PCYT2-regulated lipid biosynthesis is critical to muscle health and ageing

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.

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 labeled quadriceps from 2 days old control and Myf5Cre-Pcyt2 male mice. Images were taken under 5x magnification, and ≥2000nuclei were counted and analyzed. N=4 animals per group. Scale bar 60μm. (B) Representative images and quantification of EdU labeled primary myoblasts in cell culture isolated from control and Myf5Cre-Pcyt2 male mice. 18 biological replicate cultures from 3 independent isolations were analyzed and images were taken under 5x magnification. ≥100 nuclei counted per each culture. Each dot represents the number of EdU positive (B) cells per each culture. Scale bar 50μm. (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 50μm. 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 50μm. (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 50μm. 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 fibers 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 500μm. (C) Representative images and quantification of oxidative and glycolytic fibers 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 500μm. (D) Total number of fibers 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 500μm. 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 2μm. (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 100μm 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 analyze 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 analyze 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 50μm. (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 1μm. (I-J) Complex I and II activities of brown fat mitochondria. Paired Student t-test was used to analyze 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). (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). (E) Body weights of 8 months old control and Mnx1Cre-Pcyt2 male mice. (F) Absence of any overt clasping behavior and appearance in 8 months old Mnx1Cre-Pcyt2 male mice. (G) Schematic diagram of intestine epithelium specific Pcyt2 deficient male mice (VilinCre-Pcyt2). (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 m. (J) Schematic diagram of skin epithelium Pcyt2 deficient male mice (K14Cre-Pcyt2). Representative images of 3 animals per group are shown. (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 m. (M) Schematic diagram of mature muscle specific Pcyt2 deficient male mice (MCKCre-Pcyt2). (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 200nm. 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. 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 sarcoplasmatic reticulum (SR) Ca2+ release (d[CaTot]/dt) measured from rhod-2 Ca2+ transients in fibers from male control (n=6 mice and 23 myofibers) and Myf5Cre-Pcyt2 (n=5 mice and 21 myofibers). (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 (24h) 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 (24h) 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.

Figure 1.. Phenotypes of human PCYT2 rare disease mutations and pcyt2 mutant zebrafish.

(A) Body weight and (B) height gain of patient (male) carrying the homozygous nonsense variant 3c.1129C>T (p.Arg377Ter) in the PCYT2 gene. Controls indicate WHO standards of median weights and heights at the respective ages +/− 2 standard deviations (SD). (C) Representative appearance and quantifications of body length of control and hypomorphic pcyt2 mutant zebrafish at 14 months post fertilization. n=4 for each group. (D) Representative muscle sections and muscle myofiber sizes of control (n=4 animals and 233 myofibers in total) and hypomorphic pcyt2 (n=4 animals and 233 myofibers in total) zebrafish. Scale bar 50μm. Myofibers of the same anatomical region were analyzed with ≥ 50 myofibers per animal (E) Body weight gains of control (n=15) and Myf5Cre-Pcyt2 (n=11) littermates on standard chow diet. Two-Way ANOVA with multiple comparison followed by Bonferroni correction was used. ****p^(genotype) < 0.0001 (F) Appearance of 4 days old (P4) and 56 days old (P56) control and Myf5Cre-Pcyt2 littermates. Scale bars are 1 cm for P4 and 2 cm for P56. (G) Skeletal muscle appearance (quadriceps) isolated from 10 days control and Myf5Cre-Pcyt2 littermate mice. (H) Representative cross sections and (I) skeletal muscle myofiber diameter sizes from 6 months old control (n=4 mice and 570 myofibers) and Myf5Cre-Pcyt2 mice (n=4 mice and 640 myofibers). Myofibers were imaged using 10X magnification with ≥ 100myofibers analyzed per mouse. Scale bar 100μm. (I) Lipidomics analyses from quadriceps muscles isolated from 10 days old Myf5Cre-Pcyt2 and littermate control mice. Data are shown relative to control values. CE-cholesterol ester; Cer-Ceramides; DAG-diacylglycerols; LPC-lysophosphatidylcholines; LPE-lysophosphatidylethanolamines; PC-phosphatidylcholines; PE-phosphatidylethanolamines; PG-phosphatidylglycerols; PI-phosphatidylinositols; PS-phosphatidylserines; SM-sphingomyelins; TAG-triacylglycerols. n=4 per group. (J) Detailed analysis of PE species with different chain lengths from quadriceps muscles of Myf5Cre-Pcyt2 as compared to control mice. Data are shown as means ± SEM. *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.

