Fibroblasts sense and adapt to changes in their mechanical environment. The mechanical properties of the extracellular matrix (ECM) in particular, act as a driver for a number of cell functions including differentiation, motility, myofibroblast phenoconversion, and collagen production [1
]. Mechanical properties of the ECM can change drastically during physiological (e.g., development) and pathophysiological conditions (e.g., mechanical overload) in a number of organs [2
], including the heart. For example, the early inflammatory phase following myocardial infarction is characterized by a softening of the myocardium down to stiffnesses of a few kilopascals [3
], due to collagen degradation and cardiomyocyte death. In contrast, values exceeding 50 kilopascals have been observed in fully mature ventricular scars several months or years after injury [4
]. The current state of knowledge about the sensing of passive mechanics from cell to tissue levels in the heart has been reviewed recently in detail [8
]. The molecular mechanisms underlying cell adaptation to matrix stiffness are still ill-understood.
The main cellular components that mediate the sensing and regulation of ECM mechanics are transmembrane receptors of the integrin family, proteins associated with focal adhesions, and the actomyosin cytoskeleton [9
]. Integrins are major adhesion receptors of the cell [10
]. By physically linking the ECM to the cytoskeleton, they transmit forces and deformation between the inside of the cell and the ECM [11
Integrin signaling involves a large number of proteins, including talin and various kinases. Talin is instrumental for cell adhesion by linking integrins to the cytoskeleton. Integration of internal and external stimuli allows talin to activate integrins (i.e., convert them to high-affinity states so they can bind their substrate) “on demand” [12
]. Kinases play a central role in the transmission of signals from focal adhesions to the inside of a cell. In particular the focal adhesion kinase (FAK) is recruited to focal adhesions and then activated in response to integrin-β1 activation. This is important for adhesion turnover, Rho family guanosine triphosphatase (GTPase) activation, cell migration, and crosstalk with signals from other receptors (e.g., growth factors, [13
]). The Rho family of small GTPases and their downstream effectors such as Rho-associated protein kinase (ROCK) and myosin light chain kinases are major players in the assembly of the focal adhesion complex linking ECM to the cytoskeleton. For more detail on integrin signaling please see previous communications [14
Integrin signaling interacts with numerous additional pathways. Recently, crosstalk between mechano-sensitive ion channels and integrin signaling has been proposed. The canonical transient receptor potential channel 6 (TRPC6) binds to and activates calpain, independently of its activity as an ion channel, and regulates podocyte cytoskeleton organization, cell adhesion, and motility of podocytes [17
]. Piezo1, another cation non-selective stretch-activated channel (SAC) involved in mechano-transduction [18
], contributes to a number of physiological and pathophysiological processes, as reviewed elsewhere [20
]. Piezo1 is present in cardiac fibroblasts [24
] but its contribution to the functions of this cell type remains to be explored.
Piezo1 is a large homotrimer with more than 2500 amino acids, including 38 transmembrane helices per monomer [25
]. It is widely distributed throughout different species and cell types, and it has been reported in focal adhesions (for example in Chinese hamster ovary cells, Drosophila
glioblastoma stem cells [26
], and human neural stem cells [27
]). Similar to integrins, activation of Piezo1 can be altered by stimuli from the inside or the outside of a cell. In human neuronal stem cells, the actomyosin cytoskeleton generates sufficient forces via myosin II phosphorylation to open Piezo1 and generate Ca2+
flickers at focal adhesions [28
]. Piezo1 activity at focal adhesions has been shown to activate integrin–FAK signaling in glioblastoma and neural stem cells via Ca2+
-mediated signaling [26
]. A potential mechanism for Piezo1-mediated integrin activation has been explored in Chinese hamster ovary cells, where recruitment of the small GTPase RRas to the endoplasmic reticulum is necessary to activate the Ca2+
-activated protease calpain, increasing Ca2+
release from cytoplasmic stores [29
]. In addition, it was shown that Piezo1 is sensitized to pulling forces from the outside by binding to collagen VI in human neuroblastoma cells [30
