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European Journal of Heart Failure 2007 9(5):450-457; doi:10.1016/j.ejheart.2006.12.008
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© 2006 European society of Cardiology

Oxidative remodeling in pressure overload induced chronic heart failure{star}

Brooke C. Henderson*, Neetu Tyagi, Alexander Ovechkin, Ganesh K. Kartha, Karni S. Moshal and Suresh C. Tyagi

Department of Physiology and Biophysics University of Louisville Louisville, KY 40202, USA

* Corresponding author. 500 South Preston Road, HSC 1115A, Louisville, KY 40202, USA. Fax: +1 502 852 6239. E-mail address: bchend02{at}louisville.edu


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 
Despite extensive strides in understanding pressure overload induced heart failure, there is very little known about oxidative stress induced matrix metalloproteinase (MMP) activation, collagen degradation and remodeling in pressure overload heart failure. We hypothesize that pressure overload leads to redox imbalance causing increased expression/activity of MMP-2/9 producing collagen degradation and heart failure. To test this hypothesis, we created pressure overload heart failure by abdominal aortic stenosis (AS) in wild-type C57BL/6J and collagen mutant (Col1a1 with 129 s background) mice. At 4 weeks, post surgery, functional parameters were measured. Left ventricle (LV) tissue sections were analyzed by histology, Western Blot and PCR. The results suggest an increase in iNOS with a decrease in eNOS, an increase in nitrated protein modification and depletion of antioxidants thioredoxin and SOD in pressure overload. MMP-2/9 expression/activity and collagen degradation were increased in the AS animals. To determine whether a mutation in the collagen gene at the site of MMP cleavage mitigates cardiac hypertrophy, we used Col1a1 mice. In these mice, the AS induced LV hypertrophy (LVH) was ameliorated. In conclusion, our results suggest that AS leads to increased oxidative stress, expression/activity of MMP-2/9 and a decrease in antioxidant expression producing collagen degradation and heart failure.

Key Words: MMP • Collagen • SOD • Thioredoxin • iNOS • eNOS

Received July 19, 2006; Revised September 28, 2006; Accepted December 14, 2006


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 
Heart failure is a dynamic multifactorial process, which affects many people. Cardiac remodeling is known to be the "compensatory recovery" of the heart to an insult, i.e. increased pressure or volume, imposed on it. Pressure overload increases afterload on the heart that can elicit tissue damage [1], mechanical deformations [2] and arterial oxidative stress caused by several mechanisms. This can include direct activation of a PKC-dependant NAD(P)H oxidase pathway [3]. Mild oxidative stress may alter antioxidants by inducing or repressing proteins that participate in these systems. Depleting cellular stores of antioxidant materials [4] and/or inducing a negative correlation [5] are growing trends with the progression of heart failure because there are less antioxidants available to counteract the increasing presence of oxidative stress. Without a redox balance, there is a pro-oxidant environment, which could potentially be harmful.

Pressure overload is known to elicit hypertrophy, which is a compensatory response of the heart to a sustained increase in cardiac work [6,7]. Therefore, mechanical stretch and cellular responses [7] are thought to be two major influences ultimately leading to hypertrophy. However, the heart can only compensate for so long until the transition to decompensated heart failure is complete. This mechanism is still poorly understood.

Matrix metalloproteinases (MMPs) are well documented proteinases that can degrade collagen, which is mainly type I (85%) in the heart [8,9]. This collagen surrounds the cardiomyocytes within the heart to provide alignment and structural integrity [10]. MMP expression is regulated by a host of factors [11,12] but is mostly inducible i.e. MMP-9 [13]. Tissue inhibitors of matrix metalloproteinases (TIMPs) are known to prevent activity of MMPs by binding to them [14]. A stringent balance normally exists between proteinases and anti-proteinases in structural matrix protein turnover [14] but in pressure overload heart failure, more stretch [15] is present leading to an enhanced presence of MMP-2/9 in the tissue. Their enhanced presence and activity can lead to cell-cell interactions [11], degradation of the collagen network, and a decrease in the elastin/collagen ratio resulting in cardiac stiffness [16]. Collagen degradation is required for the creation of new integrin binding sites necessary for cell survival. However, a complete separation between the matrix and the cell leads to apoptosis, dilatation and failure [10]. Oxidative stress is thought to play a key role in this "shifting" process [17] by activating MMPs [18] which can lead to tissue and/or extracellular matrix damage. Oxidative stress accelerates heart failure [19] by regulating an increase in both collagen synthesis and MMP-2/9 [20].

