Shexiang Baoxin Pills Could Alleviate Isoproterenol-Induced Heart Failure Probably through its Inhibition of CaV1.2 Calcium Channel Currents

Heart failure (HF) affects millions of patients in the world. Shexiang Baoxin Pills (SXB) are extensively applied to treat coronary artery diseases and HF in Chinese hospitals. However, there are still no explanations for why SXB protects against HF. To assess the protective role, we created the HF model in rats by isoproterenol (ISO) subcutaneous injection, 85 milligrams per kilogram body weight for seven days. Four groups were implemented: CON (control), ISO (HF disease group), CAP (captopril, positive drug treatment), and SXB groups. Echocardiography was used to evaluate rats' HF in vivo. The human CaV1.2 (hCaV1.2) channel currents were detected in tsA-201 cells by patch clamp technique. Five different concentrations of SXB (5, 10, 30, 50, and 100 mg/L) were chosen in this study. The results showed that SXB increased cardiac systolic function and inhibited rats' cardiac hypertrophy and myocardial fibrosis induced by ISO. Subsequently, it was found that SXB was inhibited by the peak amplitudes of hCaV1.2 channel current (P < 0.01). The SXB half inhibitory dosage was 9.09 mg/L. The steady-state activation curve was 22.8 mV depolarization shifted; while the inactivation curve and the recovery from inactivation were not affected significantly. In conclusion, these results indicated that SXB inhibited ISO-induced HF in rats and inhibited the hCaV1.2 channel current. The present study paved the way for SXB to protect itself from HF.


Introduction
Heart failure (HF) causes hazardous illness globally. Tere were nearly 6.0 million Americans >20 years of age sufered HF from 2015 to 2018. [1] With the progress of society and the development of medical science and technology, more and more measurements were implemented to treat HF, for example, drug therapies including salkubatroxartan, hydrogen nitrate, β-blockers, angiotensin-converting enzyme inhibitors, and nondrug therapies, such as left ventricular assist device implantation, pacemaker implantation, and even heart transplantation. However, there was still extensive HF occur [2,3]. During the 20 years from 1995 to 2015, although there was a signifcant decrease in ischemic heart disease, HF increased from 15.5 to 16.3 people per 10,000 general population in the US [1]. Terefore, novel alternative medical treatments could beneft patients with HF.
Although SXB is efective clinically, the mechanisms of SXB treating HF were unknown yet. To clarify the roles, the model of ISO-induced HF in rats was constructed and the changes in CaV1.2 currents were detected by the patch clamp method in vitro.  -23, revised 1996). Male SD rats were ordered from the Experimental Animal Center of Guangdong Province. Each rat weighed 200-300 gram. SXB was ordered from Shanghai Hutchison Pharmaceuticals Company (batch NO: 190601, Shanghai, China). SXB was prepared in accordance with the description provided in Chinese Pharmacopeia 2020 [15].

HF Model in Rats.
We followed the methods of Qi et al. 2020 [16,17]. Briefy, isoproterenol (ISO) was subcutaneously administered in rats for 7 days with a dosage of 85 mg/ kg/day to construct an HF model. Four groups were assigned as followed: CON (control, n � 13), ISO (HF model, n � 13), CAP (captopril, n � 7), and SXB (n � 10). Saline was subcutaneously administered to the CON group; the ISO group was subcutaneously administered with ISO for 7 days. Captopril, which served as a positive control to treat HF, was subcutaneously administered at 20 mg/kg/day for 7 days after ISO stimulation in CAP rats. According to publications, our preliminary research, and conversion of the clinical humans' oral usage to rats [17], SXB powder was mixed with one-milliliter saline, at a dosage of 2.5 gram per kilogram body weight of rats, was gastric gavage daily after seven days of ISO stimulation.

Echocardiography.
Te linear array transducer (17.5 MHz) was inserted into an echocardiographic machine (Vevo 770, Visual Sonics, Toronto, Canada). Left ventricular (LV) structure and function were determined by detecting LV diastolic posterior wall thickness (LVPWd) and fraction shortening (FS). After anaesthetized by tribromoethanol intraperitoneal, rats were laid on a thermostatic plate. Firstly, at the papillary muscle level, the LV short-axis two-dimensional (2D) images were obtained, then 2D long-axis images and M-mode images were collected, and subsequently, LV end-diastolic or systolic inner dimension (LVIDd and LVIDs), LV end-diastolic anterior wall thickness (LVAWd), and LVPWd were detected. Other values could be obtained automatically by the echocardiography system, such as LV eject fraction (EF), end-diastolic/systolic volume (LVVd/s), and FS.

