Fig 2: Serum angiotensin peptides in control and MSEW mice fed a low‐fat diet (LFD) and high‐fat diet (HFD).Basal levels of the peptides were not affected by MSEW or diet in male mice: angiotensin I (AngI) (A), angiotensin II (AngII) (B), angiotensin 1‐7 (Ang1‐7) (C), angiotensin 1‐5 (Ang1‐5) (D), and angiotensin III (AngIII) (E). Data were analyzed by 2‐way ANOVA followed by Bonferroni post hoc test and reported as mean±SEM. n=5 per group in LFD‐fed mice, n=8 in HFD‐fed mice. MSEW indicates maternal separation and early weaning.

Fig 3: Renin–angiotensin–aldosterone system (RAAS) components in epididymal white adipose tissue (eWAT) and renal cortex in control and MSEW mice fed a high‐fat diet (HFD).Angiotensin II (AngII) (A), RAAS expression (B), and angiotensin‐converting enzyme (ACE) and ACE2 activity (C) in eWAT. AngII (D), RAAS components (E), and ACE and ACE2 activity (F) in renal cortex. Data were analyzed by t test. *P<0.05 vs control HFD. n=6–10 per group. AGT indicates angiotensinogen; AT1R, angiotensin type 1 receptor; MSEW, maternal separation and early weaning; Ns, no significative; and RFU, relative fluorescence units.

Fig 4: Acute blood pressure sensitivity to angiotensin II (AngII) before and after angiotensin‐converting enzyme (ACE) inhibition. A, AngII pressor response was reduced control and MSEW enalapril‐treated mice similarly. B, Blood pressure trace in response to a 10 μg/kg AngII dose. Data were analyzed by 1‐way repeated measures ANOVA followed by Tukey's multiple comparisions test and reported as mean±SEM. # P<0.05 vs untreated; & P<0.05 vs untreated, n=6 per group. ENAL indicates enalapril; MAP, mean arterial pressure; MSEW, maternal separation and early weaning; and UT, untreated.

Fig 5: Inhibition of systemic AngII by losartan exhibited no effects on cardiac diastolic function in female HFpEF mice. A) Plasma AngII concentrations in female HFpEF hepAGT+/+ and female HFpEF hepAGT‐/‐ mice were measured by ELISA (n = 10 for hepAGT+/+ CHOW group, n = 10 for hepAGT‐/‐ CHOW group, n = 9 for hepAGT+/+ HFD+L‐NAME group, n = 10 for hepAGT‐/‐ HFD+L‐NAME group). B) Experimental workflow and analysis for the effects of losartan treatment on HFpEF in female mice (n = 9 for PBS CHOW group, n = 10 for Losartan CHOW group, n = 9 for PBS HFD+L‐NAME group, n = 7 for Losartan HFD+L‐NAME group). C) Representative echocardiography images obtained from female HFpEF mice at 15w after feeding with PBS or losartan (n = 9 for PBS CHOW group, n = 10 for Losartan CHOW group, n = 9 for PBS HFD+L‐NAME group, n = 7 for Losartan HFD+L‐NAME group). D) Left ventricular ejection fraction (LVEF%) was quantified via echocardiography (n = 9 for PBS CHOW group, n = 10 for Losartan CHOW group, n = 9 for PBS HFD+L‐NAME group, n = 7 for Losartan HFD+L‐NAME group). E) Left ventricular fraction shortening (LVFS%) was quantified via echocardiography (n = 9 for PBS CHOW group, n = 10 for Losartan CHOW group, n = 9 for PBS HFD+L‐NAME group, n = 7 for Losartan HFD+L‐NAME group). F) E/A ratio was quantified via echocardiography (n = 9 for PBS CHOW group, n = 10 for Losartan CHOW group, n = 9 for PBS HFD+L‐NAME group, n = 7 for Losartan HFD+L‐NAME group). G) E/e’ ratio was quantified via echocardiography (n = 9 for PBS CHOW group, n = 10 for Losartan CHOW group, n = 9 for PBS HFD+L‐NAME group, n = 7 for Losartan HFD+L‐NAME group). H) IVRT was quantified via echocardiography (n = 9 for PBS CHOW group, n = 10 for Losartan CHOW group, n = 9 for PBS HFD+L‐NAME group, n = 7 for Losartan HFD+L‐NAME group). I) Tei index was quantified via echocardiography (n = 9 for PBS CHOW group, n = 10 for Losartan CHOW group, n = 9 for PBS HFD+L‐NAME group, n = 7 for Losartan HFD+L‐NAME group). J) mRNA abundances of cardiac atrial natriuretic peptide (ANP) and cardiac brain natriuretic peptide (BNP) were assessed in female HFpEF mice feeding with PBS or losartan (n = 9 for PBS HFD+L‐NAME group, n = 7 for Losartan HFD+L‐NAME group). Two‐ way ANOVA was used for statistical analysis in A and D–I, and Student's t test was used for statistical analysis in J.