Class I and II Histone Deacetylase Inhibitors as Therapeutic Modulators of Dilated Cardiac Tissue-Derived Mesenchymal Stem/Stromal Cells.

The prevalence of dilated cardiomyopathy (DCM) is increasing globally, highlighting the need for innovative therapeutic approaches to prevent its onset. In this study, we examined the energetic and epigenetic distinctions between dilated and non-dilated human myocardium-derived mesenchymal stem/stromal cells (hmMSCs) and assessed the effects of class I and II HDAC inhibitors (HDACi) on these cells and their cardiomyogenic differentiation. Cells were isolated from myocardium biopsies using explant outgrowth methods. Mitochondrial and histone deacetylase activities, ATP levels, cardiac transcription factors, and structural proteins were assessed using flow cytometry, PCR, chemiluminescence, Western blotting, and immunohistochemistry. The data suggest that the tested HDAC inhibitors improved acetylation and enhanced the energetic status of both types of cells, with significant effects observed in dilated myocardium-derived hmMSCs. Additionally, the HDAC inhibitors activated the cardiac transcription factors Nkx2-5, HOPX, GATA4, and Mef2C, and upregulated structural proteins such as cardiac troponin T and alpha cardiac actin at both the protein and gene levels. In conclusion, our findings suggest that HDACi may serve as potential modulators of the energetic status and cardiomyogenic differentiation of human heart hmMSCs. This avenue of exploration could broaden the search for novel therapeutic interventions for dilated cardiomyopathy, ultimately leading to improvements in heart function.

Modulation of Titin and Contraction-Regulating Proteins in a Rat Model of Heart Failure with Preserved Ejection Fraction: Limb vs. Diaphragmatic Muscle.

Heart failure with preserved ejection fraction (HFpEF) is characterized by biomechanically dysfunctional cardiomyocytes. Underlying cellular changes include perturbed myocardial titin expression and titin hypophosphorylation leading to titin filament stiffening. Beside these well-studied alterations at the cardiomyocyte level, exercise intolerance is another hallmark of HFpEF caused by molecular alterations in skeletal muscle (SKM). Currently, there is a lack of data regarding titin modulation in the SKM of HFpEF. Therefore, the aim of the present study was to analyze molecular alterations in limb SKM (tibialis anterior (TA)) and in the diaphragm (Dia), as a more central SKM, with a focus on titin, titin phosphorylation, and contraction-regulating proteins. This study was performed with muscle tissue, obtained from 32-week old female ZSF-1 rats, an established a HFpEF rat model. Our results showed a hyperphosphorylation of titin in limb SKM, based on enhanced phosphorylation at the PEVK region, which is known to lead to titin filament stiffening. This hyperphosphorylation could be reversed by high-intensity interval training (HIIT). Additionally, a negative correlation occurring between the phosphorylation state of titin and the muscle force in the limb SKM was evident. For the Dia, no alterations in the phosphorylation state of titin could be detected. Supported by data of previous studies, this suggests an exercise effect of the Dia in HFpEF. Regarding the expression of contraction regulating proteins, significant differences between Dia and limb SKM could be detected, supporting muscle atrophy and dysfunction in limb SKM, but not in the Dia. Altogether, these data suggest a correlation between titin stiffening and the appearance of exercise intolerance in HFpEF, as well as a differential regulation between different SKM groups.

Truncated titin is structurally integrated into the human dilated cardiomyopathic sarcomere.

Heterozygous (HET) truncating variant mutations in the TTN gene (TTNtvs), encoding the giant titin protein, are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here, we studied 127 clinically identified DCM human cardiac samples with next-generation sequencing (NGS), high-resolution gel electrophoresis, Western blot analysis, and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene sequence predictions were detected in the majority of the TTNtv+ samples. Full-length titin was reduced in TTNtv+ compared with TTNtv- samples. Proteomics analysis of washed myofibrils and stimulated emission depletion (STED) super-resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin was structurally integrated into the sarcomere. Sarcomere length-dependent anti-titin epitope position, shape, and intensity analyses pointed at possible structural defects in the I/A junction and the M-band of TTNtv+ sarcomeres, which probably contribute, possibly via faulty mechanosensor function, to the development of manifest DCM.

Immunological and Structural Characterization of Titin Main Immunogenic Region; I110 Domain Is the Target of Titin Antibodies in Myasthenia Gravis.

Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction (NJ) of skeletal muscles. The major MG autoantigen is nicotinic acetylcholine receptor. Other autoantigens at the NJ include MuSK, LRP4 and agrin. Autoantibodies to the intra-sarcomeric striated muscle-specific gigantic protein titin, although not directed to the NJ, are invaluable biomarkers for thymoma and MG disease severity. Thymus and thymoma are critical in MG mechanisms and management. Titin autoantibodies bind to a 30 KDa titin segment, the main immunogenic region (MIR), consisting of an Ig-FnIII-FnIII 3-domain tandem, termed I109-I111. In this work, we further resolved the localization of titin epitope(s) to facilitate the development of more specific anti-titin diagnostics. For this, we expressed protein samples corresponding to 8 MIR and non-MIR titin fragments and tested 77 anti-titin sera for antibody binding using ELISA, competition experiments and Western blots. All anti-MIR antibodies were bound exclusively to the central MIR domain, I110, and to its containing titin segments. Most antibodies were bound also to SDS-denatured I110 on Western blots, suggesting that their epitope(s) are non-conformational. No significant difference was observed between thymoma and non-thymoma patients or between early- and late-onset MG. In addition, atomic 3D-structures of the MIR and its subcomponents were elucidated using X-ray crystallography. These immunological and structural data will allow further studies into the atomic determinants underlying titin-based autoimmunity, improved diagnostics and how to eventually treat titin autoimmunity associated co-morbidities.

Small-Molecule Inhibition of MuRF1 Prevents Early Disuse-Induced Diaphragmatic Dysfunction and Atrophy.

In clinical conditions such as diaphragm paralysis or mechanical ventilation, disuse-induced diaphragmatic dysfunction (DIDD) is a condition that poses a threat to life. MuRF1 is a key E3-ligase involved in regulating skeletal muscle mass, function, and metabolism, which contributes to the onset of DIDD. We investigated if the small-molecule mediated inhibition of MuRF1 activity (MyoMed-205) protects against early DIDD after 12 h of unilateral diaphragm denervation. Wistar rats were used in this study to determine the compound's acute toxicity and optimal dosage. For potential DIDD treatment efficacy, diaphragm contractile function and fiber cross-sectional area (CSA) were evaluated. Western blotting investigated potential mechanisms underlying MyoMed-205's effects in early DIDD. Our results indicate 50 mg/kg bw MyoMed-205 as a suitable dosage to prevent early diaphragmatic contractile dysfunction and atrophy following 12 h of denervation without detectable signs of acute toxicity. Mechanistically, treatment did not affect disuse-induced oxidative stress (4-HNE) increase, whereas phosphorylation of (ser632) HDAC4 was normalized. MyoMed-205 also mitigated FoxO1 activation, inhibited MuRF2, and increased phospho (ser473) Akt protein levels. These findings may suggest that MuRF1 activity significantly contributes to early DIDD pathophysiology. Novel strategies targeting MuRF1 (e.g., MyoMed-205) have potential therapeutic applications for treating early DIDD.

Genetic Ablation of Ankrd1 Mitigates Cardiac Damage during Experimental Autoimmune Myocarditis in Mice.

Myocarditis (MC) is an inflammatory disease of the myocardium that can cause sudden death in the acute phase, and dilated cardiomyopathy (DCM) with chronic heart failure as its major long-term outcome. However, the molecular mechanisms beyond the acute MC phase remain poorly understood. The ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic stress/stretch-inducible protein, which can modulate cardiac stress response during various forms of pathological stimuli; however, its involvement in post-MC cardiac remodeling leading to DCM is not known. To address this, we induced experimental autoimmune myocarditis (EAM) in ANKRD1-deficient mice, and evaluated post-MC consequences at the DCM stage mice hearts. We demonstrated that ANKRD1 does not significantly modulate heart failure; nevertheless, the genetic ablation of Ankrd1 blunted the cardiac damage/remodeling and preserved heart function during post-MC DCM.

Empagliflozin Preserves Skeletal Muscle Function in a HFpEF Rat Model.

