Isosteviol prevents the prolongation of action potential in hypertrophied cardiomyoctyes by regulating transient outward potassium and L-type calcium channels

    Isosteviol prevents the prolongation of action potential in hypertrophied cardiomyoctyes by regulating transient outward potassium and L-type calcium channels Zhuo Fan, Nanying Lv, Xiao Luo, Wen Tan PII: DOI: Reference:

S0005-2736(17)30127-X doi:10.1016/j.bbamem.2017.04.011 BBAMEM 82476

To appear in:

BBA - Biomembranes

Received date: Revised date: Accepted date:

17 December 2016 24 March 2017 14 April 2017

Please cite this article as: Zhuo Fan, Nanying Lv, Xiao Luo, Wen Tan, Isosteviol prevents the prolongation of action potential in hypertrophied cardiomyoctyes by regulating transient outward potassium and L-type calcium channels, BBA - Biomembranes (2017), doi:10.1016/j.bbamem.2017.04.011

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ACCEPTED MANUSCRIPT Isosteviol prevents the prolongation of action potential in hypertrophied cardiomyoctyes by regulating transient outward

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potassium and L-type calcium channels

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Zhuo Fan1, Nanying Lv1, Xiao Luo1, Wen Tan2

School of Bioscience and Bioengineering, South China University of Technology,

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Guangzhou 510006, China 2

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Institute of Biomedical & Pharmaceutical Sciences, Guangdong University of Technology. NO. 100 Waihuan Xi Road, Guangzhou Higher Education Mega Center, P.R. China 510006

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Corresponding Author:

Wen Tan: Institute of Biomedical & Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China. E-mail: [email protected] (Wen Tan)

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ACCEPTED MANUSCRIPT Abstract: Cardiac hypertrophy is a thickening of the heart muscle that is associated with cardiovascular diseases such as hypertension and myocardial infarction. It occurs

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initially as an adaptive process against increased workloads and often leads to sudden arrhythmic deaths. Studies suggest that the lethal arrhythmia is attributed to

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hypertrophy-induced destabilization of cardiac electrical activity, especially the prolongation of the action potential. The reduced activity of Ito is demonstrated to be responsible for the ionic mechanism of prolonged action potential duration and

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arrhythmogeneity. Isosteviol (STV), a derivative of stevioside, plays a protective role in a variety of stress-induced cardiac diseases. Here we report effects of STV on rat

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ISO-induced hypertrophic cardiomyocytes. STV alleviated ISO-induced hypertrophy of cardiomyocytes by decreasing cell area of hypertrophied cardiomyocytes. STV

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application prevented the prolongation of action potential which was prominent in

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hypertrophied cells. The decrease and increase of current densities for Ito and ICaL observed in hypertrophied myocytes were both prevented by STV application. In

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addition, the results of qRT-PCR suggested that the changes of electrophysiological activity of Ito and ICaL are correlated to the alterations of the mRNA transcription

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level.

Keywords: Isosteviol; cardiac hypertrophy; transient outward potassium channel; L-type calcium channel.

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1. Introduction Cardiac hypertrophy often occurs in a variety of cardiovascular diseases

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including hypertension and myocardial infarction. Ventricular hypertrophy, often

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accompanied by life-threatening ventricular arrhythmias, is a major risk factor for

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sudden cardiac death [1]. This risk may result, in part, from hypertrophy-induced destabilization of cardiac electrical activity, especially the prolongation in action potential duration [2, 3]. Accumulated experimental data suggest that the alteration in

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activity of repolarizing potassium channels is a major cause of electrophysiological remodeling and arrhythmogeneity [4-7]. The transient outward potassium current (Ito),

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which activates and inactivates rapidly, is a prime repolarizing current in the ventricular myocytes of many species. Ito is initiated immediately after the upstroke of the action potential which contributes to the early phase of repolarization, thereby

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affects the shape and duration of the action potential in turn [8, 9]. In the mammalian

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heart, especially the small mammals like mice or rats, Ito current determines the action potential duration and cardiac rhythm [10, 11]. Cardiac hypertrophy has been

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identified to be associated with distinct reduction of Ito densities [12]. Voltage-gated potassium channel Kv4.2 and Kv4.3, which are thought to encode the major fraction of Ito in the myocardium of rodents, are found to be significantly reduced in the

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expression level [13-16].

