Background and Purpose: Intracellular calcium concentration ([Ca2+]i) overload occurs in myocardial ischemia and -reperfusion. The augmentation of the late sodium current (INaL) causes intracellular Na+ accumulation and subsequent [Ca2+]i overload via the reverse mode of the Na+/Ca2+ exchange current (reverse-INCX), which can lead to arrhythmia and cardiac dysfunction. Thus, inhibition of INaL is a potential therapeutic approach for ischemic heart disease. The aim of this study was to investigate the effects of thyroid hormone on augmented INaL, reverse-INCX, altered action potential duration (APD), and [Ca2+]i concentration in hypoxia/reoxygenation (H/R)-induced ventricular myocytes in vitro. Methods: The transient Na+ current (INaT), INaL, reverse-INCX, and APs were recorded using a whole-cell patch-clamp technique in neonatal mouse ventricular myocytes. [Ca2+]i concentration alteration were, respectively, observed by confocal microscopy and flow cytometry. Results: Triiodothyronine (T3) pretreatment decreased the INaL in a concentration-dependent manner. H/R injury aggravated the INaL, INaT, and reverse-INCX in cardiomyocytes and increased the continuous accumulation of [Ca2+]i (p < 0.05). The application of T3 prior to H/R injury significantly decreased the increased INaL, INaT, and reverse-INCX and blunted the [Ca2+]i increase. Furthermore, T3 pretreatment shortened the APD induced by H/R injury. Conclusion: T3 inhibited H/R-increased INaL and reverse INCX augmentation, shortened the APD, and diminished [Ca2+]i overload, indicating a potential therapeutic use of T3 as an INaL inhibitor to maintain Ca2+ homeostasis and protect cardiomyocytes against H/R injury.

Cardiomyocyte calcium concentration ([Ca2+]i) overload occurs in many pathological conditions, including hypoxia, ischemia, reperfusion, oxidative stress, cardiac hypertrophy, and heart failure [1, 2]. [Ca2+]i overload causes cellular injury and myocardial apoptosis, including mitochondrial dysfunction, reduced ATP production, and activated Ca2+-dependent protease [3]. The late sodium current (INaL), which is tetrodotoxin-sensitive, is increased in cardiomyocytes under many pathological conditions and can lead to Ca2+ overload [4, 5]. INaL also plays an important role in determining the plateau of the action potential (AP) and the AP duration (APD) under pathological conditions. The increased INaL prolongs the APD, which could cause cardiac arrhythmias [6-9]. In addition, increased INaL causes an increase in the intracellular sodium concentration and subsequently raises [Ca2+]i via the reverse mode of the Na+/Ca2+ exchanger (NCX) [7]. Inhibition of INaL could reduce the intracellular [Ca2+]i overload [8, 9], which is a potential therapeutic target for the treatment of cardiac diseases associated with intracellular [Ca2+]i overload.

Thyroid hormone (TH) signaling is essential for heart development and cardiovascular function through both genomic and nongenomic mechanisms. The classically described cellular actions of TH are mediated by triiodothyronine (T3) by binding to specific nuclear TH receptors, which function to regulate the expression of specific cardiac genes such as voltage-activated K+ channel genes such as Kv4.2, Kv4.3, and Kv1.5 [10]. T3 could increase the inward currents directly, shortening APD [13]. Previous studies suggested that T3 could rapidly and nongenomically regulate the Ca2+ ATPase enzyme, the Na+ channel, and the K+ channel and that T3 could also increase levels of sarcoplasmic reticulum Ca2+ and the contractility of myocytes [14]. Recent studies have demonstrated that altered thyroid status dramatically impacts cardiac electrical function. Hypothyroidism produces sinus bradycardia and prolongs cardiac repolarization, whereas hyperthyroidism is associated with accelerated heart rate and shortened APD [11, 12]. These results suggest potential protective effects of TH on cardiomyocytes. However, the underlying mechanisms are still unknown. Previous studies have found that the inhibition of INaL attenuates augmented INaL-induced [Ca2+]i overload [8, 9], and no study has investigated the effects of TH on INaL. Thus, this study investigated the effects of TH on INaL, Na+/Ca2+ exchange current (reverse-INCX), APD, and [Ca2+]i concentration in hypoxia/reoxygenation (H/R)-induced ventricular myocytes in vitro.

