Mitochondrial Cx43 Research Articles

Abstract 17153: Mitochondrial Cx43 and Gja1-20k Contribute to Sex-Specific Differences of Mitochondrial Response in Septic Cardiomyopathy

Introduction: Cardiac dysfunction contributes to morbidity and mortality of severe sepsis. Preserving mitochondrial (mito) activity is key to maintaining cardiac function. While epidemiological studies consistently report that male sex is an important risk factor for severe sepsis, it is unknown if male heart mito are susceptible to septic injury. We have shown that male cardiomyocyte mito were more vulnerable to TNFα- or H 2 O 2 -induced stress than females. We have also demonstrated that mito connexin 43 (Cx43 or Gja1) and its smaller isoform - Gja1-20k are implicated in sex-related heart response following myocardial ischemia. Hypothesis : In this study, we hypothesize that during sepsis, female (F) hearts exhibit preserved mito Cx43 and Gja1-20k to confer mitochondrial protection, thus improving cardiac function compared to male (M) myocardium. Methods: Age-matched adult M & F C57BL/6 mice received LPS (7.5 mg/kg BW, i.p.) or PBS (n > 6 mice/group). Cardiac function was detected 20h post LPS by 2D echocardiography. Cx43, Gja1-20k, OXPHOS, & PGC-1 were evaluated in M & F mouse heart mito 24h after LPS by Western Blot (n>3 hearts/group). Mito Cx43 was also detected by Immuno-electron microscopy (EM). Mito membrane potential (ΔΨm) was analyzed in cardiomyocytes (from adult M & F mice, n>3 hearts/group) +/- 1h LPS (1 μg/ml) using JC1 fluorescent probe. Mito respiration capacity was further assessed in H9c2 cells transfected with Gja1 siRNA upon LPS exposure by Seahorse XF Mito Stress test. Results: Decreased cardiac function was observed in M mice after LPS injection, but not in F. Higher mito levels of Cx43, Gja1-20k, OXPHOS, & PGC1 were noticed in F mouse hearts compared to M after LPS. Despite no difference in total Cx43 particles between M & F hearts by EM, more gold Cx43 particles were observed in F heart mito compared to M mito following LPS. LPS reduced ΔΨm in M cardiomyocytes, but not in F. Cx43 knockdown decreased mito maximal respiration capacity & ATP production in H9c2 cells with LPS stress. Conclusions: The mitochondria-derived protective mechanism is preserved in F mice, leading to better cardiac function than M mice following LPS-induced sepsis. F cardiac mito are more resistant to LPS challenge than M mito, partially due to preserved mito Cx43 and/or Gja1-20k.

Simvastatin Reduces Doxorubicin-Induced Cardiotoxicity: Effects beyond Its Antioxidant Activity.

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.

Mitochondrial connexin43 and mitochondrial KATP channels modulate triggered arrhythmias in mouse ventricular muscle.

Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial KATP channels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43-/-) mice, Cx43flox/flox mice were crossed with α-MHC (Myh6)-cre+/- mice. The resulting offspring, Cx43flox/flox/Myh6-cre+/- mice (cCx43-/- mice) and their littermates (cCx43+/+ mice), were used. Trabeculae were dissected from the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca2+ concentration ([Ca2+]o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2',7'-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methyl ester (TMRM), and Ca2+ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca2+ with rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial KATP. Most of cCx43-/- mice died suddenly within 8weeks. Cx43 was present in the inner mitochondrial membrane in cCx43+/+ mice but not in cCx43-/- mice. In cCx43-/- mice, the [Ca2+]o,min was lower, and Ca2+ spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43-/- mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43-/- mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca2+]o,min. These results suggest that Cx43 deficiency activates Ca2+ leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca2+, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial KATP channels in mouse hearts.

Connexin 43 hemichannels regulate mitochondrial ATP generation, mobilization, and mitochondrial homeostasis against oxidative stress.

Oxidative stress is a major risk factor that causes osteocyte cell death and bone loss. Prior studies primarily focus on the function of cell surface expressed Cx43 channels. Here, we reported a new role of mitochondrial Cx43 (mtCx43) and hemichannels (HCs) in modulating mitochondria homeostasis and function in bone osteocytes under oxidative stress. In murine long bone osteocyte-Y4 cells, the translocation of Cx43 to mitochondria was increased under H₂O₂-induced oxidative stress. H₂O₂ increased the mtCx43 level accompanied by elevated mtCx43 HC activity, determined by dye uptake assay. Cx43 knockdown (KD) by the CRISPR-Cas9 lentivirus system resulted in impairment of mitochondrial function, primarily manifested as decreased ATP production. Cx43 KD had reduced intracellular reactive oxidative species levels and mitochondrial membrane potential. Additionally, live-cell imaging results demonstrated that the proton flux was dependent on mtCx43 HCs because its activity was specifically inhibited by an antibody targeting Cx43 C-terminus. The co-localization and interaction of mtCx43 and ATP synthase subunit F (ATP5J2) were confirmed by Förster resonance energy transfer and a protein pull-down assay. Together, our study suggests that mtCx43 HCs regulate mitochondrial ATP generation by mediating K⁺, H⁺, and ATP transfer across the mitochondrial inner membrane and the interaction with mitochondrial ATP synthase, contributing to the maintenance of mitochondrial redox levels in response to oxidative stress.

