Biology subjects

Kopinke, D.

Publications and source records attributed to Kopinke, D..

12 recordsLinked to original sources

bioRxiv2026

Semaglutide promotes intramuscular fat formation after injury

Glucagon-like peptide 1 receptor agonists (GLP-1RAs) have become defining therapies in the management of type 2 diabetes and obesity. Despite recent interest in the effects of GLP-1RA therapy on skeletal muscle, their influence on muscle repair after injury remains largely untested. Because GLP-1RA use is common in populations at heightened risk for diminished regenerative capacity, a critical unanswered question is whether GLP-1R agonism supports muscle regeneration or alters the normal course of recovery after injury. Using intramuscular glycerol injection as an adipogenic injury model, we assessed whether semaglutide, a widely prescribed GLP-1RA, alters the balance between myogenesis and adipogenesis during regeneration. Surprisingly, semaglutide treatment markedly increased the formation of intramuscular adipose tissue (IMAT) and inhibited the growth of regenerated fibers. These effects were injury-dependent, as uninjured muscle showed no detectable differences in IMAT or myofiber size. Together, these findings identify a previously underappreciated context in which GLP-1RA therapy may adversely affect muscle quality.

developmental biology↗

bioRxiv2026

Primary cilia promote cardiac fibrosis and limit heart function after myocardial infarction

Cardiomyocytes die and do not regenerate after an injury such as a myocardial infarction (MI), a leading cause of mortality worldwide. Following MI, cardiac fibroblasts (CFs) proliferate and differentiate into myofibroblasts, which then produce increased collagen and extracellular matrix (ECM) leading to fibrosis. Fibrosis can weaken cardiac output via excessive stiffening and interference with electric signal transmission, but can also prevent wall rupture under load (reviewed in (1)). Thus, dampening fibrosis has been investigated as a potential therapeutic intervention. Most mammalian cells possess a single primary cilium involved in intercellular communication. We investigated the role of CF primary cilia in sensing injury signals and initiating fibrotic remodeling. We found that deleting CF cilia reduced fibrosis and improved cardiac output after MI, demonstrating that cilia act as a signaling hub that amplifies the fibrotic response in the injured heart.

physiology↗

bioRxiv2026

FAP-intrinsic Hedgehog signaling controls intramuscular adipogenesis, fibrosis, and myofiber regeneration

Fibro/adipogenic progenitors (FAPs) are multipotent stromal cells that support myofiber regeneration, but can also give rise to intramuscular adipose tissue (IMAT) and fibrotic scar tissue. While the Hedgehog pathway suppresses FAP adipogenesis and promotes myofiber repair through ligand Desert Hedgehog, the key cell type that senses this signal has remained unclear. Here, we demonstrate through FAP-specific deletion of the Hedgehog signal transducer Smoothened that FAPs are the primary Hedgehog-responding cells during muscle regeneration. Loss of Smoothened in FAPs increases IMAT, causes persistent fibrosis, reduces the Hedgehog-dependent effectors TIMP3 and GDF10, and impairs myofiber regeneration. FAPs lacking Smoothened also fail to support in vitro myoblast differentiation and fusion as efficiently as control FAPs, showing that Hedgehog signaling helps establish a pro-myogenic FAP state early after injury. Pharmacological Hedgehog activation via the Smoothened agonist SAG fails to rescue adipocyte accumulation or myofiber regeneration when FAPs lack Smoothened. Together, these findings provide direct genetic evidence that FAPs are the primary cellular mediators of Hedgehog signaling in muscle and establish FAP Hedgehog signaling competence as a key determinant of regenerative outcome and a target for restoring muscle repair in disease.

developmental biology↗

bioRxiv2026

Intramuscular Adipose Tissue Accumulation is a Key Determinant of Limb Function in Peripheral Artery Disease

