HL-1 cells were purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd. (Cat. No. HTX2129; Shenzhen, China). The authors authenticated the cells shortly before use with the short tandem repeat profiling technique. Mycoplasma testing was performed using the MycoSensor PCR Assay Kit (Agilent Technologies, Santa Clara, CA, USA). HL-1 cells were cultivated in complete Claycomb Medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA), 100 U/mL penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), 4 mM l-glutamine (Gibco), and 100 µM norepinephrine (Solarbio, Beijing, China). The cardiomyocytes were cultured on cell culture plastics coverslips coated with 0.02% gelatin (Sigma) at 37 °C in 5% CO${}_2$ [22]. The medium was refreshed every 48 h, and the cultures were split 1:3 using standard subculturing procedures once cells reached high confluence at about 3–4 days.

For cell transfection, TERT-overexpressing lentiviruses and lentiviral-based small hairpin RNA (shRNA) targeting TERT were purchased from Genechem Co., Ltd. (Shanghai, China). Empty lentivirus vector was utilized as a negative control for TERT and shTERT. The specific sequence of shRNA-TERT is provided in the supplementary materials (Supplementary Table 1); shTERT#3 was selected for subsequent experiments (Supplementary Fig. 1). When HL-1 cells reached 50% confluency, the original medium was replaced with fresh medium containing 6 µg/mL polybrene (Sigma). Next, virus suspension was added according to the manufacturer’s instructions, and the virus-containing medium was replaced with fresh complete medium after 24 h. After 72 h, lentiviral-transduced cells were selected with 3 µg/mL puromycin (Sigma) and verified by Western blot (WB) analysis. Finally, the most effective shRNA-TERT was used for the downstream functional experiments. The study design for the experiment is shown in Fig. 1.

Fig. 1.

Study design for the experiment. TERT, telomerase reverse transcriptase; PGC-1$\alpha$, peroxisome proliferator-activated receptor gamma coactivator 1-alpha; APA, action potential amplitude; RP, resting potential; APD, action potential duration; I${}_{\text{Ca,L}}$, L-type Ca${}^{2+}$ currents; MMP, mitochondrial membrane potential; OCR, oxygen consumption rate; SOD, superoxide dismutase; ROS, reactive oxygen species; WB, Western blot; shNC, negative control.

When validating the p53/PGC-1$\alpha$ pathway, the concentration of each drug was determined based on previous literature and preliminary test results [18, 23]. To investigate the effects of p53 on PGC-1$\alpha$, HL-1 cells were treated with the p53 agonist Tenovin-6 (final concentration of 15 µM; Selleck Chemicals, Houston, TX, USA) and the p53 inhibitor PFT$\alpha$ (final concentration of 15 µM; Selleck), while the control group received an equal volume of dimethyl sulfoxide (DMSO; Solarbio), and all were co-cultured for 24 h. To validate the role of PGC-1$\alpha$, HL-1 cells were treated with the PGC-1$\alpha$ agonist ZLN005 (final concentration of 20 µM; MedChemExpress [MCE], Monmouth Junction, NJ, USA) and the PGC-1$\alpha$ inhibitor SR-182923 (final concentration of 20 µM; MCE); the control group received an equal volume of DMSO (Solarbio) with a co-culture duration of 24 h.

As we previously described [24], the Axon Multiclamp 700B Amplifier (Molecular Devices, San Jose, CA, USA) and pClamp software (version 10.4; Axon Co., Scottsdale, AZ, USA) were used for the whole-cell patch-clamp study. An electrode with a tip diameter of 2–4 µm was pulled with the PP-83 Microelectrode Puller (Narishige Co., Tokyo, Japan).

