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Regulation of myocardial ßARK1 expression in catecholamine-induced cardiac hypertrophy in transgenic mice overexpressing _1B-adrenergic receptors

Guido Iaccarino, MD^* , Janelle R. Keys, PhD, Antonio Rapacciuolo, MD^*, Kyle F. Shotwell, BS, Robert J. Lefkowitz, MD^* , Howard A. Rockman, MD^* and Walter J. Koch, PhD

^* Medicine, Duke University Medical Center, Durham, North Carolina, USA
Surgery, Duke University Medical Center, Durham, North Carolina, USA
Biochemistry, Duke University Medical Center, Durham, North Carolina, USA
Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, USA

View larger version (21K): [in a new window]   Figure 1 Effects of phenylephrine (PE) treatment (100 mg/kg/day) on survival and cardiac mass in nontransgenic littermate control (NLC) mice and transgenic mice with cardiac targeted overexpression of the wild-type 1B-AR (Tg43). (A) Survival (%) in NLC (n = 15) and Tg43 (n = 27) animals after chronic PE administration. Perioperative mortality was about 10% and was not different between NLC and Tg43 mice. *p < 0.05, Tg43 versus NLC (2 test). (B) Heart-to-body weight ratio (mg/g) assessed after 3, 7 and 14 days of PE treatment in Tg43 and NLC mice. In order to simplify the graph, the data of the sham animals represent the average of each time point, as no difference was observed between them. *p < 0.05 versus sham phosphate-buffered saline treated mice; #p < 0.05 versus PE-treated NLC mice.

View larger version (93K): [in a new window]   Figure 2 Morphological and histological assessment of the cardiac effects of phenylephrine (PE) treatment in transgenic mice with cardiac targeted overexpression of the wild-type 1B-AR (Tg43) and nontransgenic littermate control (NLC) mice. (A) Morphology of two Tg43 littermates that were either phosphate-buffered saline (left) or PE (right) treated for 14 days. The heart of the sham phosphate-buffered saline treated Tg43 mouse is indistinguishable from a phosphate-buffered saline-treated NLC mouse (not shown). However, as shown on the right, PE treatment increases the size of cardiac chambers, including left and right atria in Tg43 mice. (B) Magnified section of a PE-treated Tg43 heart showing an enlarged left atria with the presence of thrombi, which was seen in >60% of the PE-treated Tg43 mice. (C) Masson trichrome stained section of a representative phosphate-buffered saline treated (sham) NLC mouse taken from the left ventricular wall toward the apex. (D) A similar section taken from a PE-treated (14 days) NLC mouse. (E) A stained section from a sham-treated Tg43 mouse showing no fibrosis. (F) Representative left ventricular section from a PE-treated (14 days) Tg43 mouse showing significant deposition of collagen and fibrosis (staining blue), which was seen in all Tg43 animals after PE.

View larger version (23K): [in a new window]   Figure 3 Effect of seven days of PE infusion on (A) myocardial ß-adrenergic receptor (AR) responsiveness and (B) ßAR kinase 1 (ßARK1) levels in nontransgenic littermate control (NLC) hearts and the hearts of transgenic mice with cardiac targeted overexpression of the wild-type 1B-AR (Tg43). (A) The activity of adenylyl cyclase from cardiac membranes was assessed basally and after isoproterenol (ISO) stimulation (10–4M). Data are expressed as the mean ± SEM of ISO-stimulated activity over the activity seen under basal conditions (n = 6 to 12). *p < 0.05 versus sham (phosphate-buffered saline) treatment; #p < 0.05 versus PE-treated NLC mice. (B) Myocardial levels of ßARK1 as measured by semiquantitative Western blotting following immunoprecipitation of cardiac extracts with a monoclonal ßARK1/2 antibody (16). Shown is the mean ± SEM of sham-treated NLC (n = 7) and Tg43 (n = 11) mice compared with PE-treated (7 days) NLC (n = 6) and Tg43 (n = 10) mice. ßARK1 levels in all mice were normalized to the levels found in sham (phosphate-buffered saline) NLC mice. Shown in the inset is a representative immunoblot of ßARK1 levels. *p < 0.05 versus sham-treated mice; #p < 0.05 versus NLC mice.

View larger version (17K): [in a new window]   Figure 4 Effects of phenylephrine (PE) treatment (7 days) on myocardial neuropeptide Y content in nontransgenic littermate control (NLC) mice and transgenic mice with cardiac targeted overexpression of the wild-type 1B-AR (Tg43). Shown is the mean ± SEM of 6 to 10 mice in each group. *p < 0.05 versus sham-treated mice; #p < 0.05 versus NLC mice. White bar = NLC; black bar = Tg43.

View larger version (44K): [in a new window]   Figure 5 Effects of phenylephrine (PE) treatment (14 days) on myocardial gene expression in nontransgenic littermate control (NLC) mice and transgenic mice with cardiac targeted overexpression of the wild-type 1B-AR (Tg43). (A) Representative Northern blot analysis of the ventricular transcript level of atrial natriuretic factor (ANF), skeletal -actin, sarcoplasmic reticulum Ca++-ATPase (SERCA2a) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the listed mice phosphate-buffered saline and PE treated. (B) Pooled quantitative data for the individual genes shown in A. Histograms represent the mean ± SEM of quantified Northern blots using the ribonucleic acid isolated from three left ventricles in each group. The data were normalized to the levels of hybridization of GAPDH and plotted as foldover GAPDH levels. *p < 0.05 versus sham-treated NLC mice.