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• W4381546844 abstract "Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Mitochondrial ATP production in ventricular cardiomyocytes must be continually adjusted to rapidly replenish the ATP consumed by the working heart. Two systems are known to be critical in this regulation: mitochondrial matrix Ca2+ ([Ca2+]m) and blood flow that is tuned by local cardiomyocyte metabolic signaling. However, these two regulatory systems do not fully account for the physiological range of ATP consumption observed. We report here on the identity, location, and signaling cascade of a third regulatory system -- CO2/bicarbonate. CO2 is generated in the mitochondrial matrix as a metabolic waste product of the oxidation of nutrients. It is a lipid soluble gas that rapidly permeates the inner mitochondrial membrane and produces bicarbonate in a reaction accelerated by carbonic anhydrase. The bicarbonate level is tracked physiologically by a bicarbonate-activated soluble adenylyl cyclase (sAC). Using structural Airyscan super-resolution imaging and functional measurements we find that sAC is primarily inside the mitochondria of ventricular cardiomyocytes where it generates cAMP when activated by bicarbonate. Our data strongly suggest that ATP production in these mitochondria is regulated by this cAMP signaling cascade operating within the inter-membrane space by activating local EPAC1 (Exchange Protein directly Activated by cAMP) which turns on Rap1 (Ras-related protein-1). Thus, mitochondrial ATP production is increased by bicarbonate-triggered sAC-signaling through Rap1. Additional evidence is presented indicating that the cAMP signaling itself does not occur directly in the matrix. We also show that this third signaling process involving bicarbonate and sAC activates the mitochondrial ATP production machinery by working independently of, yet in conjunction with, [Ca2+]m-dependent ATP production to meet the energy needs of cellular activity in both health and disease. We propose that the bicarbonate and calcium signaling arms function in a resonant or complementary manner to match mitochondrial ATP production to the full range of energy consumption in ventricular cardiomyocytes. Editor's evaluation Cardiac function is critically dependent on the homeostatic control of ATP so enough energy is provided for a given task and previous studies have described how calcium signals in the mitochondria convey a message of demand that regulates enzymes to alter ATP production accordingly. This manuscript presents a parallel mechanism that implicates CO2 and HCO3 sensors that regulate cAMP signalling. The authors have identified the putative location of the enzyme pathway following super-resolution imaging of isolated ventricular myocytes and mitochondria. Localizing sAC to the interior portion of the mitochondria is a significant advance and provides a framework for future target discovery. https://doi.org/10.7554/eLife.84204.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Cardiac ventricular myocytes work non-stop to meet the blood flow needs of the body, with the heart consuming ATP at high and widely variable rates (Boyman et al., 2020; From et al., 1986; From et al., 1990; Katz et al., 1989; Portman et al., 1989; Zhang et al., 1995; Elliott et al., 1994; Matthews et al., 1981; Massie et al., 1994; Schwartz et al., 1994; Xu et al., 1998). Because of the everchanging energy needs of the ventricular myocytes and their minimal ATP reserves (Jacobus, 1985; Wang et al., 2010), ATP production must be dynamically managed by a control system that has high gain and is rapidly responsive (Maack and O’Rourke, 2007; Murphy and Steenbergen, 2021). Recent work has shown that heart rate-dependent elevation of cytosolic Ca2+ signaling drives the ventricular myocytes to contract more frequently while elevating the mitochondrial matrix Ca2+ ([Ca2+]m) and thereby increasing ATP production (Boyman et al., 2020; Wescott et al., 2019; Garg et al., 2021). The energetic needs of ventricular myocytes are also linked to local blood supply through the newly described ‘electro-metabolic signaling (EMS)’. Through this mechanism, as [ATP] in ventricular myocytes falls with ATP consumption, small blood vessels dilate to