Residual Cystine Transport Activity for Specific Infantile and Juvenile CTNS Mutations in a PTEC-Based Addback Model.

Cystinosis is a rare, autosomal recessive, lysosomal storage disease caused by mutations in the gene CTNS, leading to cystine accumulation in the lysosomes. While cysteamine lowers the cystine levels, it does not cure the disease, suggesting that CTNS exerts additional functions besides cystine transport. This study investigated the impact of infantile and juvenile CTNS mutations with discrepant genotype/phenotype correlations on CTNS expression, and subcellular localisation and function in clinically relevant cystinosis cell models to better understand the link between genotype and CTNS function. Using CTNS-depleted proximal tubule epithelial cells and patient-derived fibroblasts, we expressed a selection of CTNSmutants under various promoters. EF1a-driven expression led to substantial overexpression, resulting in CTNS protein levels that localised to the lysosomal compartment. All CTNSmutants tested also reversed cystine accumulation, indicating that CTNSmutants still exert transport activity, possibly due to the overexpression conditions. Surprisingly, even CTNSmutants expression driven by the less potent CTNS and EFS promoters reversed the cystine accumulation, contrary to the CTNSG339R missense mutant. Taken together, our findings shed new light on CTNS mutations, highlighting the need for robust assessment methodologies in clinically relevant cellular models and thus paving the way for better stratification of cystinosis patients, and advocating for the development of more personalized therapy.

Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

Investigation on Electrospun and Solvent-Casted PCL-PLGA Blends Scaffolds Embedded with Induced Pluripotent Stem Cells for Tissue Engineering.

The design, production, and characterisation of tissue-engineered scaffolds made of polylactic-co-glycolic acid (PLGA), polycaprolactone (PCL) and their blends obtained through electrospinning (ES) or solvent casting/particulate leaching (SC) manufacturing techniques are presented here. The polymer blend composition was chosen to always obtain a prevalence of one of the two polymers, in order to investigate the contribution of the less concentrated polymer on the scaffolds' properties. Physical-chemical characterization of ES scaffolds demonstrated that tailoring of fibre diameter and Young modulus (YM) was possible by controlling PCL concentration in PLGA-based blends, increasing the fibre diameter from 0.6 to 1.0 µm and reducing the YM from about 22 to 9 MPa. SC scaffolds showed a "bubble-like" topography, caused by the porogen spherical particles, which is responsible for decreasing the contact angles from about 110° in ES scaffolds to about 74° in SC specimens. Nevertheless, due to phase separation within the blend, solvent-casted samples displayed less reproducible properties. Furthermore, ES samples were characterised by 10-fold higher water uptake than SC scaffolds. The scaffolds suitability as iPSCs culturing support was evaluated using XTT assay, and pluripotency and integrin gene expression were investigated using RT-PCR and RT-qPCR. Thanks to their higher wettability and appropriate YM, SC scaffolds seemed to be superior in ensuring high cell viability over 5 days, whereas the ability to maintain iPSCs pluripotency status was found to be similar for ES and SC scaffolds.

Botulinum Toxin Treatment of Adult Muscle Stem Cells from Children with Cerebral Palsy and hiPSC-Derived Neuromuscular Junctions.

Botulinum neurotoxin type-A (BoNT) injections are commonly used as spasticity treatment in cerebral palsy (CP). Despite improved clinical outcomes, concerns regarding harmful effects on muscle morphology have been raised, and the BoNT effect on muscle stem cells remains not well defined. This study aims at clarifying the impact of BoNT on growing muscles (1) by analyzing the in vitro effect of BoNT on satellite cell (SC)-derived myoblasts and fibroblasts obtained from medial gastrocnemius microbiopsies collected in young BoNT-naïve children (t0) compared to age ranged typically developing children; (2) by following the effect of in vivo BoNT administration on these cells obtained from the same children with CP at 3 (t1) and 6 (t2) months post BoNT; (3) by determining the direct effect of a single and repeated in vitro BoNT treatment on neuromuscular junctions (NMJs) differentiated from hiPSCs. In vitro BoNT did not affect myogenic differentiation or collagen production. The fusion index significantly decreased in CP at t2 compared to t0. In NMJ cocultures, BoNT treatment caused axonal swelling and fragmentation. Repeated treatments impaired the autophagic-lysosomal system. Further studies are warranted to understand the long-term and collateral effects of BoNT in the muscles of children with CP.

