Abstract
Aims: Caveolins (Cavs) are critical components of cholesterol-enriched plasma membrane invaginations known as caveolae and have been implicated as signalling scaffolds that regulate diverse aspects of cell function, including insulin signalling. Emerging evidence suggests that Cavs may also localise to membranes of intracellular organelles, such as the mitochondria where they may potentially influence fuel/energy metabolism. In this study, we have investigated the impact of Cav deficiency on mitochondrial integrity/function in skeletal muscle cells.
Methods: A lentiviral-based shRNA strategy was deployed to stably silence expression of Cav 1 and 3 in rat L6 skeletal muscle cells. This approach resulted in greater than 90% loss of Cav1 and Cav3 in muscle cells. L6 cells depleted of either Cav1, Cav3 or both isoforms were used for qPCR, microscopy, fluorescence-based assays as well as analysis of mitochondrial function using a Seahorse extracellular flux analyser.
Results: Mitochondrial DNA content, which serves as an indicator of mitochondrial mass, was reduced significantly in single and double Cav1 and Cav3 depleted cells. Confocal microscopy using mitotracker green to label mitochondria revealed elongation and fragmentation of mitochondria in Cav1/3 double-silenced cells. This latter observation implies changes in mitochondrial integrity and function. Consistent with this, we observed significant reductions in mitochondrial membrane potential, oxygen consumption, adenosine triphosphate production as well as a decline in production of reactive oxygen species (p < 0.05).
Summary: Our data indicate that Cav1 and Cav3 may be novel regulators in mitochondrial integrity and function in skeletal muscle.
Methods: A lentiviral-based shRNA strategy was deployed to stably silence expression of Cav 1 and 3 in rat L6 skeletal muscle cells. This approach resulted in greater than 90% loss of Cav1 and Cav3 in muscle cells. L6 cells depleted of either Cav1, Cav3 or both isoforms were used for qPCR, microscopy, fluorescence-based assays as well as analysis of mitochondrial function using a Seahorse extracellular flux analyser.
Results: Mitochondrial DNA content, which serves as an indicator of mitochondrial mass, was reduced significantly in single and double Cav1 and Cav3 depleted cells. Confocal microscopy using mitotracker green to label mitochondria revealed elongation and fragmentation of mitochondria in Cav1/3 double-silenced cells. This latter observation implies changes in mitochondrial integrity and function. Consistent with this, we observed significant reductions in mitochondrial membrane potential, oxygen consumption, adenosine triphosphate production as well as a decline in production of reactive oxygen species (p < 0.05).
Summary: Our data indicate that Cav1 and Cav3 may be novel regulators in mitochondrial integrity and function in skeletal muscle.
Original language | English |
---|---|
Article number | P59 |
Pages (from-to) | 55 |
Number of pages | 1 |
Journal | Diabetic Medicine |
Volume | 35 |
Issue number | S1 |
Early online date | 14 Mar 2018 |
DOIs | |
Publication status | Published - Mar 2018 |