Dominant Negative Suppression of Rad Leads to QT Prolongation and Causes Ventricular Arrhythmias via Modulation of L-type Ca2+ Channels in the Heart
Disorders of L-type Ca2+ channels can cause severe cardiac arrhythmias. A subclass of small GTP-binding proteins, the RGK family, regulates L-type Ca2+ current (ICa,L) in heterologous expression systems. Among these proteins, Rad (Ras associated with diabetes) is highly expressed in the heart, although its role in the heart remains unknown. Here we show that overexpression of dominant negative mutant Rad (S105N) led to an increase in ICa,L and action potential prolongation via upregulation of L-type Ca2+ channel expression in the plasma membrane of guinea pig ventricular cardiomyocytes. To verify the in vivo physiological role of Rad in the heart, a mouse model of cardiac-specific Rad suppression was created by overexpressing S105N Rad, using the α-myosin heavy chain promoter. Microelectrode studies revealed that action potential duration was significantly prolonged with visible identification of a small plateau phase in S105N Rad transgenic mice, when compared with wild-type littermate mice. Telemetric electrocardiograms on unrestrained mice revealed that S105N Rad transgenic mice had significant QT prolongation and diverse arrhythmias such as sinus node dysfunction, atrioventricular block, and ventricular extrasystoles, whereas no arrhythmias were observed in wild-type mice. Furthermore, administration of epinephrine induced frequent ventricular extrasystoles and ventricular tachycardia in S105N Rad transgenic mice. This study provides novel evidence that the suppression of Rad activity in the heart can induce ventricular tachycardia, suggesting that the Rad-associated signaling pathway may play a role in arrhythmogenesis in diverse cardiac diseases.
Rad (Ras associated with diabetes) is the prototypic member of the newly emerging RGK family of proteins, a group of Ras-related GTPases that includes Rad, Gem, and Rem.1 Rad was initially identified by subtraction cloning as an mRNA overexpressed in skeletal muscle in a subset of patients with type 2 diabetes mellitus.2 Among the RGK proteins, Rad is abundantly expressed in skeletal and cardiac muscle.2 It interacts with various signal transduction molecules such as Rho kinase, calmodulin, and calmodulin-dependent protein kinase II, leading to inhibition of their downstream signals.3–5 In epithelial or fibroblastic cells, overexpression of Rad results in stress fiber and focal adhesion disassembly, implicating an involvement in cytoskeletal regulation through the Rho kinase pathway.3 In vascular smooth muscle cells, focal gene transduction of Rad attenuates neointimal formation after balloon injury by inhibiting smooth muscle proliferation and migration activated through the Rho kinase pathway.6 Furthermore, overexpression of Rad in skeletal muscle worsens diet-induced insulin resistance and glucose intolerance, which is consistent with the observed upregulation of Rad in diabetic patients.7 Despite the critical roles of Rad in diverse biological processes, its function in the heart is still unknown.
Recently, RGK proteins were found to suppress voltage-gated L-type Ca2+ currents (ICa,L) in heterologous expression systems and insulin-secreting β cells of the pancreas.8–10 This was shown to occur via an interaction with Ca2+ channel β subunits, and the finding suggests that Rad may play an important role in cellular Ca2+ homeostasis. Indeed, overexpression of Gem in PC12 cells and MIN6 cells prevents Ca2+-triggered exocytosis via inhibition of L-type Ca2+ channels.11 In the heart, Ca2+ is essential for electrical activity and is a direct activator of the myofilaments in contraction. Among the many Ca2+ handling proteins, cardiac L-type Ca2+ channels play central roles in initiation of excitation–contraction coupling and in cardiac electrophysiological properties. Abnormal function of Rad in the heart might therefore lead to various cardiac disorders such as arrhythmias or contractile dysfunction.
We show here that dominant negative suppression of Rad led to the enhancement of ICa,L by facilitating channel expression in the plasma membrane of cardiomyocytes, resulting in prolongation of the action potential. Furthermore, transgenic mice with heart-specific overexpression of dominant negative mutant Rad (S105N) displayed ventricular arrhythmias as a consequence of QT prolongation. Our results constitute the first evidence that Rad plays an important role in regulating cardiac electrophysiological properties and that Rad could be a key molecule for understanding the mechanism of arrhythmogenesis in cardiovascular diseases.
