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The prevalence of dilated cardiomyopathy (DCM) is increasing globally, highlighting the need for innovative therapeutic approaches to prevent its onset. In this study, we examined the energetic and epigenetic distinctions between dilated and non-dilated human myocardium-derived mesenchymal stem/stromal cells (hmMSCs) and assessed the effects of class I and II HDAC inhibitors (HDACi) on these cells and their cardiomyogenic differentiation. Cells were isolated from myocardium biopsies using explant outgrowth methods. Mitochondrial and histone deacetylase activities, ATP levels, cardiac transcription factors, and structural proteins were assessed using flow cytometry, PCR, chemiluminescence, Western blotting, and immunohistochemistry. The data suggest that the tested HDAC inhibitors improved acetylation and enhanced the energetic status of both types of cells, with significant effects observed in dilated myocardium-derived hmMSCs. Additionally, the HDAC inhibitors activated the cardiac transcription factors Nkx2-5, HOPX, GATA4, and Mef2C, and upregulated structural proteins such as cardiac troponin T and alpha cardiac actin at both the protein and gene levels. In conclusion, our findings suggest that HDACi may serve as potential modulators of the energetic status and cardiomyogenic differentiation of human heart hmMSCs. This avenue of exploration could broaden the search for novel therapeutic interventions for dilated cardiomyopathy, ultimately leading to improvements in heart function.

Cartilage is an avascular tissue and sensitive to mechanical trauma and/or age-related degenerative processes leading to the development of osteoarthritis (OA). Therefore, it is important to investigate the mesenchymal cell-based chondrogenic regenerating mechanisms and possible their regulation. The aim of this study was to investigate the role of intracellular calcium (iCa 2+ ) and its regulation through voltage-operated calcium channels (VOCC) on chondrogenic differentiation of mesenchymal stem/stromal cells derived from human bone marrow (BMMSCs) and menstrual blood (MenSCs) in comparison to OA chondrocytes. The level of iCa 2+ was highest in chondrocytes, whereas iCa 2+ store capacity was biggest in MenSCs and they proliferated better as compared to other cells. The level of CaV1.2 channels was also highest in OA chondrocytes than in other cells. CaV1.2 antagonist nifedipine slightly suppressed iCa 2+ , Cav1.2 and the proliferation of all cells and affected iCa 2+ stores, particularly in BMMSCs. The expression of the CaV1.2 gene during 21 days of chondrogenic differentiation was highest in MenSCs, showing the weakest chondrogenic differentiation, which was stimulated by the nifedipine. The best chondrogenic differentiation potential showed BMMSCs ( SOX9 and COL2A1 expression); however, purposeful iCa 2+ and VOCC regulation by blockers can stimulate a chondrogenic response at least in MenSCs.

Articular cartilage is vulnerable to mechanical overload and has limited ability to restore lesions, which leads to the development of chronic diseases such as osteoarthritis (OA). In this study, the chondrogenic responses of human bone marrow mesenchymal stem cells (BMMSCs) and OA cartilage-derived chondrocytes in 3D chondroitin sulfate-tyramine/gelatin (CS-Tyr)/Gel) hydrogels with or without experimental mechanical load have been investigated. Chondrocytes were smaller in size, had slower proliferation rate and higher level of intracellular calcium (iCa 2+ ) compared to BMMSCs. Under 3D chondrogenic conditions in CS-Tyr/Gel with or without TGF-β3, chondrocytes more intensively secreted cartilage oligomeric matrix protein (COMP) and expressed collagen type II ( COL2A1 ) and aggrecan ( ACAN ) genes but were more susceptible to mechanical load compared to BMMSCs. ICa 2+ was more stably controlled in CS-Tyr/Gel/BMMSCs than in CS-Tyr/Gel/chondrocytes ones, through the expression of L-type channel subunit CaV1.2 ( CACNA1C ) and Serca2 pump ( ATP2A2 ) genes, and their balance was kept more stable. Due to the lower susceptibility to mechanical load, BMMSCs in CS-Tyr/Gel hydrogel may have an advantage over chondrocytes in application for cartilage regeneration purposes. The mechanical overload related cartilage damage in vivo and the vague regenerative processes of OA chondrocytes might be associated to the inefficient control of iCa 2+ regulating channels.

