ROLE OF MATRIX METALLOPROTEINASE-9 IN ENDOTHELIAL APOPTOSIS IN CHRONIC HEART FAILURE IN MICE*

Alexander V. Ovechkin, Neetu Tyagi,

Walter E. Rodriguez, Melvin R. Hayden, Karni S. Moshal, Suresh C. Tyagi

Department of Physiology & Biophysics, School of Medicine,

University of Louisville, Louisville, KY 40202

Running head: MMP-9-/- ameliorates endocardial apoptosis in CHF Keywords: Arteriovenous fistula, endocardial dysfunction, extracellular matrix remodeling, TUNEL, connexin-43, PAR-1, ADAM-12. *A part of this study was supported by NIH grants, HL-71010 and HL-74185. Please address for correspondence: Suresh C. Tyagi, PhD. Department of Physiology & Biophysics Health Sciences Center A-1115 School of Medicine University of Louisville Louisville, KY 40202 Tel: 502-852-3381 Fax: 502-852-6239 Email: s0tyag01@louisville.edu

Articles in PresS. J Appl Physiol (August 4, 2005). doi:10.1152/japplphysiol.00442.2005

Copyright © 2005 by the American Physiological Society.

ABSTRACT: Accumulation of oxidized extracellular matrix (ECM) between

endothelium and muscle is an important risk factor in the endothelium-myocytes

(E-M) uncoupling in congestive heart failure (CHF). Although ventricular

remodeling is accompanied by increased matrix metalloproteinase-9 (MMP-9)

activity, it is unclear whether MMP-9 plays a role in endothelial apoptosis in

chronic volume overload CHF. We tested the hypothesis that in chronic volume

overload, myocardial dysfunction involves endocardial endothelial (EE) apoptosis

in response to MMP-9 activation, ECM accumulation and E-M uncoupling.

Methods: Arteriovenous fistula (AVF) was created in control (FVB/NJ, AVF

group) and MMP-9 knockout (FVB.Cg-Mmp9tm1Tvu/J, MMP9KO+AVF group)

mice. Sham surgery was used as control. Mice were grouped as follows: WT,

n=3 (sham control), MMP9KO, n=3 (sham), AVF, n=3, and MMP9KO+AVF (n=3).

Heart function was analyzed by M-mode and Doppler echocardiography, and

with a pressure tipped Millar catheter placed in the left ventricle (LV) of

anesthetized mice 8 wks after AVF. Apoptosis was detected by measuring

caspase-3, TUNEL and CD-31 by immuno-labeling. Protease activated

receptors-1 (PAR-1), connexin-43 and a disintegrin and metalloproteinase-12

(ADAM-12) expression were measured by Western blot analyses. MMP-2 and

MMP-9 expression were measured by Q-RT-PCR. Results: Compared to

control, AVF caused an increase in LVEDP and decrease in -dP/dt. In contrast, in

MMP-9KO+AVF group, these variables were changed toward control levels.

Increased EE apoptosis (caspase-3 activation and TUNEL/CD-31 co-labeling) in

AVF mice was prevented in the MMP-9KO+AVF group. PAR-1, connexin-43 and

ADAM-12 were induced in AVF. MMP-9 gene ablation ameliorated the induction.

Conclusions: The results suggest that impaired cardiac function in volume

overload is associated with EE apoptosis, cardiac remodeling and E-M

uncoupling in response to MMP-9 activation. _______________________________________________________________ Abbreviations: AVF, arteriovenous fistula; CHF, congestive heart failure; ECM, extracellular matrix; EE, endocardial endothelium; LV, left ventricle; MMP, matrix metalloproteinase; MMP9KO, MMP-9 knock out; NO, nitric oxide; PCR, polymerase chain reaction;TUNEL, terminal deoxynucleotidyl nick end labeling; WT, wild type.