The ad hoc chemical design of random PBS-based copolymers influences the activation of cardiac differentiation while altering the HYPPO pathway target genes in hiPSCs.

Cardiac tissue engineering is a cutting-edge technology aiming to replace irreversibly damaged cardiac tissue and restore contractile functionality. However, cardiac tissue engineering porous and perfusable scaffolds to enable oxygen supply in vitro and eventually promote angiogenesis in vivo are still desirable. Two fully-aliphatic random copolymers of poly(butylene succinate) (PBS), poly(butylene succinate/Pripol), P(BSBPripol), and poly(butylene/neopentyl glycol succinate), P(BSNS), containing two different subunits, neopentyl glycol and Pripol 1009, were successfully synthesized and then electrospun in tridimentional fibrous mats. The copolymers show different thermal and mechanical behaviours as result of their chemical structure. In particular, copolymerization led to a reduction in crystallinity and consequently PBS stiffness, reaching values of elastic modulus very close to those of soft tissues. Then, to check the biological suitability, human induced Pluripotent Stem Cells (hiPSCs) were directly seeded on both PBS-based copolymeric scaffolds. The results confirmed the ability of both the scaffolds to sustain cell viability and to maintain their stemness during cell expansion. Furthermore, gene expression and immunofluorescence analysis showed that P(BSBPripol) scaffold promoted an upregulation of the early cardiac progenitor and later-stage markers with a simultaneously upregulation of HYPPO pathway gene expression, crucial for mechanosensing of cardiac progenitor cells. These results suggest that the correct ad-hoc chemical design and, in turn, the mechanical properties of the matrix, such as substrate stiffness, together with surface porosity, play a critical role in regulating the behaviour of cardiac progenitors, which ultimately offers valuable insights into the development of novel bio-inspired scaffolds for cardiac tissue regeneration.

Report and Abstracts of the 19th Meeting of the Interuniversity Institute of Myology: Assisi, October 20-23, 2022.

After two years of conferences on a virtual platform due to the COVID-19 pandemic, finally, the 19th annual meeting of the Interuniversity Institute of Myology (IIM) has returned to the heart of central Italy, in Assisi, an important cultural hub, which boasts a wide range of historic buildings and museums. This event brought together scientists from around the world providing a valuable opportunity to discuss scientific issues in the field of myology. Traditionally, the meeting particularly encourages the participation of young trainees, and the panel discussions were moderated by leading international scientists, making this a special event where young researchers had the opportunity to talk to prestigious scientists in a friendly and informal environment. Furthermore, the IIM young researchers' winners for the best oral and poster presentations, became part of the IIM Young Committee, involved in the scientific organization of sessions and roundtables and for the invitation of a main speaker for the IIM 2023 meeting. The four keynote speakers for the IIM Conference 2022 presented new insights into the role of multinucleation during muscle growth and disease, the long-range distribution of giant mRNAs in skeletal muscle, human skeletal muscle remodelling from type 2 diabetic patients and the genome integrity and cell identity in adult muscle stem cells. The congress hosted young PhD students and trainees and included 6 research sessions, two poster sessions, round tables and socio-cultural events, promoting science outreach and interdisciplinary works that are advancing new directions in the field of myology. All other attendees had the opportunity to showcase their work through poster presentations. The IIM meeting 2022 was also part of an advanced training event, which included dedicated round tables and a training session of Advanced Myology on the morning of 23 October, reserved for students under 35 enrolled in the training school, receiving a certificate of attendance. This course proposed lectures and roundtable discussions coordinated by internationally outstanding speakers on muscle metabolism, pathophysiological regeneration and emerging therapeutic approaches for muscle degenerations. As in past editions, all participants shared their results, opinions, and perspectives in understanding developmental and adult myogenesis with novel insights into muscle biology in pathophysiological conditions. We report here the abstracts of the meeting that describe the basic, translational, and clinical research and certainly contribute to the vast field of myology in an innovative and original way.

