Whether cell forces or extracellular matrix (ECM) can impact genome integrity is largely unclear. Here, acute perturbations (∼1 h) to actomyosin stress or ECM elasticity cause rapid and reversible ...changes in lamin-A, DNA damage, and cell cycle. The findings are especially relevant to organs such as the heart because DNA damage permanently arrests cardiomyocyte proliferation shortly after birth and thereby eliminates regeneration after injury including heart attack. Embryonic hearts, cardiac-differentiated iPS cells (induced pluripotent stem cells), and various nonmuscle cell types all show that actomyosin-driven nuclear rupture causes cytoplasmic mis-localization of DNA repair factors and excess DNA damage. Binucleation and micronuclei increase as telomeres shorten, which all favor cell-cycle arrest. Deficiencies in lamin-A and repair factors exacerbate these effects, but lamin-A-associated defects are rescued by repair factor overexpression and also by contractility modulators in clinical trials. Contractile cells on stiff ECM normally exhibit low phosphorylation and slow degradation of lamin-A by matrix-metalloprotease-2 (MMP2), and inhibition of this lamin-A turnover and also actomyosin contractility are seen to minimize DNA damage. Lamin-A is thus stress stabilized to mechano-protect the genome.
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•Stiff ECM, high actomyosin contractility, and low lamin-A favor nuclear rupture•Nuclear rupture causes cytoplasmic mis-localization of DNA repair factors•Repair factor depletion increases DNA damage, telomere loss, and cell-cycle arrest•Lamin-A increases during development to mechano-protect the genome
Progeroid syndromes make affected individuals appear older, and the only known causes are mutations in DNA repair factors and lamin-A. Cho et al. show that as embryonic hearts stiffen and strengthen in development, lamin-A accumulates to oppose stress-driven nuclear rupture, repair factor loss, increased DNA damage, and cell-cycle arrest.
Macrophages play an essential role in the resolution of tissue damage through removal of necrotic cells, thus paving the way for tissue regeneration. Macrophages also directly support the formation ...of new tissue to replace the injury, through their acquisition of an anti-inflammatory, or M2, phenotype, characterized by a gene expression program that includes IL-10, the IL-13 receptor, and arginase 1. We report that deletion of two CREB-binding sites from the Cebpb promoter abrogates Cebpb induction upon macrophage activation. This blocks the downstream induction of M2-specific Msr1, Il10, II13ra, and Arg-1 genes, whereas the inflammatory (M1) genes Il1, Il6, Tnfa, and Il12 are not affected. Mice carrying the mutated Cebpb promoter (βΔCre) remove necrotic tissue from injured muscle, but exhibit severe defects in muscle fiber regeneration. Conditional deletion of the Cebpb gene in muscle cells does not affect regeneration, showing that the C/EBPβ cascade leading to muscle repair is muscle-extrinsic. While βΔCre macrophages efficiently infiltrate injured muscle they fail to upregulate Cebpb, leading to decreased Arg-1 expression. CREB-mediated induction of Cebpb expression is therefore required in infiltrating macrophages for upregulation of M2-specific genes and muscle regeneration, providing a direct genetic link between these two processes.
Duchenne muscular dystrophy (DMD), the most common inherited muscular dystrophy of childhood, leads to death due to cardiorespiratory failure. Paradoxically, mdx mice with the same genetic deficiency ...of dystrophin exhibit minimal cardiac dysfunction, impeding the development of therapies. We postulated that the difference between mdx and DMD might result from differences in telomere lengths in mice and humans. We show here that, like DMD patients, mice that lack dystrophin and have shortened telomeres (mdx/mTR(KO)) develop severe functional cardiac deficits including ventricular dilation, contractile and conductance dysfunction, and accelerated mortality. These cardiac defects are accompanied by telomere erosion, mitochondrial fragmentation and increased oxidative stress. Treatment with antioxidants significantly retards the onset of cardiac dysfunction and death of mdx/mTR(KO) mice. In corroboration, all four of the DMD patients analysed had 45% shorter telomeres in their cardiomyocytes relative to age- and sex-matched controls. We propose that the demands of contraction in the absence of dystrophin coupled with increased oxidative stress conspire to accelerate telomere erosion culminating in cardiac failure and death. These findings provide strong support for a link between telomere length and dystrophin deficiency in the etiology of dilated cardiomyopathy in DMD and suggest preventive interventions.
