Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Protein expression levels of Cav1.2, Piezo1, CaM, and Src in human LAA tissues. (A) Representative western blots and densitometric analysis of Cav1.2 and Piezo1 proteins in LA tissues of AF patients and those with SR. (B) Representative western blots and densitometric analysis of CaM and Src protein in LA tissues of AF patients and those with SR. GAPDH was the internal control. ** p < 0.01. Values are presented as the mean ± standard error of the mean (SEM).

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Expressing, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effects of hypertension on the incidence of AF, I Ca,L , and Piezo1 expression in Wistar rats and SHRs with and without Val treatment. (A) Representative baseline surface ECG and intra-atrial electrocardiogram (IAEG); (B) The incidence AF in Wistar rats and SHRs treated with and without Val treatment ( n = 8). ** p < 0.01 vs. Wistar rat; ## p < < 0.01 vs. SHRs. (C) Typical surface ECG recordings of rats with AF that spontaneously reverted to SR and typical disorganized amplification of atrial waves (f wave). (D) Representative traces of AP in atrial myocytes from Wistar rats, SHRs, and SHR + Val groups and a histogram of APD in atrial myocytes from each group ( n = 12–15 myocytes from 3–4 rats). * p < 0.05 vs. Wistar rat; # p < 0.05 vs. SHRs. (E) Representative traces of I Ca,L (pulse protocol, inset), corresponding current-voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L in atrial myocytes of each group ( n = 8–14 myocytes from 3–4 rats). (F) Representative examples of immunohistochemical analysis of LA tissues from Wistar rats and SHRs treated with and without Val using Ab against Piezo1. Scale bar, 20 μm. (G) Representative western blots and densitometric analysis of Cav1.2, Piezo1, CaM, and Src in LA tissues of Wistar rats and SHRs. GAPDH was the internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Expressing, Amplification, Activation Assay, Immunohistochemical staining, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effect of HHP on the depression of I Ca,L in HL-1 cells. (A) Representative traces of AP in HL-1 cells under various hydrostatic pressures (0, 20, and 40 mmHg) for 24 h. APD 50 , APD 70 , and APD 90 of HL-1 cells were calculated ( n = 9, 10, and 7 at 0, 20, and 40 mmHg, respectively). * p < 0.05, ** p < 0.01 vs. 0 mmHg. (B) Representative traces (pulse protocol, inset), corresponding current-voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L ( n = 8–18 at 0, 20, and 40 mmHg, respectively). * p < 0.05, ** p < 0.01 vs. 0 mmHg. (C) Representative western blots and densitometric analysis of Cav1.2, Piezo1, CaM, and Src in HL-1 cells under various hydrostatic pressure (0, 20, and 40 mmHg) for 24 h. GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: The effects of Piezo1 on perceiving HHP and mediating the decrease of I Ca,L . (A,B) Representative Ca 2+ traces and Δ Ca i 2 + (ΔF/F) are shown. Ca 2+ entry was evoked by 10 μm Yoda1 in HL-1 cells stimulated by HHP in the presence or absence of the Piezo1 inhibitor GsmTx4 ( n = 50) or siRNA specifically knockdown Piezo1 ( n = 53–58). si-C, scrambled (control) siRNA; si-P, siRNA directed against Piezo1. (C) Representative traces of AP in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment (3.0 μM) and APD 50 , APD 70 , and APD 90 of HL-1 cells were calculated ( n = 9, 7, and 11 at 0, 40, and 40 mmHg + GsmTx4). * p < 0.05, ** p < 0.01 vs. 0 mmHg; # p < 0.05 vs. 40 mmHg. (D) Representative traces (pulse protocol, inset), corresponding current–voltage relationship, mean data for voltage dependence activation, inactivation, and time course of recovery current for I Ca,L in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment ( n = 9–15). ** p < 0.01 vs. 0 mmHg; ## p < 0.01 vs. 40 mmHg. (E) Representative blots and densitometry analysis of Cav1.2 in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment. (F) Representative blots and densitometry analysis of Cav1.2 in Yoda1 stimulation at different dosages (1, 3, and 10 μM) for 48 h. GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Activation Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Effect of CaM/Src on the decrease of I Ca,L induced by HHP or Yoda1 stimulation. (A) Representative blots and densitometry analysis of CaM and Src in HL-1 cells stimulated by 40 mmHg pressure with and without GsmTx4 treatment. (B) Representative blots and densitometry analysis of CaM and Src in Yoda1 stimulation at different dosages (1, 3, and 10 μM) for 48 h. (C) Representative blots and densitometry analysis of Src and p-Src ( n = 4) in HL-1 cells transfected with scrambled (control) siRNA or siRNA directed against Piezo1 for 48 h, then treated with Yoda1 at different dosages (0, 1, and 3 μM) for 15 min. (D) Representative traces of AP and histogram of APD in HL-1 cells ( n = 7–8). * p < 0.05 vs. 0 mmHg + DMSO. # p < 0.05 vs. 40 mmHg + DMSO. (E) Current–voltage relationship for I Ca,L ( n = 9–17) in HL-1 cells stimulated by 40 mmHg pressure treated with 15 μM PP1 or W7. * p < 0.05 vs. 0 mmHg + DMSO. # p < 0.05 vs. 40 mmHg + DMSO. (F) Representative blots and densitometry analysis of Cav1.2 and Src in HL-1 cells stimulated by 40 mmHg pressure treated with W7 under different concentrations (5, 10, 15, and 20 μM). (G) Representative blots and densitometry analysis of Cav1.2 in HL-1 cells stimulated by 40 mmHg pressure treated with PP1 (15 μM). (H) Current-voltage relationship for I Ca,L ( n = 8–10) in HL-1 cells stimulated by Yoda1(3 μM) treated with PP1. * p < 0.05, ** p < 0.01 vs. DMSO. # p < 0.05, ## p < 0.01 vs. Yoda1. (I) Representative blots and densitometry analysis of Cav1.2 in Yoda1(3μM) -stimulated HL-1 cells treated with PP1 (15 μM). GAPDH was used as an internal control. Values are presented as the mean ± SEM.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques: Transfection