VEGF overexpressed mesoangioblasts enhance urethral and vaginal recovery following simulated vaginal birth in rats.

Vaginal birth causes pelvic floor injury which may lead to urinary incontinence. Cell therapy has been proposed to assist in functional recovery. We aim to assess if intra-arterial injection of rat mesoangioblasts (MABs) and stable Vascular Endothelial Growth Factor (VEGF)-expressing MABs, improve recovery of urethral and vaginal function following simulated vaginal delivery (SVD). Female rats (n = 86) were assigned to either injection of saline (control), allogeneic-MABs (MABsallo), autologous-MABs (MABsauto) or allogeneic-MABs transduced to stably expressed VEGF (MABsallo-VEGF). One hour after SVD, 0.5 × 106 MABs or saline were injected into the aorta. Primary outcome was urethral (7d and 14d) and vaginal (14d) function; others were bioluminescent imaging for cell tracking (1, 3 and 7d), morphometry (7, 14 and 60d) and mRNAseq (3 and 7d). All MABs injected rats had external urethral sphincter and vaginal function recovery within 14d, as compared to only half of saline controls. Functional recovery was paralleled by improved muscle regeneration and microvascularization. Recovery rate was not different between MABsallo and MABsauto. MABsallo-VEGF accelerated functional recovery and increased GAP-43 expression at 7d. At 3d we detected major transcriptional changes in the urethra of both MABsallo and MABsallo-VEGF-injected animals, with upregulation of Rho/GTPase activity, epigenetic factors and dendrite development. MABSallo also upregulated transcripts that encode proteins involved in myogenesis and downregulated pro-inflammatory processes. MABsallo-VEGF also upregulated transcripts that encode proteins involved in neuron development and downregulated genes involved in hypoxia and oxidative stress. At 7d, urethras of MABsallo-VEGF-injected rats showed downregulation of oxidative and inflammatory response compared to MABSallo. Intra-arterial injection of MABsallo-VEGF enhances neuromuscular regeneration induced by untransduced MABs and accelerates the functional urethral and vaginal recovery after SVD.

Extracellular vesicles and Duchenne muscular dystrophy pathology: Modulators of disease progression.

Duchenne muscular dystrophy (DMD) is a devastating disorder and is considered to be one of the worst forms of inherited muscular dystrophies. DMD occurs as a result of mutations in the dystrophin gene, leading to progressive muscle fiber degradation and weakness. Although DMD pathology has been studied for many years, there are aspects of disease pathogenesis and progression that have not been thoroughly explored yet. The underlying issue with this is that the development of further effective therapies becomes stalled. It is becoming more evident that extracellular vesicles (EVs) may contribute to DMD pathology. EVs are vesicles secreted by cells that exert a multitude of effects via their lipid, protein, and RNA cargo. EV cargo (especially microRNAs) is also said to be a good biomarker for identifying the status of specific pathological processes that occur in dystrophic muscle, such as fibrosis, degeneration, inflammation, adipogenic degeneration, and dilated cardiomyopathy. On the other hand, EVs are becoming more prominent vehicles for custom-engineered cargos. In this review, we will discuss the possible contribution of EVs to DMD pathology, their potential use as biomarkers, and the therapeutic efficacy of both, EV secretion inhibition and custom-engineered cargo delivery.

Human Neuromuscular Junction on a Chip: Impact of Amniotic Fluid Stem Cell Extracellular Vesicles on Muscle Atrophy and NMJ Integrity.

