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Cardiovascular Research 2005 66(2):364-373; doi:10.1016/j.cardiores.2004.12.007
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Copyright © 2004, European Society of Cardiology

Heart function and molecular biological parameters are comparable in young adult and aged rats after chronic myocardial infarction

Alexander Deten*, Grit Marx, Wilfried Briest, Hans Christian Volz and Heinz-Gerd Zimmer

Carl-Ludwig-Institute of Physiology, Leipzig University, Liebigstr.27, D-04103 Leipzig, Germany

* Corresponding author. Tel.: +49 341 9715500; fax: +49 341 9715509. Email address: deta{at}medizin.uni-leipzig.de

Received 1 September 2004; revised 23 November 2004; accepted 2 December 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: To test the hypothesis that IL-1β and IL-6 play a pivotal role after myocardial infarction (MI) particularly in aged rats.

Methods: Chronic MI was induced in young adult (3.5 months) and aged (18 months) female Sprague–Dawley rats by ligation of the left coronary artery. Sham-operated animals of corresponding age served as controls. Heart function was measured by catheterization 4 weeks after MI. The expression of IL-1β, IL-6, TGF-β-isoforms, ANF, and components of the extracellular matrix (pro-collagen I and III, colligin, MMP-2 and TIMP2) was measured by ribonuclease protection assay.

Results: Aged control rats differed from young adult rats in that LV-developed pressure (LVDP) was higher (161 vs. 147 mm Hg, p<0.05) in response to the elevated total peripheral resistance (0.71 vs. 0.47 mm Hg ml min/kg, p<0.05). Contractility was reduced in aged controls as indicated by decreased LV dP/dt (8.106 vs. 10.606 mm Hg/s, p<0.05). LV function was severely depressed in both MI groups (reduction in LVDP by about 35% and LV dP/dt by about 30%, increase in LVEDP to 24 mm Hg) while RVP and RV dP/dt markedly increased by about 100%. This was not different between both MI groups. ANF expression as a marker of hypertrophy was induced in both MI groups, but less pronounced in the LV of aged rats. Also, the mRNA expression pattern was qualitatively comparable, but showed gradual differences.

Conclusion: These results indicate that aged rats compensate well for hemodynamic overload induced by MI. Also, the mechanisms of myocardial post-MI remodeling are comparable in young adult and aged rats.

KEYWORDS Infarction; Aging; Cytokines; Remodeling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Large myocardial infarction (MI) causes substantial cardiac remodeling including replacement of the ischemic area by scar after gradual resorption of the necrotic cells. This process is rapidly initiated to maintain structural integrity and to resist rupture. On the other hand, the preservation of cardiac function depends on the remodeling of the surviving myocardial tissue [1,2]. This cardiac remodeling is characterized by compensatory hypertrophy of cardiac myocytes and hyperplasia of non-myocytes (cardiac fibroblasts) and leads also to an adverse interstitial fibrosis at non-infarcted remote sites. Since the unwanted fibrosis contributes to development and progression of heart failure, this process has been intensively studied over the past years [3,4].

