Overexpression of M3 Muscarinic Receptor Is a Novel Strategy for Preventing Sudden Cardiac Death in Transgenic Mice
- 8 Downloads
The present study was designed to investigate the cardiac benefits of M3 muscarinic receptor (M3-mAChR) overexpression and whether these effects are related to the regulation of the inward rectifying K+ channel by microRNA-1 (miR-1) in a conditional overexpression mouse model. A cardiac-specific M3-mAChR transgenic mouse model was successfully established for the first time in this study using microinjection, and the overexpression was confirmed by both reverse transcriptase-polymerase chain reaction and Western blot techniques. We demonstrated that M3-mAChR overexpression dramatically reduced the incidence of arrhythmias and decreased the mortality in a mouse model of myocardial ischemia-reperfusion (I/R). By using whole-cell patch techniques, M3-mAChR overexpression significantly shortened the action potential duration and restored the membrane repolarization by increasing the inward rectifying K+ current. By using Western blot techniques, M3-mAChR overexpression also rescued the expression of the inward rectifying K+ channel subunit Kir2.1 after myocardial I/R injury. This result was accompanied by suppression of upregulation miR-1. We conclude that M3-mAChR overexpression reduced the incidence of arrhythmias and mortality after myocardial I/R by protecting the myocardium from ischemia in mice. This effect may be mediated by increasing the inward rectifying K+ current by downregulation of arrhythmogenic miR-1 expression, which might partially be a novel strategy for antiarrhythmias, leading to sudden cardiac death.
Sudden cardiac death is a major contributor to mortality in industrialized nations, affecting around 500,000 individuals annually in the Western world. It can also cause more deaths than acquired immunodeficiency syndrome (AIDS), lung and breast cancer and stroke combined (1). In China, preliminary data suggest that there are over 544,000 adults with sudden cardiac death each year (2). About 75% of the sudden cardiac death cases are linked to a previous myocardial infarction, and 80% show a clinical record with the history of coronary artery disease (2). Intriguingly, life-threatening cardiac arrhythmias, one of the leading causes of sudden cardiac death, have been clinically demonstrated in the vast majority of severe heart diseases, especially myocardial infarction. Unfortunately, inefficacy of conventional antiar-rhythmic drugs has also been recognized in healing the electrical remodeling process or reducing sudden cardiac death (3,4). Therefore, the primary purpose of the present study was designed to develop rational approaches to prevent lethal cardiac arrhythmias that may lead to a consequent sudden cardiac death.
Since the initial identification of the M3 subtype of the muscarinic acetylcholine receptors (M3-mAChR) in the heart, there have been increasing interest and advances in studies on the pathophysiological roles of M3-mAChR in the heart (5). Recent studies from several laboratories have provided compelling and solid evidence in support of the important roles of M3-mAChR in the regulation and maintenance of cardiac function and heart disease (6, 7, 8). M3-mAChR activates a delayed rectifying K+ current IKM3 to participate in cardiac repolarization, negative chronotropic actions and antidysrhythmic activity (suppresses ischemic dysrhythmias) (9). M3-mAChR interacts with gap-junctional channel connexin 43 to maintain cell-cell communication and excitation propagation (10). M3-mAChR regulates intracellular phosphoinositide hydrolysis to improve cardiac contraction and hemodynamic function (11). M3-mAChR also activates antiapoptotic signaling molecules, enhances endogenous antioxidant capacity and diminishes intracellular Ca2+ overload, all of which contribute to protecting the heart against ischemic injuries (12). However, it is generally difficult to predict the simultaneous involvement of two or more mAChR subtypes in a specific functional response by using antagonists of limited subtype selectivity. Development of transgenic mice overexpressing specific mAChR subtypes seems to allow a more definitive approach to defining the pathophysiological roles of mAChRs. To further explore the potential roles of M3-mAChR in myocardial ischemia and sudden cardiac death, we created a transgenic mouse model in which M3-mAChR was overexpressed conditionally.