Journal: Frontiers in Cardiovascular Medicine

Article Title: Piezo1 Participated in Decreased L-Type Calcium Current Induced by High Hydrostatic Pressure via . CaM/Src/Pitx2 Activation in Atrial Myocytes

doi: 10.3389/fcvm.2022.842885

Figure Lengend Snippet: Schematic representation of the mechanism for the decrease of I Ca,L induced by HHP. Piezo1 activated by HHP depressed I Ca,L contributing to increased AF susceptibility through the CaM/Src/Pitx2 pathway.

Article Snippet: According to standard protocols, the treated protein samples (15–30 μg) were separated by electrophoresis with 10% SDS–polyacrylamide gels and transferred to PVDF membranes, which were blocked with 5% non-fat milk for 1 h at room temperature and then incubated overnight at 4°C with primary rabbit polyclonal Abs against Piezo1, Cav1.2 (dilution, 1:1000; Alomone Labs, Jerusalem, Israel); and Src (1:1000; Abcam, Waltham, MA, USA;) and mouse polyclonal Abs against Pitx2 (1:1000; Cloud-Clone Corp., Wuhan, Hubei, China) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin (1:5000; Cell Signaling Technology, Inc., Beverly, MA, USA).

Techniques:

Journal: Biomolecules

Article Title: Functional Role of Piezo1 in the Human Eosinophil Cell Line AML14.3D10: Implications for the Immune and Sensory Nervous Systems

doi: 10.3390/biom14091157

Figure Lengend Snippet: Reverse transcription-polymerase chain reaction primer list.

