Signal transduction mechanisms in heme-based globin-coupled oxygen sensors with a focus on a histidine kinase (AfGcHK) and a diguanylate cyclase (YddV or EcDosC).

Heme is a vital cofactor of proteins with roles in oxygen transport (e.g. hemoglobin), storage (e.g. myoglobin), and activation (e.g. P450) as well as electron transfer (e.g. cytochromes) and many other functions. However, its structural and functional role in oxygen sensing proteins differs markedly from that in most other enzymes, where it serves as a catalytic or functional center. This minireview discusses the mechanism of signal transduction in two heme-based oxygen sensors: the histidine kinase AfGcHK and the diguanylate cyclase YddV (EcDosC), both of which feature a heme-binding domain containing a globin fold resembling that of hemoglobin and myoglobin.

Genome-wide meta-analysis between renal overload type and renal underexcretion type of clinically defined gout in Japanese populations.

Despite progress in understanding of the genetic basis of gout, the precise factors affecting differences in gout susceptibility among different gout subtypes remain unclear. Using clinically diagnosed gout patients, we conducted a genome-wide meta-analysis of two distinct gout subtypes: the renal overload type and the renal underexcretion type. We provide genetic evidence at a genome-wide level of significance that supports a positive association between ABCG2 dysfunction and acquisition of the renal overload type.

Disruption of the dimerization interface of the sensing domain in the dimeric heme-based oxygen sensor AfGcHK abolishes bacterial signal transduction.

The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.

Different roles of myocardial ROCK1 and ROCK2 in cardiac dysfunction and postcapillary pulmonary hypertension in mice.

Although postcapillary pulmonary hypertension (PH) is an important prognostic factor for patients with heart failure (HF), its pathogenesis remains to be fully elucidated. To elucidate the different roles of Rho-kinase isoforms, ROCK1 and ROCK2, in cardiomyocytes in response to chronic pressure overload, we performed transverse aortic constriction (TAC) in cardiac-specific ROCK1-deficient (cROCK1-/-) and ROCK2-deficient (cROCK2-/-) mice. Cardiomyocyte-specific ROCK1 deficiency promoted pressure-overload-induced cardiac dysfunction and postcapillary PH, whereas cardiomyocyte-specific ROCK2 deficiency showed opposite results. Histological analysis showed that pressure-overload-induced cardiac hypertrophy and fibrosis were enhanced in cROCK1-/- mice compared with controls, whereas cardiac hypertrophy was attenuated in cROCK2-/- mice after TAC. Consistently, the levels of oxidative stress were up-regulated in cROCK1-/- hearts and down-regulated in cROCK2-/- hearts compared with controls after TAC. Furthermore, cyclophilin A (CyPA) and basigin (Bsg), both of which augment oxidative stress, enhanced cardiac dysfunction and postcapillary PH in cROCK1-/- mice, whereas their expressions were significantly lower in cROCK2-/- mice. In clinical studies, plasma levels of CyPA were significantly increased in HF patients and were higher in patients with postcapillary PH compared with those without it. Finally, high-throughput screening demonstrated that celastrol, an antioxidant and antiinflammatory agent, reduced the expressions of CyPA and Bsg in the heart and the lung, ameliorating cardiac dysfunction and postcapillary PH induced by TAC. Thus, by differentially affecting CyPA and Bsg expressions, ROCK1 protects and ROCK2 jeopardizes the heart from pressure-overload HF with postcapillary PH, for which celastrol may be a promising agent.

Portosystemic shunt for portal hypertension after Kasai operation in patients with biliary atresia.

