Bradycardia is a condition in which pulse rate is below 60 beats/min. Coronary artery disease patients and elderly people are at a great risk of developing the abnormally slow heart rate. Currently available drugs (e.g., atropine, dopamine, isoproterenol, and epinephrine) treating bradycardia are temporizing measures only in emergency settings. If the patient does not respond to drugs, temporary or permanent cardiac pacemaker is probably indicated. However, the cost of pacing put a huge financial burden on the family. Consequently, an effective drug aiming at increasing heart rate for a long-term is in urgent demand.
The Traditional Chinese Medicines have been used to treat arrhythmia for hundreds of years and Shenxianshengmai (SXSM) is one of such medicines. It is a product consisting of eight ingredients including Radix Ginseng Rubra, Herba Epimedii Brevicornus, Fructus Psoraleae, Fructus Lycii, Herba Ephedrae Sinicae, Asarum Heterotropoides, Radix Salviae Miltiorrhizae, and Hirudo. Clinical researches demonstrated that SXSM is effective in treating bradycardia. However, it has been a mystery how SXSM plays the positive role in treatment. Therefore, we use a bradycardiac animal model to explore the molecular mechanisms of SXSM treatment. Our work will provide new insights into the mechanisms of SXSM.
Twenty-four adult rabbits (Oryctolagus cuniculus) with mean body weight of 2.5 ± 0.5 kg were used for the study. The experimental protocol was performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985) and the ARRIVE. The Care of Experimental Animals Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College approved the procedures for the care and treatment of animals. The animals were randomly divided into four groups (n = 6 in each group): sham, model, sham plus SXSM (S+SXSM), and model plus SXSM (M+SXSM) groups. Sterilized cotton bud with formaldehyde (37%, SCRC, China) was fixed on the wall of the right atrium, near the entrance of the superior vena cava until heart beat decreased 25–35%. The procedures were the same as the previous study. Then, purified water was administered orally to model group while SXSM (275 mg·kg−1·d−1) was administered to blank plus SXSM and M+SXSM groups. Lead II was used to monitor the electrocardiogram, once every week for 4 weeks. At last, animals were sacrificed 6 weeks later. The procedures in sham and S+SXSM groups were similar to model and M+SXSM groups except that formaldehyde was replaced by purified water. RR, P, PR, QRS, QT, and QTc were calculated before operation (baseline) and 6 weeks’ later, respectively.
RNA and protein preparation
Hearts of the animals were isolated and perfused with purified water. Atria and ventricle of the heart were immediately frozen in liquid nitrogen and then stored at −80°C until use for RNA extraction. MirVana™ mRNA isolation kit (Ambion-1561, USA) was used in accordance with the manufacturer's instructions to isolate total RNA. Then, NanoDrop ND-2000 (Thermo Scientific, USA) and Agilent Bioanalyzer 2100 (Agilent Technologies, USA) were used to quantify RNA and assess the RNA integrity, respectively. To minimize variations attributable to individual rabbit and maximize differences attributable to their genotype, each experiment was performed with RNA pooled from 3 atria. Ventricular tissues were homogenized in 5 volumes (v/w) of isolation buffer (300 mmol/L sucrose, 10 mmol/L Hepes/Na, 500 μmol/L ethylenediaminetetraacetic acid (EDTA)•2Na, pH 7.4, 2 mmol/L phenylmethyl sulfonyl fluoride (PMSF) and 1:1000 diluted Protease Inhibitor Cocktail (Sigma P8340, USA) using a Dounce Glass/Teflon Homogenizer according to the method of Frezza et al. Centrifugation was carried out twice, 800 ×g, 4°C for 10 min.
Gene expression profiling
Total RNA was transcribed to double-strand complementary DNA (cDNA), then synthesized into cRNAs and labeled with Cy3. Labeled cRNAs were hybridized onto Agilent Rabbit Gene Expression Chip (4*4K, Design ID: 020908, containing 43,803 probes) according to manufacturer's instructions and scanned by Agilent Scanner G2505C (Agilent Technologies). Feature Extraction software (version 10.7.1.1, Agilent Technologies) was used to analyze array images to get raw data. Data normalization was performed using GeneSpring. Differentially expressed genes were selected based on fold-change >2.0 and P < 0.05 according to two-way analysis of variance (ANOVA). Gene ontology (GO) database and KEGG were applied to determine functions of these differentially expressed messenger RNAs (mRNAs). Then, differentially expressed genes were researched by Funnet and Uniprot database (http://www.uniprot.org).
