INTRODUCTION

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HEART FAILURE (HF) is defined as the inability of the heart to pump blood at a rate commensurate with the metabolic demands of the body or can only do so at elevated filling pressures (6, 23). As such, congestive heart failure (CHF) can be attributed to multiple factors including diastolic dysfunction, systolic dysfunction, inflammation, and changes in neurohormonal regulators, which affect blood pressure and fluid retention. The complexity of the disease has contributed undoubtedly to the continued growth of the diagnosed CHF population despite the extensive arsenal of available therapies. In fact, it is estimated that by 2007 there will be 10 million cases of HF in the United States alone (30) with 6-year mortality rates for men and women at 84% and 77%, respectively (7).

It is well established that initiation of CHF pathophysiology is a multifactorial process encompassing physiological, neurohormonal, biochemical, molecular, and cellular changes, the culmination of which is the activation of compensatory mechanisms that aim to maintain heart function, but, when prolonged, evolve into maladaptive processes. For example, initial structural alterations during HF development maintain cardiac output; however, extensive left ventricular (LV) remodeling becomes deleterious and contributes to decompensation. In addition, levels of proinflammatory cytokines that initially facilitate tissue repair in CHF increase concomitantly with the severity of HF (11, 36) and have been correlated with LV dysfunction, edema, cardiomyopathy, remodeling, and mortality (11, 14, 18, 29). Collectively, these and other compensatory systems initially work to maintain cardiac function and output. However, prolonged activation of these systems results in tissue damage, organ failure, and ultimately, death.

The spontaneously hypertensive, heart failure-prone (SHHF) rat represents a congenital model of dilated cardiomyopathy with hypertension progressing to decompensated HF, which exhibits several hallmark signs of the human disease state. For example, as SHHF animals age, many of the biochemical and physiological compensatory systems observed in human patients are also activated in this model. Documented biochemical compensatory mechanisms recapitulated in the SHHF model include renin angiotensin aldosterone system activation, natriuretic peptide elevation, and tumor necrosis factor- (TNF-) elevation (2, 20). Common pathophysiological outcomes of end-stage human HF such as increased end-diastolic volume and decreased ejection fraction (EF) have also been evidenced in the SHHF animals (1, 5, 34).

Although the SHHF rat represents a commonly employed experimental model of HF, a comprehensive evaluation of the structural, functional, and molecular mediators of pathophysiology in this model has not been conducted. To further understand the underlying mechanisms of CHF progression, we temporally characterized the lean male SHHF rat model from 4 to 18 mo of age. Our results couple progressive pathophysiological changes, profiled using echocardiography, with serum and tissue biochemical markers, and the expression patterns of key cytokines, stress, and matrix molecules that contribute to CHF pathophysiology.

	METHODS

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Animals. The present study was conducted in accordance with institutional guidelines for the humane treatment of animals using both homozygous (cp/cp) and heterozygous (+/cp) lean male SHHF rats obtained from Genetic Models (Indianapolis, IN). All animals were housed in a room lit 12 h per day (6 AM-6 PM) at an ambient temperature of 22 ± 1°C. Animals were allowed 1 wk to adjust after arrival and were allowed access to rodent diet and tap water ad libitum during the experiment. SHHF rats used in the study were categorized according to age and ranged from 4 to 18 mo of age with an n ranging from 10 to 13 unless otherwise noted.

Systolic blood pressure. Systolic blood pressure (SBP) was analyzed by using the Visitech BP-2000 Blood Pressure Analysis System (Visitech Systems; Apex, NC). The BP-2000 computerized noninvasive tail-cuff system was set to measure six cycles (1 cycle every 30 s) with an upper pulse rate limit of 15 Hz and minimum average pulse amplitude at 20% of full scale. The BP-2000 system was calibrated before each data collection period by using a manometer to accurately obtain systolic pressure. Animals were randomly brought into a dimly lit quiet room, and blood pressure measurements were taken. Briefly, rats were lightly anesthetized with 0.5-1% isoflurane (AErrane, Baxter; Deerfield, IL) and placed on a warming table with tails inserted into cuffs. Six measures were taken for each animal and averaged for a mean SBP reading.

