Calcium is an important signaling molecule involved in the regulation of many cellular functions. The large free energy in the Ca2+ ion membrane gradients makes Ca2+ signaling inherently sensitive to ...the available cellular free energy, primarily in the form of ATP. In addition, Ca2+ regulates many cellular ATP-consuming reactions such as muscle contraction, exocytosis, biosynthesis, and neuronal signaling. Thus, Ca2+ becomes a logical candidate as a signaling molecule for modulating ATP hydrolysis and synthesis during changes in numerous forms of cellular work. Mitochondria are the primary source of aerobic energy production in mammalian cells and also maintain a large Ca2+ gradient across their inner membrane, providing a signaling potential for this molecule. The demonstrated link between cytosolic and mitochondrial Ca2+ concentrations, identification of transport mechanisms, and the proximity of mitochondria to Ca2+ release sites further supports the notion that Ca2+ can be an important signaling molecule in the energy metabolism interplay of the cytosol with the mitochondria. Here we review sites within the mitochondria where Ca2+ plays a role in the regulation of ATP generation and potentially contributes to the orchestration of cellular metabolic homeostasis. Early work on isolated enzymes pointed to several matrix dehydrogenases that are stimulated by Ca2+, which were confirmed in the intact mitochondrion as well as cellular and in vivo systems. However, studies in these intact systems suggested a more expansive influence of Ca2+ on mitochondrial energy conversion. Numerous noninvasive approaches monitoring NADH, mitochondrial membrane potential, oxygen consumption, and workloads suggest significant effects of Ca2+ on other elements of NADH generation as well as downstream elements of oxidative phosphorylation, including the F1FO-ATPase and the cytochrome chain. These other potential elements of Ca2+ modification of mitochondrial energy conversion will be the focus of this review. Though most specific molecular mechanisms have yet to be elucidated, it is clear that Ca2+ provides a balanced activation of mitochondrial energy metabolism that exceeds the alteration of dehydrogenases alone.
Mitochondria are key determinants of cellular health. However, the functional role of mitochondria varies from cell to cell depending on the relative demands for energy distribution, metabolite ...biosynthesis, and/or signaling. In order to support the specific needs of different cell types, mitochondrial functional capacity can be optimized in part by modulating mitochondrial structure across several different spatial scales. Here we discuss the functional implications of altering mitochondrial structure with an emphasis on the physiological trade-offs associated with different mitochondrial configurations. Within a mitochondrion, increasing the amount of cristae in the inner membrane improves capacity for energy conversion and free radical-mediated signaling but may come at the expense of matrix space where enzymes critical for metabolite biosynthesis and signaling reside. Electrically isolating individual cristae could provide a protective mechanism to limit the spread of dysfunction within a mitochondrion but may also slow the response time to an increase in cellular energy demand. For individual mitochondria, those with relatively greater surface areas can facilitate interactions with the cytosol or other organelles but may be more costly to remove through mitophagy due to the need for larger phagophore membranes. At the network scale, a large, stable mitochondrial reticulum can provide a structural pathway for energy distribution and communication across long distances yet also enable rapid spreading of localized dysfunction. Highly dynamic mitochondrial networks allow for frequent content mixing and communication but require constant cellular remodeling to accommodate the movement of mitochondria. The formation of contact sites between mitochondria and several other organelles provides a mechanism for specialized communication and direct content transfer between organelles. However, increasing the number of contact sites between mitochondria and any given organelle reduces the mitochondrial surface area available for contact sites with other organelles as well as for metabolite exchange with cytosol. Though the precise mechanisms guiding the coordinated multi-scale mitochondrial configurations observed in different cell types have yet to be elucidated, it is clear that mitochondrial structure is tailored at every level to optimize mitochondrial function to meet specific cellular demands.