To measure the action potential (AP), the patch clamp was set to “current clamp” mode, with a clamping potential of 0 mV and stimulation applied at 1500 pA for 10 ms. The AP of the cells in each group was recorded, and parameters such as AP duration (APD), AP amplitude (APA), and resting membrane potential (RMP) were calculated. The extracellular solution composition was (in mM): NaCl 140, KCl 4, CaCl${}_2$ 1, MgCl${}_2$ 1, HEPEs 10, and glucose 10, adjusted to a pH of 7.4 with NaOH. The intracellular solution consisted of (in mM): K-aspartame 120, KCl 20, Na${}_2$ATP 4, MgCl${}_2$ 1, HEPES 10, and glucose 10, and the pH was adjusted to 7.4 with KOH.

For voltage-gated L-type Ca${}^{2+}$ current (I${}_{\text{Ca,L}}$) measurements, the patch clamp was set to “voltage-clamp” mode. A holding potential of –40 mV was used, with a depolarizing pulse of 0 mV for 150 ms applied to record a slowly inactivated inward current, which was controlled with 5 µmol/L nifedipine, a Ca${}^{2+}$ channel blocker. Extracellular solution comprised (in mM): NaCl 135, CaCl${}_2$ 1, MgCl${}_2$ 5, CsCl 5.4, BaCl${}_2$ 0.3, NaH${}_2$PO${}_4$ 0.33, HEPEs 10 and glucose 10, with pH adjusted to 7.4 with NaOH. Intracellular solution included (in mM): TEA-Cl 10, CaCl${}_2$ 1, MgCl${}_2$ 5, CsCl 120, EGTA 10, Na${}_2$ATP 5, HEPEs 10 and the pH was adjusted to 7.4 with CsOH.

HL-1 cells were washed twice with Ca${}^{2+}$-free phosphate-buffered saline (PBS) (Solarbio), and incubated with Fluo-4/AM (Invitrogen) at 37 °C for 60 min at a 10 µM final concentration. The cells were digested with 0.25% trypsin-EDTA (Hyclone Laboratories, Logan, UT, USA), centrifuged at 1000 rpm for 5 min to eliminate excess dye, washed twice with Ca${}^{2+}$-free PBS, and finally resuspended to 0.3 mL with Ca${}^{2+}$-free PBS. Subsequently, Ca${}^{2+}$ fluorescence intensity was quantified using the FACS Calibur Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) at an excitation wavelength of 485 nm and emission wavelength of 520 nm. A total of 2 $\times$ 10${}^4$ cells were collected, and the intracellular Ca${}^{2+}$ concentration was expressed as mean fluorescence intensity. Data were analyzed using FlowJo software (VX10.6.2, TreeStar, Ashland, OR, USA).

The reactive oxygen species (ROS) Detection Assay Kit (Sigma) was utilized to determine the intracellular oxidative stress level. Following the manufacturer’s protocol, the cells were suspended in culture medium at a density of 5 $\times$ 10${}^5$ cells/mL. Subsequently, 1 µL ROS Detection Reagent Stock Solution was added to 1 mL culture solution, and it was incubated in a cell incubator with 5% CO${}_2$ at 37 °C, for 30–60 min in the dark. Fluorescence intensity was measured using the FACS Calibur Flow Cytometer (BD Biosciences) at an excitation wavelength of 540 nm and emission wavelength of 570 nm. Then 2 $\times$ 10${}^4$ cells were collected and the ROS concentration was expressed as mean fluorescence. Data were analyzed with FlowJo VX10 software.

Superoxide dismutase (SOD) was measured using an assay kit (Sigma). The superoxide interacts with WST-1 and an electron-coupling reagent to produce the formazan product. SOD converts superoxide to hydrogen peroxide, yielding a reduced colorimetric signal at 450 nm. A microplate reader (BioTek, Winooski, VT, USA) was used to measure the optical density value of each well at a wavelength of 450 nm and calculate the SOD (%) = ([A ${}_{\text{control 1}}$ – A ${}_{\text{control 3}}$] – [A ${}_{\text{test}}$ – A ${}_{\text{control 2}}$])/(A ${}_{\text{control 1}}$ – A ${}_{\text{control 3}}$) $\times$ 100%. The results were normalized to the protein concentration.

