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Type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) regulates the intracellular Ca2+ release from endoplasmic reticulum in human embryonic stem cells (hESCs), cardiovascular progenitor cells (CVPCs), and mammalian cardiomyocytes. However, the role of IP3R2 in human cardiac development is unknown and its function in mammalian cardiomyocytes is controversial. hESC-derived cardiomyocytes have unique merits in disease modeling, cell therapy, and drug screening. Therefore, understanding the role of IP3R2 in the generation and function of human cardiomyocytes would be valuable for the application of hESC-derived cardiomyocytes. In the current study, we investigated the role of IP3R2 in the differentiation of hESCs to cardiomyocytes and in the hESC-derived cardiomyocytes. By using IP3R2 knockout (IP3R2KO) hESCs, we showed that IP3R2KO did not affect the self-renewal of hESCs as well as the differentiation ability of hESCs into CVPCs and cardiomyocytes. Furthermore, we demonstrated the ventricular-like myocyte characteristics of hESC-derived cardiomyocytes. Under the α1-adrenergic stimulation by phenylephrine (10 μmol/L), the amplitude and maximum rate of depolarization of action potential (AP) were slightly affected in the IP3R2KO hESC-derived cardiomyocytes at differentiation day 90, whereas the other parameters of APs and the Ca2+ transients did not show significant changes compared with these in the wide-type ones. These results demonstrate that IP3R2 has minimal contribution to the differentiation and function of human cardiomyocytes derived from hESCs, thus provide the new knowledge to the function of IP3R2 in the generation of human cardiac lineage cells and in the early cardiomyocytes.

Introduction: Attenuating cardiac fibroblasts activation contributes to reducing excessive extracellular matrix deposition and cardiac structural remodeling in hypertensive hearts. Acacetin plays a protective role in doxorubicin-induced cardiomyopathy and ischemia/reperfusion injury. The aim of this study was to investigate the potential molecular mechanisms underlying the protective role of acacetin on hypertension-induced cardiac fibrosis. Methods: Echocardiography, histopathological methods, and Western blotting techniques were used to evaluate the anti-fibrosis effects in spontaneous hypertensive rat (SHR) which were daily intragastrically administrated with acacetin (10 mg/kg and 20 mg/kg) for 6 weeks. Angiotensin II (Ang II) was used to induce cellular fibrosis in human cardiac fibroblasts (HCFs) in the absence and presence of acacetin treatment for 48 h. Results: Acacetin significantly alleviated hypertension-induced increase in left ventricular (LV) posterior wall thickness and LV mass index in SHR. The expressions of collagen-1, collagen-III, and alpha-smooth muscle actin (α-SMA) were remarkedly decreased after treatment with acacetin (n = 6, p < 0.05). In cultured HCFs, acacetin significantly attenuated Ang II-induced migration and proliferation (n = 6, p < 0.05). Moreover, acacetin substantially inhibited Ang II-induced upregulation of collagen-1 and collagen-III (n = 6, p < 0.05) and downregulated the expression of alpha-SMA in HCFs. Additionally, acacetin decreased the expression of TGF-β1, p-Smad3/Smad3, and p-AKT and p-mTOR but increased the expression of Smad7 (n = 6, p < 0.05). Further studies found that acacetin inhibited TGF-β1 agonist SRI and AKT agonist SC79 caused fibrotic effect. Conclusion: Acacetin inhibits the hypertension-associated cardiac fibrotic processes through regulating TGF-β/Smad3, AKT/mTOR signal transduction pathways. [ABSTRACT FROM AUTHOR]

Highlights: [•] IP3 activates BKCa channels and exerts a coronary artery-relaxing effect. [•] IP3 enhances the STOC via IP3 receptor. [•] IP3 stimulates the Macro-current by increasing the Ca2+ sensitivity of channels. [ABSTRACT FROM AUTHOR]

