Electrophysiological evaluation of an anticancer drug gemcitabine on cardiotoxicity revealing down-regulation and modification of the activation gating properties in the human rapid delayed rectifier potassium channel.

Gemcitabine is an antineoplastic drug commonly used in the treatment of several types of cancers including pancreatic cancer and non-small cell lung cancer. Although gemcitabine-induced cardiotoxicity is widely recognized, the exact mechanism of cardiac dysfunction causing arrhythmias remains unclear. The objective of this study was to electrophysiologically evaluate the proarrhythmic cardiotoxicity of gemcitabine focusing on the human rapid delayed rectifier potassium channel, hERG channel. In heterologous hERG expressing HEK293 cells (hERG-HEK cells), hERG channel current (IhERG) was reduced by gemcitabine when applied for 24 h but not immediately after the application. Gemcitabine modified the activation gating properties of the hERG channel toward the hyperpolarization direction, while inactivation, deactivation or reactivation gating properties were unaffected by gemcitabine. When gemcitabine was applied to hERG-HEK cells in combined with tunicamycin, an inhibitor of N-acetylglucosamine phosphotransferase, gemcitabine was unable to reduce IhERG or shift the activation properties toward the hyperpolarization direction. While a mannosidase I inhibitor kifunensine alone reduced IhERG and the reduction was even larger in combined with gemcitabine, kifunensine was without effect on IhERG when hERG-HEK cells were pretreated with gemcitabine for 24 h. In addition, gemcitabine down-regulated fluorescence intensity for hERG potassium channel protein in rat neonatal cardiomyocyte, although hERG mRNA was unchanged. Our results suggest the possible mechanism of arrhythmias caused by gemcitabine revealing a down-regulation of IhERG through the post-translational glycosylation disruption possibly at the early phase of hERG channel glycosylation in the endoplasmic reticulum that alters the electrical excitability of cells.

Protein Kinase C Regulates Expression and Function of the Cav3.2 T-Type Ca2+ Channel during Maturation of Neonatal Rat Cardiomyocyte.

Two distinct isoforms of the T-type Ca2+ channel, Cav3.1 and Cav3.2, play a pivotal role in the generation of pacemaker potentials in nodal cells in the heart, although the isoform switches from Cav3.2 to Cav3.1 during the early neonatal period with an unknown mechanism. The present study was designed to investigate the molecular system of the parts that are responsible for the changes of T-type Ca2+ channel isoforms in neonatal cardiomyocytes using the whole-cell patch-clamp technique and mRNA quantification. The present study demonstrates that PKC activation accelerates the Ni2+-sensitive beating rate and upregulates the Ni2+-sensitive T-type Ca2+ channel current in neonatal cardiomyocytes as a long-term effect, whereas PKC inhibition delays the Ni2+-sensitive beating rate and downregulates the Ni2+-sensitive T-type Ca2+ channel current. Because the Ni2+-sensitive T-type Ca2+ channel current is largely composed of the Cav3.2-T-type Ca2+ channel, it is accordingly assumed that PKC activity plays a crucial role in the maintenance of the Cav3.2 channel. The expression of Cav3.2 mRNA was highly positively correlated with PKC activity. The expression of a transcription factor Nkx2.5 mRNA, possibly corresponding to the Cav3.2 channel gene, was decreased by an inhibition of PKCßII. These results suggest that PKC activation, presumably by PKCßII, is responsible for the upregulation of CaV3.2 T-type Ca2+ channel expression that interacts with a cardiac-specific transcription factor, Nkx2.5, in neonatal cardiomyocytes.

Enhanced BDNF Actions Following Acute Hypoxia Facilitate HIF-1α-Dependent Upregulation of Cav3-T-Type Ca2+ Channels in Rat Cardiomyocytes.

