1.Quantitative expression of voltage-dependent calcium channels in prostate smooth muscle cells of rats with estradiol-induced chronic nonbacterial prostatitis.
Liang ZHANG ; Chao-Zhao LIANG ; Xian-Sheng ZHANG ; Zong-Yao HAO ; Jun ZHOU ; Song FAN ; Yu LI
National Journal of Andrology 2010;16(4):295-299
OBJECTIVETo construct a rat model of chronic nonbacterial prostatitis (CP) and investigate the difference in the quantitative expression of voltage-dependent calcium channels of prostate smooth muscle cells (PSMCs) between the models and controls.
METHODSWe established a CP rat model by estrogen induction, cultured and purified the PSMCs in vitro, and extracted total RNA by Trizol. Then we measured the mRNA expression of the cal subunit in the calcium channel subtypes by reverse transcription and SYBR Green I real time RT-PCR, and compared it with that of the controls.
RESULTSThe expressions of the L-, T- and P/Q-type calcium channels were found in both the CP and control groups, and that of the CaV1.2 L-type calcium channel was significantly increased in the former as compared with the latter (0.048 +/- 0.024 versus 0.031 +/- 0.015, t = 2.846, P = 0.007), but there were no statistically significant differences in the T- and P/Q-type calcium channels between the two groups.
CONCLUSIONThe number of CaV1.2 L-type calcium channels of PSMCs and calcium influx were increased in CP patients, which may be involved in the mechanism of CP.
Animals ; Calcium Channels, L-Type ; metabolism ; Calcium Channels, Q-Type ; metabolism ; Calcium Channels, T-Type ; metabolism ; Estradiol ; pharmacology ; Male ; Myocytes, Smooth Muscle ; metabolism ; Prostate ; metabolism ; Prostatitis ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar
2.Effects of rapid atrial pacing on L-type calcium channel subunits and potassium channel Kv4.3 expressions in rabbits.
Rui-Yan MA ; Zong-Ying YANG ; Qian-Jin ZHONG
Chinese Journal of Cardiology 2007;35(12):1111-1115
OBJECTIVETo investigate rapid atrial pacing (RAP) induced atrial ultrastructural changes and mRNA and protein expression changes of L-type calcium channel subunits and potassium channel Kv4.3 in a rabbit model.
METHODSThirty-six rabbits were electrically paced at a frequency of 600 beats/min for durations ranging from 0 - 48 h via bipolar endocardial leads through surgical techniques. Ultrastructural changes of the atrium were observed through a transmission electron microscope (TEM), L-type calcium channel subunits and potassium channel Kv4.3 expressions at mRNA and protein levels were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.
RESULTSAtrial ultrastructure changes characterized by mitochondrial vacuolization, myofilament lysis, and glycogen accumulation were detected obvious at 3 h post pacing. Down-regulated mRNA expression of Ca(2+) channel beta1 and alpha1 subunits was observed 6 h post pacing, Kv4.3 mRNA down-regulation occurred 24 h post pacing, auxiliary subunit alpha2 was not affected by pacing. Protein expression of alpha1c subunit and potassium channel Kv4.3 paralleled their mRNA expression changes.
CONCLUSIONRAP induced ultrastructural changes of the atrium and down-regulated mRNA and protein expressions of L-type calcium channel subunits and potassium channel Kv4.3 occurred thereafter in response to intracellular calcium overload induced by RAP.
Animals ; Calcium Channels, L-Type ; genetics ; metabolism ; Cardiac Pacing, Artificial ; methods ; Female ; Heart Atria ; metabolism ; ultrastructure ; Male ; Patch-Clamp Techniques ; Potassium Channels ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rabbits
4.Calcium Receptor and Nitric Oxide Synthase Expression in Circular Muscle of Lower Esophagus from Patients with Achalasia.
