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Plasticity of cells, tissues, and organs is controlled by the coordinated transcription of biological programs. However, the mechanisms orchestrating such context-specific transcriptional networks mediated by the dynamic interplay of transcription factors and coregulators are poorly understood. The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a prototypical master regulator of adaptive transcription in various cell types. We now uncovered a central function of the C-terminal domain of PGC-1α to bind RNAs and assemble multiprotein complexes including proteins that control gene transcription and RNA processing. These interactions are important for PGC-1α recruitment to chromatin in transcriptionally active liquid-like nuclear condensates. Notably, such a compartmentalization of active transcription mediated by liquid-liquid phase separation was observed in mouse and human skeletal muscle, revealing a mechanism by which PGC-1α regulates complex transcriptional networks. These findings provide a broad conceptual framework for context-dependent transcriptional control of phenotypic adaptations in metabolically active tissues.

Brain-derived neurotrophic factor (BDNF) influences the differentiation, plasticity, and survival of central neurons and likewise, affects the development of the neuromuscular system. Besides its neuronal origin, BDNF is also a member of the myokine family. However, the role of skeletal muscle-derived BDNF in regulating neuromuscular physiology in vivo remains unclear. Using gain- and loss-of-function animal models, we show that muscle-specific ablation of BDNF shifts the proportion of muscle fibers from type IIB to IIX, concomitant with elevated slow muscle-type gene expression. Furthermore, BDNF deletion reduces motor end plate volume without affecting neuromuscular junction (NMJ) integrity. These morphological changes are associated with slow muscle function and a greater resistance to contraction-induced fatigue. Conversely, BDNF overexpression promotes a fast muscle-type gene program and elevates glycolytic fiber number. These findings indicate that BDNF is required for fiber-type specification and provide insights into its potential modulation as a therapeutic target in muscle diseases.

Key points Maximal endurance performance is greater in the early daytime. Timed exercise differentially alters the muscle transcriptome and (phospho)-proteome. Early daytime exercise triggers energy provisioning and tissue regeneration. Early night-time exercise activates stress-related and catabolic pathways. Scheduled training has limited effects on the muscle and liver circadian clocks. Abstract Timed physical activity might potentiate the health benefits of training. The underlying signalling events triggered by exercise at different times of day are, however, poorly understood. Here, we found that time-dependent variations in maximal treadmill exercise capacity of naive mice were associated with energy stores, mostly hepatic glycogen levels. Importantly, running at different times of day resulted in a vastly different activation of signalling pathways, e.g. related to stress response, vesicular trafficking, repair and regeneration. Second, voluntary wheel running at the opposite phase of the dark, feeding period surprisingly revealed a minimal zeitgeber (i.e. phase-shifting) effect of training on the muscle clock. This integrated study provides important insights into the circadian regulation of endurance performance and the control of the circadian clock by exercise. In future studies, these results could contribute to better understanding circadian aspects of training design in athletes and the application of chrono-exercise-based interventions in patients.

Timed physical activity might potentiate the health benefits of training. The underlying signaling events triggered by exercise at different times of the day are, however, poorly understood. Here, we found that time-dependent variations in maximal treadmill exercise capacity of naïve mice were associated with energy stores, mostly hepatic glycogen levels. Importantly, running at different times of the day resulted in a vastly different activation of signaling pathways, e.g., related to stress response, vesicular trafficking, repair, and regeneration. Second, voluntary wheel running at the opposite phase of the dark, feeding period surprisingly revealed minimal Zeitgeber (i.e., synchronizing) activity of training. This integrated study provides important insights into the circadian regulation of endurance performance and the control of the circadian clock by exercise. These results are of high importance to understand circadian aspects of training design in athletes and the application of chrono-exercise-based interventions in patients.HighlightsMaximal endurance performance is greater in the early morningTimed exercise differentially alters the muscle transcriptome and (phospho)-proteomeMorning exercise triggers energy provisioning and tissue regenerationEvening exercise activates stress-related and catabolic pathwaysTraining exerts poor Zeitgeber activity on the muscle and liver clocks

The peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α) integrates environmental cues by controlling complex transcriptional networks in various metabolically active tissues. However, it is unclear how a transcriptional coregulator coordinates dynamic biological programs in response to multifaceted stimuli such as endurance training or fasting. Here, we discovered a central function of the poorly understood C-terminal domain (CTD) of PGC-1α to bind RNAs and assemble multi-protein complexes. Surprisingly, in addition to controlling the coupling of transcription and processing of target genes, RNA binding is indispensable for the recruitment of PGC-1α to chromatin into liquid-like nuclear condensates, which compartmentalize and regulate active transcription. These results demonstrate a hitherto unsuspected molecular mechanism by which complexity in the regulation of large transcriptional networks by PGC-1α is achieved. These findings are not only essential for the basic understanding of transcriptional coregulator-driven control of biological programs, but will also help to devise new strategies to modulate these processes in pathological contexts in which PGC-1α function is dysregulated, such as type 2 diabetes, cardiovascular diseases or skeletal muscle wasting.

LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is the most frequent form of early-onset, fatal congenital muscular dystrophies. It is caused by mutations in LAMA2, the gene encoding laminin-α2, the long arm of the heterotrimeric (α2, β1, and γ1) basement membrane protein laminin-211 (Lm-211). We establish that despite compensatory expression of laminin-α4, giving rise to Lm-411 (α4, β1, and γ1), muscle basement membrane is labile in LAMA2 MD biopsies. Consistent with this deficit, recombinant Lm-411 polymerized and bound to cultured myotubes only weakly. Polymerization and cell binding of Lm-411 were enhanced by addition of two specifically designed linker proteins. One, called αLNNd, consists of the N-terminal part of laminin-α1 and the laminin-binding site of nidogen-1. The second, called mini-agrin (mag), contains binding sites for laminins and α-dystroglycan. Transgenic expression of mag and αLNNd in a mouse model for LAMA2 MD fully restored basement membrane stability, recovered muscle force and size, increased overall body weight, and extended life span more than five times to a maximum survival beyond 2 years. These findings provide a mechanistic understanding of LAMA2 MD and establish a strong basis for a potential treatment.

LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is the most frequent form of early-onset, fatal congenital muscular dystrophies. It is caused by mutations in LAMA2, the gene encoding laminin-α2, the long arm of the heterotrimeric (α2, β1, and γ1) basement membrane protein laminin-211 (Lm-211). We establish that despite compensatory expression of laminin-α4, giving rise to Lm-411 (α4, β1, and γ1), muscle basement membrane is labile in LAMA2 MD biopsies. Consistent with this deficit, recombinant Lm-411 polymerized and bound to cultured myotubes only weakly. Polymerization and cell binding of Lm-411 were enhanced by addition of two specifically designed linker proteins. One, called αLNNd, consists of the N-terminal part of laminin-α1 and the laminin-binding site of nidogen-1. The second, called mini-agrin (mag), contains binding sites for laminins and α-dystroglycan. Transgenic expression of mag and αLNNd in a mouse model for LAMA2 MD fully restored basement membrane stability, recovered muscle force and size, increased overall body weight, and extended life span more than five times to a maximum survival beyond 2 years. These findings provide a mechanistic understanding of LAMA2 MD and establish a strong basis for a potential treatment.

The basement membrane of skeletal muscle (myomatrix) consists of a complex network of highly glycosylated proteins. Important components of the myomatrix are the laminins and the collagens. During development, the myomatrix is assembled by interactions with cell surface receptors and by self-polymerization, which results in an extracellular scaffold important for skeletal muscle stability. Mutations in the α2 chain of laminin-211 impinge on myomatrix assembly and its interaction with cell surface receptors, and result in one of the most severe forms of congenital muscular dystrophy (MDC1A). Based on a detailed mechanistic understanding of the function of laminin-211, we have designed artificial linker molecules derived from agrin or perlecan with the aim to bridge the remaining myomatrix to cell surface receptors. In the conducted preclinical, proof-of-concept studies we were able to show that they can substantially ameliorate the disease phenotype in a mouse model of MDC1A. Subsequent approaches to further improve the treatment have focused on testing interventions that affect downstream pathways, such as apoptosis and inflammation, using pharmacological and genetic tools. All those treatments show additional beneficial effects on disease progression. However, semi-quantitative comparison of the treatments indicates that interventions that target early events in the disease are more efficacious than those targeting further downstream pathways. Finally, we will also report on the ongoing efforts to combine interventions that boost myomatrix assembly with those that connect it to skeletal muscle receptors. In summary, we will report on proof-of-concept studies that provide mechanistic insights and unequivocal preclinical evidence for the potential use of those therapies. As the MDC1A mice used in our studies share many hallmarks with the human disease, these preclinical data may be of high predictive value for future clinical studies.

The basement membrane surrounding skeletal muscle fibers (myomatrix) and its binding to the sarcolemma is important for the structural stability of muscle. The myomatrix consists of laminin-211 (Lm-211), collagen IV and several associated components including nidogen and perlecan. Assembly of the myomatrix is initiated by Lm-211 that binds to its cell surface receptors (alpha-dystroglycan and integrins) and then creates a primary scaffold by self-assembly. Mutations in the α2 chain of Lm-211 result in a severe form of congenital muscular dystrophy, called MDC1A. In MDC1A patients and the dyW/dyW mouse model expression of Lm-411 is increased as an attempt to compensate for the loss of Lm-211. However, Lm-411 cannot self-assemble and binds only weakly to the sarcolemma. Previous proof-of-principle studies in dyW/dyW mice have shown that transgenic expression of mini-agrin, which binds to Lm-411 and to alpha-dystroglycan, strongly ameliorates the dystrophic phenotype. We here present data that introduction of a transgene that allows self-polymerization of Lm-411 has some, although rather weak therapeutic activity in dyW/dyW mice. However, when combined with mini-agrin, this construct shows a strong synergistic effect. The improvements are seen on all levels, such as behavior, weight gain and the histology of skeletal muscles. We are currently evaluating its effect on muscle force and survival. Moreover, we analyze the signaling pathways involved in the amelioration. We also plan to test a new construct that combines the laminin polymerization and the alpha-dystroglycan binding regions. In summary, our data provide formal proof that MDC1A is caused by defects in myomatrix assembly and its connection to the sarcolemma. Our data will have significant impact in future attempts to develop new treatment strategies for MDC1A.