Besides structural alterations in the myocardium, heart failure with preserved ejection fraction (HFpEF) is also associated with molecular and physiological alterations of the peripheral skeletal muscles (SKM) contributing to exercise intolerance often seen in HFpEF patients. Recently, the use of Sodium-Glucose-Transporter 2 inhibitors (SGLT2i) in clinical studies provided evidence for a significant reduction in the combined risk of cardiovascular death or hospitalization for HFpEF. The present study aimed to further elucidate the impact of Empagliflozin (Empa) on: (1) SKM function and metabolism and (2) mitochondrial function in an established HFpEF rat model. At the age of 24 weeks, obese ZSF1 rats were randomized either receiving standard care or Empa in the drinking water. ZSF1 lean animals served as healthy controls. After 8 weeks of treatment, echocardiography and SKM contractility were performed. Mitochondrial function was assessed in saponin skinned fibers and SKM tissue was snap frozen for molecular analyses. HFpEF was evident in the obese animals when compared to lean-increased E/é and preserved left ventricular ejection fraction. Empa treatment significantly improved E/é and resulted in improved SKM contractility with reduced intramuscular lipid content. Better mitochondrial function (mainly in complex IV) with only minor modulation of atrophy-related proteins was seen after Empa treatment. The results clearly documented a beneficial effect of Empa on SKM function in the present HFpEF model. These effects were accompanied by positive effects on mitochondrial function possibly modulating SKM function.

The Effect of Heat Shock on Myogenic Differentiation of Human Skeletal-Muscle-Derived Mesenchymal Stem/Stromal Cells.

Muscle injuries, degenerative diseases and other lesions negatively affect functioning of human skeletomuscular system and thus quality of life. Therefore, the investigation of molecular mechanisms, stimulating myogenic differentiation of primary skeletal-muscle-derived mesenchymal stem/stromal cells (SM-MSCs), is actual and needed. The aim of the present study was to investigate the myogenic differentiation of CD56 (neural cell adhesion molecule, NCAM)-positive and -negative SM-MSCs and their response to the non-cytotoxic heat stimulus. The SM-MSCs were isolated from the post operation muscle tissue, sorted by flow cytometer according to the CD56 biomarker and morphology, surface profile, proliferation and myogenic differentiation has been investigated. Data show that CD56(+) cells were smaller in size, better proliferated and had significantly higher levels of CD146 (MCAM) and CD318 (CDCP1) compared with the CD56(-) cells. At control level, CD56(+) cells significantly more expressed myogenic differentiation markers MYOD1 and myogenin (MYOG) and better differentiated to the myogenic direction. The non-cytotoxic heat stimulus significantly stronger stimulated expression of myogenic markers in CD56(+) than in CD56(-) cells that correlated with the multinucleated cell formation. Data show that regenerative properties of CD56(+) SM-MSCs can be stimulated by an extracellular stimulus and be used as a promising skeletal muscle regenerating tool in vivo.

The skeletal muscle circadian clock regulates titin splicing through RBM20.

Circadian rhythms are maintained by a cell-autonomous, transcriptional-translational feedback loop known as the molecular clock. While previous research suggests a role of the molecular clock in regulating skeletal muscle structure and function, no mechanisms have connected the molecular clock to sarcomere filaments. Utilizing inducible, skeletal muscle specific, Bmal1 knockout (iMSBmal1-/-) mice, we showed that knocking out skeletal muscle clock function alters titin isoform expression using RNAseq, liquid chromatography-mass spectrometry, and sodium dodecyl sulfate-vertical agarose gel electrophoresis. This alteration in titin's spring length resulted in sarcomere length heterogeneity. We demonstrate the direct link between altered titin splicing and sarcomere length in vitro using U7 snRNPs that truncate the region of titin altered in iMSBmal1-/- muscle. We identified a mechanism whereby the skeletal muscle clock regulates titin isoform expression through transcriptional regulation of Rbm20, a potent splicing regulator of titin. Lastly, we used an environmental model of circadian rhythm disruption and identified significant downregulation of Rbm20 expression. Our findings demonstrate the importance of the skeletal muscle circadian clock in maintaining titin isoform through regulation of RBM20 expression. Because circadian rhythm disruption is a feature of many chronic diseases, our results highlight a novel pathway that could be targeted to maintain skeletal muscle structure and function in a range of pathologies.

miR-29c Increases Protein Synthesis in Skeletal Muscle Independently of AKT/mTOR.

microRNAs negatively regulate gene expression by blocking translation or increasing mRNA degradation. In skeletal muscle, these molecules play important roles in adaptive responses, and ongoing investigations are necessary to understand the fine-tune regulation of skeletal muscle mass. Herein we showed that skeletal muscle overexpression of miR-29c increased fiber size and force at 7 and 30 days after electrotransfer. At both time points, AKT/mTOR pathway components were downregulated, and, surprisingly, overall protein synthesis was strongly elevated at day 7, which normalized by day 30 after pCMVmiR-29c electrotransfer. These results indicate that miR-29c expression induces skeletal muscle hypertrophy and gain of function, which involves increased overall protein synthesis in spite of the deactivation of the AKT/mTOR pathway.