BackgroundPeripheral artery disease (PAD) and its severe form, chronic limb-threatening ischemia (CLTI), significantly impair blood flow to the lower extremities, affecting millions of adults globally. Intramuscular adipose tissue (IMAT) and fibrosis accumulation distinguish patients with CLTI from those with mild PAD, suggesting a role in CLTI pathobiology. However, the functional consequences of IMAT in CLTI remain unclear. MethodsWe compared gastrocnemius muscle samples from patients with PAD/CLTI, intermittent claudication, and non-PAD individuals. We analyzed bulk RNA sequencing, proteomic, lipidomic, and single-cell/nucleus RNA sequencing datasets. Additionally, we used murine models of hindlimb ischemia (HLI) with genetic manipulation of Ppar{gamma}, a key adipogenic transcription factor, specifically in fibro-adipogenic progenitor cells (FAPs), the cellular source of IMAT, to modulate IMAT formation and assessed the impact on limb function and pathology. ResultsPatients with CLTI exhibited significantly elevated expression of adipogenic genes and proteins in muscle specimens when compared to non-PAD controls. Murine models showed that increasing IMAT formation significantly worsened ischemic limb muscle strength and work output. In contrast, preventing IMAT formation significantly improved ischemic limb muscle strength and work output. These findings were consistent across both male and female mice, although females had greater tendency to form IMAT compared with male mice. ConclusionsIMAT accumulation is a key determinant of limb function in PAD/CLTI. Our studies demonstrate that targeting IMAT formation could improve limb function in mice with experimental PAD. Together, these findings suggest that developing strategies to limit or reduce IMAT may improve limb function and walking performance in patients with PAD/CLTI, providing a novel therapeutic avenue to address a critical unmet need. CLINICAL PERSPECTIVEO_ST_ABSWhat is new?C_ST_ABSO_LIIntramuscular adipose tissue accumulation (IMAT) distinguishes patients with chronic limb-threatening ischemia from those with milder peripheral artery disease or those without PAD and directly impairs ischemic limb muscle function. C_LIO_LIGenetic gain- and loss-of-function mouse models demonstrate that increasing IMAT worsens, while preventing IMAT formation improves, ischemic limb strength and performance independent of perfusion. C_LIO_LIAdipogenic signatures in human calf muscle negatively correlates with muscle strength and disease severity, identifying IMAT as a functional biomarker and modifiable target in PAD/CLTI. C_LI What are the clinical implications?O_LIIMAT accumulation represents an underappreciated, non-vascular mechanism contributing to leg dysfunction in PAD/CLTI. C_LIO_LITherapies aimed at limiting or reversing IMAT formation may improve leg strength and walking performance in patients with PAD/CLTI, addressing a critical unmet clinical need. C_LIO_LIIdentifying and targeting cellular pathways regulating IMAT formation from fibro-adipogenic progenitors may complement vascular interventions to enhance functional recovery after revascularization. C_LI

physiology↗

bioRxiv2025

Fibro-Adipogenic Progenitors require autocrine IGF-I in homeostatic and regenerating skeletal muscle

Fibro-Adipogenic Progenitors (FAPs) are mesenchymal stem cells that are vital for muscle homeostasis and regeneration but produce fibrosis and intramuscular fat under pathological conditions. Insulin-like Growth Factor-I (IGF-I) is a key regulator of muscle repair, satellite cell activity, macrophage polarization, and extracellular matrix (ECM) remodeling. We generated inducible FAP-specific Igf1 deficient (FID) mice to determine the necessity of FAP IGF-I. After BaCl2 injury, FID mice exhibited impaired muscle regeneration, with fewer Pax7+ cells, increased macrophage accumulation, smaller fibers, reduced ECM, and depressed FAP proliferation. Following glycerol injury, FID muscles exhibited reduced adipocyte accumulation. Primary FAPs isolated from injured FID muscles had blunted growth, upregulation of immune-regulatory genes and downregulation of ECM and cell proliferation genes, with delayed responses to fibrogenic and to adipogenic media. FAP property alterations were already present in homeostatic muscle, indicated by scRNASeq, with decreased indices of protein translation and ECM production as well as increased markers of senescence, confirmed in vivo and in vitro. Overall, FAP IGF-I is a critical autocrine factor, with further paracrine consequences for muscle regenerative capacity.

molecular biology↗

bioRxiv2024

Intramuscular adipose tissue physically restricts functional muscle recovery

With age and disease, skeletal muscle is progressively lost and replaced by fibrotic scar and intramuscular adipose tissue (IMAT). While strongly correlated, it remains unclear whether IMAT has a functional impact on muscle. In the present study, we evaluated the effects of IMAT during muscle injury by creating a mouse model where the cellular origin of IMAT, fibro/adipogenic progenitors (FAPs), are prevented from differentiating into adipocytes (FATBLOCK model). We found that blocking IMAT after an adipogenic injury allowed muscle to regenerate more efficiently, resulting in enhanced function. Our data explain why acute muscle injuries featuring IMAT infiltration, such as rotator cuff tears and acute denervation injuries, exhibit poor regeneration and lead to a loss in muscle function. It also demonstrates the therapeutic importance of preventing IMAT formation in acute injuries in order to maximize regeneration and minimize loss in muscle mass and function.