Therefore, in this study we cataloged potential changes found in the mice with pressure overload at week four. Since progression and development of heart failure parallels oxidative bursts [7] and MMP activation [18], we propose that pressure overload activates MMP-2/9 by depressing antioxidants and increasing oxidative stress leading to cardiac dysfunction. The mutation in collagen to resist collagenase activity mitigates cardiac dysfunction.


   2. Materials and methods
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 5. Limitations
 References
 
2.1. Animals
Wild-type C57BL/6J and collagen mutant (Col1a1 with 129 s background is the strain type; strain name B6;129S4-Col1a1tm1Jae/J) 8-10 week old male mice were obtained from Jackson Laboratories (Bar Harbor, Maine). The collagen mutant mice had peptide bonds between the Gly775-Ile776 of the {alpha}1(I) chain and the Gly775-Leu776 {alpha}2(I) chain that the Ile/Leu were mutated to proline. This makes the collagen resistant to collagenase digestion. Except for dermal fibrosis, these mice have normal physiological function [21]. Animal protocols were performed in accordance to the National Institutes of Health guidelines and approved by the IACUC of the University Of Louisville School Of Medicine. All animals were fed rodent chow and housed in cycle of 12 h light and 12 h dark conditions. The room was kept at 24 °C.

2.2. Pressure overload model
Pressure overload heart failure was induced by abdominal aortic banding above the left kidney. The mice were anesthetized with 2, 2, 2 tribromoethanol (0.2 ml/10 g) (Sigma-Aldrich, St. Louis, MO). A midline incision was made to expose the abdominal area. The abdominal aorta was constricted by tightening a 6-0 nylon suture against a 25-gauge blunted needle with the needle being removed after the suture was secure. The sham animals underwent the same procedure without the banding. The incision was closed in layers and the animals were allowed to recover. Animals were weighed and harvested at 2 and 4 weeks for heart, kidneys, lungs and blood. The tissues were either snap frozen in liquid nitrogen or placed in 10% formalin.

2.3. Measurement of echocardiography, electrocardiography, and diastolic function
A Hewlett-Packard Sonos 5500 echocardiographic system equipped with a 15-MHz shallow-focus 15-6 L phased-array transducer was used for measurements of LV function on anesthetized mice. This was done at 2 weeks and 4 weeks immediately prior to sacrifice. The M-mode selection allowed for the visualization of systole and diastole within the beating heart. Also from this picture LV wall thickness and diameter were assessed as well as ejection fraction (EDD-ESD/EDD).

Electrocardiography was performed using 3 leads (front and hind legs) connected to the Sinus Rhythm Analyzer (Digi-Med®). The RR intervals were collected and stored in DMSI-200 © 1995 Micro-Med, Inc Louisville, KY.

Diastolic function and arterial pressure were measured using the Heart Performance Analyzer by Digi-Med®. The data for –dP/dt were collected and stored in DMSI-200 © 1995 Micro-Med, Inc Louisville, KY.

2.4. Cardiac rings
Cardiac rings were made (to determine stretch-strain relationship) by immediately cutting the mouse heart into 1-2 mm sections and placing them on rings immersed in a bath of physiological solution (in mM) NaCl, 131.5; KCl, 1.2; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 23.5; and glucose, 11.2). Tension was increased up to 3 g for each ring and this was converted into pressure. Stretch up to 0.5 mm was measured from the increases in tension to the ring and this was converted into volume as described previously [22]. To minimize the injury time to cardiac muscle, the contractility of the heart in the cardiac rings was measured immediately after extracting the heart.