Removal of MuRF1 Increases Muscle Mass in Nemaline Myopathy Models, but Does Not Provide Functional Benefits.

Nemaline myopathy (NM) is characterized by skeletal muscle weakness and atrophy. No curative treatments exist for this debilitating disease. NM is caused by mutations in proteins involved in thin-filament function, turnover, and maintenance. Mutations in nebulin, encoded by NEB, are the most common cause. Skeletal muscle atrophy is tightly linked to upregulation of MuRF1, an E3 ligase, that targets proteins for proteasome degradation. Here, we report a large increase in MuRF1 protein levels in both patients with nebulin-based NM, also named NEM2, and in mouse models of the disease. We hypothesized that knocking out MuRF1 in animal models of NM with muscle atrophy would ameliorate the muscle deficits. To test this, we crossed MuRF1 KO mice with two NEM2 mouse models, one with the typical form and the other with the severe form. The crosses were viable, and muscles were studied in mice at 3 months of life. Ultrastructural examination of gastrocnemius muscle lacking MuRF1 and with severe NM revealed a small increase in vacuoles, but no significant change in the myofibrillar fractional area. MuRF1 deficiency led to increased weights of various muscle types in the NM models. However, this increase in muscle size was not associated with increased in vivo or in vitro force production. We conclude that knocking out MuRF1 in NEM2 mice increases muscle size, but does not improve muscle function.

Targeting MuRF1 by small molecules in a HFpEF rat model improves myocardial diastolic function and skeletal muscle contractility.

BACKGROUND: About half of heart failure (HF) patients, while having preserved left ventricular function, suffer from diastolic dysfunction (so-called HFpEF). No specific therapeutics are available for HFpEF in contrast to HF where reduced ejection fractions (HFrEF) can be treated pharmacologically. Myocardial titin filament stiffening, endothelial dysfunction, and skeletal muscle (SKM) myopathy are suspected to contribute to HFpEF genesis. We previously described small molecules interfering with MuRF1 target recognition thereby attenuating SKM myopathy and dysfunction in HFrEF animal models. The aim of the present study was to test the efficacy of one small molecule (MyoMed-205) in HFpEF and to describe molecular changes elicited by MyoMed-205. METHODS: Twenty-week-old female obese ZSF1 rats received the MuRF1 inhibitor MyoMed-205 for 12 weeks; a comparison was made to age-matched untreated ZSF1-lean (healthy) and obese rats as controls. LV (left ventricle) function was assessed by echocardiography and by invasive haemodynamic measurements until week 32. At week 32, SKM and endothelial functions were measured and tissues collected for molecular analyses. Proteome-wide analysis followed by WBs and RT-PCR was applied to identify specific genes and affected molecular pathways. MuRF1 knockout mice (MuRF1-KO) SKM tissues were included to validate MuRF1-specificity. RESULTS: By week 32, untreated obese rats had normal LV ejection fraction but augmented E/e' ratios and increased end diastolic pressure and myocardial fibrosis, all typical features of HFpEF. Furthermore, SKM myopathy (both atrophy and force loss) and endothelial dysfunction were detected. In contrast, MyoMed-205 treated rats had markedly improved diastolic function, less myocardial fibrosis, reduced SKM myopathy, and increased SKM function. SKM extracts from MyoMed-205 treated rats had reduced MuRF1 content and lowered total muscle protein ubiquitination. In addition, proteomic profiling identified eight proteins to respond specifically to MyoMed-205 treatment. Five out of these eight proteins are involved in mitochondrial metabolism, dynamics, or autophagy. Consistent with the mitochondria being a MyoMed-205 target, the synthesis of mitochondrial respiratory chain complexes I + II was increased in treated rats. MuRF1-KO SKM controls also had elevated mitochondrial complex I and II activities, also suggesting mitochondrial activity regulation by MuRF1. CONCLUSIONS: MyoMed-205 improved myocardial diastolic function and prevented SKM atrophy/function in the ZSF1 animal model of HFpEF. Mechanistically, SKM benefited from an attenuated ubiquitin proteasome system and augmented synthesis/activity of proteins of the mitochondrial respiratory chain while the myocardium seemed to benefit from reduced titin modifications and fibrosis.

