Name: protection of h9c2 myocardial cells from oxidative stress by crocetin via pink1/parkin pathway-mediated mitophagy
Uploaded: 2023-05-26T15:00:00.000+00:00
Duration: 7 min 40 s
Description: Watch this Scientific Journal Video about Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy at JoVE.com

This study was supported by the Beijing Natural Science Foundation (No. 7202119) and the National Natural Science Foundation of China (No. 82274380).

The experiments were performed in the Laboratory of Physiology at the Beijing University of Chinese Medicine, China. All study methods were performed in accordance with the relevant guidelines and regulations of Beijing University.
1. Cell culture
<ol>
	<li>Add 10% fetal bovine serum and 1% penicillin/streptomycin to Dulbecco&#39;s modified Eagle medium (DMEM) basic medium (with 4.5 g/L D-glucose, 4.g.g/L L-glutamine, and 110 mg/L sodium pyruvate; see Table of Materials) to prepare DMEM complete medium.</li>
	<li>Thaw the liquid nitrogen-frozen H9c2 myocardial cells (see Table of Materials) in warm water at 37 &#176;C with a quick and uniform stir until the ice melts.</li>
	<li>Transfer the cells to a centrifuge tube and add four times the volume of the DMEM complete medium. Centrifuge at 358 x g for 5 min at room temperature and discard the supernatant using a pipette.</li>
	<li>Dilute the obtained cell suspension with culture medium, blow gently, and inoculate the cells in a culture flask. Culture in an incubator at 37 &#176;C with 5% CO2.</li>
</ol>
2. Determination of cell viability
<ol>
	<li>Dissolve crocetin (see Table of Materials) in dimethyl sulfoxide (DMSO) to concentrations of 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 50 mM, 100 mM, and 200 mM.</li>
	<li>Dissociate the H9c2 myocardial cells by trypsin, then neutralize the mixture with DMEM complete medium.</li>
	<li>Transfer the cells to a centrifuge tube and centrifuge at 179 x g for 5 min at room temperature. Discard the supernatant and mix the cells in DMEM complete medium by gently blowing.</li>
	<li>Count the cells using a blood cell counting plate22 (see Table of Materials) and dilute them to 5 &#215; 104 cells/mL with DMEM complete medium.</li>
	<li>Divide the cells into nine equal portions. Add DMSO (diluted in a 1:1,000 ratio with DMEM complete medium) to the control group and add different concentrations of crocetin to the remaining groups at a ratio of 1:1,000.</li>
	<li>Seed the cells in 96-well plates at 100 &#181;L per well. Discard the supernatant after incubation for 24 h and wash the cells three times with PBS.</li>
	<li>After adding 100 &#181;L of DMEM basal medium, incubate the cells for 4 h and add 20 &#181;L of MTS (see Table of Materials).</li>
	<li>Incubate the cells for another 2 h, measure the absorbance at a wavelength of 490 nm, and calculate the cell viability. 
	&#8203;NOTE: Cell viability = (OD treated - OD blank) / (OD control - OD blank) &#215; 100.</li>
</ol>
3. Determination of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH Px), and catalase (CAT)
<ol>
	<li>Seed the H9c2 cells in a 6-well plate at a density of 1 &#215; 105 cells. Collect the supernatant after the intervention and detect LDH and SOD levels, according to the manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Collect the supernatant and wash it with phosphate-buffered saline (PBS) once. Add the cell lysate to the culture dish and allow it to sit on ice for 20 min. Collect the liquid from the culture dish into a centrifuge tube.</li>
	<li>Detect CK, MDA, GSH-Px, and CAT levels according to the manufacturer&#39;s instructions23 (see Table of Materials and Supplementary File 1).</li>
	<li>Detect the total protein concentration of the lysis by the bicinchoninic acid (BCA) method to correct the concentration of CK, MDA, GSH-Px, and CAT24(see Table of Materials).</li>
</ol>
4. Determination of ROS
<ol>
	<li>Seed the H9c2 cells in a 48-well plate at a density of 5 &#215; 103 cells. Dilute DCFH-DA (see Table of Materials) at a 1:1,000 ratio with the serum-free medium. Discard the cell supernatant and wash the cell twice with a serum-free medium.</li>
