux. In this study, we investigated whether TSG101 regulates mitophagy and influences the outcomes of endotoxin-induced myocardial dysfunction. TSG101 transgenic and knockdown mice underwent endotoxin/lipopolysaccharide treatment (10 μg/g) and were assessed for survival, cardiac function, systemic/local inflammation, and activity of mitophagy mediators in the heart. Upon endotoxin challenge and compared with WT mice, TSG101 transgenic mice exhibited increased survival, preserved cardiac contractile function, reduced inflammation, and enhanced mitophagy activation in the heart. By contrast, TSG101 knockdown mice displayed opposite phenotypes during endotoxemia. Mechanistically, both coimmunoprecipitation assays and coimmunofluorescence staining revealed that TSG101 directly binds to Parkin in the cytosol of myocytes and facilitates translocation of Parkin from the cytosol to the mitochondria. Our results indicate that TSG101 elevation could protect against endotoxin-triggered myocardial injury by promoting Parkin-induced mitophagy.

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 remodelling, 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 one month starting from age 2 months attenuated cardiac cell death, adverse myocardial remodelling 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.

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 signalling. This study also suggests that calpain-2/MKP-1 signaling may represent new therapeutic targets for doxorubicin-induced cardiac injury.

A novel rhodamine-based hydrazone (1) bearing morpholine as the lysosome-locating group was synthesized via Schiff base condensation. Probe 1 exhibited a remarkable fluorescent turn-on response over the pH range of 4.0–3.0, which corresponds to the acidic microenvironment of lysosomes in cancer cells. The results of fluorescence cell imaging and a subcellular localization study conducted with HeLa and HepG2 cancer cells and 293T normal cells revealed the good cell membrane permeability and potential applications of 1 in the selective differentiation of cancer cells from normal cells.

Recent studies have shown that myocardial ischemia/reperfusion (I/R)-induced necrosis can be controlled by multiple genes. In this study, we observed that both strands (5p and 3p) of miR-223 were remarkably dysregulated in mouse hearts upon I/R. Precursor miR-223 (pre-miR-223) transgenic mouse hearts exhibited better recovery of contractile performance over reperfusion period and lesser degree of myocardial necrosis than wild type hearts upon ex vivo and in vivo myocardial ischemia. Conversely, pre-miR-223 knock-out (KO) mouse hearts displayed opposite effects. Furthermore, we found that the RIP1/RIP3/MLKL necroptotic pathway and inflammatory response were suppressed in transgenic hearts, whereas they were activated in pre-miR-223 KO hearts upon I/R compared with wild type controls. Accordingly, treatment of pre-miR-223 KO mice with necrostatin-1s, a potent necroptosis inhibitor, significantly decreased I/R-triggered cardiac necroptosis, infarction size, and dysfunction. Mechanistically, we identified two critical cell death receptors, TNFR1 and DR6, as direct targets of miR-223-5p, whereas miR-223-3p directly suppressed the expression of NLRP3 and IκB kinase α, two important mediators known to be involved in I/R-induced inflammation and cell necroptosis. Our findings indicate that miR-223-5p/-3p duplex works together and cooperatively inhibits I/R-induced cardiac necroptosis at multiple layers. Thus, pre-miR-223 may constitute a new therapeutic agent for the treatment of ischemic heart disease.

The present study generated a novel DNA complex to specifically target endothelial NF-κB to inhibit cerebral vascular inflammation. This DNA complex (GS24-NFκB) contains a DNA decoy which inhibits NF-κB activity, and a DNA aptamer (GS-24), a ligand of transferrin receptor (TfR), which allows for targeted delivery of the DNA decoy into cells. The results indicate that GS24-NFκB was successfully delivered into a murine brain-derived endothelial cell line, bEND5, and inhibited inflammatory responses induced by tumor necrosis factor α (TNF-α) or oxygen-glucose deprivation/re-oxygenation (OGD/R) via down-regulation of the nuclear NF-κB subunit, p65, as well as its downstream inflammatory cytokines, inter-cellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM-1). The inhibitory effect of the GS24-NFκB was demonstrated by a significant reduction in TNF-α or OGD/R induced monocyte adhesion to the bEND5 cells after GS24-NFκB treatment. Intravenous (i.v.) injection of GS24-'NFκB (15mg/kg) was able to inhibit the levels of phoseph-p65 and VCAM-1 in brain endothelial cells in a mouse lipopolysaccharide (LPS)-induced inflammatory model in vivo. In conclusion, our approach using DNA nanotechnology for DNA decoy delivery could potentially be utilized for inhibition of inflammation in ischemic stroke and other neuro-inflammatory diseases affecting cerebral vasculature.