Connexin 43 in Mitochondria: What Do We Really Know About Its Function?

Connexins are known for their ability to mediate cell-cell communication via gap junctions and also form hemichannels that pass ions and molecules over the plasma membrane when open. Connexins have also been detected within mitochondria, with mitochondrial connexin 43 (Cx43) being the best studied to date. In this review, we discuss evidence for Cx43 presence in mitochondria of cell lines, primary cells and organs and summarize data on its localization, import and phosphorylation status. We further highlight the influence of Cx43 on mitochondrial function in terms of respiration, opening of the mitochondrial permeability transition pore and formation of reactive oxygen species, and also address the presence of a truncated form of Cx43 termed Gja1-20k. Finally, the role of mitochondrial Cx43 in pathological conditions, particularly in the heart, is discussed.

Hypoglycemia-Exacerbated Mitochondrial Connexin 43 Accumulation Aggravates Cardiac Dysfunction in Diabetic Cardiomyopathy.

BackgroundDiabetic cardiomyopathy (DCM) is a complex multifaceted disease responsible for elevated heart failure (HF) morbidity and mortality in patients with diabetes mellitus (DM). Patients with DCM exhibit subclinical diastolic dysfunction, progression toward systolic impairment, and abnormal electrophysiology. Hypoglycemia events that occur spontaneously or due to excess insulin administration threaten the lives of patients with DM—with the increased risk of sudden death. However, the molecular underpinnings of this fatal disease remain to be elucidated.Methods and ResultsHere, we used the established streptozotocin-induced DCM murine model to investigate how hypoglycemia aggravates DCM progression. We confirmed connexin 43 (Cx43) dissociation from cell–cell interaction and accumulation at mitochondrial inner membrane both in the cardiomyocytes of patients with DM and DCM murine. Here, we observed that cardiac diastolic function, induced by chronic hyperglycemia, was further aggravated upon hypoglycemia challenge. Similar contractile defects were recapitulated using neonatal mouse ventricular myocytes (NMVMs) under glucose fluctuation challenges. Using immunoprecipitation mass spectrometry, we identified and validated that hypoglycemia challenge activates the mitogen-activated protein kinase kinase (MAPK kinase) (MEK)/extracellular regulated protein kinase (ERK) and inhibits phosphoinositide 3-kinase (PI3K)/Akt pathways, which results in Cx43 phosphorylation by Src protein and translocation to mitochondria in cardiomyocytes. To determine causality, we overexpressed a mitochondrial targeting Cx43 (mtCx43) using adeno-associated virus serotype 2 (AAV2)/9. At normal blood glucose levels, mtCx43 overexpression recapitulated cardiac diastolic dysfunction as well as aberrant electrophysiology in vivo. Our findings give support for therapeutic targeting of MEK/ERK/Src and PI3K/Akt/Src pathways to prevent mtCx43-driven DCM.ConclusionDCM presents compensatory adaptation of mild mtCx43 accumulation, yet acute hypoglycemia challenges result in further accumulation of mtCx43 through the MEK/ERK/Src and PI3K/Akt/Src pathways. We provide evidence that Cx43 mislocalization is present in hearts of patients with DM hearts, STZ-induced DCM murine model, and glucose fluctuation challenged NMVMs. Mechanistically, we demonstrated that mtCx43 is responsible for inducing aberrant contraction and disrupts electrophysiology in cardiomyocytes and our results support targeting of mtCx43 in treating DCM.

