Liensinine- and Neferine-Induced Cardiotoxicity in Primary Neonatal Rat Cardiomyocytes and Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Abstract

Due to drug-induced potential congestive heart failure and irreversible dilated cardiomyopathies, preclinical evaluation of cardiac dysfunction is important to assess the safety of traditional or novel treatments. The embryos of Nelumbo nucifera Gaertner seeds are a homology of traditional Chinese medicine and food. In this study, we applied the real time cellular analysis (RTCA) Cardio system, which can real-time monitor the contractility of cardiomyocytes (CMs), to evaluate drug safety in rat neonatal CMs and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). This study showed detailed biomechanical CM contractility in vitro, and provided insights into the cardiac dysfunctions associated with liensinine and neferine treatment. These effects exhibited dose and time-dependent recovery. Neferine showed stronger blocking effect in rat neonatal CMs than liensinine. In addition, the effects of liensinine and neferine were further evaluated on hiPS-CMs. Our study also indicated that both liensinine and neferine can cause disruption of calcium homeostasis. For the first time, we demonstrated the potential cardiac side effects of liensinine or neferine. While the same inhibition was observed on hiPS-CMs, more importantly, this study introduced an efficient and effective approach to evaluate the cardiotoxicity of the existing and novel drug candidates.

Keywords: cardiotoxicity; hiPS-CM; liensinine; neferine; real-time analysis.

Figure 1

Optimization of Rat neonatal and hiPS cardiomyocytes seeding density on RTCA system. (A) Cell index; (B) Beating rate and (C) Amplitude of rat neonatal CMs within 48 h after seeding; (D) Cell index; (E) Beating rate and (F) Amplitude of hiPS-CMs. The black arrows indicated the time point of fresh medium addition. Data were presented as mean ± SD, n = 10.

Figure 1

Optimization of Rat neonatal and hiPS cardiomyocytes seeding density on RTCA system. (A) Cell index; (B) Beating rate and (C) Amplitude of rat neonatal CMs within 48 h after seeding; (D) Cell index; (E) Beating rate and (F) Amplitude of hiPS-CMs. The black arrows indicated the time point of fresh medium addition. Data were presented as mean ± SD, n = 10.

Figure 2

Representative contraction profiles of (A) rat neonatal CMs and (B) hiPS-CMs treated by reference compounds. Data were presented as mean ± SD, n = 3.

Figure 2

Representative contraction profiles of (A) rat neonatal CMs and (B) hiPS-CMs treated by reference compounds. Data were presented as mean ± SD, n = 3.

Figure 3

Typical rat neonatal CMs contraction profiles and dose responses to liensinine and neferine treatment. (A) Cell index fluctuation after various doses of liensinine treatment; (B) Temporal profiling of CMs beating status treated by various doses liensinine or neferine; (C) Dose response to liensinine and neferine of neonatal CMs 3 h after compounds treatment. Five CM beating parameters, including beating rate, amplitude, raising slop, falling slop and beating pattern, were used for evaluation; (D) Recovery time of beating rate of rat cardiomyocytes treated with various concentrations of tested compounds; (E) Chemical formula of liensinine and neferine. Data were presented as mean ± SD, n = 3.

Figure 3

Typical rat neonatal CMs contraction profiles and dose responses to liensinine and neferine treatment. (A) Cell index fluctuation after various doses of liensinine treatment; (B) Temporal profiling of CMs beating status treated by various doses liensinine or neferine; (C) Dose response to liensinine and neferine of neonatal CMs 3 h after compounds treatment. Five CM beating parameters, including beating rate, amplitude, raising slop, falling slop and beating pattern, were used for evaluation; (D) Recovery time of beating rate of rat cardiomyocytes treated with various concentrations of tested compounds; (E) Chemical formula of liensinine and neferine. Data were presented as mean ± SD, n = 3.

Figure 4

Typical contraction profiles and dose responses of rat neonatal and hiPS-CM to liensinine and neferine treatment. (A) Cell index fluctuation after various doses of liensinine treatment; (B) Temporal profiling of hiPS-CM beating functions treated by various doses of liensinine or neferine; (C) Dose response to liensinine and neferine of hiPS-CM 3 h after compounds treatment. Five hiPS-CM beating parameters, including beating rate, amplitude, raising slop, falling slop and beating pattern, were used for evaluation; (D) Recovery time of beating rate of hiPS-CM treated with various concentrations of tested compounds. Data were presented as mean ± SD, n = 3.

Figure 5

(A) A luciferase coupled ATP quantitation assay was used to determine the cytotoxicity of liensinine and neferine. Compounds were incubated in hiPS-CMs for 24 h before luminescence signal measurement; (B) Determination of LDH cytotoxicity of liensinine and neferine in hiPS-CMs. hiPS-CMs were plated in a 96-well plate in maintaining medium. Different concentrations of liensinine and neferine were added to the culture media and incubated for 24 h at 37 °C, 5% CO₂. LDH cytotoxicity was measured using the Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific, Hudson, NH, USA). Data were presented as mean ± SD, n = 3. ns, p > 0.05, **** p < 0.001.

Figure 6

Effects of liensinine and neferine on [Ca²⁺]_i in hiPS-CMs. (A) Cardiomyocytes were loaded with Fluo 4 AM and pre-incubated in Ca²⁺ free HBSS for 30 min, then exposed to HBSS containing Ca²⁺ (1.2 mM final) The medium was exchanged with maintaining medium and incubated for 15 min. Similar to calcium channel blocker verapamil, liensinine and neferine decreased [Ca²⁺]_i in hiPS-CMs. Dashed arrow indicates CaCl₂ addition and solid arrows indicate times of compound treatment. Horizontal axes in all traces show time in minutes. Measurements are indicated as a change in fluorescence after treatment divided by the initial fluorescence (F/F0); (B) Epifluorescence images show the [Ca²⁺]_i in 10 μM liensinine,1 μM liensinine, 10 μM neferine, 1 μM neferine and 5 μM verapamil addition; (C) A single calcium spark was performed within 1000 ms on hiPS-CMs. Images were shown consecutively 200 ms apart; (D) Temporal profiles of averaged calcium sparks at a typical single cardiomyocyte and the image were processed by Matrix Laboratory (MATLAB). Traces were scaled to the same peak fluorescence intensity.