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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

Cardiac TRPC3 activity modulates NCX function. NCX currents were recorded by applying depolarizing voltage steps (500 ms) from a holding potential of −40 mV and quantified as the Ni+-sensitive current components at the end of the 500 ms depolarization steps (NCX reverse mode) and 20 ms after repolarization to −40 mV (forward mode).34,35 (A) Representative Ni+-sensitive outward currents recorded during depolarizing steps as well as subsequent tail currents upon repolarization to −40 mV for 4.5 s are shown for WT cardiomyocytes (left) and TG cardiomyocytes (right) in the absence [n = 8; N = 3 (WT); n = 9; N = 3 (TG)] (top) and presence (bottom) of 1 µM GSK [n = 8; N = 3 (WT); n = 12; N = 3 (TG)]; SR function was eliminated by thapsigargin (3 µM) and l-type Ca2+ channels were blocked by nitrendipine (10 µM). (B) Mean (±SEM) I/V plots of Ni+-sensitive peak currents. Left: tail inward currents representing NCX forward-mode activity in WT and TRPC3-TG cells and in the absence or presence of 1 µM GSK. Tail currents represent Ni+-sensitive peak inward currents at 20 ms after repolarization to −40 mV with steady-state holding current subtracted. Right: mean (±SEM) peak outward currents recorded from WT and TRPC3-TG cells in the presence and absence of 1 µM GSK. * indicates significant difference (P < 0.05) to WT, WT + GSK, and TRPC3-TG; sharp (#) significant difference (P < 0.05) to WT + GSK cells. Statistical significance analysed by two-way Anova followed by Tukey's post hoc tests.
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CVV022F4: Cardiac TRPC3 activity modulates NCX function. NCX currents were recorded by applying depolarizing voltage steps (500 ms) from a holding potential of −40 mV and quantified as the Ni+-sensitive current components at the end of the 500 ms depolarization steps (NCX reverse mode) and 20 ms after repolarization to −40 mV (forward mode).34,35 (A) Representative Ni+-sensitive outward currents recorded during depolarizing steps as well as subsequent tail currents upon repolarization to −40 mV for 4.5 s are shown for WT cardiomyocytes (left) and TG cardiomyocytes (right) in the absence [n = 8; N = 3 (WT); n = 9; N = 3 (TG)] (top) and presence (bottom) of 1 µM GSK [n = 8; N = 3 (WT); n = 12; N = 3 (TG)]; SR function was eliminated by thapsigargin (3 µM) and l-type Ca2+ channels were blocked by nitrendipine (10 µM). (B) Mean (±SEM) I/V plots of Ni+-sensitive peak currents. Left: tail inward currents representing NCX forward-mode activity in WT and TRPC3-TG cells and in the absence or presence of 1 µM GSK. Tail currents represent Ni+-sensitive peak inward currents at 20 ms after repolarization to −40 mV with steady-state holding current subtracted. Right: mean (±SEM) peak outward currents recorded from WT and TRPC3-TG cells in the presence and absence of 1 µM GSK. * indicates significant difference (P < 0.05) to WT, WT + GSK, and TRPC3-TG; sharp (#) significant difference (P < 0.05) to WT + GSK cells. Statistical significance analysed by two-way Anova followed by Tukey's post hoc tests.

