Protein Kinase D Plays a Crucial Role in Maintaining Cardiac Homeostasis by Regulating Post-Translational Modifications of Myofilament Proteins.

Protein kinase D (PKD) enzymes play important roles in regulating myocardial contraction, hypertrophy, and remodeling. One of the proteins phosphorylated by PKD is titin, which is involved in myofilament function. In this study, we aimed to investigate the role of PKD in cardiomyocyte function under conditions of oxidative stress. To do this, we used mice with a cardiomyocyte-specific knock-out of Prkd1, which encodes PKD1 (Prkd1loxP/loxP; αMHC-Cre; PKD1 cKO), as well as wild type littermate controls (Prkd1loxP/loxP; WT). We isolated permeabilized cardiomyocytes from PKD1 cKO mice and found that they exhibited increased passive stiffness (Fpassive), which was associated with increased oxidation of titin, but showed no change in titin ubiquitination. Additionally, the PKD1 cKO mice showed increased myofilament calcium (Ca2+) sensitivity (pCa50) and reduced maximum Ca2+-activated tension. These changes were accompanied by increased oxidation and reduced phosphorylation of the small myofilament protein cardiac myosin binding protein C (cMyBPC), as well as altered phosphorylation levels at different phosphosites in troponin I (TnI). The increased Fpassive and pCa50, and the reduced maximum Ca2+-activated tension were reversed when we treated the isolated permeabilized cardiomyocytes with reduced glutathione (GSH). This indicated that myofilament protein oxidation contributes to cardiomyocyte dysfunction. Furthermore, the PKD1 cKO mice exhibited increased oxidative stress and increased expression of pro-inflammatory markers interleukin (IL)-6, IL-18, and tumor necrosis factor alpha (TNF-α). Both oxidative stress and inflammation contributed to an increase in microtubule-associated protein 1 light chain 3 (LC3)-II levels and heat shock response by inhibiting the mammalian target of rapamycin (mTOR) in the PKD1 cKO mouse myocytes. These findings revealed a previously unknown role for PKD1 in regulating diastolic passive properties, myofilament Ca2+ sensitivity, and maximum Ca2+-activated tension under conditions of oxidative stress. Finally, we emphasized the importance of PKD1 in maintaining the balance of oxidative stress and inflammation in the context of autophagy, as well as cardiomyocyte function.

Increased Expression of Proinflammatory Genes in Peripheral Blood Cells Is Associated with Cardiac Cachexia in Patients with Heart Failure with Reduced Ejection Fraction.

Background: Cardiac cachexia (CC) in chronic heart failure with reduced ejection fraction (HFrEF) is characterized by catabolism and inflammation predicting poor prognosis. Levels of responsible transcription factors like signal transducer and activator of transcription (STAT)1, STAT3, suppressor of cytokine signaling (SOCS)1 and SOCS3 in peripheral blood cells (PBC) are underinvestigated in CC. Expression of mediators was related to patients' functional status, body composition (BC) and metabolic gene expression in skeletal muscle (SM). Methods: Gene expression was quantified by qRT-PCR in three cohorts: non-cachectic patients (ncCHF, n = 19, LVEF 31 ± 7%, BMI 30.2 ± 5.0 kg/m2), cachectic patients (cCHF; n = 18, LVEF 27 ± 7%, BMI 24.3 ± 2.5 kg/m2) and controls (n = 17, LVEF 70 ± 7%, BMI 27.6 ± 4.6 kg/m2). BC was assessed by dual-energy X-ray absorptiometry. Blood inflammatory markers were measured. We quantified solute carrier family 2 member 4 (SLC2A4) and protein degradation by expressions of proteasome 20S subunit beta 2 and calpain-1 catalytic subunit in SM biopsies. Results: TNF and IL-10 expression was higher in cCHF than in ncCHF and controls (all p < 0.004). cCHF had a lower fat mass index (FMI) and lower fat-free mass index (FFMI) compared to ncCHF and controls (p < 0.05). STAT1 and STAT3 expression was higher in cCHF vs. ncCHF or controls (1.1 [1.6] vs. 0.8 [0.9] vs. 0.9 [1.1] RU and 4.6 [5.5] vs. 2.5 [4.8] vs. 3.0 [4.2] RU, all ANOVA-p < 0.05). The same applied for SOCS1 and SOCS3 expression (1.1 [1.5] vs. 0.4 [0.4] vs. 0.4 [0.5] and 0.9 [3.3] vs. 0.4 [1.1] vs. 0.8 [0.9] RU, all ANOVA-p < 0.04). In cCHF, higher TNF and STAT1 expression was associated with lower FMI (r = 0.5, p = 0.053 and p < 0.05) but not with lower FFMI (p > 0.4). In ncCHF, neither cytokine nor STAT/SOCS expression was associated with BC (all p > 0.3). SLC2A4 was upregulated in SM of cCHF vs. ncCHF (p < 0.03). Conclusions: Increased STAT1, STAT3, SOCS1 and SOCS3 expression suggests their involvement in CC. In cCHF, higher TNF and STAT-1 expression in PBC were associated with lower FMI. Increased SLC2A4 in cachectic SM biopsies indicates altered glucose metabolism.

