COMMENTARY
The growing importance of mitochondrial calcium in health and disease Liron Boyman1, George S. B. Williams1, and W. J. Lederer2 Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201
In Santulli et al. (1), RyR2-dependent Ca2+ leak from the sarcoplasmic reticulum (SR) is presented as a cause of mitochondrial dysfunction that contributes to heart failure (HF). The authors provide evidence in support of this provocative and important hypothesis. The strength of this paper comes primarily from the suggestions it provides that associate maladaptive changes of key physiological
signaling pathways in the heart with the development of HF. A secondary benefit of the work is the array of questions related to the findings of the authors raised by them and provoked by other recent reports (discussed below). The thinking is that RyR2-based SR Ca2+ leak increases in HF and thereby produces a pathologically elevated mitochondrial matrix
A
B
Ca2+ ([Ca2+]m) level along with excessive mitochondria reactive oxygen species (ROS) production (1). Together these elements form a causal chain in the development of HF following myocardial infarction (MI). With this argument, the paper throws down the gauntlet to the scientific community, challenging investigators to determine quantitatively how these elements interact to produce HF. Such investigations, armed with a growing arsenal of novel tools, hold an exciting potential for us to better understand cellular, organellar, and molecular dysfunctions that underlie the development of HF. Specifically, we need to understand how the transformation from healthy myocyte to dysfunctional myocyte occurs. The quantitative aspects of this process are critical because under normal conditions only modest increases in [Ca2+]m are required to regulate ATP production (2–4). Similarly, small amounts of ROS generation are a normal part of ATP generation by the electron transport chain (ETC) (5). To distinguish physiologic regulation from maladaptive pathophysiologic changes in [Ca2+]m and ROS generation in HF, molecular details of the changes must be investigated quantitatively with good temporal resolution. Furthermore, there are no a priori reasons to believe that all of the changes in cardiac function in HF are primarily mitochondrial-centric, even in the MI model of HF presented by Santulli et al. (1). Alterations in activation, production, and degradation of many parallel cytosolic proteins and processes will almost certainly occur even when HF is associated with leaky RyR2s. Santulli et al. (1) thus identify SR–mitochondrial interactions mediated by Ca2+ that contribute to HF (Fig. 1). Teasing apart the quantitative molecular and cellular details of the development of HF due to this dynamic and spatially resolved process is a huge task. Here we outline a simplified “challenge of Santulli” that arises from the PNAS paper and from a treasure trove of recent, exciting, Author contributions: L.B., G.S.B.W., and W.J.L. designed research, performed research, analyzed data, and wrote the paper.
Fig. 1. Critical observations related to mitochondrial dynamics and the etiology of HF. (A) Schematic diagram showing the spatial distribution of mitochondrial Ca2+ signaling components in heart. The ends of each intermyofibrillar mitochondrion are in close proximity (∼100 nm) to the Ca2+ release units (CRUs) that are located between the transverse-tubule (TT) and junctional SR (jsr) membranes [also see Williams et al. (24)]. (B) Venn diagram highlighting key differences and similarities in WT mice and mice with leaky RyRs (S2808D) and how these properties develop during HF. Observations related to the critical questions (outlined here) provoked by Santulli et al. (1) are bolded. 11150–11151 | PNAS | September 8, 2015 | vol. 112 | no. 36
The authors declare no conflict of interest. See companion article on page 11389. 1
L.B. and G.S.B.W. contributed equally to this work.
2
To whom correspondence should be addressed. Email: jlederer@ umaryland.edu.
