Complement C1q activates canonical Wnt signaling and promotes aging-related phenotypes.

Wnt signaling plays critical roles in development of various organs and pathogenesis of many diseases, and augmented Wnt signaling has recently been implicated in mammalian aging and aging-related phenotypes. We here report that complement C1q activates canonical Wnt signaling and promotes aging-associated decline in tissue regeneration. Serum C1q concentration is increased with aging, and Wnt signaling activity is augmented during aging in the serum and in multiple tissues of wild-type mice, but not in those of C1qa-deficient mice. C1q activates canonical Wnt signaling by binding to Frizzled receptors and subsequently inducing C1s-dependent cleavage of the ectodomain of Wnt coreceptor low-density lipoprotein receptor-related protein 6. Skeletal muscle regeneration in young mice is inhibited by exogenous C1q treatment, whereas aging-associated impairment of muscle regeneration is restored by C1s inhibition or C1qa gene disruption. Our findings therefore suggest the unexpected role of complement C1q in Wnt signal transduction and modulation of mammalian aging.

A Dietary Oxysterol, 7-Ketocholesterol, Exacerbates Imiquimod-Induced Psoriasis-like Dermatitis in Steatohepatitic Mice.

Patients with psoriasis are at a higher risk of developing nonalcoholic fatty liver disease. We previously identified an oxidized derivative of cholesterol, 7-ketocholesterol (7KC), in diet-induced steatohepatitic mice. Here, we investigated whether 7KC exacerbates psoriasis-like dermatitis by accelerating steatohepatitis in mice. A high-fat/high-cholesterol/high-sucrose/bile salt diet (nonalcoholic steatohepatitis (NASH) diet) with or without 0.0125% 7KC was fed to C57BL/6 mice (7KC or control group) for three weeks to induce steatohepatitis. A 5% imiquimod cream was then applied to the ears and dorsal skin for four days to induce psoriasis-like dermatitis. Hepatic lipid accumulation and inflammatory cell infiltration were exacerbated in the 7KC group compared with the control group after three weeks. Serum tumor necrosis factor-α (TNF-α) levels were also elevated in the 7KC group (108.5 ± 9.8 vs. 83.1 ± 13.1 pg/mL, p < 0.005). Imiquimod cream increased the psoriasis area severity index (PASI) score in mice in the 7KC group (9.14 ± 0.75 vs. 5.17 ± 1.17, p < 0.0001). Additionally, Tnfa, Il23a, Il17a, and Il22 mRNA levels in the dorsal lesion were significantly upregulated. Finally, Th17 cell differentiation and the TNF signaling pathway were enhanced in the dorsal lesions and liver of mice in the 7KC group. These data suggest that steatohepatitis and psoriasis are linked by a potent, diet-related factor.

Activation of DNA Damage Response and Cellular Senescence in Cardiac Fibroblasts Limit Cardiac Fibrosis After Myocardial Infarction.

Cardiac fibrosis plays an important role in cardiac remodeling after myocardial infarction (MI). The molecular mechanisms that promote cardiac fibrosis after MI are well studied; however, the mechanisms by which the progression of cardiac fibrosis becomes attenuated after MI remain poorly understood. Recent reports show the role of cellular senescence in limiting tissue fibrosis. In the present study, we tested whether cellular senescence of cardiac fibroblasts (CFs) plays a role in attenuating the progression of cardiac fibrosis after MI. We found that the number of γH2AX-positive CFs increased up to day 7, whereas the number of proliferating CFs peaked at day 4 after MI. Senescent CFs were also observed at day 7, suggesting that attenuation of CF proliferation occurred simultaneously with the activation of the DNA damage response (DDR) system and the appearance of senescent CFs. We next cultured senescent CFs with non-senescent CFs and showed that senescent CFs suppressed proliferation of the surrounding non-senescent CFs in a juxtacrine manner. We also found that the blockade of DDR by Atm gene deletion sustained the proliferation of CFs and exacerbated the cardiac fibrosis at the early stage after MI. Our results indicate the role of DDR activation and cellular senescence in limiting cardiac fibrosis after MI. Regulation of cellular senescence in CFs may become one of the therapeutic strategies for preventing cardiac remodeling after MI.

