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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 64  |  Issue : 2  |  Page : 72-79

Deficit of female sex hormones desensitizes rat cardiac mitophagy


1 Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand
2 Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand

Date of Submission07-Dec-2020
Date of Decision15-Mar-2021
Date of Acceptance17-Mar-2021
Date of Web Publication19-Apr-2021

Correspondence Address:
Dr. Tepmanas Bupha-Intr
Department of Physiology, Faculty of Science, Mahidol University, 272 Rama VI Road, Bangkok 10400
Thailand
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjp.cjp_102_20

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  Abstract 


Long-term deprivation of female sex hormones has been shown to mediate accumulation of damaged mitochondria in ventricular muscle leading to cardiovascular dysfunction. Therefore, the roles of female sex hormones in mitochondrial quality control are closely focused. In the present study, depletion of female sex hormones impairing mitochondrial autophagy in the heart was hypothesized. Cardiac mitophagy was therefore investigated in the heart of 10-week ovariectomized (OVX) and sham-operated (SHAM) rats. By using isolated mitochondria preparation, results demonstrated an increase in mitochondrial PTEN-induced kinase 1 accumulation in the sample of OVX rats indicating mitochondrial outer membrane dysfunction. However, no change in p62 and LC3-II translocation to mitochondria was observed between two groups indicating unresponsiveness of mitophagosome formation in the OVX rat heart. This loss might be resulted from significant decreases in Parkin and Bcl2l13 expression, but not Bnip3 activation. In summary, results suggest that mitochondrial abnormality in the heart after deprivation of female sex hormones could consequently be due to desensitization of mitophagy process.

Keywords: Bcl2l13, Bnip3, LC3-II, parkin, PTEN-induced kinase 1


How to cite this article:
Kampaengsri T, Ponpuak M, Wattanapermpool J, Bupha-Intr T. Deficit of female sex hormones desensitizes rat cardiac mitophagy. Chin J Physiol 2021;64:72-9

How to cite this URL:
Kampaengsri T, Ponpuak M, Wattanapermpool J, Bupha-Intr T. Deficit of female sex hormones desensitizes rat cardiac mitophagy. Chin J Physiol [serial online] 2021 [cited 2021 May 14];64:72-9. Available from: https://www.cjphysiology.org/text.asp?2021/64/2/72/314083




  Introduction Top


Upon menopause, many metabolic syndromes are raised especially cardiovascular disease. Increased risk of cardiovascular diseases after menopause has been attributed to a possible deficit in female sex hormone regulatory function on cardiac function.[1] Reduced ejection fraction with left ventricular hypertrophy has been demonstrated early after menopause.[2],[3] A reduction in systolic function was also confirmed in ovariectomized (OVX) rat model.[4],[5] Although some clinical trials and animal studies have demonstrated the benefits of hormone replacement therapy in preventing heart disease in postmenopausal women, others have failed to demonstrated the benefit of hormone replacement.[6],[7] Thus, it is imperative to clearly understand the mechanistic changes on deprivation of female sex hormones before recommending the replacement therapy.

In our previous study, we demonstrated that deprivation of female sex hormones in OVX rats resulted in a deterioration of cardiac mitochondrial integrity. Accumulation of mitochondria with swelling, decreased adenosine triphosphate (ATP) production and elevated reactive oxygen species (ROS) level were profoundly demonstrated after 10 weeks of ovariectomy.[8] These findings suggested that female sex hormone deficit disrupts normal cardiac mitochondrial homeostasis. Poorly functioning mitochondria not only generate inadequate supply of ATP but pose a danger to the cell by releasing pro-apoptotic factors.[9] Therefore, repairing and terminating processes of damaged mitochondria are essential, in which deprivation of female sex hormones might decrease the terminating processes.