Isosteviol is a derivative of steviosid that has been used commercially as a sugar substitute for years [17]. Studies indicated that both stevioside and isosteviol may possess

a

variety

of

biological

activities

including

anti-hypertension,

anti-hyperglycemia, anti-inflammatory and potential antitumor effects [18-22]. The myocardium protective effects of isosteviol against ischemia-reperfusion (IR) injury have also been reported [23, 24]. Isosteviol is proposed to be able to reduce the infraction area and restore the contractility in cardiac IR in vivo and in isolated hearts without introducing or even improving arrhythmia. Our previous study on its effect on sarc- and mitoKATP channels also indicated a potential cardioprotective effect of STV [25]. 3

ACCEPTED MANUSCRIPT Here we study the direct effect of isosteviol (STV) on ISO-induced hypertrophy of rat ventricular myocytes. The action potential duration (APD) was prolonged in hypertrophied myocytes and the prolongation of APD was prevented by

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STV-preincubation. In addition, STV restored the activity of Ito which were markedly down-regulated in hypertrophied myocytes. In contrast to Ito, the current density of

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ICaL channel increased firstly in hypertrophied myocytes then reduced in isosteviol-coadministration group. In addition, the channel currents, the mRNA expression of related genes at transcription level exhibited consistent results with that

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of channel currents.

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2. Materials and methods:

2.1 Isolation of rat ventricular myocytes

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Single ventricular myocytes were isolated from Sprague-Dawley rat hearts using

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a standard enzymatic technique. The protocols were approved by our institutional ethics committee. Briefly, adult Spraque Dawley rats (250-300 g) were anesthetized

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with an injection of 5% pentobarbital sodium (0.2 ml/100 g). Heparin (1000 U/Kg) was administered to prevent coagulation during heart removal. Then, hearts were rapidly removed, mounted on a Langendorff perfusion apparatus and retrogradely

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perfused with Ca2+ free tyrode solution composed of (in mM): NaCl 120, KCl 5.4, MgCl2 0.5, HEPES 25 and glucose 10 (pH 7.4 with NaOH) at 37℃. After 5 min, the perfusion solution was changed to the tyrode solution containing type II collagenase (0.6 mg/ml, Worthington), protease (0.05 mg/ml, Sigma) and Ca2+ (50 μM) for no more than 30 mins. After perfusion, the ventricular tissue was cut into small pieces in a petri dish with the same solution and was shaken gently for the dispersion of dissociated cardiac myocytes. A 250-μM mesh screen was used to separate the isolated cardiac myocytes from cardiac tissue. Cells were later collected by centrifugation at 500 rpm for 1min. Finally, the Ca2+ concentration was gradually restored to 1 mM. Cells were stored in M199 medium at a 37℃ CO2 incubator for 24 hours. A total of 35 rats were used in this study for cell isolation. 4

ACCEPTED MANUSCRIPT 2.2 Quantitative real-time RT-PCR Total RNA was extracted with the TAKARA RNAiso Plus Reagent Kit according to the manufacturer’s instructions. Reverse transcription was performed with

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TAKARA PrimeScript™RT Reagent Kit with gDNA Eraser (Perfect Real Time). The primers used for different genes are listed in table1. The real-time PCR cycling

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program involved an initial denaturation step at 95°C for 30 s, followed by 40 cycles of 10 s at 95°C and 30 s at 60°C. All quantitative real-time PCR (qRT-PCR) was performed with the ABI 7500 (Applied Biosystems, USA). Fluorescence signals of

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genes were recorded during the elongation phase of each PCR cycle. Melting curve analysis was used to confirm the amplification specificity. Each gene was quantified

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in triplicates. Real-time PCR data were analyzed by the comparative CT method. GAPDH mRNA was used as the internal control. Data were presented as

,

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where △CT=(CT gene of interest – CT internal control).

