Compromised mitochondrial quality control triggers lipin1-related rhabdomyolysis

Yamina Hamel, François-Xavier Mauvais, Marine Madrange, Perrine Renard, Corinne Lebreton, Ivan Nemazanyy, Olivier Pellé, Nicolas Goudin, Xiaoyun Tang, Mathieu P Rodero, Caroline Tuchmann-Durand, Patrick Nusbaum, David N Brindley, Peter van Endert, Pascale de Lonlay, Yamina Hamel, François-Xavier Mauvais, Marine Madrange, Perrine Renard, Corinne Lebreton, Ivan Nemazanyy, Olivier Pellé, Nicolas Goudin, Xiaoyun Tang, Mathieu P Rodero, Caroline Tuchmann-Durand, Patrick Nusbaum, David N Brindley, Peter van Endert, Pascale de Lonlay

Abstract

LPIN1 mutations are responsible for inherited recurrent rhabdomyolysis, a life-threatening condition with no efficient therapeutic intervention. Here, we conduct a bedside-to-bench-and-back investigation to study the pathophysiology of lipin1 deficiency. We find that lipin1-deficient myoblasts exhibit a reduction in phosphatidylinositol-3-phosphate close to autophagosomes and late endosomes that prevents the recruitment of the GTPase Armus, locks Rab7 in the active state, inhibits vesicle clearance by fusion with lysosomes, and alters their positioning and function. Oxidized mitochondrial DNA accumulates in late endosomes, where it activates Toll-like receptor 9 (TLR9) and triggers inflammatory signaling and caspase-dependent myolysis. Hydroxychloroquine blocks TLR9 activation by mitochondrial DNA in vitro and may attenuate flares of rhabdomyolysis in 6 patients treated. We suggest a critical role for defective clearance of oxidized mitochondrial DNA that activates TLR9-restricted inflammation in lipin1-related rhabdomyolysis. Interventions blocking TLR9 activation or inflammation can improve patient care in vivo.

Trial registration: ClinicalTrials.gov NCT04007562.

Keywords: Toll-like receptor 9; autophagosome; hydroxychloroquine; inflammation; late endosome; lipin1; mitochondrial quality control; rhabdomyolysis.

Conflict of interest statement

Y.H., F.-X.M., C.L., P.v.E., and P.d.L. have filed PCT (WO/2017/085115; EP3377095; PCT/ EP2016/077843) and US patent applications (US20180325890). Y.H., F.-X.M., M.M., P.v.E., and P.d.L. have filed PCT (WO/2019/020732; PCT/EP2018/070256) patent applications. The remaining authors declare no competing interests.

