Mitochondria inter-organelle relationships in cancer protein aggregation

Mitochondria are physically associated with other organelles, such as ER and lysosomes, forming a complex network that is crucial for cell homeostasis regulation. Inter-organelle relationships are finely regulated by both tether systems, which maintain physical proximity, and by signaling cues that induce the exchange of molecular information to regulate metabolism, Ca2+ homeostasis, redox state, nutrient availability, and proteostasis. The coordinated action of the organelles is engaged in the cellular integrated stress response. In any case, pathological conditions alter functional communication and efficient rescue pathway activation, leading to cell distress exacerbation and eventually cell death. Among these detrimental signals, misfolded protein accumulation and aggregation cause major damage to the cells, since defects in protein clearance systems worsen cell toxicity. A cause for protein aggregation is often a defective mitochondrial redox balance, and the ER freshly translated misfolded proteins and/or a deficient lysosome-mediated clearance system. All these features aggravate mitochondrial damage and enhance proteotoxic stress. This review aims to gather the current knowledge about the complex liaison between mitochondria, ER, and lysosomes in facing proteotoxic stress and protein aggregation, highlighting both causes and consequences. Particularly, specific focus will be pointed to cancer, a pathology in which inter-organelle relations in protein aggregation have been poorly investigated

Engineering muscle networks in 3D gelatin methacryloyl hydrogels: influence of mechanical stiffness and geometrical confinement

In this work, the influence of mechanical stiffness and geometrical confinement on the 3D culture of myoblast-laden gelatin methacryloyl (GelMA) photo-crosslinkable hydrogels was evaluated in terms of in vitro myogenesis. We formulated a set of cell-laden GelMA hydrogels with a compressive modulus in the range 1÷17 kPa, obtained by varying GelMA concentration and degree of cross-linking. C2C12 myoblasts were chosen as the cell model, to investigate the supportiveness of different GelMA hydrogels on myotube formation up to 2 weeks. Results showed that the hydrogels with a stiffness in the range 1÷3 kPa provided enhanced support to C2C12 differentiation in terms of myotube number, rate of formation and space distribution. Finally, we studied the influence of geometrical confinement on myotube orientation by confining cells within thin hydrogel slabs having different cross-sections: i) 2000×2000 m, ii) 1000×1000 m and iii) 500×500 m. The obtained results showed that by reducing the cross-section—i.e., by increasing the level of confinement—myotubes were more likely restrained and formed aligned myostructures that better mimicked the native morphology of skeletal muscle

High-density ZnO nanowires for cellular biointerfaces: a new role as myogenic differentiation switch

The design of artificial platforms for expanding undifferentiated stem cells is of tremendous importance for regenerative medicine [1]. We have recently demonstrated that a ZnO nanowires (NWs) decorated glass support permits to obtain a differentiation switch during proliferation for mesoangioblasts (MABs)– i.e. multipotent progenitor cells which are remarkable candidates for the therapy of muscle diseases [2]. We have optimized the ZnO NWs synthesis on glass surfaces by numerical simulations and experimental systematic investigations, considering zinc speciation and supersaturation [3]. In particular, we demonstrated by numerical simulations that the ligand ethylenediamine, at the isoelectric point of the ZnO NWs tips, can effectively control – at 1:1 stoichiometric ratio with zinc – both speciation and supersaturation of zinc in the nutrient solution. In this regard, we employed ethanolamine (a safer precursor) for in-situ producing ethylenediamine by means of a zinc-catalysed amination reaction of ethanolamine by ammonia. The obtained highquality ZnONWs-cells biointerface allows cells to maintain viability and a spherical viable undifferentiated state during the 8 days observation time. Simulations of the interface by theoretical models [4] and our experimental investigations by SEM and confocal microscopy demonstrate that NWs do not induce any damage on the cellular membrane, whilst blocking their differentiation. More specifically, the myosin heavy chain, typically expressed in differentiated myogenic progenitors, is completely absent. Interestingly, the differentiation capabilities are completely restored upon cell removal from the NW-functionalized substrate and regrowing onto a standard culture glass dish. These results open the way towards unprecedented applications of ZnO NWs for cell-based therapy and tissue engineering [5]. References [1] G. Cossu, P. Bianco, Curr. Opin. Genet. Dev. 2003, 13, 537-542. [2] V. Errico, G. Arrabito, E. Fornetti, C. Fuoco, S. Testa, G. Saggio, S. Rufini, S. M. Cannata, A. Desideri, C. Falconi, C. Gargioli, ACS Appl. Mater. Interfaces, 2018, 10, 14097- 14107. [3] G. Arrabito, V. Errico, Z. Zhang, W. Han, C. Falconi, Nano Energy, 2018, 46, 54-62. [4] N. Buch-Månson, S. Bonde, J. Bolinsson, T. Berthing, J. Nygård, K.L. Martinez, Adv. Funct. Mater. 2015, 25, 3246-3255. [5] Y. Su, I. Cockerill, Y. Wang, Y.-X. Qin, L. Chang, Y. Zheng, and D. Zhu, Trends in Biotechnology, 2019, 37, 428-441