Figure 2.. Pcyt2 deficiency affects muscle stem cell fusion and muscle hypertrophic growth.

(A) Representative images of Mf20 stained myofibers and (B) primary myoblast fusion index quantification of Control and Myf5Cre-Pcyt2 primary myoblasts after differentiation in vitro. Nine biological replicate myoblast cultures from three independent isolations were used. Each dot represents a calculated fusion index from in total n=9 cultures for each group. ≥ 300 nuclei were counted per one culture. Myofibers were imaged using 10X magnification. Scale bar 50μm. (C) Representative images and myofiber diameter quantification of Control (n=145 myofibers) and Myf5Cre-Pcyt2 (n=158 myofibers) after differentiation from primary myoblasts in vitro. Myofibers were imaged using 10X magnification. Scale bar 50μm (D) RT-PCR analysis of fusion and differentiation markers of Control and Myf5Cre-Pcyt2 myoblasts after 48h in differentiation media. N=5 cell cultures from 5 different animals per group. (E) Representative images and myoblast fusion index quantification of primary myoblasts with addition of vehicle (DMSO) and SA-Ro phosphatidylethanolamine binding peptide in differentiation media. Nine biological replicate myoblast cultures from three independent isolations were used. Each dot represents a calculated fusion index from in total n=9 cultures for each group. ≥ 300 nuclei were counted per one culture. Myofibers were imaged using 10X magnification. Scale bar 50μm. (F) Hypertrophic muscle growth in control and Myf5Cre-Pcyt2 mice. Following synergic ablation or sham surgery, M. plantaris weights were determined on the compensating limb. Each dot represents individual mice. Data are shown as means ± SEM. *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.

Figure 3.. Inactivation of Pcyt2 in mice leads to progressive weakness, muscle atrophy, inflammation and accelerated ageing.

(A) Representative images of 6 months old control and Myf5Cre-Pcyt2 male mice and (B) quantification of progressive worsening of hind limb clasping (B). Each dot represents one mouse, values are average of three measurements per mouse; scale bar 1 cm. (C) Age-dependent decline in grip strength in male control and Myf5Cre-Pcyt2 littermates. Each dot represents one mouse, values are average of three measurements per mouse. (D) Typical kyphosis appearance and kyphosis severity in 8 months old control (n=4) and Myf5Cre-Pcyt2 male mice (n=7). (E) Evident scoliosis (arrows) in a patient carrying the homozygous nonsense variant NM_001184917.2:3c.1129C>T (p.Arg377Ter) in PCYT2. (F) Representative image and quantification of relative muscle mass changes of 12 months old versus 6 months old control and Myf5Cre-Pcyt2 male littermates. QA, quadriceps; GC, gastrocnemius; TA, tibialis anterior muscles. Scale bar = 1 cm; n=7 per group. (G) Quantification of myofibers with central nuclei in quadriceps muscles from 8 months old control and Myf5Cre-Pcyt2 male mice. n=3 mice per group. Scale bar 100μm. (H) Muscle inflammation as determined by H&E staining. Data are from 12 months old male mice. Data are representative for n=4 mice per group. Scale bar 100μm. (I) Representative cross section of tibial bone in 12 months old control and Myf5Cre-Pcyt2 male mice with quantification of tibial bone cortical thickness. Randomly assigned 5 areas from n=4 mice per group were quantified. Scale bar 100μm. (J) Survival curves for control and Myf5Cre-Pcyt2 male mice. n=22 mice per group. For statistical analysis Mantel Cox test). 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.

Figure 4.. Pcyt2 deficiency severely affects muscle mitochondrial homeostasis as opposed to brown fat mitochondria.