© 2021 The Authors.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Lipin1 localizes in proximity of late endosomes, autophagosomes, and mitochondria in human myoblasts (A) The confocal images show a representative staining of endogenous lipin1 in primary myoblasts from 1 of 6 controls and 6 patients carrying LPIN1 mutations (Table S1) and in immortalized myoblasts from 1 healthy donor transduced with sh-LPIN1 or control scrambled (sh-Sc) shRNA. (B) Immunoblot analysis of endogenous lipin1 expression as in (A) in 5 patients. (C) Dot plots (mean of 3 technical replicates/dot) depict the means ± SDs of PAP activity quantified in myoblasts from 6 controls and 5 patients (Mann-Whitney U test). (D) Lipin1 distribution within myoblast organelles of 5 controls by confocal microscopy. Dot plots (50–80 cells/dot) show the means ± SDs of the percentage of lipin1 in proximity with a given protein. The images are representative of 1 of at least 3 independent experiments. Scale bars, 10 μm.
Figure 2
Figure 2
Lipin1 deficiency results in a loss of PI3P close to Rab7 structures in myoblasts (A) The confocal images show a representative 2xFYVE-GFP staining from 1 of 6 controls and patients. Dot plots (50 images/dot) show the means ± SDs number of 2xFYVE-GFP dots per patient versus control myoblast (Mann-Whitney U test). (B) As in (A), but using immortalized myoblasts from 1 healthy donor treated with sh-Sc or sh-LPIN1. The plots (30–45 cells/condition) show the number of 2xFYVE-GFP dots (Mann-Whitney U test). (C) Typical distribution of PI3P within the myoblast vesicles from 1 of 3 controls and patients. Box and whisker plots (50 images/condition) depict the percentage of proximity between PI3P and the given marker for 1 representative control and patient (Mann-Whitney U test). (D) Myoblasts from 3 controls and 3 patients were stained for PI35P2 and late endosomal or lysosomal markers. Proximity was calculated as in (C) (unpaired t test). Results are from 1 representative of at least 3 independent experiments. Scale bars, 10 μm. See also Figure S1.
Figure 3
Figure 3
Lipin1-deficient myoblasts display alterations in Rab7 positioning and activation and impaired lysosomal function (A) The distribution of endogenous Rab7 in 30 individual patient and control myoblasts was determined by calculating the intensity of Rab7 fluorescence along a longitudinal axis traversing the nucleus. (B) The interaction between Rab7 and FYCO1 determined by fluorescence resonance energy transfer (FRET) after staining with Rab7 (green) only or for Rab7 (donor) and FYCO1 (acceptor) in red was depicted by line graphs (ns, nanoseconds). (C) Myoblasts transfected with RFP-RILP were immunostained for Rab7. Box and whisker plots (30 images/condition) show the percentage of proximity of Rab7 with RFP-RILP (unpaired t test). (D and E) Myoblasts transfected with a construct encoding RFP-Armus were stained for Rab7 or PI3P. Box and whisker plots (50 images/condition) show the percentage of proximity of Rab7 (D) or PI3P (E) with RFP-Armus (Mann-Whitney U test). (F) Dot plot (mean of 2 technical replicates/dot) with lines depicts the relative level of residual EGFR as compared to β-actin upon incubation with EGF for indicated times. (G) Myoblasts were loaded with DQ-OVA, and fluorescence resulting from the cleavage of DQ-OVA substrate was analyzed by flow cytometry. Dot plots (mean of 2 technical replicates/dot) show the means ± SDs of the value of the mean fluorescence intensity (MFI) of DQ-OVA 60 and 120 min after uptake (mean effect of interaction F(1,6) = 14.43, p = 0.009, of time F(1,6) = 53.46, p = 0.0003, of subjects F(1,6) = 11.23, p = 0.0154). (H) Levels of immature and mature cathepsin D relative to β-actin expression in primary myoblasts were determined by immunoblot. (I) Myoblasts cultured in GM or EBSS were loaded with the Lysotracker Red dye, then stained for endogenous Rab7, LAMP1, and LC3. Box and whisker plots (30 images/condition) show the percentage of proximity of Rab7 (mean effect of interaction F(1,116) = 22.01, p 

Figure 4

Autophagic clearance and mitochondrial quality…

Figure 4

Autophagic clearance and mitochondrial quality and functions are impaired in human lipin1-deficient myoblasts…

Figure 4
Autophagic clearance and mitochondrial quality and functions are impaired in human lipin1-deficient myoblasts (A) Autolysosome formation (red puncta) in myoblasts transfected with the RFP-GFP-LC3 construct upon exposure to EBSS and refed or not with GM. Box and whisker plots (50 images/condition) represent the percentage of autolysosomes (mean effect of interaction F(1,196) = 44.4, p 12S mtDNA levels qPCR in cytosolic fractions from myoblasts of 4 patients and controls exposed to CCCP or EBSS (mean effect of interaction F(1,12) = 0.004867, p = 0.9455, of stimulus F(1,12) = 1.798, p = 0.2047, of subjects F(1,12) = 0.03755, p = 0.8496) and of the mtDNA motif DLOOP (mean effect of interaction F(1,12) = 0.2168, p = 0.6498, of stimulus F(1,12) = 0.3160, p = 0.5844, of subjects F(1,12) = 3.371, p = 0.0913). Dot plots (mean of 3 technical replicates/dot) depict the means ± SDs of the ratio of cycle threshold (CT) values of the given cytosolic fraction normalized to unfractioned cells. Scale bars, 10 μm (A and B). ∗p 

Figure 5

Accumulation of oxidized mitochondria enhances…

Figure 5

Accumulation of oxidized mitochondria enhances inflammation in lipin1-deficient cells through a TLR9 pathway…