Mammalian target of rapamycin inhibition impacts energy homeostasis and induces sex-specific body weight loss in humans.

BACKGROUND: Previous data from a 2-year randomized controlled trial (CRAD001ADE12) indicated that mammalian target of rapamycin (mTOR) inhibition by everolimus slowed cyst growth in patients with autosomal-dominant polycystic kidney disease (ADPKD). During the trial, we noted body weight loss in some patients, particularly in women. We hypothesized that everolimus causes body weight reduction by reduced food intake and/or metabolic changes, which could lead to cachexia. METHODS: Within a sub-analysis of the CRAD001ADE12 trial, body weight course was investigated regarding sex-specific differences in 433 adult ADPKD patients (everolimus, n = 215; placebo, n = 218). One hundred four out of 111 patients who participated in the clinical trial centre in Berlin were evaluated under everolimus/placebo therapy (on drug: everolimus, n = 48; placebo, n = 56) and after therapy (off drug: everolimus, n = 15; placebo, n = 18). Eating habits and nutrient/caloric intake were evaluated by validated questionnaires. Systemic and local metabolism was evaluated in four patients after an oral glucose load (OGL) by using calorimetry and adipose/muscle tissue microdialysis. RESULTS: Within the 2-year CRAD001ADE12 trial, a significant body weight loss was observed in female patients on everolimus versus placebo (P = 0.0029). Data of the Berlin Cohort revealed that weight loss was greater in women on everolimus versus men (P < 0.01). After 9 months, women and men had lost 2.6 ± 3.8 and 0.8 ± 1.5 kg (P < 0.05) in body weight, respectively, and after 21 months, they had lost 4.1 ± 6.6 and 1.0 ± 3.3 kg (P < 0.05), respectively. On everolimus, caloric intake was significantly lower in women versus men (1510 ± 128 vs. 2264 ± 216 kcal/day, P < 0.05), caused mainly by a lower fat and protein intake in women versus men. Cognitive restraints, disinhibition and hunger remained unchanged. In a subgroup of patients resting metabolic rate was unchanged whereas OGL-induced thermogenesis was reduced (7 ± 2 vs. 11 ± 2 kcal, P < 0.05). Fasting and OGL-induced fat oxidation was increased (P < 0.05) on versus off everolimus. In adipose tissue, fasting lipolytic activity was increased, but lipolytic activity was inhibited similarly after the OGL on versus off everolimus, respectively. In skeletal muscle, postprandial glucose uptake and aerobic glycolysis was reduced in patients on everolimus. CONCLUSIONS: mTOR inhibition by everolimus induces body weight reduction, specifically in female patients. This effect is possibly caused by a centrally mediated reduced food (fat and protein) intake and by centrally/peripherally mediated increased fat oxidation (systemic) and mobilization (adipose tissue). Glucose uptake and oxidation might be reduced in skeletal muscle. This could lead to cachexia and, possibly, muscle wasting. Therefore, our results have important implications for patients recieving immune-suppressive mTOR inhibition therapy.

SPSB1-mediated inhibition of TGF-ß receptor-II impairs myogenesis in inflammation.

BACKGROUND: Sepsis-induced intensive care unit-acquired weakness (ICUAW) features profound muscle atrophy and attenuated muscle regeneration related to malfunctioning satellite cells. Transforming growth factor beta (TGF-ß) is involved in both processes. We uncovered an increased expression of the TGF-ß receptor II (TßRII)-inhibitor SPRY domain-containing and SOCS-box protein 1 (SPSB1) in skeletal muscle of septic mice. We hypothesized that SPSB1-mediated inhibition of TßRII signalling impairs myogenic differentiation in response to inflammation. METHODS: We performed gene expression analyses in skeletal muscle of cecal ligation and puncture- (CLP) and sham-operated mice, as well as vastus lateralis of critically ill and control patients. Pro-inflammatory cytokines and specific pathway inhibitors were used to quantitate Spsb1 expression in myocytes. Retroviral expression plasmids were used to investigate the effects of SPSB1 on TGF-ß/TßRII signalling and myogenesis in primary and immortalized myoblasts and differentiated myotubes. For mechanistical analyses we used coimmunoprecipitation, ubiquitination, protein half-life, and protein synthesis assays. Differentiation and fusion indices were determined by immunocytochemistry, and differentiation factors were quantified by qRT-PCR and Western blot analyses. RESULTS: SPSB1 expression was increased in skeletal muscle of ICUAW patients and septic mice. Tumour necrosis factor (TNF), interleukin-1ß (IL-1ß), and IL-6 increased the Spsb1 expression in C2C12 myotubes. TNF- and IL-1ß-induced Spsb1 expression was mediated by NF-κB, whereas IL-6 increased the Spsb1 expression via the glycoprotein 130/JAK2/STAT3 pathway. All cytokines reduced myogenic differentiation. SPSB1 avidly interacted with TßRII, resulting in TßRII ubiquitination and destabilization. SPSB1 impaired TßRII-Akt-Myogenin signalling and diminished protein synthesis in myocytes. Overexpression of SPSB1 decreased the expression of early (Myog, Mymk, Mymx) and late (Myh1, 3, 7) differentiation-markers. As a result, myoblast fusion and myogenic differentiation were impaired. These effects were mediated by the SPRY- and SOCS-box domains of SPSB1. Co-expression of SPSB1 with Akt or Myogenin reversed the inhibitory effects of SPSB1 on protein synthesis and myogenic differentiation. Downregulation of Spsb1 by AAV9-mediated shRNA attenuated muscle weight loss and atrophy gene expression in skeletal muscle of septic mice. CONCLUSIONS: Inflammatory cytokines via their respective signalling pathways cause an increase in SPSB1 expression in myocytes and attenuate myogenic differentiation. SPSB1-mediated inhibition of TßRII-Akt-Myogenin signalling and protein synthesis contributes to a disturbed myocyte homeostasis and myogenic differentiation that occurs during inflammation.

