Recent work of a multi-national effort led by Andrés Hidalgo at CNIO in Madrid has identified an important mechanism in the healthy maintenance of cardiomyocytes (CM), extremely long-lived cells, within the myocardium.
Importantly, they show that defective or worn-out mitochondria are expelled from these energy-intensive cells, by an autophagic process, inside vesicles called exophers. These exophers are tagged with phosphatidyl serine which labels them for engulfment and rapid destruction along with their contents by cardiac macrophages (cMacs).
Failure of CM to expel these failing organelles will obviously lead to poor cardiac function while a failure of macrophage engulfment of expelled exophers may lead to inflammatory processes amongst other effects and thereby indirectly to reduced cardiac function.
In addition, the group have most recently published a detailed protocol for the isolation of exophers from cardiac tissue. In the final step of the isolation procedure exophers are purified by FACS, based on size and the exclusion of both endothelial cell-derived (via CD31-negativity) and DNA-containing (via DRAQ5-negativity).
DRAQ5™ was employed at a final concentration of 1 µM as the final step, without washing, prior to the sorting or analysis.
This novel application of the cell-permeant far-red DNA dye DRAQ5™ will enable the downstream investigation in cell-based and molecular analyses of exophers, to unpick the critical role of their expulsion and disposal by cMacs in the homeostasis of CM biology and perhaps in better treatments for heart disorders.
References:
Nicolás-Ávila, José A., et al. "A network of macrophages supports mitochondrial homeostasis in the heart." Cell 183.1 (2020): 94-109.
Nicolás-Ávila, José Ángel, María Sánchez-Diaz, and Andrés Hidalgo. "Isolation of exophers from cardiomyocyte-reporter mouse strains by fluorescence-activated cell sorting." STAR protocols 2.1 (2021): 100286.
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