Microstripe pattern substrate consisting of alternating planar and nanoprotrusive regions improved hiPSC-derived cardiomyocytes' unidirectional alignment and functional properties.

The alignment of each cell in human myocardium is considered critical for the efficient movement of cardiac tissue. We investigated 96-well microstripe-patterned plates to align human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs), which resemble fetal myocardium. The aligned CMs (ACMs) cultured on the microstripe-patterned plates exhibited pathology, motor function, gene expression, and drug response that more closely resembled those of adult cells than did unaligned CMs cultured on a flat plate (FCMs). We used these ACMs to evaluate drug side effects and efficacy, and to determine whether these were similar to adult-like responses. When CMs from patients with hypertrophic cardiomyopathy (HCMs) were seeded and cultured on the microstripe-patterned plates or layered on top of the ACMs, both sets of HCMs showed increased heart rate and synchronized contractions, indicating improved cardiac function. It is suggested that the ACMs could be used for drug screening as cells representative of adult-like CMs and be transplanted in the form of a cell sheet for regenerative treatment of heart failure.

[Electrophysiological Effects of Ionophore-induced Increases in Intracellular Na+ in Cardiomyocytes].

Na ionophores increase intracellular Na+ ([Na+]i). Membrane potentials and currents were measured using microelectrode and whole-cell patch-clamp techniques. Monensin (10-6-3×10-5 M) reduced the slope of the pacemaker potentials and shortened the action potential duration (APD) in sino-atrial nodal and Purkinje cells. Monensin (10-5 M) shortened the APD and reduced the amplitude of the plateau phase in ventricular myocytes. Monensin decreased the hyperpolarization-activated inward current (If), and it increased the transient outward potassium current (Ito) in Purkinje cells. In addition, monensin decreased the sodium current (INa), shifting the inactivation curve to the hyperpolarized direction. Moreover, monensin decreased the L-type calcium current (ICa) in ventricular myocytes. The Na+-Ca2+ exchange current (INa-Ca) was augmented particularly in the reverse mode, and the Na+-K+ pump current (INa-K) was also activated by monensin in cardiomyocytes. The ATP-activated potassium current (IK,ATP) could be induced by monensin. Notably, the inward rectifying K+ current (IK1), and the slow delayed outward K+ current (IKs) were not affected evidently by monensin. Collectively, alteration of [Na+]i can influence the activities of various ion channels and transporters. Thus, the significance of altered [Na+]i should be taken into consideration in the action of drugs affecting [Na+]i such as digitalis, Na+ channel blockers, and Na+ channel activating agents.

Monensin-Induced Increase in Intracellular Na+ Induces Changes in Na+ and Ca2+ Currents and Regulates Na+-K+ and Na+-Ca2+ Transport in Cardiomyocytes.

BACKGROUND/AIMS: Monensin, an Na ionophore, increases intracellular Na ([Na]i). Alteration of [Na]i influences ion transport through the sarcolemmal membrane. So far, the effects of monensin on ventricular myocytes have not been examined in detail. The main objective of this study was to elucidate the mechanism via which monensin-evoked increases in [Na]i affect the membrane potential and currents in ventricular myocytes of guinea pigs. METHODS: Membrane potentials and currents were measured using the whole-cell patch-clamp technique in single myocytes. The concentration of intracellular Ca ([Ca]i) was evaluated by measuring fluorescence intensity of Fluo-4. RESULTS: Monensin (10-5M) shortened the action potential duration (APD) and reduced the amplitude of the plateau phase. In addition, monensin decreased the sodium current (INa) and shifted the inactivation curve to the hyperpolarized direction. Moreover, it decreased the L-type calcium current (ICa). However, this effect was attenuated by increasing the buffering capacity of [Ca]i. The Na-Ca exchange current (INa-Ca) was activated particularly in the reverse mode. Na-K pump current (INa-K) was also activated. Notably, the inward rectifying K current (IK1) was not affected, and the change in the delayed outward K current (IK) was not evident. CONCLUSION: These results suggest that the monensin-induced shortened APD and reduced amplitude of the plateau phase are primarily due to the decrease in the ICa, the activation of the reverse mode of INa-Ca, and the increased INa-K, and second due to the decreased INa. The IK and the IK1 may not be associated with the abovementioned changes induced by monensin. The elevation of [Na]i can exert multiple influences on electrophysiological phenomena in cardiac myocytes.

Effects of 2'-3'-dihydroxy-4',6'-dimethoxychalcone derived from green perilla on auricle thickness in chronic contact dermatitis model mice.

