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Myocarditis is an inflammatory disease of the myocardium. MicroRNA-203 (miR-203) is involved in various physiological and pathological processes. In this work, we aimed to explore the roles and potential mechanisms of miR-203 in myocarditis in vitro. Cardiomyocyte H9c2 was subjected to 10 μg/mL lipopolysaccharide (LPS) for 24 hours. Real-time polymerase chain reaction analysis revealed that LPS upregulated miR-203 expression in H9c2 cells. Cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays demonstrated that inhibition of miR-203 reduced cell injury induced by LPS. The cell apoptosis rate, caspase 3 activity, caspase 3/7 activities, and the expression of cleaved-caspase 3 (c-caspase 3) were declined upon miR-203 depletion. In addition, miR-203 silencing attenuated the expression and production of inflammatory cytokines (tumor necrosis factor-α, interleukin [IL]-6, and IL-8). On the contrary, overexpression of miR-203 showed the opposite trend in cell apoptosis and inflammation. Luciferase reporter assay confirmed that miR-203 could bind with the nuclear factor interleukin-3 (NFIL3) 3'-untranslated regions (3'-UTR), and miR-203 regulated the expression of NFIL3 negatively. Moreover, NFIL3 silencing partly abolished the myocardial protective functions of miR-203 inhibitor. Herein, we suggest that miR-203 promoted cell apoptosis and inflammation induced by LPS via targeting NFIL3., (© 2018 Wiley Periodicals, Inc.)

Emodin is a natural product extracted from Rheum palmatum. There are few recent studies on emodin in the treatment of myocarditis. This study aimed to investigate the effect of emodin on lipopolysaccharide (LPS)-induced inflammatory injury in cardiomyocytes. H9c2 cells were treated with 10 μM of LPS and different concentrations (0, 1, 5, 10, 15, and 20 μM) of emodin. The expression of miR-223 was changed by transient transfection. Thereafter, cell viability, apoptosis, the expression of CyclinD1 and Jnk-associated proteins, and the release of pro-inflammatory factors were assessed by cell Counting Kit-8, flow cytometry analysis, quantitative real-time polymerase chain reaction Western blot, and enzyme-linked immunosorbent assay respectively. The results showed that 20 μM of emodin significantly decreased H9c2 cells viability. LPS significantly damaged H9c2 cells, as cell viability was reduced, CyclinD1 was down-regulated, apoptosis was induced, the release of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha were increased, and the phosphorylation of Jnk and c-Jun were promoted. Emodin protected H9c2 cells against LPS-induced inflammatory injury. miR-223 expression was significantly up-regulated by LPS exposure, while emodin lessened this up-regulation. LPS-injured H9c2 cells were attenuated by the overexpression of miR-223; emodin has protective actions on LPS-injured H9c2 cells and targets. Besides, SP600125 (an inhibitor of Jnk) eliminated miR-223-modulated inflammatory injury in H9c2 cells. These data demonstrated that emodin could attenuate LPS-induced inflammatory injury and deactivate Jnk signaling pathway through down-regulation of miR-223.

Aims: Accumulating evidence demonstrates that cardiomyocyte death contributes to the onset and progression of heart failure (HF) after myocardial injury. Recent studies revealed that immune/inflammatory reactions play important roles in cardiovascular diseases. However, it remains unclear whether immunosurveillance system, which eliminates cytopathic cells, including infected or malignant cancer cells, is involved in cardiomyocyte death, though cardiomyocytes are exposed to pathological stresses during post-infarct remodelling. The aim of this study is to clarify the pathophysiological significance of Natural Killer Group 2 member D (NKG2D)/NKG2D ligand (NKG2DL)-mediated cell death in HF after myocardial infarction (MI)., Methods and Results: MI was generated by ligating left anterior descending artery in mice. The expression of NKG2D, NKG2DLs, especially Retinoic acid early induced transcript-1ɛ (Rae-1ɛ), perforin and granzyme B was concomitantly up-regulated after MI. Immunohistological analysis revealed that Rae-1 was expressed on the membranes of injured cardiomyocytes in the infarct and border area. The MI-induced increase of Rae-1 expression was suppressed in p53-/- mice and Rae-1 was induced by the overexpression of p53. We identified p53-binding sites in Rae-1ɛ gene promoter, by chromatin immunoprecipitation assay, indicating that Rae-1 expression was mediated partially through p53. Flow cytometric analysis indicated that NKG2D-expressing immune cells in the post-infarct myocardium were mainly γδT cells. The co-culture with γδT cells increased the frequency of apoptotic cells in the cultured cardiomyocytes. The blockade of NKG2D/NKG2DL interaction by intraperitoneal injection of anti-Rae-1ɛ antibody after MI reduced the frequency of apoptotic cardiomyocytes, accompanied by suppression of cardiac fibrosis, attenuating cardiac dysfunction. Finally, tamoxifen-inducible cardiomyocyte-specific Rae-1ɛ overexpressing mice exhibited the susceptibility to post-infarct remodelling with increased cardiomyocyte apoptosis and severer cardiac dysfunction., Conclusion: The interaction between immune cells and cardiomyocytes via NKG2D/NKG2DL induces cardiomyocyte death, exacerbating cardiac remodelling after MI. The blockade of NKG2D/NKG2DL interaction could be a promising therapeutic strategy against HF., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2018. For permissions, please email: journals.permissions@oup.com.)

Background: Optimal cell type as cell-based therapies for heart failure (HF) remains unclear. We sought to compare the safety and efficacy of direct intramyocardial transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and human induced pluripotent stem cell-derived mesenchymal stem cells (hiPSC-MSCs) in a porcine model of HF., Methods: Eight weeks after induction of HF with myocardial infarction (MI) and rapid pacing, animals with impaired left ventricular ejection fraction (LVEF) were randomly assigned to receive direct intramyocardial injection of saline (MI group), 2 × 10 8 hESC-CMs (hESC-CM group), or 2 × 10 8 hiPSC-MSCs (hiPSC-MSC group). The hearts were harvested for immunohistochemical evaluation after serial echocardiography and hemodynamic evaluation and ventricular tachyarrhythmia (VT) induction by in vivo programmed electrical stimulation., Results: At 8 weeks post-transplantation, LVEF, left ventricular maximal positive pressure derivative, and end systolic pressure-volume relationship were significantly higher in the hiPSC-MSC group but not in the hESC-CM group compared with the MI group. The incidence of early spontaneous ventricular tachyarrhythmia (VT) episodes was higher in the hESC-CM group but the incidence of inducible VT was similar among the different groups. Histological examination showed no tumor formation but hiPSC-MSCs exhibited a stronger survival capacity by activating regulatory T cells and reducing the inflammatory cells. In vitro study showed that hiPSC-MSCs were insensitive to pro-inflammatory interferon-gamma-induced human leukocyte antigen class II expression compared with hESC-CMs. Moreover, hiPSC-MSCs also significantly enhanced angiogenesis compared with other groups via increasing expression of distinct angiogenic factors., Conclusions: Our results demonstrate that transplantation of hiPSC-MSCs is safe and does not increase proarrhythmia or tumor formation and superior to hESC-CMs for the improvement of cardiac function in HF. This is due to their immunomodulation that improves in vivo survival and enhanced angiogenesis via paracrine effects.

The aim of this study was to evaluate the efficacy of vaccine using replication-deficient human recombinant Type 5 replication-defective adenoviruses (AdHu5) carrying sequences of the amastigote surface protein 2 (ASP2) (AdASP2) in mice infected with the Trypanosoma cruzi ( T cruzi) Y strain. A total of 16 A/Sn mice female were distributed into four groups, as follows (n = 4 per group): Group 1 - Control Group (CTRL); Group 2 - Infected Group (TC): animals were infected by subcutaneous route with 150 bloodstream trypomastigotes of T cruzi Y strain; Group 3 - Immunized Group (AdASP-2): animals were immunized by intramuscular injection (im) route with 50 µL of AdSP-2 (2 × 10 8 plaque forming units [pfu]/cam) at day 0; Group 4-Immunized and Infected Group (AdASP-2+TC): animals were immunized by im route with 50 µL of ASP-2 (2 × 10 8  pfu/cam) and infected by T cruzi at the same day (day 0). It was observed a significant decrease of nests in the group that was immunized with AdASP-2 and infected on the same day. Tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) gene expressions showed a significant increase in the AdASP-2+TC group when compared to TC group, but it was noted that Cyclooxygenase-2 (Cox-2) was increased in TC group when compared to AdASP-2+TC group. Increase of matrix metalloproteinases-2 (MMP-2) and decrease of MMP-9 immunoexpression in the AdASP-2+TC group was noticed as well. Oxidative DNA damage was present in myocardium for AdASP-2+TC group as a result of 8-hydroxydeoxyguanosine immunoexpression. Taken together, our results highlighted an increased oxidative stress, MMP-2 activity and inflammatory host response promoted by AdASP-2 against T cruzi infection., (© 2018 Wiley Periodicals, Inc.)

