Calcium and bicarbonate signaling pathways have pivotal, resonating roles in matching ATP production to demand
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eLife assessment
The important work presented here provides findings that substantially advance our understanding of a major research question into how bicarbonate/CO2 signaling regulates cardiac mitochondrial energy supply. The methods, data, and analyses broadly support the claims with only minor weaknesses concerning the exact spatial location of the enzymes involved. The work will be of broad interest to cell biologists and biochemists interested in metabolic control.
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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 Ca 2+ ([Ca 2+ ] 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 -- CO 2 /bicarbonate. CO 2 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 ( E xchange P rotein directly A ctivated by c AMP) 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, [Ca 2+ ] 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.
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Author Response
Reviewer #1 (Public Review):
The role of HCO3 (or possibly CO2) in regulating sACs is well established yet its physiological context is less clear. The heart is indeed an excellent choice of organ to study this. Isolated mitochondria offer a tractable model for studying the model, although are not without limitations. The quality of recordings is very high, as judged by the consistency of results (i.e. lack of clustering between biological repeats). My primary concern is about distinguishing the effect of pH and HCO3. A rise in HCO3 will also raise pH unless this had been compensated by CO2. It is unclear, from the legend or results, if the bicarbonate effect is due to HCO3 or pH. Was pH controlled by matching the rise in HCO3 with an appropriate level of CO2? The swings in pH are likely to be very large and, …
Author Response
Reviewer #1 (Public Review):
The role of HCO3 (or possibly CO2) in regulating sACs is well established yet its physiological context is less clear. The heart is indeed an excellent choice of organ to study this. Isolated mitochondria offer a tractable model for studying the model, although are not without limitations. The quality of recordings is very high, as judged by the consistency of results (i.e. lack of clustering between biological repeats). My primary concern is about distinguishing the effect of pH and HCO3. A rise in HCO3 will also raise pH unless this had been compensated by CO2. It is unclear, from the legend or results, if the bicarbonate effect is due to HCO3 or pH. Was pH controlled by matching the rise in HCO3 with an appropriate level of CO2? The swings in pH are likely to be very large and, potentially, a confounding factor. Certainly, there will be an effect on the proton motive force. A more informative test would compare the effect of 0 CO2/0HCO3 at a pH set to say 7.2, 2.5% CO2/7.5 mM HCO3, and then 5% CO2/15 mM HCO3, etc. Control experiments would then repeat these observations over a range of pH (at zero CO2/HCO3) and over a range of CO2 (at constant HCO3). Data for zero bicarbonate are not informative, as this will never be a physiological setting (results claim 0-15 mM bicarb to represent physiology). Importantly, there seems to be no significant difference in 2A between 10 v 15 mM bicarb, i.e. the physiological range.
Thank you for your clear discussion and suggestions. We agree that pH must be controlled in these experiments to avoid the confounding situation you describe. In fact, pH was carefully controlled but this was not described adequately. To make this clearer the methods section was modified.
We agree that bicarbonate concentration is above 0 in living tissue. We used that value only as a reference in Figure 2A to examine which type of adenylyl cyclase (AC) is inside mitochondria, i.e., bicarbonate-activated soluble AC as opposed to transmembrane AC which is not bicarbonate activated. The wording has been corrected to better describe this. The question of what the physiological consequences are require an assay with higher signal-to-noise ratio. In effect, this is achieved in the experiments of Fig. 2B-C which show that physiologically relevant changes in bicarbonate have a large and significant influence on mitochondrial ATP production.
There is also a question on the validity of the model. A rise in respiratory rate will produce more CO2 in the matrix. This may raise matrix HCO3, and stimulate sACs therein, but the authors claim sACs are in the IMS, rather than the matrix. Since HCO3 is impermeable, it is unclear how sACs would detect HCO3 beyond the IMM. CO2 escaping the matrix will enter the continuum of the cytoplasmic space, which has finely controlled pH. Since membranes (including IMM) are highly permeable to CO2, the gradient between matrix and cytoplasm will be small (i.e. you only need a small gradient to drive a big flux, if the permeability is massive). Since CO2 can dissipate over a large volume, it is unlikely to accumulate to any degree. CO2 will be in equilibrium with HCO3 and pH (because there are carbonic anhydrases available). Since the cytoplasm has near-constant pH, [HCO3] must also be close to constancy. It is therefore difficult to imagine how HCO3 could change dramatically to meaningfully affect sACs and hence cAMP. Evidence for major changes in IMS pH in intact cells during swings of respiratory activity would be required to make this point. Indeed, for that reason, it would be more sensible to anchor sACS in the matrix, as there, HCO3 could rise to high levels, as it is impermeable, i.e. could be confined within the mitochondrion. I am therefore not convinced the numbers are favorable to the proposed mechanism to be meaningful physiologically.
