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Background: The phenomenon of intercellular mitochondrial transfer from mesenchymal stromal cells (MSCs) has shown promise for improving tissue healing after injury and has potential for treating degenerative diseases like osteoarthritis (OA). Recently MSC to chondrocyte mitochondrial transfer has been documented, but the mechanism of transfer is unknown. Full-length connexin 43 (Cx43, encoded by GJA1) and the truncated, internally translated isoform GJA1-20k have been implicated in mitochondrial transfer between highly oxidative cells, but have not been explored in orthopaedic tissues. Here, our goal was to investigate the role of Cx43 in MSC to chondrocyte mitochondrial transfer. In this study, we tested the hypotheses that (a) mitochondrial transfer from MSCs to chondrocytes is increased when chondrocytes are under oxidative stress and (b) MSC Cx43 expression mediates mitochondrial transfer to chondrocytes. Methods: Oxidative stress was induced in immortalized human chondrocytes using tert-Butyl hydroperoxide (t-BHP) and cells were evaluated for mitochondrial membrane depolarization and reactive oxygen species (ROS) production. Human bone-marrow derived MSCs were transduced for mitochondrial fluorescence using lentiviral vectors. MSC Cx43 expression was knocked down using siRNA or overexpressed (GJA1 + and GJA1-20k+) using lentiviral transduction. Chondrocytes and MSCs were co-cultured for 24 h in direct contact or separated using transwells. Mitochondrial transfer was quantified using flow cytometry. Co-cultures were fixed and stained for actin and Cx43 to visualize cell-cell interactions during transfer. Results: Mitochondrial transfer was significantly higher in t-BHP-stressed chondrocytes. Contact co-cultures had significantly higher mitochondrial transfer compared to transwell co-cultures. Confocal images showed direct cell contacts between MSCs and chondrocytes where Cx43 staining was enriched at the terminal ends of actin cellular extensions containing mitochondria in MSCs. MSC Cx43 expression was associated with the magnitude of mitochondrial transfer to chondrocytes; knocking down Cx43 significantly decreased transfer while Cx43 overexpression significantly increased transfer. Interestingly, GJA1-20k expression was highly correlated with incidence of mitochondrial transfer from MSCs to chondrocytes. Conclusions: Overexpression of GJA1-20k in MSCs increases mitochondrial transfer to chondrocytes, highlighting GJA1-20k as a potential target for promoting mitochondrial transfer from MSCs as a regenerative therapy for cartilage tissue repair in OA. [ABSTRACT FROM AUTHOR]

Background: Folate is an essential B-group vitamin and a key methyl donor with important biological functions including DNA methylation regulation. Normal neurodevelopment and physiology are sensitive to the cellular folate levels. Either deficiency or excess of folate may lead to neurological disorders. Recently, folate has been linked to tRNA cytosine-5 methylation (m5C) and translation in mammalian mitochondria. However, the influence of folate intake on neuronal mRNA m5C modification and translation remains largely unknown. Here, we provide transcriptome-wide landscapes of m5C modification in poly(A)-enriched RNAs together with mRNA transcription and translation profiles for mouse neural stem cells (NSCs) cultured in three different concentrations of folate. Results: NSCs cultured in three different concentrations of folate showed distinct mRNA methylation profiles. Despite uncovering only a few differentially expressed genes, hundreds of differentially translated genes were identified in NSCs with folate deficiency or supplementation. The differentially translated genes induced by low folate are associated with cytoplasmic translation and mitochondrial function, while the differentially translated genes induced by high folate are associated with increased neural stem cell proliferation. Interestingly, compared to total mRNAs, polysome mRNAs contained high levels of m5C. Furthermore, an integrative analysis indicated a transcript-specific relationship between RNA m5C methylation and mRNA translation efficiency. Conclusions: Altogether, our study reports a transcriptome-wide influence of folate on mRNA m5C methylation and translation in NSCs and reveals a potential link between mRNA m5C methylation and mRNA translation. [ABSTRACT FROM AUTHOR]

