Background: Monocarboxylate transporter 9 (MCT9), an orphan transporter member of the solute carrier family 16 (SLC16), possibly reabsorbs uric acid in the renal tubule and has been suggested by genome-wide association studies to be involved in the development of hyperuricemia and gout. In this study we investigated the mechanisms regulating the expression of human (h) MCT9, its degradation, and physiological functions. Methods and Results: hMCT9-FLAG was stably expressed in HEK293 cells and its degradation, intracellular localization, and urate uptake activities were assessed by pulse-chase analysis, immunofluorescence, and [14C]-urate uptake experiments, respectively. hMCT9-FLAG was localized on the plasma membrane as well as in the endoplasmic reticulum and Golgi apparatus. The proteasome inhibitors MG132 and lactacystine increased levels of hMCT9-FLAG protein expression with enhanced ubiquitination, prolonged their half-life, and decreased [14C]-urate uptake. [14C]-urate uptake was increased by both heat shock (HS) and the HS protein inducer geranylgeranylacetone (GGA). Both HS and GGA restored the [14C]-urate uptake impaired by MG132. Conclusions: hMCT9 does transport urate and is degraded by a proteasome, inhibition of which reduces hMCT9 expression on the cell membrane and urate uptake. HS enhanced urate uptake through hMCT9.

BACKGROUND Intracellular uric acid is known to increase the protein level and channel current of atrial Kv1.5; however, mechanisms of the uric acid-induced enhancement of Kv1.5 expression remain unclear. Methods and Results: The effects of uric acid on mRNA and protein levels of Kv1.5, as well as those of Akt, HSF1 and Hsp70, in HL-1 cardiomyocytes were studied by using qRT-PCR and Western blotting. The uptake of uric acid was measured using radio-labeled uric acid. The Kv1.5-mediated channel current was also measured by using patch clamp techniques. Uric acid up-taken by HL-1 cells significantly increased the level of Kv1.5 proteins in a concentration-dependent manner, with this increase abolished by an uric acid transporter inhibitor. Uric acid slowed degradation of Kv1.5 proteins without altering its mRNA level. Uric acid enhanced phosphorylation of Akt and HSF1, and thereby increased both transcription and translation of Hsp70; these effects were abolished by a PI3K inhibitor. Hsp70 knockdown abolished the uric acid-induced increases of Kv1.5 proteins and channel currents. CONCLUSIONS Intracellular uric acid could stabilize Kv1.5 proteins through phosphorylation of Akt and HSF1 leading to enhanced expression of Hsp70.

Proepicardium (PE) cells generate cardiac fibroblasts, smooth muscle cells (SMCs) and endothelial cells that form coronary arteries. T-box18 (Tbx18) is a well-known marker of PE cells and epicardium. We examined whether Tbx18-positive cells differentiated from murine embryonic stem (ES) cells serve as PE progenitors to give rise to vascular SMCs and fibroblasts. To collect Tbx18-positive cells, we established Tbx18-EGFP knock-in mouse ES cells using the CRISPR/Cas9 system. We harvested the Tbx18-EGFP-positive cells on day 8, 10 and 14 after the initiation of differentiation; Tbx18 mRNA was enriched on day 8 to 14 and Snai2 mRNA was enriched on day 8 and 10, indicating successful collection of Tbx18-positive cells. Tbx18-EGFP-positive cells expressed the PE marker WT1 on day 8 and 10. They also expressed the SMC marker Acta2 and fibroblast markers Thy1 and Fsp1 on day 8 to 14, but did not express the endothelial cell marker PECAM or the cardiac cell marker CD166 or Myh7. In conclusion, Tbx18-positive cells represent a part of PE cells in the initial phase of differentiation and subsequently include SMCs as well as fibroblasts. These results indicate that Tbx18-positive cells serve as a PE progenitor to supply a variety of cells that contribute to the formation of coronary arteries.

