Sex differences in cardiac transcriptomic response to neonatal sleep apnea

Abstract

Pediatric obstructive sleep apnea poses a significant health risk, with potential long-term consequences on cardiovascular health. This study explores the dichotomous nature of neonatal cardiac response to chronic intermittent hypoxia (CIH) between males and females, aiming to fill a critical knowledge gap in the understanding of sex-specific cardiovascular consequences of sleep apnea in early life. Neonates were exposed to CIH until p28 and underwent comprehensive in vivo physiological assessments, including whole-body plethysmography, treadmill stress-tests, and echocardiography. Results indicated that male CIH rats weighed 13.7% less than age-matched control males (p = 0.0365), while females exhibited a mild yet significant increased respiratory drive during sleep (93.94 ± 0.84 vs. 95.31 ± 0.81;p = 0.02). Transcriptomic analysis of left ventricular tissue revealed a substantial sex-based difference in the cardiac response to CIH, with males demonstrating a more pronounced alteration in gene expression compared to females (5986 vs. 3174 genes). The dysregulated miRNAs in males target metabolic genes, potentially predisposing the heart to altered metabolism and substrate utilization. Furthermore, CIH in males was associated with thinner left ventricular walls and dysregulation of genes involved in the cardiac action potential, possibly predisposing males to CIH-related arrhythmia. These findings emphasize the importance of considering sex-specific responses in understanding the cardiovascular implications of pediatric sleep apnea.

Keywords: CIH; autonomic imbalance; cardiac miRNA expression; cardiac remodeling; chronic intermittent hypoxia; transcription.

FIGURE 1

(a) Protocol schematic depicting dams with female and male pups (P1) exposed to either room air (control) or chronic intermittent hypoxia (CIH) for 28 days. Outcome measures consisted of in vivo treadmill exercise tolerance tests, whole‐body plethysmography, echocardiography, and ex vivo hematocrit and left ventricular transcriptomics. Created with BioRender.com. (b) Body weight of pups at P29 was significantly lower in males exposed to CIH than controls (n = 6–8 animals per group, one‐way ANOVA). (c) Hematocrit at P29 was significantly greater for female and male pups exposed to CIH than their sex and age matched controls (n = 4–6 animals per group, one‐way ANOVA).

FIGURE 2

(a) Treadmill exercise tolerance tests performed at P24‐P28 illustrates that both female and male rats exposed to chronic intermittent hypoxia (CIH) perform no differently than their sex and age matched controls (n = 6–8; one‐way ANOVA). (b) Whole‐body plethysmography performed at P24‐P28 identify that females exposed to CIH have increased breathing frequency during sleep than their age matched controls (n = 6–8 animals per group, one‐way ANOVA; *p = 0.02).

FIGURE 3

Transcriptome analysis of LV myocardium differentially expressed genes (DEGs). (a, d) Volcano plots of DEGs (1.4 < FC < −1.4, p < 0.05; n = 4 per group) between chronic intermittent hypoxia (CIH) and control females (a) and males (d). (b, e) Up‐ and down‐regulation of differentially expressed nucleotide sequence categories between CIH and control females (b) and males (e) show that females primarily reduce transcription while males primarily enhance transcription in response to CIH. (c, f) Top 15 biological canonical pathways represented by the DEGs in (b, e). The bar chart depicts −log significance (upper x‐axis) of the differential pathway regulation with the threshold for significance (vertical orange line) set at −log 1.3 (p < 0.05). Bar color represents either upregulation (orange) or downregulation (blue) of the pathway with more intense color gradation corresponding to a higher percentage of DEGs enriched within a given biological pathway.

FIGURE 4

miRNA analysis of LV myocardium. (a, c) Total number and up‐ or down‐regulation of differentially expressed miRNAs (2 < FC < −2, p < 0.05; n = 4 per group) in female (a) and male (c) neonatal animals exposed to chronic intermittent hypoxia (CIH) compared to age and sex matched controls. (b, d) Top 15 biological canonical pathways represented by the differentially expressed genes (DEGs) that are targets of miRNAs in (a, c). The bar chart depicts −log significance (upper x‐axis) of the differential pathway regulation with the threshold for significance (vertical orange line) set at −log 1.3 (p < 0.05). Bar color represents either upregulation (orange) or downregulation (blue) of the pathway with more intense color gradation corresponding to a higher percentage of DEGs enriched within a given biological pathway. (e) Sankey chart of miRNAs dysregulated in male neonatal CIH exposure, the DEGs (mRNA) within the dataset they are predicted to regulate and the metabolic pathways those DEGs are enriched in. Number to the right of the miRNA represents the number of DEGs within the dataset that miRNA targets. Green line leaving miRNA or mRNA illustrates that gene is upregulated; red line leaving miRNA or mRNA illustrates that gene is downregulated within the dataset.

FIGURE 5

Cardiac functional and transcriptional response to neonatal chronic intermittent hypoxia (CIH). (a) Echocardiographic measures of left ventricular posterior wall thickness in systole (LVPW;s) or diastole (LVPW;d) (n = 6–8 animals per group, one‐way ANOVA). (b) Left ventricular chamber diameter in systole and diastole (LVESD, LVEDD) is smaller in males exposed to neonatal CIH (n = 6–8 animals per group, one‐way ANOVA). (c) Expression of key cardiac contractile, ion channel and communication genes in females and males exposed to neonatal CIH, compared to their respective age and sex matched controls. Dotted vertical line indicates the fold change cut‐off for significance (1.4 < FC < −1.4, p < 0.05; n = 4 per group). The table below lists male differentially expressed miRNAs (2 < FC < −2, p < 0.05; n = 3 per group) with a symbol corresponding to the differentially expressed genes (DEGs) it is predicted to regulate in the dataset.