In this study, we analyzed functional and molecular outcomes from continuous and transient rapamycin treatment in aged, male and female C57BL/6 mice. In both sexes, rapamycin treatment replicated our previous results showing a significant improvement in cardiac diastolic function, and this effect was persistent for 2 months after rapamycin was eliminated from the diet. By focusing on molecular changes due to rapamycin treatment that persist after drug removal, we hoped to shed light on the specific mechanisms of cardiac functional rejuvenation induced by rapamycin treatment.

Work from our laboratory and others has shown that continuous rapamycin improves cardiac function, most specifically diastolic function, when administered to middle‐ or late‐aged mice (Dai et al., 2014 ; Flynn et al., 2013 ). Rapamycin can also improve cardiac structure and function in the context of various genetic and experimental conditions that promote cardiac disease (Das et al., 2014 ; Paul et al., 2014 ).

Rapamycin is an FDA‐approved drug that directly inhibits the mechanistic target of rapamycin (mTOR) Complex I (C1). Inhibition of mTORC1 has wide‐ranging effects in vivo, including altering protein synthesis, inhibiting cell growth, and stimulating stress response mechanisms and autophagy. Transient or lifelong treatment extends lifespan and/or healthspan in many organisms, ranging from nematodes to primates (Bitto, Wang, Bennett, & Kaeberlein, 2015 ). Rapamycin extends murine lifespan in both sexes, even when administered at 9 or 20 months of age in genetically heterogeneous mice (Harrison et al., 2009 ; Miller et al., 2011 ), and in C57BL/6 mice at 19 (Zhang et al., 2014 ) or 20–21 months of age (Bitto et al., 2016 ). The lifespan and healthspan extension due to rapamycin is both dose‐ and sex‐dependent (Miller et al., 2014 ). Clinically, rapamycin and so‐called “rapalogs” are used to prevent rejection after organ transplantation (Fine & Kushwaha, 2016 ) and for the prevention of restenosis after insertion of cardiac stents (Park et al., 2013 ). Major concerns in considering potential clinical translation of rapamycin treatment are potential detrimental effects that include immunomodulation, gonadal atrophy, and stomatitis (Boers‐Doets et al., 2013 ; Pallet & Legendre, 2013 ). However, these adverse effects are generally reversible, leading to the question of whether the more desirable healthspan effects of rapamycin might persist after the undesirable effects have resolved.

It is estimated that by 2030, over 8 million people will suffer from heart failure (HF) in the United States alone (Heidenreich et al., 2013 ). In the developed world, HF is the condition most responsible for poor healthspan in males (age‐standardized years lived with disability; Moran et al., 2014 ). The estimated cost of HF in the United States is estimated to be $70 billion by 2030 (Heidenreich et al., 2013 ). Historically, most attention has been focused on HF with reduced ejection fraction (HFrEF), such as may result after myocardial infarction; however, the Atherosclerosis Risk in Communities study reported that 47% of US hospitalizations due to HF were due to HF with preserved ejection fraction (HFpEF; Chang et al., 2014 ). HFpEF is generally defined clinically by signs and/or symptoms of HF combined with preserved left ventricular (LV) ejection fraction (EF). In this setting, reduced cardiac output is related to impaired diastolic LV filling, and this results in exercise intolerance and contributes to frailty. Cardiac aging in both humans and mice is characterized by a progressive decrease in diastolic function and an increase in LV hypertrophy (Chiao & Rabinovitch, 2015 ). These similarities make aging rodents a good model for study of pharmacotherapies directed toward correcting diastolic function (Dai & Rabinovitch, 2009 ). While in recent decades medical management has enjoyed substantial success in improving health and survival of patients with HFrEF, effective treatment for HFpEF has been elusive. Despite the efforts of several large randomized clinical trials designed to improve quality of life in patients with HFpEF, results have thus far been largely disappointing (Plitt, Spring, Moulton, & Agrawal, 2018 ).