Generation of Fabry cardiomyopathy model for drug screening using induced pluripotent stem cell-derived cardiomyocytes from a female Fabry patient.

BACKGROUND: Fabry disease is an X-linked disease caused by mutations in α-galactosidase A (GLA); these mutations result in the accumulation of its substrates, mainly globotriaosylceramide (Gb3). The accumulation of glycosphingolipids induces pathogenic changes in various organs, including the heart, and Fabry cardiomyopathy is the most frequent cause of death in patients with Fabry disease. Existing therapies to treat Fabry disease have limited efficacy, and new approaches to improve the prognosis of patients with Fabry cardiomyopathy are required. METHODS AND RESULTS: We generated induced pluripotent stem cell (iPSC) lines from a female patient and her son. Each iPSC clone from the female patient showed either deficient or normal GLA activity, which could be used as a Fabry disease model or its isogenic control, respectively. Erosion of the inactivated X chromosome developed heterogeneously among clones, and mono-allelic expression of the GLA gene was maintained for a substantial period in a subset of iPSC clones. Gb3 accumulation was observed in iPSC-derived cardiomyocytes (iPS-CMs) from GLA activity-deficient iPSCs by mass-spectrometry and immunofluorescent staining. The expression of ANP was increased, but the cell surface area was decreased in iPS-CMs from the Fabry model, suggesting that cardiomyopathic change is ongoing at the molecular level in Fabry iPS-CMs. We also established an algorithm for selecting proper Gb3 staining that could be used for high-content analysis-based drug screening. CONCLUSIONS: We generated a Fabry cardiomyopathy model and a drug screening system by using iPS-CMs from a female Fabry patient. Drug screening using our system may help discover new drugs that would improve the prognosis of patients with Fabry cardiomyopathy.

Cardiac hypertrophy elevates serum levels of fibroblast growth factor 23.

Several experimental studies have shown that fibroblast growth factor 23 (FGF23) induces left ventricular hypertrophy (LVH). However, the opposite directional relationship, namely a potential effect of LVH on FGF23, remains uncertain. Here we evaluated the effects of LVH on FGF23 using cardiomyocyte-specific calcineurin A transgenic mice. At six weeks, these mice showed severe LVH, with elevated levels of serum intact FGF23. FGF23 levels were elevated in cardiomyocytes, but not osteocytes, of the transgenic animals. Moreover, transverse aortic constriction also upregulated myocardial FGF23 expression in wild type mice. The promoter region of the FGF23 gene contains two putative nuclear factors of activated T cells (NFAT)-binding sites, with NFAT1 activating the promoter in a proximal NFAT-binding site dependent manner. Neither serum, urinary, or fractional excretion values of calcium and phosphate nor serum levels of 1,25(OH)2 vitamin D were different between wild type and transgenic mice. Moreover, the renal expression of FGF receptors and α-Klotho was comparable. However, plasma levels of antidiuretic hormone were significantly increased in the transgenic mice, and aquaporin-2 immunohistochemical staining was mainly positive in the apical membrane of the collecting duct, compared to a primarily cytoplasmic staining in wild type mice. Real-time PCR analyses of kidney CYP27B1 and CYP24A1 expression in wild type mice showed that exogenous antidiuretic hormone blocked FGF23's actions on these vitamin D activating or inactivating enzymes. Finally, the renal resistance of transgenic mice to FGF23 was partly overcome by tolvaptan. Thus, LVH in transgenic mice is associated with an increase in myocardial and serum intact FGF23, with the kidneys being protected against FGF23 excess by elevated antidiuretic hormone levels.