For mitochondrial membrane potential (MMP) assessment, HL-1 cells were prepared in complete Claycomb Medium at a density of 5 $\times$ 10${}^5$ cells/mL. Following the manufacturer’s instructions, 2 µL of 500X MitoTellTM Orange was added to 1 mL cell solution and incubated in a cell incubator with 5% CO${}_2$ at 37 °C, for 30 min. Fluorescence intensity was measured using the FACS Calibur Flow Cytometer (BD Biosciences) at an excitation wavelength of 540 nm and an emission wavelength of 590 nm. Then 2 $\times$ 10${}^4$ cells were collected and the MMP was expressed as mean fluorescence. Data were processed using FlowJo VX10 software.

To determine the mitochondrial oxygen consumption rate (OCR), a Seahorse analyzer (XF96; Agilent Technologies) was used [25, 26]. When cells were 70–80% confluent, OCR was assessed. Prior to measurement, cells were incubated at 37 °C without CO${}_2$ for 1 h. Basal OCR was initially measured in triplicate. Subsequently, to inhibit ATP synthase, cells were treated with 1.5 µM oligomycin, and 1.0 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was added for maximal uncoupled respiration. Next, 0.5 µM rotenone/antimycin A was used to test non-mitochondrial respiration. ATP-linked respiration was the basal OCR subtracted from the uncoupled (after the addition of oligomycin). After FCCP addition, the maximum respiratory capacity was tested, while the spare capacity was calculated by subtracting basal from FCCP-induced OCR. All Seahorse results were normalized to protein concentration, which was quantified by the Bradford assay (Solarbio).

WB analysis was conducted following previously established protocols [24]. Protein samples were equally loaded on sodium dodecyl sulfate polyacrylamide gels and transferred to nitrocellulose membranes [24]. The membranes were blocked in 5% skim milk for 1 h at room temperature and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti-actin monoclonal (1:1000, ab179467; Abcam, Cambridge, MA, USA), rabbit anti-p53 polyclonal (1:1000, ab131442; Abcam), rabbit anti-PGC-1$\alpha$ polyclonal (1:1000, ab54481; Abcam), rabbit anti-SERCA2a monoclonal (1:1000, ab150435; Abcam), rabbit anti-NCX1.1 monoclonal (1:1000, ab177952; Abcam), rabbit anti-CaV1.2 monoclonal (1:1000, ab270987; Abcam), mouse anti-TERT monoclonal (1:500, sc-377511; Santa Cruz Biotechnology, Dallas, TX, USA). The chemiluminescence detection reagent (Western Lightning Plus®ECL, N0775330; PerkinElmer, Waltham, MA, USA) showed a specific signal after 2 h of incubation with the appropriate secondary antibody (A0277, Beyotime, Shanghai, China).

For statistical evaluation, SPSS version 21.0 (IBM SPSS Statistics, Armonk, NY, USA) was employed. The results are expressed as the mean $\pm$ standard error of the mean. The Shapiro–Wilk normality test was utilized to assess the normality of the data distribution. For comparisons between two groups, the unpaired two-tailed Student’s t-test or Mann–Whitney U tests were applied for normally or non-normally distributed data, respectively. Comparisons among three groups were performed using one-way analysis of variance followed by Dunnett’s post hoc test. p 0.05 was considered statistically significant.

AP was recorded with the patch-clamp technique, and the original AP recording of the HL-1 cells is shown in Fig. 2A,B. TERT silencing significantly shortened action potential duration at 50% repolarization (APD${}_{50}$) and APD${}_{90}$ (Fig. 2E,F). In contrast to silencing, TERT overexpression significantly prolonged APD, especially APD${}_{50}$ and APD${}_{90}$ (n = 10, p 0.01) (Fig. 2C,D). Neither TERT overexpression nor silencing had a significant impact on APA and RMP compared to the respective control groups (Table 1).

Fig. 2.

