Human heart tissue mitochondria isolation: experience from patients with tetralogy of Fallot

Jefferson Kasan Hidayat; Aulia Widyaningrum; Nadila Amanda Zuliani; Franciscus Dhyanagiri Suyatna; Ratna Farida Soenarto; Muljadi Muhammad Djer

doi:10.15562/bmj.v12i2.4599

Human heart tissue mitochondria isolation: experience from patients with tetralogy of Fallot

Jefferson Kasan Hidayat ,

Aulia Widyaningrum ,

Nadila Amanda Zuliani ,

Franciscus Dhyanagiri Suyatna ,

Ratna Farida Soenarto ,

Muljadi Muhammad Djer ,

pdf |
DOI: https://doi.org/10.15562/bmj.v12i2.4599 |
Published: 2023-07-22

Abstract

Background: Mitochondria have crucial roles in human cardiomyocyte metabolism. The isolation of mitochondria from various cells and tissues has been described in previous studies. However, the isolation of human heart muscle is rarely performed because of the ethical problems inherent in obtaining fresh human heart muscle samples. Resected infundibular hypertrophy muscle in tetralogy of Fallot surgery provides a fresh sample source without causing ethical issues. This study aimed to isolate mitochondria from patients with tetralogy of Fallot.

Methods: The optimization of mitochondria isolation protocol in this in vitro study was performed to facilitate our study of ischemia and reperfusion injury. The samples of human heart tissue used in this study were resected infundibulum muscle collected during surgery to correct tetralogy of Fallot. Subjects were recruited from Cipto Mangunkusumo Hospital and Jakarta Heart Center Hospital between July 2021 and November 2022. Tissue was collected before aortic cross-clamp applied, after aortic cross-clamping applied and after aortic cross-clamping off. After collected tissue was trimmed and cleaned, mechanical disruption followed by enzymatic disruption using trypsin was performed. Homogenization was then performed with a sonicator. Mitochondria pellet was further obtained by differential fractionation of homogenate.

Results: Mean infundibular tissue weight obtained from the three periods mentioned was 64.92-109.7 mg. Our protein yield was 7.65-8.38 mg/ml. Mean JC-1 was 9945-12430 FLU/mgP. The median value of SDH activity shown by our mitochondria pellet was 17.45-18.12 nmol/min/L).

Conclusion: The fractionation method allowed us to successfully isolate mitochondria from human heart tissue with intact membrane integrity.

References

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Kurz FT, Aon MA, O’Rourke B, Armoundas AA. Functional Implications of Cardiac Mitochondria Clustering. In: Santulli G, editor. Mitochondrial dynamics in cardiovascular medicine. Switzerland: Springer; 2017. p. 1–24.
Frezza C, Cipolat S, Scorrano L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts. Nat Protoc. 2007;2(2):287–95.
Hogeboom GH, Schneider WC, Pallade GE. Cytochemical studies of mammalian tissues. Journal of Biological Chemistry. 1948;172(2):619–35.
Bossy-Wetzel E, Barsoum MJ, Godzik A, Schwarzenbacher R, Lipton SA. Mitochondrial fission in apoptosis, neurodegeneration and aging. Curr Opin Cell Biol. 2003;15(6):706–16.
Li Z, Okamoto K-I, Hayashi Y, Sheng M. The Importance of Dendritic Mitochondria in the Morphogenesis and Plasticity of Spines and Synapses. Cell. 2004;119(6):873–87.
JOHN JC ST., JOKHI RP, BARRATT CLR. The impact of mitochondrial genetics on male infertility. Int J Androl. 2005;28(2):65–73.
Kuznetsov A V., Javadov S, Margreiter R, Grimm M, Hagenbuchner J, Ausserlechner MJ. The Role of Mitochondria in the Mechanisms of Cardiac Ischemia-Reperfusion Injury. Antioxidants. 2019;8(10):454.
Sigma Aldrich. Mitochondria Isolation Kit [Package insert] [Internet]. Available from: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/200/375/mitoiso2bul-mk.pdf
Graham JM. Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation. Curr Protoc Cell Biol. 1999;4(1).
Bahnemann J, Kayo S, Wahrheit J, Heinzle E, Pörtner R, Zeng A-P. In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cells. Eng Life Sci. 2014;14(2):161–9.
Ohba M, Schatz G. Disruption of the outer membrane restores protein import to trypsin-treated yeast mitochondria. EMBO J. 1987;6(7):2117–22.
Pallotti F, Lenaz G. Isolation and Subfractionation of Mitochondria from Animal Cells and Tissue Culture Lines. In: Methods in Cell Biology. Elsevier; 2007. p. 3–44.
Tyler DD, Gonze J. The preparation of heart mitochondria from laboratory animals. In: Oxidation and Phosphorylation. Academic Press; 1967. p. 75–7. Available from: https://www.sciencedirect.com/science/article/pii/0076687967100141
Mela L, Seitz S. Isolation of mitochondria with emphasis on heart mitochondria from small amounts of tissue. In: Methods in Enzymology. Elsevier; 1979. p. 39–46.
Shinde SB, Save VC, Patil ND, Mishra KP, Tendolkar AG. Impairment of mitochondrial respiratory chain enzyme activities in tetralogy of fallot. Clinica Chimica Acta. 2007;377(1–2):138–43.
Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine [Internet]. Oxford University Press; 2015. Available from: https://doi.org/10.1093/acprof:oso/9780198717478.001.0001

