Remodeling Of T-tubule Organization And The Change Of Its Relative Proteins, JP-2 And Cav-3, In Cardiac Hypertrophy Or Heart Failure Of Mice

ObjectiveCardiac hypertrophy model or heart failure model were induced by thoracic aortic banding (TAB) surgery in mice. The following experiment was carried on those models. We aimed to evaluate whether the changes of t-tubule organization and its relative proteins, JP-2 and Cav-3, had been taken place during the cardiomyopathy.Methods1 Grouping and modeling C57BL/6 male mice (8 w, 20-25 g, clean) were used in the following experiments. Mice were divided at random into sham group (n = 6) and surgery group (n = 12). The mice were anesthetized with ketamine/xylazine (40 mg/kg and 5 mg/kg, respectively) by intrapetioneal injection. A segment of surgical silk (No. 3/0) was grasped by forceps and placed under the aorta. A gauge needle (No. 27) was placed over the aorta between brachiocephalic and left common carotid arteries. The silk was tied snugly around the aorta and the needle. Then the needle was slid out from the knot and the inside diameter of aorta was constricted to the outside diameter of the needle. The sham procedure was performed to visualize the aortic arch without any ligature.2 Echocardiography Five weeks after suegery, transthoracic echocardiograms were performed with a Sonos 5500 Imager. Ketamine HCl (25 mg/kg, subcutaneous injection) was used to induce a semiconscious state in mice. 2D images were pictured in theshort- and long-axis planes of left ventricular (LV) with an 8 MHz sector-array probe, and 100 frames per second were yielded. The mice in surgery group were divided into hypertrophy group or HF group according to the echocardiography results.3 Confocal imaging of epicardial myocyte t-tubule structure on intact heart in situ Intact mouse hearts were Langendorff-perfused at room temperature with free-Ca²⁺ Tyrode's solution, containing FM 4-64 (2.5μM), a lipophilic fluorescence indicator of membrane structure, for 20-30 min. The hearts were placed in the perfusion chamber attached on the stage of a confocal microscope, and perfused with indicator free-FM4-64/free-Ca²⁺ Tyrode's solution. Ten to fifteen images of each ventricular free wall in different locations were acquired, and power values of each ventricle were averaged to represent the global t-tubule structure of each ventricle.4 Western blotting assay The left ventricles were harvested and stored at -80°C. Frozen tissues were homogenized in a lysis buffer, containing protease inhibitors. Protein concentrations were determined using the Pierce BCA assay. After electrophoresison in a 10% sodium laurylsulfate–polyacrylamide gel, proteins were transferred onto a PVDF membrane by constant currents. The membrane was blocked in fresh filled skimmed milk for 1 h at room temperature, and then incubated with primary antibodies over night at 4°C, followed by three washes in PBS-0.1% Tween 20 solution for 15 min each. The secondary antibody and HRP-GAPDH antibody were conjuncted incubation for 1.5 h at room temperature. Immunodetection of proteins by chemiluminescence (ECL kit) was followed by exposure to X-ray film. The protein bands were analyzed using Image J software.5 statistics analysis Data were expressed as mean (x|-)±s. Unpaired t-test was used to compare averages between two groups. Chi-square was used among three or more groups. P < 0.05 was considered statistically significant. Results1 Echocardiographic Assay of Cardiac Function Mice developed hypertrophy or HF about 5 weeks after TAB surgery. Hypertrophy mice significantly increased in heart weight, ratio of heart weight to body weight and LV mass, but no difference in lung weight, end-diastolic volume and ejection fraction, in compared with those of sham-operated control mice. In contrast, HF mice not only markedly increase heart weight, ratio of heart weight to body weight and LV mass, but also significantly increased lung weight and end-diastolic volume and decreased ejection fraction.2 In situ t-tubule imaging of intact heart In the LV, t-tubule disorganization started at hypertrophy stage and worse in HF stage. The population leftward shift of t-tubule power also started at hypertrophy stage. However, the right ventricular (RV) status was different from the LV status. The significant changes of RV t-tubule were only found in HF stage, which also had a leftward shift of t-tubule power.3 Western blot analysis Compared with the sham group, both JP-2 and Cav-3 were gradually decreased in the cardiac hypertrophy group and heart failure group. At heart failure stage, both JP-2 and Cav-3 were significantly decreased in the cardiac hypertrophy group and heart failure group compared with the sham group.Conclusion1 Transformations of heart morphology and function have closed relationship with t-tubule remodeling.2 T-tubule remodeling is tightly related to the down regulation of excitation-contraction proteins, JP-2 and Cav-3.