Medaka as a model for ECG analysis and the effect of verapamil.

The heart of the medaka, a small fish native to East Asia, has electrophysiological aspects similar to mammalian hearts. We found that the heart rates of medaka were more similar to humans than mice or rats. Medaka exhibited similar electrocardiogram patterns to those of humans, suggesting a similarity in cardiac impulse formation and propagation. Their hearts also exhibited similar responsiveness to verapamil, a calcium channel antagonist; atropine, a parasympathetic nerve blocker; propranolol, a sympathetic ß-adrenergic blocker; and isoproterenol, a sympathetic ß-adrenergic agonist. We successfully analyzed action potentials and cardiac contractile forces in vivo. Verapamil affected action potential duration and reduced heart rate, suggesting the importance of voltage-dependent calcium channels in determining the heart rhythm of medaka. We also analyzed the expression of the voltage-dependent calcium channel ß2 subunit, which participates in channel formation in cardiac myocytes, and found that splice variant type-2 was the only major transcript in the heart. Our results indicate that medaka could be an appropriate animal model for studying cardiovascular pharmacology.

Aerosolized liposomes with dipalmitoyl phosphatidylcholine enhance pulmonary absorption of encapsulated insulin compared with co-administered insulin.

OBJECTIVE: We have previously shown that aerosolized liposomes with dipalmitoyl phosphatidylcholine (DPPC) enhance the pulmonary absorption of encapsulated insulin. In this study, we aimed to compare insulin encapsulated into the liposomes versus co-administration of empty liposomes and unencapsulated free insulin, where the DPCC liposomes would serve as absorption enhancer. SIGNIFICANCE: The present study provides the useful information for development of noninvasive treatment of diabetes. METHODS: Co-administration of empty DPPC liposomes and unencapsulated free insulin was investigated in vivo to assess the potential enhancement in protein pulmonary absorption. Co-administration was compared to DPPC liposomes encapsulating insulin, and free insulin. RESULTS: DPPC liposomes enhanced the pulmonary absorption of unencapsulated free insulin; however, the enhancing effect was lower than that of the DPPC liposomes encapsulating insulin. The mechanism of the pulmonary absorption of unencapsulated free insulin by DPPC liposomes involved the opening of epithelial cell space in alveolar mucosa, and not mucosal cell damage, similar to that of the DPPC liposomes encapsulating insulin. In an in vitro stability test, insulin in the alveolar mucus layer that covers epithelial cells was stable. These findings suggest that, although unencapsulated free insulin spreads throughout the alveolar mucus layer, the concentration of insulin released near the absorption surface is increased by the encapsulation of insulin into DPPC liposomes and the absorption efficiency is also increased. CONCLUSION: We revealed that the encapsulation of insulin into DPPC liposomes is more effective for pulmonary insulin absorption than co-administration of DPPC liposomes and unencapsulated free insulin.

A novel binding site between the voltage-dependent calcium channel CaV1.2 subunit and CaVß2 subunit discovered using a new analysis method for protein-protein interactions.

We developed a new method to analyze protein-protein interactions using a dual-inducible prokaryotic expression system. To evaluate protein-protein binding, a chimeric fusion toxin gene was constructed using a DNase-treated short DNA fragment (epitope library) and CcdB, which encodes a DNA topoisomerase II toxin. Protein-protein interactions would affect toxin activity, resulting in colony formation. Using this novel system, we found a new binding site in the voltage-dependent calcium channel α1 subunit (CaV1.2) for the voltage-dependent calcium channel ß2 subunit. Prokaryotic expression screening of the ß2 subunit using an epitope library of CaV1.2 resulted in two overlapping clones of the C-terminal sequence of CaV1.2. In vitro overlay and immunoprecipitation analyses revealed preferential binding of the C-terminal sequences of CaV1.2 and ß2.

A simple, dual direct expression plasmid system in prokaryotic and mammalian cells.