Drugs and Reagents

Dulbecco’s modified Eagle’s medium-F12, fetal bovine serum (FBS), and D-Hank’s solution were purchased from GIBCO-BRL (Grand Island, NE, USA). Trypsin, penicillin-streptomycin, and Fluo-3 AM kit were purchased from Beyotime Biotechnology (Beyotime, Shanghai, China). All other reagents, including T3, HEPES, 5-bromo-2-deoxyuridine, and collagenase type II, were purchased from Sigma Aldrich (St. Louis, MO, USA). T3 was stored in the dark at –20°C. On the day of experimentation, T3 was dissolved in NaOH and added to DMEM-F12 to make a stock solution at a concentration of 20 µg/L

Isolation of Ventricular Myocytes

The animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Publication No. 85-23, revised 1996), and all animal procedures were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (protocol number: 00013518).

Neonatal mice (1–3 days old) were purchased from the animal center of Hubei Province. Myocytes were prepared by enzymatic disaggregation of cardiac tissue as described previously [15]. In brief, the hearts were quickly excised and immersed in 0.25% trypsin at 4°C overnight. The heart tissues were digested with type II collagenase (Sigma; USA) at 37°C, and the isolated cells were filtered and centrifuged at 1,000 rpm for 10 min. Then, the cells were cultured for 90 min at 37°C for cardiomyocyte purification, and the cardiomyocytes were finally cultured at a concentration of 1 × 105 cells/mL in medium containing 10% heat-inactivated FBS (Gibco, USA), 1% penicillin-streptomycin, and 5-bromo-2-deoxyuridine to inhibit fibroblast growth. The cells were incubated at 37°C in a humidified incubator with 5% CO2 for 48 h prior to treatment.

Established H/R Model in vitro

For the experiment conducted under H/R conditions, cardiomyocytes were incubated with PBS in a tri-gas incubator (95% N2, 5% CO2) at 37°C for 4 h, and then the PBS was replaced with culture medium containing 10% FBS and cultured in a standard incubator (5% CO2, 37°C) for 4 h. The cells in the normal control groups were exposed to the normoxic conditions.

For T3 pretreatment, 2, 4, and 8 μL stock solutions of T3 were added into the culture medium for 24 h of pretreatment before the H/R conditions. To explore the effect of T3 pretreatment on intracellular [Ca2+]i overload under H/R injury, cultured cardiomyocytes were randomly divided into 4 groups: control group, control + T3 group, H/R group, and H/R + T3 group.

Solutions

For INaL recordings, the intracellular (pipette) solution contained the following (in mmol/L): 120 CsCl, 10 TEA-Cl, 5 Na2ATP, 5 MgCl2, 1 CaCl2, 10 EGTA, and 10 HEPES (pH 7.3, adjusted with CsOH). The bath solution contained the following (in mmol/L): 135 NaCl, 0.33 NaH2PO4, 5.4 CsCl, 1 MgCl2, 1.8 CaCl2, 0.3 BaCl2, 0.01 nifedipine, 10 HEPES, and 10 glucose (pH 7.4 adjusted with NaOH).

To record INaT, the intracellular (pipette) solution contained the following (in mmol/L): 120 CsCl, 1 CaCl2, 10 TEA-Cl, 5 Na2ATP, 5 MgCl2, 10 EGTA, and 10 HEPES (pH 7.3, adjusted with CsOH). The bath solution contained the following (in mmol/L): 105 CsCl, 30 NaCl, 1 CaCl2, 1 MgCl2, 0.01 nifedipine, 10 HEPES, and 10 glucose (pH 7.4 adjusted with CsOH).

