A robust method for assessing mitochondrial function in healthy and diseased frozen cardiac tissue
Abstract Cardiovascular diseases are often associated with impairment in mitochondrial function. However, existing respirometry measuring mitochondrial function are limited by the necessity of fresh tissue samples. This study develops a method with tailored substrate-inhibitor titration (TSIT) of mi...
Saved in:
| Main Authors: | , , , , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Nature Portfolio
2025-08-01
|
| Series: | Communications Biology |
| Online Access: | https://doi.org/10.1038/s42003-025-08608-5 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849226027456266240 |
|---|---|
| author | Liangyu Hu Alexia van Rinsum Rocco Caliandro Xi Qi Shuxiu Fan Xinyi Jiang Jur Massop Melissa Bekkenkamp-Grovenstein Christiane Ott Tilman Grune Melissa A. E. van de Wal Werner J. H. Koopman Marcel Giesbers Monika Gladka Jaap Keijer Deli Zhang |
| author_facet | Liangyu Hu Alexia van Rinsum Rocco Caliandro Xi Qi Shuxiu Fan Xinyi Jiang Jur Massop Melissa Bekkenkamp-Grovenstein Christiane Ott Tilman Grune Melissa A. E. van de Wal Werner J. H. Koopman Marcel Giesbers Monika Gladka Jaap Keijer Deli Zhang |
| author_sort | Liangyu Hu |
| collection | DOAJ |
| description | Abstract Cardiovascular diseases are often associated with impairment in mitochondrial function. However, existing respirometry measuring mitochondrial function are limited by the necessity of fresh tissue samples. This study develops a method with tailored substrate-inhibitor titration (TSIT) of mitochondrial electron transport complexes (ETC) to measure mitochondrial function in frozen cardiac samples using high-resolution respirometry. Briefly, acetyl-CoA is added to fuel the tricarboxylic acid (TCA) cycle for NADH production, enabling complex I (CI)-linked respiratory assessment. NADH is then added to measure maximum CI-linked respiratory capacity, followed by rotenone and succinate to assess complex II (CII)-linked respiratory capacity. TSIT detects mitochondrial functional differences between frozen atrial and ventricular tissue, with comparable results as measured in fresh samples. It also detects cardiac mitochondrial dysfunction across various (patho)physiological mouse models and in human frozen cardiac samples, highlighting its clinical potential. Furthermore, we provides the first evidence for SC formation between the ETC-SCs and the TCA cycle metabolon using blue native electrophoresis, underpinning why TSIT is feasible in frozen tissue. In conclusion, we establish a novel, robust, sensitive and translational method (TSIT) for assessing mitochondrial (dys)function in frozen cardiac samples from various species, enabling flexible analysis of mitochondrial function in both laboratory and clinical settings. |
| format | Article |
| id | doaj-art-e8505cfcfc5f43a0b17aec063d88cc5b |
| institution | Kabale University |
| issn | 2399-3642 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Communications Biology |
| spelling | doaj-art-e8505cfcfc5f43a0b17aec063d88cc5b2025-08-24T11:46:19ZengNature PortfolioCommunications Biology2399-36422025-08-018111410.1038/s42003-025-08608-5A robust method for assessing mitochondrial function in healthy and diseased frozen cardiac tissueLiangyu Hu0Alexia van Rinsum1Rocco Caliandro2Xi Qi3Shuxiu Fan4Xinyi Jiang5Jur Massop6Melissa Bekkenkamp-Grovenstein7Christiane Ott8Tilman Grune9Melissa A. E. van de Wal10Werner J. H. Koopman11Marcel Giesbers12Monika Gladka13Jaap Keijer14Deli Zhang15Human and Animal Physiology, Wageningen University & ResearchHuman and Animal Physiology, Wageningen University & ResearchDepartment of Medical Biology, Amsterdam Cardiovascular Sciences, Amsterdam University Medical CenterHuman and Animal Physiology, Wageningen University & ResearchHuman and Animal Physiology, Wageningen University & ResearchHuman and Animal Physiology, Wageningen University & ResearchHuman and Animal Physiology, Wageningen University & ResearchHuman and Animal Physiology, Wageningen University & ResearchDepartment of Molecular Toxicology, German Institute of Human NutritionDepartment of Molecular Toxicology, German Institute of Human NutritionDepartment of Pediatrics, Amalia Children’s Hospital, Radboud Center for Mitochondrial Medicine (RCMM), Radboud University Medical Center (Radboudumc)Human and Animal Physiology, Wageningen University & ResearchWageningen Electron Microscopy Centre, Wageningen University & ResearchDepartment of Medical Biology, Amsterdam Cardiovascular Sciences, Amsterdam University Medical CenterHuman and Animal Physiology, Wageningen University & ResearchHuman and Animal Physiology, Wageningen University & ResearchAbstract Cardiovascular diseases are often associated with impairment in mitochondrial function. However, existing respirometry measuring mitochondrial function are