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INTRODUCTION: Myocardial remodeling in congestive heart failure (CHF) is associated with

accumulation of extracellular matrix (ECM) between endothelial and muscle cells

(21) and is one of the most important factors of endocardial endothelial (EE)

dysfunction (8,14). Previous studies showed that morphological changes in

myocardium are associated with increased expression and activity of matrix

metalloproteinases (MMPs) (6, 18). MMPs degrade interstitial collagen and

elastin, but the more collagen is degraded, the more that is synthesized.

Consequently, synthesis exceeds degradation and leads to accumulation of

oxidized ECM (19, 29). ECM accumulation leads to increased endothelial to

myocyte distance (20) and, recently, we showed that activation of MMPs leads to

endothelial-myocyte uncoupling (21). There are more than 20 MMPs in the

family, but MMP-2 and MMP-9 are the most robustly increased during early and

late phase of CHF, respectively (28).

Different stimuli, including cardiac mechanical stress and overload have

been found to increase the rate of myocardial apoptosis, a process of programmed

cell death (10). It has been found that myocyte apoptosis is one of the factors in

developing of CHF, but the role of endocardial apoptosis in progression of cardiac

dysfunction remains controversial (17). The capillary endothelium, strategically

located between flowing blood and underlying cardiac muscle, plays an important

role in controlling myocardial performance (19). In fact, sixteen percent of the

myocardium consists of capillaries, including lumen and endothelium (11). We

hypothesized that in a chronic heart volume overload model and the late phase of

CHF, myocardial dysfunction involves endothelial apoptosis in response to MMP-9

activation, ECM accumulation and endothelial-myocyte (E-M) uncoupling.

Previously, we demonstrated that tissue inhibitor of metalloproteinase-4

(TIMP-4) ameliorated the endothelial cell apoptosis, in part by inhibiting MMPs

(22). To demonstrate a direct role of MMP in apoptosis, we tested the hypothesis

that ablation of MMP-9 gene can also alleviate the endothelial cell apoptosis in

heart failure.

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MATERIALS AND METHODS All mice were exposed to a similar environment and all animal procedures

were approved by the Institutional Animal Care and Use Committee of the University

of Louisville Medical Center. Male mice aged 8 to 12 weeks, MMP-9 knockout (MMP-

9KO, FVB.Cg-Mmp9tm1Tvu) and FVB/NJ controls (2), were obtained from Jackson

Laboratories (Bar Harbor, Maine). An arteriovenous fistula (AVF) was created

between the aorta and the caudal vena cava approximately 0.5 cm below the left

kidney with the use of a 30-gauge needle as described (7) in tribromoethanol-

anesthetized mice (100 mg/kg IP; n=12) (16). Mice were divided into 4 groups: 1). WT

(FVB/NJ control); 2). WT plus AVF; 3). MMP9KO; and 4). MMP-9 KO+AVF (n=3 in

each group). Because it is known that the heart became decompensated after 8-10

weeks of AVF induced chronic volume overload (21), mice were anesthetized at 8

weeks. A Millar catheter was inserted into the right common carotid artery and

advanced to the left ventricle (LV), and pressure was measured with a Micro-Med

pressure transducer. After 10 minutes of stabilization, maximum LV pressure

(LVESP), its peak time derivatives ±dP/dt, and LV end-diastolic pressure (LVEDP)

were recorded. The heart was excised under deep anesthesia. Lung, heart and body

weights were measured.

Echocardiography: A Hewlett-Packard Sonos 7770 echocardiographic

system equipped with a 15-MHz shallow-focus 15-6L phased-array transducer was

used for measurements of LV function. The transducer probe was placed on the left

hemithorax of the mice in the partial left decubitus position. Two-dimensionally

targeted M-mode echocardiograms were obtained from a short-axis view of the left

ventricle at or just below the tip of the mitral-valve leaflet, and were recorded. LV size

and the thickness of LV wall were measured. Only M-mode ECHO with well-defined

continuous interfaces of the septum and posterior wall were collected (5).

