Search Results (8)

Mitochondria are essential organelles for maintaining intracellular homeostasis. Their dysfunction can directly or indirectly affect cell functioning and is linked to multiple diseases. Donation of exogenous mitochondria is potentially a viable therapeutic strategy. For this, selecting appropriate donors of exogenous mitochondria is critical. We previously demonstrated that ultra-purified bone marrow-derived mesenchymal stem cells (RECs) have better stem cell properties and homogeneity than conventionally cultured bone marrow-derived mesenchymal stem cells. Here, we explored the effect of contact and noncontact systems on three possible mitochondrial transfer mechanisms involving tunneling nanotubes, connexin 43 (Cx43)-mediated gap junction channels (GJCs), and extracellular vesicles (Evs). We show that Evs and Cx43-GJCs provide the main mechanism for mitochondrial transfer from RECs. Through these two critical mitochondrial transfer pathways, RECs could transfer a greater number of mitochondria into mitochondria-deficient (ρ⁰) cells and could significantly restore mitochondrial functional parameters. Furthermore, we analyzed the effect of exosomes (EXO) on the rate of mitochondrial transfer from RECs and recovery of mitochondrial function. REC-derived EXO appeared to promote mitochondrial transfer and slightly improve the recovery of mtDNA content and oxidative phosphorylation in ρ⁰ cells. Thus, ultrapure, homogenous, and safe stem cell RECs could provide a potential therapeutic tool for diseases associated with mitochondrial dysfunction. Full article

This study aimed to evaluate if Simvastatin can reduce, and/or prevent, Doxorubicin (Doxo)-induced cardiotoxicity. H9c2 cells were treated with Simvastatin (10 µM) for 4 h and then Doxo (1 µM) was added, and the effects on oxidative stress, calcium homeostasis, and apoptosis were evaluated after 20 h. Furthermore, we evaluated the effects of Simvastatin and Doxo co-treatment on Connexin 43 (Cx43) expression and localization, since this transmembrane protein forming gap junctions is widely involved in cardioprotection. Cytofluorimetric analysis showed that Simvastatin co-treatment significantly reduced Doxo-induced cytosolic and mitochondrial ROS overproduction, apoptosis, and cytochrome c release. Spectrofluorimetric analysis performed by means of Fura2 showed that Simvastatin co-treatment reduced calcium levels stored in mitochondria and restored cytosolic calcium storage. Western blot, immunofluorescence, and cytofluorimetric analyses showed that Simvastatin co-treatment significantly reduced Doxo-induced mitochondrial Cx43 over-expression and significantly increased the membrane levels of Cx43 phosphorylated on Ser368. We hypothesized that the reduced expression of mitochondrial Cx43 could justify the reduced levels of calcium stored in mitochondria and the consequent induction of apoptosis observed in Simvastatin co-treated cells. Moreover, the increased membrane levels of Cx43 phosphorylated on Ser368, which is responsible for the closed conformational state of the gap junction, let us to hypothesize that Simvastatin leads to cell-to-cell communication interruption to block the propagation of Doxo-induced harmful stimuli. Based on these results, we can conclude that Simvastatin could be a good adjuvant in Doxo anticancer therapy. Indeed, we confirmed its antioxidant and antiapoptotic activity, and, above all, we highlighted that Simvastatin interferes with expression and cellular localization of Cx43 that is widely involved in cardioprotection. Full article