Echocardiography. Echocardiograms were performed by using the Agilent SONOS 5500 echocardiographic system equipped with a 15-MHz linear-array transducer (Agilent; Andover, MA). Images were obtained from rats lightly anesthetized with 1-2% isoflurane (AErrane, Baxter) lying on their left side with transducer placed on the left hemithorax. Two-dimensional parasternal long- and short-axis images of the left ventricle were obtained, and two-dimensional targeted M-mode tracings were recorded from the parasternal short-axis view at the level of the papillary muscles at a sweep speed of 150 mm/s. Doppler flow velocities were taken at the level of the mitral valve in the apical four-chamber view with the Doppler probe placed at the edge of the mitral leaflets. All measurements were performed according to the recommendations of the American Society for Echocardiography leading-edge method from three consecutive cardiac cycles (31). Measurements and calculations used are as follows: percent LV fractional shortening (FS) was calculated as follows: FS = (LVIDd  LVIDs)/LVIDd × 100, where LVIDd and LVIDs are end-diastolic and end-systolic LV internal dimensions, respectively. Relative wall thickness (RWT) was calculated as (PWd + IVSd)/LVIDd, where PWd and IVSd are end-diastolic posterior wall and interventricular septal thickness, respectively. End-diastolic (EDV) and end-systolic volumes (ESV) were calculated from LV systolic (LVAs) and diastolic (LVAd) areas via the method of discs (31). EF was calculated from systolic and diastolic volumes with the following formula: EF = (EDV  ESV)/EDV × 100. Other measurements taken include early filling velocity (E-velocity; E-vel), late filling velocity (A-velocity; A-vel), mitral valve deceleration time (Decel T), LV mass (area length method), heart rate (HR; m-mode R-R interval), stroke volume (SV; SV = EDV  ESV) and cardiac output (CO = SV × HR).

Tissue processing, staining, imaging, and analysis. Equatorial regions of the heart fixed in 10% neutral buffered formalin were routinely processed and paraffin embedded. Ten-micron sections were stained with the collagen-specific stain Picro-Sirius Red F3BA for determination of interstitial collagen volume fraction using a Videometric 150 Image Analysis System (Oncor; Gaithersburg, MD) as previously described (8). Five-micron sections were stained with hematoxylin and eosin and trichrome for histopathological analysis. Myocardial lesions of fibrosis, hypertrophy, degeneration, necrosis, and inflammatory cell infiltrates were classified and scored in five to six rats per group at 5, 9, and 16 mo of age and in nine rats at 18 mo of age. Lesions were scored as 0 (absent), 1 (minimal), 2 (mild), 3 (moderate), or 4 (marked).

Immunohistochemistry. Five-micron sections were deparaffinized, rehydrated in ethanol, and processed for antigen retrieval using citric acid. Immunohistochemistry was performed according to standard methods by using a DAKO autostainer (DAKO; Carpinteria, CA) and by using the following antibodies: osteopontin (OPN), working dilution 1:100 (mouse monoclonal, Developmental Studies Hybridoma Bank, The University of Iowa; Iowa City, IA); ED-1, working dilution 1:500 (Chemicon; Temlecula, CA); and CD-3, working dilution 1:300 (DAKO). Positive immunoreactivity was detected by using a horseradish peroxidase-conjugated secondary antibody and the substrate diaminobenzidine (DAKO). Isotype-matched IgGs at similar concentrations were used in place of the primary antibodies for negative controls. Tissues known to express these targets were used as positive controls. Immunoreactivity in the cardiac sections was scored on a scale from 0 (absent) to 4 (marked).

