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Heart failure is a common disease with poor prognosis that is associated with cardiac immune cell infiltration and dysregulated cytokine expression. Recently, the clonal expansion of hematopoietic cells with acquired (i.e., nonheritable) DNA mutations, a process referred to as clonal hematopoiesis, has been reported to be associated with cardiovascular diseases including heart failure. Mechanistic studies have shown that leukocytes that harbor these somatic mutations display altered inflammatory characteristics that worsen the phenotypes associated with heart failure in experimental models. In this review, we summarize recent epidemiological and experimental evidence that support the hypothesis that clonal hematopoiesis-mediated immune cell dysfunction contributes to heart failure and cardiovascular disease in general.

DOI： 10.1016/j.jacbts.2019.08.006

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Glutaredoxin-1 (Glrx) is a small cytosolic enzyme that removes S-glutathionylation, glutathione adducts of protein cysteine residues, thus modulating redox signaling and gene transcription. Although Glrx up-regulation prevented endothelial cell (EC) migration and global Glrx transgenic mice had impaired ischemic vascularization, the effects of cell-specific Glrx overexpression remained unknown. Here, we examined the role of EC-specific Glrx up-regulation in distinct models of angiogenesis; namely, hind limb ischemia and tumor angiogenesis. EC-specific Glrx transgenic (EC-Glrx TG) overexpression in mice significantly impaired EC migration in Matrigel implants and hind limb revascularization after femoral artery ligation. Additionally, ECs migrated less into subcutaneously implanted B16F0 melanoma tumors as assessed by decreased staining of EC markers. Despite reduced angiogenesis, EC-Glrx TG mice unexpectedly developed larger tumors compared with control mice. EC-Glrx TG mice showed higher levels of VEGF-A in the tumors, indicating hypoxia, which may stimulate tumor cells to form vascular channels without EC, referred to as vasculogenic mimicry. These data suggest that impaired ischemic vascularization does not necessarily associate with suppression of tumor growth, and that antiangiogenic therapies may be ineffective for melanoma tumors because of their ability to implement vasculogenic mimicry during hypoxia.-Yura, Y., Chong, B. S. H., Johnson, R. D., Watanabe, Y., Tsukahara, Y., Ferran, B., Murdoch, C. E., Behring, J. B., McComb, M. E., Costello, C. E., Janssen-Heininger, Y. M. W., Cohen, R. A., Bachschmid, M. M., Matsui, R. Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

DOI： 10.1096/fj.201900786R

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Therapy-related clonal hematopoiesis (t-CH) is often observed in cancer survivors. This form of clonal hematopoiesis typically involves somatic mutations in driver genes that encode components of the DNA damage response and confer hematopoietic stem and progenitor cells (HSPCs) with resistance to the genotoxic stress of the cancer therapy. Here, we established a model of TP53-mediated t-CH through the transfer of Trp53 mutant HSPCs to mice, followed by treatment with a course of the chemotherapeutic agent doxorubicin. These studies revealed that neutrophil infiltration in the heart significantly contributes to doxorubicin-induced cardiac toxicity and that this condition is amplified in the model of Trp53-mediated t-CH. These data suggest that t-CH could contribute to the elevated heart failure risk that occurs in cancer survivors who have been treated with genotoxic agents.

DOI： 10.1172/jci.insight.146076

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Clonal hematopoiesis (CH) is a phenomenon whereby somatic mutations confer a fitness advantage to hematopoietic stem and progenitor cells (HSPC) and thus facilitate their aberrant clonal expansion. These mutations are carried into progeny leukocytes leading to a situation whereby a substantial fraction of an individual's blood cells originate from the HSPC mutant clone. Although this condition rarely progresses to a hematological malignancy, circulating blood cells bearing the mutation have the potential to affect other organ systems as they infiltrate into tissues under both homeostatic and disease conditions. Epidemiological and clinical studies have revealed that CH is highly prevalent in the elderly and is associated with an increased risk of cardiovascular disease and mortality. Recent experimental studies in murine models have assessed the most commonly mutated "driver" genes associated with CH, and have provided evidence for mechanistic connections between CH and cardiovascular disease. A deeper understanding of the mechanisms by which specific CH mutations promote disease pathogenesis is of importance, as it could pave the way for individualized therapeutic strategies targeting the pathogenic CH gene mutations in the future. Here, we review the epidemiology of CH and the mechanistic work from studies using murine disease models, with a particular focus on the strengths and limitations of these experimental systems. We intend for this review to help investigators select the most appropriate models to study CH in the setting of cardiovascular disease.