Yang GAO ; Jun-Feng LIU ; Xin HE ; Xin-Bo LIU ; Ling-Ling ZHANG ; Lian-Mei ZHAO ; Chao ZHANG
Chinese Medical Journal 2018;131(23):2882-2885
Calcium Channels, L-Type
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genetics
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metabolism
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Esophageal Achalasia
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genetics
;
metabolism
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Esophagus
;
metabolism
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Humans
;
Nitric Oxide Synthase
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metabolism
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Nitric Oxide Synthase Type I
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genetics
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metabolism
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RNA, Messenger
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metabolism
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Real-Time Polymerase Chain Reaction
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Receptors, Calcium-Sensing
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genetics
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metabolism
5.Discovering L-type calcium channels inhibitors of antihypertensive drugs based on drug repositioning.
Ying-xi LIANG ; Yu-su HE ; Lu-di JIANG ; Qiao-xin YUE ; Shuai CUI ; Li BIN ; Xiao-tong YE ; Xiao-hua ZHANG ; Yang-ling ZHANG
China Journal of Chinese Materia Medica 2015;40(18):3650-3654
This study was amid to construct the pharmacophore model of L-type calcium channel antagonist in the application of screening Drugbank and TCMD. This paper repositions the approved drugs resulting from virtual screening and discusses the relocation-based drug discovery methods, screening antihypertensive drugs with L-type calcium channel function from TCMD. Qualitative hypotheses wre generated by HipHop separately on the basis of 12 compounds with antagonistic action on L-type calcium channel expressed in rabbit cardiac muscle. Datebase searching method was used to evaluate the generated hypotheses. The optimum hypothesis was used to search Drugbank and TCMD. This paper repositions the approved drugs and evaluates the antihypertensive effect of the chemical constituent of traditional Chinese medicine resulting from virtual screening by the matching score and literature. The results showed that optimum qualitative hypothesis is with six features, which were two hydrogen-bond acceptors, four hydrophobic groups, and the CAI value of 2.78. Screening Drugbank achieves 93 approved drugs. Screening TCMD achieves 285 chemical constituents of traditional Chinese medicine. It was concluded that the hypothesis is reliable and can be used to screen datebase. The approved drugs resulting from virtual screening, such as pravastatin, are potentially L-type calcium channels inhibitors. The chemical constituents of traditional Chinese medicine, such as Arctigenin III and Arctigenin are potentially antihypertensive drugs. It indicates that Drug Repositioning based on hypothesis is possible.
Animals
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Antihypertensive Agents
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chemistry
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pharmacology
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Calcium Channel Blockers
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chemistry
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pharmacology
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Calcium Channels, L-Type
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genetics
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metabolism
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Drug Repositioning
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methods
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Molecular Structure
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Myocardium
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metabolism
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Rabbits
6.Effects of microRNA-1 on negatively regulating L-type calcium channel beta2 subunit gene expression during cardiac hypertrophy.
Yang WU ; Peng GENG ; Yu-Qin WANG ; Yan LIU
Chinese Journal of Applied Physiology 2012;28(4):304-308
OBJECTIVETo investigate the negative regulation of microRNA-1 (miR-1) on L-type calcium channel beta2 subunit (Cavbeta 2) during cardiomyocyte hypertrophy and its mechanism.
METHODSCardiomyocyte hypertrophy was induced by isoproterenol (ISO). The cell surface area was measured by image analysis system (HJ2000). The targets of miR-1 were predicted by online database microCosm. The 3' untranslated region sequence of Cavbeta 2 was cloned into luciferase reporter vector and then transiently transfected into HEK293 cells. The luciferase activities of samples were measured to verify the expression of luciferase reporter vector. The expression of atrial natriuretic peptide (ANP), beta-myosin heavy chain (beta-MHC), miR-1 and the Cavbeta 2 mRNA were detected by qRT-PCR. The protein expression of Cavbeta 2 was detected by Western blot. The level of miR-1 was up-regulated by miR-1 mimic transfection and the expression level of Cavbeta 2 was down-regulated by RNAi, then effects of which on cardiomyocyte hypertrophy were investigated.