Measurements of aortic and LV blood pressure: The Millar transducer

was calibrated manually using a mercury manometer. The arterial blood pressure,

heart rate (HR), systolic and diastolic blood pressure were measured by a pressure

tipped Millar transducer (SPR-249A) inserted into the right common carotid artery.

Ten minutes after stabilization, the aortic pressure was measured by advancing the

4

catheter into the LV. The catheter was connected to a pressure transducer (Micro-

Med Corp.) positioned at the level of the heart. Ten minutes after the insertion of the

catheter, LV variables were measured (4). LVESP, LVEDP, and -dP/dt, were

measured. At the end of experiment, the anesthetized mice were prepared for the

excision of the lungs and arrested heart.

MMPs activity: Zymography using 1% gelatin gels were performed on LV

tissue homogenates as described (29).

MMP-2 and MMP-9 expression by Q-RT-PCR: DNA-free total RNA was

isolated from tissue with TRIzol reagent (GIBCO-BRL) and RNA (1µg/µl) was used for

preparation of first strand cDNA by reverse transcriptase (RT). The RNA samples

were incubated (70˚C, 5 min) with 1µl oligoprimers in a final volume of 5µl. Samples

then were incubated in 15 µl of a reaction buffer. Expression level of the RNA was

determined from 2µl of each cDNA sample. For amplification of the housekeeping

gene, glycerol aldehyde phosphate dehydrogenase (mouse GAPDH: GenBank

accession number: NM-008084). Upstream (5’-TACATTTCCCCTTCCTTACT-3’) and

down stream (3’-CCACATTTGACGTCCAGAGA-5’) primers for MMP-2 and -9 were

synthesized based on the mouse MMP-2 and -9 mRNA sequences (GenBank).

Denaturation step was set for 30s at 94°C, annealing for 60s at 60 °C and extension

for 90s cycles at 72°C. G3PDH samples were taken after 30 cycles and MMPs

samples were taken after 35 cycles. PCR reactions were performed with a Thermal

cycler (Perkin-Elmer 9600) in 50µl of 1x PCR buffer, 1.5 mM MgCl 2 , each primer at

0.2µM, 200 µM dNTP, and 1 U of Taq DNA polymerase (Invitrogen). Amplification

mixtures were analyzed by 1% agarose gel electrophoresis.

Western blot analysis of PAR-1, ADAM-12, connexin-43, and caspase-3: LV tissue homogenates were prepared, analyzed on 10% SDS-PAGE, and

transferred to PVDF membranes. Twenty five micrograms of total protein were

loaded on to each lane. The membranes were blotted with anti-caspase-3, PAR-1,

ADAM-12, or connexin-43 monoclonal antibodies (Cell signaling and Chemicon) as

previously described (4). The secondary IgG-alkaline phosphatase was used for

detection. Actin blots were used as a loading control. The bands were scanned and

5

normalized with actin intensity. The gels were stained with Commassie blue for

protein.

TUNEL and CD-31 immunohistochemical labeling: To determine whether

CHF caused endothelial and endocardial injury, 5-µm serial heart tissue sections from

snap-frozen blocks were labeled with Terminal Deoxynucleotidyl Nick End Labeling

Kit (TUNEL, Upstate cell signaling solutions, Lake Placid, NY) according to the

instructions of the manufacturer for apoptosis identification by nicked DNA labeling.

Endothelial cells were marked using FITC-labeled anti-mouse CD31/PECAM-1

antibody (Southern Biotech, Birmingham, AL) on serial tissue sections from the same

frozen blocks. Quantitative analysis was performed using Image Pro program

(IMAGE-PRO Media Cybernetics, Silver Spring, MD, USA) and data was expressed

in marker area (pixels2) averaged form five randomly selected fields per slide.

Statistical Analysis Values are reported as mean ± SEM. Differences

between groups were compared by one-way analysis of variance (ANOVA) with

repeated measurements and multiple comparisons (consideration given to within-

sample variability of observations). A probability level of p<0.05 was used to

indicate statistical significance.