Rictor/mTORC2 involves mitochondrial function in ES cells derived cardiomyocytes via mitochondrial Connexin 43

Rictor is a key component of the mammalian target of rapamycin complex 2 (mTORC2) and is required for Akt phosphorylation (Ser473). Our previous study shows that knockdown of Rictor prevents cardiomyocyte differentiation from mouse embryonic stem (ES) cells and induces abnormal electrophysiology of ES cell-derived cardiomyocytes (ESC-CMs). Besides, knockdown of Rictor causes down-expression of connexin 43 (Cx43), the predominant gap junction protein, that is located in both the sarcolemma and mitochondria in cardiomyocytes. Mitochondrial Cx43 (mtCx43) plays a crucial role in mitochondrial function. In this study, we used the model of cardiomyocyte differentiation from mouse ES cells to elucidate the mechanisms for the mitochondrial damage in ESC-CMs after knockdown of Rictor. We showed swollen and ruptured mitochondria were observed after knockdown of Rictor under transmission electron microscope. ATP production and mitochondrial transmembrane potential were significantly decreased in Rictor-knockdown cells. Furthermore, knockdown of Rictor inhibited the activities of mitochondrial respiratory chain complex. The above-mentioned changes were linked to inhibiting the translocation of Cx43 into mitochondria by knockdown of Rictor. We revealed that knockdown of Rictor inactivated the mTOR/Akt signalling pathway and subsequently decreased HDAC6 expression, resulted in Hsp90 hyper-acetylation caused by HDAC6 inhibition, thus, inhibited the formation of Hsp90-Cx43-TOM20 complex. In conclusion, the mitochondrial Cx43 participates in shRNA-Rictor-induced mitochondrial function damage in the ESC-CMs.

Abstract 17186: Molecular Mechanisms Underlying Sex-Related Differences in Mitochondrial Response to Myocardial Ischemia/Reperfusion

Introduction: Cardiac dysfunction adversely impacts patient outcomes following surgically induced myocardial ischemia/reperfusion (I/R). Maintaining mitochondrial activity is crucial to reducing myocardial I/R. Inflammation and oxidative stress triggered by I/R impair mitochondria. Our group has shown sex differences in myocardial functional recovery after I/R. Here, we determined whether sex disparity in mitochondrial response to inflammatory mediator - TNFα and to reactive oxygen species - H 2 O 2 is responsible for sex differences in cardiac dysfunction. Given the key role of mitochondrial connexin43 (Cx43) in cardiac protection initiated by ischemic preconditioning, we also investigated the implication of Cx43 in sex-related mitochondrial response. Methods: Cardiomyocytes (male & female; C57BL/6 & doxycycline inducible Tnnt2-controlled Cx43 knockout) were subjected to simulated I/R (20’ I/60’ R), TNFα (10ng/ml), or H 2 O 2 (50 μM), +/- 17β-estradiol (E2).Mitochondrial membrane potential (ΔΨm) analyzed by JC1 red to JC1 green fluorescence intensity ratio (Fig. A) and mitochondrial respiration capacity by Seahorse XF Cell Mito Stress Test were determined.Cx43 was assessed in mitochondria by Western blots and Immunoelectron microscopy. Results: Improved ΔΨm (Fig. B) and preserved mitochondrial respiratory capacity were observed in female cardiomyocytes than in male ones following simulated I/R, TNFα, or H 2 O 2 .E2 restored mitochondrial activity in cardiomyocytes (Fig. C, D). In addition, E2 increased Cx43 content and phosphorylation in cardiac mitochondria. Ablation of cardiac Cx43 abolished E2-mediated mitochondrial protection during TNFα or H 2 O 2 challenge (Fig. E). Conclusions: Female cardiomyocytes were more resistant to I/R, TNFα, or H 2 O 2 stimulation than age-matched males, with greater protective role of mitochondrial Cx43 in female hearts. E2 usage regulated mitochondrial Cx43 and preserved cardiac mitochondrial performance.

Spermine Protects Cardiomyocytes from High Glucose-Induced Energy Disturbance by Targeting the CaSR-gp78-Ubiquitin Proteasome System.

To determine the mediation of spermine on energy metabolism disorder and diabetic cardiomyopathy (DCM) development as well as the underlying mechanisms. An in vitro model of DCM was established by incubating primary cultured neonatal rat cardiomyocytes with high glucose (HG). Spermine content was assessed by RP-HPLC. The protein levels were detected by western blot. Mitochondrial functions were analyzed using the respiratory chain complex assay kit and immunofluorescence staining. The endogenous content of spermine was decreased in the HG group, and the protein levels of ornithine decarboxylase, respiratory chain complex (I-V), mitochondrial fusion-related protein (Mfn1, Mfn2), Cx43, N-cadherin, CaSR, and β-catenin (in cytomembrane) were also down-regulated by HG. In contrast, the protein levels of spermine-N1-acetyltransferase, gp78, Fis1, Drp1, and β-catenin were up-regulated by HG. Meanwhile, we observed that HG increased ubiquitination levels of Mfn1, Mfn2, and Cx43, decreased membrane potential (ΔΨm), and the opening of mitochondrial permeability transport pore (mPTP) followed by intracellular ATP leakage. The supplement of spermine or siRNA-mediated knockdown of gp78 significantly alleviated the detrimental effects of HG, while downregulation of CaSR aggravated the development of DCM. We further confirmed that the lower level of spermine by HG activates the gp78-ubiquitin-proteasome pathway via downregulation of CaSR protein level, which in turn damages mitochondrial gap junction intercellular communication and leads to reduced ATP level. The protective role of spermine on energy metabolism disorder is based on higher CaSR protein level and lower gp78 activation, pointing to the possibility that spermine can be a target for the prevention and treatment of DCM.

