Uric Acid as a Risk Factor for Chronic Kidney Disease and Cardiovascular Disease　- Japanese Guideline on the Management of Asymptomatic Hyperuricemia.

Serum uric acid (UA) is taken up by endothelial cells and reduces the level of nitric oxide (NO) by inhibiting its production and accelerating its degradation. Cytosolic and plasma xanthine oxidase (XO) generates superoxide and also decreases the NO level. Thus, hyperuricemia is associated with impaired endothelial function. Hyperuricemia is often associated with vascular diseases such as chronic kidney disease (CKD) and cardiovascular disease (CVD). It has long been debated whether hyperuricemia is causally related to the development of these diseases. The 2020 American College of Rheumatology Guideline for the Management of Gout (ACR2020) does not recommend pharmacological treatment of hyperuricemia in patients with CKD/CVD. In contrast, the Japanese Guideline on Management of Hyperuricemia and Gout (JGMHG), 3rdedition, recommends pharmacological treatment of hyperuricemia in patients with CKD. In a FREED study on Japanese hyperuricemic patients with CVD, an XO inhibitor, febuxostat, improved the primary composite endpoint of cerebro-cardio-renovascular events, providing a rationale for the use of urate-lowering agents (ULAs). Since a CARES study on American gout patients with CVD treated with febuxostat revealed increased mortality, ACR2020 recommends switching to different ULAs. However, there was no difference in the mortality of Japanese patients between the febuxostat-treated group and the placebo or allopurinol-treated groups in either the FEATHER or FREED studies.

Apoptosis induced by an uromodulin mutant C112Y and its suppression by topiroxostat.

BACKGROUND: Familial juvenile hyperuricemic nephropathy (FJHN) is an autosomal dominant disorder caused by mutations in UMOD that encodes uromodulin. Topiroxostat, a novel non-purine analog, selectively inhibits xanthine oxidase and reduces the serum uric acid levels and the urinary albuminuria. METHODS: Genomic DNA of a patient was extracted from peripheral white blood. Exons and flanking sequences of UMOD were amplified by PCR with primers. Mutation analysis was performed by direct sequencing of the PCR products. The wild-type and mutant uromodulin were expressed in HEK293 cells and analyzed by western blotting, immunoprecipitation, immunofluorescence, and flow cytometry. RESULTS: We identified an FJHN patient who carried a novel UMOD mutation G335A (C112Y). The levels of both cytosolic and secreted C112Y protein were significantly decreased compared with the wild-type, whereas the level of ubiquitination was higher in C112Y than that in the wild type. The half-life of C112Y was shortened and it was restored by a proteasome inhibitor MG132. Immunofluorescence revealed decreased levels of C112Y in the Golgi apparatus and on the plasma membrane. Expression of C112Y induced cellular apoptosis as revealed by flow cytometry. Apoptosis induced by C112Y was suppressed by topiroxostat. CONCLUSION: C112Y causes its protein instability resulting cellular apoptosis which could be suppressed with topiroxostat.

Electrophysiological properties of prion-positive cardiac progenitors derived from murine embryonic stem cells.