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RESULTS: Hemodynamic variables: Mean arterial blood pressure (MAP) and heart

rate (HR) (67.9±0.8 mmHg, n=3 and 408.8±9 beats/min, n=3, respectively) were

not significantly different between all groups (12 animals). Although the body

weight (26.7±0.5 g) was not significantly different between all groups, the heart

weight was significantly increased in AVF mice compared to controls (WT and

MMP9KO groups) (Table 1) suggesting cardiac hypertrophy in mice with chronic

volume overload. The histological data revealed biventricular hypertrophy in AVF

mice. In addition, compared to WT, the focal fibrosis was also increased in left

and right ventricles in the animals from the AVF group (Figure 1). In contrast,

there was no hypertrophy or focal fibrosis in WT, MMP-9KO and MMP-9KO+AVF

mice.

In AVF animals, compared to controls, LVESP was decreased and LVEDP

was increased. In those animals, the -dP/dt normalized by MAP, was significantly

decreased, suggesting that LV diastolic relaxation is decreased in AVF animals

compared to controls (Table1). LV systolic dysfunction in AVF animals was

shown by LVESP/HW ratio (Table1). In contrast, both diastolic and systolic

dysfunctions were prevented in MMP9KO+AVF mice (Table1).

Role of MMP-9 in cardiac remodeling: Total MMP activity in heart tissue

was significantly increased in the AVF group compared to WT. The Q-RT-PCR

data also showed increased MMP-9 mRNA expression in heart tissue from AVF

animals (Figure 2). Although the basal levels of MMP-2 were higher in MMP-9 -/-

mice then in WT, there was a significant increase in MMP-2 expression in AVF

compared to both WT and MMP-9KO groups (Figure 2). Compared to WT, the

level of PAR-1 expression was significantly increased in AVF animals, but

ablation of MMP-9, in animals that underwent AVF, ameliorated the increase

(Figure 3). Similarly, the heart tissue expressions of ADAM-12 and connexin-43,

which significantly increased in the AVF group, were attenuated in

MMP9KO+AVF animals (Figures 4 and 5). These data suggest that AVF-induced

MMP-9 is involved in PAR-1, ADAM-12, and connexin-43 over expression.

7

Cardiac function: The LV dilatation and LV wall thickness measured by

echocardiography revealed that the LVwall/LVEDD ratio was significantly decreased

in AVF group compared to controls, and ablation of MMP-9 in MMP9KO+AVF

animals prevented this decrease (Figure 6). These data suggest a role of MMP-9

in cardiac dilatation in congestive heart failure.

Endothelial and myocyte apoptosis: There was significant induction of

caspase-3 in AVF hearts as compared to WT or MMP-9KO, with or without AVF

(Figure 7). There were higher numbers of TUNEL positive cells (Figure 8) in

heart tissue of AVF animals compared to controls. Although the general

morphological appearance of the MMP-9KO/AVF heart tissue (Figure 8, panel C)

may appear to be different from WT panel, there was no quantitative or

qualitative difference when compared with MMP-9KO alone (no AVF), or WT.

The increase in caspase-3 activation and TUNEL was prevented in

MMP9KO+AVF animals. The co-labeling of TUNEL and CD-31, as a marker for

endothelial cells in serial heart tissue sections, demonstrated that endothelial

cells underwent apoptosis (Figure 9). Based on TUNEL positive and CD-31

positive versus CD-31 negative cells, we estimated that 75% TUNEL positive

cells were endothelial origin (Table II). Besides those cells, there were other

TUNEL positive cells, suggesting that endothelial, as well as myocyte apoptosis,

is involved in this chronic heart failure model (Table II). The induction of caspase-

3 and in situ TUNEL in the LV of AVF suggest involvement of MMP-9 in

transduction of apoptosis.