Figure 5
Accumulation of oxidized mitochondria enhances inflammation in lipin1-deficient cells through a TLR9 pathway (A) Circulating mtDNA was quantified in plasma by qPCR and inflammatory molecules were measured in sera by flow cytometry, from 12 healthy donors (6 for mtDNA) and 6 patients (Mann-Whitney U test). Dot plots (mean of 3 technical replicates/dot) show the means ± SDs. (B) IFN signature in peripheral blood mononuclear cells was identified by qPCR in 4 controls and 4 patients. Dot plots (mean of 3 technical replicates/dot) show the mean ± SD of the CT value of a given gene normalized to the CT value of BACT (Mann-Whitney U test). (C) DC maturation reflected by CD83 expression (mean effect of interaction F(4,70) = 2.114, p = 0.0881, of stimulus F(4,70) = 36.27, p SIGLEC1 (mean effect of interaction F(1,8) = 4.436, p = 0.0683, of stimulus F(1,8) = 9.255, p = 0.0160, of subjects F(1,8) = 20.78, p = 0.0019), ISG15 (mean effect of interaction F(1,8) = 0.3191, p = 0.5876, of stimulus F(1,8) = 21.75, p = 0.0016, of subjects F(1,8) = 41.33, p = 0.0002), and IFI27 (mean effect of interaction F(1,8) = 21.99, p = 0.0016, of stimulus F(1,8) = 25.45, p = 0.0010, of subjects F(1,8) = 91.20, p < 0.0001) expression. Dot plots (mean of 3 technical replicates/dot) show the means ± SDs of CT value calculated as in (B). (E) Production of IL-6 (ELISA) by myoblasts from 8 controls and 6 patients (mean effect of interaction F(2,36) = 3.882, p = 0.0297, of stimulus F(2,36) = 45.49, p IFNA (mean effect of interaction F(3,24) = 23.44, p < 0.0001, of stimulus F(3,24) = 74.13, p < 0.0001, of subjects F(1,24) = 42.33, p < 0.0001), and IFNB (mean effect of interaction F(3,24) = 2.207, p = 0.1133, of stimulus F(3,24) = 10.19, p = 0.0002, of subjects F(1,24) = 13.60, p = 0.0012). (F) As in (E), but using sh-Sc or sh-LPIN1-transduced immortalized myoblasts from 1 healthy donor (1 different cell vial from the same single healthy donor/dot) and primary myoblasts from 4 patients. Dot plots (mean of 4 technical replicates/dot) show the means ± SDs of the IL-6 concentration (mean effect of interaction F(2,14) = 6.234, p = 0.0116, of stimulus F(1,14) = 0.0874, p = 0.3653, of subjects F(2,14) = 4.397, p = 0.0330). (G) As in (F), but primary myoblasts from 4 controls and patients were transduced with a lentivirus expressing a plasmid encoding for a myc-DDK-tagged lipin1 protein (+LPIN1) or an empty vector (+vector) before being challenged (mean effect of interaction F(2,18) = 0.2196, p = 0.8050, of stimulus F(1,18) = 0.2906, p = 0.5965, of subjects F(2,18) = 14.80, p = 0.0002). (H) Myoblasts from 6 controls and patients were exposed to a vehicle or ethidium bromide (Et. B.). Dot plots (mean of 3 technical replicates/dot) show the means ± SDs of the ratio of the CT value for 12S mitochondrial DNA normalized to the CT value for BACT (mean effect of interaction F(1,12) = 3.186, p = 0.0996, of stimulus F(1,12) = 101.6, p < 0.0001, of subjects F(1,12) = 5.268, p = 0.0405). (I) Myoblasts from 6 controls and patients pre-treated with Et. B. for 5 days were exposed to EBSS and challenged or not with CpG-A for 16 h. Dot plots (mean of 4 technical replicates/dot) show the mean ± SD of IL-6 concentration in culture supernatants at the end of the experiment (mean effect of interaction F(3,24) = 4.989, p = 0.0079, of stimulus F(3,24) = 8.218, p = 0.0006, of subjects F(1,24) = 15.14, p = 0.0007). (J) Myoblasts were transfected with a plasmid encoding HA-tagged TLR9 and exposed to GM or EBSS and with vehicle or CpG-A before refeeding (Refed) cells exposed to EBSS and CpG-A with GM. Cells were then stained for HA-TLR9 (anti-HA antibody, red) and 8OHDG (green). Box whisker plots (25 images/condition) show the percentage of proximity of TLR9 with 8OHDG (mean effect of interaction F(4,240) = 1.712, p = 0.1481, of stimulus F(4,240) = 25.07, p 