developmental biology↗

bioRxiv2022

Hematopoietic-specific heterozygous loss of Dnmt3a exacerbates colitis-associated colon cancer

Clonal hematopoiesis (CH) is defined as clonal expansion of mutant hematopoietic stem cells absent diagnosis of a hematologic malignancy. Presence of CH in solid tumor patients, including colon cancer, correlates with shorter survival. We hypothesized that bone marrow-derived cells with heterozygous loss-of-function mutations of DNMT3A, the most common genetic alteration in CH, contribute to the pathogenesis of colon cancer. In a mouse model that combines colitis-associated colon cancer with experimental CH driven by Dnmt3a+/{Delta}, we found higher tumor penetrance and increased tumor burden compared to controls. Histopathological analysis revealed accentuated colonic epithelium injury, dysplasia and adenocarcinoma formation. Transcriptome profiling of colon tumors identified enrichment of gene signatures associated with carcinogenesis, including angiogenesis. Treatment with the angiogenesis inhibitor axitinib eliminated the colon tumor-promoting effect of experimental CH driven by Dnmt3a haploinsufficiency. This study provides conceptually novel insights into non-tumor-cell-autonomous effect of hematopoietic alterations on colon carcinogenesis and identifies potential therapeutic strategies. SUMMARYA pre-clinical mouse model demonstrates that genetic alterations in the blood system characteristic of clonal hematopoiesis (CH) contribute to an aggressive solid tumor phenotype. It further identifies cancer angiogenesis as a potential therapeutic target to mitigate adverse CH effects.

cancer biology↗

bioRxiv2022

An Optimized Approach to Study Sub-Sarcomere Structure Utilizing Super-Resolution Microscopy with Secondary VHH Nanobodies

The sarcomere is the fundamental contractile unit in skeletal muscle, and the maintenance of its structure is critical for its function. While alterations in sarcomere structure are implicated in many clinical conditions of muscle weakness this area has made limited progress due, in part, to limitations in the ability to robustly detect and measure at sub-sarcomere resolution. Classically the field has relied on approaches including confocal and electron microscopy, but there are technique-specific limitations with respect to resolution, tissue morphology, and protein specific labeling. In this study, our goal was to establish a robust and reproducible method to probe sub-sarcomere protein localization in longitudinal muscle sections. We optimized several steps from tissue preparation to antibody selection and imaging to provide the ability to quantitatively assess spatial distribution of proteins within a single sarcomere. This includes 1) in situ fixation for structural integrity, 2) use of multiple same host-species primary antibodies with Fab fragment antibody blocking to maintain specificity, and 3) the use of super-resolution structured illumination microscopy (SIM) to improve from confocal, along with use of emergent VHH secondary nanobodies to double the resolution. The combination of these methods provides a unique approach to improve visualization of sarcomere structure while simultaneously providing the ability to rigorously probe protein localization. While this study focused on assessment of skeletal muscle structure and provides an important set of tools for analysis of skeletal muscle health in disease and aging, we suggest the methods herein may prove advantageous for research outside of skeletal muscle.

physiology↗

bioRxiv2022

Hedgehog signaling acts as cell fate determinant during adult tissue repair

Successful muscle regeneration relies on the interplay of multiple cell populations. However, the signals required for this coordinated intercellular crosstalk remain largely unknown. Here, we describe how the Hedgehog (Hh) signaling pathway controls the fate of fibro/adipogenic progenitors (FAPs), the cellular origin of intramuscular fat (IMAT) and fibrotic scar tissue. Using conditional mutagenesis and pharmacological Hh modulators in vivo and in vitro, we identify DHH as the key ligand that acts as a potent adipogenic brake by preventing the adipogenic differentiation of FAPs. Hh signaling also impacts muscle regeneration, albeit indirectly through induction of myogenic factors in FAPs. Our results also indicate that ectopic and sustained Hh activation forces FAPs to adopt a fibrogenic fate resulting in widespread fibrosis. In this work, we reveal novel post-developmental functions of Hh signaling in balancing tissue regeneration and fatty fibrosis. Moreover, they provide the exciting possibility that mis-regulation of the Hh pathway with age and disease could be a major driver of pathological IMAT formation.

developmental biology↗

bioRxiv2022

A guide to examining intramuscular fat formation and its cellular origin in skeletal muscle