Table 1. Sequence specific primers used in RT-PCR

GAPDH ANP

Reserve Prime

TGGCCTCCAAGGAGTAAGAAAC

GGCCTCTCTCTTGCTCTCAGTATC

GGAGCCTGCGAAGGTCAA

TATCTTCGGTACCGGAAGCTGT

CAGAAGCTGGAGCTGATAAG

TGTAGGGCCTTGGTCCTTTG

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BNP

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Forward Prime

Kv4.2

ATGAGCTGACTGCCCAGGTTC

AATGACCAGGTGCAAGGTTAAACAA

Kv4.3

CCACGAGTTTATTGATGAGCAGAT

TGTTTTGCAGTTTGGTCTCAGTC

KChIP2

CGTGGTGACCATCGAGGAAT

GGCCTGGAGAGAGAGGGTTA

Cav1.2

TCTGCTCTGCCTGACTCTGA

CACACAATTGGCAAAAATCG

2.3 Electrophysiological recordings Whole-cell patch-clamp recordings were performed at room temperature with an EPC-10 amplifier (HEKA, Lambrecht, Germany). Cells were placed in an experimental chamber mounted on a stage of an inverted microscope (Nikon, Japan). Pipettes were pulled from borosilicate glass (Sutter, Novato, CA) and had a resistance 5

ACCEPTED MANUSCRIPT of between 2 and 4 MΩ. For Ito recording, the bath solution was (in mM): NaCl 138, KCl 4.0, MgCl2 1.0, CaCl2 2, NaH2PO4 0.33, HEPES 10, glucose 10 and CdCl2 0.3, with the pH controlled to 7.4 with NaOH. The internal solution was (in mM):

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K-aspartate 120, KCl 20, MgCl2 1.0, Mg-ATP 5, EGTA 10, HEPES 5 (pH 7.2 with

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KOH). Action potentials were measured in the Ca2+ containing Tyrode's solution

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consisting of (in mM): NaCl 137, KCl 5.4, MgCl2 1.2, CaCl2 1, NaH2PO4 1.2, HEPES 20, and glucose 10, with the pH controlled to 7.4 with NaOH .The internal solution contained (in mM): KCl 130, NaCl 10, MgCl2 5, EGTA 0.5, HEPES 10, Mg-ATP 5,

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pH adjusted to 7.2 with KOH. For L-type calcium channel recording, the bath solution was (in mM): TEA-Cl 135, CaCl2 2, Glucose 10, HEPES 10, MgCl2 1. The internal

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solution was (in mM): CsCl 110, Mg-ATP 5, EGTA 10, TEA-Cl 30 (pH 7.2 with CsOH). For Ito recording, CdCl2 (0.3 mM) was added to bath solution to block L-type

delivered

alone

or

coadministrating

with

ISO

24

hours

before

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was

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calcium channel, TTX (10μM) was added to bath solution to block Na+ current. STV

electrophysiological recording. Except in the concentration dependence experiment,

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the concentration of STV used in all experiments of this study was 10 μM. Before recording Ito, membrane potential was clamped at a holding potential of -80 mV.

Ito potassium currents was examined by a 300 ms voltage steps from -40

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mV to +50 mV in 10 mV increments. A 50 ms brief prepulse to -40 mV was used to ensure full inactivation of sodium current during the test pulse. The voltage dependence of steady-state inactivation for Ito was determined by applying a 1s conditioning pulse from a holding potential of −80 mV to a potential between −70 mV and 10 mV in steps of 10 mV, followed by a 500 ms test pulse to +60 mV. The L-type calcium currents were evoked by step depolarization to test potentials between -40 mV and +60 mV for 500 ms. In order to compensate for variations in cell size, currents were normalized to cell capacitance and reported as current densities (pA/pF). Membrane capacitance (Cm) was calculated using the automated capacitance compensation procedure of the EPC-10 amplifier. APs were measured at the current-clamp mode and evoked by applying a 5-ms depolarizing square pulse with the amplitude of 1000 pA. Action potential durations were measured at 20%, 50% and 6

ACCEPTED MANUSCRIPT 90% repolarization respectively (APD20, APD50 and APD90). Data were sampled at 10 kHz and filtered at 2.9 kHz. Cell capacitance (Cm) and series resistance (Rs) were electrically compensated in the voltage clamp experiments. The Patch Master (HEKA,

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Lambrecht,, Germany) and origin 9.0 softwares (OriginLab, Northampton, MA) were used for data acquisition and analysis respectively. All of the recordings were

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performed at room temperature (21 ~23 °C).