Figure 6

mtDNA accumulation is responsible for…

Figure 6

mtDNA accumulation is responsible for inflammation and rhabdomyolysis that can be reversed by…

Figure 6
mtDNA accumulation is responsible for inflammation and rhabdomyolysis that can be reversed by steroids or chloroquine (CLQ) treatment in vivo (A) Real-time calcium flux in myoblasts from 4 controls and patients challenged with CpG-A, after pre-incubation with ODN151, a synthetic TLR9 antagonist. Each dot plot with lines depicts the mean ± SD of the intensity of the fluo-4 calcium probe as normalized to basal intensity of 1 single individual (≥10 cells/condition). (B) Death of myoblasts was evaluated by staining cells from 4 control individuals transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein (+LPIN1) and 4 patients transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein or an empty vector, for the apoptosis marker caspase3/7 green and propidium iodide or 7-actinomycin D (7-AAD), a dye that stains both apoptotic and necrotic cells. Staurosporin (ST): positive control. Fluorescence-activated cell sorting (FACS) plots show the codistribution of the fluorescence intensity of caspase3/7 and of 7-AAD in 1 of 4 controls and patients. (C) Dot plots (≥1,000 single cells/dot) depict the mean ± SD of the proportion of dead cells (ie, cells positive for capsase3/7 and/or 7-AAD) from (B) (mean effect of interaction F(8,45) = 5.296, p = 0.0001, of stimulus F(4,45) = 70.03, p LPIN1 shRNA (mean effect of interaction F(4,20) = 6.154, p = 0.0021, of stimulus F(4,20) = 48.93, p < 0.0001, of subjects F(1,20) = 15.62, p = 0.0008). (E and F) As in (C), but myoblasts were pre-treated with Et. B. or vehicle (mean effect of interaction F(3,24) = 21.28, p 
All figures (7)
Similar articles
Cited by
References
    1. Cervellin G., Comelli I., Lippi G. Rhabdomyolysis: historical background, clinical, diagnostic and therapeutic features. Clin. Chem. Lab. Med. 2010;48:749–756. - PubMed
    1. Luck R.P., Verbin S. Rhabdomyolysis: a review of clinical presentation, etiology, diagnosis, and management. Pediatr. Emerg. Care. 2008;24:262–268. - PubMed
    1. Michot C., Hubert L., Brivet M., De Meirleir L., Valayannopoulos V., Müller-Felber W., Venkateswaran R., Ogier H., Desguerre I., Altuzarra C. LPIN1 gene mutations: a major cause of severe rhabdomyolysis in early childhood. Hum. Mutat. 2010;31:E1564–E1573. - PubMed
    1. Baba T., Kashiwagi Y., Arimitsu N., Kogure T., Edo A., Maruyama T., Nakao K., Nakanishi H., Kinoshita M., Frohman M.A. Phosphatidic acid (PA)-preferring phospholipase A1 regulates mitochondrial dynamics. J. Biol. Chem. 2014;289:11497–11511. - PMC - PubMed
    1. Donkor J., Sariahmetoglu M., Dewald J., Brindley D.N., Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns. J. Biol. Chem. 2007;282:3450–3457. - PubMed
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Figure 4
Figure 4
Autophagic clearance and mitochondrial quality and functions are impaired in human lipin1-deficient myoblasts (A) Autolysosome formation (red puncta) in myoblasts transfected with the RFP-GFP-LC3 construct upon exposure to EBSS and refed or not with GM. Box and whisker plots (50 images/condition) represent the percentage of autolysosomes (mean effect of interaction F(1,196) = 44.4, p 12S mtDNA levels qPCR in cytosolic fractions from myoblasts of 4 patients and controls exposed to CCCP or EBSS (mean effect of interaction F(1,12) = 0.004867, p = 0.9455, of stimulus F(1,12) = 1.798, p = 0.2047, of subjects F(1,12) = 0.03755, p = 0.8496) and of the mtDNA motif DLOOP (mean effect of interaction F(1,12) = 0.2168, p = 0.6498, of stimulus F(1,12) = 0.3160, p = 0.5844, of subjects F(1,12) = 3.371, p = 0.0913). Dot plots (mean of 3 technical replicates/dot) depict the means ± SDs of the ratio of cycle threshold (CT) values of the given cytosolic fraction normalized to unfractioned cells. Scale bars, 10 μm (A and B). ∗p 