	<li>Add 150 &#181;L of diluted DCFH-DA into each well and incubate at 37 &#176;C for 20 min. Wash the cells three times with a serum-free medium and capture images under a fluorescence microscope (see Table of Materials).</li>
</ol>
5. Detection of mitochondrial membrane potential
<ol>
	<li>Detect the mitochondrial membrane potential using a JC-1 mitochondrial membrane potential assay kit25 (see Table of Materials). Discard the culture medium and wash them once with serum-free medium.</li>
	<li>Add JC-1 working solution to each tube and mix them well. Incubate at 37 &#176;C for 20 min and wash the cells twice with JC-1 staining buffer. Add complete medium to each well and capture photographs under a fluorescence microscope.</li>
</ol>
6. TUNEL staining assay
<ol>
	<li>Use a TUNEL apoptosis assay kit (see Table of Materials) to determine apoptosis rates26. Discard the supernatant and wash the pellet once with serum-free medium.</li>
	<li>Add cell fixation solution and wash them with PBS once after incubating at room temperature for 30 min.</li>
	<li>Add 0.3% triton-100 to each well and incubate at room temperature for 5 min. Wash the cells with PBS twice. Add TUNEL detection working solution and incubate at 37 &#176;C in the dark for 60 min.</li>
	<li>Add an anti-fluorescence quenching sealing tablet containing 4&#8242;,6-diamidino-2-phenylindole (DAPI; see Table of Materials) and capture photographs under a fluorescence microscope.</li>
</ol>
7. Monitoring autophagic flow by transfection of mCherry GFP-LC3B adenovirus
<ol>
	<li>Replace half of the medium in each well with fresh medium. Add Ad-mCherry GFP-LC3B adenovirus (multiplicity of infection [MOI] of 2) to the culture medium and add 5 &#181;g/mL polybrene (see Table of Materials) to improve the infection efficiency.</li>
	<li>Replace the fresh medium after 24 h and observe the expression of the fluorescent protein under a confocal microscope.</li>
	<li>Culture the cells with the same conditions as section 1 after confirming successful virus infection and capture images under the confocal microscope27. 
	&#8203;NOTE: More than 20% of cells showed green fluorescent protein (GFP) fluorescence, indicating a successful infection.</li>
</ol>
8. Western blot analysis
<ol>
	<li>Collect cells for whole protein extraction and lyse them (RIPA, protease inhibitor, and phosphatase inhibitor = 100:1:1; see Table of Materials) on ice for 30 min.</li>
	<li>Add 5x protein loading buffer to the protein sample after protein quantification and boil the samples for 10 min.</li>
	<li>Electrophorese the samples that contain equal amounts of protein with sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels28. Then, transfer the samples onto polyvinylidene difluoride (PVDF) membranes.</li>
	<li>Block the PVDF membranes for 1 h with 5% skim milk powder and incubate with the primary antibody (PINK1, Parkin; see Table of Materials) at 4 &#176;C overnight and the secondary antibody at room temperature for 1 h.</li>
	<li>Detect the target bands using enhanced chemiluminescence (ECL) solution and the chemiluminescence detection system. Use Image J software to quantify the bands via densitometry28.</li>
</ol>
9. Detection of Parkin&#39;s mitochondrial translocation by immunofluorescence
<ol>
	<li>Observe the colocalization of Parkin with mitochondria by double immunofluorescence staining. Discard the cell supernatant from each group and wash them three times with PBS.</li>
	<li>Fix the cells for 10 min at room temperature with 4% paraformaldehyde and add 0.1% triton-100 in PBS.</li>
	<li>Block with animal-free block solution (see Table of Materials) for 1 h to prevent non-specific binding between proteins and antibodies and reduce the fluorescence background.</li>
	<li>Incubate with a primary antibody (Parkin and Tom20; see Table of Materials) at 4 &#176;C overnight.</li>
	<li>Add a fluorescent secondary antibody (see Table of Materials) and incubate in the dark for 1 h.</li>
	<li>Add the DAPI-containing anti-fluorescence quenching tablets and capture images under the confocal microscope.</li>
</ol>
10. Statistical analysis
<ol>
	<li>Perform statistical analysis using graphing and analysis software (see Table of Materials).</li>
	<li>Compare continuous variables between groups using one-way ANOVA. p &#60; 0.05 was considered statistically significant.</li>
</ol>