BACKGROUND: The prion protein (PrP) has been reported to serve as a surface maker for isolation of cardiomyogenic progenitors from murine embryonic stem (ES) cells. Although PrP-positive cells exhibited automaticity, their electrophysiological characteristics remain unresolved. The aim of the present study was therefore to investigate the electrophysiological properties of PrP-positive cells in comparison with those of HCN4p-or Nkx2.5-positive cells. METHODS AND RESULTS: Differentiation of AB1, HCN5p-EGFP and hcgp7 ES cells into cardiac progenitors was induced by embryoid body (EB) formation. EBs were dissociated and cells expressing PrP, HCN4-EGFP and/or Nkx2.5-GFP were collected via flow cytometry. Sorted cells were subjected to reverse transcriptase-polymerase chain reaction, immunostaining and patch-clamp experiments. PrP-positive cells expressed mRNA of undifferentiation markers, first and second heart field markers, and cardiac-specific genes and ion channels, indicating their commitment to cardiomyogenic progenitors. PrP-positive cells with automaticity showed positive and negative chronotropic responses to isoproterenol and carbamylcholine, respectively. Hyperpolarization-activated cation current (I(f)) was barely detectable, whereas Na(+) and L-type Ca(2+) channel currents were frequently observed. Their spontaneous activity was slowed by inhibition of sarcoplasmic reticulum Ca(2+) uptake and release but not by blocking I(f). The maximum diastolic potential of their spontaneous firings was more depolarized than that of Nkx2.5-GFP-positive cells. CONCLUSIONS: PrP-positive cells contained cardiac progenitors that separated from the lineage of sinoatrial node cells. PrP can be used as a marker to enrich nascent cardiac progenitors.

Trimetazidine improves left ventricular global longitudinal strain value in patients with heart failure with reduced ejection fraction due to ischemic heart disease.

Heart failure with reduced ejection fraction (HFrEF) due to ischemic heart disease (IHD) showed a progressive decline in left ventricular contractile function. However, no previous study has examined the left ventricular global longitudinal strain (LV GLS) parameter that represents LV contractile function. We investigated whether trimetazidine could improve the LV GLS value in patients with HFrEF due to IHD. We performed a double-blind, randomized controlled trial (RCT) including 26 patients with HFrEF due to stable IHD who were given modified-release trimetazidine 35 mg twice per day (n = 13) or placebo (n = 13) for 3 months in addition to standard medication. Left ventricular systolic function including GLS values was assessed at baseline and after 3 months using echocardiography. A total of 25 participants (13 control and 12 trimetazidine groups) were recruited with a baseline average age of 57.1 ± 10 years, and LV ejection fraction (LVEF) value of 34.6% ± 4.4%, and a GLS value of 7.4% ± 2.1%. Baseline clinical characteristics and echocardiogram were similar between the two groups. There was significant GLS improvement in the trimetazidine group (-6.9% ± 2.4% to -8.4% ± 2.6%, p = 0.000), but no significant differences were noted in the control group. The GLS improvement was significantly higher in the trimetazidine group than the control (1.5% + 0.9% vs. -0.7% + 1.7%, p = 0.001). No adverse drug reactions from the administration of trimetazidine in this study. Trimetazidine may improve GLS values in patients with HFrEF due to IHD.

Chemical chaperone therapy: chaperone effect on mutant enzyme and cellular pathophysiology in ß-galactosidase deficiency.

ß-Galactosidase deficiency is a group of lysosomal lipid storage disorders with an autosomal recessive trait. It causes two clinically different diseases, G(M1) -gangliosidosis and Morquio B disease. It is caused by heterogeneous mutations in the GLB1 gene coding for the lysosomal acid ß-galactosidase. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant ß-galactosidase proteins. In this study, we performed genotype analyses of patients with ß-galactosidase deficiency and identified 46 mutation alleles including 9 novel mutations. We then examined the NOEV effect on mutant ß-galactosidase proteins by using six strains of patient-derived skin fibroblast. We also performed mutagenesis to identify ß-galactosidase mutants that were responsive to NOEV and found that 22 out of 94 mutants were responsive. Computational structural analysis revealed the mode of interaction between human ß-galactosidase and NOEV. Moreover, we confirmed that NOEV reduced G(M1) accumulation and ameliorated the impairments of lipid trafficking and protein degradation in ß-galactosidase deficient cells. These results provided further evidence to NOEV as a promising chaperone compound for ß-galactosidase deficiency.

Identification, isolation and characterization of HCN4-positive pacemaking cells derived from murine embryonic stem cells during cardiac differentiation.