It has been demonstrated that estrogen attenuated lipopolysaccharide-induced autophagy in neonatal cardiomyoctes.[10] The attenuated effect of estrogen could be due to suppressed Bnip3-induced autophagy.[11] On the other hand, autophagy activity in response to lipopolysaccharide induction was found to be less in the heart of OVX rats.[12] While the role of female sex hormones in general autophagy process is controversial, mitophagic activity on deprivation of female sex hormones has never been discovered. Processes of macroautophagy begin with the induction of non-specific phagophore formation from endoplasmic membrane by the regulation of autophagy-related protein complex which is activated by unc-51 like autophagy activating kinase 1 complex.[13] By activation of autophagy-related protein complexes, phosphatidylinositol 3-phosphate and microtubule-associated protein 1 light chain 3 (LC3)-II, phagophore is elongated.[13] Elongated phagophore interacts with the target material or cargo by wrapping around and sealing to form the complete autophagosome. The formation of autophagosome requires the interaction of LC3-II to adaptor proteins on the cargo. During autophagosome formation, LC3-II serves as a receptor binding to adaptor proteins such as sequestosome-1 or p62, BCL2/adenovirus E1B interacting protein 3 (Bnip3) and BCL2-like 13 (Bcl2l13), in which later two are specific on mitochondrial membrane. In the common mitophagy pathway, mitochondrial damage leads to PTEN-induced kinase 1 (PINK1) accumulation on the outer mitochondrial membrane, and then recruitment of Parkin binding to the mitochondria. Parkin then induces polyubiquitination leading to p62 recruitment followed by LC3-II binding and so on.[14] LC3-II can also directly bind to Bnip3 and Bcl2l13 on the membrane of damaged mitochondria. During mitochondrial stress, Bnip3 is dimerized and binds to LC3-II to mediate mitophagy,[15] while Bcl2l13 on the outer mitochondrial membrane is phosphorylated upon mitochondrial damage and then bind to LC3-II to complete mitophagosome formation.[16] Subsequently, autophagosome spontaneously fuses with lysosome to become autolysosome where the cargo is degraded by lysosomal enzymes. Changes in these proteins can indicate the activation of mitophagy upon deprivation of female sex hormones.

Using an OVX rat model, we hypothesized the notion that female sex hormone deficit compromises cardiac mitophagy capacity leading to an accumulation of damaged mitochondria. An understanding of the underlying mechanism(s) regulating mitochondrial quality by female sex hormones should contribute to the development of effective therapeutic and preventive methods to alleviate cardiac diseases in the situation of a deficit of these hormones.


  Materials and Methods Top


Animals preparations

Female Sprague-Dawley rats (n = 6), 8 weeks old and weighing 180–200 g were OVX and an equal number were sham-operated (SHAM).[17] Animals were housed in an AAALAC certified facility under 12:12 light-dark cycle at 22°C ± 1°C and 30%–70% humidity and had access ad libitum to laboratory rat chow (C.P., Bangkok, Thailand) and water. Ten weeks following operation, rats were anesthetized and hearts were rapidly excised and placed in ice-cold phosphate-buffered saline (PBS) solution. Deprivation of ovarian sex hormones was confirmed by observation of marked reduction uterine weight. Animal treatment protocol was approved by the Experimental Animal Committee, Faculty of Science, Mahidol University, in accordance with the National Animal Laboratory Centre, Thailand (protocol no. MUSC59-004-337).