Brain-derived neurotrophic factor (BDNF) has recently been recognized as a cardiovascular regulator particularly in the diseased condition, including coronary artery disease, heart failure, cardiomyopathy, and hypertension. Here, we investigate the role of BDNF on the T-type Ca2+ channel, Cav3.1 and Cav3.2, in rat neonatal cardiomyocytes exposed to normoxia (21% O2) and acute hypoxia (1% O2) in vitro for up to 3 h. The exposure of cardiomyocytes to hypoxia (1 h, 3 h) caused a significant upregulation of the mRNAs for hypoxia-inducible factor 1α (Hif1α), Cav3.1, Cav3.2 and Bdnf, but not tropomyosin-related kinase receptor B (TrkB). The upregulation of Cav3.1 and Cav3.2 caused by hypoxia was completely halted by small interfering RNA (siRNA) targeting Hif1a (Hif1a-siRNA) or Bdnf (Bdnf-siRNA). Immunocytochemical staining data revealed a distinct upregulation of Cav3.1- and Cav3.2-proteins caused by hypoxia in cardiomyocytes, which was markedly suppressed by Bdnf-siRNA. These results unveiled a novel regulatory action of BDNF on the T-type Ca2+ channels expression through the HIF-1α-dependent pathway in cardiomyocytes.

Nitric oxide down-regulates voltage-gated Na+ channel in cardiomyocytes possibly through S-nitrosylation-mediated signaling.

Nitric oxide (NO) is produced from endothelial cells and cardiomyocytes composing the myocardium and benefits cardiac function through both vascular-dependent and-independent effects. This study was purposed to investigate the possible adverse effect of NO focusing on the voltage-gated Na+ channel in cardiomyocytes. We carried out patch-clamp experiments on rat neonatal cardiomyocytes demonstrating that NOC-18, an NO donor, significantly reduced Na+ channel current in a dose-dependent manner by a long-term application for 24 h, accompanied by a reduction of Nav1.5-mRNA and the protein, and an increase of a transcription factor forkhead box protein O1 (FOXO1) in the nucleus. The effect of NOC-18 on the Na+ channel was blocked by an inhibitor of thiol oxidation N-ethylmaleimide, a disulfide reducing agent disulfide 1,4-Dithioerythritol, or a FOXO1 activator paclitaxel, suggesting that NO is a negative regulator of the voltage-gated Na+ channel through thiols in regulatory protein(s) for the channel transcription.

Serum microRNA-30d is a sensitive biomarker for angiotensin II-induced cardiovascular complications in rats.

We tested the hypothesis that angiotensin II (Ang II)-induced cardiovascular complications are distinguished from what catecholamine-induced by their serum circulating biomarkers in rats. Infusion of Ang II (1.68 mg/kg/day) significantly increased systolic and diastolic blood pressure assessed at week one or later, accompanied by an increase of heart/body weight ratio. Noradrenaline infusion (5.40 mg/kg/day) produced a similar degree of hypertension, but did not increase heart weight. Ang II-, but not noradrenaline-induced hypertension was associated with a drastic upregulation of serum microRNA-30d (miR-30d) by hundreds of times, accompanied by an increase of miR-30d levels in the atrium but not in the ventricle. Ang II, but not noradrenaline, significantly increased mRNA of brain natriuretic peptide (BNP) in the atrium. Studies using rat neonatal cardiomyocytes in vitro demonstrated that BNP caused an increase of miR-30d when applied for 6 h or longer in the culture medium. In vitro application of Ang II increased the cell size, although BNP and miR-30d were unable to mimic the effect of Ang II. We conclude that serum circulating microRNA-30d is a sensitive biomarker for Ang II-induced cardiovascular complications. It is also postulated that Ang II-induced cardiomyocyte hypertrophy could be independent of miR-30d/BNP signaling pathways.

Oxytocin Downregulates the CaV1.2 L-Type Ca2+ Channel via Gi/cAMP/PKA/CREB Signaling Pathway in Cardiomyocytes.