Decreased heat shock protein (Hsp) expression in type 1 and type 2 diabetes has been implicated as a primary factor contributing to diabetes-induced organ damage. We recently showed that diabetic cardiomyocytes could release detrimental exosomes, which contain lower levels of Hsp20 than normal ones. To investigate whether such detrimental exosomes could be modified in cardiomyocytes by raising Hsp20 levels to become protective, we used a transgenic (TG) mouse model with cardiac-specific overexpression of Hsp20. TG and control wild-type (WT) mice were injected with streptozotocin (STZ) to induce diabetes. We observed that overexpression of Hsp20 significantly attenuated STZ-caused cardiac dysfunction, hypertrophy, apoptosis, fibrosis, and microvascular rarefaction. Moreover, Hsp20-TG cardiomyocytes exhibited an increased generation/secretion of exosomes by direct interaction of Hsp20 with Tsg101. Of importance, exosomes derived from TG cardiomyocytes encased higher levels of Hsp20, p-Akt, survivin, and SOD1 than WT exosomes and protected against in vitro hyperglycemia-triggered cell death, as well as in vivo STZ-induced cardiac adverse remodeling. Last, blockade of exosome generation by GW4869 remarkably offset Hsp20-mediated cardioprotection in diabetic mice. Our results indicate that elevation of Hsp20 in cardiomyocytes can offer protection in diabetic hearts through the release of instrumental exosomes. Thus, Hsp20-engineered exosomes might be a novel therapeutic agent for diabetic cardiomyopathy.

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.

Currently, most antioxidants do not show any favorable clinical outcomes in reducing myocardial ischemia-reperfusion (I/R) injury, suggesting an urgent need for exploring a new regulator of redox homeostasis in I/R hearts. Here, using heart-specific transgenic (TG) and knockdown (KD) mouse models, tumor susceptibility gene 101 (Tsg101) is defined as a novel cardiac-protector against I/R-triggered oxidative stress. RNA sequencing and bioinformatics data surprisingly reveal that most upregulated genes in Tsg101-TG hearts are transcribed by Nrf2. Accordingly, pharmacological inhibition of Nrf2 offsets Tsg101-elicited cardio-protection. Mechanistically, Tsg101 interacts with SQSTM1/p62 through its PRR domain, and promotes p62 aggregation, leading to recruitment of Keap1 for degradation by autophagosomes and release of Nrf2 to the nucleus. Furthermore, knockout of p62 abrogates Tsg101-induced cardio-protective effects during I/R. Hence, our findings uncover a previously unrecognized role of Tsg101 in the regulation of p62/Keap1/Nrf2 signaling cascades and provide a new strategy for the treatment of ischemic heart disease., Graphical abstract Image 1, Highlights • Most antioxidants are disappointing in clinically reducing cardiac oxidative damage. • Cardiac Tsg101 expression is altered during development and upon oxidative stress. • Tsg101 is a novel cardiac-protector against ischemia/reperfusion injury. • Tsg101 is new positive regulator of Nrf2 signaling pathway. • Tsg101 promotes p62 aggregation and autophagic degradation of Keap1.

Exposure to cold temperature is well known to upregulate heat shock protein (Hsp) expression and recruit and/or activate brown adipose tissue and beige adipocytes in humans and animals. However, whether and how Hsps regulate adipocyte function for energy homeostatic responses is poorly understood. Here, we demonstrate a critical role of Hsp20 as a negative regulator of adipocyte function. Deletion of Hsp20 enhances non-shivering thermogenesis and suppresses inflammatory responses, leading to improvement of glucose and lipid metabolism under both chow diet and high-fat diet conditions. Mechanistically, Hsp20 controls adipocyte function by interacting with the subunit of the ubiquitin ligase complex, F-box only protein 4 (FBXO4), and regulating the ubiquitin-dependent degradation of peroxisome proliferation activated receptor gamma (PPARγ). Indeed, Hsp20 deficiency mimics and enhances the pharmacological effects of the PPARγ agonist rosiglitazone. Together, our findings suggest a role of Hsp20 in mediating adipocyte function by linking β-adrenergic signaling to PPARγ activity.

