*3.2. Cardiac Hypertrophy*

#### 3.2.1. Heart Weight

At the end of the experimental period, the HW of 82-week-old CTRL rats was 1860 ± 93 mg. Both forms of therapy counteracted the development of cardiac hypertrophy and reduced HW significantly by 26% (CAP) and 23% (CAP + NIF). ANCOVA revealed that the treatment effects on HW did not depend on age, BW and SBP at baseline. HW/BW in untreated SHR was 4.7 ± 0.25 mg/g and was 34% higher than in the treated animals (3.5 ± 0.2 mg/g in both groups, *p* = 0.001; Figure 2a). Notably, the final BW of CTRL rats at 82 weeks of age was 378 ± 37 g and was about 15% lower than that of treated SHR (*p* = 0.08; data not shown).

**Figure 2.** (**a**): Heart weight-to-body weight ratio (HW/BW; in mg/g) expressed as mean ± SEM; (**b**): mRNA expression of atrial natriuretic peptide (ANP, in % of GAP-DH) expressed as median (line in the box) with 25th/75th percentiles (boxes) and 10th/90th percentiles (whiskers). All values were obtained from 82-week-old SHR. Significance marks: \* significant vs. CTRL (*p* ≤ 0.001).

#### 3.2.2. ANP mRNA Expression

ANP mRNA expression in the LV of untreated 82-week-old SHR was 264 [154/294] percent of GAP-DH mRNA expression. Both treatments resulted in a significant reduction of ANP mRNA expression to 60 [48/68] (CAP) and 43 [15/78] (CAP + NIF) percent of GAP-DH (*p* < 0.001; Figure 2b). ANP mRNA expression was not dependent on the covariates age, BW and SBP at baseline.

#### *3.3. Cardiac Fibrosis*

#### 3.3.1. Biochemical Markers of Cardiac Remodeling

*TGF-β:* In the LV of untreated SHR, mRNA expression of TGF-β<sup>1</sup> was 2.68 [2.10/2.74] percent of GAP-DH. Treatments with CAP and CAP + NIF decreased TGF-β<sup>1</sup> to 1.77 [1.64/2.11] and 1.19 [1.14/1.98], respectively, but this effect was significant only with CAP + NIF (*p* = 0.04). TGF-β<sup>2</sup> and TGF-β<sup>3</sup> were not significantly reduced with either treatment (Figure 3a–c). The changes in TGF-β<sup>1</sup> and TGF-β<sup>3</sup> were dependent on SBP at baseline.

**Figure 3.** (**a**–**c**): mRNA expression of TGF-β isoforms (in % of GAP-DH) in the LV. (**d**,**e**): Protein concentrations of TGF-β<sup>1</sup> and TGF-β<sup>2</sup> (in % of CTRL) in the LV. Data are given as median (line in the box) with 25th/75th percentiles (boxes) and 10th/90th percentiles (whiskers). All values were obtained from 82-week-old SHR. Significance marks: \* significant vs. CTRL; Ø significant vs. CAP.

However, both therapies significantly reduced the protein concentrations of TGF-β<sup>1</sup> and TGF-β<sup>2</sup> in the LV. TGF-β<sup>1</sup> concentration decreased by 33% (CAP) and 21% (CAP + NIF) compared to CTRL (*p* = 0.03). For TGF-β2, the combination therapy proved to be more effective than monotherapy (*p* < 0.001), and reduced the protein concentration by 41% (CAP) and by 60% (CAP + NIF) compared to CTRL (*p* < 0.001; Figure 3d,e).

*MMP-2 and TIMP-2:* The expression of MMP-2 and TIMP-2 mRNA in the LV of CTRL rats was 5.58% [4.43/8.98] and 24.9% [13.7/28.8], respectively. Both therapies resulted in a significant reduction of mRNA expressions of MMP-2 (CAP by 53%, CAP + NIF by 46% of CTRL; *p* = 0.001) and TIMP-2 (CAP by 57%, CAP by 59% of CTRL; *p* < 0.001). Both treatments also decreased the activity of MMP-2 by 41% (CAP) and 51% (CAP + NIF) of CTRL (*p* = 0.03; Figure 4). ANCOVA results showed that these changes did not depend on age, BW and SBP at baseline.