Article Snippet: The primary antibody was rabbit polyclonal anti-Piezo1 (1:25, Invitrogen, Carlsbad, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: Biomolecules

Article Title: Functional Role of Piezo1 in the Human Eosinophil Cell Line AML14.3D10: Implications for the Immune and Sensory Nervous Systems

doi: 10.3390/biom14091157

Figure Lengend Snippet: AML14.3D10 cells exhibit functional expression of the Piezo1 channel. ( a ) Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the AML14.3D10 cells shows the expression of transcripts using two different primer pairs specific for Piezo1 and Piezo2 . ( b ) Representative immunofluorescence (IF) images of AML14.3D10 cells with primary and secondary antibodies showing Alexa 555 with the indicated markers (scale bar, 20 μm). Negative controls represent AML14.3D10 cells exposed to Alexa 555 secondary antibody only (no primary antibody) in addition to DAPI. ( c ) Calcium imaging showing Ca 2+ response induced by sequential application of Yoda1 (10 μM, 30 s). ( d ) Mean normalized amplitude of sequential Yoda1-induced Ca 2+ response ( n = 56). ( e ) Representative traces showing Yoda1- (10 μM, 30 s) and Ruthenium Red-induced (10 μM, 3 min pre-treatment, Pink) Ca 2+ response. ( f ) Mean normalized amplitude of Ca 2+ response induced by sequential application of Yoda1 (10 μM, 30 s) and Ruthenium Red (10 μM, 3 min pre-treatment). ( g ) Representative traces showing Yoda1- (10 μM, 30 s) and Gd 3+ (10 μM, 3 min pre-treatment, light purple) Ca 2+ response. ( h ) Mean normalized amplitude of Ca 2+ response induced by sequential application of Yoda1 (10 μM, 30 s) and Gd 3+ (10 μM, 3 min pre-treatment). ( i ) Representative traces showing Yoda1- (10 μM, 30 s) and GsMTx4-induced (1 μM, 3 min pre-treatment, blue) Ca 2+ response. ( j ) Mean normalized amplitude of Ca 2+ response induced by sequential application of Yoda1 (10 μM, 30 s) and GsMT × 4 (1 μM, 3 min pre-treatment). ( k ) Representative traces showing Yoda1- (10 μM, 30 s) and Dooku1-induced (10 μM, 30 s, Green) Ca 2+ response. ( l ) Mean normalized amplitude of Ca 2+ response induced by sequential application of Yoda1 (10 μM, 30 s) and Dooku1 (10 μM, 3 min pre-treatment). ( m ) Mean normalized amplitude of sequential Yoda1- and Dooku1-induced Ca 2+ response in the control (n = 56), Dooku1 0.1 μM ( n = 23), 0.5 μM ( n = 26), 1 μM ( n = 49), and 10 μM ( n = 25). One-way ANOVA (Dunn’s multiple comparisons test, **** p < 0.001). ( n ) Graph showing the Ca 2+ response according to the concentration of Dooku1 on the IC 50 curve. All results are presented as the mean ± standard error of the mean (SEM). *** p < 0.001, **** p < 0.0001, Dunnett’s test following one-way analysis of variance versus the first Yoda1 treated group; ### p < 0.001, #### p < 0.0001, versus the mechanosensitive Ca 2+ channel blocker (Ruthenium Red, Gd 3+ , GsMTx4) and Dooku1 group. IC 50 : half-maximal inhibitory concentration, Gd 3+ : Gadolinium chloride. Original images of ( a ) can be found in .

Article Snippet: The primary antibody was rabbit polyclonal anti-Piezo1 (1:25, Invitrogen, Carlsbad, CA, USA).

