Abstract
Muscle degeneration is the most prevalent cause for frailty and dependency in inherited diseases and ageing. Elucidation of pathophysiological mechanisms, as well as effective treatments for muscle diseases, represents an important goal in improving human health. Here, we show that the lipid synthesis enzyme phosphatidylethanolamine cytidyltransferase (PCYT2/ECT) is critical to muscle health. Human deficiency in PCYT2 causes a severe disease with failure to thrive and progressive weakness. pcyt2-mutant zebrafish and muscle-specific Pcyt2-knockout mice recapitulate the participant phenotypes, with failure to thrive, progressive muscle weakness and accelerated ageing. Mechanistically, muscle Pcyt2 deficiency affects cellular bioenergetics and membrane lipid bilayer structure and stability. PCYT2 activity declines in ageing muscles of mice and humans, and adeno-associated virus-based delivery of PCYT2 ameliorates muscle weakness in Pcyt2-knockout and old mice, offering a therapy for individuals with a rare disease and muscle ageing. Thus, PCYT2 plays a fundamental and conserved role in vertebrate muscle health, linking PCYT2 and PCYT2-synthesized lipids to severe muscle dystrophy and ageing.
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Data availability
The original datasets used in the RNA sequencing analysis can be accessed at NCBI archived under Gene Expression Omnibus accession codes GSM6785661, GSM6785662, GSM6785663, GSM6785664, GSM6785665 and GSM6785666. Mass spectrometry lipidomics data for Figs. 1, 4 and 8 and Extended Data Fig. 7 are provided in the Supplementary Information. Further information and requests for resources and reagents should be directed to and will be fulfilled by the corresponding authors. Source data are provided with this paper.
Change history
06 April 2023
A Correction to this paper has been published: https://doi.org/10.1038/s42255-023-00791-1
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Acknowledgements
The authors thank the participants and their families for participating in the study. We thank all members of our laboratories for helpful discussions. We are grateful to Vienna BioCenter Core Facilities: Mouse Phenotyping Unit, Histopathology Unit, Bioinformatics Unit, BioOptics Unit, Electron Microscopy Unit and Comparative Medicine Unit. We are grateful to the Lipidomics Facility, and K. Klavins and T. Hannich at the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences for assistance with lipidomics analysis. We also thank T. Huan and A. Hui (UBC Vancouver) for mouse tissue and mitochondria lipidomics analysis. We thank A. Klymchenko (Laboratoire de Bioimagerie et Pathologies Université de Strasbourg, Strasbourg, France) for providing the NR12S probe. We are thankful to the Sen. Paul D. Wellstone Muscular Dystrophy Cooperative Specialized Research Center Viral Vector Core Facility for AAV6 production. We also thank K. P. Campbell and M. E. Anderson (University of Iowa, Carver College of Medicine) for advice on muscle tissue handling. We thank A. Al-Qassabi from the Sultan Qaboos University for the clinical assessment of the participants. D.C. and J.M.P. are supported by the Austrian Federal Ministry of Education, Science and Research, the Austrian Academy of Sciences, and the City of Vienna, and grants from the Austrian Science Fund (FWF) Wittgenstein award (Z 271-B19), the T. von Zastrow Foundation, and a Canada 150 Research Chairs Program (F18-01336). J.S.C. is supported by grants RO1AR44533 and P50AR065139 from the US National Institutes of Health. C.K. is supported by a grant from the Agence Nationale de la Recherche (ANR-18-CE14-0007-01). A.V.K. is supported by European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 67544, and an Austrian Science Fund (FWF; no P-33799). A.W. is supported by Austrian Research Promotion Agency (FFG) project no 867674. E.S. is supported by a SciLifeLab fellowship and Karolinska Institutet Foundation Grants. Work in the laboratory of G.S.-F. is supported by the Austrian Academy of Sciences, the European Research Council (ERC AdG 695214 GameofGates) and the Innovative Medicines Initiative 2 Joint Undertaking (grant agreement no. 777372, ReSOLUTE). S.B., M.L. and R.Y. acknowledge the support of the Spastic Paraplegia Foundation.