We introduce a simple, dual direct cloning plasmid system (pgMAX-II) for gene expression analysis in both prokaryotic (Escherichia coli) and mammalian cells. This system, which uses a prokaryotic expression unit adapted from the pgMAX system and a mammalian promoter, is effective for subcloning using the DNA topoisomerase II toxin CcdB. Given that molecular biological cloning systems broadly rely on E. coli for rapid growth, the proposed concept may have wide applicability beyond mammalian cells.

Rapid method for plasmid DNA recombination (Murakami-system).

DNA recombination is a useful technology for cloning and subsequent functional analysis, while standard techniques for plasmid DNA recombination have remained unchanged. In the present study, we introduced rapid method for plasmid DNA recombination, which we named "Murakami-system", to complete the experiments in under 33 h. For this purpose, we selected the following: PCR amplification with 25 cycles and E. coli strain with rapid growth (incubation time of 6-8 h). In addition, we selected rapid plasmid DNA purification (mini-prep; ∼10 min) and rapid restriction enzyme incubation (20 min). This recombination system enabled rapid plasmid DNA recombination within 24-33 h, which could be useful in various fields. We also established a 1-day method for competent cell preparation. Our rapid recombination system allowed several sessions of plasmid DNA recombination to be performed every week, which improves the functional analysis of various genes.•"Rapid method for plasmid DNA recombination (Murakami-system).•E. coli strain with rapid growth (incubation time of 6-8 h).•Combination of rapid protocols (PCR, electrophoresis, DNA purification, ligation, and mini-prep) enabled plasmid DNA recombination within 24-33 h.

Involvement of cholesterol membrane transporter Niemann-Pick C1-like 1 in the intestinal absorption of lutein.

PURPOSE: Lutein is a carotenoid mainly found in green leafy vegetables and is located in the macula lutea in the human eye. Since humans cannot synthesize lutein de novo, it must be digested as food. The physiological importance of an orally administered compound depends on its interaction with target tissues. It is therefore important to clarify the absorption mechanism in the intestine. Cholesterol membrane transporters Niemann-Pick C1 Like 1 (NPC1L1) and scavenger receptor class B type 1 (SR-B1) are involved in the intestinal absorption of highly lipophilic compounds including cholesterol. Ezetimibe, a selective inhibitor of intestinal NPC1L1, is the widespread lipid-lowering agent. It is important to investigate the possibility of food-drug interactions in order to prevent undesirable and harmful clinical consequences. The aim of this work was to determine whether NPC1L1, SR-B1 and other transporters are involved in absorption of lutein. METHODS: Caco-2 cells were used for accumulation and permeability study of lutein. Lutein concentration was determined by an HPLC system. The cDNA of transporters was isolated from total RNA of Caco-2 cells, and the expression of these transporters was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). RESULTS: Ezetimibe inhibited up to 40% of lutein accumulation by Caco-2 cell monolayers. Block lipid transport 1 (BLT-1), a selective chemical inhibitor of SR-B1, also inhibited lutein accumulation by Caco-2 cells. On the other hand, ATP-depletion reagents (sodium fluoride and sodium azide or carbonyl cyanide m-chlorophenylhydrazone) did not influence the accumulation or permeation of lutein significantly. CONCLUSIONS: The results show that lutein absorption is, at least in part, mediated by influx transporters NPC1L1 and SR-B1 rather than mediated by efflux transporters such as ABC (ATP-binding cassette) transporters.

The decrease in farnesoid X receptor, pregnane X receptor and constitutive androstane receptor in the liver after intestinal ischemia-reperfusion.