HW Detection and
Immunohistochemistry. Rats were sacrifced, and the body weight (BW), heart weight (HW), and tibial length (TL) of rats were determined. Ten, the whole hearts were arranged with parafn section staining. Pathological sections were cut into 5 μm slices. Myocyte hypertrophy was analyzed by staining with hematoxylineosin (HE). Cardiac fbrosis was evaluated by staining with Masson and Sirius red. Te sizes of cardiomyocytes from each rat were detected with light microscopy at ×400 magnifcation. Myocardial fbrosis was quantitatively measured by a diferent color in Masson and Sirius red at ×40 magnifcations. Te data of hypertrophied myocytes and cardiac fbrosis were obtained with Image-Pro Plus software.

Cellular Electrophysiological
Recording. PC-100 horizontal microelectrode puller (NARISHIGE Co., Japan) was used to pull the borosilicate glass electrode into the patch pipette. When flled with intracellular fuid, the tip resistance of the pipette varied between 3 and 5 MΩ. An EPC-10 amplifer, combined with Pulse software (HEKA, Lambrecht, Germany), was used to record the membrane currents in a voltage-clamp mode. Firstly, to ofset the liquid junction potentials between bath and pipette solutions. Next, to form a gigaohm seal, gentle suction, rupture the cell membrane, and stand quietly for 3 minutes to form a stable steady-state whole-cell confguration. Whole-cell capacitive currents were compensated, leak subtracted, and series resistance was compensated to 60-80%. 10 kHz and 2.9 kHz were used to sample and flter the current signal respectively (8-pole Bessel flter, 3 dB). A laptop was used to record and store the current signal. Room temperature was kept at 22-24°C for all the experiments implemented.

Drugs and Solutions.
After a gentle vortex, SXB was dissolved and fltered in sterile distilled water, with the concentration of the stock solution at 10 mg/mL. Subsequently, the targeted bath solution was obtained by diluting the stock solution with an extracellular solution. As for detecting the hCav1.2 channel currents, the pipette solution contained fve reagents as followed (in mM) 42 HEPES, 4 Mg-ATP, 120 NMDG-Cl, 5 EGTA, and 1 MgCl 2 (the pH value was adjusted to 7.3 by methane sulfonic acid). Te bath solution contained three reagents as followed (in mM) 105 Tris, 1 MgCl 2 , and 40 BaCl 2 (the pH value was adjusted to 7.3 by methane sulfonic acid) [19]. Te bath solution was changed and the tsA-201 cells were continually perfused with a constant fow rate of one to two milliliters per minute [20]. Signal currents were recorded after the whole-cell confguration formed at least three minutes so as to make the pipette solution undergo complete dialysis.

Analyses of the hCaV1.2 Channel Currents and Statics.
Origin 8.0 software (Origin Lab Corp., Northampton, MA, USA) and Patch Master (HEKA Electronics, Lambrecht/Pfalz, Germany) were used to collect and analyze the hCaV1.2 channel current data. Te Boltzmann equation was used to ft the activation curves as follows: where, V t is the test potential, V a is the half-activation potential of the hCaV1.2 channel conductance, and k a is the slope factor in the hCaV1.2 activation stage. Te curves of the steady-state inactivation with and without SXB perfusion were ftted with the equation as follows: where, I max is the maximum current from the absence of inactivation, and I c is a noninactivating current. V i , V t , and K i are the half-inactivated potential, the halfinactivated slope factor, and the test potential, respectively. For statistical analysis, data were collected as means ± standard error of the means (SEM). An unpaired t-test was used to compare two groups, while multiple groups (three or more than three groups) were compared by oneway analysis of variance plus the Bonferroni test. Signifcant diferences were accepted when P values less than 0.05.

