This study investigated whether cardiac remodeling induced by AII differed between the sexes in older C57BL/6 mice and explored underlying mechanisms. We found that 6 weeks of AII treatment caused a pronounced increase in BP in both sexes. However, AII-induced structural and functional remodeling of the ventricle differed importantly between the sexes. While AII increased signs of LV hypertrophy such as wall thickness predominantly in females, it led to LV dilation and reduced wall thickness primarily in males. Diastolic dysfunction, characterized by elevated E/A ratios and prolonged IVRT, was observed in males while females exhibited an increase in IVRT only. However, male hearts exhibited marked systolic dysfunction associated with reduced EF and a profound prolongation of IVCT. By contrast, EF was preserved above 50% and IVCT was unaffected in female hearts. Our qPCR results showed increases in mRNA for M1 (Cd80) and M2 (Mrc1) macrophages involved in inflammatory responses in AII-treated male but not female ventricles. We also found an increase in mRNA for pro-fibrotic markers (Col1a1, Col3a1, Tgfb1) in male but not in female ventricles. This male-specific increase in collagen 1 also was observed at the protein level, but AII increased collagen 3 in females when compared to AII-treated males. There was also a baseline sex difference in collagen 1 protein levels, where control females had higher levels than control males. Together, these results demonstrate that chronic exposure to AII has sex-specific adverse effects in the ventricles from older mice.

We found that chronic infusion of AII led to signs of diastolic dysfunction in hearts from older male and female mice. This included prolonged IVRT in both sexes and an increase in E/A ratios in males only. These signs are consistent with diastolic dysfunction [35] and indicate that AII slows ventricular relaxation and promotes diastolic dysfunction in the setting of aging. Previous studies report that pressor doses of AII either have no effect on IVRT or they increase IVRT in young (8–12-week-old) male mice [38, 39]. Our results extend these observations to demonstrate that AII markedly increases IVRT in the setting of aging, and that this occurs in both sexes. Unlike the increase in E/A ratios seen in our work, pressor doses of AII actually reduce E/A ratios in young male mice [38,39,40,41]. It is important to note that there are grades of diastolic dysfunction, where lower E/A ratios indicate mild diastolic dysfunction and higher ratios indicate profound dysfunction [42]. Thus, previous studies suggest that exposure to AII produces only minimal diastolic dysfunction, at least in young male mice [38,39,40,41]. However, the large increase in E/A ratios seen in our work indicates that AII infusion leads to marked diastolic dysfunction in older male mice. As diastolic dysfunction can precede systolic dysfunction [35], early diastolic changes may set the stage for systolic dysfunction in later life.

A critical finding in our study is the observation that chronic exposure to AII produced profound systolic dysfunction in hearts from aging male mice but had much less effect on systolic function in aging females. Indeed, we found that EF was markedly reduced and IVCT was increased by AII treatment in males. By contrast, IVCT was unaffected in females and EF remained above 50%. A reduction in EF and increase in IVCT are associated with poor systolic function [36, 43]. In addition, increases in IVCT predict the occurrence of HFrEF clinically [36, 43]. This suggests that AII treatment promotes severe systolic dysfunction in males but not females, and this may predispose aging male hearts towards HFrEF. In addition to the functional changes we observed, the structural changes seen in male hearts are compatible with a HFrEF phenotype. For example, the LV dilation and thinner ventricular walls seen in AII-treated male hearts is characteristic of eccentric hypertrophy, as typically seen in HFrEF [44]. Our findings are compatible with male–female differences seen clinically, where older men are much more likely to develop HFrEF than older women [27].

In contrast to males, we found few signs of systolic dysfunction in older female mice. EF declined only slightly in female mice exposed to AII and remained above 50% at the end of the study. In addition, AII had no impact on IVCT in hearts from older females. This suggests that, despite a robust increase in BP due to AII infusion, systolic function was relatively preserved in older female mice. Females also exhibited diastolic dysfunction, which is compatible with a HFpEF phenotype. The structural changes we observed in older female hearts also are consistent with this idea [44]. For example, there were signs of concentric hypertrophy in AII-treated female hearts, including thicker LV walls with no increase in LV chamber volumes, as seen in HFpEF [44]. These observations concur with clinical findings, where HFpEF is much more common in older women than in age-matched men [28].

Chronic RAS activation promotes inflammation that contributes to adverse cardiac remodeling [3, 4, 10]. Therefore, we investigated whether markers for macrophages involved in pro- (M1) and anti-inflammatory (M2) responses were affected by AII and whether this differed between the sexes. Interestingly, we found an increase in the M1 macrophage marker Cd80 in ventricles from males but not females. As M1 macrophages release pro-inflammatory cytokines and promote tissue damage [45], an increase in AII-mediated inflammation in male hearts may contribute to the adverse functional and structural remodeling observed in our study, although additional studies will be required to test this directly. Interestingly, the M2 macrophage marker Mrc1 also was elevated in ventricles from male mice. As M2 macrophages are involved in anti-inflammatory responses and repair of damaged tissues [45], it is possible that compensatory responses to reduce inflammation are activated in the aging male heart. Although M1 and M2 macrophage markers were elevated in AII-treated ventricles from older males, this was not observed in age-matched females. The reasons for this sex difference are unclear. While both sexes exhibit age-related immune dysfunction, older males exhibit more profound maladaptive changes in immune function than age-matched females [46]. In addition, the degree of systemic chronic inflammation (known as inflammaging) is higher in older males than in older females [46]. In theory, this may serve to augment male responses to immunoinflammatory challenges like exposure to AII. However, many other factors including hormonal status, the environment and overall health also can promote immune dysfunction and inflammaging, and these may also influence male–female differences in responses to AII [46, 47].

