Youan Liu

Myeloid Differentiation Factor-88 Plays a Crucial Role in the Pathogenesis of Coxsackievirus B3–Induced Myocarditis and Influences Type I Interferon Production

Background— Myeloid differentiation factor (MyD)-88 is a key adaptor protein that plays a major role in the innate immune pathway. How MyD88 may regulate host response in inflammatory heart disease is unknown. Methods and Results— We found that the cardiac protein level of MyD88 was significantly increased in the hearts of wild-type mice after exposure to Coxsackievirus B3 (CVB3). MyD88−/− mice showed a dramatic higher survival rate (86%) in contrast to the low survival (35%) in the MyD88+/+ mice after CVB3 infection (P<0.0001). Pathological examination showed a significant decrease of cardiac and pancreatic inflammation in the MyD88−/− mice. Viral concentrations in the hearts were significantly decreased in the MyD88−/− mice. Cardiac mRNA levels for interleukin (IL)-1&bgr;, tumor necrosis factor (TNF)-&agr;, interferon (IFN)-&ggr;, and IL-18 were significantly decreased in the MyD88−/− mice. Similarly, serum levels of T-helper 1 cytokines were significantly decreased in the MyD88−/− mice. In contrast, cardiac protein levels of the activated interferon regulatory factor (IRF)-3 and IFN-&bgr; were significantly increased in the MyD88−/− mice but not other usual upstream signals to IRF-3. The cardiac expression of coxsackie-adenoviral receptor and p56lck were also significantly decreased. Conclusions— MyD88 appears to be a key contributor to cardiac inflammation, mediating cytokine production and T-helper-1/2 cytokine balance, increasing coxsackie-adenoviral receptor and p56lck expression and viral titers after CVB3 exposure. Absence of MyD88 confers host protection possibly through novel direct activation of IRF-3 and IFN-&bgr;.

Curcumin prevents and reverses murine cardiac hypertrophy

Chromatin remodeling, particularly histone acetylation, plays a critical role in the progression of pathological cardiac hypertrophy and heart failure. We hypothesized that curcumin, a natural polyphenolic compound abundant in the spice turmeric and a known suppressor of histone acetylation, would suppress cardiac hypertrophy through the disruption of p300 histone acetyltransferase-dependent (p300-HAT-dependent) transcriptional activation. We tested this hypothesis using primary cultured rat cardiac myocytes and fibroblasts as well as two well-established mouse models of cardiac hypertrophy. Curcumin blocked phenylephrin-induced (PE-induced) cardiac hypertrophy in vitro in a dose-dependent manner. Furthermore, curcumin both prevented and reversed mouse cardiac hypertrophy induced by aortic banding (AB) and PE infusion, as assessed by heart weight/BW and lung weight/BW ratios, echocardiographic parameters, and gene expression of hypertrophic markers. Further investigation demonstrated that curcumin abrogated histone acetylation, GATA4 acetylation, and DNA-binding activity through blocking p300-HAT activity. Curcumin also blocked AB-induced inflammation and fibrosis through disrupting p300-HAT-dependent signaling pathways. Our results indicate that curcumin has the potential to protect against cardiac hypertrophy, inflammation, and fibrosis through suppression of p300-HAT activity and downstream GATA4, NF-kappaB, and TGF-beta-Smad signaling pathways.

Regulatory T Cells Protect Mice Against Coxsackievirus-Induced Myocarditis Through the Transforming Growth Factor β–Coxsackie-Adenovirus Receptor Pathway

Background— Coxsackievirus B3 infection is an excellent model of human myocarditis and dilated cardiomyopathy. Cardiac injury is caused either by a direct cytopathic effect of the virus or through immune-mediated mechanisms. Regulatory T cells (Tregs) play an important role in the negative modulation of host immune responses and set the threshold of autoimmune activation. This study was designed to test the protective effects of Tregs and to determine the underlying mechanisms. Methods and Results— Carboxyfluorescein diacetate succinimidyl ester–labeled Tregs or naïve CD4+ T cells were injected intravenously once every 2 weeks 3 times into mice. The mice were then challenged with intraperitoneal coxsackievirus B3 immediately after the last cell transfer. Transfer of Tregs showed higher survival rates than transfer of CD4+ T cells (P=0.0136) but not compared with the PBS injection group (P=0.0589). Interestingly, Tregs also significantly decreased virus titers and inflammatory scores in the heart. Transforming growth factor-&bgr; and phosphorylated AKT were upregulated in Tregs-transferred mice and coxsackie-adenovirus receptor expression was decreased in the heart compared with control groups. Transforming growth factor-&bgr; decreased coxsackie-adenovirus receptor expression and inhibited coxsackievirus B3 infection in HL-1 cells and neonatal cardiac myocytes. Splenocytes collected from Treg-, CD4+ T-cell–, and PBS-treated mice proliferated equally when stimulated with heat-inactivated virus, whereas in the Treg group, the proliferation rate was reduced significantly when stimulated with noninfected heart tissue homogenate. Conclusions— Adoptive transfer of Tregs protected mice from coxsackievirus B3–induced myocarditis through the transforming growth factor &bgr;–coxsackie-adenovirus receptor pathway and thus suppresses the immune response to cardiac tissue, maintaining the antiviral immune response.