**Figure 4.** (**a**,**b**): MMP-2- and TIMP2 mRNA expression (in % of GAP-DH) in the LV. (**c**): MMP-2 activity (in % of CTRL) in the LV. Data are given as median (line in the box) with 25th/75th percentiles (boxes) and 10th/90th percentiles (whiskers). All values were obtained from 82-week-old SHR. Significance marks: \* significant vs. CTRL.

*Collagens:* In the LV of CTRL rats, mRNA expression of Coll I and Coll III was 13.8% [9.7/16.7] and 24.8% [22.4/26.8], respectively. Both treatments significantly reduced Coll I to 4.5–4.8% (i.e., about 34% of CTRL, *p* < 0.001) and Coll III to 9.5–10.5% (i.e., about 40% of CTRL, *p* < 0.001; Figure 5). ANCOVA revealed that the effect of the treatments was not dependent on the values of the covariates age, BW and SBP at baseline.

**Figure 5.** Coll I (**a**) and Coll III (**b**) mRNA expression (in % of GAP-DH) in the LV. Data are given as median (line in the box) with 25th/75th percentiles (boxes) and 10th/90th percentiles (whiskers). All values were obtained from 82-week-old SHR. Significance marks: \* significant vs. CTRL (*p* < 0.001).

3.3.2. Histological Manifestation of Cardiac Fibrosis

The hearts of the untreated CTRL rats showed a marked interstitial and perivascular fibrosis (fibrosis degree 2.3 ± 0.11; Figure 6A). Both treatments significantly reduced the degree of perivascular and interstitial fibrosis. CAP therapy (fibrosis degree 1.8 ± 0.05; *p* = 0.001 vs. CTRL) proved to be slightly superior to CAP + NIF treatment (fibrosis degree 2.1 ± 0.07; *p* < 0.001 vs. CAP; Figure 6B,C). The histological degree of fibrosis did not depend on baseline age (about 60 weeks), but there was an interaction with BW and SBP at baseline.

**Figure 6.** Hearts of 82-week-old CTRL and treated SHR in trichrome staining (10-fold magnification; the scale bar in part C represents 0.1 mm). (**A**): CTRL, perivascular and marked interstitial fibrosis, fibrosis degree of the group 2.3 ± 0.11; (**B**): CAP, perivascular and slight interstitial fibrosis, fibrosis degree of the group 1.8 ± 0.05; (**C**): CAP + NIF, perivascular and interstitial fibrosis, fibrosis degree of the group 2.1 ± 0.07 (data are given as means ± SEM).

#### **4. Discussion**

In the present study, we have shown that even a late start of antihypertensive therapy can achieve a significant reduction of SBP in old SHR with the long persistence of hypertension. At that age, cardiac hypertrophy, cardiac remodeling and fibrosis, as consequences of hypertension, have already developed; they could not be prevented but their progression was delayed and attenuated by both therapies. This corresponds to the functional effects of late-onset CAP and CAP + NIF treatments in old SHR as detected by echocardiography. Compared to untreated old SHR, both types of treatment significantly delayed the increase in LV wall thickness and preserved the systolic and diastolic pumping function for a significantly longer period of time [21].

#### *4.1. Development of Hypertension in SHR in Early and Later Stages of Life*

In SHR, hypertension develops in a very early period of life. Seven-week-old SHR showed 50% higher SBP values than normotensive Wistar-Kyoto rats (WKY) of the same age, and during the following three weeks, their SBP increased significantly to almost 170% of WKY [14]. During this early stage, SHR developed functional and morphological vascular changes [22,23] which are associated with vascular hypertrophy and increasing vascular resistance, thus leading to cardiac pressure overload [24]. The development of cardiac hypertrophy in SHR starts between four [25] and twelve weeks of age [26]. Our previous study on young SHR showed that mild cardiac hypertrophy and fibrosis were already present at 10 weeks of age [14]. As hypertension progresses, SHR develops increasing structural changes in the heart between the sixth and 24th months of life, which are associated with a further increase in cardiac hypertrophy [27]. In the context of hypertrophy and remodeling, numerous fetal genes are re-expressed, such as ANP [28,29].

