Selective activation of adrenoceptors potentiates IKs current in pulmonary vein cardiomyocytes through the protein kinase A and C signaling pathways.

Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The IKs current in PVC was markedly enhanced by both ß1- and ß2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by ß2-adrenoceptor stimulation than ß1-adrenoceptor stimulation. Both ß-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective IKs blocker. However, HMR-1556 markedly reduced the ß-adrenoceptor-potentiated firing rate. The stimulatory effects of ß- and α1-adrenoceptor on IKs in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under ß-adrenoceptor activation, suggesting that the functional role of IKs might increase during sympathetic excitation under in vivo conditions.

Characterization and functional role of rapid- and slow-activating delayed rectifier K+ currents in atrioventricular node cells of guinea pigs.

The atrioventricular (AV) node is the only conduction pathway where electrical impulse can pass from atria to ventricles and exhibits spontaneous automaticity. This study examined the function of the rapid- and slow-activating delayed rectifier K+ currents (IKr and IKs) in the regulation of AV node automaticity. Isolated AV node cells from guinea pigs were current- and voltage-clamped to record the action potentials and the IKr and IKs current. The expression of IKr or IKs was confirmed in the AV node cells by immunocytochemistry, and the positive signals of both channels were localized mainly on the cell membrane. The basal spontaneous automaticity was equally reduced by E4031 and HMR-1556, selective blockers of IKr and IKs, respectively. The nonselective ß-adrenoceptor agonist isoproterenol markedly increased the firing rate of action potentials. In the presence of isoproterenol, the firing rate of action potentials was more effectively reduced by the IKs inhibitor HMR-1556 than by the IKr inhibitor E4031. Both E4031 and HMR-1556 prolonged the action potential duration and depolarized the maximum diastolic potential under basal and ß-adrenoceptor-stimulated conditions. IKr was not significantly influenced by ß-adrenoceptor stimulation, but IKs was concentration-dependently enhanced by isoproterenol (EC50: 15 nM), with a significant negative voltage shift in the channel activation. These findings suggest that both the IKr and IKs channels might exert similar effects on regulating the repolarization process of AV node action potentials under basal conditions; however, when the ß-adrenoceptor is activated, IKs modulation may become more important.

Postnatal developmental changes in the sensitivity of L-type Ca2+ channel to inhibition by verapamil in a mouse heart model.

BackgroundIn the clinical setting, verapamil is contraindicated in neonates and infants, because of the perceived risk of hypotension or bradyarrhythmia. However, it remains unclear whether there is an age-dependent difference in the sensitivity of cardiac L-type Ca2+ channel current (ICa,L) to inhibition by verapamil.MethodsVentricular myocytes were enzymatically dissociated from the hearts of six different age groups (0, 7, 14, 21, 28 days, and 10-15 weeks) of mice, using a similar Langendorff-perfusion method. Whole-cell patch-clamp technique was applied to examine the sensitivity of ICa,L to inhibition, by three classes of structurally different L-type Ca2+ channel antagonists.ResultsVerapamil, nifedipine, and diltiazem concentration-dependently blocked the ventricular ICa,L in all six age groups. However, although nifedipine and diltiazem blocked ventricular ICa,L with a similar potency in all age groups, verapamil more potently blocked ventricular ICa,L in day 0, day 7, day 14, and day 21 mice, than in day 28, and 10-15-week mice.ConclusionIn a mouse heart model, ventricular ICa,L before the weaning age (~21 days of age) exhibited a higher sensitivity to inhibition by verapamil than that after the weaning age, which may explain one possible mechanism associated with the development of verapamil-induced hypotension in human neonates and infants.

Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells.