Canonical and Non-Canonical Roles of Connexin43 in Cardioprotection.

Since the mid-20th century, ischemic heart disease has been the world’s leading cause of death. Developing effective clinical cardioprotection strategies would make a significant impact in improving both quality of life and longevity in the worldwide population. Both ex vivo and in vivo animal models of cardiac ischemia/reperfusion (I/R) injury are robustly used in research. Connexin43 (Cx43), the predominant gap junction channel-forming protein in cardiomyocytes, has emerged as a cardioprotective target. Cx43 posttranslational modifications as well as cellular distribution are altered during cardiac reperfusion injury, inducing phosphorylation states and localization detrimental to maintaining intercellular communication and cardiac conduction. Pre- (before ischemia) and post- (after ischemia but before reperfusion) conditioning can abrogate this injury process, preserving Cx43 and reducing cell death. Pre-/post-conditioning has been shown to largely rely on the presence of Cx43, including mitochondrial Cx43, which is implicated to play a major role in pre-conditioning. Posttranslational modifications of Cx43 after injury alter the protein interactome, inducing negative protein cascades and altering protein trafficking, which then causes further damage post-I/R injury. Recently, several peptides based on the Cx43 sequence have been found to successfully diminish cardiac injury in pre-clinical studies.

Trastuzumab-induced cardiotoxicity and role of mitochondrial connexin43 in the adaptive response

Trastuzumab, the humanized monoclonal antibody specific for HER2 receptor, is the gold standard in the treatment of HER2+ breast cancer. Despite its high therapeutic efficacy, cardiotoxicity has emerged as a significant side effect. The molecular mechanisms involved are not well understood, but all converge on mitochondria. Mitochondrial Cx43 can confer cardioprotection by regulating mitochondrial calcium homeostasis, ROS production and propagation of apoptotic signals, and studies report that it is overexpressed both in ischemic preconditioning and in Doxorubicin-induced cardiotoxicity. This study was designed to evaluate whether mitochondrial Cx43 (mCx43) is also involved in Trastuzumab-induced cardiotoxicity. Here we demonstrated that mCx43 is overexpressed in Trastuzumab-treated H9c2 cells. Our data showed that inhibition of Cx43 translocation to mitochondria, obtained by radicicol pre-treatment, significantly increases cytosolic and mitochondrial superoxide formation, mitochondrial membrane depolarization and the consequent apoptosis induced by Trastuzumab. Our results support the hypothesis that disruption of mitochondrial function is the principal mechanism by which Trastuzumab elicits its cardiotoxicity and mCx43 appears to counteract the Trastuzumab-induced mitochondrial damage.

Inhibition of connexin 43 attenuates oxidative stress and apoptosis in human umbilical vein endothelial cells

BackgroundPrevious studies demonstrated an important role for connexin 43 (Cx43) in the regulation of apoptosis by influencing mitochondrial functions. This study aimed to investigate the relationship between Cx43 and lipopolysaccharide (LPS)-induced oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs).MethodsWestern blot was performed to determine mitochondrial Cx43 (MtCx43) protein level and phosphorylation (p-MtCx43). Gap19, a selective Cx43 inhibitor, was used to examine the effects of Cx43 on LPS-induced oxidative stress and apoptosis in HUVECs. Expression of regulatory genes associated with oxidative stress was examined by quantitative polymerase chain reaction (qPCR) and Western blot. Apoptosis was assessed by flow cytometry.ResultsLPS stimulation resulted in increased levels of MtCx43 and p-MtCx43. Interestingly, Gap19 antagonized the upregulation of glutathione S-transferase Zeta 1 (GSTZ1) and cytochrome b alpha beta (CYBB), and the downregulation of antioxidant 1 (ATOX1), glutathione synthetase (GSS) and heme oxygenase 1 (HMOX1) induced by LPS or Cx43 overexpression. Moreover, the increased production of reactive oxygen species (ROS) and apoptosis elicited by LPS or Cx43 overexpression were reduced following treatment with Gap19.ConclusionsSelective inhibition of Cx43 hemichannels protects HUVECs from LPS-induced apoptosis and this may be via a reduction in oxidative stress production.

Mitochondrial connexin 43 in sex-dependent myocardial responses and estrogen-mediated cardiac protection following acute ischemia/reperfusion injury.