Neuromuscular junctions (NMJs) are specialized synapses, crucial for the communication between spinal motor neurons (MNs) and skeletal muscle. NMJs become vulnerable in degenerative diseases, such as muscle atrophy, where the crosstalk between the different cell populations fails, and the regenerative ability of the entire tissue is hampered. How skeletal muscle sends retrograde signals to MNs through NMJs represents an intriguing field of research, and the role of oxidative stress and its sources remain poorly understood. Recent works demonstrate the myofiber regeneration potential of stem cells, including amniotic fluid stem cells (AFSC), and secreted extracellular vesicles (EVs) as cell-free therapy. To study NMJ perturbations during muscle atrophy, we generated an MN/myotube co-culture system through XonaTM microfluidic devices, and muscle atrophy was induced in vitro by Dexamethasone (Dexa). After atrophy induction, we treated muscle and MN compartments with AFSC-derived EVs (AFSC-EVs) to investigate their regenerative and anti-oxidative potential in counteracting NMJ alterations. We found that the presence of EVs reduced morphological and functional in vitro defects induced by Dexa. Interestingly, oxidative stress, occurring in atrophic myotubes and thus involving neurites as well, was prevented by EV treatment. Here, we provided and validated a fluidically isolated system represented by microfluidic devices for studying human MN and myotube interactions in healthy and Dexa-induced atrophic conditions-allowing the isolation of subcellular compartments for region-specific analyses-and demonstrated the efficacy of AFSC-EVs in counteracting NMJ perturbations.

State of the Art Procedures for the Isolation and Characterization of Mesoangioblasts.

Adult skeletal muscle is a dynamic tissue able to regenerate quite efficiently, thanks to the presence of stem cell machinery. Besides the quiescent satellite cells that are activated upon injury or paracrine factors, other stem cells are described to be directly or indirectly involved in adult myogenesis. Mesoangioblasts (MABs) are vessel-associated stem cells originally isolated from embryonic dorsal aorta and, at later stages, from the adult muscle interstitium expressing pericyte markers. Adult MABs entered clinical trials for the treatment of Duchenne muscular dystrophy and the transcriptome of human fetal MABs has been described. In addition, single cell RNA-seq analyses provide novel information on adult murine MABs and more in general in interstitial muscle stem cells. This chapter provides state-of-the-art techniques to isolate and characterize murine MABs, fetal and adult human MABs.

Cardiomyocyte differentiation from human induced pluripotent stem cells is delayed following knockout of Bcl-2.

Anti-apoptotic B-cell lymphoma 2 (Bcl-2) regulates a wide array of cellular functions involved in cell death, cell survival and autophagy. Less known is its involvement in the differentiation of cardiomyocytes. As a consequence, mechanisms by which Bcl-2 contributes to cardiac differentiation remain to be elucidated. To address this, we used CRISPR/Cas9 to knockout (KO) BCL2 in human induced pluripotent stem cells (hiPSCs) and investigated the consequence of this KO for differentiation towards cardiomyocytes. Our results indicate that differentiation of hiPSCs to cardiomyocytes was delayed following BCL2 KO. This was not related to the canonical anti-apoptotic function of Bcl-2. This delay led to reduced expression and activity of the cardiomyocyte Ca2+ toolkit. Finally, Bcl-2 KO reduced c-Myc expression and nuclear localization in the early phase of the cardiac differentiation process, which accounts at least in part for the observed delay in the cardiac differentiation. These results suggest that there is a central role for Bcl-2 in cardiomyocyte differentiation and maturation.

Extracellular vesicle-derived miRNAs improve stem cell-based therapeutic approaches in muscle wasting conditions.

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells.

PGC-1α in the myofibers regulates the balance between myogenic and adipogenic progenitors affecting muscle regeneration.

Skeletal muscle repair is accomplished by satellite cells (MuSCs) in cooperation with interstitial stromal cells (ISCs), but the relationship between the function of these cells and the metabolic state of myofibers remains unclear. This study reports an altered proportion of MuSCs and ISCs (including adipogenesis-regulatory cells; Aregs) induced by the transgenic overexpression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in the myofibers (MCK-PGC-1α mice). Although PGC-1α-driven increase of MuSCs does not accelerate muscle regeneration, myogenic progenitors isolated from MCK-PGC-1α mice and transplanted into intact and regenerating muscles are more prone to fuse with recipient myofibers than those derived from wild-type donors. Moreover, both young and aged MCK-PGC-1α animals exhibit reduced perilipin-positive areas when challenged with an adipogenic stimulus, demonstrating low propensity to accumulate adipocytes within the muscle. Overall, these results unveil that increased PGC-1α expression in the myofibers favors pro-myogenic and anti-adipogenic cell populations in the skeletal muscle.