Fowl adenovirus serotype 4 (FAdV-4) is the causative agent of hydropericardium syndrome (HPS), which is characterized by the accumulation of a clear, straw-colored fluid in the pericardial sac, and high mortality rates. In order to explore the mechanism of FAdV-4-induced cardiac damage, dynamic pathology, apoptosis, and inflammatory reactions were analyzed in vivo. Moreover, we detected viral proliferation, and ultrastructure, inflammation and apoptosis of cardiomyocytes (CM) after FAdV-4 infection in vitro. The results showed that FAdV-4 impaired cardiac integrity and function by causing apoptosis and inflammation in vivo. Flow cytometry showed that CM infected with FAdV-4 did not show apoptosis in vitro. In addition, the mRNA expression of four inflammatory cytokines (interleukin (il)1B, il6, il8, and tumor necrosis factor), and activity of three myocardial enzymes were significantly different between FAdV-4 and control groups. However, in vitro, these indexes showed no significant difference between the groups. These observations collectively indicated that the heart was not the target organ of FAdV-4, and the virus may not directly lead to the occurrence of CM apoptosis and inflammation. To explore the source of pericardial effusion, we measured total protein, albumin, aspartate aminotransferase, creatine kinase isoenzyme, lactate dehydrogenase, potassium, sodium, and chloride ions in serum and pericardial effusion. Pericardial effusion was derived from vascular exudation rather than CM degeneration. Further studies are needed to investigate the exudation mechanism of vascular endothelial cells in FAdV-4 infection then weakened or eliminated pericardial effusion to minimize heart injury and/or restore damaged CM., (© 2018 Poultry Science Association Inc.)

Background: An association between Tako-Tsubo cardiomyopathy (TTC) and underlying malignancies has been observed, suggesting that TTC might be the consequence of paraneoplastic phenomena. This study investigates the presence of autoantibodies against cardiomyocytes as well as adrenergic (β 1 , β 2 ) and muscarinic (M2) receptors in patients with TTC., Methods and Results: Serum from 20 TTC patients and 20 controls with ischemic heart disease was obtained. Indirect immunofluorescence testing for intracellular autoantibodies against cardiomyocytes showed a homogenous distribution, as in both groups 9 of 20 sera displayed a characteristic binding pattern of antibodies including vascular walls and intracellular structures. Flow cytometry analysis revealed no difference between TTC and controls in the binding of autoantibodies to the surface antigens of cardiomyocyte HL-1 cells (p = 0.569, t-test). Flow cytometry analysis of nontransfected wild type cells (p = 0.633, t-test), M2 receptor-transfected cells (p = 0.687, t-test), β 1 receptor-transfected cells (p = 0.444, t-test) and β 2 receptor-transfected cells (p = 0.632, t-test) showed similar results for control and TTC sera. Likewise, the binding pattern of TTC patients with a history of neoplasia compared to those without or to controls did not differ significantly (p > 0.05, u-test)., Conclusion: Findings suggest that the presumed paraneoplastic etiology of TTC cannot be attributed to the formation of these antibodies., (Copyright © 2018. Published by Elsevier GmbH.)

Immune responses contribute to a large extent to heart diseases. However, it is still not clear how the key inflammatory mediator interferon-γ (IFNγ) plays a role in doxorubicin (DOX)-induced cardiomyopathy. We report here that DOX-induced heart dysfunction involves IFNγ signaling in mice. The IFNγ receptor was found to be highly expressed on cardiomyocytes, and its downstream signaling was activated in heart tissues upon DOX treatment. In vitro, IFNγ strongly aggravated the injury of cardiomyocytes exposed to DOX. Although not affecting DOX-induced cell death, IFNγ disrupted mitochondrial respiration and fatty acid oxidation in DOX-exposed cardiomyocytes. IFNγ extended the suppression of the AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) axis by DOX to a p38-dependent branch. Activation of AMPK or inhibition of p38 inhibited the enhancing effect of IFNγ on the DOX-induced cardiotoxicity and prolonged the survival time in DOX-treated mice. Taken together, our results indicate that reprogramming of cardiac metabolism by IFNγ represents a previously unidentified key step for DOX-induced cardiomyopathy. This unavoidable impact of IFNγ on cardiomyocyte metabolism during chemotherapy redirects our attention to the balance between beneficial immunosurveillance of cancer cells and unwanted toxic side-effects. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Among the different cardiovascular disease complications, atherosclerosis-induced myocardial infarction (MI) is the major contributor of heart failure (HF) and loss of life. This review presents short- and long-term features of post-MI in human hearts and animal models. It is known that the heart does not regenerate, and thus loss of cardiac cells after an MI event is permanent. In survivors of a heart attack, multiple neurohumoral adjustments as well as simultaneous remodeling in both infarcted and noninfarcted regions of the heart help sustain pump function post-MI. In the early phase, migration of inflammatory cells to the infarcted area helps repair and remove the cell debris, while apoptosis results in the elimination of damaged cardiomyocytes, and there is an increase in the antioxidant response to protect the survived myocardium against oxidative stress (OS) injury. However, in the late phase, it appears that there is a relative increase in OS and activation of the innate inflammatory response in cardiomyocytes without any obvious inflammatory cells. In this late stage in survivors of MI, a progressive slow activation of these processes leads to apoptosis, fibrosis, cardiac dysfunction, and HF. Thus, this second phase of an increase in OS, innate inflammatory response, and apoptosis results in wall thinning, dilatation, and consequently HF. It is important to note that this inflammatory response appears to be innate to cardiomyocytes. Blunting of this innate immune cardiomyocyte response may offer new hope for the management of HF.

Patients affected by Duchenne muscular dystrophy (DMD) develop a progressive dilated cardiomyopathy characterized by inflammatory cell infiltration, necrosis, and cardiac fibrosis. Standard treatments consider the use of β-blockers and angiotensin-converting enzyme inhibitors that are symptomatic and unspecific toward DMD disease. Medications that target DMD cardiac fibrosis are in the early stages of development. We found immunoproteasome dysregulation in affected hearts of mdx mice (murine animal model of DMD) and cardiomyocytes derived from induced pluripotent stem cells of patients with DMD. Interestingly, immunoproteasome inhibition ameliorated cardiomyopathy in mdx mice and reduced the development of cardiac fibrosis. Establishing the immunoproteasome inhibition-dependent cardioprotective role suggests the possibility of modulating the immunoproteasome as new and clinically relevant treatment to rescue dilated cardiomyopathy in patients with DMD., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Increasing evidence has indicated that lncRNAs and miRNAs play important roles in the pathogenesis of myocardial ischemic and reperfusion (I/R) injury. This study investigated the potential roles and underlying molecular mechanisms of lncRNA H19 and H19-derived miR-675 in regulating myocardial I/R injury in vitro and in vivo. The results showed that expression of H19 and H19-derived miR-675 was upregulated in cardiomyocytes exposed to oxygen-glucose deprivation and reperfusion. Knockdown of H19 increased cell viability, reduced cell apoptosis, decreased inflammatory cytokines (IL-1β, TNF-α and IL-6), inhibited oxidative stress, downregulated p-IκB-α and p-p65, and upregulated expression of Nrf2 and HO-1. All of these effects were partly reversed by overexpression of miR-675. Furthermore, we found that PPARα was a target gene of miR-675 and that H19 negatively regulated PPARα expression via miR-675. By inhibiting PPARα, the biological effects of miR-675 or H19 inhibition on cellular functions (apoptosis, inflammation and oxidative stress) were at least partially reversed. Moreover, knockdown of H19 significantly reduced infarct size, increased left ventricular systolic pressure, and decreased left ventricular end-diastolic pressure in a mouse model of myocardial I/R. Taken together, these data indicate that H19 inhibition protects the heart against myocardial I/R injury, which may be partly attributed to regulation of the miR-675/PPARα axis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Autoantibodies directed against G-protein-coupled receptors (GPCR-AAB), an autoantibody type discovered in the 1970s, affect functionally their targets and are therefore called functional autoantibodies. GPCR-AAB are increasingly accepted as the origin or amplifier of various diseases. Here, we describe the present "gold standard" for measurement of GPCR-AAB in human blood. This bioassay monitors the chronotropic activity of GPCR-AAB by recording the spontaneous beating of cultured neonatal rat cardiomyocytes. The construction of this bioassay and its procedure and standardization for GPCR-AAB measurement are described in detail and also include the application of the bioassay for GPCR-AAB differentiation related to first the targeted receptors and IgG subclasses carrying the GPCR-AAB and second the extracellular receptor-binding site and specific epitopes targeted by the GPCR-AAB.