Preserving mitochondrial activity is crucial in rescuing cardiac function following acute myocardial ischemia/reperfusion (I/R). The sex difference in myocardial functional recovery has been observed after I/R. Given the key role of mitochondrial connexin43 (Cx43) in cardiac protection initiated by ischemic preconditioning, we aimed to determine the implication of mitochondrial Cx43 in sex-related myocardial responses and toexamine the effect of estrogen (17β-estradiol, E2) on Cx43, particularly mitochondrial Cx43-involved cardiac protection following I/R. Mouse primary cardiomyocytes and isolated mouse hearts (from males, females, ovariectomized females, and doxycycline-inducible Tnnt2-controlled Cx43 knockout without or with acute post-ischemic E2 treatment) were subjected to simulated I/R in culture or Langendorff I/R (25-min warm ischemia/40-min reperfusion), respectively. Mitochondrial membrane potential and mitochondrial superoxide production were measured in cardiomyocytes. Myocardial function and infarct size were determined. Cx43 and its isoform, Gja1-20k, were assessed in mitochondria. Immunoelectron microscopy and co-immunoprecipitation were also used to examine mitochondrial Cx43 and its interaction with estrogen receptor-α by E2 in mitochondria, respectively. There were sex disparities in stress-induced cardiomyocyte mitochondrial function. E2 partially restored mitochondrial activity in cardiomyocytes following acute injury. Post-ischemia infusion of E2 improved functional recovery and reduced infarct size with increased Cx43 content and phosphorylation in mitochondria. Ablation of cardiac Cx43 aggravated mitochondrial damage and abolished E2-mediated cardiac protection during I/R. Female mice were more resistant to myocardial I/R than age-matched males with greater protective role of mitochondrial Cx43 in female hearts. Post-ischemic E2 usage augmented mitochondrial Cx43 content and phosphorylation, increased mitochondrial Gja1-20k, and showed cardiac protection.

Connexin-43 Is a Negative Regulator of Mitochondrial Fission, Mitophagy and Apoptosis of Dividing Hematopoietic Stem Cells through the Drp1-Pink1 Axis

Lifelong production of blood by hematopoietic stem and progenitor cells (HSPC) depends on stem cells self-renewal and their maintenance by bone marrow (BM) microenvironment. BM chemotherapy or whole body irradiation frequently causes hematological abnormalities in cancer patients. HSC within the BM exhibit quiescent state with low mitochondrial reactive oxygen species (ROS) and hyperpolarizing membrane potential, however upon stress hematopoiesis HSC exit quiescence and either self-renew or differentiate in mature cells. Understanding the mechanism controlling hematopoiesis regeneration upon replicative stress is expected to provide molecular targets for amelioration of chemotherapy induced toxicity. Mitochondria recognize and respond to many stresses by modifying mitochondrial dynamics and macroscopic autophagy (mitophagy), and maintains HSC function both at intrinsic and extrinsic level (Ho et al., Nature 2017). Upon myeloablative stress, hematopoietic Connexin 43 (H-Cx43) preserves HSPC survival and efficient blood formation by transfer of damaging excess ROS to BM stromal cells (BMSC), preventing HSC apoptosis and lethal hematology failure (Taniguchi-Ishikwawa E et al., PNAS 2012), and its intermediate C-loop domain has been shown to act as negative regulator of the beclin complex of microautophagy (Eloy Bejarano et al, Nat Cell Biol. 2014). Since HSC apoptosis depends on mitochondrial activation and mitochondrial homeostasis is maintained by coordinated regulation of mitochondrial fission/fusion and mitophagy, we hypothesized that mitochondrial Cx43 may exert a cell-autonomous activity over the mitochondrial turnover upon cell division in the regeneration phase after myelotoxic chemotherapy. To analyze the role of Cx43 in HSC mitochondrial dynamics and fate, we created HSC mitochondrial reporter mice by crossing Cox8 mitochondrial signal localization peptide-Dendra2 fusion protein transgenic (mito-Dendra2) mice with Vav-Cre; Cx43f/f (H-Cx43Δ/Δ)mice. While quiescent Cx43Δ/Δ HSC do not demonstrate any major phenotype, snapshot high resolution microscopy demonstrated that Cx43 deficiency enhances mitochondrial superoxide levels, depolarizes membrane potential and results in larger mitochondria accumulation (~2 fold) after forced division of HSC. In addition, time lapsed cinematography of red-fluorescent, photo-converted mito-Dendra2 demonstrated that Cx43-deficient mitochondria in cycling HSC split into multiple smaller fragments. Phosphorylation of Drp1 (Ser616) and its accumulation with mitochondria is higher in dividing Cx43Δ/ΔHSC, suggesting that Cx43 is a negative regulator of mitochondrial fission and its deficiency facilitates mitochondrial fragmentation. Inhibition of mitochondrial fission with overexpression of the dominant-negative Drp1 mutant (K38A) or the Drp1 inhibitor Mdivi1 prevents the effect of Cx43 deficiency on mitochondrial hyper-fragmentation. The fate of the smaller fragments is mitophagy as demonstrated by increased co-localization of Cx43 deficient mitochondria with the ubiquitin kinase Pink1, the autophagy reporter's p62 and LC3 punctae, and the lysosomal membrane proteins Lamp1/2 in HSC. Genetic silencing of Atg7 or addition of the autophagolysosome inhibitor bafilomycin A1 to dividing HSC prevents the induction of mitophagy in dividing Cx43Δ/Δ HSC. Overexpression of structure-function mutants in Cx43Δ/Δ HSC (Cys-less, with 6 point mutations in Cysteine residues replaced by Alanine, resulting in impaired hemichannel docking or the truncated C-terminus mutant D257 resulting in impaired signaling and intramolecular interactions needed for channel gating) demonstrated that both the formation of whole intercellular channel and the presence of C-terminus once Cx43 channel formed are indispensable for the negative regulatory role of Cx43 on mitophagy. Our results thus identify gap junction protein Cx43 as an important regulator of mitochondrial fission and mitophagy in dividing HSC which may be a target for preservation of hematopoietic stem cell regenerative capacity. DisclosuresNo relevant conflicts of interest to declare.