increase local blood flow (Zhao et al., 2020; Grainger and Santana, 2020). EMS thereby will increase the supply of oxygen and nutrient substrates and speed up removal of metabolic waste products by increased flow through local end-arterioles and capillaries. Together, EMS and [Ca2+]m signaling provide important feedback controls to increase ATP production in working ventricular myocytes. There is, however, still a huge gap in accounting for the full physiological scale of ATP consumption by ventricular myocytes (Boyman et al., 2020; From et al., 1986; From et al., 1990; Katz et al., 1989; Portman et al., 1989; Zhang et al., 1995; Elliott et al., 1994; Matthews et al., 1981; Massie et al., 1994; Schwartz et al., 1994; Xu et al., 1998). There is no characterized feedback signal yet that provides a mechanism for ventricular myocytes to scale-up ATP production by their mitochondria when increased work is being done by the heart at constant [Ca2+]m. This kind of situation arises routinely when the heart must pump at the same rate but against a greater afterload. This occurs, for example, when blood pressure rises. The investigation presented in this paper seeks to fill this major gap in our understanding. Here, we report on an understudied and poorly understood signaling pathway in cardiac mitochondria in which CO2 and soluble adenylyl cyclase (sAC) play pivotal roles. CO2 is generated in mitochondria largely as a waste product originating from processing of energy substrates by the Krebs cycle, therefore reflecting the extent of energy metabolism. This CO2 is dissolved in the local aqueous fraction and is in dynamic aqueous equilibrium with bicarbonate (HCO3−). Importantly, sAC is activated by bicarbonate but unlike ‘transmembrane’ adenylyl cyclase (tmAC), sAC is found in solution not in the cellular membranes. When sAC is activated, like tmAC, it generates cAMP as the second messenger (Buck et al., 1999; Litvin et al., 2003). The 10-member family of signaling proteins known as adenylyl cyclases (ACs) is exquisitely adaptable and transduces a wide array of biological signals by generating the second messenger cAMP (Zaccolo et al., 2001; Di Benedetto et al., 2021). The family is best known by the nine members that are incorporated in the plasma membranes of almost every cell type via transmembrane (tm) protein domains. While tmACs are broadly regulated by G proteins in response to hormonal stimuli (Oldham and Hamm, 2008; Syrovatkina et al., 2016), they are able to maintain specificity and high signal-to-noise ratio through proximity to their targets docked at A-kinase anchoring proteins (AKAPs) (Scott et al., 2013; Zaccolo and Pozzan, 2002). Further signal compartmentalization is gained by nearby ‘firewalls’ of phosphodiesterases (PDEs) that hydrolyze cAMP to AMP (Lomas and Zaccolo, 2014; Baillie et al., 2019; Mongillo et al., 2004; Burdyga et al., 2018). In contrast, there is only one mammalian member of the sAC subset, and it is much less studied or understood. Mammalian sACs are structural homologues of the bacterial sACs and also lack a transmembrane domain. Instead, they are confined inside organelles like mitochondria, centrioles, and nuclei (Zippin et al., 2003; Feng et al., 2006; Acin-Perez et al., 2009). Unlike tmACs, sACs achieve high local signal-to-noise ratio by producing cAMP inside the small subcellular volumes that also contain their targets (Tresguerres et al., 2011; Rossetti et al., 2021), namely protein kinase A (PKA) and ‘Exchange Proteins Activated by cAMP’ or EPACs (Schmid et al., 2007; Sample et al., 2012; Parker et al., 2019). A number of recent papers investigated sAC and its activation by bicarbonate seeking to identify what was sensed by sAC, where in the cell the reactions took place and what the purpose of this signaling system was (Acin-Perez et al., 2009; Valsecchi et al., 2013; Valsecchi et al., 2014; Acin-Perez et al., 2011). This work by Manfredi et al., although widely cited, is controversial (Di Benedetto et al., 2021; Covian et al., 2014; Lefkimmiatis et al., 2013; Wang et al., 2016). These publications concluded that the bicarbonate-activated sAC works within the mitochondrial matrix to produce cAMP, which activates PKA, which in turn increases ATP production. It was also reported that the