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MMP in the failing heart: a potential therapeutic target. Heart failure. Heart failure is a major public health problem (1% of the total population in France).

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MMP in the failing heart:

a potential therapeutic target

Heart failure

Heart failure is a major public health problem (1% of the total population in France).

It is is a common sequel to many forms of heart diseases, with various causes including pulmonary disease, myocardial infarction, arrhythmias, anemia, renal failure, nephrotic syndrome, and thyroid disease.

It is a complex disease, with many phenotypes, but which displays two main characteristics: cardiac growth and cardiac remodeling.


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Cardiac remodeling in heart failure

Normal heart.

Hypertrophic heart.

Failing heart.

Large left ventricular chamber, thin left ventricular wall, loss of pumping capacity.

Thick walls, small ventricular chambers, increased pumping capacity.

(Freeman et al. JCI, 2001, 107:967; Limbird and Vaughan, PNAS, 1999, 96: 7125)

The remodeling process is not only due to changes in the cardiac cells but also to changes in the extracellular matrix.


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Extracellular matrix (ECM) in the heart

Scanning electron micrograph, longitudinal and transversal sections, showing collagen architecture in the left pig heart ventricle.(Zenat et al. Am J Physiol (2000) 279:H602)

The myocardial ECM contains fibrillar collagens (types 1 and 3) which ensure structural integrity of the adjoining myocytes and alignment which imparts mechanical support to the myocardium and govern tissue stiffness.

Disruption within the ECM network will lead to loss of a normal structural support and abnormal stress for cardiomyocytes during contraction. This will result in changes in myocardial geometry and function.


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Matrix metalloproteases (MMPs) in the heart

MMP-2 , MMP-9 , MMP-3, MMP-1 , MMP-13 and MT1-MMP have been identified in the human heart, and are responsible for ECM degradation.

A cause/effect relationship between MMPs and the left ventricular remodeling proces in the course of heart failure has been documented by several studies using different approaches:

- expression and activity of the cardiac MMPs in animal models of heart failure, and human failing hearts;

- studies in knocked-out and transgenic animal models;

- pharmacological studies with MMP inhibitors.


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Rapid pacing leads to left ventricular dysfunction.

Expression and activity of the cardiac MMPs increase time-dependently, together with the progression of heart failure.

Immuno blotting

Gelatin zymography

Expression and activity of MMPs in heart failure (1).

Rapid pacing in pig.

(Spinale et al. Circ.Res., 1998,82:482)

In this model, collagen content in the heart decreases.


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Non infarcted

infarcted

Expression and activity of MMPs in heart failure (2).

The infarcted mouse.

In the infarcted mouse, cardiac MMPs expression is increased.

Ducharme et al. JCI, 2000, 106:55

Increased MMPs surpass TIMPs and this occurs together with an augmentation in the collagen content.

Immunoblotting


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6 weeks

11 weeks

15 weeks

Iwanaga et al. JACC, 2002, 39:1384

DS: Dahl salt-sensitive rat

DR: Dahl salt-resistant rat

Gelatin zymography

Immunoblotting

Expression and activity of MMPs in heart failure (3).

The Dahl salt-sensitive rat.

The Dahl salt-sensitive rat develops hypertension associated with a rapidly developing heart failure when it is given a diet supplied with sodiumsalt.

In the DS rats, MMP-2 activation surpasses that of TIMPs. Myocardial fibrosis appears focally.


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Expression and activity of MMPs in heart failure (4).

The human failing heart.

Li et al. Circulation 1998, 98:1728

Thomas et al. Circulation 1998, 97:1708

Immunoblottings

Gelatin zymographies

MMP-3 and MMP-9 are increased. TIMP-1, -3 and -4 are reduced. Such an imbalance favors persistent MMP activity.


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Heling et al. Circ Res 2000, 86:846-853

Expression and activity of MMPs in heart failure (5).

The human failing heart.

The increased content in MMPs is associated with alterations of the ECM, and fibrosis.


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Immunoblottings

Total collagen was unchanged but the ratio of undenatured collagens to total soluble collagens was increased.

Expression and activity of MMPs in heart failure (6).

The human failing heart.

Support with left ventricular assist devices downregulates matrix metalloproteases, increases TIMPs and reduces collagen damage.

Li et al. Circulation 2001, 104: 1152


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Infarcted

MMP-9 KO mouse

Infarcted control mouse

Studies with knocked out animal models (1).

MMP-9 KO mouse

In the mouse, targeted deletion of cardiac MMP-9attenuates left ventricular enlargement and collagen accumulation after experimental infarction.


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At 6 weeks, increase in type 3 collagen content.

At 6 months, pathological hypertrophy with disruption of the sarcomeric architecture. However, the left ventricular pressure is increased. At 12 months, the contractile function is deteriorated.

Control heart

Transgenic MMP-1 heart

Studies with transgenic animal models (2).

Transgenic MMP-1 mouse

A mouse that constituvely expresses human MMP-1

Kim et al. JCI, 2000, 106:857

Direct disruption of the extracellular matrix, induced by the overexpression of MMP-1, reproduces changes observed in the progression of human heart failure: initial adaptative response followed by progressive loss of function.


