• Title, Summary, Keyword: Myocytes

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Endothelin Receptor Overexpression Alters Diastolic Function in Cultured Rat Ventricular Myocytes

  • Kang, Mi-Suk;Walker, Jeffery W.;Chung, Ka-Young
    • Biomolecules & Therapeutics
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    • v.20 no.4
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    • pp.386-392
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    • 2012
  • The endothelin (ET) signaling pathway controls many physiological processes in myocardium and often becomes upregulated in heart diseases. The aim of the present study was to investigate the effects of ET receptor upregulation on the contractile function of adult ventricular myocytes. Primary cultured adult rat ventricular myocytes were used as a model system of ET receptor overexpression in the heart. Endothelin receptor type A ($ET_A$) or type B ($ET_B$) was overexpressed by Adenoviral infection, and the twitch responses of infected ventricular myocytes were measured after ET-1 stimulation. Overexpression of $ET_A$ exaggerated positive inotropic effect (PIE) and diastolic shortening of ET-1, and induced a new twitch response including twitch broadening. On the contrary, overexpression of $ET_B$ increased PIE of ET-1, but did not affect other two twitch responses. Control myocytes expressing endogenous receptors showed a parallel increase in twitch amplitude and systolic $Ca^{2+}$ in response to ET-1. However, intracellular $Ca^{2+}$ did not change in proportion to the changes in contractility in myocytes overexpressing $ET_A$. Overexpression of $ET_A$ enhanced both systolic and diastolic contractility without parallel changes in $Ca^{2+}$. Differential regulation of this nature indicates that upregulation of $ET_A$ may contribute to diastolic myocardial dysfunction by selectively targeting myofilament proteins that regulate resting cell length, twitch duration and responsiveness to prevailing $Ca^{2+}$.

Depression of L-type $Ca^{2+}$ and Transient Outward $K^+$ Currents in Endotoxin-treated Rat Cardiac

  • Park, Kyu-Sang;Lee, Boo-Soo;Kong, In-Deok;Lee, Joong-Woo
    • The Korean Journal of Physiology and Pharmacology
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    • v.3 no.6
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    • pp.623-630
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    • 1999
  • Decreased cardiac contractility occurs in endotoxicosis, but little is known about the ionic mechanism responsible for myocardial dysfunction. In this study, we examined the changes in $Ca{2+}$ and $K^+$ currents in cardiac myocytes from endotoxin-treated rat. Ventricular myocytes were isolated from normal and endotoxemic rats (ex vivo), that were treated for 10 hours with Salmonella enteritidis lipopolysaccharides (LPS; 1.5 mg/kg) intravenously. Normal cardiac myocytes were also incubated for 6 hours with 200 ng/ml LPS (in vitro). L-type $Ca{2+}$ current $(I_{Ca,L})$ and transient outward $K^+$ current $(I_{to})$ were measured using whole cell patch clamp techniques. Peak $I_{Ca,L}$ was reduced in endotoxemic myocytes (ex vivo; 6.00.4 pA/pF, P<0.01) compared to normal myocytes (control; 10.90.6 pA/pF). Exposure to endotoxin in vitro also attenuated $I_{Ca,L}$ (8.40.4 pA/pF, P<0.01). The amplitude of $(I_{to})$ on depolarization to 60 mV was reduced in endotoxin treated myocytes (16.51.5 pA/pF, P<0.01, ex vivo; 20.00.9 pA/pF, P<0.01 , in vitro) compared to normal myocytes (control; 24.71.0 pA/pF). There was no voltage shift in steady-state inactivation of $I_{Ca,L}$ and $(I_{to})$ between groups. These results suggest that endotoxin reduces $Ca{2+}$ and $K^+$ currents of rat cardiac myocytes, which may lead to cardiac dysfunction.