To record INCX, the intracellular (pipette) solution contained the following (in mmol/L): 115 CsCl, 10 NaCl, 1 CaCl2, 5 Mg-ATP, 20 TEA-Cl, 10 EGTA, and 10 HEPES (pH 7.25, adjusted with CsOH). The bath solution contained the following (in mmol/L): 140 NaCl, 5 CsCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 5 glucose (pH 7.35, adjusted with CsOH). In addition, 1.0 mmol/L BaCl2 and 1.0 µmol/L nicardipine were added to block the potassium current and ICaL, respectively.

To record AP, the intracellular (pipette) solution contained the following (in mmol/L): 110 L-aspartic acid potassium salt, 20 KCl, 8 NaCl, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES, and 40 sucrose (pH 7.2, adjusted with KOH). The bath solution contained the -following (in mmol/L): 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 10 HEPES, and 10 glucose (pH 7.4, adjusted with NaOH).

Whole-Cell Patch-Clamp Technique

The conventional whole-cell patch-clamp technique using an EPC-9 amplifier (HEKA Electronic, Lambrecht, Germany) was applied to record transmembrane potentials and ion currents. The patch electrode resistance (when filled with pipette solution) was 3–5 MΩ. Cell capacitance and series resistances were electronically compensated by 60–80%. Currents were filtered at 2 kHz and digitized at 10 kHz. All experiments were performed at room temperature (22–25°C).

INaT current–voltage (I–V) relation and activation properties were determined by applying depolarizing steps 100 ms in duration from holding potential (Vh) –120 mV in 10 mV increments, ranging from –90 to +50 mV. The maximum value of I INaT was measured. For inactivation properties, INaT was determined using 100 ms conditioning pulses from a holding potential of –120 mV, ranging from –120 to –45 mV in 10 mV increments, followed by a test pulse to –10 mV for 100 ms.

INaL was recorded by a 300 ms depolarizing pulse to –20 mV from a holding potential of –90 at a frequency of 0.2 Hz. The amplitude of INaL was determined from the average current measured during a time interval of 190–210 ms after initiation of the depolarizing pulse to eliminate any contribution of INaT [2]. To record the current-voltage relationship of INaL, 300-ms depolarizing pulses to membrane potentials from –80 to +40 mV were applied at 0.5 Hz from a potential of –90 mV.

INCX was elicited by a 10 ms prepulse to +60 mV, followed by a 2 s ramp pulse protocol ranging from +60 to –120 mV (with a speed of –90 mV/s) from a holding potential of –40 mV. INCX was measured as the current sensitive to 5 mmol/L Ni2+ at +50 and –100 mV.

AP was evoked by 5 ms duration of 1.5 times diastolic threshold current pulses at 0.25 Hz. The AP amplitude and APD at 50 and 90% repolarization (APD50 and APD90) were analyzed.

Measurement of [Ca2+]i Levels by Flow Cytometry and Confocal Microscopy

Ca2+ indicators Fluo-3/AM was employed to measure the [Ca2+]i concentrations. The cardiomyocytes were seeded in circular discs and treated in the manner described above. Then washed with D-Hank’s 3 times and incubated with 5 µmol/L Fluo-3/AM for 40 min at 37°C in dark in D-Hank’s solution. After loading, the cells were incubated for 20 min at room temperature to ensure the complete transformation of fluo-3 AM into fluo-3. The [Ca2+]i concentration was evaluated by measuring the fluorescence intensity using flow cytometer (Becton Dickinson, San Jose, CA, USA) and the Confocal Microscopy System (Carl Zeiss, Berlin, Germany). The excitation wavelength used in the detection is 488 nm, and the emission is 525 nm. In flow cytometry experiments, at least 10,000 events were obtained.

Data Analysis

All current amplitudes were normalized to cell capacitance and expressed as pA/pF. All data were presented as the mean ± SD and were analyzed using FitMaster (v2 x 32, HEKA) and SPSS 22.0 software. Figures were plotted with Origin (version 8.0, OriginLab Co., MA, USA). Student t test was used to determine the difference between 2 groups of data. One-way analyses of variance were followed by the Scheffé test for multiple comparisons. Values of p < 0.05 were considered significant.