Aims Impaired sarcoplasmic reticulum (SR) Ca2+ cycling and depressed contractility, a hallmark of human and experimental heart failure, has been partially attributed to increased protein phosphatase 1 (PP-1) activity, associated with down-regulation of its endogenous inhibitor-1. The levels and activity of inhibitor-1 are reduced in failing hearts, contributing to dephosphorylation and inactivation of key calcium cycling proteins. Therefore, we investigated the mechanisms that mediate decreases in inhibitor-1 by post-transcriptional modification. Methods and Results Bioinformatics revealed that 17 human microRNAs may serve as modulators of inhibitor-1. However, real-time PCR analysis identified only one of these microRNAs, miR-765, as being increased in human failing hearts concomitant with decreased inhibitor-1 levels. Expression of miR-765 in HEK293 cells or mouse ventricular myocytes confirmed suppression of inhibitor-1 levels through binding of this miR-765 to the 3'-untranslated region of inhibitor-1 mRNA. To determine the functional significance of miR-765 in Ca2+ cycling, pri-miR-765 as well as a non-translated nucleotide sequence (miR-Ctrl) were expressed in adult mouse ventricular myocytes. The inhibitor-1 expression levels were decreased, accompanied by enhanced PP-1 activity in the miR-765 cardiomyocytes, and these reflected depressed contractile mechanics and Ca2+ transients, compared with the miR-Ctrl group. The depressive effects were associated with decreases in the phosphorylation of phospholamban and SR Ca2+ load. These miR-765 negative inotropic effects were abrogated in inhibitor-1-deficient cardiomyocytes, suggesting its apparent specificity for inhibitor-1. Conclusions miR-765 levels are increased in human failing hearts. Such increases may contribute to depressed cardiac function through reduced inhibitor-1 expression and enhanced PP-1 activity, associated with reduced SR Ca2+ load.

The expression of annexin A2 (ANXA2) in nasopharyngeal carcinoma (NPC) cells induces the immunosuppressive response in dendritic cells; however, the oncogenic effect and clinical significance of ANXA2 have not been fully investigated in NPC cells. Immunohistochemical staining for ANXA2 was performed in 61 patients and the association with clinicopathological status was determined. Short hairpin (sh)RNA knockdown of ANXA2 was used to examine cellular effects of ANXA2, by investigating alterations in cell proliferation, migration, invasion, adhesion, tube-formation assay, and chemo- and radiosensitivity assays were performed. RT-qPCR, Western blotting, and immunofluorescence were applied to determine molecular expression levels. Clinical association studies showed that the expression of ANXA2 was significantly correlated with metastasis (p = 0.0326) and poor survival (p = 0.0256). Silencing of ANXA2 suppressed the abilities of cell proliferation, adhesion, migration, invasion, and vascular formation in NPC cell. ANXA2 up-regulated epithelial-mesenchymal transition associated signal proteins. Moreover, ANXA2 reduced sensitivities to irradiation and chemotherapeutic drugs. These results define ANXA2 as a novel prognostic factor for malignant processes, and it can serve as a molecular target of therapeutic interventions for NPC.

The heart is a very complex conglomeration of organized interactions between various different cell types that all aid in facilitating myocardial function through contractility, sufficient perfusion, and cell-to-cell reception. In order to make sure that all features of the heart work effectively, it is imperative to have a well-controlled communication system among the different types of cells. One of the most important ways that the heart regulates itself is by the use of extracellular vesicles, more specifically, exosomes. Exosomes are types of nano-vesicles, naturally released from living cells. They are believed to play a critical role in intercellular communication through the means of certain mechanisms including direct cell-to-cell contact, long-range signals as well as electrical and extracellular chemical molecules. Exosomes contain many unique features like surface proteins/receptors, lipids, mRNAs, microRNAs, transcription factors and other proteins. Recent studies indicate that the exosomal contents are highly regulated by various stress and disease conditions, in turn reflective of the parent cell status. At present, exosomes are well appreciated to be involved in the process of tumor and infection disease. However, the research on cardiac exosomes is just emerging. In this review, we summarize recent findings on the pathologic effects of exosomes on cardiac remodeling under stress and disease conditions, including cardiac hypertrophy, peripartum cardiomyopathy, diabetic cardiomyopathy and sepsis-induced cardiovascular dysfunction. In addition, the cardio-protective effects of stress-preconditioned exosomes and stem cell-derived exosomes are also summarized. Finally, we discuss how to epigenetically reprogram exosome contents in host cells which makes them beneficial for the heart.