BACKGROUND: Development of biological pacemaker is a potential treatment for bradyarrhythmias. Pacemaker cells could be extracted from differentiated embryonic stem (ES) cells based on their specific cell marker hyperpolarization-activated cyclic nucleotide-gated (HCN)4. The goal of this study was to develop a method of identification, isolation, and characterization of pacemaking cells derived from differentiated ES cells with GFP driven by HCN4 promoter. METHODS AND RESULTS: Polymerase chain reaction (PCR) screening and southern blot analysis revealed that HCN4p-EGFP trans-gene was stably integrated into the chromosome of mouse AB1 ES cells. RT-PCR and immunostaining results showed similar expression of the specific cardiac pacemaker markers of the HCN4p-EGFP ES cells and its parental AB1 ES cell lines. Although HCN4p-EGFP trans-gene may have slight effect on the general mesodermal differentiation, it had no effect on the pluripotency of ES cells, on the transcription of cardiac specific factors and cardiac contractile proteins, and on the capability of ES cells to differentiate into pacemaker cells. Electrophysiological study indicated that HCN4p-GFP-positive cells revealed the spontaneous action potential, which was slowed by the treatment with 2 mM Cs(+), and expressed the hyperpolarization-activeted cation current I(f) encoded by HCN4 gene. CONCLUSION: By the approach of using stable transfectant of HCN4p-EGFP gene, the identification, isolation, and characterization of ES cell-derived pacemaking cells could be carried out.

Recurrent SARS-CoV-2 RNA positivity after COVID-19: a systematic review and meta-analysis.

Present study aimed to estimate the incidence of recurrent SARS-CoV-2 RNA positivity after recovery from COVID-19 and to determine the factors associated with recurrent positivity. We searched the PubMed, MedRxiv, BioRxiv, the Cochrane Library, ClinicalTrials.gov, and the World Health Organization International Clinical Trials Registry for studies published to June 12, 2020. Studies were reviewed to determine the risk of bias. A random-effects model was used to pool results. Heterogeneity was assessed using I2. Fourteen studies of 2568 individuals were included. The incidence of recurrent SARS-CoV-2 positivity was 14.8% (95% confidence interval [CI] 11.44-18.19%). The pooled estimate of the interval from disease onset to recurrence was 35.4 days (95% CI 32.65-38.24 days), and from the last negative to the recurrent positive result was 9.8 days (95% CI 7.31-12.22 days). Patients with younger age and a longer initial illness were more likely to experience recurrent SARS-CoV-2 positivity, while patients with diabetes, severe disease, and a low lymphocyte count were less likely to experience. Present study concluded that the incidence of recurrent SARS-CoV-2 positivity was 14.8% suggesting further studies must be conducted to elucidate the possibility of infectious individuals with prolonged or recurrent RNA positivity.

Functional stabilization of Kv1.5 protein by Hsp70 in mammalian cell lines.

The aim of this study was to elucidate the mechanisms for regulations of cardiac Kv1.5 channel expression. We particularly focused on the role of heat shock proteins (Hsps). We tested the effects of Hsps on the stability of Kv1.5 channels using biochemical and electrophysiological techniques: co-expression of Kv1.5 and Hsp family proteins in mammalian cell lines, followed by Western blotting, immunoprecipitation, pulse-chase analysis, immunofluorescence and whole-cell patch clamp. Hsp70 and heat shock factor 1 increased the expression of Kv1.5 protein in HeLa and COS7 cells, whereas either Hsp40, 27 or 90 did not. Hsp70 prolonged the half-life of Kv1.5 protein. Hsp70 was co-immunoprecipitated and co-localized with Kv1.5-FLAG. Hsp70 significantly increased the immunoreactivity of Kv1.5 in the endoplasmic reticulum, Golgi apparatus and on the cell membrane. Hsp70 enhanced Kv1.5 current of transfected cells, which was abolished by pretreatment with brefeldin A or colchicine. Thus, Hsp70, but not other Hsps, stabilizes functional Kv1.5 protein.

Optimization of PCR Condition: The First Study of High Resolution Melting Technique for Screening of APOA1 Variance.