The transition to heart failure in SHR is associated with the development of fibrosis resulting from the increased expression of genes encoding extracellular matrix (ECM) proteins and from the increased turnover of matrix proteins through increased MMP activity [20]. TGF-β as an important regulator of MMPs and TIMPs plays a crucial role in ECM remodeling [30,31]. The upregulation of TGF-β in the LV, particularly of TGF-β1, is associated with increased activity of MMP-2 and enhanced synthesis of collagens and other extracellular matrix proteins [4,8]. Of note, activation of the RAAS upregulates TGFβ<sup>1</sup> synthesis and results in myocardial fibrosis. Correspondingly, RAAS antagonization attenuates these processes [8], which was also confirmed by the present results.

Significant collagen deposition in the heart of SHR has been found as early as at 8 weeks of age [32], and its amount further increased during adulthood [33]. The remodeling processes led to reduced contractile myocardial function, ventricular fibrosis and heart failure at an advanced age (>20 months) in untreated SHR [4,34]. This was also confirmed

in an echocardiography study on old SHR. Both echocardiographic examination and heart catheterization showed marked LV dysfunction in untreated animals [21]. We assume that the loss in BW observed in the SHR CTRL group of the present study might be a consequence of this severe cardiac dysfunction. A comparison of the present results with those of the previous study in young SHR [14] reveals significant increases both in SBP and in biochemical and histological markers of ECM remodeling in the old SHR CTRL group (Table 1). In particular, it confirms a significant increase in markers of cardiac hypertrophy (HW/BW, ANP mRNA expression). Note that these markers did not depend on age, BW and SBP at the beginning of the experiment. The expression of TGF-β isoforms and TIMP-2 was also significantly higher in old than in young SHR, while MMP-2 expression was only slightly elevated. Coll I and III mRNA were equal in old and young SHR. However, considering the significantly higher histological degree of fibrosis in old SHR and the marked effects of treatment both in young and old SHR, this finding may suggest that collagen mRNA expression in SHR is elevated from a young age and does not adequately reflect the degree of collagen deposition in the heart. This corresponds with our observation that collagen mRNA in 82-week-old SHR is not dependent on age, BW, and SBP at baseline.

**Table 1.** Comparison of systolic blood pressure (SBP) and parameters of cardiac hypertrophy and ECM remodeling in young (10-week-old) and old (82-week-old) SHR.


Data from young SHR are from [14]. Normally distributed parameters are given as mean ± SEM, and statistics were performed using a t-test. Parameters that were not normally distributed are given as median [25th/75th percentile], statistics were calculated using Mann-Whitney's U test. HW/BW heart weight-to-body weight ratio; ANP atrial natriuretic peptide; TGF-β transforming growth factor-β; MMP-2 matrix metalloproteinase 2; TIMP-2 tissue inhibitor of metalloproteinases 2; Coll I, Coll III collagen types I and III.

#### *4.2. Effects of Antihypertensive Therapies*

The degree of BP reduction is associated with the age at which treatment is implemented. The early start of antihypertensive therapy does not only reduce BP but has also cardioprotective effects, which are well-documented [2,7,13]. Early antihypertensive treatment can lead to normotension, whereas late therapy-onset results only in incomplete blood pressure regulation [35]. Moreover, early antihypertensive therapy can prevent or, at least, minimize secondary damage such as cardiac hypertrophy and remodeling, which again maintain and increase hypertension [2,3]. This was fully confirmed in our previous study on young SHR [14]. Harrap and co-workers [36] suspected a critical phase with an increased sensitivity to pharmacological influences between the 6th and 10th week of life in the development of hypertension in SHR. Intervention in the blood pressure regulation during this time may change the long-term course of disease development [36]. In young rats, RAAS antagonists are particularly effective, even if the therapy is stopped after a defined period of treatment [3,35,36]. In contrast, therapy onset at a higher age cannot completely reverse the development of hypertension. A study of Kost et al. [35] showed