www.pnas.org/cgi/doi/10.1073/pnas.1514284112
Question 1: How do leaky RyR2 channels lead to elevated [Ca2+]m content in the steadystate? Overall, cellular Ca2+ balance depends on Ca2+ pump-leak balance across the sarcolemmal membrane (14) and not the leakiness of internal organelles. If only the leakiness of RyR2s were to increase, then the global cellwide effect would be to deplete the cell of Ca2+, shifting Ca2+ from the SR to the extracellular volume, including the circulation. If mitochon drial [Ca2+]m increases when RyR2s become leaky, additional changes must be occurring and these need to be identified and measured quantitatively. Question 2. How and why does mitochondrial ROS increase in HF? To determine this, a host of things must be measured including metabolic substrate, redox state in mitochondria and in the cytosol, partial pressure of O2, ATP and ADP levels, the potential across the inner membrane (ΔΨm), and more. Additionally, nonmitochondrial ROS sources should be measured. By knowing what cellular processes change and when they change in relationship to the ROS production, it should be possible to understand mechanistically HF changes and the possible role of increases in ROS production. Question 3. What is the cellular [ATP]i level before, during the onset of HF, and during
Boyman et al.
terminal HF? [ATP]i can now be measured in live functioning cells using fluorescence sensors (15, 16). It would be valuable to measure [ATP]i in the context of SR Ca2+ leak and changes of [Ca2+]m at different stages of HF. Also see question 5. Question 4. Are the mitochondrial dysfunctions observed in HF associated with activation of mPTP? Do changes in mitochondrial function correlate with changes in [ATP]i, [Ca2+]m, and ROS generation, and, if so, how tightly correlated are the changes? Because [Ca2+]m and ROS are known activators of mPTP (17), are the S2808D cardiomyocytes more prone to mPTP activation and the corresponding cell injury? Alternatively, under such conditions, does mPTP activation have an inverse (i.e., ameliorating) (18) effect by purging the mitochondria of excess Ca2+, and therefore sustaining mitochondrial metabolism? Furthermore, can these measurements help us to determine the molecular identity of mPTP?
the stress created by the loss of previously healthy and functional tissue. Just as “orphan RyR2s” develop due to the reorganization of the transverse tubules in pressure-overload HF (19–21), so too does this happen following MI in human and model disease (22). Thus, the development of cellular and mitochondrial dysfunction following MI seems to depend on the consequences of the initiating insult, which include changes in Ca2+ signaling as well as changes to the cellular cytoskeleton, both of which may alter cellular structure and mitochondrial behavior (23). The cellular and molecular understanding developed by answering question 5 should inform our view of the response of SR Ca2+ leakiness and mitochondrial behavior to MI.
Although we appreciate that there are many other critical features in HF and mitochondrial biology that cannot be addressed here, this very brief perspective may help to clarify how an exciting set of present (1) and future Question 5. What cellular changes exacer- work may be linked and thereby contribute bate the damage caused by MI and drive to a broader understanding of heart and cellular maladaptation leading to terminal mitochondrial biology. HF? The cellular manifestations of HF develop as a maladaptation by the noninfarcted heart cells following the initial insult. Presumably, this response is a consequence of
1 Santulli G, Xie W, Reiken SR, Marks AR (2015) Mitochondrial calcium overload is a key determinant in heart failure. Proc Natl Acad Sci USA 112:11389–11394. 2 Williams GS, Boyman L, Lederer WJ (2015) Mitochondrial calcium and the regulation of metabolism in the heart. J Mol Cell Cardiol 78:35–45. 3 Glancy B, Balaban RS (2012) Role of mitochondrial Ca2+ in the regulation of cellular energetics. Biochemistry 51(14):2959–2973. 4 Boyman L, et al. (2014) Calcium movement in cardiac mitochondria. Biophys J 107(6):1289–1301. 5 Murphy MP (2009) How mitochondria produce reactive oxygen species. Biochem J 417(1):1–13. 6 Luongo TS, et al. (2015) The mitochondrial calcium uniporter matches energetic supply with cardiac workload during stress and modulates permeability transition. Cell Reports 12(1):23–34. 7 Kwong JQ, et al. (2015) The mitochondrial calcium uniporter selectively matches metabolic output to acute contractile stress in the heart. Cell Reports 12(1):15–22. 8 Pan X, et al. (2013) The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter. Nat Cell Biol 15(12):1464–1472. 9 Boyman L, Williams GS, Khananshvili D, Sekler I, Lederer WJ (2013) NCLX: The mitochondrial sodium calcium exchanger. J Mol Cell Cardiol 59:205–213. 10 Palty R, et al. (2010) NCLX is an essential component of mitochondrial Na+/Ca2+ exchange. Proc Natl Acad Sci USA 107(1):436–441. 11 Alavian KN, et al. (2014) An uncoupling channel within the c-subunit ring of the F1FO ATP synthase is the mitochondrial permeability transition pore. Proc Natl Acad Sci USA 111(29):10580–10585. 12 Kwong JQ, Molkentin JD (2015) Physiological and pathological roles of the mitochondrial permeability transition pore in the heart. Cell Metab 21(2):206–214. 13 Giorgio V, et al. (2013) Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Natl Acad Sci USA 110(15):5887–5892.
