Fig 2: Expression of alpha-cardiac actin (ACTC1) and cardiac troponin T (TNNT2) genes in healthy and pathological/dilated myocardium-derived hmMSC during exposure to 1 µM SAHA for 3, 7, and 14 days. (A) Morphology of healthy and pathological hmMSC during 14 days of exposure to 1 µM SAHA on Col, Col-MPC, Col-HA hydrogels and plastic (Control). (B) Expression of ACTC1 (upper panels) and TNNT2 (lower panels) in healthy and pathological hmMSC grown on Col, Col-MPC, Col-HA hydrogels and plastic (Control). Relative cardiac gene expression is shown as 2-?Ctx2,000,000 and was normalized to the ACTB gene. Data of 3 replicates (n = 3) of two patients in each group are shown with ±SD. Data are significant at * p < 0.05, ** p < 0.01 comparing expression of ACTC1 and TNNT2 cultivated on the hydrogels with the cells cultivated on plastic. The arrows indicate the longitudinal growth pattern of healthy hmMSC.

Fig 3: Hypothesized role of RYBP during in vitro cardiomyogenesis.Schematic representation showing possible regulatory functions of RYBP during in vitro cardiac differentiation. RYBP can exert both repression (a) and activation (b, c) functions during cardiac morphogenesis. RYBP as the member of ncPRC1s along with RING1 can repress key lineage genes such as ion channel genes during the ES cell stage (a). As a repressor RYBP can repress ion channel genes such as Hcn2, Hcn3, Kcnj8 and Cacna1g. As an activator, RYBP can activate key cardiac transcription factors, like Shh, Isl1, Nkx2-5, Mef2c and Tbx5 and that are required for normal cardiac progenitor formation (b). RYBP and RING1 can target together Tpm4 and Myh7, and as they were described as essential components for proper cardiac morphogenesis (c), whilst other sarcomeric genes such as Actc1, Myom1, Tnnt2 and Ttn could be regulated by NKX2-5, MEF2C and TBX5.

Fig 4: The effect of HDAC inhibitor SAHA on HDAC activity and cardiomyogenic differentiation of healthy and pathological/dilated myocardium-derived hmMSC. (A) HDAC activity in the healthy and pathological hmMSC. (B) The effect of SAHA on HDAC activity during 3 days of incubation. Expression of cardiomyogenic differentiation-specific genes: alpha-cardiac actin (ACTC1) (C) and cardiac troponin T (TNNT2). (D) The relative cardiac gene expression is shown as 2-?Ctx2,000,000 and was normalized to the ACTB gene. Data are shown as mean ± SD and are significant at * p = 0.05, ** p = 0.01 levels from not less than three repeats (n = 3) of three patients from each group as calculated using the GraphPad Prism 6 software.

Fig 5: The sarcomeric thin and thick filament associated proteins are disorganized in the lack of RYBP during in vitro cardiac differentiation.(A) Immunocytochemical analysis of thin filament markers ACTC1(A a-d), CTNT (A f-i), CTNI (A k-n) and TM (A p-s) in wild type (A a, c, f, h, k, m, p, r) and Rybp null mutant CMCs (A b, d, g, i, l, n, q, s). Vertical axis indicates the intensity of the fluorescence signal of the immunopositive cells (A e, j, o, t). (B) Immunocytochemical analysis of thick filament markers MYOSIN (B a-d), MYOM (B f-i) and TTN (B k-n) in wild type and Rybp null mutant CMCs. Vertical axis indicates the intensity of the fluorescence signal of the immunopositive cells (B e, j, o). Semi-quantification of the signals was performed using the RGB measuring tool in ImageJ program for both the thin and thick-filament proteins. Abbreviations: ACTC1: Actin alpha cardiac muscle 1; CTNT: Cardiac troponin T; CTNI: Cardiac troponin I; TM: Tropomyosin; MYOM: Myomesin; TTN: Titin; d: day; CMC: cardiomyocyte. Olympus Confocal IX 81, Obj.: 60x; Scale bar: Myosin 20 µm, Myom 100 µm/ 50 µm, Ttn 20 µm/ 30 µm. Means are standard deviation ± SD. Values of p < 0.05 were accepted as significant (* p < 0.05; ** p < 0.01; *** p < 0.001). Statistical method: t test type 3.