Intracellular energy distribution has attracted much interest and has been proposed to occur in skeletal muscle via metabolite-facilitated diffusion; however, genetic evidence suggests that ...facilitated diffusion is not critical for normal function. We hypothesized that mitochondrial structure minimizes metabolite diffusion distances in skeletal muscle. Here we demonstrate a mitochondrial reticulum providing a conductive pathway for energy distribution, in the form of the proton-motive force, throughout the mouse skeletal muscle cell. Within this reticulum, we find proteins associated with mitochondrial proton-motive force production preferentially in the cell periphery and proteins that use the proton-motive force for ATP production in the cell interior near contractile and transport ATPases. Furthermore, we show a rapid, coordinated depolarization of the membrane potential component of the proton-motive force throughout the cell in response to spatially controlled uncoupling of the cell interior. We propose that membrane potential conduction via the mitochondrial reticulum is the dominant pathway for skeletal muscle energy distribution.
Mitochondrial structures were probably observed microscopically in the 1840s, but the idea of oxidative phosphorylation (OXPHOS) within mitochondria did not appear until the 1930s. The foundation for ...research into energetics arose from Meyerhof's experiments on oxidation of lactate in isolated muscles recovering from electrical contractions in an O2 atmosphere. Today, we know that mitochondria are actually reticula and that the energy released from electron pairs being passed along the electron transport chain from NADH to O2 generates a membrane potential and pH gradient of protons that can enter the molecular machine of ATP synthase to resynthesize ATP. Lactate stands at the crossroads of glycolytic and oxidative energy metabolism. Based on reported research and our own modelling in silico, we contend that lactate is not directly oxidized in the mitochondrial matrix. Instead, the interim glycolytic products (pyruvate and NADH) are held in cytosolic equilibrium with the products of the lactate dehydrogenase (LDH) reaction and the intermediates of the malate‐aspartate and glycerol 3‐phosphate shuttles. This equilibrium supplies the glycolytic products to the mitochondrial matrix for OXPHOS. LDH in the mitochondrial matrix is not compatible with the cytoplasmic/matrix redox gradient; its presence would drain matrix reducing power and substantially dissipate the proton motive force. OXPHOS requires O2 as the final electron acceptor, but O2 supply is sufficient in most situations, including exercise and often acute illness. Recent studies suggest that atmospheric normoxia may constitute a cellular hyperoxia in mitochondrial disease. As research proceeds appropriate oxygenation levels should be carefully considered.
figure legend Credit for the discovery of what would become known as mitochondria is given to Rudolf Albrecht von Kölliker in 1857; these structures were subsequently described in greater detail by Richard Altmann. In 1898, Benda used a derivation of the Greek words for ‘thread’ and ‘granule’ to name these structures ‘mitochondria’. In 1907, Fletcher and Hopkins reported the disappearance of lactate in the presence of O2 in previously stimulated muscles. Approximately two decades later, Meyerhof's work on O2 consumption and lactate (La−) resynthesis into glycogen during the recovery of isolated skeletal muscles from prior contractions was an early hint at the intersection of glycolysis and aerobic phosphorylation. Warburg related these phenomena to the metabolic physiology of cancer. Research by both Meyerhof and Emden led to discovery of the glycolytic pathway. In the 1930s, the work of Lundsgaard, Krebs, Kalckar, the Coris, Belitzer and Szent‐Györgi, and subsequently Lipmann, Ochoa, Bensley & Hoerr and Claude in the 1940s led to establishing the bioenergetics of glycolysis and the TCA cycle and compounds of high phosphoryl transfer potential. The 1950s heralded the age of research using isolated, functioning mitochondria to explore bioenergetics, and featured prominently the work of Lehninger, Estabrook & Saktor, and Chance & Williams. In the 1960s, Peter Mitchell first proposed the chemiosmotic theory of oxidative phosphorylation, for which he was awarded the Nobel Prize. During this same decade, work by Borst clarified the malate‐aspartate shuttle, wherein the exchange of anionic aspartate for undissociated glutamate (one negative charge exported from the matrix per exchange) is driven by the membrane potential (ΔΨ). Work by Skulachev in this decade and beyond further clarified mitochondrial bioenergetics and mitochondrial morphology. Boyer elucidated the nature of the ATP synthase, ultimately winning the Nobel Prize for his work. In the 1980s, David Nicholls further clarified mitochondrial bioenergetics, and the work of George Brooks initiated the era of the cell‐to‐cell lactate shuttle. Starting in the 1990s, research emerged suggesting that mitochondria are capable of transporting La− across the inner membrane and oxidizing it without the support of the cytosolic‐mitochondrial electron shuttles (i.e. the malate‐aspartate and glycerol‐3‐phosphate shuttles). The ultimate combustion of La− obviously takes place in the mitochondria; there is no question about that simple conclusion. However, our view is that La− is not directly oxidized by LDH in the mitochondrial matrix, but rather La− must first be converted to pyruvate (Pyr−) in the cytosol or intermembrane space. Rationale for this view includes the high activity of the near‐equilibrium enzyme LDH, which exceeds glycolytic capacity, the highly oxidized cytosolic NAD+/NADH ratio relative to the mitochondrial matrix, and the thermodynamic necessity for an energy‐driven accumulation of shuttle species (e.g. ΔΨ‐dependent aspartate‐glutamate exchanger). Modelling in silico demonstrates that an active LDH in the matrix would render mitochondria nearly incapable of oxidizing Pyr−, a result which is inconsistent with decades of studies from hundreds of laboratories using both isolated mitochondria and permeabilized cells in which the mitochondrial reticulum remains intact. Healthy mitochondria function well, even at low O2 levels such that dysoxia is rare and low O2 is likely to be a minor factor in the increasing concentrations of La− typical with exercise or even many acute critical care situations.