Biochemical assays. TNF-, TNF receptors 1 and 2 (TNFR1 and TNFR2), and IL-6 were quantitated according to standard procedures. Serum TNF- was analyzed by ELISA utilizing an Ultra Sensitive rat TNF- kit (Biosource International; Camarillo, CA). Plasma TNFR1 and TNFR2 levels were determined using Quantikine M mouse sTNFR1 and sTNFR2 Immunoassay kits (R & D Systems; Minneapolis, MN). Plasma IL-6 was determined by using a rat IL-6 ELISA (Biosource; Camarillo, CA). Urinary proteinuria was determined by using the Bio-Rad protein dye reagent (Hercules, CA). The assay was modified to a 96-well plate format according to the manufacturer's instructions. Matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) activity was examined by zymography in heart extracts. Briefly, LV tissue samples were homogenized in 25 ml ice-cold extraction buffer containing 1% Triton X-100, 25 mM HEPES, 0.15 M NaCl, 2 mM EDTA, and a complete protease inhibitor cocktail (Roche; Indianapolis, IN) The homogenates were centrifuged (4°C, 8,000 g, 20 min), protein concentrations were assessed with the use of a bicinchoninic acid assay (Pierce; Rockford, IL), and equivalent amounts were separated by SDS-PAGE. After electrophoresis, gels were washed and allowed to renature for 1 h. Gels were incubated at 37°C for 16-18 h in developing buffer containing 1 mM Tris base, 40 mM Tris · HCl, 200 nM NaCl, 5 mM CaCl₂ and 0.2% Brij 35 and stained with Coomassie blue, and proteases were visualized by the absence of staining indicating substrate cleavage.

RNA isolation. RNA was extracted from frozen heart tissue by using the Totally RNA Isolation Kit (Ambion; Austin, TX) as previously described (19). Tissues were crushed, homogenized, and denatured according to the manufacturer's instructions. RNA was further purified by DNase digestion to remove genomic DNA and LiCl precipitation to remove carbohydrates. Purified RNA was precipitated with 7.5 M LiCl/50 mM EDTA and incubated overnight at 80°C. All samples were diluted and analyzed spectrophotometrically for concentration and purity and stored at 80°C.

Taqman quantitative RT-PCR. All primers and probes were designed by using Primer Express software supplied with the 7700 Sequence Detection System based on known rat sequences and synthesized by Applied Biosystems (Foster City, CA) as previously described (35). Standard curves were performed to determine the efficiency of each primer/probe set in the Taqman reaction before analysis of the experimental samples. All target gene results were normalized to the housekeeping gene cyclophilin. Total, purified RNA (200 ng) was added to an RT-PCR reaction mix, which contained the following: 12.5 µl of 2× One-Step PCR Master Mix without uracil-N-glycosylase, 0.625 µl of a 40× MultiScribe and RNase Inhibitor Mix, 0.625 µl of 20 mM forward primer, 0.625 µl of 20 mM reverse primer, 0.5 ml of 5 mM Taqman probe, and 0.125 ml of DNase/RNase-free water. All samples were analyzed in duplicate. The following protocol was applied to all reactions: 30 min at 48°C (reverse transcription), 10 min at 95°C (inactivation of reverse transcriptase), 40 cycles of 15 s at 95°C and 1 min at 60°C (PCR). Data analysis was performed using the Sequence Detection System software from Applied Biosystems. Primer probe sets for cyclooxygenase (COX-2), IL-1, IL-6, OPN, TNF-, inducible nitric oxide synthase, ICAM-1, VCAM-1, atrial natriuretic peptide, brain natriuretic peptide, endothelin-1 (ET-1), angiotensin-converting enzyme (ACE), transforming growth factor (TGF-), myosin heavy chain- and - (MHC- and MHC-), collagenase- type I (COL-I), collagenase- type III (COL-III), fibronectin (FN), MMP-2, MMP-3, MMP-8, tissue inhibitor of MMP-1 (TIMP-1), TIMP-2, TIMP-4, and membrane type 1 MMP (MT1-MMP) are listed in Table 1. All oligonucleotides are written 5'-3'. Primers were unlabeled and all probes were labeled at the 5' end with 6-carboxyfluorescein reporter dye and at the 3' end with 6-carboxy-N,N,N',N'-tetramethylrhodamine quencher dye. Expression levels in SHHF animals were compared with message levels in control 4-mo-old Lewis rats.

