The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability

Abstract

Mitochondrial calcium (mCa2+) has a central role in both metabolic regulation and cell death signalling, however its role in homeostatic function and disease is controversial1. Slc8b1 encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), which is proposed to be the primary mechanism for mCa2+ extrusion in excitable cells2,3. Here we show that tamoxifen-induced deletion of Slc8b1 in adult mouse hearts causes sudden death, with less than 13% of affected mice surviving after 14 days. Lethality correlated with severe myocardial dysfunction and fulminant heart failure. Mechanistically, cardiac pathology was attributed to mCa2+ overload driving increased generation of superoxide and necrotic cell death, which was rescued by genetic inhibition of mitochondrial permeability transition pore activation. Corroborating these findings, overexpression of NCLX in the mouse heart by conditional transgenesis had the beneficial effect of augmenting mCa2+ clearance, preventing permeability transition and protecting against ischaemia-induced cardiomyocyte necrosis and heart failure. These results demonstrate the essential nature of mCa2+ efflux in cellular function and suggest that augmenting mCa2+ efflux may be a viable therapeutic strategy in disease.

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Figure 1: Deletion of Slc8b1 from adult cardiomyocytes causes left ventricular remodelling and heart failure followed by sudden death.
Figure 2: Deletion of Slc8b1 in adult mice ablates mCa2+ efflux resulting in mitochondrial-dependent necrotic cell death.
Figure 3: Cardiomyocyte overexpression of NCLX increases mCa2+ efflux capacity.
Figure 4: Overexpression of NCLX protects against myocardial ischaemic reperfusion injury and ischaemic heart failure.

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Acknowledgements

We thank T. Tierney, N. Shah, and P. Kelly for technical assistance in the Elrod Laboratory. We thank S. Modla for her expertise in TEM sample processing (Delaware Biotechnology Institute). This study was supported by grants to J.W.E. from the NIH (R01 HL123966, P01 DA037830 sub-8614) and AHA (14SDG18910041) and (15PRE25080299 to T.S.L.), (16PRE31030038 to A.A.L.), and (17PRE33460423 to J.P.L.).

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J.W.E. and T.S.L. contributed to study design, data analysis and writing the paper; T.S.L. was involved with all assays and data collection; J.P.L. performed western blots and NADH assays; P.G. performed ACM iCa2+ transient analysis; M.N. performed myocardial infarction data acquisition; A.A.L. assisted with the ischaemia reperfusion study and mitochondrial membrane potential assays; S.S. and M.M. assisted with efflux analysis in permeabilized cells; A.C.C. performed qPCR assays; D.K. performed histology; E.G. performed ischaemia reperfusion and myocardial infarction surgeries; J.H.v.B. and J.D.M. aided mutant mouse generation; E.J.T. supplied human heart samples; X.C. performed radiotelemeter implantation and assisted with electrocardiogram analysis; J.W.E., J.H.v.B, T.S.L., S.R.H., J.P.L., A.C.C. and A.A.L. assisted with data interpretation and edited the manuscript.

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Correspondence to John W. Elrod.

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Reviewer Information: Nature thanks M. Murphy and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 shRNA knockdown of NCLX and biophysical characterization in non-excitable cells.

a, qPCR analysis of SLC8B1 mRNA expression in shRNA stable knockdown HeLa cell lines versus scramble shRNA control cell line (scr. con). b, mCa2+ transients recorded in cells expressing the genetic mCa2+ sensor, mitycam, during histamine treatment (100 μM). Mean intensity shown as solid lines, thin dashed lines display s.e.m. c, mCa2+ peak amplitude. d, Per cent mCa2+ efflux versus scramble shRNA control. n = 42–49 cells per group. eg, HeLa cells were loaded with the Ca2+ sensor, Fura-FF, and the Δψ sensor, JC-1, permeabilized with digitonin (dg) and treated with SERCA inhibitor, thapsigargin (thaps) for simultaneous ratiometric monitoring during repetitive additions of 5 μM Ca2+ (black arrows). FCCP was used to collapse Δψ at the conclusion of each experiment. h, mCa2+ uptake capacity versus scramble shRNA control cells following 20 μM Ca2+ (fourth pulse). i, JC-1-derived Δψ before FCCP addition. n = 3 experiments per assay; *P < 0.05, ***P < 0.001 versus scramble shRNA control.

Extended Data Figure 2 Characterization of Slc8b1 conditional knockout following tamoxifen administration.

a, Images of chimeric founder mice and estimated per cent chimerism, black coat colour correlates with mutant ES cell contribution to development. b, Image of gel with PCR genotyping results for Slc8b1 loxP targeted mice. c, Left ventricular end-systolic dimension (LVESD) three days after tamoxifen administration. d, Curve fitting of mitochondrial swelling traces after addition of 500 μM Ca2+. e, Representative images of MitoSOX Red-stained ACMs (scale bar, 40 μm). f, Fold change in MitoSOX Red intensity three days after tamoxifen administration. n = 42–52 ACMs per group. g, Representative ECG recordings of Slc8b1fl/fl × MCM mice (n = 4–7) at baseline, three days and five days after tamoxifen treatment, and day of sinus arrest. h, Quantitation of PR interval. i, Quantitation of QRS interval. j, Quantitation of heart rate (HR). BPM, beats per minute. k, Western blot of proposed MPTP components from heart protein lysate: CypD and ANT, VDAC shown in Fig. 1d. l, Size distribution (perimeter) of mitochondria analysed in electron microscopy images three days after tamoxifen (minimum 200 mitochondria quantified per mouse, n = 3–4 mice per group). m, Quantification of NAD+/NADH ratio three days after tamoxifen administration (fold change versus control). n = 6–7 per group. n, Western blots of total pyruvate dehydrogenase (PDH E1α) and phosphorylated-PDH (p-PDH) from heart protein lysate. o, Fold change in the ratio of p-PDH/total PDH E1α versus control. n = 3 per group; *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. Source data

