Upregulation of transient receptor potential melastatin 4 (TRPM4) in ventricular fibroblasts from heart failure patients

Abstract

The transient receptor potential melastatin 4 (TRPM4) is a Ca²⁺-activated nonselective monovalent cation channel belonging to the TRP channel superfamily. TRPM4 is widely expressed in various tissues and most abundantly expressed in the heart. TRPM4 plays a critical role in cardiac conduction. Patients carrying a gain-of-function or loss-of-function mutation of TRPM4 display impaired cardiac conduction. Knockout or over-expression of TRPM4 in mice recapitulates conduction defects in patients. Moreover, recent studies have indicated that TRPM4 plays a role in hypertrophy and heart failure. Whereas the role of TRPM4 mediated by cardiac myocytes has been well investigated, little is known about TRPM4 and its role in cardiac fibroblasts. Here we show that in human left ventricular fibroblasts, TRPM4 exhibits typical Ca²⁺-activation characteristics, linear current-voltage (I-V) relation, and monovalent permeability. TRPM4 currents recorded in fibroblasts from heart failure patients (HF) are more than 2-fold bigger than those from control individuals (CTL). The enhanced functional TRPM4 in HF is not resulted from changed channel properties, as TRPM4 currents from both HF and CTL fibroblasts demonstrate similar sensitivity to intracellular calcium activation and extracellular 9-phenanthrol (9-phen) blockade. Consistent with enhanced TRPM4 activity, the protein level of TRPM4 is about 2-fold higher in HF than that of CTL hearts. Moreover, TRPM4 current in CTL fibroblasts is increased after 24 hours of TGFβ1 treatment, implying that TRPM4 in vivo may be upregulated by fibrogenesis promotor TGFβ1. The upregulated TRPM4 in HF fibroblasts suggests that TRPM4 may play a role in cardiac fibrogenesis under various pathological conditions.

Keywords: Calcium signaling; Heart failure; Human ventricular fibroblasts; TRP channels; TRPM4.

Fig. 1

Functional expression of TRPM4 in human ventricular fibroblasts. a Time-dependent activation of TRPM4 recorded in a HF fibroblast, and inhibition by 100 μM 9-phen. Inward and outward currents were measured at −100 mV and +100 mV. TRPM4 currents were elicited by a ramp protocol ranging from −120 to +100 mV with the recording condition of 100 μM Ca²⁺ in the pipette solution. NMDG was applied to make sure that there was no leak current throughout the recording time. Isotonic Ca²⁺ solution was used to detect whether there was any Ca²⁺ permeation. b Representative recordings of TRPM4 in Tyrode solution and after blockade by 9-phen. Note that inward current was eliminated by NMDG and isotonic Ca²⁺ solution, indicating that there was no leak current and no Ca²⁺ permeability. c Representative TRPM4 currents recorded in CTL fibroblasts treated with TRPM4-shRNA or SC-shRNA. d Average current amplitude of TRPM4 in fibroblasts treated with TRPM4-shRNA (4.1±0.4 pA/pF, n=11) or SC-shRNA (21.7±2.2, n=12). Note that TRPM4 currents were largely eliminated by TRPM4-shRNA (**p<0.01)

Fig. 2

Comparison of TRPM4 currents recorded from fibroblasts of control individuals (CTL) and heart failure patients (HF). a Representative recordings of TRPM4 before and after activation, as well as after NMDG application at the corresponding time points (a, b, c) as shown in b from CTL fibroblasts. b Time-dependent activation of TRPM4 from CTL fibroblasts. Inward and outward current were measured at −100 and +100 mV respectively. c Representative traces of TRPM4 before and after activation, as well as after NMDG application at the corresponding time points (a, b, c) as shown in d from an HF fibroblast. d Time-dependent activation of TRPM4 from HF fibroblasts. Inward and outward currents were measured at −100 and +100 mV, respectively. e, f Comparison of TRPM4 current density from representative cells (c), and average current density of CTL (n=50) and HF (n=38) fibroblasts isolated from 6 CTL and HF hearts. Current density from HF fibroblasts is significantly bigger than that of CTL fibroblasts (***p<0.001)

Fig. 3

Ca²⁺-sensitivity of TRPM4 from CTL and HF fibroblasts. a, c Representative TRPM4 currents recorded with various concentrations of intracellular Ca²⁺ in CTL (a) and HF (c) fibroblasts. b, d Concentration-dependent effects of intracellular Ca²⁺ on TRPM4. Dose–response curves yielded EC₅₀ of 15.5±0.6 μM for CTL fibroblasts (b) and 14.4±0.8 μM for HF fibroblasts (d), respectively. No significant difference in EC₅₀ was observed (n=6 for each Ca²⁺ concentration in CTL and HF groups)

Fig. 4

Concentration-dependent effects of 9-phen on TRPM4 currents recorded from CTL and HF fibroblasts. a, c Representative recordings of TRPM4 current elicited by voltage ramp ranging from −120 to +100 mV. Effects of 9-phen at 0.1, 1, 10, 30, and 100 μM were tested in CTL (a) and HF (c) fibroblasts. The effect of 9-phen was reversal. b, d Dose–response curves constructed from normalized current amplitude yielded IC₅₀ of 20.7±2.9 μM for CTL fibroblasts (b) and 19.5 ±3.7 μM for HF fibroblasts (d), respectively. No significant difference in IC₅₀ was observed (n=6 for each group)

Fig. 5

TRPM4 expression detected by western blot (WB) in CTL and HF left ventricle. a WB results of TRPM4 and loading control using GAPDH. b Average ratio of TRPM4 versus GAPDH in CTL and HF hearts. Expression of TRPM4 was significantly bigger in HF hearts than that in CTL hearts (**p<0.01, n=6 for each group). c qPCR quantification of TRPM4 expression levels in fibroblasts of CTL and HF. RNA samples were extracted from the same batches of fibroblasts used for patch-clamp experiments (**p<0.01, n=6 for each group)

Fig. 6

Effect of TGFβ1 on TRPM4 in CTL fibroblasts. a Representative TRPM4 recorded from CTL fibroblasts 24 h after treatment with 10 ng/ml TGFβ1 (blue trace) in comparison with current recorded from nontreated control fibroblasts. b Average current density of TRPM4 in TGFβ1-treated (n=16) and nontreated (n=14) fibroblasts (**p<0.01). c TRPM4 currents recorded from CTL fibroblasts before and after perfusion with 10 ng/ml TGFβ1. NMDG was used to monitor leak current. d Mean current density of TRPM4 before and after TGFβ1 perfusion. No statistical difference was observed (n=8)