Advances of Traditional Chinese Medicine Regulating Connexin43 in the Prevention and Treatment of Myocardial Infarction

Gap junctions are the main form of interaction between cardiomyocytes, through which the electrochemical activities between cardiomyocytes can be synchronized to maintain the normal function of the heart. Connexins are the basis of gap junctions. Changes in the expression, structural changes (e.g., phosphorylation and dephosphorylation), and distribution of connexins can affect the normal electrophysiological activities of the heart. Myocardial infarction (MI) and concurrent arrhythmia, shock, or heart failure can endanger life. The structural and functional damage of connexin (Cx) 43 in cardiomyocytes is a central part of the pathological progression of MI and is one of the main pathological mechanisms of arrhythmia after MI. Therefore, increasing Cx43 expression has become one of the main measures to prevent MI. Also, intervention in Cx43 expression can improve the structural and electrical remodeling of the myocardium to improve MI prognosis. Here, research progress of Cx43 in MI and its prevention and treatment using Traditional Chinese Medicine formulations is reviewed.

Primary Osteocyte Supernatants Metabolomic Profiling of Two Transgenic Mice With Connexin43 Dominant Negative Mutants

Osteocytes could release some small molecules (≤ 1 kDa) through gap junctions and hemichannels to extracellular environment, such as prostaglandin E2 (PGE2), nitric oxide (NO) and adenosine triphosphate (ATP), which play key roles in transferring signals between bone cells and other tissue cells. Connexin (Cx) 43 is the most abundant connexin in osteocytes. To further discover molecules released by osteocytes through Cx43 channels and better understand the regulatory function of Cx43 channels in osteocytes, we performed non-targeted global metabolomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) on conditioned medium collected from osteocytes isolated from two transgenic mouse models with Cx43 dominant negative mutants driven by a 10 kb-DMP1 promoter: R76W (gap junctions are blocked, whereas hemichannels are promoted) and Δ130-136 (both gap junctions and hemichannels are blocked). The results revealed that several new categories of molecules, such as “fatty acyls” and “carboxylic acids and derivatives”, could be released through osteocytic Cx43 channels. In addition, alteration of Cx43 channel function affected the release of metabolites related to inflammatory reaction and oxidative stress. Pathway analysis further showed that citric acid cycle was the most differential metabolic pathway regulated by Cx43 channels. In sum, these results isolated new potential metabolites released by osteocytes through Cx43 channels, and offered a novel perspective to understand the regulatory mechanisms of osteocytes on themselves and other cells as well.

Activation of Cx-43 Hemichannels Induces the Generation of Ca2+-Oscillations in White Adipocytes and Stimulates Lipolysis.

With fluorescence microscopy, it was revealed that a decrease in the concentration of extracellular calcium ([Ca2+]ex) results in two types of Ca2+-responses in white adipocytes: Ca2+-oscillations and transient Ca2+-signals. Activation of connexin hemichannels is involved in the mechanism of generation of Ca2+-oscillations, since the blockers of connexin hemichannels - carbenoxolone, octanol and proadifen, as well as Cx43 gene knock-down lead to complete suppression of these signals. TIRF microscopy confirmed activation of Cx-43 in response to the reduction of [Ca2+]ex. In response to the activation of Cx-43, the secretion of ATP-containing vesicles from adipocytes occurs. And this ATP release is suppressed in adipocytes along with the Cx43 gene knock-down and is inhibited by Bafilomycin A1, a vacuolar ATPase inhibitor. At the level of intracellular signaling, the generation of Ca2+-oscillations in white adipocytes in response to a decrease in [Ca2+]ex takes place due to the mobilization of Ca2 + ions from the thapsigargin-sensitive Ca2+-pool in the endoplasmic reticulum via IP3R as a result of the activation of P2Y1 purinergic receptors and the phosphoinositide signaling pathway. Such paracrine activation of white adipose tissue in response to the opening of Cx43 hemichannels leads to local signal propagation and regulation of gene expression. At 24 hours after activation of Cx-43 and generation of Ca2+-oscillations in white adipocytes, there is a change in the expression of key genes involved in the regulation of lipolysis, which is accompanied by a decrease in the number of adipocytes that contained lipid droplets. Meanwhile, inhibition or knock-down of Cx-43 leads to inhibition of lipolysis and accumulation of lipid droplets. In this study, we elucidate and research the mechanism of generation of Ca2+-oscillations in white adipocytes in response to decreased concentration of Ca2 + ions in the external environment and show the correlation between periodic Ca2+-modes and lipolysis/lipogenesis balance regulation.