DOI： 10.1093/cvr/cvab215

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Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.

DOI： 10.3791/61875

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Dopamine (DA) activates mitogen-activated protein kinase (MAPK) via protein kinase A (PKA)/Rap1 in medium spiny neurons (MSNs) expressing the dopamine D1 receptor (D1R) in the nucleus accumbens (NAc), thereby regulating reward-related behavior. However, how MAPK regulates reward-related learning and memory through gene expression is poorly understood. Here, to identify the relevant transcriptional factors, we perform proteomic analysis using affinity beads coated with cyclic AMP response element binding protein (CREB)-binding protein (CBP), a transcriptional coactivator involved in reward-related behavior. We identify more than 400 CBP-interacting proteins, including Neuronal Per Arnt Sim domain protein 4 (Npas4). We find that MAPK phosphorylates Npas4 downstream of PKA, increasing the Npas4-CBP interaction and the transcriptional activity of Npas4 at the brain-derived neurotrophic factor (BDNF) promoter. The deletion of Npas4 in D1R-expressing MSNs impairs cocaine-induced place preference, which is rescued by Npas4-wild-type (WT), but not by a phospho-deficient Npas4 mutant. These observations suggest that MAPK phosphorylates Npas4 in D1R-MSNs and increases transcriptional activity to enhance reward-related learning and memory.

DOI： 10.1016/j.celrep.2019.10.116

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BACKGROUND: Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. The RHOA-associated protein kinase (ROCK/Rho-kinase), an effector regulated by the small GTPase RHOA, causes pathological phosphorylation of proteins, resulting in cardiovascular diseases. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiovascular diseases remains unclear. METHODS: To explore the role of PKNs in heart failure, we generated tamoxifen-inducible, cardiomyocyte-specific PKN1- and PKN2-knockout mice by intercrossing the αMHC-CreERT2 line with Pkn1flox/flox and Pkn2flox/flox mice and applied a mouse model of transverse aortic constriction- and angiotensin II-induced heart failure. To identify a novel substrate of PKNs, we incubated GST-tagged myocardin-related transcription factor A (MRTFA) with recombinant GST-PKN-catalytic domain or GST-ROCK-catalytic domain in the presence of radiolabeled ATP and detected radioactive GST-MRTFA as phosphorylated MRTFA. RESULTS: We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 (cmc-PKN1/2 DKO) did not affect basal heart function but protected mice from pressure overload- and angiotensin II-induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. We confirmed that endogenous MRTFA phosphorylation in the heart was induced by pressure overload- and angiotensin II-induced cardiac dysfunction in wild-type mice, whereas cmc-PKN1/2 DKO mice suppressed transverse aortic constriction- and angiotensin II-induced phosphorylation of MRTFA. Although RHOA-mediated actin polymerization accelerated MRTFA-induced gene transcription, PKN1 and PKN2 inhibited the interaction of MRTFA with globular actin by phosphorylating MRTFA, causing increased serum response factor-mediated expression of cardiac hypertrophy- and fibrosis-associated genes. CONCLUSIONS: Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.