PURPOSE: Intestinal ischemia-reperfusion (I/R) damages remote organs, including the liver, and promotes multi-organ failure (MOF). However, the molecular mechanisms underlying acute liver injury after intestinal I/R have not been completely elucidated. Farnesoid X receptor (FXR), pregnane X receptor (PXR) and constitutive androstane receptor (CAR) regulate metabolizing enzymes and transporters, and coordinately prevent hepatotoxicity reflecting an inability of appropriate excretion of endogenous toxic compounds. In this study, we assessed FXR, PXR and CAR expression levels and their localization levels in nuclei in the liver after intestinal I/R. We also investigated the effect of IL-6 on FXR, PXR and CAR expression levels and their localization levels in nuclei in in vitro experiments. METHODS: We used intestinal I/R model rats. Moreover, HepG2 cells were used in in vitro study. Real-time PCR and Western blotting were used to assess mRNA and protein expression levels. Nuclear receptor localization in nuclei was analyzed by Western blotting using nuclear extracts. RESULTS: FXR and PXR expression levels began to be decreased at 3 h, and FXR, PXR and CAR expression levels were decreased at 6 h after intestinal I/R. The localization levels of FXR, PXR and CAR in nuclei began to be decreased at 3 h, and all of them were decreased at 6 h after intestinal I/R. In HepG2 cells, FXR, PXR and CAR expression levels were decreased by 0.5-1 ng/mL, 0.5-100 ng/mL and 100 ng/mL IL-6 treatment for 24 h, respectively. FXR, PXR and CAR localization levels in nuclei were suppressed by 0.5-10 ng/mL, 10-100 ng/mL and 10-100 ng/mL IL-6 treatment for 24 h, respectively. CONCLUSIONS: FXR, PXR and CAR expression levels are decreased in the liver after intestinal I/R. IL-6 is one of main causes the decreases in expressions of these receptors.

Intestinal ischemia-reperfusion increases efflux for uric acid via paracellular route in the intestine, but decreases that via transcellular route mediated by BCRP.

PURPOSE: Uric acid is thought to be one of the most important antioxidants in human biological fluids. Intestinal ischemia-reperfusion (I/R) is an important factor associated with high rates of morbidity and mortality. Reactive oxygen species (ROS) are responsible for intestinal I/R injury. The aim of this study was to clarify the efflux for uric acid from the intestine after intestinal I/R. METHODS: We used intestinal ischemia-reperfusion (I/R) model rats. Serosal to mucosal flux for [¹4C]-uric acid was assessed by using Ussing-type diffusion chambers. BCRP/Bcrp expression was assessed by Western blot analysis. Caco-2 cells were used for a model of the intestinal epithelium, and rotenone was used as a mitochondrial dysfunction inducer. RESULTS: Serosal to mucosal flux for uric acid was increased after intestinal I/R, and that for mannitol was also increased. Ko143, which is a BCRP inhibitor, did not affect the uric acid transport. The decreasing uric acid transport mediated by Bcrp was caused by decrease in the level of Bcrp homodimer, bridged by an S-S bond. The suppression of Bcrp S-S bond formation was associated with mitochondrial dysfunction. Moreover, BCRP S-S bond formation activity was decreased by rotenone in Caco-2 cells. CONCLUSIONS: Serosal to mucosal flux for uric acid is significantly increased via the paracelluler route, but that via the transcellular route mediated by Bcrp is decreased after intestinal I/R. The decreasing uric acid flux mediated by Bcrp is caused by suppression of Bcrp S-S bond formation. This suppression of Bcrp S-S bond formation may be related to mitochondrial dysfunction.

Enhanced ß-adrenergic response in mice with dominant-negative expression of the PKD2L1 channel.

Polycystic kidney disease (PKD) is the most common genetic cause of kidney failure in humans. Among the various PKD-related molecules, PKD2L1 forms cation channels, but its physiological importance is obscure. In the present study, we established a transgenic mouse line by overexpressing the dominant-negative form of the mouse PKD2L1 gene (i.e., lacking the pore-forming domain). The resulting PKD2L1del-Tg mice exhibited supraventricular premature contraction, as well as enhanced sensitivity to ß-adrenergic stimulation and unstable R-R intervals in electrocardiography. During spontaneous atrial contraction, PKD2L1del-Tg atria showed enhanced sensitivity to isoproterenol, norepinephrine, and epinephrine. Action potential recording revealed a shortened action potential duration in PKD2L1del-Tg atria in response to isoproterenol. These findings indicated increased adrenergic sensitivity in PKD2L1del-Tg mice, suggesting that PKD2L1 is involved in sympathetic regulation.