Immunofluorescence analysis

To evaluate amount of autophagosome formation, cardiac tissue was processed for immunofluorescence study. After harvested the heart, a piece of left ventricle was fixed in 4%s (w/v) paraformaldehyde for 8 h followed by incubated with 15% (w/v) sucrose in PBS for 4 h and 30% (w/v) sucrose overnight at 4°C. The sample was embedded in optimal cutting temperature compound and stored at −80°C. The sample was cut at 7 μm thickness with a cryostat (Leica, CM1850). Tissue section was mounted on poly-l-lysine coated slide and air-dried for 20 min. Section was blocked and permeabilized by solution containing 3% (v/v) normal donkey serum and 5% (w/v) saponin in PBS for 1 h at 37°C. Primary antibodies rabbit anti-LC3B (L7543; Sigma-Aldrich) (1:2,000 dilution) and mouse anti-cardiac troponin I (cTnI) (ab10231; Abcam) (1:50 dilution) were applied to the section for overnight at 4°C. After washing, section was then incubated with secondary antibodies of donkey anti-rabbit immunoglobulin G (IgG) HL (Alexa Fluor® 488, Abcam) (1:200 dilution) and donkey anti-mouse IgG HL (Alexa Fluor® 647, Abcam) (1:200 dilution) and 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (D9542; Sigma-Aldrich) (1:400 dilution) for nuclear staining for 1 h in the dark. Five fields per heart section were obtained at 300 magnification using olympus FV10i-DOC confocal laser-scanning microscope. The counted dot size was bigger-than or equal to 0.3 μm diameter as previously described.[18]

Mitochondria isolation

The left ventricular tissue (0.3 g) was minced in a homogenizing buffer (0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl), homogenized in a glass-Teflon homogenizer (15 strokes at 800 rpm), centrifuged at 500 g for 5 min at 4°C, supernatant re-centrifuged at 15,000 g for 10 min at 4°C, and pellet (isolated mitochondria) suspended in homogenizing buffer at 4°C. Mitochondria suspension was adjusted to 36% (w/v) iodixanol (Axis-Shield Diagnostics Ltd.), transferred to an ultracentrifuge tube and overlaid with 26% (w/v) followed by 20% (w/v) iodixanol solution and centrifuged at 100,000 g for 4 h at 4°C resulting in separation into four fractions (bands). The fraction between 20 and 26% iodixanol was harvested and placed on ice as mitochondrial rich fraction. The fraction was diluted with homogenizing buffer and centrifuged at 15,000 g for 15 min at 4°C, and pellet was suspended in buffer solution (50 mM sucrose, 200 mM mannitol, 5 mM KH2PO4, 5 mM 3-(N-Morpholino) propanesulfonic acid (MOPS), and 2 mM taurine) (modified from OptiPrepTM Application Sheet S14).

Immunoblot analyses

Whole heart homogenate or mitochondrial fraction was dissolved in extracting buffer containing 50 mM Tris pH 6.8, 2.5% (w/v) sodium dodecyl sulfate (SDS), 10% (w/v) glycerol, 1 mM dithiothreitol, 1 μg/mL leupeptin, 1 μg/mL pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and separated by SDS polyacrylamide gel electrophoresis, transferred into nitrocellulose membrane, incubated with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20, followed by primary antibodies; rabbit anti-LC3B (L7543; Sigma-Aldrich) (1:2,000 dilution), mouse anti-VDAC1/Porin (ab14734; Abcam) (1:5,000 dilution), rabbit anti-β-actin (4967; Cell Signaling Technology) (1:5,000 dilution), rabbit anti-Parkin (ab179812; Abcam) (1:1,000 dilution), rabbit anti-PINK1 (P0076; Sigma-Aldrich) (1:2,000 dilution), rabbit anti-Bnip3 (3769; Cell Signaling Technology) (1:2,000 dilution) or rabbit anti-Bcl2l13 (ab27795; Abcam) (1:1,000 dilution), and then goat secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology) or donkey HRP-conjugated anti-mouse IgG (Fitzgerald). Immunoreactive bands were recorded by a CanoScan LiDE 120 Scanner (Tokyo, Japan) and quantified using ImageJ software version 1.50i (Bethesda, MD, USA).

Statistical analysis

All values are presented as the mean ± standard error of the mean. Difference between two experimental groups was analyzed using independent t-test in SPSS statistic software version 18.0 (SPSS Inc., Chicago, IL, USA). Difference is considered statistically significant at P < 0.05.


