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 Ca 2+ 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.

Our aim was to further elucidate the cardiac lineage development of bone marrow-derived mesenchymal stem cells (MSC) and to identify cells which had the potential for cardiac myogenic differentiation when compared to skeletal muscle satellite (Sk-sat) myogenesis. Unlike Sk-sat, MSC expressed the early cardiac markers Nkx2.5 and GATA4. Their expression was significantly increased by culturing MSC with Bone Morphogenetic Protein 4 (BMP4). Enhanced cardiac myogenic lineage differentiation and loss of stem cell characteristics induced by BMP4 were further confirmed by flow cytometry of cells stained for Nkx2.5 and Sca-1 expression. MSC also expressed skeletal genes (MyoG, ssTnI, Sk-Act) early in culture but their expression was suppressed when BMP4 was added from day 0–6 (p < 0.05). BMP4 treated MSC also exhibited a 6-fold increase in cTnI expression by day 12 in culture. The average MSC action potential time duration at 90% (APD90) was 32.3 ± 4 ms, with some cells exhibiting action potentials closer to Sk-sat APD90 of 13.7 ± 0.9 ms. After treatment with BMP4, MSC significantly increased their APD90 to 54.4 ± 7.6 ms, shifting from the shorter skeletal-like signature, towards a longer action potential duration more characteristic of a cardiomyocyte signature. Our results show that MSC and Sk-sat exhibit similarities in myogenic lineage development early in culture but that BMP4 clearly enhances cardiac myogenic development, suppresses skeletal myogenesis, and leads to loss of “stemness” in MSC. These findings provide novel information regarding the use of BMP4 to accelerate cardiac myogenic development in harvested MSC and further support the use of MSC in cardiac regenerative therapy.

Satellite cells can maintain or repair muscle because they possess stem cell properties, making them a valuable option for cell therapy. However, cell transplants into skeletal muscle of patients with muscular dystrophy are limited by donor cell attachment, migration, and survival in the host tissue. Cells used for therapy are selected based on specific markers present in the plasma membrane. Although many markers have been identified, there is a need to find a marker that is expressed at different states in satellite cells, activated, quiescent, or differentiated cell. Furthermore, the marker has to be present in human tissue. Recently we reported that the plasma membrane α2δ1 protein is involved in cell attachment and migration in myoblasts. The α2δ1 subunit forms a part of the L-type voltage-dependent calcium channel in adult skeletal muscle. We found that the α2δ1 subunit is expressed in the majority of newly isolated satellite cells and that it appears earlier than the α1 subunits and at higher levels than the β or γ subunits. We also found that those cells that expressed α2δ1 would differentiate into muscle cells. This evidence indicates that the α2δ1 may be used as a marker of satellite cells that will differentiate into muscle.

Bone marrow-derived mesenchymal stem cells (MSC) can be differentiated into myocytes, as well as adipocytes, chondrocytes, and osteocytes in culture. Calcium channels mediate excitation-contraction coupling and are essential for the function of muscle. However, little is known about the expression of calcium channel subunits and calcium handling in stem cells. We examined whether the expression of calcium channel subunits in MSC is similar to that of skeletal muscle satellite cells and if their levels of expression are modified after treatment with bone morphogenetic protein-4 (BMP4). We found that during myogenic differentiation, MSC first express the α2δ1 subunit and the cardiac channel subunit Cav1.2. In contrast to the α2δ1 subunit levels, the Cav1.2 subunit decreases rapidly with time. The skeletal channel subunit Cav1.1 is detected at day 3 but its expression increases considerably, resembling more closely the expression of the subunits in satellite cells. Treatment of MSC with BMP4 caused a significant increase in expression of Cav1.2, a delay in expression of Cav1.1, and a reduction in the duration of calcium transients when extracellular calcium was removed. Calcium currents and transients followed a pattern related to the expression of the cardiac (Cav1.2) or skeletal (Cav1.1) α1subunits. These results indicate that differentiation of untreated MSC resembles differentiation of skeletal muscle and that BMP4 reduces skeletal muscle calcium channel expression and promotes the expression of cardiac calcium channels during myogenic differentiation.

The calcium channel a 2 /d 1 subunit is expressed prior to Ca v 1.1 and is localized at the leading edges of young skeletal myotubes, suggesting that it may interact with other cellular components. To address this issue, multi protein complexes were isolated from skeletal muscle of P0 mice and analyzed with blue native PAGE and Western blots. These studies showed that the a 2 /d 1 subunit is found in a large complex of ~670 kDa, which was also present in cultures of skeletal myotubes and C2C12 cells. This complex was isolated and further analyzed with mass spectroscopy. Mascot search results identified the b subunit of the ATP synthase (ATP5b) in the ~670 kDa complex, which is normally present in the mitochondrial membrane. However, the presence of ATP5b and a 2 /d 1 subunit in the plasma membrane was confirmed by biotin labeling of membrane proteins. Immunocytochemistry analysis of skeletal myotubes in culture showed co-localization of a 2 /d 1 subunit and ATP5b in some cells. To determine a functional significance of the interaction between a 2 /d 1 subunit and ATP5b, calcium transients were electrically evoked and recorded from myotubes using confocal microscopy. Exposure of myotubes to an ATP5b monoclonal antibody resulted in a significantly faster decay of calcium transients after a train of stimuli. This effect was less pronounced when myotubes were stimulated with a single pulse. These experiments provide the first direct evidence suggesting that the a 2 /d 1 subunit may be able to form complexes with proteins other than the Cav1.1 and suggest new signaling roles of a 2 /d 1 in muscle cells. In addition, they show that a molecule involved in the synthesis of ATP in the inner mitochondrial membrane can be also localized in the plasma membrane. Supported by the Muscular Dystrophy Association.