Aims: Inadequate healing after myocardial infarction (MI) leads to heart failure and fatal ventricular rupture, while optimal healing requires timely induction and resolution of inflammation. This study tested the hypothesis that heat shock protein B1 (HSPB1), which limits myocardial inflammation during endotoxemia, modulates wound healing after MI., Methods and Results: To test this hypothesis, cardiomyocyte-specific HSPB1 knockout (Hspb1-/-) mice were generated using the Cre-LoxP recombination system. MI was induced by ligation of the left anterior descending coronary artery in Hspb1-/- and wild-type (WT) littermates. HSPB1 was up-regulated in cardiomyocytes of WT animals in response to MI, and deficiency of cardiomyocyte HSPB1 increased MI-induced cardiac rupture and mortality within 21 days after MI. Serial echocardiography showed more aggravated remodelling and cardiac dysfunction in Hspb1-/- mice than in WT mice at 1, 3, and 7 days after MI. Decreased collagen deposition and angiogenesis, as well as increased MMP2 and MMP9 activity, were also observed in Hspb1-/- mice compared with WT controls after MI, using immunofluorescence, polarized light microscopy, and zymographic analyses. Notably, Hspb1-/- hearts exhibited enhanced and prolonged leucocyte infiltration, enhanced expression of inflammatory cytokines, and enhanced TLR4/MyD88/NFκB activation compared with WT controls after MI. In-depth molecular analyses in both mice and primary cardiomyocytes demonstrated that cardiomyocyte-specific knockout of HSPB1 increased nuclear factor-κB (NFκB) activation, which promoted the expression of proinflammatory mediators. This led to increased leucocyte recruitment, thereby to excessive inflammation, ultimately resulting in adverse remodelling, cardiac dysfunction, and cardiac rupture following MI., Conclusion: These data suggest that HSPB1 acts as a negative regulator of NFκB-mediated leucocyte recruitment and the subsequent inflammation in cardiomyocytes. Cardiomyocyte HSPB1 is required for wound healing after MI and could be a target for myocardial repair in MI patients.

Despite incremental improvements in the field of tissue engineering, no technology is currently available for producing completely autologous implants where both the cells and the scaffolding material are generated from the patient, and thus do not provoke an immune response that may lead to implant rejection. Here, a new approach is introduced to efficiently engineer any tissue type, which its differentiation cues are known, from one small tissue biopsy. Pieces of omental tissues are extracted from patients and, while the cells are reprogrammed to become induced pluripotent stem cells, the extracellular matrix is processed into an immunologically matching, thermoresponsive hydrogel. Efficient cell differentiation within a large 3D hydrogel is reported, and, as a proof of concept, the generation of functional cardiac, cortical, spinal cord, and adipogenic tissue implants is demonstrated. This versatile bioengineering approach may assist to regenerate any tissue and organ with a minimal risk for immune rejection., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims: CD1d is a member of the cluster of differentiation 1 (CD1) family of glycoproteins expressed on the surface of various antigen-presenting cells, which is recognized by natural killer T (NKT) cells. CD1d-dependent NKT cells play an important role in immune-mediated diseases; but the role of these cells in regulating cardiac remodelling remains unknown., Methods and Results: Cardiac remodelling was induced by angiotensin (Ang) II infusion for 2 weeks. Ang II-induced increase in hypertension, cardiac performance, hypertrophy and fibrosis, inflammatory response, and activation of the NF-kB and TGF-β1/Smad2/3 pathways was significantly aggravated in CD1d knockout (CD1dko) mice compared with wild-type (WT) mice, but these effects were markedly abrogated in WT mice treated with α-galactosylceramide (αGC), a specific activator of NKT cells. Adoptive transfer of CD1dko bone marrow cells to WT mice further confirmed the deleterious effect of CD1dko. Moreover, IL-10 expression was significantly decreased in CD1dko hearts but increased in αGC-treated mice. Co-culture experiments revealed that CD1dko dendritic cells significantly reduced IL-10 mRNA expression from NKT cells. Administration of recombinant murine IL-10 to CD1dko mice improved hypertension, cardiac performance, and adverse cardiac remodelling induced by Ang II, and its cardioprotective effect was possibly associated with activation of STAT3, and inhibition of the TGF-β1 and NF-kB pathways., Conclusion: These findings revealed a previously undefined role for CD1d-dependent NKT cells in Ang II-induced cardiac remodelling, hence activation of NKT cells may be a novel therapeutic target for hypertensive cardiac disease.

The transcription factor STAT3 has a protective function in the heart. Until recently, the role of STAT3 in hypertension-induced cardiac hypertrophy was unsettled. Earlier studies revealed that global reduction of STAT3 activity reduced cardiac hypertrophy with hypertension, but caused a disruption of myofilaments and increased contractile dysfunction. However, newer studies with cardiomyocyte-specific deletion of STAT3 indicate that STAT3 does not cause cardiac hypertrophy with increased blood pressure. Rather, cardiac STAT3 is important for maintaining metabolic homeostasis, and loss of STAT3 in cardiomyocytes makes the heart more susceptible to chronic pathological insult, for example by disrupting glucose metabolism and protective signaling networks via the upregulation of certain microRNAs. This scenario has implications for understanding peripartum cardiomyopathy as well. In viral myocarditis, STAT3 opposes the initiation of the dilated phenotype by maintaining membrane integrity via the expression of dystrophin. STAT3 signaling was also found to attenuate myocarditis by polarizing macrophages to a less inflammatory phenotype. On the other hand, STAT3 contributes to immune-mediated myocarditis due to IL-6-induced complement component C3 production in the liver, as well as the differentiation of Th17 cells, which play a role in initiation and development of myocarditis. Besides canonical signaling pathways, unphosphorylated STAT3 (U-STAT3) and redox-activated STAT3 have been shown to couple to transcription in the heart. In addition, tissue signaling cytokines such as IL-22 and IL-17 have been proposed to have actions on the heart that involve STAT3, but are not fully defined. Understanding the novel and often protective aspects of STAT3 in the myocardium could lead to new therapeutic approaches to treat heart disease.

The auxiliary β 4 subunit of the cardiac Ca v 1.2 channel plays a poorly understood role in gene transcription. Here, we characterized the regulatory effects of the β 4 subunit in H9c2 rat cardiac cells on the abundances of Ifnb mRNA [which encodes interferon-β (IFN-β)] and of the IFN-β-related genes Ddx58 , Ifitm3 , Irf7 , Stat2 , Ifih1 , and Mx1 , as well as on the abundances of the antiviral proteins DDX58, IRF7, STAT2, and IFITM3. Knocking down the β 4 subunit in H9c2 cells reduced the expression of IFN-β-stimulated genes. In response to inhibition of the kinase JAK1, the abundances of β 4 subunit mRNA and protein were decreased. β 4 subunit abundance was increased, and it translocated to the nucleus, in cells treated with IFN-β, infected with dengue virus (DENV), or transfected with poly(I:C), a synthetic analog of double-stranded RNA. Cells that surrounded the virus-infected cells showed translocation of β 4 subunit proteins to nuclei in response to spreading infection. We showed that the β 4 subunit interacted with the transcriptional regulator IRF7 and that the activity of an Irf7 promoter-driven reporter was increased in cells overexpressing the β 4 subunit. Last, overexpressing β 4 in undifferentiated and differentiated H9c2 cells reduced DENV infection and decreased the abundance of the viral proteins NS1, NS3, and E-protein. DENV infection and poly(I:C) also increased the concentration of intracellular Ca 2+ in these cells. These findings suggest that the β 4 subunit plays a role in promoting the expression of IFN-related genes, thereby reducing viral infection., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Heart failure due to pressure overload is frequently associated with inflammation. In addition to inflammatory responses of the innate immune system, autoimmune reactions of the adaptive immune system appear to be triggered in subgroups of patients with heart failure as demonstrated by the presence of autoantibodies against myocardial antigens. Moreover, T cell-deficient and T cell-depleted mice have been reported to be protected from heart failure induced by transverse aortic constriction (TAC) and we have shown recently that CD4 + -helper T cells with specificity for an antigen in cardiomyocytes accelerate TAC-induced heart failure. In this study, we set out to investigate the potential contribution of CD8 + -cytotoxic T cells with specificity to a model antigen (ovalbumin, OVA) in cardiomyocytes to pressure overload-induced heart failure. In 78% of cMy-mOVA mice with cardiomyocyte-specific OVA expression, a low-grade OVA-specific cellular cytotoxicity was detected after TAC. Adoptive transfer of OVA-specific CD8 + -T cells from T cell receptor transgenic OT-I mice before TAC did not increase the risk of OVA-specific autoimmunity in cMy-mOVA mice. After TAC, again 78% of the mice displayed an OVA-specific cytotoxicity with on average only a three-fold higher killing of OVA-expressing target cells. More CD8 + cells were present after TAC in the myocardium of cMy-mOVA mice with OT-I T cells (on average 17.5/mm 2 ) than in mice that did not receive OVA-specific CD8 + -T cells (3.6/mm 2 ). However, the extent of fibrosis was similar in both groups. Functionally, as determined by echocardiography, the adoptive transfer of OVA-specific CD8 + -T cells did not significantly accelerate the progression from hypertrophy to heart failure in cMy-mOVA mice. These findings argue therefore against a major impact of cytotoxic T cells with specificity for autoantigens of cardiomyocytes in pressure overload-induced heart failure.