Effects of TERT on APD. (A,B) Original recording of the action potential (AP) in TERT overexpression (A) and TERT-silenced (B) HL-1 cells. (C) TERT overexpression significantly prolonged APD${}_{50}$. (D) APD${}_{90}$ of the TERT overexpression group was clearly prolonged compared with the Vector group. (E) TERT silencing significantly shortened APD${}_{50}$. (F) TERT silencing shortened APD${}_{90}$. **p 0.01 vs. Vector group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. shNC group. TERT, telomerase reverse transcriptase; APD, action potential duration; APD${}_{50}$, action potential duration at 50% repolarization; APD${}_{90}$, action potential duration at 90% repolarization.

Previous studies have indicated that a decrease in Cav1.2, accompanied by reduced I${}_{\text{Ca,L}}$, is a primary pathological change in APD shortening [27, 28]. As demonstrated in Fig. 3A, I${}_{\text{Ca,L}}$ amplitude in the TERT-silenced group was markedly lower compared to the shNC group. Conversely, TERT overexpression significantly increased I${}_{\text{Ca,L}}$ amplitude in HL-1 cells. Furthermore, when the current amplitude was replaced by current density, we found that silencing TERT led to a decrease in peak I${}_{\text{Ca,L}}$ density from –9.31 $\pm$ 0.5 pA/pF to –7.35 $\pm$ 0.8 pA/pF (n = 10, p 0.05; Fig. 3C). For the opposite condition, the peak I${}_{\text{Ca,L}}$ density of the TERT overexpression group (–16.3 $\pm$ 1.2 pA/pF) was significantly greater than that of the control group (–9.35 $\pm$ 0.6 pA/pF) at 0 mV depolarization (n = 10, p 0.01; Fig. 3B).

Fig. 3.

Effects of TERT on I${}_{\mathbf{\text{Ca,L}}}$. (A) Original recording of the effects of TERT on the amplitude of I${}_{\text{Ca,L}}$ in HL-1 cells. (B) TERT overexpression significantly increased the peak I${}_{\text{Ca,L}}$ density. (C) Silencing TERT caused decreased the peak I${}_{\text{Ca,L}}$ density. (D,E) Original recording of the voltage-dependent effect of TERT overexpression (D) and TERT silencing (E) on I${}_{\text{Ca,L}}$. (F,G) I-V curve of TERT overexpression (F) and TERT silencing (G) in HL-1 cells. (H,I) The steady-state activation curve of I${}_{\text{Ca,L}}$ in TERT overexpression (H) and TERT silencing (I) group. (J,K) The steady-state inactivation curve of I${}_{\text{Ca,L}}$ in TERT overexpression (J) and TERT silencing (K) groups. (L,M) I${}_{\text{Ca,L}}$ recovery curve after inactivation in TERT overexpression (L) and TERT silencing (M) groups. **p 0.01 vs. Vector group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. shNC group. TERT, telomerase reverse transcriptase; I-V curve, current density–voltage curve.

I${}_{\text{Ca,L}}$ was elicited by continuous stimulation, and the changes in I${}_{\text{Ca,L}}$ at each stimulation voltage were observed at the same time (Fig. 3D,E). The I-V curve was obtained by plotting the current density and stimulation voltage, which showed a typical “inverted bell” Ca${}^{2+}$ current characteristic (Fig. 3F,G), indicating that the role of TERT on I${}_{\text{Ca,L}}$ is voltage-dependent.

The steady-state activation curve of I${}_{\text{Ca,L}}$ showed that the half-activation voltage shifted to the right in the TERT-silenced group (Fig. 3I), while the slope of the curve increased in the TERT overexpression group (Fig. 3H), but neither was statistically significant (p $>$ 0.05). These findings indicated a limited effect of TERT on the steady-state activation of I${}_{\text{Ca,L}}$.

Compared with the control group, the steady-state inactivation curve of I${}_{\text{Ca,L}}$ in TERT-silenced cells shifted to the left, which means that it moved to the hyperpolarization direction, indicating that channel steady-state inactivation increased near the resting potential (RP) (Fig. 3K). However, when overexpressing TERT, the above curve changed to the opposite direction. The steady-state inactivation curve of I${}_{\text{Ca,L}}$ in the TERT overexpression group shifted to the right from the control group (Fig. 3J), suggesting that the steady-state inactivation of Ca${}^{2+}$ channels was slowed; that is, the channel inactivation process was weakened at the same stimulation pulse, which may be the reason for the increase in current density. These results indicate that TERT may mediate current changes by altering channel inactivation.