Current tracings were fitted using Hill or Boltzmann functions with Origin 8.0. Steady-state activation data and inactivation relationships of INaT were fitted to the Boltzmann equation as follows: Y = 1/(1 + exp [Vm –V1/2]/k), where Vm was the membrane potential, V1/2 was the half-activation or half-inactivation potential, and k was the slope factor. For the steady-state activation and inactivation curve, Y is the relative conductance (G/Gmax) and relative current (I/Imax), respectively.

T3 Inhibited INaL in Neonatal Mouse Ventricular Cardiomyocytes Induced by H/R Injury

Previous reports show that INaL is increased under hypoxic conditions; thus, we studied the effects of T3 on INaL after exposure to H/R injury. INaL was recorded with 300 ms voltage steps from a holding potential of –120 to –20 mV at 0.5 Hz. The recorded sodium current includes INaT in depolarization and INaL in repolarization. Mexiletine, a blocker of the late Na+ current as previously reported, minimally affects the INaT and the peak INa [16]. We use mexiletine to separate INaL from the sodium current in cardiomyocytes. After the application of 10 µmol/L mexiletine, INaL was almost completely blocked with current densities decreasing from 0.79 ± 0.07 pA/pF to 0.056 ± 0.014 pA/pF (n = 10, p < 0.01; Fig. 1), with no significant change in INaT, the results were consistent with the above conclusions. In addition, the increased INaL induced by H/R injury (from 0.7 ± 0.07 to 1.85 ± 0.05 pA/pF) was completely blocked by mexiletine (0.071 ± 0.014 pA/pF; n = 10, p < 0.01). These results showed that the increased recorded currents were INaL.

Fig. 1.

Effects of T3 on control INaL in cultured neonatal mouse cardiomyocytes. a The blockade of 10 μmol/L mexiletine on control and H/R-increased INaL. b The peak INaL on cardiomyocytes in control group and T3 (20, 40, and 80 ng/mL) treatment group, n= 10 in each group. c Typical whole-cell INaL recorded in the control group and presence of 20, 40, and 80 ng/mL T3. Data were expressed as mean ± SD, # p < 0.05, ## p < 0.01 vs. control. INaL, late sodium current; H/R, hypoxia/reoxygenation; T3, triiodothyronine.

Fig. 1.

Effects of T3 on control INaL in cultured neonatal mouse cardiomyocytes. a The blockade of 10 μmol/L mexiletine on control and H/R-increased INaL. b The peak INaL on cardiomyocytes in control group and T3 (20, 40, and 80 ng/mL) treatment group, n= 10 in each group. c Typical whole-cell INaL recorded in the control group and presence of 20, 40, and 80 ng/mL T3. Data were expressed as mean ± SD, # p < 0.05, ## p < 0.01 vs. control. INaL, late sodium current; H/R, hypoxia/reoxygenation; T3, triiodothyronine.

Close modal

T3 inhibited INaL in a concentration-dependent -manner; the results showed that T3 (20, 40, 80 ng/mL) decreased the INaL current density from 0.76 ± 0.07 to 0.68 ± 0.014 pA/pF (p > 0.05), 0.57 ± 0.031 pA/pF (n = 10, p < 0.05), and 0.49 ± 0.023 pA/pF (n = 10, p < 0.01; Fig. 1), respectively. Then, 80 ng/mL T3 was chosen for the subsequent experiments.

The INaL current/voltage (I/V) relation curves of cardiomyocytes in each group are shown in Figure 2. T3 pretreatment decreased the INaL current density of cardiomyocytes (n = 10, p < 0.05). After H/R treatment, the current density of INaL was significantly increased from 0.7 ± 0.07 pA/pF to 1.85 ± 0.05 pA/pF (n = 10, p < 0.0001). However, this increase was effectively inhibited by T3 pretreatment, T3 diminished the downward shift of the I/V curve in H/R-induced cardiomyocytes, and decreased the INaL to 1.16 ± 0.079 pA/pF (n = 10, p < 0.0001).