Both advancing age and estrogen loss exacerbate acute myocardial infarction in the female heart. However, the mechanistic underpinnings of age-related differences in cell death after ischemia-reperfusion (I/R) injury in female subjects and reductions in cardioprotective reserve capacity remain largely unexplored. The aim of the present study was to determine the efficacy of programmed necrosis inhibition on infarct size reduction and preservation of left ventricular (LV) function after I/R injury with female aging. Fischer 344 rats were ovariectomized (OVX) at 15 mo and studied at 24 mo (MO OVX) versus adult rats with intact ovaries (6 mo). After in vivo coronary artery ligation (55-min ischemia and 2- or 6-h reperfusion), necrostatin-1 (Nec-1; 3.5 or 5.7 mg/kg) delivered upon reperfusion significantly reduced infarct size by 37% and improved LV function in the MO OVX group ( P < 0.01). Although age-associated elevations in cyclophilin D and mitochondrial acetylation ( P < 0.001) were unaffected by Nec-1, profound reductions in IL-1, IL-6, and TNF-α ( P < 0.05) as well as cardiac immune cell infiltration were observed in MO OVX but not adult rats. We conclude that chronic inflammation and postmenopausal estrogen deficiency conspire to exacerbate acute infarction through a mechanism involving exaggerated mitochondria-mediated programmed necrosis through receptor-interacting protein 1 signaling. Modulatory effects of programmed necrosis inhibition on proinflammatory cytokine production after I/R reveal a potentially important mechanistic target to restore and preserve cardiac function in the OVX aged female heart. NEW & NOTEWORTHY Myocardial infarct size reduction by inhibition of programmed necrosis in aged female subjects suggests a dominant cell death pathway. Alterations in mitochondrial protein levels and acetylation underscore a mitochondria-dependent mechanism, whereas the profound cytokine reduction in aged subjects alone points to a divergent role for immune modulation of programmed necrosis and viable therapeutic target.

Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. However, very little is known about the underlying mechanisms that initiate and modulate the associated protein aggregation and deposition. Model systems have been established to investigate these disease-associated processes. One of these systems comprises two 114 amino acid light-chain variable domains of the kappa 4 IgG family, SMA and LEN. Despite high sequence identity (93%), SMA is amyloidogenic in vivo, but LEN adopts a stable dimer, displaying amyloidogenic properties only under destabilising conditions in vitro. We present here a refined and reproducible periplasmic expression and purification protocol for SMA and LEN that improves on existing methods and provides high yields of pure protein (10-50mg/L), particularly suitable for structural studies that demand highly concentrated and purified proteins. We confirm that recombinant SMA and LEN proteins have structure and dimerization capabilities consistent with the native proteins and employ fluorescence to probe internalization and cellular localization within cardiomyocytes. We propose periplasmic expression and simplified chromatographic steps outlined here as an optimized method for production of these and other variable light chain domains to investigate the underlying mechanisms of light chain amyloidosis. We show that SMA and LEN can be internalised within cardiomyocytes and were observed to localise to the perinuclear area, assessed by confocal microscopy as a possible mechanism for underlying cytotoxicity and pathogenesis associated with amyloidosis., Competing Interests: The authors have declared that no competing interests exist.

Cardiac regeneration, that is, restoration of the original structure and function in a damaged heart, differs from tissue repair, in which collagen deposition and scar formation often lead to functional impairment. In both scenarios, the early-onset inflammatory response is essential to clear damaged cardiac cells and initiate organ repair, but the quality and extent of the immune response vary. Immune cells embedded in the damaged heart tissue sense and modulate inflammation through a dynamic interplay with stromal cells in the cardiac interstitium, which either leads to recapitulation of cardiac morphology by rebuilding functional scaffolds to support muscle regrowth in regenerative organisms or fails to resolve the inflammatory response and produces fibrotic scar tissue in adult mammals. Current investigation into the mechanistic basis of homeostasis and restoration of cardiac function has increasingly shifted focus away from stem cell-mediated cardiac repair towards a dynamic interplay of cells composing the less-studied interstitial compartment of the heart, offering unexpected insights into the immunoregulatory functions of cardiac interstitial components and the complex network of cell interactions that must be considered for clinical intervention in heart diseases.

The functions of macrophages in healthy tissues extend beyond their well-established roles as immune sentinels and effectors. Among tissues, cells of the brain and heart possess unique excitatory properties that likely demand special support. Accordingly, existing evidence demonstrates that microglia in the brain has an active role in synaptic organization, control of neuronal excitability, phagocytic removal of debris, and trophic support during brain development. In the heart, recent studies suggest that cardiac macrophages are involved in the regulation of heart homeostasis by phagocytosis, production of trophic, and immune-related factors, and by forming direct contacts with cardiomyocytes to regulate electrical conduction. In this review, we discuss mechanisms associated with the high degree of specialization of resident macrophages in both tissues, their origin and heterogeneity, and their contributions in regulating homeostasis under steady-state and pathological conditions., (©2018 Society for Leukocyte Biology.)

We determined the roles of TLR3 and TLR9 in adverse events of polymicrobial sepsis, with a focus on development of septic cardiomyopathy, progression of which we have recently shown to be complement- and histones-dependent. So Wt, TLR3-knocked out (K.O.), and TLR9-K.O. mice were subjected to polymicrobial sepsis following cecal ligation and puncture (CLP). In the absence of either TLR3 or TLR9, the intensity of echocardiogram (Echo)-Doppler dysfunction during development of cardiomyopathy was substantially reduced in the K.O. mice. Based on our prior studies emphasizing the adverse effects of plasma C5a and histones in the cardiomyopathy of sepsis, in TLR3- and TLR9-K.O. mice, there were striking reductions in plasma levels of C5a and histones as well as reduced levels of cytokines in plasma and heart tissue after CLP. Since we know that histones cause cardiac dysfunction, rat cardiomyocytes (CMs) were exposed in vitro to the histones (purified from calf thymus), which caused bleb formation on the surfaces of CMs, suggesting histones may perturb the cell membrane of CMs. In vitro , exposure of CMs to the histones for 3 hours caused lactate dehydrogenase release from CMs. These data indicate that sepsis-induced cardiac dysfunction requires presence of TLR3 and TLR9 and may be linked to histone-induced damage of CMs.