2.4 Chemicals and drugs

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The sodium salt of isosteviol was provided by Key Pharma Biomedical Inc. All chemicals for tyrode’s solution, external and internal solution of patch-clamp

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recording were bought from Sigma Chemical Co. Collagenase was purchased from Worthington. Pentobarbital sodium was obtained from Sangon Biotech (Shanghai)

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and heparin was obtained from Aladdin Reagent.

2.5 Data acquisition and analysis

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Data were acquired with the Pulse/PulseFit program (HEKA, Lambrecht, Germany) and further analyzed using Origin 7.0 (OriginLab, Northampton, MA). The activation time constant (τact) and inactivation time constant (τinact) were obtained by

trace

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using a single exponential function fitted to the rising and decaying curves of current recorded

at

+50

mV

respectively.

Bolzmann

equations

G/Gmax=1/[1+exp(V1/2−V)/k] and I/Imax=1/[1+exp(V1/2−V)/k] were used to fit to the voltage-dependence of activation and inactivation curves respectively. Data were shown as means ± SD. Statistical analysis was carried out by one-way ANOVA using a Bonferroni test. The criterion for a significant difference was p < 0.05.

3. Results: 3.1 STV attenuated ISO-induced hypertrophy of cardiomyocytes. Acutely isolated ventricular myocytes were pretreated with isoproterenol (ISO, 5 μM) for 24 hours to introduce hypertrophy in cardiomyocytes. For ISO+STV group, 10 μM STV was co-administrated to investigate the effect of STV on ISO-induced 7

ACCEPTED MANUSCRIPT myocytes hypertrophy. For control+STV group, 10 μM STV alone was administrated to investigate the effect of STV on control rat myocytes. The four panels of figure 1A showed cells for Control, ISO, ISO+STV, and control+STV groups respectively

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captured by an inverted microscope. Randomly selected 100 cells in each group were used to calculate the mean cell surface area. Figure 1B showed that the mean cell

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surface area was enlarged prominently by ISO treatment, which was reduced again significantly by STV-coadministration. STV alone has no significant effect on cell surface area of cardiomyocytes. The mean cell surface area was 1694.67 ± 103.50

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μm2, 2084.45 ± 186.85 μm2 and 1803.41 ± 155.87 μm2 and 1699.56 ± 178.28 for Control, ISO,ISO+STV and Control+STV groups respectively. Besides cell area,

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myocyte viability was also measured. ISO decreased cell viability revealed by Typan Blue staining to 57.1% of control level, which was raised to 84.5% by STV

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co-administration.

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Atrial natriuretic peptide (ANP) and brain (B-type) natriuretic peptide (BNP) are upregulated in hypertrophied myocardial cells and are often used as bio-makers for

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cardiac hypertrophy. ANP and BNP are tested to compare the mRNA expression level for Control, ISO and ISO+STV groups, which were normalized to GAPDH (Fig. 2). The mRNA expression of ANP and BNP at transcription level were both increased

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significantly for ISO group whereas decreased when co-administrated with STV. The relative mRNA expression levels of ANP were 0.01498 ± 0.00156, 0.02411 ± 0.00158 and 0.01898 ± 0.00175 for Control, ISO and ISO+STV groups respectively. For BNP, the relative mRNA expression levels of the three groups were 0.01936 ± 0.00310, 0.05341 ± 0.00852 and 0.03308 ± 0.00153 respectively.

3.2 STV prevented the prolongation of action potential duration in ISO-induced hypertrophic cardiomyocytes. To investigate how ISO-induced hypertrophy and STV-coadministration affect the electrophysiology properties of cardiomyocytes, the action potentials were recorded by whole cell patch clamp method for control, ISO, ISO+STV and Control+STV groups. The action potential duration at 20%, 50% and 90% of 8

ACCEPTED MANUSCRIPT repolarization (APD90) were compared among groups in figure 3B. APD20, APD50 and APD90 were all lengthened in ISO-treated hypertrophied cells. Co-administration with STV (10 μM) prevented these prolongations of AP durations introduced by ISO (Fig.