In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration have been proposed, localized in different subcellular organelles. However, visualizing thermogenesis directly in intact organelles has been challenging. Here we have developed genetically encoded, GFP-based thermosensors (tsGFPs) that enable visualization of thermogenesis in discrete organelles in living cells. In tsGFPs, a tandem formation of coiled-coil structures of the Salmonella thermosensing protein TlpA transmits conformational changes to GFP to convert temperature changes into visible and quantifiable fluorescence changes. Specific targeting of tsGFPs enables visualization of thermogenesis in the mitochondria of brown adipocytes and the endoplasmic reticulum of myotubes. In HeLa cells, tsGFP targeted to mitochondria reveals heterogeneity in thermogenesis that correlates with the electrochemical gradient. Thus, tsGFPs are powerful tools to noninvasively assess thermogenesis in living cells.

Phosphatidylinositol4-phosphate 5-kinase prevents the decrease in the HERG potassium current induced by Gq protein-coupled receptor stimulation.

The human ether-a-go-go-related gene (HERG) potassium current (IHERG) has been shown to decrease in amplitude following stimulation with Gq protein-coupled receptors (GqRs), such as α1-adrenergic and M1-muscarinic receptors (α1R and M1R, respectively), at least partly via the reduction of membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The present study was designed to investigate the modulation of HERG channels by PI(4,5)P2 and phosphatidylinositol4-phosphate 5-kinase (PI(4)P5-K), a synthetic enzyme of PI(4,5)P2. Whole-cell patch-clamp recordings were used to examine the activity of HERG channels expressed heterologously in Chinese Hamster Ovary cells. The stimulation of α1R with phenylephrine or M1R with acetylcholine decreased the amplitude of IHERG accompanied by a significant acceleration of deactivation kinetics and the effects on IHERG were significantly attenuated in cells expressing PI(4)P5-K. The density of IHERG in cells expressing GqRs alone was significantly increased by the coexpression of PI(4)P5-K without significant differences in the voltage dependence of activation and deactivation kinetics. The kinase-deficient substitution mutant, PI(4)P5-K-K138A did not have these counteracting effects on the change in IHERG by M1R stimulation. These results suggest that the current density of IHERG is closely dependent on the membrane PI(4,5)P2 level, which is regulated by PI(4)P5-K and GqRs and that replenishing PI(4,5)P2 by PI(4)P5-K recovers IHERG.

Sevoflurane protects ventricular myocytes against oxidative stress-induced cellular Ca2+ overload and hypercontracture.

BACKGROUND: Oxidative stress is implicated in pathogenesis of cardiac reperfusion injury, characterized by cellular Ca overload and hypercontracture. Volatile anesthetics protect the heart against reperfusion injury primarily by attenuating Ca overload. This study investigated electrophysiological mechanisms underlying cardioprotective effects of sevoflurane against oxidative stress-induced cellular injury. METHODS: The cytosolic Ca levels and cell morphology were assessed in mouse ventricular myocytes, using confocal fluo-3 fluorescence imaging, whereas membrane potentials and L-type Ca current (ICa,L) were recorded using whole-cell patch-clamp techniques. Phosphorylation of Ca/calmodulin-dependent protein kinase II was examined by Western blotting. RESULTS: Exposure to H2O2 (100 µM) for 15 min evoked cytosolic Ca elevation and hypercontracture in 56.8% of ventricular myocytes in 11 experiments, which was partly but significantly reduced by nifedipine, tetracaine, or SEA0400. Sevoflurane prevented H2O2-induced cellular Ca overload in a concentration-dependent way (IC50 = 1.35%). Isoflurane (2%) and desflurane (10%) also protected ventricular myocytes by a degree similar to sevoflurane (3%). Sevoflurane suppressed H2O2-induced electrophysiological disturbances, including early afterdepolarizations, voltage fluctuations in resting potential, and abnormal automaticities. H2O2 significantly enhanced ICa,L by activating Ca/calmodulin-dependent protein kinase II, and subsequent addition of sevoflurane, isoflurane, or desflurane similarly reduced ICa,L to below baseline levels. Phosphorylated Ca/calmodulin-dependent protein kinase II increased after 10-min incubation with H2O2, which was significantly prevented by concomitant administration of sevoflurane. CONCLUSIONS: Sevoflurane protected ventricular myocytes against H2O2-induced Ca overload and hypercontracture, presumably by affecting multiple Ca transport pathways, including ICa,L, Na/Ca exchanger and ryanodine receptor. These actions appear to mediate cardioprotection against reperfusion injury associated with oxidative stress.