Fibro-adipogenic progenitors (FAPs) are mesenchymal stromal cells that play a crucial role during skeletal muscle homeostasis and regeneration. FAPs build and maintain the extracellular matrix that acts as a molecular myofiber scaffold. In addition, FAPs are indispensable for myofiber regeneration as they secrete a multitude of beneficial factors sensed by the muscle stem cells (MuSCs). In diseased states, however, FAPs are the cellular origin of intramuscular fat and fibrotic scar tissue. This fatty fibrosis is a hallmark of sarcopenia and neuromuscular diseases, such as Duchenne Muscular Dystrophy. One significant barrier in determining why and how FAPs differentiate into intramuscular fat is effective preservation of adipocytes, especially in frozen tissue sections. Conventional methods of skeletal muscle tissue processing, such as snap-freezing, do not properly preserve the morphology of individual adipocytes, thereby preventing accurate visualization and quantification. Here, we describe a protocol that provides robust preservation of adipocyte morphology in skeletal muscle sections allowing visualization, imaging, and quantification of intramuscular fat. We also outline how to process a portion of muscle tissue for RT-qPCR, enabling users to confirm observed changes in fat formation by viewing differences in expression of adipogenic genes. Additionally, we will describe how our protocol can be adopted to visualize adipocytes by whole mount immunofluorescence of muscle samples. Finally, we will outline how to combine this protocol with genetic lineage tracing of Pdgfr-expressing FAPs to study the adipogenic conversion of FAPs. Our protocol consistently yields high-resolution and morphologically accurate immunofluorescent images of adipocytes that, along with confirmation by RT-qPCR, allows for robust, rigorous, and reproducible visualization and quantification of intramuscular fat. Together, our analysis pipeline is the first step to improve our understanding of how FAPs differentiate into intramuscular fat and provides a framework to validate novel interventions to prevent fat formation.

developmental biology↗

bioRxiv2021

Automatic and unbiased segmentation and quantification of myofibers in skeletal muscle

Skeletal muscle has the remarkable ability to regenerate. However, with age and disease muscle strength and function decline. Myofiber size, which is affected by injury and disease, is a critical measurement to assess muscle health. Here, we test and apply Cellpose, a recently developed deep learning algorithm, to automatically segment myofibers within murine skeletal muscle. We first show that tissue fixation is necessary to preserve cellular structures such as primary cilia, small cellular antennae, and adipocyte lipid droplets. However, fixation generates heterogeneous myofiber labeling, which impedes intensity-based segmentation. We demonstrate that Cellpose efficiently delineates thousands of individual myofibers outlined by a variety of markers, even within fixed tissue with highly uneven myofiber staining. We created a novel ImageJ plugin (LabelsToRois) that allows processing multiple Cellpose segmentation images in batch. The plugin also contains a semi-automatic erosion function to correct for the area bias introduced by the different stainings, identifying myofibers as accurately as human experts. We successfully applied our segmentation pipeline to uncover myofiber size differences between two different muscle injury models, cardiotoxin and glycerol. Thus, Cellpose combined with LabelsToRois allows for fast, unbiased, and reproducible myofiber quantification for a variety of staining and fixation conditions.

bioinformatics↗

bioRxiv2019

Mechanistic Elucidation of the Antitumor Properties of a Novel Death Receptor 5 Activator

Disulfide bond Disrupting Agents (DDAs) are a new chemical class of agents recently shown to have activity against breast tumors in animal models. However, it is unknown how DDAs trigger cancer cell death without affecting nontransformed cells. As demonstrated here, DDAs are the first compounds identified that upregulate the TRAIL receptor DR5 through both transcriptional and posttranscriptional mechanisms. At the protein level, DDAs alter DR5 disulfide bonding to increase steady-state DR5 levels and oligomerization, leading to downstream Caspase 8 and 3 activation. DDAs and TRAIL synergize to kill cancer cells and are cytotoxic to HER2+ cancer cells with acquired resistance to the EGFR/HER2 tyrosine kinase inhibitor. Investigation of the mechanisms responsible for DDA selectivity for cancer cells reveals that DDA-induced upregulation of DR5 is enhanced in the context of EGFR overexpression, and DDA-induced cytotoxicity is strongly amplified by MYC overexpression. Together, the results demonstrate selective DDA lethality against oncogene-transformed cells, DDA-mediated DR5 upregulation and protein stabilization, and DDAs against drug-resistant and metastatic cancer cells. DDAs thus represent a new therapeutic approach to cancer therapy.

cancer biology↗