Background: Although urate impaired the endothelial function, its underlying mechanism remains unknown. We hypothesized that urate impaired nitric oxide (NO) production in human umbilical vein endothelial cells (HUVECs) via activation of uric acid transporters (UATs).Purpose and method: In the present study, we studied effects of urate on NO production and eNOS protein expression in HUVEC cells in the presence and absence of urate lowering agents using molecular biological and biochemical assays.Results: HUVECs expressed the 4 kinds of UATs, URATv1, ABCG2, MRP4 and MCT9. Exposure to urate at 7mg/dl for 24h significantly reduced production of NO. Pretreatment with benzbromarone, losartan or irbesartan normalized NO production. The same exposure resulted in dephosphorylation of endothelial NO synthase (eNOS) in HUVECs. Again pretreatment with benzbromarone, losartan or irbesartan abolished this effect.Conclusion: Urate reduced NO production by impaired phosphorylation of eNOS in HUVEC via activation of UATs, which could be normalized by urate lowering agents.

BACKGROUND Long QT syndrome 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). Most of its mutations give rise to unstable hERG proteins degraded by the proteasome. Recently, carbachol was reported to stabilize the wild-type hERG-FLAG via activation of the muscarinic type 3 receptor (M3-mAChR). Its action on mutant hERG-FLAG, however, remains uninvestigated.Methods and Results:A novel mutant hERG-FLAG carried 2 mutations: an amino acid substitution G572S and an in-frame insertion D1037_V1038insGD. When expressed in HEK293 cells, this mutant hERG-FLAG was degraded by the proteasome and failed to be transported to the cell surface. Carbachol restored stability of the mutant hERG-FLAG and facilitated cell-surface expression. Carbachol activated PKC, augmented phosphorylation of heat shock factor 1 (HSF1) and enhanced expression of heat shock proteins (hsps), hsp70 and hsp90. Both a M3-mAChR antagonist, 4-DAMP, and a PKC inhibitor, bisindolylmaleimide, abolished carbachol-induced stabilization of the mutant hERG-FLAG. CONCLUSIONS M3-mAChR activation leads to enhancement of hsp expression via PKC-dependent phosphorylation of HSF1, thereby stabilizing the mutant hERG-FLAG protein. Thus, M3-mAChR activators may have a therapeutic value for patients with LQT2. (Circ J 2016; 80: 2443-2452).

BACKGROUND:The prion protein (PrP) might be useful as a tool to collect cardiac progenitor cells derived from embryonic stem (ES) cells. It is also possible that PrP(+) cells include undifferentiated cells with a capacity to develop into tumors.METHODS:PrP(+) cells isolated from embryoid bodies (EB) formed by mouse AB1 ES cells were examined using RT-PCR analysis and clonogeneic cell assay. To assess their potential to differentiate into cardiomyocytes, Nkx2.5(GFP/+) (hcgp7) cells, another ES cell line that carries the GFP reporter gene in the Nkx2.5 loci, were used.RESULTS:PrP(+) cells isolated from EB of day 7 and 14 did not express pluripotency markers, but expressed cardiac cell markers, while PrP(+) cells isolated from EB of day 21 expressed pluripotency markers. Cultured PrP(+) cells isolated from EB of day 21 expressed pluripotency markers to form colonies, whereas those isolated from EB of day 7 and 14 did not. To exclude proliferating cells from PrP(+) cells, stage specific embryo antigen 1 (SSEA1) was employed as a second marker. PrP(+)/SSEA1(-) cells did not proliferate and expressed cardiac cell markers, while PrP(+)/SSEA1(+) did proliferate.CONCLUSION:PrP(+) cells isolated from EB included undifferentiated cells in day 21. PrP(+)/SSEA1(-) cells included cardiomyoctes, suggesting PrP and SSEA1 may be useful as markers to enrich the fraction of cardiomyocytes.

BACKGROUND:Besides its antiarrhythmic action, carvedilol has an activity to suppress cardiac tissue damage. However, it is unknown whether it has any effect on cellular apoptosis and ion channel remodelling.PURPOSE:To know whether carvedilol has any effect on apoptosis and ion channel remodeling of HL-1 cells expressing E334K MyBPC, and comparing it with bisoprolol.METHOD:We examined effects of carvedilol and bisoprolol on the levels of pro- and anti-apoptotic proteins and ion channels as well as apoptosis of HL-1 cells transfected with E334K MyBPC using Western blot and flow cytometry.RESULTS:Carvedilol decreased the protein levels of p53, Bax and cytochrome c and increased that of Bcl-2 in HL-1 cells expressing E334K MyBPC. Bisoprolol failed to affect the protein levels. Both carvedilol and bisoprolol increased the protein levels of Cav1.2 but not that of Nav1.5. Carvedilol was stronger than bisoprolol at decreasing the number of annexin-V positive cells in HL-1 cells expressing E334K MyBPC.CONCLUSION:Carvedilol suppressed apoptosis of HL-1 cells expressing E334K MyBPC through modification of pro- and anti-apoptotic proteins, whose was associated with an increase of Cav 1.2 protein expression.