Mentions: Initial characterization of the effects of AngII on NCX1 forward-mode activity by tail current analysis revealed that AngII increased NCX-inward currents only in TRPC-TG myocytes (see Supplementary material online, Figure S2C). To test whether TRPC3 expression and activity indeed determines exchanger operation during cyclic de- and repolarization, we performed patch-clamp experiments in TRPC3-TG cardiomyocytes to delineate cyclic NCX function as the Ni2+-sensitive current component (Figure 4). These experiments were performed in the absence of functional sarcoplasmic reticulum (SR) using a voltage-clamp protocol to quantify reverse- and forward-mode NCX activity during depolarization and subsequent repolarization, respectively.34,35 SR function was disrupted with thapsigargin (Tg, 3 µmol/L), and voltage-gated Ca2+ entry was blocked with nitrendipine (10 µM). TRPC3-TG myocytes displayed elevated NCX outward as well as consecutive inward currents compared with wild-type myocytes when applying strong depolarizing voltage steps (+100 mV; Figure 4). Moreover, NCX currents were significantly amplified by TRPC3 overexpression in the presence of GSK. In WT myocytes, GSK promoted NCX currents only slightly to a level observed constitutively in TRPC3-TG myocytes. Thus, activation of TRPC3 at elevated expression levels significantly enhanced NCX currents in this voltage-clamp protocol, indicating that TRPC3 may dynamically affect ion concentration at the exchanger. Local, TRPC3-mediated Na+ loading at rest (holding potential) is expected to promote reverse-mode currents upon depolarization. The resulting Ca2+ accumulation during depolarization is, in turn, likely to facilitate Ca2+-induced Ca2+ release and drive forward-mode currents upon subsequent repolarization. We hypothesized that these alterations in Ca2+ cycling may promote spontaneous Ca2+ mobilization from the SR representing a potential trigger of arrhythmic events. We tested this concept by Ca2+ imaging experiments using confocal line scanning fluorescence microscopy. Ca2+ mobilization was visualized during a short subsequent, quiescent period in the absence of transmembrane Ca2+ and Na+ fluxes after an equilibrating train of stimuli in physiologic conditions as illustrated in Supplementary material online, Figure S8. Ca2+ spark frequency was significantly higher in TRPC3-TG than in WT myocytes already at basal levels of TRPC activity (Figure 5). Activation of TRPC3 by GSK (1 µM) clearly increased the frequency of Ca2+ sparks in TRPC3-TG and promoted Ca2+ discharge in WT myocytes to a level slightly below that observed in unstimulated TRPC3-TG myocytes. Interestingly, experiments designed to evaluate SR Ca2+ content did not indicate differences between TRPC3-TG and WT myocytes. Details of the Ca2+ transients measured in WT and TG myocytes are given in the supplemental information section (Table 1).Table 1


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)