Postnatal developmental decline in IK1 in mouse ventricular myocytes isolated by the Langendorff perfusion method: comparison with the chunk method.

Expression and function of cardiac ion channels exhibit postnatal developmental changes, which, however, has not yet been proven in ventricular myocytes isolated using similar techniques. In this study, ventricular myocytes were enzymatically dissociated from mouse heart at different postnatal ages (including postnatal day 0) by similar techniques using Langendorff perfusion. Whole-cell patch-clamp experiments were performed to record action potentials, I (K1), I (Kr), I (Kur), I (ss), and I (Ca,L), in ventricular myocytes freshly isolated from postnatal days 0, 7, and 14 and adult mice. Viable ventricular myocytes of day-0 mouse heart exhibited spindle-shaped appearance having cell length of approximately 50 µm, which gradually developed to a rod-shaped one having clear cross striation with cell length of approximately 120 µm (adult). The action potential duration markedly shortened, while the resting membrane potential depolarized to a small but significant extent during postnatal development. I (K1) density was maximal in postnatal day-0 ventricular myocytes and gradually decreased during development, which was accompanied by postnatal depolarization of resting membrane potential. However, I (K1) density was markedly decreased by approximately 80% in postnatal day-0 ventricular myocytes, when isolated by the chunk method. Quantitative real-time polymerase chain reaction (PCR) and western blot analyses demonstrated higher Kir2.3 expression but lower expression levels of Kir2.1 and Kir2.2 in day-0 mouse ventricles, compared with those of day-14 and adult mouse ventricles. Whereas I (Kr) exhibited marked decrease during postnatal development, I (Kur), I (ss), and I (Ca,L) exhibited postnatal developmental increase. The present cell isolation method using the Langendorff perfusion thus found that, in mouse ventricles, I (K1) exhibited postnatal developmental decrease, associated with depolarization of resting potential.

Atypically Shaped Cardiomyocytes (ACMs): The Identification, Characterization and New Insights into a Subpopulation of Cardiomyocytes.

In the adult mammalian heart, no data have yet shown the existence of cardiomyocyte-differentiable stem cells that can be used to practically repair the injured myocardium. Atypically shaped cardiomyocytes (ACMs) are found in cultures of the cardiomyocyte-removed fraction obtained from cardiac ventricles from neonatal to aged mice. ACMs are thought to be a subpopulation of cardiomyocytes or immature cardiomyocytes, most closely resembling cardiomyocytes due to their spontaneous beating, well-organized sarcomere and the expression of cardiac-specific proteins, including some fetal cardiac gene proteins. In this review, we focus on the characteristics of ACMs compared with ventricular myocytes and discuss whether these cells can be substitutes for damaged cardiomyocytes. ACMs reside in the interstitial spaces among ventricular myocytes and survive under severely hypoxic conditions fatal to ventricular myocytes. ACMs have not been observed to divide or proliferate, similar to cardiomyocytes, but they maintain their ability to fuse with each other. Thus, it is worthwhile to understand the role of ACMs and especially how these cells perform cell fusion or function independently in vivo. It may aid in the development of new approaches to cell therapy to protect the injured heart or the clarification of the pathogenesis underlying arrhythmia in the injured heart.

Propofol, an Anesthetic Agent, Inhibits HCN Channels through the Allosteric Modulation of the cAMP-Dependent Gating Mechanism.

