Estrogen but not testosterone preserves myofilament function from doxorubicin-induced cardiotoxicity by reducing oxidative modifications

Abstract

Here, we aimed to explore sex differences and the impact of sex hormones on cardiac contractile properties in doxorubicin (DOX)-induced cardiotoxicity. Male and female Sprague-Dawley rats were subjected to sham surgery or gonadectomy and then treated or untreated with DOX (2 mg/kg) every other week for 10 wk. Estrogen preserved maximum active tension (T_max) with DOX exposure, whereas progesterone and testosterone did not. The effects of sex hormones and DOX correlated with both altered myosin heavy chain isoform expression and myofilament protein oxidation, suggesting both as possible mechanisms. However, acute treatment with oxidative stress (H₂O₂) or a reducing agent (DTT) indicated that the effects on T_max were mediated by reversible myofilament oxidative modifications and not only changes in myosin heavy chain isoforms. There were also sex differences in the DOX impact on myofilament Ca²⁺ sensitivity. DOX increased Ca²⁺ sensitivity in male rats only in the absence of testosterone and in female rats only in the presence of estrogen. Conversely, DOX decreased Ca²⁺ sensitivity in female rats in the absence of estrogen. In most instances, this mechanism was through altered phosphorylation of troponin I at Ser²³/Ser²⁴. However, there was an additional DOX-induced, estrogen-dependent, irreversible (by DTT) mechanism that altered Ca²⁺ sensitivity. Our data demonstrate sex differences in cardiac contractile responses to chronic DOX treatment. We conclude that estrogen protects against chronic DOX treatment in the heart, preserving myofilament function. NEW & NOTEWORTHY We identified sex differences in cardiotoxic effects of chronic doxorubicin (DOX) exposure on myofilament function. Estrogen, but not testosterone, decreases DOX-induced oxidative modifications on myofilaments to preserve maximum active tension. In rats, DOX exposure increased Ca²⁺ sensitivity in the presence of estrogen but decreased Ca²⁺ sensitivity in the absence of estrogen. In male rats, the DOX-induced shift in Ca²⁺ sensitivity involved troponin I phosphorylation; in female rats, this was through an estrogen-dependent mechanism.

Fig. 1.

Effects of sex hormone deprivation and doxorubicin (DOX) treatment on percent survival. A: percent survival of female DOX-treated sham-operated (sham) control and ovarectomized (OVX) rats with and without estrogen (E)/progesterone (P) supplementation. B: percent survival of male DOX-treated sham control and orchidectomized (ORX) rats with and without testosterone (T) supplementation. *P < 0.05 from the DOX-treated sham control group by Kaplan-Meier analysis.

Fig. 2.

Effects of sex hormone deprivation and doxorubicin (DOX) treatment on skinned papillary function. A: mean active tension as a function of pCa and fitted curves from skinned left ventricular (LV) fibers from female sham-operated (sham) and ovarectomized (OVX) rats with and without DOX treatment. B: mean active tension as a function of pCa and fitted curves from skinned LV fibers from male sham and orchidectomized (ORX) rats with and without DOX treatment. C: mean active tension as a function of pCa and fitted curves from skinned LV fibers from female sham, OVX + DOX, and OVX + DOX groups with estrogen treatment (OVX + DOX + E₂). D: mean active tension as a function of pCa and fitted curves from skinned LV fibers from male sham, ORX + DOX, and ORX + DOX groups with testosterone treatment (ORX + DOX + T). E and F: summary data for maximum active tension (T_max). G and H: summary data for Ca²⁺ sensitivity (pCa₅₀). Data are means ± SE of 10–16 fibers from 7–8 hearts/group. *P < 0.05 from the sham control groups of the same treatment; #P < 0.05 from the DOX-untreated sham groups using a Student-Newman-Keuls test after two-way ANOVA.

Fig. 3.

