1. Date of download: 7/3/2016 Copyright © The American College of Cardiology. All rights reserved. From: Targeted Inhibition of β-Adrenergic Receptor Kinase-1–Associated Phosphoinositide-3 Kinase Activity Preserves β-Adrenergic Receptor Signaling and Prolongs Survival in Heart Failure Induced by Calsequestrin Overexpression J Am Coll Cardiol. 2005;45(11):1862-1870. doi:10.1016/j.jacc.2005.02.062 Catalytically inactive phosphoinositide-3 kinase γ (PI3Kγ inact ) overexpression normalizes βAR kinase-1 (βARK1)-associated PI3K activity in failing hearts from calsequestrin (CSQ) transgenic mice. (a) βARK1-associated PI3K activity in cardiac membranes from wild type (WT) (n = 8), CSQ (n = 8), and CSQ/PI3Kγ inact (n = 8) mouse hearts. *p < 0.001 CSQ vs. WT or CSQ/PI3Kγ inact (analysis of variance with Neuman-Keuls correction); PI3Kγ (b) or α (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 PI3Kα over WT levels in both CSQ or CSQ/PI3Kγ inact (in CSQ 1.0 ± 0.3-fold, in CSQ/PI3Kγ inact 0.7 ± 0.1-fold compared with WT). Ori = origin; PIP = phosphatidylinositol-mono-phosphate; PIP2 = phosphatidylinositol-bis-phosphate. Figure Legend:

2. Date of download: 7/3/2016 Copyright © The American College of Cardiology. All rights reserved. From: Targeted Inhibition of β-Adrenergic Receptor Kinase-1–Associated Phosphoinositide-3 Kinase Activity Preserves β-Adrenergic Receptor Signaling and Prolongs Survival in Heart Failure Induced by Calsequestrin Overexpression J Am Coll Cardiol. 2005;45(11):1862-1870. doi:10.1016/j.jacc.2005.02.062 Inhibition of receptor-localized phosphoinositide 3-kinase (PI3K) activity does not affect downstream signaling pathways. (a) Northern blotting analysis showing β-adrenergic receptor (β 1 AR) levels in 12-week-old wild type (WT), calsequestrin (CSQ), and CSQ/catalytically inactive PI3Kγ (CSQ/PI3Kγ inact ) 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/PI3Kγ inact 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 p38β (e). Western blotting was carried out to evaluate total protein levels of each kinase (b to d). *p < 0.01 for CSQ or CSQ/PI3Kγ inact 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/PI3Kγ inact mice. IB = immunoblotting; IP = immunoprecipitation. Figure Legend:

3. Date of download: 7/3/2016 Copyright © The American College of Cardiology. All rights reserved. From: Targeted Inhibition of β-Adrenergic Receptor Kinase-1–Associated Phosphoinositide-3 Kinase Activity Preserves β-Adrenergic Receptor Signaling and Prolongs Survival in Heart Failure Induced by Calsequestrin Overexpression J Am Coll Cardiol. 2005;45(11):1862-1870. doi:10.1016/j.jacc.2005.02.062 Cardiac specific overexpression of catalytically inactive phosphoinositide-3 kinase γ (PI3Kγ inact ) improves cardiac function of calsequestrin (CSQ) transgenic mice. (a) Representative echocardiograms from CSQ or CSQ/PI3Kγ inact 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/PI3Kγ inact (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/PI3Kγ inact mice. *p < 0.05 CSQ or CSQ/PI3Kγ inact vs. respective eight weeks measurement; †p < 0.05 CSQ/PI3Kγ inact vs. CSQ age-matched mice (repeated measures analysis of variance); ‡p < 0.0001 WT vs. CSQ or CSQ/PI3Kγ inact at all time points. Figure Legend:

4. Date of download: 7/3/2016 Copyright © The American College of Cardiology. All rights reserved. From: Targeted Inhibition of β-Adrenergic Receptor Kinase-1–Associated Phosphoinositide-3 Kinase Activity Preserves β-Adrenergic Receptor Signaling and Prolongs Survival in Heart Failure Induced by Calsequestrin Overexpression J Am Coll Cardiol. 2005;45(11):1862-1870. doi:10.1016/j.jacc.2005.02.062 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 γ (PI3Kγ inact ) transgenic mice. *p < 0.0001 WT vs. CSQ or CSQ/PI3Kγ inact ; †p < 0.01 CSQ/PI3Kγ inact vs. CSQ (analysis of variance with Neuman- Keuls correction). Figure Legend:

5. Date of download: 7/3/2016 Copyright © The American College of Cardiology. All rights reserved. From: Targeted Inhibition of β-Adrenergic Receptor Kinase-1–Associated Phosphoinositide-3 Kinase Activity Preserves β-Adrenergic Receptor Signaling and Prolongs Survival in Heart Failure Induced by Calsequestrin Overexpression J Am Coll Cardiol. 2005;45(11):1862-1870. doi:10.1016/j.jacc.2005.02.062 Restoration of β-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 γ (PI3Kγ inact ) (n = 20) mice; CSQ mice had a mean survival age of days 151.9 ± 18.6, whereas CSQ/PI3Kγ inact mice reached a mean survival age of 234.2 ± 14.3 days. *p < 0.001 for WT vs. CSQ or CSQ/PI3Kγ inact ; †p < 0.001 CSQ/PI3Kγ inact vs. CSQ, Kaplan-Meier analysis. (b) Kaplan-Meier survival curves according to gender in CSQ (females, n = 11; males, n = 8) and CSQ/PI3Kγ inact mice (females, n = 11; males, n = 9). In the analysis censored at 150 days, *p < 0.05 for PI3Kγ inact, p = 0.3 for gender; in the analysis censored at 300 days, †p < 0.01 for PI3Kγ inact and female gender (Cox proportional hazard analysis). Figure Legend:

6. Date of download: 7/3/2016 Copyright © The American College of Cardiology. All rights reserved. From: Targeted Inhibition of β-Adrenergic Receptor Kinase-1–Associated Phosphoinositide-3 Kinase Activity Preserves β-Adrenergic Receptor Signaling and Prolongs Survival in Heart Failure Induced by Calsequestrin Overexpression J Am Coll Cardiol. 2005;45(11):1862-1870. doi:10.1016/j.jacc.2005.02.062 Equal expression levels of calsequestrin (CSQ) and catalytically inactive phosphoinositide 3-kinase γ (PI3Kγ inact ) 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 PI3Kγ inact 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 β-adrenergic receptor kinase 1 (βARK1) in the cytoplasm of the same animals were also unchanged. Figure Legend: