Organisms are constantly exposed to the influence of adverse environmental factors

der of events is still largely unknown. Previously, we associated CnA activation with reduced expression of Cx43 and NaV1.5 and with increased susceptibility for polymorphic ventricular tachyarrhythmias in 4-week old hypertrophic MHC-CnA hearts. This model therefore may serve as an accelerated model for the pathophysiological changes seen in heart failure patients. 1 Cardiac Remodeling in CnA-Induced Hypertrophy In the current study, we have exploited the ventricles of this mouse model further to investigate PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19650784 calcineurin-dependent changes in three key conductional parameters during postnatal development, in order to visualize the order of adverse events ultimately leading to arrhythmias. fibrosis was calculated as a percentage of the area of each image. Immunoblotting Total cellular protein lysates were isolated from the ventricles and analyzed by immunoblotting as described before. Briefly, 20 mg of protein lysate was separated by 7% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently electrotransferred onto a nitrocellulose membrane. Equal loading of protein was assessed by Ponceau S staining. Proteins were detected after incubation with specific primary and 2883-98-9 biological activity secondary antibodies using a standard ECL procedure. Protein levels were expressed as the ratio of protein of interest/correspondent Ponceau S staining and both were quantified using Image Lab 3.0.1 software. Materials and Methods Ethics Statement The experimental protocol was performed in accordance with the national guidelines and approved by the local Ethical Animal Experimental Committee of the University of Utrecht. All efforts were made to minimize suffering. Animals Male and female MHC-CnA mice, kindly provided by Dr. E. Olson, were compared with age-matched WT littermates at week 0, 1, 2, 3 and 4 after birth. Genotype of mice was determined by polymerase chain reaction on DNA isolated from ear or tail biopsy specimens, as described before. Mice were housed under standard conditions with food and water given ad libitum and maintained on a 12 h light/dark cycle with controlled temperature and humidity. Antibodies As primary antibodies, the following antibodies were used: mouse monoclonal antibodies against Cx43, Cx43-NP and N-cadherin; rabbit polyclonal antibodies against Cx43, NaV1.5 and CnA; goat polyclonal antibodies against connective tissue growth factor . Alexa Fluor 594 and fluorescein isothiocyanate -conjugated anti-mouse or anti-rabbit whole IgG were used as secondary antibodies for immunohistochemistry; horseradish peroxidase -conjugated donkey anti-mouse, anti-rabbit or anti-goat as secondary antibodies for immunoblotting. Heart Sampling Mice were sacrificed directly or 1, 2, 3 and 4 weeks after birth and hearts were rapidly excised and rinsed in cold phosphate buffered saline. Subsequently the hearts were either immediately frozen in liquid nitrogen or the atria were discarded and the ventricles frozen in liquid nitrogen and immunoblotting). Only ventricles were analyzed in all experiments. Tissue samples were stored at 80uC until further use. Immunohistochemistry and Histology For immunohistochemistry and collagen detection, cryosections of 4 different WT and MHC-CnA hearts were prepared from different levels of the heart. Immunolabeling was performed as previously described, antibodies are listed below. After immunolabeling, sections were analyzed with a widefield microscope with epifluorescence equipment. To evaluate the amount of