Effects of sex hormone deprivation and DOX treatment on expression of myosin heavy chain (MHC) isoforms. A: relative amount of α-MHC as a percentage of total (α+β) MHC of left ventricular homogenates from each female group. Inset: example gel for each of the eight groups. B: same as in A but for male groups. E, estrogen; P, progesterone; T, testosterone; OVX, ovarectomized; ORX, orchidectomized. Data are means ± SE from 4–5 hearts/group. *P < 0.05 from the sham control group of the same treatment; #P < 0.05 from the doxorubicin (DOX)-untreated sham group using a Student-Newman-Keuls test after two-way ANOVA.

Fig. 4.

Effects of sex hormone deprivation and doxorubicin (DOX) treatment on carbonylation levels of cardiac myofilament proteins. A: representative OxyBlot (left) and Coomassie-stained (right) gels demonstrating carbonylation of various myofilament proteins in left ventricular homogenates from each group of female rats. B: summary data showing the relative amount of carbonylated proteins to total protein from each group of female rats. C and D: same as in A and B but for male rat groups. MW, molecular weight standard; E, estrogen; P, progesterone; T, testosterone; OVX, ovarectomized; ORX, orchidectomized; MHC, myosin heavy chain; MyBP, myosin-binding protein; TnT, troponin T; Tm, tropomyosin; MLC, myosin light chain. Data are means ± SE from 4–5 hearts/group. *P < 0.05 from the sham control group of the same treatment; #P < 0.05 from the DOX-untreated sham group using a Student-Newman-Keuls test after two-way ANOVA.

Fig. 5.

Direct effects of H₂O₂ and DTT on maximum active contraction in rat hearts. A: maximum tension (T_max) in skinned left ventricular fibers in the absence and presence of 5 mM H₂O₂ from each group of female rats. B: same as in A but for the male rat groups. DOX, doxorubicin; E, estrogen; P, progesterone; T, testosterone; OVX, ovarectomized; ORX, orchidectomized. Data are means ± SE of 10–16 fibers from 7–8 hearts/group. *P < 0.05 from the H₂O₂-untreated group; #P < 0.05 from the sham control group of the same treatment; $P < 0.05 from the DOX-untreated sham group using a Student-Newman-Keuls test after two-way ANOVA. C: T_max in skinned cardiac cells from sham groups of female and male rats in the absence/presence of 1 mM DTT treatment. Data are means ± SE of 15–16 single skinned cells from 5 hearts/group. *P < 0.05 from the DOX-untreated sham control group using a Student-Newman-Keuls test after two-way ANOVA.

Fig. 6.

Effects of H₂O₂ and DTT on myofilament Ca²⁺ sensitivity in rat hearts. A: pCa₅₀ in skinned left ventricular fibers in the absence and presence of 5 mM H₂O₂ from each group of female rats. B: same as in A but for the male rat groups. DOX, doxorubicin; E, estrogen; P, progesterone; T, testosterone; OVX, ovarectomized; ORX, orchidectomized. Data are means ± SE; n = 12–16 fibers from 7–8 hearts/group. #P < 0.05 from the DOX-untreated sham group using a Student-Newman-Keuls test after two-way ANOVA. C: pCa₅₀ of skinned cardiac cells from sham groups of female and male rats with and without 1 mM DTT. Data are means ± SE of 15–16 cells from 5 hearts/group. *P < 0.05 from the sham control group of the same treatment using a Student-Newman-Keuls test after two-way ANOVA.

Fig. 7.

Effects of sex hormone deprivation and doxorubicin (DOX) treatment on cardiac troponin I (TnI) phosphorylation. A: quantification and representative immunoblot (above) of phosphorylated TnI at Ser²³/Ser²⁴ (pS23/24 TnI) and total TnI in female groups. B: same as in A but for the male groups. p/T TnI, phosphorylated to total TnI; E, estrogen; P, progesterone; T, testosterone; OVX, ovarectomized; ORX, orchidectomized. Data are means ± SE from 5 hearts/group. *P < 0.05 from sham control group of the same treatment; #P < 0.05 from the DOX-untreated sham group using a Student-Newman-Keuls test after two-way ANOVA.