Oxytocin (OT) and its receptor (OTR) are expressed in the heart and are involved in the physiological cardiovascular functional system. Although it is known that OT/OTR signaling is cardioprotective by reducing the inflammatory response and improving cardiovascular function, the role of OT in the cardiac electrical excitation modulation has not been clarified. This study investigates the molecular mechanism of the action of OT on cardiomyocyte membrane excitation focusing on the L-type Ca2+ channel. Our methodology uses molecular biological methods and a patch-clamp technique on rat cardiomyocytes with OT, combined with several signal inhibitors and/or activators. Our results show that long-term treatment of OT significantly decreases the expression of Cav1.2 mRNA, and reduces the L-type Ca2+ channel current (ICa.L) in cardiomyocytes. OT downregulates the phosphorylated component of a transcription factor adenosine-3',5'-cyclic monophosphate (cAMP) response element binding protein (CREB), whose action is blocked by OTR antagonist and pertussis toxin, a specific inhibitor of the inhibitory GTP-binding regulators of adenylate cyclase, Gi. On the other hand, the upregulation of Cav1.2 mRNA expression by isoproterenol is halted by OT. Furthermore, inhibition of phospholipase C (PLC) was without effect on the OT action to downregulate Cav1.2 mRNA-which suggests a signal pathway of Gi/protein kinase A (PKA)/CREB mediated by OT/OTR. These findings indicate novel signaling pathways of OT contributing to a downregulation of the Cav1.2-L-type Ca2+ channel in cardiomyocytes.

Manifestations of gene expression profiles in human right atrial myocardium caused by mechanical stretch.

This investigation was aimed to identify gene profiles in human atrial myocardium in response to chronic mechanical stretch. Right atrial appendages from 21 patients were divided into 2 groups based on the size of right atrial volume. The microarray DATA analyses differentially identified 335 genes (> 2.0-fold, corrected P < 0.05) including "functionally unknown genes". This study identified 26 up-regulated genes (natriuretic peptide B, G protein subunit gamma 13, thyroid stimulating hormone beta, etc.) and 23 down-regulated genes (oligodendrocyte transcription factor 1, carbonic anhydrase 12, etc.), which could be responsible for chronic stretch-mediated structural remodeling in the atrium.

Binge Alcohol Exposure Triggers Atrial Fibrillation Through T-Type Ca2+ Channel Upregulation via Protein Kinase C (PKC) / Glycogen Synthesis Kinase 3ß (GSK3ß) / Nuclear Factor of Activated T-Cells (NFAT) Signaling　- An Experimental Account of Holiday Heart Syndrome.

BACKGROUND: The association between binge alcohol ingestion and atrial fibrillation (AF), often termed "holiday heart syndrome", has long been recognized. However, the underlying cellular and molecular mechanisms are unknown.Methods and Results:An experimental model of binge alcohol-induced AF was developed to elucidate the mechanisms linking acute ethanol exposure to changes in ion channel transcription and AF susceptibility. AF-susceptibility during transesophageal electrical stimulation was enhanced 8 h after, but not immediately or 24 h after, acute alcohol intake. T-type calcium channel (TCC) blockade and calcineurin inhibition diminished the AF-promoting effect of ethanol. Long-term (8-24 h) exposure to ethanol augmented TCC isoform-expression (Cav3.1 and Cav3.2) and currents in cardiomyocytes, accompanied by upregulation of the transcription factors, Csx/Nkx2.5 and nuclear factor of activated T-cells (NFAT), in the nucleus, and of phospho-glycogen synthesis kinase 3ß (GSK3ß) in the cytosol. Inhibition of protein kinase C (PKC) during the 7- to 8-h period following ethanol exposure attenuated susceptibility to AF, whereas acute exposure did not. GSK3ß inhibition itself upregulated TCC expression and increased AF susceptibility. CONCLUSIONS: The present study results suggest a crucial role for TCC upregulation in the AF substrate following binge alcohol-drinking, resulting from ethanol-induced PKC-activation that hyperphosphorylates GSK3ß to cause enhanced calcineurin-NFAT-Csx/Nkx2.5 signaling. These observations elucidate for the first time the potential mechanisms underlying the clinically well-recognized, but mechanistically enigmatic, "holiday heart syndrome".

Magnesium Deficiency Causes Transcriptional Downregulation of Kir2.1 and Kv4.2 Channels in Cardiomyocytes Resulting in QT Interval Prolongation.