Bone Marrow Hematopoietic Connexin 43 Is Required for Mitotransfer and AMPK Dependent Mesenchymal Microenvironment Regeneration after Irradiation

Hematopoietic stem cell/progenitor (HSCP) transplantation (HSCT) is routinely used for the treatment of cancer and inborn hematopoietic defects. The bone marrow (BM) microenvironment (ME) is a major regulator of hematopoietic function and fate. Clinical data supports osteoblastic regeneration after HSCT despite the inability of BM mesenchymal stem cells (BM-MSC) to engraft. Therefore, understanding the hematopoietic-dependent mechanisms controlling ME mesenchymal regeneration is expected to provide molecular targets for intervention in the context of HSCT. Hematopoietic connexin-43 (H-Cx43) mediates HSCP survival and efficient blood formation by scavenging damaging excess reactive oxygen species (ROS) through transfer to BM mesenchymal stromal cells (BM-MSC) after chemotherapy, preventing lethal hematopoietic failure (Taniguchi-Ishikwawa E et al., PNAS 2012), while the expression of Cx43 on BM-MSC regulates CXCL12 secretion and HSCP homeostasis (Schajnovitz A et al., Nat. Immunol., 2011). Since Cx43 is expressed in mitochondria, we hypothesized that H-Cx43 mediated ROS transfer upon stress depends on hematopoietic mitochondria transfer and uptake by the BM-MSC. We created chimeric mice by transplanting Vav1-CreTg/-, Cox8 mitochondrial localization signal-Dendra2Tg/- wild-type (mDendra2/WT) or Cx43fl/fl(mDendra2/Cx43Δ/Δ) HSCP to lethally irradiated, congenic WT mice and assessed the recovery of stromal cell regeneration via transfer of mitochondria to BM-MSC. H-Cx43Δ/Δchimeric mice have delayed lympho-hematopoietic recovery after irradiation or chemotherapy which can be reversed by restoration of hematopoietic Cx43 expression. H-Cx43Δ/Δchimeric mice exhibit decreased (~60-80%) and delayed colony-forming-unit-fibroblast (CFU-F) and osteoblast (CFU-Ob) regeneration and hematopoietic recovery. The delayed hematopoietic response in H-Cx43Δ/Δchimeras associated with ~40% reduction in mitochondrial transfer from HSCP to Lin-/CD45-/PDGFRα+/Sca1- BM stromal cells (MSC/P). Reverse transplantation experiments indicate that stromal Cx43 is dispensable for mitochondrial transfer from BM stroma to HSCP. Impaired mitochondrial uptake in H-Cx43Δ/Δchimeras associated with ~30-40% decreased mitochondrial ROS (mROS), membrane potential (MMP) and proliferation (assessed by in vivo BrdU uptake) of recipient MSC/P, suggesting that the transferred mitochondria reprogram the recipient mesenchymal progenitor metabolism. Defects of mitotransfer from H-Cx43Δ/ΔHSCP to BM MSC/P and in recipient BM MSC/P mitochondrial activity were recapitulated in in vitro co-cultures. Interestingly, intracellular [ATP] is upregulated (~2 fold) in MSC/P from chimeric H-Cx43Δ/ΔBM that received donor-derived mitochondria, as compared to donor mitochondria containing MSC/P from WTchimeras. Hemichannel opening causes loss of ATP, we therefore speculated that ATP released from MSC/P upon irradiation and transplantation is uptaken by HSPC, activating mitochondrial transfer as part of BM regeneration. Forced glycolysis-dependent restoration of [ATP] in MSC/P but not in HSCP enhances transfer of mitochondria from HSCP to MSC/P, suggesting that BM stromal [ATP] is an irradiation-responsive positive regulator of mitochondria transfer. Hemichannel-derived exogenous ATP suppresses AMPK activation, which regulates cellular metabolic homeostasis by modulating mitochondrial ROS, mitochondria dynamics and the fate of mitochondria. We found that MSC/P recipient of H-Cx43Δ/Δ mitochondria have increased AMPK activity as assessed by increased phosphorylation of AMPK and its downstream effectors ULK1 and ACC (~2-fold) when compared with MSC/P recipient of H-WT mitochondria, whereas MSC/P containing no donor-derived mitochondria from either chimeric mice are insensitive to the effect of Cx43 deficiency. In vivo administration of the AMPK inhibitor BML-275 dramatically increased the mitochondria transfer from HSCP to MSC/P in WT and H-Cx43Δ/Δ chimeras, and completely restores the negative effect of H-Cx43 deficiency on BM mesenchymal and hematopoietic regeneration. Our data indicate that hematopoietic mitochondrial Cx43 is required to control both mitochondrial transfer and BM ME energetic balance and regeneration after myeloablative irradiation. DisclosuresNo relevant conflicts of interest to declare.

