Ivo analyses of mice as described previously [8,19]. On day 28 after TAC surgery, Title Loaded From File echocardiographic studies were performed under anesthesia with a mixture of medetomidine (0.3 mg/kg), midazolam (4 mg/kg) and butorphanol tartrate (5 mg/kg), and spontaneous respiration. A 2D parasternal shortaxis view of the LV was obtained at the level of the papillary muscles by applying the transducer lightly to the mid-upper leftTwinkle and Pressure Overloadanterior chest wall. After ensuring that the image was on axis (based on roundness of the LV cavity), 2D targeted M-mode tracings were recorded at a paper speed of 50 mm/s. Anterior, posterior end diastolic 15481974 wall thickness and LV internal dimensions were measured. While under anesthesia, a 1.4 Fr micromanometer-tipped catheter (Millar Instruments) was inserted into the right carotid artery and advanced into the LV to measure pressures for the assessment of aortic pressure and LV end 16574785 diastolic pressure.In vitro ExperimentsPrimary culture of neonatal rat cardiac fibroblasts was prepared from the ventricles of neonatal Sprague-Dawley rats as described previously [15,21]. Briefly, neonatal rats were euthanized by decapitation under anesthesia with isoflurane, after which the hearts were rapidly excised and digested. Anesthesia depth was monitored by limb withdrawal in response to toe pinching. After digesting the myocardial tissue with trypsin (Wako) and collagenase type 2 (Worthington), cells were suspended in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) containing 10 fetal bovine serum (Thermo SCIENTIFIC), penicillin (Invitrogen) and streptomycin (Invitrogen), and plated in 100 mm culture dishes for 70 minutes to remove non-adherent cardiac myocytes. Adherent cardiac fibroblasts were maintained at 37uC in humidified air with 5 CO2. Considering the possibility that cardiac fibroblasts may lose the original characteristics after prolonged culture, cells were used within 2 passages in all experiments. Cells were infected with AxCAhTwinkle, AxCAsirTwinkle or AxCALacZ (multiplicity of infection; 1) in serum-free DMEM for 1 hour, and cultured for another 72 hours in DMEM containing 5 fetal bovine serum. Then the cells were stimulated with angiotensin II (AngII, Sigma-Aldrich, 1 mM) for 24 hours, and collected for mRNA analyses.Histopathological StudiesAfter in vivo echocardiographic and hemodynamic studies, the heart was excised and weighed, and dissected into the right and left ventricles, including the septum. The heart tissues were fixed in 6 formaldehyde, embedded in paraffin, and cut into 5 mm thick sections. Sections were stained with hematoxylin-eosin and Masson’s trichrome for assessments of myocyte cross-sectional area and collagen volume fraction [13]. To measure the myocyte cross-sectional area, each section was photographed under a microscope (DMD108, Leica Microsystems) at a final magnification of 2006. The profiles of 30 to 40 myocytes cut in crosssections were Title Loaded From File traced manually and digitized. The digitized profiles were transferred to a personal computer that calculated the area. Three to 4 fields were randomly selected from 3 coronal sections of each heart. Thus, 100 to 200 myocytes were measured for each animal, and the mean myocyte cross-sectional area was calculated. Collagen volume fraction was measured in 6 fields randomly selected from each coronal section (basal, mid and apical sections) in each animal. Each field was photographed under a microscope at a final magnificat.Ivo analyses of mice as described previously [8,19]. On day 28 after TAC surgery, echocardiographic studies were performed under anesthesia with a mixture of medetomidine (0.3 mg/kg), midazolam (4 mg/kg) and butorphanol tartrate (5 mg/kg), and spontaneous respiration. A 2D parasternal shortaxis view of the LV was obtained at the level of the papillary muscles by applying the transducer lightly to the mid-upper leftTwinkle and Pressure Overloadanterior chest wall. After ensuring that the image was on axis (based on roundness of the LV cavity), 2D targeted M-mode tracings were recorded at a paper speed of 50 mm/s. Anterior, posterior end diastolic 15481974 wall thickness and LV internal dimensions were measured. While under anesthesia, a 1.4 Fr micromanometer-tipped catheter (Millar Instruments) was inserted into the right carotid artery and advanced into the LV to measure pressures for the assessment of aortic pressure and LV end 16574785 diastolic pressure.In vitro ExperimentsPrimary culture of neonatal rat cardiac fibroblasts was prepared from the ventricles of neonatal Sprague-Dawley rats as described previously [15,21]. Briefly, neonatal rats were euthanized by decapitation under anesthesia with isoflurane, after which the hearts were rapidly excised and digested. Anesthesia depth was monitored by limb withdrawal in response to toe pinching. After digesting the myocardial tissue with trypsin (Wako) and collagenase type 2 (Worthington), cells were suspended in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma-Aldrich) containing 10 fetal bovine serum (Thermo SCIENTIFIC), penicillin (Invitrogen) and streptomycin (Invitrogen), and plated in 100 mm culture dishes for 70 minutes to remove non-adherent cardiac myocytes. Adherent cardiac fibroblasts were maintained at 37uC in humidified air with 5 CO2. Considering the possibility that cardiac fibroblasts may lose the original characteristics after prolonged culture, cells were used within 2 passages in all experiments. Cells were infected with AxCAhTwinkle, AxCAsirTwinkle or AxCALacZ (multiplicity of infection; 1) in serum-free DMEM for 1 hour, and cultured for another 72 hours in DMEM containing 5 fetal bovine serum. Then the cells were stimulated with angiotensin II (AngII, Sigma-Aldrich, 1 mM) for 24 hours, and collected for mRNA analyses.Histopathological StudiesAfter in vivo echocardiographic and hemodynamic studies, the heart was excised and weighed, and dissected into the right and left ventricles, including the septum. The heart tissues were fixed in 6 formaldehyde, embedded in paraffin, and cut into 5 mm thick sections. Sections were stained with hematoxylin-eosin and Masson’s trichrome for assessments of myocyte cross-sectional area and collagen volume fraction [13]. To measure the myocyte cross-sectional area, each section was photographed under a microscope (DMD108, Leica Microsystems) at a final magnification of 2006. The profiles of 30 to 40 myocytes cut in crosssections were traced manually and digitized. The digitized profiles were transferred to a personal computer that calculated the area. Three to 4 fields were randomly selected from 3 coronal sections of each heart. Thus, 100 to 200 myocytes were measured for each animal, and the mean myocyte cross-sectional area was calculated. Collagen volume fraction was measured in 6 fields randomly selected from each coronal section (basal, mid and apical sections) in each animal. Each field was photographed under a microscope at a final magnificat.