]. Further connecting Piezo1 with outside-in signaling, channel expression has been shown to increase (≈1.4 fold) in stem cells cultured on stiffer polyacrylamide gels (5 kPa vs. 0.1 kPa, [26
]). Along similar lines, recruitment of Piezo1-expressing monocytes (required for vascularization of implanted, hydrogel-based cardiac tissue patches) depends on physiological hydrogel stiffness in mice [31
Piezo1 expression is altered in a number of diseases, for example in amyloid-responsive cells in Alzheimer’s disease [32
] or in red blood cells in hereditary xerocytosis [33
]. Piezo1 upregulation contributes to stiffening of aggressive gliomas in Drosophila
]. Furthermore, Piezo1 expression in human atrial fibroblasts was reported to contribute to enhanced secretion of interleukin-6 (IL-6), a profibrotic cytokine [24
]. In mouse cardiac fibroblasts, it was shown that the secretome is modulated by pro-fibrotic stimuli, including stiff growth matrices and transforming growth factor β [34
]. Our own data suggest that Piezo1 expression and activity are increased in fibroblasts in the context of atrial fibrillation (AF, [35
]). To explore the role of Piezo1 in the control of cell mechanical properties and cell adaptation to changes of matrix stiffness, the present study combines human cell culture systems and hydrogels of different stiffness with nanoindentation and imaging. Our results demonstrate that Piezo1 expression contributes to: (i) cytoskeleton organization, (ii) cell mechanical properties, and (iii) cellular adaptation to changes in matrix stiffness. These effects can be transmitted to other cells via secreted IL-6.
2. Materials and Methods
2.1. Cell Culture
2.1.1. Cell Types and Maintenance
Human embryonic kidney cells (HEK 293T/17, ATCC-LGC Standards, Manassas, Virginia, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with low glucose (D6046, Sigma-Aldrich, Hamburg, Germany) supplemented with 10% fetal calf serum (F9665, Sigma-Aldrich, Hamburg, Germany) and 1% penicillin/streptomycin (P4333, Sigma-Aldrich, Hamburg, Germany). HEK cells offer the advantage of being easy to transfect even with large constructs and thus represent a widely used cell culture model for overexpression experiments. We use HEK cells to overexpress the 2521 amino acid protein Piezo1 (Uniprot entry Q92508, Section 2.1.2
) and to test the effects of various compounds on Piezo1-induced cell stiffening (Section 2.1.3
A human atrial fibroblast line (HAF, [36
]) was cultured in DMEM supplemented with 2 mM L-alanyl-L-glutamine (GlutaMAX, 31966021, LifeTechnologies, Darmstadt, Germany), 10% fetal calf serum and 1% penicillin/streptomycin. At ≈90% confluence, cells were detached using Trypsin-ethylene-diamine-tetraacetic acid (59418C, Sigma-Aldrich, Hamburg, Germany) and seeded in fresh polystyrene flasks (Z707538, TPP, Trasadingen, Switzerland) for maintenance, or on various substrates for experiments (see below).
For transient overexpression of enhanced green fluorescent protein (EGFP), of EGFP and Piezo1, or of EGFP and TRPC6, cells were transfected with the respective plasmids (vector backbone: pIRES2_EGFP, 6029-1, Addgene, Watertown, MA, USA) 24 h after seeding, using JetPEI transfection reagent (101-10N, Polyplus transfections, Illkirch, France) according to manufacturer’s instructions and as described previously [37
]. For each 35 mm diameter well, 1 µL JetPEI transfection reagent and 0.5 µg of plasmid DNA were mixed in 100 µL NaCl solution (150 mM) and incubated for 20 min at room temperature (≈21 °C) before being added to the wells. Successfully transfected cells were identified by cytosolic EGFP fluorescence. Cells transfected with Piezo1 and EGFP will further be referred to as “Piezo1”, cells with TRPC6 and EGFP as “TRPC6”, and EGFP-expressing control cells with neither Piezo1 nor TRPC6 as “EGFP”.
Knock-down of target genes was performed using previously validated SMARTpool siRNA (L-020870-03 against Piezo1 (siPiezo1), D-001810-10-05 as a non-targeting control (siNT), Horizon Discovery, Cambridge, United Kingdom [24
]). The day after cell seeding, HAF were transfected with the respective siRNA using HiPerFect (301704, Qiagen, Hilden, Germany). For each 35 mm diameter well, 12 µL of transfection reagent and 8 nM of siRNA were diluted in 187.2 µL of DMEM + GlutaMAX in 15 mL tubes. After 15 min of incubation at room temperature, the transfection mix was filled up to 2 mL with complete culture medium (DMEM + GlutaMAX + 10% fetal calf serum + 1% penicillin/streptomycin) and carefully mixed. The old culture medium was removed from the cells and replaced by the transfection mix.