Quiescent skeletal muscle stem cells (MuSCs) are morphologically and functionally heterogeneous and exhibit different lengths of cellular extensions, which we call protrusions. Here, we present a ...protocol for ex vivo two-photon imaging of MuSCs in their native environment. We describe steps for muscle dissection, fixation, embedding, imaging, and analysis of datasets. This protocol allows the examination of MuSC morphology and protrusions at the single-cell level as well as stem cell numbers.
For complete details on the use and execution of this protocol, please refer to Ma et al. (2022).1
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•Whole-mount skeletal muscle tissue for two-photon imaging•Workflow to evaluate muscle stem cell morphology and analysis of cellular protrusions•This protocol can be adapted to other muscle-resident cells
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Quiescent skeletal muscle stem cells (MuSCs) are morphologically and functionally heterogeneous and exhibit different lengths of cellular extensions, which we call protrusions. Here, we present a protocol for ex vivo two-photon imaging of MuSCs in their native environment. We describe steps for muscle dissection, fixation, embedding, imaging, and analysis of datasets. This protocol allows the examination of MuSC morphology and protrusions at the single-cell level as well as stem cell numbers.
NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of ...natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function.
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•Mice with ∼85% NAD depletion in skeletal muscle are grossly normal as young adults•Reduced NAD content impairs mitochondrial function and fiber integrity over time•Progressive muscle dysfunction can be reversed by the NAD precursor NR•Preventing muscle NAD loss during aging partially preserves exercise performance
NAD levels decline in multiple tissues with age or in disease. Frederick et al. show that impaired intramuscular NAD synthesis compromises skeletal muscle mass and strength over time but can be quickly restored with an oral NAD precursor. Upregulation of the NAD salvage pathway preserves exercise performance in aged mice.
Telomere length has been correlated with various diseases, including cardiovascular disease and cancer. The use of currently available telomere-length measurement techniques is often restricted by ...the requirement of a large amount of cells (Southern-based techniques) or the lack of information on individual cells or telomeres (PCR-based methods). Although several methods have been used to measure telomere length in tissues as a whole, the assessment of cell-type-specific telomere length provides valuable information on individual cell types. The development of fluorescence in situ hybridization (FISH) technologies enables the quantification of telomeres in individual chromosomes, but the use of these methods is dependent on the availability of isolated cells, which prevents their use with fixed archival samples. Here we describe an optimized quantitative FISH (Q-FISH) protocol for measuring telomere length that bypasses the previous limitations by avoiding contributions from undesired cell types. We have used this protocol on small paraffin-embedded cardiac-tissue samples. This protocol describes step-by-step procedures for tissue preparation, permeabilization, cardiac-tissue pretreatment and hybridization with a Cy3-labeled telomeric repeat complementing (CCCTAA)
peptide nucleic acid (PNA) probe coupled with cardiac-specific antibody staining. We also describe how to quantify telomere length by means of the fluorescence intensity and area of each telomere within individual nuclei. This protocol provides comparative cell-type-specific telomere-length measurements in relatively small human cardiac samples and offers an attractive technique to test hypotheses implicating telomere length in various cardiac pathologies. The current protocol (from tissue collection to image procurement) takes ∼28 h along with three overnight incubations. We anticipate that the protocol could be easily adapted for use on different tissue types.