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D.C. and J.M.P. designed and supervised the mouse study and wrote the manuscript with input from the co-authors. All experiments were performed and established by D.C. with the following exceptions: K.E. performed BLSM measurements. E.S. performed GPMV isolation and image analysis with assistance from D.C. E.K. performed synergic ablation assay and analysis under supervision form Z.R. F.T. performed treadmill experiment with assistance from D.C. and under supervision from Z.R. R.Y. collected and analysed zebrafish models under supervision from M.L. L.X.H. and V.S. performed lipidomics analysis under supervision of G.S.-F. A.T. and S.G. performed Pcyt2 enzyme activity analysis under supervision of M.B. A.A. performed in vivo brown fat activity and Ucp1 RT–PCR analysis under supervision from C. Knauf. A.W. performed respiration analysis under supervision from A. Kozlov. M.O. performed and analysed mouse calorimetry experiments. C. Kutchukian. and C.S. performed myofibre calcium kinetics experiment, with analysis and supervision under V.J. S.J.F.C. assisted with western blot experiments. M.N. performed bioinformatic analysis of RNA sequencing and of efficiency of Pcyt2 deletion in mice. A. Kavirayani performed myositis scoring. N.D.M. performed atomic force microscopy measurements and data analysis with assistance from D.C. T.S. and B.H. assisted in histological analysis. L.H. performed AAV6 intravenous injections. A.H. assisted in tissue sampling for western blot experiments. S.J. provided Pcyt2-floxed mice. E.R. and T.G. collected human muscle biopsy samples. J.S.C. generated the AAV6 vector and provided guidance with all AAV experiments. J.M., F.A.-M. and S.B. identified PCYT2 human mutant carriers, collected growth data and generated growth curves.
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Extended data
Extended Data Fig. 1 PE synthesis pathways and EPT1 rare disease mutation carriers.
(A) Schematic diagram of phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylserine (PS) phospholipids synthesis. EK-Ethanolamine kinase; PCYT2- CTP:phosphoethanolamine cytidylyltransferase; EPT1- ethanolaminephosphotransferase 1; PSS2- Phosphatidylserine Synthase 2; PSD- Phosphatidylserine decarboxylase; CK- Choline kinase; PCYT1- Choline-phosphate cytidylyltransferase; CEPT1- Choline/ethanolaminephosphotransferase 1; PSS1- Phosphatidylserine Synthase 1. (B) Height and weight gains of three patients (#1 male, #2 female, #3 male) carrying the homozygous missense variant c.335 G > C (p.Arg112Pro) in the EPT1 gene. Controls indicate WHO standards of median weights and heights at the respective ages +/− 2 standard deviations (SD).
Extended Data Fig. 2 Analysis of Pcyt2 deletion in mice.
(A) Schematic diagram of exon 2 deletion in Myf5Cre-Pcyt2 male mice and confirmation by RNA sequencing. Exon and introns structures as well as LoxP sites targeted to exon 2 and loss of exon 2 upon Cre-mediated recombination are shown for the murine Pcyt2 locus. n = 3 animals per group.
Extended Data Fig. 3 Characterization of Myf5Cre-Pcyt2 mice.
(A) Body weights of control and Myf5Cre-Pcyt2 male mice at P1 and P4. (B) Body length gains of control and Myf5Cre-Pcyt2 male mice. n = 6 per group for body length analysis. (C) Body weights of 2 months old control and Myf5Cre-Pcyt2 female mice. (D) Body lengths of 2 months old control and Myf5Cre-Pcyt2 female mice. (E) Skeletal muscle and tissue weight isolated from (E) 10 day old control (n = 6) and Myf5Cre-Pcyt2 (n = 8) and (F) 2 months old (P56) control (n = 8) and Myf5Cre-Pcyt2 (n = 7) littermate male mice. QA, quadriceps; GC, gastrocnemius; TA, tibialis anterior muscles. Liver and spleen weights are shown as controls. Scale bars 1 cm. (G, H) Gross skeletal muscle appearance of 56 days old control and Myf5Cre-Pcyt2 male littermates. (I) mRNA TPM levels of enzymes from the PE and PC branch of the phospholipid synthesis Kennedy pathway. n = 3 mice per group. Data are shown as means ± SEM. Data are shown as means ± SEM. Each dot represents data point from individual mice unless stated otherwise. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, n.s. not significant (unpaired Student t-test).