Congestive heart failure has been associated with local and systemic elevation of pro-inflammatory cytokines, including interleukin (IL)-1β and IL-6 [5,6]. In previous work, we and others have shown that myocardial expression as well as plasma levels of pro-inflammatory cytokines are elevated after acute MI [7,8]. However, cytokine expression was not significantly elevated at later times after chronic MI, when cellular inflammatory infiltration had abated but signs of heart failure had developed [7]. Others, however, showed increased myocardial cytokine expression [9]. Since it is also well recognized that the plasma levels of the pro-inflammatory cytokines increase with age [10], this study was conducted to test the hypothesis that cardiac cytokine expression increased in aged compared to young adult rats after chronic MI. Besides measuring heart function with ultraminiature tip pressure transducers, we have focused on synthetic and degradative processes of the extracellular matrix (ECM) turnover by measuring mRNA expression of type I and type III collagen, matrix metalloproteinase (MMP)-2, and tissue inhibitor of matrix metalloproteinase (TIMP)-2. The regulation of collagen expression was studied by measuring the mRNA expression of the transforming growth factor (TGF)-β isoforms. The expression of the atrial natriuretic factor (ANF) served as marker for myocyte hypertrophy.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Animal model
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the appropriate State agency of Saxony. Myocardial infarction was induced in female young adult (3.5 months of age and 251 ± 6 g of body weight at the beginning of the study) and aged (18 months of age and 374 ± 9 g of body weight at the beginning of the study) Sprague–Dawley rats (Charles River) by ligation of the left anterior descending coronary artery (LAD) under ether anesthesia [3,7]. Briefly, the fourth intercostal space was opened, the heart was exteriorized, and the pericardium was cut. The LAD was ligated between the left auricle and the pulmonary outflow tract with a monofil thread (Ethicon USP 6/0, Johnson+Johnson) while holding the apex of the heart with forceps. Thereafter the chest was closed and the rats were allowed to recover. Sham-operated animals underwent the same procedure except that no ligation was performed.

2.2. Hemodynamic measurements
Heart function was measured 4 weeks after surgery in closed-chest spontaneously breathing rats anesthetized with thiopental sodium (Trapanal® 60 mg/kg i.p., Byk Gulden) using ultraminiature catheter pressure-transducers (3 F, Millar Instruments) [3,7]. Briefly, the RV catheter (model SPR-291) was inserted into the right jugular vein and advanced into the RV via the right atrium. After collection of the RV data, the LV catheter (model SPR-249) was placed in the right carotid artery and advanced upstream to the aorta and into the LV. Heart rate (HR), right and left ventricular (RV and LV, respectively) pressure and the rate in rise and fall of ventricular pressure (dP/dtmax and dP/dtmin, respectively) were recorded continuously on a PC at a sampling rate of 2 kHz using DASYLab V7.00 software (National Instruments) for 10–15 min. Cardiac output was measured by the thermodilution method (Cardiomax IIR, Columbus Instruments). For data analysis IOX 1.7.0.18 software was additionally used (EMKA technologies).

2.3. Tissue collection and infarct size measurement
After the hemodynamic measurements had been obtained, the hearts were arrested in diastole by a 10% KCl injection, rapidly excised, and cut at mid-papillary level. From the basal part, the RV free wall was trimmed away and the infarct area was cut from the non-infarcted LV additionally leaving a border zone of about 2 mm in width. The tissue pieces were snap frozen in liquid nitrogen for RNA isolation. The apical parts of the hearts were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 7 µm. A hemalaun and eosin or Mason trichrome staining was performed, and the sections were analyzed with a Zeiss Axioskop microscope and photographed. The MI size was calculated as ratio of the infarcted segment to the total LV perimeter at mid-papillary level averaged between endocardial and epicardial measurements using the ImageJ 1.33 k software (NIH), and the average MI of three sections was expressed as a percentage of total LV perimeter [11].

2.4. RNase protection assay (RPA)
Total RNA was isolated using the Trizol®-Reagent (Gibco BRL) according to the protocol supplied by the manufacturer. For the RNase protection assay (RPA), 2.5 µg, 5 µg or 7.5 µg of total RNA were used for the ECM-, TGF- or rCK1-template set, respectively. The probe template set rCK1 was obtained from PharMingen while the template sets for ECM components and TGF-β-isoforms were generated by RT-PCR [3,7]. Each probe template set was labeled with [{alpha}-32P]-UTP (3000 Ci/mmol, Amersham) by means of RiboQuant® In Vitro Transcription Kit (PharMingen) as described by the manufacturer. After hybridization (final concentration: 8 x 103 cpm/µl for each probe in template set) at 56 °C for 12–16 h the unhybridized riboprobe was digested with a mixture of RNases A and T1 (RiboQuant® RPA Kit, PharMingen), according to the manufacturers instructions. Protected probes were electrophoresed on a denaturing gel containing 5% polyacrylamide/8 M urea and visualized and quantified using the Molecular Imager® FX and Quantity One 4.4 software (BioRad). The signals of specific mRNAs were normalized to those of L32 or ARPP mRNA for the rCK1- or the ECM- and the TGF-template set, respectively.