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M., Yanagawa, T., Iijima, M., Sesaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SQSTM1/p62+promotes+mitochondrial+ubiquitination+independently+of+PINK1+and+PRKN/parkin+in+mitophagy.">SQSTM1/p62 promotes mitochondrial ubiquitination independently of PINK1 and PRKN/parkin in mitophagy.</a> Autophagy. 15 (11), 2012-2018 (2019).</li><li>Klionsky, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+(4th+edition).">Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).</a> Autophagy. 17 (1), 1 (2021).</li></ol>

The authors have no conflicts of interest to declare.

The exploration of effective ingredients from complex compounds of natural drugs through advanced technology has been a hotspot of TCM research29, and can provide laboratory evidence for future drug development after verification. Safflower is a representative drug in the treatment of &#34;promoting blood circulation and minimizing blood stasis&#34; and is widely used in the treatment of myocardial infarction30,31. Saffron is believed to have similar effects to safflower, and its effect in promoting blood circulation and removing blood stasis is significantly better than safflower31,32. Crocetin is one of the main active components of saffron33, so it was used in the study of this experiment.
H2O2 can cause oxidative stress injury of cardiomyocytes and simulate the myocardial infarction state of cardiomyocytes, which is established as a model of myocardial infarction in vitro34. In this study, a low concentration of crocetin could promote the cell viability of cardiomyocytes, which may be closely related to the activation of mitochondrial energy metabolism. Crocetin can restore the decreased viability of cardiomyocytes induced by H2O2 and has a dose-dependent effect, suggesting that crocetin can improve the oxidative stress damage of cardiomyocytes. Meanwhile, crocetin can effectively reverse myocardial damage and oxidative stress indexes caused by H2O2, further confirming its myocardial protective effect.
The decline in mitochondrial membrane potential is one of the hallmark events in the early stages of apoptosis35. JC-1 dyes aggregate in a potential-dependent manner within the mitochondria. In the normal mitochondrial matrix, JC-1 forms a fluorescent polymer in red. When the mitochondrial membrane potential collapses, JC-1 emits green fluorescence as monomers36. TUNEL detects nuclear DNA strand breaks in the late stages of apoptosis37. Apoptotic cells activate DNA endonuclease enzymes that cut genomic DNA between nucleosomes, and exposed 3&#39;-OH can be detected with green fluorescent probe fluorescein (FITC)-labeled dUTP catalyzed by terminal deoxynucleotidyl transferases37. The results of this study show that the mitochondrial membrane potential decreased and the apoptosis of cardiomyocytes appeared after H2O2 modeling; the changes could be reversed by crocetin to a certain extent, suggesting that crocetin could effectively inhibit the apoptosis of cardiomyocytes caused by oxidative stress.
PINK1/Parkin is a classical pathway that mediates mitophagy38. PINK1 accumulates on the outer mitochondrial membrane after mitochondrial damage and Parkin is recruited to ubiquitinate the extramitochondrial membrane protein, which binds to autophagy-related receptor proteins to form autophagosomes, marking the occurrence of mitophagy39,40,41. The results show that the protein levels of PINK1 and Parkin were increased in cardiomyocytes after H2O2 modeling, suggesting excessive autophagy. After the intervention of crocetin, the protein levels of PINK1 and Parkin in cardiomyocytes treated with H2O2 were reversed to a certain extent, suggesting that it may play a therapeutic role by inhibiting excessive mitochondrial autophagy. In this study, crocetin reduced H2O2-induced oxidative damage and the apoptosis of H9c2 cardiomyocytes, and it is speculated that this effect might be achieved by affecting the PINK1/Parkin pathway to inhibit excessive mitophagy.
This experiment demonstrated the intervention of herbal lyophilized powder on cellular oxidative stress and mitophagy. Using mCherry-GFP-LC3B adenovirus to observe mitochondrial autophagy was a crucial step in the experiment. The key to the success of this step was to increase the infection rate of the cells, and the proviral infection reagent polybrene was added to the medium in advance to increase the infection efficiency greatly. This was achieved by neutralizing the electrostatic repulsion between the cell surface sialic acid and the viral particles, thus facilitating adsorption. It is also worth noting that, as a relatively safe virus, although the adenovirus genome does not integrate into the host cell genome after infection and does not replicate in the cell, it is still potentially biologically dangerous. Therefore, conducting the experiments under strict compliance with regulatory requirements is recommended.
Fluorescent labeling is a common method for detecting mitophagy, but due to its poor specificity, other organelles may be incorrectly labeled and thus interfere with the experimental results41. As technology advances continue, we can expect the development of more precise and reliable fluorescent probes to further explore the mechanism of mitophagy.

Acute myocardial infarction (AMI) is a life-threatening myocardial necrosis caused by severe and persistent ischemia and hypoxia to coronary arteries1,2. Percutaneous coronary intervention (PCI) is one of the first-line therapeutic strategies for AMI, and usually protects cardiomyocytes from ischemic damage3,4. The distal myocardium will lack blood and oxygen supply if not promptly and effectively treated after AMI, which leads to ischemic necrosis and further cardiovascular complications5,6. Promoting cardiomyocyte recovery and minimizing irreversible myocardial damage after missing the PCI surgical opportunity has been a research hotspot. After AMI, cardiomyocytes are in a state of ischemia and hypoxia, resulting in the inhibition of mitochondrial oxidative phosphorylation, reduction of NAD+ to NADPH, and increased single electron reduction7. As a result, the incomplete reduction reaction of oxygen generates an excess of reactive oxygen species (ROS) and ultimately leads to oxidative stress damage to cardiomyocytes8. An excessive accumulation of ROS triggers lipid peroxidation, further disrupting the structure and function of mitochondrial membranes. The result is a continuous opening of mitochondrial permeability transition pores and a decrease in mitochondrial membrane potential, inducing apoptosis and necrosis.
Angiotensin-converting enzyme (ACE) inhibitors, angiotensin-receptor blockers (ARBs), the inhibitors of &#946;-adrenoceptors, aldosterone antagonists, and other standard drugs in AMI can help enhance heart function after myocardial infarction and prevent the occurrence of malignant events, such as arrhythmias and left ventricular remodeling9. However, postinfarction survival and prognosis are greatly affected by infarct size, and satisfactory results have not been achieved for reducing cardiomyocyte apoptosis10,11. Thus, the development of drugs to promote cardiomyocyte recovery after myocardial infarction has become an urgent issue.
Traditional medicine has been a source of inspiration for modern pharmaceutical research for many years12,13,14,15. Traditional Chinese medicine (TCM) has a long history in the treatment of AMI, and a series of randomized control trials in recent years have confirmed that TCM can indeed improve the prognosis of patients16,17. According to TCM theory, AMI is caused by blood stasis18,19, so drugs for promoting blood circulation are usually used for the treatment of AMI in the acute phase20. Among them, saffron is believed to have a powerful effect on blood activation and stasis, and is often used in the acute treatment of AMI. Crocetin, a major component of saffron, may play a key role in protecting cardiomyocytes21.
In this study, H9c2 myocardial cells were induced by H2O2 to simulate myocardial ischemia/reperfusion, which causes a cardiomyocyte injury of AMI, and crocetin was used as an intervention to investigate its protective effect against oxidative stress-induced myocardial injury. The mechanism of crocetin protecting cardiomyocytes was further explored through mitophagy. More importantly, this article provides a reference for the technical approach to the study of mitophagy and describes the entire experimental procedure in detail.