Extended Data Fig. 4 Myoblast proliferation assessment in Myf5Cre-Pcyt2 mice.
(A) Representative images and quantification of BrdU labelled quadriceps from 2 days old control and Myf5Cre-Pcyt2 male mice. Images were taken under 5x magnification, and ≥2000nuclei were counted and analysed. N = 4 animals per group. Scale bar 60 µm. (B) Representative images and quantification of EdU labelled primary myoblasts in cell culture isolated from control and Myf5Cre-Pcyt2 male mice. 18 biological replicate cultures from 3 independent isolations were analysed and images were taken under 5x magnification. ≥100 nuclei counted per each culture. Each dot represents the number of EdU positive cells per each culture. Scale bar 50 µm. (C) Number of Pax7 positive nuclei in quadriceps from 6 months old male control (n = 5) and Myf5Cre-Pcyt2 mice (n = 4). ≥100 nuclei per each individual section from each mouse were counted. Scale bar 50 µm. Data are shown as means ± SEM. Unpaired Student t-test with Welch correction was used for statistical analysis. Each dot represents the number of EdU positive cells per each culture. Scale bar 50 µm. (C) Number of Pax7 positive nuclei in quadriceps from 6 months old control (n = 5) and Myf5Cre-Pcyt2 mice (n = 4). ≥100 nuclei per each individual section from each mouse were counted. Scale bar 50 µm. Data are shown as means ± SEM. Unpaired Student t-test with Welch correction was used for statistical analysis.
Extended Data Fig. 5 Myofiber type distribution in skeletal muscle of Myf5Cre-Pcyt2 mice.
(A) Western blot analysis of critical regulators of protein synthesis and translation S6K1 and 4E-BP1 in overloaded M. plantaris from Control and Myf5Cre-Pcyt2 male mice. Each lane represents individual mice. Two-Way ANOVA with multiple comparison followed by Bonferroni correction was used for statistical analysis. (B) Representative images and quantification of MyHC!, MyhCIIA and MyHCIIB fibres in skeletal muscle (quadriceps) from 6 months old control and Myf5Cre-Pcyt2 male mice. Images were taken under 5x magnification, and ≥100 myofibers were counted at 3 different matching histological areas. N = 4 animals per group. Scale bar 500 µm. (C) Representative images and quantification of oxidative and glycolytic fibres in skeletal muscle (quadriceps) from 6 months old control and Myf5Cre-Pcyt2 male mice. Images were taken under 10x magnification, and ≥1000 myofibers were counted at matching histological areas. N = 5 animals per group. Scale bar 500 µm. (D) Total number of fibres in skeletal muscle (quadriceps) from 6 months old control and Myf5Cre-Pcyt2 male mice. Images were taken under 2.5x magnification. N = 5 animals per group. Scale bar 500 µm. Data are shown as means ± SEM. Unless otherwise stated, unpaired Student t-test with Welch correction was used for statistical analysis.
Extended Data Fig. 6 Muscle inflammation and metabolic assessment of Myf5Cre-Pcyt2 mice.
(A) Grip strength of 6 months old control and Myf5Cre-Pcyt2 females. Each dot represents one mouse, values are average of three measurements per mouse. (B) Representative electron microscopy images of quadriceps of 15 months old control and Myf5Cre-Pcyt2 male mice. Note accumulation of tubular aggregates in the mutant animals (red arrows). Representative images of 3 animals per group are shown. Scale bar 2 µm. (C) Characterization of muscle inflammation in 12 months old Myf5Cre-Pcyt2 male mice. Helper T cells (CD4+) and cytotoxic T cells (CD8+) are shown. Scale bar 100 µm for H&E stained and 50 µm for immune cell staining. Representative staining of 3 animals per group are shown. (D) Inflammatory cytokine levels in the quadriceps of 12 months old Myf5Cre-Pcyt2 male mice. (E) Fed blood glucose levels on normal chow diet of 8 months old control and Myf5Cre-Pcyt2 male mice. (F) Food consumption analysis of 6 month and 8 months old control and Myf5Cre-Pcyt2 mice (G) Cage activity of 6 months old male control and Myf5Cre-Pcyt2 mice (n = 12 per group). Multiple ANOVA was used to analyse the data. (H) Energy expenditure of 6 months old male control and Myf5Cre-Pcyt2 mice during the resting (light) and active (dark) phases. (I) Cage activity under thermoneutrality of 6 months old male control and Myf5Cre-Pcyt2 mice (n = 6 per group). Multiple ANOVA was used to analyse the data. (J) Energy expenditure of 6 months old male control and Myf5Cre-Pcyt2 mice during the resting (light) and active (dark) phases under thermoneutrality. (K) Grip strength assessment of 6 months old male control and Myf5Cre-Pcyt2 mice under thermoneutrality. Data are shown as means ± SEM. Each dot represents data point from individual mice unless stated otherwise. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, n.s. not significant. Unpaired Student t-test with Welch correction was used for statistical analysis unless stated otherwise.