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DISCUSSION

We and others suggested the role of endocardial apoptosis in the cardiac

dysfunction in congestive heart failure (27, 31). However, the mechanism of this

phenomenon is still unknown (1, 18). The results of the present study show that

impaired cardiac function in chronic volume overload CHF is associated with

endothelial and myocyte apoptosis and is accompanied by endothelial-myocyte

uncoupling in response to MMP-9 activation. MMPs are potential mediators of

cardiac remodeling and progression to heart failure. Previously, we showed that

during the compensatory phase of CHF, MMP-2 is increased and MMP-9 is over

expressed (23). Others have suggested that targeted deletion of MMP genes in

mice attenuates cardiac remodeling in acute (6, 9, 26) and in LV pressure

overload heart failure models (10). In the present study we demonstrated

molecular and cellular mechanisms that mediate the pathogenesis of chronic

heart failure, and the involvement of MMPs in cardiac remodeling and

dysfunction.

The histological and gravimetrical data suggested that cardiac hypertrophy

in mice with chronic volume overload heart failure were associated with ECM

accumulation (Figure 1). We obtained histological data on MMP-9KO similar to

the data obtained by others (6) and found that there was no significant difference

in MMP-9KO (control and AVF). The total MMP-2 and MMP-9 expressions were

significantly increased in animals from the AVF group (Figure 2). We measured

total MMP and MMP-9 expression in MMP-9KO animals (control and AVF), and

found that there was no expression of MMP-9 in control and AVF. However,

there was increased MMP-2 expression (Figure 2). This finding is in part due to a

compensatory response to MMP-9 ablation (6). In addition, the lung weight from

the AVF animals was significantly higher then controls, suggesting pulmonary

edema (Table 1). A significant decrease in the LVwall/LVEDD ratio, changes in

LVESP and LVEDP, decrease in LV contraction and relaxation suggested that

cardiac function in those animals was also impaired (Table 1 and Figure 6). The

ablation of MMP-9 in animals with AVF significantly normalized LV parameters,

and clearly indicates that MMP-9 plays a crucial role in pathogenesis of chronic

9

heart failure. These results are consistent with previous reports that MMP-9

inhibition, or targeted deletion of the MMP-9 gene in mice, attenuates LV dilation

and decreases collagen accumulation in the infarcted area after experimental MI

(6) or after AVF (3) or after pressure overload (10).

Our hypothesis was that impaired cardiac function in CHF could be the

consequence of endothelial apoptosis in response to E-M uncoupling due to

ECM accumulation in the myocardium (Figure 10). We found that the levels of

caspase-3 activation and numbers of TUNEL positive cells in heart tissue of AVF

animals were significantly higher than in controls (Figures 7 and 8). Serial heart

tissue sections were immunochemically developed with CD-31 antibody as a

label for endothelial cells and TUNEL for apoptosis. This TUNEL/CD-31 co-

labeling showed that endothelial, as well as myocyte apoptosis, is involved in

pathogenesis of CHF (Figure 9). Previously, we reported that activated PAR-1

lead to the cardiac remodeling (21) and inhibition of MMPs prevented connexin-

43 degradation and ameliorated heart failure (13). In the present study, we found

that besides increased expression of PAR-1, ADAM-12 (a disintegrin and

metalloprotease), enzyme involved in cell adhesion and myoblast differentiation

and fusion (15), was also over expressed in AVF animals. This finding suggests

that increased activity of PAR-1 could lead activation of several proteolytic

events. It was shown previously that reduced Connexin-43 expression produces

uncoupling between myocytes (24). In our model, increased expression of

connexin-43 might be associated with E-M uncoupling due to a compensatory

response to cell disintegration by ADAM-12. However, further studies are needed

to confirm the relationship between PAR-1 and connexin-43 remodeling by

ADAM-12. The results of our experiments show that in animals from

MMP9KO+AVF, the increase in the levels of apoptosis was prevented (Figures 8

and 9). In addition, PAR-1, ADAM-12 and connexin-43 expressions were also

normalized (Figures 3-5).