Quantitative polymerase chain reaction
SYBR Green quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed on the genes ATP2A1, ERP27, FKBP1B, MBIP, and 18S rRNA (as an internal control) to confirm the results of gene expression chip. Genes were selected from interesting functional groups revealed by GO analysis [Table 1]. Primers were designed with LightCycler Probe Design software 2.0 (Roche Applied Bioscience, Swiss). The cDNA was synthesized at 37°C for 15 min in a 10 μl reaction containing 0.5 μg total RNA, 2 μl PrimerScript Buffer, 0.5 μl oligo dT, 0.5 μl random 6mers and 0.5 μl PrimerScript RT Enzyme Mix I (TaKaRa, Japan). Real-time RT-PCR reactions included 1 μl of cDNA, 5 μl 2 × LightCycler® 480 SYBR Green I master mix (Roche), 0.2 μl forward primer, 0.2 μl reverse primer, and 3.6 μl water of nuclease-free. All PCR reactions were carried out in triplicate with the following conditions: 95°C for 10 min, followed by 40 cycles of 10 s at 95°C, 30 s at 60°C in the LightCycler® 480 II Real-time PCR Instrument (Roche). For each selected gene, melting curve analysis was performed to validate the specific generation of the expected PCR product. The expression of each gene was normalized as ΔCt (Ct of target gene – Ct of internal control gene) using 18S rRNA as the control. Relative quantification using the ΔΔCt method was applied to compare the amounts of mRNA in sham versus model groups and model versus SXSM groups.
Protein preparation and isobaric tags for elative and absolute quantitation labeling
Protein preparation from rabbits was performed following the published method with some modifications. Briefly, ventricles from three rabbits were pooled and homogenized in 5 volumes (v/w) of isolation buffer (0.3 mol/L sucrose, 10 mmol/L Hepes-Na, pH 7.0, 0.5 mmol/L EDTA, 2 mmol/L PMSF, and 1:1000 diluted Protease Inhibitor Cocktail [Sigma P8340]) using a Dounce Glass/Teflon Homogenizer. Centrifugations were carried out twice to discard nuclear and cell debris, 800 ×g, 4°C for 10 min. The supernatant was then collected and stored at −80°C.
To minimize variations attributable to individual rabbit and maximize differences attributable to their genotype, each experiment was performed with RNA pooled from three ventricles. Equal amounts of protein (75 μg) from each pooled sample were digested with trypsin (0.5 μg/μl) at 37°C for 16h and labeled with unique isobaric tags for elative and absolute quantitation (iTRAQ) reagent (114 for model group, 115 for SXSM group). Labeled samples were pooled and dried in a vacuum centrifuge.
Isobaric tags for elative and absolute quantitation proteomic analysis
The labeled dried peptides were dissolved in mobile phases A (2% acetonitrile [ACN], pH 10.0). Then, samples were loaded on the reversed phase column (Agela, 5 μm, 150 Ε, 4.6 mm × 250 mm,) and separated on an L-3000 HPLC system (Rigol, China) at a flow rate of 1 ml/min. Mobile phase A consisted of 2% ACN and mobile phase B consisted of 98% ACN. Both of them were adjusted pH to 10.0 using NH3•H2O. The gradient used was described as following: 5–8% B, 2 min; 8–18% B, 11 min; 18–32% B, 9 min; 32–95% B, 1 min; 95% B, 1 min; 95–5% B, 2 min. The temperature of Column Oven was set as 60°C. Fractions were collected every minute and then dried in a vacuum centrifuge. Forty fractions were collected and desalted. Then, fractions were combined into twelve fractions and vacuum-dried until analyzed by LC/MS/MS.