role of this signaling system is to sense and respond to ‘nutrient availability’ (Acin-Perez et al., 2009; Valsecchi et al., 2013; Valsecchi et al., 2014; Acin-Perez et al., 2011). Despite seeming straightforward findings, all key mechanistic findings have been disputed by other investigators (Di Benedetto et al., 2021; Lefkimmiatis et al., 2013; Covian et al., 2014; Wang et al., 2016). While almost all published investigations agree that elevated bicarbonate leads to an increase in ATP production, they disagree on how this happens. The role of PKA in the matrix is disputed (Di Benedetto et al., 2021; Lefkimmiatis et al., 2013; Covian et al., 2014; Wang et al., 2016) as is the matrix localization of native sAC (Covian et al., 2014; Wang et al., 2016). Furthermore, key pharmacological tools used in the original study were later found to cause acute mitochondrial toxicity rather than selectively inhibit sAC (Wang et al., 2016; Di Benedetto et al., 2013). In light of these major points of disagreement, we investigated sAC quantitatively with high spatial resolution to determine where sAC was located, how it worked, where the intermediate signals were generated, and importantly, what its overall role in cellular metabolism may be. Additionally, we decided to use fresh and functional heart tissue as our source of mitochondria because it is one of the most metabolically dynamic tissues. Additionally, we reasoned that by discovering how sAC worked in cardiac mitochondria, we were likely to provide a solid physiological framework to understand how sAC worked in other tissues. In contrast, the original investigations (Acin-Perez et al., 2009; Valsecchi et al., 2013; Valsecchi et al., 2014; Acin-Perez et al., 2011) of sAC used mitochondria from cells in which mitochondrial ATP production normally operates over a very narrow range (van Dyke et al., 1983; Bracht et al., 2016; Shadrin et al., 2015; do Nascimento et al., 2018; de Medeiros et al., 2015; Colturato et al., 2012) and sAC signaling itself has a low signal-to-noise ratio. To augment the signal, transgenic sAC was overexpressed in the model cells thereby possibly obscuring the role of native sAC. When each element was studied, we found that all the key conclusions of the initial investigation by Manfredi et al. linking sAC and PKA in the matrix to ATP production were disputed by our data and those of other investigators (see Table in Supplementary file 1). Additionally, we found that sAC signaling reports on nutrient consumption not on nutrient ‘availability’ (Acin-Perez et al., 2009; Valsecchi et al., 2013; Valsecchi et al., 2014; Acin-Perez et al., 2011). Furthermore, a major finding of the work presented here is that mitochondrial sAC signaling works independently of, yet in conjunction with, mitochondrial Ca2+ signaling system. Together they report on the full scale of ATP needs in health and disease. This resonant or complementary signaling between the two systems enables the mitochondria in cardiac ventricular myocytes to produce ATP continuously at levels appropriate for the physiological demands of the heart. Results An investigation is presented that centers on where sAC is located within the ventricular myocyte, how that position affects its function and how bicarbonate increases ATP production through sAC. That investigation requires high-resolution imaging and quantitative biochemical investigations to determine where sAC resides and what its product, cAMP, controls. This work then examines how the local potential effectors of cAMP – specifically EPAC1 and PKA – may be connected to the dynamic control of ATP production by mitochondria. Under these conditions, sAC signaling is examined in context of Ca2+ signaling to examine their interactions. To place the results in a physiological context they are discussed in the setting of the working heart. Localization of sAC in ventricular myocytes Immunofluorescence localization of sAC at high resolution was undertaken using the Zeiss Airyscan 880 super-resolution microscope and the R21 monoclonal anti-sAC antibody (Zippin et al., 2003; Wang et al., 2016; Zippin et al., 2013; Fazal et al., 2017; Liu et al., 2019) validated in sAC knockout mice (Chen et al., 2013). Figure 1A shows simultaneously acquired images of a