(A) Total PE levels and (B) global lipidomics analyses of purified mitochondria isolated from 2 months old Control and Myf5Cre-Pcyt2 male mice. N=6 mice per group (C) Pathway enrichment analysis of differentially expressed genes in Control and Myf5Cre-Pcyt2 quadriceps isolated from 10 days old male pups. Evident enrichment of mitochondrial dysfunction linked genes specifically in the muscle of Myf5Cre-Pcyt2 mice. N=4 mice per group. (D) Muscle mitochondrial function assessed by measurements of complex I linked activity on isolated mitochondria from 2 months and 6 months old control and Myf5Cre-Pcyt2 male mice respectively. Paired Student t-test. (E) Measurement and quantification of myofiber mitochondrial reactive oxygen species (mtROS) in isolated myofibers (EDL muscle) from 6 months old male control (n=3 mice and 61 myofiber) and Myf5Cre-Pcyt2 mice (n=3 mice and 59 myofibers) as detected by MitoSox staining. Each dot represents relative amount of mtROS from a single myofiber. Scale bar 25μm. (F) Evidence of increased protein oxidative damage in quadriceps muscles isolated from 6 months old male Myf5Cre-Pcyt2 mice. Representative blots are shown for n=3 mice per group. (G) Catalase anti-oxidant activity in quadriceps muscles from 6 months old control and Myf5Cre-Pcyt2 male mice. (H) Increased levels of phospho-JNK (pJNK) and FoxO1 in quadriceps muscles from 6 months old male Myf5Cre-Pcyt2 mice (n=3) as compared to controls (n=3). 4 animals per group were analyzed in total, representative blot from 3 animals per group is shown (I) Increased levels of myofiber wasting markers in muscles of 8 months old male Myf5Cre-Pcyt2 mice. 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.

Figure 5.. Loss of Pcyt2 results in altered muscle membrane architectures.

(A) Scheme of GPMV isolation from primary myoblasts. (B) Polarization microscopy analysis of NR12S dye-stained myoblast-derived GPMVs from male control (n=71) and Myf5Cre-Pcyt2 (n=71) myoblasts. Each dot represents GP values of a single GPMV. GPMVs were derived from two independent isolations Scale bar 10μm. (C) Scheme and representative example of GPMVs (arrows) immediately after isolation from skeletal myofibers. Images are taken at 0 and 30 minutes under GPMV conditions. Scale bar 50μm. (D) Polarization microscopy of NR12S-stained GPMVs from control (n=58) and Myf5Cre-Pcyt2 (n=99) from primary myofibers (as shown in C). from two independent isolations. Representative images and quantifications are shown. Each dot represents GP values of a single GPMV. Scale bar 10μm. (E) Scheme of Brillouin light scattering microscopy for freshly isolated myofibers. (F) Surface stiffness analysis measured by Brillouin frequency shift (BFS) from isolated myofibers for male control (n=3 mice and 60 myofibers in total) and Myf5Cre-Pcyt2 (n=3 mice and 60 myofibers in total). Left panels indicate representative Brillouin images. Each data point in the right panel represents a BFS peak value of the individual myofiber surface. (G) Representative qualitative membrane stiffness data of male control and Myf5Cre-Pcyt2 myofibers assessed by atomic force microscopy. Displayed by curve angles in the approach (0 to −1000nm) and retraction phase (−1000 to 0nm), the cantilever bends less for Myf5Cre-Pcyt2 myofibers, indicating lower surface stiffness. In the prolonged part of retraction phase (0 to 400nm) the cantilever remains deeper within the Myf5Cre-Pcyt2 myofibers, indicating higher degree of surface deformity upon pressure. (H) Quantitative myofiber membrane stiffness as assessed by atomic force microscopy (Young’s modulus scale in kilopascal, kPa). For each myofiber we collected ≥4000 measurements (5μm X 5μm area). Matlab’s Randsample function was used to uniformly sample each myofiber measurements. Each dot represents 500 data points per each myofiber, from control (n=20) and Myf5Cre-Pcyt2 (n=26) myofibers. Data are shown as means ± SEM. Data are shown as means ± SEM. *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.

Figure 6.. Pcyt2 is essential for muscle membrane integrity and strain tolerance.