Protective effect of soy isoflavone genistein on ischemia-reperfusion in the rat small intestine.

Ischemia-reperfusion (I/R) injury of the intestine is an important factor associated with high rates of morbidity and mortality. Intestinal I/R is a common clinical problem in the settings of severe burns, circulatory shock and strangulation ileus. Intestinal I/R damages remote organs and promotes multi-organ failure. It has been shown that enteral feeding before ischemic insults is beneficial for reducing organ injury and improving survival after intestinal I/R. In that study, the authors used a standard complex enteral diet and they suggested that it is important to find new nutrient formulas. Since reactive oxygen species are responsible for intestinal I/R injury, we focused on a dietary polyphenol, the soy isoflavone genistein. Genistein has a wide spectrum of biochemical and pharmacological activities. However, the possibility of a protective effect of genistein as enteral nutrition on I/R injury has not been investigated. We therefore investigated the protective effect of genistein on oxidative injury using intestinal I/R model rats. We found that genistein, which has combined antioxidant activity from radical scavenging, xanthine oxidase inhibition and chain-breaking effects, exhibits a protective effect on intestinal I/R injury. The results suggest that genistein, a soy isoflavone, has the possibility as a new nutrient formula of enteral feeding.

Protective effect of lutein after ischemia-reperfusion in the small intestine.

Lutein is a carotenoid mainly found in green leafy vegetables and is located in the macula lutea in the human eye. Since humans cannot synthesise lutein de novo, it must be digested as food. The physiological importance of an orally administered compound depends on its interaction with target tissues. There is little information about the effects of intake of lutein in tissues other than the eyes. The aim of this study was to clarify the protective effect of lutein against oxidative injury using ischemia-reperfusion (I/R) model rats and to determine the relationship between pharmacokinetics and antioxidant activity of lutein. Intestinal I/R was induced by 30-min occlusion of the superior mesenteric artery followed by 60-min reperfusion. After 60min of reperfusion, intestinal tissue was used for analysis of Evans blue dye extravasation, lipid peroxidation and myeloperoxidase activity. Lutein administered before I/R had a significant protective effect against oxidative injury.

Pharmacokinetic properties of lutein emulsion after oral administration to rats and effect of food intake on plasma concentration of lutein.

Lutein is a carotenoid found mainly in green leafy vegetables and is located in the macula lutea in the human eye. An intake of lutein as food is needed since humans cannot synthesize it de novo. Although lutein has received much attention recently due to its antioxidant activities, little information about the pharmacokinetic properties of lutein is available. Lutein emulsion formulation was used and the pharmacokinetics of lutein emulsion after oral administration to rats was investigated. The bioavailability of lutein using this formulation was calculated to be 5.20%. It was found that a large amount of lutein was accumulated in the intestinal mucosa. The absorption of orally administered compounds in the intestine can be enhanced by interaction with food or food components. Thus, the effect of food intake on the intestinal absorption of lutein was investigated. The plasma concentration of lutein after oral administration of the emulsion formulation was improved significantly by food intake. It is possible that the absorption of lutein in the intestine is improved significantly by some food components. Bile acids may also play important roles in the intestinal absorption of lutein since the absorption of lipophilic compounds such as cholesterol is related to bile acids. The results of these studies should contribute to an improvement of lutein absorption and provide important information for obtaining more effective pharmacological effects of lutein.

Alteration of P-gp expression after intestinal ischemia-reperfusion following 16-h fasting in rats.