Fig. 2.

Effects of T3 on increased INaL induced by H/R injury in cultured neonatal mouse cardiomyocytes. a Typical whole-cell INaL recorded in each group of cardiomyocytes. b The current–voltage relationship for INaL in each group. c The peak INaL on cardiomyocytes in each group, n = 10 in each group. Data were -expressed as mean ± SD, # p < 0.05, #### p < 0.0001 vs. control; **** p< 0.0001 vs. H/R. T3, triiodothyronine; H/R, hypoxia/reoxygenation; INaL, late sodium current.

Fig. 2.

Effects of T3 on increased INaL induced by H/R injury in cultured neonatal mouse cardiomyocytes. a Typical whole-cell INaL recorded in each group of cardiomyocytes. b The current–voltage relationship for INaL in each group. c The peak INaL on cardiomyocytes in each group, n = 10 in each group. Data were -expressed as mean ± SD, # p < 0.05, #### p < 0.0001 vs. control; **** p< 0.0001 vs. H/R. T3, triiodothyronine; H/R, hypoxia/reoxygenation; INaL, late sodium current.

Close modal

T3 Decreased INaT of Ventricular Cardiomyocytes Induced by H/R Injury

The INaT current/voltage (I/V) relation curves of cardiomyocytes in each group are shown in Figure 3. At the concentration of 80 ng/mL T3, the magnitudes of INaT were decreased from –45.093 ± 2.198 to –22.456 ± 1.58 pA/pF (n = 10, p < 0.01). During H/R injury, the current density of INaT was significantly increased from –45.093 ± 2.198 to –89.319 ± 9.08 pA/pF (n = 10, p < 0.01 vs. control). However, this increase was effectively offset by T3 pretreatment; T3 diminished the downward shift of the I/V curve in H/R induced cardiomyocytes, and the INaT was decreased to –54.403 ± 8.774 pA/pF (n = 10, p < 0.05 vs. H/R).

Fig. 3.

Effects of T3 on INaT in cultured neonatal mouse cardiomyocytes of each group. a Typical whole-cell INaT recorded in each group of cardiomyocytes. b The current–voltage relationship for INaT in cardiomyocytes of each group. c The peak INaT on cardiomyocytes in each group, n = 10 in each group. d The steady-state activation and inactivation current recordings of INaT on cardiomyocytes in each group. Data were expressed as mean ± SD, ## p < 0.01 vs. control; ** p < 0.01 vs. H/R. T3, triiodothyronine; H/R, hypoxia/reoxygenation.

Fig. 3.

Effects of T3 on INaT in cultured neonatal mouse cardiomyocytes of each group. a Typical whole-cell INaT recorded in each group of cardiomyocytes. b The current–voltage relationship for INaT in cardiomyocytes of each group. c The peak INaT on cardiomyocytes in each group, n = 10 in each group. d The steady-state activation and inactivation current recordings of INaT on cardiomyocytes in each group. Data were expressed as mean ± SD, ## p < 0.01 vs. control; ** p < 0.01 vs. H/R. T3, triiodothyronine; H/R, hypoxia/reoxygenation.

Close modal

The affinity of T3 for the activated or inactivated states of the channel was determined by assessing the shifts in the steady-state availability curves. The steady-state activation curve was obtained from Boltzmann equation G/Gmax = 1/(1 + exp [(V1/2 – Vm)/k]), and the steady state inactivation curve was obtained from Boltzmann equation I/Imax = 1/(1 + exp [(Vm – V1/2)/k]). The steady-state activation of INaT was examined, and the half-maximal activation potential of INaT (V1/2) values were –20.74 ± 1.569 and –17.61 ± 1.911 mV (n = 10, p < 0.01), and the values of k were 7.61 ± 0.249 and 7.5 ± 0.80979 (p > 0.05) before and after application of 80 ng/mL T3, respectively (Fig. 3). The activation curve of INaT shifted toward a more positive potential with T3 pretreatment. During H/R treatment, the results showed a left shift of the activation curve of INaT with a decreased V1/2 from –20.74 ± 1.569 to –28.82 ± 1.327 mV (n = 10, p < 0.01), and the value of k showed no significant differences between the 2 groups. However, the effect in activation potential was diminished with T3 pretreatment, with an increased value of V1/2 (–21.51 ± 2.197 mV, n = 10, p < 0.05), and the value of k showed no significant differences between the 2 groups.