2.5. Western blot
Collagen, nitrotyrosine, SOD, and TIMP-4 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), eNOS and iNOS from BD Biosciences (Franklin Lakes, NJ) and elastin antibody from Chemicon (Temecula, CA). The LV extracts were prepared in an overnight cacodylic acid extracellular buffer (10 mM/L cacodylic acid pH 5.0, 8.76 g/L NaCl, 5 mM/L ZnCl2, 2.22 g/L CaCl2, 0.098 g/L NaN3, 100 µL/L Triton X-100). The samples were assessed for protein concentration with a Bio-Rad assay (Philadelphia, PA) using the Bradford method [23]. 20 µg of the protein were mixed with equal volumes of reducing 1xSDS sample buffer. They were electrophoresed on 10% SDS-PAGE gels. The gel was then blotted onto a polyvinylidene difluoride membrane. After being transferred, blots were washed with Tris-buffered saline (TBS) for 5 min at room temperature and incubated in blocking buffer of 5% milk in Tris-buffered saline with 1% tween (TBST) for 1 h at room temperature. The blots were then incubated with the indicated primary antibodies (3% milk in TBST) overnight at 4 °C using slow rocking. The blots were washed three times (for 15-min each) with TBST and incubated with HRP-conjugated secondary antibody (3% milk in TBST). After being washed three times (for 15-min each) with TBST, the proteins of interest were detected using an ECL plus kit (Amersham Biosciences, Piscataway, NJ). The membranes were then stripped using 0.2 M NaOH/0.1% SDS solution for 30 min at room temperature and reprobed for β-actin. Band densities were normalized to the untreated controls and presented as percentage changes.

2.6. Gelatin zymography
MMP activity was measured using gelatin zymography. The LV extracts were prepared as previously mentioned. Protein concentration was measured using the Bradford method [23]. 20 µg of the protein was electrophoresed on 8% SDS-PAGE gels containing 15 mg/ml gelatin as a substrate at constant voltage. After electrophoresis, the gels were rinsed in renaturation buffer (2.5% Triton X-100) on a shaker for 30 min to remove SDS and then incubated overnight at 37 °C in a water bath with activation buffer (in mM 50 Tris.HCl, pH 7.4, and 5 CaCl2). Gels were stained using 0.5% Coomassie blue R-250, followed by appropriate destaining. MMP activity was detected as a white band on a dark blue background and quantitated densitometrically using Un-Scan-It software (Silk Scientific, Orem, UT).

2.7. Semi-quantitative RT-PCR
Frozen tissue was homogenized in a microdismembrator and mRNA extracted using TRIzol reagent (Invitrogen, USA) following the manufacturer's procedure. The cDNA was synthesized from 1 µg total RNA in a final reaction volume of 20 µl by reverse transcriptase (RT). The RNA samples were incubated (70 °C, 5 min) with 1 µl oligo (deoxythymidine) primers in a final volume of 5 µl. Samples were then incubated (42 °C, 60 min) in 15 µl of reaction buffer (1x) containing MgCl2; 5 mM, deoxynu-cleotide triphosphates (dNTPs; 10 mM) and Im-Prom-II– reverse transcriptase (1 µl/15 µl reaction) and rRNasin (Promega, Madison ) as RNase-inhibitor (20 U) 15 µl. The reaction volumes were brought up to 15 µl with autoclaved water. A negative control was included.