Phenotypic Screening Using Patient-Derived Induced Pluripotent Stem Cells Identified Pyr3 as a Candidate Compound for the Treatment of Infantile Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic disorder that is characterized by hypertrophy of the myocardium. Some of the patients are diagnosed for HCM during infancy, and the prognosis of infantile HCM is worse than general HCM. Nevertheless, pathophysiology of infantile HCM is less investigated and remains largely unknown. In the present study, we generated induced pluripotent stem cells (iPSCs) from two patients with infantile HCM: one with Noonan syndrome and the other with idiopathic HCM. We found that iPSC-derived cardiomyocytes (iPSC-CMs) from idiopathic HCM patient were significantly larger and showed higher diastolic intracellular calcium concentration compared with the iPSC-CMs from healthy subject. Unlike iPSC-CMs from the adult/adolescent HCM patient, arrhythmia was not observed as a disease-related phenotype in iPSC-CMs from idiopathic infantile HCM patient. Phenotypic screening revealed that Pyr3, a transient receptor potential channel 3 channel inhibitor, decreased both the cell size and diastolic intracellular calcium concentration in iPSC-CMs from both Noonan syndrome and idiopathic infantile HCM patients, suggesting that the target of Pyr3 may play a role in the pathogenesis of infantile HCM, regardless of the etiology. Further research may unveil the possibility of Pyr3 or its derivatives in the treatment of infantile HCM.

Generation of Induced Pluripotent Stem Cells From Patients With Duchenne Muscular Dystrophy and Their Induction to Cardiomyocytes.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene which encodes dystrophin protein. Dystrophin defect affects cardiac muscle as well as skeletal muscle. Cardiac dysfunction is observed in all patients with DMD over 18 years of age, but there is no curative treatment for DMD cardiomyopathy. To establish novel experimental platforms which reproduce the cardiac phenotype of DMD patients, here we established iPS cell lines from T lymphocytes donated from two DMD patients, with a protocol using Sendai virus vectors. We successfully conducted the differentiation of the DMD patient-specific iPS cells into beating cardiomyocytes. DMD patient-specific iPS cells and iPS cell-derived cardiomyocytes would be a useful in vitro experimental system with which to investigate DMD cardiomyopathy.

Twin Study: The Factors Affecting the Serum LDL-C and HDL-C Levels and an RNA-Seq Analysis in Mononuclear Cells in Monozygotic Twins.

AIM: A twin study is a valuable tool for elucidating the acquired factors against lifestyle diseases such as dyslipidemia, diabetes mellitus, and obesity. We aimed 1. to investigate the factors that affect low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in monozygotic (MZ) twins, and 2. to identify genes which expression levels changed in pairs with large differences in LDL-C or HDL-C levels. METHODS: The registered database at the Center for Twin Research, Osaka University, containing 263 pairs of MZ twins, was analyzed. 1. The effects of smoking, exercise, nutritional factors, and anthropometric and biochemical parameters on LDL-C or HDL-C levels were examined in MZ twins. 2. RNA sequencing in the peripheral blood mononuclear cells of 59 pairs was analyzed for large differences of LDL-C or HDL-C groups. RESULTS: 1. The ΔLDL-C levels were significantly associated with an older age, the ΔTG levels, and ΔBMI. ΔHDL-C levels were associated with the ΔBMI, ΔTG, ΔTP, and ΔLDL-C levels. The HDL-C levels were affected by smoking and exercise habits. The intakes of cholesterol and saturated fatty acids were not associated with the LDL-C or HDL-C levels. 2. An RNA sequencing analysis revealed that the expression of genes related to the TLR4 and IFNG pathways was suppressed in accordance with the HDL-C levels in the larger ΔHDL-C group among the 59 pairs. CONCLUSION: We identified the factors affecting the LDL-C or HDL-C levels in monozygotic twins. In addition, some types of inflammatory gene expression in peripheral blood mononuclear cells were suppressed in accordance with the HDL-C levels, thus suggesting the importance of weight management and exercise habits in addition to dietary instructions to control the LDL-C or HDL-C levels.

