Abstract 11240: Factors-based Human Cardiomyocytes Differentiation Exhibits Defective Maturation and Excitation Through Aberrant Calcium Handling Proteins
Rationale- Recent breakthrough in direct cardiac reprogramming in mice provided a new platform towards the development of regenerative therapy; however, the underlying mechanisms controlling human cardiomyogenesis remain unclear. Here, we investigate the role of calcium-dependent cardiac maturation during the process of factors-based human cardiomyocytes differentiation.
Methods and Results- Human cardiac progenitor cells (CPCs) were isolated and transduced with lentiviral vectors encoding Tbx5, GATA4, and Mef2C (TGM). Differentiated cardiomyocytes were identified by alpha-MHC promoter-driven reporter gene and mitochondrial-labeling fluorescence. Calcium oscillation imaging, FACS analysis using signal-regulatory protein alpha (SIRPA), and cardiac structural as well as ion channel regulatory proteins were examined to verify cardiac maturation. We found that TGM transduction in CPCs gave rise to substantial cardiomyocytes addressed by a significantly augmented SIRPA and mitochondrial expressing cell population, those were confirmed as striated cardiac muscles by immunostaining of alpha-MHC and cTnT; however, FACS analysis showed that functionally mature cardiomyocytes expressing alpha-MHC, alpha-actinin, and cTnT were modest. Spontaneous calcium oscillation was identified in undifferentiated CPCs, we then evaluated the functional important of cardiac ion channels in factors-based differentiation. Although TGM induction markedly upregulated the expressions of sodium and potassium channels modulators include SCN5A, Kir2.2, HERG, KvLQT1, and Kv4.3, altered calcium oscillation frequency was only functionally coupled with the upregulation of ryanodine receptors and L-type calcium-channel alpha 1C. Electrical stimulation had no effects on calcium oscillation in TGM-induced CPCs, as also shown by reduced expressions of sarcolemmal calcium-cycling regulatory proteins, NCX1, SERCA2, and inositol 1,4,5-triphosphate receptors in endoplasmic reticulum.
Conclusions- Our data suggest that factors-based cardiac-lineage induction in human CPCs has critical deficits towards functional maturation and excitation, consistent with a repression in calcium handling proteins necessary to complete the processes.
- © 2012 by American Heart Association, Inc.