Cellular and Molecular Mechanisms of Heterotopic Ossification in Fibrodysplasia Ossificans Progressiva.

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 ACVR1 gene (activin A receptor type I), an important signaling receptor involved in endochondral ossification. The ACVR1R206H 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 ACVR1R206H 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 ACVR1R206H 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 ACVR1R206H FAPs in muscle regeneration and therapeutic targeting of these cells to reduce HO formation in FOP.

Motor neurons and endothelial cells additively promote development and fusion of human iPSC-derived skeletal myocytes.

BACKGROUND: Neurovascular cells have wide-ranging implications on skeletal muscle biology regulating myogenesis, maturation, and regeneration. Although several in vitro studies have investigated how motor neurons and endothelial cells interact with skeletal myocytes independently, there is limited knowledge about the combined effect of neural and vascular cells on muscle maturation and development. METHODS: Here, we report a triculture system comprising human-induced pluripotent stem cell (iPSC)-derived skeletal myocytes, human iPSC-derived motor neurons, and primary human endothelial cells maintained under controlled media conditions. Briefly, iPSCs were differentiated to generate skeletal muscle progenitor cells (SMPCs). These SMPCs were seeded at a density of 5 × 104 cells/well in 12-well plates and allowed to differentiate for 7 days before adding iPSC-derived motor neurons at a concentration of 0.5 × 104 cells/well. The neuromuscular coculture was maintained for another 7 days in coculture media before addition of primary human umbilical vein endothelial cells (HUVEC) also at 0.5 × 104 cells/well. The triculture was maintained for another 7 days in triculture media comprising equal portions of muscle differentiation media, coculture media, and vascular media. Extensive morphological, genetic, and molecular characterization was performed to understand the combined and individual effects of neural and vascular cells on skeletal muscle maturation. RESULTS: We observed that motor neurons independently promoted myofiber fusion, upregulated neuromuscular junction genes, and maintained a molecular niche supportive of muscle maturation. Endothelial cells independently did not support myofiber fusion and downregulated expression of LRP4 but did promote expression of type II specific myosin isoforms. However, neurovascular cells in combination exhibited additive increases in myofiber fusion and length, enhanced production of Agrin, along with upregulation of several key genes like MUSK, RAPSYN, DOK-7, and SLC2A4. Interestingly, more divergent effects were observed in expression of genes like MYH8, MYH1, MYH2, MYH4, and LRP4 and secretion of key molecular factors like amphiregulin and IGFBP-4. CONCLUSIONS: Neurovascular cells when cultured in combination with skeletal myocytes promoted myocyte fusion with concomitant increase in expression of various neuromuscular genes. This triculture system may be used to gain a deeper understanding of the effects of the neurovascular niche on skeletal muscle biology and pathophysiology.

Ex vivo two-photon imaging of whole-mount skeletal muscles to visualize stem cell behavior.

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.

Impacts of radiation exposure, hindlimb unloading, and recovery on murine skeletal muscle cell telomere length.

Astronauts are exposed to harsh conditions, including cosmic radiation and microgravity. Spaceflight elongates human telomeres in peripheral blood, which shorten upon return to Earth and approach baseline levels during postflight recovery. Astronauts also encounter muscle atrophy, losing up to 20% loss of muscle mass on spaceflights. Telomere length changes in muscle cells of astronauts remain unexplored. This study investigates telomere alterations in grounded mice experiencing radiation exposure and muscle atrophy, via a hindlimb unloading spaceflight mimicking model. We find telomere lengthening is present in muscle stem cells and in myofiber nuclei, but not in muscle-resident endothelial cells. We further assessed telomere length in the model following hindlimb unloading recovery. We find that telomere length failed to return to baseline values. Our results suggest a role for telomeres in muscle acclimatization, which is relevant for the well-being of astronauts in space, and upon their return to Earth.

CD206+ tendon resident macrophages and their potential crosstalk with fibroblasts and the ECM during tendon growth and maturation.

