TRPC4α and TRPC4β Similarly Affect Neonatal Cardiomyocyte Survival during Chronic GPCR Stimulation

Abstract

The Transient Receptor Potential Channel Subunit 4 (TRPC4) has been considered as a crucial Ca2+ component in cardiomyocytes promoting structural and functional remodeling in the course of pathological cardiac hypertrophy. TRPC4 assembles as homo or hetero-tetramer in the plasma membrane, allowing a non-selective Na+ and Ca2+ influx. Gαq protein-coupled receptor (GPCR) stimulation is known to increase TRPC4 channel activity and a TRPC4-mediated Ca2+ influx which has been regarded as ideal Ca2+ source for calcineurin and subsequent nuclear factor of activated T-cells (NFAT) activation. Functional properties of TRPC4 are also based on the expression of the TRPC4 splice variants TRPC4α and TRPC4β. Aim of the present study was to analyze cytosolic Ca2+ signals, signaling, hypertrophy and vitality of cardiomyocytes in dependence on the expression level of either TRPC4α or TRPC4β. The analysis of Ca2+ transients in neonatal rat cardiomyocytes (NRCs) showed that TRPC4α and TRPC4β affected Ca2+ cycling in beating cardiomyocytes with both splice variants inducing an elevation of the Ca2+ transient amplitude at baseline and TRPC4β increasing the Ca2+ peak during angiotensin II (Ang II) stimulation. NRCs infected with TRPC4β (Ad-C4β) also responded with a sustained Ca2+ influx when treated with Ang II under non-pacing conditions. Consistent with the Ca2+ data, NRCs infected with TRPC4α (Ad-C4α) showed an elevated calcineurin/NFAT activity and a baseline hypertrophic phenotype but did not further develop hypertrophy during chronic Ang II/phenylephrine stimulation. Down-regulation of endogenous TRPC4α reversed these effects, resulting in less hypertrophy of NRCs at baseline but a markedly increased hypertrophic enlargement after chronic agonist stimulation. Ad-C4β NRCs did not exhibit baseline calcineurin/NFAT activity or hypertrophy but responded with an increased calcineurin/NFAT activity after GPCR stimulation. However, this effect was not translated into an increased propensity towards hypertrophy but rather less hypertrophy during GPCR stimulation. Further analyses revealed that, although hypertrophy was preserved in Ad-C4α NRCs and even attenuated in Ad-C4β NRCs, cardiomyocytes had an increased apoptosis rate and thus were less viable after chronic GPCR stimulation. These findings suggest that TRPC4α and TRPC4β differentially affect Ca2+ signals, calcineurin/NFAT signaling and hypertrophy but similarly impair cardiomyocyte viability during GPCR stimulation.

Fig 1. Enhanced expression of TRPC4α and TRPC4β after pressure overload-induced cardiac hypertrophy.

A, Expression levels of TRPC4α and TRPC4β were analyzed in mouse heart homogenates from transverse aortic constriction (TAC) or sham treated mice (3 mice/group). B, quantification of TRPC4α and TRPC4β expression levels relative to sham controls. *P<0.05 vs Sham. C. Detection of TRPC4α and TRPC4β in lysates from neonatal rat cardiomyocytes with TRPC4 or myc-tag antibodies. D, Average quantified values of TRPC4α and TRPC4β signals expressed relative to Ad-βgal for n = 3 experiments. *P<0.05 vs Ad-βgal CON. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control.

Fig 2. Spontaneous Ca²⁺ influx in TRPC4α and TRPC4β overexpressing neonatal rat cardiomyocytes.

A, Representative Fura-2 Ca²⁺ recordings of neonatal rat cardiomyocytes (NRCs) infected with either Ad-βgal, Ad-TRPC4α or Ad-TRPC4β. Cells were perfused with a nominally Ca²⁺-free Tyrode solution including verapamil (10 μM). After 50 s, Ca²⁺ influx was challenged by perfusing the cells with a solution containing 1.8 mM Ca²⁺. B, Mean Δ ratio values were calculated by subtracting the peak value at 1.8 mM from the baseline value at 0 mM Ca²⁺.*P<0.05 vs Ad-βgal. Cells from three different experiments were analyzed. N-number of cells is indicated in the bars.

Fig 3. TRPC4β overexpression is connected with an increased Ca²⁺ influx after angiotensin II stimulation of cardiomyocytes.

A, Representative Fura-2 Ca²⁺ traces in neonatal rat cardiomyocytes (NRCs) infected with either Ad-βgal (A), Ad-TRPC4α (B) or Ad-TRPC4β (C). Quiescent cells were perfused with a normal Tyrode solution containing 1 mM Ca²⁺. Verapamil (10 μM) was included throughout the recordings. After 50 s, angiotensin II (Ang II; 1 μM) was applied. D, Mean Δ ratio values were calculated by subtracting the peak value after Ang II addition from the baseline value. *P<0.05 vs Ad-βgal and Ad-TRPC4α. Cells from three different experiments were analyzed. N-number of cells is indicated in the bars.

Fig 4. Overexpression of TRPC4α increases the Ca²⁺ transient amplitude at baseline and TRPC4β elevates the Ca²⁺ transient amplitude at baseline and during Ang II stimulation.

