Taku Sakai

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.

Complement C1q-induced activation of β-catenin signalling causes hypertensive arterial remodelling.

Hypertension induces structural remodelling of arteries, which leads to arteriosclerosis and end-organ damage. Hyperplasia of vascular smooth muscle cells (VSMCs) and infiltration of immune cells are the hallmark of hypertensive arterial remodelling. However, the precise molecular mechanisms of arterial remodelling remain elusive. We have recently reported that complement C1q activates β-catenin signalling independent of Wnts. Here, we show a critical role of complement C1-induced activation of β-catenin signalling in hypertensive arterial remodelling. Activation of β-catenin and proliferation of VSMCs were observed after blood-pressure elevation, which were prevented by genetic and chemical inhibition of β-catenin signalling. Macrophage depletion and C1qa gene deletion attenuated the hypertension-induced β-catenin signalling, proliferation of VSMCs and pathological arterial remodelling. Our findings unveil the link between complement C1 and arterial remodelling and suggest that C1-induced activation of β-catenin signalling becomes a novel therapeutic target to prevent arteriosclerosis in patients with hypertension.

Wnt/β-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 &bgr;-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.

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.

Activation of endothelial β-catenin signaling induces heart failure

Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-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/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure.

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.

Author Correction: Activation of endothelial β-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.

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.

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.