Metabolic remodeling in cardiac hypertrophy and heart failure with reduced ejection fraction occurs independent of transcription factor EB in mice.

Background: A metabolic shift from fatty acid (FAO) to glucose oxidation (GO) occurs during cardiac hypertrophy (LVH) and heart failure with reduced ejection fraction (HFrEF), which is mediated by PGC-1α and PPARα. While the transcription factor EB (TFEB) regulates the expression of both PPARGC1A/PGC-1α and PPARA/PPARα, its contribution to metabolic remodeling is uncertain. Methods: Luciferase assays were performed to verify that TFEB regulates PPARGC1A expression. Cardiomyocyte-specific Tfeb knockout (cKO) and wildtype (WT) male mice were subjected to 27G transverse aortic constriction or sham surgery for 21 and 56 days, respectively, to induce LVH and HFrEF. Echocardiographic, morphological, and histological analyses were performed. Changes in markers of cardiac stress and remodeling, metabolic shift and oxidative phosphorylation were investigated by Western blot analyses, mass spectrometry, qRT-PCR, and citrate synthase and complex II activity measurements. Results: Luciferase assays revealed that TFEB increases PPARGC1A/PGC-1α expression, which was inhibited by class IIa histone deacetylases and derepressed by protein kinase D. At baseline, cKO mice exhibited a reduced cardiac function, elevated stress markers and a decrease in FAO and GO gene expression compared to WT mice. LVH resulted in increased cardiac remodeling and a decreased expression of FAO and GO genes, but a comparable decline in cardiac function in cKO compared to WT mice. In HFrEF, cKO mice showed an improved cardiac function, lower heart weights, smaller myocytes and a reduction in cardiac remodeling compared to WT mice. Proteomic analysis revealed a comparable decrease in FAO- and increase in GO-related proteins in both genotypes. A significant reduction in mitochondrial quality control genes and a decreased citrate synthase and complex II activities was observed in hearts of WT but not cKO HFrEF mice. Conclusions: TFEB affects the baseline expression of metabolic and mitochondrial quality control genes in the heart, but has only minor effects on the metabolic shift in LVH and HFrEF in mice. Deletion of TFEB plays a protective role in HFrEF but does not affect the course of LVH. Further studies are needed to elucidate if TFEB affects the metabolic flux in stressed cardiomyocytes.

The Transcription Factor EB (TFEB) Sensitizes the Heart to Chronic Pressure Overload.

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.

Riociguat attenuates the changes in left ventricular proteome and microRNA profile after experimental aortic stenosis in mice.

BACKGROUND AND PURPOSE: Development and progression of heart failure involve endothelial and myocardial dysfunction as well as a dysregulation of the NO-sGC-cGMP signalling pathway. Recently, we reported that the sGC stimulator riociguat has beneficial effects on cardiac remodelling and progression of heart failure in response to chronic pressure overload. Here, we examined if these beneficial effects of riociguat were also reflected in alterations of the myocardial proteome and microRNA profiles. EXPERIMENTAL APPROACH: Male C57BL/6N mice underwent transverse aortic constriction (TAC) and sham-operated mice served as controls. TAC and sham animals were randomised and treated with either riociguat or vehicle for 5 weeks, starting 3 weeks after surgery, when cardiac hypertrophy was established. Afterwards, we performed mass spectrometric proteome analyses and microRNA sequencing of proteins and RNAs, respectively, isolated from left ventricles (LVs). KEY RESULTS: TAC-induced changes of the LV proteome were significantly reduced by treatment with riociguat. Bioinformatics analyses revealed that riociguat improved TAC-induced cardiovascular disease-related pathways, metabolism and energy production, for example, reversed alterations in the levels of myosin heavy chain 7, cardiac phospholamban and ankyrin repeat domain-containing protein 1. Riociguat also attenuated TAC-induced changes of microRNA levels in the LV. CONCLUSION AND IMPLICATIONS: The sGC stimulator riociguat exerted beneficial effects on cardiac structure and function during pressure overload, which was accompanied by a reversal of TAC-induced changes of the cardiac proteome and microRNA profile. Our data support the potential of riociguat as a novel therapeutic agent for heart failure.

