Pura Muñoz-Cánoves

Spatial chromatin organization dictates cellular function and resilience, yet scalable imaging methods to quantify chromatin states in situ across aging and interventions are lacking. Although ATAC-see can visualize accessible chromatin, its broader application is hindered by protocol variability, low throughput and incompatibility with complex tissues. Here, we systematically optimize the ATAC-see workflow for robust, high-throughput quantitative imaging in fixed, adherent mammalian cells and fresh frozen tissues. We validate the sensitivity of the platform to pharmacologic remodeling and apply it to replicative, chronological and pathological aging in primary human fibroblasts, revealing progressive age-associated chromatin opening and heterochromatin remodeling. Furthermore, we demonstrate that our optimized ATAC-see captures rapid, reversible chromatin reorganization during OSK(M)-driven partial reprogramming of aged fibroblasts. Finally, we extend a cost-effective and accessible protocol to murine tissue sections, quantifying in situ age-dependent remodeling. This standardized framework establishes chromatin accessibility as a highly scalable, sequencing-compatible imaging biomarker for evaluating aging and rejuvenation.

Here, we present targeted ATAC-see (tATAC-see), a visual genomics assay for site-specific chromatin profiling. By integrating the in situ visualization of ATAC-see with antibody-tethered tagmentation, tATAC-see captures chromatin states at defined protein-occupied domains with the simplicity of standard immunofluorescence. Modulating the spatial interaction radius of Tn5 via salt titration enables extended tagmentation of local chromatin environments near the target. We validated this imaging-based method by capturing the expanded chromatin neighborhoods of active euchromatin (H3K27ac and H3K4me3) alongside the dense structural lamina-associated domains (LADs) of lamin A/C and lamin B1. Using an HDAC inhibitor, we tested the sensitivity of the assay to detect dynamic structural remodeling. We not only tracked chromatin decompaction but also demonstrated the robust structural resistance of LADs. As a proof-of-principle biological application, we applied tATAC-see to models of replicative, chronological and pathological (Hutchinson–Gilford progeria syndrome) aging to assess its ability to detect well-characterized peripheral heterochromatin and LAD remodeling. We observed divergent chromatin trajectories at the nuclear envelope, reflective of the distinct roles for the lamins. Lamin B1 domains exhibit increased local accessibility consistent with age-associated heterochromatin erosion, whereas lamin A/C-associated domains physically detach from their scaffold. Ultimately, tATAC-see provides a robust and accessible platform for mechanistic and population-level studies to uncover spatial epigenome dynamics.

Skeletal muscle atrophy and weakness resulting from loss of motor neuron innervation are shared hallmarks of amyotrophic lateral sclerosis (ALS) and age-associated sarcopenia. However, muscle wasting in ALS is rapid and severe, whereas sarcopenia progresses gradually over decades, peaking at an advanced age. The molecular mechanisms underlying muscle wasting in these conditions remain incompletely defined. Here, we used the SOD1 ^G93A ALS mouse model and wild-type mice across the lifespan to generate an integrated single-nucleus transcriptomic atlas of skeletal muscle, mapping changes associated with ALS and aging. This atlas is complemented by structural and functional analyses. Our results identify transcriptionally distinct condition-specific myonuclear states, including disease-responsive programs within synaptic myonuclei at the neuromuscular junction, that are shared or distinct between the two conditions. Notably, at early stages, muscle from ALS mice exhibits molecular hallmarks of aged muscle at the myofiber level, indicating premature aging and reinforcing aging as a risk factor in ALS. At advanced disease stages and in old age, myonuclear subpopulations follow similar degenerative pathways in fast glycolytic fibers, consistent with their loss in both conditions. However, a distinct ALS-enriched myonuclei subpopulation displays uniquely regulated genes not observed in aged muscle, potentially underlying the more rapid and severe pathology observed in ALS. This atlas maps shared and divergent mechanisms of muscle degeneration in ALS and sarcopenia and provides a resource for identifying therapeutic targets tailored to each condition.

Stem cells are central for tissue homeostasis and regeneration after stress, and their function depends on interactions with neighboring niche cells. In skeletal muscle, the resident stem cells (also called satellite cells [SCs]) decline in number and function with age, leading to loss of regenerative capacity and, eventually, deterioration of muscle integrity and physiology. Aged SCs lose the capacity to preserve quiescence or to activate and expand upon injury, in part due to changes in the local and systemic environment. Here, we focus on the age-associated intrinsic changes of SCs and the causes of their cellular and molecular decline. We discuss results from single-cell omics technologies that have shed light on the interactions between SCs and niche cells and how far-reaching signals impact SC function. We present future perspectives on rejuvenating interventions to counter age-dependent regenerative decline and the open questions in muscle stem cell aging.

Removal of cellular waste from the extracellular space is fundamental for tissue health. Because the rate of material ejected by parenchymal cells varies across tissues, we searched for mechanisms that couple waste production and removal. Here, we show that the uptake of parenchyma-released mitochondria by macrophages is prominent across organs that rely on oxidative respiration—including heart, skeletal muscle, and brown adipose tissue—and that macrophage numbers closely align with the mitochondrial activity of each of these organs. We found that the mitochondrial activity of myofibers dictates the abundance of macrophages by modulating colony-stimulating factor 1 (CSF1) availability and the number of CSF1-producing fibroblasts in the tissue. Consequently, inhibition of CSF1–CSF1 receptor (CSF1R) signaling depleted macrophages and collapsed the mitochondrial activity of skeletal muscles. We propose that, by coupling macrophage abundance to the mitochondrial activity of their parenchyma, tissues ensure efficient waste disposal and fitness.

