Sergio De La Fuente

Increased mitochondrial nanotunneling activity, induced by calcium imbalance, affects intermitochondrial matrix exchanges.

Significance Nanotunnels are long, thin mitochondrial extensions that have been implied in direct long-distance (1 to >5 µm) communication between mitochondria of cardiac myocytes. The engineered RyR2A4860G+/− mutation, resulting in loss of function of the sarcoplasmic reticulum calcium release channel and arrhythmia, induces a striking increase in the frequency of long-distance intermitochondrial communication via nanotunnels without involvement of obvious mitochondrial migration. We use this model for exploring the significance of mitochondrial nanotunneling in myocardium and the contribution of microtubules to the formation of these unusual organelle extensions using EM tomography and live confocal imaging. This study constitutes an approach to arrhythmia investigations that focuses on a new target: the mitochondria. Exchanges of matrix contents are essential to the maintenance of mitochondria. Cardiac mitochondrial exchange matrix content in two ways: by direct contact with neighboring mitochondria and over longer distances. The latter mode is supported by thin tubular protrusions, called nanotunnels, that contact other mitochondria at relatively long distances. Here, we report that cardiac myocytes of heterozygous mice carrying a catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutation (A4860G) show a unique and unusual mitochondrial response: a significantly increased frequency of nanotunnel extensions. The mutation induces Ca2+ imbalance by depressing RyR2 channel activity during excitation–contraction coupling, resulting in random bursts of Ca2+ release probably due to Ca2+ overload in the sarcoplasmic reticulum. We took advantage of the increased nanotunnel frequency in RyR2A4860G+/− cardiomyocytes to investigate and accurately define the ultrastructure of these mitochondrial extensions and to reconstruct the overall 3D distribution of nanotunnels using electron tomography. Additionally, to define the effects of communication via nanotunnels, we evaluated the intermitochondrial exchanges of matrix-targeted soluble fluorescent proteins, mtDsRed and photoactivable mtPA-GFP, in isolated cardiomyocytes by confocal microscopy. A direct comparison between exchanges occurring at short and long distances directly demonstrates that communication via nanotunnels is slower.

Spatial Separation of Mitochondrial Calcium Uptake and Extrusion for Energy-Efficient Mitochondrial Calcium Signaling in the Heart

SUMMARY Mitochondrial Ca2+ elevations enhance ATP production, but uptake must be balanced by efflux to avoid overload. Uptake is mediated by the mitochondrial Ca2+ uniporter channel complex (MCUC), and extrusion is controlled largely by the Na+/Ca2+ exchanger (NCLX), both driven electrogenically by the inner membrane potential (Δ𝚿m). MCUC forms hotspots at the cardiac mitochondria-junctional SR (jSR) association to locally receive Ca2+ signals; however, the distribution of NCLX is unknown. Our fractionation-based assays reveal that extensively jSRassociated mitochondrial segments contain a minor portion of NCLX and lack Na+-dependent Ca2+ extrusion. This pattern is retained upon in vivo NCLX overexpression, suggesting extensive targeting to non-jSR-associated submitochondrial domains and functional relevance. In cells with non-polarized MCUC distribution, upon NCLX overexpression the same given increase in matrix Ca2+ expends more Δ𝚿m. Thus, cardiac mitochondrial Ca2+ uptake and extrusion are reciprocally polarized, likely to optimize the energy efficiency of local calcium signaling in the beating heart.