Hidden Agenda - The Involvement of Endoplasmic Reticulum Stress and Unfolded Protein Response in Inflammation-Induced Muscle Wasting.

Critically ill patients at the intensive care unit (ICU) often develop a generalized weakness, called ICU-acquired weakness (ICUAW). A major contributor to ICUAW is muscle atrophy, a loss of skeletal muscle mass and function. Skeletal muscle assures almost all of the vital functions of our body. It adapts rapidly in response to physiological as well as pathological stress, such as inactivity, immobilization, and inflammation. In response to a reduced workload or inflammation muscle atrophy develops. Recent work suggests that adaptive or maladaptive processes in the endoplasmic reticulum (ER), also known as sarcoplasmic reticulum, contributes to this process. In muscle cells, the ER is a highly specialized cellular organelle that assures calcium homeostasis and therefore muscle contraction. The ER also assures correct folding of proteins that are secreted or localized to the cell membrane. Protein folding is a highly error prone process and accumulation of misfolded or unfolded proteins can cause ER stress, which is counteracted by the activation of a signaling network known as the unfolded protein response (UPR). Three ER membrane residing molecules, protein kinase R-like endoplasmic reticulum kinase (PERK), inositol requiring protein 1a (IRE1a), and activating transcription factor 6 (ATF6) initiate the UPR. The UPR aims to restore ER homeostasis by reducing overall protein synthesis and increasing gene expression of various ER chaperone proteins. If ER stress persists or cannot be resolved cell death pathways are activated. Although, ER stress-induced UPR pathways are known to be important for regulation of skeletal muscle mass and function as well as for inflammation and immune response its function in ICUAW is still elusive. Given recent advances in the development of ER stress modifying molecules for neurodegenerative diseases and cancer, it is important to know whether or not therapeutic interventions in ER stress pathways have favorable effects and these compounds can be used to prevent or treat ICUAW. In this review, we focus on the role of ER stress-induced UPR in skeletal muscle during critical illness and in response to predisposing risk factors such as immobilization, starvation and inflammation as well as ICUAW treatment to foster research for this devastating clinical problem.

Muscle Mass and Inflammation in Older Adults: Impact of the Metabolic Syndrome.

BACKGROUND: Inflammatory processes are a cause of accelerated loss of muscle mass. Metabolic syndrome (MetS) is a highly prevalent age-related condition, which may promote and be promoted by inflammation. However, whether inflammation in MetS (metaflammation) is associated with lower muscle mass is still unclear. METHODS: Complete cross-sectional data on body composition, MetS, and the inflammatory markers interleukin (IL)-1ß, IL-6, IL-10, tumor necrosis factor (TNF), and C-reactive protein (CRP) were available for 1,377 BASE-II participants (51.1% women; 68 ± 4 years old). Appendicular lean mass (ALM) was assessed by dual-energy X-ray absorptiometry. Low muscle mass (low ALM-to-BMI ratio [ALMBMI]) was defined according to the Foundation for the National Institutes of Health (FNIH) Sarcopenia Project. Regression models, adjusted for an increasing number of confounders (sex, age, physical activity, morbidities, diabetes mellitus type II, TSH, albumin, HbA1c, smoking habits, alcohol intake, education, and energy intake/day), were used to calculate the association between low ALMBMI and high inflammation (tertile 3) according to MetS. RESULTS: MetS was present in 36.2% of the study population, and 9% had low ALMBMI. In the whole study population, high CRP (odds ratio [OR]: 2.7 [95% CI: 1.6-4.7; p = 0.001]) and high IL-6 (OR: 2.1 [95% CI: 1.2-1.9; p = 0.005]) were associated with low ALMBMI. In contrast, no significant association was found between TNF, IL-10, or IL-1ß with low ALMBMI. When participants were stratified by MetS, results for IL-6 remained significant only in participants with MetS. CONCLUSIONS: Among BASE-II participants, low ALMBMI was associated with inflammation. Low-grade inflammation triggered by disease state, especially in the context of MetS, might favor loss of muscle mass, so a better control of MetS might help to prevent sarcopenia. Intervention studies to test whether strategies to prevent MetS might also prevent loss of muscle mass seem to be promising.

Skeletal muscle derived Musclin protects the heart during pathological overload.