Propofol is a broadly used intravenous anesthetic agent that can cause cardiovascular effects, including bradycardia and asystole. A possible mechanism for these effects is slowing cardiac pacemaker activity due to inhibition of the hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels. However, it remains unclear how propofol affects the allosteric nature of the voltage- and cAMP-dependent gating mechanism in HCN channels. To address this aim, we investigated the effect of propofol on HCN channels (HCN4 and HCN2) in heterologous expression systems using a whole-cell patch clamp technique. The extracellular application of propofol substantially suppressed the maximum current at clinical concentrations. This was accompanied by a hyperpolarizing shift in the voltage dependence of channel opening. These effects were significantly attenuated by intracellular loading of cAMP, even after considering the current modification by cAMP in opposite directions. The differential degree of propofol effects in the presence and absence of cAMP was rationalized by an allosteric gating model for HCN channels, where we assumed that propofol affects allosteric couplings between the pore, voltage-sensor, and cyclic nucleotide-binding domain (CNBD). The model predicted that propofol enhanced autoinhibition of pore opening by unliganded CNBD, which was relieved by the activation of CNBD by cAMP. Taken together, these findings reveal that propofol acts as an allosteric modulator of cAMP-dependent gating in HCN channels, which may help us to better understand the clinical action of this anesthetic drug.

Expression and functional maintenance of volume-regulated anion channels in myometrial smooth muscles of pregnant mice.

Pregnancy causes changes in the uterus, such as increased cell volume and altered water content. However, the mechanisms that protect the structure and maintain the function of uterine smooth muscle cells against these changes during pregnancy have not been clarified. This study focused on the volume-regulated anion channel (VRAC), which opens with cell swelling under low osmotic pressure and releases Cl- ions and various organic osmolytes to resist cell swelling and regulates a wide range of biological processes such as cell death. In this study, myometrial smooth muscle (MSM) tissues and cells (MSMCs) were collected from non-pregnant and pregnant mice. Using western blotting and immunocytochemistry, leucine-rich repeat containing protein 8A (LRRC8A), an essential membrane protein that constitutes part of the VRAC, was determined to be diffused throughout MSMCs including in the cell membrane. Patch-clamp experiments were performed to investigate the electrophysiology of swelling-induced Cl- currents (ICl, swell) mediated by the VRAC. No significant changes between non-pregnancy and pregnancy groups were observed in either the expression density of LRRC8A or the current density of ICl, swell, however the presence of LRRC8A on the cell membrane was significantly increased in the third trimester of pregnancy compared to the non-pregnancy. This study suggests that the VRAC may play a role, such as maintaining cellular homeostasis in the pregnant MSM.

Sevoflurane protects ventricular myocytes from Ca2+ paradox-mediated Ca2+ overload by blocking the activation of transient receptor potential canonical channels.

BACKGROUND: Volatile anesthetics produce cardioprotective action by attenuating cellular Ca2+ overload. The Ca2+ paradox is an important model for studying the mechanisms associated with Ca2+ overload-mediated myocardial injury, and was recently found to be mediated by Ca2+ entry through the transient receptor potential canonical channels upon Ca2+ repletion. This study investigated the effect of sevoflurane on cellular mechanisms underlying the Ca2+ paradox. METHODS: The Ca2+ paradox was examined in fluo-3 or mag-fluo-4-loaded mouse ventricular myocytes using confocal laser scanning microscope, upon Ca2+ repletion after 15 min of Ca2+ depletion in the absence and presence of sevoflurane. RESULTS: The Ca2+ paradox was evoked in approximately 65% of myocytes upon Ca2+ repletion, as determined by an abrupt elevation of cytosolic Ca2+ accompanied by hypercontracture. The Ca2+ paradox was significantly suppressed by sevoflurane administered for 3 min before and during Ca2+ repletion (Post) or during Ca2+ depletion and repletion (Postlong), and Postlong was more beneficial than Post application. The sarcoplasmic reticulum Ca2+ levels gradually decreased during Ca2+ depletion, and the Ca2+ paradox was readily evoked in myocytes with reduced sarcoplasmic reticulum Ca2+ levels. Postlong but not Post application of sevoflurane prevented decrease in sarcoplasmic reticulum Ca2+ levels by blocking Ca2+ leak through ryanodine receptors. Whole cell patch-clamp recordings revealed that sevoflurane rapidly blocked thapsigargin-induced transient receptor potential canonical currents. CONCLUSIONS: Sevoflurane protects ventricular myocytes from Ca2+ paradox-mediated Ca2+ overload by blocking transient receptor potential canonical channels and by preventing the decrease in sarcoplasmic reticulum Ca2+ levels, which is associated with less activation of transient receptor potential canonical channels.

