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TRPC3 contributes to regulation of cardiac contractility and arrhythmogenesis by dynamic interaction with NCX1.

Doleschal B, Primessnig U, Wölkart G, Wolf S, Schernthaner M, Lichtenegger M, Glasnov TN, Kappe CO, Mayer B, Antoons G, Heinzel F, Poteser M, Groschner K - Cardiovasc. Res. (2015)

Bottom Line: GSK1702934A induced a transient, non-selective conductance and prolonged action potentials in TRPC3-overexpressing myocytes but lacked significant electrophysiological effects in wild-type myocytes.Excessive activation of TRPC3 is associated with transient cellular Ca2+ overload, spatial uncoupling between TRPC3 and NCX1, and arrhythmogenesis.We propose TRPC3-NCX micro/nanodomain communication as determinant of cardiac contractility and susceptibility to arrhythmogenic stimuli.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria.

No MeSH data available.


Related in: MedlinePlus

GSK disrupts TRPC3 and NCX colocalization in adult mouse cardiomyocytes. (A) Confocal fluorescence images of freshly isolated cardiac myocytes (n = 15; N = 3) without (top) and after 5-min exposure to GSK (bottom) and stained for NCX1 (FITC, green) and TRPC3 (TRITC, red) were obtained using a Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany). Immunostaining was performed as described previously.21 Magnified parts of the images depicting cell regions used for line scan analysis shown in B. (B) Overlap of FITC-NCX and TRITC-TRPC3 fluorescence intensities along a line scan (white line in micrograph) in the absence (top) and after 5-min exposure to GSK (bottom). Well-correlated intensity peaks are indicated by arrows, non-correlating peaks by x. (C) Mean (±SEM) pearson correlation coefficients (left) and non-linear correlation coefficients (right) of FITC-NCX and TRITC-TRPC3 fluorescence intensity as gained from line scans (n = 6) recorded in myocytes with and without exposure to 1 µM GSK as indicated. (D) Fluorescence microscopic images of HL-1 cells transfected with CFP-NCX split exchanger (CFP-NCX-265 + NCX-272, cyan), YFP-TRPC3 (green), overlay of YFP-TRPC3, and CFP-NCX split exchanger fluorescence (overlay) and net FRET after bleedthrough- and colocalization correction (nFRET) as described in Ref. 39. (E) Representative time course of rawFRET intensity recorded from a cell expressing YFP-TRPC3 and CFP-NCX-265 + NCX-272 (background subtracted). Image showing non-normalized FRET (rawFRET) recorded at the initial phase of the timeline. After 1min, 1 µM GSK was added to the chamber. Insert showing mean normalized rawFRET intensities (±SEM) recorded at indicated times from six independent experiments. Statistical significance was analysed by t-test.
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CVV022F6: GSK disrupts TRPC3 and NCX colocalization in adult mouse cardiomyocytes. (A) Confocal fluorescence images of freshly isolated cardiac myocytes (n = 15; N = 3) without (top) and after 5-min exposure to GSK (bottom) and stained for NCX1 (FITC, green) and TRPC3 (TRITC, red) were obtained using a Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany). Immunostaining was performed as described previously.21 Magnified parts of the images depicting cell regions used for line scan analysis shown in B. (B) Overlap of FITC-NCX and TRITC-TRPC3 fluorescence intensities along a line scan (white line in micrograph) in the absence (top) and after 5-min exposure to GSK (bottom). Well-correlated intensity peaks are indicated by arrows, non-correlating peaks by x. (C) Mean (±SEM) pearson correlation coefficients (left) and non-linear correlation coefficients (right) of FITC-NCX and TRITC-TRPC3 fluorescence intensity as gained from line scans (n = 6) recorded in myocytes with and without exposure to 1 µM GSK as indicated. (D) Fluorescence microscopic images of HL-1 cells transfected with CFP-NCX split exchanger (CFP-NCX-265 + NCX-272, cyan), YFP-TRPC3 (green), overlay of YFP-TRPC3, and CFP-NCX split exchanger fluorescence (overlay) and net FRET after bleedthrough- and colocalization correction (nFRET) as described in Ref. 39. (E) Representative time course of rawFRET intensity recorded from a cell expressing YFP-TRPC3 and CFP-NCX-265 + NCX-272 (background subtracted). Image showing non-normalized FRET (rawFRET) recorded at the initial phase of the timeline. After 1min, 1 µM GSK was added to the chamber. Insert showing mean normalized rawFRET intensities (±SEM) recorded at indicated times from six independent experiments. Statistical significance was analysed by t-test.

Mentions: A close spatial relation between TRPC3 and NCX1 has previously been demonstrated21,38 and is considered prerequisite for functional consequences of cardiac TRPC3 activation. We hypothesized that alterations in spatial coupling betweenTRPC3 and NCX1 may be involved in prolongation of functional consequences of TRPC3 activation. In a first set of experiments, we analysed the localization of TRPC3 and NCX1 in TRPC3-TG myocytes by immunocytochemistry (Figure 6). Our results corroborated colocalization of the two transport molecules at basal conditions. Interestingly, activation of TRPC3 channels by GSK effectively disrupted colocalization of the signalling proteins (Figure 6) and a similar effect was initiated by AngII (see Supplementary material online, Figure S9).Figure 6


TRPC3 contributes to regulation of cardiac contractility and arrhythmogenesis by dynamic interaction with NCX1.

Doleschal B, Primessnig U, Wölkart G, Wolf S, Schernthaner M, Lichtenegger M, Glasnov TN, Kappe CO, Mayer B, Antoons G, Heinzel F, Poteser M, Groschner K - Cardiovasc. Res. (2015)

GSK disrupts TRPC3 and NCX colocalization in adult mouse cardiomyocytes. (A) Confocal fluorescence images of freshly isolated cardiac myocytes (n = 15; N = 3) without (top) and after 5-min exposure to GSK (bottom) and stained for NCX1 (FITC, green) and TRPC3 (TRITC, red) were obtained using a Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany). Immunostaining was performed as described previously.21 Magnified parts of the images depicting cell regions used for line scan analysis shown in B. (B) Overlap of FITC-NCX and TRITC-TRPC3 fluorescence intensities along a line scan (white line in micrograph) in the absence (top) and after 5-min exposure to GSK (bottom). Well-correlated intensity peaks are indicated by arrows, non-correlating peaks by x. (C) Mean (±SEM) pearson correlation coefficients (left) and non-linear correlation coefficients (right) of FITC-NCX and TRITC-TRPC3 fluorescence intensity as gained from line scans (n = 6) recorded in myocytes with and without exposure to 1 µM GSK as indicated. (D) Fluorescence microscopic images of HL-1 cells transfected with CFP-NCX split exchanger (CFP-NCX-265 + NCX-272, cyan), YFP-TRPC3 (green), overlay of YFP-TRPC3, and CFP-NCX split exchanger fluorescence (overlay) and net FRET after bleedthrough- and colocalization correction (nFRET) as described in Ref. 39. (E) Representative time course of rawFRET intensity recorded from a cell expressing YFP-TRPC3 and CFP-NCX-265 + NCX-272 (background subtracted). Image showing non-normalized FRET (rawFRET) recorded at the initial phase of the timeline. After 1min, 1 µM GSK was added to the chamber. Insert showing mean normalized rawFRET intensities (±SEM) recorded at indicated times from six independent experiments. Statistical significance was analysed by t-test.
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CVV022F6: GSK disrupts TRPC3 and NCX colocalization in adult mouse cardiomyocytes. (A) Confocal fluorescence images of freshly isolated cardiac myocytes (n = 15; N = 3) without (top) and after 5-min exposure to GSK (bottom) and stained for NCX1 (FITC, green) and TRPC3 (TRITC, red) were obtained using a Leica SP5 confocal microscope (Leica Microsystems, Mannheim, Germany). Immunostaining was performed as described previously.21 Magnified parts of the images depicting cell regions used for line scan analysis shown in B. (B) Overlap of FITC-NCX and TRITC-TRPC3 fluorescence intensities along a line scan (white line in micrograph) in the absence (top) and after 5-min exposure to GSK (bottom). Well-correlated intensity peaks are indicated by arrows, non-correlating peaks by x. (C) Mean (±SEM) pearson correlation coefficients (left) and non-linear correlation coefficients (right) of FITC-NCX and TRITC-TRPC3 fluorescence intensity as gained from line scans (n = 6) recorded in myocytes with and without exposure to 1 µM GSK as indicated. (D) Fluorescence microscopic images of HL-1 cells transfected with CFP-NCX split exchanger (CFP-NCX-265 + NCX-272, cyan), YFP-TRPC3 (green), overlay of YFP-TRPC3, and CFP-NCX split exchanger fluorescence (overlay) and net FRET after bleedthrough- and colocalization correction (nFRET) as described in Ref. 39. (E) Representative time course of rawFRET intensity recorded from a cell expressing YFP-TRPC3 and CFP-NCX-265 + NCX-272 (background subtracted). Image showing non-normalized FRET (rawFRET) recorded at the initial phase of the timeline. After 1min, 1 µM GSK was added to the chamber. Insert showing mean normalized rawFRET intensities (±SEM) recorded at indicated times from six independent experiments. Statistical significance was analysed by t-test.
Mentions: A close spatial relation between TRPC3 and NCX1 has previously been demonstrated21,38 and is considered prerequisite for functional consequences of cardiac TRPC3 activation. We hypothesized that alterations in spatial coupling betweenTRPC3 and NCX1 may be involved in prolongation of functional consequences of TRPC3 activation. In a first set of experiments, we analysed the localization of TRPC3 and NCX1 in TRPC3-TG myocytes by immunocytochemistry (Figure 6). Our results corroborated colocalization of the two transport molecules at basal conditions. Interestingly, activation of TRPC3 channels by GSK effectively disrupted colocalization of the signalling proteins (Figure 6) and a similar effect was initiated by AngII (see Supplementary material online, Figure S9).Figure 6

Bottom Line: GSK1702934A induced a transient, non-selective conductance and prolonged action potentials in TRPC3-overexpressing myocytes but lacked significant electrophysiological effects in wild-type myocytes.Excessive activation of TRPC3 is associated with transient cellular Ca2+ overload, spatial uncoupling between TRPC3 and NCX1, and arrhythmogenesis.We propose TRPC3-NCX micro/nanodomain communication as determinant of cardiac contractility and susceptibility to arrhythmogenic stimuli.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria.

No MeSH data available.


Related in: MedlinePlus