RESULTS(1) The expression of miR-1 was significantly reduced in cardiomyocyte hypertrophy. Upregulating the miR-1 level could suppress the increase of cell surface area, the expression of ANP and beta-MHC mRNA (P < 0.05). (2) Cavbeta 2 was the one of potential targets of miR-1 by prediction using online database microCosm. The luciferase activities of HEK293 cells with the plasmid containing miR-1 and wide type Cavbeta 3' UTR sequence was significantly decreased when compared with that of control group (P < 0.01). Up-regulation of the miR-1 level could suppress the protein expression of Cavbeta 2. (3) The expression of Cavbeta 2 was significantly increased in cardiomyocyte hypertrophy induced by ISO. Downregulation of Cavbeta by RNAi could markedly inhibit the increase of cell surface area, the expression of ANP and beta-MHC mRNA.
CONCLUSIONCavbeta2 is one of potential targets of miR-1 by bioinformatics prediction. The experiment data confirms that Cavbeta2 is truly the target of miR-1. MiR-1 can negatively regulate the expression of Cavbeta 2, resulting in the decrease of intracellular Ca2+ content and the attenuation of cardiomyocyte hypertrophy.
Animals ; Atrial Natriuretic Factor ; metabolism ; Calcium Channels, L-Type ; genetics ; Cardiomegaly ; genetics ; Gene Expression Regulation ; HEK293 Cells ; Humans ; MicroRNAs ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection ; Ventricular Myosins ; metabolism
7.TRPC3 cation channel plays an important role in proliferation and differentiation of skeletal muscle myoblasts.
Jin Seok WOO ; Chung Hyun CHO ; Do Han KIM ; Eun Hui LEE
Experimental & Molecular Medicine 2010;42(9):614-627
During membrane depolarization associated with skeletal excitation-contraction (EC) coupling, dihydropyridine receptor [DHPR, a L-type Ca2+ channel in the transverse (t)-tubule membrane] undergoes conformational changes that are transmitted to ryanodine receptor 1 [RyR1, an internal Ca2+-release channel in the sarcoplasmic reticulum (SR) membrane] causing Ca2+ release from the SR. Canonical-type transient receptor potential cation channel 3 (TRPC3), an extracellular Ca2+-entry channel in the t-tubule and plasma membrane, is required for full-gain of skeletal EC coupling. To examine additional role(s) for TRPC3 in skeletal muscle other than mediation of EC coupling, in the present study, we created a stable myoblast line with reduced TRPC3 expression and without alpha1SDHPR (MDG/TRPC3 KD myoblast) by knock-down of TRPC3 in alpha1SDHPR-null muscular dysgenic (MDG) myoblasts using retrovirus-delivered small interference RNAs in order to eliminate any DHPR-associated EC coupling-related events. Unlike wild-type or alpha1SDHPR-null MDG myoblasts, MDG/TRPC3 KD myoblasts exhibited dramatic changes in cellular morphology (e.g., unusual expansion of both cell volume and the plasma membrane, and multi-nuclei) and failed to differentiate into myotubes possibly due to increased Ca2+ content in the SR. These results suggest that TRPC3 plays an important role in the maintenance of skeletal muscle myoblasts and myotubes.
Animals
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Calcium/metabolism
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Calcium Channels/metabolism
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Calcium Channels, L-Type/genetics/metabolism
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Cations/metabolism
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*Cell Differentiation
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*Cell Proliferation
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Cells, Cultured
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Excitation Contraction Coupling
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Gene Knockdown Techniques
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Membrane Potentials
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Mice
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Muscle Fibers, Skeletal/*metabolism
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Muscle Proteins/metabolism
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Myoblasts, Skeletal/*metabolism
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Ryanodine Receptor Calcium Release Channel/metabolism
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Sarcoplasmic Reticulum/*physiology
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Synaptophysin/metabolism
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TRPC Cation Channels/genetics/*metabolism
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Transient Receptor Potential Channels/metabolism
8.Effect of chronic electrical stimulation of phrenic nerve at different frequencies on mRNA and protein expression of skeletal DHPR(alpha1) and RyRs in the diaphragm muscle of rabbits.