Limitations: TUNEL detection alone is not enough to confirm apoptotic

death (12, 25, and 30). Therefore, we measured the levels of caspase-3, and

showed that a caspase-3 inhibitor ameliorated the endothelial cell apoptosis (22).

10

Here, we suggested that caspase-3 was significantly induced in AVF hearts as

compared with the sham or the MMP-9-/- mouse hearts with and without AVF

(Figure 7). These results suggested apoptosis in the AVF hearts, in part, due to

activation of caspase-3. The connection between PAR-1 and connexin-43 may not be strong.

However, in light of our human heart end-stage failure data, in which we showed

that connexin-43 synthesis and degradation both were increased, and MMPs

were activated in CHF (22). It is a logical extension of the idea that volume

overload stress activated the MMP that in turn activated PAR-1, leading to

induction of connexin-43 in AVF.

In summary, the results of the present study demonstrate that in chronic

volume overload model of CHF in mice, impaired cardiac function is associated

with endothelial and myocyte apoptosis. The mechanism of these phenomena

involves activation of MMP-9, leading to cardiac remodeling and E-M uncoupling.

Our study also shows that MMP-9 has a major role in ventricular remodeling

associated with endothelial and myocyte apoptosis (Figure 10).

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Table I:

Gravimetric and hemodynamic data

WT (n=3)

MMP9KO (n=3)

AVF (n=3)

MMP9KO+AVF (n=3)

Body weight, g 27.3 ± 0.4 26.2±0.5 27.1±0.6 26.3±0.3

Heart weight, g 0.15 ± 0.012 0.16±0.013 0.232±0.025* 0.18±0.014

Lungs weight, g 0.32 ± 0.04 0.31±0.05 0.54±0.07* 0.39±0.06

Heart rate, b/min 409.2 ± 9.5 428.3 ±8.9 398.5 ±10.1 399.2 ±7.4

LVEDP, mmHg 1.2 ± 0.6 1.4 ±1.0 8.4 ±2.9* 2.6 ±0.8

LVESP, mmHg 112.7 ± 1.6 110.9 ±2.0 96.4 ±1.8* 99.6 ±1.3

LVESP/Heart Weight 746.7 ± 133.3

687.5 ± 153.9 414.4 ± 72* 559.7 ± 92.3

MAP, mmHg 69.8 ± 0.7 67.7 ± 0.6 71.8 ± 1.1 62.3 ± 0.7

-dP/dt, mmHg/s 5491 ± 201 4838 ± 288 3156 ± 243* 4391 ± 298

+dP/dt, mmHg/s 9050 ± 435 8236 ± 378 7666 ± 443 7997 ± 478

(-dP/dt)/MAP(sec-1) 81.7 ± 6.3 82.4 ± 3.2 40.4 ± 4.1* 68.9 ± 8.3

LVEDd (mm) 2.81 ± 0.08 2.78 ± 0.07 3.91 ± 0.11* 2.91 ± 0.08

LVEDs (mm) 1.69 ± 0.07 1.65 ± 0.07 2.79 ± 0.12* 1.83 ± 0.09

LVPWd (mm) 0.74 ± 0.03 0.72 ± 0.04 0.85 ± 0.05* 0.79 ± 0.05

LVPWs (mm) 1.12 ± 0.03 1.08 ± 0.03 1.29 ± 0.04* 1.08 ± 0.03

FS, % 39.86 ± 0.85 40.65 ± 1.1 28.65 ± 1.03* 37.45 ± 1.33 Values are means ± SEM; n=number of animals. LVEDP and LVESP, left

ventricular end-diastolic and end-systolic pressure, respectively; MAP, mean

arterial pressure; LVEDd and LVEDd, left ventricular end-diastolic and end-

systolic diameter, respectively; LVPWd and LVPWs, left ventricular end-diastolic

and end-systolic wall thickness, , respectively; FS, fractional shortening. *

indicates p<0.05. Note that there were no significant differences between the

data obtained from WT, MMP9KO and MMP9KO+AVF groups.

