The current data focus on inward left ventricular hypertrophy and associated factors. Our prior work reports group measures from the FST and GTT [25]. Therefore, while main effects are stated below, sex- and stress-specific differences are outlined in the supplemental material.

Heart size was determined by 3 measurements: left ventricular diameter, interior wall thickness, and free wall thickness (Fig. 2). Two-way ANOVA comparison of ventricular diameter found a main effect of stress [F(1, 111) = 11.62, p = 0.0009, η2 = 9.326] with post-hoc analysis indicating the No CVS females had larger ventricle diameter than No CVS males (Fig. 2A; p = 0.0437). Interior (Fig. 2B) and free (Fig. 2C) wall measurements both exhibited main effects of sex [F(1, 110) = 13.71, p = 0.0003, η2 = 11.0] and [F(1, 111) = 19.08, p < 0.0001, η2 = 14.58], respectively. Post-hoc analysis of the interior wall measurements indicated CVS females had thicker interior walls than CVS males (p = 0.0144). This was reflected in the free wall analysis with CVS females (p = 0.0082) and No CVS females (p = 0.0177) having significantly thicker free walls than their male counterparts. There were no significant differences within-sex for ventricle measurements.

Heart and hypertrophy measurements. The left ventricle (A), interior wall (B), and free wall (FW) (C) were measured and corrected for body weight (BW) at the time of tissue collection. Concentric ventricular hypertrophy (VH) was represented by a ratio of the free wall to the left ventricular diameter (LVD) (D). Each group was then separated into subgroups by chronic stress (CVS) and sex (n = 8/sex No CVS and n = 12/sex CVS each for low, mid, and high), and these were compared to determine differences between subpopulations (E). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Concentric left VH was determined by a ratio of the free wall thickness to the left ventricular diameter (Fig. 2D). Analysis indicated a main effect of sex [F(1, 111) = 14.39, p = 0.0002, η2 = 11.0] where CVS females had increased inward remodeling compared to CVS males (p = 0.0009). An internal comparison of tertiles (n = 8/sex No CVS and n = 12/sex CVS) representing low, mid, and high VH within each sex and stress condition (Fig. 2E) indicated main effects of hypertrophy [F(2, 103) = 118.1, p < 0.0001, η2 = 50.77], sex [F(1, 103) = 49.89, p < 0.0001, η2 = 10.72], and stress [F(1, 103) = 4.717, p = 0.0322, η2 = 1.014]. Additionally, interactions of hypertrophy X sex [F(2,103) = 5.932, p = 0.0036, η2 = 2.550] and sex X stress [F(1, 103) = 5.520, p = 0.0207, η2 = 1.186] were present with the high hypertrophy group greater than mid and low in all conditions (Table 1). All within-sex and -stress subgroup comparisons were significant except for mid vs. low in males and No CVS females. Tertiles were defined through equal division of subjects in terms of FW: LVD and not based on a priori criteria for VH. This approach may have affected statistical separation of the low and mid VH animals in some subgroups. However, the high VH groups were significantly different from all other subgroups in each population (Fig. 2E). Overall, these data indicate that females had larger body weight-corrected heart size that culminated in increased inward hypertrophic remodeling after CVS. Further, the distribution of individuals into subgroups based on VH indicated significant population differences in VH that were moderated by sex, stress, and sex x stress interactions.

Behavioral coping style (Fig. 3) was impacted by subsequent VH depending on the age of the animals. When separated into VH subpopulations, young passive coping (Fig. 3A), represented by immobility during FST immediately following CVS, had main effects of sex [F(1,103) = 31.18, p < 0.0001, η2 = 20.26], stress [F(1, 103) = 4.641, p = 0.0335, η2 = 3.015] and future VH [F(2, 103) = 4.786, p = 0.0103, η2 = 6.218] with an interaction between VH and stress [F(2, 103) = 3.148, p = 0.0471, η2 = 4.09]. Young active coping, represented by swimming during FST, was not significantly impacted by VH (Fig. 3B). However, analysis found main effects of sex [F(1, 103) = 17.82, p < 0.0001, η2 = 12.88] and stress [F(1, 103) = 10.04, p = 0.002, η2 = 7.258], with the VH-susceptible CVS males showing less active coping than No CVS counterparts (p = 0.0462).

Coping behavior. Animals were challenged with a forced swim test immediately following chronic variable stress (CVS) and after aging. Coping style was assessed as passive (A, C) or active (B, D). These were then compared across ventricular hypertrophy (VH) subpopulations (n = 8/sex No CVS and n = 12/sex CVS each for low, mid, and high). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

In aged animals, immobility (Fig. 3C) was not impacted by VH but maintained a main sex effect [F(1, 102) = 30.75, p < 0.0001, η2 = 22.15]. However, within the VH-resilient animals, aged CVS males had more passive coping than CVS females (p = 0.0467). Active coping in aged animals (Fig. 3D) was affected by sex [F(1, 102) = 14.52, p = 0.0002, η2 = 11.36] and the interaction between stress exposure and hypertrophy [F(2, 102) = 3.565, p = 0.0319, η2 = 5.583]. Hormonal responses to FST (Fig. S1) did not have main effects or interactions that involved hypertrophy. Instead, the sex and stress effects previously reported for CVS to modify glucose mobilization and corticosterone secretion in a sex-specific manner [25] largely persisted after tertile separation. Taken together, stress coping behaviors across the lifespan were mediated by sex, stress history, VH susceptibility, and interactions between CVS and VH. Specific subgroup differences indicate that the young CVS-exposed males most susceptible to VH had reduced active coping compared to controls. Alternatively, the aged CVS-exposed females least susceptible to VH had less passive coping than chronically-stressed males.