Characterization of proteome-size scaling by integrative omics reveals mechanisms of proliferation control in cancer.

Almost all living cells maintain size uniformity through successive divisions. Proteins that over and underscale with size can act as rheostats, which regulate cell cycle progression. Using a multiomic strategy, we leveraged the heterogeneity of melanoma cell lines to identify peptides, transcripts, and phosphorylation events that differentially scale with cell size. Subscaling proteins are enriched in regulators of the DNA damage response and cell cycle progression, whereas super-scaling proteins included regulators of the cytoskeleton, extracellular matrix, and inflammatory response. Mathematical modeling suggested that decoupling growth and proliferative signaling may facilitate cell cycle entry over senescence in large cells when mitogenic signaling is decreased. Regression analysis reveals that up-regulation of TP53 or CDKN1A/p21CIP1 is characteristic of proliferative cancer cells with senescent-like sizes/proteomes. This study provides one of the first demonstrations of size-scaling phenomena in cancer and how morphology influences the chemistry of the cell.

Pemafibrate suppresses NLRP3 inflammasome activation in the liver and heart in a novel mouse model of steatohepatitis-related cardiomyopathy.

Although patients with nonalcoholic fatty liver disease have been reported to have cardiac dysfunction, and appropriate model has not been reported. We established a novel mouse model of diet-induced steatohepatitis-related cardiomyopathy and evaluated the effect of pemafibrate. C57Bl/6 male mice were fed a (1) chow diet (C), (2) high-fat, high-cholesterol, high-sucrose, bile acid diet (NASH diet; N), or (3) N with pemafibrate 0.1 mg/kg (NP) for 8 weeks. In the liver, macrophage infiltration and fibrosis in the liver was observed in the N group compared to the C group, suggesting steatohepatitis. Free cholesterol accumulated, and cholesterol crystals were observed. In the heart, free cholesterol similarly accumulated and concentric hypertrophy was observed. Ultrahigh magnetic field magnetic resonance imaging revealed that the left ventricular (LV) ejection fraction (EF) was attenuated and LV strain was focally impaired. RNA sequencing demonstrated that the NOD-like receptor and PI3 kinase-Akt pathways were enhanced. mRNA and protein expression of inflammasome-related genes, such as Caspase-1, NLRP3, and IL-1ß, were upregulated in both the liver and heart. In the NP compared to the N group, steatohepatitis, hepatic steatosis, and cardiac dysfunction were suppressed. Sequential administration of pemafibrate after the development of steatohepatitis-related cardiomyopathy recovered hepatic fibrosis and cardiac dysfunction.

Homogeneous 2D and 3D alignment of cardiomyocyte in dilated cardiomyopathy revealed by intravital heart imaging.

In contrast to hypertrophic cardiomyopathy, there has been reported no specific pattern of cardiomyocyte array in dilated cardiomyopathy (DCM), partially because lack of alignment assessment in a three-dimensional (3D) manner. Here we have established a novel method to evaluate cardiomyocyte alignment in 3D using intravital heart imaging and demonstrated homogeneous alignment in DCM mice. Whilst cardiomyocytes of control mice changed their alignment by every layer in 3D and position twistedly even in a single layer, termed myocyte twist, cardiomyocytes of DCM mice aligned homogeneously both in two-dimensional (2D) and in 3D and lost myocyte twist. Manipulation of cultured cardiomyocyte toward homogeneously aligned increased their contractility, suggesting that homogeneous alignment in DCM mice is due to a sort of alignment remodelling as a way to compensate cardiac dysfunction. Our findings provide the first intravital evidence of cardiomyocyte alignment and will bring new insights into understanding the mechanism of heart failure.

Cardiac dopamine D1 receptor triggers ventricular arrhythmia in chronic heart failure.