<table><tbody><tr><td>0.25% trypsin</td><td>Gibco</td><td>2323363</td><td></td></tr><tr><td>1% Penicillin-streptomycin</td><td>Sigma</td><td>V900929</td><td></td></tr><tr><td>5x protein loading buffer</td><td>Beijing Pulilai Gene Technology</td><td>B1030-5</td><td></td></tr><tr><td>Ad-mCherry GFP-LC3B adenovirus</td><td>Beyotime</td><td>C3011</td><td></td></tr><tr><td>Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L)&amp;nbsp;</td><td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td><td>ZF-0514</td><td></td></tr><tr><td>Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L)</td><td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td><td>ZF-0513</td><td></td></tr><tr><td>Animal-free blocking solution</td><td>CST</td><td>15019s</td><td></td></tr><tr><td>Anti-Parkin antibody</td><td>Santa Cruz</td><td>sc-32282</td><td></td></tr><tr><td>Anti-PINK1 antibody</td><td>ABclonal</td><td>A11435</td><td></td></tr><tr><td>Anti-TOM20 antibody</td><td>ABclonal</td><td>A19403</td><td></td></tr><tr><td>Anti-&amp;beta;-actin&amp;nbsp; antibody</td><td>ABclonal</td><td>AC026</td><td></td></tr><tr><td>BCA protein assay kit</td><td>KeyGEN Biotech</td><td>KGP902</td><td></td></tr><tr><td>Blood cell counting plate</td><td>Servicebio</td><td>WG607</td><td></td></tr><tr><td>CAT assay kits</td><td>Nanjing Jiancheng Bioengineering Institute</td><td>A007-1-1</td><td></td></tr><tr><td>Chemiluminescence detection system</td><td>Shanghai Qinxiang Scientific Instrument Factory</td><td>ChemiScope 6100</td><td></td></tr><tr><td>CK assay kits</td><td>Nanjing Jiancheng Bioengineering Institute</td><td>A032-1-1</td><td></td></tr><tr><td>Coenzyme Q10 (CoQ 10)</td><td>Macklin</td><td>C6129</td><td></td></tr><tr><td>Crocetin</td><td>Chengdu Ruifensi Biotechnology Co., Ltd.</td><td>RFS-Z01802006012</td><td></td></tr><tr><td>DAPI-containing antifluorescence quenching tablets</td><td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td><td>ZLI-9557</td><td></td></tr><tr><td>DCFH-DA</td><td>Beyotime</td><td>S0033S</td><td></td></tr><tr><td>DMSO</td><td>Solarbio</td><td>D8371</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle medium (DMEM)</td><td>Gibco</td><td>8122091</td><td></td></tr><tr><td>Enhanced Chemiluminescence (ECL) solution</td><td>NCM Biotech</td><td>P10100</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Corning-Cellgro</td><td>35-081-CV</td><td></td></tr><tr><td>GraphPad Prism 7.0&amp;nbsp;</td><td>https://www.graphpad.com/</td><td></td><td></td></tr><tr><td>GSH-Px assay kits</td><td>Nanjing Jiancheng Bioengineering Institute</td><td>A005-1-2</td><td></td></tr><tr><td>H9c2 myocardial cells</td><td>Beijing Dingguochangsheng Biotech Co., Ltd.</td><td>CS0062</td><td></td></tr><tr><td>Horseradish peroxidase (HRP)-conjugated goat anti-goat IgG (H+L)&amp;nbsp;</td><td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td><td>ZB-2305</td><td></td></tr><tr><td>Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L)&amp;nbsp;</td><td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td><td>ZB-2301</td><td></td></tr><tr><td>JC-1 mitochondrial membrane potential assay kit</td><td>LABLEAD</td><td>J22202</td><td></td></tr><tr><td>LDH assay kits</td><td>Nanjing Jiancheng Bioengineering Institute</td><td>A020-2-2</td><td></td></tr><tr><td>MDA assay kits</td><td>Nanjing Jiancheng Bioengineering Institute</td><td>A003-2-2</td><td></td></tr><tr><td>Methanol</td><td>Aladdin</td><td>A2114057</td><td></td></tr><tr><td>MTS assay</td><td>Promega</td><td>G3581</td><td></td></tr><tr><td>Perhydrol</td><td>G-clone</td><td>CS7730</td><td></td></tr><tr><td>Phosphatase inhibitor</td><td>CWBIO</td><td>CW2383</td><td></td></tr><tr><td>Polybrene</td><td>Beyotime</td><td>C0351</td><td></td></tr><tr><td>Polyvinylidene difluoride (PVDF) membranes</td><td>Millipore</td><td>ISEQ00010</td><td></td></tr><tr><td>Radioimmunoprecipitation assay (RIPA) lysis buffer</td><td>Solarbio</td><td>R0010</td><td></td></tr><tr><td>SDS-PAGE gels</td><td>Shanghai Epizyme Biomedical Technology</td><td>PG112</td><td></td></tr><tr><td>SDS-PAGE running buffer powder</td><td>Servicebio</td><td>G2018-1L</td><td></td></tr><tr><td>SDS-PAGE transfer buffer powder</td><td>Servicebio</td><td>G2017-1L</td><td></td></tr><tr><td>SOD assay kits</td><td>Nanjing Jiancheng Bioengineering Institute</td><td>A001-2-2</td><td></td></tr><tr><td>Tris-buffered saline powder</td><td>Servicebio</td><td>G0001-2L</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>SLCC9172</td><td></td></tr><tr><td>TUNEL apoptosis assay kit</td><td>Beyotime</td><td>C1086</td><td></td></tr><tr><td>Tween-20</td><td>Solarbio</td><td>T8220</td><td></td></tr></tbody></table>

protection of h9c2 myocardial cells from oxidative stress by crocetin via pink1/parkin pathway-mediated mitophagy

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 &#181;M) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.