Myoblast Myogenic Differentiation but Not Fusion Process Is Inhibited via MyoD Tetraplex Interaction

The presence of tetraplex structures in the promoter region of the myogenic differentiation 1 gene (MyoD1) was investigated with a specific tetraplex-binding porphyrin (TMPyP4), to test its influence on the expression of MyoD1 itself and downstream-regulated genes during myogenic differentiation. TMPyP4-exposed C2C12 myoblasts, blocking MyoD1 transcription, proliferated reaching confluence and fused forming elongated structures, resembling myotubes, devoid of myosin heavy chain 3 (MHC) expression. Besides lack of MHC, upon MyoD1 inhibition, other myogenic gene expressions were also affected in treated cells, while untreated control cell culture showed normal myotube formation expressing MyoD1, Myog, MRF4, Myf5, and MHC. Unexpectedly, the myomaker (Mymk) gene expression was not affected upon TMPyP4 exposure during C2C12 myogenic differentiation. At the genomic level, the bioinformatic comparison of putative tetraplex sites found that three tetraplexes in MyoD1 and Myog are highly conserved in mammals, while Mymk and MHC did not show any conserved tetraplexes in the analysed regions. Thus, here, we report for the first time that the inhibition of the MyoD1 promoter function, stabilizing the tetraplex region, affects downstream myogenic genes by blocking their expression, while leaving the expression of Mymk unaltered. These results reveal the existence of two distinct pathways: one leading to cell fusion and one guaranteeing correct myotube differentiation

Myoblast Myogenic Differentiation but Not Fusion Process Is Inhibited via MyoD Tetraplex Interaction

The presence of tetraplex structures in the promoter region of the myogenic differentiation 1 gene (MyoD1) was investigated with a specific tetraplex-binding porphyrin (TMPyP4), to test its influence on the expression of MyoD1 itself and downstream-regulated genes during myogenic differentiation. TMPyP4-exposed C2C12 myoblasts, blocking MyoD1 transcription, proliferated reaching confluence and fused forming elongated structures, resembling myotubes, devoid of myosin heavy chain 3 (MHC) expression. Besides lack of MHC, upon MyoD1 inhibition, other myogenic gene expressions were also affected in treated cells, while untreated control cell culture showed normal myotube formation expressing MyoD1, Myog, MRF4, Myf5, and MHC. Unexpectedly, the myomaker (Mymk) gene expression was not affected upon TMPyP4 exposure during C2C12 myogenic differentiation. At the genomic level, the bioinformatic comparison of putative tetraplex sites found that three tetraplexes in MyoD1 and Myog are highly conserved in mammals, while Mymk and MHC did not show any conserved tetraplexes in the analysed regions. Thus, here, we report for the first time that the inhibition of the MyoD1 promoter function, stabilizing the tetraplex region, affects downstream myogenic genes by blocking their expression, while leaving the expression of Mymk unaltered. These results reveal the existence of two distinct pathways: one leading to cell fusion and one guaranteeing correct myotube differentiation