2 RESULTS

2.1 Rapamycin persistently improves diastolic function In both humans and mice, diastolic function is measured by comparing the relative proportion of LV filling that takes place in early diastole by LV relaxation (Ea) versus that takes place in late diastole, secondary to atrial contraction (Aa). In healthy hearts, the early component is greater than the late component, and diastolic dysfunction is conventionally ascribed when there is a reversal of this ratio, that is, an early‐to‐late filling ratio below 1.0. At the start of the study, the 24‐month‐old male and 22‐month‐old female mice (both at 75th percentile of lifespan; Turturro et al., 1999) demonstrated an Ea/Aa ratio averaging close to 1 (approximately half the mice above and half below 1.0), which is typical of this age in C57BL/6NIA mice. In the control group, this ratio stayed steady, but animals exposed to rapamycin for 8 weeks improved their diastolic function significantly (Figure 1a). As noted previously, this level of improvement brought the Ea/Aa ratio back approximately halfway to that of young mice (Chiao et al., 2016; Dai et al., 2014). After cessation of treatment, rapamycin‐induced improvement persisted for an additional 8 weeks, with cardiac performance maintained at levels near those of mice receiving 16‐week continuous rapamycin treatment (diastolic function, 82% persistent in females and 78% in males at 16 weeks). Figure 1 Open in figure viewer PowerPoint Rapamycin persistently improves diastolic function and reverses cardiac hypertrophy. (a) Ea/Aa ratios of female and male mice over the course of treatment (average ± SEM). Continuous rapamycin treatment (rapa, dotted line), persistence (pers, dashed line), and aged control (control, solid line). Both rapa and persistence groups are statistically significantly higher than controls for weeks 8, 12, and 16 by one‐way ANOVA followed by Tukey's post hoc for all groups at each time point per sex. Rapa and persistence groups’ Ea/Aa increased significantly by one‐way ANOVA with repeated measures for each group over time. (b) Systolic function parameters measured by echocardiography at 16 weeks. EF: ejection fraction; FS: fractional shortening. Black bars, %EF; white bars, %FS; C = control, P = persistence, R = rapa, Y = young. *significant by t test between old control and young groups, †significant by t test between pers and young groups. (c) Heart mass in grams normalized to tibia length (mm) for all groups at 16 weeks. p‐values from Tukey's post hoc tests when sex‐specific one‐way ANOVA was significant. * versus young, # versus control, and versus rapa. (d) Body mass in grams for all groups over time. *p < .05, **p < .01, ***p < .001 Our previous work in 24‐ to 26‐month‐old female C57BL/6NIA mice showed no significant change in systolic functional measures (fractional shortening, FS and EF) during 10 weeks of rapamycin treatment (Dai et al., 2014). Concordantly, there were no measurable differences in FS or EF in the female mice at 16 weeks in this study (Figure 1b). However, males showed a small, but statistically significant, reduced FS and EF in old compared to young control mice. While rapamycin‐treated old mouse EF and FS were intermediate between young and old values for both sexes, these differences did not reach significance. We quantitated cardiac hypertrophy by measuring cardiac weight normalized to tibia length at necropsy (Figure 1c). Female mice at 16 weeks showed a decrease in cardiac hypertrophy after rapamycin treatment, and this effect trended toward persistent (p = .086) by t test. Males also showed a reduction in hypertrophy with rapamycin treatment (p = .012), and again, this difference approached significance in the persistence group (p = .081) by t test. Combining both sexes by scaling all data to the same‐sex control average did yield a significant difference between old and persistence groups (Figure S1). The reduction in cardiac hypertrophy cannot be explained by reduction in overall body size, as the body weight over time in all groups was similar and relatively stable (Figure 1d). Young animals were smaller for both sexes (mean ± SEM F: 21.01 ± 0.60, M: 29.57 ± 0.39).

2.2 Passive stiffness of the left ventricle is decreased with rapamycin To examine whether the change in diastolic function could be due to passive rather than active relaxation of the left ventricle, we extracted LV multicellular preparations from our control, rapamycin, persistence, and young animals (n = 5~8 per group) and tested how much force it took to passively stretch the demembranated myocardium (Figure 2a). This stiffness generally increases with age in mice, dogs, and humans (Asif et al., 2000; Campbell & Sorrell, 2015). We found that rapamycin treatment significantly and persistently reversed the age‐related increase in passive stiffness of the preparations. We did not detect a significant difference in the passive stiffness of rapamycin‐treated groups compared to persistence groups. These data suggest that myocardial passive stiffness is a significant contributor to the diastolic dysfunction seen with aging and that rapamycin can persistently reverse this effect. One potential cause of increased passive stiffness is increased fibrosis of the extracellular matrix of the ventricular wall (Jalil et al., 1989). We examined this by staining with picrosirius red and quantifying percent of collagen deposition detected under polarizing light. We observed no significant differences between old groups (control, rapamycin, and persistence; Figure 2b). Figure 2 Open in figure viewer PowerPoint Passive stiffness increases with age and is persistently decreased with rapamycin. (a) Passive force for each mouse in each condition is shown for six length changes. All data were normalized to the control animals at 24% length change. Mean ± SEM is shown for each group at each length change. p‐values are from linear modeling of log‐transformed data. N = 3~12 mice per group. (b) Percent positive picrosirius red staining in ventricle slices. Each dot is median % fibrosed area for one mouse. C: control; R: rapamycin; P: persistence; Y: young