PURPOSE: Many biliary atresia (BA) patients will eventually develop liver failure even after a successful Kasai portoenterostomy. A common complication of long-term BA survivors with their native liver is problematic portal hypertension. The aim of this study was to defend the view that portosystemic shunts can delay or negate the need for transplantation in these children. METHODS: A retrospective single center review of the efficacy of portosystemic shunts in BA patients after a successful Kasai portoenterostomy was conducted. RESULTS: From 1991 to 2017, 11 patients received portosystemic shunts. Median age of Kasai operation was 48 (36-61) days. Shunts were performed at the median age of 6.2 (4.1-6.8) years. Three of these eleven patients required subsequent liver transplantation. OS at 5 and 10 years were 90.9% and 81.8%, respectively. TFS at 5 and 10 years were 90.9% and 72.7%, respectively. Long-term complications included mild encephalopathy in 2 patients, hypersplenism in 3, and cholestasis in 1. CONCLUSION: Portosystemic shunt for the treatment of portal hypertension in carefully selected BA patients is an effective option in delaying or negating the need for liver transplantation.

Subtype-specific gout susceptibility loci and enrichment of selection pressure on ABCG2 and ALDH2 identified by subtype genome-wide meta-analyses of clinically defined gout patients.

OBJECTIVES: Genome-wide meta-analyses of clinically defined gout were performed to identify subtype-specific susceptibility loci. Evaluation using selection pressure analysis with these loci was also conducted to investigate genetic risks characteristic of the Japanese population over the last 2000-3000 years. METHODS: Two genome-wide association studies (GWASs) of 3053 clinically defined gout cases and 4554 controls from Japanese males were performed using the Japonica Array and Illumina Array platforms. About 7.2 million single-nucleotide polymorphisms were meta-analysed after imputation. Patients were then divided into four clinical subtypes (the renal underexcretion type, renal overload type, combined type and normal type), and meta-analyses were conducted in the same manner. Selection pressure analyses using singleton density score were also performed on each subtype. RESULTS: In addition to the eight loci we reported previously, two novel loci, PIBF1 and ACSM2B, were identified at a genome-wide significance level (p<5.0×10-8) from a GWAS meta-analysis of all gout patients, and other two novel intergenic loci, CD2-PTGFRN and SLC28A3-NTRK2, from normal type gout patients. Subtype-dependent patterns of Manhattan plots were observed with subtype GWASs of gout patients, indicating that these subtype-specific loci suggest differences in pathophysiology along patients' gout subtypes. Selection pressure analysis revealed significant enrichment of selection pressure on ABCG2 in addition to ALDH2 loci for all subtypes except for normal type gout. CONCLUSIONS: Our findings on subtype GWAS meta-analyses and selection pressure analysis of gout will assist elucidation of the subtype-dependent molecular targets and evolutionary involvement among genotype, phenotype and subtype-specific tailor-made medicine/prevention of gout and hyperuricaemia.

Heme: emergent roles of heme in signal transduction, functional regulation and as catalytic centres.

Protoporphyrin IX iron complex (heme) is an important cofactor for oxygen transfer, oxygen storage, oxygen activation, and electron transfer when bound to the heme proteins hemoglobin, myoglobin, cytochrome P450 and cytochrome c, respectively. In addition to these prototypical heme proteins, there are emergent, critical roles of exchangeable/labile heme in signal transduction. Specifically, it has been shown that association/dissociation of heme to/from heme-responsive sensors regulates numerous functions, including transcription, DNA binding, microRNA splicing, translation, protein kinase activity, protein degradation, heme degradation, K+ channel function, two-component signal transduction, and many other functions. In this review, we provide a comprehensive overview of structure-function relationships of heme-responsive sensors and describe new, additional roles of exchangeable/labile heme as functional inhibitors and activators. In order to complete the description of the various roles of heme in heme-bound proteins, we also mention heme as a novel chemical reaction centre for aldoxime dehydratase, cis-trans isomerase, N-N bond formation, hydrazine formation and S-S formation, and other functions. These unprecedented functions of exchangeable/labile heme and heme proteins should be of interest to biological chemists. Insight into underlying molecular mechanisms is essential for understanding the new role of heme in important physiological and pathological processes.

Genome-wide association study revealed novel loci which aggravate asymptomatic hyperuricaemia into gout.