2.1.3. Compounds and Treatments
The compounds listed in Table 1
(or the respective solvents for controls) were added to the culture medium 24 h after transfection. Functional experiments were performed 2 days after exposure to compounds (i.e., 3 days post-transfection).
Unless explicitly stated otherwise, cells were cultured in the presence of antibiotics (172 µM streptomycin and 200 µM penicillin). Streptomycin is a hydrophilic, and thus non-cell-permeable [38
], blocker of cation non-selective SAC, including Piezo1. For experiments without streptomycin, the culture medium was replaced by medium without antibiotics (DMEM, low glucose with 10% fetal calf serum) 24 h after transfection. Piezo1 has also been detected in the endoplasmic reticulum membrane [29
], which is not accessible to hydrophilic streptomycin. As the endoplasmic reticulum constitutes a major reservoir for Ca2+
, we further assessed the role of the Ca2+
-activated protease calpain by directly blocking it, using the inhibitory peptide N-acetyl-leucin-leucin-norleucinal (ALLN, [40
]). Cell morphology and detachment in response to ALLN treatment were assessed by light microscopy (10× magnification, Nikon Eclipse TS100 inverted microscope equipped with Leica EC3 camera). Cell detachment was quantified as percentage of rounded-up cells present on the bottom of the dish in a field of view.
To interfere directly with integrin-β1 signaling, conformation-specific antibodies, stabilizing either the active (P5D2) or inactive (HMβ1.1) conformation of integrin-β1 were used [41
]. Further, integrin-β1 downstream effectors FAK and ROCK were inhibited using the small molecules PF-00562271 and Y-27632, respectively. A monoclonal antibody was used to neutralize secreted IL-6 in the culture medium.
2.2. Cell Culture Matrices with Different Stiffness
Hydrogels with light-tunable mechanical properties (CyPhyGels) were prepared as described previously [42
]. In short, the cyanobacterial photoreceptor-1 was recombinantly expressed, purified and covalently coupled to 8-arm polyethylene glycol. For casting, 30 µL of CyPhyGel solution was spread on square (22 by 22 mm) coverslips, resulting in CyPhyGels with ≈100 µm thickness, tunable by illumination with light of different wavelengths to a stiffness between 2.7 and 4.6 kPa [42
For cell experiments, coverslips with CyPhyGels were placed into plastic culture dishes with 35 mm diameter, HAF were seeded on top at a density of 2750 cells/cm2 and transfected 24 h after seeding (for detail see description of individual experiments). All experiments using CyPhyGels were performed under green light illumination to prevent unintended changes in mechanical properties of the culture substrate.
For nanoindentation experiments, HEK cells were seeded at a density of 5500 cells/cm2 and HAF at 2750 cells/cm2 on CyPhyGels (as described above) or in plastic tissue culture dishes with 35 mm diameter (93040, TPP; 2 mL of culture medium/dish) and transfected and/or treated as indicated for individual experiments. Nanoindentation was performed 3 days post-transfection. Before nanoindentation experiments, the culture medium was replaced by phosphate buffered saline (containing (in mM): NaCl 137, KCl 2.7, Na2HPO4 10, and KH2PO4 1.8; pH 7.4, 300 mOsm/L) at room temperature and experiments were performed within 1 h of taking cells out of the incubator.
