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. 2022 Dec 23:13:1056657.
doi: 10.3389/fphys.2022.1056657. eCollection 2022.

TRPC1 channels underlie stretch-modulated sarcoplasmic reticulum calcium leak in cardiomyocytes

Molly E Streiff  1   2 , Andrea C Corbin  1   2 , Azmi A Ahmad  1   2 , Chris Hunter  1 , Frank B Sachse  1   2
Affiliations

TRPC1 channels underlie stretch-modulated sarcoplasmic reticulum calcium leak in cardiomyocytes

Molly E Streiff et al. Front Physiol. .

Abstract

Transient receptor potential canonical 1 (TRPC1) channels are Ca2+-permeable ion channels expressed in cardiomyocytes. An involvement of TRPC1 channels in cardiac diseases is widely established. However, the physiological role of TRPC1 channels and the mechanisms through which they contribute to disease development are still under investigation. Our prior work suggested that TRPC1 forms Ca2+ leak channels located in the sarcoplasmic reticulum (SR) membrane. Prior studies suggested that TRPC1 channels in the cell membrane are mechanosensitive, but this was not yet investigated in cardiomyocytes or for SR localized TRPC1 channels. We applied adenoviral transfection to overexpress or suppress TRPC1 expression in neonatal rat ventricular myocytes (NRVMs). Transfections were evaluated with RT-qPCR, western blot, and fluorescent imaging. Single-molecule localization microscopy revealed high colocalization of exogenously expressed TRPC1 and the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2). To test our hypothesis that TRPC1 channels contribute to mechanosensitive Ca2+ SR leak, we directly measured SR Ca2+ concentration ([Ca2+]SR) using adenoviral transfection with a novel ratiometric genetically encoded SR-targeting Ca2+ sensor. We performed fluorescence imaging to quantitatively assess [Ca2+]SR and leak through TRPC1 channels of NRVMs cultured on stretchable silicone membranes. [Ca2+]SR was increased in cells with suppressed TRPC1 expression vs. control and Transient receptor potential canonical 1-overexpressing cells. We also detected a significant reduction in [Ca2+]SR in cells with Transient receptor potential canonical 1 overexpression when 10% uniaxial stretch was applied. These findings indicate that TRPC1 channels underlie the mechanosensitive modulation of [Ca2+]SR. Our findings are critical for understanding the physiological role of TRPC1 channels and support the development of pharmacological therapies for cardiac diseases.

Keywords: TRPC1; calcium; cardiomyocyte; mechanosensitivity; sarcoplasmic reticulum.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Expression of TagBFP2 adenoviral constructs in NRVMs. Fluorescent imaging of TagBFP2 in cells expressing (A) TRPC1-TagBFP2, (B) TagBFP2, (C) scrambled shRNA-TagBFP2, and (D) shRNA-TRPC1-TagBFP2. Scale bars = 20 µm.
FIGURE 2
FIGURE 2
Mean fold change of TRPC1 mRNA expression of infected NRVMs from WT NRVMs quantified by RT-qPCR with primers targeting rat TRPC1 (A) exon 3-4 (Rn00677552_m1) (B) exon 6-7 (Rn00677554_m1) (C) exon 8-9 (Rn00585625_m1) (D) exon 10-11 (Rn00677549_g1) and (E) exon 11-12 (Rn01447000_m1) (n = 3 litters). Brackets mark significant differences (p < 0.05).
FIGURE 3
FIGURE 3
Super-resolution images of NRVMs infected with TRPC1-TagBFP2 (A–F) and TagBFP2 (G–L). TRPC1-TagBFP2 cells exhibit a striated arrangement of both (A) TagBFP2 and (B) SERCA2 fluorescence. (C) Colocalization of TagBFP2 and SERCA2 in TRPC1-TagBFP2 cells is high, shown in white. (D–F) Zoomed in region from (A–C) depicted by white box in (C). (G) TagBFP2 fluorescence in control cells is distributed throughout the cell, with more dense concentration near sarcomere structures. (H) SERCA2 fluorescence in TagBFP2 control cells follows a network, with (I) minimal colocalization with TagBFP2. (J–L) Zoomed in region from (G–I) depicted by white box in (I). Scale bars in (C,I) = 10 µm. Scale bars in (F,L) = 3 µm.
FIGURE 4
FIGURE 4
Quantification of colocalization from super-resolution images of NRVMs infected with TRPC1-TagBFP2 or TagBFP2. (A) Average distribution of nearest neighbor distances (NND) of SERCA2 from TagBFP2 in TRPC1-TagBFP2 cells and TagBFP2 control cells. (B) Mean NND in TRPC1-TagBFP2 cells (98.08 ± 4.98 nm) is significantly lower than in TagBFP2 control cells (145.93 ± 5.55 nm) (n = 8 cells). Bracket marks significant difference (p < 0.001).
FIGURE 5
FIGURE 5
Super-resolution images of GCEPIAer-SNAP construct in TRPC1-TagBFP2 cells. (A) SNAP-Cell 647-SiR signal exhibits a network pattern strikingly similar to the (B) tRFP signal corresponding to TRPC1-TagBFP2. (C) The patterns exhibited from markers for both the GCEPIAer-SNAP construct and TRPC1-TagBFP2 construct reveal abundant overlap. Scale bar = 5 µm.
FIGURE 6
FIGURE 6
Calibrated [Ca2+]SR from fluorescent calcium imaging of NRVMs. Representative ratiometric images of the GCEPIAer signal over the SNAP signal in NRVMs infected with (A) TRPC1-TagBFP2 under 0% strain, (B) TagBFP2 under 0% strain, (C) shRNA-TRPC1-TagBFP2 under 0% strain, (D) TRPC1-TagBFP2 under 10% strain, (E) TagBFP2 under 10% strain, and (F) shRNA-TRPC1-TagBFP2 under 10% strain. Scale bars = 20 µm. (G) Example [Ca2+]SR transients from TRPC1-TagBFP2, TagBFP2 and shRNA-TRPC1-TagBFP2 infected un stretched and stretched NRVMs. (H) Scatter plot of diastolic [Ca2+]SR of each infection group with 0 or 10% stretch averaged by membrane; points correspond to the average value of all cells on each membrane and bars represent mean and SEM determined by multilevel mixed-effects model. Diastolic [Ca2+]SR in unstretched NRVMs is significantly lower in TRPC1-TagBFP2 and TagBFP2 cells compared to shRNA-TRPC1-TagBFP2 cells. Sustained 10% stretch significantly lowered [Ca2+]SR in TRPC1-TagBFP2 infected NRVMs. Brackets mark significant differences (p < 0.05).
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