Materials and Methods
All experimental procedures and protocols were approved by the Animal Care and Use Committee of Keio University and conformed to the NIH Guide for the Care and Use of Laboratory Animals. An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Myocyte Isolation and Cultures
Myocytes were isolated from the left ventricles of adult guinea pigs and mice using enzymatic digestions as previously described, with slight modifications.12,13 After isolation, ventricular myocytes were cultured in DMEM containing 10% FBS and 1% penicillin–streptomycin (all from Invitrogen, Carlsbad, Calif) for 24 hours.
Production of S105N Rad Transgenic Mouse
The complete mutant mouse S105N Rad (with the serine at the 105-aa position substituted to arginine, to inhibit GTP binding) cDNA construct was subcloned into the region downstream of the α-myosin heavy chain promoter14 previously subcloned into the PBS2 SK+ plasmid. The complete transgene was isolated using NotI digestion of the PBS2 SK+ plasmid. Transgenic mice were generated by the Animal Laboratory Center of Keio University by cDNA microinjection of fertilized C57BL/6×SJL oocytes using standard techniques.
All data are shown as means±SEM. Statistical differences were determined using repeated-measures ANOVA, and P<0.05 was considered significant.
Rad Interacts With L-Type Ca2+ Channel β Subunits
Previous reports have shown that Rad interacts with Ca2+ channel β subunits in heterologous expression systems, resulting in the inhibition of ICa,L.15,16 To examine whether Rad physically interacts with the β subunits, glutathione S-transferase (GST) pull-down assays were performed. As shown in Figure 1A, recombinant GST wild-type (WT) Rad bound to the β subunits in whole-cell extracts derived from transfected Cos7 cells, whereas GST-S105N Rad did not. Furthermore, interaction between Rad and β subunits was confirmed in the context of the cellular environment. HEK293 cells were transiently transfected with Flag-tagged β2a subunits and hemagglutinin-tagged WT Rad or S105N Rad. After 24 hours in culture, coimmunoprecipitation assays were performed. Consistent with the previous report,15 the β2a subunits showed an interaction with WT Rad (Figure 1B), but no interaction with S105N Rad was detected. Because S105N Rad, which could bind GDP but not GTP, is known to function as a dominant negative mutant in the regulation of neointimal formation after vascular injury,6 we examined whether S105N Rad showed dominant negative inhibition of the interaction between WT Rad and β subunits. A plasmid encoding S105N Rad fused with Myc tag at its N terminus was cotransfected with WT Rad and β2a-subunit plasmids into HEK293 cells. As shown in Figure 1C, Myc-tagged S105N Rad suppressed the interaction between WT Rad and β2a subunits in a dose-dependent manner, indicating that S105N Rad did function as a dominant negative mutant for the interaction between WT Rad and β2a subunits.
Based on these results, we next studied whether the interaction between Rad and the β subunit affected the trafficking of Ca2+ channel α subunit to the plasma membrane. To do this, green fluorescent protein (GFP)-fused Cav1.2 (cardiac α1c subunit) and β2a subunit were coimmunostained with WT Rad or S105N Rad in HEK293 cells and visualized by confocal microscopy. Both WT Rad and S105N Rad were localized at the plasma membrane, when expressed alone (Figure 2A). Although individually expressed α1c subunit was localized mainly in the cytoplasm (Figure 2B), cotransfection with β2a subunits resulted in the translocation of α1c subunits from the cytoplasm to plasma membrane, confirming that the β subunit plays a chaperon-like role with the α1c subunit (Figure 2B). When WT Rad was expressed together with both the α1c and β2a subunits, the α1c subunit showed a cytoplasmic distribution, whereas WT Rad and the β2a subunit were still colocalized at the plasma membrane, indicating that WT Rad disrupted the binding of α1c subunit to β2a subunit (Figure 2C). In contrast, S105N Rad did not affect the localization of the α1c and β2a subunits (Figure 2D). These results indicated that the interaction between Rad and the β subunit regulated the trafficking of the α1c subunit to the plasma membrane.