Fibrodysplasia ossificans progressiva (FOP) is a debilitating genetic disorder characterized by recurrent episodes of heterotopic ossification (HO) formation in muscles, tendons, and ligaments. FOP ...is caused by a missense mutation in the
gene (activin A receptor type I), an important signaling receptor involved in endochondral ossification. The
mutation induces increased downstream canonical SMAD-signaling and drives tissue-resident progenitor cells with osteogenic potential to participate in endochondral HO formation. In this article, we review aberrant ACVR1
signaling and the cells that give rise to HO in FOP. FOP mouse models and lineage tracing analyses have been used to provide strong evidence for tissue-resident mesenchymal cells as cellular contributors to HO. We assess how the underlying mutation in FOP disrupts muscle-specific dynamics during homeostasis and repair, with a focus on muscle-resident mesenchymal cells known as fibro-adipogenic progenitors (FAPs). Accumulating research points to FAPs as a prominent HO progenitor population, with
FAPs not only aberrantly differentiating into chondro-osteogenic lineages but creating a permissive environment for bone formation at the expense of muscle regeneration. We will further discuss the emerging role of
FAPs in muscle regeneration and therapeutic targeting of these cells to reduce HO formation in FOP.
Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle beyond the inherent regenerative capacity of the body, generally leading to severe functional deficit. Formation of ...appropriate somato-motor innervations remains one of the biggest challenges for both autologous grafts as well as tissue-engineered muscle constructs. We aim to address this challenge by developing pre-innervated tissue-engineered muscle comprised of long aligned networks of spinal motor neurons and skeletal myocytes on aligned nanofibrous scaffolds. Motor neurons led to enhanced differentiation and maturation of skeletal myocytes in vitro. These pre-innervated tissue-engineered muscle constructs when implanted in a rat VML model significantly increased satellite cell density, neuromuscular junction maintenance, graft revascularization, and muscle volume over three weeks as compared to myocyte-only constructs and nanofiber scaffolds alone. These pro-regenerative effects may enhance functional neuromuscular regeneration following VML, thereby improving the levels of functional recovery following these devastating injuries.
Background
Telomere defects are thought to play a role in cardiomyopathies, but the specific cell type affected by the disease in human hearts is not yet identified. The aim of this study was to ...systematically evaluate the cell type specificity of telomere shortening in patients with heart failure in relation to their cardiac disease, age, and sex.
Methods and Results
We studied cardiac tissues from patients with heart failure by utilizing telomere quantitative fluorescence in situ hybridization, a highly sensitive method with single‐cell resolution. In this study, total of 63 human left ventricular samples, including 37 diseased and 26 nonfailing donor hearts, were stained for telomeres in combination with cardiomyocyte‐ or α‐smooth muscle cell‐specific markers, cardiac troponin T, and smooth muscle actin, respectively, and assessed for telomere length. Patients with heart failure demonstrate shorter cardiomyocyte telomeres compared with nonfailing donors, which is specific only to cardiomyocytes within diseased human hearts and is associated with cardiomyocyte DNA damage. Our data further reveal that hypertrophic hearts with reduced ejection fraction exhibit the shortest telomeres. In contrast to other reported cell types, no difference in cardiomyocyte telomere length is evident with age. However, under the disease state, telomere attrition manifests in both young and older patients with cardiac hypertrophy. Finally, we demonstrate that cardiomyocyte‐telomere length is better sustained in women than men under diseased conditions.
Conclusions
This study provides the first evidence of cardiomyocyte‐specific telomere shortening in heart failure.
Muscle stem cells (MuSCs) are essential for tissue homeostasis and regeneration, but the potential contribution of MuSC morphology to in vivo function remains unknown. Here, we demonstrate that ...quiescent MuSCs are morphologically heterogeneous and exhibit different patterns of cellular protrusions. We classified quiescent MuSCs into three functionally distinct stem cell states: responsive, intermediate, and sensory. We demonstrate that the shift between different stem cell states promotes regeneration and is regulated by the sensing protein Piezo1. Pharmacological activation of Piezo1 is sufficient to prime MuSCs toward more responsive cells. Piezo1 deletion in MuSCs shifts the distribution toward less responsive cells, mimicking the disease phenotype we find in dystrophic muscles. We further demonstrate that Piezo1 reactivation ameliorates the MuSC morphological and regenerative defects of dystrophic muscles. These findings advance our fundamental understanding of how stem cells respond to injury and identify Piezo1 as a key regulator for adjusting stem cell states essential for regeneration.
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