DOI： 10.1161/CIRCULATIONAHA.119.041019

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Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

DOI： 10.3791/59977

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Janus kinase 2 (valine to phenylalanine at residue 617) (JAK2 V617F ) mutations lead to myeloproliferative neoplasms associated with elevated myeloid, erythroid, and megakaryocytic cells. Alternatively these same mutations can lead to the condition of clonal hematopoiesis with no impact on blood cell counts. Here, a model of myeloid-restricted JAK2 V617F expression from lineage-negative bone marrow cells was developed and evaluated. This model displayed greater cardiac inflammation and dysfunction following permanent left anterior descending artery ligation and transverse aortic constriction. These data suggest that JAK2 V617F mutations arising in myeloid progenitor cells may contribute to cardiovascular disease by promoting the proinflammatory properties of circulating myeloid cells.

DOI： 10.1016/j.jacbts.2019.05.013

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BACKGROUND: Although the complex roles of macrophages in myocardial injury are widely appreciated, the function of neutrophils in nonischemic cardiac pathology has received relatively little attention. METHODS: To examine the regulation and function of neutrophils in pressure overload-induced cardiac hypertrophy, mice underwent treatment with Ly6G antibody to deplete neutrophils and then were subjected to transverse aortic constriction. RESULTS: Neutrophil depletion diminished transverse aortic constriction-induced hypertrophy and inflammation and preserved cardiac function. Myeloid deficiency of Wnt5a, a noncanonical Wnt, suppressed neutrophil infiltration to the hearts of transverse aortic constriction-treated mice and produced a phenotype that was similar to the neutropenic conditions. Conversely, mice overexpressing Wnt5a in myeloid cells displayed greater hypertrophic growth, inflammation, and cardiac dysfunction. Neutrophil depletion reversed the Wnt5a overexpression-induced cardiac pathology and eliminated differences in cardiac parameters between wild-type and myeloid-specific Wnt5a transgenic mice. CONCLUSIONS: These findings reveal that Wnt5a-regulated neutrophil infiltration has a critical role in pressure overload-induced heart failure.

DOI： 10.1161/CIRCULATIONAHA.118.038820

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A long-standing question in neurodevelopment is how neurons develop a single axon and multiple dendrites from common immature neurites. Long-range inhibitory signaling from the growing axon is hypothesized to prevent outgrowth of other immature neurites and to differentiate them into dendrites, but the existence and nature of this inhibitory signaling remains unknown. Here, we demonstrate that axonal growth triggered by neurotrophin-3 remotely inhibits neurite outgrowth through long-range Ca2+ waves, which are delivered from the growing axon to the cell body. These Ca2+ waves increase RhoA activity in the cell body through calcium/calmodulin-dependent protein kinase I. Optogenetic control of Rho-kinase combined with computational modeling reveals that active Rho-kinase diffuses to growing other immature neurites and inhibits their outgrowth. Mechanistically, calmodulin-dependent protein kinase I phosphorylates a RhoA-specific GEF, GEF-H1, whose phosphorylation enhances its GEF activity. Thus, our results reveal that long-range inhibitory signaling mediated by Ca2+ wave is responsible for neuronal polarization.Emerging evidence suggests that gut microbiota influences immune function in the brain and may play a role in neurological diseases. Here, the authors offer in vivo evidence from a Drosophila model that supports a role for gut microbiota in modulating the progression of Alzheimer's disease.

DOI： 10.1038/s41467-017-00044-2

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Identifying the substrates of protein kinases to understand their modes of action has been undertaken by various approaches and remains an ongoing challenge. Phosphoproteomic technologies have accelerated the accumulation of data concerning protein phosphorylation and have uncovered vast numbers of phosphorylation sites in vivo. In this unit, a novel in vitro screening approach for protein kinase substrates is presented, based on protein-protein interaction and mass spectrometry-based phosphoproteomic technology. © 2016 by John Wiley & Sons, Inc.