Pathophysiological roles of cardiac dopamine system remain unknown. Here, we show the role of dopamine D1 receptor (D1R)-expressing cardiomyocytes (CMs) in triggering heart failure-associated ventricular arrhythmia. Comprehensive single-cell resolution analysis identifies the presence of D1R-expressing CMs in both heart failure model mice and in heart failure patients with sustained ventricular tachycardia. Overexpression of D1R in CMs disturbs normal calcium handling while CM-specific deletion of D1R ameliorates heart failure-associated ventricular arrhythmia. Thus, cardiac D1R has the potential to become a therapeutic target for preventing heart failure-associated ventricular arrhythmia.

Dietary Oxysterol, 7-Ketocholesterol Accelerates Hepatic Lipid Accumulation and Macrophage Infiltration in Obese Mice.

Non-alcoholic fatty liver disease is strongly associated with obese and type 2 diabetes. It has been reported that an oxidized cholesterol, 7-ketocholesterol (7KC), might cause inflammatory response in macrophages and plasma 7KC concentration were higher in patients with cardiovascular diseases or diabetes. Therefore, we have decided to test whether small amount of 7KC in diet might induce hepatic steatosis and inflammation in two types of obese models. We found that addition of 0.01% 7KC either in chow diet (CD, regular chow diet with 1% cholesterol) or western type diet (WD, high fat diet with 1% cholesterol) accelerated hepatic neutral lipid accumulation by Oil Red O staining. Importantly, by lipid extraction analysis, it has been recognized that triglyceride rather than cholesterol species was significantly accumulated in CD+7KC compared to CD as well as in WD+7KC compared to WD. Immunostaining revealed that macrophages infiltration was increased in CD+7KC compared to CD, and also in WD+7KC compared to WD. These phenotypes were accompanied by inducing inflammatory response and downregulating fatty acid oxidation. Furthermore, RNA sequence analysis demonstrated that 7KC reduced expression of genes which related to autophagy process. Levels of LC3-II protein were decreased in WD+7KC compared to WD. Similarly, we have confirmed the effect of 7KC on acceleration of steatohepatitis in db/db mice model. Collectively, our study has demonstrated that small amount of dietary 7KC contributed to accelerate hepatic steatosis and inflammation in obese mice models.

Adeno-associated virus-mediated gene delivery promotes S-phase entry-independent precise targeted integration in cardiomyocytes.

Post-mitotic cardiomyocytes have been considered to be non-permissive to precise targeted integration including homology-directed repair (HDR) after CRISPR/Cas9 genome editing. Here, we demonstrate that direct delivery of large amounts of transgene encoding guide RNA (gRNA) and repair template DNA via intra-ventricular injection of adeno-associated virus (AAV) promotes precise targeted genome replacement in adult murine cardiomyocytes expressing Cas9. Neither systemic injection of AAV nor direct injection of adenovirus promotes targeted integration, suggesting that high copy numbers of single-stranded transgenes are required in cardiomyocytes. Notably, AAV-mediated targeted integration in cardiomyocytes both in vitro and in vivo depends on the Fanconi anemia pathway, a key component of the single-strand template repair mechanism. In human cardiomyocytes differentiated from induced pluripotent stem cells, AAV-mediated targeted integration fluorescently labeled Mlc2v protein after differentiation, independently of DNA synthesis, and enabled real-time detection of sarcomere contraction in monolayered beating cardiomyocytes. Our findings provide a wide range of applications for targeted genome replacement in non-dividing cardiomyocytes.

Author Correction: Activation of endothelial ß-catenin signaling induces heart failure.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure.