The steady-state inactivation curve of INaT was also examined, the half-maximal inactivation potential of INaT (V1/2) value was changed from –82.21 ± 1.406 to –76.0 ± 2.577 mV (n = 10, p < 0.0001) after application of T3, and the value of k showed no significant differences between the 2 groups. The inactivation curve of INaT shifted toward a more negative potential with T3 pretreatment. During H/R treatment, the V1/2 changed to –101.9 ± 1.678 mV, leading to a right shift of the inactivation curve, while no significant difference was observed in the k values between the 2 groups. However, the right shift in the INaT inactivation curve in H/R group was reversed by T3 pretreatment, with an increased value of V1/2 (–86.05 ± 6.07 mV, n = 10, p < 0.05); the value of k showed no significant differences between the 2 groups (Fig. 3).

Effects of T3 on the Increased Reverse INCX Induced by H/R Injury

As shown in Figure 4, at a concentration of 80 ng/mL T3, the forward INCX current density was decreased from –0.831 ± 0.165 to –0.39 ± 0.079 pA/pF (n = 10, p < 0.05), and the reverse INCX current density was decreased from 1.555 ± 0.119 to 0.94 ± 0.034 pA/pF (n = 10, p < 0.001). H/R injury increased the forward INCX to –1.264 ± 0.102 pA/pF (n = 10, p < 0.05), and the reverse INCX current density was increased from to 3.829 ± 0.145 pA/pF (n = 10, p < 0.0001), compared with that of the control group. However, T3 pretreatment decreased these values to –0.832 ± 0.16 pA/pF (n = 10, p < 0.05) and 3.829 ± 0.145 pA/pF (n = 10, p < 0.0001), T3 diminished the downward shift of I/V curve induced by H/R injury.

Fig. 4.

Effects of T3 on INCX in cultured neonatal mouse cardiomyocytes of each group. a Representative trace of Na+-Ca2+ exchange current from cardiomyocytes conducted under conditions of control and 5 mmol/L Ni2+ treated. b Ni2+-sensitive INCX obtained in each group, data were obtained by subtracting the data in trace Ni2+-treated from the data in traces control, control + T3, H/R, and H/R + T3, respectively. c Mean current densities of forward INCX and reverse INCX under different conditions. Data were expressed as mean ± SD, # p < 0.05, ### p < 0.001, #### p < 0.0001 vs. control; * p < 0.05, **** p < 0.0001 vs. H/R. INCX, Na+/Ca2+ exchange; T3, triiodothyronine; H/R, hypoxia/reoxygenation.

Fig. 4.

Effects of T3 on INCX in cultured neonatal mouse cardiomyocytes of each group. a Representative trace of Na+-Ca2+ exchange current from cardiomyocytes conducted under conditions of control and 5 mmol/L Ni2+ treated. b Ni2+-sensitive INCX obtained in each group, data were obtained by subtracting the data in trace Ni2+-treated from the data in traces control, control + T3, H/R, and H/R + T3, respectively. c Mean current densities of forward INCX and reverse INCX under different conditions. Data were expressed as mean ± SD, # p < 0.05, ### p < 0.001, #### p < 0.0001 vs. control; * p < 0.05, **** p < 0.0001 vs. H/R. INCX, Na+/Ca2+ exchange; T3, triiodothyronine; H/R, hypoxia/reoxygenation.