A, Ca²⁺ transients were measured in Ad-βgal, Ad-C4α or Ad-C4β neonatal rat cardiomyocytes (NRCs) in a normal Tyrode solution with angiotensin II (Ang II; 1 μM) or without Ang II during 1 Hz of pacing. B, The Ca²⁺ amplitude under steady state pacing was significantly increased in Ad-C4α and Ad-C4β cardiomyocytes. Ang II stimulation resulted in an elevation of the Ca²⁺ peak in Ad-βgal and Ad-C4β but not Ad-C4α cardiomyocytes as analyzed after 30–60 s of Ang II application. *P<0.05 vs Ad-βgal CON and Ad-C4β CON; ^#P<0.05 vs Ad-βgal CON; ^§P<0.05 vs Ad-βgal and Ad-C4β CON; ^$P<0.05 vs Ad-βgal Ang II/PE. C, Diastolic [Ca²⁺]_i levels measured with Fura-2 were not different between Ad-βgal, Ad-C4α or Ad-C4β NRCs. Cells from at least three different experiments were analyzed. N-number of cells is indicated in the bars.

Fig 5. NFATc1 nuclear translocation is promoted by TRPC4α at baseline and by TRPC4β upon agonist stimulation.

A, Neonatal rat cardiomyocytes (NRCs) were co-infected with Ad-βgal, Ad-TRPC4α or Ad-TRPC4β/ NFATc1-GFP. Nuclear localization of NFATc1-GFP was used as an indicator for its activation. Green: NFATc1-GFP. Scale bar: 20 μm. B, Quantification of NFATc1 localized in the nucleus of NRCs that were stimulated with angiotensin II/phenylephrine (Ang II/PE; 1 μM/50 μM) or vehicle (CON). *P<0.05 vs unstimulated; ^#P<0.05 vs Ad-TRPC4α or Ad-TRPC4β CON, respectively; ^$P<0.05 vs Ad-βgal and Ad-TRPC4α Ang II/PE. Cells from at least three different experiments were analyzed. N-number of cells is indicated in the bars.

Fig 6. TRPC4α increases cardiomyocyte hypertrophy at baseline but not after GPCR stimulation.

A, B Hypertrophic growth of cardiomyocytes was measured as an increase of the cell surface after angiotensin II/phenylephrine (Ang II/PE) or vehicle (CON) treatment for 24 h. Red: α-actinin; Blue: DAPI. *P<0.05 vs Ad-βgal CON; ^#P<0.05 vs Ad-βgal CON. Cells from at least three different experiments were analyzed. Scale bar: 20 μm. N-number of cells is indicated in the bars.

Fig 7. Down-regulation of endogenous TRPC4α decreases baseline cardiomyocyte size and promotes cardiomyocyte hypertrophy after chronic agonist stimulation.

A, TRPC4α was down-regulated in neonatal rat cardiomyocytes (NRCs) by a siRNA-dependent approach. Down-regulation of TRPC4α was verified by Western blotting. B, Hypertrophic growth of NRCs transfected with control siRNA (con siRNA) or TRPC4α (C4α) siRNA was measured as an increase of the cell surface after angiotensin II/phenylephrine (Ang II/PE) or vehicle (CON) treatment for 24 h. Red: α-actinin; Blue: DAPI. *P<0.05 vs con siRNA and C4α siRNA CON; ^#P<0.05 vs con siRNA CON; ^$P<0.05 vs con siRNA Ang II/PE. Cells from three different experiments were analyzed. Scale bar: 20 μm. N-number of cells is indicated in the bars.

Fig 8. TRPC4β attenuates hypertrophic growth in cardiomyocytes after GPCR stimulation.

A, Hypertrophic growth of cardiomyocytes was measured as an increase of the cell surface after angiotensin II/phenylephrine (Ang II/PE) or vehicle (CON) treatment for 24 h. Red: α-actinin; Blue: DAPI. *P<0.05 vs Ad-βgal CON; ^$P<0.05 vs Ad-C4β AngII/PE. Cells from three different experiments were analyzed. Scale bar: 20 μm. N-number of cells is indicated in the bars. B, Expression and phosphorylation of phospholamban (PLN) and histone deacetylase 4 (HDAC4) in Ad-βgal and Ad-TRPC4β NRCs after stimulation with Ang II/PE or vehicle (CON) for 24 h. phospho-PLN: PLN PT17; phospho-HDAC4: HDAC4 PS632; glyceraldehyde 3-phosphate dehydrogenase: GAPDH. Shown are representative immunoblots from three different experiments. C, Average quantified values of PLN PT17 signals expressed relative to Ad-βgal CON for n = 3 experiments. D, Average quantified values of HDAC4 PS632 signals expressed relative to Ad-βgal CON for n = 3 experiments.

Fig 9. Increased apoptotic rate in cardiomyocytes overexpressing TRPC4α or TRPC4β.

A, Cleaved caspase-3 expression levels were detected in neonatal rat cardiomyocytes (NRCs) after Ang II/PE or vehicle (CON) treatment. GAPDH: loading control. Myc: detection of TRPC4α and TRPC4β. Shown are representative immunoblots from three independent experiments. B, Average quantified values of cleaved caspase-3 expression levels relative to Ad-βgal CON for n = 3 experiments. C, Capase-3 activity was compared between Ad-βgal, Ad-TRPC4α and Ad-TRPC4β in NRCs. Caspase-3 assays were performed in duplicate and average values were quantified relative to Ad-βgal CON; n = 3 experiments.

Fig 10. TRPC4α and TRPC4β lower cardiomyocyte viability after chronic agonist stimulation.

The vitality of cardiomyocytes expressing βgal (Ad- βgal), TRPC4α (Ad-C4α) or TRPC4β (Ad-C4β) was determined after angiotensin II/phenylephrine (Ang II/PE) or vehicle (CON) treatment for 24 h by applying the trypan blue exclusion assay. The assay was performed in duplicate and average values were quantified relative to Ad-βgal CON; n = 3 experiments. *P<0.05 vs Ad-C4α and Ad-C4β Ang II/PE. B. ^#P<0.05 vs Ad-C4α and Ad-C4β CON.