Figure 5

Accumulation of oxidized mitochondria enhances…

Figure 5

Accumulation of oxidized mitochondria enhances inflammation in lipin1-deficient cells through a TLR9 pathway…

Figure 5
Accumulation of oxidized mitochondria enhances inflammation in lipin1-deficient cells through a TLR9 pathway (A) Circulating mtDNA was quantified in plasma by qPCR and inflammatory molecules were measured in sera by flow cytometry, from 12 healthy donors (6 for mtDNA) and 6 patients (Mann-Whitney U test). Dot plots (mean of 3 technical replicates/dot) show the means ± SDs. (B) IFN signature in peripheral blood mononuclear cells was identified by qPCR in 4 controls and 4 patients. Dot plots (mean of 3 technical replicates/dot) show the mean ± SD of the CT value of a given gene normalized to the CT value of BACT (Mann-Whitney U test). (C) DC maturation reflected by CD83 expression (mean effect of interaction F(4,70) = 2.114, p = 0.0881, of stimulus F(4,70) = 36.27, p SIGLEC1 (mean effect of interaction F(1,8) = 4.436, p = 0.0683, of stimulus F(1,8) = 9.255, p = 0.0160, of subjects F(1,8) = 20.78, p = 0.0019), ISG15 (mean effect of interaction F(1,8) = 0.3191, p = 0.5876, of stimulus F(1,8) = 21.75, p = 0.0016, of subjects F(1,8) = 41.33, p = 0.0002), and IFI27 (mean effect of interaction F(1,8) = 21.99, p = 0.0016, of stimulus F(1,8) = 25.45, p = 0.0010, of subjects F(1,8) = 91.20, p < 0.0001) expression. Dot plots (mean of 3 technical replicates/dot) show the means ± SDs of CT value calculated as in (B). (E) Production of IL-6 (ELISA) by myoblasts from 8 controls and 6 patients (mean effect of interaction F(2,36) = 3.882, p = 0.0297, of stimulus F(2,36) = 45.49, p IFNA (mean effect of interaction F(3,24) = 23.44, p < 0.0001, of stimulus F(3,24) = 74.13, p < 0.0001, of subjects F(1,24) = 42.33, p < 0.0001), and IFNB (mean effect of interaction F(3,24) = 2.207, p = 0.1133, of stimulus F(3,24) = 10.19, p = 0.0002, of subjects F(1,24) = 13.60, p = 0.0012). (F) As in (E), but using sh-Sc or sh-LPIN1-transduced immortalized myoblasts from 1 healthy donor (1 different cell vial from the same single healthy donor/dot) and primary myoblasts from 4 patients. Dot plots (mean of 4 technical replicates/dot) show the means ± SDs of the IL-6 concentration (mean effect of interaction F(2,14) = 6.234, p = 0.0116, of stimulus F(1,14) = 0.0874, p = 0.3653, of subjects F(2,14) = 4.397, p = 0.0330). (G) As in (F), but primary myoblasts from 4 controls and patients were transduced with a lentivirus expressing a plasmid encoding for a myc-DDK-tagged lipin1 protein (+LPIN1) or an empty vector (+vector) before being challenged (mean effect of interaction F(2,18) = 0.2196, p = 0.8050, of stimulus F(1,18) = 0.2906, p = 0.5965, of subjects F(2,18) = 14.80, p = 0.0002). (H) Myoblasts from 6 controls and patients were exposed to a vehicle or ethidium bromide (Et. B.). Dot plots (mean of 3 technical replicates/dot) show the means ± SDs of the ratio of the CT value for 12S mitochondrial DNA normalized to the CT value for BACT (mean effect of interaction F(1,12) = 3.186, p = 0.0996, of stimulus F(1,12) = 101.6, p < 0.0001, of subjects F(1,12) = 5.268, p = 0.0405). (I) Myoblasts from 6 controls and patients pre-treated with Et. B. for 5 days were exposed to EBSS and challenged or not with CpG-A for 16 h. Dot plots (mean of 4 technical replicates/dot) show the mean ± SD of IL-6 concentration in culture supernatants at the end of the experiment (mean effect of interaction F(3,24) = 4.989, p = 0.0079, of stimulus F(3,24) = 8.218, p = 0.0006, of subjects F(1,24) = 15.14, p = 0.0007). (J) Myoblasts were transfected with a plasmid encoding HA-tagged TLR9 and exposed to GM or EBSS and with vehicle or CpG-A before refeeding (Refed) cells exposed to EBSS and CpG-A with GM. Cells were then stained for HA-TLR9 (anti-HA antibody, red) and 8OHDG (green). Box whisker plots (25 images/condition) show the percentage of proximity of TLR9 with 8OHDG (mean effect of interaction F(4,240) = 1.712, p = 0.1481, of stimulus F(4,240) = 25.07, p 