Autologous iPSC-Derived Human Neuromuscular Junction to Model the Pathophysiology of Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) is a heterogeneous group of genetic neurodegenerative disorders, characterized by progressive lower limb spasticity and weakness resulting from retrograde axonal degeneration of motor neurons (MNs). Here, we generated in vitro human neuromuscular junctions (NMJs) from five HSP patient-specific induced pluripotent stem cell (hiPSC) lines, by means of microfluidic strategy, to model disease-relevant neuropathologic processes. The strength of our NMJ model lies in the generation of lower MNs and myotubes from autologous hiPSC origin, maintaining the genetic background of the HSP patient donors in both cell types and in the cellular organization due to the microfluidic devices. Three patients characterized by a mutation in the SPG3a gene, encoding the ATLASTIN GTPase 1 protein, and two patients with a mutation in the SPG4 gene, encoding the SPASTIN protein, were included in this study. Differentiation of the HSP-derived lines gave rise to lower MNs that could recapitulate pathological hallmarks, such as axonal swellings with accumulation of Acetyl-α-TUBULIN and reduction of SPASTIN levels. Furthermore, NMJs from HSP-derived lines were lower in number and in contact point complexity, denoting an impaired NMJ profile, also confirmed by some alterations in genes encoding for proteins associated with microtubules and responsible for axonal transport. Considering the complexity of HSP, these patient-derived neuronal and skeletal muscle cell co-cultures offer unique tools to study the pathologic mechanisms and explore novel treatment options for rescuing axonal defects and diverse cellular processes, including membrane trafficking, intracellular motility and protein degradation in HSP.

Long-term culture of patient-derived cardiac organoids recapitulated Duchenne muscular dystrophy cardiomyopathy and disease progression.

Duchenne Muscular Dystrophy (DMD) is an X-linked neuromuscular disease which to date is incurable. The major cause of death is dilated cardiomyopathy however, its pathogenesis is unclear as existing cellular and animal models do not fully recapitulate the human disease phenotypes. In this study, we generated cardiac organoids from patient-derived induced pluripotent stem cells (DMD-COs) and isogenic-corrected controls (DMD-Iso-COs) and studied if DMD-related cardiomyopathy and disease progression occur in the organoids upon long-term culture (up to 93 days). Histological analysis showed that DMD-COs lack initial proliferative capacity, displayed a progressive loss of sarcoglycan localization and high stress in endoplasmic reticulum. Additionally, cardiomyocyte deterioration, fibrosis and aberrant adipogenesis were observed in DMD-COs over time. RNA sequencing analysis confirmed a distinct transcriptomic profile in DMD-COs which was associated with functional enrichment in hypertrophy/dilated cardiomyopathy, arrhythmia, adipogenesis and fibrosis pathways. Moreover, five miRNAs were identified to be crucial in this dysregulated gene network. In conclusion, we generated patient-derived cardiac organoid model that displayed DMD-related cardiomyopathy and disease progression phenotypes in long-term culture. We envision the feasibility to develop a more complex, realistic and reliable in vitro 3D human cardiac-mimics to study DMD-related cardiomyopathies.

Shared and Divergent Epigenetic Mechanisms in Cachexia and Sarcopenia.

Significant loss of muscle mass may occur in cachexia and sarcopenia, which are major causes of mortality and disability. Cachexia represents a complex multi-organ syndrome associated with cancer and chronic diseases. It is often characterized by body weight loss, inflammation, and muscle and adipose wasting. Progressive muscle loss is also a hallmark of healthy aging, which is emerging worldwide as a main demographic trend. A great challenge for the health care systems is the age-related decline in functionality which threatens the independence and quality of life of elderly people. This biological decline can also be associated with functional muscle loss, known as sarcopenia. Previous studies have shown that microRNAs (miRNAs) play pivotal roles in the development and progression of muscle wasting in both cachexia and sarcopenia. These small non-coding RNAs, often carried in extracellular vesicles, inhibit translation by targeting messenger RNAs, therefore representing potent epigenetic modulators. The molecular mechanisms behind cachexia and sarcopenia, including the expression of specific miRNAs, share common and distinctive trends. The aim of the present review is to compile recent evidence about shared and divergent epigenetic mechanisms, particularly focusing on miRNAs, between cachexia and sarcopenia to understand a facet in the underlying muscle wasting associated with these morbidities and disclose potential therapeutic interventions.