ventricular myocyte co-labeled for sAC, ATP synthase (complex-V or CV of the Electron Transport Chain, or ETC), and F-actin. ATP synthase is a component of the inner mitochondrial membrane (IMM) while F-actin filaments are extramitochondrial. Figure 1A (left panel) shows the location of sAC (yellow), with a zoomed-in region in the upper right corner. The sAC labeling clearly localizes to the mitochondria, which appear with punctate features (roughly 1 μm across) occurring in rows between the F-actin-containing (blue) myofilaments (see Figure 1A; Barth et al., 1992). This conclusion is strengthened by the similarity of the distribution of sAC to that of ATP synthase (red in Figure 1A). This point is well illustrated in the merged image. The mitochondrial localization of sAC is also supported by colocalization analysis measured by the Pearson’s correlation coefficient between CV and sAC (Figure 1B). Likewise, in Figure 1C, D, it is clear that neither protein is colocalized with the contractile filament (F-actin). This examination of the transverse axis of the ventricular myocytes shows that sAC and CV occur at the same location (Figure 1C) and the same frequency in the myocyte. While F-actin is also found at the same frequency as sAC and CV, F-actin is only found between them (Figure 1D, E). While sAC and ATP synthase are co-localizing to mitochondria, the question remains, where in the mitochondria is sAC located. There are four possible locations to consider: the outer mitochondrial membrane (OMM), the intermembrane space (IMS), the IMM, and the matrix. Figure 1 with 1 supplement see all Download asset Open asset Mitochondrial localization of soluble adenylyl cyclase (sAC). (A) Airyscan super-resolution fluorescence images of a cardiomyocyte (left to right) immuno-labeled for sAC (yellow) and ATP synthase (red), and loaded with Alexa Fluor 488 phalloidin (1 M) to label the F-actin within the contractile filaments (cyan). Merged image (far right) shows superimposed labeling of sAC, ATP synthase, and F-actin. (B) Pearson’s correlation analysis of subcellular colocalization of sAC with ATP synthase (yellow), sAC with F-actin filaments (cyan), and ATP synthase with F-actin filaments (red), (n = 12 cells). (C) Fluorescence profile of sAC (yellow) and ATP synthase (red). (D) Fluorescence profile of F-actin (cyan) and ATP synthase (red). (E) Mean of peak interval for data as in (C, D) (n = 13 cells). (F) Airyscan super-resolution images of isolated cardiac mitochondria immuno-labeled for sAC (yellow, left), Tom20 (blue, center), and merged images (right). (G) Fluorescence profile of sAC (yellow) and Tom20 (blue) for individual mitochondrial images as in F (n = 36). Mean peak intervals (inμm) are indicated (n = 36 mitochondria). Data in (B, E and G) are mean ± standard error of the mean (SEM). One-way two-tailed analysis of variance (ANOVA) with Bonferroni correction in B, E. ***p < 0.001. NS = not significant (p > 0.05). Figure 1—source data 1 Numeric source data for Figure 1. https://cdn.elifesciences.org/articles/84204/elife-84204-fig1-data1-v2.xlsx Download elife-84204-fig1-data1-v2.xlsx To narrow down the possibilities, super-resolution microscopy was applied to isolated mitochondria (Figure 1F, G), using immunolabeling for sAC and Tom20 (Translocase of the Outer Membrane 20), an abundant OMM protein (Omura, 1998). The resolution achieved clearly distinguished between OMMs of adjacent mitochondria and showed that sAC is located within the perimeter of the OMM but is not co-localized with Tom20. Since sAC does not contain any membrane-spanning sequences, it is unlikely to be embedded in the IMM. This then suggests that sAC is in the IMS or in the matrix or in both compartments. Bicarbonate sensing in isolated cardiac mitochondria There are two important functional distinctions between sACs and tmACs. First, sAC is directly activated by HCO3− to produce cAMP while the tmACs are not (Buck et al., 1999; Litvin et al., 2003). Second, sAC is not activated by forskolin to produce cAMP (Buck et al., 1999; Litvin et al., 2003), while all nine types of tmACs are (Zhang et al., 1997). We used these established features of the different kinds of ACs to determine the identity of the ACs in cardiac mitochondria. As indicated by the experiments