Role of mitophagy and innate immune system in the febrile decompensations of inherited rhabdomyolysis: Therapeutic perspectives – MetabInf

Rhabdomyolysis (RM) results from a impairment in ATP metabolism and a deregulation of ion channels, leading to a rapid increase of cytoplasmic ionized calcium concentration and the destruction of muscle cells. Although rare, the prognosis of RM related to Inherited Metabolic Diseases (IMD) is poor, in the absence of a specific treatment. Importantly, RM is precipitated by febrile illnesses and fasting.
In the case of the prototype syndrome caused by LPIN1 mutations, we have identified the pathogenic sequence linking lipid metabolism and RM in myoblasts, with reduced Vps34 PI-3K activity and Phospho-Inositide-3 Phosphate (PI3P) levels in late endosomes, resulting in decreased recruitment of the Rab7 GTPase-activating protein (GAP) Armus, perturbation in late endosomal architecture and functions including lysosomal degradation and mitophagic elongation. As a consequence, TLR9 is recruited to, and trapped into late endosomes where it remains activated upon nutrient refeeding. In myoblasts of patients exposed to a TLR9 ligand and cultured in minimal medium in vitro, a setting mimicking febrile illness, we observed an accumulation of mtDNA, a TLR9 hyperactivity with an increase in inflammatory cytokine production, an aberrant calcium release into the cytosol, and cell death, reproducing RM in vitro. Pretreating cells with a TLR9 antagonist or an inhibitor (Hydroxychloroquine) corrected the phenotype in vitro and improved the clinical course of patients treated by Hydroxychloroquine.
Mitophagy, which is highly developped in cells with high energy demand such as heart and skeletal muscle, is the key mechanism preventing the release of highly immune-stimulatory mtDNA by removing damaged mitochondria through autophagolysosomal activity. A defect in mitophagy results in endogenous mtDNA accumulation, which can trigger inflammation via activation of TLR9, NLRP3 inflammasome or stimulator of interferon genes (STING) signaling. Numerous perturbations of the interplay between autophagy, lipids and energy metabolism can result in altered mitophagy, including ATP depletion, ROS production, or alterations in structural membrane lipids of the organelles involved in mitophagy such as endoplasmic reticulum (ER) and Golgi apparatus.
We suspect that mitophagy and inflammatory response can be altered in a wide range of inherited RM, and propose to test this hypothesis in myoblasts and plasma of 18 patients suffering from fever-related RM due to one of five monogenic diseases: mitochondrial fatty acid oxidation deficiencies (FAOD) including LCHAD, VLCAD and CPT2 deficiencies, TANGO2 and FDX1L mutations, in order to find a treatment like in lipin-1 deficiency. All these diseases result in ATP depletion and mitochondrial dysfunction. Moreover accumulation of metabolites in FAOD upregulate the Akt/mTORC1 pathway that inhibits mitophagy while TANGO2 regulates the organization of the Golgi apparatus and the ER, both playing a crucial role in mitophagy. A collection of myoblasts and plasma for all of these patients and control individuals has been established with ethical authorizations. The project will consist in 4 main tasks that will allow us to: i) establish inflammatory profiles in myoblasts and plasma related to the diseases under investigation; ii) examine the consequences of the mutations on mitochondrial functions, structure and integrity and organelle dynamics, iii) identify metabolic and/or inflammatory stress responsible for abnormal calcium flux and suspcetibility to cell death, which are the hallmarks of RM; iv) identify and test the efficacy of candidate drugs in vitro.
We anticipate that perturbations in mitophagy and innate immune system could be a common denominator in energetic diseases to explain RM decompensations. Identification of pathogenic molecular mechanisms will facilitate the identification of novel therapeutic approaches that are missing today, and could be proposed to cure heterogeneous pathologies.