SXB Inhibited HF After ISO Stimulation in Vivo.
To further ensure the role of SXB efects on HF, microscopic anatomic analyses were performed on HE, Sirius red, and Masson-stained thin sections of hearts. Moreover, the heart, lung, and liver after the rats were sacrifced, were weighed and statistically compared to the CON, ISO, CAP, and SXB groups. Figure 2(a) shows that the cross-section area (CSA) of the ISO group was more augmented than CON rats ( Figure 2(e), ISO, 198.60 ± 1.46 μm 2 vs. CON, 109.30 ± 0.78 μm 2 , P < 0.001), while it was reduced in CAP and SXB rats (CAP, 161.50 ± 0.76 μm 2 vs. SXB, 137.80 ± 0.57 μm 2 , respectively). Figures 2(b) and 2(c) are the long-axis and short-axis of Sirius-stained hearts sections of the 4 groups respectively (cardiac fbrosis was indicated by the arrow). Figure 2(d) is the typical Masson-stained heart section of the CON, ISO, CAP, and SXB groups. As shown in Figure 2(f ), the area of myocardial fbrosis in the ISO group are more increased than in the CON rats' (12.12 ± 0.42% vs. 0.58 ± 0.29%, P < 0.001), while it is decreased in CAP and SXB rats (8.54 ± 0.42% vs. 5.41 ± 0.54%, respectively). Together, the study showed that SXB reversed pathological eccentric hypertrophy and myocardial fbrosis in rats with heart failure.

SXB Inhibited the CaV1.2 Calcium Channel Current in a Dose-Denpendent Manner.
To explore the unveiled nature of SXB's prevention from HF, the L-type calcium channel, the vital molecule in the calcium-induced calcium release (CICR) process, which participated in HF, were detected [21]. By whole cell patch clamp recording, we detected the hCaV1.2 channel currents, which is the main α-subunit of the L-type calcium channel. Te voltage protocol is listed in Figure 4(a), which holding potential kept at −60 mV, depolarized to +20 mV for 500-ms. Figure 4(b) shows the representative plots of the hCaV1.2 current curves with the fve diferent SXB concentrations (5 mg/L, 10 mg/L, 30 mg/L, 50 mg/L, and 100 mg/L). As shown in Figure 4(c), the four SXB groups reduced the hCaV1.2 peak amplitude signifcantly, compared with the 5 mg/L SXB group (P < 0.001). Figure 4(d) shows the dose-response curve of SXB affected by the hCaV1.2 current, with the half inhibitory concentration (IC 50 ) as 9.09 ± 0.33 mg/L.

SXB Efects on the Gating Kinetics of the hCaV1.2 Channel
Current. To further reveal the electrophysiological properties of SXB on the hCaV1.2 channel current, we analyzed the activation and inactivation curves. Figure 5(a) shows the voltage protocol. Te holding potential was −60 mV, the depolarizing pulses lasted for 400-ms, enhanced from −60 mV to +60 mV, with 10 mV increments per pulse, followed with a 2 ms interval, kept at −60 mV, then the test pulse was set to +10 mV for 100 ms, the total protocol was evoked every 15 seconds. [22,23] Figures 5(c) and 5(d) are the typical hCaV1.2 currents curves before and after the SXB perfusion. Figure 5(b) shows that the peak current of the hCaV1.2 channel was signifcantly reduced in the 30 mg/L SXB group, compared with the control (Ipeak, Control, −199.82 ± 30.39 pA, SXJ, −62.36 ± 10.67 pA, P < 0.01) in tsA-201 cells. Te activation gating kinetics were compared and shown in Figure 6(a), a clear inhibition of hCaV1.2 currents was observed with 30 mg/L SXB. Te characteristics of the I-V relationship showed that 30 mg/L SXB reduced the peak values of hCaV1.2 currents, which were reduced to 31.21% of the control, SXB changed the voltage of peak activation of the hCaV1.2 currents (the normalized I-V curves, Figure 6(b)). Te activation curve of the current was shifted by approximately 23 mV toward the depolarized potential during cell exposure to SXB; however, no apparent change in the slope factor (i.e., Ka) was demonstrated in its presence ( Figure 6(c)), which might be due to no altering the hCaV1.2 activation gating kinetics.
Te inactivation curves were obtained with the above protocol ( Figure 6(a)). Te curves were obtained before and after 30 mg/L SXB perfusion (Figure 6(d)). Te smooth curve was ftted to the Boltzmann equation (Methods. 2.8).
No signifcant changes were shown between CON and SXB treating groups (P > 0.05). Together, the study showed that SXB did not infuence the inactivated channels signifcantly in the present study.