The template sets contained the following cDNA probes (probe length in bp/protected):

(1) rCK1: IL-1{alpha} (432/403), IL-1β (390/361), TNF-β (351/322), IL-3 (315/286), IL-4 (285/256), IL-5 (255/226), IL-6 (231/202), IL-10 (210/181), TNF-{alpha} (189/160), IL-2 (171/142), Interferon {gamma} (156/127), L32 (141/112), and GAPDH (126/97);
(2) TGF: TGF-β1 (442/412), TGF-β3 (288/230), TGF-β2 (282/209), ARPP (143/113), and GAPDH (128/82);
(3) ECM: collagen type {alpha}1 (I) (504/449), TIMP-2 (404/326), MMP-2 (360/269), ANF (309/234), collagen type {alpha}1 (III) (310/211), colligin (225/161), ARPP (143/113), and GAPDH (128/82).

2.5. Statistical analysis
The data are expressed as mean ± S.E.M. A Kruskall–Wallis ANOVA on ranks was used for multigroup comparison subsequently utilizing multiple comparison procedure according to Dunn's method (Statgraphics 4.1, Statistical Graphics). A value of p<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. Hemodynamic measurements and infarct size
Measurements of LV function revealed a higher LV systolic pressure (LVSP) and a higher total peripheral resistance (TPR) in the aged rats while heart rate (HR), cardiac output (CO), and LV end-diastolic pressure (LVEDP) were comparable in young adult and aged control rats (Table 1). The rates of rise and fall in LV pressure (LV dP/dtmax and LV dP/dtmin, respectively) were significantly lower in the aged rats compared with the young adult rats (Fig. 1). This was accompanied by an increase in the time constant of relaxation {tau} in the aged animals. RV parameters were significantly lower in aged control rats when compared with young adult animals 4 weeks after sham operation with the exception that there were no differences in RVEDP (Table 1).


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  		Table 1 Hemodynamic data in young adult and aged rats 4 weeks after sham operation or myocardial infarction (MI)

 

Figure 1
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  		Fig. 1 Original recordings of left and right heart function in young adult (left) and aged (right) rats 4 weeks after sham operation (upper panels) or MI (lower panels).

 
The infarct sizes were 47.5 ± 1.4% and 46.7 ± 1.6% in the young adult and aged rats, respectively (n=7 in each group; p=NS). Severe contractile dysfunction was overt in both young adult and aged rats as evidenced by a reduction of LV developed pressure to about 100 mm Hg while LVEDP strongly increased to 25 mm Hg 4 weeks after MI (Table 1). Also LV dP/dtmax and LV dP/dtmin were severely depressed 4 weeks after MI (Fig. 1). This was accompanied by an increase in the time constant of relaxation {tau}. However, the impairment in LV function was comparable between young adult and aged rats 4 weeks after MI indicating a comparable capability of compensation in young adult and aged rats after MI. Likewise, cardiac output was well maintained in both the young adult and the aged rats 4 weeks after MI (Table 1). A more pronounced decrease in CO (p=0.113 and 0.087 MI vs. sham-operated controls for the young adult and aged rats, respectively) was presumably prevented by a concomitant decrease in TPR. In contrast, parameters of RV function strongly increased 4 weeks after MI (Table 1). While RVSP and RVEDP were comparable in young adult and aged rats 4 weeks after MI, RV dP/dtmax and RV dP/dtmin were significantly lower in the aged rats.