Gelsolin Regulates Cardiac Remodeling After Myocardial Infarction Through DNase I–Mediated Apoptosis

Gelsolin, a calcium-regulated actin severing and capping protein, is highly expressed in murine and human hearts after myocardial infarction and is associated with progression of heart failure in humans. The biological role of gelsolin in cardiac remodeling and heart failure progression after injury is not defined. To elucidate the contribution of gelsolin in these processes, we randomly allocated gelsolin knockout mice (GSN−/−) and wild-type littermates (GSN+/+) to left anterior descending coronary artery ligation or sham surgery. We found that GSN−/− mice have a surprisingly lower mortality, markedly reduced hypertrophy, smaller late infarct size, less interstitial fibrosis, and improved cardiac function when compared with GSN+/+ mice. Gene expression and protein analysis identified significantly lower levels of deoxyribonuclease (DNase) I and reduced nuclear translocation and biological activity of DNase I in GSN−/− mice. Absence of gelsolin markedly reduced DNase I–induced apoptosis. The association of hypoxia-inducible factor (HIF)-1α with gelsolin and actin filaments cleaved by gelsolin may contribute to the higher activation of DNase. The expression pattern of HIF-1α was similar to that of gelsolin, and HIF-1α was detected in the gelsolin complex by coprecipitation and HIF-1α bound to the promoter of DNase I in both gel-shift and promoter activity assays. Furthermore, the phosphorylation of Akt at Ser473 and expression of Bcl-2 were significantly increased in GSN−/− mice, suggesting that gelsolin downregulates prosurvival factors. Our investigation concludes that gelsolin is an important contributor to heart failure progression through novel mechanisms of HIF-1α and DNase I activation and downregulation of antiapoptotic survival factors. Gelsolin inhibition may form a novel target for heart failure therapy.

Survival and Cardiac Remodeling After Myocardial Infarction Are Critically Dependent on the Host Innate Immune Interleukin-1 Receptor-Associated Kinase-4 Signaling A Regulator of Bone Marrow-Derived Dendritic Cells

Background— The innate immune system greatly contributes to the inflammatory process after myocardial infarction (MI). Interleukin-1 receptor–associated kinase-4 (IRAK-4), downstream of Toll/interleukin-1 receptor signaling, has an essential role in regulating the innate immune response. The present study was designed to determine the mechanism by which IRAK-4 is responsible for the cardiac inflammatory process, which consequently affects left ventricular remodeling after MI. Methods and Results— Experimental MI was created in IRAK-4−/− and wild-type mice by left coronary ligation. Mice with a targeted deletion of IRAK-4 had an improved survival rate at 4 weeks after MI. IRAK-4−/− mice also demonstrated attenuated cardiac dilation and decreased inflammation in the infarcted myocardium, which was associated with less proinflammatory and Th1 cytokine expression mediated by suppression of nuclear factor-&kgr;B and c-Jun N-terminal kinase activation. IRAK-4−/− mice had fewer infiltrations of CD45+ leukocytes and CD11c+ dendritic cells, inhibition of apoptosis, and reduced fibrosis and nitric oxide production. Cardiac dendritic cells in IRAK-4−/− mice were relatively immature or functionally naïve after MI in that they demonstrated less cytokine and costimulatory molecule gene expression. Furthermore, IRAK-4−/− dendritic cells have less mobilization capacity. Transfer of wild type–derived bone marrow dendritic cells into IRAK-4−/− mice for functional dendritic cell reconstitution negated the survival advantage and reduced the cardiac dilation observed with IRAK-4−/− mice at 28 days after MI. Conclusions— Deletion of IRAK-4 has favorable effects on survival and left ventricular remodeling after MI through modification of the host inflammatory process by blunting the detrimental bone marrow dendritic cells mobilization after myocardial ischemia.