Resident macrophages exist in a variety of tissues, including tendon, and play context-specific roles in their tissue of residence. In this study, we define the spatiotemporal distribution and phenotypic profile of tendon resident macrophages and their crosstalk with neighboring tendon fibroblasts and the extracellular matrix (ECM) during murine tendon development, growth, and homeostasis. Fluorescent imaging of cryosections revealed that F4/80+ tendon resident macrophages reside adjacent to Col1a1-CFP+ Scx-GFP+ fibroblasts within the tendon fascicle from embryonic development (E15.5) into adulthood (P56). Through flow cytometry and qPCR, we found that these tendon resident macrophages express several well-known macrophage markers, including Adgre1 (F4/80), Mrc1 (CD206), Lyve1, and Folr2, but not Ly-6C, and express the Csf1r-EGFP ("MacGreen") reporter. The proportion of Csf1r-EGFP+ resident macrophages in relation to the total cell number increases markedly during early postnatal growth, while the density of macrophages per mm2 remains constant during this same time frame. Interestingly, proliferation of resident macrophages is higher than adjacent fibroblasts, which likely contributes to this increase in macrophage proportion. The expression profile of tendon resident macrophages also changes with age, with increased pro-inflammatory and anti-inflammatory cytokine expression in P56 compared to P14 macrophages. In addition, the expression profile of limb tendon resident macrophages diverges from that of tail tendon resident macrophages, suggesting differential phenotypes across anatomically and functionally different tendons. As macrophages are known to communicate with adjacent fibroblasts in other tissues, we conducted ligand-receptor analysis and found potential two-way signaling between tendon fibroblasts and resident macrophages. Tendon fibroblasts express high levels of Csf1, which encodes macrophage colony stimulating factor (M-CSF) that acts on the CSF1 receptor (CSF1R) on macrophages. Importantly, Csf1r-expressing resident macrophages preferentially localize to Csf1-expressing fibroblasts, supporting the "nurturing scaffold" model for tendon macrophage patterning. Lastly, we found that tendon resident macrophages express high levels of ECM-related genes, including Mrc1 (mannose receptor), Lyve1 (hyaluronan receptor), Lair1 (type I collagen receptor), Ctss (elastase), and Mmp13 (collagenase), and internalize DQ Collagen in explant cultures. Overall, our study provides insights into the potential roles of tendon resident macrophages in regulating fibroblast phenotype and the ECM during tendon growth.

Fission impossible: Mitochondrial dynamics direct muscle stem cell fates.

Muscle stem cells (MuSCs) exhibit different metabolic profiles depending on their activity, however the mechanisms by which mitochondria affect MuSC fate has been understudied. In this issue of Cell Stem Cell, Hong et al. (2022) and Baker et al. (2022) demonstrate that defects in mitochondrial dynamics hinder proper MuSC activation and impair muscle regeneration.

Piezo1 regulates the regenerative capacity of skeletal muscles via orchestration of stem cell morphological states.

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.

Dynamics of skeletal muscle-resident stem cells during myogenesis in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease in which extraskeletal (heterotopic) bone forms within tissues such as skeletal muscles, often in response to injury. Mutations in the BMP type I receptor ACVR1/ALK2 cause FOP by increasing BMP pathway signaling. In contrast to the growing understanding of the inappropriate formation of bone tissue within the muscle in FOP, much is still unknown about the regenerative capacity of adult diseased muscles. Utilizing an inducible ACVR1R206H knock-in mouse, we found that injured Acvr1R206H/+ skeletal muscle tissue regenerates poorly. We demonstrated that while two resident stem cell populations, muscle stem cells (MuSCs) and fibro/adipogenic progenitors (FAPs), have similar proliferation rates after injury, the differentiation potential of mutant MuSCs is compromised. Although MuSC-specific deletion of the ACVR1R206H mutation does not alter the regenerative potential of skeletal muscles in vivo, Acvr1R206H/+ MuSCs form underdeveloped fibers that fail to fuse in vitro. We further determined that FAPs from Acvr1R206H/+ mice repress the MuSC-mediated formation of Acvr1R206H/+ myotubes in vitro. These results identify a previously unrecognized role for ACVR1R206H in myogenesis in FOP, via improper interaction of tissue-resident stem cells during skeletal muscle regeneration.

