Nox-Dependent Mechanisms of Cardiomyocyte Dysfunction in a Model of Pressure Overload Giovanna Frazziano, PhD Vascular Medicine Institute Department of Pharmacology and Chemical Biology University of Pittsburgh
Background PAH • Pulmonary Arterial Hypertension 4,2% 0,6% 6,8 % (PAH) is characterized by a sustained 9,7 % elevation in pulmonary arterial pressure (>25 mmHg). Simonneau G et al., J Am Coll Cardiol. 2009 Jun 30;54(1Suppl):S43-54. • The prevalence of PAH ranges from 78,7% 26 to 52 case per milion adults. Peacock et al., Eur Respir J 2007; 30:104 – 109 Groupe 1: PAH. • Mortality of 37.2% at 3 years after Groupe 2: associated with left-sided heart disease. diagnosis with aggressive treatment. Groupe 3: associated with lung diseases or hypoxemia. Hemnes AR, Champion HC. Int J Clin Pract Suppl. Groupe 4: due to chronic thrombotic or embolic disease. 2008(160):11-19 . Groupe 5: with unclear multifactorial mechanisms.
Pathophysiology of PAH 1. Vasoconstriction 2. Vascular Remodeling 3. Thrombosis Decreased arterial lumen Increased pulmonary vascular resistance RV Failure
Existing Therapies & Clinical Needs Bosentan, Ca 2+ channel blockers Oxygen sitaxsentan (-) (+) Vasoconstriction Vasodilation (+) Iloprost, Sildenafil PGI 2 Beraprost ? Vardenafil
Reactive oxygen species (ROS) • Increases in reactive oxygen species (ROS) production are implicated in cardiac remodeling. Zhang M et al., Proc Natl Acad Sci U S A. 2010 Oct 19;107(42):18121-6. • Sources of ROS in the stressed heart include NADPH oxidase, uncoupled NO synthase, xanthine oxidase, and mitochondria. Santos CX et al., . Free Radic Biol Med. 2011 Apr 1;50(7):777-93. • Reported NADPH oxidase isoforms in the heart are Nox2 and Nox4. Nabeebaccus A et al., Heart Fail Rev. 2011 Jan;16(1):5-12.
NADPH oxidase (Nox) SOD .- O 2 H 2 O 2 p22 phox Nox2 p22 phox Nox2 Rac2 p47 phox p67 phox p40 phox .- or H 2 O 2 O 2 Nox4 p22 phox Al Ghouleh et al. Free Radic Biol Med. 2011
Hypothesis Pressure overload (PO) induces Nox4 activity and, in turn, Nox2 activation leading to RV failure. PO Nox4 Initiating ROS Nox2 NOS uncoupling Right ventricle failure
Model: Pulmonary Artery Banding (PAB) • Pulmonary vascular Baseline PAB resistance, is mechanically controlled Hemostatic titanium clip size 26G needle • Pure hemodynamic stress on the right ventricle • Independent of changes in the pulmonary vasculature
Experimental Plan Acute Phase Chronic Phase 0 1h 3h 6 h 1day 7 days 21 days • • Biochemical studies Hemodynamic studies • • ROS production Hypertrophy • mRNA and protein expression
H 2 O 2 Levels in Heart Homogenates in Acute PAB Right Ventricle (RV) Left Ventricle (LV) Sham 200 200 RFU (% of Respective Sham for Each RFU (% of Respective Sham for Each PAB 30 min Sham * PAB 1h PAB 30 min PAB 3h PAB 1h ** PAB 6h 150 PAB 3h 150 * p< 0.01 ** p<0.005 n= 4-6 PAB 1 day PAB 6h Time Point) PAB 1 day Time Point) 100 100 50 50 0 0 30 min 1h 3h 6h 1 day 30 min 1h 3h 6h 1 day * p< 0.01 vs sham, ** p<0.005 vs sham