Abstract: High conductance Ca2+ activated K+ channels (BKCa) in vascular smooth muscles play important roles in controlling the vascular tone by determining the level of membrane potential and Ca2+ influx through voltage gated Ca2+ channels. Agents that alter the activity of Ca2+ channels or BKCa thus affect the vascular tone in both physiological and pathological conditions. Danshen, the dried root of Salvia miltiorrhiza, is a commonly used traditional Chinese medicine and is widely used as an effective remedy for cardiovascular and cerebral vascular diseases partly by its vasodilatation. Sodium tanshinoneII-A sulfonate (DS-201) is a water-soluble derivative of Tanshinone IIA, the main active component of Danshen. The purpose of this study was to explore possible mechanisms of vasodilative effects of DS-201 using porcine coronary artery smooth muscle. DS-201 induced relaxation of the coronary smooth muscle which had been contracted with 30 mM KCl, and the relaxation was inhibited by 100 nM iberiotoxin (IbTX), a specific BKCa channel blocker. Using perforated whole-cell recordings and single channel recordings, effects of DS-201 on BKCa were examined. The results showed that DS-201 activated BKCa. Extracellular application of DS-201 at 40, 80 µM under the whole-cell configuration induced increases of the BKCa macroscopic currents by 43.6%, 42.1% respectively, and the spontaneous transient outward K+ currents (STOCs) by 48.7%, 47.4% respectively. In inside-out patches, bath application of 20–150 μM of DS-201 activated BKCa by 5.4–173.2 fold. These results indicate that the vasodilatation by DS-201 is related to activation of BKCa. [Copyright &y& Elsevier]

Type 2 inositol 1,4,5-trisphosphate receptor (IP 3 R2) regulates the intracellular Ca 2+ release from endoplasmic reticulum in human embryonic stem cells (hESCs), cardiovascular progenitor cells (CVPCs), and mammalian cardiomyocytes. However, the role of IP 3 R2 in human cardiac development is unknown and its function in mammalian cardiomyocytes is controversial. hESC-derived cardiomyocytes have unique merits in disease modeling, cell therapy, and drug screening. Therefore, understanding the role of IP 3 R2 in the generation and function of human cardiomyocytes would be valuable for the application of hESC-derived cardiomyocytes. In the current study, we investigated the role of IP 3 R2 in the differentiation of hESCs to cardiomyocytes and in the hESC-derived cardiomyocytes. By using IP 3 R2 knockout (IP 3 R2KO) hESCs, we showed that IP 3 R2KO did not affect the self-renewal of hESCs as well as the differentiation ability of hESCs into CVPCs and cardiomyocytes. Furthermore, we demonstrated the ventricular-like myocyte characteristics of hESC-derived cardiomyocytes. Under the α 1 -adrenergic stimulation by phenylephrine (10 μmol/L), the amplitude and maximum rate of depolarization of action potential (AP) were slightly affected in the IP 3 R2KO hESC-derived cardiomyocytes at differentiation day 90, whereas the other parameters of APs and the Ca 2+ transients did not show significant changes compared with these in the wide-type ones. These results demonstrate that IP 3 R2 has minimal contribution to the differentiation and function of human cardiomyocytes derived from hESCs, thus provide the new knowledge to the function of IP 3 R2 in the generation of human cardiac lineage cells and in the early cardiomyocytes.

Background: The incidence of atrial fibrillation (AF) in rheumatic heart diseases (RHD) is very high and increases with age. Occurrence and maintenance of AF are very complicated process accompanied by many different mechanisms. Ion-channel remodeling, including the voltage-gated potassium channel Kv1.5, plays an important role in the pathophysiology of AF. However, the changes of Kv1.5 channel expression in Han Chinese patients with RHD and AF remain poorly understood. The aim of the present study was to investigate whether the Kv1.5 channels of the right atria may be altered with RHD, age, and sex to contribute to AF., Material/methods: Right atrial appendages were obtained from 20 patients with normal cardiac functions who had undergone surgery, and 26 patients with AF. Subjects were picked from 4 groups: adult and aged patients in normal sinus rhythm (SR) and AF. Patients were divided into non-RHD and RHD groups or men and women groups in normal SR and AF, respectively. The expression of Kv1.5 protein and messenger RNA (mRNA) were measured using Western blotting and polymerase chain reaction (PCR) method, respectively., Results: Compared with the SR group, the expression of Kv1.5 protein decreased significantly in the AF group. However, neither Kv1.5 protein nor KCNA5 mRNA had significant differences in adult and aged groups, non-RHD and RHD group, and men and women group of AF., Conclusions: The expression of Kv1.5 channel protein changes with AF but not with age, RHD, and sex in AF.

Objective: To establish a perforated patch-clamp technology with amphotericin B and beta-escin and to research the regulation of small conductance calcium-activated potassium channel SK2 currents by calcium ions., Methods: Single human atrial myocytes were enzymatically isolated from the right atrial appendage. Amphotericin B and / or beta-escin were used by perforated electrode liquid. The regulation of SK2 current by calcium ions in human atrial myocytes was performed with the perforated patch-clamp technique. The intracellular calcium changes were measured by the intracellular calcium test system., Results: Mixed perforated electrode liquid compared with 150 microg/ml amphotericin B or 6.88 microg/ml beta-escin alone, it was easy to seal cells and activate SK2 current by the former method. Moreover, the ration of F340/380 was consistent with the change of intracellular free calcium ion concentration increase after the formation of perforation. The ration of F340/380 was measured by intracellular calcium test system., Conclusion: The appropriate concentration of amphotericin B mixed with beta-escin can form a stable whole-cell patch recording technology that is appropriate for the research of SK2 current regulation by intracellular calcium.

Objective: To compare the amplitude of the SK2 current (small conductance calcium-activated potassium channel) in human atrial myocytes with or without persistent atrial fibrillation (AF)., Methods: Right atrial appendage was obtained from 15 patients with sinus rate (SR) and 7 patients with AF underwent surgical valve replacement. Single myocyte was isolated by enzymatic dissociation method and the SK2 channel current density was recorded using whole-cell patch clamp techniques to detect the changes. Immunofluorescence was used to observe SK2 channel protein distribution on right atrial appendage., Results: Using the whole cell patch-clamp recording techniques, an inward rectifier K(+) mix currents could be obtained from both SR (n = 15) and AF (n = 7) samples, I(K1) mix currents density in single myocyte of AF group was significantly increased than in SR group [(-16.42 ± 5.32) pA/pF vs (-6.59 ± 2.24) pA/pF, P < 0.01], which could be partially inhibited by apamin (100 nmol/L). The apamin-sensitive current was obtained by subtraction of the currents before and after treatment with apamin. SK2 current density was significantly increased in AF group than that of SR group [(-9.81 ± 2.54) pA/pF vs (-3.67 ± 0.37) pA/pF, P < 0.01]. SK2 channel protein was evidenced with immunofluorescence method in right atrial appendage from AF group and SR group., Conclusion: SK2 channel protein and current were present in atrial myocytes. The SK2 current density was significantly increased in AF group than in SR group suggesting that the increase of SK2 current might contribute to the electrical remodeling in AF patients.

Objective: To test the effects of combined use of dofetilide and verapamil on the action potential of papillary muscles in guinea pigs., Methods: Under the stimulation of different frequencies (0.2, 0.5, 1.0, 1.25 or 2.0 Hz), the action potential of papillary muscles in guinea pigs was recorded with the standard microelectrode techniques. The impacts of different concentrations of amiodarone (1, 5 or 10 micromol/L), dofetilide (10, 50, 100 nmol/L), and a combination of 100 nmol/L dofetilide and 1 micromol/L verapamil on the action potential were tested., Results: Amiodarone prolonged the action potential duration (APD) significantly, measured both at 50% (APD50) and 90% (APD90) of repolarization, in a concentration-dependent manner independent from stimulation frequencies. Dofetilide prolonged APD in a concentration-dependent manner, which was negatively dependent on stimulation frequencies. The frequency-dependent effect was ameliorated by adding 1 micromol/L verapamil to dofetilide., Conclusion: Both amiodarone and dofetilide prolong APD in a concentration-dependent manner. The class III antiarrhythmic compounds, amiodarone, has less frequency-dependent effect than the pure class III antiarrhythmic drug. A combined use of potassium and calcium antagonists may reduce the frequency-dependence of the pure class III antiarrhythmic drug.