Ovarian cancer is the deadliest gynecological cancer in women, with a survival rate of less than 30% when the cancer has spread throughout the peritoneal cavity. Aggregation of cancer cells increases their viability and metastatic potential; however, there are limited studies that correlate these functional changes to specific phenotypic alterations. In this study, we investigated changes in mitochondrial morphology and dynamics during malignant transition using our MOSE cell model for progressive serous ovarian cancer. Mitochondrial morphology was changed with increasing malignancy from a filamentous network to single, enlarged organelles due to an imbalance of mitochondrial dynamic proteins (fusion: MFN1/OPA1, fission: DRP1/FIS1). These phenotypic alterations aided the adaptation to hypoxia through the promotion of autophagy and were accompanied by changes in the mitochondrial ultrastructure, mitochondrial membrane potential, and the regulation of reactive oxygen species (ROS) levels. The tumor-initiating cells increased mitochondrial fragmentation after aggregation and exposure to hypoxia that correlated well with our previously observed reduced growth and respiration in spheroids, suggesting that these alterations promote viability in non-permissive conditions. Our identification of such mitochondrial phenotypic changes in malignancy provides a model in which to identify targets for interventions aimed at suppressing metastases. [ABSTRACT FROM AUTHOR]

Myocardial ischemia leads to conduction slowing, cell-to-cell uncoupling, and arrhythmias. We previously demonstrated that varying perfusate sodium (Na+) and calcium (Ca2+) attenuates conduction slowing and arrhythmias during simulated ischemia with continuous perfusion. Cardioprotection was selectively associated with widening of the perinexus, a gap junction adjacent nanodomain important to ephaptic coupling. It is unknown whether perfusate composition affects the perinexus or ischemic conduction during nonsimulated ischemia, when coronary flow is reduced or halted. We hypothesized that altering preischemic perfusate composition could facilitate perinexal expansion and attenuate conduction slowing during global ischemia. To test this hypothesis, ex vivo guinea pig hearts (n = 49) were Langendorff perfused with 145 or 153 mM Na+ and 1.25 or 2.0 mM Ca2+ and optically mapped during 30 min of no-flow ischemia. Altering Na+ and Ca2+ did not substantially affect baseline conduction. Increasing Na+ and decreasing Ca2+ both lowered pacing thresholds, whereas increasing Ca2+ narrowed perinexal width (Wp). A least squares mean estimate revealed that reduced perfusate Na+ and Ca2+ resulted in the most severe conduction slowing during ischemia. Increasing Na+ alone modestly attenuated conduction slowing, yet significantly delayed the median time to conduction block (10 to 16 min). Increasing both Na+ and Ca2+ selectively widened Wp during ischemia (22.7 vs. 15.7 nm) and attenuated conduction slowing to the greatest extent. Neither repolarization nor levels of total or phosphorylated connexin43 correlated with conduction slowing or block. Thus, perfusate-dependent widening of the perinexus preserved ischemic conduction and may be an adaptive response to ischemic stress. NEW & NOTEWORTHY Conduction slowing during acute ischemia creates an arrhythmogenic substrate. We have shown that extracellular ionic concentrations can alter conduction by modulating ephaptic coupling. Here, we demonstrate increased extracellular sodium and calcium significantly attenuate conduction slowing during no-flow ischemia. This effect was associated with selective widening of the perinexus, an intercalated disc nanodomain and putative cardiac ephapse. These findings suggest that acute changes in ephaptic coupling may serve as an adaptive response to ischemic stress. [ABSTRACT FROM AUTHOR]

We previously demonstrated that altering extracellular sodium (Nao) and calcium (Cao) can modulate a form of electrical communication between cardiomyocytes termed "ephaptic coupling" (EpC), especially during loss of gap junction coupling. We hypothesized that altering Nao and Cao modulates conduction velocity (CV) and arrhythmic burden during ischemia. Electrophysiology was quantified by optically mapping Langendorff-perfused guinea pig ventricles with modified Nao (147 or 155 mM) and Cao (1.25 or 2.0 mM) during 30 min of simulated metabolic ischemia (pH 6.5, anoxia, aglycemia). Gap junction- adjacent perinexal width (WP), a candidate cardiac ephapse, and connexin (Cx)43 protein expression and Cx43 phosphorylation at S368 were quantified by transmission electron microscopy and Western immunoblot analysis, respectively. Metabolic ischemia slowed CV in hearts perfused with 147 mM Nao and 2.0 mM Cao; however, theoretically increasing EpC with 155 mM Nao was arrhythmogenic, and CV could not be measured. Reducing Cao to 1.25 mM expanded WP, as expected during ischemia, consistent with reduced EpC, but attenuated CV slowing while delaying arrhythmia onset. These results were further supported by osmotically reducing WP with albumin, which exacerbated CV slowing and increased early arrhythmias during ischemia, whereas mannitol expanded WP, permitted conduction, and delayed the onset of arrhythmias. Cx43 expression patterns during the various interventions insufficiently correlated with observed CV changes and arrhythmic burden. In conclusion, decreasing perfusate calcium during metabolic ischemia enhances perinexal expansion, attenuates conduction slowing, and delays arrhythmias. Thus, perinexal expansion may be cardioprotective during metabolic ischemia. NEW & NOTEWORTHY This study demonstrates, for the first time, that modulating perfusate ion composition can alter cardiac electrophysiology during simulated metabolic ischemia. [ABSTRACT FROM AUTHOR]

