Immunohistochemistry Hearts from young and aged mice were co

Immunohistochemistry: Hearts from young and aged mice were collected at the indicated times, fixed overnight in 10% formalin, and embedded in paraffin. Serial 5-μm heart sections from each group were analyzed. Samples were stained with H&E for routine histologic examination. Immunohistochemical staining was performed as described previously [20]. The histological sections were stained with primary buy something online against Mac-2 (1:200, Abcam) at 4 °C overnight. The bound antibodies were labeled using a second antibody (Vectastain ABC Kit, VECTOR Laboratories, Inc., Burlingame, CA). Images were captured using a Zeiss Axioimager Motorized fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Isolation of murine cardiomyocytes: Mice were given 100 units of heparin i.p. (Sagent Pharmaceuticals, Schaumburg, IL) for anticoagulation before anaesthetized with 100 mg/kg sodium pentobarbital i.p. (Sigma, St. Louis, MO). The heart was excised and fastened onto the cardiomyocyte perfusion apparatus (Radnoti, Monrovia, CA) and perfusion was initiated in the Langendorff mode. Hearts were perfused at 37 °C with a Ca2+-free Krebs-Henseleit based buffer (pH 7.3) containing: 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 10 mM HEPES, 14.7 mM KCl, 1.7 mM MgSO4, 120.3 mM NaCl, 4.6 mM NaHCO3, 30 mM taurine, 10 mM glucose, and 10 mM 2,3-butanedione monoxime that was bubbled with 95% O2/5% CO2. After a few minutes of stabilization, the heart was then digested with the same perfusion buffer containing 0.067 mg/mL Liberase Blendzyme 4 (Roche, Indianapolis, IN). After digestion, the heart was removed and minced. Extracellular Ca2+ was added back to the cells to reach a final concentration of 1 mM. Cell shortening/relengthening: The mechanical properties of cardiomyocytes were assessed by using a SoftEdge MyoCam system (IonOptix Corporation, Milton, MA) [21]. Cardiomyocytes were placed in a chamber and stimulated with a suprathreshold voltage at a frequency of 0.5 Hz. IonOptix SoftEdge software was used to capture changes in sarcomere length during shortening and re-lengthening. Cell shortening and re-lengthening were assessed using the following indices: peak shortening (PS), the amplitude myocytes shortened on electrical stimulation, which is indicative of peak ventricular contractility; time-to-90% relengthening (TR90), the duration of myocytes to reach 90% relengthening, an indicative of diastolic duration; and maximal velocities of shortening and re-lengthening (±dL/dt); time to peak shortening (TPS). Intracellular Cafluorescence measurement: Intracellular Ca2+ was measured by using a dual-excitation, single emission photomultiplier system (IonOptix). Cardiomyocytes were loaded with fura 2-AM(2 μM) and were exposed to light emitted by a 75 W halogen lamp through either a 340- or 380-nm filter while being stimulated to contract at a frequency of 0.5 Hz. Fluorescence emissions were then detected [21]. Western blot analysis: The protein concentrations of all samples were measured by using Bradford dye-binding method (Dye Reagent Concentrate, Bio-Rad Protein Assay). Proteins from the heart were separated by SDS–PAGE, transferred to nitrocellulose membranes (Millipore, Bedford, MA), and probed with primary antibodies against p-AMPK, p-ACC, PP2A, PP2Cα, p62, atg5, LC3 I/II, p-mTOR, p-S6 and followed by incubation with horseradish peroxidase (HRP)-coupled anti-rabbit secondary antibody (Cell Signaling Technology Inc, Beverly, MA). Blue x-ray film (Phenix, Candler, NC) was used for photon detection and image development. Films were scanned with the Bio-Rad GS-700 scanner in the core facility of the SMBS and the relative density of the bands on the film was determined by Image J software. Statistical analysis: Data were Mean ± SEM and analyzed by using GraphPad Prism 7.0 statistical analysis software. Difference was assessed using analysis of variance (ANOVA) followed by a Tukey's post hoc test or Student's t-test. A p value < 0.05 was considered statistically significant.