OBJECTIVE: The first ever genome-wide association study (GWAS) of clinically defined gout cases and asymptomatic hyperuricaemia (AHUA) controls was performed to identify novel gout loci that aggravate AHUA into gout. METHODS: We carried out a GWAS of 945 clinically defined gout cases and 1003 AHUA controls followed by 2 replication studies. In total, 2860 gout cases and 3149 AHUA controls (all Japanese men) were analysed. We also compared the ORs for each locus in the present GWAS (gout vs AHUA) with those in the previous GWAS (gout vs normouricaemia). RESULTS: This new approach enabled us to identify two novel gout loci (rs7927466 of CNTN5 and rs9952962 of MIR302F) and one suggestive locus (rs12980365 of ZNF724) at the genome-wide significance level (p<5.0×10-8). The present study also identified the loci of ABCG2, ALDH2 and SLC2A9. One of them, rs671 of ALDH2, was identified as a gout locus by GWAS for the first time. Comparing ORs for each locus in the present versus the previous GWAS revealed three 'gout vs AHUA GWAS'-specific loci (CNTN5, MIR302F and ZNF724) to be clearly associated with mechanisms of gout development which distinctly differ from the known gout risk loci that basically elevate serum uric acid level. CONCLUSIONS: This meta-analysis is the first to reveal the loci associated with crystal-induced inflammation, the last step in gout development that aggravates AHUA into gout. Our findings should help to elucidate the molecular mechanisms of gout development and assist the prevention of gout attacks in high-risk AHUA individuals.

Beneficial Effects of Imatinib in a Patient with Suspected Pulmonary Veno-Occlusive Disease.

Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension (PH). The prognosis of PVOD patients remains poor, since no effective medical therapy is yet available. Imatinib is a tyrosine kinase inhibitor specific for platelet-derived growth factor receptor and is expected as a treatment option for pulmonary arterial hypertension (PAH). Recently, it has been reported that imatinib improved functional capacity of a patient with PVOD. We here report a patient with suspected PVOD who has been successfully treated with imatinib and is alive for 6 years after diagnosis. A 57-year-old woman was admitted to a hospital for severe dyspnea. Echocardiography suggested the presence of PH, because tricuspid regurgitation pressure gradient was elevated. The patient was then transferred to our hospital by an ambulance ahead of schedule due to fever and worsening dyspnea. Because the patient had no left heart disease, we diagnosed that she had PAH associated with severe right heart failure. We immediately started treatment with nitric oxide (NO) for her severe hypoxia; however, it caused pulmonary edema. We suspected PVOD from CT characteristics and pulmonary edema after PAH-targeted vasodilator therapy, and then started oral imatinib treatment. In response to imatinib, her pulmonary edema gradually improved. Since then, the patient has been alive for 6 years with imatinib and pulmonary vasodilators. At present, lung transplantation is the only effective therapy for PVOD with limited availability. We therefore propose that imatinib may be a treatment option for PVOD and a bridge to lung transplantation.

Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.

The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length AfGcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. We conclude that AfGcHK functions as an ensemble of molecules sampling at least two conformational states.

Sex differences in hemodynamic responses and long-term survival to optimal medical therapy in patients with pulmonary arterial hypertension.

It is widely known that the incidence of pulmonary arterial hypertension (PAH) is higher in female, whereas prognosis is poorer in male patients. However, sex differences in hemodynamic response to and long-term prognosis with PAH-targeted treatment in the modern era remain to be fully elucidated. We examined the long-term prognosis of 129 consecutive PAH patients (34 males and 95 females) diagnosed in our hospital from April 1999 to October 2014, and assessed hemodynamic changes in response to PAH-targeted therapy. Female patients had better 5-year survival compared with male patients (74.0 vs. 53.4%, P = 0.003); however, higher age quartiles in females were associated with poor outcome. Follow-up examination after medical treatment showed significant decreases in mean pulmonary arterial pressure (mPAP), pulmonary vascular resistance (PVR), and pulmonary arterial capacitance (PAC) in both sexes (both P < 0.05), whereas only females had a significant improvement in right ventricular end-diastolic pressure (RVEDP), right atrial pressure (RAP), cardiac index, and mixed venous oxygen saturation (SvO2) (all P < 0.05). Baseline age significantly correlated with the hemodynamic changes only in female patients; particularly, there were significant sex interactions in RVEDP and RAP (both P < 0.10). The multivariable analysis showed that SvO2 at baseline and mPAP and SvO2 at follow-up were significant prognostic factors in males, whereas the changes in mPAP, PVR, and PAC and use of endothelin-receptor antagonist in females. These results indicate that female PAH patients have better long-term prognosis than males, for which better improvements of right ventricular functions and hemodynamics may be involved.

