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

Abstract

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 (I(j)) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage (V(j)) dependence. I(j) records from hMSC-HeLaCx43 and hMSC-HeLaCx40 cell pairs also showed symmetrical and asymmetrical V(j) dependence, while hMSC-HeLaCx45 pairs always produced asymmetrical I(j) with pronounced V(j) gating when the Cx45 side was negative. Symmetrical I(j) suggests that the dominant functional channel is homotypic, while the asymmetrical I(j) suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (gamma(j)) of 30-80 pS. The macroscopic I(j) obtained from hMSC-cardiac myocyte cell pairs exhibited asymmetrical V(j) dependence, while single channel events revealed gamma(j) of the size range 40-100 pS. 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.

Figure 1. Identification of connexins in gap junctions of hMSCs

Immunostaining of Cx43 (A), Cx40 (B) and Cx45 (C). D, immunoblot analysis of Cx43 in canine ventricular myocytes and hMSCs. Whole cell lysates (120μg) from ventricular cells or hMSCs were resolved by SDS, transferred to membranes, and blotted with Cx43 antibodies. Migration of molecular weight markers is indicated to the right of the blot.

Figure 2. Macroscopic and single channel properties of gap junctions between hMSC pairs

Gap junction currents (I_j) elicited from hMSCs using a symmetrical bipolar pulse protocol (10 s, from ±10mV to ±110mV, V_h= 0mV) showed two types of voltage-dependent current deactivation: symmetrical (A) and asymmetrical (B).C, summary plots of normalized instantaneous (○) and steady-state (•) g_jversusV_j. Left panel, quasi-symetrical relationship from 5 pairs; continuous line, Boltzmann fit: V_j,0=−70/65mV, g_j,min= 0.29/0.34, g_j,max= 0.99/1.00, z= 2.2/2.3 for negative/positive V_j. Right panel, asymmetrical relationship from 6 pairs; Boltzmann fit for negative V_j: V_j,0=−72mV, g_j,min= 0.25, g_j,max= 0.99, z= 1.5. D and E, single channel recordings from pairs of hMSCs. Pulse protocol (V₁ and V₂) and associated multichannel currents (I₂) recorded from a cell pair during maintained V_j of ±80mV. The discrete current steps indicate the opening and closing of single channels. Dashed line: zero current level. The all points current histograms on the right-hand side reveal a conductance of ∼50pS.

Figure 3. Macroscopic junctional currents in cell pairs between a hMSC and HeLa cell expressing only Cx40, Cx43 or Cx45

In all cases hMSC to HeLa cell coupling was tested 6–12h after initiating coculture. A, I_j elicited in response to a series of 5 s voltage steps (V_j) in hMSC-HeLaCx43 pairs. Top, symmetrical current deactivation; bottom, asymmetrical current–voltage dependence. B, macroscopic I_j recordings from hMSC–HeLaCx40 pairs exhibit symmetrical (top panel) and asymmetrical (bottom panel) voltage-dependent deactivation. C, asymmetrical I_j from an hMSC–HeLaCx45 pair exhibits voltage-dependent gating when the Cx45 side is relative negative. I_j recorded from hMSC. D, g_j,ss plots versusV_j from pairs between hMSC and transfected HeLa cells. Left panel, hMSC-HeLaCx43 pairs, quasi-symmetrical relationship (•) and asymmetrical relationship (○); continuous and dashed lines are Boltzmann fits (see text for details). Middle panel, symmetrical (•) and asymmetrical (○) relationships from hMSC–HeLaCx40 pairs; the continuous and dashed lines correspond to Boltzmann fits (see text for details). Right panel, asymmetrical relationship from hMSC–HeLaCx45 cell pairs; continuous line, Boltzmann fit for positive V_j (see text for details). E, cell-to-cell Lucifer Yellow (LY) spread in cell pairs: from an hMSC to an hMSC (upper panel), from a HeLaCx43 to an hMSC (middle panel) and from an hMSC to a HeLaCx43 (bottom panel). In all cases a pipette containing 2 mm LY was attached to the left-hand cell in the whole-cell configuration. Epifluorescent micrographs taken at 12 min after dye injection show LY spread to the adjacent (right-hand) cell. The simultaneously measured junctional conductance revealed g_j of ∼13nS, ∼16nS and ∼18nS of the pairs, respectively.

Figure 4. Macroscopic and single channel properties of gap junctions between hMSC–canine ventricle cell pairs

Myocytes were plated between 12 and 72h and cocultured with hMSCs for 6–12h before measuring coupling. A, localization of Cx43 for hMSC-canine ventricle cell pairs. Most of Cx43 was localized to the ventricular cell ends and a small amount of Cx43 was present along the lateral borders. The intensive Cx43 staining was detected between the end of the rod-shaped ventricular cell (middle cell) and the hMSC (right cell). There is no detectable Cx43 staining between the ventricular cell and the hMSC on the left side. B, top, phase-contrast micrograph of a hMSC–canine ventricular myocyte pair. Bottom, monopolar pulse protocol (V₁ and V₂) and associated macroscopic junctional currents (I₂) exhibiting asymmetrical voltage dependence. C, top, multichannel current elicited by symmetrical biphasic 60mV pulse. Dashed line, zero current level; dotted lines, represent discrete current steps indicative of opening and closing of channels. The current histograms yielded a conductance of ∼40–50pS. Bottom, multichannel recording during maintained V_j of 60mV. The current histograms revealed several conductances of 48–64pS with several events with conductance of 84pS to 99pS (arrows) which resemble operation of Cx43, heterotypic Cx40–Cx43 and/or homotypic Cx40 channels.