The question of how CO2/bicarbonate signaling can work in the intermembrane space (IMS) is explicitly addressed in the revisions in the results and discussion sections. CO2, a membrane permeable gas, can easily cross the IMM (permeability coefficient of 0.01 to 0.33 cm/s). Once in the IMS, CO2 can combine with water to produce bicarbonate, a fast reaction in a physiological context (i.e. mM/s in physiological saline) that can be accelerated to nearly diffusion limits by carbonic anhydrase if present. The assumption that all the “CO2 escaping the matrix will enter the continuum of the cytoplasmic space” is not supported by the structure of cardiomyocyte mitochondria. As illustrated in new Fig 2H, only a small fraction (9-15%) of the IMS occurs along the mitochondrial periphery adjacent to the outer membrane and cytosol. Most of the IMS is contained inside the cristae (the intracristal space or ICS), which in interfibrillar mitochondrion are composed of closely packed extended flat membranes that create scores of alternating layers of matrix and ICS ideal for rapid gas exchange between compartments. The cristae are connected to the peripheral IMS through narrow “crista junction” openings that restrict solute diffusion between the peripheral IMS and ICS. Thus, rather than being dominated by the ionic equilibria of the cytosol, the crista compartments are functionally distinct from the peripheral IMS region and cytosol. We cite recent publications using super-resolution light microscopy in which gradients of 0.3-0.4 pH units (dependent on metabolic state) have been detected along the cristae of respiring cells and between the peripheral IMS and crista interiors. These diffusion effects would likely be even more pronounced for cardiac muscle mitochondria, which have larger, more densely packed lamellar cristae than other cell types. Thus, the microenvironment of the cristae provide confined spaces in close communication with the matrix CO2 production, and ideal for operation of a sAC signaling system.
Reviewer #2 (Public Review):
The authors explore the role of bicarbonate-regulated soluble adenylate cyclase in modulating cardiac mitochondrial energy supply. In isolated rat mitochondria, they show that cyclic AMP (but not the permeable cAMP analog 8-Br-cAMP) increases ATP production via a Ca-independent mechanism at a location in the intermembrane space of the mitochondria, rather than in the matrix, as previously reported. Moreover, they show that inhibition of EPAC, but not PKA, inhibits the response. The effect required supplementing the mitochondria with GTP and GDP to facilitate activation of the EPAC effector GTPase Rap1. The study provides interesting new information about how the heart might adapt to changes in energy supply and demand through complementary regulatory processes involving both Ca and cyclic AMP.
The authors nicely demonstrate that soluble adenylate cyclase is localized to mitochondria. They argue, based on the effects of cyclic AMP, which is accessible to the mitochondrial intermembrane space (IMS) but not the matrix, that the signalling pathway is located in the IMS. They also find that EPAC/Rap1 is the likely downstream effector of cyclic AMP, through yet unknown targets regulating oxidative phosphorylation.
A weakness is that the components of signaling (sAC, EPAC, and rap1) are not definitively localized to a specific mitochondrial compartment using the superresolution imaging methods employed.
Thank you for the concise summary of key findings. While the super-resolution data indicates sAC and CA are localized to the interior of the mitochondria (possibly co-localized in the same subspace), identification of the particular microcompartment is not possible from the imaging data alone. We explain clearly in the manuscript that the functional experiments are critical to the conclusion that the sAC signaling pathway most likely operates in the IMS.
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eLife assessment
The important work presented here provides findings that substantially advance our understanding of a major research question into how bicarbonate/CO2 signaling regulates cardiac mitochondrial energy supply. The methods, data, and analyses broadly support the claims with only minor weaknesses concerning the exact spatial location of the enzymes involved. The work will be of broad interest to cell biologists and biochemists interested in metabolic control.