Cathepsin-L Ameliorates Cardiac Hypertrophy Through Activation of the Autophagy–Lysosomal Dependent Protein Processing Pathways

Background Autophagy is critical in the maintenance of cellular protein quality control, the final step of which involves the fusion of autophagosomes with lysosomes. Cathepsin‐L (CTSL) is a key member of the lysosomal protease family that is expressed in the murine and human heart, and it may play an important role in protein turnover. We hypothesized that CTSL is important in regulating protein processing in the heart, particularly under pathological stress. Methods and Results Phenylephrine‐induced cardiac hypertrophy in vitro was more pronounced in CTSL‐deficient neonatal cardiomyocytes than in in controls. This was accompanied by a significant accumulation of autophagosomes, increased levels of ubiquitin‐conjugated protein, as well as impaired protein degradation and decreased cell viability. These effects were partially rescued with CTSL1 replacement via adeno‐associated virus–mediated gene transfer. In the in vivo murine model of aortic banding (AB), a deficiency in CTSL markedly exacerbated cardiac hypertrophy, worsened cardiac function, and increased mortality. Ctsl−/− AB mice demonstrated significantly decreased lysosomal activity and increased sarcomere‐associated protein aggregation. Homeostasis of the endoplasmic reticulum was also altered by CTSL deficiency, with increases in Bip and GRP94 proteins, accompanied by increased ubiquitin–proteasome system activity and higher levels of ubiquitinated proteins in response to AB. These changes ultimately led to a decrease in cellular ATP production, enhanced oxidative stress, and increased cellular apoptosis. Conclusions Lysosomal CTSL attenuates cardiac hypertrophy and preserves cardiac function through facilitation of autophagy and proteasomal protein processing.

Cathepsin-L contributes to cardiac repair and remodelling post-infarction

AIMS Cathepsin-L (CTSL) is a member of the lysozomal cysteine protease family, which participates in remodelling of various tissues. Herein, we sought to examine the potential regulation of CTSL in cardiac remodelling post-infarction. METHODS AND RESULTS Experimental myocardial infarction (MI) was created in CTSL-deficient (Ctsl(-/-)) mice (B6 × FSB/GnEi a/a Ctsl(fs)/J) and wild-type littermates (Ctsl(+/+)) by left coronary artery ligation. At days 3, 7, 14, and 28 post-MI, we monitored survival rate and evaluated cardiac function, morphology, and molecular endpoints of repair and remodelling. Survival was 56% in Ctsl(-/-) mice in contrast to 80% (P < 0.05) in Ctsl(+/+) mice post-MI by day 28. The Ctsl(-/-) mice exhibited greater scar dilatation, wall thinning, and worse cardiac dysfunction when compared with Ctsl(+/+) mice. Cardiac matrix metallopeptidase-9 (MMP-9) activity was also diminished, and c-kit-positive cells, natural killer cells, fibrocytes, and monocytes mobilized to peripheral blood and deposited to the infarcted myocardium were significantly decreased in Ctsl(-/-) mice. Furthermore, the local inflammatory response, and granulocyte-colony stimulating factor, stem cell factor (SCF), and stromal cell-derived factor-1 (SDF-1α) expression, as well as cell proliferation, revascularization, and myofibroblast deposition were significantly decreased in Ctsl(-/-) mice compared with Ctsl(+/+) mice. CONCLUSION Our data indicate that CTSL regulates cardiac repair and remodelling post-MI through a mechanism with multiple pathways.

Innate Immune Interleukin-1 Receptor–Associated Kinase 4 Exacerbates Viral Myocarditis by Reducing CCR5+CD11b+ Monocyte Migration and Impairing Interferon Production

Background— Viral myocarditis follows a fatal course in ≈30% of patients. Interleukin-1 receptor–associated kinase 4 (IRAK4), a major nodal signal transducer in innate immunity, can play a pivotal role in host inflammatory response. We sought to determine how IRAK4 modulates inflammation and outcome in a mouse model of viral myocarditis. Methods and Results— Myocarditis was induced after intraperitoneal inoculation of coxsackievirus B3 into C57Bl/6 IRAK4-deficient mice and their littermate controls. Mortality and viral proliferation were markedly reduced in IRAK4−/− mice compared with their IRAK4+/+ littermates. Disease resistance of IRAK4−/− mice paralleled increased amounts of protective heart-infiltrating CCR5+ monocytes/macrophages and enhanced interferon-&agr; and interferon-&ggr; production 2 days after infection. Competitive bone marrow chimera demonstrated that intact IRAK4 function inhibited heart-specific migration of bone marrow–derived CCR5+ cells. Mechanistically, lack of IRAK4 resulted in interferon regulatory factor 5 homodimerization via reduced melanoma differentiation-associated protein 5 degradation and enhanced Stat1 and Stat5 phosphorylation. Consequently, antiviral interferon-&agr; and interferon-&ggr; production, as well as CCR5+ cell recruitment, increased, whereas the overall proinflammatory response was drastically reduced in the absence of IRAK4. Conclusions— Innate immunity signal transducer IRAK4 exacerbates viral myocarditis through inhibition of interferon production and reduced mobilization of protective CCR5+ monocytes/macrophages to the heart. The combination of IRAK4 inhibitors and antiviral adjuvants may become an attractive therapeutic approach against viral myocarditis in the future.