Isoproterenol-induced Cardiac Dysfunction in Male and Female C57Bl/6 Mice

Sex-related differences in cardiovascular diseases are complex and context-dependent. The objective of this work was to comprehensively determine key sex differences in the response to acute and chronic adrenergic stimulation in C57Bl/6 mice. Cardiac function was assessed by trans-thoracic echocardiography before and after acute adrenergic stimulation (a single sub-cutaneous dose of isoproterenol 10 mg/kg) in male and female C57Bl/6 mice. Thereafter, chronic adrenergic stimulation was achieved by sub-cutaneous injections of isoproterenol 10 mg/kg/day for 14 days in male and female mice. Cardiac function and morphometry were assessed by trans-thoracic echocardiography on the 15th day. Thereafter, the mice were euthanized and the hearts were collected. Histopathological analysis of myocardial tissue was performed after staining with hematoxylin & eosin, trichrome, and MAC-2 antibody. Gene expression of remodeling and fibrotic markers was assessed by real-time PCR. Cardiac function and morphometry were also measured before and after isoproterenol 10 mg/kg/day for 14 days in groups of gonadectomized male and female mice and sham-operated controls. In the current work, there were no statistically significant differences in key echocardiographic parameters between male and female C57Bl/6 mice in response to acute adrenergic stimulation. After chronic adrenergic stimulation, there was similar degree of cardiac dysfunction, cardiac hypertrophy, and myocardial fibrosis in male and female mice. Similarly, chronic isoproterenol administration induced hypertrophic and fibrotic genes in hearts of male and female mice to the same extent. Intriguingly, gonadectomy of male and female mice did not have a significant impact on isoproterenol-induced cardiac dysfunction as compared to sham-operated animals. The current work demonstrated lack of significant sex-related differences in isoproterenol-induced cardiac hypertrophy, dysfunction, and fibrosis in C57Bl/6 mice. This study suggests that female sex may not be sufficient to protect the heart against a severe pathologic stimulus and underscores the notion that sexual dimorphism in cardiovascular diseases is highly model-dependent.

C57Bl/6 mice. This study suggests that female sex may not be sufficient to protect the heart 41 against a severe pathologic stimulus and underscores the notion that sexual dimorphism in

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Cardiovascular diseases remain the leading cause of mortality in both men and women globally [1], despite the conventional dogma that women are more protected against 48 cardiovascular diseases than men. Indeed, the incidence of cardiovascular diseases in post-49 menopausal women is equal to those in age-matched men [2]. Furthermore, there are a number 50 of cardiovascular diseases that are more prevalent in young pre-menopausal women than in men,

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Statistical analysis for histopathologic grading and MAC-2 staining was performed using the non-

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parametric Kruskal-Wallis test. A p value of < 0.05 was taken to indicate statistical significance.

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Acute effects of isoproterenol administration on echocardiographic parameters in intact induced a significant increase in heart rate by 22% in male and 13% in female mice (Fig. 1G).

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Matched two-way ANOVA revealed a significant effect of isoproterenol on all the measured 160 parameters, while sex effect was significant only on the cardiac output. There was no significant 161 interaction between isoproterenol and sex in all the measured parameters (Supplementary Table   162 2). 178 Isoproterenol-treated mice exhibited a significantly greater heart weight/tibia length ratio (HW/TL) 179 compared to saline-treated mice (Fig. 2G). Two-way ANOVA revealed a significant isoproterenol 180 effect on all the measured parameters, while sex effect was significant only in LV Mass and the 181 heart weight/tibia length ratio (HW/TL). There was no significant interaction between isoproterenol 182 and sex in any of the measured parameters (Supplementary Table 3).   Table 4).

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Effect of castration of male mice on isoproterenol-induced cardiac dysfunction.

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Isoproterenol treatment for 14 days resulted in similar functional changes in sham and castrated  Table 5).

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Effect of ovariectomy of female mice on isoproterenol-induced cardiac dysfunction.

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Isoproterenol treatment for 14 days resulted in similar functional changes in sham and echocardiographic images obtained in M-Mode from each group are displayed in Fig. 7A. Ejection 219 fraction and fractional shortening were significantly decreased following isoproterenol treatment 220 in sham (23% and 28%, respectively) and ovariectomized (35% and 42% respectively) mice ( Fig.   221 7B and 7C). ISO treatment resulted in significantly greater end-systolic and diastolic volumes in 222 both sham (99% and 46%, respectively) and ovariectomized (128% and 31%, respectively) mice 223 ( Fig. 7D and 7E). Cardiac output was unaltered by isoproterenol treatment in either group (Fig.   224  7F). Isoproterenol administration caused a significant increase in LV mass in both sham and 225 ovariectomized mice (Fig. 7G). Matched two-way ANOVA revealed a significant isoproterenol 226 effect on all the measured parameters except the cardiac output. However, the ovariectomy effect 227 and the interaction between isoproterenol and ovariectomy were not statistically significant

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Sex-related differences have been described in a number of animal models of cardiac shown that MAC-2 positive cells were equally elevated in the hearts of male and female mice.

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MAC-2 expression has been shown to be highly associated with myocardial fibrosis [34,35], 284 although its causative role in inducing myocardial fibrosis is still controversial [36].

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In order to identify the potential role of male sex hormones, we determined the response 286 to chronic isoproterenol administration in castrated male mice compared to sham-operated 287 animals. Intriguingly, there was no significant difference in the isoproterenol-induced cardiac 288 dysfunction between castrated and sham-operated mice. In agreement with our results, castration 289 had no effect on LV dilation produced by chronic isoproterenol administration 0.015 mg/kg/day 290 for 6 months in Sprague Dawley rats [37]. In contrast, castration prevented cardiac hypertrophy,

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LV dilation, and increased interstitial collagen produced by chronic isoproterenol administration 0.04 mg/kg/day for 6 months male SHRs [10]. These effects may be attributed to the protective 293 effect of castration on the underlying cardiovascular pathology in male SHR, as previously 294 reported [38], rather than the response to isoproterenol.

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In order to identify the role of female sex hormones, we studied the effects of isoproterenol

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prevented cardiac hypertrophy and dysfunction caused by pressure overload, but not by 306 myocardial infarction [41]. Although the results of these gonadectomy experiments corroborated 307 our previous findings in intact animals, the small sample size and the lack of saline-treated control 308 mice are limitations to the current work. Therefore, future studies are planned to specifically 309 determine the effects of gonadectomy on isoproterenol-induced cardiac dysfunction and 310 remodeling and to identify the molecular determinants of these effects.

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Values are shown as means ± SEM. *p<0.05, compared to control treatment of the same sex.