of Figure 2A, treating isolated mitochondria with bicarbonate robustly activates cAMP production (from 0 to physiological bicarbonate of 15 mM) while treatment with forskolin (25 µM) had no effect on cAMP production. The simple conclusion from these experiments is that the cardiac mitochondria contain functional sAC and no detectable tmACs. Figure 2 with 2 supplements see all Download asset Open asset Mitochondrial function of soluble adenylyl cyclase (sAC). (A) Quantitative ELISA measurements of cAMP inside isolated mitochondria (pmol/mg). cAMP is measured following treatment with indicated concentrations of bicarbonate (HCO3−), which activates sAC, and forskolin, which activates transmembrane adenylyl cyclases (tmACs) (n = 6, 6, 5 for 0, 10, and 15 mM [HCO3−], respectively, mitochondria are isolated independently from four hearts). (B) The dependence of ATP production on [HCO3−] and [IBMX]. Sigmoidal fits to the data are shown. (C) The sensitivity of ATP production to phosphodiesterase inhibition (with 50 μM IBMX) at different concentrations of [HCO3−]. (D) Airyscan super-resolution images of isolated cardiac mitochondria immuno-labeled for CAXIV (red, left), Tom20 (blue, center), and merged images (right). (E) Fluorescence profile of CAXIV (red) and Tom20 (blue) for individual mitochondrial images as in D. Mean peak intervals (in μm) are indicated (n = 32 mitochondria). (F) Airyscan super-resolution images of isolated cardiac mitochondria immuno-labeled for CAXIV (red, left), sAC (yellow, center), and merged images (right). (G) Fluorescence profile of CAXIV (red) and sAC (yellow) for individual mitochondrial images as in F (n = 32). For (B, C), n = 12–16 independent experiments per group, mitochondria are isolated independently from four hearts. Data in (A–C) are mean ± standard error of the mean (SEM). One-way two-tailed analysis of variance (ANOVA) with Bonferroni correction in (A, C). *p < 0.05, **p < 0.01, ***p < 0.001. (H) Structure of a typical mitochondria from a rat cardiomyocyte showing the intimate arrangement and packing of the cristae and matrix and the IMS. The image is a slice from an electron microscopic tomogram with membranes traced for clarity and quantitation. As shown in the accompanying video of a similar tomogram (Figure 2—video 1), crista compartments are formed by closely spaced parallel membranes that extend out of the plane of this image. The functional ‘intermembrane space’ (IMS) consists of the narrow peripheral space between the inner boundary membrane (IBM) and the outer mitochondrial membrane (OMM) plus the spaces inside the cristae that connect to the peripheral space through narrow (20–40 nm) crista junctions (represented by small white circles) (Frey and Mannella, 2000). Stereological analysis (Smith and Page, 1976) indicates the cristae contain 85% of the total IMS in this mitochondrion, a value that increases to 91% for a mitochondrion with similar crista packing and an area ×2.5 larger. Scale bar is 250 nm. Figure 2—source data 1 Numeric source data for Figure 2. https://cdn.elifesciences.org/articles/84204/elife-84204-fig2-data1-v2.xlsx Download elife-84204-fig2-data1-v2.xlsx Further definition of the behavior of the mitochondrial sAC system was provided by use of the membrane permeable PDE inhibitor IBMX, 3-isobutyl-1-methylxanthine (Wells and Miller, 1988). PDEs are plentiful in nearly all cells that use cAMP and are distributed to help focus the action of the cyclic nucleotide to a local region (Baillie et al., 2019; Maurice et al., 2014). The PDEs limit the diffusion of the cAMP away from their intended target. This has often been likened to a ‘firewall’ against the excessive signaling of cAMP within a region of a cell or within an organelle (Lomas and Zaccolo, 2014; Baillie et al., 2019; Mongillo et al., 2004; Burdyga et al., 2018). Conversely, inhibition of endogenous PDEs within mitochondrial compartments would be expected to enhance cAMP signaling within those compartments. The effects of the PDE inhibitor IBMX on the action of sAC in isolated mitochondria are presented in Figure 2B, C. Specifically, physiological concentrations of HCO3− (10 and 15 mM) significantly increase ATP production, and IBMX elevates it further, for a combined effect of doubling ATP output. These