3.2. Myocardial mRNA expression of cytokines
Weak but detectable signals of mRNA expression were obtained for only 4 out of the 11 cytokine probes analyzed with the rCK1 set (Fig. 2, middle panel). In addition, there were only few differences 4 weeks after surgery. The mRNA expression of IL-1β was significantly lower in the LV, but not in the RV of aged control rats compared with young adults. It increased in the infarct area of both the young adult and the aged rats 4 weeks after MI (Fig. 3, bottom left). This increase, however, was more pronounced in the aged rats. In the non-infarcted LV, IL-1β and IL-6 slightly but significantly increased only in the aged rats (Fig. 4, bottom left). There were no further changes in cytokine mRNA expression detectable 4 weeks after MI.


Figure 2
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  		Fig. 2 Representative autoradiographs from RNase protection assays of ECM-set (left), rCK1-set (middle), and TGF-set (right) in the infarct area of young adult (y) or aged rats 4 weeks after sham operation (S) or myocardial infarction (MI) as indicated. The rCK1-RPA additionally shows samples 6 h after coronary artery occlusion. Each lane was loaded with 2.5 µg (ECM-set), 5 µg (TGF-set) or 7.5 µg (rCK1-set) total RNA.

 

Figure 3
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  		Fig. 3 Relative mRNA abundance (upper panels) and changes of mRNA expression from corresponding sham-operated controls (lower panels) in the infarct area 4 weeks after sham operation or MI. Number of experiments as in Table 1. {dagger}p<0.05 aged vs. young adult rats *p<0.05 MI vs. sham-operated controls.

 

Figure 4
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  		Fig. 4 Relative mRNA abundance (upper panels) and changes of mRNA expression from corresponding sham-operated controls (lower panels) in the non-infarcted LV 4 weeks after sham operation or MI. Number of experiments as in Table 1. {dagger}p<0.05 aged vs. young adult rats *p<0.05 MI vs. sham-operated controls.

 
3.3. Myocardial mRNA expression pattern of TGF-β isoforms
The mRNA expression pattern of TGF-β isoforms changed in a typical manner that was different in the infarct scar when compared with the non-infarcted myocardium. The most pronounced increase was observed for TGF-β3 in the infarct scar (Fig. 2, right panel). Also the mRNA expression of TGF-β1 and-β2 was upregulated, but less pronounced than that of TGF-β3 (Fig. 3, bottom left). Interestingly, the relative changes for all three TGF-β-isoforms were comparable between young adult and aged rats, although TGF-β1 as well as TGF-β3 mRNA, but not TGF-β2 mRNA was more abundant in the aged rats (Fig. 3, upper left). In the non-infarcted LV, the mRNA expression of all three isoforms was higher in all samples of the aged animals and significantly increased. The most pronounced induction occurred for the mRNA expression of TGF-β2. This, however, was not different between young adult and aged rats (Fig. 4, bottom left). In contrast, the increase in the mRNA expression of TGF-β1 and TGF-β2 was significantly more pronounced in the RV of aged rats when compared with young adult rats 4 weeks after MI (Fig. 5, bottom left). Furthermore, TGF-β3 was upregulated only in the aged rats, but not the young adults. Noteworthy, the mRNA expression of all TGF-β-isoforms was not different between young adult and aged rats in the RV 4 weeks after sham operation (Fig. 5, upper left).


Figure 5
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  		Fig. 5 Relative mRNA abundance (upper panels) and changes of mRNA expression from corresponding sham-operated controls (lower panels) in the non-infarcted RV 4 weeks after sham operation or MI. Number of experiments as in Table 1. {dagger}p<0.05 aged vs. young adult rats *p<0.05 MI vs. sham-operated controls.