Telomere length assessments of muscle stem cells in rodent and human skeletal muscle sections.

Measurements of telomere length in skeletal muscle stem cells (MuSCs), a rare cell population within muscles, provide insights into cellular dysfunction in diseased conditions. Here, we describe a protocol (cryosection muscle quantitative fluorescent in situhybridization) using skeletal muscle cryosections for assessments of telomere length in MuSCs, in their native environment. Using a free software, telomere length measurements are assessed on a single-cell level. We also provide methodology to perform data analyses in several ways. For complete details on the use and execution of this protocol, please refer to Tichy et al. (2021).

Persistent NF-κB activation in muscle stem cells induces proliferation-independent telomere shortening.

During the repeated cycles of damage and repair in many muscle disorders, including Duchenne muscular dystrophy (DMD), the muscle stem cell (MuSC) pool becomes less efficient at responding to and repairing damage. The underlying mechanism of such stem cell dysfunction is not fully known. Here, we demonstrate that the distinct early telomere shortening of diseased MuSCs in both mice and young DMD patients is associated with aberrant NF-κB activation. We find that prolonged NF-κB activation in MuSCs in chronic injuries leads to shortened telomeres and Ku80 dysregulation and results in severe skeletal muscle defects. Our studies provide evidence of a role for NF-κB in regulating stem-cell-specific telomere length, independently of cell replication, and could be a congruent mechanism that is applicable to additional tissues and/or diseases characterized by systemic chronic inflammation.

Gli1 Defines a Subset of Fibro-adipogenic Progenitors that Promote Skeletal Muscle Regeneration With Less Fat Accumulation.

Skeletal muscle has remarkable regenerative ability after injury. Mesenchymal fibro-adipogenic progenitors (FAPs) are necessary, active participants during this repair process, but the molecular signatures of these cells and their functional relevance remain largely unexplored. Here, using a lineage tracing mouse model (Gli1-CreER Tomato), we demonstrate that Gli1 marks a small subset of muscle-resident FAPs with elevated Hedgehog (Hh) signaling. Upon notexin muscle injury, these cells preferentially and rapidly expanded within FAPs. Ablation of Gli1+ cells using a DTA mouse model drastically reduced fibroblastic colony-forming unit (CFU-F) colonies generated by muscle cells and impaired muscle repair at 28 days. Pharmacologic manipulation revealed that Gli1+ FAPs rely on Hh signaling to increase the size of regenerating myofiber. Sorted Gli1+ FAPs displayed superior clonogenicity and reduced adipogenic differentiation ability in culture compared to sorted Gli1- FAPs. In a glycerol injury model, Gli1+ FAPs were less likely to give rise to muscle adipocytes compared to other FAPs. Further cell ablation and Hh activator/inhibitor treatments demonstrated their dual actions in enhancing myogenesis and reducing adipogenesis after injury. Examining single-cell RNA-sequencing dataset of FAPs from normal mice indicated that Gli1+ FAPs with increased Hh signaling provide trophic signals to myogenic cells while restrict their own adipogenic differentiation. Collectively, our findings identified a subpopulation of FAPs that play an essential role in skeletal muscle repair. © 2021 American Society for Bone and Mineral Research (ASBMR).

Innervation: the missing link for biofabricated tissues and organs.

Innervation plays a pivotal role as a driver of tissue and organ development as well as a means for their functional control and modulation. Therefore, innervation should be carefully considered throughout the process of biofabrication of engineered tissues and organs. Unfortunately, innervation has generally been overlooked in most non-neural tissue engineering applications, in part due to the intrinsic complexity of building organs containing heterogeneous native cell types and structures. To achieve proper innervation of engineered tissues and organs, specific host axon populations typically need to be precisely driven to appropriate location(s) within the construct, often over long distances. As such, neural tissue engineering and/or axon guidance strategies should be a necessary adjunct to most organogenesis endeavors across multiple tissue and organ systems. To address this challenge, our team is actively building axon-based "living scaffolds" that may physically wire in during organ development in bioreactors and/or serve as a substrate to effectively drive targeted long-distance growth and integration of host axons after implantation. This article reviews the neuroanatomy and the role of innervation in the functional regulation of cardiac, skeletal, and smooth muscle tissue and highlights potential strategies to promote innervation of biofabricated engineered muscles, as well as the use of "living scaffolds" in this endeavor for both in vitro and in vivo applications. We assert that innervation should be included as a necessary component for tissue and organ biofabrication, and that strategies to orchestrate host axonal integration are advantageous to ensure proper function, tolerance, assimilation, and bio-regulation with the recipient post-implant.