Each reaction was run in a volume of 25 µl. Primers were added to a final concentration of 0.2 µmol/l to the reaction mixture consisting of cDNA (1 µl per reaction), dNTP(6.25 mmol/l), MgCl2 (1.5 mmol/l), 2.5 µl 10x PCR buffer and 0.02 UTaq polymerase (all promega). The primer for MMP9 and MMP2, forward 5'-TACAGGGCCCCTTCCTTACT-3' and reverse 5'-CCACATTTGACGTCCAGAGA-3' forward 5'-AGGTGGACCATGAGGTGAAC-3' and reverse 5'-CGGTTGAAGCAAAGAAGGAG-3' primers gave amplification product of 116 and 98 base pairs respectively, while the thioredoxin primer, forward 5'-TGGACTTCTCTGCTACGTGGT-3'' reverse5'-CCTTGTTAGCACCGGAGAAC-3'' gave a product of 210 base pairs. The GAPDH forward primer 5'-ACAACTTTGGCATTGTGGAA-3') and reverse 5'-GATGCAGGGATGATGTTCTG-3' gave a product of 133 base pairs. The PCR reaction was performed with hot start at 94 °C for 2 min, followed by cycles of 30 s at 94 °C, 30 s at 57 °C and 1 min at 72 °C with final extension at 72 °C for 5 min. PCR reaction was analyzed in a 1% agarose gel electrophoresis containing ethidium bromide. GAPDH was used as a loading control.

2.8. Histology
The heart section was placed in 10% formalin. The section then underwent a graded alcohol dehydration process. It was then fixed to a holder using paraplast and left overnight. Sections were then cut into 3 mm sections and stained with Sirius red to detect for hypertrophy. Optical light microscopy was performed at 40x magnification.

2.9. Statistical analysis
A sample size of n=6 was used in each group. The data are presented as the mean+/–SE. The difference was calculated statistically with a significance level of p<0.05 using ANOVA followed by Bonferroni post hoc test.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 5. Limitations
 References
 
3.1. Gravimetric and hemodynamic parameters, and diastolic function
The lung weight and heart weight (HW)/body weight (BW) ratio were increased in AS groups as compared to sham, suggesting LVH and congestive heart failure in AS groups (Table 1). To determine the diastolic rate of relaxation in these animals, we measured (–dP/dt/MAP). This index of relaxation was significantly suppressed in the AS animal compared to control (Table 1). These results suggest an impairment of diastolic function in the AS animals.


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  		Table 1 Mean gravimetric and echocardiographic parameters and mean arterial pressure in sham and aortic stenosis (AS) model at 4weeks

 
3.2. Echocardiography, EKG and oxidative stress
To determine whether an increase in MMP and a decrease in TIMP-4 are associated with LV dysfunction, cardiac function was monitored by both echocardiography and EKG. The echocardiogram showed an increase in left ventricle diameter (Table 1) for the AS animals in systole as well as a deviated wave pattern that was not present in the sham animals (Fig. 1A). The EKG remained normal for the sham group at 4 weeks; however, the AS animals showed a decrease in RR intervals and an arrhythmogenic heart rate compared to sham (Fig. 1B, Table 1). In addition, there was an increase in total nitrated protein levels in the AS compared to sham (Fig. 1C), suggesting more oxidative stress and reactive nitrogen species (RNS) generation due to the pressure overload.


Figure 01
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  		Fig. 1 A: Echocardiography of sham and AS. B: EKG analysis of sham and AS, showing arrhythmogenesis (arrow) in AS. C: Western blot of total nitrated proteins: lane 1, sham; lane 2, AS; β-actin for equal loading. D: Graph represents scan data for band intensities (A–E at 4weeks); *p<0.05; n=6.

 
3.3. Oxidative stress
The levels of thioredoxin and Cu/Zn SOD were decreased in the AS groups compared to sham (Fig. 2A-B). Also the levels of eNOS (Fig. 2C) was found to be decreased while there was an increase in iNOS expression (Fig. 2D) in the AS group compared to sham.


Figure 02
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  		Fig. 2 A: PCR of thioredoxin (TRX): lane 1, molecular weight marker (Mr); lane 2, sham; lane 3, aortic stenosis (AS). B: Western blot of Cu/Zn superoxide dismutase (Cu/ZnSOD). C: Western blot of eNOS. D: Western blot of iNOS. For B–D: lane 1, sham; lane 2, AS (B–D at 4weeks); GAPDH and β-actin for equal loading; graphs represent scan data for band intensities; *p<0.05; n=6.