Regarding the effects of TERT on the kinetics of recovery after I${}_{\text{Ca,L}}$ inactivation, we found that when the TERT gene was silenced, the recovery curve after channel inactivation shifted to the right and the recovery time was prolonged, indicating that TERT changed the current density by affecting the recovery kinetics of Ca${}^{2+}$ channels (Fig. 3M). However, the process of recovery after Ca${}^{2+}$ current inactivation was accelerated in the TERT overexpression group, suggesting that it may have been another factor for the current increase (Fig. 3L).

CaV1.2 is linked to Ca${}^{2+}$ inward flow. The expression of CaV1.2 in TERT-silenced cells was significantly decreased, while TERT overexpression led to CaV1.2 upregulation (Fig. 4E–H).

Fig. 4.

Effects of TERT on intracellular Ca${}^{\mathrm{2}\mathrm{+}}$ and Ca${}^{\mathrm{2}\mathrm{+}}$ transporters. (A) Representative images of Ca${}^{2+}$ concentration in TERT overexpression HL-1 cells. (B) Intracellular Ca${}^{2+}$ concentration quantified by mean fluorescence intensity in TERT overexpression HL-1 cells. (C) Representative images of Ca${}^{2+}$ concentration in TERT-silenced HL-1 cells. (D) Intracellular Ca${}^{2+}$ concentration quantified by mean fluorescence intensity in TERT-silenced HL-1 cells. (E,F) WB analyses of the Ca${}^{2+}$ transporter in TERT overexpression HL-1 cells. (G,H) WB analyses of the Ca${}^{2+}$ transporter in TERT-silenced HL-1 cells. *p 0.05, **p 0.01 vs. Vector group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. shNC group. TERT, telomerase reverse transcriptase.

Ca${}^{2+}$ is the most important second messenger in cardiomyocytes, and abnormally increased intracellular Ca${}^{2+}$ can lead to electrical remodeling, APD shortening, and a decrease in I${}_{\text{Ca,L}}$. As depicted in Fig. 4C,D, TERT silencing led to intracellular Ca${}^{2+}$ overload (n = 5, p 0.01). However, TERT overexpression significantly reduced the intracellular Ca${}^{2+}$ concentration compared to the control (Fig. 4A,B).

The maintenance of intracellular Ca${}^{2+}$ homeostasis is co-regulated by the sarcoplasmic reticulum (SR) Ca${}^{2+}$ transporter, cell membrane Ca${}^{2+}$ transporter, and mitochondrial Ca${}^{2+}$ transporter [29]. The concentration of extracellular Ca${}^{2+}$ is much higher than that of intracellular Ca${}^{2+}$. When cells are stimulated, extracellular Ca${}^{2+}$ flows into the cell through the CaV1.2 Ca${}^{2+}$ channel on the cell membrane, activating the cardiac ryanodine receptor, RyR2, to release Ca${}^{2+}$ and eventually leading to the contraction of cardiomyocytes. Subsequently, the vast majority of intracellular free Ca${}^{2+}$ is recovered by SERCA2a in the SR, and the remaining Ca${}^{2+}$ is transported extracellularly by NCX1.1. Mitochondria act as intracellular Ca${}^{2+}$ pools providing a buffering effect on intracellular Ca${}^{2+}$, and proteins such as mitochondrial calcium uniporter, mitochondrial calcium uptake 1, and voltage-dependent anion-selective channel 1 are involved in mitochondrial Ca${}^{2+}$ uptake [29, 30].

