Angiotensin II Overstimulation Leads to an Increased Susceptibility to Dilated Cardiomyopathy and Higher Mortality in Female Mice

Figures

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  Figure 1. Survival curves reveals an early onset and higher rate of mortality in female AT1R mice compared to males. The graph represents the percentage of survival of CTL and AT1R mice of both sexes (M, F) between 0 and 15 months of age (*p = 0.0013 vs. F CTL, †p = 0.0036 vs. M AT1R, Cox regression analysis) (M: CTL N = 107, AT1R N = 91, F: CTL N = 91, AT1R N = 100).

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  Figure 2. Ventricular myocyte ionic currents are similarly reduced in male and female AT1R mice in comparison to CTL. (a) Representative ventricular INa recorded in CTL and AT1R myocytes of both sexes. Mean data were plotted in a current-voltage (IV) relationships graph demonstrating that the reduction caused by AT1R overexpression is similar in male (M) (CTL: n = 13, AT1R: n = 16) and female (F) (CTL: n = 21, AT1R n = 18). (b) Typical ICaL traces are presented and mean IV curves show that ICaL is reduced to the same extent in male and female AT1R mice (M: n = 8, F: n = 10) in comparison to CTL (M: n = 6, F: n = 21). (c) Representative Ipeak recordings from all groups are displayed. Mean IV curves also demonstrate that AT1R overexpression similarly reduces total K+ current (Ipeak) in males (CTL: n = 15, AT1R n = 11) and females (CTL: n = 13, AT1R n = 15). Voltage protocols used for the 3 types of ionic current are shown in insets. Male data for INa and Ipeak presented in this figure represent a subset of the data presented in22,24.

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  Figure 3. Female AT1R mice display more severe signs of cellular and ventricular hypertrophy in comparison to male AT1R mice. (a) Comparison of the cell capacitance measured in CTL and AT1R ventricular myocytes from male and female mice. (b,c) Comparison of the length (b) and width (c) of the ventricular myocytes isolated from CTL and AT1R mice of both sexes. (d) M-mode echocardiography was used to calculate the left ventricular (LV) mass for CTL and AT1R mice of both sexes. (e) Body weight (BW) and (f) LV mass/BW ratio is reported for each groups (*p < 0.05 vs. sex-matched CTL, †p < 0.05 vs. M AT1R, ‡p < 0.05 vs. M CTL) (numbers above the bar graphs represent the number of cells or mice examined).

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  Figure 4. Echocardiographic data reveal that female AT1R mice develop more severe left ventricular eccentric hypertrophy and dilation than AT1R males. (a) Typical examples of an M-mode echocardiography recordings in female CTL and AT1R mice. (b,c) The left ventricular internal diameter at end-diastole (LVIDd) and end-systole (LVIDs) were calculated from the echocardiography data obtained from CTL and AT1R mice of both sexes. (d) The LV fractional shortening representing the systolic function was calculated as follows: LV FS (%) = [(LVIDd − LVIDs)/LVIDd] × 100. (e,f) The echocardiographic data were also used to assess the LV posterior wall thickness (LVPWd) (e) and the interventricular septum (IVSd) (f) for each groups. (g) The relative wall thickness was determined as follows: LV RWT = (IVSd + LVPWd)/LVIDd. These data indicate that AT1R stimulation leads to more pronounced eccentric hypertrophy and dilation in females than males (*p < 0.005 vs. sex-matched CTL, †p = 0.05 vs. M AT1R) (M: CTL N = 7, AT1R N = 7; F: CTL N = 5, AT1R N = 8). Echocardiography values of male CTL and AT1R mice presented in this figure represent a subset of the data published in22.

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  Figure 5. Alterations in Ca2+ homeostasis are greater in female than male AT1R myocytes. (a) Typical Ca2+ transients in ventricular myocytes of male and female AT1R and control mice are shown. The dashed line indicates 0 nM Ca2+. Ca2+ transients (b), diastolic Ca2+ (c), time to 50% decay (d), time to 90% decay (e) and tau (f) were measured in CTL and AT1R myocytes of both sexes (*p < 0.05 vs. sex-matched CTL, †p = 0.005 vs. M AT1R, ‡p = 0.01 vs. M CTL) (M CTL n = 18, M AT1R n = 28, F CTL n = 28, F AT1R, n = 18).

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  Figure 6. Caffeine-induced Ca2+ transients are differently affected in male and female AT1R myocytes. (a) Representative recordings of caffeine-induced Ca2+ transients in ventricular myocytes of male and female CTL and AT1R mice are shown. (b) Mean data for caffeine-induced Ca2+ transients show that female have lower SR load than male in CTL conditions (‡p = 4 × 10−5) and that SR load is only reduced in male AT1R myocytes (*p = 0.0004). (c) Fractional release defined as the ratio of Ca2+ transient amplitude over the caffeine-induced Ca2+ transient is increased in female CTL in comparison to males (‡p = 0.01). Time to 50% decay (d) and time to 90% decay (e) are presented (*p < 0.0005 vs. sex-matched CTL, †p = 2 × 10−5 vs. M AT1R, ‡p≤0.01 vs. M CTL) (M CTL n = 18, M AT1R n = 22, F CTL n = 28, F AT1R n = 14).

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  Figure 7. Spontaneous SR Ca2+ release in AT1R mice. (a) Typical example of a spontaneous Ca2+ release recorded following a stimulated Ca2+ transient in a female AT1R myocyte. (b) Bar graphs presenting the incidence of spontaneous Ca2+ release in myocytes from male and female CTL and AT1R mice. AT1R females have an increase in spontaneous Ca2+ release compared to sex-matched CTL and AT1R males although the difference was not statistical significance (p = 0.149, GLIMMIX procedure). Values above the bar graphs represent the number of cells.

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  Figure 8. Ca2+ sparks frequency is increased in female AT1R myocytes in comparison to female CTL. (a) Representative Ca2+ sparks recorded in ventricular myocytes isolated from female CTL and AT1R mice. (b) AT1R overexpression caused no change in sparks frequency in male myocytes (CTL n = 31, AT1R n = 13) whereas the frequency significantly increased in female AT1R (n = 20) in comparison to female CTL (n = 22) (*p = 0.029). (c) Sparks amplitude decreased in male AT1R (n = 11) compared to CTL (n = 24) (*p = 0.025) but did not differ between females (CTL n = 12, AT1R n = 18). (d–f) AT1R overexpression did not affect tau (d), FDHM (e) or FWHM (f).