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3B). The values of APD20 were 5.33 ± 0.58 ms, 7.40 ± 1.74 ms and 5.35 ± 0.61 ms for Control, ISO and ISO+STV groups. The APD50 for Control, ISO and ISO+STV were

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13.51 ± 3.63, 20.23 ± 5.29 and 14.71 ± 1.66 ms respectively. The values of APD90 were 52.00 ± 5.94 ms, 68.23 ± 4.13 ms and 56.62 ± 10.16 ms for Control, ISO and ISO+STV groups respectively. Administration of STV alone (Control+STV) did not

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introduce any change for APD at every repolarization stage. In contrast to APD, the resting membrane potentials were not changed by ISO or STV-preincubation (Data

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not shown).

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3.3 STV re-enlarged Ito currents which were decreased in ISO-induced

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hypertrophied cardiomyocytes.

Ito current is a major repolarizing current in the ventricular myocytes of rodents

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and determines the shape and duration of action potential. Ito current was elicited by step potentials from -40 mV to +50 mV with 10mV increments and the current at +50 mV depolarizing potential was used for comparison among groups. Fig. 4A showed

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that the current amplitude of Ito at +50 mV for ISO group was decreased significantly compared with that of control group. Co-administrating cells with STV (10 μM) significantly re-enlarged the Ito amplitude decreased by ISO-induced hypertrophy. STV alone (Control+STV group) caused no significant effect on the amplitude of Ito density. Fig. 4B showed the current density/voltage relationships for Control, ISO, ISO+STV and Control+STV groups. The mean Ito densities at +50 mV for Control, ISO, ISO + STV and Control+STV were 5.24 ± 1.00 pA/pF (n=6), 2.39 ± 0.46 pA/pF (n=7), 6.99 ± 1.44 pA/pF (n=5) and 4.72 ± 0.72 pA/pF (n=5) groups. Membrane capacitance (Cm) calculated by the automated capacitance compensation procedure of the EPC-10 amplifier was also compared among groups. ISO increased membrane capacitance of myoctyes to 145.49 ± 17.86 pF from 118.64 ± 13.49 pF of control. STV coadministration reduced the membrane capacitance of cardiomyocytes again to 9

ACCEPTED MANUSCRIPT 120.60 ± 9.83 pF.

3.4 Concentration dependence of STV for the recovery effect on Ito density.

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As STV co-administration prevented the decrease of Ito density induced by ISO, we next investigated if the effect of STV on Ito was concentration dependent. The

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concentration of ISO used to introduce cell hypertrophy was 5 μM. Different concentrations of STV from 100 nM to 100 μM were co-administrated with ISO for ISO + STV group. Figure 5 showed that with the concentration increasing, the current

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density of Ito, which was decreased by ISO treatment, was restored gradually. The mean values of Ito density with higher STV concentrations (>5 μM) were even larger

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than that of control group although these differences did not reach a significant level. For STV concentration of 1 μM, 5 μM, 10 μM and 100 μM, the Ito densities were 5.06

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± 0.29 pA/pF, 6.18 ± 1.27 pA/pF, 6.99 ± 1.44 pA/pF and 6.86 ± 1.36 pA/pF, all of

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which were significantly larger than that of ISO group (2.39 ± 0.46 pA/pF). 100 nM

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STV did not significantly increase the Ito density compared with ISO-treated group.

3.5 Effect of ISO and ISO + STV on the kinetic properties of Ito. The kinetic properties of Ito were also examined. Fig 6A and B exhibited the

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activation and inactivation time constant fitted by single exponential functions. Data showed that the activation time constant (τact) was increased significantly by ISO-induced cell hypertrophy, whereas co-administration with STV did not introduce further alteration compared with ISO group (Fig. 6A). The activation time constants (τact) for Control, ISO and ISO+STV were 21.95± 1.90, 43.60 ± 4.92 and 39.48 ± 1.66 ms respectivity. Similarly, the inactivation time constant was increased by ISO treatment, which was not further changed by STV co-administration (Fig. 6B). The inactivation time constants (τinact) were 354.18 ± 38.82, 419.35 ± 18.20 and 434.75 ± 12.85 ms for Control, ISO and ISO+STV groups.