To examine whether Rad regulates the function of L-type Ca2+ channels, we recorded Ba2+ currents in HEK293 cells using the whole-cell patch clamp technique. Currents conducted by L-type Ca2+ channels were recorded with 4 mmol/L Ba2+ in the external solution as a charge carrier. The currents in cells in which S105N Rad was coexpressed with GFP-α1c subunit and β2a subunit were similar to those in control cells (average current densities for GFP-α1c subunit, β2a subunit, and S105N Rad: 9.4±0.9 pA/pF [n=6]; for GFP-α1c subunit, β2a subunit, and blank: 8.8±0.9 pA/pF [n=7]; not significant), whereas in cells expressing the GFP-α1c subunit, β2a subunit, and WT Rad, the currents were very small (0.20±0.03 pA/pF [n=5] versus control; P<0.01), and similar to those in cells expressing GFP-α1c subunit alone (0.19±0.04 pA/pF [n=5]), indicating the association of WT Rad-mediated suppression with the β subunit (Figure 2E and 2F). These results confirmed that WT Rad dramatically suppressed the function of the L-type Ca2+ channel, whereas S105N Rad did not.
Rad Regulates ICa,L via Inhibition of α subunit trafficking to Plasma Membrane in Guinea Pig Ventricular Cardiomyocytes
To investigate the physiological role of Rad in the heart, we transduced adenovirus (Ad) encoding WT Rad or S105N Rad into guinea pig ventricular cardiomyocytes and recorded ICa,L using the whole-cell patch clamp technique. Interestingly, the peak ICa,L was larger in the cells overexpressing S105N Rad than in controls (12.0±1.5 pA/pF at 0 mV [n=7] in Ad-S105N Rad–transduced cells versus 5.4±1.0 pA/pF at 0 mV [n=8] in Ad-GFP transduced controls; P<0.05), whereas ICa,L was dramatically smaller in cells overexpressing WT Rad (1.4±0.1 pA/pF at 0 mV [n=6] in Ad-WT Rad–transduced cells versus control; P<0.01) (Figure 3A and 3B). Because S105N Rad itself does not affect the ICa,L, as shown in Figure 2F, enhancement of ICa,L by S105N Rad in cardiomyocytes might be attributable to the dominant negative suppression of endogenous Rad activity. Because the L-type Ca2+ channel contributes to the ion influx and plateau phase of the cardiac action potential, we investigated whether Rad-mediated regulation of ICa,L might affect the action potential in heart cells. Action potentials were recorded in guinea pig ventricular cells transduced with Ad-GFP (control), Ad-WT Rad, and Ad-S105N Rad. As expected, overexpression of WT Rad resulted in a dramatic shortening of the action potential duration (APD) and abolished the robust action potential plateau (APD90 128±7.76 ms [n=5] in Ad-WT Rad–transduced cells versus 267±26.3 ms [n=8] in Ad-GFP–transduced control cells; P<0.01), whereas S105N Rad significantly prolonged the APD without changing the action potential configuration (538±70.0 ms [n=6] in Ad-S105N Rad–transduced cells versus control; P<0.01) (Figure 3C and 3D).
To investigate the mechanisms of Rad-mediated regulation of ICa,L in native guinea pig cardiomyocytes, we performed cell fractionation and used Western blot analysis to quantify the expression of α1c subunit in the plasma membrane of cells after 24 hours in culture. The amount of α1c subunit protein extracted from the membrane fraction was significantly lower in Ad-WT Rad–transduced cells than in Ad-GFP–transduced control cells, whereas the overexpression of S105N Rad resulted in a significantly greater extraction of α1c subunit protein from the membrane fraction (Figure 4). These findings show that dominant negative suppression of endogenous Rad by S105N Rad increased Ca2+ channel expression at the plasma membrane, thereby implicating endogenous Rad in the regulation of cardiac L-type Ca2+ channel expression in the physiological context.