  Results Top


Characteristics of test rats

A significant decrease in uterine weight of OVX rats confirmed deprivation of ovarian hormones following 10 weeks of ovariectomy compared to the SHAM control [Table 1] consistently to a previous study.[8] Ovariectomy also resulted in increases in body and heart weights.
Table 1: Characteristics of 10-week ovariectomized and sham-operated rats

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Suppression of cardiac autophagy in ovariectomized rats

To determine cardiac autophagic capacity, the number of autophagosome formation was evaluated by immunofluorescence study [Figure 1]a and [Figure 1]b. The number of LC3 accumulation was lower in the heart of OVX rats than that in the heart of SHAM controls. This finding suggested that lack of ovarian sex hormones decreased the autophagic activity. Although the deprivation of female sex hormones had no effect on the expressions of LC3 in the whole heart [Figure 1]c and [Figure 1]d, a decrease in LC3-II: total LC3 ratio [Figure 1e] suggested a possible decrease in phagophore formation in the heart of OVX rats. These results suggest that lack of female sex hormones associated with a reduction of general autophagic activation.
Figure 1: Effect of female sex hormone deprivation on cardiac autophagy. (a) Representative immunofluorescence staining of LC3, cTnI and nucleus (DAPI) in left ventricular slices of sham-operated rats (SHAM) and ovariectomized rats (OVX). (b) Scatter plot with bar graph demonstrates the number of LC3 accumulative dots from immunofluorescence images representing the level of autophagy. (c) Representative immunoblot bands of LC3 and ACTB in the heart of SHAM and OVX groups. (d and e) Scatter plot with bar graphs demonstrate the expression level of LC3 to ACTB ratio and percentage of LC3-II to total LC3 (LC3-I plus LC3-II), respectively. Data are mean ± standard error of the mean from 6 to 7 hearts (5 fields per heart) in each group. *Significant difference (P < 0.05) between two groups using independent t-test.

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Expression of mitophagy-associated proteins

To quantify if any change in mitophagy-associated proteins, protein expression of PINK1, Parkin and p62 in cardiac muscle was analyzed using immunoblot analysis. Whole left ventricular preparation demonstrated a significant decrease in PINK1 expression in the heart of OVX rats as compared to SHAM controls [Figure 2]a. Parkin expression was also significantly decreased in the left ventricular homogenate from OVX rats [Figure 2]b. However, there was no change in p62 protein expression in the heart of OVX rats [Figure 2]c. Downregulation of both PINK1 and Parkin suggested a possibility that the heart of OVX rats might had a decrease in mitochondrial autophagosome formation.
Figure 2: Effect of 10.week ovariectomy on the expression of mitophagy-associated proteins in the heart. Expression levels of PINK1 (a), Parkin (b) and p62 (c) in whole heart homogenate from sham-operated (SHAM) and ovariectomized (OVX) rats were demonstrated as normalization with ACTB. Data are mean ± standard error of the mean from 6 to 7 hearts in each group. *Significant difference (P < 0.05) between two groups using independent t-test.

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Mitochondrial damage and autophagosome formation

[Figure 3] compares the level of mitochondrial damage and autophagosome formation by evaluating the localization of specific adaptor protein in isolated mitochondrial preparation. Results demonstrated that 10-week deprivation of ovarian sex hormones caused a significant increase in PINK1 translocation to mitochondrial membrane [Figure 3]a indicating high mitochondrial membrane damage. On the other hand, less amount of Parkin translocation to the mitochondria from the heart of OVX rats was observed as compared to that of SHAM [Figure 3]b. Interestingly, there was no change in the amount of p62 and LC3-II binding to the mitochondria in the OVX preparation [Figure 3]c and [Figure 3]d. When the relationship between PINK1 translocation and LC3-II binding on mitochondria was compared [Figure 3]e, a decrease in LC3-II: PINK1 ratio in the preparation of OVX rats indicated desensitization of mitochondrial autophagosome formation.
Figure 3: Effect of 10-week ovariectomy on translocation of mitophagy adaptor protein in the heart. Expression levels of PINK1 (a), Parkin (b), p62 (c) and LC3-II (d) in isolated mitochondrial preparation from sham-operated (SHAM) and ovariectomized (OVX) rats were demonstrated as normalization with porin, and the ratio of mitochondrial LC3-II: PINK1 (e). Data are mean ± standard error of the mean from 6 to 7 hearts in each group. *Significant difference (P < 0.05) between two groups using independent t-test.

