*4.2. Cardioprotective Effects*

The therapy effects were not confined to antihypertensive effects but also included cardioprotective effects. With C+N treatment, cardiac hypertrophy and remodeling were differentially attenuated in young and old SHRs.

#### 4.2.1. Effects on Cardiac Hypertrophy and Remodeling in Young SHRs

In genetic forms of hypertension such as in SHRs or in humans with essential hypertension, vascular hypertrophy, and consequently elevated vascular resistance, cause pressure load to the LV, which in turn induces hypertrophy and remodeling of the LV myocardium. In a previous study on 6-month-old SHRs, both total peripheral resistance (TPR) and HW/BW were significantly higher than in age-matched WKY rats [35]. A similar observation has been made in the ySHR study: TPR of yCtrl rats was also significantly higher than in age matched WKY rats (0.20 ± 0.01 and 0.12 ± 0.02 mmHg·min·kg·mL−1, respectively; *p* = 0.01; unpublished data).

In SHR, development of cardiac hypertrophy starts after the prehypertensive stage between the 4th and 12th week of life [36,37]. At this stage, HW/BW of SHRs is about 13% higher than that of age-matched WKY [24]. Several studies showed that antihypertensive therapy with RAS antagonists started between the 4th and 14th week of life reduced HW/BW or LVW/BW by 19–27% [24,29,30,38,39]. Our study on young SHRs provided similar results: three weeks of C+N treatment reduced HW/BW by about 14%, but not to the level of normotensive WKY rats [26]. Without treatment, cardiac hypertrophy progresses over time, but even when treatment with RAS antagonists is started in adulthood (between week 24 and 34 of life), significant attenuation of cardiac hypertrophy can be achieved [24,40].

However, with progress of cardiac hypertrophy, remodeling processes including profibrotic processes will develop. Perrucci and co-workers demonstrated cardiac fibrosis and significantly increased collagen deposition in the hearts of 8-week-old untreated SHRs [41]. The progredient increase in blood pressure and the development of LV hypertrophy and fibrosis are accompanied by a deterioration of LV function. Echocardiographic examination demonstrated significant systolic and diastolic dysfunction in untreated SHRs even at 2–3 months of age [37]. With enalapril treatment over 14 weeks, deterioration of ejection fraction and fractional shortening as well as development of cardiac fibrosis were prevented or at least attenuated [42]. This is in line with our observations on ySHRs; at 10 weeks of age, they presented mild cardiac fibrosis, which was significantly reduced by more than 50% with three weeks of C+N treatment [26].

#### 4.2.2. Effects on Cardiac Hypertrophy and Remodeling in Old SHRs

With progress of age, the HW/BW differences between SHRs and WKY increased up to 29% at week 83 [24]. In the advanced stage of hypertension at week 82, cardiac hypertrophy of our untreated oSHRs had achieved a markedly higher degree than in young animals, as reflected by the significantly higher values of ANP mRNA expression (20 times higher than in yCtrls) and HW/BW (more than 30% higher than in yCtrls). Angiotensin II exerts many prohypertrophic effects on the heart. Consequently, prevention or attenuation of cardiac hypertrophy and transition into heart failure is a major goal of ACE inhibitor therapy [43]. Even in normotensive WKY rats, RAS antagonists can significantly reduce HW/BW [24]. In senescent SHRs at more than 80 weeks of age, short-term treatment with RAS antagonists has only weak effects: after 8 weeks of losartan therapy, HW/BW was only reduced by 7% [24]. In contrast, chronic captopril treatment administered from 14 to 24 months of age-reduced LVW/BW by about 30% [44], which is similar to the results of the present study. Direct antihypertrophic effects on cardiomyocytes have also been demonstrated for nifedipine [45]. Thus, cardioprotective effects of C+N treatment are not only based on antihypertensive effects, but in addition on direct antihypertrophic effects. This is also reflected in the decrease in HW related to the decrease in SBP, which was even higher in oSHRs than in ySHRs.

As cardiac hypertrophy progresses, remodeling and accumulation of extracellular matrix (ECM) advance too. Increased mRNA levels of ECM molecules such as transforming growth factor-beta and tissue inhibitor of metalloproteinases 2 have been found in 82-weekold untreated SHRs [27]. Of note, the levels of collagen mRNA and protein develop with aging in a nonsynchronous way: in young rats, collagen mRNA in the heart is relatively high, but does not result in collagen accumulation. In contrast, collagen mRNA in hearts of old rats is hardly elevated, but induces a two-fold increase in collagen protein content [46]. This may explain the similar levels of collagen mRNA in old and young untreated SHRs in the present study, which were associated with different histological degrees of fibrosis. While C+N treatment in ySHRs did not decrease collagen mRNA expression, but significantly reduced fibrosis, the opposite effect was achieved in oSHRs. Analysis of mRNA expression can only reflect a moment within a process developing over a longer period of time. In particular, accumulation of collagen is not linearly related to elevated levels of collagen mRNA. Hence, we consider the histological degree of fibrosis to be the more meaningful parameter with respect to cardiac remodeling. Even though the treatment effect on collagen mRNA was much greater in oSHRs compared with ySHRs, attenuation of fibrosis was only weak as the intervention was initiated in an advanced stage of cardiac remodeling. An early start of antihypertensive treatment may attenuate and delay the process of remodeling more effectively.

The transition into fibrosis is associated with further functional deterioration and finally leads to cardiac failure. At 80 weeks of age, diastolic function and compliance of the LV in untreated SHRs were impaired compared with age-matched WKY [47]. Treatment significantly improved ejection fraction index, but not to the level of normotensive WKY [44]. An echocardiographic study on old SHRs demonstrated a deterioration of both systolic and diastolic LV function between week 60 and 82 without treatment. Moreover, LV catheterization under anesthesia revealed signs of cardiac failure and LV decompensation. All of these changes were attenuated and delayed, but not completely prevented by antihypertensive treatment [48]. These results are in line with the weak antifibrotic effect of a late-onset treatment.

#### 4.2.3. Early-Onset versus Late-Onset Treatment

Similar to its effects on SBP, early-onset antihypertensive treatment also has long-lasting effects on cardiac hypertrophy and remodeling. Antagonization of prohypertrophic Angiotensin II effects in a sensitive period of life (up to age 20 weeks) makes even a transient treatment with ACE inhibitors effective with regard to prevention of LV hypertrophy [29,39].

The duration of treatment is an important factor with regard to the treatment effect. This is well illustrated by a comparison of our results with those of Demirci and co-workers: They applied losartan at the same dose over the same period of time to young and old SHRs and observed a lower treatment effect in the old animals [24]. In contrast, we administered the same medication (C+N) to young and old SHRs, but a more than sevenfold treatment interval was necessary to achieve a similar antihypertrophic treatment effect in oSHRs [26,27]. Lifelong antihypertensive treatment started at one month of age doubled

the lifespan of stroke-prone SHRs to 30 months, which is identical to that of normotensive WKY. In contrast, 80% of untreated SHRs had died after 15 months [49]. These findings emphasize the importance of a long-lasting antihypertensive therapy, and this is the more important the later in life treatment is initiated.