(A) Penetrance of Evans blue into the quadriceps muscle of 6 months old male control and Myf5Cre-Pcyt2 mice after i.p. injection. Gross morphologies and histological sections are shown. Scale bars are 1cm and 100μm. (B) Quantification of Evans blue after extraction from the muscle. n=3 per group. (C) Laser induced damage of isolated myofibers from male 6 months old control (n=9) and Myf5Cre-Pcyt2 (n=12) myofibers. The injured membrane areas of the myofibers are indicated by arrows. Right panel shows quantification of fm43 influx over the indicated time n=9–12 myofibers per group from two independent isolations. Scale bar 50μm (D) Running distance during eccentric exercise regime of 6 months old male control (n=6) and Myf5Cre-Pcyt2 (n=4) mice. (E) Representative histological analysis (H&E staining) of quadriceps muscles isolated from untrained (no training) 6 months old male control (n=4) or Myf5Cre-Pcyt2 mice (n=4) and from 6 months old control (n=6) or Myf5Cre-Pcyt2 mice (n=4) after eccentric exercise (training). Black and blue arrows show inflammation and ectopic fat deposits. Scale bars 100μm (F) Myopathy scores in 6 months old male control (n=6) and Myf5Cre-Pcyt2 (n=4) mice following eccentric exercise. The following parameters were used: inflammation, myofiber necrosis, atrophy, interstitial fibrosis, loss of membrane integrity, regenerating myofibers. Each was scored with 1–4 depending of the severity, and summed. (G) Blood muscle creatine kinase levels inferred from muscle creatine kinase activity from 6 months old sedentary and immediately after eccentric exercise of male control and Myf5Cre-Pcyt2 mice. (H) F-actin staining of skeletal muscle tissue isolated from 6 months old male control and Myf5Cre-Pcyt2 mice after eccentric exercise. Images of quadriceps cross-sections were taken using 20x magnifications. ≥100 myofibers were counted. n=3 mice per group. Scale bar 15μm. 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.

Figure 7.. Adenovirus based Pcyt2 gene therapy in mice is efficient for treatment of Pcyt2 deficiency-induced muscle pathology.

(A) Scheme of Pcyt2 muscle specific gene therapy. (B) Grip strength of male control (saline) Myf5Cre-Pcyt2 (saline) and Myf5Cre-Pcyt2 (AAV6-CK8-Pcyt2HA) mice. (C) Muscle weight isolated from 6 months old male control, Myf5Cre-Pcyt2 saline treated and Myf5Cre-Pcyt2 AAV6-Pcyt2 treated mice. Each dot represents single mice. QA, quadriceps; GC, gastrocnemius; TA, tibialis anterior muscles. Scale bars 1 cm. (D) Assessment of Pcyt2HA expression in Myf5Cre-Pcyt2 AAV6-Pcyt2 treated male mice as determined by quadriceps lysate anti-HA immunoprecipitation, followed by anti-Pcyt2 blot, 6 months after the gene delivery. (E) Total phosphatidylethanolamine levels from quadriceps of control, Myf5Cre-Pcyt2 saline treated and Myf5Cre-Pcyt2 AAV6-Pcyt2 treated male mice, 6 months after the treatment. (F) Myofiber diameter sizes from 6 months old control (n=5 mice and ≥ 60 myofibers analyzed per mouse), Myf5Cre-Pcyt2 saline treated (n=5 mice and ≥ 60 myofibers analyzed per mouse), and Myf5Cre-Pcyt2 AAV6-Pcyt2 mice (n=5 mice and ≥ 60 myofibers analyzed per mouse). Myofibers were imaged using 10X magnification. Scale bar 100μm. (G) Polarization microscopy of NR12S-stained muscle-derived GPMVs from control (n=84), Myf5Cre-Pcyt2 saline (n=93) and Myf5Cre-Pcyt2 AAV6-Pcyt2 treated (n=88) 6 months old male mice. GPMVs were derived from three independent isolations. Each dot represents values of a single GPMV. Scale bar 5μm. (H) Surface stiffness analysis by Brillouin frequency shift (BFS) from isolated myofibers of control, Myf5Cre-Pcyt2 saline and Myf5Cre-Pcyt2 AAV6-Pcyt2 treated 6 months old male mice. Each data point in the right panel represents a BFS peak value of the myofiber surface. 20 myofibers from n=4 male mice per group. (I) Muscle mitochondrial respiration of control, Myf5Cre-Pcyt2 saline and Myf5Cre-Pcyt2 AAV6-Pcyt2 treated mice as assessed by complex I linked activity on muscle lysates isolated from 6 months old male mice. N=5 mice per group. Paired Student t-test was used for statistical analysis. Dashed line indicates the average value of mitochondrial activities measured from 5 individual control male mice. 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. Unless otherwise indicated, Multiple comparison One-Way ANOVA with Dunnett correction was used for statistical analysis unless stated otherwise.