Mapping biological circuit connectivity has revolutionized our understanding of structure-function relationships. Although connectomic analyses have primarily focused on neural systems, electrical ...connectivity within muscle mitochondrial networks was recently demonstrated to provide a rapid mechanism for cellular energy distribution. However, tools to evaluate organelle connectivity with high spatial fidelity within single cells are currently lacking. Here, we developed a framework to quantitatively assess mitochondrial network connectivity and interactions with cellular sites of energy storage, utilization, and calcium cycling in cardiac, oxidative, and glycolytic muscle. We demonstrate that mitochondrial network configuration, individual mitochondrial size and shape, and the junctions connecting mitochondria within each network are consistent with the differing contraction demands of each muscle type. Moreover, mitochondria-lipid droplet interaction analyses suggest that individual mitochondria within networks may play specialized roles regarding energy distribution and calcium cycling within the cell and reveal the power of connectomic analyses of organelle interactions within single cells.
The specific cellular role of mitochondria is influenced by the surrounding environment because effective mitochondrial function requires the delivery of inputs (e.g., oxygen) and export of products ...(e.g., signaling molecules) to and from other cellular components, respectively. Recent technological developments in mitochondrial imaging have led to a more precise and comprehensive understanding of the spatial relationships governing the function of this complex organelle, opening a new era of mitochondrial research. Here, I highlight current imaging approaches for visualizing mitochondrial form and function within complex cellular environments. Increasing clarity of mitochondrial behavior within cells will continue to lend mechanistic insights into the role of mitochondria under normal and pathological conditions and point to spatially regulated processes that can be targeted to improve cellular function.
Advances in super-resolution microscopy now enable the visualization of thousands of individual mitochondria with molecular precision throughout large tissues, as well as unprecedented views of the dynamic nature of internal mitochondrial structures.Expansion of our ability to simultaneously visualize multiple mitochondrial structures and proteins together with other organelles has provided novel mechanistic insights into the intra- and interorganelle interactions of mitochondrial networks.Spatially resolved measures of mitochondrial energetic flux provide a promising avenue for evaluating the impact of interventions into cellular energy metabolism within heterogeneous cells and tissues.Accompaniment of high-throughput image analysis platforms with big data-generating microscopy approaches now enables systems-level evaluations of how mitochondria behave within the cellular environment.