There is a growing body of evidence showing that the dysfunction of the potassium channel is closely related to cardiac diseases (13). Among them, the Kir family, which expresses two transmembrane-spanning segments flanking a conserved pore region, is structurally and functionally different from other ion channels. Kir2.1 subunits, encoded by the potassium inwardly-rectifying channel, subfamily J, member 2 (KCNJ2) gene, is the main K+ channel subunit that mediates inward rectifier potassium channel current (IK1) in cardiomyocytes (14), which plays a key role in setting up the resting membrane potentials and in the repolarization of cardiac myocytes, especially affecting the action potential duration (APD) at 90% repolarization (APD90) (15). Many studies have demonstrated that IK1 is dysfunctional in several cardiac diseases such as cardiac ischemia-reperfusion (I/R) injury, arrhythmia and heart failure (16, 17, 18).
Since the discovery of microRNA, a single-stranded noncoding RNA of approximately 22 nucleotides long and >700 miRNAs have been identified in humans (19), which regulates up to 20–30% of gene expression by binding to 3′ untranslated regions (UTRs) of mRNAs through inexact sequence matching, targeting numerous mRNAs for translational inhibition or degradation (20). For example, the overexpressed microRNA-1 (miR-1) is arrhythmogenic either in ischemic or normal hearts, which could slow the conduction and depolarized the cytoplasmic membrane by posttranscriptionally repressing KCNJ2 (21).
This study was designed to investigate the cardiac beneficial effects of M3-mAChR overexpression and whether they are related to the regulation of inward rectifying K+ channel by miR-1 and how the molecular processes within the cardiac cell are controlled after the overexpression of M3-mAChR. These can definitely help us better understanding the pathology of myocardial ischemia that are associated with the regulation of gene expression and transcriptional activity, which also might be a novel strategy for preventing sudden cardiac death.
Materials and Methods
Vector Construction and Generation of M3-mAChR Transgenic Mouse
Mouse Model of Myocardial Ischemia Reperfusion
The 562-L male M3 transgenic (M3Tg) mice and their wild-type (WT) littermates, weighing approximately 25 g, were subjected to the sham or myocardial I/R model as previously described (23). The ECG and mortality were observed in each group to evaluate the severity of arrhythmia. Then, the hearts were removed for ventricular cardiomyocyte isolation, Western blot and realtime reverse transcriptase-polymerase chain reaction (RT-PCR) assay. This study was approved and supervised by the Institutional Animal Care and Use Committee of Harbin Medical University. Use of animals was in accordance with the regulations of the ethic committees of Harbin Medical University and was confirmed with the Guide for the Care and Use of Laboratory Animals, published by the U.S. National Institutes of Health (NIH publication no. 85–23, revised 1996) (see the online supplementary materials).
The incidence of arrhythmias and the mortality rate were registered and evaluated for a continuous period of 2 h after myocardial I/R injury or sham surgery. An arrhythmia score was used to include the incidence and duration of arrhythmias by giving a grade to the animals (24) (see the online supplementary materials).
Arrhythmic Model Induced by Aconitine in Mice
Aconitine (5 mg/mL, 2 µg/10 g mice; Sigma, St. Louis, MO, USA) was pumped at a constant speed using Longerpump (LSP02-1B; Baoding Longer Precision Pump Company, Baoding, China) to induce the ventricular arrhythmias within 4 min. In each group, the arrhythmia was observed until the death of the mice. The survival time was estimated in WT and M3Tg mice.
Ventricular Cardiomyocyte Isolation and Patch-Clamp Recording
The dissociation procedures for ventricular cardiomyocytes were similar to that described previously (24). The cardiomyocytes were transferred to a chamber mounted on an inverted microscope (Nikon Diaphot; Nikon, Tokyo, Japan) for electrophysiological recording. Action potential (AP) and potassium currents (K+) were recorded under current and voltage-clamp configuration, respectively, using the whole-cell patch-clamp technique with an Ax-opatch 200B amplifier (Axon Instruments, Foster City, CA, USA) (see the online supplementary materials).