Reticulon-1C Involvement in Muscle Regeneration

Skeletal muscle is a very dynamic and plastic tissue, being essential for posture, locomotion and respiratory movement. Muscle atrophy or genetic muscle disorders, such as muscular dystrophies, are characterized by myofiber degeneration and replacement with fibrotic tissue. Recent studies suggest that changes in muscle metabolism such as mitochondrial dysfunction and dysregulation of intracellular Ca2+ homeostasis are implicated in many adverse conditions affecting skeletal muscle. Accumulating evidence also suggests that ER stress may play an important part in the pathogenesis of inflammatory myopathies and genetic muscle disorders. Among the different known proteins regulating ER structure and function, we focused on RTN-1C, a member of the reticulon proteins family localized on the ER membrane. We previously demonstrated that RTN-1C expression modulates cytosolic calcium concentration and ER stress pathway. Moreover, we recently reported a role for the reticulon protein in autophagy regulation. In this study, we found that muscle differentiation process positively correlates with RTN-1C expression and UPR pathway up-regulation during myogenesis. To better characterize the role of the reticulon protein alongside myogenic and muscle regenerative processes, we performed in vivo experiments using either a model of muscle injury or a photogenic model for Duchenne muscular dystrophy. The obtained results revealed RTN-1C up-regulation in mice undergoing active regeneration and localization in the injured myofibers. The presented results strongly suggested that RTN-1C, as a protein involved in key aspects of muscle metabolism, may represent a new target to promote muscle regeneration and repair upon injury

Skeletal Muscle-Derived Human Mesenchymal Stem Cells: Influence of Different Culture Conditions on Proliferative and Myogenic Capabilities

Skeletal muscle tissue is characterized by restrained self-regenerative capabilities, being ineffective in relation to trauma extension both in time span (e.g., chronic diseases) and in size (e.g., large trauma). For these reasons, tissue engineering and/or cellular therapies represent a valuable solution in the cases where the physiological healing process failed. Satellite cells, the putative skeletal muscle stem cells, have been the first solution explored to remedy the insufficient self-regeneration capacity. Nevertheless, some limitation related to donor age, muscle condition, expansion hitch, and myogenic potentiality maintenance have limited their use as therapeutic tool. To overcome this hindrance, different stem cells population with myogenic capabilities have been investigated to evaluate their real potentiality for therapeutic approaches, but, as of today, the perfect cell candidate has not been identified yet. In this work, we analyze the characteristics of skeletal muscle-derived human Mesenchymal Stem Cells (hMSCs), showing the maintenance/increment of myogenic activity upon differential culture conditions. In particular, we investigate the influence of a commercial enriched growth medium (Cyto-Grow), and of a medium enriched with either human-derived serum (H.S.) or human Platelet-rich Plasma (PrP), in order to set up a culture protocol useful for employing this cell population in clinical therapeutic strategies. The presented results reveal that both the enriched medium (Cyto-Grow) and the human-derived supplements (H.S. and PrP) have remarkable effects on hMSCs proliferation and myogenic differentiation compared to standard condition, uncovering the real possibility to exploit these human derivatives to ameliorate stem cells yield and efficacy

3D bioprinting of hydrogel constructs with cell and material gradients for the regeneration of full-thickness chondral defect using a microfluidic printing head

Osteochondral tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human articular chondrocytes (hACs). Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of realtime quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of osteochondral defect when compared to untreated control

Engineering human-scale artificial bone grafts for treating critical-size bone defects

The manufacturing of artificial bone grafts can potentially circumvent the issues associated with current bone grafting treatments for critical-size bone defects caused by pathological disorders, trauma, or massive tumor ablation. In this study, we report on a potentially patient-specific fabrication process in which replicas of bone defects, in particular zygomatic and mandibular bones and phalanxes of a hand finger, were manufactured by laser stereolithography an used as templates for the creation of PDMS molds. Gas-in-water foams were cast in the molds, rapidly frozen, freeze-dried, and cross-linked. Since bone matrix consists essentially of collagen and hydroxyapatite, biomimetic scaffolds were fabricated using gelatin and hydroxyapatite in a ratio very similar to that found in bone. The obtained composite scaffolds were excellent replicas of the original bone defects models and presented both a superficial and internal porous texture adequate for cellular and blood vessels infiltration. In particular, scaffolds exhibited a porous texture consisting of pores and interconnects with average size of about 300 and 100 μm, respectively, and a porosity of 90%. In vitro culture tests using hMSCs demonstrated scaffold biocompatibility and capacity in inducing differentiation toward osteoblasts progenitors. In vivo cellularized implants showed bone matrix deposition and recruitment of blood vessels. Overall, the technique/materials combination used in this work led to the fabrication of promising mechanically stable, bioactive, and biocompatible composite scaffolds with well-defined architectures potentially valuable in the regeneration of patient-specific bone defects