Figure 6

mtDNA accumulation is responsible for…

Figure 6

mtDNA accumulation is responsible for inflammation and rhabdomyolysis that can be reversed by…

Figure 6
mtDNA accumulation is responsible for inflammation and rhabdomyolysis that can be reversed by steroids or chloroquine (CLQ) treatment in vivo (A) Real-time calcium flux in myoblasts from 4 controls and patients challenged with CpG-A, after pre-incubation with ODN151, a synthetic TLR9 antagonist. Each dot plot with lines depicts the mean ± SD of the intensity of the fluo-4 calcium probe as normalized to basal intensity of 1 single individual (≥10 cells/condition). (B) Death of myoblasts was evaluated by staining cells from 4 control individuals transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein (+LPIN1) and 4 patients transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein or an empty vector, for the apoptosis marker caspase3/7 green and propidium iodide or 7-actinomycin D (7-AAD), a dye that stains both apoptotic and necrotic cells. Staurosporin (ST): positive control. Fluorescence-activated cell sorting (FACS) plots show the codistribution of the fluorescence intensity of caspase3/7 and of 7-AAD in 1 of 4 controls and patients. (C) Dot plots (≥1,000 single cells/dot) depict the mean ± SD of the proportion of dead cells (ie, cells positive for capsase3/7 and/or 7-AAD) from (B) (mean effect of interaction F(8,45) = 5.296, p = 0.0001, of stimulus F(4,45) = 70.03, p LPIN1 shRNA (mean effect of interaction F(4,20) = 6.154, p = 0.0021, of stimulus F(4,20) = 48.93, p < 0.0001, of subjects F(1,20) = 15.62, p = 0.0008). (E and F) As in (C), but myoblasts were pre-treated with Et. B. or vehicle (mean effect of interaction F(3,24) = 21.28, p 
All figures (7)
Similar articles
Cited by
References
    1. Cervellin G., Comelli I., Lippi G. Rhabdomyolysis: historical background, clinical, diagnostic and therapeutic features. Clin. Chem. Lab. Med. 2010;48:749–756. - PubMed
    1. Luck R.P., Verbin S. Rhabdomyolysis: a review of clinical presentation, etiology, diagnosis, and management. Pediatr. Emerg. Care. 2008;24:262–268. - PubMed
    1. Michot C., Hubert L., Brivet M., De Meirleir L., Valayannopoulos V., Müller-Felber W., Venkateswaran R., Ogier H., Desguerre I., Altuzarra C. LPIN1 gene mutations: a major cause of severe rhabdomyolysis in early childhood. Hum. Mutat. 2010;31:E1564–E1573. - PubMed
    1. Baba T., Kashiwagi Y., Arimitsu N., Kogure T., Edo A., Maruyama T., Nakao K., Nakanishi H., Kinoshita M., Frohman M.A. Phosphatidic acid (PA)-preferring phospholipase A1 regulates mitochondrial dynamics. J. Biol. Chem. 2014;289:11497–11511. - PMC - PubMed
    1. Donkor J., Sariahmetoglu M., Dewald J., Brindley D.N., Reue K. Three mammalian lipins act as phosphatidate phosphatases with distinct tissue expression patterns. J. Biol. Chem. 2007;282:3450–3457. - PubMed
Show all 71 references
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Cite
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Figure 5
Figure 5