Effects of Scavengers on H 2 O 2 in RV in Acute PAB * * * * * * p < 0.005 versus Sham or PAB without inhibitors *
Effects of Inhibitors on H 2 O 2 in RV in Acute PAB * * * * * * * p < 0.005 versus PAB without inhibitors
Effects of Inhibitors on H 2 O 2 in RV in Acute PAB 200 RFU (% of Sham without inhibitors) 150 Without inibitors DPI 20 µM 100 DPI 20 µM * 50 * * * m 0 Sham PAB 1h PAB 3h PAB 6h * * p < 0.005 versus Sham or PAB without inhibitors
H 2 O 2 Levels in Right Ventricle Homogenates (RV) in Acute PAB in Nox2 null mice * * * * * * p < 0.005 versus Sham without inhibitors
.- Production in RV Homogenates O 2 During Acute PAB Sham PAB 1h PAB 3h PAB 6h PAB 1 day Sham Nox2 null PAB Nox2 null
mRNA expression of Nox2 and Nox4 in RV
Time-Dependent Antioxidant Protein Expression in Response to PAB Sham PAB 1h PAB 3h PAB 6h
Catalase Activity in RV Homogenates in Response to Acute PAB * Sham PAB 1h PAB 3h PAB 6h p < 0.005 versus Sham
Experimental Plan Acute Phase Chronic Phase 0 1h 3h 6 h 1day 7 days 21 days • • Biochemical studies Hemodynamic studies • • ROS production Hypertrophy • mRNA and protein expression
Hypertrophy in Chronic PAB Fulton Index ( RV/LV +S) 0.5 60 * 50 0.4 * RV EDV (µL) * 40 0.3 30 0.2 20 0.1 10 0 0 Control PAB PAB Sham Control PAB PAB Sham 1 wk 3 wk 1 wk 3 wk * p < 0.005 versus Sham
Effects of Nox2ds tat on PAB-induced hypertrophy Fulton Index (RV/LV+S) 80 0.5 0.4 60 ** RV EDV (µL) ** 0.3 40 0.2 20 0.1 0 0 Control PAB PAB Sham Control Sham PAB PAB Nox2ds-tat Nox2ds-tat Nox2ds-tat: Nox2ds modified for tissue permeation * p < 0.005 versus Sham ** p< 0.005 versus PAB
Effects of Nox2 absence on PAB-induced hypertrophy 0.5 60 150 * * Fulton Index (RV/LV+S) Contractility index (1/s) 50 0.4 ** ** RV EDV ( µL) ** 40 100 * 0.3 30 0.2 20 50 0.1 10 0 0 0 Sha Control PAB PAB Sham Control PAB PAB Sham Control Sham PAB PAB WT Nox2 null m WT Nox2 null WT Nox2 null * p < 0.005 versus Sham ** p< 0.005 versus PAB WT
Summary and Conclusions Nox-derived H 2 O 2 increase at 6 hrs consistent with an early role for ROS in RV dysfunction. Nox2 is not transcriptionally upregulated at early time points whereas Nox4 is. An earlier upregulation of Nox4 could indicate a primary role for Nox4 in RV failure. Nox2 absence or inhibition prevent PAB-induced hypertrophy, suggesting a Nox2 role in PAB chronic phase.
Future Directions PAB ? Nox 2 and or Nox 4 • CamKII ? • AKT • p38-ERK Cardiac hypertrophy
2-D Gel for New RV Nox2 Target Discovery Sham 6h RV PAB 6h RV PAB 6h Nox2-/- RV
2Dige gel 150 kDa 110 kDa 1 96 kDa 2 4 5 6 84 kDa 3 13 14 12 15 17 75 kDa 7 9 21 10 67 kDa 16 18 22 8 11 20 24 19 28 50 kDa 23 25 26 27 29 30 32 33 40 kDa 31 34 35 37 38 36 30 kDa 42 39 43 41 40 44 45 46 20 kDa 47 48 49 14 kDa 5 52 57 58 4 50 51 55 60 53 56 59 pH 4.0 5.0 5.5 6.0 6.5 7.0 8.0
Acknowledgements Ri.MED Foundation NIH
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