that SHR, which received captopril at the age of 24 weeks, had higher BP values than SHR treated with captopril at the age of four weeks. These results are in line with the findings of the present study: In old SHR, therapy with CAP or CAP + NIF over 22 weeks significantly reduced SBP by about 20%. However, these values were with 183 and 194 mmHg, respectively, about 25% higher than in 10-week-old SHR after only three weeks with CAP or CAP + NIF treatment [14]. Of note, in that study on young SHR, the relative SBP reduction (in % of baseline) was with 18% and 3%, respectively, lower than in the present old SHR study with the same doses of CAP and CAP + NIF. This clearly shows that the doses of antihypertensive drugs are also effective in old SHR indicating that their final SBP remains elevated mainly due to their higher baseline SBP. An interesting question is whether increased doses of antihypertensive drugs might induce a stronger SBP reduction.

There are only a few therapy studies in old SHR with late onset of antihypertensive treatment. Ito et al. [37], Susic et al. [38] and Mukherjee and Sen [39] examined old SHR at the age between 12 to 22 months during treatment with RAAS antagonists over different time intervals (between 4 and 12 weeks) and showed reduced signs for hypertrophy. Only Brooks et al. [40] treated SHR aged 12, 18 and 21 months with captopril 2 g/L in drinking water over a very long period of four to 12 months. They showed attenuated signs of heart failure and decreased ventricular hypertrophy even when heart failure had already started to develop.

In the present study, antihypertensive therapy with CAP or CAP + NIF over 22 weeks significantly reduced HW and HW/BW. Correspondingly, mRNA expression of the hypertrophy marker ANP in the LV decreased by 77 and 84%, respectively, compared to untreated CTRL (Figure 2). However, HW/BW was still about 12–14% above the values of 10-week-old SHR treated with CAP and CAP + NIF over three weeks. ANP mRNA of treated old SHR was even about tenfold of the values in corresponding young animals [14].

In addition, both types of treatment diminished ECM remodeling in the heart of old SHR. AT II plays an important role in the development of hypertension-induced cardiac fibrosis [8,41] by stimulating the production of IL-6 and TGF-β<sup>1</sup> in cardiac fibroblasts [42]. A significant reduction of TGF-β<sup>1</sup> expression by CAP was also shown by Brooks and co-workers [40] in 12 to 24-month-old SHR. As a result of the reduced TGF-β production, which was predominantly due to the effect of CAP, we observed a significant reduction of mRNA expressions of MMP-2, TIMP-2, Coll I and Coll III, as well as of MMP-2 activity by about 50–60% in the LV of old SHR (Figures 3–5). Consequently, a significant decrease of histological signs of fibrosis was found in CAP and CAP + NIF animals (Figure 6). However, this effect was much lower than in young SHR: in our previous study on young SHR, fibrosis was attenuated by 60% with CAP + NIF treatment and completely abrogated by CAP treatment [14]. In contrast, in old SHR the fibrosis degree only decreased by 9% and 22%, respectively, despite a much longer period of treatment. As indicated by ANCOVA results, the high SBP at the beginning of treatment might have contributed to this reduced therapy effect. Consequently, functional deterioration due to fibrotic impediment could be delayed but not completely prevented.

An echocardiographic study in old SHR demonstrated that with antihypertensive treatment, systolic and diastolic pump function in treated SHR was maintained for a longer period of time. In these animals, a slight deterioration of cardiac function began around the 76th week of life, while untreated rats showed a progressive decline of systolic and diastolic function throughout the experimental period [21]. Once structural changes in LV have occurred, therapy with inhibitors of AT converting enzyme or calcium antagonists can no longer reverse them but delay and attenuate the deterioration of cardiac function [43]. Nevertheless, even in long-standing hypertension, which may be diagnosed late, immediate therapy can significantly lower SBP, delay the development of hypertrophy and significantly reduce fibrosis, as demonstrated in the present study. Thus, the onset of heart failure can be prevented or at least delayed.