Pressure overload induces a transition from cardiac hypertrophy to heart failure, but its underlying mechanisms remain elusive. Here we reconstruct a trajectory of cardiomyocyte remodeling and clarify distinct cardiomyocyte gene programs encoding morphological and functional signatures in cardiac hypertrophy and failure, by integrating single-cardiomyocyte transcriptome with cell morphology, epigenomic state and heart function. During early hypertrophy, cardiomyocytes activate mitochondrial translation/metabolism genes, whose expression is correlated with cell size and linked to ERK1/2 and NRF1/2 transcriptional networks. Persistent overload leads to a bifurcation into adaptive and failing cardiomyocytes, and p53 signaling is specifically activated in late hypertrophy. Cardiomyocyte-specific p53 deletion shows that cardiomyocyte remodeling is initiated by p53-independent mitochondrial activation and morphological hypertrophy, followed by p53-dependent mitochondrial inhibition, morphological elongation, and heart failure gene program activation. Human single-cardiomyocyte analysis validates the conservation of the pathogenic transcriptional signatures. Collectively, cardiomyocyte identity is encoded in transcriptional programs that orchestrate morphological and functional phenotypes.

Targeted Genome Replacement via Homology-directed Repair in Non-dividing Cardiomyocytes.

Although high-throughput sequencing can elucidate the genetic basis of hereditary cardiomyopathy, direct interventions targeting pathological mutations have not been established. Furthermore, it remains uncertain whether homology-directed repair (HDR) is effective in non-dividing cardiomyocytes. Here, we demonstrate that HDR-mediated genome editing using CRISPR/Cas9 is effective in non-dividing cardiomyocytes. Transduction of adeno-associated virus (AAV) containing sgRNA and repair template into cardiomyocytes constitutively expressing Cas9 efficiently introduced a fluorescent protein to the C-terminus of Myl2. Imaging-based sequential evaluation of endogenously tagged protein revealed that HDR occurs in cardiomyocytes, independently of DNA synthesis. We sought to repair a pathological mutation in Tnnt2 in cardiomyocytes of cardiomyopathy model mice. An sgRNA that avoided the mutated exon minimized deleterious effects on Tnnt2 expression, and AAV-mediated HDR achieved precise genome correction at a frequency of ~12.5%. Thus, targeted genome replacement via HDR is effective in non-dividing cardiomyocytes, and represents a potential therapeutic tool for targeting intractable cardiomyopathy.

DNA single-strand break-induced DNA damage response causes heart failure.

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.

Activation of endothelial ß-catenin signaling induces heart failure.

Activation of ß-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/ß-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of ß-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of ß-catenin, which activates Wnt/ß-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/ß-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/ß-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure.

Wnt/ß-Catenin Signaling Contributes to Skeletal Myopathy in Heart Failure via Direct Interaction With Forkhead Box O.

BACKGROUND: There are changes in the skeletal muscle of patients with chronic heart failure (CHF), such as volume reduction and fiber type shift toward fatigable type IIb fiber. Forkhead box O (FoxO) signaling plays a critical role in the development of skeletal myopathy in CHF, and functional interaction between FoxO and the Wnt signal mediator ß-catenin was previously demonstrated. We have recently reported that serum of CHF model mice activates Wnt signaling more potently than serum of control mice and that complement C1q mediates this activation. We, therefore, hypothesized that C1q-induced activation of Wnt signaling plays a critical role in skeletal myopathy via the interaction with FoxO. METHODS AND RESULTS: Fiber type shift toward fatigable fiber was observed in the skeletal muscle of dilated cardiomyopathy model mice, which was associated with activation of both Wnt and FoxO signaling. Wnt3a protein activated FoxO signaling and induced fiber type shift toward fatigable fiber in C2C12 cells. Wnt3a-induced fiber type shift was inhibited by suppression of FoxO1 activity, whereas Wnt3a-independent fiber type shift was observed by overexpression of constitutively active FoxO1. Serum of dilated cardiomyopathy mice activated both Wnt and FoxO signaling and induced fiber type shift toward fatigable fiber in C2C12 cells. Wnt inhibitor and C1-inhibitor attenuated FoxO activation and fiber type shift both in C2C12 cells and in the skeletal muscle of dilated cardiomyopathy mice. CONCLUSIONS: C1q-induced activation of Wnt signaling contributes to fiber type shift toward fatigable fiber in CHF. Wnt signaling may be a novel therapeutic target to prevent skeletal myopathy in CHF.