Alteration of P-glycoprotein (P-gp) expression influences the pharmacokinetics of P-gp substrates after intestinal ischemia-reperfusion (I/R). Fasting before intestinal I/R affects intestinal I/R injury. However, the effect of fasting on the alteration of P-gp expression after intestinal I/R has not been clarified. We previously reported that P-gp expression was altered after intestinal I/R following feeding. In the present study, we investigated the expression of P-gp after intestinal I/R following 16-h fasting. The mdr1a levels were decreased at 6 h in the jejunum, at 1 h in the ileum, at 6 and 24 h in the liver and at 6 h in the kidney. The mdr1b levels were decreased at 6 h in the ileum and at 1 and 6 h in the kidney. The mdr1b level in the liver was increased at 1 and 6 h. The expression of P-gp was decreased at 6 h in the jejunum, at 1, 6 and 24 h in the ileum, and at 6 h in the kidney. These alterations were different to those after intestinal I/R following feeding. In particular, the decrease in P-gp expression in the 16-h fasting I/R rats occurred at an early time during reperfusion compared with that in the feeding I/R rats. In conclusion, feeding condition affects the alteration of P-gp expression after intestinal I/R. Therefore, it is important to understand the patient's feeding condition for safe drug therapy.

A dual prokaryotic (E. coli) expression system (pdMAX).

In this study, we introduced an efficient subcloning and expression system with two inducible prokaryotic expression promoters, arabinose and lac, in a single plasmid in Escherichia coli. The arabinose promoter unit allows for the expression of a FLAG-tagged protein, while the isopropyl-ß-D-thiogalactoside (IPTG)-inducible unit allows for the expression of a Myc-tagged protein. An efficient subcloning (DNA insertion) system (iUnit) follows each promoter. The iUnit, based on a toxin that targets DNA topoisomerase of E. coli, allows for effective selection with arabinose or IPTG induction. With the dual promoter plasmid (pdMAX) system, expressed lacZ (ß-galactosidase) activity was significantly decreased compared with the original solo expression system. Despite this disadvantage, we believe that the pdMAX system remains useful. A recombinant plasmid (pdMAX/ara/DsRed/IPTG/EGFP; pdMAX/DsRed/EGFP) with DsRed in the arabinose expression unit and EGFP in the IPTG expression unit showed fluorescent protein expression following additional low-temperature incubation. Thus, the novel pdMAX system allowed efficient subcloning of two different genes and can be used to induce and analyze the expression of two distinct genes. The proposed system can be applied to various types of prokaryotic gene expression analysis.

Attenuated ß-adrenergic response in calcium/calmodulin-dependent protein kinase IV-knockout mice.

In the present study, we examined the importance of Ca2+/calmodulin-dependent protein kinase IV (CaMKIV) in the regulation of cardiac function using genetically modified CaMKIV-null mice. RT-PCR analysis revealed decreased expression of voltage-dependent calcium channels in the cardiac myocytes of CaMKIV-null mice compared with wild-type mice. CaMKIV-null mice showed shortened QT time on electrocardiograms. Pharmacological analysis revealed decreased responsiveness to the ß-adrenergic blocker propranolol in CaMKIV-null mice, whereas the plasma norepinephrine level was not affected. CaMKIV-null mice showed decreased baroreflex on electrocardiograms. Heart rate variability analysis showed unstable R-R intervals, a decreased low frequency power/high frequency power (LF/HF) ratio, and increased standard deviation of the normal to normal R-R intervals (SDNN) in CaMKIV-null mice, suggesting decreased responsiveness to ß-adrenergic stimulation in CaMKIV-null mice. Atrial contraction analysis and cardiac action potential recording showed a decreased response to the ß-adrenoceptor agonist isoproterenol in CaMKIV-null mice. Furthermore, fluorescence imaging in a CRE-hrGFP assay revealed a decreased response to isoproterenol in CaMKIV-null cardiac myocytes. Taken together, our data strongly suggest a significant effect of CaMKIV gene ablation on cardiac ß-adrenergic signal transduction.

Contribution of organic anion transporting polypeptide OATP2B1 to amiodarone accumulation in lung epithelial cells.

