Delayed Enrichment of Mesenchymal Cells Promotes Cardiac Lineage and Calcium Transient Development

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can be induced to differentiate into myogenic cells. Despite their potential, previous studies have not been successful in producing a high percentage of cardiac-like cells with a muscle phenotype. We hypothesized that cardiac lineage development in BM-MSC is related to cell passage, culture milieu, and enrichment for specific cell subtypes before and during differentiation. Our study demonstrated that Lin− BM-MSC at an intermediate passage (IP; P8–P12) expressed cardiac troponin T (cTnT) after 21 days in culture. Cardiac TnT expression was similar whether IP cells were differentiated in media containing 5-azacytidine+2% FBS (AZA; 14%) or 2% FBS alone (LS; 12%) and both were significantly higher than AZA+5% FBS. This expression was potentiated by first enriching for CD117/Sca-1 cells followed by differentiation (AZA, 39% and LS, 28%). A second sequential enrichment for the dihydropyridine receptor subunit α2δ1 (DHPR-α2) resulted in cardiac TnT expressed in 54% of cultured cells compared to 28% of cells after CD117/Sca-1+ enrichment. Cells enriched for CD117/Sca-1 and subjected to differentiation displayed spontaneous intracellular Ca2+ transients with an increase in transient frequency and a 60% decrease in the transient duration amplitude between days 14 and 29. In conclusion, IP CD117/ Sca-1+ murine BM-MSCs display robust cardiac muscle lineage development that can be induced independent of AZA but is diminished under higher serum concentrations. Furthermore, temporal changes in calcium kinetics commensurate with increased cTnT expression suggest progressive maturation of a cardiac muscle lineage. Enrichment with CD117/Sca-1 to establish lineage commitment followed by DHPR- α2 in lineage developing cells may enhance the therapeutic potential of these cells for transplantation

The ‘Reverse FDUF’ Mechanism of Atrial Excitation–Contraction Coupling Sustains Calcium Alternans—A Hypothesis

Cardiac calcium alternans is defined as beat-to-beat alternations of Ca transient (CaT) amplitude and has been linked to cardiac arrhythmia, including atrial fibrillation. We investigated the mechanism of atrial alternans in isolated rabbit atrial myocytes using high-resolution line scan confocal Ca imaging. Alternans was induced by increasing the pacing frequency until stable alternans was observed (1.6–2.5 Hz at room temperature). In atrial myocytes, action potential-induced Ca release is initiated in the cell periphery and subsequently propagates towards the cell center by Ca-induced Ca release (CICR) in a Ca wave-like fashion, driven by the newly identified ‘fire-diffuse-uptake-fire’ (FDUF) mechanism. The development of CaT alternans was accompanied by characteristic changes of the spatio-temporal organization of the CaT. During the later phase of the CaT, central [Ca]i exceeded peripheral [Ca]i that was indicative of a reversal of the subcellular [Ca]i gradient from centripetal to centrifugal. This gradient reversal resulted in a reversal of CICR propagation, causing a secondary Ca release during the large-amplitude alternans CaT, thereby prolonging the CaT, enhancing Ca-release refractoriness and reducing Ca release on the subsequent beat, thus enhancing the degree of CaT alternans. Here, we propose the ‘reverse FDUF’ mechanism as a novel cellular mechanism of atrial CaT alternans, which explains how the uncoupling of central from peripheral Ca release leads to the reversal of propagating CICR and to alternans

Ischemia/Reperfusion Injury Protection by Mesenchymal Stem Cell Derived Antioxidant Capacity

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 Ca(2+), and significantly attenuated cell shortening. Reperfusion of VMs with Ctrl tyrode or MSC-conditioned tyrode (ConT) resulted in a transient increase of the Ca(2+) 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 Ca(2+) release