Effects of crocetin on cell viability 
Crocetin at 0.1 &#181;M, 0.5 &#181;M, 1 &#181;M, 5 &#181;M, 10 &#181;M, 50 &#181;M, and 100 &#181;M had a significant proliferative effect on cells, while crocetin at concentrations above 200 &#181;M significantly inhibited the proliferation of H9c2 cells (Figure 1A). After 4 h of treatment with 400 &#181;M H2O2, the cell viability was reduced considerably, and crocetin could reverse this change to a certain extent (Figure 1B). Since no significant difference between 10 &#181;M and 100 &#181;M crocetin was observed on the H2O2-induced H9c2 cell viability, 10 &#181;M crocetin was chosen as the high concentration, and 1 &#181;M and 0.1 &#181;M were used as the medium and low dose groups, respectively.
Effects of crocetin on LDH, CK, MDA, SOD, GSH-Px, and CAT in H9c2 cells 
After 4 h of treatment with 400 &#181;M H2O2, the levels of LDH, CK, and MDA increased appreciably, while the levels of SOD, GSH-Px, and CAT decreased. Pretreatment of 10 &#181;M crocetin for 24 h can reverse the above changes and shows an obvious dose-dependent effect. As a positive control drug, coenzyme Q10 can only change the levels of CK, MDA, and SOD (Figure 2).
The effect of crocetin on ROS in H9c2 cardiomyocytes 
As a blank control, H9c2 cardiomyocytes expressed almost no ROS. At the same time, 400 &#181;M H2O2 for 4 h treatment could enhance the ROS level notably, which can be reversed by 10 &#181;M crocetin to some extent (Figure 3). The fluorescence results showed that green fluorescence was very weak in the normal group. In comparison, 400 &#181;M H2O2 for 4 h treatment could enhance the green fluorescence signal, and this enhancement could be reduced by 10 &#181;M crocetin (Figure 3).
Effects of crocetin on H2O2-induced mitochondrial membrane potential and apoptosis 
JC-1 staining showed more red fluorescence and less green fluorescence in the blank control group. After 4 h of treatment of 400 &#181;M H2O2, more green fluorescence and less red fluorescence were observed, and 10 &#181;M crocetin could reverse this change to some extent (Figure 4A). TUNEL staining results showed that apoptosis-related signaling was not detected in the blank control group, while the apoptosis-related signaling was appreciably enhanced after 400 &#181;M H2O2 for 4 h of treatment, which could be reversed by 10 &#181;M crocetin to some extent (Figure 4B).
Effects of crocetin on H2O2-induced excessive autophagy 
H9c2 cardiomyocytes in the blank control group showed no obvious autophagy flow. Fluorescence showed the appearance of punctate yellow spots in H9c2 cardiomyocytes pretreated with 400 &#181;M H2O2 for 4 h, indicating an obvious over-activation of autophagy. However, this change was reversed after the 10 &#181;M crocetin pretreatment. In the control group, the Ad-mCherry GFP-LC3B virus could only be observed as a weak diffuse yellow background by fluorescence. However, punctate yellow spots were observed after 400 &#181;M H2O2 for 4 h of treatment, and this change was reversed after the 10 &#181;M crocetin pretreatment (Figure 5).
Detection of crocetin on the expression of mitophagy-related proteins 
Western blot results showed that in the control group, the expression levels of PINK1 and Parkin were lower. In 4 h H2O2-stimulated H9c2 cardiomyocytes, the expression levels of PINK1 and Parkin increased, while the 10 &#181;M crocetin pretreatment could reduce the increase of PINK1 and Parkin&#160;(Figure 6).
Detection of the effect of crocetin on the translocation of Parkin mitochondria 
The immunofluorescence results showed that the red fluorescence signal representing Parkin in the blank control group was very weak; however, after 400 &#181;M H2O2 for 4 h of treatment, the red fluorescence signal was enhanced, and colocalization with the green fluorescence representing Tom20 increased. After the pretreatment of 10 &#181;M crocetin, the red fluorescence signal was weakened, and colocalization with the green fluorescence signal was reduced (Figure 7).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65105/65105fig01.jpg" /> 
Figure 1: Detection of cell viability by the MTT assay. (A) Effects of crocetin at different concentrations on cell viability (n = 6). (B) Effects of crocetin at different concentrations on cell viability after H202 intervention (n = 6). *p &#60; 0.05 versus control group, #p &#60; 0.05 versus H2O2 treatment group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65105/65105fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65105/65105fig02.jpg" /> 
Figure 2: Detection of LDH, CK, MDA, SOD, GSH-Px, and CAT levels. (A) LDH level in cell supernatant (n = 6). (B) CK level in cell lysate (n = 6). (C) MDA level in cell lysate (n = 6). (D) SOD level in cell supernatant (n = 6). (E) GSH-Px level in cell lysate (n = 6). (F) CAT level in cell lysate (n = 6). *p &#60; 0.05 versus blank control group, #p &#60; 0.05 versus H2O2 treatment group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65105/65105fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65105/65105fig03.jpg" /> 
Figure 3: ROS determined by DCFH-DA. (A)&#160;DCFH-DA was used to measure ROS levels in H9c2 cardiomyocytes. ROS: green. (B) Quantification data of ROS. (a) H9c2 cardiomyocytes without treatment. (b) H9c2 cardiomyocytes stimulated with 400 &#181;M H2O2 for 4 h. (c) H9c2 cells pretreated with 10 &#181;M crocetin for 24 h and then stimulated with 400 &#181;M H2O2 for 4 h. Scale bar = 24 &#181;m. *p &#60; 0.05 versus blank control group, #p &#60; 0.05 versus H2O2 treatment group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65105/65105fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65105/65105fig04.jpg" /> 
Figure 4: Detection of mitochondrial membrane potential and apoptosis. (A) Mitochondrial membrane potential was determined by JC-1 staining. JC-1 aggregates: red; JC-1 monomers: green. (B) Apoptosis was detected by TUNEL staining in H9c2 cells. TUNEL: green; DAPI: blue. (a) H9c2 cardiomyocytes without treatment. (b) H9c2 cardiomyocytes stimulated with 400 &#181;M H2O2 for 4 h. (c) H9c2 cells pretreated with 10 &#181;M crocetin for 24 h and then stimulated with 400 &#181;M H2O2 for 4 h. Scale bars = 72 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65105/65105fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65105/65105fig05.jpg" /> 
Figure 5: Autophagic flux detected by mCherry-GFP-LC3B adenovirus. mCherry: red; GFP: green. (a) H9c2 cardiomyocytes without treatment. (b) H9c2 cardiomyocytes stimulated with 400 &#181;M H2O2 for 4 h. (c) H9c2 cells pretreated with 10 &#181;M crocetin for 24 h and then stimulated with 400 &#181;M H2O2 for 4 h. Scale bars = 50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65105/65105fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65105/65105fig06.jpg" /> 
Figure 6: The content of mitophagy-related proteins was detected by western blotting. (A) Representative western blot illustrating PINK1 and Parkin expression. &#946;-actin was adopted as an internal reference. (B) Relative PINK1 expression (n = 3). (C) Relative Parkin expression (n = 3). *p &#60; 0.05 versus control group, #p &#60; 0.05 versus H2O2 treatment group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65105/65105fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/65105/65105fig07.jpg" /> 
Figure 7: Detection of Parkin&#39;s mitochondrial translocation by immunofluorescence double staining. Red fluorescence-labeled Parkin&#39;s protein and green fluorescence labeled-Tom20 protein. Parkin: red; Tom20: green; DAPI: blue). (a) H9c2 cardiomyocytes without treatment. (b) H9c2 cardiomyocytes stimulated with 400 &#181;M H2O2 for 4 h. (c) H9c2 cells pretreated with 10 &#181;M crocetin for 24 h and then stimulated with 400 &#181;M H2O2 for 4 h. Scale bars = 24 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65105/65105fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: The working instructions of LDH, CK, MDA, SOD, GSH-Px, and CAT assays. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65105/65105_Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy at JoVE.com