Cachexia is associated with poor prognosis in chronic heart failure patients, but the underlying mechanisms of cachexia triggered disease progression remain poorly understood. Here, we investigate whether the dysregulation of myokine expression from wasting skeletal muscle exaggerates heart failure. RNA sequencing from wasting skeletal muscles of mice with heart failure reveals a reduced expression of Ostn, which encodes the secreted myokine Musclin, previously implicated in the enhancement of natriuretic peptide signaling. By generating skeletal muscle specific Ostn knock-out and overexpressing mice, we demonstrate that reduced skeletal muscle Musclin levels exaggerate, while its overexpression in muscle attenuates cardiac dysfunction and myocardial fibrosis during pressure overload. Mechanistically, Musclin enhances the abundance of C-type natriuretic peptide (CNP), thereby promoting cardiomyocyte contractility through protein kinase A and inhibiting fibroblast activation through protein kinase G signaling. Because we also find reduced OSTN expression in skeletal muscle of heart failure patients, augmentation of Musclin might serve as therapeutic strategy.

Stimulation of soluble guanylyl cyclase (sGC) by riociguat attenuates heart failure and pathological cardiac remodelling.

BACKGROUND AND PURPOSE: Heart failure is associated with an impaired NO-soluble guanylyl cyclase (sGC)-cGMP pathway and its augmentation is thought to be beneficial for its therapy. We hypothesized that stimulation of sGC by the sGC stimulator riociguat prevents pathological cardiac remodelling and heart failure in response to chronic pressure overload. EXPERIMENTAL APPROACH: Transverse aortic constriction or sham surgery was performed in C57BL/6N mice. After 3 weeks of transverse aortic constriction when heart failure was established, animals receive either riociguat or its vehicle for 5 additional weeks. Cardiac function was evaluated weekly by echocardiography. Eight weeks after surgery, histological analyses were performed to evaluate remodelling and the transcriptome of the left ventricles (LVs) was analysed by RNA sequencing. Cell culture experiments were used for mechanistically studies. KEY RESULTS: Transverse aortic constriction resulted in a continuous decrease of LV ejection fraction and an increase in LV mass until week 3. Five weeks of riociguat treatment resulted in an improved LV ejection fraction and a decrease in the ratio of left ventricular mass to total body weight (LVM/BW), myocardial fibrosis and myocyte cross-sectional area. RNA sequencing revealed that riociguat reduced the expression of myocardial stress and remodelling genes (e.g. Nppa, Nppb, Myh7 and collagen) and attenuated the activation of biological pathways associated with cardiac hypertrophy and heart failure. Riociguat reversed pathological stress response in cultivated myocytes and fibroblasts. CONCLUSION AND IMPLICATIONS: Stimulation of the sGC reverses transverse aortic constriction-induced heart failure and remodelling, which is associated with improved myocardial gene expression. LINKED ARTICLES: This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Sepsis induces interleukin 6, gp130/JAK2/STAT3, and muscle wasting.

BACKGROUND: Sepsis and inflammation can cause intensive care unit-acquired weakness (ICUAW). Increased interleukin-6 (IL-6) plasma levels are a risk factor for ICUAW. IL-6 signalling involves the glycoprotein 130 (gp130) receptor and the JAK/STAT-pathway, but its role in sepsis-induced muscle wasting is uncertain. In a clinical observational study, we found that the IL-6 target gene, SOCS3, was increased in skeletal muscle of ICUAW patients indicative for JAK/STAT-pathway activation. We tested the hypothesis that the IL-6/gp130-pathway mediates ICUAW muscle atrophy. METHODS: We sequenced RNA (RNAseq) from tibialis anterior (TA) muscle of cecal ligation and puncture-operated (CLP) and sham-operated wildtype (WT) mice. The effects of the IL-6/gp130/JAK2/STAT3-pathway were investigated by analysing the atrophy phenotype, gene expression, and protein contents of C2C12 myotubes. Mice lacking Il6st, encoding gp130, in myocytes (cKO) and WT controls, as well as mice treated with the JAK2 inhibitor AG490 or vehicle were exposed to CLP or sham surgery for 24 or 96 h. RESULTS: Analyses of differentially expressed genes in RNAseq (≥2-log2-fold change, P < 0.01) revealed an activation of IL-6-signalling and JAK/STAT-signalling pathways in muscle of septic mice, which occurred after 24 h and lasted at least for 96 h during sepsis. IL-6 treatment of C2C12 myotubes induced STAT3 phosphorylation (three-fold, P < 0.01) and Socs3 mRNA expression (3.1-fold, P < 0.01) and caused myotube atrophy. Knockdown of Il6st diminished IL-6-induced STAT3 phosphorylation (-30.0%; P < 0.01), Socs3 mRNA expression, and myotube atrophy. JAK2 (- 29.0%; P < 0.01) or STAT3 inhibition (-38.7%; P < 0.05) decreased IL-6-induced Socs3 mRNA expression. Treatment with either inhibitor attenuated myotube atrophy in response to IL-6. CLP-operated septic mice showed an increased STAT3 phosphorylation and Socs3 mRNA expression in TA muscle, which was reduced in septic Il6st-cKO mice by 67.8% (P < 0.05) and 85.6% (P < 0.001), respectively. CLP caused a loss of TA muscle weight, which was attenuated in Il6st-cKO mice (WT: -22.3%, P < 0.001, cKO: -13.5%, P < 0.001; WT vs. cKO P < 0.001). While loss of Il6st resulted in a reduction of MuRF1 protein contents, Atrogin-1 remained unchanged between septic WT and cKO mice. mRNA expression of Trim63/MuRF1 and Fbxo32/Atrogin-1 were unaltered between CLP-treated WT and cKO mice. AG490 treatment reduced STAT3 phosphorylation (-22.2%, P < 0.05) and attenuated TA muscle atrophy in septic mice (29.6% relative reduction of muscle weight loss, P < 0.05). The reduction in muscle atrophy was accompanied by a reduction in Fbxo32/Atrogin-1-mRNA (-81.3%, P < 0.05) and Trim63/MuRF1-mRNA expression (-77.6%, P < 0.05) and protein content. CONCLUSIONS: IL-6 via the gp130/JAK2/STAT3-pathway mediates sepsis-induced muscle atrophy possibly contributing to ICUAW.