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Table II:

Morphological data

WT (n=3)

MMP9KO (n=3)

AVF (n=3)

MMP9KO+AVF (n=3)

CD31 positive cells/field 2456 ± 232 2277 ± 256 1845 ± 296 1978 ± 273

TUNEL positive cells/field 103 ± 23 87 ± 26 978 ± 32* 198 ± 39†

CD31/TUNEL co- localized cells/field 63 ± 15 56 ± 18 645 ± 22* 135 ± 26†

Values are means ± SEM; n=number of animals. * indicates p<0.05 compared to

all groups. † indicates p<0.05 compared to WT or MMP9KO group.

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Legend to Figures: Figure 1: Histological analysis of left ventricle (LV) at 8 wks after AVF.

Frozen tissue sections were labeled with trichrome for collagen. A, WT; B, AVF

mice. Arrows indicate deposition of collagen (blue). Note that the heart with AVF

is hypertrophic. Regions shown in inset boxes were observed using 20 X

magnifications. C, WT; D, AVF mice. Note greater collagen expression in over

loaded heart.

Figure 2: Effects of AVF on total MMP activity (A). MMP-9 (B) and MMP-2

gene expression (C) in the LV by Q-RT-PCR analysis; Mean±SEM; *, p<0.05

compared to all groups; †, p<0.05 compared to all groups except MMP9KO

group.

Figure 3: Comparison of effects of AVF on PAR-1 expression in LV by

Western blot analysis; Mean±SEM; n=3 in each group; *, p<0.05 compared to all

groups. ∧, p<0.05 compared to all groups except AVF. Note that increased PAR-

1 expression in AVF group was prevented in MMP9KO+AVF group.

Figure 4: Comparison of effects of AVF on ADAM-12 expression in LV by

Western blot analysis; Mean±SEM; n=3 in each group; *, p<0.05. Note that

increased ADAM-12 expression in AVF group was prevented in MMP9KO+AVF

group.

Figure 5: Comparison of effects of AVF on Connexin-43 expression in LV

by Western blot analysis; Mean±SEM; n=3 in each group; *, p<0.05. Note that

increased Connexin-43 expression in AVF group was prevented in

MMP9KO+AVF group.

Figure 6: Two-dimensional M-mode echocardiogram of LV (A, WT group;

B, AVF group; C, MMP9KO+AVF group) and comparison of effects of AVF on LV

function (D). Mean±SEM; *, p=0.005. Note that increase in LV end-diastolic

diameter (LVEDD) in AVF animal (B) was prevented in MMP9KO+AVF animal

(C). Figure 7: Comparison of effects of AVF on Caspase-3 expression in LV

by Western blot analysis; Mean±SEM; n=3 in each group; *, p<0.05. Note that

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increased caspase-3 expression in AVF group was prevented in MMP9KO+AVF

group.

Figure 8: Effect of AVF on endocardial apoptosis (TUNEL assay) in LV

(A, WT group; B, AVF group; C, MMP9KO+AVF group). D, quantitative

presentation of TUNEL marker expression; n=3 in each group. Scale bars

indicate means ± SEM. * Indicates p<0.0025 compared to all groups.

Figure 9: Comparison of immunofluorescence staining for apoptotic cells

(TUNEL assay) in LV. A, in AVF group; B, in MMP9KO+AVF group. Regions

shown were enlarged 6X. Bright green spots show positive immunostaining for

apoptotic cells. Red spots show positive immunostaining for CD-31 (endothelial

marker). Note greater EE apoptosis in AVF group (A) compared to

MMP9KO+AVF group (B).

Figure 10: Overall hypothesis: stress and progression of CHF are

associated with activation of latent MMP which activates PAR receptors and

subsequent activate ADAM-12. This, in turn, induces MMP-9 and ADAM-12

dependent oxidized ECM accumulation, leading to E-M uncoupling and

endothelial apoptosis.

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