Lipopolysaccharide Activates Calcineurin in Ventricular Myocytes
Jun Suzuki, MD, PhD*, ,*,
Evelyn Bayna, PhD*,
Hai Ling Li, PhD*,
Erminia Dalle Molle, BS*, and
Wilbur Y.W. Lew, MD, FACC*,
* Cardiology Section, Department of Medicine, V.A. San Diego Healthcare System, San Diego, California
University of California, San Diego, San Diego, California
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Figure 1 Time Course and LPS Dose Dependence of Calcineurin Activity in Cardiac Myocytes
(A) Calcineurin activity in cardiac myocytes (mean ± SE, n = 4) increased with lipopolysaccharide (LPS) (10 ng/ml) compared with control (vehicle) after 1 to 24 h, with t 1/2 of 4.8 h (p < 0.05). (B) Lipopolysaccharide dose-calcineurin activity relationship (mean ± SE, n = 4). After 16 h incubation with LPS, calcineurin activity increased in cardiac myocytes with an EC50 of 0.80 ng/ml LPS (p < 0.05).
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Figure 2 Calcineurin Activity in Cardiac Myocytes With LPS and/or Ang II Inhibited by Losartan
Calcineurin activity in cardiac myocytes (mean + SE, n = 6) increased after 16-h incubation with lipopolysaccharide (LPS) (10 ng/ml) and/or angiotensin II (Ang II) (100 nmol/l) without cumulative effects (p < 0.05). This was blocked by pre-treating myocytes with losartan (1 µmol/l, Ang II type 1 receptor inhibitor) 1 h before LPS or Ang II, while losartan alone had no effect.
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Figure 3 Cyclosporin A, BAPTA-AM, TG, Ryanodine, or Nicotine Inhibit LPS-Induced Calcineurin Activity
Calcineurin activity in cardiac myocytes (mean + SE) increased after 16-h incubation with lipopolysaccharide (LPS) (10 ng/ml) compared with control (vehicle) (p < 0.05). (A) This was blocked by pre-treating myocytes (1 h before LPS) with either cyclosporin A (0.5 µmol/l), 1,2-Bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA-AM) (0.1 µmol/l), or thapsigargin (TG) (1 µmol/l) (n = 5). (B) Lipopolysaccharide effects were blocked by pre-treating myocytes with ryanodine (1 µmol/l, 1 h before LPS), or nicotine (15 ng/ml, 4 h before LPS) (n = 6).
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Figure 4 LPS- or Ang II-Induced TUNEL Staining in Cardiac Myocytes Inhibited by Cyclosporin A
Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining of cardiac myocytes (mean % + SE) increased after 24 h with lipopolysaccharide (LPS) (10 ng/ml, n = 7) (A) or angiotensin II (Ang II) (100 nmol/l, n = 9) (B) compared with control (vehicle) (p < 0.05). In both cases, this was blocked by the cyclosporin A (0.5 µmol/l, 1 h before LPS or Ang II), while cyclosporin A alone had no effect (p < 0.05).
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Figure 5 LPS- or Ang II-Induced TUNEL Staining of Cardiac Myocytes Inhibited by BAPTA-AM or Thapsigargin
Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining of cardiac myocytes (mean % + SE) increased after 24 h with lipopolysaccharide (LPS) (10 ng/ml) or angiotensin II (Ang II) (100 nmol/l) compared with control (vehicle) (p < 0.05). This was blocked by pre-treating myocytes 1 h before LPS or Ang II with: (A) 1,2-Bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA-AM) (0.1 µmol/l), while BAPTA-AM alone had no effect (n = 5); (B) Thapsigargin (TG) (1 µmol/l), while TG alone had no effect (n = 9).
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Figure 6 Time Course for Caspase-3 Activity in Left Ventricle After LPS In Vivo
Caspase-3 activity in the left ventricle increased from control (time = 0) 4 to 24 h after injecting lipopolysaccharide (LPS) (1 mg/kg intravenously) in vivo (p < 0.05, mean + SE, each bar n = 7 to 19).
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Figure 7 Left Ventricle Myocyte TUNEL Staining After LPS In Vivo Inhibited by Pre-Treating Rats With Cyclosporin A
Terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining in the left ventricle (mean + SE, n = 5) increased 24 h after in vivo intravenous injection (tail vein) of lipopolysaccharide (LPS) (1 mg/kg) compared with control (saline injection) (p < 0.05, 2-way analysis of variance). This was blocked by pre-treating rats with cyclosporin A (20 mg/kg/day subcutaneously for 3 days before LPS or saline injections), while cyclosporin A treatment alone had no effect.
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Figure 8 Proposed Cell Signaling Pathways for LPS-Induced Activation of Cardiac Calcineurin
Proposed cell signaling pathways for lipopolysaccharide (LPS) induced activation of calcineurin. Inhibitory effects are shown with red lines. Ang II = angiotensin II; AT1 = angiotensin II type 1 receptor; BAPTA-AM = 1,2-Bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester; SR = sarcoplasmic reticulum; TLR-4 = Toll-like receptor-4.
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