Metastatic bone tumor-induced changes in gene transcription and translation in pain-related regions of the nervous system may participate in the development and maintenance of bone cancer pain. Epigenetic modifications including DNA methylation regulate gene transcription. Here, we report that intrathecal injection of decitabine, a DNA methyltransferase (DNMT) inhibitor, dose dependently attenuated the development and maintenance of bone cancer pain induced by injecting prostate cancer cells into the tibia. The level of the de novo DNMT3a, but not DNMT3b, time dependently increased in the ipsilateral L4/5 dorsal horn (not L4/5 dorsal root ganglion) after prostate cancer cells injection. Blocking this increase through microinjection of recombinant adeno-associated virus 5 (AAV5) expressing Dnmt3a shRNA into dorsal horn rescued prostate cancer cells-induced downregulation of dorsal horn Kv1.2 expression and impaired prostate cancer cells-induced pain hypersensitivity. In turn, mimicking this increase through microinjection of AAV5 expressing full-length Dnmt3a into dorsal horn reduced dorsal horn Kv1.2 expression and produced pain hypersensitivity in the absence of prostate cancer cells injection. Administration of neither decitabine nor virus affected locomotor function and acute responses to mechanical, thermal, or cold stimuli. Given that Dnmt3a mRNA is co-expressed with Kcna2 mRNA (encoding Kv1.2) in individual dorsal horn neurons, our findings suggest that increased dorsal horn DNMT3a contributes to bone cancer pain through silencing dorsal horn Kv1.2 expression. DNMT3a may represent a potential new target for cancer pain management.

Septic shock increases vascular permeability, leading to multiple organ failure including cardiac dysfunction, a major contributor to septic death. Podosome, an actin-based dynamic membrane structure, plays critical roles in extracellular matrix degradation and angiogenesis. However, whether podosome contributes to endothelial barrier dysfunction during septic shock remains unknown. In this study, we found that the endothelial hyperpermeability, stimulated by phorbol 12-myristate 13-acetate and thrombin, was accompanied by increased formation of podosome clusters at the cell periphery, indicating a positive correlation between podosome clusters and endothelial leakage. Interestingly, we observed that circulating exosomes collected from septic mice were able to stimulate podosome cluster formation in cardiac endothelial cells, together with increased permeability in vitro/in vivo and cardiac dysfunction. Mechanistically, we identified that septic exosomes contained higher levels of reactive oxygen species (ROS) than normal ones, which were effectively transported to endothelial cells (ECs). Depletion of ROS in septic exosomes significantly reduced their capacity for promoting podosome cluster formation and thereby dampened vascular leakage. Finally, we elucidated that podosome cluster-induced endothelial hyperpermeability was associated with fragmentation/depletion of zonula occludens-1 (ZO-1) at the cell periphery. Our results demonstrate that septic exosomes were enriched with high amounts of ROS, which can be transported to ECs, leading to the generation of podosome clusters in target ECs and thereby, causing ZO-1 relocation, vascular leakage, and cardiac dysfunction.

Diabetic subjects are at risk of developing cardiovascular disease, which accounts for 60–80% of diabetes-related mortality. Atherosclerosis is still considered as a leading cause of heart failure in diabetic patients, but it could also be an intrinsic and long-term effect of contractile cardiac cells malfunction, known as diabetic cardiomyopathy (DCM). Pathologically, this cardiac dysfunction is manifested by inflammation, apoptosis, fibrosis, hypertrophy and altered cardiomyocytes metabolism. However, the underlying molecular mechanisms of DCM pathophysiology are not clearly understood. Recent and several studies have suggested that exosomes are contributed to the regulation of cell-to-cell communication. Therefore, their in-depth investigation can interpret the complex pathophysiology of DCM. Structurally, exosomes are membrane-bounded vesicles (10–200 nm in diameter), which are actively released from all types of cells and detected in all biological fluids. They carry a wide array of bioactive molecules, including mRNAs, none-coding RNAs (e.g., microRNAs, lncRNAs, circRNAs, etc), proteins and lipids. Importantly, the abundance and nature of loaded molecules inside exosomes fluctuate with cell types and pathological conditions. This chapter summarizes currently available studies on the exosomes’ role in the regulation of diabetic cardiomyopathy. Specifically, the advances on the pathological effects of exosomes in diabetic cardiomyopathy as well as the therapeutic potentials and perspectives are also discussed.

Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells. Thus, our study provides a novel mechanism underlying diabetes mellitus-induced myocardial vascular deficiency which may be caused by secretion of anti-angiogenic exosomes from cardiomyocyes.

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.

Cardiac hypertrophy is a complex pathological process that involves multiple factors including inflammation and apoptosis. Interferon regulatory factor 7 (IRF7) is a multifunctional regulator that participates in immune regulation, cell differentiation, apoptosis, and oncogenesis. However, the role of IRF7 in cardiac hypertrophy remains unclear. We performed aortic banding in cardiac-specific IRF7 transgenic mice, IRF7 knockout mice, and the wild-type littermates of these mice. Our results demonstrated that IRF7 was downregulated in aortic banding–induced animal hearts and cardiomyocytes that had been treated with angiotensin II or phenylephrine for 48 hours. Accordingly, heart-specific overexpression of IRF7 significantly attenuated pressure overload–induced cardiac hypertrophy, fibrosis, and dysfunction, whereas loss of IRF7 led to opposite effects. Moreover, IRF7 protected against angiotensin II–induced cardiomyocyte hypertrophy in vitro. Mechanistically, we identified that IRF7-dependent cardioprotection was mediated through IRF7 binding to inhibitor of κB kinase-β, and subsequent nuclear factor-κB inactivation. In fact, blocking nuclear factor-κB signaling with cardiac-specific inhibitors of κBα S32A/S36A super-repressor transgene counteracted the adverse effect of IRF7 deficiency. Conversely, activation of nuclear factor-κB signaling via a cardiac-specific conditional inhibitor of κB kinase-β S177E/S181E (constitutively active) transgene negated the antihypertrophic effect of IRF7 overexpression. Our data demonstrate that IRF7 acts as a novel negative regulator of pathological cardiac hypertrophy by inhibiting nuclear factor-κB signaling and may constitute a potential therapeutic target for pathological cardiac hypertrophy.

Pathological cardiac hypertrophy is a major risk factor for developing heart failure, the leading cause of death in the world. Growth/differentiation factor 1 (GDF1), a transforming growth factor-β family member, is a regulator of cell growth and differentiation in both embryonic and adult tissues. Evidence from human and animal studies suggests that GDF1 may play an important role in cardiac physiology and pathology. However, a critical role for GDF1 in cardiac remodelling has not been investigated. Here, we performed gain-of-function and loss-of-function studies using cardiac-specific GDF1 knockout mice and transgenic mice to determine the role of GDF1 in pathological cardiac hypertrophy, which was induced by aortic banding (AB). The extent of cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. Our results demonstrated that cardiac specific GDF1 overexpression in the heart markedly attenuated cardiac hypertrophy, fibrosis, and cardiac dysfunction, whereas loss of GDF1 in cardiomyocytes exaggerated the pathological cardiac hypertrophy and dysfunction in response to pressure overload. Mechanistically, we revealed that the cardioprotective effect of GDF1 on cardiac remodeling was associated with the inhibition of the MEK–ERK1/2 and Smad signaling cascades. Collectively, our data suggest that GDF1 plays a protective role in cardiac remodeling via the negative regulation of the MEK–ERK1/2 and Smad signaling pathways.

Aims Dickkopf-3 (DKK3), a secreted protein in the Dickkopf family, is expressed in various tissues, including the heart, and has been shown to play an important role in tissue development. However, the biological function of DKK3 in the heart remains largely unexplored. This study aimed to examine the role of DKK3 in pathological cardiac hypertrophy. Methods and results We performed gain-of-function and loss-of-function studies using DKK3 cardiac-specific transgenic (TG) mice and DKK3 knockout (KO) mice (C57BL/6J background). Cardiac hypertrophy was induced by aortic banding. Cardiac hypertrophy was evaluated by echocardiographic, haemodynamic, pathological, and molecular analyses. Our results demonstrated that the loss of DKK3 exaggerated pressure overload-induced cardiac hypertrophy, fibrosis, and dysfunction, whereas the overexpression of DKK3 protected the heart against pressure overload-induced cardiac remodelling. These beneficial effects were associated with the inhibition of the ASK1-JNK/p38 (apoptosis signal-regulating kinase 1-c-Jun N-terminal kinase/p38) signalling cascade. Parallel in vitro experiments confirmed these in vivo observations. Co-immunoprecipitation experiments suggested that physical interactions occurred between DKK3 and ASK1. Moreover, rescue experiments indicated that, in DKK3 TG mice, the activation of ASK1 using a cardiac-specific conditional ASK1 transgene reduced the functionality of DKK3 in response to pressure overload; furthermore, the inactivation of ASK1 by dominant-negative ASK1 rescued pressure overload-induced cardiac abnormalities in DKK3 KO mice. Conclusion Taken together, our findings indicate that DKK3 acts as a cardioprotective regulator of pathological cardiac hypertrophy and that this function largely occurs via the regulation of ASK1-JNK/p38 signalling.