ACKNOWLEDGMENTS. This work was supported by American Heart Association Grant 15SDG22100002 (to L.B.) and National Heart Lung and Blood Institute Grants K25HL125762 (to G.S.B.W.) and U01HL116321 (to W.J.L.).
14 Eisner D, Bode E, Venetucci L, Trafford A (2013) Calcium flux balance in heart. J Mol Cell Cardiol 58:110–117. 15 Yaginuma H, et al. (2014) Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging. Sci Rep 4:6522. 16 Nakano M, Imamura H, Nagai T, Noji H (2011) Ca2+ regulation of mitochondrial ATP synthesis visualized at the single cell level. ACS Chem Biol 6(7):709–715. 17 Halestrap AP (2010) A pore way to die: The role of mitochondria in reperfusion injury and cardioprotection. Biochem Soc Trans 38(4): 841–860. 18 Elrod JW, Molkentin JD (2013) Physiologic functions of cyclophilin D and the mitochondrial permeability transition pore. Circ J 77(5):1111–1122. 19 Song LS, et al. (2006) Orphaned ryanodine receptors in the failing heart. Proc Natl Acad Sci USA 103(11):4305–4310. 20 Wei S, et al. (2010) T-tubule remodeling during transition from hypertrophy to heart failure. Circ Res 107(4):520–531. 21 Wagner E, et al. (2012) Stimulated emission depletion live-cell superresolution imaging shows proliferative remodeling of T-tubule membrane structures after myocardial infarction. Circ Res 111(4):402–414. 22 Zhang C, et al. (2014) Microtubule-mediated defects in junctophilin-2 trafficking contribute to myocyte transverse-tubule remodeling and Ca2+ handling dysfunction in heart failure. Circulation 129(17):1742–1750. 23 Li S, et al. (2015) Transient assembly of F-actin on the outer mitochondrial membrane contributes to mitochondrial fission. J Cell Biol 208(1):109–123. 24 Williams GS, Boyman L, Chikando AC, Khairallah RJ, Lederer WJ (2013) Mitochondrial calcium uptake. Proc Natl Acad Sci USA 110(26):10479–10486.
PNAS | September 8, 2015 | vol. 112 | no. 36 | 11151
COMMENTARY
perplexing, and sometimes contradictory reports on the molecular and cellular nature of mitochondrial Ca2+ signaling in heart and elsewhere. These studies involve the mitochondrial Ca2+ uniporter (6–8), the mitochondrial sodium–calcium exchanger (NCLX or mNCX) (9, 10), the mitochondrial permeability-transition pore (mPTP) (11–13), and an array of mitochondrialcentric functions (ranging from the ETC to ATP synthase and more), each of which may be involved with or contribute to the development of HF. Here we present five questions that if addressed quantitatively may link Ca2+ signaling and RyR2 leakiness to cardiac metabolic regulation and HF. Fig. 1A outlines graphically the location and function of key players in mitochondrial dynamics. Fig. 1B shows changes that may occur in WT control heart cells vs. experimental S2808D cells with leaky RyR2s. Critical aspects of the transition from control through MI to HF are highlighted. With the issues now identified and the availability of new tools, the time is ripe for these questions to be addressed.