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Reviewer #1 (Public Review):
The role of HCO3 (or possibly CO2) in regulating sACs is well established yet its physiological context is less clear. The heart is indeed an excellent choice of organ to study this. Isolated mitochondria offer a tractable model for studying the model, although are not without limitations. The quality of recordings is very high, as judged by the consistency of results (i.e. lack of clustering between biological repeats). My primary concern is about distinguishing the effect of pH and HCO3. A rise in HCO3 will also raise pH unless this had been compensated by CO2. It is unclear, from the legend or results, if the bicarbonate effect is due to HCO3 or pH. Was pH controlled by matching the rise in HCO3 with an appropriate level of CO2? The swings in pH are likely to be very large and, potentially, a confounding …
Reviewer #1 (Public Review):
The role of HCO3 (or possibly CO2) in regulating sACs is well established yet its physiological context is less clear. The heart is indeed an excellent choice of organ to study this. Isolated mitochondria offer a tractable model for studying the model, although are not without limitations. The quality of recordings is very high, as judged by the consistency of results (i.e. lack of clustering between biological repeats). My primary concern is about distinguishing the effect of pH and HCO3. A rise in HCO3 will also raise pH unless this had been compensated by CO2. It is unclear, from the legend or results, if the bicarbonate effect is due to HCO3 or pH. Was pH controlled by matching the rise in HCO3 with an appropriate level of CO2? The swings in pH are likely to be very large and, potentially, a confounding factor. Certainly, there will be an effect on the proton motive force. A more informative test would compare the effect of 0 CO2/0HCO3 at a pH set to say 7.2, 2.5% CO2/7.5 mM HCO3, and then 5% CO2/15 mM HCO3, etc. Control experiments would then repeat these observations over a range of pH (at zero CO2/HCO3) and over a range of CO2 (at constant HCO3). Data for zero bicarbonate are not informative, as this will never be a physiological setting (results claim 0-15 mM bicarb to represent physiology). Importantly, there seems to be no significant difference in 2A between 10 v 15 mM bicarb, i.e. the physiological range.
There is also a question on the validity of the model. A rise in respiratory rate will produce more CO2 in the matrix. This may raise matrix HCO3, and stimulate sACs therein, but the authors claim sACs are in the IMS, rather than the matrix. Since HCO3 is impermeable, it is unclear how sACs would detect HCO3 beyond the IMM. CO2 escaping the matrix will enter the continuum of the cytoplasmic space, which has finely controlled pH. Since membranes (including IMM) are highly permeable to CO2, the gradient between matrix and cytoplasm will be small (i.e. you only need a small gradient to drive a big flux, if the permeability is massive). Since CO2 can dissipate over a large volume, it is unlikely to accumulate to any degree. CO2 will be in equilibrium with HCO3 and pH (because there are carbonic anhydrases available). Since the cytoplasm has near-constant pH, [HCO3] must also be close to constancy. It is therefore difficult to imagine how HCO3 could change dramatically to meaningfully affect sACs and hence cAMP. Evidence for major changes in IMS pH in intact cells during swings of respiratory activity would be required to make this point. Indeed, for that reason, it would be more sensible to anchor sACS in the matrix, as there, HCO3 could rise to high levels, as it is impermeable, i.e. could be confined within the mitochondrion. I am therefore not convinced the numbers are favorable to the proposed mechanism to be meaningful physiologically.
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Reviewer #2 (Public Review):
The authors explore the role of bicarbonate-regulated soluble adenylate cyclase in modulating cardiac mitochondrial energy supply. In isolated rat mitochondria, they show that cyclic AMP (but not the permeable cAMP analog 8-Br-cAMP) increases ATP production via a Ca-independent mechanism at a location in the intermembrane space of the mitochondria, rather than in the matrix, as previously reported. Moreover, they show that inhibition of EPAC, but not PKA, inhibits the response. The effect required supplementing the mitochondria with GTP and GDP to facilitate activation of the EPAC effector GTPase Rap1. The study provides interesting new information about how the heart might adapt to changes in energy supply and demand through complementary regulatory processes involving both Ca and cyclic AMP.
The authors …
Reviewer #2 (Public Review):
The authors explore the role of bicarbonate-regulated soluble adenylate cyclase in modulating cardiac mitochondrial energy supply. In isolated rat mitochondria, they show that cyclic AMP (but not the permeable cAMP analog 8-Br-cAMP) increases ATP production via a Ca-independent mechanism at a location in the intermembrane space of the mitochondria, rather than in the matrix, as previously reported. Moreover, they show that inhibition of EPAC, but not PKA, inhibits the response. The effect required supplementing the mitochondria with GTP and GDP to facilitate activation of the EPAC effector GTPase Rap1. The study provides interesting new information about how the heart might adapt to changes in energy supply and demand through complementary regulatory processes involving both Ca and cyclic AMP.
The authors nicely demonstrate that soluble adenylate cyclase is localized to mitochondria. They argue, based on the effects of cyclic AMP, which is accessible to the mitochondrial intermembrane space (IMS) but not the matrix, that the signalling pathway is located in the IMS. They also find that EPAC/Rap1 is the likely downstream effector of cyclic AMP, through yet unknown targets regulating oxidative phosphorylation.
A weakness is that the components of signaling (sAC, EPAC, and rap1) are not definitively localized to a specific mitochondrial compartment using the superresolution imaging methods employed.
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