Background: Tumor Necrosis Factor a (TNFa) upregulation during acute inflammatory response has been associated with numerous cardiac effects including modulating Connexin43 and vascular permeability. This may in turn alter cardiac gap junctional (GJ) coupling and extracellular volume (ephaptic coupling) respectively. We hypothesized that acute exposure to pathophysiological TNFa levels can modulate conduction velocity (CV) in the heart by altering electrical coupling: GJ and ephaptic. Methods and Results: Hearts were optically mapped to determine CV from control, TNFa and TNFa + high calcium(2.5 vs. 1.25 mM) treated guinea pig hearts over 90 mins. Transmission electron microscopy was performed to measure changes in intercellular separation in the gap junction-adjacent extracellular nanodomain--perinexus (WP). Cx43 expression and phosphorylation were determined by Western blotting and Cx43 distribution by confocal immunofluorescence. At 90 mins, longitudinal and transverse CV (CVL and CVT, respectively) increased with control Tyrode perfusion but TNFa slowed CVT alone relative to control and anisotropy of conduction increased, but not significantly. TNFa increased WP relative to control at 90 mins, without significantly changing GJ coupling. Increasing extracellular calcium after 30 mins of just TNFa exposure increased CVT within 15 mins. TNFa + high calcium also restored CVT at 90mins and reduced WP to control values. Interestingly, TNFa + high calcium also improved GJ coupling at 90mins, which along with reduced WP may have contributed to increasing CV. Conclusions: Elevating extracellular calcium during acute TNFa exposure reduces perinexal expansion, increases ephaptic, and GJ coupling, improves CV and may be a novel method for preventing inflammation induced CV slowing. [ABSTRACT FROM AUTHOR]

Our laboratory previously demonstrated that perfusate sodium and potassium concentrations can modulate cardiac conduction velocity (CV) consistent with theoretical predictions of ephaptic coupling (EpC). EpC depends on the ionic currents and intercellular separation in sodium channel rich intercalated disk microdomains like the perinexus. We suggested that perinexal width (WP) correlates with changes in extracellular calcium ([Ca2+]o). Here, we test the hypothesis that increasing [Ca2+]o reduces WP and increases CV. Mathematical models of EpC also predict that reducing WP can reduce sodium driving force and CV by self-attenuation. Therefore, we further hypothesized that reducing WP and extracellular sodium ([Na+]o) will reduce CV consistent with ephaptic self-attenuation. Transmission electron microscopy revealed that increasing [Ca2+]o (1 to 3.4 mM) significantly decreased WP. Optically mapping wild-type (WT) (100% Cx43) mouse hearts demonstrated that increasing [Ca2+]o increases transverse CV during normonatremia (147.3 mM), but slows transverse CV during hyponatremia (120 mM). Additionally, CV in heterozygous (~50% Cx43) hearts was more sensitive to changes in [Ca2+]o relative to WT during normonatremia. During hyponatremia, CV slowed in both WT and heterozygous hearts to the same extent. Importantly, neither [Ca2+]o nor [Na+]o altered Cx43 expression or phosphorylation determined by Western blotting, or gap junctional resistance determined by electrical impedance spectroscopy. Narrowing WP, by increasing [Ca2+]o, increases CV consistent with enhanced EpC between myocytes. Interestingly, during hyponatremia, reducing WP slowed CV, consistent with theoretical predictions of ephaptic self-attenuation. This study suggests that serum ion concentrations may be an important determinant of cardiac disease expression. [ABSTRACT FROM AUTHOR]

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14-3-3 proteins can regulate protein trafficking, and a 14-3-3 mode-1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C-terminus. We hypothesized that Cx43Ser373 phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14-3-3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14-3-3τ in cell lines increases gap junction plaque size at cell-cell borders. Cx43S373A mutation prevents Cx43/14-3-3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff-perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no-flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen-activated protein kinase has previously been implicated in Cx43 internalization. The Cx43S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium. [ABSTRACT FROM AUTHOR]