Inhibition of the NLRP3/IL-1ß axis protects against sepsis-induced cardiomyopathy.

BACKGROUND: Septic cardiomyopathy worsens the prognosis of critically ill patients. Clinical data suggest that interleukin-1ß (IL-1ß), activated by the NLRP3 inflammasome, compromises cardiac function. Whether or not deleting Nlrp3 would prevent cardiac atrophy and improve diastolic cardiac function in sepsis was unclear. Here, we investigated the role of NLRP3/IL-1ß in sepsis-induced cardiomyopathy and cardiac atrophy. METHODS: Male Nlrp3 knockout (KO) and wild-type (WT) mice were exposed to polymicrobial sepsis by caecal ligation and puncture (CLP) surgery (KO, n = 27; WT, n = 33) to induce septic cardiomyopathy. Sham-treated mice served as controls (KO, n = 11; WT, n = 16). Heart weights and morphology, echocardiography and analyses of gene and protein expression were used to evaluate septic cardiomyopathy and cardiac atrophy. IL-1ß effects on primary and immortalized cardiomyocytes were investigated by morphological and molecular analyses. IonOptix and real-time deformability cytometry (RT-DC) analysis were used to investigate functional and mechanical effects of IL-1ß on cardiomyocytes. RESULTS: Heart morphology and echocardiography revealed preserved systolic (stroke volume: WT sham vs. WT CLP: 33.1 ± 7.2 µL vs. 24.6 ± 8.7 µL, P < 0.05; KO sham vs. KO CLP: 28.3 ± 8.1 µL vs. 29.9 ± 9.9 µL, n.s.; P < 0.05 vs. WT CLP) and diastolic (peak E wave velocity: WT sham vs. WT CLP: 750 ± 132 vs. 522 ± 200 mm/s, P < 0.001; KO sham vs. KO CLP: 709 ± 152 vs. 639 ± 165 mm/s, n.s.; P < 0.05 vs. WT CLP) cardiac function and attenuated cardiac (heart weight-tibia length ratio: WT CLP vs. WT sham: -26.6%, P < 0.05; KO CLP vs. KO sham: -3.3%, n.s.; P < 0.05 vs. WT CLP) and cardiomyocyte atrophy in KO mice during sepsis. IonOptix measurements showed that IL-1ß decreased contractility (cell shortening: IL-1ß: -15.4 ± 2.3%, P < 0.001 vs. vehicle, IL-1RA: -6.1 ± 3.3%, P < 0.05 vs. IL-1ß) and relaxation of adult rat ventricular cardiomyocytes (time-to-50% relengthening: IL-1ß: 2071 ± 225 ms, P < 0.001 vs. vehicle, IL-1RA: 564 ± 247 ms, P < 0.001 vs. IL-1ß), which was attenuated by an IL-1 receptor antagonist (IL-1RA). RT-DC analysis indicated that IL-1ß reduced cardiomyocyte size (P < 0.001) and deformation (P < 0.05). RNA sequencing showed that genes involved in NF-κB signalling, autophagy and lysosomal protein degradation were enriched in hearts of septic WT but not in septic KO mice. Western blotting and qPCR disclosed that IL-1ß activated NF-κB and its target genes, caused atrophy and decreased myosin protein in myocytes, which was accompanied by an increased autophagy gene expression. These effects were attenuated by IL-1RA. CONCLUSIONS: IL-1ß causes atrophy, impairs contractility and relaxation and decreases deformation of cardiomyocytes. Because NLRP3/IL-1ß pathway inhibition attenuates cardiac atrophy and cardiomyopathy in sepsis, it could be useful to prevent septic cardiomyopathy.

Energy Metabolites as Biomarkers in Ischemic and Dilated Cardiomyopathy.