Myoblast 3D bioprinting to burst in vitro skeletal muscle differentiation.

Skeletal muscle regeneration is one of the major areas of interest in sport medicine as well as trauma centers. Three-dimensional (3D) bioprinting (BioP) is nowadays widely adopted to manufacture 3D constructs for regenerative medicine but a comparison between the available biomaterial-based inks (bioinks) is missing. The present study aims to assess the impact of different hydrogels on the viability, proliferation, and differentiation of murine myoblasts (C2C12) encapsulated in 3D bioprinted constructs aided to muscle regeneration. We tested three different commercially available hydrogels bioinks based on: (1) gelatin methacrylate and alginate crosslinked by UV light; (2) gelatin methacrylate, xanthan gum, and alginate-fibrinogen; (3) nanofibrillated cellulose (NFC)/alginate-fibrinogen crosslinked with calcium chloride and thrombin. Constructs embedding the cells were manufactured by extrusion-based BioP and C2C12 viability, proliferation, and differentiation were assessed after 24 h, 7, 14, 21, and 28 days in culture. Although viability, proliferation, and differentiation were observed in all the constructs, among the investigated bioinks, the best results were obtained by using NFC/alginate-fibrinogen-based hydrogel from 7 to 14 days in culture, when the embedded myoblasts started fusing, forming at day 21 and day 28 multinucleated myotubes within the 3D bioprinted structures. The results revealed an extensive myotube alignment all over the linear structure of the hydrogel, demonstrating cell maturation, and enhanced myogenesis. The bioprinting strategies that we describe here denote a strong and endorsed approach for the creation of in vitro artificial muscle to improve skeletal muscle tissue engineering for future therapeutic applications.

Diabetes-Induced Cellular Senescence and Senescence-Associated Secretory Phenotype Impair Cardiac Regeneration and Function Independently of Age.

Diabetes mellitus (DM) affects the biology of multipotent cardiac stem/progenitor cells (CSCs) and adult myocardial regeneration. We assessed the hypothesis that senescence and senescence-associated secretory phenotype (SASP) are main mechanisms of cardiac degenerative defect in DM. Accordingly, we tested whether ablation of senescent CSCs would rescue the cardiac regenerative/reparative defect imposed by DM. We obtained cardiac tissue from nonaged (50- to 64-year-old) patients with type 2 diabetes mellitus (T2DM) and without DM (NDM) and postinfarct cardiomyopathy undergoing cardiac surgery. A higher reactive oxygen species production in T2DM was associated with an increased number of senescent/dysfunctional T2DM-human CSCs (hCSCs) with reduced proliferation, clonogenesis/spherogenesis, and myogenic differentiation versus NDM-hCSCs in vitro. T2DM-hCSCs showed a defined pathologic SASP. A combination of two senolytics, dasatinib (D) and quercetin (Q), cleared senescent T2DM-hCSCs in vitro, restoring their expansion and myogenic differentiation capacities. In a T2DM model in young mice, diabetic status per se (independently of ischemia and age) caused CSC senescence coupled with myocardial pathologic remodeling and cardiac dysfunction. D + Q treatment efficiently eliminated senescent cells, rescuing CSC function, which resulted in functional myocardial repair/regeneration, improving cardiac function in murine DM. In conclusion, DM hampers CSC biology, inhibiting CSCs' regenerative potential through the induction of cellular senescence and SASP independently from aging. Senolytics clear senescence, abrogating the SASP and restoring a fully proliferative/differentiation-competent hCSC pool in T2DM with normalization of cardiac function.