Autophagy is constitutively active in normal mouse sino-atrial nodal cells.

This study was designed to examine the autophagy in sino-atrial (SA) nodal cells from the normal adult mouse heart. Autophagy is the cellular process responsible for the degradation and recycling of long-lived and/or damaged cytoplasmic components by lysosomal digestion. In the heart, autophagy is known to occur at a low level under physiological conditions, but to become upregulated when cells are exposed to certain stresses, such as ischemia. We examined whether the basal level of autophagy in SA nodal cells was different from that in ventricular or atrial myocytes. An ultrastructural analysis revealed that the SA nodal cells contained a number of autophagic vacuoles (autophagosomes) with various stages of degradation by lysosomal digestion, whereas the number of those in ventricular or atrial myocytes was either negligible or very small. The immunostaining of autophagosome marker microtubule-associated protein 1 light chain 3 (LC3) and lysosome marker lysosome-associated membrane protein 1 (LAMP1) indicated that the content of both autophagosomes and lysosomes were much greater in SA nodal cells than in ordinary cardiomyocytes. Our results provide evidence that the autophagy is active in normal SA nodal cells, which is not a stress-activated process but a constitutive event in the mouse heart.

An Antegrade Perfusion Method for Cardiomyocyte Isolation from Mice.

In basic research using mouse heart, isolating viable individual cardiomyocytes is a crucial technical step to overcome. Traditionally, isolating cardiomyocytes from rabbits, guinea pigs or rats has been performed via retrograde perfusion of the heart with enzymes using a Langendorff apparatus. However, a high degree of skill is required when this method is used with a small mouse heart. An antegrade perfusion method that does not use a Langendorff apparatus was recently reported for the isolation of mouse cardiomyocytes. We herein report a complete protocol for the improved antegrade perfusion of the excised heart to isolate individual heart cells from adult mice (8 - 108 weeks old). Antegrade perfusion is performed by injecting perfusate near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. All procedures are carried out on a pre-warmed heater mat under a microscope, which allows for the injection and perfusion processes to be monitored. The results suggest that ventricular and atrial myocytes, and fibroblasts can be well isolated from a single adult mouse simultaneously.

Elevation of propofol sensitivity of cardiac IKs channel by KCNE1 polymorphism D85N.

BACKGROUND AND PURPOSE: The slowly activating delayed rectifier K+ channel (IKs ), composed of pore-forming KCNQ1 α-subunits and ancillary KCNE1 ß-subunits, regulates ventricular repolarization in human heart. Propofol, at clinically used concentrations, modestly inhibits the intact (wild-type) IKs channels and is therefore unlikely to appreciably prolong QT interval in ECG during anaesthesia. However, little information is available concerning the inhibitory effect of propofol on IKs channel associated with its gene variants implicated in QT prolongation. The KCNE1 single nucleotide polymorphism leading to D85N is associated with drug-induced QT prolongation and therefore regarded as a clinically important genetic variant. This study examined whether KCNE1-D85N affects the sensitivity of IKs to inhibition by propofol. EXPERIMENTAL APPROACH: Whole-cell patch-clamp and immunostaining experiments were conducted in HEK293 cells and/or mouse cardiomyocyte-derived HL-1 cells, transfected with wild-type KCNQ1, wild-type or variant KCNE1 cDNAs. KEY RESULTS: Propofol inhibited KCNQ1/KCNE1-D85N current more potently than KCNQ1/KCNE1 current in HEK293 cells and HL-1 cells. Immunostaining experiments in HEK293 cells revealed that pretreatment with propofol (10 µM) did not appreciably affect cell membrane expression of KCNQ1 and KCNE1 proteins in KCNQ1/KCNE1 and KCNQ1/KCNE1-D85N channels. CONCLUSION AND IMPLICATIONS: The KCNE1 polymorphism D85N significantly elevates the sensitivity of IKs to inhibition by propofol. This study detects a functionally important role of KCNE1-D85N polymorphism in conferring genetic susceptibility to propofol-induced QT prolongation and further suggests the possibility that the inhibitory action of anaesthetics on ionic currents becomes exaggerated in patients carrying variants in genes encoding ion channels.