The growing importance of mitochondrial calcium in health and disease Liron Boyman1, George S. B. Williams1, and W. J. Lederer2 Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine, Baltimore, MD 21201
In Santulli et al. (1), RyR2-dependent Ca2+ leak from the sarcoplasmic reticulum (SR) is presented as a cause of mitochondrial dysfunction that contributes to heart failure (HF). The authors provide evidence in support of this provocative and important hypothesis. The strength of this paper comes primarily from the suggestions it provides that associate maladaptive changes of key physiological
signaling pathways in the heart with the development of HF. A secondary benefit of the work is the array of questions related to the findings of the authors raised by them and provoked by other recent reports (discussed below). The thinking is that RyR2-based SR Ca2+ leak increases in HF and thereby produces a pathologically elevated mitochondrial matrix
A
B
Ca2+ ([Ca2+]m) level along with excessive mitochondria reactive oxygen species (ROS) production (1). Together these elements form a causal chain in the development of HF following myocardial infarction (MI). With this argument, the paper throws down the gauntlet to the scientific community, challenging investigators to determine quantitatively how these elements interact to produce HF. Such investigations, armed with a growing arsenal of novel tools, hold an exciting potential for us to better understand cellular, organellar, and molecular dysfunctions that underlie the development of HF. Specifically, we need to understand how the transformation from healthy myocyte to dysfunctional myocyte occurs. The quantitative aspects of this process are critical because under normal conditions only modest increases in [Ca2+]m are required to regulate ATP production (2–4). Similarly, small amounts of ROS generation are a normal part of ATP generation by the electron transport chain (ETC) (5). To distinguish physiologic regulation from maladaptive pathophysiologic changes in [Ca2+]m and ROS generation in HF, molecular details of the changes must be investigated quantitatively with good temporal resolution. Furthermore, there are no a priori reasons to believe that all of the changes in cardiac function in HF are primarily mitochondrial-centric, even in the MI model of HF presented by Santulli et al. (1). Alterations in activation, production, and degradation of many parallel cytosolic proteins and processes will almost certainly occur even when HF is associated with leaky RyR2s. Santulli et al. (1) thus identify SR–mitochondrial interactions mediated by Ca2+ that contribute to HF (Fig. 1). Teasing apart the quantitative molecular and cellular details of the development of HF due to this dynamic and spatially resolved process is a huge task. Here we outline a simplified “challenge of Santulli” that arises from the PNAS paper and from a treasure trove of recent, exciting, Author contributions: L.B., G.S.B.W., and W.J.L. designed research, performed research, analyzed data, and wrote the paper.
Fig. 1. Critical observations related to mitochondrial dynamics and the etiology of HF. (A) Schematic diagram showing the spatial distribution of mitochondrial Ca2+ signaling components in heart. The ends of each intermyofibrillar mitochondrion are in close proximity (∼100 nm) to the Ca2+ release units (CRUs) that are located between the transverse-tubule (TT) and junctional SR (jsr) membranes [also see Williams et al. (24)]. (B) Venn diagram highlighting key differences and similarities in WT mice and mice with leaky RyRs (S2808D) and how these properties develop during HF. Observations related to the critical questions (outlined here) provoked by Santulli et al. (1) are bolded. 11150–11151 | PNAS | September 8, 2015 | vol. 112 | no. 36
The authors declare no conflict of interest. See companion article on page 11389. 1
L.B. and G.S.B.W. contributed equally to this work.
2
To whom correspondence should be addressed. Email: jlederer@ umaryland.edu.
www.pnas.org/cgi/doi/10.1073/pnas.1514284112
Question 1: How do leaky RyR2 channels lead to elevated [Ca2+]m content in the steadystate? Overall, cellular Ca2+ balance depends on Ca2+ pump-leak balance across the sarcolemmal membrane (14) and not the leakiness of internal organelles. If only the leakiness of RyR2s were to increase, then the global cellwide effect would be to deplete the cell of Ca2+, shifting Ca2+ from the SR to the extracellular volume, including the circulation. If mitochon drial [Ca2+]m increases when RyR2s become leaky, additional changes must be occurring and these need to be identified and measured quantitatively. Question 2. How and why does mitochondrial ROS increase in HF? To determine this, a host of things must be measured including metabolic substrate, redox state in mitochondria and in the cytosol, partial pressure of O2, ATP and ADP levels, the potential across the inner membrane (ΔΨm), and more. Additionally, nonmitochondrial ROS sources should be measured. By knowing what cellular processes change and when they change in relationship to the ROS production, it should be possible to understand mechanistically HF changes and the possible role of increases in ROS production. Question 3. What is the cellular [ATP]i level before, during the onset of HF, and during
Boyman et al.
