Mesenchymal stem cells improve cardiac conduction by upregulation of connexin 43 through paracrine signaling

Abstract

Mesenchymal stem cells (MSCs) were shown to improve cell survival and alleviate cardiac arrhythmias when transplanted into cardiac tissue; however, little is known about the mechanism by which MSCs modify the electrophysiological properties of cardiac tissue. We aimed to distinguish the influence of cell-cell coupling between myocytes and MSCs from that of MSC-derived paracrine factors on the spontaneous activity and conduction velocity (θ) of multicellular cardiomyocyte preparations. HL-1 cells were plated on microelectrode arrays and their spontaneous activity and θ was determined from field potential recordings. In heterocellular cultures of MSCs and HL-1 cells the beating frequency was attenuated (t(0h): 2.26 ± 0.18 Hz; t(4h): 1.98 ± 0.26 Hz; P < 0.01) concomitant to the intercellular coupling between MSCs and cardiomyocytes. In HL-1 monolayers supplemented with MSC conditioned media (ConM) or tyrode (ConT) θ significantly increased in a time-dependent manner (ConT: t(0h): 2.4 cm/s ± 0.2; t(4h): 3.1 ± 0.4 cm/s), whereas the beating frequency remained constant. Connexin (Cx)43 mRNA and protein expression levels also increased after ConM or ConT treatment over the same time period. Enhanced low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation after ConT treatment implicates the Wnt signaling pathway. Suppression of Wnt secretion from MSCs (IWP-2; 5 μmol/l) reduced the efficacy of ConT to induce phospho-LRP6 and to increase θ. Inhibition of β-catenin (cardamonin; 10 μmol/l) or GSK3-α/β (LiCl; 5 mmol/l) also suppressed changes in θ, further supporting the hypothesis that MSC-mediated Cx43 upregulation occurs in part through secreted Wnt ligands and activation of the canonical Wnt signaling pathway.

Fig. 1.

Mesenchymal stem cells (MSCs) establish intercellular coupling with cardiomyocytes and modify their electrophysiological properties. Addition of MSCs to a monolayer of spontaneously beating HL-1 cells attenuates their beating frequency over time (A), whereas no change in θ was determined (B). Three-dimensional reconstruction of z-stack images (C, left) obtained by confocal microscopy shows calcein/AM-loaded MSCs on top of an HL-1 monolayer (C, right). Dye transfer through gap junction channels was determined between the 2 cell types. Heterocellular coupling between MSCs and HL-1 cells occurs rapidly over the first hours of coculture (D; solid line) and is suppressed by carbenoxolone (dotted line).

Fig. 2.

Paracrine factors secreted by MSCs increase the conduction velocity of HL-1 monolayers. Conditioned culture medium from MSCs (ConM; A) as well as tyrode conditioned in the presence of mouse MSCs (ConT; B) and human MSCs (h-ConT; B) increased θ in HL-1 monolayers in a time-dependent manner (*P < 0.05 and &P < 0.05 compared with Ctrl). Contour plots of an HL-1 monolayer at 0.5 h (C) and 4 h (D) after addition of ConT are also shown.

Fig. 3.

MSC-conditioned medium increases connexin (Cx)43 expression in cardiomyocytes. mRNA content of Cx43 and Cx45 determined by quantitative RT-PCR in HL-1 cells after 4 h of treatment with ConM showed a significant increase in Cx43 but not Cx45 (A). Densitometric quantitation of Cx43 blots (B) shown (C and D) revealed a significant increase in the ratio of phosphorylated vs. nonphosphorylated Cx43 in both ConM- and ConT-treated groups (* and &P < 0.05 compared with respective Ctrl). Western blotting results of ConM (C)- and ConT (D)-treated HL-1 cells probed for Cx43 and Na_v1.5 protein levels are shown. GAPDH is shown as a loading control (n = 3 for each group).

Fig. 4.

β-Catenin inhibition prevents the ConT-mediated increase in Cx43 protein levels. Western blotting analysis of Cx43 protein levels (A) after 4 h of ConT or Ctrl treatment in the presence or absence of cardamonin (Carda: 10 μmol/l; n = 3 for each) is shown. Cardamonin prevented the ConT-mediated increase of θ in HL-1 monolayers (B; *P < 0.05 compared with Ctrl).

Fig. 5.

Lithium inhibition of GSK-3β mimicks the effect of ConT on θ and Cx43 protein expression. Supplementation of Ctrl tyrode with LiCl (4 h) led to an increase in Cx43 protein levels in HL-1 monolayers (A). A concomitant increase in θ (B) was seen in spontaneously beating HL-1 cells. The LiCl-induced increase in θ was not additive to the effect of ConT (*P < 0.05 compared with Ctrl; &P < 0.05 compared with Ctrl + Li).

Fig. 6.

A ConM-mediated increase in θ does not depend on phosphatidylinositol 3-kinase/Akt signaling. Western blotting analysis of ConM-mediated (4 h) changes in p-Akt and Cx43 in the presence and absence of wortmannin (WM; 50 and 100 nM; A) is shown. ConM (4 h) also increased p-ERK 1/2 whereas changes in p-Akt were close to baseline (B). GAPDH is shown as a loading control for both experiments. The change of θ in Ctrl and ConT-treated HL-1 cells with and without the addition of an anti-VEGF receptor (VEGF-R) antibody (*P < 0.05 compared with Ctrl; C) is shown.

Fig. 7.

ConT-mediated changes depend upon the activation of low-density lipoprotein receptor-related protein 6 (LRP6) through agonists of the canonical Wnt-signaling pathway. Changes in p-LRP6, p-ERK1/2, and Cx43 after 30 min (A) or 4 h (C) of incubation with ConT that was generated from control MSCs or MSCs treated with IWP-2 (ConT_IWP-2) are shown. Densitometric quantitation (B) of the blot shown in A revealed significantly reduced p-LRP-6 (n = 3), without significant reduction in Cx43 protein. At both time points p-ERK1/2 was increased well above control levels (n = 3). GAPDH was used as a loading control. The ConT_IWP-2-treated HL-1 monolayer showed an attenuated increase in θ (D) compared with ConT-treated cultures (*P < 0.05 between treatment groups indicated). Rel, relative.

Fig. 8.

Canonical Wnt signaling and ERK1/2 signaling pathways affect Cx43 expression. Treatment of HL-1 monolayers with media obtained from Wnt3a overexpressing L1 cells significantly increased the θ after 4 h (A; *P < 0.05 compared with Ctrl). PD98058, an ERK1/2 inhibitor, attenuated the ConT-mediated increase of θ in HL-1 monolayers (B; *P < 0.05 compared with Ctrl; &P < 0.05 compared with Ctrl + PD98059; #P < 0.05 compared with ConT). A schematic summary of the experimental results illustrates the proposed signal transduction pathway (C). RTK, receptor tyrosine kinase.