ESC CMs can be subdivided into atrial and ventricular CMs

ESC-CMs can be subdivided into atrial and ventricular CMs (Burridge et al., 2012). It has been documented that the expression of the α-isoform of myosin heavy chain (α-MHC), MLC2a, and MLC1a become restricted to the atrium during murine development (Zammit et al., 2000). Here, we employed MLC2a and MLC2v immunocytological staining to identify murine and human CM subtypes and demonstrated that Raman microspectroscopy can identify atrial and ventricular CM subpopulations derived from pluripotent murine and human cells. Atrial and ventricular CMs exhibit differences in the expression and organization of structural proteins (Bird et al., 2003; Zammit et al., 2000). We identified structural protein signals (938, 1265, 1658 cm−1) as highly relevant for the spectral-based differentiation of atrial and ventricular CMs. Muscle fibers, including myosin proteins, contain predominantly alpha-helical conformations, which can be identified using wavenumbers at 938 and 1660 cm−1 (Pézolet et al., 1988). These signals are consistent with those identified in purified myosin and tropomyosin (Pascut et al., 2011; Pézolet et al., 1988). The spectral area of 1380–1490 cm−1 was more prominent in ventricular CMs than in atrial CMs. Pascut et al. (2011) showed that this area exhibits specific signals that can be used to discriminate CMs from non-CMs. The wavenumber region of 1380–1490 cm−1 refers to CH2 and CH3 vibrations and could signify differences in the organization of intracellular proteins (Table 1). In murine fetal development, it was demonstrated that atrial-ventricular specification is initiated before E15.5 at the fetal stage (Zammit et al., 2000). However, less is known about these developmental stages in human heart development. Notably, the Raman data we recorded from human fetal heart tissues and human cardiac p2y inhibitor attest a higher complexity than in the respective data from murine cells. Previously, MLC2a and MLC2v double-positive CMs were derived from hESC cultures (Mummery et al., 2012), which are also seen during normal human heart development. These CM subpopulations, which are not yet fully committed to an atrial or ventricular fate, were also noted in our study. In future studies, Raman microspectroscopy could be further correlated to gene and protein marker expression profiles and functional measurements of these CM subpopulations. The technology could thereby help to resolve early cell fate decisions made on the path toward atrial and ventricular cardiac phenotypes (Sylva et al., 2014). By characterizing ESC-CMs, fetal CMs, and murine adult CMs using Raman microspectroscopy, we confirmed a fetal cardiac cell phenotype profile of ESC-CMs, which is in accordance with investigations in which comparable gene expression profiles of ESC-CMs and fetal CMs were reported (Cao et al., 2008). We further showed that lipid- and protein-related Raman bands at 1266 cm−1, 1437 cm−1, 1658 cm−1 were more prominent in adult CMs than in ESC-CMs or fetal CMs. Immunofluorescence staining confirmed the presence of densely packed sarcomeric myosin in adult CMs versus more diffusely organized sarcomeric structures with shorter fragments in fetal CMs, which could be the possible reason for the spectroscopic differences that were detected in our study. Different stages of sarcomeric organization and increasing myofibril densities were also seen when employing standard methods in developing CMs (Pohjoismaki et al., 2013). In adult CMs, a lower nuclear density, higher mitochondrial density, and a metabolic shift toward β-oxidation of fatty acids was previously reported (Pohjoismaki et al., 2013). In addition, it was shown that fetal and adult CMs express differential isoforms of troponin C (Siedner et al., 2003), which might also have an impact on protein-related Raman signals at wavenumbers 1266 and 1658 cm−1. Many features that are characteristic for fetal cardiac phenotypes have been described in stressed and pathological CMs (Gilsbach et al., 2014). Relapse of the MHC-isoform shift and the downregulation of fatty acid oxidation were reported as hallmarks of heart failure (Rajabi et al., 2007). Our results suggest that Raman microspectroscopy could potentially detect pathological CMs and therefore represents a very promising tool for the monitoring and diagnosis of pathological or stress-induced remodeling processes in the heart.