Mitochondrial fusion dynamics is robust in the heart and depends on calcium oscillations and contractile activity

Abstract

Mitochondrial fusion is thought to be important for supporting cardiac contractility, but is hardly detectable in cultured cardiomyocytes and is difficult to directly evaluate in the heart. We overcame this obstacle through in vivo adenoviral transduction with matrix-targeted photoactivatable GFP and confocal microscopy. Imaging in whole rat hearts indicated mitochondrial network formation and fusion activity in ventricular cardiomyocytes. Promptly after isolation, cardiomyocytes showed extensive mitochondrial connectivity and fusion, which decayed in culture (at 24-48 h). Fusion manifested both as rapid content mixing events between adjacent organelles and slower events between both neighboring and distant mitochondria. Loss of fusion in culture likely results from the decline in calcium oscillations/contractile activity and mitofusin 1 (Mfn1), because (i) verapamil suppressed both contraction and mitochondrial fusion, (ii) after spontaneous contraction or short-term field stimulation fusion activity increased in cardiomyocytes, and (iii) ryanodine receptor-2-mediated calcium oscillations increased fusion activity in HEK293 cells and complementing changes occurred in Mfn1. Weakened cardiac contractility in vivo in alcoholic animals is also associated with depressed mitochondrial fusion. Thus, attenuated mitochondrial fusion might contribute to the pathogenesis of cardiomyopathy.ardiac contractions require a constant energy supply, which is provided by mitochondrial metabolism. ATP is needed for excitation-contraction coupling (ECC) for both contraction and relaxation in each cycle (1, 2). ECC-associated cytoplasmic Ca 2+ transients ([Ca 2+ ] c ) are propagated to the mitochondrial matrix (3) to regulate Ca 2+ -dependent mitochondrial dehydrogenases, ATP synthesis, and intracellular Ca 2+ homeostasis (4). Thus, cardiac mitochondria are forced to work permanently and likely require quality control mechanisms to keep them in a functioning state.In many tissues, mitochondria are permanently rebuilt through evolutionarily conserved cyclic processes of fusion and fission. Fusion involves content exchange, allowing complementation of mitochondrial solutes, proteins, and DNA. Fission allows segregation of damaged components (5, 6). Both fusion and fission are promoted by mitochondrial movements that can bring distant mitochondria close to one another and can also separate interacting structures (7). However, the spatial arrangements and mitochondrial morphology are determined by sarcomers in ventricular myocytes, the contractile units of the adult mammalian heart. The gaps among densely packed parallel myofibrils are inhabited by bullet-like mitochondria that run longitudinally, interacting intimately with the junctional sarcoplasmic reticulum (SR), in a conformation that facilitates Ca 2+ exchange and supports ECC (4,8). Despite the spatial restrictions intrinsic to adult cardiomyocyte mitochondria, interactions among these organelles have been proposed based on functional observations. Reactive oxygen species (ROS)...

Alterations in fatty acid metabolism and sirtuin signaling characterize early type-2 diabetic hearts of fructose-fed rats

“…Generally speaking, mitochondria can quickly adapt to changes in metabolic flux (Eisner et al. 2017), but clearly much more work is necessary to map morphological changes and fusion/fission events in diabetic mitochondria.…”

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

“…The live-imaging approach follows seminal experimental guidelines developed and implemented by Veronica Eisner (Department de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile) (32). Imaging of isolated cells was performed within 12 h after cardiomyocyte isolation in 0.25% BSA with the addition of 10 mM BDM to inhibit contraction using a laser-scanning microscope (LSM 780, Carl Zeiss), exciting 488 nm (mtPA-GFP) and 568 nm (mtDsRed) with 63×/1.4 N.A.…”

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