Sex-dependent alterations in cardiac cytochrome P450-mediated arachidonic acid metabolism in pressure overload-induced cardiac hypertrophy in rats

Abstract

Cardiac hypertrophy is a risk factor for heart failure and is usually less common in young women than in men. Cytochrome P450 (CYP) enzymes in the heart metabolize arachidonic acid into hydroxyeicosatetraenoic acids (HETEs), which generally have hypertrophic effects, and epoxyeicosatrienoic acids, which have cardioprotective effects. In this study, we aimed to investigate sex-specific differences in cardiac hypertrophy and cardiac CYP, HETE, and epoxyeicosatrienoic acid levels in response to pressure overload. Adult male and female Sprague-Dawley rats were subject to sham or abdominal aortic constriction (AAC) surgeries. Five weeks postsurgery, cardiac function was assessed by echocardiography. The mRNA and protein levels of hypertrophic markers and CYP enzymes were measured by real-time polymerase chain reaction and Western blot. Heart tissue HETE levels and microsomal formation of HETEs and epoxyeicosatrienoic acids were measured by liquid chromatography-tandem mass spectrometry. Our results show significant sex-specific differences in AAC-induced cardiac hypertrophy. Echocardiography and ventricular wall measurements showed more hypertrophy in male rats. Some hypertrophic markers were significantly upregulated only in male AAC rats and were significantly higher in the hearts of male rats compared to female AAC rats. Different CYP hydroxylases such as CYP1B1, CYP4A, and CYP4F and epoxygenases such as CYP2C and CYP2J10 were significantly upregulated in the hearts of male AAC rats only. The heart level of 12(R)-HETE and the microsomal formation of several HETEs were also significantly increased only in male rats. In conclusion, male rats developed stronger AAC-induced cardiac hypertrophy compared to female rats, which was accompanied by a significant increase in cardiac CYP enzymes and HETEs. SIGNIFICANCE STATEMENT: Previous studies demonstrated that male rats experience more severe cardiac hypertrophy compared to female rats. To our knowledge, this research is the first to investigate and compare the expression of cytochrome P450 enzymes and arachidonic acid metabolites in male and female rat hearts following pressure overload-induced hypertrophy. This study highlights significant sex-specific differences in cytochrome P450-mediated metabolism during hypertrophy, providing valuable insights into the molecular mechanisms underlying these responses and identifying potential targets for sex-specific therapies in cardiac diseases.

Keywords: Arachidonic acid; Cardiac hypertrophy; Cytochrome P450; Epoxyeicosatrienoic acids; Hydroxyeicosatetraenoic acids.

Fig. 1

Effect of abdominal aortic constriction (AAC)-induced hypertrophy on echocardiography, HW:TL ratio, and the mRNA expression of hypertrophic markers. Adult male and female Sprague–Dawley rats were subject to sham or AAC surgeries. Five weeks after the surgery, 2D ultrasonic echocardiography was conducted (A), the heart was isolated and HW:TL ratio was calculated (B), and the mRNA expression of the hypertrophic markers ACTA1, β/α-MHC, ANP, and BNP (C) was determined by real-time PCR. M-mode slices were taken in parasternal long axis mode. The results of all groups were normalized to the GAPDH housekeeping gene. Results are represented as mean ± SD, n = 4–7. HW:TL data were analyzed using unpaired Student’s t test, ∗P < .05 compared to same-sex sham, whereas hypertrophic markers data were analyzed using two-way ANOVA followed by Tukey’s multiple comparison post hoc analysis, ∗P < .05 compared to same-sex sham; ^#P < .05 compared to male rats of the same surgical group. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 2

Effect of abdominal aortic constriction (AAC)-induced hypertrophy on the mRNA expression of CYP hydroxylases and CYP ω-hydroxylases. Adult male and female Sprague–Dawley rats were subject to sham or AAC surgeries. Five weeks after the surgery, the heart was isolated, and the mRNA expression of CYP1A1 (A), CYP1A2 (B), CYP1B1 (C), CYP2E1 (D), CYP3A2 (E), CYP4A1 (F), CYP4A2 (G), CYP4A3 (H), CYP4F1 (I), CYP4F4 (J), CYP4F5 (K), and CYP4F6 (L) was determined by real-time PCR. The results of all groups were normalized to the GAPDH housekeeping gene. Results are represented as mean ± SD, n = 4–8. Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparison post hoc analysis, ∗P < .05 compared to the same-sex sham; ^#P < .05 compared to male rats of the same surgical group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 3

