Corticosteroids Are Essential for Maintaining Cardiovascular Function in Male Mice

Abstract

Activation of the hypothalamic-pituitary-adrenal axis results in the release of hormones from the adrenal glands, including glucocorticoids and mineralocorticoids. The physiological association between corticosteroids and cardiac disease is becoming increasingly recognized; however, the mechanisms underlying this association are not well understood. To determine the biological effects of corticosteroids on the heart, we investigated the impact of adrenalectomy in C57BL/6 male mice. Animals were adrenalectomized (ADX) at 1 month of age and maintained for 3-6 months after surgery to evaluate the effects of long-term adrenalectomy on cardiac function. Morphological evaluation suggested that ADX mice showed significantly enlarged hearts compared with age-matched intact controls. These changes in morphology correlated with deficits in left ventricular (LV) function and electrocardiogram (ECG) abnormalities in ADX mice. Correlating with these functional defects, gene expression analysis of ADX hearts revealed aberrant expression of a large cohort of genes associated with cardiac hypertrophy and arrhythmia. Combined corticosterone and aldosterone replacement treatment prevented the emergence of cardiac abnormalities in ADX mice, whereas corticosterone replacement prevented the effects of adrenalectomy on LV function but did not block the emergence of ECG alterations. Aldosterone replacement did not preserve the LV function but prevented ECG abnormalities. Together, the data indicate that adrenal glucocorticoids and mineralocorticoids either directly or indirectly have selective effects in the heart and their signaling pathways are essential in maintaining normal cardiac function.

Figure 1.. Plasma levels of corticosterone (A), aldosterone (B), and epinephrine (C) in control (intact adrenal glands) and ADX mice.

Hormone levels were measured at 1, 3, and 6 months after adrenalectomy. Data are mean ± SEM (n = 10 mice per group). *, P < .05; **, P < .01; ***, P < .001 vs control mice.

Figure 2.. Long-term adrenalectomy leads to a significant decrease in cardiac performance.

A, Representative M-mode images from age-matched intact (control) and ADX mice at 6 months after surgery. B, Transthoracic echocardiography on conscious male C57BL/6 ADX and age-matched controls at 6 months after ADX. IVSTD, anterior wall thickness at diastole; IVSTS, anterior wall thickness at systole; PWTD, PWT at diastole; PWTS, PWT at systole; LV VolD, LV volume in diastole; LV VolS, LV volume in systole; TL, tibia length; HW, heart weight; BW, body weight; HR, heart rate. Data are ± SEM (n = 15 mice per group). A Student's t test was performed to determine statistical significance (P < .05).

Figure 3.. Adrenalectomy leads to changes in heart morphology and cardiomyocyte size.

A, Representative images of intact hearts from age-matched control (intact) and long-term ADX male mice (6 mo after surgery). B, Representative images of H&E-stained sections of control and long-term ADX mice. C, Representative images of TRITC-lectin staining of cardiomyocytes from control and long-term ADX mice. D, Morphometric analysis of cardiomyocyte cross-sectional area. Data are mean ± SEM (n = 10 mice per group). Scale bar, 50 μm. A Student's t test was performed to determine statistical significance (*, P < .05).

Figure 4.. Dysregulation of genes associated to cardiac hypertrophy in long-term ADX mice.

A–C, RT-PCR analysis of βMHC, SKA, and BNP mRNA from hearts of control (intact) and long-term (6 mo) ADX mice. Values are normalized to cyclophilin (PPIB) mRNA. Data are mean ± SEM (n = 6 mice per group). **, P < .01 vs control mice. This PCR study is independent from the microarray data.

Figure 5.. Global gene expression profiles of long-term ADX hearts.

Microarray analyses were performed on RNA from the hearts of age-matched male controls (intact) and ADX mice (6 mo after surgery). A, Total number of genes differentially expressed in the hearts of ADX mice compared with aged-matched controls: of the 2400 significant altered genes identified, 40% (971 genes) were induced, and 60% (1469 genes) were repressed in ADX hearts. Red and green colors correspond to induced and repressed expression, respectively. B, Differentially expressed genes were analyzed by IPA software for each age group. Shown are the top 10 biological functions most significantly associated with the genes differentially regulated in the hearts of ADX mice. C, Most significant disorders or functions associated with the cardiovascular disease dysregulated genes in ADX hearts.

Figure 6.. IPA identified that genes involved in cardiovascular disease were associated with ventricular tachycardia (13 genes), heart dysfunction (50 genes), and failure (12 genes).

Among the dysregulated genes in these 3 categories we found RYR2, CACNB2, CACNA1C, CACNA2D1, CACNA2D2, SLC8A1, and a Ca++ transporting, cardiac muscle, slow twitch ATPase (ATP2A2 or SERCA2). These genes are marked by a red asterisk (*). Red and green colors correspond to induced and repressed expression, respectively.

Figure 7.. Down-regulation of genes involved in calcium signaling the hearts of ADX mice.

Total RNA was isolated from whole hearts of control and ADX mice (6 mo after surgery). CACNB2, CACNA1C1, CACNA2D1, CACNA2D2, RyR2, cardiac troponin T type 2, and SERCA2 mRNA levels were measured by qPCR. Data are mean ± SEM (n = 6 mice per group). *, P < .05; **, P < .01; ***, P < .001 vs control mice.

Figure 8.. Hormone replacement effects on cardiac function in ADX mice.

A, Representative M-mode images from age-matched intact (control), ADX, and corticosterone-ADX (cort, 25 μg/mL)-treated mice, aldosterone-ADX-treated mice (aldo, 0.3 μg/mL), corticosterone/aldosterone-treated mice, and betamethasone-ADX (Beta, 2.5 μg/mL)-treated mice. B, Transthoracic echocardiography on conscious C57BL/6 hormone-treated ADX mice, ADX, and age-matched controls at 3 months after ADX. Data are mean ± SEM (n = 8–12 mice per group). IVSTD, anterior wall thickness at diastole; IVSTS, anterior wall thickness at systole; PWTD, PWT at diastole; PWTS, PWT at systole; LV VolD, LV volume in diastole; LV VolS, LV volume in systole; TL, tibia length; HW, heart weight; BW, body weight; HR, heart rate. One-way ANOVA with multiple comparisons was performed to determine significance between groups (P < .05).

Figure 9.. RT-PCR analysis of βMHC, SKA, CACNA1C1, CACNA2D1, and RYR2 mRNA from hearts of control, ADX, and corticosterone-treated ADX mice.

Values are normalized to cyclophilin (PPIB) mRNA. Data are mean ± SEM (n = 6 mice per group). One-way ANOVA was performed to determine significance between groups. *, P < .05; **, P < .01; ***, P < .001; n.s., not significant.