Statistical analysis. For all parameters excluding histology and gene expression data, there were four distinct groups. Each group was composed of SHHF rats at different ages: 4, 9, 15, and 18 mo of age. For histological parameters, the groups were 5, 9, 16, and 18 mo of age. The gene expression analysis was performed on SHHF animals aged 4, 9, 12, and 15 mo of age with the inclusion of a 4-mo-old Lewis rat control animal. Data were analyzed by using one-way ANOVA. Taqman statistics were performed on the rank transforms of the raw data (nonparametric analysis) to account for any inequality of variance. The age group means for the gene expression variables were compared with a Lewis rat control group by using the least significant differences means comparison procedure. The age group means for the physiological, echocardiographic, and biochemical variables were compared with all other group means using two-tailed Tukey's Studentized range test (HSD). Data means that were found to be significantly different from other means with Tukey's HSD were assigned different letters. Data were analyzed by using PROC GLM in the SAS statistical software package (SAS PC, version 6.12, SAS Institute; Cary, NC). All data are reported as means ± SE.

	RESULTS

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Physiological analysis. The comparison of body weight (BW), SBP, and proteinuria in the SHHF model over time is shown in Table 2. SHHF animals were hypertensive compared with normal Sprague-Dawley rats [SBP; 110-130 mmHg, (2)] as early as 4 mo of age, and SBP continued to rise as HF developed. Animals entering into the mid- and late-stage HF (~16-18 mo of age) demonstrated decreases in SBP concurrent with a large increase in urinary protein levels.

Echocardiographic analysis. A comprehensive temporal echocardiographic evaluation of SHHF pathophysiology demonstrated progressive, marked changes in cardiac structure and function (Table 3). All echocardiographic parameters changed with age, and several notable changes are described in detail. At 4 and 9 mo of age, RWT was significantly increased compared with the 15- and 18-mo-old SHHF animals (RWT: 45.4 ± 1.5, 4 mo and 45.7 ± 1.3, 9 mo vs. 37.5 ± 1.6, 15 mo and 29.6 ± 1.1, 18 mo), whereas older animals exhibited significant increases in LV end-diastolic and end-systolic volume and area compared with earlier time points, accompanied by diminishing RWT. Significant loss of cardiac function was also apparent beginning at 15 mo of age. EF and FS were decreased from 45.3 ± 1.4 at 15 mo to 29.1 ± 2.5 at 18 mo and 28.8 ± 1.0 at 15 mo to 16.1 ± 0.9 at 18 mo, respectively. Moreover, Doppler echocardiographic analysis revealed progressive diastolic dysfunction characterized by augmented early E-vel and diminished A-vel with age, which is indicative of abnormal diastolic filling. Furthermore, Decel T was attenuated and paralleled by shorter isovolumetric relaxation times. Consistent with age-dependent changes in blood pressure and body weight, temporal echocardiographic analysis indicated progressive cardiac dysfunction concomitant with deleterious LV remodeling as SHHF animals transitioned from a compensated, hypertrophic state to decompensated HF.

Histopathology and immunohistochemistry. Myocardial lesions and inflammatory infiltrates were assessed in 5-, 9-, 16-, and 18-mo-old SHHF rats. Lesions were absent in 5-mo-old SHHF rats. In older SHHF rats, there was a progressive increase in the incidence and/or severity of myocardial fibrosis and degeneration (Table 4).

View larger version (131K): [in this window] [in a new window]   Fig. 1.   Interstitial and perivascular fibrosis. A: collagen volume fraction. Image analysis demonstrated a progressive increase in interstitial collagen over time. B: perivascular collagen (blue) in a left ventricular (LV) intramyocardial arteriole at 5 mo. C: perivascular collagen (blue) in a right ventricular (RV) intramyocardial artery at 5 mo. D: LV perivascular and intersititial fibrosis (blue) at 16 mo. E: RV perivascular and intersititial fibrosis at 16 mo.

View larger version (71K): [in this window] [in a new window]   Fig. 2.   ED-1 immunohistochemistry. A: rare ED-1-positive cell (brown, arrowhead) in LV myocardium at 5 mo. B: multiple ED-1-positive cells (brown) in LV myocardium at 16 mo. Macrophages are located in areas of myocyte loss and interstitial fibrosis.