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Based on in vitro experiments, this study revealed the mechanism of crocetin in repairing oxidative stress damage of cardiomyocytes by influencing mitophagy, in which the PINK1/Parkin signaling pathway plays an important role.

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and ...

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Nanyang Technological University

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1.0K Views.  Beijing University of Chinese Medicine. Dit protocol biedt een referentie voor de technische benadering van de studie van autofaag en het beschrijft de volledige experimentele procedures in detail. Het belangrijkste voordeel van deze techniek is dat het de lengtewaarneming van de verandering in autofagische flux mogelijk maakt. Voordat u met het experiment begint, bepaalt u de effectieve infectieconcentratie en neemt u de nodige veiligheidsmaatregelen.Begin met het centrifugeren van geneutraliseerde H9C2 myocardiale cellen b...

Video: Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Enhancing the Engraftment of Human Induced Pluripotent Stem Cell-derived Cardiomyocytes via a Transient Inhibition of Rho Kinase Activity

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. Y-27632 is a highly potent inhibitor of Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) and is used to prevent dissociation-induced cell apoptosis (anoikis). We demonstrate that Y-27632 pretreatment for human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs+RI) prior to implantation results in a cell engraftment rate improvement in a mouse model of acute myocardial infarction (MI). Here, we describe a complete procedure of hiPSC-CMs differentiation, purification, and cell pretreatment with Y-27632, as well as the resulting cell contraction, calcium transient measurements, and transplantation into mouse MI models. The proposed method provides a simple, safe, effective, and low-cost method which significantly increases the cell engraftment rate. This method cannot only be used in conjunction with other methods to further enhance the cell transplantation efficiency but also provides a favorable basis for the study of the mechanisms of other cardiac diseases.

In this protocol, we demonstrate and elaborate on how to use human induced pluripotent stem cells for cardiomyocyte differentiation and purification, and further, on how to improve its transplantation efficiency with Rho-associated protein kinase inhibitor pretreatment in a mouse myocardial infarction model.

A crucial factor in improving cellular therapy effectiveness for myocardial regeneration is to safely and efficiently increase the cell engraftment rate. ...