[1] Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K, et al. Energy Conversion: Mitochondria and Chloroplasts. In: Wilson J, Hunt T, editors. Molecular Biology of the Cell. 6th Ed. New York: W.W. Norton & Company; 2015. p. 753–812.

[2] Kurz FT, Aon MA, O’Rourke B, Armoundas AA. Functional Implications of Cardiac Mitochondria Clustering. In: Santulli G, editor. Mitochondrial dynamics in cardiovascular medicine. Switzerland: Springer; 2017. p. 1–24.

[3] Frezza C, Cipolat S, Scorrano L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts. Nat Protoc. 2007;2(2):287–95.

[4] Hogeboom GH, Schneider WC, Pallade GE. Cytochemical studies of mammalian tissues. Journal of Biological Chemistry. 1948;172(2):619–35.

[5] Bossy-Wetzel E, Barsoum MJ, Godzik A, Schwarzenbacher R, Lipton SA. Mitochondrial fission in apoptosis, neurodegeneration and aging. Curr Opin Cell Biol. 2003;15(6):706–16.

[6] Li Z, Okamoto K-I, Hayashi Y, Sheng M. The Importance of Dendritic Mitochondria in the Morphogenesis and Plasticity of Spines and Synapses. Cell. 2004;119(6):873–87.

[7] JOHN JC ST., JOKHI RP, BARRATT CLR. The impact of mitochondrial genetics on male infertility. Int J Androl. 2005;28(2):65–73.

[8] Kuznetsov A V., Javadov S, Margreiter R, Grimm M, Hagenbuchner J, Ausserlechner MJ. The Role of Mitochondria in the Mechanisms of Cardiac Ischemia-Reperfusion Injury. Antioxidants. 2019;8(10):454.

[9] Sigma Aldrich. Mitochondria Isolation Kit [Package insert] [Internet]. Available from: https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/200/375/mitoiso2bul-mk.pdf

[10] Graham JM. Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation. Curr Protoc Cell Biol. 1999;4(1).

[11] Bahnemann J, Kayo S, Wahrheit J, Heinzle E, Pörtner R, Zeng A-P. In search of an effective cell disruption method to isolate intact mitochondria from Chinese hamster ovary cells. Eng Life Sci. 2014;14(2):161–9.

[12] Ohba M, Schatz G. Disruption of the outer membrane restores protein import to trypsin-treated yeast mitochondria. EMBO J. 1987;6(7):2117–22.

[13] Pallotti F, Lenaz G. Isolation and Subfractionation of Mitochondria from Animal Cells and Tissue Culture Lines. In: Methods in Cell Biology. Elsevier; 2007. p. 3–44.

[14] Tyler DD, Gonze J. The preparation of heart mitochondria from laboratory animals. In: Oxidation and Phosphorylation. Academic Press; 1967. p. 75–7. Available from: https://www.sciencedirect.com/science/article/pii/0076687967100141

[15] Mela L, Seitz S. Isolation of mitochondria with emphasis on heart mitochondria from small amounts of tissue. In: Methods in Enzymology. Elsevier; 1979. p. 39–46.

[16] Shinde SB, Save VC, Patil ND, Mishra KP, Tendolkar AG. Impairment of mitochondrial respiratory chain enzyme activities in tetralogy of fallot. Clinica Chimica Acta. 2007;377(1–2):138–43.

[17] Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine [Internet]. Oxford University Press; 2015. Available from: https://doi.org/10.1093/acprof:oso/9780198717478.001.0001

Human heart tissue mitochondria isolation: experience from patients with tetralogy of Fallot

Abstract

References

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