With more than 25 million people affected, heart failure (HF) is a global threat. As energy production pathways are known to play a pivotal role in HF, we sought here to identify key metabolic changes in ischemic- and non-ischemic HF by using a multi-OMICS approach. Serum metabolites and mRNAseq and epigenetic DNA methylation profiles were analyzed from blood and left ventricular heart biopsy specimens of the same individuals. In total we collected serum from n = 82 patients with Dilated Cardiomyopathy (DCM) and n = 51 controls in the screening stage. We identified several metabolites involved in glycolysis and citric acid cycle to be elevated up to 5.7-fold in DCM (p = 1.7 × 10-6). Interestingly, cardiac mRNA and epigenetic changes of genes encoding rate-limiting enzymes of these pathways could also be found and validated in our second stage of metabolite assessment in n = 52 DCM, n = 39 ischemic HF and n = 57 controls. In conclusion, we identified a new set of metabolomic biomarkers for HF. We were able to identify underlying biological cascades that potentially represent suitable intervention targets.

Serum amyloid A1 mediates myotube atrophy via Toll-like receptors.

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.

Ninjurin1 regulates striated muscle growth and differentiation.

Chronic pressure overload due to aortic valve stenosis leads to pathological cardiac hypertrophy and heart failure. Hypertrophy is accompanied by an increase in myocyte surface area, which requires a proportional increase in the number of cell-cell and cell-matrix contacts to withstand enhanced workload. In a proteomic analysis we identified nerve injury-induced protein 1 (Ninjurin1), a 16kDa transmembrane cell-surface protein involved in cell adhesion and nerve repair, to be increased in hypertrophic hearts from patients with aortic stenosis. We hypothesised that Ninjurin1 is involved in myocyte hypertrophy. We analyzed cardiac biopsies from aortic-stenosis patients and control patients undergoing elective heart surgery. We studied cardiac hypertrophy in mice after transverse aortic constriction and angiotensin II infusions, and performed mechanistic analyses in cultured myocytes. We assessed the physiological role of ninjurin1 in zebrafish during heart and skeletal muscle development. Ninjurin1 was increased in hearts of aortic stenosis patients, compared to controls, as well as in hearts from mice with cardiac hypertrophy. Besides the 16kDa Ninjurin1 (Ninjurin1-16) we detected a 24kDa variant of Ninjurin1 (Ninjurin1-24), which was predominantly expressed during myocyte hypertrophy. We disclosed that the higher molecular weight of Ninjurin1-24 was caused by N-glycosylation. Ninjurin1-16 was contained in the cytoplasm of myocytes where it colocalized with stress-fibers. In contrast, Ninjurin1-24 was localized at myocyte membranes. Gain and loss-of-function experiments showed that Ninjurin1-24 plays a role in myocyte hypertrophy and myogenic differentiation in vitro. Reduced levels of ninjurin1 impaired cardiac and skeletal muscle development in zebrafish. We conclude that Ninjurin1 contributes to myocyte growth and differentiation, and that these effects are mainly mediated by N-glycosylated Ninjurin1-24.

HDAC4 mutations cause diabetes and induce ß-cell FoxO1 nuclear exclusion.

BACKGROUND: Studying patients with rare Mendelian diabetes has uncovered molecular mechanisms regulating ß-cell pathophysiology. Previous studies have shown that Class IIa histone deacetylases (HDAC4, 5, 7, and 9) modulate mammalian pancreatic endocrine cell function and glucose homeostasis. METHODS: We performed exome sequencing in one adolescent nonautoimmune diabetic patient and detected one de novo predicted disease-causing HDAC4 variant (p.His227Arg). We screened our pediatric diabetes cohort with unknown etiology using Sanger sequencing. In mouse pancreatic ß-cell lines (Min6 and SJ cells), we performed insulin secretion assay and quantitative RT-PCR to measure the ß-cell function transfected with the detected HDAC4 variants and wild type. We carried out immunostaining and Western blot to investigate if the detected HDAC4 variants affect the cellular translocation and acetylation status of Forkhead box protein O1 (FoxO1) in the pancreatic ß-cells. RESULTS: We discovered three HDAC4 mutations (p.His227Arg, p.Asp234Asn, and p.Glu374Lys) in unrelated individuals who had nonautoimmune diabetes with various degrees of ß-cell loss. In mouse pancreatic ß-cell lines, we found that these three HDAC4 mutations decrease insulin secretion, down-regulate ß-cell-specific transcriptional factors, and cause nuclear exclusion of acetylated FoxO1. CONCLUSION: Mutations in HDAC4 disrupt the deacetylation of FoxO1, subsequently decrease the ß-cell function including insulin secretion, resulting in diabetes.

Muscle wasting and function after muscle activation and early protocol-based physiotherapy: an explorative trial.