View larger version (88K): [in this window] [in a new window]   Fig. 3.   Osteopontin (OPN) immunohistochemistry. There was minimal OPN immunostaining (brown) of myocytes in the midmyocardium at 5 mo (A). Number of stained myoctes and intensity of staining increased at 9 mo (B) such that a distinct band of positive staining was evident in the midmyocardium. At 16 mo (C), immunostaining was intense in myocytes and macrophages (arrowhead).

Biochemical analysis. The mechanisms underlying the developing SHHF pathophysiology were investigated by monitoring circulating markers of inflammation and remodeling in animals at 4, 9, 16, and 18 mo of age (Table 5). Circulating levels of TNFR1, TNFR2, TNF-, and IL-6 significantly increased in 18-mo-old animals compared with earlier time points. Similar to the human disease state, these inflammatory markers are significantly elevated in more advanced HF. Finally, MMP zymographic activity analysis demonstrated peak gelatinase activity at 18 mo of age (Fig. 4).

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Time-course analysis of total gelatinolytic activity measured by zymography. Gels (n = 3/time point) were quantified with ImageQuant Software on the Personal Densitometer SI (Molecular Dynamics; Sunnyvale, CA). SHHF, spontaneously hypertensive heart failure. Values are expressed as means ± SE. Means with same letter are not significantly different at P < 0.05. Means with different letters are statistically different from each other, P < 0.05.

Transcriptional profiling analysis. To further characterize the molecular mechanisms underlying the progression from hypertension to HF in 4- to 15-mo-old rats, we evaluated the expression of multiple genes in heart tissue by using quantitative RT-PCR. Consistent with histological evidence of infiltrating inflammatory cells, several proinflammatory markers were modulated over time with disease progression in the SHHF rat (Table 6). OPN gene expression was markedly elevated by 4 mo of age (~11-fold) and further increased up to ~15-fold by 15 mo of age compared with control. COX-2, IL-1, IL-6, and ICAM-1 exhibited a different expression pattern where expression was highest at 9 mo followed by a downregulation of expression at 12 and 15 mo of age. By 9 mo, COX-2 mRNA expression increased (~5-fold), IL-1 (~4-fold), IL-6 (~14-fold), and ICAM-1 (~1.4-fold). VCAM-1 expression was modestly elevated (~1.3-fold) at 4 and 9 mo of age, followed by normalization of expression by 12 mo of age. In contrast, gene expression of inducible nitric oxide synthase and TNF- was somewhat attenuated over time as the pathology persisted with the lowest expression level exhibited at 15 mo of age.

	DISCUSSION

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The objective of the current study is to provide a foundation for further studies with standard therapies. The applicability of studies in untreated animals to clinical findings is limited by the fact that nearly all treated HF patients are receiving multiple pharmaceutical interventions. However, it is paramount to the understanding of the impact and mechanisms of action of therapeutic agents that the characteristics of HF be defined. By contributing a comprehensive echocardiographic and molecular evaluation of HF pathophysiology to the knowledge base, it is anticipated that a greater appreciation of the impact of treatment on the drivers of HF will be achieved. Thus a novel comprehensive evaluation of the structural, functional, and molecular progression of HF was conducted in the lean male SHHF rat model. In the compensated state, animals were hypertensive as early as 4 mo of age with accompanying myocardial hypertrophy clearly evidenced by 9 mo of age as demonstrated by increased expression of molecular hypertrophic/stress markers, increased relative wall thickness, and preserved function. As described previously, animals began to transition to a decompensated state around 15 mo of age (21), and many hallmark features of late-stage HF such as myocardial inflammation, increased chamber size, and diminishing EF were evidenced.

Previous M-mode echocardiographic analysis of SHHF animals demonstrated an increase in LV mass and LVIDd with diminishing FS at 18 mo of age relative to age-matched controls (2, 13). These studies were confirmed and extended using two-dimensional M-mode and Doppler echocardiographic analysis, which elucidated a progressive increase in LV mass, diastolic and systolic chamber area, and volume concomitant with a reduction in EF and FS. Comprehensive echocardiographic evaluation of SHHF disease progression reveals multiple commonalities between human and experimental HF. For example, mid- and end-stage HF in the SHHF model is accompanied by large increases in EDV and marked reductions in EF, which parallel clinical observations in decompensated HF patients (33).