Accumulation of oxidized mitochondria enhances inflammation in lipin1-deficient cells through a TLR9 pathway (A) Circulating mtDNA was quantified in plasma by qPCR and inflammatory molecules were measured in sera by flow cytometry, from 12 healthy donors (6 for mtDNA) and 6 patients (Mann-Whitney U test). Dot plots (mean of 3 technical replicates/dot) show the means ± SDs. (B) IFN signature in peripheral blood mononuclear cells was identified by qPCR in 4 controls and 4 patients. Dot plots (mean of 3 technical replicates/dot) show the mean ± SD of the CT value of a given gene normalized to the CT value of BACT (Mann-Whitney U test). (C) DC maturation reflected by CD83 expression (mean effect of interaction F(4,70) = 2.114, p = 0.0881, of stimulus F(4,70) = 36.27, p SIGLEC1 (mean effect of interaction F(1,8) = 4.436, p = 0.0683, of stimulus F(1,8) = 9.255, p = 0.0160, of subjects F(1,8) = 20.78, p = 0.0019), ISG15 (mean effect of interaction F(1,8) = 0.3191, p = 0.5876, of stimulus F(1,8) = 21.75, p = 0.0016, of subjects F(1,8) = 41.33, p = 0.0002), and IFI27 (mean effect of interaction F(1,8) = 21.99, p = 0.0016, of stimulus F(1,8) = 25.45, p = 0.0010, of subjects F(1,8) = 91.20, p < 0.0001) expression. Dot plots (mean of 3 technical replicates/dot) show the means ± SDs of CT value calculated as in (B). (E) Production of IL-6 (ELISA) by myoblasts from 8 controls and 6 patients (mean effect of interaction F(2,36) = 3.882, p = 0.0297, of stimulus F(2,36) = 45.49, p IFNA (mean effect of interaction F(3,24) = 23.44, p < 0.0001, of stimulus F(3,24) = 74.13, p < 0.0001, of subjects F(1,24) = 42.33, p < 0.0001), and IFNB (mean effect of interaction F(3,24) = 2.207, p = 0.1133, of stimulus F(3,24) = 10.19, p = 0.0002, of subjects F(1,24) = 13.60, p = 0.0012). (F) As in (E), but using sh-Sc or sh-LPIN1-transduced immortalized myoblasts from 1 healthy donor (1 different cell vial from the same single healthy donor/dot) and primary myoblasts from 4 patients. Dot plots (mean of 4 technical replicates/dot) show the means ± SDs of the IL-6 concentration (mean effect of interaction F(2,14) = 6.234, p = 0.0116, of stimulus F(1,14) = 0.0874, p = 0.3653, of subjects F(2,14) = 4.397, p = 0.0330). (G) As in (F), but primary myoblasts from 4 controls and patients were transduced with a lentivirus expressing a plasmid encoding for a myc-DDK-tagged lipin1 protein (+LPIN1) or an empty vector (+vector) before being challenged (mean effect of interaction F(2,18) = 0.2196, p = 0.8050, of stimulus F(1,18) = 0.2906, p = 0.5965, of subjects F(2,18) = 14.80, p = 0.0002). (H) Myoblasts from 6 controls and patients were exposed to a vehicle or ethidium bromide (Et. B.). Dot plots (mean of 3 technical replicates/dot) show the means ± SDs of the ratio of the CT value for 12S mitochondrial DNA normalized to the CT value for BACT (mean effect of interaction F(1,12) = 3.186, p = 0.0996, of stimulus F(1,12) = 101.6, p < 0.0001, of subjects F(1,12) = 5.268, p = 0.0405). (I) Myoblasts from 6 controls and patients pre-treated with Et. B. for 5 days were exposed to EBSS and challenged or not with CpG-A for 16 h. Dot plots (mean of 4 technical replicates/dot) show the mean ± SD of IL-6 concentration in culture supernatants at the end of the experiment (mean effect of interaction F(3,24) = 4.989, p = 0.0079, of stimulus F(3,24) = 8.218, p = 0.0006, of subjects F(1,24) = 15.14, p = 0.0007). (J) Myoblasts were transfected with a plasmid encoding HA-tagged TLR9 and exposed to GM or EBSS and with vehicle or CpG-A before refeeding (Refed) cells exposed to EBSS and CpG-A with GM. Cells were then stained for HA-TLR9 (anti-HA antibody, red) and 8OHDG (green). Box whisker plots (25 images/condition) show the percentage of proximity of TLR9 with 8OHDG (mean effect of interaction F(4,240) = 1.712, p = 0.1481, of stimulus F(4,240) = 25.07, p 