BACKGROUND: Early mobilization improves physical independency of critically ill patients at hospital discharge in a general intensive care unit (ICU)-cohort. We aimed to investigate clinical and molecular benefits or detriments of early mobilization and muscle activating measures in a high-risk ICU-acquired weakness cohort. METHODS: Fifty patients with a SOFA score ≥9 within 72 h after ICU admission were randomized to muscle activating measures such as neuromuscular electrical stimulation or whole-body vibration in addition to early protocol-based physiotherapy (intervention) or early protocol-based physiotherapy alone (control). Muscle strength and function were assessed by Medical Research Council (MRC) score, handgrip strength and Functional Independence Measure at first awakening, ICU discharge, and 12 month follow-up. Patients underwent open surgical muscle biopsy on day 15. We investigated the impact of muscle activating measures in addition to early protocol-based physiotherapy on muscle strength and function as well as on muscle wasting, morphology, and homeostasis in patients with sepsis and ICU-acquired weakness. We compared the data with patients treated with common physiotherapeutic practice (CPP) earlier. RESULTS: ICU-acquired weakness occurs within the entire cohort, and muscle activating measures did not improve muscle strength or function at first awakening (MRC median [IQR]: CPP 3.3 [3.0-4.3]; control 3.0 [2.7-3.4]; intervention 3.0 [2.1-3.8]; P > 0.05 for all), ICU discharge (MRC median [IQR]: CPP 3.8 [3.4-4.4]; control 3.9 [3.3-4.0]; intervention 3.6 [2.8-4.0]; P > 0.05 for all), and 12 month follow-up (MRC median [IQR]: control 5.0 [4.3-5.0]; intervention 4.8 [4.3-5.0]; P = 0.342 for all). No signs of necrosis or inflammatory infiltration were present in the histological analysis. Myocyte cross-sectional area in the intervention group was significantly larger in comparison with the control group (type I +10%; type IIa +13%; type IIb +3%; P < 0.001 for all) and CPP (type I +36%; type IIa +49%; type IIb +65%; P < 0.001 for all). This increase was accompanied by an up-regulated gene expression for myosin heavy chains (fold change median [IQR]: MYH1 2.3 [1.1-2.7]; MYH2 0.7 [0.2-1.8]; MYH4 5.1 [2.2-15.3]) and an unaffected gene expression for TRIM63, TRIM62, and FBXO32. CONCLUSIONS: In our patients with sepsis syndrome at high risk for ICU-acquired weakness muscle activating measures in addition to early protocol-based physiotherapy did not improve muscle strength or function at first awakening, ICU discharge, or 12 month follow-up. Yet it prevented muscle atrophy.

Excitotoxic inactivation of constitutive oxidative stress detoxification pathway in neurons can be rescued by PKD1.

Excitotoxicity, a critical process in neurodegeneration, induces oxidative stress and neuronal death through mechanisms largely unknown. Since oxidative stress activates protein kinase D1 (PKD1) in tumor cells, we investigated the effect of excitotoxicity on neuronal PKD1 activity. Unexpectedly, we find that excitotoxicity provokes an early inactivation of PKD1 through a dephosphorylation-dependent mechanism mediated by protein phosphatase-1 (PP1) and dual specificity phosphatase-1 (DUSP1). This step turns off the IKK/NF-κB/SOD2 antioxidant pathway. Neuronal PKD1 inactivation by pharmacological inhibition or lentiviral silencing in vitro, or by genetic inactivation in neurons in vivo, strongly enhances excitotoxic neuronal death. In contrast, expression of an active dephosphorylation-resistant PKD1 mutant potentiates the IKK/NF-κB/SOD2 oxidative stress detoxification pathway and confers neuroprotection from in vitro and in vivo excitotoxicity. Our results indicate that PKD1 inactivation underlies excitotoxicity-induced neuronal death and suggest that PKD1 inactivation may be critical for the accumulation of oxidation-induced neuronal damage during aging and in neurodegenerative disorders.

Deletion of Nlrp3 protects from inflammation-induced skeletal muscle atrophy.

BACKGROUND: Critically ill patients develop atrophic muscle failure, which increases morbidity and mortality. Interleukin-1ß (IL-1ß) is activated early in sepsis. Whether IL-1ß acts directly on muscle cells and whether its inhibition prevents atrophy is unknown. We aimed to investigate if IL-1ß activation via the Nlrp3 inflammasome is involved in inflammation-induced atrophy. METHODS: We performed an experimental study and prospective animal trial. The effect of IL-1ß on differentiated C2C12 muscle cells was investigated by analyzing gene-and-protein expression, and atrophy response. Polymicrobial sepsis was induced by cecum ligation and puncture surgery in Nlrp3 knockout and wild type mice. Skeletal muscle morphology, gene and protein expression, and atrophy markers were used to analyze the atrophy response. Immunostaining and reporter-gene assays showed that IL-1ß signaling is contained and active in myocytes. RESULTS: Immunostaining and reporter gene assays showed that IL-1ß signaling is contained and active in myocytes. IL-1ß increased Il6 and atrogene gene expression resulting in myocyte atrophy. Nlrp3 knockout mice showed reduced IL-1ß serum levels in sepsis. As determined by muscle morphology, organ weights, gene expression, and protein content, muscle atrophy was attenuated in septic Nlrp3 knockout mice, compared to septic wild-type mice 96 h after surgery. CONCLUSIONS: IL-1ß directly acts on myocytes to cause atrophy in sepsis. Inhibition of IL-1ß activation by targeting Nlrp3 could be useful to prevent inflammation-induced muscle failure in critically ill patients.