WB analysis revealed that silencing TERT significantly downregulated the expression of SERCA2a (Fig. 4G), and TERT overexpression upregulated the expression of SERCA2a (Fig. 4E). The primary role of NCX1.1 in the heart is to extrude Ca${}^{2+}$ from the cell, countering the Ca${}^{2+}$ that enters the cytoplasm during systole through CaV1.2 [31]. In this study, we found that silencing the TERT gene significantly downregulated the expression of NCX1.1, while TERT overexpression had the opposite effect (Fig. 4E–H). The above results suggest that telomere shortening decreases Ca${}^{2+}$ uptake and reduces Ca${}^{2+}$ efflux, ultimately leading to intracellular Ca${}^{2+}$ accumulation.

In this study, flow cytometry was used to detect intracellular ROS. As indicated in Fig. 5C,D, compared with the shNC group, silencing of TERT in HL-1 cells caused a significant increase in ROS (n = 5, p 0.01). Conversely, ROS levels in TERT overexpression HL-1 cells was significantly lower compared with control cells (Fig. 5A,B). SOD is an important member of the intracellular antioxidant system, and can scavenge harmful ROS to relieve the damage caused by oxidative stress. Compared with the respective control groups, SOD activity in the TERT overexpression group was increased, while it was significantly decreased in TERT-silenced cells (Fig. 5I,J). These findings demonstrate that TERT silencing can lead to an increase in ROS content and decrease in SOD activity, consistent with the finding that telomere shortening can lead to increased oxidative stress in cardiomyocytes [32, 33].

Fig. 5.

Effects of TERT on mitochondrial function. (A) Representative images of ROS detected by flow cytometry in TERT overexpression HL-1 cells. (B) ROS were quantified and found to be significantly lower in TERT overexpression HL-1 cells than in the controls. (C) Representative images of ROS detected by flow cytometry in TERT-silenced HL-1 cells. (D) ROS were quantified and found to be significantly higher in TERT-silenced HL-1 cells than in the controls. (E) Representative images of MMP in the TERT overexpression group. (F) Quantification of MMP in TERT overexpression HL-1 cells. (G) Representative images of MMP in TERT-silenced HL-1 cells. (H) Quantification of MMP in TERT-silenced HL-1 cells. (I) SOD activity in TERT overexpression HL-1 cells was higher than that in the control group. (J) Silencing of TERT significantly decreased SOD activity. (K) OCR curve of TERT overexpression HL-1 cells. (L) The OCR of TERT overexpression HL-1 cells. (M) OCR curve of TERT-silenced HL-1 cells. (N) The OCR of TERT-silenced HL-1 cells. *p 0.05, **p 0.01 vs. Vector group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. the shNC group. TERT, telomerase reverse transcriptase; ROS, reactive oxygen species; MMP, mitochondrial membrane potential; SOD, superoxide dismutase; OCR, oxygen consumption rate; ATP, adenosine triphosphate; FCCP, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone.

The MMP is a crucial indicator of mitochondrial function. A decrease in MMP is often observed in the early stages of apoptosis. As shown in Fig. 5G,H, silencing of TERT caused a significant decrease in MMP (n = 5, p 0.01); however, in TERT overexpression HL-1 cells, the MMP was higher than that in the control cells (Fig. 5E,F). In this study, TERT silencing resulted in a decrease in OCR in HL-1 cells, with a significant decrease in basal OCR, ATP-linked OCR, maximal OCR, and reserve OCR (Fig. 5M,N). Conversely, the OCR of TERT overexpression HL-1 cells was higher than that of the control, and the basal OCR, ATP-linked OCR, maximal OCR, and reserve OCR were significantlyincreased (p 0.01). Besides, proton leak OCR and non-mitochondrial OCR were also increased, with no statistical difference compared with the control group (Fig. 5K,L). In summary, TERT silencing led to impaired mitochondrial function, increased ROS production, and decreased OCR, MMP, and SOD activity.