However, ISO did not introduce

any significant changing on half activation or inactivation voltages. The half activation potentials (V1/2act) were 6.08 ± 1.85 and 6.09 ± 2.77 mV for Control and ISO groups. The slopes of steady-state activation curves (kact) for Control and ISO 10

ACCEPTED MANUSCRIPT groups were 10.39 ± 0.65 and 12.72 ± 2.68 respectively. For steady-state inactivation, V1/2inact were -26.83±0.55 and -25.48±1.49 mV for Control and ISO groups. The slopes (kinact) of Control and ISO groups were 5.56 ± 0.41 and 6.20 ± 0.41

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respectively.

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3.6 Effect of ISO and ISO + STV on mRNA expression of Kv4.2, Kv4.3 and KChIP2.

Kv4.2, Kv4.3 and the accessory subunit KChIP2 are the major components of Ito.

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So we investigated if their expressions at transcription level are changed by ISO-induced hypertrophy and STV preincubation. The mRNA level of Kv4.2, Kv4.3

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and KChIP2 were all decreased in ISO-treated hypertrophied cells (Fig. 7). Co-administration with STV (10 μM) attenuated the reduction in mRNA level for

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Kv4.2, Kv4.3 and KChIP2. In Fig. 7A, the mRNA expression levels for Kv4.2 were

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0.00160 ± 0.00012, 0.00034 ± 0.00018 and 0.0014 ± 0.00014 for Control, ISO and ISO+STV groups. In Fig. 7B, the mRNA expression levels for Kv4.3 were 0.00038 ±

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0.00004, 0.00023 ± 0.00003 and 0.00035 ± 0.00003 for Control, ISO and ISO+STV groups. The mRNA expression levels for KChIP2 in Fig. 7C were 0.03183 ± 0.00210, 0.01019 ± 0.00217 and 0.02148 ± 0.00189 for Control, ISO and ISO+STV groups

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respectively.

3.7 Effect of ISO and ISO+STV on L-type Ca2+ current and mRNA expression of Cav1.2. We finally investigated if L-type Ca2+ current was also affected by ISO-induced cell hypertrophy and STV preincubation. Fig. 8A showed the representative current curves at +10 mV of ICaL for Control, ISO and ISO+STV groups.

The current

density of ICaL at +10 mV was prominently enlarged in ISO-treated hypertrophied cells. Co-administration with STV significantly attenuated the enlargement induced by ISO. The current densities of ICaL were -4.95 ± 0.45 pA/pF, 7.29 ± 1.30pA/pF and 5.05 ± 0.98 pA/pF for Control, ISO and ISO + STV groups respectively. The mRNA expression level of Cav1.2 was also examined by qRT-PCR. Fig. 9 11

ACCEPTED MANUSCRIPT showed that the mRNA level of Cav1.2 was increased significantly in ISO group and STV co-administration attenuated the increasement caused by ISO-induced cell hypertrophy, which was consistent with the result of ICaL. The mRNA levels were

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0.00596 ± 0.00057, 0.01017 ± 0.00168 and 0.00640 ± 0.00042 for Control, ISO and

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ISO + STV groups respectively.

Discussion:

Ventricular hypertrophy is major risk factor for sudden cardiac death. The

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transient outward potassium current (Ito) has been identified to be decreased in hypertrophied myocardium, which is a main cause of electrophysiological remodeling

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and arrhythmogeneity through altering the shape and duration of action potential. This study mainly investigated the alterations of cell electrophysiology and mRNA

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transcription of some relative genes in ISO-induced hypertrophic cardiomyocytes and

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how these changes were affected by STV preincubation. Data showed that STV inhibited the increase of cell surface area and transcription level of hypertrophy

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indicators (ANP and BNP) in ISO-induced hypertrophic cardiomyocytes significantly. At electrophysiology level, STV prevented the prolongation of action potential in hypertrophic cells. STV modulated Ito and ICaL currents by regulating the