2.3 Rapamycin dramatically alters protein abundance in both sexes; however, the persistence of these changes varies by sex In our previous work, we applied proteomics to detect many differences in protein abundances due to 10‐week rapamycin treatment beginning at 24 months in female C57BL/6NIA mice (Dai et al., 2014). Thus, an important question was whether the changes in proteome abundance with rapamycin treatment were persistent after drug removal. Figure 3a shows heatmaps of the set of all proteins in each sex which had a significant difference between control and continuous rapamycin treatment at the 16‐week time point (by Student's t test, adjusted for multiple comparisons, as described in Methods). (Figure S2 shows principal component analysis for males and females using the same data as Figure 3a) In the comparison between continuous treatment and persistence groups, females had close similarities between these two groups, while the males showed persistence group protein abundances that were intermediate between old control and continuous rapamycin. Qiagen Ingenuity Pathway Analysis (IPA) software was used to identify significantly changed canonical pathways. This revealed that eight of the top 10 pathways were conserved between sexes. The five most significantly changed pathways in each sex are shown in the heatmap of Figure 3b, four of which are conserved between sexes; again, it is apparent that the rapamycin and persistence groups are similar in the females, but in the male cohort, the persistence group is more intermediate. Gene names and z‐scores for all heatmaps are listed in Tables S1–S4. The distribution of percent persistence of the proteins within each IPA category is plotted in Figure 3c. It can be seen that for all top six pathways but mitochondrial dysfunction, the median protein abundance in the female persistence groups is actually a larger change (120%–125% effect in the same direction) than in the continuous rapamycin treatment group, whereas the median mitochondrial dysfunction pathway persistence is ~86% persistent in females (Table S5). As predicted by the overall proteomics, persistence within IPA pathways was appreciably lower in males than females and differences between pathways were less apparent. Figure 3 Open in figure viewer PowerPoint z‐scores of protein abundance differences by protein, with red indicating greater abundance and blue meaning less abundance (−1.15 > z > 1.15). (b) Z‐score heatmaps of protein abundance, organized into the five most significantly altered pathways (by IPA) for each sex—females on the left and males on the right. (c) Asymmetrical beanplots show the range of the percent persistence for proteins in each IPA category (y‐axis), with females (light gray) on the left side of each bean and males (dark gray) on the right. Black bars denote the median of the range for each sex/category. The data range was limited to 0%–200% for easier visualization. All data shown are from tissue collected at the 16‐week time point in the old mice. C—untreated control, R—continuous rapamycin treatment, and P—persistence 8 weeks after cessation of 8‐week treatment. Protein names are shown in Tables S4 Persistence of abundance changes in proteins in top IPA pathways differs by sex. (a) Dendrograms and heatmaps showing all significantly altered protein abundances due to rapamycin for each sex. Dendrograms show Spearman's distance as a measure of relatedness. Color shows‐scores of protein abundance differences by protein, with red indicating greater abundance and blue meaning less abundance (−1.15 >> 1.15). (b)‐score heatmaps of protein abundance, organized into the five most significantly altered pathways (by IPA) for each sex—females on the left and males on the right. (c) Asymmetrical beanplots show the range of the percent persistence for proteins in each IPA category (‐axis), with females (light gray) on the left side of each bean and males (dark gray) on the right. Black bars denote the median of the range for each sex/category. The data range was limited to 0%–200% for easier visualization. All data shown are from tissue collected at the 16‐week time point in the old mice. C—untreated control, R—continuous rapamycin treatment, and P—persistence 8 weeks after cessation of 8‐week treatment. Protein names are shown in Tables S1 The mitochondrial dysfunction pathway is a larger set of proteins (females n = 27, males n = 55) compared to the other top five pathways (females n = 6–18, males n = 19–29), and was more heterogeneous. When electron transport chain (ETC) proteins alone were examined, persistence was varied between the sexes and the individual complexes of the ETC (Table 1), with the females again generally showing greater persistence in each complex than the males. Interestingly, the mean persistence of proteins in Complex V of the respiratory chain (ATP synthase) was very high in both sexes. Many of the proteins found to be altered significantly in both sexes for the mitochondrial dysfunction category were associated with Complex I (NADH:ubiquinone oxidoreductase) of the ETC; persistence of proteins in this complex was 76.90% in females and 26.34% in males. Table 1. Average percent persistence per individual complex of the respiratory chain by sex Females Males % SD n % SD n CI 76.90 41.55 41 26.34 38.27 39 CII 67.61 22.01 3 19.00 49.78 4 CIII 94.25 78.93 9 −6.54 68.64 9 CIV 92.70 85.36 14 32.24 54.82 16 CV 145.75 108.29 8 143.24 238.64 7 One explanation for variation in the degree of persistence of proteins might be a relationship with protein half‐life, in that proteins with faster turnover might show less persistent changes after withdrawal of rapamycin treatment. However, we did not find any significant correlation between persistence of protein abundance changes due to rapamycin and our previously measured half‐lives of the same proteins in the heart (Dai et al., 2014; data not shown).