Sui-Yang ZHANG ; Gang LIU ; Dong-Lin WANG ; Xian-Jian GUO ; Gui-Sheng QIAN
Acta Physiologica Sinica 2004;56(2):198-203
The mRNA and protein expression of skeletal dihydropyridine receptor isoform alpha1 subunit (DHPR(alpha1)) and ryanodine receptor(1-3) (RyR(1-3)) during chronic electrical stimulation (CES) of phrenic nerve have rarely been explored. In the present study, we explored the signal translation mode of calcium release unit in diaphragm muscle of rabbits after CES. Thirty rabbits were used and randomly divided into the normal, 10, 20, 50 and 100 Hz groups. Phrenic nerve was continuously (5 weeks, 2x 2 h/d) stimulated at 10, 20, 50 and 100 Hz respectively (impulse width 0.2 ms, 3~6 waves/time, 45 times/min, 10~20 V). Reverse transcription PCR and immunohistochemical methods were employed. The results showed that mRNA and protein expressions of DHPR(alpha1) and RyR(1) in 10 and 20 Hz groups were more significantly lower than those in the control group (P<0.01), but mRNA and protein expressions of DHPR(alpha1) and RyR(1) were significantly higher in 50 and 100 Hz groups than those in the control group (P<0.01); a lower level of mRNA expression of RyR(2) was found in 10 and 20 Hz groups. It is suggested that the calcium release unit and the signal transduction mode between DHPR and RyRs were altered from conformational changes of linked proteins to Ca(2+)-induced Ca(2+) release (CICR) in the diaphragmatic muscle of rabbits after chronic low-frequency electrical stimulation of phrenic nerve for 5 weeks.
Animals
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Calcium
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metabolism
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Calcium Channels, L-Type
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biosynthesis
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genetics
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Diaphragm
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metabolism
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physiology
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Electric Stimulation
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Female
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Male
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Muscle, Skeletal
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metabolism
;
physiology
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Phrenic Nerve
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metabolism
;
physiology
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RNA, Messenger
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biosynthesis
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genetics
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Rabbits
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Random Allocation
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Ryanodine Receptor Calcium Release Channel
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biosynthesis
;
genetics
9.Transient receptor potential A1 is involved in cold-induced contraction in the isolated rat colon smooth muscle.
Yang DONG ; Hai-Lian SHI ; Jian-Rong SHI ; Da-Zheng WU
Acta Physiologica Sinica 2010;62(4):349-356
Transient receptor potential (TRP) A1, a member of TRP channel family, is activated by noxious cold. The aims of this study were to determine if TRPA1 contributed to cold-induced contractions in the isolated rat colon preparations and explore the potential mechanisms. The colon smooth muscle layers were surgically isolated from the male Wistar rats and changes in isotonic tension of longitudinal muscle under various treatments were recorded as colonic motilities. Cold stimuli were obtained by the reperfusion with Krebs-Henseleit solution at given temperature using Constant Flow Pump. The mRNA expressions of TRPA1, TRPV1 and TRPM8 in rat colon smooth muscle layer were examined by using reverse transcription-polymerase chain reaction (RT-PCR) techniques. The results showed that the contractions induced by cold stimuli (from 37 degrees C to 12 degrees C stepwise) were inversely proportional to the temperature with a maximum contraction at 17 degrees C in both proximal and distal colons (P<0.01). RT-PCR analysis revealed the expression of TRPA1, but not TRPM8 and TRPV1, in the rat proximal and distal colon smooth muscle layers. Cold-induced colonic contractions were specially inhibited by TRPA1 blocker, ruthenium red (30 μmol/L), in the proximal and distal colon (P<0.05). The cold-induced contractions of proximal (P<0.01, P<0.05) and distal colons (both P<0.001) were almost abolished or inhibited by the pretreatments of TRPA1 agonists, Allyl isothiocyanate (AITC, 300 μmol/L) and cinnamaldehyde (CA, 1 mmol/L). Extracellular calcium removal (EGTA, 1 mmol/L), PLC blocker (U73122, 10 μmol/L) and IP(3) receptor blocker (2-aminoethoxydiphenyl borate, 2-APB, 30 μmol/L) all decreased the contractions evoked by the cooling at 17 degrees C in the proximal and distal colon (P<0.001, P<0.05, P<0.001). Atropine (1 μmol/L) had no effects on these contractions. L-type Ca(2+) channels blocker nifedipine (1 μmol/L) and neurotoxin tetrodotoxin (TTX, 2 μmol/L) decreased the contractile response in the distal colon (P<0.01, P<0.05), but not in the proximal colon. In conclusion, TRPA1 contributes to cold-induced contractions of the rat colon smooth muscle, and the mechanism of TRPA1 activation involves PLC/IP(3)/Ca(2+) pathway. L-type Ca(2+) channel and neurogenic mechanism other than muscarinic receptor might be partially involved in cold-induced contraction of the distal colon, which probably resulted in higher contraction of distal colon compared with that of proximal colon.
Animals
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Calcium Channels, L-Type
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metabolism
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Cold Temperature
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Colon
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metabolism
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physiology
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In Vitro Techniques
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Male
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Muscle Contraction
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physiology
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Muscle, Smooth
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metabolism
;
physiology
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Physical Stimulation
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RNA, Messenger
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genetics
;
metabolism
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Rats
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Rats, Wistar
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TRPA1 Cation Channel
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TRPC Cation Channels
;
genetics
;
metabolism
10.Reduced Ca2+ current in rat cardiomyocytes transfected with troponin I R145W mutation gene.
Heng-Fang WU ; Xiang-Jian CHEN ; Di YANG
Chinese Journal of Cardiology 2007;35(11):1000-1004
OBJECTIVETo investigate the effects of cardiac troponin I R145W mutation, detected in Chinese patients with hypertrophic cardiomyopathy, on Ca(2+) current modulation.
METHODSR146W mutation (resemble R145W in human) was introduced into rat cardiac troponin I cDNA by site-directed mutagenesis. With EGFP as a reporter gene, replication-defective adenovirus containing the wild or mutant cTnI gene was constructed. Adult rat cardiomyocytes, were isolated by Langendorff perfusion and cultured with serum-free medium and transduced with the recombinant adenoviruses. Western blot was used to determine the recombinant proteins. Whole cell patch clamp was employed to record L-type Ca(2+) currents on cultured myocytes. Intracellular free Ca(2+) and caffeine-induced sarcoplasmic reticulum (SR) Ca(2+) release were determined after the cells incubated with Fura-2/AM.
RESULTSDNA sequencing confirmed that R146W mutation was generated in rat cTnI cDNA. Bright green fluorescence was observed in the cultured cardiomyocytes at 48 h after transduction. The recombinant proteins could be identified with cTnI or GFP monoclonal antibody. The peak current of L-type Ca(2+) channel in cells transduced with cTnI R146W was significantly decreased compared to control cells and cells transfected with wild cTnI. Intracellular free Ca(2+) concentrations and caffeine-induced SR Ca(2+) release determined by Fura-2/AM were similar among various cells.
CONCLUSIONReduced peak current of L-type Ca(2+) channel in cells transduced with cTnI R146W might contribute to the disease-causing mechanism of this mutation in patients with hypertrophic cardiomyopathy.
Animals ; Calcium ; metabolism ; Calcium Channels, L-Type ; metabolism ; Cardiomyopathy, Hypertrophic ; genetics ; metabolism ; physiopathology ; Cells, Cultured ; Female ; Mutagenesis, Site-Directed ; Mutation ; Myocytes, Cardiac ; metabolism ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Transfection ; Troponin I ; genetics