 
3.4. Extracellular matrix activity
The zymographic analysis revealed an increase in MMP-2/9 activities at 2 and 4 weeks in the AS animals compared to sham animals (Fig. 3A). These levels of MMP-2 and -9 were also induced and increased at the mRNA expression levels for the AS animals (Fig. 3B-C). The levels of cardiospecific TIMP (TIMP-4) was decreased significantly in the AS groups compared to sham (Fig. 3D), suggesting matrix degradation in pressure overload animals.


Figure 03
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  		Fig. 3 A: Zymography of MMP activity: lane 1, sham; lane 2, AS (at 2weeks); lane 3, AS (at 4weeks). B & C: PCR of MMP-2 and -9, respectively. D: Western blot of TIMP-4. For B–D: lane 1, sham; lane 2, AS (B–D at 4weeks); GAPDH and β-actin for equal loading; graphs represent scan data for band intensities; *p<0.05; n=6.

 
3.5. Extracellular matrix composition
To determine whether an increase in MMP activation causes collagen degradation, we measured collagen and elastin protein expression. Collagen degradation was noticed at week 2 but more pronounced at week 4 in the AS animals compared to sham (Fig. 4A). Concomitantly, the elastin expression was decreased in week 4 AS animals compared to sham (Fig. 4B).


Figure 04
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  		Fig. 4 A: Western blot of collagen: lane 1, sham; lane 2, AS animal (at 2weeks); lane 3, AS animal (at 4weeks). B: Western blot of elastin: lane 1, sham; lane 2, AS (at 4weeks). For A–B: β-actin for equal loading; graphs represent scan data for band intensities; *p<0.05; n=6.

 
3.6. Heart structure and function
Histological data suggest biventricular hypertrophy in the AS hearts as compared to sham hearts (Fig. 5A). Although we used Sirius Red to show histological evidence of collagen fibrosis, we quantified collagen type I and elastin contents by Western blot analysis in hearts of sham mice and AS mice (Fig. 4A-B). Also, there was a converse relationship between the levels of collagen (a stiffer protein) and levels of elastin (a flexible protein). To determine compliance (passive diastolic function) in these animals, we measured cardiac stretch-strain relationship. The results suggested that cardiac stiffness is present in the AS model and reduced in the sham animals (Fig. 5C). However, there was a rightward shift in the sham with collagen mutant and collagen mutant with AS. This suggests that the collagen mutant, with and without an increased pressure, have improved diastolic function compared to both the wild-type sham and AS animals. To determine whether a mutation in collagen mitigates the cardiac wall stress, we measured this relationship in all four groups. There was an increase in stress with the AS animals compared to sham, sham with collagen mutant and collagen mutant with AS (Fig. 5D). This suggests that the collagen mutant with the site of MMP cleavage being altered has intact collagen, minimal left ventricle hypertrophy and wall stress.


Figure 05
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  		Fig. 5 A: Formalin-fixed tissue section analysis by histological staining with Sirius red for sham and AS at week 4. B: HW/BW graph for sham, AS, transgene (TG-collagen mutant)+sham and TG+AS groups. C: Passive diastolic function for sham, AS, TG+sham, and TG+AS. D: LV systolic wall stress for sham, AS, TG+sham, and TG+AS. Each bar (B, D) and point (C) represent 4week animals with mean±standard error; *p<0.05; n=6.

 

   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
5. Limitations
 References
 
The hypothesis that pressure overload leads to increased oxidative stress and increased expression/activity of MMP-2/9, leading to myocardial hypertrophy and heart failure has been suggested previously [20,24-26]; however, the present study extends the notion that oxidative stress MMP activation with collagen degradation is a pre-requisite for LVH and heart failure. To differentiate LV dysfunction from heart failure, we showed evidence for higher lung weights due to pulmonary edema in mice with pressure overload. Echocardiography also showed increases in LV diameter in these animals. However, in the echocardiographic data obtained 2 weeks after banding, we did not find any significant changes.