BACKGROUND: Mechanisms for QT interval prolongation and cardiac arrhythmogenesis in hypomagnesemia are poorly understood. This study investigated the potential molecular mechanism for QT prolongation caused by magnesium (Mg) deficiency in rats by using the patch clamp technique and molecular biology.Methods and Results:Male Wistar rats were fed an Mg-free diet or a normal diet for up to 12 weeks. There was QT prolongation in the ECG of Mg-deficient rats, and cardiomyocytes from these rats showed prolongation of action potential duration. Electrophysiological studies showed that inward-rectifying K+current (IK1) and transient outward K+current (Ito) were decreased in Mg-deficient cardiomyocytes, and these findings were consistent with the downregulation of mRNA, as well as protein levels of Kir2.1 and Kv4.2. In Mg-deficient cardiomyocytes, transcription factors, GATA4 and NFAT, were upregulated, whereas CREB was downregulated. In contrast to Mg deficiency, cellular Mg2+overload in cultured cardiomyocytes resulted in the upregulation of Kir2.1 and Kv4.2, which was accompanied by the downregulation of GATA4 and NFATc4, and the upregulation of CREB. Activation of NFAT and inhibition of CREB reduced Kv4.2-Ito, whereas Kir2.1-IK1was reduced by CREB inhibition but not by NFTA activation. CONCLUSIONS: Intracellular Mg deficiency downregulates IK1and Itoin cardiomyocytes, and this is mediated by the transcription factors, NFAT and CREB. These results provide a novel mechanism for the long-term QT interval prolongation in hypomagnesemia.

Cardiomyocytes cultured on mechanically compliant substrates, but not on conventional culture devices, exhibit prominent mitochondrial dysfunction due to reactive oxygen species and insulin resistance under high glucose.

RATIONALE: Diabetes causes cardiac dysfunction, and understanding of its mechanism is still incomplete. One reason could be limitations in modeling disease conditions by current in vitro cardiomyocyte culture. Emerging evidence suggests that the mechanical properties of the microenvironment affect cardiomyocyte function. Nevertheless, the impact of high glucose on cardiomyocytes cultured on substrates whose stiffness matches that of the heart (approximately 15 kPa) is untested. OBJECTIVE: To test the hypothesis that cardiomyocytes cultured in microenvironments that mimic the mechanical properties of those for cardiomyocytes in vivo may reproduce the pathophysiology characteristics of diabetic cardiomyocytes ex vivo, such as the morphological appearance, ROS accumulation, mitochondrial dysfunction, apoptosis and insulin-stimulated glucose uptake. METHODS AND RESULTS: Isolated neonatal rat cardiomyocytes were seeded on 15 kPa polyacrylamide (PAA) gels, whose stiffness mimics that of heart tissues, or on glass coverslips, which represent conventional culture devices but are unphysiologically stiff. Cells were then cultured at 5 mM glucose, corresponding to the normal blood glucose level, or at high glucose levels (10 to 25 mM). Cytoskeletal disorganization, ROS accumulation, attenuated mitochondrial membrane potential and attenuated ATP level caused by high glucose and their reversal by a ROS scavenger were prominent in cells on gels, but not in cells on coverslips. The lack of response to ROS scavenging could be attributable to enhanced apoptosis in cells on glass, shown by enhanced DNA fragmentation and higher caspase 3/7 activity in cells on glass coverslips. High-glucose treatment also downregulated GLUT4 expression and attenuated insulin-stimulated glucose uptake only in cells on 15 kPa gels. CONCLUSION: Our data suggest that a mechanically compliant microenvironment increases the susceptibility of primary cardiomyocytes to elevated glucose levels, which enables these cells to serve as an innovative model for diabetic heart research.

Testosterone-mediated upregulation of delayed rectifier potassium channel in cardiomyocytes causes abbreviation of QT intervals in rats.

Men have shorter rate-corrected QT intervals (QTc) than women, especially at the period of adolescence or later. The aim of this study was to elucidate the long-term effects of testosterone on cardiac excitability parameters including electrocardiogram (ECG) and potassium channel current. Testosterone shortened QT intervals in ECG in castrated male rats, not immediately after, but on day 2 or later. Expression of Kv7.1 (KCNQ1) mRNA was significantly upregulated by testosterone in cardiomyocytes of male and female rats. Short-term application of testosterone was without effect on delayed rectifier potassium channel current (IKs), whereas IKs was significantly increased in cardiomyocytes treated with dihydrotestosterone for 24 h, which was mimicked by isoproterenol (24 h). Gene-selective inhibitors of a transcription factor SP1, mithramycin, abolished the effects of testosterone on Kv7.1. Testosterone increases Kv7.1-IKs possibly through a pathway related to a transcription factor SP1, suggesting a genomic effect of testosterone as an active factor for cardiac excitability.