Carboxylated phytosterol derivative-introduced liposomes for skin environment-responsive transdermal drug delivery system.

Transdermal drug delivery systems are a key technology for skin-related diseases and for cosmetics development. The delivery of active ingredients to an appropriate site or target cells can greatly improve the efficacy of medical and cosmetic agents. For this study, liposome-based transdermal delivery systems were developed using pH-responsive phytosterol derivatives as liposome components. Succinylated phytosterol (Suc-PS) and 2-carboxy-cyclohexane-1-carboxylated phytosterol (CHex-PS) were synthesized by esterification of hydroxy groups of phytosterol. Modification of phytosterol derivatives on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes was confirmed by negatively zeta potentials at alkaline pH and the change of zeta potentials with decreasing pH. In response to acidic pH and temperatures higher than body temperature, Suc-PS-containing and CHex-PS-containing liposomes exhibited content release at intracellular acidic compartments of the melanocytes at the basement membrane of the skin. Phytosterol-derivative-containing liposomes were taken up by murine melanoma-derived B16-F10 cells. These liposomes delivered their contents into endosomes and cytosol of B16-F10 cells. Furthermore, phytosterol-derivative-containing liposomes penetrated the 3 D skin models and reached the basement membrane. Results show that pH-responsive phytosterol-derivative-containing DMPC liposomes are promising for use in transdermal medical or cosmetic agent delivery to melanocytes.

GWAS of clinically defined gout and subtypes identifies multiple susceptibility loci that include urate transporter genes.

OBJECTIVE: A genome-wide association study (GWAS) of gout and its subtypes was performed to identify novel gout loci, including those that are subtype-specific. METHODS: Putative causal association signals from a GWAS of 945 clinically defined gout cases and 1213 controls from Japanese males were replicated with 1396 cases and 1268 controls using a custom chip of 1961 single nucleotide polymorphisms (SNPs). We also first conducted GWASs of gout subtypes. Replication with Caucasian and New Zealand Polynesian samples was done to further validate the loci identified in this study. RESULTS: In addition to the five loci we reported previously, further susceptibility loci were identified at a genome-wide significance level (p<5.0×10-8): urate transporter genes (SLC22A12 and SLC17A1) and HIST1H2BF-HIST1H4E for all gout cases, and NIPAL1 and FAM35A for the renal underexcretion gout subtype. While NIPAL1 encodes a magnesium transporter, functional analysis did not detect urate transport via NIPAL1, suggesting an indirect association with urate handling. Localisation analysis in the human kidney revealed expression of NIPAL1 and FAM35A mainly in the distal tubules, which suggests the involvement of the distal nephron in urate handling in humans. Clinically ascertained male patients with gout and controls of Caucasian and Polynesian ancestries were also genotyped, and FAM35A was associated with gout in all cases. A meta-analysis of the three populations revealed FAM35A to be associated with gout at a genome-wide level of significance (p meta =3.58×10-8). CONCLUSIONS: Our findings including novel gout risk loci provide further understanding of the molecular pathogenesis of gout and lead to a novel concept for the therapeutic target of gout/hyperuricaemia.

Structural characterization of the heme-based oxygen sensor, AfGcHK, its interactions with the cognate response regulator, and their combined mechanism of action in a bacterial two-component signaling system.

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX-MS studies on the AfGcHK:RR complex also showed that the N-side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the ß-strand B2 area of the RR protein's Rec1 domain, and that the C-side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and ß-strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375-1389. © 2016 Wiley Periodicals, Inc.