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Knock-down of Type 2 Inositol 1,4,5-Trisphosphate Receptors using Adenovirus in Adult Ventricular Myocytes (아데노바이러스를 이용한 성체 심실 근세포 이노시톨 1,4,5-삼인산 수용체 제 2 아형의 발현 억제)

  • Son, Min-Jeong;Subedi, Krishna P.;Woo, Sun-Hee
    • YAKHAK HOEJI
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    • v.54 no.1
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    • pp.8-12
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    • 2010
  • Inositol 1,4,5-trisphosphate ($IP_3$) receptor ($IP_3R$)-mediated signaling pathway is involved in many cellular processes including fertilization, apoptosis and neuronal function. Although cardiac myocytes express the $IP_3R$, its pathophysiological role has not been clearly understood because of limited selectivity of currently available pharmacological blockers. In the present study we constructed shRNA-expressing adenovirus to knock-down the type 2 $IP_3R$ ($IP_3R2$), a major subtype in cardiac ventricular myocytes, and demonstrated that the virus successfully eliminated the expression and localization of the $IP_3R2$. These results may provide a reliable tool for probing pathophysiological roles of the $IP_3R2$ in isolated intact cardiac myocytes.

Decrease of Intracellular pH and Activation of $Na^+-H^+$ Exchanger by Fluid Pressure in Rat Ventricular Myocytes (유체 압력에 의한 흰쥐 심실근세포 pH의 감소 및 $Na^+-H^+$ 교환체의 활성화)

  • Kim, Joon-Chul;Woo, Sun-Hee
    • YAKHAK HOEJI
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    • v.55 no.3
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    • pp.247-250
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    • 2011
  • An increase in ventricular pressure can alter cardiac excitation and contraction. Recent report has demonstrated that fluid pressure (FP) suppresses L-type $Ca^{2+}$ current with acceleration of the current inactivation in ventricular myocytes. Since the L-type $Ca^{2+}$ channels known to be regulated by intracellular pH ($pH_i$), this study was designed to explore whether pressurized fluid flow affects pHi in isolated rat ventricular myocytes. A flow of pressurized (~16 dyne/$cm^2$) fluid, identical to that bathing the myocytes, was applied onto single myocytes, and intracellular $H^+$ concentration was monitored using confocal $H^+$ imaging. FP significantly decreased $pH_i$ by $0.07{\pm}0.01$ pH units (n=16, P<0.01). Intracellular acidosis enhances the activity of $Na^+-H^+$ exchanger (NHE). Therefore, we examined if the NHE activity is increased by FP using the NHE inhibitor, HOE642. Although HOE642 did not alter $pH_i$ in control conditions, it decreased $pH_i$ in cells pre-exposed to FP, suggesting enhancement of NHE activity by FP. In addition, FP-induced intracellular acidosis was larger in cells pre-treated with HOE642 than in cells under the control conditions. These results suggest that FP induces intracellular acidosis and that NHE may contribute to extrude $H^+$ during the FP-induced acidosis in rat ventricular myocytes.

Increase of L-type Calcium Current by cGMP-dependent Protein Kinase Regulates in Rabbit Ventricular Myocytes

