https://doi.org/10.1007/s00392-025-02625-4
1Kerckhoff Klinik GmbH Abteilung für Kardiologie Bad Nauheim, Deutschland; 2Max-Planck-Institut für Herz- und Lungenforschung Bad Nauheim, Deutschland; 3IPPMed - Institut für Pharmakologie und Präventive Medizin GmbH Cloppenburg, Deutschland; 4Universitätsklinikum Gießen und Marburg GmbH Medizinische Klinik I - Kardiologie und Angiologie Gießen, Deutschland
Objectives: Arrythmia, a cardiac disorder characterized by irregular impulse generation and ventricular rhythm, occurs in between 1.5-5% of the population and can lead to cardiomyopathy and heart failure (HF). As we observed that a growth factor (GF) cocktail (GFC) derived from cardiac microvascular endothelial cells (EC) modulates contraction and remodeles cultured adult rat ventricular cardiomyocytes (ARC), we aimed to identify these GF and their effects.
Methods: GFC was obtained from 100L of serum and supplement free medium 199 exposed to cultured confluent EC for 2 days. GF were separated by ion exchange, heparin and reversed-phase chromatography. GF were identified by MALDI-TOF mass spectrometry (Bruker) and analysed in ARC supplemented with 2% selected human serum. Effects of GF on cell surface area (SA; ImageJ), protein synthesis (PS; [3H] phenylalamine incorporation), total protein content (PC), number and contraction rate (BPM, beats per minute, video microscopy) and O2-consumption (Seahorse, Agilent), were analysed after 7days.
After 7 and 12 days, Western blot analysis and phase-contrast microscopy were performed. Remodeling was monitored by re-expression of non-muscle α-actin (Acta 2), α-actinin 1 and 4 (Actn1, Actn4). Control cultures (100%) were supplemented with 2% serum. The number of experiments is 4, and FGF2 significance of p<0.05 versus control and versus TGFβ is indicated by p* and P*, respectively.
Results: Mass spectrometry revealed effective concentrations of various cytokines and growth factors in GFC such as LIF, oncostatin M, fibroblast growth factor 2 (FGF2), and transforming growth factor-β (TGFβ). Because FGF2 (20 ng/ml) and TGFβ (2 ng/ml) had opposite effects on cultured ARC, we focused here on the analysis of these two GFs. FGF-2 stimulation enlarged SA (134±8%; p*), PS (207±18%; p*) and PC (121±4%; p*). Simultaneous treatment of FGF2 stimulated cardiomyocytes with TGFβ downregulated increases in SA (113±3%; P*) and PS (140±8%; P*), but not PC, suggesting that protein degradation is inhibited by TGFβ in ARC to the same extent as PS.
Most surprisingly, FGF-2 potentiated the number of contracting ARC (from 7±2 to 89±9 cells per field; p*), their contraction rate (from 6±3 to 70±17 BPM; p*) and their oxygen consumption (from 100±9% to 210±14%, p*). These effects were downregulated by TGFβ to near control levels of 4±1, 7±2 and 81±5%, respectively (all P*). FGF2 treated cultures started to form a confluent synchroneous contracting ARC monolayer from day 7. These cells were able to induce contractile responses of freshly added isolated cardiomyocytes indicating pacemake-like capabilities. Dedifferentiation, a key feature of cardiac remodeling, was determined by re-expression of Acta2 (286±16%; p*), Actn1 (562±64%; p*) and Actn4 (706±171%; p*) and was downregulated by TGFβ (185±12%, 233±47%, 229±33%; all P*).
Conclusions and outlook: We may speculate that cardiac arrhythmia is based on an imbalance of growth factors such as high FGF-2 and low TGF. FGF-2 is known to promote dedifferentiation, while TGF does the opposite. So, blocking FGF-2 or adding TGF may tip the balance into the right direction.