BACKGROUND: High resolution melting (HRM) is a post-PCR technique for variant screening and genotyping based on the different melting points of DNA fragments. The advantages of this technique are that it is fast, simple, and efficient and has a high output, particularly for screening of a large number of samples. APOA1 encodes apolipoprotein A1 (apoA1) which is a major component of high density lipoprotein cholesterol (HDL-C). This study aimed to obtain an optimal quantitative polymerase chain reaction (qPCR)-HRM condition for screening of APOA1 variance. METHODS: Genomic DNA was isolated from a peripheral blood sample using the salting out method. APOA1 was amplified using the RotorGeneQ 5Plex HRM. The PCR product was visualized with the HRM amplification curve and confirmed using gel electrophoresis. The melting profile was confirmed by looking at the melting curve. RESULTS: Five sets of primers covering the translated region of APOA1 exons were designed with expected PCR product size of 100-400 bps. The amplified segments of DNA were amplicons 2, 3, 4A, 4B, and 4C. Amplicons 2, 3 and 4B were optimized at an annealing temperature of 60 °C at 40 PCR cycles. Amplicon 4A was optimized at an annealing temperature of 62 °C at 45 PCR cycles. Amplicon 4C was optimized at an annealing temperature of 63 °C at 50 PCR cycles. CONCLUSION: In addition to the suitable procedures of DNA isolation and quantification, primer design and an estimated PCR product size, the data of this study showed that appropriate annealing temperature and PCR cycles were important factors in optimization of HRM technique for variant screening in APOA1.

Inhibitory effects of local anesthetics on the proteasome and their biological actions.

Local anesthetics (LAs) inhibit endoplasmic reticulum-associated protein degradation, however the mechanisms remain elusive. Here, we show that the clinically used LAs pilsicainide and lidocaine bind directly to the 20S proteasome and inhibit its activity. Molecular dynamic calculation indicated that these LAs were bound to the ß5 subunit of the 20S proteasome, and not to the other active subunits, ß1 and ß2. Consistently, pilsicainide inhibited only chymotrypsin-like activity, whereas it did not inhibit the caspase-like and trypsin-like activities. In addition, we confirmed that the aromatic ring of these LAs was critical for inhibiting the proteasome. These LAs stabilized p53 and suppressed proliferation of p53-positive but not of p53-negative cancer cells.

Impairment of ubiquitin-proteasome system by E334K cMyBPC modifies channel proteins, leading to electrophysiological dysfunction.

Cardiac arrhythmogenesis is regulated by channel proteins whose protein levels are in turn regulated by the ubiquitin-proteasome system (UPS). We have previously reported on UPS impairment induced by E334K cardiac myosin-binding protein C (cMyBPC), which causes hypertrophic cardiomyopathy (HCM) accompanied by arrhythmia. We hypothesized that UPS impairment induced by E334K cMyBPC causes accumulation of cardiac channel proteins, leading to electrophysiological dysfunction. Wild-type or E334K cMyBPC was overexpressed in HL-1 cells and primary cultured neonatal rat cardiac myocytes. The expression of E334K cMyBPC suppressed cellular proteasome activities. The protein levels of K(v)1.5, Na(v)1.5, Hcn4, Ca(v)3.2, Ca(v)1.2, Serca, RyR2, and Ncx1 were significantly higher in cells expressing E334K cMyBPC than in wild type. They further increased in cells pretreated with MG132 and had longer protein decays. The channel proteins retained the correct localization. Cells expressing E334K cMyBPC exhibited higher Ca(2+) transients and longer action potential durations (APDs), accompanied by afterdepolarizations and higher apoptosis. Those augments of APD and Ca(2+) transients were recapitulated by a simulation model. Although a Ca(2+) antagonist, azelnidipine, neither protected E334K cMyBPC from degradation nor affected E334K cMyBPC incorporation into the sarcomere, it normalized the APD and Ca(2+) transients and partially reversed the levels of those proteins regulating apoptosis, thereby attenuating apoptosis. In conclusion, UPS impairment caused by E334K cMyBPC may modify the levels of channel proteins, leading to electrophysiological dysfunction. Therefore, UPS impairment due to a mutant cMyBPC may partly contribute to the observed clinical arrhythmias in HCM patients.

