The mitochondrial metabolic reprogramming agent trimetazidine as an 'exercise mimetic' in cachectic C26-bearing mice.

BACKGROUND: Cancer cachexia is characterized by muscle depletion and exercise intolerance caused by an imbalance between protein synthesis and degradation and by impaired myogenesis. Myofibre metabolic efficiency is crucial so as to assure optimal muscle function. Some drugs are able to reprogram cell metabolism and, in some cases, to enhance metabolic efficiency. Based on these premises, we chose to investigate the ability of the metabolic modulator trimetazidine (TMZ) to counteract skeletal muscle dysfunctions and wasting occurring in cancer cachexia. METHODS: For this purpose, we used mice bearing the C26 colon carcinoma as a model of cancer cachexia. Mice received 5 mg/kg TMZ (i.p.) once a day for 12 consecutive days. A forelimb grip strength test was performed and tibialis anterior, and gastrocnemius muscles were excised for analysis. Ex vivo measurement of skeletal muscle contractile properties was also performed. RESULTS: Our data showed that TMZ induces some effects typically achieved through exercise, among which is grip strength increase, an enhanced fast-to slow myofibre phenotype shift, reduced glycaemia, PGC1α up-regulation, oxidative metabolism, and mitochondrial biogenesis. TMZ also partially restores the myofibre cross-sectional area in C26-bearing mice, while modulation of autophagy and apoptosis were excluded as mediators of TMZ effects. CONCLUSIONS: In conclusion, our data show that TMZ acts like an 'exercise mimetic' and is able to enhance some mechanisms of adaptation to stress in cancer cachexia. This makes the modulation of the metabolism, and in particular TMZ, a suitable candidate for a therapeutic rehabilitative protocol design, particularly considering that TMZ has already been approved for clinical use

CD19-targeting CAR T cells protect from ANCA-induced acute kidney injury

OBJECTIVES: Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV) are life-threatening systemic autoimmune diseases manifesting in the kidneys as necrotizing crescentic glomerulonephritis (NCGN). ANCA antigens are myeloperoxidase (MPO) or proteinase 3. Current treatments include steroids, cytotoxic drugs and B cell-depleting antibodies. The use of chimeric antigen receptor (CAR) T cells in autoimmune diseases is a promising new therapeutic approach. We tested the hypothesis that CAR T cells targeting CD19 deplete B cells, including MPO-ANCA-producing B cells, thereby protecting from ANCA-induced NCGN. METHODS: We tested this hypothesis in a preclinical MPO-AAV mouse model. NCGN was established by immunisation of MPO(-/-) mice with murine MPO, followed by irradiation and transplantation with haematopoietic cells from wild-type mice alone or together with either CD19-targeting CAR T cells or control CAR T cells. RESULTS: CD19 CAR T cells efficiently migrated to and persisted in bone marrow, spleen, peripheral blood and kidneys for up to 8 weeks. CD19 CAR T cells, but not control CAR T cells, depleted B cells and plasmablasts, enhanced the MPO-ANCA decline, and most importantly protected from NCGN. CONCLUSION: Our proof-of-principle study may encourage further exploration of CAR T cells as a treatment for ANCA-vasculitis patients with the goal of drug-free remission

Muscle RING-finger 2 and 3 maintain striated-muscle structure and function

Background: The Muscle-specific RING-finger (MuRF) protein family of E3 ubiquitin ligases is important for maintenance of muscular structure and function. MuRF proteins mediate adaptation of striated muscles to stress. MuRF2 and MuRF3 bind to microtubules and are implicated in sarcomere formation with noticeable functional redundancy. However, if this redundancy is important for muscle function in vivo is unknown. Our objective was to investigate cooperative function of MuRF2 and MuRF3 in the skeletal muscle and the heart in vivo. Methods: MuRF2 and MuRF3 double knockout mice (DKO) were generated and phenotypically characterized. Skeletal muscle and the heart were investigated by morphological measurements, histological analyses, electron microscopy, immunoblotting, and real-time PCR. Isolated muscles were subjected to in vitro force measurements. Cardiac function was determined by echocardiography and working heart preparations. Function of cardiomyocytes was measured in vitro. Cell culture experiments and mass-spectrometry were used for mechanistic analyses. Results: DKO mice showed a protein aggregate myopathy in skeletal muscle. Maximal force development was reduced in DKO soleus and extensor digitorum longus. Additionally, a fibre type shift towards slow/type I fibres occurred in DKO soleus and extensor digitorum longus. MuRF2 and MuRF3-deficient hearts showed decreased systolic and diastolic function. Further analyses revealed an increased expression of the myosin heavy chain isoform beta/slow and disturbed calcium handling as potential causes for the phenotype in DKO hearts. Conclusions: The redundant function of MuRF2 and MuRF3 is important for maintenance of skeletal muscle and cardiac structure and function in vivo