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Mitophagosome formation: Bnip3 and Bcl2l13 pathways

It has been recently reported that phagophore also interacts with damaged mitochondria through a direct interaction of LC3-II with various mitochondrial membrane proteins including Bnip3 and Bcl2l13, to form mitophagosome in cardiomyocytes.[14] Immunoblot analysis of Bnip3 indicates no difference in the left ventricular homogenate between SHAM and OVX rats in both homodimer and monomer isoforms [Figure 4]a, [Figure 4]b, [Figure 4]c. Mitochondrial preparations also revealed no difference between SHAM and OVX in both the monomer and the homodimer forms [Figure 4]d, [Figure 4]e, [Figure 4]f.
Figure 4: Effect of 10-week ovariectomy on the expression of mitochondrial LC3-II-binding protein Bnip3 on the mitochondrial membrane in the heart. Monomer and homodimer expression levels of Bnip3 in whole heart homogenate (a-c) and in isolated mitochondrial preparation (d-f) from sham-operated (SHAM) and ovariectomized (OVX) rats were demonstrated as normalization with ACTB and porin, respectively. Data are mean ± standard error of the mean from 6 to 7 hearts in each group. No significant difference (P < 0.05) was found among all parameters between them using independent t-test.

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In addition, the expression of Bcl2l13 was significantly lower in OVX rats compared to that in SHAM rats in both whole left ventricular homogenate and isolated mitochondrial preparation [Figure 5]. Downregulation of Bcl2l13 may be another mechanistic signal giving rise to the unresponsiveness of mitophagosome formation in the heart after female sex hormone deprivation.
Figure 5: Effect of 10-week ovariectomy on the expression of mitochondrial LC3-II-binding protein Bcl2l13 on the mitochondrial membrane in the heart. Expression levels of Bcl2l13 in whole heart homogenate (a) and in isolated mitochondrial preparation (b) from sham-operated (SHAM) and ovariectomized (OVX) rats were demonstrated as normalization with ACTB and porin, respectively. Data are mean ± standard error of the mean from 6 to 7 hearts in each group. *Significant difference (P < 0.05) between two groups using independent t-test.

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  Discussion Top


A major finding from the present study is the demonstration that there was an impairment of mitophagosome formation in response to elevated abnormal mitochondria in the ventricle of 10-week OVX rat hearts. Increased PINK1 mitochondrial translocation confirmed the dysfunction of mitochondria after deprivation of female sex hormones. However, without any change in LC3-II-mitochondria interaction, it could imply that the level of mitochondrial autophagosome formation was sustained in the OVX rat heart though increased damaged mitochondria. The desensitization in mitochondrial autophagosome formation in the heart of OVX rats might partly be due to the downregulation of two adaptor proteins, Parkin and Bcl2l13.