The aim of the present study was to investigate the effects of inositol 1,4,5-trisphosphate (IP(3))-generating agonist UTP on spontaneous transient outward currents (STOCs), and explore the role of intracellular Ca(2+) release in the current response mediated by IP(3) in porcine coronary artery smooth muscle cells (CASMCs). The coronary artery was excised from the fresh porcine heart and cut into small segments (2 mm × 5 mm) and then transferred to enzymatic dissociation solution for incubation. Single CASMCs were obtained by two-step enzyme digestion at 37 °C. STOCs were recorded and characterized using the perforated whole-cell patch-clamp configuration in freshly isolated porcine CASMCs. The currents were amplified and filtered by patch-clamp amplifier (Axopatch 200B), and then the digitized data were recorded by pClamp 9.0 software and further analyzed by MiniAnalysis 6.0 program. The results were as follows: (1) UTP led to conspicuous increases in STOC amplitude by (57.54±5.34)% and in frequency by (77.46±8.42)% (P<0.01, n=38). (2) The specific blocker of phospholipase C (PLC) - U73122 (5 μmol/L) remarkably reduced STOC amplitude by (31.04±7.46)% and frequency by (41.65±16.59)%, respectively (P<0.05, n=10). In the presence of U73122, UTP failed to reactivate STOCs (n=7). (3) Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two blockers of L-type voltage-dependent Ca(2+) channels, had little effects on STOCs initiated by UTP (n=8). (4) 1 μmol/L bisindolylmaleimide I (BisI), a potent blocker of protein kinase C (PKC), significantly increased STOC amplitude by (65.44±24.66)% and frequency by (61.35±21.47)% (P<0.01, n=12); UTP (40 μmol/L), applied in the presence of 1 μmol/L BisI, could further increase STOC activity (P<0.05, P<0.01, n=12). Subsequent application of ryanodine (50 μmol/L) abolished STOC activity. (5) In the presence of UTP (40 μmol/L), inhibition of IP(3) receptors (IP(3)Rs) by 2-aminoethoxydiphenyl borate (2-APB, 40 μmol/L) reduced STOC amplitude by (24.08±3.97)% (P<0.05, n=8), but had little effect on STOC frequency (n=8). While application of 2-APB (80 μmol/L) significantly reduced STOC amplitude by (31.43±6.34)% and frequency by (40.59±19.01)%, respectively (P<0.05, P<0.01, n=6). Subsequent application of ryanodine (50 μmol/L) completely blocked STOC activity. Pretreatment of cells with 2-APB (40 μmol/L) or ryanodine (50 μmol/L), UTP (40 μmol/L) failed to reactivate STOCs. The results suggest that UTP activates STOCs mainly via PLC and IP(3)-dependent mechanisms. Complex Ca(2+)-mobilization pathways are involved in UTP-mediated STOC activation in porcine CASMCs.

To approach the method of isolation of tolerant human atrial myocytes, single myocytes were isolated by modified procedure of enzymatic dissociation with protease (type XXIV) and collagenase (type V). L-type calcium channel current (I(Ca-L)), sodium current (I(Na)), transient outward potassium current (I(to1)), and inward rectifier potassium current (I(K1)) in isolated atrial myocytes were recorded by using whole-cell patch-clamp techniques. Single cardiocytes isolated by this method were smooth, well-striated and rod-shaped. The yields of recordable myocytes, which viable and calcium-tolerant for electrophysiological studies, were 50%-60% of the total isolated cells. Compared with other isolation methods, this method was simple and steady, but with yield of a great number of qualified myocytes. The currents recorded in these cells were functional and active. Our research suggests that the myocytes isolated by the described method in this paper have normal electrophysiological function and are appropriate for patch-clamp experiments.