The E3 ubiquitin ligase TRIM62 and inflammation-induced skeletal muscle atrophy

Introduction: Intensive care unit (ICU)-acquired weakness (ICUAW) complicates the disease course of critically ill patients. Inflammation and acute-phase response occur directly within myocytes and contribute to ICUAW. We observed that TRIM62, an E3-ubiquitin ligase and modifier of inflammation, is increased in skeletal muscle of ICUAW patients. We investigated regulation and function of muscular TRIM62 in critical illness. Methods: Twenty-six critically ill patients with Sequential Organ Failure Assessment scores more than or equal to 8 underwent two skeletal muscle biopsies from the vastus lateralis at median days 5 and 15 in ICU. Four patients undergoing elective orthopedic surgery served as controls. TRIM62 expression and protein content was analyzed in these biopsies. Kinetics of Trim62, Atrogin1 and MuRF1 expression were determined in the gastrocnemius/plantaris and tibialis anterior from mouse models of inflammation, denervation and starvation induced muscle atrophy to differentiate between these contributors of ICUAW. Cultured myocytes were used for mechanistic analyses. Results: TRIM62 expression and protein content was increased early and remained elevated in muscle from critically ill patients. In all three animal models muscular Trim62 expression was early and continuously increased. Trim62 was expressed in myocytes and its overexpression activated the atrophy-inducing activator protein 1 signal transduction pathway. Knockdown of Trim62 by siRNA inhibited lipopolysaccharide induced interleukin-6 expression. Conclusions: TRIM62 is activated in muscle of critically ill patients. It could play a role in the pathogenesis of ICUAW by activating and maintaining inflammation in myocytes. Trial registration: Current Controlled Trials, ISRCTN77569430. Registered 13 February 2008

CSF2-dependent monocyte education in the pathogenesis of ANCA-induced glomerulonephritis

OBJECTIVES: Myeloid cell activation by antineutrophil cytoplasmic antibody (ANCA) is pivotal for necrotising vasculitis, including necrotising crescentic glomerulonephritis (NCGN). In contrast to neutrophils, the contribution of classical monocyte (CM) and non-classical monocyte (NCM) remains poorly defined. We tested the hypothesis that CMs contribute to antineutrophil cytoplasmic antibody-associated vasculitis (AAV) and that colony-stimulating factor-2 (CSF2, granulocyte-macrophage colony-stimulating factor (GM-CSF)) is an important monocyte-directed disease modifier.METHODS: Myeloperoxidase (MPO)-immunised MPO(-/-) mice were transplanted with haematopoietic cells from wild-type (WT) mice, C-C chemokine receptor 2 (CCR2)(-/-) mice to abrogate CM, or transcription factor CCAAT-enhancer-binding protein beta (C/EBPß)(-/-) mice to reduce NCM, respectively. Monocytes were stimulated with CSF2, and CSF2 receptor subunit beta (CSF2rb)-deficient mice were used. Urinary monocytes and CSF2 were quantified and kidney expression was analysed. CSF2-blocking antibody was used in the nephrotoxic nephritis (NTN) model. RESULTS: Compared with WT mice, CCR2(-/-) chimeric mice showed reduced circulating CM and were protected from NCGN. C/EBPß(-/-) chimeric mice lacked NCM but developed NCGN similar to WT chimeric mice. Kidney and urinary CSF2 were upregulated in AAV mice. CSF2 increased the ability of ANCA-stimulated monocytes to generate interleukin-1ß and to promote T17(H) effector cell polarisation. CSF2rb(-/-) chimeric mice harboured reduced numbers of kidney T17(H) cells and were protected from NCGN. CSF2 neutralisation reduced renal damage in the NTN model. Finally, patients with active AAV displayed increased urinary CM numbers, CSF2 levels and expression of GM-CSF in infiltrating renal cells. CONCLUSIONS: CMs but not NCMs are important for inducing kidney damage in AAV. CSF2 is a crucial pathological factor by modulating monocyte proinflammatory functions and thereby T17(H) cell polarisation

Circulating Extracellular Vesicles as Putative Mediators of Cardiovascular Disease in Paediatric Chronic Kidney Disease