Extended Data Figure 3 Examination of Ca2+ flux following αMHC-Cre-mediated deletion of Slc8b1.

a, Adult heart protein lysate isolated from: Slc8b1fl/fl × αMHC-Cre mice and controls were examined by western blot for NCLX expression and various other proteins involved in mCa2+ exchange: MCU, MCUb, MICU1, EMRE, LETM1, VDAC; and OXPHOS components: ATP synthase subunit α (CV-Sα), complex III subunit core 2 (CIII-C2), complex IV subunit I (CIV-SI), and complex I subunit NDUFB8 (CI-SIV). n = 3 hearts per group. b, c, Series of mitycam mCa2+ transients recorded during pacing (0.1 Hz). d, mCa2+ time to peak amplitude. e, Per cent mCa2+ efflux 10 s after pacing. f, Tetramethylrhodamine, ethyl ester (TMRE) (Δψ) in intact ACM. n = 23–24 ACMs per group. g, h, Representative recordings of iCa2+ transients (Fluo-4) in ACMs paced at 1 Hz with or without isoproterenol (Iso, 100 nM). i, iCa2+ peak amplitude. j, iCa2+ time to peak amplitude. k, iCa2+ time to 50% decay. l, Time to 90% decay. n = 10 cells per group. m, n, Permeabilized ACM mCa2+ uptake rate (m) and per cent uptake (n) after Ca2+ addition. *P < 0.05, **P < 0.01, ***P < 0.001.

Extended Data Figure 4 Characterization of NCLX transgenic mice.

a, b, Series of mitycam mCa2+ transients recorded during pacing (0.1 Hz). c, Tetramethylrhodamine, ethyl ester (TMRE) (Δψ) in intact ACMs. n = 23–36 ACMs per group. d, e, Representative traces of iCa2+ transients (Fluo-4) in ACMs paced at 1 Hz with or without isoproterenol (100 nM). f, iCa2+ peak amplitude. g, iCa2+ time to peak amplitude. h, Time to 50% decay. i, Time to 90% decay (n = 15 cells per group). j, Recording of transients in Digitonin (dig)-permeabilized ACMs loaded with Fura-FF, treated with thapsigargin (thaps) and placed in 20 μM bath Ca2+ before treatment with the MCU inhibitor, Ru360, to quantify the rate of mCa2+ efflux independent of uptake. kq, Seahorse analysis of mitochondrial oxygen consumption rates (OCR) in ACMs in the presence of pyruvate or palmitate. m, Basal OCR. n, ATP-linked respiration after addition of ATP synthase inhibitor, oligomycin. o, Maximal respiration after addition of protonophore, FCCP. p, Spare respiratory capacity (max − basal). q, Proton leak (post-oligomycin OCR – non-mitochondrial OCR). n = 11–15 per condition. r, Quantification of NAD+/NADH ratio three days after tamoxifen (fold change versus control). n = 4–5 per group. s, Western blot of total pyruvate dehydrogenase (PDH E1α) and phosphorylated-PDH (p-PDH) from heart protein lysate. *P < 0.05, ***P < 0.001. Source data

Extended Data Figure 5 Analysis of NCLX-Tg mice 24 h after ichaemia reperfusion and 4 weeks after myocardial infarction.

a, Quantification of TUNEL+ interstitial cells after ischaemia reperfusion. be, B-mode speckle tracking analysis of left ventricular function 4 weeks after myocardial infarction: longitudinal strain (b), longitudinal strain rate (c), radial strain (d), radial strain rate (e). n = 20 tTA and n = 18 NCLX-Tg. f, Lung oedema (wet − dry lung weight). n = 7 per sham group, n = 17 per myocardial infarction group. g, Kaplan–Meier survival curves post myocardial infarction. n = 28 tTA and n = 27 NCLX-Tg. hl, qPCR quantification of mRNA expression in hearts from sham-treated mice or 4 weeks after myocardial infarction. Nppa, atrial natriuretic peptide; Nppb, brain natriuretic peptide; Spp1, osteopontin; Postn1, periostin; Acta2, smooth muscle α-actin (sham, n = 3 group; myocardial infarction, n = 7 per group). m, Representative images of H&E-stained heart sections from the infarct border zone 4 weeks post myocardial infarction. Scale bar, 400 μm. n, o, qPCR analysis of mRNA expression for inflammatory cytokines 4 weeks post myocardial infarction. Il1b, interleukin-1β; Il6, interleukin-6. n = 3 per sham group, n = 7 per myocardial infarction group. p, Western blot of pyruvate dehydrogenase subunits and phosphorylated-PDH (p-PDH) from heart protein lysate 4 weeks post myocardial infarction. q, Fold-change in the ratio of p-PDH/total PDH E1α versus tTA control. n = 4 per group; *P < 0.05, **P < 0.01, ***P < 0.001, versus αMHC tTA post injury; #P < 0.05, ##P < 0.01 versus sham control. Source data

Extended Data Table 1 Electrocardiography measurements of Slc8b1fl/fl × MCM mice (n = 7) before and after the delivery of tamoxifen
Extended Data Table 2 Genotype distribution of Slc8b1 × αMHC-Cre mice

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Luongo, T., Lambert, J., Gross, P. et al. The mitochondrial Na+/Ca2+ exchanger is essential for Ca2+ homeostasis and viability. Nature 545, 93–97 (2017). https://doi.org/10.1038/nature22082

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