| Objective:This study investigated the effect of exercise stress on L-type calcium current(ICa,L) and the putative intracellular cascade responsible for the effects.Methods:20Wister rats( The Laboratory Animal Center Of Hebei Medical University,1502056),weight 200~250g were randomly divided into 3 groups for treatment: sedentary(without exercise), exercised to exhaustion and salubrinal percutaneously injected 30 minutes before every exhaustive exercise.Rats of the exercise group were forced to swim until exhaustion each time for 9 days with 5% body weight attached to the head. Salubrinal(1mg/kg) or an equivalent volume of placebo solution(dimethyl sulfoxide) were injected via the subcutaneous daily for the first 3 days, followed by subcutaneous injections(0.5mg/kg) daily for 9 days(starting at 30 min before exercise), control rats received placebo solution. After 1 day recovery period, we used whole cell patch-clamp technique to investigate ICa,L, sedentary rats served as controls. In addition, expressions of endoplasmic reticulum(ER)chaperone protein levels were analyzed.Myocardiocytes Isolation:Wister rats, unfractionated heparin 5000U/Kg intraperitoneal injection 20 minutes before 1% pentobarbital.When anesthesia was done. Isolated the heart of the rat. Then put it into the 4℃ tyrode solution. Tidy the root of the arterial、insert the arterial pipe and then hung it into the Langendorff system. Perfused with none calcium tyrode solution 8~10ml/min. After5~7minutesreplaced the liquide with collagenase for 20 minutes,maintain the tempreture at 37℃.As the procedure was done,put the heart into the 37℃ KB solution.Cut off the other tissues except for the left ventricular tissues. Scissor the ventricular as 2mm×2mm×2mm pieces.Sucked it to isolated as a single unit. 30 minutes still in the KB solution at room tempreture then transfer it into 4℃ for using.Measurement of the survival myocytes:1ml cardiomyocytes KB solution with one drop 0.2% Trypan blue,the death cells were become blue while the survival myocytes were bright、smooth and transparent. Survival rate(%)=survival myocytes/total cells×100%.Western blot:After being rinsed in cold PBS for 3 times, cells were homogenized in RIPA buffer, when required, the membrane fractions were prepared as described previously. Thus, the supernatant was then centrifuged at 120 000 g for 15 min at 4°C. Samples(10–20 mg) were run on SDS-PAGE gels, transferred to PVDF filter membranes, and Western blotted with monoclonal antibody against C/EBP homologous protein(CHOP), phospho-e IF2α(p-e IF2α), e IF2α and caspase-12(cell signaling technology). PVDF membranes were then incubated with HRP-conjugated antirabbit immunoglobulin G antibody(Santa Cruz Biotechnology, Inc) for 1 hour. The blot was developed with an ECL-Plus chemiluminescence reagent kit and visualized with UVP Bio-Imaging Systems. Blot densities were analyzed using Image J software.Recording techniques:Perforated and conventional(ruptured) whole-cell patch-clamp techniques were used to record the membrane potentials and ICa L in the current and voltage clamp modes, respectively. An EPC-8 patch-clamp amplifier(HEKA, Lambrecht, Germany) was used for these recordings. Fire-polished pipettes pulled from borosilicate glass capillaries(Narishige Scientific Instrument Lab., Tokyo, Japan). Micropipette resistance had a resistance of 2.0–3.5 m?, when filled with the pipette solution, and the patch-clamp experiments were performed at room temperature.Results: We discovered that exhaustive exercise triggered ER stress, demonstrated by elevated expression of phospho-e IF2α(1±0.50 Vs 2.31±0.87,P=0.04),CCAT/enhancer-binding homologous protein(CHOP)( 1±0.30 Vs 3.13±1.20,P=0.034) and caspase-12(1±0.44 Vs 2.6±0.84,P=0.014), which are markers of ER stress. When compared to controls(n=12), ICa,L current was inhibited by exhaustive exercise(n=9),(-3.80±1.26 Vs-2.24±1.11,P=0.009), this inhibitory effect was blocked by salubrinal(n=7),(-3.80±1.26 Vs-3.82±1.49,P=0.979), a selective e IF2α dephosphorylation inhibitor, was used to inhibit ER stress, suggesting that ER stress participates in the regulation of ICa,L. However, exhaustive exercise didn’t change the voltage dependence of steady-state activation and inactivation of ICa,L, and salubrinal infusion also showed no difference in voltage dependence of steady-state activation(-21.06±1.07 m V Vs-19.39±1.64 m V Vs-17.69±1.26 m V,P=0.96) and inactivation(-29.51± 0.18 m V Vs-27.01±0.40 m V Vs-27.21± 0.44 m V,P=0.98)of ICa,L.Conclusions:1 Exercise stress inhibits ICa-L.2 Exercise stress activates ER stress.3 ER stress may participate the mechanism to regulate the L-type calcium ion. |
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