  • Han, Jin;Kim, Na-Ri;Kim, Eui-Yong;Ho, Won-Kyung;Earm, Yung-E;Kim, Han-Kyoun
    • The Korean Journal of Physiology and Pharmacology
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    • v.2 no.6
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    • pp.733-742
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    • 1998
  • Background: We have previously reported that not only cGMP but also 8-Br-cGMP or 8-pCPT-cGMP, specific and potent stimulators of cGMP-dependent protein kinase (cGMP-PK), increased basal L-type calcium current $(I_{Ca})$ in rabbit ventricular myocytes. Our findings in rabbit ventricular myocytes were entirely different from the earlier findings in different species, suggesting that the activation of cGMP-PK is involved in the facilitation of $I_{Ca}}$ by cGMP. However, there is no direct evidence that cGMP-PK can stimulate $I_{Ca}}$ in rabbit ventricular myocytes. In this report, we focused on the direct effect of cGMP-PK on $I_{Ca}}$ in rabbit ventricular myocytes. Methods and Results: We isolated single ventricular myocytes of rabbit hearts by using enzymatic dissociation. Regulation of $I_{Ca}}$ by cGMP-PK was investigated in rabbit ventricular myocytes using whole-cell voltage clamp method. $I_{Ca}}$ was elicited by a depolarizing pulse to +10 mV from a holding potential of -40 mV. Extracellular 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP), potent stimulator of cGMP-dependent protein kinase (cGMP-PK), increased basal $I_{Ca}}$. cGMP-PK also increased basal $I_{Ca}}$. The stimulation of basal $I_{Ca}}$ by cGMP-PK required both 8-Br-cGMP in low concentration and intracellular ATP to be present. The stimulation of basal $I_{Ca}}$ by cGMP-PK was blocked by heat inactivation of the cGMP-PK and by bath application of 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer (Rp-pCPT-cGMP), a phosphodiesterase-resistant cGMP-PK inhibitor. When $I_{Ca}}$ was increased by internal application of cGMP-PK, IBMX resulted in an additional stimulation of $I_{Ca}}$. In the presence of cGMP-PK, already increased $I_{Ca}}$ was potentiated by bath application of isoprenaline or forskolin or intracellular application of cAMP. Conclusions: We present evidence that cGMP-PK stimulated basal $I_{Ca}}$ by a direct phosphorylation of L-type calcium channel or associated regulatory protein in rabbit ventricular myocytes.

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Stretch-activated $K^+$ Channels in Rat Atrial Myocytes

  • Youm, Jae-Boum
    • The Korean Journal of Physiology and Pharmacology
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    • v.7 no.6
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    • pp.341-348
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    • 2003
  • Mechanical stimuli to the cardiac myocytes initiate many biochemical and physiological events. Stretch-activated cation channels have been suggested to mediate these events. In this study, cell-attached and inside-out excised-patch clamp methods were used to identify stretch-activated cation channels in adult rat atrial myocytes. Channel openings were increased in cell-attached configuration when negative pressure was applied to the pipette, and also in inside-out excised patches by negative pressure. The channel was not permeable to $Cl^-$, $Na^+$ and $Cs^+$, but selectively permeable to $K^+$, and the degree of activation was dependent on the magnitude of negative pressure (full activation at ${\sim} -50 mmHg). In symmetrical 140 mM KCl, the slope conductance was $51.2{\pm}3$ pS between the potentials of -80 and 0 mV and $55{\pm}6$ pS between 0 and +80 mV (n=5). Glibenclamide ($100{mu}M$) or ATP (2 mM) failed to block the channel openings, indicating that it is not ATP-sensitive $K^+$ channel. Arachidonic acid ($30{mu}M$), which has been shown to activate a $K^+$ channel cooperatively with membrane stretch, did not affect the channel activity. $GdCl_3$ ($100{mu}M$) also did not alter the activity. These results demonstrate that the mechanical stretch in rat atrial myocytes activates a novel $K^+$-selective cation channel, which is not associated with other $K^+$ channels such as ATP-sensitive and arachidonic acid-activated $K^+$ channel.

Effect of Fluid Pressure on L-type $Ca^{2+}$ Current in Rat Ventricular Myocytes (백서 심실 근세포 L형 $Ca^{2+}$ 전류에 대한 유체압력의 효과)

  • Lee Sun-Woo;Woo Sun-Hee
    • YAKHAK HOEJI
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    • v.50 no.2
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    • pp.111-117
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    • 2006
  • Cardiac chambers serve as mechanosensory systems during the haemodynamic or mechanical disturbances. To examine a possible role of fluid pressure (FP) in the regulatien of atrial $Ca^{2+}$ signaling we investigated the effect of FP on L-type $Ca^{2+}$ current $(I_{Ca})$ in rat ventricular myocytes using whole-cell patch-clamp technique. FP $(\sim40cm\;H_2O)$ was applied to whole area of single myocytes with electronically controlled micro-jet system. FP suppressed the magnitude of peak $I_{Ca}$ by $\cong25\%$ at 0 mV without changing voltage dependence of the current-voltage relationship. FP significantly accelerated slow component in inactivation of $I_{Ca}$, but not its fast component. Analysis of steady-state inactivation curve revealed a reduction of the number of $Ca^{2+}$ channels available for activity in the presence of FP. Dialysis of myocytes with high concentration of immobile $Ca^{2+}$ buffer partially attenuated the FP-induced suppression of $I_{Ca}$. In addition, the intracellular $Ca^{2+}$ buttering abolished the FP-induced acceleration of slow component in $I_{Ca}$ inactivation. These results indicate that FP sup-presses $Ca^{2+}$ currents, in part, by increasing cytosolic $Ca^{2+}$ concentration.