Biochemistry

Analyzing Mitochondrial Physiology and Bioenergetics in Fruit Flies

Analyzing Mitochondrial Function in a Drosophila melanogaster PINK1B9-Null Mutant Using High-resolution Respirometry

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy metabolism with impairment of mitochondrial functions. Mitochondria are organelles that control both energy metabolism and cellular processes involved in cell survival and death. For this reason, approaches to evaluate mitochondrial function can offer important insights into cellular conditions in pathological and physiological processes. In this regard, high-resolution respirometry (HRR) protocols allow evaluation of the whole mitochondrial respiratory chain function or the activity of specific mitochondrial complexes. Furthermore, studying mitochondrial physiology and bioenergetics requires genetically and experimentally tractable models such as Drosophila melanogaster.
This model presents several advantages, such as its similarity to human physiology, its rapid life cycle, easy maintenance, cost-effectiveness, high throughput capabilities, and a minimized number of ethical concerns. These attributes collectively establish it as an invaluable tool for dissecting complex cellular processes. The present work explains how to analyze mitochondrial function using the Drosophila melanogaster PINK1B9-null mutant. The pink1 gene is responsible for encoding PTEN-induced putative kinase 1, through a process recognized as mitophagy, which is crucial for the removal of dysfunctional mitochondria from the mitochondrial network. Mutations in this gene have been associated with an autosomal recessive early-onset familial form of PD. This model can be used to study mitochondrial dysfunction involved in the pathophysiology of PD.

Author Spotlight: Exploring Mitochondrial Function and Chemical Toxicity Using Drosophila melanogaster 

Here we present a high-resolution respirometry protocol to analyze bioenergetics in PINK1B9-null mutant fruit flies. The method uses the Substrate-Uncoupler-Inhibitor-Titration (SUIT) protocol.

Neurodegenerative diseases, including Parkinson&#39;s Disease (PD), and cellular disturbances such as cancer are some of the disorders that disrupt energy ...

A Flow Cytometry-based Assay for Measuring Mitochondrial Membrane Potential in Cardiac Myocytes After Hypoxia/Reoxygenation

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment elevated myocardial infarction. However, reperfusion per se can result in further cardiomyocyte death, a phenomenon known as reperfusion injury. The opening of the mitochondrial permeability transition pore (mPTP), with the decrease of the mitochondrial membrane potential (MMP), or mitochondrial depolarization, is universally recognized as the final step of reperfusion injury and is responsible for mitochondrial and cardiomyocyte death. JC-1 is a lipophilic cationic dye that accumulates in mitochondria depending on the value of MMP. The higher the MMP is, the more JC-1 accumulates in the mitochondria. The increasing amounts of JC-1 in mitochondria can be reflected by a fluorescence emission shift from green (~530 nm) to red (~590 nm). Therefore, the reduction of the red/green fluorescence intensity ratio can indicate the depolarization of mitochondria. Here, we take advantage of JC-1 to measure MMP, or the opening of mPTP in human cardiac myocytes after hypoxia/reoxygenation, detected by flow cytometry.

Here, we present a protocol that uses JC-1 dye to assess the mitochondrial membrane potential of cells after being exposed to hypoxia/reoxygenation with or without a protective agent.

Timely and efficient reperfusion of the occluded coronary artery is the best strategy for decreasing myocardial infarct size in patients with a ST-segment ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...

Time-Lapse Video Microscopy for Assessment of EYFP-Parkin Aggregation as a Marker for Cellular Mitophagy

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time points. Time intervals can be set through a computer interface that controls the microscope-integrated camera. This kind of microscopy requires both the ability to acquire very rapid events and the signal generated by the observed cellular structure during these events. After the images have been collected, a movie of the entire experiment is assembled to show the dynamic of the molecular events of interest. Time-lapse video microscopy has a broad range of applications in the biomedical research field and is a powerful and unique tool for following the dynamics of the cellular events in real time. Through this technique, we can assess cellular events such as migration, division, signal transduction, growth, and death. Moreover, using fluorescent molecular probes we are able to mark specific molecules, such as DNA, RNA or proteins and follow them through their molecular pathways and functions. Time-lapse video microscopy has multiple advantages, the major one being the ability to collect data at the single-cell level, that make it a unique technology for investigation in the field of cell biology. However, time-lapse video microscopy has limitations that can interfere with the acquisition of high quality images. Images can be compromised by both external factors; temperature fluctuations, vibrations, humidity and internal factors; pH, cell motility. Herein, we describe a protocol for the dynamic acquisition of a specific protein, Parkin, fused with the enhanced yellow fluorescent protein (EYFP) in order to track the selective removal of damaged mitochondria, using a time-lapse video microscopy approach.

Herein, we describe in detail a time-lapse video microscopy approach to measuring the temporal recruitment of EYFP-Parkin during the selective removal of damaged mitochondria. This dynamic process of EYFP-Parkin-dependent removal of damaged mitochondria can be used as an indicator of cellular health under different experimental conditions.

Time-lapse video microscopy can be defined as the real time imaging of living cells. This technique relies on the collection of images at different time ...

Biology

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between ...