Cardiovascular disease (CVD) is the leading cause of mortality in chronic kidney disease (CKD). However, the pathogenesis of CVD in CKD remains incompletely understood. Endothelial extracellular vesicles (EC‐EVs) have previously been associated with CVD. We hypothesized that CKD alters EV release and cargo, subsequently promoting vascular remodelling. We recruited 94 children with CKD, including patients after kidney transplantation and healthy donors, and performed EV phenotyping and functional EV analyses in the absence of age‐related comorbidities. Plasma EC‐EVs were increased in haemodialysis patients and decreased after kidney transplantation. Thirty microRNAs were less abundant in total CKD plasma EVs with predicted importance in angiogenesis and smooth muscle cell proliferation. In vitro, CKD plasma EVs induced transcriptomic changes in angiogenesis pathways and functionally impaired angiogenic properties, migration and proliferation in ECs. High shear stress, as generated by arterio‐venous fistulas, and uremic toxins were considered as potential drivers of EV release, but only the combination increased EV generation from venous ECs. The resulting EVs recapitulated miRNA changes observed in CKD in vivo. In conclusion, CKD results in the release of EVs with altered miRNA profiles and anti‐angiogenic properties, which may mediate vascular pathology in children with CKD. EVs and their miRNA cargo may represent future therapeutic targets to attenuate CVD in CKD

Circulating extracellular vesicles as putative mediators of cardiovascular disease in paediatric chronic kidney disease

Cardiovascular disease (CVD) is the leading cause of mortality in chronic kidney disease (CKD). However, the pathogenesis of CVD in CKD remains incompletely understood. Endothelial extracellular vesicles (EC-EVs) have previously been associated with CVD. We hypothesized that CKD alters EV release and cargo, subsequently promoting vascular remodelling. We recruited 94 children with CKD, including patients after kidney transplantation and healthy donors, and performed EV phenotyping and functional EV analyses in the absence of age-related comorbidities. Plasma EC-EVs were increased in haemodialysis patients and decreased after kidney transplantation. Thirty microRNAs were less abundant in total CKD plasma EVs with predicted importance in angiogenesis and smooth muscle cell proliferation. In vitro, CKD plasma EVs induced transcriptomic changes in angiogenesis pathways and functionally impaired angiogenic properties, migration and proliferation in ECs. High shear stress, as generated by arterio-venous fistulas, and uremic toxins were considered as potential drivers of EV release, but only the combination increased EV generation from venous ECs. The resulting EVs recapitulated miRNA changes observed in CKD in vivo. In conclusion, CKD results in the release of EVs with altered miRNA profiles and anti-angiogenic properties, which may mediate vascular pathology in children with CKD. EVs and their miRNA cargo may represent future therapeutic targets to attenuate CVD in CKD

Charakterisierung der MuRF2/MuRF3-Doppelknockout-Mauslinie hinsichtlich ihres Herz- und Skelettmuskel-Phänotyps