Nanoindentation experiments were performed using the Chiaro system (Optics11, Amsterdam, Netherlands). Optical and geometrical calibrations were performed according to the manufacturer’s instructions. Spherical glass tips with 3.0–3.4 µm radius, attached to cantilevers with a spring constant between 0.012 N/m and 0.030 N/m were used to indent cells (Figure 1
A). The cell surface was identified manually by approaching it in 1 µm steps. After touching the cell, the tip was lifted by 5 µm and a displacement of 10 µm was initiated with a speed of 5 µm/s [36
]. The probe was then held at maximal motor displacement for 2 s before being retracted at the same speed (Figure 1
B). Each cell was indented once at 3 different positions (excluding the region containing the nucleus). The effective Young’s modulus (Eeff
) was derived from force vs. indentation curves, using a Hertzian model for contact mechanics (Figure 1
]) under the assumption of a Poisson ratio of 0.5 which is customarily used for mechanical testing of cells [46
]. The height of HEK cells was 20.0 ± 0.9 µm (n = 16), that of HAF was 9.2 ± 0.4 µm (n = 14). As the Hertzian model is only valid for deformations up to 10% of sample thickness [47
], no more than the first 2 µm of indentation were considered for HEK cells and no more than 0.9 µm for HAF. For such small deformations, the body of mildly-structured cells (such as cultured HEK or HAF) is usually considered homogeneous [48
2.4. Cytoskeleton: Staining, Image Acquisition, and Data Analysis
For imaging of the actin cytoskeleton, HAF were seeded onto borosilicate glass coverslips (#1.5, 631-0151, VWR, Ismaning, Germany) inside 24-well plates (662160, Greiner Bio-One, Frickenhausen, Germany) at a density of 1500 cells/cm2 (0.5 mL of culture medium/well), followed by transfection 24 h later. Then, 3 days post-transfection, cells were chemically fixed using a 4% para-formaldehyde solution. F-actin was stained using Phalloidin-iFluor-647 (ab176759, Abcam, Berlin, Germany), nuclear counterstain was performed using 4′,6-diamidin-2-phenylindol (DAPI, D1306, ThermoFisher, Dreieich, Germany), and coverslips were mounted using PermaFluor mounting medium (60085968, ThermoFisher, Dreieich, Germany).
Imaging was performed on a Leica TCS SP8 X laser scanning confocal microscope using a 63× glycerol immersion objective with a numerical aperture of 1.3. Z-stacks with a step size of 1 µm covering the whole height of a single cell were acquired with an optical thickness of 0.33 µm/plane. For analysis, all planes were background subtracted using the corresponding function in Fiji [51
], and maximal intensities were projected onto a single plane. Average fluorescence intensity and area occupied by each cell were calculated after manually outlining cell borders based on the F-actin image. Spatial organization and apparent thickness of actin bundles were analyzed spectrally using Cytospectre [52
]. In fluorescent microscopy, fine structures appear broadened due to diffraction. The size of the point spread function is in the order of half the wavelength. Assessing differences in spatial extent below this range can be problematic but larger changes can be easily detected. The apparent bundle thickness is a reliable parameter for characterizing cytoskeletal reorganization in the microscale. However, due to the limited resolution it is not possible to obtain detailed information on nano-structural rearrangements. Actin bundle orientation parameters used in this study are angular standard deviation (SD), which describes the variation of actin bundle orientations, and circular kurtosis, a measure for the peakedness of the distribution of orientations.
2.5. Gene Expression Analysis
Three days post-transfection, isolation of mRNA from cultured HAF was performed using a commercial RNA isolation kit (RNeasy Micro Kit, Cat. no.: 74004). In brief, HAF were washed once with phosphate buffered saline and subsequently overlaid with “RLT” lysis buffer (40 µL/cm2) supplemented with 2-mercaptoethanol (1:100). After incubation for 10 min at room temperature, cell lysates were scraped off the plates and transferred to 1.5 mL reaction tubes. For CyPhyGels, cell lysate and CyPhyGel were scraped off and transferred to 1.5 mL reaction tubes together. The lysate was then cleared from CyPhyGel by centrifugation (5 min at 5000× g). RNA isolation was performed according to manufacturer’s instructions. RNA concentration was determined spectrometrically. Per sample, 100 ng of RNA were utilized to generate complementary DNA (cDNA) using TaqMan Reverse Transcription Reagents (N8080234, ThermoFisher, Dreieich, Germany).
Relative mRNA expression levels were determined by qPCR. cDNA was amplified in TaqMan Fast Advanced Master Mix (4444556, ThermoFisher, Dreieich, Germany) for a total of 40 cycles, using the assays listed in Table 2
. Levels of mRNA expression of Piezo1 and Piezo2 were normalized to the expression level of glyceraldehyde-3-phosphate dehydrogenase as internal reference.