BACKGROUNDHyperuricemia induces endothelial dysfunction, oxidative stress and inflammation, increasing cardiovascular morbidities. It also raises the incidence of atrial fibrillation; however, underlying mechanisms are unknown.METHODSANDRESULTSThe effects of urate on expression of Kv1.5 in cultured mouse atrial myocytes (HL-1 cells) using reverse transcriptase-PCR, immunoblots, flow cytometry and patch-clamp experiments were studied. Treatment with urate at 7 mg/dl for 24 h increased the Kv1.5 protein level, enhanced ultra-rapid delayed-rectifier K(+)channel currents and shortened action potential duration in HL-1 cells. HL-1 cells expressed the influx uric acid transporter (UAT), URATv1, and the efflux UATs, ABCG2 and MRP4. An inhibitor against URATv1, benzbromarone, abolished the urate effects, whereas an inhibitor against ABCG2, KO143, augmented them. Flow cytometry showed that urate induced an increase in reactive oxygen species, which was abolished by the antioxidant, N-acetylcysteine (NAC), and the NADPH-oxidase inhibitor, apocynin. Both NAC and apocynin abolished the enhancing effects of urate on Kv1.5 expression. A urate-induced increase in the Kv1.5 proteins was accompanied by phosphorylation of extracellular signal-regulated kinase (ERK), and was abolished by an ERK inhibitor, PD98059. NAC abolished phosphorylation of ERK by urate.CONCLUSIONSIntracellular urate taken up by UATs enhanced Kv1.5 protein expression and function in HL-1 atrial myocytes, which could be attributable to ERK phosphorylation and oxidative stress derived from nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase.

7 mg/dl.4 It is caused by an imbalance between urate synthesis and its renal excretion; the predominant cause is reduced excretion due to altered expression of uric acid transporters (UATs) in the kidney. UATs in renal proximal tubular cells play a pivotal role in the regulation of serum urate levels.5,6 Hyperuricemia has been reported to be associated with various kinds of diseases such as hypertension, metabolic synompared to other mammals whose serum urate levels are 0.5–1 mg/dl or less, humans have a higher serum urate level due to loss of uricase activity.1 Hyperuricemia, defined as a condition with the serum urate level exceeding 6.8 mg/dl, is a risk factor for gout, kidney stone and renal failure.2,3 Recently, the guideline in Japan has indicated that hyperuricemia is defined as the serum urate level of more than C

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.

Besides its antiarrhythmic effect on atrial fibrillation, bepridil protects tissue, yet its effect on apoptosis has never been fully tested. We examine the effect of bepridil on apoptosis of HL-1 cells expressing E334K myosin-binding protein C (MyBPC), a model cell of apoptosis. Bepridil was compared with amiodarone, and its effects on the expression of pro-and anti-apoptotic protein and apoptosis of HL-1 cells expressing mutant E334K MyBPC- green fluorescent protein (GFP) was analyzed using Western blot and a flow cytometer. Bepridil decreased the protein levels of both Bax and cytochrome c of cells expressing E334K MyBPC-GFP with no changes in p53 and Bcl-2, while amiodarone decreased cytochrome c but did not influence Bax except in its highest concentration. It also decreased the number of Annexin-V positive cells of HL-1 cells expressing E334K MyBPC-GFP, and decreased apoptosis of HL-1 cells expressing E334K MyBPC-GFP.