Objective: To investigate the feasibility of cultured rat ventricular myocytes taking the place of acutely enzymatic isolated myocardia for study in the pacemaker current electrophysiology., Methods: By using patch-clamp technique, the whole-cell pacemaker currents were recorded to study the difference of pacemaker current electrophysiology between neonatal rat ventricular myocytes isolated acutely and maintained in cell culture., Results: The cell membrane capacitances (Cm) were significantly decreased in myocardia even upon short term of cell culture. The Cm of myocardia cultured 1-3 day were (43 +/- 3) pF, with decreased 23% compared to acute enzymatic isolated cells C(56+/-7) pF, P<0. 013. It seemed like the Cm increased slightly upon cell cultured time, but no significant difference appearing among various culture stages (P > 0. 05) . No significant difference of pacemaker current electrophysiological character, such as reverse potential, current density, active threshold and V0.5, was detected from different cell group (P>0. 05)., Conclusion: The cultured myocardium can take place of acute enzymatic isolated myocardium used to study in pacemaker current. The cells cultured no more than one week may be more suitable to this kind of electrophysiological experiment.

Spontaneous transient outward currents (STOCs) play an important role in the myogenic regulation of small artery tone, such as coronary artery. In the present study, we investigated the electrophysiological properties and the regulation of STOCs in vascular smooth muscle cells (VSMCs) of porcine coronary artery by perforated patch-clamp technique. Our data showed that STOCs were dependent on voltage and extracellular calcium and they were highly variable in amplitudes and frequencies. STOCs superimposed stochastically onto whole-cell K(+) currents induced by step and ramp protocols. STOCs were completely abolished by ChTX [inhibitor of large-conductance Ca(2+)-activated potassium (BK(Ca)) channels], removal of extracellular Ca(2+), or addition of ryanodine (50 mumol/L) respectively. In contrast, CdCl2 and verapamil, inhibitors of voltage-dependent L-type Ca(2+) channels, had little effect on STOCs. Caffeine (5 mmol/L) transiently increased STOCs (hump), followed by a temporary inhibition. Ca(2+) ionophore A23187 increased both amplitude and frequency of STOCs. Na(+) ionophore monensin increased the frequency of STOCs. STOCs were strongly inhibited by KB-R7943, a selective inhibitor of the reverse mode of the Na(+)/Ca(2+) exchanger. Based on these observations, we conclude that STOCs are mediated by BK(Ca) channels. The generation and activation of STOCs depend upon Ca(2+) influx through Na(+)/Ca(2+) exchange and release of Ca(2+) from sarcoplasmic reticulum (SR) via ryanodine receptors. This suggests that Na(+)/Ca(2+) exchange determines calcium store refilling. Recycling of entering Ca(2+) from superficial SR may locally elevate Ca(2+) concentration at the plasma membrane, thereby activating BK(Ca) channels and then initiating STOCs.

Objective: To investigate the changes of L-type Ca(2+) channel current (I(Ca-L)) and its voltage-dependent activation and inactivation in atrial myocytes of patients with atrial fibrillation (AF)., Methods: The specimens of right atrial appendage were obtained from 18 patients with normal sinus rhythm (NSR) and 12 patients with chronic atrial fibrillation (CAF). Single myocytes were isolated by enzymatic dissociation with two-step method and the ionic currents were recorded using whole-cell patch clamp techniques to detect the changes of I(Ca-L) density and kinetic properties., Results: (1) I(Ca-L) density was (-1.32 +/- 0.19) pA/pF in CAF group (n = 12) and (-4.58 +/- 0.39) pA/pF in NSR group (n = 21) at the test potential from -40 mV to 0 mV. I(Ca-L) density of CAF group was significantly reduced (P < 0.01), compared with the NSR group. (2) No significant differences were noted between the two groups in the voltage-dependent activation parameters (V(1/2), K) and inactivation parameters (V(1/2), K)., Conclusions: I(Ca-L) density of CAF group was significantly decreased whereas it's voltage-dependent kinetic properties had no change. This phenomenon may be one of the mechanisms of atrial electrophysiological remodeling in chronic atrial fibrillation.

