The Effect of Estrogen on Intracellular Ca2+ and Na+ Regulation in Heart Failure

Figures

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  Figure 1. The Effect of Heart Failure and Long-Term Absence of Estrogen on Ca2+ Transient Amplitude (A) Representative Ca2+ transient traces from Fluo-4-loaded myocytes. (B) All AC groups had significantly lower transient amplitudes compared with the Sham group. The Ca2+ transient amplitude was further reduced by 10% in ACOV compared with the AC group (Sham, n = 44/3 cells AC, n = 47/3; ACOV, n = 31/3; ACOV+E, n = 21/2; 1-way analysis of variance (ANOVA) with a Tukey’s multiple comparisons test), ∗∗∗p < 0.001. (C) Measurement of total SR Ca2+ content through integration of the caffeine-induced inward NCX current. Myocytes from failing hearts were characterized by a significant decrease in SR Ca2+ content compared with Sham (p < 0.001). The long-term absence of ovarian hormones further reduced SR Ca2+ (ACOV). (Sham, n = 33/3; AC, n = 34/4; ACOV, n = 42/3; ACOV+E, n = 29/3; 1-way ANOVA with a Fisher post hoc test: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Bars show mean (95% confidence interval). AC = aortic constriction; ACOV = aortic constriction with ovariectomy; ACOV+E = aortic constriction with ovariectomy supplemented with 17β-estradiol.

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  Figure 2. Quantification of Ca2+ Spark Frequency and Morphology (A) The frequency of Ca2+ sparks increased in myocytes from the AC groups and was highest in the ACOV group compared with Sham. The frequency in ACOV+E group is more comparable with the Sham group. (B) The amplitude of the Ca2+ spark was similar between the Sham and the AC group. Myocytes from ACOV had the largest Ca2+ spark amplitude between groups. (C) Spark mass in ACOV+E group is lower compared with ACOV. (D) The spark-mediated SR leak (spark mass x frequency) increased in all AC groups compared with Sham, with ACOV having the highest leak.(Sham, n = 57/5; AC, n = 43/3; ACOV, n = 56/4; ACOV+E, n = 47/3; 1-way ANOVA with a Fisher post hoc test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001). Bars show mean (95% confidence interval). Abbreviations as in Figure 1.

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  Figure 3. Action Potential Duration Time to 90% Repolarization (APD90) (A) Representative action potential traces. (B) APD90 was prolonged in all AC groups compared with Sham. The ACOV mean APD90 was 23.6% longer than Sham. Sham n = 44/5, AC, n = 46/5, ACOV n = 47/3, ACOV+E n = 35/3; 1-way ANOVA with Tukey’s multiple comparisons test, ∗∗p = 0.01, ∗∗∗p < 0.001. Bars show mean (95% confidence interval). Abbreviations as in Figure 1.

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  Figure 4. Evaluation of Na+ Influx and Efflux Kinetics (A) Representative trace of Na+/K+ ATPase current alterations when the superfusate was changed from 6 mM K+ to a K+-free solution to inhibit the Na+/K+ ATPase and then following the reintroduction of 6 mM K+ to reactivate the pump. (B) The Na+/K+ ATPase–mediated Na+ extrusion rate following pump reactivation and (C) the total Na+/K+ ATPase current density. The Na+ efflux through Na+/K+ ATPase current density and Na+ extrusion rate were reduced in AC groups. The long-term absence of ovarian hormones resulted in a further reduction in Na+ extrusion rate compared with the AC group. (Sham, n = 22/2; AC, n = 19/3; ACOV, n = 25/2; ACOV+E, n = 27/2; 1-way ANOVA with a Tukey’s multiple comparisons test, ∗p < 0.05, ∗∗∗p < 0.001). (D) Na+ influx through the ranolazine-sensitive late Na+ current (INa,L) in myocytes isolated from AC groups. (E) The INa,L was increased in AC groups, ACOV had a 15 % greater INa,L-related Na+ influx compared with AC. The estradiol-supplemented group had similar Na+ influx compared with AC. (Sham, n = 31/3; AC, n = 25/3; ACOV, n = 23/3; ACOV+E, n = 21/3; 1-way ANOVA with a Tukey’s multiple comparisons test, ∗p < 0.05,…

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  Figure 5. Na+/K+ ATPase and Its Regulatory Subunit Expression (A) A sample Western blot of Na+/K+ ATPase (NKA) α1 expression in Sham and AC groups with the grouped data (C). The Na+/K+ ATPase α1 relative expression was unchanged in the AC groups compared with Sham, except for the ACOV+E group, in which the ratio was 25.9% greater. (B) A sample Western blot of phospholemman (PLM/FXYD1) expression in Sham and AC groups with the grouped data (D). Phospholemman expression decreases in the ACOV group, but supplementation with estradiol increases expression to match Sham levels, with the ACOV+E ratio 36% greater than ACOV expression. (E) The ratio of the expression levels of the 2 proteins (Na+/K+ ATPase: PLM) in the AC and ACOV cohorts increased compared with Sham. N = 6 for each group; 1-way ANOVA with a Fisher post hoc test, ∗p < 0.05, ∗∗p < 0.01. Bars show mean ± SD. Abbreviations as in Figure 1.