terminal HF? [ATP]i can now be measured in live functioning cells using fluorescence sensors (15, 16). It would be valuable to measure [ATP]i in the context of SR Ca2+ leak and changes of [Ca2+]m at different stages of HF. Also see question 5. Question 4. Are the mitochondrial dysfunctions observed in HF associated with activation of mPTP? Do changes in mitochondrial function correlate with changes in [ATP]i, [Ca2+]m, and ROS generation, and, if so, how tightly correlated are the changes? Because [Ca2+]m and ROS are known activators of mPTP (17), are the S2808D cardiomyocytes more prone to mPTP activation and the corresponding cell injury? Alternatively, under such conditions, does mPTP activation have an inverse (i.e., ameliorating) (18) effect by purging the mitochondria of excess Ca2+, and therefore sustaining mitochondrial metabolism? Furthermore, can these measurements help us to determine the molecular identity of mPTP?
the stress created by the loss of previously healthy and functional tissue. Just as “orphan RyR2s” develop due to the reorganization of the transverse tubules in pressure-overload HF (19–21), so too does this happen following MI in human and model disease (22). Thus, the development of cellular and mitochondrial dysfunction following MI seems to depend on the consequences of the initiating insult, which include changes in Ca2+ signaling as well as changes to the cellular cytoskeleton, both of which may alter cellular structure and mitochondrial behavior (23). The cellular and molecular understanding developed by answering question 5 should inform our view of the response of SR Ca2+ leakiness and mitochondrial behavior to MI.
Although we appreciate that there are many other critical features in HF and mitochondrial biology that cannot be addressed here, this very brief perspective may help to clarify how an exciting set of present (1) and future Question 5. What cellular changes exacer- work may be linked and thereby contribute bate the damage caused by MI and drive to a broader understanding of heart and cellular maladaptation leading to terminal mitochondrial biology. HF? The cellular manifestations of HF develop as a maladaptation by the noninfarcted heart cells following the initial insult. Presumably, this response is a consequence of
1 Santulli G, Xie W, Reiken SR, Marks AR (2015) Mitochondrial calcium overload is a key determinant in heart failure. Proc Natl Acad Sci USA 112:11389–11394. 2 Williams GS, Boyman L, Lederer WJ (2015) Mitochondrial calcium and the regulation of metabolism in the heart. J Mol Cell Cardiol 78:35–45. 3 Glancy B, Balaban RS (2012) Role of mitochondrial Ca2+ in the regulation of cellular energetics. Biochemistry 51(14):2959–2973. 4 Boyman L, et al. (2014) Calcium movement in cardiac mitochondria. Biophys J 107(6):1289–1301. 5 Murphy MP (2009) How mitochondria produce reactive oxygen species. Biochem J 417(1):1–13. 6 Luongo TS, et al. (2015) The mitochondrial calcium uniporter matches energetic supply with cardiac workload during stress and modulates permeability transition. Cell Reports 12(1):23–34. 7 Kwong JQ, et al. (2015) The mitochondrial calcium uniporter selectively matches metabolic output to acute contractile stress in the heart. Cell Reports 12(1):15–22. 8 Pan X, et al. (2013) The physiological role of mitochondrial calcium revealed by mice lacking the mitochondrial calcium uniporter. Nat Cell Biol 15(12):1464–1472. 9 Boyman L, Williams GS, Khananshvili D, Sekler I, Lederer WJ (2013) NCLX: The mitochondrial sodium calcium exchanger. J Mol Cell Cardiol 59:205–213. 10 Palty R, et al. (2010) NCLX is an essential component of mitochondrial Na+/Ca2+ exchange. Proc Natl Acad Sci USA 107(1):436–441. 11 Alavian KN, et al. (2014) An uncoupling channel within the c-subunit ring of the F1FO ATP synthase is the mitochondrial permeability transition pore. Proc Natl Acad Sci USA 111(29):10580–10585. 12 Kwong JQ, Molkentin JD (2015) Physiological and pathological roles of the mitochondrial permeability transition pore in the heart. Cell Metab 21(2):206–214. 13 Giorgio V, et al. (2013) Dimers of mitochondrial ATP synthase form the permeability transition pore. Proc Natl Acad Sci USA 110(15):5887–5892.
