Identifying the role of connexin 43 in transport of microRNAs

Abstract

Cardiovascular disease (CVD) is the leading cause of mortality worldwide. Present treatments for CVD rely heavily on surgical interventions and drug therapies that relieve symptoms rather than cure the disease. Therefore, there is active research to find alternative therapeutic approaches for CVD. The small gene regulating molecules, microRNAs (miRNAs) are known to mediate structural changes in cardiovascular diseases. Therapy using miRNAs is a novel approach to restoring function of damaged cardiac tissue in CVD. Global tissue expression of miRNAs is regulated via movement of miRNAs through multiple vesicular transport pathways and gap junctions. Connexin 43 (Cx43) which is the protein constituting the gap junctions specifically between ventricular cardiomyocytes, may provide a potential therapeutic target for regulating the expression of miRNAs in cardiac tissue. The aim of this study was to explore miRNA transport via human Cx43 in a single cell model and then in human cardiomyocytes. I hypothesised that Cx43 will have a role in intercellular miRNA transport and will thus mediate miRNA expression in cardiac cells.

To characterise the function of Cx43 in miRNA transport, without interference from vesicular transport pathways seen in cardiomyocytes, a single cell model of human-Cx43 mRNA injected Xenopus laevis oocytes was constructed. The function of exogenously expressed human Cx43 was determined using in vitro electrophysiology. No significant increase in current was observed in connexin activating Ca2+ free solution for the human-Cx43 mRNA injected oocytes (n=6, N=3)1 in comparison to the control non-Cx43 expressing oocytes (n=6, N=3). However, consistent with the previous studies, current across the cell membrane of all oocytes from both groups was reduced by 100% or 50% on re-application of Ca2+ (2 mM) or non-specific connexin blocker, lanthanum (0.05 mM), respectively. The Ca2+ and lanthanum sensitivity confirms that the observed current is mediated by connexins. Western blot analysis on the native oocyte suggests the presence of endogenous connexin, which could be contributing to the lack of significant functional difference for the human cRNA injected oocyte. Hence, an endogenous connexin knockdown model of oocytes must be considered for testing the permeability of Cx43 to miRNA.

The possibility of transport in adult human cardiomyocytes was tested using AC16 cells derived from human ventricular cardiomyocytes. The transport of transfected miRNA-1 from the cells into its extracellular environment (cell media) was tested using lanthanum (2 mM). No significant difference in miRNA transport was seen upon blocking connexin transport by lanthanum (n=3).

The findings in this thesis represent a first step towards a better understanding of the possibility that miRNA moves through gap junctions in adult human cardiomyocytes.

1n = number of oocytes whereas N= number of frogs oocytes were extracted from.