Localization and distribution of connexin 43 in mitochondria of cervical cancer HeLa cells

Objective To study the distribution of connexin 43 (Cx43) in cervical cancer HeLa cells, and to verify the localization of Cx43 in mitochondria. Methods HeLa cells were segregated into cytoplasm, cell nucleus, mitochondria and supernatant after segregation by using the method of homogenate and centrifuge. Immunoelectron microscope was used to observe the morphology of mitochondria and the localization as well as the distribution of Cx43 in HeLa cells. Voltage-dependent anion channel 1 (VDAC1) was used to confirm the localization of mitochondria. Immunofluorescence was used to costain HeLa cells with Cx43 and mitochondrial marker VDAC1 to verify mitochondria localization of Cx43 in cervical cancer HeLa cells. Then Western blot was used to quantify the expression of Cx43 in fractions (cytoplasmic fraction, nuclear, mitochondria and post mitochondrial supernatant). Mitochondrial markers including VDAC1 and cytochrome c oxidase Ⅳ (COX Ⅳ) were used to confirm mitochondria. Plasma membrane marker (LHR) was used to confirm plasma membrane. Results Immunoelectron microscope confirmed that the normal mitochondria or cystic swollen one could be seen in the complete HeLa cells and the detached HeLa cells mitochondria, with the presence of Cx43 and VDAC1 in detached mitochondria. Immunofluorescence showed Cx43 colocalized with VDAC1. There was a significant difference in the Cx43 expressions of the subcellular structure in the HeLa cells [cytoplasm (1.23±0.11), cell nucleus (0.39±0.09), mitochondria (3.67±0.59), supernatant after segregation (0.16±0.06); F = 84.17, P < 0.05]. It also showed that the relative amount of Cx43 in mitochondria was enriched. Conclusions Cx43 is enriched in mitochondria in cervical cancer HeLa cells. Therefore, Cx43 in mitochondria might be a potential target in diagnosis, therapy and prognosis of cervical cancer. Key words: HeLa cells; Connexin 43; Mitochondria; Localization; Distribution

Inhibition of Connexin 43 translocation on mitochondria accelerates CoCl2-induced apoptotic response in a chemical model of hypoxia

Hypoxia is the basis of many myocardial conditions, and it can initiate cell death programs, among which apoptosis is well-known. Connexin 43 (Cx43), the major component of cardiomyocyte Gap Junctions, is important in cardioprotection. Cx43is involved in ischemia/reperfusion injury and ischemic preconditioning's protection in vitro; however, its role on cardiomyocyte under hypoxia is still unclear. Reports have shown that Cx43 is also located at the inner membrane of mitochondria where it has been implicated in enhanced ischemic preconditioning response.In this study, to evaluate the role of mitochondrial Cx43 in hypoxia, we used an in vitro model of chemical hypoxia induced by Cobalt chloride (CoCl2) in H9c2 cell line. CoCl2 (50–100–150μM) was administered for 3 or 6h, alone or combined with Radicicol, that inhibits Cx43 translocation on mitochondria, to demonstrate the crucial role of mitochondrial Cx43 in the cardioprotection. In fact, reduction of Cx43 translocation on mitochondria increases mitochondrial ROS production, cytosolic and mitochondrial calcium overload and mitochondrial membrane depolarization, thus resulting in an increase of the triggering apoptotic pathway.In conclusion, our study demonstrates the involvement of mitochondrial Cx43 in the apoptotic process in a chemical hypoxia model and suggests that mitochondrial Cx43 plays a crucial role in cytoprotection.

