Enhanced antioxidant capacity prevents epitranscriptomic and cardiac alterations in adult offspring gestationally-exposed to ENM

Abstract

Maternal engineered nanomaterial (ENM) exposure during gestation has been associated with negative long-term effects on cardiovascular health in progeny. Here, we evaluate an epitranscriptomic mechanism that contributes to these chronic ramifications and whether overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mPHGPx) can preserve cardiovascular function and bioenergetics in offspring following gestational nano-titanium dioxide (TiO₂) inhalation exposure. Wild-type (WT) and mPHGPx (Tg) dams were exposed to nano-TiO₂ aerosols with a mass concentration of 12.01 ± 0.50 mg/m³ starting from gestational day (GD) 5 for 360 mins/day for 6 nonconsecutive days over 8 days. Echocardiography was performed in pregnant dams, adult (11-week old) and fetal (GD 14) progeny. Mitochondrial function and global N⁶-methyladenosine (m⁶A) content were assessed in adult progeny. MPHGPx enzymatic function was further evaluated in adult progeny and m⁶A-RNA immunoprecipitation (RIP) was combined with RT-qPCR to evaluate m⁶A content in the 3'-UTR. Following gestational ENM exposure, global longitudinal strain (GLS) was 32% lower in WT adult offspring of WT dams, with preservation in WT offspring of Tg dams. MPHGPx activity was significantly reduced in WT offspring (29%) of WT ENM-exposed dams, but preserved in the progeny of Tg dams. M⁶A-RIP-qPCR for the SEC insertion sequence region of mPHGPx revealed hypermethylation in WT offspring from ENM-exposed WT dams, which was thwarted in the presence of the maternal transgene. Our findings implicate that m⁶A hypermethylation of mPHGPx may be culpable for diminished antioxidant capacity and resultant mitochondrial and cardiac deficits that persist into adulthood following gestational ENM inhalation exposure.

Keywords: Environmental exposure; GPx4; M6A; N6-methyladenosine; mitochondria.

Figure 1.

MPHGPx mouse breeding strategy. (A) Schematic of mPHGPx breeding strategies with transgene effect on offspring from maternal (left) or paternal (right) transgene; (B) Schematic of exposure paradigm that was implemented for each group, representing all groups that were utilized in the study; (C) A timeline of the study. MPHGPx: mitochondrial phospholipid hydroperoxide glutathione peroxidase.

Figure 2.

Maternal nano-TiO₂ inhalation exposure characteristics. (A) Real-time (black line) aerosol mass concentration measurements with the red line indicating the target concentration (12 mg/m³) of a typical 360 min maternal nano-TiO₂ inhalation exposure; (B) Count size distribution of the nano-TiO₂ aerosols measured with a high resolution electric low-pressure impactor (ELPI+). The red line designates the log normal distribution obtained with the log probability plot method (CMD = 0.163 μm, with a GSD = 1.77); (C) Count size distribution of nano-TiO₂ aerosols measured with a scanning mobility particle sizer (SMPS) with the red line representing a log normal fit of the data (CMD = 0.190 μm, GSD = 1.97); (D) Mass size distribution of nano-TiO₂ aerosols measured with a nano micro-orifice uniform deposit impactor (MOUDI). The red line represents a log normal fit of the data indicating a mass median aerodynamic diameter (MMAD) of 0.968 μm and a GSD of 2.56. CMD: count median diameter; GSD: geometric standard deviation.

Figure 3.

Assessment of fetal pup cardiac function in utero. (A) Graphical depiction of the uterine horn and the pup identification system; (B) Representative M-mode echocardiographic scan of a fetal pup with the short-axis left ventricular trace (blue); (C–D) Representative B-mode scan of a fetal pup with the left and right ventricles identified by blue arrows at end diastole (C) and end systole (D); (E–F) Representative B-mode scan in the short-axis of a fetal pup at with green outlines indicating tracking of the epicardium and endocardium borders through (E) end diastole and (F) end systole. R: right; L: left.

Figure 4.

Electron transport chain (ETC) complex activities of adult PPME and MPME offspring. (A–D) Electron transport chain (ETC) activities assessed in cardiac protein lysate of adult offspring for (A) complex I, (B) complex III, (C) complex IV, and (D) complex V (ATP Synthase). PPME WT Sham, n = 7; PPME WT TiO₂, n = 6; PPME Tg Sham, n = 6; PPME Tg TiO₂, n = 5; MPME WT Sham, n = 4; MPME WT TiO₂, n = 5; MPME Tg Sham, n = 7; MPME Tg TiO₂, n = 4. Adult = 11 weeks old. Statistical difference was defined by p ≤ 0.05. * = group difference determined by a two-way ANOVA and ns=no statistical difference. A letter above a group denotes statistical significance between those groups based on a Tukey’s multiple-comparisons test. A dagger (†) above a group indicates statistical significance between WT Sham and WT TiO₂ or Tg Sham and Tg TiO₂ of the same maternal genetic group (MPME or PPME) based on a Student’s t-test. All data are presented as the mean ± the standard error of the mean (SEM). Adult = 11 weeks of age, mPHGPx=mitochondrial phospholipid hydroperoxide glutathione peroxidase, PPME = Paternal mPHGPx maternal exposure, MPME = Maternal mPHGPx maternal exposure, WT Sham = wild-type offspring whose dam was exposed to control air, Tg Sham = mPHGPx transgenic offspring whose dam was exposed to control air, WT TiO₂ = wild-type offspring whose dam was exposed to nano-TiO₂, Tg TiO₂ = mPHGPx transgenic offspring whose dam was exposed to nano-TiO₂, Unit = nanomoles of substrate oxidized (minute⁻¹).