Osteoporosis and sarcopenia (termed “Osteosarcopenia”), the twin-aging diseases, are major contributors to reduced bone mass and muscle weakness in the elderly population. Connexin 43 (Cx43) in osteocytes has been previously reported to play vital roles in bone homeostasis and muscle function in mature mice. The Cx43-formed gap junctions (GJs) and hemichannels (HCs) in osteocytes are important portals for the exchange of small molecules in cell-to-cell and cell-to-extracellular matrix, respectively. However, the roles of Cx43-based GJs and HCs in both bone and muscle aging are still unclear. Here, we used two transgenic mouse models with overexpression of the dominant negative Cx43 mutants primarily in osteocytes driven by the 10-kb Dmp1 promoter, R76W mice (inhibited gap junctions but enhanced hemichannels) and Δ130–136 mice (both gap junction and hemichannels are inhibited), to determine the actions of Cx43-based hemichannels (HCs) and gap junctions (GJs) in the regulation of bone and skeletal muscle from aged mice (18 months) as compared with those from adult mice (10 months). We demonstrated that enhancement of Cx43 HCs reduces bone mass due to increased osteoclast surfaces while the impairment of Cx43 HCs increases osteocyte apoptosis in aged mice caused by reduced PGE₂ levels. Furthermore, altered mitochondrial homeostasis with reduced expression of Sirt-1, OPA-1, and Drp-1 resulted in excessive ROS level in muscle soleus (SL) of aged transgenic mice. In vitro, the impairment of Cx43 HCs in osteocytes from aged mice also promoted muscle collagen synthesis through activation of TGFβ/smad2/3 signaling because of reduced PGE₂ levels in the PO CM. These findings indicate that the enhancement of Cx43 HCs while GJs are inhibited reduces bone mass, and the impairment of Cx43 HCs inhibits PGE₂ level in osteocytes and this reduction promotes muscle collagen synthesis in skeletal muscle through activation of TGFβ/smad2/3 signaling, which together with increased ROS level contributes to reduced muscle force in aged mice. Full article

Diabetic retinopathy (DR) is one of the most common causes of vision loss and blindness among the working-age population. High glucose (HG)-induced decrease in mitochondrial connexin 43 (mtCx43) level is known to promote mitochondrial fragmentation, cytochrome c release, and apoptosis in retinal endothelial cells associated with DR. In this study, we investigated whether counteracting HG-induced decrease in mtCx43 level would preserve mitochondrial integrity and prevent apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM glucose) or HG (30 mM glucose) medium for 7 days. In parallel, cells grown in HG were transfected with Cx43 plasmid, or empty vector (EV), as control. Western blot (WB) analysis showed a significant decrease in mtCx43 level concomitant with increased cleaved caspase-3, Bax, cleaved PARP, and mitochondrial fragmentation in cells grown in HG condition compared to those grown in N medium. When cells grown in HG were transfected with Cx43 plasmid, mtCx43 level was significantly increased and resulted in reduced cleaved caspase-3, Bax, cleaved PARP and preservation of mitochondrial morphology with a significant decrease in the number of TUNEL-positive cells compared to those grown in HG alone. Findings from the study indicate a novel role for mtCx43 in regulating apoptosis and that maintenance of mtCx43 level could be useful in preventing HG-induced apoptosis by reducing mitochondrial fragmentation associated with retinal vascular cell loss in DR. Full article

Connexin 43 (Cx43) is a gap junction protein that assembles at the cell border to form intercellular gap junction (GJ) channels which allow for cell–cell communication by facilitating the rapid transmission of ions and other small molecules between adjacent cells. Non-canonical roles of Cx43, and specifically its C-terminal domain, have been identified in the regulation of Cx43 trafficking, mitochondrial preconditioning, cell proliferation, and tumor formation, yet the mechanisms are still being explored. It was recently identified that up to six truncated isoforms of Cx43 are endogenously produced via alternative translation from internal start codons in addition to full length Cx43, all from the same mRNA produced by the gene GJA1. GJA1-11k, the 11kDa alternatively translated isoform of Cx43, does not have a known role in the formation of gap junction channels, and little is known about its function. Here, we report that over expressed GJA1-11k, unlike the other five truncated isoforms, preferentially localizes to the nucleus in HEK293FT cells and suppresses cell growth by limiting cell cycle progression from the G₀/G₁ phase to the S phase. Furthermore, these functions are independent of the channel-forming full-length Cx43 isoform. Understanding the apparently unique role of GJA1-11k and its generation in cell cycle regulation may uncover a new target for affecting cell growth in multiple disease models. Full article