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The possible role of MMPs in the development of cardiac fibrosis

1.MMPs are increased in late-stage experimental and human heart failure.

2.TIMPs are decreased in the failing human heart

3. A paradoxal end-result is increased MMPs accompanied with increased fibrosis.

Cardiac MMPs not only ensure degradation of matrix components, but also modulate collagen synthesis.: digestion of matrix components leads to the release of biological factors (TGFß, IGF, FGF, TNFalpha..) from the extracellular matrix which initiate neo-synthesis of collagens.

Cardiac fibrosis is not only anincrease in matrix collagen, but also changes in collagen type, organization and cross-links.


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PD-166793 fibrosis (Park Davis/ Pfizer)

Rapid pacing

(S)-2-(4’-bromo-biphenyl-4-sulfonylamino-

3-methyl butyric acid) does not inhibit TACE contrarily to Batimastat and Marimastat.

Pharmacological studies (1)

Effect of PD166793 in the pig exposed to rapid pacing

Spinale et al. Circ. Res. 1999, 85: 364


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Parameters fibrosis

Effect of PD-166793

Ventricular remodeling

Contraction

Collagen content

Attenuation

Improvement

Decrease

Pharmacological studies (2).

Effect of PD166793 in the rat with progressive heart failure.

Peterson et al. Circulation 2001, 103:2303

PD-166793 administration was initiated in 9 month-old rats and continued for 4 months.

PD-166793 treatment attenuates the degree of left ventricular remodeling in Spontaneously Hypersentive Heart Failure (SHHF).


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CP-471,474 (Pfizer) fibrosis

A broad spectrum inhibitor

Echocardiography in the infarcted mouse

Pharmacological studies (3)

Effect of CP-471,474 in the infarcted mouse

Rohde et al. Circulation 1999, 99:3063


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Pharmacological studies (4) fibrosis

Effect of CP-471,474 in the infarcted rabbit

Lindsey et al. Circulation 2002, 105:753

CP-471,474 (Pfizer) a broad spectrum inhibitor but that does not inhibit MMP-1 in rabbits which like humans, but not rodents, express MMP-1 as a major collagenase.


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Our objectives in the RTN project fibrosis

  • 1. Definition of experimental animal models of heart failure that display characteristics of the human disease, i.e., activation of MMP-3 and 9 and fibrosis.

  • - Dahl salt sensitive rats and L-NAME rats, two animal models of heart failure associated with hypertension;

    • - Infarcted rats.

2. Measurements of cardiac MMP activity and abundance in cardiac tissue fractions in the animals treated or not with MMP inhibitor;

- Fluorescent substrates and zymography;

- Immunoblotting.

  • 3. Measurements of functional parameters and disease markers in the animals treated or not with MMP inhibitor

  • - follow up of the cardiac function parameters by echocardiography;

    • - collagen cleavage assessed in tissue sections by immunostaining;

    • - oxidative stress assessed in tissue sections by immunostaining;

    • - studies of calcium cycle and contraction in isolated, electrically stimulated cardiomyocytes.


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- Echocardiographic parameters fibrosis

LVW/ BW

d PWLV

FS

(mg/g)

(mm)

(%)

DR rats (11 w)

3,5 ± 0,3

43,0 ± 0,9

0,09 ± 0,01

DS rats (11 w)

3,5 ± 0,1

0,13 ± 0,01*

50,0 ± 1,4*

DR rats (15 w)

3,3 ± 0,1

0,09 ± 0,01

42,9 ± 1,7

DS rats (15 w)

4,1 ± 0,1*

0,11 ± 0,01

39,8 ± 2,3†

- Survival

1

0.5

Animal Survival

DR rats

DS rats

0

10

12

14

Weeks

The Dahl salt sensitive rat model (1)

Functional parameters

- Blood Pressure: 220 mmHg in the Dahl salt sensitive (DS) rat as young as 11 weeks, to compare with 152 mmHg in the Dahl salt resistant (DR) rat.


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11 weeks fibrosis

Collagen staining with red sirius

DS 11 weeks

DR 11 weeks

15 weeks

DR

DS

Endogenous MMP activity

16

Collagénase activity (nU)

8

0

6

11

15

Age of the rats (weeks)

MMP-3

DS rats

DR rats

Immunoblottings

The Dahl salt sensitive rat model (2)

Fibrosis and MMPs


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Isolated cardiomyocytes fibrosisloaded with Fura-2 , electrically stimulated and exposed to a positive inotropic agent

Ca transients

0.75

F360 / F380

0.55

1200

1260

600

660

0

60

1800

1860

Contraction

8

FS %

0

0

60

1200

1260

600

660

1800

1860

Time (s)

Isolated adult rat cardiomyocytes

Immunostaining with phalloidin


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Participants fibrosis

Sylviane Adubeiro

Marie Claude Bourin

Michel Cailleret

Gabriele Candiani

Nicole Defer

Catherine Pavoine

Françoise Pecker


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