Aims: β1-adrenergic receptor autoantibodies (β1-AAs) and β2-adrenergic receptor autoantibodies (β2-AAs) are present in patients with heart failure (HF); however, their interrelationship with cardiac structure and function remains unknown. This study explored the effects of the imbalance between β1-AAs and β2-AAs on cardiac structure and its underlying mechanisms in HF., Methods and Results: Patients with left systolic HF who suffered from coronary heart disease (65.9%) or dilated cardiomyopathy (34.1%) were divided into New York Heart Association Classes I-II (n = 51) and Classes III-IV (n = 37) and compared with healthy volunteers as controls (n = 41). Total immunoglobulin G from HF patient serum comprising β1-AAs and/or β2-AAs were determined and purified for in vitro studies from neonatal rat cardiomyocytes (NRCMs). In addition, HF was induced by doxorubicin in mice. We observed that the increased ratio of β1-AAs/β2-AAs was associated with worsening HF in patients. Moreover, β2-AAs from patients with HF suppressed the hyper-shrinking and apoptosis of NRCMS induced by β1-AAs from some patients. Finally, β2-AAs alleviated both myocardial damage and β1-AAs production induced by doxorubicin in mice., Conclusion: β2-AAs were capable of antagonizing the effects imposed by β1-AAs both in vitro and in vivo. The imbalance of β1-AAs and β2-AAs in patients with HF is a mechanism underlying HF progression, and the increasing ratio of β1-AAs/β2-AAs should be considered a clinical assessment factor for the deterioration of cardiac function in patients with HF.

Chronic heart failure (CHF) is a syndrome characterized by shortness of breath, fluid retention, and a progressive reduction in cardiac function. More than 60% of the cases are ischemic in origin (i.e., due to myo-cardial infarction) and about 30% are caused by non-ischemic myocardial damage (i.e., due to genetic or non-genetic causes like myocardial inflammation). Because of alterations in both cellular and humoral immunity patients with non-ischemic CHF often develop abnormal or misled immune responses, including cross-reacting antibodies and/or autoantibodies to various cardiac anti-gens. Non-ischemic myo-cardial damage was found to progress to CHF particularly, when associated (a) with the generation of autoantibodies directed against distinct myocyte membrane proteins critically involved in cardiac function - like G-protein coup-led membrane receptors (GPCRs), or (b) with virus persistence in the myocardium. This article will review current knowledge on the pathophysiological relevance of GPCR-autoreactivity in CHF by giving an overview on the so far available evidence from pre-clinical, clinical and epidemiological studies on the CHF-inducing potential of GPCR-autoantibodies and thereon based novel therapeutic approaches in GPCR autoantibody-associated CHF.

Patients suffering from chronic heart failure (CHF) caused or promoted by autoantibodies against cardiac beta 1 -adrenergic receptors (beta 1 AR) could benefit from specific therapies aimed at tolerance induction, removal or neutralisation of beta 1 AR autoantibodies, provided the patients can be selected for these therapies by reliable detection and quantitation of beta 1 AR autoantibodies in their circulation and by a valid assessment of the autoantibodies's putative cardio-pathogenic potential. Here, we discuss the current state of knowledge regarding the effects of CHF-associated (auto)antibodies on beta 1 AR function and beta 1 AR-mediated signal tranduction and discuss the presumed role of these effects in the development and progression of CHF. Identification of disease-relevant functional autoantibody effects and their specific assessment in medical diagnostic will be a prerequesite for the implementation of novel specific therapies not only for CHF caused or promoted by beta 1 AR autoantibodies but alos for most other diseases involving autoantibodies that target G-protein coupled receptors.

Background and Aims: The aberrant immune responses play a critical role in the pathogenesis of myocarditis. Vitamin D receptor (VDR) has immune regulatory functions. This study aims to investigate the role of VDR in restricting the immune inflammation in the heart., Methods and Results: The human heart samples were obtained from the heart transplantation. T helper (Th)2 and Th1 responses in the heart tissue were characterized by histology and immune assay. VDR-/- mice and recombination activating gene 2-/- mice were used in the experiments to test the role of VDR in maintaining the homeostasis in the heart. The results showed that, besides tissue damage, lower expression of VDR, high frequency of Th2 cells and increase in Th2 cytokines in the hearts of patients with myocarditis at the end stage of heart failure. The spontaneous Th2-biased inflammation was observed in the hearts of VDR-/- mice. CD4+ T cells from the VDR-/- mouse hearts were at highly activating status. The naïve VDR-/- CD4+ T cells and naïve CD4+ T cells from human hearts with myocarditis were prone to differentiate into Th2 cells. VDR formed complexes with GATA3, the interleukin (IL)-4 transcription factor, to prevent the Il4 gene transcription. Transplantation with VDR-/-CD4+ T cells induced the Th2-biased inflammation in the hearts of Rag2-/- mice. Reconstitution of VDR in CD4+ T cells inhibited the Th2-biased inflammation in the heart., Conclusions: VDR-deficiency contributes to the pathogenesis of myocarditis. To enhance the VDR expression in CD4+, T cells haves the therapeutic potential for the treatment of myocarditis.

Dynamin-related peptide 1 (Drpl)-mediated mitochondrial fission is an important process associated with cardiac dysfunction under different pathological conditions. The aim of the present study was to investigate the expression of Drpl during inflammatory myocardial injury. Sprague‑Dawley rats were treated intraperitoneally with lipopolysaccharides (LPS). Furthermore, cultured H9C2 cardiomyocytes were treated with LPS, interleukin‑6 (IL‑6) and tumor necrosis factor‑α (TNF‑α). Total and mitochondrial proteins were isolated from the heart tissue of rats and from the H9C2 cardiomyocytes. Expression levels of Drp1 and RhoA were analyzed by western blotting. Mitochondrial morphology was determined using confocal laser microscopy. The levels of mitochondrial Drp1 and phosphorylated‑Drp1 (p‑Drp1) Ser616 were revealed to be increased in rats 6 h after injection with LPS (5, 10 or 20 mg/kg). Furthermore, treatment with LPS and IL‑6 did not demonstrate a significant effect on the expression of total and mitochondrial Drp1 in H9C2 cardiomyocytes in vitro; however, treatment with TNF‑α (20 ng/ml) significantly enhanced the levels of mitochondrial Drp1 and p‑Drp1 Ser616. Following TNF‑α treatment, the expression of Ras homolog gene family member A (RhoA) was also revealed to increase. Treatment with both Y‑27632 and fasudil, [Rho kinase (ROCK) inhibitors], was demonstrated to attenuate the otherwise TNF‑α‑induced increase in p‑Drp1 Ser616 and mitochondrial Drp1. In addition, it was revealed that Y‑27632 and fasudil may also attenuate the TNF‑α‑induced increase in mitochondrial fragmentation and cell viability. Therefore, the findings of the present study suggest that TNF‑α is the predominant inducer of Drp1 S616 phosphorylation during sepsis. The results of the present study also suggest that the RhoA/ROCK pathway may be involved in the phosphorylation and mitochondrial translocation of Drp1, which leads to mitochondrial fragmentation.

Previous studies have suggested the involvement of CD4 + T lymphocytes in cardiac remodelling. T-bet can direct Th1 lineage commitment. This study aimed to investigate the functional significance of T-bet in cardiac remodelling induced by pressure overload using T-bet global knockout rats. Increased T-bet levels were observed in rodent and human hypertrophied hearts. T-bet deficiency resulted in a less severe hypertrophic phenotype in rats. CD4 + T-lymphocyte reconstitution in T-bet-/- rats resulted in aggravated cardiac remodelling. T-cell homing molecule expression and cytokine secretion were altered in T-bet-deficient rat hearts. Administration of exogenous interferon-γ (IFN-γ) offset T-bet deficiency-mediated cardioprotection. Cardiomyocytes cultured in T-bet-/- CD4 + T-cell-conditioned media showed a reduced hypertrophic response after hypertrophic stimuli, which was abolished by an IFN-γ-neutralizing antibody. Taken together, our findings show that T-bet deficiency attenuates pressure overload-induced cardiac remodelling in rats. Specifically, targeting T-bet in T cells may be of great importance for the treatment of pathological cardiac remodelling and heart failure.

Aims: The aim of our study was to set up a simple and reliable isolation method of living ventricular cardiomyocytes (vCMs) for molecular and biological studies., Methods and Results: A standard technique for the retrograde perfusion of an enzymatic solution was used to isolate cardiac cells from adult mouse heart. Fluorescence-activated cell sorting (FACS) on adult murine cardiac ventricle cells was performed, comparing the intrinsic autofluorescence in the FITC channel and the forward scatter (FSC) parameter in order to isolate highly fluorescent cells. The expression of cell-specific mRNAs was assessed with real-time PCR in cells sorted on the basis of their FITC and FSC characteristics. We identified two distinct subpopulations of cells harvested after retrograde perfusion of wild-type heart: FITChigh/FSCdim and FITCdim/FSChigh. Immunophenotyping and mRNA analysis (qPCR and RNA sequencing) revealed that only FITChigh/FSCdim cells were highly enriched in CM markers. Genes with high expression in endothelial cells and fibroblasts were enriched in the FITCdim/FSChigh subpopulation. With the use of tdTomatofl/fl-α-myosin heavy chain MerCreMer+/-mouse heart, we found that tdTomato-positive vCMs were present in the FITChigh/FSCdim region but were only rare in the FITCdim/FSChigh fraction., Conclusion: We have developed a simple and reliable method for the isolation of highly purified vCMs from the adult murine myocardium, avoiding fixation and permeabilization steps. These isolated vCMs can be used in particular for detailed molecular studies, avoiding contamination with other myocardial cell types., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2017. For Permissions, please email: journals.permissions@oup.com.)