Results of this study suggest an increase in oxidative stress (OS) in animals with pressure overload induced heart failure at week four. The presence of increased OS has been documented to be caused by several factors in vivo and in vitro, such as up regulation of the NADPH pro-oxidant system [3] and disruption of mitochondrial electron transport chain components [27]. However, no definite trigger of OS has been found, only potential sources of increased oxidative stress. Our data suggest that there is a dynamic shift in expression of potential mediators of OS. First, there was a decrease in the protein expression of both thioredoxin and SOD that was consistent with work done by Kishmoto [5] and Thomas [4]. This antioxidant depletion suggests an imbalance in the normal redox turnover. Ma et al. [28] suggested that treatment with an anti-ischemic agent not only increased levels of SOD but also improved systolic function. Such work has also been done with ACE inhibitors and produced similar findings [29]. Both studies suggest that treatment with drugs alleviates OS production, but the underlying mechanism which triggers this increased reactive oxygen species production remains unknown. Secondly, there was an increase in protein modifications by nitration that could be potentially due to the decrease in eNOS and increase in iNOS activity. This suggests a depletion of bioavailable NO that is needed to promote diastolic relaxation and/or endothelial function [27]. Moreover, this could potentiate a vicious paradigm of increased OS with decreased NO that supports a shift to failure in the heart. Studies by Kodja et al. [27] concluded that peroxynitrite, a byproduct of NO and reactive oxygen species, can inhibit mitochondrial electron transport and enhance production of hydrogen peroxide.

Pressure overload induced increase in oxidative stress parallels increase in expression and activity of MMP-2/9 and hypertrophy. It has been well documented that OS is present in the failing heart [17] and can activate MMPs [30]. Also, in pressure overloaded animal there is a presence of left ventricular hypertrophy [6,7]. MMPs, especially MMP-2/9, are known to digest collagen [21]. We found that collagen degradation happened as early as week 2 and the elasticity of the ventricle decreased as early as week 4, thus decreasing the elastin/collagen ratio and compliance of the heart. Tyagi et al. [10], described the need for collagen-cardiomyocyte alignment for proper functioning of the heart. If this alignment is impaired or compromised, this can lead to cardiac dysfunction and eventually to heart failure. Potential misalignment and systolic and/or diastolic failure was observed in the animals in our study.

Previously, we have shown a relationship between myocardial stiffness, MMP activation and protein nitration [17]. Therefore, we created pressure overload in collagen mutant mice to determine a cause and effect relationship between myocardial collagen degradation, stiffness, oxidative stress, hypertrophy, MMP activation, altered expression of NOS, and protein nitration. Although there was depressed cardiac function in collagen mutant mice as compared to wild-type sham and AS animals, in the collagen mutant mice with AS, pressure overload did not induce LVH or cardiac dysfunction. The endpoint of maintained diastolic function was assessed by ex vivo passive length-tension relationship. This data suggests that although diastolic function was decreased in the collagen mutant mice, there was no further deterioration of diastolic function after AS. These data provide evidence that collagen degradation, in part, causes cardiac diastolic dysfunction in pressure overload hypertrophy.

Echocardiography showed increases in ventricular diameter in the AS mice by week four, suggesting the onset of heart failure and also revealed that the AS animals were not ejecting as much blood and the aortic flow was decreased. EKG showed a depressed QR wave and extended ST wave in the AS animals. This extended ST wave is thought to be a characteristic of impaired repolarization, which was confirmed by the decrease in –dP/dt/MAP. Also, in order to maintain adequate blood flow into the circulation, heart rate (HR) in the AS animals was increased, suggesting compensation for the increase in pressure and decrease in ejection fraction. In this case, the echo parameters revealed a decrease in aortic flow with the AS animals.


