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.

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

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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 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.

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.

Disseminated intravascular coagulation with acutely-thrombosed popliteal aneurysm.

Disseminated intravascular coagulation (DIC) is an extremely rare complication of acute thrombosis in popliteal aneurysms and makes it difficult to restore the blood flow with thrombolytic therapy or surgical repair. A 75-year-old man with a history of hypertension presented to the emergency department with complaints of right leg pain and bleeding tendency over a 5-day period. The laboratory findings and multislice computed tomography were suggestive of overt DIC caused by acute thrombosis in the right popliteal aneurysm. Successfully treated with medication, he could discharge without surgical or thrombolytic recanalization of the aneurysm.