Genome-wide association study of clinically defined gout identifies multiple risk loci and its association with clinical subtypes.

OBJECTIVE: Gout, caused by hyperuricaemia, is a multifactorial disease. Although genome-wide association studies (GWASs) of gout have been reported, they included self-reported gout cases in which clinical information was insufficient. Therefore, the relationship between genetic variation and clinical subtypes of gout remains unclear. Here, we first performed a GWAS of clinically defined gout cases only. METHODS: A GWAS was conducted with 945 patients with clinically defined gout and 1213 controls in a Japanese male population, followed by replication study of 1048 clinically defined cases and 1334 controls. RESULTS: Five gout susceptibility loci were identified at the genome-wide significance level (p<5.0×10(-8)), which contained well-known urate transporter genes (ABCG2 and SLC2A9) and additional genes: rs1260326 (p=1.9×10(-12); OR=1.36) of GCKR (a gene for glucose and lipid metabolism), rs2188380 (p=1.6×10(-23); OR=1.75) of MYL2-CUX2 (genes associated with cholesterol and diabetes mellitus) and rs4073582 (p=6.4×10(-9); OR=1.66) of CNIH-2 (a gene for regulation of glutamate signalling). The latter two are identified as novel gout loci. Furthermore, among the identified single-nucleotide polymorphisms (SNPs), we demonstrated that the SNPs of ABCG2 and SLC2A9 were differentially associated with types of gout and clinical parameters underlying specific subtypes (renal underexcretion type and renal overload type). The effect of the risk allele of each SNP on clinical parameters showed significant linear relationships with the ratio of the case-control ORs for two distinct types of gout (r=0.96 [p=4.8×10(-4)] for urate clearance and r=0.96 [p=5.0×10(-4)] for urinary urate excretion). CONCLUSIONS: Our findings provide clues to better understand the pathogenesis of gout and will be useful for development of companion diagnostics.

Basigin mediates pulmonary hypertension by promoting inflammation and vascular smooth muscle cell proliferation.

RATIONALE: Cyclophilin A (CyPA) is secreted from vascular smooth muscle cells (VSMCs) by oxidative stress and promotes VSMC proliferation. However, the role of extracellular CyPA and its receptor Basigin (Bsg, encoded by Bsg) in the pathogenesis of pulmonary hypertension (PH) remains to be elucidated. OBJECTIVE: To determine the role of CyPA/Bsg signaling in the development of PH. METHODS AND RESULTS: In the pulmonary arteries of patients with PH, immunostaining revealed strong expression of CyPA and Bsg. The pulmonary arteries of CyPA(±) and Bsg(±) mice exposed to normoxia did not differ in morphology compared with their littermate controls. In contrast, CyPA(±) and Bsg(±) mice exposed to hypoxia for 4 weeks revealed significantly reduced right ventricular systolic pressure, pulmonary artery remodeling, and right ventricular hypertrophy compared with their littermate controls. These features were unaltered by bone marrow reconstitution. To further evaluate the role of vascular Bsg, we harvested pulmonary VSMCs from Bsg(+/+) and Bsg(±) mice. Proliferation was significantly reduced in Bsg(±) compared with Bsg(+/+) VSMCs. Mechanistic studies demonstrated that Bsg(±) VSMCs revealed reduced extracellular signal-regulated kinase 1/2 activation and less secretion of cytokines/chemokines and growth factors (eg, platelet-derived growth factor-BB). Finally, in the clinical study, plasma CyPA levels in patients with PH were increased in accordance with the severity of pulmonary vascular resistance. Furthermore, event-free curve revealed that high plasma CyPA levels predicted poor outcome in patients with PH. CONCLUSIONS: These results indicate the crucial role of extracellular CyPA and vascular Bsg in the pathogenesis of PH.

Rho-Kinase Inhibition During Early Cardiac Development Causes Arrhythmogenic Right Ventricular Cardiomyopathy in Mice.