After dissolved in 0.2% fatty acid and 5% methanol, the dried tryptic peptides loaded and trapped on a precolumn (C18, 100 μm × 20 mm, 5 μm particle size), then separated on an analytical column (C18, 75 μm × 150 mm, 3 μm particle size). Peptides were eluted from the C18 analytical column with 40-min gradient at a flow rate of 350 nL/min on Eksigent Ultra HPLC (AB Sciex). The MS conditions for TripleTOF 5600 were set as the followings: the spray voltage was set of 2.5 kv and the temperature of heater was 150°C. The MS scan range was set at 350 to 1250 m/z and the MS/MS scan range was 100–1500 m/z. Data-dependent acquisition was performed and top 50 precursor ions were selected to fragment using collision induced dissociation (CID). The collision-induced dissociation energy was automatically adjusted by the rolling CID function.
Database search and bioinformatics
The resulting MS/MS data were then compared against data in the NCBI database (Rabbit.protein-20150201) using ProteinPilot™ Software Beta (version 4.5, AB, USA). For protein identification and quantification, peptide mass tolerance and fragment tolerance were each set at 0.3 Da. Only one missed tryptic cleavage was allowed. The false positive rates were controlled below 1%. The following criteria were used to select differentially expressed proteins: (1) proteins including at least one unique high-scoring peptide; (2) P < 0.05; and (3) fold-changes needed to be >2 or <0.5. The UniProt knowledge base (Swiss-Prot/TrEMBL, http://www.uniprot.org/) and GO database were applied to further classify these differentially expressed proteins.
Samples were prepared in SDS sample buffer, separated on 10% SDS gel, and transferred to 0.45 μm polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies for ATPB, and NDUFS1, and with AP-conjugated secondary antibodies. Proteins were detected by BCIP/NBT method following the instruction for the Western blot kit.
Electrocardiogram data were expressed as mean ± standard error (SE). Two-way ANOVA was used to test difference of basic parameters between groups. Independent sample t-test was used to estimate difference between groups, with P < 0.05 considered statistically significant. Analyses were performed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA) software.
Effect of long-term Shenxianshengmai treatment on slow heart rate
Representative electrocardiography recordings of sham, model, and SXSM-treated rabbits are illustrated and analyzed in Figure 1 and Table 2, respectively. No difference was observed among baselines of all groups (t = 1.459, P > 0.05). As is evident, chemical injury of sinoatrial node decreased the mean heart rate by 32% (RR interval from 253 ± 10 ms in sham group to 406 ± 35 ms in model group, t = 10.296, P < 0.05, n = 6, respectively) after six weeks. This effect was partially reversed by 4-week SXSM treatment (275 mg·kg−1·d−1, RR interval from 406 ± 35 ms in model group to 251 ± 3 ms in M+SXSM group, t = 10.491, P < 0.05). In addition, SXSM also increased heart rate of sham rabbits (RR interval from 406 ± 35 ms in model group to 186 ± 8 ms in S+SXSM group, P < 0.05). Except for RR interval, SXSM had no significant effect on atrial, atrioventricular, and ventricular conduction parameters, since the P, PR, QRS, and QT interval were not modified [Table 2].
Effects of long-term Shenxianshengmai treatment on cardiac transcripts
To explore the gene expression changes induced by chemical lesions of SA node, we compared sham and model group and identified 102 altered genes, among which 72 genes were downregulated and 30 were upregulated [Supplementary Table 1]. To follow the changes induced by SXSM treatment, we compared model and M+SXSM rabbits and found 109 differentially expressed genes [60 downregulated and 49 upregulated, Supplementary Table 2]. Among these altered genes, a total of 11 genes (ATP2A1, ERP27, FKBP1B, MBIP, PPIC, PRKCZ, VIPR1, PRLR, NIM1K, LOC100355813, and MMP1) were appeared in both model and SXSM originated differentially expressed genes [Supplementary Figure 1]. Moreover, the expressions of nine of them (ATP2A1, ERP27, FKBP1B, MBIP, PPIC, PRKCZ, VIPR1, PRLR, and NIM1K) were restored by SXSM in model rabbits [Table 1].
Altered genes: A total of 11 genes were appeared in both model and SXSM originated differentially expressed genes.
Our results revealed that SXSM increased heart rate by inhibiting heart parasympathetic transmission based on the decreased CHRNA2 (encodes nicotinic acetylcholine receptor) and increased ACE-1 (encodes acetylcholinesterase) [Table 1]. They all indicate that parasympathetic synaptic transmission in heart was inhibited by SXSM. Therefore, sympathetic nerve was relatively stimulated and the heart rate increased.
In addition, restored calcium handling also plays an essential role in the increased heartbeat. Both ATP2A1 (encodes calcium ATPase, SERCA2a) and FKBP1B (encodes FKBP12.6 protein, an inhibitor of calcium release channel [RyR2]) were downregulated in model group and upregulated in M+SXSM group. Therefore, restored Ca2+ stores induced by restored expression of ATP2A1and FKBP1B contribute directly to the increased heart rate through functioning similarly to sympathetic stimulation.
Restored signaling also plays an important role in the effect of SXSM due to the restored MBIP, PPIC, PRKCZ, vasoactive intestinal peptide receptor (VIPR), PRLR.
Confirmation of altered gene expression by quantitative real-time reverse transcription-polymerase chain reaction
Quantitative real-time RT-PCR was performed to confirm the results from gene expression chip. Four altered genes – ATP2A1, ERP27, FKBP1B, and MBIP – were selected. The relative mRNA expression level of each selected gene was normalized to 18S rRNA. As demonstrated in Figure 2, the expression trend of mRNA expression changes as verified by real-time RT-PCR was in agreement with that detected by gene expression chip.
Effects of long-term Shenxianshengmai treatment on cardiac proteins
A total of 125 proteins were altered after SXSM treatment [Supplementary Table 3]. As displayed in Figure 3, the most altered proteins were those participate in oxidative phosphorylation and tricarboxylic acid (TCA) cycle. SXSM-enhanced TCA cycle due to increased aconitate hydratase, succinyl-CoA ligase (GDP-forming) subunit alpha and beta (SUCLG1/2) and succinyl-CoA ligase (ADP-forming) subunit beta (SUCLGA2), isocitrate dehydrogenase2, and dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (DLAT, PDHB). SXSM also increased NADH dehydrogenase. The upregulated proteins include NADH dehydrogenase (complex I) core subunit (NDUFS1/3, NDUFV2), accessory subunit (NDUFS4/5), alpha (NDUFA2/7/8/9/12/13), and beta (NDUFB6) subcomplex. In addition, two subunits of mitochondrial ATP synthase (encoded by ATP5B and ATP5H) were also increased. These increased proteins may lead to enhanced mitochondrial membrane respiratory chain and increased ATP generation. All these results demonstrated that SXSM could improve the energy supplement of ventricular myocardium.
Confirmation of altered proteins by Western blot
Western blot was performed to confirm the results from iTRAQ. Considering the biological function, we selected two increased proteins: ATP synthase subunit beta (encoding by ATP5B) and complex I subunit (encoding by NDUFS1). β-actin was also detected as an internal control. As demonstrated in Figure 4, beta subunit of ATP synthase and subunit of complex I also increased after four weeks treatment with SXSM. This result was in agreement with that detected by iTRAQ.
The present study shows directly or indirectly mRNA remodeling of bradycardia for the first time and demonstrates that SXSM is effective in treating bradycardia. However, it is not possible to assume that all changes in gene expression are coupled to the development of bradycardia. In fact, the present data did not exclude the possibility that the part of the gene expression modifications was associated with the SA lesion, or secondary to the development of bradycardia.
Our results revealed that SXSM increased heart rate by inhibiting heart parasympathetic transmission based on the decreased CHRNA2 (encodes nicotinic acetylcholine receptor) and increased ACE-1 (encodes acetylcholinesterase). Reduced nicotinic acetylcholine receptors (encoded by CHRNA2), which form acetylcholine (ACh)-gated ion channels on the presynaptic and postsynaptic sides of the neuromuscular junction, suggested the inhibition of heart parasympathetic transmission. Moreover, it is well known that acetylcholinesterase (encode by ACE-1) locates at mainly neuromuscular junctions and serves to terminate parasympathetic synaptic transmission by hydrolyzing the neurotransmitter ACh. The increased expression of ACE-1 after SXSM treatment also indicates that parasympathetic synaptic transmission in heart was inhibited by SXSM. Therefore, sympathetic nerve was relatively stimulated. In addition to an increased force of heartbeat, this stimulation also causes the increase in heart rate.
Moreover, restored calcium handling also plays an essential role in the increased heartbeat. Bramich et al. reported that increases in force and heart rate evoked by sympathetic nerve stimulation resulted from the release of Ca2+ from intracellular Ca2+ stores-endoplasmic reticulum (ER). FKBP12.6 inhibits basal RyR2 activity. PKA-dependent RyR2 phosphorylation interrupt FKBP12.6-RyR2 association and activate RyR2 in myocytes. Sarcoplasmic reticulum (SR)/ER calcium ATPases (SERCAs) are calcium pumps that couple ATP hydrolysis with calcium transport across the SR/ER membrane. As a consequence of this activity, they maintain a level of resting intra-ER free calcium that is three to four orders of magnitude higher than the cytosolic Ca2+ concentration. Reduced SR Ca2+ release is due to diminished SR Ca2+ content directly related to a depressed expression of SERCA2a protein. Enhancing SERCA2a expression may improve SR Ca2+ handling in failing human myocardium. From our results, both ATP2A1 and FKBP1B were downregulated in model group and upregulated in M+SXSM group. Therefore, restored Ca2+ stores induced by restored expression of SERCA2a and FKBP12.6 contributed directly to increased heart rate.
Previous studied suggested that reduced ACE (encodes angiotensin I converting enzyme) may contribute to the improvement of heart function. Hence, reduced ACE after SXSM treatment may also play a positive role in heart.
In addition, restored signaling also play an important role in the effect of SXSM due to the restored MBIPth, PPIC, PRKCZ, VIPR, and PRLR. MBIP interacts with MUK/DLK/ZPK (a MAPKKK class protein kinase) and inhibits the activity of it to induce JNK/SAPK activation. The protein encoded by PPIC is a member of the PPIase family. PPIases catalyze the cis-trans isomerization of proline imidic peptide bonds in oligopeptides and accelerate the folding of proteins. Along with PPIB, PPIC localizes to the ER, where it maintains redox homeostasis. Increasing evidence from studies using in vitro and in vivo systems points to PKC zeta (PRKCZ) as a key regulator of critical intracellular signaling pathways such as mitogen-activated protein kinase cascade, transcriptional factor nuclear factor-kappa B activation, ribosomal S6-protein kinase signaling, and cell polarity. VIPR is a receptor for vasoactive intestinal peptide. The activity of it is mediated by G proteins which activate adenylyl cyclase. The PRLR is a cytokine receptor, and second messenger cascades include the JAK-STAT pathway, JAK-RUSH pathway, Ras-Raf-MAPK, and PI3K/AKT/mTOR pathway.
In ventricular myocardium, SXSM increased the supply of ATP by enhancing TCA cycle and oxidation-respiratory chain. Upregulated proteins ranged from enzymes of TCA cycle to subunits of complex I and ATP synthase. It was well known that mitochondrial ATP synthase catalyzes ATP synthesis. It included two complexes: the soluble catalytic core, F1, and the membrane-spanning component, F0, which comprises the proton channel. The F1 complex consists of 5 different subunits (alpha, beta, gamma, delta, and epsilon). The F0 seems to have nine subunits (a, b, c, d, e, f, g, and F6 and 8). According to our results, the increased ATP5B and ATP5H encode the beta subunit of F1 and d subunit of the F0 complex, respectively. Thus, ATP generation was effectively enhanced in ventricular myocardium.
In conclusion, our bradycardia model showed that long-term SXSM stimulate sympathetic transmission by increasing the expression of acetylcholinesterase and reduce the expression of nicotinic receptor to increase heart rate. SXSM also restored the calcium handling genes and altered genes involved in signaling. In addition, SXSM improves the ATP supply of ventricular myocardium by increasing proteins involved in TCA cycle and oxidation-respiratory chain. These data provide insights for the future study of SXSM.
Supplementary information is linked to the online version of the paper on the Chinese Medical Journal website.
Financial support and sponsorship
This work was supported by the grant from the International S&T Cooperation Program of China (No. 2013DFA31620).
Conflicts of interest
There are no conflicts of interest.
The authors greatly appreciate Peng Peng, Hui-Dong Zhang, Liu-Jun Jia and Jia-Fei Luo from Animal Center of Fuwai Hospital.
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Edited by: Li-Shao Guo