Molecular Mechanisms behind Persistent Presence of Parvovirus B19 in Human Dilated Myocardium.

The role of parvovirus B19 (PVB19) in the pathogenesis of idiopathic dilated cardiomyopathy (DCM) remains poorly understood. Therefore, we have measured the levels of inflammation, fibrosis, apoptosis, and necrosis in endomyocardial biopsies (EMBs) and sera of nonischemic PVB19-positive (n = 14) and PVB19-negative (n = 18) DCM patients. Chronic persistence of PVB19 in myocardium did not induce significant infiltration of T cells (CD3 and CD45Ro) and macrophages (CD68), and did not secrete TNFα, IL-6, and CRB. The fibrosis in PVB19-positive EMBs was also lower compared to the virus-negative ones, while ECM degrading matrix metalloproteinase MMP1 and gelatinase MMP2 were significantly (by twofold) upregulated. In addition, there was no activation of neither apoptotic nor necrotic pathways. However, levels of antiapoptotic mitochondrial Bcl-2 and heat shock protein 60 (Hsp60) in PVB19-positive biopsies were almost threefold lower than in PVB19-negative ones revealing impairment of mitochondria. Altogether, data indicate that persistence of PVB19 in myocardiums of nonischemic DCM patients can cause myocardial ECM remodeling through the MMPs, such as MMP1 and MMP2, and mitochondrial impairment. The correlative analysis of measured biomarkers suggested likely further activation of apoptotic cell death pathways rather than fibrosis. Data also suggest that antiviral therapy could be beneficial for PVB19-positive DCM patients by managing further pathological myocardial remodeling.

MuRF1 deficiency prevents age-related fat weight gain, possibly through accumulation of PDK4 in skeletal muscle mitochondria in older mice.

Recent studies show that muscle mass and metabolic function are interlinked. Muscle RING finger 1 (MuRF1) is a critical muscle-specific ubiquitin ligase associated with muscle atrophy. Yet, the molecular target of MuRF1 in atrophy and aging remains unclear. We examined the role of MuRF1 in aging, using MuRF1-deficient (MuRF1-/- ) mice in vivo, and MuRF1-overexpressing cell in vitro. MuRF1 deficiency partially prevents age-induced skeletal muscle loss in mice. Interestingly, body weight and fat mass of more than 7-month-old MuRF1-/- mice were lower than in MuRF1+/+ mice. Serum and muscle metabolic parameters and results of indirect calorimetry suggest significantly higher energy expenditure and enhanced lipid metabolism in 3-month-old MuRF1-/- mice than in MuRF1+/+ mice, resulting in suppressed adipose tissue gain during aging. Pyruvate dehydrogenase kinase 4 (PDK4) is crucial for a switch from glucose to lipid metabolism, and the interaction between MuRF1 and PDK4 was examined. PDK4 protein levels were elevated in mitochondria from the skeletal muscle in MuRF1-/- mice. In vitro, MuRF1 interacted with PDK4 but did not induce degradation through ubiquitination. Instead, SUMO posttranscriptional modification (SUMOylation) of PDK4 was detected in MuRF1-overexpressing cells, in contrast to cells without the RING domain of MuRF1. MuRF1 deficiency enhances lipid metabolism possibly by upregulating PDK4 localization into mitochondrial through prevention of SUMOylation. Inhibition of MuRF1-mediated PDK4 SUMOylation is a potential therapeutic target for age-related dysfunction of lipid metabolism and muscle atrophy.

Cardiomyogenic Differentiation Potential of Human Dilated Myocardium-Derived Mesenchymal Stem/Stromal Cells: The Impact of HDAC Inhibitor SAHA and Biomimetic Matrices.