Circulating cytokines and their receptors are now being established as markers of human HF severity and patient survival (11, 28, 29). Consistent with human CHF, circulating TNF- was found to be progressively elevated in SHHF rats with age and disease advancement. In contrast, myocardial TNF- expression is modestly decreased with age. The observed dissociation between circulating TNF- levels and myocardial expression is not surprising because even an 11-fold overexpression of myocardial TNF- does not result in elevated circulating TNF- in transgenic mouse models (4). In addition to TNF-, both IL-6 and the soluble forms of TNF- receptors (R1 and R2) were also found to be elevated in NYHA class III and IV HF patients and SHHF rats with late-stage HF (11, 28). Indeed, IL-6 has been demonstrated as a prognosticator of mortality (29), and elevated IL-6 levels have been positively correlated to increases in end-diastolic and end-systolic volumes (28).

In addition to IL-6, the expression of levels of other inflammatory markers, including IL-1, COX-2, and ICAM-1, were all highest at 9 mo of age before decompensation. These molecules are established mediators of the inflammatory response. Thus decompensation in the SHHF rat may be preceded by an inflammatory burst that initiates the recruitment of cellular infiltrates and activation of signaling pathways that may initially be reparative but eventually become maladaptive with long exposure.

Rapidly emerging as a key contributor to myocardial remodeling and inflammation, the cytokine and potent macrophage chemoattractant OPN is expressed by a number of cell types, including cardiomyoctyes and participates in cell adhesion, chemotaxis, and cellular signaling (9, 10). Although OPN expression has been shown to be elevated in spontaneously hypertensive rats and cardiomyopathic hamsters with CHF (32, 39), the current study is the first to describe the role of OPN in the SHHF rat. Myocardial remodeling resulting from hypertension or infarction is positively correlated with elevated cardiac expression of OPN, suggesting a function for this adhesion molecule in myocardial structural alterations that accompany external stress (12, 37). Thus in this model it is likely that this adhesion molecule serves a role in both monocyte/macrophage recruitment and adherence and myocardial remodeling that typifies HF progression.

In addition to inflammation, components and modulators of the extracellular matrix serve key functions in compensatory and maladaptive LV remodeling that accompanies human CHF development (38). Structural changes were also evident in aging SHHF rats. Although collagen and FN message remained unchanged from 4 to 18 mo of age, myocardial fibrosis was progressively elevated. These findings parallel previous studies by Li et al. (17), which demonstrated an increase in collagen volume fraction in SHHF animals in the absence of increased COL-I or COL-III expression. The apparent disparity between message and protein levels for these structural molecules is most likely explained by posttranslational processing (15). As the principle regulators of the extracellular matrix, MMPs and TIMPs have been shown to be selectively modulated with CHF progression. Indeed, both MMP and TIMP mRNA levels have been shown to be upregulated in patients and obese SHHF male rats, and inhibition of MMPs attenuated LV remodeling in SHHF and postmyocardial infarction rat models of HF (17, 26, 27). In the present study, lean male SHHF animals also demonstrated augmented levels of MMP and TIMP expression with the exception of MMP-3. These data are consistent with published studies in obese, male SHHF rats where MMP-3 message was not elevated in the SHHF animals at age 9 and 13 mo (17). Collectively, these data suggest that LV remodeling over the course of CHF progression is a deliberate and highly orchestrated molecular process integrating a selective inflammatory response with extracellular matrix adaptations.

SHHF rats progress from a spontaneously hypertensive, compensated phenotype to overt, late-stage CHF. During CHF development, a multitude of maladaptive molecular and cellular systems are initiated culminating with the advancement to the end-stage disease. Although experimental models are recognized approximations of human pathophysiology, the SHHF model shares many common features with the human disease state. In this regard, the SHHF rat offers a unique opportunity to dissect the complex molecular and cellular and physiological network of compensatory and decompensatory drivers of HF.