Pre-innervated tissue-engineered muscle promotes a pro-regenerative microenvironment following volumetric muscle loss.

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.

Elevated BMP and Mechanical Signaling Through YAP1/RhoA Poises FOP Mesenchymal Progenitors for Osteogenesis.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by the formation of extraskeletal bone, or heterotopic ossification (HO), in soft connective tissues such as skeletal muscle. All familial and sporadic cases with a classic clinical presentation of FOP carry a gain-of-function mutation (R206H; c.617 G > A) in ACVR1, a cell surface receptor that mediates bone morphogenetic protein (BMP) signaling. The BMP signaling pathway is recognized for its chondro/osteogenic-induction potential, and HO in FOP patients forms ectopic but qualitatively normal endochondral bone tissue through misdirected cell fate decisions by tissue-resident mesenchymal stem cells. In addition to biochemical ligand-receptor signaling, mechanical cues from the physical environment are transduced to activate intracellular signaling, a process known as mechanotransduction, and can influence cell fates. Utilizing an established mesenchymal stem cell model of mouse embryonic fibroblasts (MEFs) from the Acvr1R206H/+ mouse model that mimics the human disease, we demonstrated that activation of the mechanotransductive effectors Rho/ROCK and YAP1 are increased in Acvr1R206H/+ cells. We show that on softer substrates, a condition associated with low mechanical signaling, the morphology of Acvr1R206H/+ cells is similar to the morphology of control Acvr1+/+ cells on stiffer substrates, a condition that activates mechanotransduction. We further determined that Acvr1R206H/+ cells are poised for osteogenic differentiation, expressing increased levels of chondro/osteogenic markers compared with Acvr1+/+ cells. We also identified increased YAP1 nuclear localization in Acvr1R206H/+ cells, which can be rescued by either BMP inhibition or Rho antagonism. Our results establish RhoA and YAP1 signaling as modulators of mechanotransduction in FOP and suggest that aberrant mechanical signals, combined with and as a result of the increased BMP pathway signaling through mutant ACVR1, lead to misinterpretation of the cellular microenvironment and a heightened sensitivity to mechanical stimuli that promotes commitment of Acvr1R206H/+ progenitor cells to chondro/osteogenic lineages.

Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest.

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.

ACVR1R206H FOP mutation alters mechanosensing and tissue stiffness during heterotopic ossification.

An activating bone morphogenetic proteins (BMP) type I receptor ACVR1 (ACVR1R206H) mutation enhances BMP pathway signaling and causes the rare genetic disorder of heterotopic (extraskeletal) bone formation fibrodysplasia ossificans progressiva. Heterotopic ossification frequently occurs following injury as cells aberrantly differentiate during tissue repair. Biomechanical signals from the tissue microenvironment and cellular responses to these physical cues, such as stiffness and rigidity, are important determinants of cell differentiation and are modulated by BMP signaling. We used an Acvr1R206H/+ mouse model of injury-induced heterotopic ossification to examine the fibroproliferative tissue preceding heterotopic bone and identified pathologic stiffening at this stage of repair. In response to microenvironment stiffness, in vitro assays showed that Acvr1R206H/+ cells inappropriately sense their environment, responding to soft substrates with a spread morphology similar to wild-type cells on stiff substrates and to cells undergoing osteoblastogenesis. Increased activation of RhoA and its downstream effectors demonstrated increased mechanosignaling. Nuclear localization of the pro-osteoblastic factor RUNX2 on soft and stiff substrates suggests a predisposition to this cell fate. Our data support that increased BMP signaling in Acvr1R206H/+ cells alters the tissue microenvironment and results in misinterpretation of the tissue microenvironment through altered sensitivity to mechanical stimuli that lowers the threshold for commitment to chondro/osteogenic lineages.