Figure 5.

Antioxidant activity and H₂O₂ production in adult offspring following gestational inhalation exposure. (A) Total GPx activity in cardiac protein lysate determined by cumene hydroperoxide as substrate and normalized to protein content; (B) Mitochondrial GPx (mPHGPx) activity in cardiac protein lysate determined by phosphatidylcholine hydroperoxide (PCOOH) as substrate and normalized to protein content; (C) Total hydrogen peroxide (H₂O₂) concentration in cardiac protein lysate normalized to protein content; (D) Hydrogen peroxide (H₂O₂) concentration in cardiac isolated mitochondria of adult offspring normalized to protein content. PPME WT Sham, n = 7 (protein) and n = 6 (mitochondria); PPME WT TiO₂, n = 6; PPME Tg Sham, n = 6 (protein) and n = 5 (mitochondria); PPME Tg TiO₂, n = 5; MPME WT Sham, n = 4; MPME WT TiO₂, n = 5; MPME Tg Sham, n = 7; MPME Tg TiO₂, n = 4. Statistical difference was defined by p ≤ 0.05. * = group difference determined by a two-way ANOVA and ns = no statistical difference. A letter above a group denotes statistical significance between those groups based on a Tukey’s multiple-comparisons test. A dagger (†) above a group indicates statistical significance between WT Sham and WT TiO₂ or Tg Sham and Tg TiO₂ of the same maternal genetic group (MPME or PPME) based on a Student’s t-test. All data are presented as the mean ± the standard error of the mean (SEM). Adult = 11 weeks of age, mPHGPx = Mitochondrial phospholipid hydroperoxide glutathione peroxidase, PPME = Paternal mPHGPx maternal exposure, MPME = Maternal mPHGPx maternal exposure, WT Sham = wild-type offspring whose dam was exposed to control air, Tg Sham = mPHGPx transgenic offspring whose dam was exposed to control air, WT TiO₂ = wild-type offspring whose dam was exposed to nano-TiO₂, Tg TiO₂ = mPHGPx transgenic offspring whose dam was exposed to nano-TiO₂.

Figure 6.

Epitranscriptomic mechanism contributing to diminished antioxidant scavenging ability following maternal nano-TiO₂ inhalation exposure. (A) Global m⁶A content was determined as a percentage (%) of total RNA isolated from adult offspring cardiac tissue. PPME WT Sham, n = 6; PPME WT TiO₂, n = 5; PPME Tg Sham, n = 6; PPME Tg TiO₂, n = 5; MPME WT Sham, n = 4; MPME WT TiO₂, n = 5; MPME Tg Sham, n = 6; MPME Tg TiO₂, n = 4; (B) Isolated RNA with 3 min fragmentation utilized to achieve ~130 nt fragments (top) compared to no fragmentation (bottom) (C) Schematic of the predicted m⁶A site in the 3′-UTR of the mouse mPHGPx mRNA, specifically within the SECIS region; (D) M⁶A-RIP-qPCR for mPHGPx (3′-UTR region including potential m⁶A site). N = 4 per group (ran in triplicate). Statistical difference was defined by P ≤ 0.05 (ns = no significance) based on a one or two-way ANOVA, where appropriate. A letter above a group denotes statistical significance between those groups based on a Tukey’s multiple-comparisons test. A dagger (†) above a group indicates statistical significance between WT Sham and WT TiO₂ or Tg Sham and Tg TiO₂ of the same maternal genetic group (MPME or PPME) based on a Student’s t-test. All data are presented as the mean ± the standard error of the mean (SEM). Adult = 11 weeks old, mPHGPx = mitochondrial phospholipid hydroperoxide glutathione peroxidase, M⁶A = N⁶-Methyladenosine, PPME = Paternal mPHGPx maternal exposure, MPME = Maternal mPHGPx maternal exposure, WT Sham = wild-type offspring whose dam was exposed to control air, Tg Sham = mPHGPx transgenic offspring whose dam was exposed to control air, WT TiO₂ = wild-type offspring whose dam was exposed to nano-TiO₂, Tg TiO₂ = mPHGPx transgenic offspring whose dam was exposed to nano-TiO₂, RIN = RNA integrity number, SECIS = selenocysteine insertion sequence.

Figure 7.

Physiological and molecular consequences of gestational nano-TiO₂ inhalation exposure in adult offspring. Following gestational exposure, elevated ROS levels in the progeny are concomitant with cardiac functional alterations that can be mitigated by overexpression of maternal mPHGPx. Augmented mitochondrial ROS plays a key role in the persistence of deficits into adulthood (11 weeks of age) characterized by diminished GLS and ETC complex activities. A mechanism that could promote the sustained consequences is initiated by high oxidative stress, which increases m⁶A methylation at the SECIS in the 3′-UTR of mPHGPx. Ultimately, this may tamper with the SECIS binding region for the SBP2 that is vital for the incorporation of selenocysteine and the catalytic activity of mPHGPx. Decreased mPHGPx activity then propagates mitochondrial bioenergetic deficits, limiting overall cardiac performance in the adult offspring. MPHGPx = mitochondrial phospholipid hydroperoxide glutathione peroxidase, ROS = reactive oxygen species, GLS = global longitudinal strain, ETC = electron transport chain, m⁶A = N⁶-methyladenosine, SECIS = Selenocysteine insertion sequence, SBP2 = SECIS binding protein 2.