Therapies intended to mitigate cardiovascular complications cannot be applied in practice without detailed knowledge of molecular mechanisms. Mitochondria, as the end-effector of cardioprotection, represent one of the possible therapeutic approaches. The present review provides an overview of factors affecting the regulation processes of mitochondria at the level of mitochondrial permeability transition pores (mPTP) resulting in comprehensive myocardial protection. The regulation of mPTP seems to be an important part of the mechanisms for maintaining the energy equilibrium of the heart under pathological conditions. Mitochondrial connexin 43 is involved in the regulation process by inhibition of mPTP opening. These individual cardioprotective mechanisms can be interconnected in the process of mitochondrial oxidative phosphorylation resulting in the maintenance of adenosine triphosphate (ATP) production. In this context, the degree of mitochondrial membrane fluidity appears to be a key factor in the preservation of ATP synthase rotation required for ATP formation. Moreover, changes in the composition of the cardiolipin’s structure in the mitochondrial membrane can significantly affect the energy system under unfavorable conditions. This review aims to elucidate functional and structural changes of cardiac mitochondria subjected to preconditioning, with an emphasis on signaling pathways leading to mitochondrial energy maintenance during partial oxygen deprivation. Full article

It is becoming clear that in addition to gap junctions playing a role in cell–cell communication, gap junction proteins (connexins) located in cytoplasmic compartments may have other important functions. Mitochondrial connexin 43 (Cx43) is increased after ischemic preconditioning and has been suggested to play a protective role in the heart. How Cx43 traffics to the mitochondria and the interactions of mitochondria with other Cx43-containing structures are unclear. In this study, immunocytochemical, super-resolution, and transmission electron microscopy were used to detect cytoplasmic Cx43-containing structures and to demonstrate their interactions with other cytoplasmic organelles. The most prominent cytoplasmic Cx43-containing structures—annular gap junctions—were demonstrated to form intimate associations with lysosomes as well as with mitochondria. Surprisingly, the frequency of associations between mitochondria and annular gap junctions was greater than that between lysosomes and annular gap junctions. The benefits of annular gap junction/mitochondrial associations are not known. However, it is tempting to suggest, among other possibilities, that the contact between annular gap junction vesicles and mitochondria facilitates Cx43 delivery to the mitochondria. Furthermore, it points to the need for investigating annular gap junctions as more than only vesicles destined for degradation. Full article

Doxorubicin (DOXO) administration induces alterations in Connexin 43 (Cx43) expression and localization, thus, inducing alterations in chemical and electrical signal transmission between cardiomyocytes and in intracellular calcium homeostasis even evident after a single administration. This study was designed to evaluate if Diazoxide (DZX), a specific opener of mitochondrial K_ATP channels widely used for its cardioprotective effects, can fight DOXO-induced cardiotoxicity in a short-time mouse model. DZX (20 mg/kg i.p.) was administered 30 min before DOXO (10 mg/kg i.p.) in C57BL/6j female mice for 1–3 or seven days once every other day. A recovery of cardiac parameters, evaluated by Echocardiography, were observed in DZX+DOXO co-treated mice. Western blot analysis performed on heart lysates showed an increase in sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCAII) and a reduction in phospholamban (PLB) amounts in DZX+DOXO co-treated mice. A contemporary recovery of intracellular Ca²⁺-signal, detected spectrofluorometrically by means of FURA-2AM, was observed in these mice. Cx43 expression and localization, analyzed by Western blot and confirmed by immunofluorescence analysis, showed that DZX co-treatement increases Cx43 amount both on sarcoplasmic membrane and on mitochondria. In conclusion, our data demonstrate that, in a short-time mouse model of DOXO-induced cardiotoxicity, DZX exerts its cardioprotective effects also by enhancing the amount Cx43. Full article