 
3.4. Myocardial mRNA expression of extracellular matrix components
Sustained activation of metabolism of the extracellular matrix (ECM) was indicated by the persistent increase in the mRNA expression of colligin, type I and type III collagen, MMP-2, and TIMP2 in the infarct scar of both the young adult and the aged rats 4 weeks after permanent coronary artery occlusion (Fig. 2, left panel). Interestingly, type I as well as type III collagen mRNA were less abundant, while TIMP2 mRNA was more abundant in the aged rats when compared with the young adult rats 4 weeks after sham operation. The relative increases in type I and in type III collagen, however, were more pronounced in the aged rats (Fig. 3, bottom right). Also in the non-infarcted LV and RV, all analyzed components of the ECM significantly increased 4 weeks after MI. Although all but TIMP2 mRNA was less abundant in both the LV and the RV of aged rats when compared with young adult rats 4 weeks after sham operation (Figs. 4 and 5Go, each upper right), the relative changes in the LV were not significantly different of young adult and aged rats 4 weeks after MI (Fig. 4, bottom right). In the RV, on the other hand, the induction in the mRNA expression of colligin as well as of type I and type III collagen was significantly more pronounced in the myocardium of the aged rats (Fig. 5, bottom right).

3.5. Myocardial mRNA expression of ANF
Myocardial mRNA expression of ANF was higher in the LV than in the RV (Fig. 6) and higher in the LV of aged rats when compared with young adult rats 4 weeks after sham operation. It was strongly induced in both the non-infarcted LV and RV 4 weeks after MI. This increase was comparable in the RV, but significantly less pronounced in the LV of aged rats 4 weeks after MI.


Figure 6
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  		Fig. 6 Relative ANF mRNA abundance in the non-infarcted RV (left) and LV (right) 4 weeks after sham operation or MI. Number of experiments as in Table 1. {dagger}p<0.05 aged vs. young adult rats *p<0.05 MI vs. sham-operated controls.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
In the aged control rats, LVSP and TPR were higher while LV contractility (LV dP/dtmax) and relaxation (LV dP/dtmin, {tau}) were lower when compared with young adult control rats. After MI, LV function was altered to a similar degree in the aging rats as in the young adult rats (Table 1). Also, the mRNA expression of cytokines, TGF-β-isoforms, and components of the ECM was qualitatively similar, but showed gradual differences. These differences included a more pronounced increase of IL-1β and collagen expression in the infarct scar (Fig. 3) as well as a more pronounced increase of TGF-β and collagen expression in the RV (Fig. 5) of the aged rats 4 weeks after MI. The increase in ANF mRNA expression was less pronounced in the non-infarcted LV of aged rats, but similar in the RV when compared with young adult rats 4 weeks after MI (Fig. 6).

4.1. Molecular biological alterations in the aged control hearts
ECM metabolism in the aged control hearts was characterized by reduced mRNA expression of collagen and MMP-2 while TIMP2 expression was unchanged (Figs. 4 and 5Go). This is in good agreement with previous studies that show a decline in collagen synthesis per se in the aged heart by significant reductions in collagen type I and type III mRNA levels and [3H]proline incorporation [12–14]. Interestingly, the alterations in ECM metabolism were associated with elevated TGF-β expression in the LV but not the RV of the aged when compared with young adult control hearts (Figs. 4 and 5Go, upper panels). This further points to age-associated alterations in the metabolism of cardiac fibroblasts as previously suggested [15,16]. Interestingly, cytokine expression was not increased in the aged control hearts, but IL-1β rather was decreased in the LV (Fig. 4, upper left). The elevated ANF mRNA expression in the LV (Fig. 6) is in good accordance with the well-known myocyte hypertrophy in the aging heart [17,18].

4.2. Infarct size and heart function
Cardiovascular diseases typically occur in the elderly, and MI in that group is associated with increased morbidity and mortality [19,20]. Thus it was supposed that the alterations of the aging heart may compromise the cardiac adaptation to hemodynamic overload and could have major implications in the age-related decrease in cardiac tolerance to ischemia [17,19]. Studies have demonstrated worsening of myocardial stunning, decreased recoveries in pump function and high-energy phosphates, and greater tissue damage following acute ischemic insult in senescent than in adult heart [21,22]. Evidence also suggested that the benefits of preconditioning wane with increasing age and are lost in the hearts of senescent animals [21–24]. Interestingly, however, in our study there was no significant difference in infarct size between the young adult and aging rats as also in previous studies [23,24].