Dilated cardiomyopathy (DCM) is the most common type of nonischemic cardiomyopathy characterized by left ventricular or biventricular dilation and impaired contraction leading to heart failure and even patients' death. Therefore, it is important to search for new cardiac tissue regenerating tools. Human mesenchymal stem/stromal cells (hmMSCs) were isolated from post-surgery healthy and DCM myocardial biopsies and their differentiation to the cardiomyogenic direction has been investigated in vitro. Dilated hmMSCs were slightly bigger in size, grew slower, but had almost the same levels of MSC-typical surface markers as healthy hmMSCs. Histone deacetylase (HDAC) activity in dilated hmMSCs was 1.5-fold higher than in healthy ones, which was suppressed by class I and II HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) showing activation of cardiomyogenic differentiation-related genes alpha-cardiac actin (ACTC1 ) and cardiac troponin T (TNNT2 ). Both types of hmMSCs cultivated on collagen I hydrogels with hyaluronic acid (HA) or 2-methacryloyloxyethyl phosphorylcholine (MPC) and exposed to SAHA significantly downregulated focal adhesion kinase ( PTK2 ) and activated ACTC1 and TNNT2 . Longitudinal cultivation of dilated hmMSC also upregulated alpha-cardiac actin. Thus, HDAC inhibitor SAHA, in combination with collagen I-based hydrogels, can tilt the dilated myocardium hmMSC toward cardiomyogenic direction in vitro with further possible therapeutic application in vivo.

Background: In this study the effect of histone deacetylase (HDAC) inhibitor suberoylanilide hydroxamic acid (SAHA) on the energetic status and cardiomyogenic differentiation of human healthy and dilated myocardium-derived mesenchymal stromal cells (hmMSC) have been investigated., Methods: The hmMSC were isolated from the healthy and dilated post-operation heart biopsies by explant outgrowth method. Cell proliferation, HDAC activity, mitochondrial membrane potential, and level of adenosine triphosphate (ATP) were evaluated. The effect of SAHA on mitochondrial parameters has been investigated also by Seahorse XF analyzer and cardiomyogenic differentiation was confirmed by the expression of transcription factor NK2 Homeobox 5 (Nkx2.5), cardiac troponin T and alpha cardiac actin at gene and protein levels., Results: Dilated myocardium-derived hmMSC had almost 1.5 folds higher HDAC activity compared to the healthy cells and significantly lower mitochondrial membrane potential and ATP level. HDAC class I and II inhibitor SAHA improved energetic status of mitochondria in dilated myocardium-isolated hmMSC and increased expression of cardiac specific proteins during 14 days of exposure of cells to SAHA., Conclusions: HDAC inhibitor SAHA can be a promising therapeutic for dilated cardiomyopathy (DCM). Dilated hmMSC exposed to SAHA improved energetic status and, subsequently, cardiomyogenic differentiation. Data suggest that human dilated myocardium-derived MSC still have cardio tissue regenerative potential, which might be stimulated by HDAC inhibitors.

Background: The transfection of human mesenchymal stem cells (hMSCs) with the hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2) gene has been demonstrated to provide biological pacing in dogs with complete heart block. The mechanism appears to be the generation of the ion current (If) by the HCN2-expressing hMSCs. However, it is not clear how the transfection process and/or the HCN2 gene affect the growth functions of the hMSCs. Therefore, we investigated survival, proliferation, cell cycle, and growth on a Kapton® scaffold of HCN2-expressing hMSCs., Methods: hMSCs were isolated from the bone marrow of healthy volunteers applying a selective cell adhesion procedure and were identified by their expression of specific surface markers. Cells from passages 2-3 were transfected by electroporation using commercial transfection kits and a pIRES2-EGFP vector carrying the pacemaker gene, mouse HCN2 (mHCN2). Transfection efficiency was confirmed by enhanced green fluorescent protein (EGFP) fluorescence, quantitative real-time polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). After hMSCs were transfected, their viability, proliferation, If generation, apoptosis, cell cycle, and expression of transcription factors were measured and compared with non-transfected cells and cells transfected with pIRES2-EGFP vector alone., Results: Intracellular mHCN2 expression after transfection increased from 22.14 to 62.66 ng/mg protein (p < 0.05). Transfection efficiency was 45 ± 5 %. The viability of mHCN2-transfected cells was 82 ± 5 %; they grew stably for more than 3 weeks and induced If current. mHCN2-transfected cells had low mitotic activity (10.4 ± 1.24 % in G2/M and 83.6 ± 2.5 % in G1 phases) as compared with non-transfected cells (52-53 % in G2/M and 31-35 % in G1 phases). Transfected cells showed increased activation of nine cell cycle-regulating transcription factors: the most prominent upregulation was of AMP-dependent transcription factor ATF3 (7.11-fold, p = 0.00056) which regulates the G1 phase. mHCN2-expressing hMSCs were attached and made anchorage-dependent connection with other cells without transmigration through a 12.7-μm thick Kapton® HN film with micromachined 1-3 μm diameter pores., Conclusions: mHCN2-expressing hMSCs preserved the major cell functions required for the generation of biological pacemakers: high viability, functional activity, but low proliferation rate through the arrest of cell cycle in the G1 phase. mHCN2-expressing hMSCs attached and grew on a Kapton® scaffold without transmigration, confirming the relevance of these cells for the generation of biological pacemakers.