Glaucoma is a frequent ocular disease that may lead to blindness. Primary open-angle glaucoma (POAG) and ocular hypertension (OHT) are common diseases with increased intraocular pressure (IOP), which are mainly responsible for these disorders. Their pathogenesis is widely unknown. We screened the sera of patients with POAG and OHT for the prevalence of autoantibodies (AAb) against G protein-coupled receptors (GPCRs) in comparison to controls. Employing frequency modulation of spontaneously contracting neonatal rat cardiomyocytes in vitro , agonistic GPCR AAb were to be detected in roughly 75% of the patients with POAG and OHT, however, not in controls. Using inhibitory peptides the AAb' target was identified as β2 adrenergic receptor (β2AR). The AAb interact with the second extracellular loop of β2AR. The peptides 181-187 and 186-192 were identified as binding sites of the AAb within the extracellular loop II. The binding of the AAb to β2ARs was verified by surface-plasmon-resonance analysis. The isotype of the AAb was (immunoglobulin) IgG3. In an additional pilot principal-of-proof study, including four patients with POAG, the removal of the AAb against the β2AR and other immunoglobulins G by immunoadsorption resulted in a transient reduction of IOP. These findings might indicate a possible role of agonistic AAb directed against β2ARs in the dynamics of aqueous humor and might support a contribution of adaptive autoimmunity in the etiopathogenesis of POAG and OHT.

Cell-based analytics for the detection of the beta1-adrenoceptor autoantibody (beta1-AAB) are functional, yet difficult to handle, and should be replaced by easily applicable, routine lab methods. Endeavors to develop solid-phase-based assays such as ELISA to exploit epitope moieties for trapping autoantibodies are ongoing. These solid-phase-based assays, however, are often unreliable when used with human patient material, in contrast to animal derived autoantibodies. We therefore tested an immunogen peptide-based ELISA for the detection of beta1-AAB, and compared commercially available goat antibodies against the 2nd extracellular loop of human beta1-adrenoceptor (ADRB1-AB) to autoantibodies enriched from patient material. The functionality of these autoantibodies was tested in a cell based assay for comparison and their structural appearance was investigated using 2D gel electrophoresis. The ELISA showed a limit of detection for ADRB1-AB of about 1.5 nmol antibody/L when spiked in human control serum and only about 25 nmol/L when spiked in species identical (goat) matrix material. When applied to samples of human origin, the ELISA failed to identify the specific beta1-AABs. A low concentration of beta1-AAB, together with structural inconsistency of the patient originated samples as seen from the 2D Gel appearance, might contribute to the failure of the peptide based ELISA technology to detect human beta1-AABs.

Sarcoplasmic/endoplasmic reticulum Ca 2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971-990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971-990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IA k rather than IE k By creating IA k /and IE k /SERCA2a 971-990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971-990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971-990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971-990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971-990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Phagocytosis after myocardial infarction (MI) is a prerequisite to cardiac repair. Recruited monocytes clear necrotic cardiomyocytes and differentiate into cardiac macrophages. Some studies have linked apoptotic cell receptors on cardiac macrophages to tissue repair; however, the contribution of precursor monocyte phagocytic receptors, which are the first to interact with the cardiac parenchyma, is unclear. The scavenger receptor cluster of differentiation (CD)36 protein was detected on cardiac Ly6cHI monocytes, and bone marrow-derived Cd36 was essential for both early phagocytosis of dying cardiomyocytes and for smaller infarct sizes in female and male mice after permanent coronary ligation. Cd36 deficiency led to reduced expression of phagocytosis receptor Mertk and nuclear receptor Nr4a1 in cardiac macrophages, the latter previously shown to be required for phagocyte survival. Nr4a1 was required for phagocytosis-induced Mertk expression, and Nr4a1 protein directly bound to Mertk gene regulatory elements. To test the overall contribution of the Cd36-Mertk axis, MI was induced in Cd36 -/- Mertk -/- double-knockout mice and led to increases in myocardial rupture. These data implicate monocyte CD36 in the mitigation of early infarct size and transition to Mertk- dependent macrophage function. Increased myocardial rupture in the absence of both Cd36 and Mertk underscore the physiologic significance of phagocytosis during tissue injury.-Dehn, S., Thorp, E. B. Myeloid receptor CD36 is required for early phagocytosis of myocardial infarcts and induction of Nr4a1-dependent mechanisms of cardiac repair., (© FASEB.)

The signal transducer and activator of transcription 3 (STAT3) is known to be involved in hypertrophy and fibrosis in cardiac dysfunction. The activation of STAT3 via the phosphorylation of STAT3 is required for the production of functional activity. It has been established that lipopolysaccharide (LPS)‑induced phosphorylation of STAT3 in cardiomyocytes primarily occurs through a direct receptor‑mediated action. This effect is demonstrated to be produced rapidly. STAT3 in cardiac fibrosis of diabetes is induced by high glucose through promotion of the STAT3‑associated signaling pathway. However, the time schedule for STAT3 activation between LPS and high glucose appears to be different. Therefore, the difference in STAT3 activation between LPS and hyperglycemia in cardiomyocytes requires elucidation. The present study investigated the phosphorylation of STAT3 induced by LPS and hyperglycemia in the rat cardiac cell line H9c2. Additionally, phosphorylation of STAT3 induced by erythropoietin (EPO) via receptor activation was compared. Then, the downstream signals for fibrosis, including the connective tissue growth factor (CTGF) and matrix metalloproteinase (MMP)‑9, were determined using western blotting, while the mRNA levels were quantified. LPS induced a rapid elevation of STAT3 phosphorylation in H9c2 cells within 30 min, similar to that produced by EPO. However, LPS or EPO failed to modify the mRNA level of STAT3, and/or the downstream signals for fibrosis. High glucose increased STAT3 phosphorylation to be stable after a long period of incubation. Glucose incubation for 24 h may augment the STAT3 expression in a dose‑dependent manner. Consequently, fibrosis‑associated signals, including CTGF and MMP‑9 protein, were raised in parallel. In the presence of tiron, an antioxidant, these changes by hyperglycemia were markedly reduced, demonstrating the mediation of oxidative stress. Therefore, LPS‑ or EPO‑induced STAT3 phosphorylation is different compared with that caused by high glucose in H9c2 cells. Sustained activation of STAT3 by hyperglycemia may promote the expression of fibrosis‑associated signals, including CTGF and MMP‑9, in H9c2 cells. Therefore, regarding the cardiac dysfunctions associated with diabetes and/or hyperglycemia, the identification of nuclear STAT3 may be more reliable compared with the assay of phosphorylated STAT3 in cardiac cells.