Extended Data Fig. 7 Characterization of the brown adipose tissue from Myf5Cre-Pcyt2 mice.
(A, B) Lipidomics analyses from brown fat isolated from 10-day old Myf5Cre -Pcyt2 and control male mice. n = 4 per group. (C) Brown fat differentiation in lipid free conditions from 2-day old primary pre-adipocytes isolated from control and Myf5Cre -Pcyt2 male mice. Scale bar 50 µm. (D, E) Brown fat activity as addressed by exposure of 6-month-old control and Myf5Cre-Pcyt2 male mice to cold (4C) or during fasting. (F) Ucp1 mRNA levels in brown fat of 6-month-old control and Myf5Cre-Pcyt2 male mice. (G) Mitochondrial content in brown adipose tissue. (H) BAT mitochondrial structure of 6-month-old Myf5Cre-Pcyt2 male mice. Representative images of 3 animals per group are shown. Scale bar 1 µm. (I, J) Complex I and II activities of brown fat mitochondria. Paired Student t-test was used to analyse the data. Data are shown as means ± SEM. Each dot represents data point from individual mice unless stated otherwise. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, n.s. not significant (unpaired Student t-test, unless otherwise stated).
Extended Data Fig. 8 Specific inactivation of Pcyt2 in multiple mouse tissues.
(A) Schematic diagram to generate adipose tissue specific Pcyt2 deficient male mice (AdipoQCre-Pcyt2). Credit: IMP-IMBA Graphics Department. (B) Body weights and appearances of 6 months old control and AdipoQCre-Pcyt2 male mice. (C) Fasting blood glucose of 6 months old control and AdipoQCre-Pcyt2 male littermates fed a chow diet. (D) Schematic diagram of motor neuron specific Pcyt2 deficient male mice (Mnx1Cre-Pcyt2). Credit: IMP-IMBA Graphics Department. (E) Body weights of 8 months old control and Mnx1Cre-Pcyt2 male mice. (F) Absence of any overt clasping behaviour and appearance in 8 months old Mnx1Cre-Pcyt2 male mice. (G) Schematic diagram of intestine epithelium specific Pcyt2 deficient male mice (VilinCre-Pcyt2). Credit: IMP-IMBA Graphics Department. (H) Body weights of 6 months old control and VilinCre-Pcyt2 littermates. (I) Histological sections of intestine isolated from 12 months old control and VilinCre-Pcyt2 male mice. Scale bar 100 μm. (J) Schematic diagram of skin epithelium Pcyt2 deficient male mice (K14Cre-Pcyt2). Representative images of 3 animals per group are shown. Credit: IMP-IMBA Graphics Department. (K) Body weights and appearances of 6 months old control and K14Cre-Pcyt2 male mice. (L) Histological sections of skin isolated from 12 months old control and K14Cre-Pcyt2 male littermates. Representative images of 3 animals per group are shown. Scale bar 100 μm. (M) Schematic diagram of mature muscle specific Pcyt2 deficient male mice (MCKCre-Pcyt2). Credit: IMP-IMBA Graphics Department. (N) Grip strength of 18 months old control and muscle specific MckCre-Pcyt2 male mice. Data are shown as means ± SEM. Each dot represents individual mice, each mouse was tested in triplicates. Mean values ± SEM are displayed. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, n.s. not significant (unpaired Student t-test).
Extended Data Fig. 9 Assessment of mitochondrial homeostasis and SS-31 treatment.