ACKNOWLEDGMENTS. This work was supported by American Heart Association Grant 15SDG22100002 (to L.B.) and National Heart Lung and Blood Institute Grants K25HL125762 (to G.S.B.W.) and U01HL116321 (to W.J.L.).
14 Eisner D, Bode E, Venetucci L, Trafford A (2013) Calcium flux balance in heart. J Mol Cell Cardiol 58:110–117. 15 Yaginuma H, et al. (2014) Diversity in ATP concentrations in a single bacterial cell population revealed by quantitative single-cell imaging. Sci Rep 4:6522. 16 Nakano M, Imamura H, Nagai T, Noji H (2011) Ca2+ regulation of mitochondrial ATP synthesis visualized at the single cell level. ACS Chem Biol 6(7):709–715. 17 Halestrap AP (2010) A pore way to die: The role of mitochondria in reperfusion injury and cardioprotection. Biochem Soc Trans 38(4): 841–860. 18 Elrod JW, Molkentin JD (2013) Physiologic functions of cyclophilin D and the mitochondrial permeability transition pore. Circ J 77(5):1111–1122. 19 Song LS, et al. (2006) Orphaned ryanodine receptors in the failing heart. Proc Natl Acad Sci USA 103(11):4305–4310. 20 Wei S, et al. (2010) T-tubule remodeling during transition from hypertrophy to heart failure. Circ Res 107(4):520–531. 21 Wagner E, et al. (2012) Stimulated emission depletion live-cell superresolution imaging shows proliferative remodeling of T-tubule membrane structures after myocardial infarction. Circ Res 111(4):402–414. 22 Zhang C, et al. (2014) Microtubule-mediated defects in junctophilin-2 trafficking contribute to myocyte transverse-tubule remodeling and Ca2+ handling dysfunction in heart failure. Circulation 129(17):1742–1750. 23 Li S, et al. (2015) Transient assembly of F-actin on the outer mitochondrial membrane contributes to mitochondrial fission. J Cell Biol 208(1):109–123. 24 Williams GS, Boyman L, Chikando AC, Khairallah RJ, Lederer WJ (2013) Mitochondrial calcium uptake. Proc Natl Acad Sci USA 110(26):10479–10486.
PNAS | September 8, 2015 | vol. 112 | no. 36 | 11151
COMMENTARY
perplexing, and sometimes contradictory reports on the molecular and cellular nature of mitochondrial Ca2+ signaling in heart and elsewhere. These studies involve the mitochondrial Ca2+ uniporter (6–8), the mitochondrial sodium–calcium exchanger (NCLX or mNCX) (9, 10), the mitochondrial permeability-transition pore (mPTP) (11–13), and an array of mitochondrialcentric functions (ranging from the ETC to ATP synthase and more), each of which may be involved with or contribute to the development of HF. Here we present five questions that if addressed quantitatively may link Ca2+ signaling and RyR2 leakiness to cardiac metabolic regulation and HF. Fig. 1A outlines graphically the location and function of key players in mitochondrial dynamics. Fig. 1B shows changes that may occur in WT control heart cells vs. experimental S2808D cells with leaky RyR2s. Critical aspects of the transition from control through MI to HF are highlighted. With the issues now identified and the availability of new tools, the time is ripe for these questions to be addressed.