2.6. IL-6 Measurements by Enzyme-Linked Immunosorbent Assay
Cell-free supernatants from HAF, transfected with either the EGFP or Piezo1 expression plasmid, were collected just before the start of nanoindentation experiments (3 days post-transfection) and stored at −20 °C. IL-6 was detected (in Nunc MaxiSorp 96-well plates (11530627, Invitrogen, Karlsruhe, Germany)) using a colorimetric assay based on the IL-6 Human Uncoated ELISA Kit (88-7066-88, Invitrogen) according to manufacturer’s instructions. Absorbance was measured with a Microplate Reader (Tecan Infinite 200) and analyzed with Magellan data analysis software.
2.7. Piezo1 Protein Level Detected by Western Blot
Three days after transfecting HEK cells and HAF with the respective plasmids or siRNAs, cells were lysed in radio immunoprecipitation buffer (containing (in mM): Tris-HCl 50 and NaCl 150; and, (in % w/v) NP-40 1, sodium deoxycholate 1, and sodium dodecyl sulphate 0.1) supplemented with protease inhibitors (1:200, 539134, Calbiochem, Darmstadt, Germany) for 15 min on ice, followed by centrifugation (15 min at 15,000× g, 4 °C). Subsequently, cleared lysates (supernatants) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis using 7% polyacrylamide gels and transferred to nitrocellulose membranes. These were saturated by incubation in 5% bovine serum albumin in phosphate buffered saline for 1 h at room temperature. Primary antibodies against Piezo1 (generated in rabbit, 15939-1-AP, Proteintech, Manchester, United Kingdom) and glyceraldehyde-3-phosphate dehydrogenase (generated in mouse, G8795, Sigma-Aldrich, Hamburg, Germany) were applied 1:1000 in 5% bovine serum albumin overnight at 4 °C with gentle agitation. Horseradish peroxidase-coupled secondary antibodies (anti-rabbit, 7074S, Cell Signaling Technologies, Danvers, MA, USA, and anti-mouse, HAF007, R&D Systems, Minneapolis, MN, USA) were applied 1:5000. Membranes were visualized using SuperSignal West Pico PLUS Chemiluminescence Substrate or Femto Maximum Sensitivity Substrate (34580 or 34096, ThermoFisher) and recorded using a Fusion-Fx gel documentation system (Vilber, Eberhardzell, Germany).
2.8. Patch-Clamp Recording of Piezo1 Activity
The patch-clamp technique was used to evaluate the presence of functional Piezo1 at the plasma membrane. Cell-attached patch-clamp recordings were performed using bath and pipette solutions previously described for characterizing Piezo1 channels [37
]. The pipette medium contained (in mM): NaCl 150, KCl 5, CaCl2
2, and HEPES 10; pH 7.4 with NaOH, 310 mOsm/L and the bath medium contained (in mM): KCl 155, EGTA 5, MgCl2
3, and HEPES 10; pH 7.2 with KOH, 310 mOsm/L. Pressure pulses of increasing amplitude (from 0 to −60 mmHg, in −10 mmHg increments) were applied through the recording electrode using a pressure-clamp device (ALA High Speed Pressure Clamp-1 system; ALA Scientific, Farmingdale, NY, USA). Experiments were performed at room temperature (20 °C), using a patch-clamp amplifier (200B, Axon Instruments, San Jose, CA, USA) and a Digidata 1440A interface (Axon Instruments). Recorded currents were digitized at 3 kHz, low-pass filtered at 1 kHz, and analyzed with pCLAMP10.3 software (Axon Instruments) and OriginPro 2019 (OriginLabCorporation, Northampton, MA, USA).
2.9. Statistical Analyses
Individual data points deviating by more than 3 standard deviations from the mean of the raw data set were defined as outliers and removed from further analysis (in total 1.69% of cells). Normal distribution of the data was assessed for each group using Shapiro–Wilk test and had to be rejected for most groups. Thus, to determine statistical significance of differences between experimental groups, non-parametric Kruskal–Wallis–ANOVA has been performed, followed by post-hoc Dunn’s comparison of the means. Groups were considered significantly different with a p-value <0.05. Data is presented as mean ± standard error to the mean, and as single data points. All statistical analyses were performed in OriginPro 2019 (OriginLabCorporation). Graphical summary of the main results was created with BioRender.com.
With exception of Figure 4, all experiments on HAF reported in this study were performed side-by-side with controls (i.e., all conditions in an individual figure panel were tested on the same day) to account for variations between experiments.