The accumulation mechanisms of amiodarone (AMD) involving transporters in lung alveolar epithelial type II cells were studied. The uptake of AMD was examined using human alveolar epithelial-derived cell line A549 as a model. AMD was transported by the carrier-mediated system, and the apparent K(m) and V(max) values were 66.8+/-30.3 muM and 49.7+/-9.7 nmol/mg protein/5 min, respectively. The uptake of AMD by A549 cells was Na(+)-independent and was inhibited by substrates of human organic anion transporting polypeptide (OATP). The inhibition profiles were similar to the inhibitory effects of several compounds on OATP2B1-mediated E-3-S transport, and RT-PCR analysis showed mRNA expression of OATP2B1 and 1B3 in A549 cells. SiRNAs targeted to the OATP2B1 gene decreased the OATP2B1 mRNA expression level in A549 cells up to about 50% and reduced the uptake of AMD up to about 40%. These results indicate that AMD uptake mediated by carriers, including OATP2B1, might lead to accumulation of AMD in the lung and AMD-induced pulmonary toxicity (AIPT).

Liver X receptor regulates expression of MRP2 but not that of MDR1 and BCRP in the liver.

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. Multidrug resistance-associated protein 2 (MRP2), multidrug resistance 1 (MDR1) and breast cancer resistance protein (BCRP) play an important role in the efflux of a broad range of endogenous and xenobiotic compounds from hepatocytes. Since the effects of LXR activation on there transporters have been obscure, we investigated the effects of LXR agonists, TO901317 and 25-hydroxycholesterol, on MRP2, MDR1, BCRP expression in HepG2 cells and the rat liver. In an in vitro study, TO901317 increased ABCA1, an LXR target gene, and MRP2 mRNA and protein levels. On the other hand, TO901317 had little effect on MDR1 and BCRP mRNA levels. In an in vivo study, Abca1 and Mrp2 mRNA and protein levels were increased by TO901317, but TO901317 had no effect on Mdr1a and Bcrp mRNA levels in the rat liver. Moreover, TO901317-induced MRP2 mRNA expression was blocked by LXRalpha knockdown. In this study, we demonstrated that LXR activation induced expression of MRP2 but not that of MDR1 and BCRP in hepatocytes. The results suggest that agonists for LXR activate transcription of the MRP2 gene in order to promote excretion of endogenous and xenobiotic compounds from hepatocytes into bile.

Pharmacokinetics of oral and intravenous administration of digoxin after intestinal ischemia-reperfusion.

Intestinal ischemia-reperfusion (I/R) causes gut dysfunction characterized by decreased basement membrane integrity and decreased barrier function. Indeed, it has been reported that the absorption of several drugs is altered after intestinal I/R. Intestinal I/R also promotes multi-organ failure (MOF). The liver and kidney can be affected by MOF after intestinal I/R. However, little is known about the alteration of pharmacokinetics after intravenous administration in intestinal I/R injury. In the present study, we investigated pharmacokinetics of digoxin after oral administration and intravenous administration in intestinal I/R injury. Plasma digoxin concentration in I/R rats after oral administration was not significantly altered at any time compared with that in sham-operated rats. Plasma digoxin concentration in rats reperfused for 1 h after intravenous administration was significantly higher than that in sham-operated rats. Plasma digoxin concentrations in rats reperfused for 6 and 24 h were the same as those in sham-operated rats. The area under the concentration.time curve after intravenous administraion (AUC(i.v.)) and total clearance (CL(tot)) in rats reperfused for 1 h was 1.89- and 0.57-fold higher than that in sham-operated rats. However, elimination rate (k(e)) and half-life (t(1/2)) in rats reperfused for 1 h were not altered. Distribution volume (V(d)) in rats reperfused for 1 h was decreased than that in sham-operated rats, but there was not statistical difference. These results suggest that intestinal I/R affected the V(d) of digoxin, and plasma concentration of digoxin was increased. The present study suggests that understanding pharmacokinetics of drug after intravenous administration in intestinal I/R injury is important to provide valuable information for safe drug therapy for intestinal I/R patients.

