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

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.