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RationaleThe ubiquitin–proteasome system (UPS) is responsible for skeletal muscle atrophy. We showed earlier that the transcription factor EB (TFEB) plays a role by increasing E3 ubiquitin ligase muscle really interesting new gene-finger 1(MuRF1)/tripartite motif-containing 63 (TRIM63) expression. MuRF 1 ubiquitinates structural proteins and mediates their UPS-dependent degradation. We now investigated how TFEB-mediated TRIM63 expression is regulated.
ObjectiveBecause protein kinase D1 (PKD1), histone deacetylase 5 (HDAC5), and TFEB belong to respective families with close structural, regulatory, and functional properties, we hypothesized that these families comprise a network regulating TRIM63 expression.
Methods and ResultsWe found that TFEB and transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3) activate TRIM63 expression. The class IIa HDACs HDAC4, HDAC5, and HDAC7 inhibited this activity. Furthermore, we could map the HDAC5 and TFE3 physical interaction. PKD1, PKD2, and PKD3 reversed the inhibitory effect of all tested class IIa HDACs toward TFEB and TFE3. PKD1 mediated nuclear export of all HDACs and lifted TFEB and TFE3 repression. We also mapped the PKD2 and HDAC5 interaction. We found that the inhibitory effect of PKD1 and PKD2 toward HDAC4, HDAC5, and HDAC7 was mediated by their phosphorylation and 14-3-3 mediated nuclear export.
ConclusionTFEB and TFE3 activate TRIM63 expression. Both transcription factors are controlled by HDAC4, HDAC5, HDAC7, and all PKD-family members. We propose that the multilevel PKD/HDAC/TFEB/TFE3 network tightly controls TRIM63 expression.
Abstract
Background
Critically ill patients frequently develop muscle atrophy and weakness in the intensive‐care‐unit setting [intensive care unit‐acquired weakness (ICUAW)]. Sepsis, systemic inflammation, and acute‐phase response are major risk factors. We reported earlier that the acute‐phase protein serum amyloid A1 (SAA1) is increased and accumulates in muscle of ICUAW patients, but its relevance was unknown. Our objectives were to identify SAA1 receptors and their downstream signalling pathways in myocytes and skeletal muscle and to investigate the role of SAA1 in inflammation‐induced muscle atrophy.
Methods
We performed cell‐based in vitro and animal in vivo experiments. The atrophic effect of SAA1 on differentiated C2C12 myotubes was investigated by analysing gene expression, protein content, and the atrophy phenotype. We used the cecal ligation and puncture model to induce polymicrobial sepsis in wild type mice, which were treated with the IкB kinase inhibitor Bristol‐Myers Squibb (BMS)‐345541 or vehicle. Morphological and molecular analyses were used to investigate the phenotype of inflammation‐induced muscle atrophy and the effects of BMS‐345541 treatment.
Results
The SAA1 receptors Tlr2, Tlr4, Cd36, P2rx7, Vimp, and Scarb1 were all expressed in myocytes and skeletal muscle. Treatment of differentiated C2C12 myotubes with recombinant SAA1 caused myotube atrophy and increased interleukin 6 (Il6) gene expression. These effects were mediated by Toll‐like receptors (TLR) 2 and 4. SAA1 increased the phosphorylation and activity of the transcription factor nuclear factor ‘kappa‐light‐chain‐enhancer' of activated B‐cells (NF‐κB) p65 via TLR2 and TLR4 leading to an increased binding of NF‐κB to NF‐κB response elements in the promoter region of its target genes resulting in an increased expression of NF‐κB target genes. In polymicrobial sepsis, skeletal muscle mass, tissue morphology, gene expression, and protein content were associated with the atrophy response. Inhibition of NF‐κB signalling by BMS‐345541 increased survival (28.6% vs. 91.7%, P < 0.01). BMS‐345541 diminished inflammation‐induced atrophy as shown by a reduced weight loss of the gastrocnemius/plantaris (vehicle: −21.2% and BMS‐345541: −10.4%; P < 0.05), tibialis anterior (vehicle: −22.7% and BMS‐345541: −17.1%; P < 0.05) and soleus (vehicle: −21.1% and BMS‐345541: −11.3%; P < 0.05) in septic mice. Analysis of the fiber type specific myocyte cross‐sectional area showed that BMS‐345541 reduced inflammation‐induced atrophy of slow/type I and fast/type II myofibers compared with vehicle‐treated septic mice. BMS‐345541 reversed the inflammation‐induced atrophy program as indicated by a reduced expression of the atrogenes Trim63/MuRF1, Fbxo32/Atrogin1, and Fbxo30/MuSA1.