AII-induced inflammation can convert cardiac fibroblasts to myofibroblasts, which increases synthesis of extracellular matrix proteins (e.g., collagens) leading to cardiac fibrosis [6,7,8,9, 48]. We therefore investigated markers of fibrosis including Col1a1, Col3a1, Tgfb1, and Fn1 to determine if they were affected by AII. Levels of mRNA for Fn1, an extracellular matrix component that is activated before collagen deposition [49], were not affected by AII in either sex. However, mRNA for Tgfb1, a growth factor that converts fibroblasts to myofibroblasts, and mRNA for the primary cardiac Col1a1 and Col3a1 [48] was increased in hearts from male but not female AII-treated mice. This suggests that markers of cardiac fibrosis are increased by AII treatment in male but not female hearts.

The corresponding collagen 1 protein levels also were increased by AII treatment in male hearts when but not in female hearts. In terms of impact on the aging male heart, the AII-induced increase in collagen 1 would be expected to increase ventricular fibrosis and promote systolic dysfunction [48, 50, 51] as we observed in this study. We also found that AII treatment increased collagen 3 protein in females compared to collagen 3 protein levels in males. As collagen 3 is deposited earlier than collagen 1 during cardiac fibrosis [52], it is possible that AII would increase collagen 1 over a longer time frame in females. On the other hand, overexpression of collagen 3 prevents systolic dysfunction, LV dilatation, and ventricular fibrosis in a rodent model of ischemic heart disease [53]. Thus, this may be a beneficial change in the female heart that helps mitigate adverse effects of AII. We also found that the levels of collagen 1 protein were higher in control females than in control males. Interestingly, previous work has shown that, while young female hearts have less collagen 1 than males, collagen 1 increases with age due to an increase in fibroblast proliferation in female but not male hearts [54]. This may account for the higher levels of collagen 1 protein in female control mice when compared to male controls. However, it is evident from our findings that collagen 1 does not further increase in response to AII in older female hearts. We also saw an increase in mRNA for Tgfb1 in treated males, with no corresponding increase in protein. Perhaps if we had waited longer, we would have observed an increase in TGF-β protein.

There are limitations to the work presented here. The results of our qPCR and Western blot studies clearly indicate that AII induced markers of fibrosis in aged male but not female mice; however, it would be of interest to quantify the extent of fibrosis with Picrosirius red or Trichrome staining in future work. We used 16-month-old mice [55] to approximate the age of onset of hypertension (e.g., mean age of onset of 46 years) seen clinically [29]. In future work, it would be interesting to include younger mice to determine if the observed sex difference emerges at earlier ages. It would also be of interest to determine whether adjusting the AII dose to a more extended treatment period would exacerbate these male–female differences in cardiac remodeling. AII had little effect on heart rate, although it caused a small decrease in older males. It is important to note that heart rate was measured in anesthetized mice undergoing echocardiography and isoflurane can affect heart rate in C57Bl/6 mice [56]. Direct electrocardiogram recordings in unanesthetized mice would be of interest. We also found that AII caused a slightly larger increase in BP in males (1.5-fold) than in females (1.4-fold). Although it is possible that some sex-specific effects of AII could relate to this small difference in BP, this seems unlikely given the small magnitude of the difference in BP compared to the large sex differences in cardiac remodeling reported here. Finally, some echocardiography measurements were variable, especially in females. For example, the IVRT is longer in control females than males at the end of the study but not at baseline. In addition, AII increased LVPWd in females only. While this observation should be interpreted cautiously, the increase in LVAWs is convincing and supports the idea that LV wall thickness increases in response to chronic AII in females. A larger sample size for these parameters would have been helpful.

Our study has evaluated the impact of AII on cardiac remodeling in relevant preclinical models (e.g., older mice of both sexes). As previous preclinical studies of AII remodeling have used young, mostly male animals, our findings help fill an important knowledge gap. It seems clear that both age and sex are critical in setting the stage for the expression of late life illnesses like cardiovascular diseases. Preclinical studies of chronic conditions, like cardiovascular diseases, should include older animals of both sexes to better mimic the individuals who are most likely to develop these diseases as they age. Our findings have potential clinical significance. We showed that chronic exposure to AII at this age resulted in adverse cardiac remodeling featuring both structural (hypertrophy, dilation, inflammation, and fibrosis) and functional (systolic and diastolic dysfunction) impairment that differed in many respects between the sexes. For example, aged male mice have increased E/A ratios and prolonged IVRT suggestive of diastolic dysfunction in response to after AII infusion, while older females exhibit only prolonged IVRT. In addition, AII induces profibrotic responses in males only. This suggest that the molecular mechanism involved in diastolic dysfunction may differ between the sexes. Future studies should explore whether there are female-specific impairments in calcium handling and/or myofilament proteins, both of which are implicated in diastolic dysfunction [57]. A greater understanding of these underlying mechanisms may help identify new sex-specific therapeutic strategies for HF treatment in older people. At present, treatment of HFrEF with sacubitril–valsartan (neprilysin inhibitor/AII receptor blocker) is recommended for both sexes, but it is not yet clear that this treatment is effective in HFpEF, regardless of sex [58]. As collagen levels were higher in males, therapeutic strategies targeting cardiac fibrosis may be beneficial in older men. Additional studies to explore this concept would be of interest.

In summary, older male mice exposed to chronic AII infusion exhibit profound systolic dysfunction with LV dilation and thinning of the ventricular walls, changes that are not seen in older females. This appears to be mediated, at least in part, by enhanced profibrotic signaling in older male hearts. In contrast, AII-treated females exhibit increased LV wall thickness, with EF preserved at values well above 50%. These findings are important as they provide mechanistic insights into the higher prevalence of HFrEF in men and increased prevalence of HFpEF in women [27]. These observations may help explain sex differences in HF in later life.