Structural and Functional Relations between the Connective Tissue and Epithelium of Enamel Organ and their Role during Enamel Maturation

The morphological and possible functional interactions between the connective tissue and epithelial elements of enamel organ were examined during the maturation phase, using immunohistochemical techniques. Decalcified mandibular sections (10µm) including incisors from Wistar rats ages 10–12 weeks were used. Sections were incubated with one or two primary antibodies targeting cell cytoskeleton (vimentin, α-actin, α-tubulin), dendritic marker (OX6), gap junctions (cx-43), enzymes (nitric-oxide synthase (nos1) and cyclooxygenase (cox1)), and ion transporters (Na+/H+ exchanger (NHE1) and Na+/Ca2+ exchanger (NCX)) for 24h, before incubation with the appropriate conjugated fluorescent secondary antibodies. Sections were then examined by fluorescence microscopy. Haematoxylin-eosin slides were also employed. Cellular heterogeneity and morphological modulations were identified within the epithelial and connective tissue elements of the enamel organ suggesting complex cellular interactions and indicating the use of enamel organ term to represent all these regions. Also, some ion transportation activity, and nos1 and cox1 signalling pathways have been identified, indicating intercellular communication between these regions. A hypothesis was suggested, to explain the morphological modulation of ameloblasts and papillary cells during enamel maturation aimed to increase the transporting membrane surface area to accomplish faster and bulker ion transportation to achieve controlled pH and to direct Ca2+ towards enamel. Connective tissue covering epithelial cells of the enamel organ showed morphological and physiological interaction during enamel maturation, suggesting new functional concepts.

Abstract 465: A 3D Bioprinted Human Cardiac Cell Platform to Model the Pathophysiology of Diabetes

Type-II diabetes (T2D) patients affected by underlying hyperglycemic (high glucose/blood sugar) conditions often suffer from cardiac atrophy, resulting in tissue mass reduction and debilitating cardiac health. To understand pathophysiological mechanisms during progression of cardiac atrophy, a 3D bioprinted organoid platform was developed from a mixture of hydrogels containing human cardiac cells, including cardiomyocytes (CM), fibroblasts (CF) and endothelial cells (EC), to mimic the functionality of the in-vivo tissue. The organoids were cultured using normoglycemic- or hyperglycemic-conditions. The expression of essential biomarkers in these organoids, for myocardin (Myocd), troponin-I (TRP-I), fibroblast protein-1 (FSP-1) and endothelin-1 (ET-1) was confirmed. To assess the physiological cellular connections during hyperglycemia, the presence of Connexin-43 (CX-43) was assessed in the presence of a CX-43 blocker, gap26. Epigenomic tools were used to simultaneously interrogate histone-modifications by histone 3 lysine 9 mono-methylation (H3K9me1) along with the co-regulation of inflammatory mediators, such as the high mobility group box 1 (HMGB1) and toll like receptor 4 (TLR4) in the cardiac organoids cultured using normal versus hyperglycemic conditions. Organoids exposed to high glucose showed an increased expression of H3K9me1 as well as inflammatory mediators HMGB1 and TLR4. Hyperglycemia also exhibited alterations in expression of Myocd and FSP-1 in the organoids, compared to normoglycemic conditions. Treatment with gap26 affected the CX-43 expression significantly, in organoids cultured under hyperglycemia suggesting that high glucose conditions associated with prolonged diabetes may lead to compromised CM-CF coupling, essential for maintenance of cardiac functionality. Increased levels of H3K9me1 suggest decreased expression of Myocd, which may lead to CM degeneration. Epigenetic modifications including alterations in histone methylation in regulation of the myocardial genes and gap junction proteins under hyperglycemic conditions, may lead to cardiac atrophy. We expect to establish an actual T2D patient iPSC cell derived cardiac platform, to offer new therapeutic opportunities within the field.

Expression of “Connexin 43” in Colorectal Carcinomas: Histopathological and Immunohistochemical Study

BACKGROUND: Colorectal cancer is one of the most common cancers worldwide and leading cause of cancer related deaths. Connexins are integral membrane proteins that form channels between adjacent cells. Gap junction intercellular communication plays essential roles in tissue homoeostasis and regulation of cell growth and differentiation. Connexins can act as either tumor suppressors or tumor promoters. The human connexin protein family contains 21 members, of which the most widely studied is connexin 43 (Cx 43). OBJECTIVES: Investigation of immunohistochemical expression of Cx 43 in cases of colorectal adenoma and carcinoma and correlation of this expression with the clinico-pathological aspects of the tumors. MATERIALS AND METHODS: Seventy formalin fixed paraffin embedded BC tissue sections were randomly collected. All the available data were collected from the patients’ reports. The paraffin blocks were sectioned and stained with hematoxylin and eosin stains for histologic evaluation. Additional sections were immunostained with Cx 43. RESULTS: Cx 43 expression was negative in all studied cases. CONCLUSION: Cx 43 is a tumor suppressor that is lost early in colorectal carcinogenesis and can be considered as potential target for cancer chemoprevention and chemotherapy aiming at restoration of normal connexin expression and functional gap junctions.