Interleukin-6 (IL-6) signaling is critical for cardiomyocyte hypertrophy, while the role of IL-6 in the pathogenesis of myocardium hypertrophy remains controversial. To determine the essential role of IL-6 signaling for the cardiac development during AngII-induced hypertension, and to elucidate the mechanisms, wild-type (WT) and IL-6 knockout (IL-6 KO) mice were infused subcutaneously with either vehicle or AngII (1.5 μg/h/mouse) for 1 week. Immunohistological and serum studies revealed that the extents of cardiac fibrosis, inflammation and apoptosis were reduced in IL-6 KO heart during AngII-stimulation, while cardiac hypertrophy was obviously induced. To investigate the underlying mechanisms, by using myocardial tissue and neonatal cardiomyocytes, we observed that IL-6/STAT3 signaling was activated under the stimulation of AngII both in vivo and in vitro. Further investigation suggested that STAT3 activation enhances the inhibitory effect of EndoG on MEF2A and hampers cardiomyocyte hypertrophy. Our study is the first to show the important role of IL-6 in regulating cardiac pathogenesis via inflammation and apoptosis during AngII-induced hypertension. We also provide a novel link between IL-6/STAT3 and EndoG/MEF2A pathway that affects cardiac hypertrophy during AngII stimulation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Nuclear factor of activated T cells 5 (NFAT5)/Tonicity enhancer binding protein (TonEBP) is a transcription factor induced by hypertonic stress in the kidney. However, the function of NFAT5 in other organs has rarely been studied, even though it is ubiquitously expressed. Indeed, although NFAT5 was reported to be critical for heart development and function, its role in infectious heart diseases has remained obscure. In this study, we aimed to understand the mechanism by which NFAT5 interferes with infection of Coxsackievirus B3 (CVB3), a major cause of viral myocarditis. Our initial results demonstrated that although the mRNA level of NFAT5 remained constant during CVB3 infection, NFAT5 protein level decreased because the protein was cleaved. Bioinformatic prediction and verification of the predicted site by site-directed mutagenesis experiments determined that the NFAT5 protein was cleaved by CVB3 protease 2A at Glycine 503. Such cleavage led to the inactivation of NFAT5, and the 70-kDa N-terminal cleavage product (p70-NFAT5) exerted a dominant negative effect on the full-length NFAT5 protein. We further showed that elevated expression of NFAT5 to counteract viral protease cleavage, especially overexpression of a non-cleavable mutant of NFAT5, significantly inhibited CVB3 replication. Ectopic expression of NFAT5 resulted in elevated expression of inducible nitric oxide synthase (iNOS), a factor reported to inhibit CVB3 replication. The necessity of iNOS for the anti-CVB3 effect of NFAT5 was supported by the observation that inhibition of iNOS blocked the anti-CVB3 effect of NFAT5. In a murine model of viral myocarditis, we observed that treatment with hypertonic saline or mannitol solution upregulated NFAT5 and iNOS expression, inhibited CVB3 replication and reduced tissue damage in the heart. Taken together, our data demonstrate that the anti-CVB3 activity of NFAT5 is impaired during CVB3 infection due to 2A-mediated cleavage of NFAT5. Thus induction of NFAT5 by hypertonic agents may be a promising strategy for the development of anti-CVB3 therapeutics.

We investigated whether CD4 + -T cells with specificity for an antigen in cardiomyocytes promote the progression from hypertrophy to heart failure in mice with increased pressure load due to transverse aortic constriction (TAC). OT-II mice expressing a transgenic T cell receptor (TCR) with specificity for ovalbumin (OVA) on CD4 + -T cells and cMy-mOVA mice expressing OVA on cardiomyocytes were crossed. The resulting cMy-mOVA-OT-II mice did not display signs of spontaneous autoimmunity despite the fact that their OVA-specific CD4 + -T cells were not anergic. After TAC, progression to heart failure was significantly accelerated in cMy-mOVA-OT-II compared to cMy-mOVA mice. No OVA-specific antibodies were induced in response to TAC in cMy-mOVA-OT-II mice, yet more CD3 + T cells infiltrated their myocardium when compared with TAC-operated cMy-mOVA mice. Systemically, the proportion of activated CD4 + -T cells with a Th 1 and Th 17 cytokine profile was increased in cMy-mOVA-OT-II mice after TAC. Thus, T helper cells with specificity for an antigen in cardiomyocytes can directly promote the progression of heart failure in response to pressure overload independently of autoantibodies.

Spleen tyrosine kinase (SYK), a non-receptor protein tyrosine kinase, is reported to be related to cell survival after A/H (anoxia/hypoglycemia) insult. However, the role of SYK in cardiocyte survival under A/H injury remains unclear. In this study, we aimed to gain insight into the role and molecular mechanism of SYK in cardiocytes exposed to A/H stress. The mRNA and protein expressions of SYK in H9c2 cardiocytes exposed to A/H injury, separately detected by real-time quantitative PCR and Western blot, were both robustly up-regulated. Then we overexpressed SYK in H9c2 with A/H injury, and found that cell viability was significantly increased and LDH leakage was decreased. Moreover, apoptosis measured by annexin V-fluorescein isothiocyanate/propidium iodide and reactive oxygen species (ROS) identified by 2', 7'-dichlorofluorescin diacetate were markedly inhibited in H9c2 with A/H injury following SYK overexpression. Furthermore, we observed that SYK could induce HO-1 expression by regulating the Akt phosphorylation level in H9c2 with A/H injury, protecting H9c2 from the injury induced by A/H treatment., (Copyright © 2017. Published by Elsevier Ltd.)

The immune inflammatory response plays a crucial role in many cardiac pathophysiological processes, including ischaemic cardiac injury and the post-infarction repair process. MicroRNAs (miRNAs) regulate the development and function of dendritic cells (DCs), which are key players in the initiation and regulation of immune responses; however, the underlying regulatory mechanisms remain unclear. Here, we used the supernatants of necrotic primary cardiomyocytes (Necrotic-S) to mimic the myocardial infarction (MI) microenvironment to investigate the role of miRNAs in the regulation of DC-mediated inflammatory responses. Our results showed that Necrotic-S up-regulated the DC maturation markers CD40, CD83 and CD86 and increased the production of inflammatory cytokines, concomitant with the up-regulation of miR-181a and down-regulation of miR-150. Necrotic-S stimulation activated the JAK/STAT pathway and promoted the nuclear translocation of c-Fos and NF-κB p65, and silencing of STAT1 or c-Fos suppressed Necrotic-S-induced DC maturation and inflammatory cytokine production. The effects of Necrotic-S on DC maturation and inflammatory responses, its activation of the JAK/STAT pathway and the induction of cardiomyocyte apoptosis under conditions of hypoxia were suppressed by miR-181a or miR-150 overexpression. Taken together, these data indicate that miR-181a and miR-150 attenuate DC immune inflammatory responses via JAK1-STAT1/c-Fos signalling and protect cardiomyocytes from cell death under conditions of hypoxia., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Cardiac arrhythmias confer a considerable burden of morbidity and mortality in industrialized countries. Although coronary artery disease and heart failure are the prevalent causes of cardiac arrest, in 5-15% of patients, structural abnormalities at autopsy are absent. In a proportion of these patients, mutations in genes encoding cardiac ion channels are documented (inherited channelopathies), but, to date, the molecular autopsy is negative in nearly 70% of patients. Emerging evidence indicates that autoimmunity is involved in the pathogenesis of cardiac arrhythmias. In particular, several arrhythmogenic autoantibodies targeting specific calcium, potassium, or sodium channels in the heart have been identified. Experimental and clinical studies demonstrate that these autoantibodies can promote conduction disturbances and life-threatening tachyarrhythmias by inducing substantial electrophysiological changes. In this Review, we propose the term 'autoimmune cardiac channelopathies' to define this novel pathogenic mechanism of cardiac arrhythmias, which could be more frequent and clinically relevant than previously appreciated. Indeed, pathogenic autoantibodies against ion channels are detectable not only in patients with manifest autoimmune disease, but also in apparently healthy individuals, which suggests a causal role in some cases of unexplained arrhythmias and cardiac arrest. Considering this possibility and performing specific testing in patients with 'idiopathic' rhythm disturbances could create novel treatment opportunities.