Atrial Fibrillation-Mediated Upregulation of miR-30d Regulates Myocardial Electrical Remodeling of the G-Protein-Gated K(+) Channel, IK.ACh.

BACKGROUND: Atrial fibrillation (AF) begets AF in part due to atrial remodeling, the molecular mechanisms of which have not been completely elucidated. This study was conducted to identify microRNA(s) responsible for electrical remodeling in AF. METHODS AND RESULTS: The expression profiles of 1205 microRNAs, in cardiomyocytes from patients with persistent AF and from age-, gender-, and cardiac function-matched control patients with normal sinus rhythm, were examined by use of a microRNA microarray platform. Thirty-nine microRNAs differentially expressed in AF patients' atria were identified, including miR-30d, as a candidate responsible for ion channel remodeling by in silico analysis. MiR-30d was significantly upregulated in cardiomyocytes from AF patients, whereas the mRNA and protein levels ofCACNA1C/Cav1.2 andKCNJ3/Kir3.1, postulated targets of miR-30d, were markedly reduced.KCNJ3/Kir3.1 expression was downregulated by transfection of the miR-30 precursor, concomitant with a reduction of the acetylcholine-sensitive inward-rectifier K(+)current (IK.ACh).KCNJ3/Kir3.1 (but notCACNA1C/Cav1.2) expression was enhanced by the knockdown of miR-30d. The Ca(2+)ionophore, A23187, induced a dose-dependent upregulation of miR-30d, followed by the suppression ofKCNJ3mRNA expression. Blockade of protein kinase C signaling blunted the [Ca(2+)]i-dependent downregulation of Kir3.1 via miR-30d. CONCLUSIONS: The downward remodeling ofIK.AChis attributed, at least in part, to deranged Ca(2+)handling, leading to the upregulation of miR-30d in human AF, revealing a novel post-transcriptional regulation ofIK.ACh. (Circ J 2016; 80: 1346-1355).

Short- and long-term inhibition of cardiac inward-rectifier potassium channel current by an antiarrhythmic drug bepridil.

Bepridil is an effective antiarrhythmic drug on supraventricular and ventricular arrhythmias, and inhibitor of calmodulin. Recent investigations have been elucidating that bepridil exerts antiarrhythmic effects through its acute and chronic application for patients. The aim of this study was to identify the efficacy and the potential mechanism of bepridil on the inward-rectifier potassium channel in neonatal rat cardiomyocytes in acute- and long-term conditions. Bepridil inhibited inward-rectifier potassium current (I K1) as a short-term effect with IC50 of 17 µM. Bepridil also reduced I K1 of neonatal cardiomyocytes when applied for 24 h in the culture medium with IC50 of 2.7 µM. Both a calmodulin inhibitor (W-7) and an inhibitor of calmodulin-kinase II (KN93) reduced I K1 when applied for 24 h as a long-term effect in the same fashion, suggesting that the long-term application of bepridil inhibits I K1 more potently than that of the short-term application through the inhibition of calmodulin kinase II pathway in cardiomyocytes.

Hypomagnesemic down-regulation of L-type Ca(2+) channel in cardiomyocyte as an arrhythmogenic substrate in rats.

The present study was designed to investigate the effect of magnesium (Mg) depletion on the expression of voltage-gated calcium (Ca(2+)) channels and Ca(2+) currents in the heart and thereby on hypomagnesemic arrhythmogenesis in adult male rats. Male Wistar rats were fed an Mg-free diet or a normal diet for up to 16 weeks. Serum Mg concentrations were significantly reduced at week 4 or later with an Mg-free diet, which experimentally represents hypomagnesemia. Myocardial Mg contents were also reduced at week 16 accompanied by myocardial hypertrophy. Telemetric ECG recordings revealed a long-term changes of ECG parameters in hypomagnesemic rats; RR shortening, QT prolongation and appreciable PR prolongation. At the same time, hypomagnesemic rats demonstrate various bradycardiac arrhythmias including ventricular premature beats, atrioventricular blocks and sinus arrest, which were never recoded in rats fed by a normal diet. Electrophysiological studies elucidated that the L-type Ca(2+) channel current was decreased in Mg-deficient cardiomyocytes, and these findings were consistent with down-regulation of CaV1.2-mRNA but not in levels of CaV1.3, CaV3.1 or CaV3.2. These findings provide novel insights into hypomagnesemic electrophysiological disorders in the heart, and should be considered when assessing the design of effective antiarrhythmic treatments in patients with hypomagnesemia.