Human Skeletal Stem Cells: The Markers Provide Some Clues in the Hunt for Hidden Treasure.

Little is known about skeletal stem cell populations in vivo. Recently in Cell, Chan et al. (2018) identified a human skeletal stem cell population that can be isolated from multiple human bone locations and is capable of self-renewal and differentiation into bone, cartilage, and stroma, but not fat.

Engineered DNA plasmid reduces immunity to dystrophin while improving muscle force in a model of gene therapy of Duchenne dystrophy.

In gene therapy for Duchenne muscular dystrophy there are two potential immunological obstacles. An individual with Duchenne muscular dystrophy has a genetic mutation in dystrophin, and therefore the wild-type protein is "foreign," and thus potentially immunogenic. The adeno-associated virus serotype-6 (AAV6) vector for delivery of dystrophin is a viral-derived vector with its own inherent immunogenicity. We have developed a technology where an engineered plasmid DNA is delivered to reduce autoimmunity. We have taken this approach into humans, tolerizing to myelin proteins in multiple sclerosis and to proinsulin in type 1 diabetes. Here, we extend this technology to a model of gene therapy to reduce the immunogenicity of the AAV vector and of the wild-type protein product that is missing in the genetic disease. Following gene therapy with systemic administration of recombinant AAV6-microdystrophin to mdx/mTRG2 mice, we demonstrated the development of antibodies targeting dystrophin and AAV6 capsid in control mice. Treatment with the engineered DNA construct encoding microdystrophin markedly reduced antibody responses to dystrophin and to AAV6. Muscle force in the treated mice was also improved compared with control mice. These data highlight the potential benefits of administration of an engineered DNA plasmid encoding the delivered protein to overcome critical barriers in gene therapy to achieve optimal functional gene expression.

A robust Pax7EGFP mouse that enables the visualization of dynamic behaviors of muscle stem cells.

BACKGROUND: Pax7 is a transcription factor involved in the specification and maintenance of muscle stem cells (MuSCs). Upon injury, MuSCs leave their quiescent state, downregulate Pax7 and differentiate, contributing to skeletal muscle regeneration. In the majority of regeneration studies, MuSCs are isolated by fluorescence-activated sorting (FACS), based on cell surface markers. It is known that MuSCs are a heterogeneous population and only a small percentage of isolated cells are true stem cells that are able to self-renew. A strong Pax7 reporter line would be valuable to study the in vivo behavior of Pax7-expressing stem cells. METHODS: We generated and characterized the muscle properties of a new transgenic Pax7EGFP mouse. Utilizing traditional immunofluorescence assays, we analyzed whole embryos and muscle sections by fluorescence microscopy, in addition to whole skeletal muscles by 2-photon microscopy, to detect the specificity of EGFP expression. Skeletal muscles from Pax7EGFP mice were also evaluated in steady state and under injury conditions. Finally, MuSCs-derived from Pax7EGFP and control mice were sorted and analyzed by FACS and their myogenic activity was comparatively examined. RESULTS: Our studies provide a new Pax7 reporter line with robust EGFP expression, detectable by both flow cytometry and fluorescence microscopy. Pax7EGFP-derived MuSCs have identical properties to that of wild-type MuSCs, both in vitro and in vivo, excluding any positional effect due to the transgene insertion. Furthermore, we demonstrated high specificity of EGFP to label MuSCs in a temporal manner that recapitulates the reported Pax7 expression pattern. Interestingly, immunofluorescence analysis showed that the robust expression of EGFP marks cells in the satellite cell position of adult muscles in fixed and live tissues. CONCLUSIONS: This mouse could be an invaluable tool for the study of a variety of questions related to MuSC biology, including but not limited to population heterogeneity, polarity, aging, regeneration, and motility, either by itself or in combination with mice harboring additional genetic alterations.

Cardiomyocyte-Specific Telomere Shortening is a Distinct Signature of Heart Failure in Humans.

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.