Western Blot Analysis
The procedures for membrane protein extraction from ventricular tissue and immunoblotting are described in detail elsewhere (12). The primary antibodies of anti-Kir2.1, anti-Kv4.2 (Alomone Labs, Jerusalme, Israel), and anti-Cav1.2, anti-M3 (Santa Cruz Technology, Santa Cruz, CA, USA), were used, with GAPDH (anti-GAPDH antibody from Kangcheng, Shanghai, China) as an internal control. Western blot bands were quantified using Odyssey v1.2 software by measuring the band intensity (area × optical density [OD]) for each group (see the online supplementary materials).
Determination of M3-mAChR mRNA, KCNJ2, KCND2, CACNA1C and miR-1 Levels
RT-PCR assay was carried out to quantify the levels of M3-mAChR mRNA, KCNJ2, KCND2 (potassium voltage-gated channel, Shal-related subfamily, member 2), CACNA1C (calcium channel, voltage-dependent, L type, alpha 1C subunit) transcripts and miR-1. The total RNA samples were isolated with a mirVana™ miRNA Isolation Kit (Ambion Inc., Austin, TX, USA) from mouse myocardial I/R. The miR-1 levels were measured using the mirVana™ qRT-PCR miRNA Detection Kit (Ambion), which is a kind of quantitative RT-PCR kit and was used in conjunction with real-time PCR with SYBR Green I for quantification of miR-1 transcripts (see the online supplementary materials).
Average data were expressed as mean ± standard error of the mean (SEM). Statistical comparisons (performed using analysis of variance [ANOVA]) were carried out using SPSS 13.0. A two-tailed P value <0.05 indicated a statistically significant difference. Nonlinear least-square curve fitting was performed with CLAMPFIT in pCLAMP 10.0 or GraphPad Prism.
All supplementary materials are available online at www.molmed.org .
Characterization of M3-mAChR in Tg Mice
Our data showed that the mRNA levels of M3-mAChR were higher in M3-overexpressing mice than in their WT counterparts (Figure 1B; P < 0.01). Meanwhile, the protein expression of M3-mAChR in M3-overexpressing mice (1.56 ± 0.17) was also higher than that in WT mice (Figure 1C; P < 0.05). To further confirm the successful establishment of M3-mAChR overexpression, whole-cell patch-clamp experiments were performed using ventricular cardiomyocytes in the presence of 2 mmol/L 4-aminopyridine (4-AP) in the perfusate to antecedently block the transient outward rapid K+ current (Ito). As shown in Figures 1D and E, 5 mmol/L choline (an agonist of M3 muscarinic receptor; Sigma) was applied to the bath. The voltage-clamp data showed that choline activated a delayed rectifier type of current (IKM3) when depolarizing voltage steps were applied. The IKM3 density in ventricular cardiomyocytes of M3Tg mice at +50 mV (46.3 ± 5.8 pA/pF) was higher than that of WT mice (15.4 ± 2.5 pA/pF, P < 0.01 WT versus M3Tg mice).
Cardiac Beneficial Effects of M3-mAChR Overexpression against Mice Myocardial I/R and Aconitine Injury
Aconitine is a highly poisonous alkaloid derived from various aconite species and is used for creating models of cardiac arrhythmia (25). The arrhythmia was also induced by aconitine, and the survival time was obviously prolonged in M3Tg mice compared with WT mice (Figure 2G; P < 0.01).
M3-mAChR Overexpression Shortened APD90 of Ventricular Cardiomyocytes
AP characteristics of cardiac myocytes in WT and M3-overexpressing (M3Tg) mice from both control and myocardial I/R injury.