The gap junction modifier ZP1609 decreases cardiomyocyte hypercontracture following ischaemia/reperfusion independent from mitochondrial connexin 43.

Dysregulation of gap junction-mediated cell coupling contributes to development of arrhythmias and myocardial damage after ischaemia/reperfusion (I/R). Connexin 43 (Cx43) is present at ventricular gap junctions and also in the mitochondria of cardiomyocytes. The dipeptide (2S, 4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid (ZP1609) has antiarrhythmic properties and reduces infarct size when given at reperfusion. However, it is unclear, whether ZP1609 targets Cx43-containing mitochondria and affects cardiomyocyte hypercontracture following I/R. We studied the effects of ZP1609 on the function of murine sub-sarcolemmal mitochondria (SSM, containing Cx43) and interfibrillar mitochondria (IFM, lacking Cx43). Murine isolated cardiomyocytes were subjected to simulated I/R without and with ZP1609 (applied during I/R or at the onset of reperfusion only), and the number of cardiomyocytes undergoing hypercontracture was quantified. Biochemical pathways targeted by ZP1609 in cardiomyocytes were analysed. ZP1609 inhibited ADP-stimulated respiration and ATP production in SSM and IFM. ROS formation and calcium retention capacities in SSM and IFM were not affected by ZP1609, whereas potassium uptake was enhanced in IFM. The number of rod-shaped cardiomyocytes was increased by ZP1609 (10μM) when administered either during I/R or reperfusion. ZP1609 altered the phosphorylation of proteins contributing to the protection against I/R injury. ZP1609 reduced mitochondrial respiration and ATP production, but enhanced potassium uptake of IFM. Additionally, ZP1609 reduced the extent of cardiomyocytes undergoing hypercontracture following I/R. The protective effect was independent of mitochondrial Cx43, as ZP1609 exerts its effects in Cx43-containing SSM and Cx43-lacking IFM.

Mitochondrial Cx43 hemichannels contribute to mitochondrial calcium entry and cell death in the heart.

Mitochondrial connexin 43 (Cx43) plays a key role in cardiac cytoprotection caused by repeated exposure to short periods of non-lethal ischemia/reperfusion, a condition known as ischemic preconditioning. Cx43 also forms calcium (Ca2+)-permeable hemichannels that may potentially lead to mitochondrial Ca2+ overload and cell death. Here, we studied the role of Cx43 in facilitating mitochondrial Ca2+ entry and investigated its downstream consequences. To that purpose, we used various connexin-targeting peptides interacting with extracellular (Gap26) and intracellular (Gap19, RRNYRRNY) Cx43 domains, and tested their effect on mitochondrial dye- and Ca2+-uptake, electrophysiological properties of plasmalemmal and mitochondrial Cx43 channels, and cell injury/cell death. Our results in isolated mice cardiac subsarcolemmal mitochondria indicate that Cx43 forms hemichannels that contribute to Ca2+ entry and may trigger permeability transition and cell injury/death. RRNYRRNY displayed the strongest effects in all assays and inhibited plasma membrane as well as mitochondrial Cx43 hemichannels. RRNYRRNY also strongly reduced the infarct size in ex vivo cardiac ischemia-reperfusion studies. These results indicate that Cx43 contributes to mitochondrial Ca2+ homeostasis and is involved in triggering cell injury/death pathways that can be inhibited by RRNYRRNY peptide.

Connexin 43 and Mitochondria in Cardiovascular Health and Disease.

Connexin 43 (Cx43) is the major connexin protein in ventricular cardiomyocytes. Six Cx43 proteins assemble into so-called hemichannels at the sarcolemma and opposing hemichannels form gap junctions, which allow the passage of small molecules and electrical current flow between adjacent cells. Apart from its localization at the plasma membrane, Cx43 is also present in cardiomyocyte mitochondria, where it is important for mitochondrial function in terms of oxygen consumption and potassium fluxes. The expression of gap junctional and mitochondrial Cx43 is altered under several pathophysiological conditions among them are hypertension, hypertrophy, hypercholesterolemia, ischemia/reperfusion injury, post-infarction remodeling, and heart failure. The present review will focus on the role of Cx43 in cardiovascular diseases and will highlight the importance of mitochondrial Cx43 in cardioprotection.