The aim of the present study was to examine the effects of tetramethylpyrazine (TMP) on large-conductance Ca(2+)-activated potassium channels (BK(Ca) channels) in porcine coronary artery smooth muscle cells, in order to provide the experimental evidence for expounding the mechanism of TMP in dilating coronary artery. Cell-attached and inside-out single channel recording techniques were used to observe the effects of TMP on BK(Ca) channels as well as the effects after the cells were treated by protein kinase A (PKA) inhibitor or protein kinase G (PKG) inhibitor. In inside-out patch, TMP activated BK(Ca) channels by increasing open-state probability (N(Po)) and decreasing close time (Tc) in a concentration-dependent manner. TMP (0.73~8.07 mmol/L) in the bath solution increased N(Po) from (0.01+/-0.003) to (0.03+/-0.01)~(1.21+/-0.18) (P<0.01, n=10), and decreased Tc from (732.33+/-90.67) ms to (359.67+/-41.30) ~ (2.96+/-0.52) ms (P<0.01, n=10). These actions of TMP occurred even when the free Ca(2+) concentration in the bath was reduced to ~ 0 mmol/L. The specific inhibitors of PKA (H-89, 3 mumol/L) and PKG (KT-5823, 1 mumol/L) had no influence on the activation of TMP on BK(Ca) channels. These findings suggest that TMP can directly activate BK(Ca) channels in coronary artery smooth muscle, which probably is an important mechanism in dilating coronary artery.

Objective: To compare the changes of both inward rectifying K(+) (Kir) current(I(k1)) density and mRNA expression level of Kir2.1, a major subfamily of Kir in chronic human atrial fibrillation (CAF) with those in normal sinus rhythm (NSR)., Methods: I(k1) density was measured with whole-cell patch clamp technique in single myocyte isolated by an enzymatic dissociation method from right atrial appendages in patients with CAF (n = 8) and those with NSR (n = 12). The mRNA expression levels of Kir2.1 was determined in right atrial appendages from CAF (n = 19) and NSR (n = 18) by semiquantitative reverse-transcription polymerase chain reaction (RT-PCR)., Result: The average resting membrane potentials were similar between CAF and NSR (-78.95 mV +/- 4.67 mV and -70.22 mV +/- 11.08 mV, P>0.05). I(k1) density in single myocyte significantly increased at hyperpolarized potential level (-100 mV) in CAF compared to that in NSR (-9.59 pA/pF +/- 2.47 pA/pF vs. -5.58 pA/pF +/- 2.52 pA/pF, P<0.01). The mRNA level of Kir2.1 was also significantly higher in CAF than that of NSR (0.50+/-0.16 vs. 0.34+/-0.09, P<0.05)., Conclusion: The data suggest that Kir2.1 up-regulation and I(k1) current increase might contribute to the electrical remodeling in CAF patients.

D-myo-inositol 1,4,5-trisphosphate (IP(3)) plays an important role in signal transduction. It releases Ca(2+) from intracellular sites, which activates the Ca(2+)-dependent channels such as large-conductance Ca(2+)-activated potassium channels (BK channels). The present study was therefore designed to determine if the activity of BK channels in porcine coronary artery smooth muscle cells was increased by IP(3). Using the inside-out patch-clamp technique, the activity of single BK channels was recorded in porcine coronary artery smooth muscle cells. In excised inside-out membrane patches, IP(3) (10-50 micromol/L) enhanced the open probability (Po) of BK channels in a dose-dependent manner in the intracellular side of inside-out patches and its effect was almost completely abolished by washout. The open-state probability of the BK channels increased from a control level of 0.0402+/-0.0152 to 0.1365+/-0.0212 (20 micromol/L IP(3)) and 0.1865+/-0.0175 (30 micromol/L IP(3)). IP(3) decreased the mean close time markedly, but had no effect on the amplitude of BK channels. The activation of IP(3) on BK channels did not decline. The metabolite of IP(3) had no obvious effect on BK channels. This study provides evidence that IP(3) activates BK channels in porcine coronary artery smooth muscle cells in a dose-dependence manner.