Striction. After de-airing, the thorax is closed with layered 6-0 Dexon sutures to minimize the risk of pneumothorax. Post-operative analgesia is immediately provided with Buprenorphine 0.1 mL, which is continued twice daily and as needed for an additional 72 hours. Mice were allowed to survive for 7 days or 3 weeks after pulmonary artery constriction (PAC) and 7 days or 10 weeks after thoracic aortic constriction (TAC). Two groups of controls (n = 6/group) were studied 7 days and 10 weeks after sham surgery.MedChemExpress 223488-57-1 Biventricular Conductance Catheter InstrumentationAll animals underwent LY2409021 manufacturer terminal hemodynamic evaluation. Biventricular catheterization was performed at the time of sacrifice in all animals. Mice were anesthetized with 2.0 isoflurane administered via a non-invasive nose-cone. Body temperature was monitored by a rectal thermistor probe and maintained at 37.5uC with heating pads and a cycling heat lamp. In the supine position, the right common carotid and right external jugular vein were surgically isolated. Silk ties were placed at the distal ends of both vessels while overhand loopsHistologic Quantification of Cardiac Hypertrophy and FibrosisRV and LV collagen abundance by picrosirius red staining were quantified as percent fibrosis of the total RV and LV respectively. Cardiomyocyte cross-sectional area was quantified as previously described [28?0].Biventricular RemodelingFigure 1. Biventricular conductance catheterization in a closed-chest, non-invasively ventilated mouse. A) Hemodynamic tracings illustrating pressure volume (PV) catheters tracking from the right atrium (RA) to right ventricle (RV) via the right external jugular vein and aorta (Ao) to left ventricle (LV) via the right carotid artery via a suprasternal incision in a closed-chest mouse (*, salivary gland). B) Representative steady-state PV loops in mouse models of (i) primary and (ii) secondary right ventricular pressure overload (RVPO) (blue loops represent sham operated animals). doi:10.1371/journal.pone.0070802.gReal-time Quantitative Polymerase Chain Reaction (RTPCR)For RT-PCR, total RNA was extracted from RV and LV tissues directly using Trizol (Invitrogen), converted to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For all RT-PCR experiments, samples were quantified in triplicate using 40 cycles performed at 94uC for 30 sec., 60uC for 45 sec, 72uC for 45 sec using an ABI PrismH 7900 Sequence Detection System using appropriate primers as previously described [28?0].Immunoblot Analysis (Western)Total protein was extracted and quantified from tissue homogenates as described (28?0). Immunoblot analysis was then performed as previously described using antibodies for mouse targeted proteins including: Type I collagen (Santa Cruz Inc), calcineurin (Cell Signaling), pERK (Millipore), total ERK (Cell Signaling), pSmad-3 (Santa Cruz Inc), and total Smad-3 (Cell Signaling).Statistical AnalysisResults are presented as mean 6 standard deviation. Intergroup comparisons were made with a Student’s t-test and two-factor ANOVA. All statistical analyses were performed using SigmaStat Version 3.1 (Systat Software, Inc). An 23977191 alpha level of P,0.05 was considered to indicate a significant effect or between-groups difference.secondary RVPO (Figure S1A). Compared to sham-controls, peak RV systolic pressure was increased with no change in RV enddiastolic pressure in both 7-day primary and 10-week secondary RVPO. RV end-diastolic volume was significantly.Striction. After de-airing, the thorax is closed with layered 6-0 Dexon sutures to minimize the risk of pneumothorax. Post-operative analgesia is immediately provided with Buprenorphine 0.1 mL, which is continued twice daily and as needed for an additional 72 hours. Mice were allowed to survive for 7 days or 3 weeks after pulmonary artery constriction (PAC) and 7 days or 10 weeks after thoracic aortic constriction (TAC). Two groups of controls (n = 6/group) were studied 7 days and 10 weeks after sham surgery.Biventricular Conductance Catheter InstrumentationAll animals underwent terminal hemodynamic evaluation. Biventricular catheterization was performed at the time of sacrifice in all animals. Mice were anesthetized with 2.0 isoflurane administered via a non-invasive nose-cone. Body temperature was monitored by a rectal thermistor probe and maintained at 37.5uC with heating pads and a cycling heat lamp. In the supine position, the right common carotid and right external jugular vein were surgically isolated. Silk ties were placed at the distal ends of both vessels while overhand loopsHistologic Quantification of Cardiac Hypertrophy and FibrosisRV and LV collagen abundance by picrosirius red staining were quantified as percent fibrosis of the total RV and LV respectively. Cardiomyocyte cross-sectional area was quantified as previously described [28?0].Biventricular RemodelingFigure 1. Biventricular conductance catheterization in a closed-chest, non-invasively ventilated mouse. A) Hemodynamic tracings illustrating pressure volume (PV) catheters tracking from the right atrium (RA) to right ventricle (RV) via the right external jugular vein and aorta (Ao) to left ventricle (LV) via the right carotid artery via a suprasternal incision in a closed-chest mouse (*, salivary gland). B) Representative steady-state PV loops in mouse models of (i) primary and (ii) secondary right ventricular pressure overload (RVPO) (blue loops represent sham operated animals). doi:10.1371/journal.pone.0070802.gReal-time Quantitative Polymerase Chain Reaction (RTPCR)For RT-PCR, total RNA was extracted from RV and LV tissues directly using Trizol (Invitrogen), converted to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For all RT-PCR experiments, samples were quantified in triplicate using 40 cycles performed at 94uC for 30 sec., 60uC for 45 sec, 72uC for 45 sec using an ABI PrismH 7900 Sequence Detection System using appropriate primers as previously described [28?0].Immunoblot Analysis (Western)Total protein was extracted and quantified from tissue homogenates as described (28?0). Immunoblot analysis was then performed as previously described using antibodies for mouse targeted proteins including: Type I collagen (Santa Cruz Inc), calcineurin (Cell Signaling), pERK (Millipore), total ERK (Cell Signaling), pSmad-3 (Santa Cruz Inc), and total Smad-3 (Cell Signaling).Statistical AnalysisResults are presented as mean 6 standard deviation. Intergroup comparisons were made with a Student’s t-test and two-factor ANOVA. All statistical analyses were performed using SigmaStat Version 3.1 (Systat Software, Inc). An 23977191 alpha level of P,0.05 was considered to indicate a significant effect or between-groups difference.secondary RVPO (Figure S1A). Compared to sham-controls, peak RV systolic pressure was increased with no change in RV enddiastolic pressure in both 7-day primary and 10-week secondary RVPO. RV end-diastolic volume was significantly.