Amplitude of action (mV)
−54.3 ± 0.8
40.9 ± 3.2
95.3 ± 3.7
182.7 ± 16.0
−63.3 ± 1.5b
40.7 ± 3.1
104.1 ± 3.4
130.2 ± 16.7c
−54.5 ± 0.9
32.1 ± 2.6
86.7 ± 3.5
161.1 ± 10.4
−56.1 ± 1.2
34.8 ± 2.6
90.9 ± 3.5
131.6 ± 7.9d
M3-mAChR Overexpression Reduced IK1 but Not Ito and ICa,L in Ventricular Cardiomyocytes
M3-mAChR Overexpression Altered Protein and mRNA Levels of Potassium and Calcium Channel Subunits in Myocardium
M3-mAChR Overexpression Suppressed Upregulated miR-1 Induced by Myocardial I/R Injury
Here we demonstrate that cardiac overexpression of M3-mAChR plays a critical role in the reduction of the incidence of arrhythmias and mortality in a mouse model by protecting the myocardium from ischemia, increasing the survival time after aconitine treatment and decreasing the arrhythmic score after myocardial I/R. Our patch-clamp data also demonstrate that M3-mAChR overexpression significantly shortened the APD by increasing the IK1 and consequently enhancing the membrane stability of ventricular cardiomyocytes without affecting Ito. More importantly, our molecular study clearly showed that the Kir2.1 was upregulated and miR-1 expression was downregulated in the myocardium from M3-overexpressing mice. This molecular evidence further confirmed our electrophysiological study. These findings not only help us understand the mechanisms underlying the cardioprotective effects of M3-mAChR from ischemia, but also conceptually advance our view of miRNAs that may serve as potential therapeutic and drug targets.
There are important fundamental implications of our findings. First, a large body of evidence indicates that muscarinic acetylcholine receptors, especially M3-mAChR, play critical roles in regulating the activity of many important functions of cardiovascular systems (26). Previous studies have demonstrated that M3-mAChR may possess a potential role in protecting the heart against ischemia through multiple mechanisms, including the interaction with gap junction channel connexin 43 to maintain cell-cell communication (10), activation of antiapoptotic signal molecule Bcl-2 and p38 mitogen-activated protein kinase and a decrease in Ca2+ overload induced by myocardial ischemia in ventricular cardiomyocytes (12), which has been known to improve cardiac contraction and hemodynamics by activating intracellular phosphoinositide hydrolysis via a Gq pathway (27).
Second, our study is the first to our knowledge to demonstrate that an M3Tg, specifically expressed in cardiac tissues, was successfully established and it is a useful animal model to study the M3-mAChR functions on cardiac diseases. The successful establishment of M3Tg was confirmed by its positive genotype of M3-mAChR by PCR data confirming much higher mRNA and protein expressions, and more importantly, the current density of IKM3 mediated by M3-mAChR was increased by an M3-mAChR agonist in the ventricular cardiomyocytes (Figure 1). Most importantly, the overexpression of M3-mAChR significantly reduced the incidence of arrhythmias induced by myocardial I/R, sequentially decreased the mortality of myocardial I/R and improved the survival time (Figure 2). These data significantly enhanced knowledge of the potential roles of M3-mAChR in myocardium against myocardial I/R injury, and this knowledge has been demonstrated by many researchers (28, 29, 30, 31).
Myocardial ischemia results in a dramatic disorder of automaticity, in an increased heterogeneity of conduction and structural remodeling and in alterations of ion channel protein expression and distribution (32). These results contribute to the development of an electrical disturbance that leads to life-threatening cardiac arrhythmias. To explore the underlying ion channel mechanisms of M3-mAChR in its cardiac protection against myocardial I/R injury, the APD and some currents responsible for the characteristic differences in AP wave shapes were also examined. Our results indicate that M3-mAChR overexpression shortened the APD and increased the RMP. Effects of 4-diphenylacetoxy-N-methylpiperidine-methiodide (4-DAMP), a M3 receptor blocker, on the RMP between WT and M3Tg mice were also detected in our recent experiments. However, a significant difference of the RMP between WT and M3Tg mice was not detected in the presence of either 4.0 or 8.0 nmol/L 4-DAMP (data not shown), suggesting that 4-DAMP does not affect/reverse an increased inward rectifying K+ current (especially the upregulation of Kir2.1) nor the hyperpolarized RPM mediated by M3-mAChR overexpression. Although the activation of M3-mAChR can be blocked by 4-DAMP, but the channel protein of Kir2.1, a downstream product of M3-mAChR overexpression, was upregulated already. Blocking the receptor itself will not affect the Kir2.1 functional expression, and that is the reason why the RMP was not changed accordingly.