Techniques: Functional Assay, Expressing, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Imaging, Control, Concentration Assay

Journal: Biomolecules

Article Title: Functional Role of Piezo1 in the Human Eosinophil Cell Line AML14.3D10: Implications for the Immune and Sensory Nervous Systems

doi: 10.3390/biom14091157

Figure Lengend Snippet: Piezo1 activation within AML14.3D10 cells induces various changes in cytokine expression and secretion. ( a ) AML14.3D10 cells were seeded at a density of 2 × 10 4 cells per well in 100 μL of culture medium in 96-well transparent plates with 0.1% DMSO, Yoda1 5, 10, and 50 μM for 24 h. The WST-1 activity of 0.1% DMSO and Yoda1 at 5 μM, 10 μM, and 50 μM, normalized to the WST-1 activity of the control, is 0.957, 0.897, 0.857, and 0.459, respectively. Results are presented as the mean ± standard error of the mean (SEM). Dunnett’s test following one-way analysis of variance **** p < 0.001 compared with control group (n = 5). ( b ) Reference map for cytokine array, adapted from the manufacturer’s information. Complete array images from ( c ) lysates and ( d ) supernatants of eosinophils. The human cytokine array of lysate proteins from eosinophils cultured in 0.1% DMSO and Yoda1 media. The human cytokine array of supernatants from eosinophils cultured in 0.1% DMSO and Yoda1 media. BLANK (BLK) represents an uncultured RPMI1640 medium. ( e ) Heat map showing the relative cytokine concentration with selected inflammatory cytokines, anti-inflammatory cytokines, and growth factors in lysate proteins from stimulated eosinophils after 24 h. ( f ) Heat map showing the relative concentration of cytokines with selected inflammatory cytokines, anti-inflammatory cytokines, and growth factors in supernatants from stimulated eosinophils after 24 h. All results are presented as the mean ± standard error of the mean (SEM). **** p < 0.0001, ns, Dunnett’s test following one-way analysis of variance versus the control group. Ns indicates no significant difference. DMSO: dimethyl sulfoxide.

Article Snippet: The primary antibody was rabbit polyclonal anti-Piezo1 (1:25, Invitrogen, Carlsbad, CA, USA).

Techniques: Activation Assay, Expressing, Activity Assay, Control, Cell Culture, Concentration Assay

Journal: Biomolecules

Article Title: Functional Role of Piezo1 in the Human Eosinophil Cell Line AML14.3D10: Implications for the Immune and Sensory Nervous Systems

doi: 10.3390/biom14091157

Figure Lengend Snippet: Piezo1 activation within AML14.3D10 cells induces various changes in cytokine expression over different time intervals. Quantitative real-time polymerase chain reaction analysis was performed for the following. ( a ) Pro-inflammatory cytokines: IL-1α (24 h, n = 3), IL-1β (24 h, n = 3), IL-6 (24 h, n = 3), IL-8 (CXCL8) (24 h, n = 3), IL-12B (2 h, n = 3), CCL5 (RANTES) (12 h, n = 3); Inflammasome: Caspase-1 (24 h, n = 3), Caspase-3 (24 h, n = 5), and NLRP3 (12 h, n = 3). ( b ) Anti-inflammatory cytokines: IL-4 (2 h, n = 3), IL-10 (4 h, n = 3), IL-13 (2 h, n = 3), TGF-β1 (12 h, n = 3). ( c ) Time course of gene expression induced by Piezo1 activation in AML14.3D10 cells. Each time point value is an average of data: 2-h time point (Blue, IL-4, IL-13, IL-12B), 4-h time point (Green, IL-10), 12-h time point (Pink, NLRP3, CCL5 (RANTES), TGF-β1), 24-h time point (Red, IL-1α, IL-1β, IL-6, IL-8 (CXCL8), Caspase-1, Caspase-3). Data are presented as the mean ± standard error of the mean. * p < 0.05, ** p < 0.01, *** p < 0.001, Dunnett’s test following one-way analysis of variance versus the control group; # p < 0.05, ## p < 0.01, versus the Yoda1 group. CXCL: C-X-C motif ligand, CCL: C-C motif ligand, NLRP3: nucleotide-binding domain, leucine-rich–containing family, pyrin domain–containing-3, TGF: transforming growth factor, IL: interleukin.

Article Snippet: The primary antibody was rabbit polyclonal anti-Piezo1 (1:25, Invitrogen, Carlsbad, CA, USA).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Control, Binding Assay