Immunohistochemical and Ultrastructural Features of the Seasonal Changes in the Epididymal Epithelium of Camel (Camelus dromedarius)

In order to evaluate the influence of reproductive activity on the functional role of the epididymal epithelium in the Egyptian dromedary camel, Connexin-43 (Cx-43), vascular endothelial growth factor (VEGF), and androgen receptor (AR) immunoreactivity in the epididymal epithelium and the fine structure of the principal, dark, basal, apical, and halo cells were investigated. The secretory activity of the principal cells was amplified in the breeding season, while its endocytotic function became more active in the nonbreeding season. This was evidenced by punctate strong immunoreactive signals for Cx-43, which appeared to be more intense in the apical region of these epithelial cells, and the extremely long slender stereocilia (microvilli) with multiple junctional complexes. The nonbreeding principal cells revealed granular immunoreactive signals for VEGF scattered in the apical and basal cytoplasm. Ultrastructurally, both extreme vacuolation and several multivesicular inclusion bodies were observed in their cytoplasm. Dark cell size greatly diminished in the nonbreeding season and their nuclear morphology greatly changed from oval to lobulated shape. The plasma membrane of the apical cells expressed several infoldings (microvilli) in the breeding season. However, it was almost smooth in the nonbreeding season except for a small microvillus that appeared as a bleb-like projection. In some regions, a strong dense immunoreactivity for VEGF could be recognized in the cytoplasm of the apical cells and some basal ones. Halo cells with numerous multivesicular inclusions occupying most of the cytoplasm and a lobulated eccentric nucleus were detected in the nonbreeding season. In conclusion, these findings indicate that the reproductive activity has a significant impact on the immunohistochemical and ultrastructural profiles of the epithelial cells lining the Egyptian dromedary camel epididymis.

Distinct preterm labor phenotypes have unique inflammatory signatures and contraction associated protein profiles†

Preterm labor (PTL) is the predominant cause of childhood morbidity and mortality. It has several phenotypes, each with a distinct etiology often involving inflammation. Here, in samples of reproductive tissues obtained in early PTL from women with phenotypically defined PTL, we examined the presence and distribution of inflammation and its relationship with prolabor gene expression. In chorioamnionitis (CA-PTL), cytokine protein concentrations were increased across all tissues; in idiopathic (I-PTL), the inflammatory changes were limited to the choriodecidua; inflammation was not a feature of placental abruption (PA-PTL). CA-PTL was associated with activation of p65 in the myometrium and AP-1 in the choriodecidua, and PA-PTL with CREB in the choriodecidua. In the myometrium, PGHS-2 mRNA level was increased in CA- and I-PTL; in the amnion, PGHS-2 mRNA level was higher in PA- and I-PTL, while in CA-PTL, OT, OTR mRNA, and CX-43 expression were increased. In the choriodecidua, PGHS-2 mRNA level was unchanged, but in CA and I-PTL, OT mRNA level were increased and OTR was reduced. These data show that CA-PTL is associated with widespread inflammation and prolabor gene expression. In contrast, in I-PTL, inflammation is limited to the choriodecidua, with discrete increases in PGHS-2 in the amnion and OT in the choriodecidua. Inflammation is not a feature of PA-PTL, which is associated with increased OT and OTR in the amnion.

Modulating cardiac conduction during metabolic ischemia with perfusate sodium and calcium in guinea pig hearts

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.

β-Carotene gingival mucoadhesive patch on Cx-43 and 8-Oxo-dg expression to prevent micronucleus formation due to panoramic radiography exposure

Introduction: β-carotene as a lipid-soluble antioxidant substance, expected to prevents the oxidative reaction and to improve the junctional communication detected by Cx-43. Micronucleus is known as one of the early markers of DNA damage which might be related to carcinogenesis mechanism that increased by panoramic radiography radiation exposure. The objective of this research was to analyse the effect of the β-carotene gingival mucoadhesive patch on Cx-43 expression and 8-oxo-dG to prevent micronucleus formation at the gingival mucosa of New Zealand rabbit due to panoramic radiography radiation exposure. Methods: β-carotene gingival mucoadhesive patch was applied to gingival mucosa of the experimental rabbits. In Group I, the rabbits without patch was a control group. Group II has applied the patch before radiography exposures, Group III during the radiography exposures, and Group IV after radiography exposures. Micronucleus samples were taken from the rabbit’s swabbed gingival mucosa, then stained using modified Feulgen-Rossenbeck. The 8-oxo-dG and Cx-43 expressions were detected using immunohistochemistry technique. The results were then analysed using two-way ANOVA. Results: The increasing number of micronucleus in Group I had a different mean (p < 0.05) with Group III and IV. The expression of 8-oxo-dG and Cx-43 had significant differences (p < 0.05) between Group I and III. This results indicated that β-carotene gingival mucoadhesive patch prevents the increasing number of micronucleus especially when it applied before and during panoramic radiography radiation exposure by the increasing Cx-43 and decreasing 8-oxo-dG expression. Conclusion: The β-carotene gingival mucoadhesive patch can be utilised to prevent the panoramic radiography radiation exposure’s effect. Keywords: β-carotene, gingival-mucoadhesive patch, micronucleus, radiation exposure