Based on our previous report showing accumulation of swollen mitochondria, lowering of ATP synthesis, and increasing in ROS production in the heart of 10-week OVX rats,[8] it is then possible that deprivation of female sex hormones might harm the mitochondrial quality, either through biogenesis or mitophagy. The present findings demonstrate that lack of female sex hormones induced an unresponsive formation of mitophagosome to handle the higher level of damaged mitochondria. Thus, an impairment of mitophagy activation is, in part, an answer underlying the accumulation of mitochondria in OVX rat myocardium. In contrast to our finding, one previous study demonstrated that estrogen treatment at the onset of reperfusion could decrease mitophagy and mitochondria dysfunction.[19] This contradicted action of estrogen on mitophagy between the OVX rat heart and the heart with ischemic-reperfusion injury may relate between genomic and nongenomic activation. Previous reports in other cell types have also shown conflicting actions of ovarian sex hormones especially estrogen on mitophagy.[20],[21],[22] In insulin-secreting β-cell line estradiol suppressed the mitophagy of the cell.[20] In bone cells, however, while estradiol enhanced the viability of the cell through reduced mitophagy in chondrocytes (ATDC5),[21] a stimulating effect on mitophagy to improve cell survival was the case in osteoblast cell line (MC3T3-E1).[22] These opposed effects of female sex hormones, either enhancing or repressing, on the mitophagy in various cell types indicate a tissue-specific action of the sex hormones in maintaining cellular homeostasis as the final outcome.

The mechanistic signals by which deficit of female sex hormones impairs the cardiac mitophagy of unhealthy mitochondria were, in part, associated with PINK1/Parkin and Bnip3/Bcl2l13. A variety of signals and pathways have been reported to activate mitophagy in the heart.[14] To date, the classical well-characterized mitophagy mechanism has been presented to involve the PINK1/Parkin pathway,[14] which has also been proposed to be the pathway underlying the regulatory impact of female sex hormones. Despite a higher accumulation of PINK1, it is the suppression of Parkin translocation to mitochondria in sex hormone-deficit group that influences the outcome.

In addition to PINK1/Parkin pathway, female sex hormones may regulate cardiac mitophagy via other mediators like Bnip3/Bcl2l13, acting as mitophagy receptors that can bind through LC3-binding motifs to clear mitochondria in a variety of cell types, including cardiomyocytes.[14] Especially, Bcl2l13 has been shown to induce mitochondrial fragmentation and mitophagy in mammalian cells.[16] In the present study, we proposed that female sex hormones play a significant role in regulating the expression of Bcl2l13, but not Bnip3.

The agreement of our results to the hypothesis that deficit of female sex hormones impairs mitophagy in the heart undeniably raises questions concerning the cardiac functional impact. The deterioration of mitochondrial quality and subsequent cardiac dysfunction due to mitophagy impairment has also been evidenced in many metabolic diseases including aging, vascular occlusion injury, and diabetic cardiomyopathy.[23],[24],[25] Decreased mitophagy activity has been shown to be a mechanistic process underlying accumulation of damaged mitochondria-associated cardiac dysfunction in aging mouse.[23] In PINK1-knockout mice, development of age-dependent cardiac hypertrophy was observed together with falling of cardiac mitochondrial integrity and increasing oxidative stress.[26] Furthermore, a larger size of myocardial infarction was induced in the heart of PINK1-knockout mice than in the wild-type control after facing to ischemia reperfusion injury.[24] Impaired mitophagy also contributes to diabetic cardiomyopathy induction due to reduced clearance of dysfunctional mitochondria.[25] In addition, knock-down of Bnip3 could prevent doxorubicin-induced cell death in postnatal rat cardiomyocytes.[27] Further investigations to search for potential approaches to improve mitophagy and consequent increased healthy mitochondria in both physiological and pathological interferences will certainly help maintain cardiovascular homeostasis.


  Conclusion Top


In conclusion, the work described in this study showed for the first time that long-term deficit of female sex hormones impairs cardiac mitophagy activation in response to increased damaged mitochondria. Signaling defects underlying this impaired mitophagy involve alterations in PINK1, Parkin, and Bcl2l13 mediators. The maintenance of healthy mitochondrial quality could therefore be another essential preventive and therapeutic direction toward normal physiology of the heart in postmenopausal women.

Financial support and sponsorship

The study was supported by grants from Thailand Research Fund (RSA6080064), Science Achievement Scholarship of Thailand and Central Instrument Facility (CIF) grant, Faculty of Science, Mahidol University.

Conflicts of interest

There are no conflicts of interest.



 
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