(A, B) Muscle mitochondrial function assessed by measurements of complex II linked activity on isolated mitochondria from 2 months and from (C) 6 months old control and Myf5Cre-Pcyt2 male mice respectively. Paired Student t-test was used for statistical analysis. (D, E) Ultrastructure and total numbers of muscle mitochondria from 8 months -old control (n = 6) and Myf5Cre-Pcyt2 male mice (n = 6). Scale bar 200 nm. Unpaired Student t-test with Welch correction was used for statistical analysis (F) Function of muscle mitochondria under increasing concentrations of phosphoethanolamine, as assessed by measurements of complex I linked activity on isolated mitochondria from 2 months old control and Myf5Cre-Pcyt2 male mice. N = 3 mice per group. Two-Way ANOVA with multiple comparison followed by Bonferroni correction was used for statistical analysis. (G, H) Grip strength and organ weight measurements of 6 months old control (vehicle) and Myf5Cre-Pcyt2 male mice that have been treated with either vehicle or ss-31 compound for two months. Credit: IMP-IMBA Graphics Department. Data are shown as means ± SEM. Multiple comparison One-Way ANOVA with Dunnett correction was used for statistical analysis.
Extended Data Fig. 10 Assessment of calcium handling, and autophagy markers in skeletal muscle.
(A) Representative images of SR ultrastructure in skeletal muscles from 6 months old male control and Myf5Cre-Pcyt2 mice. Scale bar 200 nm. (B) Voltage-dependence of the peak rate of sarcoplasmic reticulum (SR) Ca2+ release (d[CaTot]/dt) measured from rhod-2 Ca2+ transients in fibres from male control (n = 6 mice and 23 myofibres) and Myf5Cre-Pcyt2 (n = 5 mice and 21 myofibres). (C) Decline of voltage-activated fluo-4FF Ca2+ transients in muscle fibers from control (n = 2 mice and 6 myofibers) and Myf5Cre-Pcyt2 (n = 3 mice and 8 myofibers) in response to an exhausting voltage stimulation protocol. (D) LC3 I/II and p62 levels in quadriceps from 8 months old control and Myf5Cre-Pcyt2 male mice under fed and fasting (24 h) conditions. N = 3 mice per group. (E) LC3 I/II and p62 levels in diaphragm from 8 months old control and Myf5Cre-Pcyt2 male mice under fed and fasting (24 h) conditions. N = 3 mice per group. (F, G) Quantification of p62 levels under fed and fasting conditions from quadriceps and diaphragm muscle respectively. Each dot represents individual mice. Data are shown as means ± SEM. Unpaired Student t-test with Welch correction was used for statistical analysis.
Supplementary information
Supplementary Information
Supplementary Methods and Supplementary Figs. 1–8
Laser-induced myofiber damage assessment on myofibres isolated 6-month-old control mice.
Laser-induced myofibre damage assessment on myofibres isolated 6-month-old control mice.
Laser-induced myofibre damage assessment on myofibres isolated 6-month-old Myf5Cre Pcyt2 mice.
Laser-induced myofibre damage assessment on myofibres isolated 6-month-old Myf5Cre Pcyt2 mice.
Video of the last bout of eccentric exercise (day 13) of 6-month-old control and Myf5Cre-Pcyt2 mice. First, mice were adjusted to a lower speed (9 m min−1; t = 0–10 s), followed by a higher speed bout (20 m min−1; t = 10–40 s).
Supplementary Data 1
Statistical source data for Supplementary Fig. 4.
Supplementary Data 2
Statistical source data for Supplementary Fig. 5.
Supplementary Data 3
Statistical source data for Supplementary Fig. 7.
Supplementary Data 4
Statistical source data for Supplementary Fig. 8.
Supplementary Data 5
Untargeted lipidome analysis of skeletal muscle at P10.
Supplementary Data 6
Untargeted lipidome analysis of BAT at P10.
Supplementary Data 7
Untargeted lipidome analysis of skeletal muscle mitochondria.
Supplementary Data 8
Untargeted lipidome analysis of young and old skeletal muscle.
Source data
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Cikes, D., Elsayad, K., Sezgin, E. et al. PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing. Nat Metab 5, 495–515 (2023). https://doi.org/10.1038/s42255-023-00766-2
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DOI: https://doi.org/10.1038/s42255-023-00766-2
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