E3-Ubiquitin-Ligasen übertragen Ubiquitin auf die von ihnen gebundenen Substratproteine. Durch diese Ubiquitinierung werden Proteine für den kontrollierten Abbau im Ubiquitin-Proteasom-System markiert. Dieser Prozess beeinflusst aber auch die Aktivität verschiedener Signalwege, die Lokalisation von Proteinen oder die strukturelle Integrität zellulärer Komponenten. MuRF1, MuRF2 und MuRF3 sind E3-Ubiquitin-Ligasen, die hauptsächlich in quergestreifter Muskulatur exprimiert werden. Von MuRF1 ist bereits bekannt, dass es u. a. über die Ubiquitinierung von Myosinen und deren anschließender Degradation an der Entwicklung der Herz- und Skelettmuskelatrophie beteiligt ist. Da das Wissen über MuRF2 und MuRF3 in diesem Zusammenhang noch begrenzt ist, sollte die Auswirkung der kombinierten Keimbahndeletion von MuRF2 und MuRF3 in einem Mausmodell untersucht werden. Der Doppelknockout (DKO) von MuRF2 und MuRF3 führte zu Veränderungen der Morphologie und der Funktionsfähigkeit der Skelett- und Herzmuskulatur. In Skelettmuskelfasern kam es zur Ablagerung myosinhaltiger Proteinaggregate, zu einer Zunahme an langsam kontrahierenden Muskelfasern sowie zum Auftreten von Myozyten mit zentral gelegenen Nuclei als Anzeichen von Regenerationsprozessen. Isolierte Skelettmuskeln von DKO-Mäusen entwickelten eine geringere maximale spezifische Kraft als Muskeln aus Kontrolltieren. Ihre Herzen waren morphologisch unauffällig. Dennoch waren die Kontraktion des linken Ventrikels und das Schlagvolumen reduziert. Darüber hinaus zeigten isolierte Kardiomyozyten Beeinträchtigungen der Kontraktionsfähigkeit und der Kalziumströme in vitro. Eine massenspektrometrische Untersuchung ergab, dass in den Muskeln der MuRF2/3-DKO-Mäuse im Vergleich zu den Kontrollmäusen 12 Proteine in erhöhter Menge vorhanden waren. Eine Anreicherung von MAPKAP-K3, einem dieser Proteine, und von MAPKAP-K2 konnte im Western Blot von Proteinlysaten aus Skelettmuskeln und dem Herz der MuRF2/3-DKO-Mäuse detektiert werden.E3 ubiquitin ligases attach the small modifier ubiquitin to their substrate proteins. This ubiquitin-tag not only marks proteins for the proteasome dependent degradation, but also influences the activity of signalling pathways, the localisation of proteins or the structural integrity of cellular components. MuRF1, MuRF2, and MuRF3 are E3 ubiquitin ligases predominantly expressed in striated muscles. MuRF1 is involved in cardiac and skeletal muscle atrophy by mediating proteasome-dependent degradation of myosins. The knowledge about MuRF2 and MuRF3 in this context is limited. Therefore, a mouse model was used to analyse the impact of the combined deletion of MuRF2 and MuRF3. The double knockout (DKO) of MuRF2 and MuRF3 influenced the structure and function of skeletal and cardiac muscle. Skeletal muscle fibres exhibited myosin-containing protein aggregates, a fibre-type shift towards slow fibres, and myoycytes with central nuclei which is an indication of regeneration. Maximal force development was reduced in isolated hindlimb muscles M. soleus and M. extensor digitorum longus of MuRF2/3-DKO mice. Hearts were morphologically normal. No protein aggregates or signs of fibrosis were detected. However, heart performance was impaired. The contractibility of the left ventricle and the ejection fraction were reduced. Isolated cardiomyocytes showed a diminished contractibility. Furthermore, their speed of contraction and relaxation was reduced and they had impaired calcium transients. Mass spectrometric analysis of muscle lysates identified 12 enriched proteins in MuRF2/3-DKO muscles. Western Blot analysis confirmed that MAPKAP-K3, one of these proteins, and MAPKAP-K2 were enriched in lysates of skeletal muscles and left ventricles of MuRF2/3-DKO mice. Further investigations will show how MAPKAP-K2- and MAPKAP-K3-signalling pathways are involved in the development of the MuRF2/3-DKO-phenotype

Endothelial but not systemic ferroptosis inhibition protects from antineutrophil cytoplasmic antibody-induced crescentic glomerulonephritis

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are systemic autoimmune diseases featuring small blood vessel inflammation and organ damage, including necrotizing crescentic glomerulonephritis (NCGN). Persistent vascular inflammation leads to endothelial and kidney cell necrosis. Ferroptosis is a regulated cell death pathway executed by reactive oxygen species and iron-dependent lipid peroxidation culminating in cell membrane rupture. Here we show that ANCA-activated neutrophils induced endothelial cell (EC) death in vitro that was prevented by ferroptosis inhibition with Ferrostatin-1, Liproxstatin-1 and small inhibiting RNA against the enzyme AcylCoA Synthetase Long Chain Family Member 4 (ACSL4). In contrast, neither necroptosis nor apoptosis inhibition affected EC death. Moreover, both ferroptosis inhibitors alleviated lipid peroxide accumulation in EC. Increased lipid peroxidation was detected in kidney sections of AAV mice by immunohistochemistry. We generated MPO(-/-) ACSL4(flox) Tie2-Cre(+) mice lacking ACSL4 specifically in EC (ACSL4(ΔEC)) to study the significance of endothelial ferroptosis in vivo. ACSL4(ΔEC) chimeric mice, but not control mice (ACSL4(WT)), were protected from NCGN in a MPO-AAV bone-marrow transplantation model. These data establish that EC ferroptosis contributes to ANCA-induced glomerulonephritis. However, systemic pharmacological ferroptosis inhibition with Ferrostatin-1 or Liproxstatin-1 did not protect from NCGN in a murine AAV model. Ferrostatin-1 treatment both directly activated T cell proliferation and indirectly myeloid-mediated T cell proliferation and polarization in vitro. Conceivably, both effects may cancel the beneficial effect of endothelial ferroptosis inhibition. Mechanistically, we describe the importance of EC ferroptosis for the development of AAV. However, the lack of protection with systemic pharmacological ferroptosis inhibition should discourage clinicians from evaluating this treatment strategy in clinical AAV studies