OBJECTIVE: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by fibrofatty changes of the right ventricle, ventricular arrhythmias, and sudden death. Though ARVC is currently regarded as a disease of the desmosome, desmosomal gene mutations have been identified only in half of ARVC patients, suggesting the involvement of other associated mechanisms. Rho-kinase signaling is involved in the regulation of intracellular transport and organizes cytoskeletal filaments, which supports desmosomal protein complex at the myocardial cell-cell junctions. Here, we explored whether inhibition of Rho-kinase signaling is involved in the pathogenesis of ARVC. APPROACH AND RESULTS: Using 2 novel mouse models with SM22α- or αMHC-restricted overexpression of dominant-negative Rho-kinase, we show that mice with Rho-kinase inhibition in the developing heart (SM22α-restricted) spontaneously develop cardiac dilatation and dysfunction, myocardial fibrofatty changes, and ventricular arrhythmias, resulting in premature sudden death, phenotypes fulfilling the criteria of ARVC in humans. Rho-kinase inhibition in the developing heart results in the development of ARVC phenotypes in dominant-negative Rho-kinase mice through 3 mechanisms: (1) reduction of cardiac cell proliferation and ventricular wall thickness, (2) stimulation of the expression of the proadipogenic noncanonical Wnt ligand, Wnt5b, and the major adipogenic transcription factor, PPARγ (peroxisome proliferator activated receptor-γ), and inhibition of Wnt/ß-catenin signaling, and (3) development of desmosomal abnormalities. These mechanisms lead to the development of cardiac dilatation and dysfunction, myocardial fibrofatty changes, and ventricular arrhythmias, ultimately resulting in sudden premature death in this ARVC mouse model. CONCLUSIONS: This study demonstrates a novel crucial role of Rho-kinase inhibition during cardiac development in the pathogenesis of ARVC in mice.

Rho Kinases and Cardiac Remodeling.

Hypertensive cardiac remodeling is characterized by left ventricular hypertrophy and interstitial fibrosis, which can lead to heart failure with preserved ejection fraction. The Rho-associated coiled-coil containing kinases (ROCKs) are members of the serine/threonine protein kinase family, which mediates the downstream effects of the small GTP-binding protein RhoA. There are 2 isoforms: ROCK1 and ROCK2. They have different functions in different types of cells and tissues. There is growing evidence that ROCKs contribute to the development of cardiovascular diseases, including cardiac fibrosis, hypertrophy, and subsequent heart failure. Recent experimental studies using ROCK inhibitors, such as fasudil, have shown the benefits of ROCK inhibition in cardiac remodeling. Mice lacking each ROCK isoform also exhibit reduced myocardial fibrosis in a variety of pathological models of cardiac remodeling. Indeed, clinical studies with fasudil have suggested that ROCKs could be potential novel therapeutic targets for cardiovascular diseases. In this review, we summarize the current understanding of the roles of ROCKs in the development of cardiac fibrosis and hypertrophy and discuss their therapeutic potential for deleterious cardiac remodeling. (Circ J 2016; 80: 1491-1498).

Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor AfGcHK.

AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O2 or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H2S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O2, CO, and NO. To address this knowledge gap, we investigated the effects of H2S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O2-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na2S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na2S, the initially formed Fe(III)-SH heme complex was quickly converted into Fe(II) and Fe(II)-O2 complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)-SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)-OH complex), whereas in the case of EcDOS the changes in coordination caused by Na2S treatment led to remarkable increases in catalytic activity.

Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity.

The globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction's kinetics. The kcat values of the heme Fe(III)-OH(-), Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1-1.2 min(-1), and their Km(ATP) values were 18.9-35.4 µM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min(-1) but a very high Km(ATP) value of 357 µM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and Km(ATP) values of 0.4 min(-1) and 78 µM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with kcat and Km(ATP) values of 0.3 min(-1) and 33.6 µM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme's catalytic activity because they modulate its ATP binding affinity and thus change its kcat/Km(ATP) value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed.