Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Feb 14;11:44.
doi: 10.3389/fphys.2020.00044. eCollection 2020.

Modulation of Calcium Transients in Cardiomyocytes by Transient Receptor Potential Canonical 6 Channels

Affiliations
Free PMC article

Modulation of Calcium Transients in Cardiomyocytes by Transient Receptor Potential Canonical 6 Channels

Azmi A Ahmad et al. Front Physiol. .
Free PMC article

Abstract

Transient receptor potential canonical 6 (TRPC6) channels are non-selective cation channels that are thought to underlie mechano-modulation of calcium signaling in cardiomyocytes. TRPC6 channels are involved in development of cardiac hypertrophy and related calcineurin-nuclear factor of activated T cells (NFAT) signaling. However, the exact location and roles of TRPC6 channels remain ill-defined in cardiomyocytes. We used an expression system based on neonatal rat ventricular myocytes (NRVMs) to investigate the location of TRPC6 channels and their role in calcium signaling. NRVMs isolated from 1- to 2-day-old animals were cultured and infected with an adenoviral vector to express enhanced-green fluorescent protein (eGFP) or TRPC6-eGFP. After 3 days, NRVMs were fixed, immunolabeled, and imaged with confocal and super-resolution microscopy to determine TRPC6 localization. Cytosolic calcium transients at 0.5 and 1 Hz pacing rates were recorded in NRVMs using indo-1, a ratio-metric calcium dye. Confocal and super-resolution microscopy suggested that TRPC6-eGFP localized to the sarcolemma. NRVMs infected with TRPC6-eGFP exhibited higher diastolic and systolic cytosolic calcium concentration as well as increased sarcoplasmic reticulum (SR) calcium load compared to eGFP infected cells. We applied a computer model comprising sarcolemmal TRPC6 current to explain our experimental findings. Altogether, our studies indicate that TRPC6 channels play a role in sarcolemmal and intracellular calcium signaling in cardiomyocytes. Our findings support the hypothesis that upregulation or activation of TRPC6 channels, e.g., in disease, leads to sustained elevation of the cytosolic calcium concentration, which is thought to activate calcineurin-NFAT signaling and cardiac hypertrophic remodeling. Also, our findings support the hypothesis that mechanosensitivity of TRPC6 channels modulates cytosolic calcium transients and SR calcium load.

Keywords: calcium transient; cardiomyocyte; mechanosensitivity; transient receptor potential canonical 6; transient receptor potential canonical channels.

Figures

Figure 1
Figure 1
Confocal microscopic images of living NRVMs infected with (A) eGFP at 20 MOI, (B) TRPC6-eGFP at 10 MOI, and (C) shRNA-TRPC6-eGFP at 100 MOI. eGFP and shRNA-TRPC6-eGFP infected cells exhibit a diffuse cytosolic signal. In TRPC6-eGFP cells, signal is localized to sarcolemma and nuclear membrane. Colormap for each signal is added for comparison of fluorescence intensities between groups. (D) Western blotting with anti-eGFP antibody (AB6556) of NRVMs expressing TRPC6-eGFP (lane 1), eGFP (lane 2), and shRNA-TRPC6-eGFP (lane 3). Expression of eGFP at ~30 kDa band was detected in eGFP and shRNA-TRPC6-eGFP cells. (E) Longer exposure of lane 1 confirms TRPC6-eGFP expression at the expected 135 kDa molecular weight of the construct.
Figure 2
Figure 2
Confocal microscopic images of fixed infected NRVMs. (A) NRVM infected with eGFP at 20 MOI present an intracellular eGFP distribution. (B) TRPC6 labeling with LS-C19628 antibody shows marginal signal. (C) DAPI used as a marker for the nucleus. (D) Overlay of (A–C). (E) NRVMs infected with TRPC6-eGFP at 10 MOI present signal at the sarcolemma. (F) TRPC6 labeling shows high fluorescence signal at the sarcolemma and nuclear membrane. (G) DAPI as marker for the nucleus. (H) Overlay of (E–G) shows high colocalization of TRPC6-eGFP and anti-TRPC6 antibody labeling, suggesting successful detection of our vector by the antibody. (I) NRVM infected with shRNA-TRPC6-eGFP at 100 MOI present diffuse, very bright intracellular eGFP signal. (J) TRPC6 labeling shows marginal fluorescence. (K) DAPI as a marker for the nucleus. (L) Overlay of (I–K). Imaging of eGFP, TRPC6, and DAPI in the three experimental groups was done with identical settings. Colormap for each signal is added for comparison of fluorescence intensities between groups. TRPC6 labeling had much higher fluorescence in TRPC6-eGFP than eGFP and shRNA-TRPC6-eGFP cells. Scale bar in (A) applies to (B–L).
Figure 3
Figure 3
Super-resolution microscopy of fixed NRVM infected with TRPC6-eGFP at 10 MOI and labeled with anti-eGFP nanobody. (A) Wide-field reference image of eGFP signal. (B) Super-resolution image of TRPC6-eGFP signal from box in (A). The TRPC6-eGFP pattern suggests sarcolemmal localization.
Figure 4
Figure 4
Indo-1 calibration protocol in NRVMs bathed in 10 μM ionomycin. Example trace shows (A) F405, (B) F485, and (C) F405/F485 during application of a bath solution with 0 and 2 mM Ca2+. (D) Fmin and Fmax were calculated from F405/F485 for 0 and 2 mM Ca2+ application, respectively. Fmin and Fmax for all groups were not significantly different. (E) Average Sf2/Sb2 was calculated as the ratio from the F485 emission during the 0 Ca2+ and 2 mM Ca2+. Sf2/Sb2 in all groups was not significantly different.
Figure 5
Figure 5
Measurement and analysis of [Ca2+]i. Example traces for NRVMs expressing eGFP or TRPC6-eGFP and paced at (A) 0.5 and (B) 1 Hz. Expression of TRPC6-eGFP caused a positive shift in [Ca2+]i transients vs. eGFP at both pacing rates. (C) Diastolic and (D) systolic [Ca2+]i increased in TRPC6-eGFP vs. eGFP cells for both pacing rates. (E) TDecay decreased in TRPC6-eGFP vs. eGFP cells for 0.5 Hz pacing. (F) SR load increased in NRVMs expressing TRPC6-eGFP vs. eGFP.
Figure 6
Figure 6
Computer model of NRVM with currents through TRPC6 channels modeled as sarcolemmal leak with GTRPC6 at 0, 3, and 6 μS/μF. [Ca2+]i at (A) 0.5 and (B) 1 Hz pacing. Increased GTRPC6 caused a positive shift in [Ca2+]i transients at both 0.5 and 1 Hz pacing. (C) Diastolic and (D) systolic [Ca2+]i at 0.5 and 1 Hz increased for increased GTRPC6. (E) TDecay decreased with increased GTRPC6. (F) SR load increased with increasing GTRPC6.

Similar articles

See all similar articles

References

    1. Ahmad A. A., Streiff M., Hunter C., Hu Q., Sachse F. B. (2017). Physiological and pathophysiological role of transient receptor potential canonical channels in cardiac myocytes. Prog. Biophys. Mol. Biol. 130, 254–263. 10.1016/j.pbiomolbio.2017.06.005, PMID: - DOI - PubMed
    1. Aires V., Hichami A., Boulay G., Khan N. A. (2007). Activation of TRPC6 calcium channels by diacylglycerol (DAG)-containing arachidonic acid: a comparative study with DAG-containing docosahexaenoic acid. Biochimie 89, 926–937. 10.1016/j.biochi.2006.10.016, PMID: - DOI - PubMed
    1. Bon R. S., Beech D. J. (2013). In pursuit of small molecule chemistry for calcium-permeable non-selective TRPC channels – mirage or pot of gold? Br. J. Pharmacol. 170, 459–474. 10.1111/bph.12274, PMID: - DOI - PMC - PubMed
    1. Cayouette S., Lussier M. P., Mathieu E. L., Bousquet S. M., Boulay G. (2004). Exocytotic insertion of TRPC6 channel into the plasma membrane upon Gq protein-coupled receptor activation. J. Biol. Chem. 279, 7241–7246. 10.1074/jbc.M312042200, PMID: - DOI - PubMed
    1. Dibb K. M., Eisner D. A., Trafford A. W. (2007). Regulation of systolic [Ca2+]i and cellular Ca2+ flux balance in rat ventricular myocytes by SR Ca2+, L-type Ca2+ current and diastolic [Ca2+]i. J. Physiol. 585, 579–592. 10.1113/jphysiol.2007.141473, PMID: - DOI - PMC - PubMed
Feedback