The data of this study indicate that the functional adaptation to chronic MI by permanent coronary artery occlusion for 4 weeks in the aging rat heart is similar to those of the young adult (Table 1). These results are in accordance with an earlier report, in which heart function was not significantly different between mature and aged rats (initial age 7 and 18 months, respectively) 5 months after MI [25]. In another study, a slightly reduced developed pressure was observed in aged rats (16 months) compared with young rats (4 months), but there were essentially no differences in LVEDP and LV dP/dtmax (parameters for relaxation not reported) 1 week after large MI [26]. Both studies, however, reported a limited compensatory hypertrophic growth of myocytes in the viable LV, even though the molecular myocyte growth control, at least in part, was preserved in the aged rats. Also in our present study, a limited myocyte hypertrophy may be reflected by the marked, but less pronounced increase in ANF expression in the non-infarcted LV of the aged rats (Fig. 6). Both limited hypertrophy and concomitant less ANF expression have been described in aging animals with experimental pressure [27] and volume [28] overload. In another study, however, there were no differences in the hypertrophic response to aortic constriction for 4 weeks between 9 months and 27 months old rats [29]. A lack of increased LV mass after renovascular hypertension for 3 months in aged rats (24 months) was associated with increased myocyte dropout, but also cellular myocyte hypertrophy [30]. This myocyte hypertrophy was even more pronounced in the aged rats when compared with middle-aged rats (15 months). Since it was also shown that the hypertrophic response to thyroxin is not limited by age [31], it may be suggested that the reactive myocyte growth is stimuli-dependent. It was also suggested that the extent to which the heart, with time, can compensate is not age-dependent [25]. The nearly preserved heart function as well as the marked increase in ANF expression, as observed in this study, supports the conclusion that aging rats do well compensate for the hemodynamic overload induced by large MI.

4.3. Wound healing and inflammatory response in the infarct area
In animal models of wound repair in the aged, there is a 20% to 60% delay in the rate of healing as compared to young animals [32]. The consensus is that the effect of aging on wound repair is primarily a temporal delay and not an actual impairment in the quality of healing [33]. This is in good accordance with the data of this study, since there were essentially no qualitative differences in the mRNA abundance of cytokines between young adult and aged animals 4 weeks after MI (Fig. 3, upper left). During the final resolution phase of wound healing, collagen synthesis and turnover continue, and fibroblasts differentiate into myofibroblasts, allowing further wound contraction. Fibroblasts from aged donors have been shown to exhibit an altered responsiveness to IL-1β [15] as well as diminished response to growth factors and diminished replicative capacity [34,35]. This is in agreement with the data of this study, consistently showing increased TGF-β mRNA abundance in the infarct samples of the aged rats (Fig. 3, upper left). The more pronounced relative increase in collagen I and III mRNA expression in the aged animals (Fig. 3, bottom right), on the other hand, was associated with a more pronounced relative increase in IL-1β in the infarct area (Fig. 3, bottom left). IL-1β is a pleiotropic cytokine which has a profound influence on the regulation of connective tissue metabolism [36]. It is a potent inducer of interstitial collagenase [37], but can exert a positive but also a negative regulatory effect on collagen expression and protein synthesis [38,39]. It may, therefore, be suggested that IL-1β, possibly together with the high TGF-β levels, contributes to the more pronounced relative increase in collagen mRNA expression in the infarct area of the aged animals (Fig 3, bottom left).