Calcium is believed to regulate mitochondrial oxidative phosphorylation, thereby contributing to the maintenance of cellular energy homeostasis. Skeletal muscle, with an energy conversion dynamic ...range of up to 100-fold, is an extreme case for evaluating the cellular balance of ATP production and consumption. This study examined the role of Ca2+ in the entire oxidative phosphorylation reaction network in isolated skeletal muscle mitochondria and attempted to extrapolate these results back to the muscle, in vivo. Kinetic analysis was conducted to evaluate the dose–response effect of Ca2+ on the maximal velocity of oxidative phosphorylation (V maxO) and the ADP affinity. Force-flow analysis evaluated the interplay between energetic driving forces and flux to determine the conductance, or effective activity, of individual steps within oxidative phosphorylation. Measured driving forces extramitochondrial phosphorylation potential (ΔG ATP), membrane potential, and redox states of NADH and cytochromes b H, b L, c 1, c, and a,a 3 were compared with flux (oxygen consumption) at 37 °C; 840 nM Ca2+ generated an ∼2-fold increase in V maxO with no change in ADP affinity (∼43 μM). Force-flow analysis revealed that Ca2+ activation of V maxO was distributed throughout the oxidative phosphorylation reaction sequence. Specifically, Ca2+ increased the conductance of Complex IV (2.3-fold), Complexes I and III (2.2-fold), ATP production/transport (2.4-fold), and fuel transport/dehydrogenases (1.7-fold). These data support the notion that Ca2+ activates the entire muscle oxidative phosphorylation cascade, while extrapolation of these data to the exercising muscle predicts a significant role of Ca2+ in maintaining cellular energy homeostasis.
Mitochondrial network connectivity enables rapid communication and distribution of potential energy throughout the cell. However, this connectivity puts the energy conversion system at risk, because ...damaged elements could jeopardize the entire network. Here, we demonstrate the mechanisms for mitochondrial network protection in heart and skeletal muscle (SKM). We find that the cardiac mitochondrial reticulum is segmented into subnetworks comprising many mitochondria linked through abundant contact sites at highly specific intermitochondrial junctions (IMJs). In both cardiac and SKM subnetworks, a rapid electrical and physical separation of malfunctioning mitochondria occurs, consistent with detachment of IMJs and retraction of elongated mitochondria into condensed structures. Regional mitochondrial subnetworks limit the cellular impact of local dysfunction while the dynamic disconnection of damaged mitochondria allows the remaining mitochondria to resume normal function within seconds. Thus, mitochondrial network security is comprised of both proactive and reactive mechanisms in striated muscle cells.
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•Mitochondrial networks are functionally linked through intermitochondrial junctions•Regional mitochondrial subnetworks proactively limit spread of local dysfunction•Dysfunctional mitochondria are electrically separated from the network in seconds•Physical network separation involves mitochondrial retraction
Network connectivity allows information sharing and distribution but also enables propagation of localized dysfunction. Glancy et al. demonstrate the existence of both proactive and reactive network protection mechanisms designed to minimize the spread of dysfunction throughout the coupled mitochondrial networks in heart and skeletal muscle cells.
Abstract
Human movement occurs through contraction of the basic unit of the muscle cell, the sarcomere. Sarcomeres have long been considered to be arranged end-to-end in series along the length of ...the muscle into tube-like myofibrils with many individual, parallel myofibrils comprising the bulk of the muscle cell volume. Here, we demonstrate that striated muscle cells form a continuous myofibrillar matrix linked together by frequently branching sarcomeres. We find that all muscle cells contain highly connected myofibrillar networks though the frequency of sarcomere branching goes down from early to late postnatal development and is higher in slow-twitch than fast-twitch mature muscles. Moreover, we show that the myofibrillar matrix is united across the entire width of the muscle cell both at birth and in mature muscle. We propose that striated muscle force is generated by a singular, mesh-like myofibrillar network rather than many individual, parallel myofibrils.
Mitochondrial networks provide coordinated energy distribution throughout muscle cells. However, pathways specifying mitochondrial networks are incompletely understood and it is unclear how they ...might affect contractile fiber-type. Here, we show that natural energetic demands placed on Drosophila melanogaster muscles yield native cell-types among which contractile and mitochondrial network-types are regulated differentially. Proteomic analyses of indirect flight, jump, and leg muscles, together with muscles misexpressing known fiber-type specification factor salm, identified transcription factors H15 and cut as potential mitochondrial network regulators. We demonstrate H15 operates downstream of salm regulating flight muscle contractile and mitochondrial network-type. Conversely, H15 regulates mitochondrial network configuration but not contractile type in jump and leg muscles. Further, we find that cut regulates salm expression in flight muscles and mitochondrial network configuration in leg muscles. These data indicate cell type-specific regulation of muscle mitochondrial network organization through evolutionarily conserved transcription factors cut, salm, and H15.
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