Summary: During each heartbeat, intercellular electrical coupling via connexin43 (Cx43) gap junctions enables synchronous cardiac contraction. In failing hearts, impaired Cx43 trafficking reduces gap junction coupling, resulting in arrhythmias. Here we report that internal translation within Cx43 (GJA1) mRNA occurs, resulting in truncated isoforms that autoregulate Cx43 trafficking. We find that at least four truncated Cx43 isoforms occur in the human heart, with a 20 kDa isoform predominating. In-frame AUG codons within GJA1 mRNA are the translation initiation sites and their ablation arrests trafficking of full-length Cx43. The 20 kDa isoform is sufficient to rescue this trafficking defect in trans, suggesting it as a trafficking chaperone for Cx43. Limiting cap-dependent translation through inhibition of mTOR enhances truncated isoform expression, increasing Cx43 gap junction size. The results suggest that internal translation is a mechanism of membrane protein autoregulation and a potent target for therapies aimed at restoring normal electrical coupling in diseased hearts. [Copyright &y& Elsevier]

In cancer progression, carcinoma cells gain invasive behavior through a loss of epithelial characteristics and acquisition of mesenchymal properties, a process that can lead to epithelial-mesenchymal transition (EMT). TGF-β is a potent inducer of EMT, and increased TGF-β signaling in cancer cells is thought to drive cancer-associated EMT. Here, we examine the physiological requirement for mTOR complex 2 (mTORC2) in cells undergoing EMT. TGF-β rapidly induces mTORC2 kinase activity in cells undergoing EMT, and controls epithelial cell progression through EMT. By regulating EMT-associated cytoskeletal changes and gene expression, mTORC2 is required for cell migration and invasion. Furthermore, inactivation of mTORC2 prevents cancer cell dissemination in vivo. Our results suggest that the mTORC2 pathway is an essential downstream branch of TGF-β signaling, and represents a responsive target to inhibit EMT and prevent cancer cell invasion and metastasis. [ABSTRACT FROM AUTHOR]

Rapid electrical conduction in the His-Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His-Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction-forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. lrx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for lrx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart. [ABSTRACT FROM AUTHOR]

The BAR domain protein superfamily is involved in membrane invagination and endocytosis, but its role in organizing membrane proteins has not been explored. In particular, the membrane scaffolding protein BIN1 functions to initiate Ttubule genesis in skeletal muscle cells. Constitutive knockdown of BIN1 in mice is perinatal lethal, which is associated with an induced dilated hypertrophic cardiomyopathy. However, the functional role of BIN1 in cardiomyocytes is not known. An important function of cardiac T-tubules is to allow L-type calcium channels (Cav1.2) to be in close proximity to sarcoplasmic reticulum-based ryanodine receptors to initiate the intracellular calcium transient. Efficient excitation-contraction (EC) coupling and normal cardiac contractility depend upon Cav1.2 localization to T-tubules. We hypothesized that BIN1 not only exists at cardiac T-tubules, but it also localizes Cav1.2 to these membrane structures. We report that BIN1 localizes to cardiac T-tubules and clusters there with Cav1.2. Studies involve freshly acquired human and mouse adult cardiomyocytes using complementary immunocytochemistry, electron microscopy with dual immunogold labeling, and co-immunoprecipitation. Furthermore, we use surface biotinylation and live cell confocal and total internal fluorescence microscopy imaging in cardiomyocytes and cell lines to explore delivery of Cav1.2 to BIN1 structures. We find visually and quantitatively that dynamic microtubules are tethered to membrane scaffolded by BIN1, allowing targeted delivery of Cav1.2 from the microtubules to the associated membrane. Since Cav1.2 delivery to BIN1 occurs in reductionist non-myocyte cell lines, we find that other myocyte-specific structures are not essential and there is an intrinsic relationship between microtubulebased Cav1.2 delivery and its BIN1 scaffold. In differentiated mouse cardiomyocytes, knockdown of BIN1 reduces surface Cav1.2 and delays development of the calcium transient, indicating that Cav1.2 targeting to BIN1 is functionally important to cardiac calcium signaling. We have identified that membrane-associated BIN1 not only induces membrane curvature but can direct specific antegrade delivery of microtubule-transported membrane proteins. Furthermore, this paradigm provides a microtubule and BIN1-dependent mechanism of Cav1.2 delivery to T-tubules. This novel Cav1.2 trafficking pathway should serve as an important regulatory aspect of EC coupling, affecting cardiac contractility in mammalian hearts. [ABSTRACT FROM AUTHOR]