Skeletal muscle is a central determinant of organismal health. Preserving muscle quality is therefore critical for preventing disease and sustaining quality of life across the lifespan. Despite its central role, the field lacks a unifying framework that defines the core properties of skeletal muscle health. Here, we propose a conceptual framework for muscle homeostasis built around seven interconnected hallmarks—metabolism and bioenergetics, proteostasis, genomics, excitability, structure, regeneration and cross-talk—that collectively govern muscle integrity, adaptability and resilience. Each hallmark is mechanistically grounded, quantifiable and potentially modifiable. This framework provides a unifying blueprint for the next generation of precision diagnostics and targeted therapies for preserving skeletal muscle health.

We present targeted ATAC-see (tATAC-see), a visual genomics assay for site-specific chromatin profiling. By integrating the in situ visualization of ATAC-see with antibody-tethered tagmentation, tATAC-see captures chromatin states at defined protein-occupied domains with the simplicity of standard immunofluorescence. Modulating the spatial interaction radius of Tn5 via salt titration enables extended tagmentation of local chromatin environments near the target. We validated this imaging-based method by capturing the expanded chromatin neighborhoods of active euchromatin (H3K27ac, H3K4me3) alongside the dense structural lamina-associated domains (LADs) of lamin A/C and lamin B1. Using an HDAC inhibitor, we tested the assay's sensitivity to detect dynamic structural remodeling. We not only tracked chromatin decompaction but also demonstrated the robust structural resistance of LADs. As a proof-of-principle biological application, we applied tATAC-see to models of replicative, chronological, and pathological (Hutchinson-Gilford progeria syndrome) aging to assess its ability to detect well-characterized peripheral heterochromatin and LAD remodeling. We observed divergent chromatin trajectories at the nuclear envelope, reflective of the lamins' distinct roles. lamin B1 domains exhibit increased local accessibility consistent with age-associated heterochromatin erosion, while lamin A/C-associated domains physically detach from their scaffold. Ultimately, tATAC-see provides a robust, accessible platform for mechanistic and population-level studies to uncover spatial epigenome dynamics.

Spatial chromatin organization dictates cellular function and resilience, yet scalable imaging methods to quantify chromatin states in situ across aging and interventions are lacking. While ATAC-see can visualize accessible chromatin, its broader application is hindered by protocol variability, low throughput, and incompatibility with complex tissues. Here, we systematically optimize the ATAC-see workflow for robust, high-throughput quantitative imaging in fixed, adherent mammalian cells and fresh frozen tissues. We validate the platform’s sensitivity to pharmacologic remodeling and apply it to replicative, chronological, and pathological aging in primary human fibroblasts, revealing progressive age-associated chromatin opening and heterochromatin remodeling. Furthermore, we demonstrate that our optimized ATAC-see captures rapid, reversible chromatin reorganization during OSK(M)-driven partial reprogramming of aged fibroblasts. Finally, we extend a cost-effective and accessible protocol to murine tissue sections, quantifying in situ age-dependent remodeling. This standardized framework establishes chromatin accessibility as a highly scalable, sequencing-compatible imaging biomarker for evaluating aging and rejuvenation. Summary Statement: ATAC-see was optimized for scalable, quantitative imaging of chromatin remodeling during aging and cellular reprogramming, and extended to characterize age-associated epigenetic changes across organs.

Background: Duchenne muscular dystrophy (DMD) is a severe X-linked disorder marked by progressive muscle degeneration and regeneration, inflammation and fibrosis. Cellular senescence has emerged as a potential driver of chronic muscle damage, yet its temporal dynamics and therapeutic relevance remain unclear. Methods: We analyzed senescent cell burden in skeletal and cardiac muscles of the DBA/2-mdx mouse model, which closely mimics features of human DMD. The senolytic combination of dasatinib and quercetin (D + Q) was administered during early or late disease phases to evaluate the impact of senescent cell clearance. Skeletal muscle strength was measured by grip strength and ex vivo force assays, while cardiac function was assessed by echocardiography. Fibrosis and senescence markers were quantified histologically, and transcriptional changes associated with senolysis were identified using bulk RNA sequencing (RNA-seq). Results: In skeletal muscle, senescent cells appear and peak during early stages of disease progression (3–5 months), coinciding with high degeneration and regeneration activity, and then decline with age as fibrosis increases. In contrast, in the heart, senescent cells emerge at late stages of disease progression (around 12 months), correlating with heart fibrogenesis. Notably, senolytic intervention in the DBA/2-mdx mice promotes a regenerative and antifibrotic gene signature in both tissues. However, the timing of senolytic therapy determines its efficacy: early treatment with D + Q reduces senescent cell burden, decreases fibrosis, and improves fiber size and contractile performance in skeletal muscle, while later treatment reduces cardiac senescence and fibrosis but does not improve skeletal muscle pathology. Conclusions: Cellular senescence is a dynamic and targetable feature in DMD, with tissue- and age-specific patterns. It represents a potential modifiable therapeutic target, and temporally optimized senolytic strategies could serve as effective adjuncts to current and emerging DMD treatments.