   5. Limitations
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
References
 
Ideally to test the hypothesis that hypertension leads to an increase in OS, (and thus to an increase in MMP-2/9 expression which leads to heart failure), we needed to show a direct connection between OS and MMP-2/9. This could have been achieved by using p47phox (–/–) mice, and showing that in these mice reduced OS leads to reduced MMP-2/9 expression and collagen degradation after pressure overload. Furthermore, one would expect experiments using cardiac fibroblasts to show that OS leads to reduced collagen degradation in col1a1-mutant mice. This would have provided a mechanistic link between OS, MMP2/9, and collagen degradation. However, our hypothesis proves that in vivo pressure overload in collagen mutant mice leads to increased oxidative stress causing MMP activation and collagen degradation, which instigates LVH and heart failure. Therefore, the col1a1 mouse model was used to prove this hypothesis.

This study does not explain whether collagen degradation indeed occurs downstream of oxidant stress or if it plays a significant role not only in myocardial stiffness but also in myocardial hypertrophy and remodeling. The goal of this study was to determine the role of collagen degradation in cardiac hypertrophy, and we therefore measured LV function and LVH in collagen mutant mice. It would be interesting to examine the effect of pressure overload on antioxidants, extracellular matrix activity, extracellular matrix composition, cardiac function and remodeling in collagen transgenic animals. Moreover to determine whether collagen mutation mitigates mortality and morbidity, the mortality curve comparing WT and col1a1 mice would be of interest, to show, that observed differences in hypertrophy and collagen degradation translate into mortality differences. These studies are currently in progress.

Although at present we do not see an apparent connection of thioredoxin and SOD to eNOS, it is possible that a decrease in thioredoxin and SOD may decrease generalized anti-oxidants and lead to eNOS uncoupling and inactivity.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 
{star} A part of this study was supported by NIH supplement grant HL-74185. Back


   References
Top
 Notes
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 5. Limitations
 References
 