data are consistent with a cAMP signaling system inside mitochondria that responds to bicarbonate by activating sAC, that is modulated by PDEs that reduce cAMP levels, and that regulates the primary function of these mitochondria, the generation of ATP. The HCO3− that activates sAC in mitochondria is produced spontaneously by hydration of CO2, a reaction that is greatly accelerated by the enzyme carbonic anhydrase (CA). As shown in Figure 2D, E, we find that cardiac mitochondria contain CA-XIV (also see Figure 2—figure supplement 1). As with sAC (Figure 1F, G), co-labeling for TOM20 indicates that CA-XIV is localized to an interior compartment of the mitochondria. Thus, the two critical upstream components of the CO2/bicarbonate signaling pathway reside inside the mitochondria of ventricular myocytes. Nevertheless, with the resolution achieved, it cannot be determined whether sAC and CA-XIV are both inside the matrix, the IMS, or both compartments. If sAC and CA-XIV both reside inside the mitochondrial matrix, where CO2 is produced, it would enable sAC to rapidly track changes in CO2 production in terms of [HCO3−]. However, the alternative possibility in which sAC is in the IMS will also enable effective tracking of CO2 production by sAC despite the low permeability of the IMM to the bicarbonate anion (Arias-Hidalgo et al., 2016). Mitochondria in the interfibrillar regions of cardiomyocytes have cristae with extended flat surfaces packed closely together, creating stacks of thin alternating layers of intracristal and matrix spaces (Figure 2I and Picard et al., 2013). This ‘lamellar’ membrane morphology combined with the very high permeability of the IMM to CO2 (Arias-Hidalgo et al., 2016; Itel et al., 2012) is ideal for gas exchange between the two compartments (Noble, 1983). In fact, most (85% or more) of the IMS volume in these lamellar mitochondria is in the crista layers adjacent to matrix and not in the peripheral IMS abutting the OMM and cytosol (see legend, Figure 2I). Thus, the level of CO2 inside cristae should rapidly adjust in parallel with that in the matrix where it is generated, and spontaneous CO2 hydration should generate steady-state levels of HCO3− that track energy consumption. The presence of CA in the same subcompartment as sAC would speed up the response time of the metabolic tracking, but steady-state bicarbonate levels inside cristae should vary with matrix CO2 even in the absence of CA. Mitochondrial cAMP signaling The above results indicate that the cAMP generated inside mitochondria by sAC and regulated by PDEs directly modulates ATP production by mitochondria. To investigate this process in more detail, the characteristics of the sAC signaling system aid us in the design of experiments to quantitatively assess the contributions of sAC to metabolic regulation of the heart. A critical characteristic is that the sAC output signal following activation is cAMP, which cannot readily cross a membrane due to its hydrophilicity (Lefkimmiatis et al., 2013; Di Benedetto et al., 2013). We can use this information to determine in which compartment sAC signaling is working in ventricular myocytes. In the special case of an isolated mitochondrial preparation, nucleotides like cAMP applied to the extra-mitochondrial space gain entry to the mitochondrial IMS through the numerous VDAC pores in the OMM (Rostovtseva and Colombini, 1996; Rostovtseva and Colombini, 1997; Rostovtseva et al., 2002). However, cAMP has been shown to be excluded from the matrix because the IMM is impermeable to it (Lefkimmiatis et al., 2013; Di Benedetto et al., 2013). The experiments shown in Figure 3 use this understanding to investigate sAC signaling in more detail. Figure 3 Download asset Open asset cAMP control of mitochondrial ATP production. (A) Left, sensitivity of mitochondrial ATP production to cAMP and phosphodiesterase inhibition (with 50 μM IBMX). Measurements are carried out at low [Ca2+]m (<200 nM). Right, sensitivity of mitochondrial ATP production to cAMP, EPAC1 inhibition (25 μM CE3F4), and protein kinase A (PKA) inhibition (1 μM H89). Measurements were carried out at low [Ca2+]m (<200 nM). (B) Same as A but at high [Ca2+]m (>2 μM). (C) Pull-down and immunoblot analysis for the active form of Rap1 (GTP-bound Rap1) in