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.