Recent studies revealed that relatively small changes in perfusate sodium ([Na+]o) composition significantly affect cardiac electrical conduction and stability in contraction arrested ex vivo Langendorff heart preparations before and during simulated ischemia. Additionally, [Na+]o modulates cardiomyocyte contractility via a sodium-calcium exchanger (NCX) mediated pathway. It remains unknown, however, whether modest changes to [Na+]o that promote electrophysiologic stability similarly improve mechanical function during baseline and ischemia–reperfusion conditions. The purpose of this study was to quantify cardiac mechanical function during ischemia–reperfusion with perfusates containing 145 or 155 mM Na+ in Langendorff perfused isolated rat heart preparations. Relative to 145 mM Na+, perfusion with 155 mM [Na+]o decreased the amplitude of left-ventricular developed pressure (LVDP) at baseline and accelerated the onset of ischemic contracture. Inhibiting NCX with SEA0400 abolished LVDP depression caused by increasing [Na+]o at baseline and reduced the time to peak ischemic contracture. Ischemia–reperfusion decreased LVDP in all hearts with return of intrinsic activity, and reperfusion with 155 mM [Na+]o further depressed mechanical function. In summary, elevating [Na+]o by as little as 10 mM can significantly modulate mechanical function under baseline conditions, as well as during ischemia and reperfusion. Importantly, clinical use of Normal Saline, which contains 155 mM [Na+]o, with cardiac ischemia may require further investigation. [ABSTRACT FROM AUTHOR]

Vascular pericytes provide critical contributions to the formation and integrity of the blood vessel wall within the microcirculation. Pericytes maintain vascular stability and homeostasis by promoting endothelial cell junctions and depositing extracellular matrix (ECM) components within the vascular basement membrane, among other vital functions. As their importance in sustaining microvessel health within various tissues and organs continues to emerge, so does their role in a number of pathological conditions including cancer, diabetic retinopathy, and neurological disorders. Here, we review vascular pericyte contributions to the development and remodeling of the microcirculation, with a focus on the local microenvironment during these processes. We discuss observations of their earliest involvement in vascular development and essential cues for their recruitment to the remodeling endothelium. Pericyte involvement in the angiogenic sprouting context is also considered with specific attention to crosstalk with endothelial cells such as through signaling regulation and ECM deposition. We also address specific aspects of the collective cell migration and dynamic interactions between pericytes and endothelial cells during angiogenic sprouting. Lastly, we discuss pericyte contributions to mechanisms underlying the transition from active vessel remodeling to the maturation and quiescence phase of vascular development. [ABSTRACT FROM AUTHOR]

Connexin43 (Cx43; gene name GJA1) is the most ubiquitously expressed gap junction protein, and understanding of its regulation largely falls under transcription and post-translational modification. In addition to Cx43, Gja1 mRNA encodes internally translated isoforms regulating gap junction formation, whose expression is modulated by TGF-β. Here, using RLM-RACE, we identify distinct Gja1 transcripts differing only in 5′ UTR length, of which two are upregulated during TGF-β exposure and hypoxia. Introduction of these transcripts into Gja1 −/− cells phenocopies the response of Gja1 to TGF-β with reduced internal translation initiation. Inhibiting pathways downstream of TGF-β selectively regulates levels of Gja1 transcript isoforms and translation products. Reporter assays reveal enhanced translation of full-length Cx43 from shorter Gja1 5′ UTR isoforms. We also observe a correlation among UTR selection, translation, and reduced gap junction formation in aged heart tissue. These data elucidate a relationship between transcript isoform expression and translation initiation regulating intercellular communication. • In aged hearts and during hypoxia or TGF-β treatment, the GJA1 mRNA 5′ UTR is truncated • Activation of P38 and ERK signal transduction pathways regulate GJA1 5′ UTR length • Truncated GJA1 5′ UTRs are sufficient to suppress internal translation of GJA1-20k • Reduced GJA1-20k expression is correlated with P38 activation in aged hearts Connexin43 gap junctions enable direct intercellular communication facilitating action potential propagation. Internal translation of connexin43 mRNA generates the truncated isoform GJA1-20k, which promotes gap junction formation. During aging, Zeitz et al. find that activation of stress-response pathways shortens connexin43 mRNA UTRs to limit GJA1-20k translation coincident with gap junction loss. [ABSTRACT FROM AUTHOR]