Conclusions
SAA1 activates the TLR2/TLR4//NF‐κB p65 signalling pathway to cause myocyte atrophy. Systemic inhibition of the NF‐κB pathway reduced muscle atrophy and increased survival of septic mice. The SAA1/TLR2/TLR4//NF‐κB p65 atrophy pathway could have utility in combatting ICUAW.
Abstract
Fatty acid products derived from cytochromes P450 (CYP) monooxygenase and lipoxygenase (LOX)/CYP ω/(ω‐1)‐hydroxylase pathways are a superclass of lipid mediators with potent bioactivities. Whether or not the chronic kidney disease (CKD) and hemodialysis treatments performed on end‐stage renal disease (ESRD) patients affect RBC epoxy fatty acids profiles remains unknown. Measuring the products solely in plasma is suboptimal. Since such determinations invariably ignore red blood cells (RBCs) that make up 3 kg of the circulating blood. RBCs are potential reservoirs for epoxy fatty acids that regulate cardiovascular function. We studied 15 healthy persons and 15 ESRD patients undergoing regular hemodialysis treatments. We measured epoxides derived from CYP monooxygenase and metabolites derived from LOX/CYP ω/(ω‐1)‐hydroxylase pathways in RBCs by LC–MS/MS tandem mass spectrometry. Our data demonstrate that various CYP epoxides and LOX/CYP ω/(ω‐1)‐hydroxylase products are increased in RBCs of ESRD patients, compared to control subjects, including dihydroxyeicosatrienoic acids (DHETs), epoxyeicosatetraenoic acids (EEQs), dihydroxydocosapentaenoic acids (DiHDPAs), and hydroxyeicosatetraenoic acids (HETEs). Hemodialysis treatment did not affect the majority of those metabolites. Nevertheless, we detected more pronounced changes in free metabolite levels in RBCs after dialysis, as compared with the total RBC compartment. These findings indicate that free RBC eicosanoids should be considered more dynamic or vulnerable in CKD.
Factors causing the increased cardiovascular morbidity and mortality in hemodialysis (HD) patients are largely unknown. Oxylipins are a superclass of lipid mediators with potent bioactivities produced from oxygenation of polyunsaturated fatty acids. We previously assessed the impact of HD on oxylipins in arterial blood plasma and found that HD increases several oxylipins. To study the phenomenon further, we now evaluated the differences in arterial and venous blood oxylipins from patients undergoing HD. We collected arterial and venous blood samples in upper extremities from 12 end-stage renal disease (ESRD) patients before and after HD and measured oxylipins in plasma by LC-MS/MS tandem mass spectrometry. Comparison between cytochrome P450 (CYP), lipoxygenase (LOX), and LOX/CYP ω/(ω-1)-hydroxylase metabolites levels from arterial and venous blood showed no arteriovenous differences before HD but revealed arteriovenous differences in several CYP metabolites immediately after HD. These changes were explained by metabolites in the venous blood stream of the upper limb. Decreased soluble epoxide hydrolase (sEH) activity contributed to the release and accumulation of the CYP metabolites. However, HD did not affect arteriovenous differences of the majority of LOX and LOX/CYP ω/(ω-1)-hydroxylase metabolites. The HD treatment itself causes changes in CYP epoxy metabolites that could have deleterious effects in the circulation.