Effect of abdominal aortic constriction (AAC)-induced hypertrophy on the mRNA expression of CYP epoxygenases. Adult male and female Sprague–Dawley rats were subject to sham or AAC surgeries. Five weeks after the surgery, the heart was isolated, and the mRNA expression of CYP2B1 (A), CYP2B2 (B), CYP2C11 (C), CYP2C13 (D), CYP2J3 (E), CYP2J4 (F), and CYP2J10 (G) was determined by real-time PCR. The results of all groups were normalized to the GAPDH housekeeping gene. Results are represented as mean ± SD, n = 5–7. Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparison post hoc analysis, ∗P < .05 compared to the same-sex sham; ^#P < .05 compared to male rats of the same surgical group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 4

Effect of abdominal aortic constriction (AAC)-induced hypertrophy on the protein expression of CYP enzymes. Adult male and female Sprague–Dawley rats were subject to sham or AAC surgeries. Five weeks after the surgery, heart microsomes were isolated, and the protein expression of CYP1A2, CYP1B1, CYP4A1, CYP4F, and CYP2J was determined in male (A) and female (B) rats by Western blot. The results of all groups were normalized to the GAPDH housekeeping protein. Results are represented as mean ± SD, n = 5–6. Data were analyzed using unpaired Student’s t test, ∗P < .05 compared to the same-sex sham. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 5

Effect of abdominal aortic constriction (AAC)-induced hypertrophy on the formation of HETEs in the heart microsomes. Adult male and female Sprague–Dawley rats were subject to sham or AAC surgeries. Five weeks after the surgery, heart microsomes were isolated and incubated with arachidonic acid, and the formation of 5(R)-HETE (A), 5(S)-HETE (B), 8(R)-HETE (C), 8(S)-HETE (D), 9(R)-HETE (E), 9(S)-HETE (F), 11(R)-HETE (G), 11(S)-HETE (H), 12(R)-HETE (I), 12(S)-HETE (J), 15(R)-HETE (K), 15(S)-HETE (L), 16(R)-HETE (M), 16(S)-HETE (N), 19(S)-HETE (O), and 20-HETE (P) was determined by LC-MS/MS. Results are represented as mean ± SD, n = 4–6. Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparison post hoc analysis, ∗P < .05 compared to the same-sex sham; ^#P < .05 compared to male rats of the same surgical group.

Fig. 6

Effect of abdominal aortic constriction (AAC)-induced hypertrophy on the formation of EETs in the heart microsomes. Adult male and female Sprague–Dawley rats were subject to sham or AAC surgeries. Five weeks after the surgery, heart microsomes were isolated and incubated with arachidonic acid, and the formation of 5,6(R/S)-EET (A), 5,6(S/R)-EET (B), 8,9(R/S)-EET (C), 8,9(S/R)-EET (D), 11,12(R/S)-EET (E), 11,12(S/R)-EET (F), 14,15(R/S)-EET (G), and 14,15(S/R)-EET (H) was determined by LC-MS/MS. Results are represented as mean ± SD, n = 4-6. Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparison post hoc analysis. LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Fig. 7

Effect of abdominal aortic constriction (AAC)-induced hypertrophy on the basal heart levels of HETEs. Adult male and female Sprague–Dawley rats were subject to sham or AAC surgeries. Five weeks after the surgery, HETEs were extracted from the hearts by liquid–liquid extraction, and the levels of 5(R)-HETE (A), 5(S)-HETE (B), 11(R)-HETE (C), 11(S)-HETE (D), 12(R)-HETE (E), 15(R)-HETE (F), and 15(S)-HETE (G) were determined by LC-MS/MS. Results are represented as mean ± SD, n = 4-6. Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparison post hoc analysis, ∗P < .05 compared to the same-sex sham; ^#P < .05 compared to male rats of the same surgical group. LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Fig. 8

Effect of abdominal aortic constriction (AAC)-induced hypertrophy on the mRNA expression of inflammatory cytokines and sEH. Adult male and female Sprague–Dawley rats were subject to sham or AAC surgeries. Five weeks after the surgery, the heart was isolated, and the mRNA expression of TNF-α (A), IL-1β (B), IL-6 (C), and sEH (D) was determined by real-time PCR. The results of all groups were normalized to the GAPDH housekeeping gene. Results are represented as mean ± SD, n = 6–8. Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparison post hoc analysis, ∗P < .05 compared to the same-sex sham; ^#P < .05 compared to male rats of the same surgical group. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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