limited by the necessity of fresh tissue samples. This study develops a method with tailored substrate-inhibitor titration (TSIT) of mitochondrial electron transport complexes (ETC) to measure mitochondrial function in frozen cardiac samples using high-resolution respirometry. Briefly, acetyl-CoA is added to fuel the tricarboxylic acid (TCA) cycle for NADH production, enabling complex I (CI)-linked respiratory assessment. NADH is then added to measure maximum CI-linked respiratory capacity, followed by rotenone and succinate to assess complex II (CII)-linked respiratory capacity. TSIT detects mitochondrial functional differences between frozen atrial and ventricular tissue, with comparable results as measured in fresh samples. It also detects cardiac mitochondrial dysfunction across various (patho)physiological mouse models and in human frozen cardiac samples, highlighting its clinical potential. Furthermore, we provides the first evidence for SC formation between the ETC-SCs and the TCA cycle metabolon using blue native electrophoresis, underpinning why TSIT is feasible in frozen tissue. In conclusion, we establish a novel, robust, sensitive and translational method (TSIT) for assessing mitochondrial (dys)function in frozen cardiac samples from various species, enabling flexible analysis of mitochondrial function in both laboratory and clinical settings.https://doi.org/10.1038/s42003-025-08608-5 |
| spellingShingle | Liangyu Hu Alexia van Rinsum Rocco Caliandro Xi Qi Shuxiu Fan Xinyi Jiang Jur Massop Melissa Bekkenkamp-Grovenstein Christiane Ott Tilman Grune Melissa A. E. van de Wal Werner J. H. Koopman Marcel Giesbers Monika Gladka Jaap Keijer Deli Zhang A robust method for assessing mitochondrial function in healthy and diseased frozen cardiac tissue Communications Biology |
| title | A robust method for assessing mitochondrial function in healthy and diseased frozen cardiac tissue |
| title_full | A robust method for assessing mitochondrial function in healthy and diseased frozen cardiac tissue |
| title_fullStr | A robust method for assessing mitochondrial function in healthy and diseased frozen cardiac tissue |
| title_full_unstemmed | A robust method for assessing mitochondrial function in healthy and diseased frozen cardiac tissue |
| title_short | A robust method for assessing mitochondrial function in healthy and diseased frozen cardiac tissue |
| title_sort | robust method for assessing mitochondrial function in healthy and diseased frozen cardiac tissue |
| url | https://doi.org/10.1038/s42003-025-08608-5 |
| work_keys_str_mv | AT liangyuhu arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT alexiavanrinsum arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT roccocaliandro arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT xiqi arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT shuxiufan arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT xinyijiang arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT jurmassop arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT melissabekkenkampgrovenstein arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT christianeott arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT tilmangrune arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT melissaaevandewal arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT wernerjhkoopman arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT marcelgiesbers arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT monikagladka arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT jaapkeijer arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT delizhang arobustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT liangyuhu robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT alexiavanrinsum robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT roccocaliandro robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT xiqi robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT shuxiufan robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT xinyijiang robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT jurmassop robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT melissabekkenkampgrovenstein robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT christianeott robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT tilmangrune robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT melissaaevandewal robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT wernerjhkoopman robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT marcelgiesbers robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT monikagladka robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT jaapkeijer robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue AT delizhang robustmethodforassessingmitochondrialfunctioninhealthyanddiseasedfrozencardiactissue |