Telomere shortening leads to intracellular Ca${}^{2+}$ overload and dysfunction of mitochondria via the p53/PGC-1$\alpha$ pathway [7, 8, 18, 19]. To validate this pathway, we intervened the molecules on upstream of the regulatory axis. It is noteworthy that the expression of p53 was upregulated and the level of PGC-1$\alpha$ was downregulated in TERT-silenced cells (Fig. 6B,E). Conversely, TERT overexpression decreased p53 expression and increased PGC-1$\alpha$ expression in HL-1 cells (Fig. 6A,D). Subsequently, we treated HL-1 cells with the p53 agonist Tenovin-6 and inhibitor PFT$\alpha$. The results showed that treatment with PFT$\alpha$ upregulated the expression of PGC-1$\alpha$, while Tenovin-6 downregulated expression (Fig. 6C,F). These results suggest that TERT regulates the p53/PGC-1$\alpha$ pathway in atrial myocytes.

Fig. 6.

TERT regulates the p53/PGC-1$\alpha$ pathway. (A,D) WB in TERT-overexpressed cells. (B,E) WB in TERT-silenced cells. (C,F) WB after treatment with the p53 agonist Tenovin-6 and inhibitor PFT$\alpha$. **p 0.01 vs. Vector group; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs. shNC group; p 0.01 vs. Control group. TERT, telomerase reverse transcriptase; PGC-1$\alpha$, peroxisome proliferator-activated receptor gamma coactivator 1-alpha.

PGC-1$\alpha$ is known for its role in mitochondrial biosynthesis, but its effect on intracellular Ca${}^{2+}$ has been less studied. In this study, we treated HL-1 cells with the PGC-1$\alpha$ agonist ZLN005 and PGC-1$\alpha$ inhibitor SR-18292 to observe their effects on cellular electrophysiology, intracellular Ca${}^{2+}$, and Ca${}^{2+}$ transporters. The results showed that the SR-18292 led to decreased I${}_{\text{Ca,L}}$, intracellular Ca${}^{2+}$ overload, increased ROS, and decreased MMP, OCR, and SOD activity. In addition, the expression of SERCA2a, CaV1.2, and NCX1.1 was downregulated (Figs. 7,8). However, ZLN005 treatment increased I${}_{\text{Ca,L}}$ (Fig. 7A,B) and decreased intracellular Ca${}^{2+}$ levels (Fig. 7C,D). Additionally, ZLN005 upregulated the expression of SERCA2a, CaV1.2, and NCX1.1 (Fig. 7E,F). Furthermore, we also found that ZLN005 reduced ROS levels and increased SOD activity, MMP, and OCR compared with the control group (Fig. 8). These results indicate that ZLN005 has similar effects as TERT overexpression, and coculture with SR-18292 resulted in similar effects as TERT silencing. Therefore, PGC-1$\alpha$ may be a new target for the treatment of cardiovascular diseases.

Fig. 7.

Effects of PGC-1$\alpha$ on I${}_{\mathbf{\text{Ca,L}}}$, intracellular Ca${}^{\mathrm{2}\mathrm{+}}$, and Ca${}^{\mathrm{2}\mathrm{+}}$ transporters. (A) Original record of I${}_{\text{Ca,L}}$. (B) Role of PGC-1$\alpha$ on I${}_{\text{Ca,L}}$ current density. (C) Quantification of intracellular Ca${}^{2+}$. (D) Representative images of Ca${}^{2+}$ concentration detected by flow cytometry. (E,F) WB analyses of Ca${}^{2+}$ transporters. *p 0.05, **p 0.01 vs. Control group.

Fig. 8.

Effects of PGC-1$\alpha$ on mitochondrial function. (A) Role of PGC-1$\alpha$ on ROS. (B) Role of PGC-1$\alpha$ on SOD activity. (C) Role of PGC-1$\alpha$ on MMP. (D) The OCR curve of HL-1 cells treated with ZLN005 and SR-18292. (E) Effects of PGC-1$\alpha$ on the OCR. *p 0.05, **p 0.01 vs. Control group. ROS, reactive oxygen species; SOD, superoxide dismutase; MMP, mitochondrial membrane potential; OCR, oxygen consumption rate; FCCP, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; ATP, adenosine triphosphate.