Cardiac TRPC3 activity modulates NCX function. NCX currents were recorded by applying depolarizing voltage steps (500 ms) from a holding potential of −40 mV and quantified as the Ni+-sensitive current components at the end of the 500 ms depolarization steps (NCX reverse mode) and 20 ms after repolarization to −40 mV (forward mode).34,35 (A) Representative Ni+-sensitive outward currents recorded during depolarizing steps as well as subsequent tail currents upon repolarization to −40 mV for 4.5 s are shown for WT cardiomyocytes (left) and TG cardiomyocytes (right) in the absence [n = 8; N = 3 (WT); n = 9; N = 3 (TG)] (top) and presence (bottom) of 1 µM GSK [n = 8; N = 3 (WT); n = 12; N = 3 (TG)]; SR function was eliminated by thapsigargin (3 µM) and l-type Ca2+ channels were blocked by nitrendipine (10 µM). (B) Mean (±SEM) I/V plots of Ni+-sensitive peak currents. Left: tail inward currents representing NCX forward-mode activity in WT and TRPC3-TG cells and in the absence or presence of 1 µM GSK. Tail currents represent Ni+-sensitive peak inward currents at 20 ms after repolarization to −40 mV with steady-state holding current subtracted. Right: mean (±SEM) peak outward currents recorded from WT and TRPC3-TG cells in the presence and absence of 1 µM GSK. * indicates significant difference (P < 0.05) to WT, WT + GSK, and TRPC3-TG; sharp (#) significant difference (P < 0.05) to WT + GSK cells. Statistical significance analysed by two-way Anova followed by Tukey's post hoc tests.
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CVV022F4: Cardiac TRPC3 activity modulates NCX function. NCX currents were recorded by applying depolarizing voltage steps (500 ms) from a holding potential of −40 mV and quantified as the Ni+-sensitive current components at the end of the 500 ms depolarization steps (NCX reverse mode) and 20 ms after repolarization to −40 mV (forward mode).34,35 (A) Representative Ni+-sensitive outward currents recorded during depolarizing steps as well as subsequent tail currents upon repolarization to −40 mV for 4.5 s are shown for WT cardiomyocytes (left) and TG cardiomyocytes (right) in the absence [n = 8; N = 3 (WT); n = 9; N = 3 (TG)] (top) and presence (bottom) of 1 µM GSK [n = 8; N = 3 (WT); n = 12; N = 3 (TG)]; SR function was eliminated by thapsigargin (3 µM) and l-type Ca2+ channels were blocked by nitrendipine (10 µM). (B) Mean (±SEM) I/V plots of Ni+-sensitive peak currents. Left: tail inward currents representing NCX forward-mode activity in WT and TRPC3-TG cells and in the absence or presence of 1 µM GSK. Tail currents represent Ni+-sensitive peak inward currents at 20 ms after repolarization to −40 mV with steady-state holding current subtracted. Right: mean (±SEM) peak outward currents recorded from WT and TRPC3-TG cells in the presence and absence of 1 µM GSK. * indicates significant difference (P < 0.05) to WT, WT + GSK, and TRPC3-TG; sharp (#) significant difference (P < 0.05) to WT + GSK cells. Statistical significance analysed by two-way Anova followed by Tukey's post hoc tests.
Mentions: Initial characterization of the effects of AngII on NCX1 forward-mode activity by tail current analysis revealed that AngII increased NCX-inward currents only in TRPC-TG myocytes (see Supplementary material online, Figure S2C). To test whether TRPC3 expression and activity indeed determines exchanger operation during cyclic de- and repolarization, we performed patch-clamp experiments in TRPC3-TG cardiomyocytes to delineate cyclic NCX function as the Ni2+-sensitive current component (Figure 4). These experiments were performed in the absence of functional sarcoplasmic reticulum (SR) using a voltage-clamp protocol to quantify reverse- and forward-mode NCX activity during depolarization and subsequent repolarization, respectively.34,35 SR function was disrupted with thapsigargin (Tg, 3 µmol/L), and voltage-gated Ca2+ entry was blocked with nitrendipine (10 µM). TRPC3-TG myocytes displayed elevated NCX outward as well as consecutive inward currents compared with wild-type myocytes when applying strong depolarizing voltage steps (+100 mV; Figure 4). Moreover, NCX currents were significantly amplified by TRPC3 overexpression in the presence of GSK. In WT myocytes, GSK promoted NCX currents only slightly to a level observed constitutively in TRPC3-TG myocytes. Thus, activation of TRPC3 at elevated expression levels significantly enhanced NCX currents in this voltage-clamp protocol, indicating that TRPC3 may dynamically affect ion concentration at the exchanger. Local, TRPC3-mediated Na+ loading at rest (holding potential) is expected to promote reverse-mode currents upon depolarization. The resulting Ca2+ accumulation during depolarization is, in turn, likely to facilitate Ca2+-induced Ca2+ release and drive forward-mode currents upon subsequent repolarization. We hypothesized that these alterations in Ca2+ cycling may promote spontaneous Ca2+ mobilization from the SR representing a potential trigger of arrhythmic events. We tested this concept by Ca2+ imaging experiments using confocal line scanning fluorescence microscopy. Ca2+ mobilization was visualized during a short subsequent, quiescent period in the absence of transmembrane Ca2+ and Na+ fluxes after an equilibrating train of stimuli in physiologic conditions as illustrated in Supplementary material online, Figure S8. Ca2+ spark frequency was significantly higher in TRPC3-TG than in WT myocytes already at basal levels of TRPC activity (Figure 5). Activation of TRPC3 by GSK (1 µM) clearly increased the frequency of Ca2+ sparks in TRPC3-TG and promoted Ca2+ discharge in WT myocytes to a level slightly below that observed in unstimulated TRPC3-TG myocytes. Interestingly, experiments designed to evaluate SR Ca2+ content did not indicate differences between TRPC3-TG and WT myocytes. Details of the Ca2+ transients measured in WT and TG myocytes are given in the supplemental information section (Table 1).Table 1

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