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Targeted Inhibition of ß-Adrenergic Receptor Kinase-1-Associated Phosphoinositide-3 Kinase Activity Preserves ß-Adrenergic Receptor Signaling and Prolongs Survival in Heart Failure Induced by Calsequestrin Overexpression

Cinzia Perrino, MD^_*, Sathyamangla V. Naga Prasad, PhD^_*, Mrinali Patel^_*, Matthew J. Wolf, MD, PhD^_* and Howard A. Rockman, MD^_*,,_*

^_* Department of Medicine, Duke University Medical Center, Durham, North Carolina
Department of Cell Biology and Molecular Genetics, Duke University Medical Center, Durham, North Carolina

View larger version (40K): [in a new window]   Figure 1 Catalytically inactive phosphoinositide-3 kinase (PI3Kinact) overexpression normalizes ßAR kinase-1 (ßARK1)-associated PI3K activity in failing hearts from calsequestrin (CSQ) transgenic mice. (a) ßARK1-associated PI3K activity in cardiac membranes from wild type (WT) (n = 8), CSQ (n = 8), and CSQ/PI3Kinact (n = 8) mouse hearts. *p < 0.001 CSQ vs. WT or CSQ/PI3Kinact (analysis of variance with Neuman-Keuls correction); PI3K (b) or (c) activities were assayed from the cytosolic extracts of the same hearts. Left panels show representative PI3K assays; right panels show summary data. There was no significant increase in the activity of PI3K over WT levels in both CSQ or CSQ/PI3Kinact (in CSQ 1.0 ± 0.3-fold, in CSQ/PI3Kinact 0.7 ± 0.1-fold compared with WT). Ori = origin; PIP = phosphatidylinositol-mono-phosphate; PIP2 = phosphatidylinositol-bis-phosphate.

View larger version (37K): [in a new window]   Figure 2 Inhibition of receptor-localized phosphoinositide 3-kinase (PI3K) activity does not affect downstream signaling pathways. (a) Northern blotting analysis showing ß-adrenergic receptor (ß1AR) levels in 12-week-old wild type (WT), calsequestrin (CSQ), and CSQ/catalytically inactive PI3K (CSQ/PI3Kinact) hearts (upper panel); equal loading of the different RNA samples was confirmed by methylene blue staining of nylon membranes (bottom panel). (b to e) Mitogen-activated protein kinase activation was determined in WT, CSQ, and CSQ/PI3Kinact hearts by the ability to in vitro phosphorylate myelin binding protein (MBP) or recombinant glutathione S transferase-proto-oncogene c-Jun (GST-cJun). Representative kinase assays and relative densitometric evaluation of at least six independent experiments are shown for extracellular signal-related kinase (ERK) (b), c-Jun N-terminal kinase (JNK) (c), p38 (d), and p38ß (e). Western blotting was carried out to evaluate total protein levels of each kinase (b to d). *p < 0.01 for CSQ or CSQ/PI3Kinact versus WT (analysis of variance with Neuman-Keuls correction). (f) Western blotting analysis showing similar activation of protein kinase B (PKB) and glycogen synthase kinase (GSK) under basal conditions in single CSQ and binary CSQ/PI3Kinact mice. IB = immunoblotting; IP = immunoprecipitation.

View larger version (54K): [in a new window]   Figure 3 Cardiac specific overexpression of catalytically inactive phosphoinositide-3 kinase (PI3Kinact) improves cardiac function of calsequestrin (CSQ) transgenic mice. (a) Representative echocardiograms from CSQ or CSQ/PI3Kinact mice at 8, 12, and 16 weeks of age. Absolute values of left ventricular end-diastolic diameter (LVEDD, b), left ventricular end-systolic diameter (LVESD, c), and % fractional shortening (%FS, d) in age-matched wild type (WT) (striped circle), CSQ (solid circle), and CSQ/PI3Kinact (open circle) mice are shown in the left panels. In the right panels percent change variation over time is also shown for CSQ and CSQ/PI3Kinact mice. *p < 0.05 CSQ or CSQ/PI3Kinact vs. respective eight weeks measurement; p < 0.05 CSQ/PI3Kinact vs. CSQ age-matched mice (repeated measures analysis of variance); p < 0.0001 WT vs. CSQ or CSQ/PI3Kinact at all time points.

View larger version (22K): [in a new window]   Figure 4 Targeted inhibition of phosphoinositide-3 kinase (PI3K) reduces cardiac chamber size. Bar graphs showing (a) body weight (BW), (b) left atrium (LA) weight, (c) left ventricular weight/body weight (LV/BW), (d) heart weight/body weight (H/BW) in wild type (WT) mice, single calsequestrin (CSQ) transgenic, and binary CSQ/catalytically inactive phosphoinositide 3-kinase (PI3Kinact) transgenic mice. *p < 0.0001 WT vs. CSQ or CSQ/PI3Kinact; p < 0.01 CSQ/PI3Kinact vs. CSQ (analysis of variance with Neuman-Keuls correction).

View larger version (21K): [in a new window]   Figure 5 Restoration of ß-adrenergic receptor function improves survival in mice with heart failure. (a) Kaplan-Meier survival curves of wild type (WT) (n = 18), calsequestrin (CSQ) (n = 19), and CSQ/catalytically inactive phosphoinositide 3-kinase (PI3Kinact) (n = 20) mice; CSQ mice had a mean survival age of days 151.9 ± 18.6, whereas CSQ/PI3Kinact mice reached a mean survival age of 234.2 ± 14.3 days. *p < 0.001 for WT vs. CSQ or CSQ/PI3Kinact; p < 0.001 CSQ/PI3Kinact vs. CSQ, Kaplan-Meier analysis. (b) Kaplan-Meier survival curves according to gender in CSQ (females, n = 11; males, n = 8) and CSQ/PI3Kinact mice (females, n = 11; males, n = 9). In the analysis censored at 150 days, *p < 0.05 for PI3Kinact, p = 0.3 for gender; in the analysis censored at 300 days, p < 0.01 for PI3Kinact and female gender (Cox proportional hazard analysis).

View larger version (32K): [in a new window]   Figure 6 Equal expression levels of calsequestrin (CSQ) and catalytically inactive phosphoinositide 3-kinase (PI3Kinact) transgenes in young and old male and female mice. Representative immunoblottings (two animals/group) and relative quantitative densitometric analysis (three to four animals/group) showing similar expression levels of the alpha-myosin-heavy-chain-driven transgenes CSQ and PI3Kinact in the cytosolic fraction of hearts from female and male mice at 12 weeks of age (a, 84 days, 12 weeks) or older (b, 160 to 252 days, >22 weeks). Expression levels of ß-adrenergic receptor kinase 1 (ßARK1) in the cytoplasm of the same animals were also unchanged.