Figure 6

mtDNA accumulation is responsible for…

Figure 6

mtDNA accumulation is responsible for inflammation and rhabdomyolysis that can be reversed by…

Figure 6
mtDNA accumulation is responsible for inflammation and rhabdomyolysis that can be reversed by steroids or chloroquine (CLQ) treatment in vivo (A) Real-time calcium flux in myoblasts from 4 controls and patients challenged with CpG-A, after pre-incubation with ODN151, a synthetic TLR9 antagonist. Each dot plot with lines depicts the mean ± SD of the intensity of the fluo-4 calcium probe as normalized to basal intensity of 1 single individual (≥10 cells/condition). (B) Death of myoblasts was evaluated by staining cells from 4 control individuals transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein (+LPIN1) and 4 patients transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein or an empty vector, for the apoptosis marker caspase3/7 green and propidium iodide or 7-actinomycin D (7-AAD), a dye that stains both apoptotic and necrotic cells. Staurosporin (ST): positive control. Fluorescence-activated cell sorting (FACS) plots show the codistribution of the fluorescence intensity of caspase3/7 and of 7-AAD in 1 of 4 controls and patients. (C) Dot plots (≥1,000 single cells/dot) depict the mean ± SD of the proportion of dead cells (ie, cells positive for capsase3/7 and/or 7-AAD) from (B) (mean effect of interaction F(8,45) = 5.296, p = 0.0001, of stimulus F(4,45) = 70.03, p LPIN1 shRNA (mean effect of interaction F(4,20) = 6.154, p = 0.0021, of stimulus F(4,20) = 48.93, p < 0.0001, of subjects F(1,20) = 15.62, p = 0.0008). (E and F) As in (C), but myoblasts were pre-treated with Et. B. or vehicle (mean effect of interaction F(3,24) = 21.28, p 
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    1. Cervellin G., Comelli I., Lippi G. Rhabdomyolysis: historical background, clinical, diagnostic and therapeutic features. Clin. Chem. Lab. Med. 2010;48:749–756. - PubMed
    1. Luck R.P., Verbin S. Rhabdomyolysis: a review of clinical presentation, etiology, diagnosis, and management. Pediatr. Emerg. Care. 2008;24:262–268. - PubMed
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Figure 6
Figure 6
mtDNA accumulation is responsible for inflammation and rhabdomyolysis that can be reversed by steroids or chloroquine (CLQ) treatment in vivo (A) Real-time calcium flux in myoblasts from 4 controls and patients challenged with CpG-A, after pre-incubation with ODN151, a synthetic TLR9 antagonist. Each dot plot with lines depicts the mean ± SD of the intensity of the fluo-4 calcium probe as normalized to basal intensity of 1 single individual (≥10 cells/condition). (B) Death of myoblasts was evaluated by staining cells from 4 control individuals transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein (+LPIN1) and 4 patients transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein or an empty vector, for the apoptosis marker caspase3/7 green and propidium iodide or 7-actinomycin D (7-AAD), a dye that stains both apoptotic and necrotic cells. Staurosporin (ST): positive control. Fluorescence-activated cell sorting (FACS) plots show the codistribution of the fluorescence intensity of caspase3/7 and of 7-AAD in 1 of 4 controls and patients. (C) Dot plots (≥1,000 single cells/dot) depict the mean ± SD of the proportion of dead cells (ie, cells positive for capsase3/7 and/or 7-AAD) from (B) (mean effect of interaction F(8,45) = 5.296, p = 0.0001, of stimulus F(4,45) = 70.03, p LPIN1 shRNA (mean effect of interaction F(4,20) = 6.154, p = 0.0021, of stimulus F(4,20) = 48.93, p < 0.0001, of subjects F(1,20) = 15.62, p = 0.0008). (E and F) As in (C), but myoblasts were pre-treated with Et. B. or vehicle (mean effect of interaction F(3,24) = 21.28, p 
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