Computational protocol: Acute exhaustive aerobic exercise training impair cardiomyocyte function and calcium handling in Sprague-Dawley rats

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Protocol publication

[…] Samples were prepared with LDS protein loading buffer and 0.2 M 1,4-dithiothreitol and heated for 10 min at 70°C. Equal amounts of protein were loaded onto each well of NuPage Novex Bis-Tris 10% gels (Life Technologies, Carlsbad, CA, USA). Chameleon Duo Pre-Stained Protein Ladder (LI-COR Biosciences, Lincoln, NE, USA) was used as a standard. Gels were run in BOLT MES Running buffer (Life Technologies, Carlsbad, CA, USA) for 80 minutes at 150 V, 4°C. Proteins were then transferred onto a methanol activated Immobilon-FL PVDF membranes (Merck Millipore Ltd., Carrigtwohill, Ireland) at 20 V, 90 minutes, 4°C, in BOLT Transfer buffer (Life Technologies, Carlsbad, CA, USA). After the transfer, membranes were washed, dehydrated and cut according to the protein ladder to separate SERCA2a, β-tubulin and phospholamban with its phosphorylated states (serine 16 and threonine 17).Membranes were rehydrated and incubated in Odyssey TBS Blocking buffer (LI-COR Biosciences, Lincoln, NE, USA) for 1 hour at room temperature prior to incubation with primary antibodies diluted in Odyssey TBS Blocking buffer supplemented with 0.2% Tween over night at 4°C, constant shaking. Primary antibodies used were commercially available and included polyclonal rabbit anti-SERCA2a (#A010-20) 1:10,000, monoclonal mouse anti-phospholamban (#A010-14) 1:5,000, polyclonal rabbit anti-serine 16 phosphorylated phospholamban (pPLB Ser16; #A010-12) 1:2,500, and polyclonal rabbit anti-threonine 17 phosphorylated phospholamban (pPLB Thr17; #A010-13) 1:2,500, all from Badrilla Ltd. (Leeds, UK). Polyclonal anti-β-tubulin (#AB6046, Abcam, Cambridge, UK) 1:10,000 was used to measure protein expression levels of β-tubulin as a loading control.Membranes were washed in TBS Tween buffer before incubation with fluorescent dye-conjugated secondary antibodies for 1 hour at room temperature, constant shaking. Secondary antibodies: Donkey anti-rabbit IRDye 680LT 1:40,000 and goat anti-mouse IRDye 800CW 1:15,000 (both from LI-COR Biosciences, Lincoln, NE, USA), diluted in Odyssey TBS Blocking buffer supplemented with 0.2% Tween and 0.01% SDS. Membranes were washed in TBS Tween, followed by TBS, air dried and scanned using the Odyssey FC Imaging system (LI-COR Biosciences, Lincoln, NE, USA). Protein bands were analyzed using Image Studio Ver3.1 (LI-COR Biosciences, Lincoln, NE, USA). For PLB, pPLB Ser16 and pPLB Thr17 the monomer band was quantified. Band signals were corrected for local background and SERCA, PLB, pPLB Ser16 and pPLB Thr17 signals were normalized to β-tubulin and a common sample that was run on all gels. Samples were analyzed with four technical replicates. Normalized signals were converted to fold change for exhaustively exercised rats compared to sedentary controls. [...] Statistics were performed using IBM SPSS Statistics 21 (IBM Corporation, Armonk, NY). All data were tested with Shapiro-Wilk test of normality. Measures of contractility, Ca2+ handling and mitochondrial respiration of RV and LV were compared between exhaustively exercised animals and control animals using Levene’s test of equality of variances followed by independent Student’s t-tests. Measurements of protein expression levels of SERCA2a, PLB, pPLB Ser16 and pPLB Thr17 in RV and LV were compared between exhaustively exercised and sedentary control animals. Fold change data were Log2 transformed prior to statistical testing with independent samples Kruskal-Wallis test. P ≤ 0.05 was considered statistically significant. Data are presented as means ± SD. […]

Pipeline specifications

Software tools Image Studio, SPSS
Applications Miscellaneous, DNA fingerprinting
Organisms Rattus norvegicus, Homo sapiens
Diseases Heart Diseases
Chemicals Calcium