P19 embryonal carcinoma (EC) cells are pluripotent stem cells and have numerous morphological and biochemical properties in common with embryonic stem (ES) cells. However, P19 cells differentiate very ineffectively as embryoid bodies (EBs) without the specific chemical inducers whereas ES cells exhibit spontaneous differentiation to the three germ layers. Recently the heterochromatin protein 1 (HP1) family protein HP1γ, which is an epigenetic modulator that binds histone H3 methylated at lysine 9, is shown to be associated with the progression from pluripotent to differentiated status in ES cells. Therefore, to study the role of HP1γ in the differentiation capacity of P19 cells, we have established a HP1γ-overexpressing P19 cell line (HPlγ-P19). Similar to the parental P19 cells, undifferentiated HP1γ-P19 cells continued to express pluripotency marker genes. However, HP1γ-P19 cells exhibited significant morphological differentiation including beating cardiomyocytes, as well as Tuj1-positive neuronal cells and Sox17-positive endodermal cells after EB formation under a normal culture condition. Moreover, real-time RT-qPCR analysis revealed that HP1γ-P19 EB cells expressed various differentiation marker genes. Thus, HP1γ-P19 cells could give rise to all three germ layers in EBs without any drug treatment. Therefore, HP1γ affects the spontaneous differentiation potential of P19 cells, and might play major roles in the decision of cell fates in pluripotent stem cells.

BACKGROUND:Apoptosis appears to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM). We have previously reported 3 HCM patients carrying the E334K MYBPC3, and that heterologous expression of E334K cMyBPC in cultured cells induced apoptosis. The purpose of this study was to identify pharmacological agents that would inhibit apoptosis in HL-1 cardiomyocytes expressing E334K cMyBPC.METHODS AND RESULTS:E334K cMyBPC expression in cells increased levels of pro-apoptosis (p53, Bax and cytochrome c) and decreased levels of anti-apoptosis (Bcl-2 and Bcl-XL). While the beta blocker carvedilol (1 μM) normalized the level of p53 and Bcl-2 and the calcium channel blocker (CCB) bepridil (0.5 μM) normalized that of Bcl-2, both the CCB azelnidipine (1 μM) and the angiotensin receptor blocker (ARB) olmesartan (10 μM) normalized those of p53, Bax, cytochrome c, and Bcl-XL. Among those proteins, cytochrome c was the one which showed the highest degree of change. Both azelnidipine (0.1 μM) and olmesartan (1 μM) reduced the level of cytochrome c by 40.2 ± 4.3% and 31.3 ± 5.1%, respectively. The CCB amlodipine and the ARB valsartan reduced it only by 19.1 ± 2.1% and 20.1 ± 5.2%, respectively. Flow cytometric analysis and annexin V staining showed that treatment of cells with azelnidipine (0.1 μM) plus olmesartan (0.3 μM) or that with amlodipine (0.1 μM) plus valsartan (0.3 μM) reduced the number of apoptotic cells by 35.8 ± 10.5% and 18.4 ± 3.2%, respectively.CONCLUSION:Azelnidipine plus olmesartan or amlodipine plus valsartan inhibited apoptosis of HL-1 cells expressing E334K cMyBPC, and the former combination was more effective than the latter.

While bepridil has been reported to alter the stability of ion channel proteins, the precise mechanism of action remains unclear. We examined the effect of bepridil on the stability of Kv1.5 channel proteins expressed in COS7 cells. Bepridil at 0.3-30 μM increased the protein level of Kv1.5 channels in a concentration-dependent manner. Chase experiments showed that bepridil delayed the degradation process of Kv1.5 channel proteins in the same manner as a proteasomal inhibitor, MG132, did. Bepridil increased the immunofluorescent signal of Kv1.5 channel proteins in the endoplasmic reticulum (ER) and Golgi apparatus and on the cell surface. The cell fraction experiment also showed bepridil-induced increases in Kv1.5 in the ER, Golgi apparatus, and the cell membrane. Bepridil at a lower concentration of 1 μM had no effect on the proteasome activity in vitro. A blocker of the ultrarapid delayed-rectifier K(+) channel current, 4-aminopyridine (4AP), abolished bepridil-induced increases in Kv1.5. Kv1.5-medicated membrane currents measured as 4AP-sensitive currents were increased by bepridil. Taken together, we conclude that bepridil stabilizes Kv1.5 proteins at the ER through an action as a chemical chaperone, thereby increasing the density of Kv1.5 channels in the cell membrane.

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.