Original articleIntrafibrillar and perinuclear mitochondrial heterogeneity in adult cardiac myocytes
Introduction
Mitochondria localized in different regions of a cell, interact with local intracellular structures and may display distinct morphological and biochemical properties. These mitochondrial subsets may also exhibit different responses to substrates, inhibitors and may vary in their energetics, sensitivity to stimulation, resistance to apoptosis, and other characteristics. In cardiac mycoytes, mitochondria are spatially separated into different populations. One just beneath the surface sarcolemma (subsarcolemmal mitochondria, SSM), the major population which are between the myofibrils (interfibrillar mitochondria, IFM), and a third population that is perinuclear (PNM). The IFM are organized in a lattice of parallel rows surrounding and surrounded by the contractile myofilament and these IFM may be restricted in their positions. This type of organization may provide bioenergetic support for contraction and mitochondrial interactions with the cytoskeleton [1,2] and sarcoplasmic reticulum (SR) [[3], [4], [5]]. The heart performs energetically demanding work (contraction, rapid cycles of ion transport) that require large amounts of ATP every second and that work can dynamically regulate gene expression and protein synthesis to adapt to different chronic workloads.
In adult cardiac myocytes, the precise matching of energy supply with demand are critical in maintaining cardiac function at different workloads and substrate supply. Heterogeneity of mitochondrial energetics can cause complex dynamic behavior to deal with these changes in cellular workload or environmental conditions [6]. This includes local metabolic transients, cell-wide coordinated redox transitions, and propagated metabolic waves within myocytes [7]. There is growing evidence for functional heterogeneity of mitochondria with respect to their biochemical, respiratory or enzymatic activities, Ca2+ handling and membrane potential. However most prior characterizations have relied on isolated mitochondria that are separated from their normal in-situ environment [[8], [9], [10], [11], [12]], which may limit conclusions. Confocal imaging allows spatial analysis of different mitochondrial populations in live myocytes with respect to redox potentials [6] and mitochondrial Ca2+ handling [13,14]. However, these florescence sensors can be complicated by contaminating cytosolic signals. Previous cardiac myocyte studies have mostly focused on comparing isolated SSM and IFM [[8], [9], [10], [11], [12]], but less is known about how IFM and PNM differ in myocytes. Here we use real-time confocal imaging on live cardiac myocytes to examine morphological and functional differences between IFM and PNM.
In many cell types mitochondria are described as “highly dynamic”, including frequent mitochondrial fission/fusion events and translocation within cells [[15], [16], [17], [18]]. Indeed, mitochondria in many cell types are dynamically rebuilt through continuous fusion and fission process in response to various cellular signals. Mitochondrial fission is required to create new mitochondria, but it also allows segregation of damaged mitochondria for degradation. Mitochondrial fusion produces elongated mitochondria and allows exchange of materials between mitochondria. Hence, mitochondrial fusion and fission appear to occur in a constant and balanced manner to allow the mitochondrial network to adapt to metabolic needs of the cell.
We hypothesize that in cardiac myocytes PNM exhibit dynamic fission/fusion and translocation similar to that described in other cell types, while IFM may be much more static, constrained in part by their rigid organization in a “lattice” of parallel rows surrounded by contractile myofilaments. The myofilament and Z-line structures could severely limit both movement and dynamic interaction between IFM. Despite these spatial restrictions cardiac IFM may exhibit dynamic interaction via nanotunnels and fusion/fission, for which the key proteins are all expressed in cardiac myocytes [[19], [20], [21]]. However, there is limited direct comparison of mitochondrial motility differences in IFM vs. PNM. Here, we use multiple approaches to examine the morphology and functional dynamics of IFM vs. PNM in adult cardiac myocytes. Our data indicated that IFM and PNM vary substantially in morphology, Ca2+ uptake, permeability transition pore (PTP) opening, motility, fission/fusion events and mitochondrial turnover via mitophagy.
Section snippets
Mitochondrial distribution and morphology (IFM vs. PNM)
We studied mitochondrial organization and function using confocal imaging and electron microscopy (EM) of intact and permeabilized rabbit ventricular myocyte. As classically described in EM images [22] and in Fig. 1A, IFM in adult ventricular myocytes are aligned in longitudinal chains between the myofibrils, and seem to be squeezed into these tracks by the myofilaments which perforce are longitudinally continuous. This may contribute to the typically oval shape of IFM, and further lateral
Discussion
Mitochondria are crucial determinants in the life and death of cells and changes in their morphology and function underlie processes [30,31]. Unlike many cell types, in cardiac myocytes the rigid myofilament and Z-line structures may cause morphological difference between IFM and PNM. The proximity of IFM to the intense energy demands placed on the myofilaments may also contribute to functional differences in mitochondrial Ca2+handling. Here, we compared these two mitochondrial sub-populations
Cardiac Myocyte isolation, dye loading and permeabilization
Cardiac myocytes were isolated from New Zealand white rabbits and C57BL6 mice using retrograde Langendorff perfusion using Liberase TM (0.075 mg/mL, Roche) and Trypsin (0.0138%, Gibco) (37 °C) as previously described [53]. All procedures were approved by the University of California Davis Institutional Animal Care and Use Committee (IACUC) in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Freshly isolated myocytes were plated on laminin-coated glass cover slip for
Funding
The study was supported by National Institutes of Health grants R01-HL132831 and R01-HL030077 and National Natural Science Foundation of China grants 81741050 and 8187350.
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