It is well recognized that the plasma levels of the pro-inflammatory cytokines TNF-{alpha}, IL-1β and IL-6 increase with age and after MI [10]. In addition, inflammation is a causative factor in age-related delayed healing. The inflammatory response may be inappropriately excessive in aged subjects [40]. The influx of leukocytes may occur unabated, leading to enhanced cytokine and chemokine production, further leukocyte recruitment, and ultimately tissue destruction. However, there was essentially no change in the expression of TNF-{alpha} and IL-6 and only a moderate increase in IL-1β in both the young adult as well as the aged rats 4 weeks after MI (Figs. 2 and 3Go, bottom left). Therefore, the inflammatory response seemed not to escalate as also remodeling and function were not exacerbated in the aged rats. For comparison, each five additional animals were analyzed 6 h after coronary artery occlusion (Fig. 2). There was a strong induction in IL-1β and IL-6 expression. This, however, showed essentially no differences between young adult and aged rats, further emphasizing that inflammatory response and wound repair is generally comparable in both groups.

4.4. Remodeling of the non-infarcted myocardium
The well known general postinfarction remodeling process of the surviving myocardium is, in part, an expected adaptation to abnormal stress and strain that induce autocrine/paracrine responses, and comprises progressive structural and functional adaptations in viable cardiac myocytes and in the interstitial matrix [41]. In accordance with the observation that aging rats do well compensate for the hemodynamic overload induced by large MI, it is not surprising that also the analyzed molecular biological parameters of the ECM remodeling in the non-infarcted LV were comparable in young adult and aged rats (Fig. 4). This elucidates a previous report, showing no difference in fibrosis between adult and aged rats 5 months after MI [25]. However, as for the myofibroblasts of the infarct scar, it may be concluded that the regulation of fibroblast metabolism is altered in aged rats. This may also include increased collagen cross-linking, as reported for aged rats compared with young rats after small MI [42]. Also in that study there was no difference in gross collagen content neither in the scar nor in the viable LV between young and old rats. Therefore, it seems that the development of myocardial fibrosis in the non-ischemic myocardium is an obligatory phenomenon following permanent myocardial ischemia regardless of the size of the injury and independent of hemodynamic change and age.

Interestingly, the situation appeared to be somewhat different in the RV (Figs. 5 and 6Go), in that the ANF expression was comparable in young adult and aged rats 4 weeks after MI, also suggesting comparable myocyte hypertrophy. Interestingly, the increase in TGF-β isoforms as well as in collagen expression was more pronounced in the RV of aged rats (Fig. 5 bottom panels). Since this was accompanied by a significantly less pronounced increase in RV dP/dtmax and RV dP/dtmin in the aged animals (Table 1), it may be speculated that this resulted in a more pronounced fibrosis and, also, stiffness of the RV.

The cytokine expression, on the other hand, was completely unchanged in the RV of both the young adult and the aged rats 4 weeks after MI. Since also in the non-infarcted LV the cytokine expression was only slightly increased compared to sham-operated controls, but not different from young adult rats 4 weeks after MI, these data do not support the hypothesis that the pro-inflammatory cytokines play a pivotal role especially in the aged rats after chronic MI.

4.5. Limitations of the study
There are some limitations of this study. First, it should be kept in mind that gene expression not necessarily directly translates into identical changes in protein levels and, therefore, might not be directly related to cardiac function or remodeling. Second, female rats were used in this study although it is well known that there are gender-dependent differences regarding different aspects of heart function and wound healing after MI. However, it is also well recognized today that in post-menopausal females the risk rapidly increases to levels comparable with males. Nevertheless, we cannot rule out the possibility that there might be different results of our study if conducted in males.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the Deutsche Forschungsgemeinschaft (ZI 199/10-3, ZI 199/10-4), by a grant of the Medical Faculty of the University of Leipzig (formel.1-19) and by a grant of BMBF (NBL-3-Förderung; Kennzeichen 01ZZ0106). The excellent technical assistance of Brigitte Mix is gratefully appreciated.


   Notes
 
Time for primary review 21 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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