Decreased Expression of Gap Junction Delta-2 (GJD2) Messenger RNA and Connexin 36 Protein in Form-Deprivation Myopia of Guinea Pigs
Introduction
Myopia, commonly known as nearsightedness, has emerged as a significant global health concern over the past three decades. In certain urban areas of Southeast Asia, the prevalence of myopia among adolescents has reached alarming rates of 80% to 90%. Among myopic individuals, approximately 10% to 20% develop high myopia, which can lead to severe complications such as retinal detachment, macular retinoschisis, and myopic choroidal neovascularization, potentially causing irreversible vision loss. The etiology of myopia is multifactorial, with both environmental and genetic factors playing crucial roles in its development.
Numerous genome-wide association studies (GWAS) have identified various susceptible genes associated with myopia. Among these, the gap junction delta-2 (GJD2) gene, which encodes the connexin 36 (Cx36) protein, has been consistently linked to myopia. The association between GJD2 and myopia was first identified in 2010 by Solouki et al., and since then, more than ten independent replication studies and GWAS have confirmed this association. Cx36 is a prominent gap junction protein expressed in cones, AII amacrine cells, bipolar cells, and ganglion cells in the mammalian retina. It plays a critical role in electrical synaptic transmission, contributing to signal averaging and noise reduction in vision processing.
Despite the strong genetic evidence linking GJD2 to myopia, functional studies to confirm this association have been lacking. This study aimed to investigate the changes in mRNA and protein expression levels of GJD2 and Cx36 in a form-deprivation myopia (FDM) animal model using guinea pigs. The findings provide valuable insights into the potential role of Cx36 in the development of myopia and offer a foundation for future research into the underlying mechanisms.
Methods
Ethical approval for this study was obtained from the Animal Care and Use Committee of Sichuan University, and all experimental procedures adhered to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.
Animal Model
Four-week-old guinea pigs were obtained from the Animal Laboratory Center of Sichuan University. The animals were randomly divided into two groups: the FDM group and the control group, each consisting of 12 guinea pigs. The FDM group had their right eyes covered with opaque hemispherical plastic lenses for three weeks to induce form-deprivation myopia. The control group wore transparent plano lenses on their right eyes. The left eyes of both groups remained untreated. The lenses were made from polymethyl methacrylate and were specifically designed to fit the guinea pigs’ eyes. The optical area of the lenses in the FDM group was heavily scratched to cause visual form deprivation.
Ocular Biometry Assessment
Ocular biometric measurements were taken at baseline (0-week) and after three weeks of form deprivation. These measurements included refractive status, axial length (AL), and corneal radius of curvature. Refractive status was evaluated using a streak retinoscope, with spherical equivalence (SE) calculated as the spherical degree plus half the cylindrical degree. Axial length was measured using A-scan ultrasonography, and corneal curvature was determined using a portable automatic keratometer. All measurements were performed independently by two optometrists to ensure accuracy.
Tissue Extraction
After three weeks, the guinea pigs were deeply anesthetized, and their eyes were enucleated. The retinas were carefully peeled from the eyes and stored at -80°C until further analysis.
Real-Time Reverse Transcription Quantitative Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from the retinal tissues using TRIzol reagent. Reverse transcription was performed using a Gold Script cDNA kit, and qRT-PCR was conducted using SYBR Green with an ABI 7500 Real-Time PCR System. The relative mRNA expression of GJD2 was calculated using the 2-ΔΔCt method, with β-actin serving as an internal control.
Western Blotting Analysis
Protein was extracted from the retinal tissues using radio-immunoprecipitation assay lysis buffer and quantified using a bicinchoninic acid protein assay kit. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were probed with primary antibodies against Cx36 and β-actin, followed by horseradish peroxidase-conjugated secondary antibodies. The protein bands were visualized using an Odyssey Western blot platform, and the optical densities were analyzed using Alpha Ease FC software.
Statistical Analysis
The data were analyzed using SPSS version 20. The normality of the data was assessed using the Kolmogorov-Smirnov test. Comparisons between the FDM and control groups were made using independent t-tests or Mann-Whitney U tests, as appropriate. A significance level of P < 0.05 was used for all statistical analyses.
Results
Optic and Biometric Parameters
At baseline, there were no significant differences in SE, axial length, or corneal curvature between the FDM and control groups. After three weeks of form deprivation, the FDM group exhibited a significant myopic shift, with SE changing from 4.03 ± 1.45 D to -2.03 ± 0.49 D, representing a shift of approximately -6.75 D. In contrast, the control group remained hyperopic, with only a minor shift of -0.50 D. The axial length increased by 0.74 mm in the FDM group compared to 0.10 mm in the control group. No significant changes in corneal curvature were observed between the two groups.
Relative mRNA and Protein Expression of Cx36
The relative mRNA expression of GJD2 in the FDM group decreased by 31.58% compared to the control group. Similarly, the protein expression of Cx36 in the FDM group was reduced by 37.72% compared to the controls. These findings indicate a significant downregulation of both GJD2 mRNA and Cx36 protein in the retinas of guinea pigs with form-deprivation myopia.
Discussion
This study provides compelling evidence for the involvement of Cx36 in the development of myopia. The significant reduction in both mRNA and protein levels of GJD2 and Cx36 in the FDM model suggests that Cx36 plays a crucial role in the pathogenesis of myopia. These findings align with previous GWAS results that identified GJD2 as a susceptible gene for myopia and offer a functional basis for this association.
Cx36 is a key component of gap junctions in the retina, facilitating electrical coupling between neurons. It is widely expressed in various retinal cells, including cones, AII amacrine cells, bipolar cells, and ganglion cells. The electrical coupling mediated by Cx36 is essential for signal averaging and noise reduction in vision processing. Disruptions in this coupling could potentially impair the quality of the retinal image, leading to abnormal refractive development.
The ON and OFF signaling pathways in the retina are critical for normal vision. An imbalance in these pathways, particularly a defective ON pathway, has been linked to increased susceptibility to form-deprivation myopia. Cx36 is known to play a role in the ON pathway, and its downregulation could disrupt the balance between ON and OFF signaling, contributing to myopia development.
The activity of Cx36 is regulated by light exposure and dopamine levels, both of which are known to influence eye growth and myopia control. Additionally, adenosine has been shown to modulate the phosphorylation state of Cx36, affecting photoreceptor coupling. The cyclic adenosine monophosphate (cAMP) level, which is influenced by adenosine, plays a role in scleral collagen remodeling, a critical step in the development of myopia.
This study has several limitations. The expression changes of Cx36 were only measured at a single time point (three weeks), and capturing the dynamic changes throughout the experimental period could provide more detailed insights. Additionally, the study did not investigate the specific mechanisms by which Cx36 influences myopia development. Future research could explore the effects of Cx36 overexpression or inhibition, as well as the use of Cx36 knockout animal models, to further elucidate its role in myopia.
Conclusion
The findings of this study demonstrate a significant decrease in the expression of GJD2 mRNA and Cx36 protein in the retinas of guinea pigs with form-deprivation myopia. These results provide functional evidence supporting the association between GJD2 and myopia, as identified by previous GWAS. The role of Cx36 in electrical synaptic transmission and its regulation by light and dopamine suggest that it may be a key player in the development of myopia. Further research into the mechanisms underlying Cx36’s involvement in myopia could lead to new therapeutic strategies for preventing and treating this prevalent condition.
doi.org/10.1097/CM9.0000000000000319
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