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.

Mesenchymal stem cell (MSC) transplantation after ischemia/reperfusion (I/R) injury reduces infarct size and improves cardiac function. We used mouse ventricular myocytes (VMs) in an in vitro model of I/R to determine the mechanism by which MSCs prevent reperfusion injury by paracrine signaling. Exposure of mouse VMs to an ischemic challenge depolarized their mitochondrial membrane potential (Ψ_mito), increased their diastolic Ca²⁺, and significantly attenuated cell shortening. Reperfusion of VMs with Ctrl tyrode or MSC-conditioned tyrode (ConT) resulted in a transient increase of the Ca²⁺ transient amplitudes in all cells. ConT-reperfused cells exhibited a decreased number early after depolarization (EADs) (ConT: 6.3% vs. Ctrl: 28.4%) and prolonged survival (ConT: 58% vs. Ctrl: 33%). Ψ_mito rapidly recovered in Ctrl as well as ConT-treated VMs on reperfusion; however, in Ctrl solution, an exaggerated hyperpolarization of Ψ_mito was determined that preceded the collapse of Ψ_mito. The ability of ConT to attenuate the hyperpolarization of Ψ_mito was suppressed on inhibition of the PI3K/Akt signaling pathway or I_K,ATP. However, protection of Ψ_mito was best mimicked by the reactive oxygen species (ROS) scavenger mitoTEMPO. Analysis of ConT revealed a significant antioxidant capacity that was linked to the presence of extracellular superoxide dismutase (SOD3) in ConT. In conclusion, MSC ConT protects VMs from simulated I/R injury by its SOD3-mediated antioxidant capacity and by delaying the recovery of Ψ_mito through Akt-mediated opening of I_K,ATP. These changes attenuate reperfusion-induced ROS production and prevent the opening of the permeability transition pore and arrhythmic Ca²⁺ release.