  1. Cardiovascular Disease and Oxidative Stress. 1998-2002, Great Smokies Diagnostic Laboratory.
  2. MacKenna D., Summerour S.R., Villarreal F.J. Role of mechanical factors in modulating cardiac fibroblast function and extracellular matrix synthesis. Cardiovasc Res (2000) 46(2):257–263.[Abstract/Free Full Text]
  3. Ungvari Z., et al. Increased superoxide production in coronary arteries in hyperhomocysteinemia: role of tumor necrosis factor-alpha, NAD(P)H oxidase, and inducible nitric oxide synthase. Arterioscler Thromb Vasc Biol (2003) 23(3):418–424.[Abstract/Free Full Text]
  4. Thomas J. Oxidative stress: including gluthathione, a peptide for cellular defense against oxidative stress. (1999) Iowa State University.
  5. Kishimoto C., et al. Serum thioredoxin (TRX) levels in patients with heart failure. Jpn Circ J (2001) 65(6):491–494.[CrossRef][Medline]
  6. Akhter S.A., et al. Targeting the receptor-Gq interface to inhibit in vivo pressure overload myocardial hypertrophy. Science (1998) 280(5363):574–577.[Abstract/Free Full Text]
  7. Byrne J.A., et al. Oxidative stress and heart failure. Arch Mal Coeur Vaiss (2003) 96(3):214–221.[Web of Science][Medline]
  8. D'Armiento J. Matrix metalloproteinase disruption of the extracellular matrix and cardiac dysfunction. Trends Cardiovasc Med (2002) 12(3):97–101.[CrossRef][Web of Science][Medline]
  9. Weber K.T. Cardiac interstitium in health and disease: the fibrillar collagen network. J Am Coll Cardiol (1989) 13(7):1637–1652.[Abstract]
  10. Tyagi S.C., et al. Differential gene expression of extracellular matrix components in dilated cardiomyopathy. J Cell Biochem (1996) 63(2):185–198.[Web of Science][Medline]
  11. Nagase H., Woessner J.F. Jr. Matrix metalloproteinases. J Biol Chem (1999) 274(31):21491–21494.[Free Full Text]
  12. Springman E.B., et al. Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a "cysteine switch" mechanism for activation. Proc Natl Acad Sci U S A (1990) 87(1):364–368.[Abstract/Free Full Text]
  13. Nagase H. Zinc metalloproteinases in health and disease. (1996) Taylor and Francis London. 153–204.
  14. Tyagi S.C., et al. Co-expression of tissue inhibitor and matrix metalloproteinase in myocardium. J Mol Cell Cardiol (1995) 27(10):2177–2189.[CrossRef][Web of Science][Medline]
  15. Tyagi S.C., et al. Stretch-induced membrane type matrix metalloproteinase and tissue plasminogen activator in cardiac fibroblast cells. J Cell Physiol (1998) 176(2):374–382.[CrossRef][Web of Science][Medline]
  16. Mujumdar V.S., Tyagi S.C. Temporal regulation of extracellular matrix components in transition from compensatory hypertrophy to decompensatory heart failure. J Hypertens (1999) 17(2):261–270.[Web of Science][Medline]
  17. Hunt M.J., et al. Induction of oxidative stress and disintegrin metalloproteinase in human heart end-stage failure. Am J Physiol Lung Cell Mol Physiol (2002) 283(2):L239–L245.[Abstract/Free Full Text]
  18. Tyagi S.C., Ratajska A., Weber K.T. Myocardial matrix metalloproteinase(s): localization and activation. Mol Cell Biochem (1993) 126(1):49–59.[CrossRef][Web of Science][Medline]
  19. Peterson J.T., et al. Matrix metalloproteinase inhibition attenuates left ventricular remodeling and dysfunction in a rat model of progressive heart failure. Circulation (2001) 103(18):2303–2309.[Abstract/Free Full Text]
  20. Siwik D.A., Pagano P.J., Colucci W.S. Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts. Am J Physiol Cell Physiol (2001) 280(1):C53–C60.[Abstract/Free Full Text]
  21. Liu X., et al. A targeted mutation at the known collagenase cleavage site in mouse type I collagen impairs tissue remodeling. J Cell Biol (1995) 130(1):227–237.[Abstract/Free Full Text]
  22. Tyagi S.C., Smiley L.M., Mujumdar V.S. Homocyst(e)ine impairs endocardial endothelial function. Can J Physiol Pharm (1999) 77(12):950–957.[CrossRef][Web of Science][Medline]
  23. Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem (1976) 72:248–254.[CrossRef][Web of Science][Medline]
  24. Kameda K., et al. Correlation of oxidative stress with activity of matrix metalloproteinase in patients with coronary artery disease. Possible role for left ventricular remodelling. Eur Heart J (2003) 24(24):2180–2185.[Abstract/Free Full Text]
  25. Moshal K.S., et al. Protease-activated receptor and endothelial-myocyte uncoupling in chronic heart failure. Am J Physiol Heart Circ Physiol (2005) 288(6):H2770–H2777.[Abstract/Free Full Text]
  26. Takimoto E., et al. Oxidant stress from nitric oxide synthase-3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load. Clin Investig (2005) 115(5):1221–1231.
  27. Kojda G., Harrison D. Interactions between NO and reactive oxygen species: pathophysiological importance in atherosclerosis, hypertension, diabetes and heart failure. Cardiovasc Res (1999) 43(3):562–571.[CrossRef][Web of Science][Medline]
  28. Ma Q.L., Xie Y., Zhang S.D. Effects of trimetazidine on serum oxygen free radicals in congestive heart failure. Hunan Yi Ke Da Xue Xue Bao (2002) 27(6):527–529.[Medline]
  29. Cannella G., et al. Prolonged therapy with ACE inhibitors induces a regression of left ventricular hypertrophy of dialyzed uremic patients independently from hypotensive effects. Am J Kidney Dis (1997) 30(5):659–664.[Web of Science][Medline]
  30. Rajagopalan S., et al. Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability. J Clin Invest (1996) 98(11):2572–2579.[Web of Science][Medline]

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