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corresponding channel mRNA transcription levels. These data indicate that STV could be used as a potential drug in treatment of cardiovascular diseases considering its ability to attenuate hypertrophy and modulate the electrophysiologic properties of cardiomyocytes. Accumulated experimental data suggest that the life-threatening ventricular arrhythmia in cardiac hypertrophy is attributed to abnormal remodeling of the Kv4-family of transient potassium channels [11-13]. However, the specific molecular mechanism of how the transient potassium channels are regulated during cardiac hypertrophy remains unknown. Ito density is identified to be modulated physiologically by adrenergic receptor activation [26]. The effects of myocardial α-adrenergic and β-adrenergic receptor stimulation are mediated through the activation of intracellular second messenger cascades, which then lead to activation of 12

ACCEPTED MANUSCRIPT protein kinases and the downstream effectors including the transient outward potassium channel through phosphorylation and dephosphorylation processes [27]. Multiple consensus phosphorylation sites for PKA and PKC have been identified in

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Kv4 α subunits [28].

which

plays

a

critical

role

in

regulating

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(LTCC),

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In addition to the transient outward potassium channel, the L-type Ca2+ channel Ca2+

dependent

excitation-contraction coupling, is also associated with hypertrophic signaling. An increase in LTCC activity has been observed in various models of cardiac hypertrophy

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[29]. The changing activity of LTCC could be attributed to the regulation of phosphorylation/dephosphorylation state of the channel α subunit and/or the protein

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expression on cell membrane [30, 31]. Li et al. in their study identified that myocardin and NFATc4 synergistically up-regulated the expression of LTCC α subunit in

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endothelin-1-induced cardiomyocyte hypertrophy [32]. Han et al. found that Trpc3

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mediates pathologic cardiac hypertrophy elevation via direct regulation of CaV1.2 expression [33]. Another study of Chen et al. showed that the Chinese drug Wenxin

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Keli could shorten the prolonged AP duration in hypertrophied myocytes by regulating the gating properties of L-type calcium channel [34]. All these study indicated LTCC as a regulating target for drugs or endogenous signaling molecules to

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exert their role in hypertrophic signaling. In this study, we found that ICaL was increased in ISO-induced cardiomyocytes and that STV could attenuate hypertrophy by reducing the channel activity, which is consistent with the above investigations. ISO, as a β-AR agonist, has been widely used to induce cardiac hypertrophy both in vivo and in vitro [35, 36]. We used ISO as a hypertrophic inducer to examine the effect of STV on hypertrophied cardiomyoctyes. We found that STV attenuated ISO-induced cardiomyocytes hypertrophy by regulating activity of Ito and ICaL. The electrophysiological modulation of Ito and ICaL by STV could be attributed to changes at the mRNA expression level of their corresponding genes since the kinetic properties of activation and inactivation were not changed by STV application. Under physiological condition, we showed that STV had no significant effect on either action potential duration or Ito currents. These may indicate no direct role of STV in 13

ACCEPTED MANUSCRIPT cardiomyocytes without ISO stimulation. Although we speculate that STV regulate Ito and ICaL by changing the relative mRNA expression level, STV could also regulate channel activity by a modulation at protein level through direct interaction or some

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other signal transduction pathways.

Besides adrenergic receptor signaling pathway, several other signaling factors

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have been found to be associated with the progression of myocardial hypertrophy and be chosen as regulating target for the anti-hypertrophic effect of drugs. For example, the mitogen-activated protein kinase (MAPK) signaling pathway has been identified

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to play an important role in the anti-hypertrophic effect of Danhong injection [36]. Aalpha-difluoromethylornithine (DFMO) can attenuate cardiomyocyte hypertrophy

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by inhibiting the NO/cGMP-dependent protein kinase-1 pathway [37]. Furthermore, oxidative stress is also a major contributor to pathophysiology of cardiomyocytes.

