Nuclear-mitochondrial communication involving miR-181c plays an important role in cardiac dysfunction during obesity

Abstract

Aims: In cardiomyocytes, there is microRNA (miR) in the mitochondria that originates from the nuclear genome and matures in the cytoplasm before translocating into the mitochondria. Overexpression of one such miR, miR-181c, can lead to heart failure by stimulating reactive oxygen species (ROS) production and increasing mitochondrial calcium level ([Ca²⁺]_m). Mitochondrial calcium uptake 1 protein (MICU1), a regulatory protein in the mitochondrial calcium uniporter complex, plays an important role in regulating [Ca²⁺]_m. Obesity results in miR-181c overexpression and a decrease in MICU1. We hypothesize that lowering miR-181c would protect against obesity-induced cardiac dysfunction.

Methods and results: We used an in vivo mouse model of high-fat diet (HFD) for 18 weeks and induced high lipid load in H9c2 cells with oleate-conjugated bovine serum albumin in vitro. We tested the cardioprotective role of lowering miR-181c by using miR-181c/d^-/- mice (in vivo) and AntagomiR against miR-181c (in vitro). HFD significantly upregulated heart levels of miR-181c and led to cardiac hypertrophy in wild-type mice, but not in miR-181c/d^-/- mice. HFD also increased ROS production and pyruvate dehydrogenase activity (a surrogate for [Ca²⁺]_m), but the increases were alleviated in miR-181c/d^-/- mice. Moreover, miR-181c/d^-/- mice fed a HFD had higher levels of MICU1 than did wild-type mice fed a HFD, attenuating the rise in [Ca²⁺]_m. Overexpression of miR-181c in neonatal ventricular cardiomyocytes (NMVM) caused increased ROS production, which oxidized transcription factor Sp1 and led to a loss of Sp1, thereby slowing MICU1 transcription. Hence, miR-181c increases [Ca²⁺]_m through Sp1 oxidation and downregulation of MICU1, suggesting that the cardioprotective effect of miR-181c/d^-/- results from inhibition of Sp1 oxidation.

Conclusion: This study has identified a unique nuclear-mitochondrial communication mechanism in the heart orchestrated by miR-181c. Obesity-induced overexpression of miR-181c increases [Ca²⁺]_m via downregulation of MICU1 and leads to cardiac injury. A strategy to inhibit miR-181c in cardiomyocytes can preserve cardiac function during obesity by improving mitochondrial function. Altering miR-181c expression may provide a pharmacologic approach to improve cardiomyopathy in individuals with obesity/type 2 diabetes.

Keywords: MICU1; Mitochondria; Mitochondrial calcium; Obesity; miR-181c; microRNA.

Figure 1.. Effect of high fat diet on cardiac mitochondrial miR-181c expression.

Quantitative PCR (SYBR) was used to assess (A) miR-181 expression in heart tissues, and (B) miR-181c expression in the mitochondrial fraction of heart tissues, from mice after 18 weeks of being fed normal chow (N Chow) or the 60% high fat diet (HFD). SNO-RD61 was used to normalize miR-181-a/b/c/d expression from the whole heart homogenate, and mitochondrial gene-encoded 12S rRNA was used to normalize the miR-181c expression from isolated mitochondrial fraction. Finally, miRNA expressions were normalized to corresponding N Chow groups (n=6–7). Data were analyzed by 2-way ANOVA with repeated measures. *p<0.05 vs. N Chow, and **p<0.001 vs. N. Chow. (n=5).

Figure 2.. miR-181c plays an important role in obesity-induced cardiac hypertrophy.

(A) Hypertrophy was measured as the ratio between the wet weight of the whole heart and the corresponding tibia length of each mouse (n=8–9). Western blot analysis of hypertrophic marker (B) B-type natriuretic peptide (BNP) (upper bands), (C) atrial natriuretic peptide (ANP) (upper bands), and (D) β-myosin heavy chain (β -MHC) (upper bands) expression in the total heart homogenate from WT and c/d KO mice fed a normal chow or HFD for 18 weeks. α-tubulin (lower bands in all three panels) was used to normalize BNP, ANP and β-MHC expression (n = 5–7). Data were analyzed by 2-way ANOVA *p<0.05 vs. WT-N Chow, †p<0.001 vs. WT-HFD.

Figure 3.. Lack of miR-181c can protect cardiac mitochondria from consequences of diet-induced obesity.

(A) Amplex red assay was performed to measure mitochondrial ROS production (n=5–7). (B) pyruvate dehydrogenase (PDH) enzyme activity was measured in the mitochondrial fractions of WT and c/d KO mice after the 18-week diet regimen (n=5–7). (C) Western blot analysis of PDH expression in the total heart homogenate from WT and c/d KO mice fed a normal or HFD for 18 weeks. PDH (upper bands) expression was normalized to α-tubulin (lower bands). A.U., arbitrary units (n = 5–7). Data were analyzed by both 2-way ANOVA, and two-tailed unpaired t-test. *p<0.05 vs. WT-N Chow; **p<0.001 vs. WT-N Chow; †p<0.05 vs. WT-HFD.

