Mitochondria from striate muscles: Novel techniques to isolate and characterise the subsarcolemmal and intermyofibrillar fractions

• Published in: Vascular pharmacology Vol. 103-105; pp. 61 - 62
• Main Authors: Popara, J., Femminò, S., Zicola, E., Abbadessa, G., Racca, S., Mancardi, D.
• Format: Journal Article
• Language: English
• Published: Philadelphia Elsevier Inc 01-04-2018; Elsevier Science Ltd
• Subjects: Background noise; Cells; Electrophoresis; Flow cytometry; Gel electrophoresis; Heart attacks; Immunohistochemistry; Ion channels; Mass spectrometry; Mass spectroscopy; Mitochondria; Muscles; Myocardium; Noise reduction; Organelles; Physical exercise; Physical properties; Proteomics; Relative abundance; Two dimensional analysis
• ISSN: 1537-1891; 1879-3649
• DOI: 10.1016/j.vph.2017.12.039

Objective: It has been shown that striate muscle mitochondria can be classified in two fractions depending on their structure and distribution in the cell: subsarcolemmal (SSM) and interfibrillar (IFM). It has also been reported that different factors can selectively affect relative abundance of IFM and SSM. Thus, developing novel techniques to distinguish and separate these fractions is of great importance. Methods: In order to quantity and characterize IFM and SSM, we isolated gastrocnemius and myocardium from physically trained and sedentary rats and we tested different techniques. Longitudinal and transversal sections of a striate muscle have been used for immunohistochemistry analysis. Mitochondria were labeled with anti-voltage dependent anion channel (VDAC) antibody. 8 um thick slices were examined with LSM-800 confocal microscope. Furthermore, mitochondria have been extracted following a modified protocol from Frezza's et al. in order to perform studies on functional organelles. This protocol allows us to obtain intact and functional mitochondria used to perform functional assays. To analyze physical properties of extracted mitochondria, Beckman coulter CYAN flow cytometer has been used. Moreover, isolated mitochondria were processed and analyzed by 2D gel electrophoresis with 7 cm 3-10 pH strips, followed by MALDI-TOF mass spectrometry analysis. Results: Immunohistochemistry analysis suggests equal distribution of mitochondria inside muscle fiber. Though, we find that longitudinal sections can provide better insights on 3D structure and arrangement within the fibers. Flow cytometry analysis can provide significant data. However, due to mitochondrial size, particular attention should be paid on background noise reduction. Proteomics analysis highlighted some differences between mitochondria extracted from muscles which did or did not undergo intense physical exercise. Conclusions: Mitochondria are involved in a plethora of physiological and pathophysiological processes. Therefore, creating new tools to understand their structure in relation to the intracellular distribution is of pivotal importance.