Oxidative stress has been implicated in the pathogenesis of allergic contact dermatitis. The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway, an in vivo antioxidant system, induces antioxidant enzymes. In our previous studies, we isolated 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC) from green perilla and identified it as a novel activator of the Nrf2-ARE pathway. We also discovered that it exerted cytoprotective effects against oxidative stress in PC12 cells. However, its effects on skin disease model animals in vivo remain unclear. In the present study, auricular thickness time-dependently increased with the repeated application of picryl chloride, and significant increases were observed from Day 2 in chronic contact hypersensitivity (cCHS) model mice. Histological changes, such as higher numbers of cells in the epidermis, were observed with increases in auricular thickness. The administration of DDC every two days from Day 6 suppressed the increases in auricular thickness and the number of scratching events in a dose-dependent manner. The expression levels of antioxidant enzymes increased in the mouse auricle 24 h after the administration of DDC. These results presume that DDC inhibits increases in auricular thickness in cCHS mice by up-regulating the expression of antioxidative enzymes through the activation of the Nrf2-ARE pathway.

Protective Effects of 2',3'-Dihydroxy-4',6'-dimethoxychalcone Derived from Green Perilla Leaves against UV Radiation-Induced Cell Injury in Human Cultured Keratinocytes.

Skin exposure to UV rays causes the production of reactive oxygen species (ROS), and it is a major risk factor for various skin disorders and diseases. In particular, exposure to UV-A is a major cause of photoaging. We have previously isolated 2',3'-dihydroxy-4',6'-dimethoxychalcone (DDC) from green perilla leaves as an activator of the nuclear factor erythroid 2-related factor-2 (Nrf2)-antioxidant response element (ARE) and demonstrated the protective effects of DDC both in vitro and in vivo in PC12 cells and Parkinson's disease models, respectively. In this study, we used HaCaT cells to examine the effects of DDC on ROS production and cell damage induced by UV-A. Our results indicated that UV-A irradiation in HaCaT cells increased ROS production in an energy-dependent manner. In addition, cell viability decreased in an energy-dependent manner 24 h after UV-A irradiation. However, treatment with DDC 24 h prior to UV-A irradiation significantly suppressed UV-A radiation-induced ROS production. In addition, DDC showed cytoprotective effects when used 24 h before and after UV-A irradiation. Treatment with DDC for 24 h also increased the expression levels of heme oxygenase-1 (HO-1) in a concentration-dependent manner. Pretreatment with the HO-1 inhibitor followed by DDC treatment before UV-A irradiation for 24 h reduced ROS production and the cytoprotective effect. These results suggest that DDC increases the expression levels of HO-1 and protects HaCaT cells through the suppression of UV radiation-induced ROS production.

Donepezil modulates amyloid precursor protein endocytosis and reduction by up-regulation of SNX33 expression in primary cortical neurons.

Donepezil, a therapeutic drug for Alzheimer's disease, ameliorates cognitive dysfunction through selective inhibition of acetylcholinesterase. However, recent studies have also reported off-target effects of donepezil that likely contribute to its therapeutic effects. In this study, we investigated the (i) role of donepezil in amyloid precursor protein (APP) processing and (ii) involvement of sorting nexin protein 33 (SNX33), a member of the sorting nexin protein family, in this processing. Results showed that donepezil induces an increase in SNX33 expression in primary cortical neurons. The secretion of sAPPα in culture media increased, whereas the expression of full-length APP in the cell lysate remained unchanged. Exposure of cortical cultures to donepezil led to a decrease in amyloid ß (Aß) protein levels in a concentration- and time-dependent manner. This decrease was not affected by concomitant treatment with acetylcholine receptor antagonists. SNX33 knockdown by target-specific morpholino oligos inhibited the effects of donepezil. Donepezil treatment increased cell membrane surface expression of APP in SNX33 expression-dependent manner. These results suggested that donepezil decreases the level of Aß by increasing SNX33 expression and APP cleavage by α-secretase in cortical neurons.

An inotropic action caused by muscarinic receptor subtype 3 in canine cardiac purkinje fibers.

Objective. The objective of this study was to investigate the inotropic mechanisms and the related muscarinic receptor subtype of acetylcholine (ACh) in canine cardiac Purkinje fibers. Materials and Methods. Isolated Purkinje fiber bundles were used for the measurement of contraction. The receptor subtype was determined using PCR and real-time PCR methods. Results. ACh evoked a biphasic response with a transient negative inotropic effect followed by a positive inotropic effect in a concentration-dependent manner. The biphasic inotropic actions of ACh were inhibited by the pretreatment with atropine. Caffeine inhibited the positive inotropic effect of ACh. ACh increased inositol-1,4,5-trisphosphate content in the Purkinje fibers, which was abolished by atropine. Muscarinic subtypes 2 (M2) and 3 (M3) mRNAs were detected in the canine Purkinje fibers albeit the amount of M3 mRNA was smaller than M2 mRNA. M1 mRNA was not detected. Conclusion. These results suggest that the positive inotropic action of ACh may be mediated by the activation of IP3 receptors through the stimulation of M3 receptors in the canine cardiac Purkinje fibers.