Enhancement of endothelial function inhibits left atrial thrombi development in an animal model of spontaneous left atrial thrombosis.

BACKGROUND: Left atrial (LA) thrombosis is an important cause of systemic embolization. The SPORTS rat model of LA thrombi (Spontaneously-Running Tokushima-Shikoku), which have a unique characteristic of high voluntary wheel running, was previously established. The aim of the present study was to investigate how SPORTS rats develop LA thrombi. METHODS AND RESULTS: Nitric oxide (NO) produced from cardiovascular endothelial cells plays an important protective role in the local regulation of blood flow, vascular tone, and platelet aggregation. No evidence of atrial fibrillation or hypercoagulability in SPORTS rats regardless of age was found; however, SPORTS rats demonstrated endothelial dysfunction and a decrease of NO production from a young age. In addition, endothelial NO synthase activity was significantly decreased in the LA and thoracic aorta endothelia of SPORTS rats. While voluntary wheel running was able to intermittently increase NO levels, running did not statistically decrease the incidence of LA thrombi at autopsy. However, L-arginine treatment significantly increased NO production and provided protection from the development of LA thrombi in SPORTS rats. CONCLUSIONS: They present study results indicate that NO has an important role in the development of LA thrombus, and endothelia pathways could provide new targets of therapy to prevent LA thrombosis.

Establishment of a model of spontaneously-running-Tokushima-shikoku rats with left atrial thrombosis.

Studies that investigate the underlying mechanisms of disease and treatment options typically require the use of a suitable animal model. Few suitable animal models exist for left atrial thrombosis. Here, we demonstrated that the Spontaneously-Running-Tokushima-Shikoku (SPORTS) rat - a Wistar strain known for its running ability-is predisposed to the development of thrombi in the left atrium. We investigated the incidence of left atrial thrombosis in male (n = 16) and female (n = 17) SPORTS rats and observed organized atrial thrombosis in 57% and 38% of males and female rats, respectively. In the male rats, systolic blood pressures and heart rates were significantly higher in SPORTS rats than in control Wistar rats. We could not find any evidence of arrhythmias, such as atrial fibrillation, during electrocardiographic examination of SPORTS rats. We believe that the SPORTS rat could serve as a new research model for left atrial thrombosis; further, it may be suitable for research investigating the development of new antithrombotic approaches for the control of atrial thrombosis or familial thrombophilia in humans.

Beta-adrenergic-AMPK pathway phosphorylates acetyl-CoA carboxylase in a high-epinephrine rat model, SPORTS.

We established a new animal model called SPORTS (Spontaneously-Running Tokushima-Shikoku) rats, which show high-epinephrine (Epi) levels. Recent reports show that Epi activates adenosine monophosphate (AMP)-activated protein kinase (AMPK) in adipocytes. Acetyl-CoA carboxylase (ACC) is the rate-limiting enzyme in fatty acid synthesis, and the enzymatic activity is suppressed when its Ser-79 is phosphorylated by AMPK. The aim of this study was to investigate the in vivo effect of Epi on ACC and abdominal visceral fat accumulation. We divided both 6-week male control and SPORTS rats into two groups, which were fed either normal diet or high fat and sucrose (HFS) diet for 16 weeks. At the end of diet treatment, retroperitoneal fat was collected for western blotting and histological analysis. Food intake was not different among the groups, but SPORTS rats showed significantly lower weight gain than control rats in both diet groups. After 10 weeks of diet treatment, glucose tolerance tests (GTTs) revealed that SPORTS rats had increased insulin sensitivity. Furthermore, SPORTS rats had lower quantities of both abdominal fat and plasma triglyceride (TG). In abdominal fat, elevated ACC Ser-79 phosphorylation was observed in SPORTS rats and suppressed by an antagonist of beta-adrenergic receptor (AR), propranolol, or an inhibitor of AMPK, Compound C. From these results, high level of Epi induced ACC phosphorylation mediated through beta-AR and AMPK signaling pathways in abdominal visceral fat of SPORTS rats, which may contribute to reduce abdominal visceral fat accumulation and increase insulin sensitivity. Our results suggest that beta-AR-regulated ACC activity would be a target for treating lifestyle-related diseases, such as obesity.