DOI： 10.1002/cpcb.8

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Cell Biology  

<p>Protein phosphorylation plays an important role in the physiological regulation of cardiac function. Myocardial contraction and pathogenesis of cardiac diseases have been reported to be associated with adaptive or maladaptive protein phosphorylation; however, phosphorylation signaling in the heart is not fully elucidated. We recently developed a novel kinase-interacting substrate screening (KISS) method for exhaustive screening of protein kinase substrates, using mass spectrometry and affinity chromatography. First, we examined protein phosphorylation by extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), which has been relatively well studied in cardiomyocytes. The KISS method showed that ERK and PKA mediated the phosphorylation of known cardiac-substrates of each kinase such as Rps6ka1 and cTnI, respectively. Using this method, we found about 330 proteins as Rho-kinase-mediated substrates, whose substrate in cardiomyocytes is unknown. Among them, CARP/Ankrd1, a muscle ankyrin repeat protein, was confirmed as a novel Rho-kinase-mediated substrate. We also found that non-phosphorylatable form of CARP repressed cardiac hypertrophy-related gene Myosin light chain-2v (MLC-2v) promoter activity, and decreased cell size of heart derived H9c2 myoblasts more efficiently than wild type-CARP. Thus, focused proteomics enable us to reveal a novel signaling pathway in the heart.</p>

DOI： 10.1247/csf.16011

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Language：Japanese   Publishing type：Research paper (scientific journal)  

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.

DOI： 10.1083/jcb.201412008

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DOI： 10.1016/j.ijcard.2015.03.379

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Cell Biology  

Protein kinase A (PKA) is a serine/threonine kinase whose activity depends on the levels of cyclic AMP (cAMP). PKA plays essential roles in numerous cell types such as myocytes and neurons. Numerous substrate screens have been attempted to clarify the entire scope of the PKA signaling cascade, but it is still underway. Here, we performed a comprehensive screen that consisted of immunoprecipitation and mass spectrometry, with a focus on the identification of PKA substrates. The lysate of HeLa cells treated with Forskolin (FSK)/3-isobutyl methyl xanthine (IBMX) and/or H-89 was subjected to immunoprecipitation using anti-phospho-PKA substrate antibody. The identity of the phosophoproteins and phosphorylation sites in the precipitants was determined using liquid chromatography tandem mass spectrometry (LC/MS/MS). We obtained 112 proteins as candidate substrates and 65 candidate sites overall. Among the candidate substrates, Rho-kinase/ROCK2 was confirmed to be a novel substrate of PKA both <i>in vitro</i> and <i>in vivo</i>. In addition to Rho-kinase, we found more than a hundred of novel candidate substrates of PKA using this screen, and these discoveries provide us with new insights into PKA signaling.

DOI： 10.1247/csf.14014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japan Society for Cell Biology  

Protein phosphorylation plays a key role in regulating nearly all intracellular biological events. However, poorly developed phospho-specific antibodies and low phosphoprotein abundance make it difficult to study phosphoproteins. Cellular protein phosphorylation data have been obtained using phosphoproteomic approaches, but the detection of low-abundance or fast-cycling phosphorylation sites remains a challenge. Enrichment of phosphoproteins together with phosphopeptides may greatly enhance the spectrum of low-abundance but biologically important phosphoproteins. Previously, we used 14-3-3ζ to selectively enrich for HeLa cell lysate phosphoproteins. However, because 14-3-3 does not isolate phosphoproteins lacking the 14-3-3-binding motif, we looked for other domains that could complementarily enrich for phosphoproteins. We here assessed and characterized the phosphoprotein binding domains Pin1-WW, CHEK2-FHA, and DLG1-GK. Using a strategy based on affinity chromatography, phosphoproteins were collected from the lysates of HeLa cells treated with phosphatase inhibitor or cAMP activator. We identified different subsets of phosphoproteins associated with WW or FHA after calyculin A, okadaic acid, or forskolin treatment. Our Kinase-Oriented Substrate Screening (KiOSS) method, which used phosphoprotein-binding domains, showed that WW and FHA are applicable and useful for the identification of novel phospho-substrates for kinases and can therefore be used as biological filters for comprehensive phosphoproteome analysis.

DOI： 10.1247/csf.15004

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Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.1038/hr.2013.101

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Language：English   Presentation type：Poster presentation  

Venue：Boston, Massachusetts   Country：United States  

Language：English   Presentation type：Oral presentation (general)