Characterization of the rapidly activating delayed rectifier potassium current, I (Kr), in HL-1 mouse atrial myocytes.

HL-1 is the adult murine cardiac cell line that can be passaged repeatedly in vitro without losing differentiated phenotype. The present study was designed to characterize the rapidly activating delayed rectifier potassium current, I (Kr), endogenously expressed in HL-1 cells using the whole-cell patch-clamp technique. In the presence of nisoldipine, depolarizing voltage steps applied from a holding potential of -50 mV evoked the time-dependent outward current, followed by slowly decaying outward tail current upon return to the holding potential. The amplitude of the current increased with depolarizations up to 0 mV but then progressively decreased with further depolarizations. The time-dependent outward current as well as the tail current were highly sensitive to block by E-4031 and dofetilide (IC(50) of 21.1 and 15.1 nM, respectively) and almost totally abolished by micromolar concentrations of each drug, suggesting that most of the outward current in HL-1 cells was attributable to I (Kr). The magnitude of I (Kr) available from HL-1 cells (18.1 +/- 1.5 pA pF(-1)) was sufficient for reliable measurements of various gating parameters. RT-PCR and Western blot analysis revealed the expression of alternatively spliced forms of mouse ether-a-go-go-related genes (mERG1), the full-length mERG1a and the N-terminally truncated mERG1b isoforms. Knockdown of mERG1 transcripts with small interfering RNA (siRNA) dramatically reduced I (Kr) amplitude, confirming the molecular link of mERG1 and I (Kr) in HL-1 cells. These findings demonstrate that HL-1 cells possess I (Kr) with properties comparable to those in native cardiac I (Kr) and provide an experimental model suitable for studies of I (Kr) channels.

Angiotensin II type 1 receptor mediates partially hyposmotic-induced increase of I (Ks) current in guinea pig atrium.

A repolarizing conduction in the heart augmented by hyposmotic or mechanically induced membrane stretch is the slow component of delayed rectifier K(+) current (I (Ks)). I (Ks) upregulation is recognized as a factor promoting appearance of atrial fibrillation (AF) since gain-of-function mutations of the channel genes have been detected in congenital AF. Mechanical stretch activates angiotensin II type 1 (AT(1)) receptor in the absence of its physiological ligand angiotensin II. We investigated the functional role of AT(1) receptor in I (Ks) enhancement in hyposmotically challenged guinea pig atrial myocytes using the whole-cell patch-clamp method. In atrial myocytes exposed to hyposmotic solution with osmolality decreased to 70% of the physiological level, I (Ks) was enhanced by 84.1%, the duration of action potential at 90% repolarization (APD(90)) was decreased by 16.8%, and resting membrane potential was depolarized (+4.9 mV). The hyposmotic-induced effects on I (Ks) and APD(90) were significantly attenuated by specific AT(1) receptor antagonist candesartan (1 and 5 muM). Pretreatment of atrial myocytes with protein tyrosine kinase inhibitors tyrphostin A23 and A25 suppressed but the presence of tyrosine phosphatase inhibitor orthovanadate augmented hyposmotic stimulation of I (Ks). The above results implicate AT(1) receptor and tyrosine kinases in the hyposmotic modulation of atrial I (Ks) and suggest acute antiarrhythmic properties of AT(1) antagonists in the settings of stretch-related atrial tachyarrhythmias.

A novel type of self-beating cardiomyocytes in adult mouse ventricles.