Telmisartan, an angiotensin II type 1 receptor antagonist, attenuates T-type Ca2+ channel expression in neonatal rat cardiomyocytes.

Recently, it has been revealed that angiotensin II type 1 receptor (AT(1)) antagonists act as antiarrhythmic agents and that the T-type Ca2+ channel plays an important role in arrhythmia. However, it remains unclear how the T-type Ca2+ channel expression system is involved in angiotensin II-mediated arrhythmogenesis in cardiomyocytes. In this study, we investigated the effect of telmisartan, an AT(1) receptor antagonist, on transcriptional regulation of T-type Ca2+ channel isoform (Ca(v)3.1 and Ca(v)3.2) expression and cardiac contractility using rat neonatal cardiomyocytes. Cultured cardiomyocytes were stimulated with telmisartan and/or angiotensin II for 24 h. T-type Ca2+ currents (I(Ca.T)) were then measured with the patch clamp technique, while Ca(v)3.1 and Ca(v)3.2 mRNA expression were assessed by real-time PCR. Expression of Ca(v)3.1 and Ca(v)3.2 mRNA as well as I(Ca.T) current density in cardiomyocytes increased significantly after long-term application of angiotensin II (24 h), which was accompanied by extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) phosphorylation. In contrast, telmisartan decreased Ca(v)3.1 and Ca(v)3.2 mRNA expression as well as I(Ca.T) in a dose-dependent manner in the absence of angiotensin II. In addition, the basal phosphorylation level of p38MAPK but not ERK1/2 was decreased by telmisartan in the absence of angiotensin II. Valsartan, an AT(1) receptor antagonist, did not mimic the action of telmisartan, while the action of telmisartan was completely blocked by valsartan. These results indicate that telmisartan attenuates T-type Ca2+ channel expression likely through p38MAPK activity in an agonist-independent manner, which suggests a novel pharmacological action of telmisartan.

17 beta-estradiol modulates expression of low-voltage-activated Ca(V)3.2 T-type calcium channel via extracellularly regulated kinase pathway in cardiomyocytes.

T-type Ca(2+) channel current (I(Ca,T)) plays an important role for spontaneous pacemaker activity and is involved in the progression of structural heart diseases. Estrogens are of importance for the regulation of growth and differentiation and function in a wide array of target tissues, including those in the cardiovascular system. The aim of this study was to elucidate the short-term and long-term effects of 17beta-estradiol (E(2)) on I(Ca,T) in cardiomyocytes. We employed in vivo and in vitro techniques to clarify E(2)-mediated modulation of heart rate (HR) in ovariectomized rats and I(Ca,T) in cardiomyocytes. Ovariectomy increased HR and E(2) supplement reduced HR in ovariectomized rats. Slowing of E(2)-induced HR was consistent with the deceleration of automaticity in E(2)-treated neonatal cardiomyocytes. Short-term application of E(2) did not have significant effects on I(Ca,T), whereas in cardiomyocytes treated with 10 nm E(2) for 24 h, estrogen receptor-independent down-regulation of peak I(Ca,T) and declination of Ca(V)3.2 mRNA were observed. Expression of a cardiac-specific transcription factor Csx/Nkx2.5 was also suppressed by E(2) treatment for 24 h. On the other hand, expression of Ca(V)3.1 mRNA was unaltered by E(2) treatment in this study. An ERK-1/2, 5 inhibitor, PD-98059, abolished the effects of E(2) on I(Ca,T) and Ca(V)3.2 mRNA as well as Csx/Nkx2.5 mRNA. These findings indicate that E(2) decreases Ca(V)3.2 I(Ca,T) through activation of ERK-1/2, 5, which is mediated by the suppression of Csx/Nkx2.5-dependent transcription, suggesting a genomic effect of E(2) as a negative chronotropic factor in the heart.