Close modal

Effects of T3 on AP of Cardiomyocytes

As shown in Table 1, the APD50 and APD90 of cardiomyocytes were shortened with T3 pretreatment, the APD50 decreased from 18.83 ± 1.64 to 11.65 ± 0.98 ms (n = 15, p < 0.01), and the APD90 decreased from 106.3 ± 9.85 to 58.02 ± 3.97 ms (n = 15, p < 0.0001), respectively, compared with the control group. After the H/R condition, the APD50 and APD90 of cardiomyocytes were increased to 28.03 ± 1.85 and 162.6 ± 14.42 ms (n = 15, both p < 0.01 vs. control). However, this increase was significantly inhibited with T3 pretreatment; the APD50 decreased to 15.58 ± 1.37 ms (n= 15, p < 0.0001), and the APD90 decreased to 101.1 ± 11.86 ms (n = 15, p < 0.01), compared with that of the control. In all groups, T3 had no effect on the AP amplitude.

Table 1.

The effects of T3 on action potential in neonatal mouse cardiomyocytes

The effects of T3 on action potential in neonatal mouse cardiomyocytes
The effects of T3 on action potential in neonatal mouse cardiomyocytes

T3 Blunted the [Ca2+]i Increase Induced by H/R Injury

As shown in Figure 5, there was no statistical difference in Fluo-3 fluorescence intensity of flow cytometry between the control group and control + T3 group (n = 3, p > 0.05). H/R injury significantly potentiated Fluo-3 fluorescence compared with the control group, suggesting an elevation in [Ca2+]i concentration. The levels of Ca2+ concentration in H/R group were increased by 58.1% (n = 6, p < 0.05). However, T3 pretreatment greatly blunted the increase of the [Ca2+]i, thus the levels of Ca2+ concentration in H/R+T3 group were increased by 31.2% compared with the control (n = 6, p < 0.05), which indicated that T3 can diminish H/R-induced [Ca2+]i overload and maintain [Ca2+]i homeostasis. The fluorescence intensity changes of the confocal microscopy were consistent with the flow cytometry.

Fig. 5.

Effects of T3 on [Ca2+]i concentration in cultured neonatal mouse cardiomyocytes of each group. a The effects of calcium overload were analyzed by flow cytometry using the fluorescent probe Fluo-3/AM. b Fluorescence intensity of flow cytometry in each group. c The effects of [Ca2+]i concentration were analyzed by the confocal microscopy using the fluorescent probe Fluo-3/AM. Data were expressed as mean ± SD, # p < 0.05 vs. control; * p < 0.05 vs. H/R. T3, triiodothyronine; H/R, hypoxia/reoxygenation.

Fig. 5.

Effects of T3 on [Ca2+]i concentration in cultured neonatal mouse cardiomyocytes of each group. a The effects of calcium overload were analyzed by flow cytometry using the fluorescent probe Fluo-3/AM. b Fluorescence intensity of flow cytometry in each group. c The effects of [Ca2+]i concentration were analyzed by the confocal microscopy using the fluorescent probe Fluo-3/AM. Data were expressed as mean ± SD, # p < 0.05 vs. control; * p < 0.05 vs. H/R. T3, triiodothyronine; H/R, hypoxia/reoxygenation.

Close modal

TH is considered a major regulator of cardiovascular function, including regulation of contractile and calcium-handling protein expression, ion channels, and sympathetic tonus in cardiovascular system homeostasis [17, 18]. Recent study has shown that low-dose T3 therapy could protect post infarct cardiac tissue from dysfunction and arrhythmias without adverse effects [17], although the mechanisms of this process remain unknown. This study investigated the cardioprotection effect of T3 in maintaining calcium homeostasis by regulating the NCX, late sodium channels and levels of [Ca2+]i concentration in H/R-induced cardiomyocytes.