Regulation of monocarboxylate transporter 1 in skeletal muscle cells by intracellular signaling pathways.

Skeletal muscle is the major producer of lactic acid in the body, but its oxidative fibers also use lactic acid as a respiratory fuel. Monocarboxylate transporter (MCT) 1 has been suggested to play a major role in influx of L-lactic acid for oxidation. The regulation mechanism of MCT1 was characterized utilizing rhabdomyosarcoma cells as an in vitro skeletal muscle model. The uptake of L-lactic acid via MCT1 was studied in the presence of various intracellular regulatory pathways, including pathways mediated by protein kinases A, C and G (PKA, PKC and PKG), protein tyrosine kinase (PTK), and Ca2+/calmodulin modulators. The results showed that PKG-, PTK-, and Ca2+/calmodulin-mediated regulatory pathways play no role in the regulation of L-lactic acid uptake, but a role for PKC- and PKA-mediated pathways was apparent. Uptake of L-lactic acid appeared to be stimulated by phorbol 12-myristate 13-acetate (PMA, a PKC activator) via an increase in Vmax of transport processes with no alteration in Km. In parallel, PMA treatment also resulted in an increase in the level of MCT1 expression. On the other hand, exposure to 8-Br-cAMP, a cAMP analog, and to forskolin, an adenylyl cyclase activator, resulted in a significant decrease in L-lactic acid uptake. Additionally, 8-Br-cAMP reduced Vmax but not Km values. Parallel to the decrease in Vmax of L-lactic acid uptake, the level of MCT1 expression was decreased in response to incubation with 8-Br-cAMP. These results indicate the possible involvement of a PKC- and PKA-mediated pathway associated with expression of MCT1 and lactate transport.

Decreased cardiac pacemaking and attenuated ß-adrenergic response in TRIC-A knockout mice.

Changes in intracellular calcium levels in the sinus node modulate cardiac pacemaking (the calcium clock). Trimeric intracellular cation (TRIC) channels are counterion channels on the surface of the sarcoplasmic reticulum and compensate for calcium release from ryanodine receptors, which play a major role in calcium-induced calcium release (CICR) and the calcium clock. TRIC channels are expected to affect the calcium clock in the sinus node. However, their physiological importance in cardiac rhythm formation remains unclear. We evaluated the importance of TRIC channels on cardiac pacemaking using TRIC-A-null (TRIC-A-/-) as well as TRIC-B+/-mice. Although systolic blood pressure (SBP) was not significantly different between wild-type (WT), TRIC-B+/-, and TRIC-A-/-mice, heart rate (HR) was significantly lower in TRIC-A-/-mice than other lines. Interestingly, HR and SBP showed a positive correlation in WT and TRIC-B+/-mice, while no such correlation was observed in TRIC-A-/-mice, suggesting modification of the blood pressure regulatory system in these mice. Isoproterenol (0.3 mg/kg) increased the HR in WT mice (98.8 ±â€…15.1 bpm), whereas a decreased response in HR was observed in TRIC-A-/-mice (23.8 ±â€…5.8 bpm), suggesting decreased sympathetic responses in TRIC-A-/-mice. Electrocardiography revealed unstable R-R intervals in TRIC-A-/-mice. Furthermore, TRIC-A-/-mice sometimes showed sinus pauses, suggesting a significant role of TRIC-A channels in cardiac pacemaking. In isolated atrium contraction or action potential recording, TRIC-A-/-mice showed decreased response to a ß-adrenergic sympathetic nerve agonist (isoproterenol, 100 nM), indicating decreased sympathetic responses. In summary, TRIC-A-/-mice showed decreased cardiac pacemaking in the sinus node and attenuated responses to ß-adrenergic stimulation, indicating the involvement of TRIC-A channels in cardiac rhythm formation and decreased sympathetic responses.