isolated mitochondria stimulated with cAMP. γ-GTP is used to maximally activate Rap1. (D) The relative amounts of active Rap1 (mitochondria isolated independently from n = 9 hearts). (E) Schematic diagram showing the likely locations of key proteins in soluble adenylyl cyclase (sAC) mitochondrial signaling. While sAC is clearly located within the intermembrane space (IMS), and both EPAC and Rap1 have signaling domains in the IMS, it is uncertain how the ‘target’ protein(s) are activated to increase ATP production. For (A, B), n = 12–16 independent experiments per group, mitochondria are isolated independently from four hearts. In (D), mitochondria are isolated independently from nine hearts. Data in (A, B) and (D) are mean ± standard error of the mean (SEM). One-way two-tailed analysis of variance (ANOVA) with Bonferroni correction in (A, B). One-sample t-test for (D). *p < 0.05, **p < 0.01, ***p < 0.001. Figure 3—source data 1 Western blot analysis in Figure 3C, D (anti-Rap1). https://cdn.elifesciences.org/articles/84204/elife-84204-fig3-data1-v2.zip Download elife-84204-fig3-data1-v2.zip Figure 3—source data 2 Original file No. 1 for western blot analysis in Figure 3C, D (anti-Rap1). https://cdn.elifesciences.org/articles/84204/elife-84204-fig3-data2-v2.zip Download elife-84204-fig3-data2-v2.zip Figure 3—source data 3 Original file No. 2 for western blot analysis in Figure 3C, D (anti-Rap1). https://cdn.elifesciences.org/articles/84204/elife-84204-fig3-data3-v2.zip Download elife-84204-fig3-data3-v2.zip Figure 3—source data 4 Original file No. 3 for western blot analysis in Figure 3C, D (anti-Rap1). https://cdn.elifesciences.org/articles/84204/elife-84204-fig3-data4-v2.zip Download elife-84204-fig3-data4-v2.zip Figure 3—source data 5 Original file No. 4 for western blot analysis in Figure 3C, D (anti-Rap1). https://cdn.elifesciences.org/articles/84204/elife-84204-fig3-data5-v2.zip Download elife-84204-fig3-data5-v2.zip Figure 3A shows that ATP production in isolated mitochondria with low [Ca2+]m is small, about 2 nM ATP/mg/s and is not significantly increased by the PDE inhibitor IBMX. These data suggest that there is little or no cAMP present in the mitochondrial compartment in which sAC is located under these conditions. However, when cAMP is added extra-mitochondrially to volume that also contains the mitochondria, there is a clear increase of approximately 50% in ATP production that is further augmented to 100% (doubling) upon addition of IBMX. This finding re-emphasizes that the sAC is in the mitochondria and, moreover, that it is located in the IMS, that is, accessible to externally added cAMP. While nucleotides can enter the IMS through VDAC (Rostovtseva and Colombini, 1996; Rostovtseva and Colombini, 1997; Rostovtseva et al., 2002), it has been shown that cAMP cannot cross the IMM (Lefkimmiatis et al., 2013; Di Benedetto et al., 2013). Thus, the production of cAMP by sACs activates a target in the IMS which, in turn, produces a signaling cascade that can activate one or more targets in the IMM and/or matrix space. There are two possible direct targets for this locally elevated cAMP – namely, PKA and/or EPAC. To examine these targets, experiments were conducted under conditions where matrix Ca2+ levels ([Ca2+]m) were measured quantitatively and kept low (under 200 nM). Figure 3A (right panel – black bars) shows that, in the absence of added extra-mitochondrial cAMP, blocking PKA (with its inhibitor H89) does not affect ATP production nor does the blocking of EPAC1 by the inhibitor CE3F4. However, when cAMP is applied extra-mitochondrially in low [Ca2+]m (green bars) there is an increase in ATP production that is inhibited by CE3F4 but not H89. From these experiments, we conclude that mitochondrial EPAC1 is the target protein activated by cAMP applied to isolated mitochondria, and" @default.
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• W4381546844 date "2023-05-16" @default.
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• W4381546844 title "Author response: Calcium and bicarbonate signaling pathways have pivotal, resonating roles in matching ATP production to demand" @default.
• W4381546844 doi "https://doi.org/10.7554/elife.84204.sa2" @default.
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