Nox-Dependent Mechanisms of Cardiomyocyte Dysfunction in a Model of - - PowerPoint PPT Presentation

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

• Pulmonary

Arterial Hypertension (PAH) is characterized by a sustained 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

26 to 52 case per milion adults.

Peacock et al., Eur Respir J 2007; 30:104–109

• Mortality of 37.2% at 3 years after

diagnosis with aggressive treatment.

Hemnes AR, Champion HC. Int J Clin Pract Suppl. 2008(160):11-19.

Groupe 1: PAH. Groupe 2: associated with left-sided heart disease. Groupe 3: associated with lung diseases or hypoxemia. Groupe 4: due to chronic thrombotic or embolic disease. Groupe 5: with unclear multifactorial mechanisms. 4,2% 78,7% 9,7 % 0,6% 6,8 %

PAH

Pathophysiology of PAH

Decreased arterial lumen Increased pulmonary vascular resistance

RV Failure

• 2. Vascular Remodeling
• 1. Vasoconstriction
• 3. Thrombosis

Existing Therapies & Clinical Needs

Vasoconstriction

Bosentan, sitaxsentan (-)

Oxygen Ca2+ channel blockers PGI2 Sildenafil Vardenafil Iloprost, Beraprost

Vasodilation

(+) (+)

?

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.

Nox4 p22phox

NADPH oxidase (Nox)

Nox2 p22phox Nox2 O2

.-

H2O2

SOD

p67phox p40phox p47phox Rac2 O2

.- or H2O2

Al Ghouleh et al. Free Radic Biol Med. 2011

p22phox

Hypothesis

Pressure overload (PO) induces Nox4 activity and, in turn, Nox2 activation leading to RV failure.

PO

Initiating ROS NOS uncoupling

Right ventricle failure

Nox4 Nox2

Model: Pulmonary Artery Banding (PAB)

Hemostatic titanium clip size 26G needle

Baseline

PAB

• Pulmonary vascular

resistance, is mechanically controlled

• Pure hemodynamic

stress on the right ventricle

• Independent of changes

in the pulmonary vasculature

Experimental Plan

0 1h 3h 6 h 1day 7 days 21 days

• Biochemical studies
• ROS production
• mRNA and protein expression
• Hemodynamic studies
• Hypertrophy

Acute Phase Chronic Phase

H2O2 Levels in Heart Homogenates in Acute PAB

50 100 150 200 30 min 1h 3h 6h 1 day

RFU (% of Respective Sham for Each Time Point)

Right Ventricle (RV)

**

*

PAB 1h PAB 30 min Sham PAB 3h PAB 6h PAB 1 day

* p< 0.01 ** p<0.005 n= 4-6

50 100 150 200 30 min 1h 3h 6h 1 day

RFU (% of Respective Sham for Each Time Point)

Left Ventricle (LV)

PAB 1h PAB 30 min Sham PAB 3h PAB 6h PAB 1 day

* p< 0.01 vs sham, ** p<0.005 vs sham

Effects of Scavengers on H2O2 in RV in Acute PAB

p < 0.005 versus Sham or PAB without inhibitors

* * * * * * *

Effects of Inhibitors on H2O2 in RV in Acute PAB

* * * * * *

p < 0.005 versus PAB without inhibitors

*

50 100 150 200 Sham PAB 1h PAB 3h PAB 6h RFU (% of Sham without inhibitors) DPI 20 µM

Effects of Inhibitors on H2O2 in RV in Acute PAB

m Without inibitors DPI 20 µM

p < 0.005 versus Sham or PAB without inhibitors

* * * * * *

H2O2 Levels in Right Ventricle Homogenates (RV) in Acute PAB in Nox2 null mice

* * *

p < 0.005 versus Sham without inhibitors

* * *

O2

.- Production in RV Homogenates

During Acute PAB

Sham Nox2 null PAB Nox2 null Sham PAB 1h PAB 3h PAB 6h PAB 1 day

mRNA expression of Nox2 and Nox4 in RV

Time-Dependent Antioxidant Protein Expression in Response to PAB

PAB 6h PAB 1h Sham PAB 3h

Catalase Activity in RV Homogenates in Response to Acute PAB

*

PAB 6h PAB 1h Sham PAB 3h

p < 0.005 versus Sham

Experimental Plan

0 1h 3h 6 h 1day 7 days 21 days

• Biochemical studies
• ROS production
• mRNA and protein expression
• Hemodynamic studies
• Hypertrophy

Acute Phase Chronic Phase

Hypertrophy in Chronic PAB

0.1 0.2 0.3 0.4 0.5

Control PAB 1 wk PAB 3 wk

Fulton Index (RV/LV +S)

10 20 30 40 50 60

Control PAB 1 wk PAB 3 wk

RV EDV (µL)

Sham Sham

* * *

p < 0.005 versus Sham

*

Nox2ds-tat: Nox2ds modified for tissue permeation

Effects of Nox2ds tat on PAB-induced hypertrophy

p < 0.005 versus Sham p< 0.005 versus PAB

*

**

20 40 60 80

Control PAB PAB Nox2ds-tat

RV EDV (µL)

**

0.1 0.2 0.3 0.4 0.5

Control PAB PAB Nox2ds-tat

Fulton Index (RV/LV+S)

**

Sham Sham

0.1 0.2 0.3 0.4 0.5 Control PAB WT PAB Nox2 null Fulton Index (RV/LV+S)

** *

50 100 150 Control PAB WT PAB Nox2 null Contractility index (1/s)

** *

10 20 30 40 50 60 Control PAB WT PAB Nox2 null RV EDV (µL)

* **

Effects of Nox2 absence on PAB-induced hypertrophy

p < 0.005 versus Sham p< 0.005 versus PAB WT

*

**

Sha m

Sham Sham Sham

Summary and Conclusions

• Nox-derived H2O2 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

Cardiac hypertrophy ?

• CamKII
• AKT
• p38-ERK

?

2-D Gel for New RV Nox2 Target Discovery

PAB 6h RV Sham 6h RV PAB 6h Nox2-/- RV

2Dige gel

1 2 3 4 5 6 7 8 9 10 12 11 14 13 16 15 17 19 18 20 21 22 23 24 25 26 27 28 29 30 31 32 34 33 35 36 37 38 39 40 41 42 44 43 45 46 47 48 49 50 51 52 5 4 53 55 56 57 58 59 60

110 kDa 96 kDa 84 kDa 75 kDa 67 kDa 50 kDa 40 kDa 30 kDa 20 kDa 14 kDa pH 4.0 5.0 5.5 6.0 6.5 7.0 8.0 150 kDa

Acknowledgements

Ri.MED Foundation NIH