Involvement of the Phospholipase C β1 Pathway in Desensitization of the Carbachol-activated Nonselective Cationic Current in Murine Gastric Myocytes

  • Kim, Byung Joo;So, Insuk;Kim, Ki Whan
    • Molecules and Cells
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    • v.22 no.1
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    • pp.65-69
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    • 2006
  • In murine gastrointestinal myocytes muscarinic stimulation activates nonselective cation channels via a G-protein and $Ca^{2+}$-dependent pathway. We recorded inward cationic currents following application of carbachol ($I_{CCh}$) to murine gastric myocytes held at -60 mV, using the whole-cell patch-clamp method. The properties of the inward cationic currents were similar to those of the nonselective cation channels activated by muscarinic stimulation in other gastrointestinal smooth muscle cells. CCh-induced $I_{CCh}$ and spontaneous decay of $I_{CCh}$ (desensitization of $I_{CCh}$) occurred. Unlike the situation in guinea pig gastric myocytes, desensitization was not affected by varying $[EGTA]_i$. Pretreatment with the PLC inhibitor (U73122) blocked the activation of $I_{CCh}$, and desensitization of $I_{CCh}$ was attenuated in PLC ${\beta}_1$ knock-out mice. These results suggest that the desensitization of $I_{CCh}$ in murine gastric myocytes is not due to a pathway dependent on intracellular $Ca^{2+}$ but to the PLC ${\beta}_1$ pathway.

Modulation of Subcellular Ca2+ Signal by Fluid Pressure in Rat Atrial Myocytes

  • Woo Sun-Hee;Morad Martin
    • Biomolecules & Therapeutics
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    • v.14 no.1
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    • pp.19-24
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    • 2006
  • Atrial chambers serve as mechanosensory systems during the haemodynamic or mechanical disturbances, which initiates arrhythmia. Atrial myocytes, lacking t-tubules, have two functionally separate sarcoplasmic reticulums (SRs): those at the periphery close to the surface membrane, and those at the cell interior (center) not associated with the membrane. To explore possible role of fluid pressure (FP) in the regulation of atrial local $Ca^{2+}$ signaling we investigated the effect of FP on subcellular $Ca^{2+}$ signals in isolated rat atrial myocytes using confocal microscopy. FP was applied to whole area of single myocyte with pressurized automatic micro-jet (200-400 $mmH_2O$) positioned close to the cell. Application of FP enhanced spontaneous occurrences of peripheral and central $Ca^{2+}$ sparks with larger effects on the peripheral release sites. Unitary properties of single sparks were not altered by FP. Exposure to higher FP often triggered longitudinal $Ca^{2+}$ wave. These results suggest that fluid pressure may directly alter excitability of atrial myocytes by activating $Ca^{2+}$-dependent ionic conductance in the peripheral membrane and by enhancing spontaneous activation of central myofilaments.

Background Non-Selective Cation Channels in Rat Atrial Myocytes

  • Youm, Jae-Boum;Zhang, Yin-Hua;Ho, Won-Kyung;Earm, Yung-E
    • Proceedings of the Korean Biophysical Society Conference
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    • pp.38-38
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    • 1998
  • Resting membrane potential of atrial myocytes is less negative than K+ equilibrium potential, suggesting the presence of ion channels carrying inward currents. We investigated the background Na$\^$+/ current in rat atrial myocytes using both conventional whole cell voltage clamp technique and single channel recording.(omitted)

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