Figure 4.. In vitro obese condition induces miR-181c expression in the heart.

Quantitative PCR (SYBR) was performed with the RNA fraction of H9c2 cells after 24, 30, 38, and 48 hrs of lipid-load for (A) miR-181c expression and (B) MICU1 expression. SNORD61 and β-actin were used to normalize miR-181c and MICU1 expression, respectively. Data were analyzed by 2-way ANOVA with repeated measures, n = 9–12/group, *p<0.05 vs. BSA-treated group.

Figure 5.. Loss of miR-181c protects H9c2 cells from lipid-load through a mitochondrial pathway.

(A) The Amplex red assay was performed to measure the ROS production rate in H9c2 cells cultured with a high lipid load or bovine serum albumin (BSA, control) at 30 and 48 hrs after transfection with either scramble sequence or AntagomiR-181c (n=6–8/group). (B) The pyruvate dehydrogenase (PDH) activity assay was performed as a measure of [Ca²⁺]_m in H9c2 cells grown under conditions of lipid-load or BSA at 30 and 48 hrs after transfection with either scramble sequence or AntagomiR-181c (n=5–6/group). Data were analyzed by 2-way ANOVA with repeated measures, *p<0.05 vs. BSA; #p<0.05 vs. BSA-treated Scr; †p<0.05 vs. AntagomiR-181c.

Figure 6.. miR-181c generates [Ca²⁺]_m overload through mitochondrial ROS production.

NMVMs were isolated from MCU^flox/flox mouse pups. (A) Western blot analysis was performed to compare the expression of MCU in MCU^flox/flox-NMVMs infected with the Cre adenovirus (MCU^−/−) to that in non-infected controls (MCU^fl/fl). Next, NMVMs from the MCU^flox/flox Cre-infected group were transfected with either scramble sequence (Scr) or miR-181c (miR-181c overexpression [OE]) for 48 hrs. (B) Pyruvate dehydrogenase (PDH) activity (surrogate measure of [Ca²⁺]_m) was measured in the transfected NMVMs. Data presented in panel B were analyzed by both 2-way ANOVA, and two-tailed unpaired t-test, n=5–6/group. (C) Amplex red assay was used to measure mitochondrial ROS in the transfected NMVMs. Data were analyzed by 2-way ANOVA with repeated measures, n=7–8/group. **p<0.001 vs. WT and control (not transfected; MCU^fl/fl); #p<0.0001 vs. Scr.

Figure 7.. miR-181c overexpression-induced ROS production leads to oxidation of mitochondrial Sp1 at cysteine residues.

(A) Nuclear (Nuc), mitochondrial (Mito), and cytoplasmic (Cyto) proteins were isolated from C57BL6 mouse hearts by subcellular fractionation. Western blots were probed with anti-Sp1, anti-histone 3 (His3; nuclear marker), anti-voltage-dependent anion channel (VDAC; mitochondrial marker), and anti-α-tubulin (cytosolic marker). (B) Sp1 oxidation at the cysteine residue (Ox-Sp1) was measured in NMVMs that were isolated from C57BL6 pups, transfected with either scramble sequence (Scr) or miR-181c (miR-181c overexpression [OE]), and treated with or without Mito-TEMPO (25 μM for 48 h). Total Sp1 expression was used to normalize Ox-Sp1. miR-181c overexpression increased Sp1 oxidation, which was significantly diminished with Mito-TEMPO treatment. Data were analyzed by 2-way ANOVA with repeated measures, n=4–5/group, *p<0.05 vs. Scr without Mito-TEMPO; †p<0.05 vs. miR-181c OE without Mito-TEMPO treatment.

Figure 8.. Mitochondrial Sp1 oxidation plays an important role in obesity-induced [Ca²⁺]_m overload.

Two groups of animals, C57BL6 (WT) and miR-181c/d^−/− (c/d KO), were fed either normal chow (N Chow) or a 60% high-fat diet (HFD) for 18 weeks. (A) Total heart homogenate was used to measure Sp1 oxidation at the cysteine residue (Ox-Sp1) by Western blot. Total Sp1 expression was used to normalize Ox-Sp1. (B, C) Western blot analysis of Sp1 and MICU1. α-Tubulin was used to normalize Sp1 and MICU1 expression (n=5–7/group). Data were analyzed by 2-way ANOVA with repeated measures, *p<0.05 vs. WT-N Chow; †p<0.001 vs. WT-HFD