2.4 Rapamycin differentially alters respiratory chain complex activity by sex Since rapamycin did persistently alter ETC Complex I (CI) peptide abundances for both sexes, we hypothesized that there may be a connection between ETC complex activity and diastolic function—that is, rapamycin might persistently alter the function of the ETC in both sexes and more specifically that rapamycin might change the ratio of complex activity to complex abundance, thereby changing the flux through the ETC. Electron flux is largely responsible for determining the mitochondrial membrane potential (ΔΨ M ), by creating a proton gradient across in the inner mitochondrial membrane. An increase in ΔΨ M has been correlated with an increase in the production of ROS in myocardium and in isolated cardiac mitochondria (Aon, Cortassa, & O'Rourke, 2008; Chen & Zweier, 2014). Rapamycin has previously been shown to lower ΔΨ M , through a mechanism independent of its well‐known cellular targets (Schieke et al., 2006). Thus, changing the ratio of complex activity to complex abundance might reduce the ΔΨ M from a more pathological level in the aging heart, to a more physiological level. The impact of this could be reduced ROS, while maintaining appropriate ATP production, as needed for cardiac cycling activity. We therefore compared activity levels of the individual ETC complexes to the abundance of Complex proteins in the ETC, thereby obtaining a measure of activity/quantity of ETC protein. In the female cohorts, we were unable to detect significant differences in activity of CI, CII, and CIV between treatment groups (by ANOVA; Figure 4). There was, however, an age‐related increase in CIII activity in females, which was reduced persistently by rapamycin. Males showed a significant increase in CI activity with aging, which was partially reversed, again persistently, with rapamycin (Figure 4). Males also showed a significant increase in CII activity with rapamycin, though this was not persistent eight weeks after drug removal. All complex activity data were normalized to mitochondrial content, as determined by citrate synthase (CS) activity (see Figure S3). We also measured mitochondrial:nuclear DNA ratios in all samples and determined that mitochondrial content by this metric was similar between all old groups within each sex (Figure S3). Figure 4 Open in figure viewer PowerPoint Rapamycin alters ETC Complex activity differentially by sex. (a) Boxplots of the activity in nmol/min mg−1 of each of the complexes, normalized to the activity of citrate synthase for each sample, then as a ratio against the control average activity per complex. Each data point is one mouse (average from technical triplicates). N per group is 8 to 16. C—control at 16 weeks, P—persistence, R—rapa, Y—young. Stars indicate significance, by Tukey's post hoc after an ANOVA of all groups per sex/complex which had an ANOVA p < .05, compared to control. *p < .05, **p < .01, ***p < .001 While there was sexual dimorphism in the abundance and activity of components of ETC CI and CIII, both sexes consistently demonstrated a much greater ratio of activity:abundance in old controls than in young animals (Figure 5; see Discussion). The 16‐week rapamycin and the persistence treatment groups both showed a significant decline in the ratio of activity:abundance of CI, bringing levels toward that of the young animals. Inclusion of accessory components in addition to core components of CI did not substantially alter this effect. In CIII, females, but not males, showed a reversal of the age‐dependent increase in activity:abundance ratio in rapamycin and persistence groups (Figure 5). Consistent changes were not found in CII or CIV.