Effects of acetaldehyde on action potentials and Ca2+ currents in single atrial myocytes from the bullfrog.

AIM: The purpose of the present study was to examine the effects of acetaldehyde on the contractile force and membrane potentials and currents in the bullfrog heart. METHODS: Contractile force was recorded using right atrial tissues, and membrane potentials and currents were measured by using whole cell patch clamp methods in right atrial myocytes. RESULTS: Acetaldehyde at 500 µmol/l and 1 mmol/l increased the contractile force significantly. Acetaldehyde at 300 and 500 µmol/l increased the overshoot and the plateau of electrically induced action potentials in a concentration-dependent and reversible manner, while the resting membrane potential did not change. The duration of the action potential (APD(90)) measured at the 90% repolarization level was shortened. The L-type Ca(2+) current (I(Ca)) increased significantly when 300 and 500 µmol/l were applied. The fast transient inward current, the inward rectifying potassium current and the outward delayed-rectifier potassium current were not changed following acetaldehyde application (500 µmol/l or 1 mmol/l). CONCLUSION: These results suggest that acetaldehyde increased the I(Ca), thereby increased the contractile force, the overshoot and the plateau of action potentials. The shortening of APD(90) may be due to the acceleration of the current decay during the I(Ca) inactivation phase.

Inhibitory effects of alprostadil (prostaglandin E1) incorporated in lipid microspheres of soybean oil on intimal hyperplasia following balloon injury in rabbits.

The present study was performed to assess the inhibitory effects of alprostadil (CAS 745-65-3, prostaglandin E1, PGE1) incorporated in lipid microspheres (here-in-after referred to as lipo PGE1; Palux inj.) on intimal thickening following balloon injury in the carotid artery of normal rabbits. Lipo PGE1 was given intravenously to animals twice a day at doses of 20 or 40 micrograms/kg/day from ballooning (day 1) until day 3, and at half these doses from day 4 to day 20. The carotid artery was removed for histopathological staining on the next day (day 21) after the last administration. Lipo PGE1 significantly reduced both the intimal/medial are (I/M) ratio and stenosis ratio by about half in the 40 micrograms/kg/day on day 21 after ballooning, compared with the vehicle group. Infiltration of macrophage, expression of proliferating cell nuclear antigen (PCNA)-positive cells was inhibited by the administration of lipo PGE1 on day 3 after ballooning. Adhesion of platelets to injured arterial walls was also inhibited on day 3. Lipo PGE1 at 40 micrograms/kg/day exerted more potent inhibitory effects on I/M and stenosis ratios and histopathological changes such as infiltration of macrophage and expression of PCNA-positive cells than at 20 micrograms/kg/day. These findings suggest that lipo PGE1 inhibits the intimal hyperplasia after balloon injury in rabbit carotid artery, possibly by inhibiting platelet functions.

Cyclic AMP-mediated increase in L-type calcium current (ICa,L) by nitroglycerin in guinea-pig ventricular myocytes.

The effects of nitroglycerin (NG) on the L-type Ca2+ current (ICa,L) were investigated in guinea-pig ventricular myocytes. NG did not affect the basal ICa,L significantly. The ICa,L was increased by NG when the ICa,L was augmented by the pre-treatment with isoproterenol (Iso), and this increase was abolished by the pretreatment with methylene blue (MB), a guanylate cyclase inhibitor. NG also increased ICa,L in the myocytes undergoing the pretreatment with isobutylmethylxanthine (IBMX). The NG-induced increase in ICa,L was also observed in the myocytes intracellularly dialyzed with cyclic adenosine monophosphate (cAMP). The order of the Iso-induced increase in ICa,L was Iso, IBMX and cAMP. Relatively a lower concentration of the extracellularly applied 8bromo-cyclic guanosine monophosphate (8Br-cGMP) increased ICa,L in the myocytes dialyzed with cAMP, whereas the higher concentration of 8Br-cGMP decreased ICa,L. NG produced a marked increase in cGMP levels, and a slight increase in cAMP levels in the ventricular tissues. These results suggest that the stimulatory effect of NG on the ICa,L is due to an inhibition of cGMP-inhibitable cAMP-phosphodiesterase (PDE) and a possible activation of cGMP-dependent protein kinase via the NG-induced increase in cGMP levels in guinea-pig ventricular myocytes.