Effect of losartan and benzbromarone on the level of human urate transporter 1 mRNA.

Both an angiotensin II receptor blocker, losartan (CAS 124750-99-8) and a serum urate lowering agent, benzbromarone (CAS 3562-84-3) exert a uricosuric action by inhibiting urate transporter 1 (URAT1). A recent clinical trial indicated that losartan could reduce the level of serum urate in hypertensive patients treated with urate lowering agents, suggesting the different mode of action of losartan from benzbromarone. In the present study, the effect of losartan and benzbromarone on the level of URAT1 mRNA was determined in transfected HEK293 cells. Losartan caused a significant reduction of its mRNA level, whereas it was not affected by benzbromarone. These results indicate that losartan decreases the level of human URAT1 mRNA, which may underlie the uricosuric action of losartan in hypertensive patients treated with serum urate lowering agents.

Stabilizing effects of eicosapentaenoic acid on Kv1.5 channel protein expressed in mammalian cells.

We investigated the effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the stability of Kv1.5 channel protein. The expression and function of Kv1.5 (Kv1.5-FLAG) in transfected African green monkey kidney fibroblast cells as well as rat atrium were estimated by immunoblotting, immunoprecipitation, immunofluorescence and patch-clamp techniques. Both EPA and DHA immediately blocked Kv1.5 channel current in a dose-dependent manner, accompanied by reduction of their phosphorylation. Chronic treatment (for 12 h) with EPA at lower concentrations (0.3-10 muM) increased the level of Kv1.5-FLAG protein as well as Kv1.5 channel current without changes in its gating kinetics, prolonging its half-life; in contrast, both EPA and DHA at higher concentrations (30-100 muM) decreased the expression of Kv1.5-FLAG. EPA at the higher concentrations also decreased mRNA of Kv1.5 and synapse-associated protein 97 expression. EPA at the lower concentrations increased Kv1.5 expression in the endoplasmic reticulum, Golgi apparatus and cell membrane. EPA-induced increase of Kv1.5 channel expression and current was abolished by pretreatment with the protein transport inhibitor brefeldin A or colchicines, and by the Kv1.5 channel blocker 4-aminopyridine. Oral administration of EPA (30 mg/kg) increased the level of endogenous Kv1.5 in rat atria. These results indicate that chronic treatment with EPA at lower concentrations stabilizes Kv1.5 channel protein in the endoplasmic reticulum and Golgi apparatus thereby enhancing the Kv1.5 channel current on the cell membrane.

Ubiquitin-proteasome system impairment caused by a missense cardiac myosin-binding protein C mutation and associated with cardiac dysfunction in hypertrophic cardiomyopathy.

The ubiquitin-proteasome system is responsible for the disappearance of truncated cardiac myosin-binding protein C, and the suppression of its activity contributes to cardiac dysfunction. This study investigated whether missense cardiac myosin-binding protein C gene (MYBPC3) mutation in hypertrophic cardiomyopathy (HCM) leads to destabilization of its protein, causes UPS impairment, and is associated with cardiac dysfunction. Mutations were identified in Japanese HCM patients using denaturing HPLC and sequencing. Heterologous expression was investigated in COS-7 cells as well as neonatal rat cardiac myocytes to examine protein stability and proteasome activity. The cardiac function was measured using echocardiography. Five novel MYBPC3 mutations -- E344K, DeltaK814, Delta2864-2865GC, Q998E, and T1046M -- were identified in this study. Compared with the wild type and other mutations, the E334K protein level was significantly lower, it was degraded faster, it had a higher level of polyubiquination, and increased in cells pretreated with the proteasome inhibitor MG132 (50 microM, 6 h). The electrical charge of its amino acid at position 334 influenced its stability, but E334K did not affect its phosphorylation. The E334K protein reduced cellular 20 S proteasome activity, increased the proapoptotic/antiapoptotic protein ratio, and enhanced apoptosis in transfected Cos-7 cells and neonatal rat cardiac myocytes. Patients carrying the E334K mutation presented significant left ventricular dysfunction and dilation. The conclusion is the missense MYBPC3 mutation E334K destabilizes its protein through UPS and may contribute to cardiac dysfunction in HCM through impairment of the ubiquitin-proteasome system.