Dilated cardiomyopathy (DCM) is the most common type of nonischemic cardiomyopathy characterized by left ventricular or biventricular dilation and impaired contraction leading to heart failure and even patients' death. Therefore, it is important to search for new cardiac tissue regenerating tools. Human mesenchymal stem/stromal cells (hmMSCs) were isolated from post-surgery healthy and DCM myocardial biopsies and their differentiation to the cardiomyogenic direction has been investigated in vitro. Dilated hmMSCs were slightly bigger in size, grew slower, but had almost the same levels of MSC-typical surface markers as healthy hmMSCs. Histone deacetylase (HDAC) activity in dilated hmMSCs was 1.5-fold higher than in healthy ones, which was suppressed by class I and II HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) showing activation of cardiomyogenic differentiation-related genes alpha-cardiac actin (ACTC1) and cardiac troponin T (TNNT2). Both types of hmMSCs cultivated on collagen I hydrogels with hyaluronic acid (HA) or 2-methacryloyloxyethyl phosphorylcholine (MPC) and exposed to SAHA significantly downregulated focal adhesion kinase (PTK2) and activated ACTC1 and TNNT2. Longitudinal cultivation of dilated hmMSC also upregulated alpha-cardiac actin. Thus, HDAC inhibitor SAHA, in combination with collagen I-based hydrogels, can tilt the dilated myocardium hmMSC toward cardiomyogenic direction in vitro with further possible therapeutic application in vivo.

Titin kinase ubiquitination aligns autophagy receptors with mechanical signals in the sarcomere.

Striated muscle undergoes remodelling in response to mechanical and physiological stress, but little is known about the integration of such varied signals in the myofibril. The interaction of the elastic kinase region from sarcomeric titin (A168-M1) with the autophagy receptors Nbr1/p62 and MuRF E3 ubiquitin ligases is well suited to link mechanosensing with the trophic response of the myofibril. To investigate the mechanisms of signal cross-talk at this titin node, we elucidated its 3D structure, analysed its response to stretch using steered molecular dynamics simulations and explored its functional relation to MuRF1 and Nbr1/p62 using cellular assays. We found that MuRF1-mediated ubiquitination of titin kinase promotes its scaffolding of Nbr1/p62 and that the process can be dynamically down-regulated by the mechanical unfolding of a linker sequence joining titin kinase with the MuRF1 receptor site in titin. We propose that titin ubiquitination is sensitive to the mechanical state of the sarcomere, the regulation of sarcomere targeting by Nbr1/p62 being a functional outcome. We conclude that MuRF1/Titin Kinase/Nbr1/p62 constitutes a distinct assembly that predictably promotes sarcomere breakdown in inactive muscle.

Muscle ankyrin repeat protein 1 (MARP1) locks titin to the sarcomeric thin filament and is a passive force regulator.

Muscle ankyrin repeat protein 1 (MARP1) is frequently up-regulated in stressed muscle, but its effect on skeletal muscle function is poorly understood. Here, we focused on its interaction with the titin-N2A element, found in titin's molecular spring region. We show that MARP1 binds to F-actin, and that this interaction is stronger when MARP1 forms a complex with titin-N2A. Mechanics and super-resolution microscopy revealed that MARP1 "locks" titin-N2A to the sarcomeric thin filament, causing increased extension of titin's elastic PEVK element and, importantly, increased passive force. In support of this mechanism, removal of thin filaments abolished the effect of MARP1 on passive force. The clinical relevance of this mechanism was established in diaphragm myofibers of mechanically ventilated rats and of critically ill patients. Thus, MARP1 regulates passive force by locking titin to the thin filament. We propose that in stressed muscle, this mechanism protects the sarcomere from mechanical damage.

Regulation of Glucose Metabolism by MuRF1 and Treatment of Myopathy in Diabetic Mice with Small Molecules Targeting MuRF1.

The muscle-specific ubiquitin ligase MuRF1 regulates muscle catabolism during chronic wasting states, although its roles in general metabolism are less-studied. Here, we metabolically profiled MuRF1-deficient knockout mice. We also included knockout mice for MuRF2 as its closely related gene homolog. MuRF1 and MuRF2-KO (knockout) mice have elevated serum glucose, elevated triglycerides, and reduced glucose tolerance. In addition, MuRF2-KO mice have a reduced tolerance to a fat-rich diet. Western blot and enzymatic studies on MuRF1-KO skeletal muscle showed perturbed FoxO-Akt signaling, elevated Akt-Ser-473 activation, and downregulated oxidative mitochondrial metabolism, indicating potential mechanisms for MuRF1,2-dependent glucose and fat metabolism regulation. Consistent with this, the adenoviral re-expression of MuRF1 in KO mice normalized Akt-Ser-473, serum glucose, and triglycerides. Finally, we tested the MuRF1/2 inhibitors MyoMed-205 and MyoMed-946 in a mouse model for type 2 diabetes mellitus (T2DM). After 28 days of treatment, T2DM mice developed progressive muscle weakness detected by wire hang tests, but this was attenuated by the MyoMed-205 treatment. While MyoMed-205 and MyoMed-946 had no significant effects on serum glucose, they did normalize the lymphocyte-granulocyte counts in diabetic sera as indicators of the immune response. Thus, small molecules directed to MuRF1 may be useful in attenuating skeletal muscle strength loss in T2DM conditions.