A disturbed inflammatory response following myocardial infarction (MI) is associated with poor prognosis and increased tissue damage. Monocytes are key players in healing after MI, but little is known about the role of the cardiac niche in monocyte activation. This study investigated microenvironment-dependent changes in inflammatory monocytes after MI RNA sequencing analysis of murine Ly6C high monocytes on day 3 after MI revealed differential regulation depending on location. Notably, the local environment strongly impacted components of the WNT signaling cascade. Analysis of WNT modulators revealed a strong upregulation of WNT Inhibitory Factor 1 (WIF1) in cardiomyocytes-but not fibroblasts or endothelial cells-upon hypoxia. Compared to wild-type (WT) littermates, WIF1 knockout mice showed severe adverse remodeling marked by increased scar size and reduced ejection fraction 4 weeks after MI While FACS analysis on day 1 after MI revealed no differences in neutrophil numbers, the hearts of WIF1 knockouts contained significantly more inflammatory monocytes than hearts from WT animals. Next, we induced AAV-mediated cardiomyocyte-specific WIF1 overexpression, which attenuated the monocyte response and improved cardiac function after MI, as compared to control-AAV-treated animals. Finally, WIF1 overexpression in isolated cardiomyocytes limited the activation of non-canonical WNT signaling and led to reduced IL-1β and IL-6 expression in monocytes/macrophages. Taken together, we investigated the cardiac microenvironment's interaction with recruited monocytes after MI and identified a novel mechanism of monocyte activation. The local initiation of non-canonical WNT signaling shifts the accumulating myeloid cells toward a pro-inflammatory state and impacts healing after myocardial infarction., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Studies on inflammatory disorders elucidated the pivotal role of the CX3CL1/CX3CR1 axis with respect to the pathophysiology and diseases progression. Coxsackievirus B3 (CVB3)-induced myocarditis is associated with severe cardiac inflammation, which may progress to heart failure. We therefore investigated the influence of CX3CR1 ablation in the model of acute myocarditis, which was induced by inoculation with 5x105 plaque forming units of CVB3 (Nancy strain) in either CX3CR1-/- or C57BL6/j (WT) mice. Seven days after infection, myocardial inflammation, remodeling, and titin expression and phosphorylation were examined by immunohistochemistry, real-time PCR and Pro-Q diamond stain. Cardiac function was assessed by tip catheter. Compared to WT CVB3 mice, CX3CR1-/- CVB3 mice exhibited enhanced left ventricular expression of inflammatory cytokines and chemokines, which was associated with an increase of immune cell infiltration/presence. This shift towards a pro-inflammatory immune response further resulted in increased cardiac fibrosis and cardiomyocyte apoptosis, which was reflected by an impaired cardiac function in CX3CR1-/- CVB3 compared to WT CVB3 mice. These findings demonstrate a cardioprotective role of CX3CR1 in CVB3-infected mice and indicate the relevance of the CX3CL1/CX3CR1 system in CVB3-induced myocarditis.

The cardiac extracellular matrix (ECM) not only provides mechanical support, but also transduces essential molecular signals in health and disease. Following myocardial infarction, dynamic ECM changes drive inflammation and repair. Early generation of bioactive matrix fragments activates proinflammatory signaling. The formation of a highly plastic provisional matrix facilitates leukocyte infiltration and activates infarct myofibroblasts. Deposition of matricellular proteins modulates growth factor signaling and contributes to the spatial and temporal regulation of the reparative response. Mechanical stress due to pressure and volume overload and metabolic dysfunction also induce profound changes in ECM composition that contribute to the pathogenesis of heart failure. This manuscript reviews the role of the ECM in cardiac repair and remodeling and discusses matrix-based therapies that may attenuate remodeling while promoting repair and regeneration.

The DNA damage response (DDR) plays a pivotal role in maintaining genome integrity. DNA damage and DDR activation are observed in the failing heart, however, the type of DNA damage and its role in the pathogenesis of heart failure remain elusive. Here we show the critical role of DNA single-strand break (SSB) in the pathogenesis of pressure overload-induced heart failure. Accumulation of unrepaired SSB is observed in cardiomyocytes of the failing heart. Unrepaired SSB activates DDR and increases the expression of inflammatory cytokines through NF-κB signalling. Pressure overload-induced heart failure is more severe in the mice lacking XRCC1, an essential protein for SSB repair, which is rescued by blocking DDR activation through genetic deletion of ATM, suggesting the causative role of SSB accumulation and DDR activation in the pathogenesis of heart failure. Prevention of SSB accumulation or persistent DDR activation may become a new therapeutic strategy against heart failure.

It is well known that cardiac dysfunction develops during sepsis in both humans and in rodents (rats, mice). These defects appear to be reversible, since after "recovery" from sepsis, cardiac dysfunction disappears and the heart returns to its function that was present before the onset of sepsis. Our studies, using in vivo and in vitro models, have demonstrated that C5a and its receptors (C5aR1 and C5aR2) play key roles in cardiac dysfunction developing during sepsis. Use of a neutralizing antibody to C5a largely attenuates cardiac dysfunction and other adverse events developing during sepsis. The molecular basis for cardiac dysfunctions is linked to generation of C5a and its interaction with C5a receptors present on surfaces of cardiomyocytes (CMs). It is established that C5a interactions with C5a receptors leads to significant reductions involving faulty contractility and relaxation in CMs. In addition, C5a interactions with C5a receptors on CMs results in reductions in Na + /K + -ATPase in CMs. This ATPase is essential for intact action potentials in CMs. The enzymatic activity and protein for this ATPase were strikingly reduced in CMs during sepsis by unknown mechanisms. In addition, C5a interactions with C5aRs also caused reductions in CM homeostatic proteins that regulate cytosolic [Ca 2+ ]i in CMs: sarco/endoplasmic reticulum Ca 2+ -ATPase2 (SERCA2) and Na + /Ca 2+ exchanger (NCX). In the absence of C5a receptors, defects in SERCA2 and NCX in CMs after sepsis are strikingly attenuated. These observations suggest new strategies to protect the heart from dysfunction developing during sepsis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Despite the use of rabbits in biomedical research, including regulatory toxicology and cardiovascular studies, little data exist on heart findings in this species. This study was designed to document myocardial findings in female rabbits and the impact of study-related procedures typical for vaccine toxicology studies. One hundred and forty 6- to 8-month-old female New Zealand White rabbits were divided equally into 2 groups, high and low study procedure groups (group 1 and group 2, respectively). All animals received intramuscular (IM) injections of sterile saline every 2 weeks for 5 times and were necropsied 2 days after the final IM injection. Clinical chemistry, hematology, and urinalysis were evaluated. Blood for stress biomarkers (norepinephrine, epinephrine, cortisol, and corticosterone), C-reactive protein, cardiac troponin I, and creatine kinase were collected at time 0 (just before dose administration) and then at 4, 24, and 48 hr after dose administration in group 1 only. Hearts were assessed histologically. Focal to multifocal minimal inflammatory cell infiltrates were common (∼80%), particularly in the left ventricle and interventricular septum, and were similar to the types of infiltrates identified in other laboratory animal species. Additionally, study-related procedures elevated serum stress biomarkers and exacerbated the frequency and severity of myocardial inflammatory cell infiltrates.

Background/aims: The expression of a novel lncRNA, myocardial infarction associated transcript 1(Mirt1), has been shown to be upregulated in acute myocardial infarction (AMI). However, the role of Mirt1 in AMI is not clear., Methods: In this study, we analyzed the level of Mirt1 in cardiomyocytes and cardiac fibroblasts in AMI mice. Moreover, adenovirus mediated knockdown of Mirt1 was employed to clarify its roles in AMI mice or cultured cardiac fibroblasts. The cardiac functions and infarct size of AMI mice were examined, and tissues and cultured cells were collected and processed for histology and biochemical examination., Results: We demonstrated that Mirt1 was mainly expressed in cardiac fibroblasts, and that knockdown of Mirt1 improved cardiac functions, decreased cardiomyocytes apoptosis and attenuated inflammatory cell infiltration in vivo. Furthermore, knockdown of Mirt1 in cardiac fibroblasts not only attenuated the apoptosis of cardiomyocytes, but also suppressed the migration of macrophages under hypoxia in vitro. NF-κB signaling pathway, activated under hypoxia, was also inhibited by Mirt1 knockdown in fibroblasts., Conclusions: Knockdown of Mirt1 attenuates AMI injury presumably by decreasing cardiomyocytes apoptosis and reducing inflammatory cell infiltration. These effects could be attributed, at least partly, to inhibition of the NF-κB pathway, resulting in decreased expression of inflammatory factors., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

The cardiac myocyte differs strikingly from the specialized cells of the immune system, which has two different responses to invading organisms and tissue damage. Adaptive or acquired immunity generates highly specific antibodies in response to threats and is an essential component of immunity; however, adaptive immunity can take 4-7 days to mobilize, and a more primitive response, innate immunity, fills the gap. Innate immunity is expressed in complex and in primitive life forms. Specialized receptors, Toll-like receptors (TLRs), which are widely distributed throughout different tissues recognize danger signals and rapidly respond with the release of noxious substances, such as TNFα. The problem is that many endogenous molecules have been found to act as ligands for specific TLRs, and when these molecules are released into the extracellular environment, they can cause problems by activating innate immunity and an inflammatory response. In cardiac myocytes heat shock protein (HSP)60 can activate TLR4, as can HMGB1, and this type of response can amplify the response to ischemia/reperfusion leading to increased cell and tissue injury. Activation of TLRs can potentially amplify chronic, inflammatory diseases, such as ischemic heart failure. Thus, it is important to understand the regulation of the TLRs and their downstream effects. This chapter will focus on the TLRs and cardiac myocytes.

Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1β in DM mice. IL-1β causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1β-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1β axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1β as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.