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Microfluidic‐based technologies enable the development of cell culture systems that provide tailored microenvironmental inputs to mammalian cells. Primary myoblasts can be induced to differentiate into multinucleated skeletal muscle cells, myotubes, which are a relevant model system for investigating skeletal muscle metabolism and physiology in vitro. However, it remains challenging to differentiate primary myoblasts into mature myotubes in microfluidics devices. Here we investigated the effects of integrating continuous (solid) and intermittent (dashed) walls in microfluidic channels as topological constraints in devices designed to promote the alignment and maturation of primary myoblast‐derived myotubes. The topological constraints caused alignment of the differentiated myotubes, mimicking the native anisotropic organization of skeletal muscle cells. Interestingly, dashed walls facilitated the maturation of skeletal muscle cells, as measured by quantifying myotube cell area and the number of nuclei per myotube. Together, our results suggest that integrating dashed walls as topographic constraints in microfluidic devices supports the alignment and maturation of primary myoblast‐derived myotubes. [ABSTRACT FROM AUTHOR]

Abstract: Most antidiabetic drugs treat disease symptoms rather than adipose tissue dysfunction as a key pathogenic cause in the metabolic syndrome and type 2 diabetes. Pharmacological targeting of adipose tissue through the nuclear receptor PPARg, as exemplified by glitazone treatments, mediates efficacious insulin sensitization. However, a better understanding of the context‐specific PPARg responses is required for the development of novel approaches with reduced side effects. Here, we identified the transcriptional cofactor Cited4 as a target and mediator of rosiglitazone in human and murine adipocyte progenitor cells, where it promoted specific sets of the rosiglitazone‐dependent transcriptional program. In mice, Cited4 was required for the proper induction of thermogenic expression by Rosi specifically in subcutaneous fat. This phenotype had high penetrance in females only and was not evident in beta‐adrenergically stimulated browning. Intriguingly, this specific defect was associated with reduced capacity for systemic thermogenesis and compromised insulin sensitization upon therapeutic rosiglitazone treatment in female but not male mice. Our findings on Cited4 function reveal novel unexpected aspects of the pharmacological targeting of PPARg. [ABSTRACT FROM AUTHOR]

Key points Transcriptional co-activator PGC-1α1 has been shown to regulate energy metabolism and to mediate metabolic adaptations in pathological and physiological cardiac hypertrophy but other functional implications of PGC-1α1 expression are not known., Transgenic PGC-1α1 overexpression within the physiological range in mouse heart induces purposive changes in contractile properties, electrophysiology and calcium signalling but does not induce substantial metabolic remodelling., The phenotype of the PGC-1α1 transgenic mouse heart recapitulates most of the functional modifications usually associated with the exercise-induced heart phenotype, but does not protect the heart against load-induced pathological hypertrophy., Transcriptional effects of PGC-1α1 show clear dose-dependence with diverse changes in genes in circadian clock, heat shock, excitability, calcium signalling and contraction pathways at low overexpression levels, while metabolic genes are recruited at much higher PGC-1α1 expression levels., These results imply that the physiological role of PGC-1α1 is to promote a beneficial excitation-contraction coupling phenotype in the heart., Abstract The transcriptional coactivator PGC-1α1 has been identified as a central factor mediating metabolic adaptations of the heart. However, to what extent physiological changes in PGC-1α1 expression levels actually contribute to the functional adaptation of the heart is still mostly unresolved. The aim of this study was to characterize the transcriptional and functional effects of physiologically relevant, moderate PGC-1α1 expression in the heart. In vivo and ex vivo physiological analysis shows that expression of PGC-1α1 within a physiological range in mouse heart does not induce the expected metabolic alterations, but instead induces a unique excitation-contraction (EC) coupling phenotype recapitulating features typically seen in physiological hypertrophy. Transcriptional screening of PGC-1α1 overexpressing mouse heart and myocyte cultures with higher, acute adenovirus-induced PGC-1α1 expression, highlights PGC-1α1 as a transcriptional coactivator with a number of binding partners in various pathways (such as heat shock factors and the circadian clock) through which it acts as a pleiotropic transcriptional regulator in the heart, to both augment and repress the expression of its target genes in a dose-dependent fashion. At low levels of overexpression PGC-1α1 elicits a diverse transcriptional response altering the expression state of circadian clock, heat shock, excitability, calcium signalling and contraction pathways, while metabolic targets of PGC-1α1 are recruited at higher PGC-1α1 expression levels. Together these findings demonstrate that PGC-1α1 elicits a dual effect on cardiac transcription and phenotype. Further, our results imply that the physiological role of PGC-1α1 is to promote a beneficial EC coupling phenotype in the heart. [ABSTRACT FROM AUTHOR]

