Unravelling the effects of mechanical physiological conditioning on cardiac adipose tissue-derived progenitor cells in vitro and in silico

Mechanical conditioning is incompletely characterized for stimulating therapeutic cells within the physiological range. We sought to unravel the mechanism of action underlying mechanical conditioning of adipose tissue-derived progenitor cells (ATDPCs), both in vitro and in silico. Cardiac ATDPCs, grown on 3 different patterned surfaces, were mechanically stretched for 7 days at 1 Hz. A custom-designed, magnet-based, mechanical stimulator device was developed to apply ~10% mechanical stretching to monolayer cell cultures. Gene and protein analyses were performed for each cell type and condition. Cell supernatants were also collected to analyze secreted proteins and construct an artificial neural network. Gene and protein modulations were different for each surface pattern. After mechanostimulation, cardiac ATDPCs increased the expression of structural genes and there was a rising trend on cardiac transcription factors. Finally, secretome analyses revealed upregulation of proteins associated with both myocardial infarction and cardiac regeneration, such as regulators of the immune response, angiogenesis or cell adhesion. To conclude, mechanical conditioning of cardiac ATDPCs enhanced the expression of early and late cardiac genes in vitro. Additionally, in silico analyses of secreted proteins showed that mechanical stimulation of cardiac ATDPCs was highly associated with myocardial infarction and repair.


Human ATDPCs isolation and culture
Informed consent was obtained from all patients, the study protocol was approved by the local Ethics Committee (Germans Trias i Pujol University Hospital Ethics Committee), and it conformed to the principles of the Declaration of Helsinki. Tissues were obtained from a total of 37 patients (cardiac adipose tissue samples) and 6 patients (subcutaneous adipose tissue samples). Cells isolated from each tissue source were pooled and used for experiments.
Subcutaneous ATDPCs were used as control cells.
The following cardiac markers were evaluated: transcription factors (Tbx5, MEF2A, GATA-4), structural genes (α-actinin, cTnI, β-MyHC), and calcium-handling related genes (Cx43, SERCA2). Data were collected and examined on the Light Cycler ® 480 Real-Time PCR System (Roche); each sample was analysed in duplicate. The Livak method (2) was used to quantify the absolute (2 -ΔΔCT ) and relative (2 -ΔCT ) expression of each gene between mechanically conditioned and control samples, using GAPDH as an endogenous reference. The ratio of the 2 conditions (control and stimulated) were collected and calculated as the fold expression (stimulated/control).

Proteome obtainment
Starting from a volume of 3 mL, secretome samples were concentrated using VivaSpin The mass spectrometer was operated in positive ionization mode with nanospray voltage set at 2.5 kV and source temperature at 200 °C. Ultramark 1621 for the FT mass analyzer was used for external calibration prior the analyses. Moreover, an internal calibration was also performed using the background polysiloxane ion signal at m/z 445.1200. The instrument was operated in DDA mode and full MS scans with 1 micro scans at resolution of 60,000 were used over a mass range of m/z 350-1500 with detection in the Orbitrap. Auto gain control (AGC) was set to 1E6, and dynamic exclusion (60 seconds) and charge state filtering disqualifying singly charged peptides were both activated. In each cycle of DDA analysis, following each survey scan the top ten most intense ions with multiple charged ions above a threshold ion count of 5000 were selected for fragmentation at normalized collision energy of 35%. Fragment ion spectra produced via collision-induced dissociation (CID) were acquired in the ion trap, AGC was set to 5E4, isolation window of 2.0 m/z and maximum injection time of 50 ms was used.

In silico functional and topological analyses of the secretome
The topological analysis evaluates direct and indirect links between the proteins that define the secretome profile of each treatment and the proteins included in the description of the following processes: myocardial infarction (MI), cardiac regeneration and Gene Ontology (GO) terms (4) associated (myoblast differentiation, cardiac muscle cell proliferation, cardiomyocyte proliferation, cardiomyocyte apoptosis, and cardiomyocyte differentiation). and green circles indicate proteins or effectors linked to the secretome proteins. comparisons between mechanically stimulated (MS) and control (Con) samples. Relative expression (2 -ΔCT ) and fold-changes in expression (MS/Con) are shown for cardiomyogenic genes. Values were normalized to GAPDH expression and represent the mean ± SEM for at least 4 independent experiments. *P < 0.05 (significant) and #P < 0.10 (trend).

Supplementary Table 1. Cardiac markers relative expression for each condition in subcutaneous ATDPCs culture.
Gene expressions were analysed in duplicate for comparisons between mechanically stimulated (MS) and control (Con) samples. Relative expression (2 -ΔCT ) and fold-changes in expression (MS/Con) are shown for cardiomyogenic genes. Values were normalized to GAPDH expression and represent the mean ± SEM for at least 4 independent experiments. *P < 0.05 (significant) and #P < 0.10 (trend).