Herbal Munziq Ameliorates Myocardial Ischemia-Reperfusion Injury by Inhibiting Inflammation

The objective of this study was to investigate the cardioprotective effects of Munziq on abnormal body fluid myocardial ischemia-reperfusion injury (MIRI) and its underlying mechanism.Normal rats and rats with abnormal body fluid (ABF) were pre-treated with Munziq for 21 days. Following this, MIRI models were established. Histopathological changes and myocardial ultrastructure changes were observed by Hematoxylin and Eosin (HE)staining and transmission electron microscopy to observe pathological manifestations of myocardial injury. Serum CK-MB, cTn-T, and ICAM-1 levels were detected by Enzyme-Linked Immunosorbent Assay (ELISA) to observe myocardial injury-related markers. The levels of IL-1&#946;, IL-6, and TNF-&#945; in serum and myocardial tissue were also detected by ELISA to observe the anti-inflammatory effect. The expression levels of NF-&#954;B signaling pathway-related proteins NIK, IKK&#945;, Pik&#945;, and p65 were detected by Western blot analysis. The results showed that myocardial injury in the ABF MIRI group was more severe compared to the control MIRI group. Munziq pretreatment has the potential to mitigate the pathological changes induced by ischemia-reperfusion injury and could protect cardiac function. Protein levels of the NF-&#954;B pathway and downstream effectors IL-1&#946;, IL-6, and TNF-&#945; were significantly up-regulated in the MIRI group while down-regulated in the Munziq group. Interestingly, there was more activation of the NF-&#954;B signaling pathway and higher levels of downstream inflammatory cytokines in the ABF MIRI group. The results suggest that MIRI was more severe in ABF. Munziq has cardioprotective effects in ischemia and reperfusion injury. This protective effect may be acted by suppressing the NF-&#954;B signaling pathway.

This study investigates the cardioprotective effects of Munziq, a traditional Uyghur herbal preparation, on myocardial ischemia-reperfusion injury (MIRI) in rats with abnormal body fluid. Through comprehensive experimental methods, we demonstrate Munziq's potential to mitigate MIRI by suppressing the NF-&#954;B signaling pathway.

The objective of this study was to investigate the cardioprotective effects of Munziq on abnormal body fluid myocardial ischemia-reperfusion injury (MIRI) ...

Quantification of Mitochondrial Membrane Potential and Superoxide Levels

Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, various mitochondrial quality control mechanisms cooperate to maintain a healthy mitochondrial network. One such pathway is mitophagy, where PTEN-induced kinase 1 (PINK1) and Parkin phospho-ubiquitination of damaged mitochondria facilitate autophagosome sequestration and subsequent removal from the cell via lysosome fusion. Mitophagy is important for cellular homeostasis, and mutations in Parkin are linked to Parkinson's disease (PD). Due to these findings, there has been a significant emphasis on investigating mitochondrial damage and turnover to understand the molecular mechanisms and dynamics of mitochondrial quality control. Here, live-cell imaging was used to visualize the mitochondrial network of HeLa cells, to quantify the mitochondrial membrane potential and superoxide levels following treatment with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a mitochondrial uncoupling agent. In addition, a PD-linked mutation of Parkin (ParkinT240R) that inhibits Parkin-dependent mitophagy was expressed to determine how mutant expression impacts the mitochondrial network compared to cells expressing wild-type Parkin. The protocol outlined here describes a simple workflow using fluorescence-based approaches to quantify mitochondrial membrane potential and superoxide levels effectively.

Author Spotlight: Fluorescence-Based Quantification of Mitochondrial Membrane Potential and Superoxide Levels Using Live Imaging in HeLa Cells  

This technique describes an effective workflow to visualize and quantitatively measure mitochondrial membrane potential and superoxide levels within HeLa cells using fluorescence-based live imaging.

Mitochondria are dynamic organelles critical for metabolic homeostasis by controlling energy production via ATP synthesis. To support cellular metabolism, ...

Therapeutic Effects of Huazhuojiedu Decoction on Precancerous Lesions of Gastric Cancer by Regulating Mitophagy

This research aims to explore the therapeutic effect and potential mechanisms of Huazhuojiedu decoction (HZJD) for alleviating precancerous lesions of gastric cancer (PLGC) both in vivo and in vitro. HZJD is a traditional Chinese herbal formula consisting of 11 herbs. Sprague-Dawley (SD) rats were randomly divided into four subgroups: control group, model group, positive drug group, and HZJD group. Hematoxylin-eosin (H&amp;E) staining, high iron diamine-alcian blue (HID-AB) staining, alcian blue-periodic acid Schiff (AB-PAS) staining, immunohistochemistry, immunofluorescence, RT-qPCR, and Western blot assays were performed after 10 weeks of HZJD treatment. In vitro, the cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assays were used to detect cell proliferation. RT-qPCR and Western blot assays were performed to evaluate mitophagy levels. The results indicated that HZJD could retard the pathological progression in PLGC rats and reduce PLGC cell proliferation. Treatment with HZJD significantly increased the mRNA and protein expression levels of Sirt3, Foxo3a, Parkin, and LC3 II/I, while decreasing the mRNA and protein expression levels of p62 and Tomm20. HZJD was found to have the ability to reverse the decline in mitophagy activity both in vivo and in vitro. In conclusion, the study assessed the impact of HZJD and provided evidence regarding its potential molecular mechanism.

Here, we introduce a protocol that demonstrates the therapeutic efficacy of Huazhuojiedu decoction (HZJD) in alleviating precancerous lesions of gastric cancer using mitophagy regulation.

This research aims to explore the therapeutic effect and potential mechanisms of Huazhuojiedu decoction (HZJD) for alleviating precancerous lesions of ...

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

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