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Studies have shown that increased reactive oxygen species (ROS) in cardiomyocytes

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were involved in the development of cardiac hypertrophy and heart failure [38-41]. Activation of KATP channel has also been identified to attenuate the degree of

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myocardial hypertrophy by modulating the expression level and spatial distribution of Cx43 [42]. Our previous study investigated the effect of STV on sarc- and mitoKATP channels and showed that STV could sensitize these KATP channels in a ROS

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dependent manner [25]. We speculate that the ROS-mediated regulation of KATP channel could also be a potential mechanism by which STV exert the anti-cardiac hypertrophic effect. In conclusion, this study evaluated the effect of STV on ISO-induced hypertrophic cardiomyocytes by examining the action potential duration and comparing the current density and the mRNA expression level of transient outward potassium and L-type calcium channels. The results showed that STV prevented the prolongation of action potential duration in hypertrophic cardiomyocytes by regulating Ito and L-type calcium channel currents. However the specific regulating mechanism and signaling pathway remains unknown.

ACKNOWLEDGEMENTS 14

ACCEPTED MANUSCRIPT This work was supported by the Fundamental Research Funds for the Central Universities (No. 2015ZM165) and grant from the National Natural Science

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Foundation of China (Grant No. 31300940).

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DISCLOSURE STATEMENT

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None.

A.W. Haider, M.G. Larson, E.J. Benjamin, D. Levy, Increased left ventricular mass and

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1.

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Fig 1. Comparison of cell area among Control, ISO, ISO+STV and Control+STV groups. Images of myocytes are acquired by Nikon inverted microscope. 100 cells of randomly selected for each

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group were analyzed and cell areas were calculated by image pro plus 6.0. ** means p < 0.01.

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Fig 2. mRNA expression of ANP and BNP for Control, ISO and ISO + STV groups. The mRNA expression levels of ANP and BNP were assessed by qRT-PCR method and were both normalized to GAPDH. The RT-PCR experiments were repeated for 3 times for each group. * means p < 0.05

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and ** means p < 0.01. (NS) means no significant difference.

Fig 3. Effect of ISO and ISO+STV on action potential duration of cardiomyocytes. (A)

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Representative AP traces of Control, ISO, ISO+STV and Control+STV groups. (B) Effect of ISO and ISO+STV on action potential duration at 20% ( APD20), 50% (APD50) and 90% (APD90) of

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repolarization . * means p < 0.05. (NS) means no significant difference.

Fig 4. Effect of STV on Ito of ISO-induced hypertrophic and normal cardiomyocytes. (A) Representative Ito traces at +50 mV depolarizing potential for Control, ISO, ISO+STV and Control+STV groups. (B) Current-voltage relationships of Ito for Control, ISO, ISO+STV and Control+STV groups. Currents were normalized to cell capacitance.

Fig 5. Concentration dependence for the recovery effect of STV on Ito current density. ISO was pretreated 24 hours before recording for ISO group. STV with the concentrations from 100 nM to 100 μM were added at the same time with ISO. ** means p < 0.01, (NS) means no significant difference.

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recorded at +50 mV. ** means p < 0.01, (NS) means no significant difference.

Fig 7. mRNA expression of Kv4.2, Kv4.3 and KChIP2 for Control, ISO and ISO+STV groups.

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The mRNA expression levels for each gene were assessed by qRT-PCR method and all were normalized to GAPDH. The RT-PCR experiments were repeated for 3 times for each group. *

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means p < 0.05 and ** means p < 0.01.

Fig 8. Effect of ISO and ISO+STV preincubation on L-type Ca2+ current of cardiomyocytes. (A)

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Representative ICaL traces at +10 mV potential for Control, ISO and ISO+STV groups. (B) Current-voltage relationships of ICaL for Control, ISO and ISO+STV groups. Currents were

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Fig 9. mRNA expression of Cav1.2 for Control, ISO and ISO+STV groups. The mRNA

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expression level was assessed by qRT-PCR method and all normalized to GAPDH. The RT-PCR

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experiments were repeated for 3 times for each group. * means p < 0.05.

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Graphical abstract

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ACCEPTED MANUSCRIPT Highlights 1. STV alleviated cell hypertrophy by decreasing cell area of hypertrophied cardiomyocytes.

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2. STV prevented the prolongation of action potential in hypertrophied cardiomyocytes.

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3. STV prevented the decrease of current densities of Ito in hypertrophied cardiomyocytes.

4. STV prevented the increase of current densities of ICaL in hypertrophied

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cardiomyocytes.

5. The effect of STV on mRNA transcription of related genes showed consistent

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results.

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