This study was designed to investigate the presence of resident heart cells that are distinct from terminally-differentiated cardiomyocytes. Adult mouse heart was coronary perfused with collagenase, and ventricles were excised and further digested. After spinning cardiomyocyte-containing fractions down, the supernatant fraction was collected and cultured without adding any chemicals. Two to five days after plating, some of rounded cells adhered to the culture dish, gradually changed their shape and then started self-beating. These self-beating cells did not appreciably proliferate but underwent a further morphological maturation process to form highly branched shapes with many projections. These cells were mostly multinucleated, well sarcomeric-organized and expressed cardiac marker proteins, defined as atypically-shaped cardiomyocytes (ACMs). Patch-clamp experiments revealed that ACMs exhibited spontaneous action potentials arising from the preceding slow diastolic depolarization. We thus found a novel type of resident heart cells in adult cardiac ventricles that spontaneously develop into self-beating cardiomyocytes.

A simple antegrade perfusion method for isolating viable single cardiomyocytes from neonatal to aged mice.

The aim of this study was to establish a simple and reproducible antegrade perfusion method for isolating single viable mouse heart cells and to determine the standard practical protocols that are appropriate for mice of various ages. Antegrade perfusion was performed by injecting perfusate from near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. This could thoroughly perfuse the myocardium through the coronary circulation. All procedures were carried out on a prewarmed heater mat under a microscope, which allows for the processes of injection and perfusion to be monitored. With appropriate adjustment of the size of the injection needle, the composition and amount of enzyme solution and the perfusion flow rate, this antegrade perfusion method could be applied to the hearts of neonatal to aged mice. We examined the morphological characteristics and electrophysiological properties of the isolated ventricular and atrial myocytes and found that these cells were mostly identical to those obtained with the traditional Langendorff-based retrograde perfusion method. Interstitial nonmyocytes, such as cardiac progenitor cells, were also isolated simultaneously from the supernatant fraction of the centrifugation, similar to the retrograde perfusion method. The results suggest that single heart cells can be well isolated with high degree of quality by the present antegrade perfusion method, regardless of the age of the mouse.

Novel intracellular transport-refractory mutations in KCNH2 identified in patients with symptomatic long QT syndrome.

BACKGROUND: Missense mutations in KCNH2, a gene encoding the Kv11.1 channel, cause long QT syndrome (LQTS) type 2 primarily by disrupting the intracellular transport of Kv11.1 to the plasma membrane. The present study aimed to clarify the functional changes by two novel KCNH2 missense mutations. METHODS: We performed genetic screening of three unrelated symptomatic LQTS probands with family histories of cardiac symptoms. Chinese hamster ovary cells were transfected with wild-type (WT) and/or mutant KCNH2 plasmid and examined by patch-clamp technique. Immunostaining and confocal microscopy were performed to evaluate the intracellular localization of WT and homozygous mutant Kv11.1 in human embryonic kidney cells. For the study of trafficking rescue, we used low-temperature incubation (30°C). We also examined pharmacological rescue of homozygous mutant Kv11.1 current in cells treated with E-4031 or dofetilide. RESULTS: We identified two novel KCNH2 missense mutations, G785D and T826I. Electrophysiological study showed that both mutant channels were nonfunctional in homozygous condition and reduced current densities by half in heterozygous condition compared with WT Kv11.1. Heterozygous Kv11.1-G785D produced a significant positive shift in activation and a significant negative shift in inactivation, whereas heterozygous Kv11.1-T826I caused no kinetic changes. Immunostaining revealed that both were transport-refractory mutations. Incubation at 30°C rescued plasma membrane expression of Kv11.1-T826I but not G785D. We confirmed low-temperature-induced restoration of homozygous Kv11.1-T826I transport by functional current measurements. In contrast, incubation with E-4031 or dofetilide failed to produce measurable currents in both homozygous mutant channels. CONCLUSIONS: Two novel KCNH2 mutations disrupted the intracellular transport of Kv11.1. Low-temperature incubation rescued plasma membrane expression of Kv11.1-T826I but not G785D. Both mutations exerted loss-of-function effects on Kv11.1 and explained the phenotypes of the mutation carriers.