SXB Efects on the Recovery from Inactivation (RFI) of the hCav1.2 Channel.
Te RFI of hCav1.2 was detected with a double-pulse RFI protocol before and after SXB perfusion (Figure 7(a)). A 1s-pulse rose to +10 mV and then, decreased to a holding potential (−60 mV), followed by time intervals, changing from 50 ms to 10 s. After that, the potential again rose to +10 mV for 50 ms. Figures 7(c) and 7(d) were the typical RFI curves with or without SXB perfusion, which did not show signifcant diferences between both the groups (Figure 7(b)). Tus, SXB did not regulate the extent of calcium channel recovery, regardless of the duration of pulse stimulation.

Discussion
Te present study frst explored the cardio-protective efects of SXB against ISO-induced HF in rats. Te results demonstrate that SXB markedly reversed eccentric cardiac hypertrophy, decreased cardiac fbrosis, and increased cardiac systolic function in ISO-induced HF rats. Furthermore, SXB might regulate the hCaV1.2 currents.
Tere are various methods to construct animal HF models, such as transverse aortic constriction (TAC), spontaneously hypertensive rats, and ISO stimulation [24]. Constructing an HF model by using spontaneously hypertensive rats is time-consuming. Although it is commonly used to induce eccentric cardiac hypertrophy by the TAC method due to a high-pressure overload [25], it activates multiple signaling pathways, complicating mechanistic studies In contrast, ISO stimulation-induced HF is more maneuverable and can provide a more distinct mechanism. In our study, ISO-induced pathological myocardial hypertrophy is feasible. Although this method is not in common use, our studies revealed that ISO stimulation can be a convenient and stable method to construct a rat's model of decompensate cardiac hypertrophy.
We demonstrated that administration of ISO for 7 days induced heart failure, which was evaluated by an increase in LVVs in echocardiography (Figure 1(g)) as well as lung edema in anatomic data (Figures 2(d)-2(f )), SXB could reverse pathological eccentric cardiac hypertrophy and alleviate heart failure.
Te HF mechanisms are complex and far more to elucidate clearly due to thousands of molecular pathways, such as a β-adrenergic receptor, L-type calcium channel, protein kinases (PK) A, PKC, Ca 2+ /calmodulin-dependent kinase II, calcineurin, and phosphodiesterase [26]. β-adrenergic  stimulation can lead to HF, while the β-adrenergic antagonists can signifcantly alleviate heart failure [27,28]. Te mechanism underlying the altered CaV1.2 kinetics in HF is multilevel and multifactorial. Te CaV1.2 channel is infuenced by lots of molecules, activation of β-adrenergic receptors increases CaV1.2 current [29], increased sarcoplasmic reticulum [Ca 2+ ], and augmented cardiac contractility. Tere are a lot of diseases occurring due to dysfunction of the CaV1.2 current, such as HF, cardiac hypertrophy, atrial fbrillation, and ischemic heart disease [30,31]. It is vital for the L-type calcium channel current to activate the CICR in response to membrane depolarization in the heart. At least three subunits are essential to form the cardiac CaV1.2 channel: the pore-forming subunit (α1c) and two accessory subunits (β2a and α2/δ). Ca 2+ infux was increased in transgenic mice overexpression of CaV1.2 α1C, which resulted in blunting of β-adrenergic modulation, cardiac hypertrophy, and HF [32][33][34]. Overexpressing the β2a subunit of CaV1.2 current, also augmented murine cardiac CaV1.2 activity, pathological hypertrophy, and increased death [35]. Terefore, over CaV1.2 current is harmful and can promote heart failure. Inhibiting the over CaV1.2 current is a strategy to treat HF. In our study, it was revealed that SXB could block the CaV1.2 channel current so as to alleviate HF. Since SXB is made up of multiple compositions of herbs, such as artifcial bezoar, artifcial musk, borneol, cinnamon, ginseng extract, styrax, and venenum bufonis, it is difcult to elucidate the precise mechanism of each component of SXB. Furthermore, whether SXB infuences other potassium or sodium channels, is still a mystery. Future works might be focused on the efects of SXB on other ion channels if it is possible.
In general, our study showed, for the frst time, that SXB preferentially binds to the pore gate domain of hCaV1.2 so as to block the hCaV1.2 channels current, reversing the eccentric pathological hypertrophy, reducing myocardial fbrosis, and augmenting the cardiac function so as to treat HF. Terefore, the present mechanistic study paved the way for SXB protection from HF in clinics.