Figure 8.. Pcyt2 activity is reduced in aged muscles from humans and mice and Pcyt2 gene delivery ameliorates age-related atrophy in sarcopenic mice.

(A) PCYT2 activity in quadriceps from young (20–30yr) and middle aged (45–62yr) healthy human volunteers. Each dot represents individual human. (B) Pcyt2 activity in quadriceps from young (6 month) and pre-sarcopenic (24 months old) C57B6/J male mice. (C) Lipidomics analyses from quadriceps isolated from young (6 months old) and pre-sarcopenic (24 months old) C57B6/J male mice. mice. Data are shown relative to control values. CE-cholesterol ester; Cer-Ceramides; DAG-diacylglycerols; LPC-lysophosphatidylcholines; LPE-lysophosphatidylethanolamines; PC-phosphatidylcholines; PE-phosphatidylethanolamines; PG-phosphatidylglycerols; PI-phosphatidylinositols; PS-phosphatidylserines; SM-sphingomyelins; TAG-triacylglycerols. n=5 mice per group. (D) Scheme of adenovirus based, muscle-specific delivery of Pcyt2 to pre-sarcopenic 24 months old C57B6/J male mice. (E) Assessment of Pcyt2HA expression as determined by anti-HA immunoprecipitation, followed by an anti-Pcyt2 blot, from quadriceps isolated 2 months after the gene delivery. (F) Total phosphatidylethanolamine levels from quadriceps of young (6 months old), and aged (26 months old) control (saline) and AAV6-CK8-Pcyt2HA transduced C57B6/J male mice two months after AAV6 injection. (G) Polarization microscopy of NR12S-stained GPMVs isolated from aged (26 months old) control (n=63) (saline) and AAV6-CK8-Pcyt2HA transduced (n=79) C57B6/J male mice two months after AAV6 injection. GPMVs were derived from three independent isolations. Each dot represents values of a single GPMV. Scale bar 10μm. (H) Surface stiffness analysis as measured by Brillouin frequency shift (BFS) from isolated myofibers of aged (26 months old) control (saline) and AAV6-CK8-Pcyt2HA transduced C57B6/J male mice two months after AAV6 injection. Dashed line indicates the average value of Brillouin frequency shift (BFS) measured separately from five 6 month old male mice. Each data point in the right panel represents a BFS peak value of the myofiber surface. 7 myofibers from n=7 mice per group were analyzed. (I) Muscle mitochondrial function of aged (26 months old) control (saline) and AAV6-CK8-Pcyt2HA transduced C57B6/J male mice two months after the treatment as measured by complex I linked activity. Paired Student t-test was used for statistical analysis. Dashed line indicates the average value of mitochondrial activities measured separately from five 6 month old male mice. (J) Myofiber diameter sizes from aged (26 months old) control (saline) (n=5 mice and ≥ 180 myofibers analyzed per mouse.) and AAV6-CK8-Pcyt2HA transduced C57B6/J male mice (n=5 mice and ≥ 180 myofibers analyzed per mouse) two months after the AAV6 injection. Dashed line indicates the average value of myofiber diameter sizes measured separately from five 6 months old male mice (≥ 60 myofibers analyzed per mouse). Scale bar 100μm. (K) Grip strength measurements on aged (26 months old) control (saline; n=15) and AAV6-CK8-Pcyt2HA transduced (n=11) C57B6/J male mice one and two months after AAV6 injection. Repeated Measures Two-Way ANOVA with Bonferroni correction was used for statistical analysis. 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.