Long-chain fatty acids (LCFAs) serve as energy sources, components of cell membranes, and precursors for signaling molecules. Uremia alters LCFA metabolism so that the risk of cardiovascular events in chronic kidney disease (CKD) is increased. End-stage renal disease (ESRD) patients undergoing dialysis are particularly affected and their hemodialysis (HD) treatment could influence blood LCFA bioaccumulation and transformation. We investigated blood LCFA in HD patients and studied LCFA profiles in vivo by analyzing arterio–venous (A–V) LFCA differences in upper limbs. We collected arterial and venous blood samples from 12 ESRD patients, before and after HD, and analyzed total LCFA levels in red blood cells (RBCs) and plasma by LC–MS/MS tandem mass spectrometry. We observed that differences in arterial and venous LFCA contents within RBCs (RBC LCFA A–V differences) were affected by HD treatment. Numerous saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) n-6 showed negative A–V differences, accumulated during peripheral tissue perfusion of the upper limbs, in RBCs before HD. HD reduced these differences. The omega-3 quotient in the erythrocyte membranes was not affected by HD in either arterial or venous blood. Our data demonstrate that A–V differences in fatty acids status of LCFA are present and active in mature erythrocytes and their bioaccumulation is sensitive to single HD treatment.
Aging is an independent risk factor for hypertension, cardiovascular morbidity, and mortality. However, detailed mechanisms linking aging to cardiovascular disease are unclear. We studied the aging effects on the role of perivascular adipose tissue and downstream vasoconstriction targets, voltage-dependent KV7 channels, and their pharmacological modulators (flupirtine, retigabine, QO58, and QO58-lysine) in a murine model. We assessed vascular function of young and old mesenteric arteries in vitro using wire myography and membrane potential measurements with sharp electrodes. We also performed bulk RNA sequencing and quantitative reverse transcription-polymerase chain reaction tests in mesenteric arteries and perivascular adipose tissue to elucidate molecular underpinnings of age-related phenotypes. Results revealed impaired perivascular adipose tissue-mediated control of vascular tone particularly via KV7.3–5 channels with increased age through metabolic and inflammatory processes and release of perivascular adipose tissue-derived relaxation factors. Moreover, QO58 was identified as novel pharmacological vasodilator to activate XE991-sensitive KCNQ channels in old mesenteric arteries. Our data suggest that targeting inflammation and metabolism in perivascular adipose tissue could represent novel approaches to restore vascular function during aging. Furthermore, KV7.3–5 channels represent a promising target in cardiovascular aging.
The transcription factor EB (TFEB) promotes protein degradation by the autophagy and lysosomal pathway (ALP) and overexpression of TFEB was suggested for the treatment of ALP-related diseases that often affect the heart. However, TFEB-mediated ALP induction may perturb cardiac stress response. We used adeno-associated viral vectors type 9 (AAV9) to overexpress TFEB (AAV9-Tfeb) or Luciferase-control (AAV9-Luc) in cardiomyocytes of 12-week-old male mice. Mice were subjected to transverse aortic constriction (TAC, 27G; AAV9-Luc: n = 9; AAV9-Tfeb: n = 14) or sham (AAV9-Luc: n = 9; AAV9-Tfeb: n = 9) surgery for 28 days. Heart morphology, echocardiography, gene expression, and protein levels were monitored. AAV9-Tfeb had no effect on cardiac structure and function in sham animals. TAC resulted in compensated left ventricular hypertrophy in AAV9-Luc mice. AAV9-Tfeb TAC mice showed a reduced LV ejection fraction and increased left ventricular diameters. Morphological, histological, and real-time PCR analyses showed increased heart weights, exaggerated fibrosis, and higher expression of stress markers and remodeling genes in AAV9-Tfeb TAC compared to AAV9-Luc TAC. RNA-sequencing, real-time PCR and Western Blot revealed a stronger ALP activation in the hearts of AAV9-Tfeb TAC mice. Cardiomyocyte-specific TFEB-overexpression promoted ALP gene expression during TAC, which was associated with heart failure. Treatment of ALP-related diseases by overexpression of TFEB warrants careful consideration.