Key points Using an experimental rat intensive care unit (ICU) model, not limited by early mortality, we have previously shown that passive mechanical loading attenuates the loss of muscle mass and force-generation capacity associated with the ICU intervention., Mitochondrial dynamics have recently been shown to play a more important role in muscle atrophy than previously recognized., In this study we demonstrate that mitochondrial dynamics, as well as mitophagy, is affected by mechanosensing at the transcriptional level, and muscle changes induced by unloading are counteracted by passive mechanical loading., The recently discovered ubiquitin ligases Fbxo31 and SMART are induced by mechanical silencing, an induction that similarly is prevented by passive mechanical loading., Abstract The complete loss of mechanical stimuli of skeletal muscles, i.e. loss of external strain related to weight bearing and internal strain related to activation of contractile proteins, in mechanically ventilated, deeply sedated and/or pharmacologically paralysed intensive care unit (ICU) patients is an important factor triggering the critical illness myopathy (CIM). Using a unique experimental ICU rat model, mimicking basic ICU conditions, we have recently shown that mechanical silencing is a dominant factor triggering the preferential loss of myosin, muscle atrophy and decreased specific force in fast- and slow-twitch muscles and muscle fibres. The aim of this study is to gain improved understanding of the gene signature and molecular pathways regulating the process of mechanical activation of skeletal muscle that are affected by the ICU condition. We have focused on pathways controlling myofibrillar protein synthesis and degradation, mitochondrial homeostasis and apoptosis. We demonstrate that genes regulating mitochondrial dynamics, as well as mitophagy are induced by mechanical silencing and that these effects are counteracted by passive mechanical loading. In addition, the recently identified ubiquitin ligases Fbxo31 and SMART are induced by mechanical silencing, an induction that is reversed by passive mechanical loading. Thus, mechano-cell signalling events are identified which may play an important role for the improved clinical outcomes reported in response to the early mobilization and physical therapy in immobilized ICU patients. [ABSTRACT FROM AUTHOR]

Apelin and its G-protein-coupled receptor APJ have various beneficial effects on cardiac function and blood pressure. The mechanisms that regulate apelin gene expression are not known. Because apelin gene expression has been shown to increase in cardiac ischemia, we investigated if apelin (Apln) gene expression was sensitive to hypoxia. Here we show that hypoxia increases the apelin expression in rat myocardium and in cultured cardiomyocytes. Pharmacological activation of hypoxia inducible factor by desferrioxamine (DFO) or expression of a constitutively active form of HIF-1α increased apelin expression in cardiomyocyte cultures. The induction of apelin by hypoxia was abolished on transient expression of the HIF inhibitory PAS protein in cardiomyocytes. Increased apelin expression induced by hypoxia or DFO was accompanied by the processing of the cellular storage form proapelin into smaller apelin peptides and increased secretion of these biologically active forms of apelin. In a rat in vivo model, acute myocardial infarction (24 h) led to a transient increase in ventricular apelin mRNA levels. Our results indicate that apelin gene is regulated by hypoxia in cardiac myocytes via the HIF pathway, suggesting a role for apelin as a potential marker for acute cardiac hypoxia with a possible compensatory role in myocardial tissue suffering from oxygen deprivation. [ABSTRACT FROM AUTHOR]

Senescence is an important cellular program occurring in development, tissue repair, cancer, and aging. Increased senescence is also associated with disease states, including obesity and Type 2 diabetes (T2D). Characterizing and quantifying senescent cells at a single cell level has been challenging and particularly difficult in large primary cells, such as human adipocytes. In this study, we present a novel approach that utilizes reflected light for accurate senescence‐associated beta‐galactosidase (SABG) staining measurements, which can be integrated with immunofluorescence and is compatible with primary mature adipocytes from both human and mouse, as well as with differentiated 3T3‐L1 cells. This technique provides a more comprehensive classification of a cell's senescent state by incorporating multiple criteria, including robust sample‐specific pH controls. By leveraging the precision of confocal microscopy to detect X‐gal crystals using reflected light, we achieved superior sensitivity over traditional brightfield techniques. This strategy allows for the capture of all X‐gal precipitates in SABG‐stained samples, revealing diverse X‐gal staining patterns and improved detection sensitivity. Additionally, we demonstrate that reflected light outperforms western blot analysis for the detection and quantification of senescence in mature human adipocytes, as it offers a more accurate representation of SABG activity. This detection strategy enables a more thorough investigation of senescent cell characteristics and specifically a deeper look at the relationship between adipocyte senescence and obesity associated disorders, such as T2D. [ABSTRACT FROM AUTHOR]