Calcium homeostasis is essential for maintaining cardiac function. [Ca2+]i overload occurs in many pathological conditions, including hypoxia, ischemia, reperfusion, oxidative stress, cardiac hypertrophy, and heart failure [1-2]. [Ca2+]i overload leads to cellular injury, cell death, electrical activity disorders, and ventricular systolic dysfunction, which would eventually aggravate myocardial dysfunction and induce cardiac arrhythmias [5, 8, 19, 20]. Inhibiting [Ca2+]i and maintaining calcium homeostasis are important factors in protecting cardiac myocytes against I/R injury. Under pathological conditions, increased INaL could cause [Ca2+]i overload by increasing reverse INCX [7]. Extensive studies have reported that inhibition of NCX can prevent cardiac diseases through inhibition of INaL, which was associated with intracellular [Ca2+]i overload [8, 9]. Previous studies show that INaL is increased in many pathological conditions such as hypoxia and ischemia. Past studies have reported that low T3 treatment could promote cardiac function and prevent cardiac remodeling by inhibiting I/R-induced cardiomyocyte apoptosis [21, 22]. In this study, T3 pretreatment decreased the elevation of INaL and greatly blunted the increase of the [Ca2+]i concentration induced by H/R injury, and this effect was concentration-dependent. These data suggest that T3 may protect cardiomyocytes against H/R-induced injury by maintaining intracellular calcium homeostasis. T3 could rapidly, and nongenomically regulate the Ca2+ ATPase enzyme, the Na+ channel, and the K+ channel [10], suggesting that T3 contributes to Ca2+ homeostasis in cardiomyocytes.

NCX plays an important role in regulating [Ca2+]i and maintaining Ca2+ homeostasis in the heart, and the expression level of NCX is high in the fetal heart but gradually decreases after birth [23]. Previous study has indicated that the reverse INCX could be suppressed under normal or hypoxic conditions by inhibiting INaL, which shows that an increased INaL causes this change [24]. Thus, the inhibition of INaL is expected to decrease the augmented reverse INCX. The present study showed that H/R-induced injury caused an increase in reverse INCX on cardiomyocytes, while pretreatment of T3 inhibited this increase. Inhibition of INaL could result in a decrease in reverse INCX, inhibition of cell shortening, and [Ca2+]i overload amelioration [25], Thus, we hypothesized that T3 could protect cardiomyocytes from H/R injury by preventing abnormal Ca2+ influx by decreasing reverse INCX.

In the present study, the effect of T3 on ventricular cardiomyocyte APs was also studied. A previous study reported that T3 could regulate the expression of specific cardiac genes such as voltage-activated K+ channel genes and increase the inward currents directly, which shortened APD [13]. In this study, T3 shortened the prolonged APD50 and APD90 in H/R-induced cardiomyocytes, thus we speculate that T3 may have potential antiventricular arrhythmia effects, but further researches from larger studies are needed to verify this link. In addition, the normal and H/R-augmented INaT were also inhibited by T3 pretreatment, and the increased INaT current density induced by H/R injury was significantly diminished upon T3 pretreatment. Moreover, the steady-activation curve of INaT was shifted toward a more positive voltage, and the steady-inactivation curve of INaT was shifted toward a more negative voltage, which indicated that T3 can inhibit the rate of activation and accelerate the rate of inactivation of sodium channels to keep the Na+ channels stable.

In conclusion, T3-inhibited H/R-increased INaL and decreased the reverse INCX augmentation, shortened the APD, and diminished [Ca2+]i overload, indicating a potential therapeutic use of T3 as an INaL inhibitor to maintain Ca2+ homeostasis and protect cardiomyocytes against H/R injury.

This work was supported by Department of Cardiology, Renmin Hospital of Wuhan University, Cardiovascular Research Institute, Wuhan University, Hubei Key Laboratory of Cardiology.

All the authors do not have any possible conflicts of interest.

This work was supported by the Chinese National Nature Science Foundation (30900609, 81270271, and 81570333).

B.Z. and L.L. designed the experiment. L.L. and X.L. carried out the experiments. C.Z. and H.R. conceived and coordinated the study. L.L. wrote the manuscript. All authors reviewed the results and approved the final version.

1.
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