Consistent with our previous study (21), although individuals surviving from myocardial I/R presented a RMP depolarization, IK1 density reduction and Kir2.1 protein downregulation, interestingly, IK1 density and Kir2.1 expression were significantly higher in myocardial I/R hearts of M3Tg mice compared with WT mice (Figures 4 and 5). These results suggest that M3-mAChR overexpression plays, at least in part, an important role in cardioprotection. Under normal conditions, cardiac IK1 dominates the K+ conductance near the RMP. In accordance with this theory, our current-clamp data demonstrate that the greater the outward currents of IK1, the shorter the APD. It is also well known that IK1 stabilizes the RMP and determines the cell input resistance, which is the resistance of the membrane in the subthreshold voltage range (33). Other researches have reported that IK1 is downregulated in several other cardiac disorders such as heart failure and long QT syndrome (34,35). The downregulation of IK1 is also observed in the models of cardiac hypertrophy and in the patients with cardiomyopathies (36). This downregulation of IK1 has greater impacts on life-threatening arrhythmias due to membrane depolarization, APD prolongation, and both early and delayed after depolarizations (17).
It has been known that KCNJ2, which encodes the cardiac IK1 channels, is responsible for setting and maintaining the cardiac RMP. Although the protein level for Kir2.1 was obviously different, there were no detectable changes of KCNJ2 among the four experimental groups (Figure 5). This discrepancy between mRNA and protein levels may come from the function of microRNA, such as miR-1, which displayed opposite trends compared with the Kir2.1 protein level. As many studies have reported (21,37), microRNA can control the translation of mRNA by targeting its 3′UTR but without affecting the mRNA expression. The increased Kir2.1 protein function and expression may have resulted from the low expression of miR-1. Our data show that the mRNA level of CACNA1C was much higher in M3Tg I/R mice than in WT I/R mice. However, the protein levels of Cav1.2 were not significantly different. The reasons for this may be as follows: first, the translational level was affected by many factors posttranscription, which may be related to the regulator control system in different organs; and, second, the intensity of protein function correlates closely with the protein expression level but not its mRNA level, which was certificated with no differences of ICa,L among these groups.
Our previous study indicated that Kir2.1 was downregulated by miR-1, which was overexpressed in patients with coronary artery disease. Furthermore, repression of these genes by miR-1 contributes significantly to the arrhythmogenic potential of miR-1 during myocardial injury (21). Interestingly, our data demonstrated that miR-1 was downregulated in M3Tg mice to further lead to the increase of IK1 functional expression (Figure 6). Here, we conclude that the overexpression of M3-mAChR significantly suppressed upregulated miR-1 after myocardial I/R injury and restored the impaired KCNJ2 gene expression and function. These changes may be a novel molecular and cellular mechanism underlying the ischemic cardioprotective effects of M3-mAChR.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This study was supported in part by the National Basic Research Program (973 Program) of China (2007CB512000/2007CB512006) and the National Natural Science Foundation of China (81072639, 30973531). We thank J Robbins for the gift of the α-MHC vector.
- 7.Shi H, Wang H, Li D, Nattel S, Wang Z. (2004) Differential alterations of receptor densities of three muscarinic acetylcholine receptor subtypes and current densities of the corresponding K+ channels in canine atria with atrial fibrillation induced by experimental congestive heart failure. Cell. Physiol. Biochem. 14:31–40.CrossRefGoogle Scholar
- 27.Shi H, Wang H, Yang B, Xu D, Wang Z. (2004) The M3 receptor-mediated K(+) current (IKM3), a G(q) protein-coupled K(+) channel. J. Biol. Chem. 279:1774–2178.Google Scholar