Laima Valiuniene

Connexin‐specific cell‐to‐cell transfer of short interfering RNA by gap junctions

The purpose of this study was to determine whether oligonucleotides the size of siRNA are permeable to gap junctions and whether a specific siRNA for DNA polymerase β (pol β) can move from one cell to another via gap junctions, thus allowing one cell to inhibit gene expression in another cell directly. To test this hypothesis, fluorescently labelled oligonucleotides (morpholinos) 12, 16 and 24 nucleotides in length were synthesized and introduced into one cell of a pair using a patch pipette. These probes moved from cell to cell through gap junctions composed of connexin 43 (Cx43). Moreover, the rate of transfer declined with increasing length of the oligonucleotide. To test whether siRNA for pol β was permeable to gap junctions we used three cell lines: (1) NRK cells that endogenously express Cx43; (2) Mβ16tsA cells, which express Cx32 and Cx26 but not Cx43; and (3) connexin‐deficient N2A cells. NRK and Mβ16tsA cells were each divided into two groups, one of which was stably transfected to express a small hairpin RNA (shRNA), which gives rise to siRNA that targets pol β. These two pol β knockdown cell lines (NRK‐kcdc and Mβ16tsA‐kcdc) were co‐cultured with labelled wild type, NRK‐wt or Mβ16tsA‐wt cells or N2A cells. The levels of pol β mRNA and protein were determined by semiquantitative RT‐PCR and immunoblotting. Co‐culture of Mβ16tsA‐kcdc cells with Mβ16tsA‐wt, N2A or NRK‐wt cells had no effect on pol β levels in these cells. Similarly, co‐culture of NRK‐kcdc with N2A cells had no effect on pol β levels in the N2A cells. In contrast, co‐culture of NRK‐kcdc with NRK‐wt cells resulted in a significant reduction in pol β in the wt cells. The inability of Mβ16tsA‐kcdc cells to transfer siRNA is consistent with the fact that oligonucleotides of the 12 nucleotide length were not permeable to Cx32/Cx26 channels. This suggested that Cx43 but not Cx32/Cx26 channels allowed the cell‐to‐cell movement of the siRNA. These results support the novel hypothesis that non‐hybridized and possible hybridized forms of siRNA can move between mammalian cells through connexin‐specific gap junctions.

Human mesenchymal stem cells make cardiac connexins and form functional gap junctions

Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell‐to‐cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell‐to‐cell coupling to each other, to HeLa cells transfected with Cx40, Cx43 and Cx45 and to acutely isolated canine ventricular myocytes. The junctional currents (Ij) recorded between hMSC pairs exhibited quasi‐symmetrical and asymmetrical voltage (Vj) dependence. Ij records from hMSC–HeLaCx43 and hMSC–HeLaCx40 cell pairs also showed symmetrical and asymmetrical Vj dependence, while hMSC–HeLaCx45 pairs always produced asymmetrical Ij with pronounced Vj gating when the Cx45 side was negative. Symmetrical Ij suggests that the dominant functional channel is homotypic, while the asymmetrical Ij suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (γj) of 30–80pS. The macroscopic Ij obtained from hMSC–cardiac myocyte cell pairs exhibited asymmetrical Vj dependence, while single channel events revealed γj of the size range 40–100pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.

Coupling an HCN2‐expressing cell to a myocyte creates a two‐cell pacing unit

We examined whether coupling of a ventricular myocyte to a non‐myocyte cell expressing HCN2 could create a two‐cell syncytium capable of generating sustained pacing. Three non‐myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2‐induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell–cell coupling between heterologous pairs increased with time in co‐culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2‐expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell–cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of ∼0.6 to 1.7 Hz (1.09 ± 0.05 Hz). Addition of carbenoxolone (200 μm), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 μm 9‐amino‐1,2,3,4‐tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2‐expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell‐based biological pacemakers in vivo once an understanding of delivery and target cell geometry is defined.

A comparison of two cellular delivery mechanisms for small interfering RNA.

Cellular delivery of small interfering RNAs to target cells of a tissue has the potential to travel by two intercellular pathways. For intimately apposed cells gap junctions allow transport exclusive of the extracellular space. For cells not in intimate contact, exocytotic release of vesicular contents and subsequent retrieval via endocytosis of exosomes and other vesicular contents represent an alternative intercellular delivery system that utilizes the extracellular space. Previous studies have shown siRNA/miRNA transfer from a delivery cell to a target cell via gap junction channels. We hypothesized that siRNA can be delivered via gap junctions and downregulate the expression of a reporter gene, the cyclic nucleotide‐gated cation channel gene (mHCN2), in the recipient cells of cell pairs. Whole‐cell patch clamp was used to measure the mHCN2‐induced current and junctional conductance. The target cells were HEK293 cells that endogenously express Cx43 or HeLaCx43 cells, both transfected with mHCN2. The source cells were HEK293 or HeLaCx43 cells transfected with fluorescent‐labeled siRNA targeting mHCN2. We found that siRNA targeting mHCN2 resulted in significant downregulation of mHCN2 currents both in single cells and the recipient cell of a cell pair. In addition we also documented downregulation in target cells that were not in contact with source cells suggesting an extracellular‐mediated delivery. To test further for extracellular delivery HEK293/HCN2 or HeLaCx43/HCN2 cells were cultured in medium collected from HEK293 or HeLaCx43 cells transfected with fluorescent‐labeled siRNA or fluorescent‐labeled morpholino designed to target HCN2. After 24 h single HEK293/HCN2 or HeLaCx43cells showed accumulation of siRNA. The mHCN2 currents were also down regulated in cells with siRNA uptake. Application of 200 nmol/L Bafilomycin A1, which has been shown to affect endosome acidification and endocytotic activity, resulted in a smaller accumulation of fluorescent‐labeled siRNA in single target cells. In distinction to siRNA, morpholinos targeting HCN2 exhibited greatly reduced extracellularly mediated transfer while in cell pairs, target cells exhibited reduced HCN2 currents consistent with effective gap junction‐mediated delivery.

Altered conductance and permeability of Cx40 mutations associated with atrial fibrillation

Alterations in the permeability and conductance of mutant atrial connexins may contribute to reentry arrhythmias.

Gap Junction Channels Exhibit Connexin-specific Permeability to Cyclic Nucleotides

Kanaporis et al. 2008. J. Gen. Physiol. doi:10.1085/jgp.200709934 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1085%252Fjgp.200709934%26rft_id%253Dinfo%253Apmid%252F18378798%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%