#### *4.3. Comparison of Monotherapy CAP with Combination Therapy CAP + NIF*

Previous studies in rats have shown that RAAS antagonists have superior antihypertensive effects compared to other therapies, e.g., calcium antagonists [44]. RAAS antagonists play an important role in lowering HW, preventing cardiac hypertrophy and mitigating structural remodeling of the cardiovascular system and terminal organ damage in young SHR [2,7,13]. In our previous study on young SHR, a monotherapy with the calcium antagonist NIF proved to be effective, but was less effective than CAP [14].

In the present study on old SHR, monotherapy with CAP and the combination of CAP + NIF proved to be equivalent. Previous results of preliminary examinations in young SHR showed a particularly high antihypertensive and cardioprotective effect of therapies with RAAS antagonists [14]. Two therapies from that study were applied in the present study to old SHR (CAP and CAP + NIF). These therapeutic regimens induced a significant reduction in SBP and an attenuated and delayed development of cardiac hypertrophy. An echocardiographic examination in old SHR demonstrated that rats treated with CAP or CAP + NIF exhibited a stable LV function, while untreated SHR showed clear signs of LV insufficiency [21].

#### *4.4. Limitations of the Study*

The main limitation of this study is the low number of experimental animals. The group size was calculated for a medium effect size of SBP reduction (with Cohen's f = 0.25). However, clinical symptoms of decompensated heart failure and also the BW reduction of CTRL rats showed high interindividual differences. These characteristics correspond to observations in human pathology. The number of animals to be examined should be kept as low as possible, since only as little SHR as necessary should be exposed to the suffering caused by the consequences of hypertension. The calculated sample size of 21 animals was considered representative.

A further limitation is that we did not measure tibia length. Treated SHR slightly increased their body weight over the experimental period, while untreated CTRL animals showed a weight loss of 11% relative to baseline. Thus, the ratio of heart weight to tibia length would be more adequate than HW/BW in reflecting cardiac hypertrophy.

With respect to the similar efficacy of CAP monotherapy and CAP + NIF combination therapy, further studies should include a direct comparison of monotherapies with CAP and with NIF in old SHR. Further research might also examine the question of whether a late-onset therapy with higher doses of antihypertensive drugs may result in the further reduction of SBP and better cardioprotective effects.

#### **5. Conclusions**

Two therapy regimes, a monotherapy with CAP and a combination therapy with CAP + NIF, proved to be equally effective at reducing SBP and delaying the development of cardiac secondary damage in old SHR. Long-term-treatment significantly reduced SBP as well as cardiac hypertrophy and fibrosis. However, the levels of young SHR with earlystarting treatment were not achieved. These findings are of importance with regard to the late diagnosis of arterial hypertension in humans, which is often made in the elderly. Consequent antihypertensive therapy, even if it starts at an advanced age, can reduce cardiac stress and thus cardiac secondary diseases.

**Author Contributions:** B.R., H.-G.Z., C.H. and J.B. contributed conception and design of the study; C.H., J.B. and B.R. performed the animal experiments including blood pressure measurements. C.H. prepared the histological slices; C.H., K.S. and P.G. evaluated and took photographs from the histological slices. C.H. performed the molecular biological analyses; C.H. and A.S. evaluated the molecular biological data. C.H. and B.R. performed the statistics. C.H., J.B. and P.G. created figures and tables. C.H. and B.R. drafted the original manuscript; A.S. and H.-G.Z. reviewed and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** This manuscript does not contain clinical studies or patient data. The study was conducted according to the guidelines of the Declaration of Helsinki. All animal protocols were approved by the state agency (Landesdirektion Sachsen, number and date of approval: TVV 36/08; 13 May 2009) in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and with the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" [17].

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We gratefully appreciate the valuable technical assistance of Brigitte Mix, Ursula Vogt and Grit Marx. The authors acknowledge support from the German Research Foundation (DFG) and Universität Leipzig within the program of Open Access Publishing.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.

#### **Abbreviations**


#### **References**