Glucose clearance and glucocorticoid responses during hyperglycemia were not impacted by VH in young rats (Fig. S2). However, after aging, baseline corticosterone and glucocorticoid responses to metabolic stress were impacted by VH in a sex-dependent manner (Fig. 4). Baseline corticosterone (Fig. 4A) in aged animals was affected by sex [F(1, 85) = 4.912, p = 0.0293, η2 = 0.0293] and an interaction of sex, stress, and VH [F(2, 85) = 3.283, p = 0.0423, η2 = 0.0423]. Moreover, peak corticosterone levels 30 min after intraperitoneal glucose injection (Fig. 4B) had a main effect of sex [F(1, 86) = 6.772, p = 0.0109, η2 = 6.818] and total glucocorticoid release after hyperglycemia (Fig. 4C) was impacted by an interaction of VH and sex [F(2, 35) = 5.042, p = 0.0119, η2 = 20.66]. There were no significant differences in cumulative glucose clearance (Fig. 4D). Given the complex interaction between sex, stress, and VH on baseline corticosterone, additional regressive analysis is described below to isolate individual associations.

Glucose tolerance. Aged animals were metabolically challenged following chronic variable stress (CVS). Baseline corticosterone was measured at 0 min (T0) taken prior to intraperitoneal glucose injection (A). Peak corticosterone was measured 30 min following glucose injection (B). Total plasma corticosterone (C) and blood glucose (D) were calculated from an AUC analysis. Groups were analyzed according to ventricular hypertrophy (VH) subpopulations (n = 8/sex No CVS and n = 12/sex CVS each for low, mid, and high). Data are expressed as mean ± SEM. * p < 0.05. AUC: area under the curve

Measures of both visceral (mesenteric) and subcutaneous (inguinal) adiposity were significantly impacted by VH. Mesenteric adiposity (Fig. 5A) had a main effect of VH [F(2, 101) = 3.916, p = 0.023, η2 = 6.755]. Moreover, inguinal adiposity (Fig. 5B) was affected by sex [F(1, 100) = 94.98, p < 0.0001, η2 = 41.97] and an interaction between VH, sex, and stress [F(2, 100) = 3.09, p = 0.499, η2 = 2.731]. Among the VH-resilient groups, CVS males had greater inguinal adiposity than CVS females (p = 0.0038). Additionally, male rats in the mid VH group had greater inguinal adiposity than females regardless of stress (No CVS: p < 0.0001; CVS: p = 0.0005). In the VH-susceptible groups, CVS males had higher adiposity than CVS females (p < 0.0001). Additional somatic measures (Fig. S3) identified primary sex effects that did not relate to VH across spleen and adrenal indices, plasma triglycerides and cholesterol immediately following CVS, as well as body weight after CVS and aging. Collectively, subcutaneous adiposity was regulated by interactions between sex, stress history, and VH where chronically-stressed males had greater subcutaneous adiposity than CVS females across risk/resilience subpopulations.

Adiposity. Following aging, mesenteric white adipose (mWAT) (A) and inguinal white adipose (iWAT) (B) tissues were measured. Groups were analyzed according to ventricular hypertrophy (VH) subpopulations within chronic variable stress (CVS) and sex (n = 8/sex No CVS and n = 12/sex CVS each for low, mid, and high). Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. BW: body weight

In the cases where ANOVA found main or interaction effects with VH, regressive analyses were performed within stress (No CVS/CVS) and sex (female/male) conditions to examine individual associations with VH susceptibility. This approach revealed that VH positively correlated with baseline corticosterone only in aged No CVS male rats (r = 0.449, p = 0.041; Fig. 6A). Furthermore, VH positively correlated with visceral adiposity (r = 0.387, p = 0.026) only in aged males that were previously exposed to CVS (Fig. 6B). Interestingly, there were no significant correlations with VH in females across any measure, regardless of stress history. Ultimately, aged males specifically had endocrine and metabolic associations with VH susceptibility that related to early-life stress history.

Correlations. Regressive analysis within chronic variable stress (CVS) and sex (No CVS n = 24/sex; CVS n = 36/sex) identified ventricular hypertrophy (VH) associations of aged No CVS male baseline corticosterone (A) and aged CVS male visceral adiposity (B). GTT: glucose tolerance test, mWAT: mesenteric white adipose tissue, BW: body weight