Changes of HCN gene expression and I(f) currents in Nkx2.5-positive cardiomyocytes derived from murine embryonic stem cells during differentiation.

Changes in the expression of hyperpolarization-activated cyclic nucleotide (HCN)-gated channels and I(f) currents during the differentiation of embryonic stem cells into cardiac cells remain unknown. We examined changes of HCN genes in expression and function during the differentiation of Nkx2.5-positive cardiac precursor cells derived from mouse ES cells using cell sorting, RTPCR, immunofluorescence and whole cell patch-clamp techniques. Cs(+)-induced inhibition of automaticity and transcription of HCN genes increased during differentiation. Expressions of Nkx2.5, a marker of cardiac progenitor cell, and Flk1, a marker of hemangioblast, were mutually exclusive. Messenger RNA and proteins encoded by HCN1 and 4 genes were predominantly observed in Nkx2.5-positive cells on day 15, although Flk1-positive cells did not express genes of the HCN family on that day. Cs(+)-induced prolongation of the cycle of spontaneous action potentials and I(f) currents were predominantly observed on day 15. These results suggested that a fraction of Nkx2.5-positive cardiac precursor cells was committed to pacemaking cells expressing I(f) channels predominantly encoded by HCN 1 and 4 genes.

Inhibition of beta-adrenergic signaling by intracellular AMP is independent of cell-surface adenosine receptors in rat cardiac cells.

We report a novel action of intracellular adenosine monophosphate (AMP) to inhibit beta-adrenergic signaling in isolated rat ventricular myocytes. Extracellular application of adenosine or AMP suppressed isoproterenol (Iso)-induced prolongation of action potential duration (APD). This effect was completely abolished by an A(1)-receptor antagonist, DPCPX. Intracellular application of AMP, but not adenosine, attenuated Iso-induced APD prolongation. Iso-induced increases in the L-type Ca(2+) current (I(Ca,L)) were also inhibited by intracellular AMP. These inhibitory effects were not affected by either DPCPX or glibenclamide. In vitro, AMP directly inhibited PKA activity via binding to its regulatory subunit. These results suggest that intracellular AMP attenuates beta-adrenergic signaling by directly inhibiting PKA activity, independently of A(1)-purinergic receptor.

Delayed onset of beating and decreased expression of T-type Ca2+ channel in mouse ES cell-derived cardiocytes carrying human chromosome 21.

The mouse ES cell line hcgp7/#21, which carries a human chromosome 21 (hChr.21), was used as an in vitro model to examine the effects of hChr.21 on cardiomyocyte differentiation. Cardiomyocytes derived from hcgp7/#21 showed a significant delay in the onset of spontaneous beating. The number of Nkx2.5/GFP(+) cardiac progenitor cells was comparable to that in control ES cells and they also expressed comparable mRNA levels for mesodermal markers, cardiac specific transcription factors, contractile proteins, and L-type Ca(2+) channels. However, cells from hcgp7/#21 expressed significantly reduced levels of mRNA for Cav3.1 and Cav3.2, which was consistent with the decreased number of cells expressing T-type Ca(2+) channels and decreased T-type Ca(2+) channel currents. These findings suggest that the presence of human chromosome 21 suppresses expression of T-type Ca(2+) channels in cardiomyocytes during differentiation, which may be responsible for delayed onset of spontaneous beating.