Identification of Bovine miRNAs with the Potential to Affect Human Gene Expression.

Milk and other products from large mammals have emerged during human evolution as an important source of nutrition. Recently, it has been recognized that exogenous miRNAs (mRNA inhibited RNA) contained in milk and other tissues of the mammalian body can enter the human body, which in turn have the ability to potentially regulate human metabolism by affecting gene expression. We studied for exogenous miRNAs from Bos taurus that are potentially contain miRNAs from milk and that could act postprandially as regulators of human gene expression. The interaction of 17,508 human genes with 1025 bta-miRNAs, including 245 raw milk miRNAs was studied. The milk bta-miR-151-5p, bta-miR-151-3p, bta-miRNA-320 each have 11 BSs (binding sites), and bta-miRNA-345-5p, bta-miRNA-614, bta-miRNA-1296b and bta-miRNA-149 has 12, 14, 15 and 26 BSs, respectively. The bta-miR-574-5p from cow's milk had 209 human genes in mRNAs from one to 25 repeating BSs. We found 15 bta-miRNAs that have 100% complementarity to the mRNA of 13 human target genes. Another 12 miRNAs have BSs in the mRNA of 19 human genes with 98% complementarity. The bta-miR-11975, bta-miR-11976, and bta-miR-2885 BSs are located with the overlap of nucleotide sequences in the mRNA of human genes. Nucleotide sequences of BSs of these miRNAs in 5'UTR mRNA of human genes consisted of GCC repeats with a total length of 18 nucleotides (nt) in 18 genes, 21 nt in 11 genes, 24 nt in 14 genes, and 27-48 nt in nine genes. Nucleotide sequences of BSs of bta-miR-11975, bta-miR-11976, and bta-miR-2885 in CDS mRNA of human genes consisted of GCC repeats with a total length of 18 nt in 33 genes, 21 nt in 13 genes, 24 nt in nine genes, and 27-36 nt in 11 genes. These BSs encoded polyA or polyP peptides. In only one case, the polyR (SLC24A3 gene) was encoded. The possibility of regulating the expression of human genes by exogenous bovine miRNAs is discussed.

In silico Prediction of miRNA Interactions With Candidate Atherosclerosis Gene mRNAs.

The involvement of genes and miRNAs in the development of atherosclerosis is a challenging problem discussed in recent publications. It is necessary to establish which miRNAs affect the expression of candidate genes. We used known candidate atherosclerosis genes to predict associations. The quantitative characteristics of interactions of miRNAs with mRNA candidate genes were determined using the program, which identifies the localization of miRNA binding sites in mRNA, the free energy interaction of miRNA with mRNA. In mRNAs of GAS6 and NFE2L2 candidate genes, binding sites of 21 miRNAs and of 15 miRNAs, respectively, were identified. In IRS2 mRNA binding sites of 25 miRNAs were located in a cluster of 41 nt. In ADRB3, CD36, FASLG, FLT1, PLA2G7, and PPARGC1A mRNAs, clusters of miR-466, ID00436.3p-miR, and ID01030.3p-miR BS were identified. The organization of overlapping miRNA binding sites in clusters led to their compaction and caused competition among the miRNAs. The binding of 53 miRNAs to the mRNAs of 14 candidate genes with free energy interactions greater than -130 kJ/mole was determined. The miR-619-5p was fully complementary to ADAM17 and CD36 mRNAs, ID01593.5p-miR to ANGPTL4 mRNA, ID01935.5p-miR to NFE2L2, and miR-5096 to IL18 mRNA. Associations of miRNAs and candidate atherosclerosis genes are proposed for the early diagnosis of this disease.