In Vivo Phenotype of Dominant Negative Suppression of Rad in Mouse Heart
To investigate the physiological role of endogenous Rad in vivo, we took advantage of the dominant negative mutant Rad to engineer an in vivo model of Rad disruption, obtained by overexpressing the S105N Rad under the control of the α-myosin heavy chain promoter. In transgenic (TG) mice aged 12 weeks, the total Rad protein expression in whole heart was 6 times greater than in their WT littermates (Figure 5A), indicating that the expression level of S105N Rad was approximately 5 times that of endogenous Rad. This level of exogenous S105N Rad expression in TG mice should have been sufficient to suppress the endogenous Rad/β subunit interaction, as shown in Figure 1B. Thus, we performed coimmunoprecipitation assays to confirm the effect of S105N Rad overexpression on the endogenous Rad/β subunit interaction in whole hearts. As shown in Figure 5B, the total amount of Rad that bound to the β subunits was much less in S105N TG mouse heart than that in WT mouse heart. Furthermore, we used Western blot analysis to compare α1c protein expression in the plasma membranes of S105N Rad TG and WT mice. The α1c protein expression was significantly greater in S105N Rad TG mice than WT mice, supporting the proposition that dominant negative suppression of Rad facilitates α1c subunit expression at the plasma membrane (Figure 5C). Accordingly, immunohistological analysis revealed that the T tubules of ventricular cardiomyocytes isolated from S105N Rad TG mouse hearts were more intensely immunoreactive for α1c subunits than those of WT mice (Figure 5D). The relative mean fluorescence of α1c subunits in the T-tubule areas to that in non–T-tubule area (between T tubules) were significantly greater in the S105N Rad TG mouse cells than in the WT mouse cells, indicating that S105N Rad facilitated the α1c subunit trafficking to the T tubules in cardiomyocytes (Figure 5D).
Next, action potentials were recorded from left ventricular papillary muscles using conventional microelectrode techniques. The APD was measured at 3 different pacing cycle lengths (100 ms, 150 ms, and 200 ms). As shown in Figure 6A, the APD was prolonged, and a subtle plateau phase was observed in S105N Rad TG mice. Consistent with the in vitro data in guinea pig cells, the APD50 and APD90 were both longer in S105N Rad TG mice than in WT mice at each pacing cycle length (Figure 6B; APD90 at 100 ms pacing cycle length, 33.9±2.5 ms [n=8] in S105N TG mice, versus 20.9±2.6 ms [n=8] in WT mice; P<0.05). There were no significant differences in resting membrane potential or Vmax between WT and TG mice (Figure 6C and 6D), suggesting that dominant negative suppression of Rad did not affect the inward rectifying K+ currents and Na+ currents. We also examined the transient outward potassium current (Ito), which mainly affects repolarization in the mouse action potential. There were no significant differences between the WT and S105N Rad TG mice in either peak or sustained Ito current densities, implying that Rad did not affect Ito function (Figure 6E).
As expected from the APD data, surface electrocardiograms showed QT prolongation in S105N Rad TG mice compared with WT mice (Figure 7A). Both QT and QTc intervals in S105N Rad TG mice were significantly longer than those in WT mice (QTc, 60.1±3.0 ms [n=8] in S105N Rad TG mice, versus 47.3±3.3 ms [n=8] in WT mice; P<0.05; Figure 7B). No significant differences were detected in other ECG parameters, such as RR, PR, and QRS intervals, as described in Table I in the online data supplement. Given the importance of QT prolongation as a cause of lethal ventricular arrhythmias, we investigated whether the dominant negative suppression of endogenous Rad activity produced arrhythmias in S105N Rad TG mice. In vivo ECGs were recorded from freely moving mice for 24 hours. No arrhythmias were observed in the recordings from WT mice (n=8). In contrast, among the TG mice (n=8), we recorded transient sinus arrest and second-degree atrioventricular block in 6 mice each, and ventricular extrasystoles in 5 mice (Figure 7C). In some patients with long QT syndrome, fatal ventricular arrhythmias occur under physical exertion and emotional stress.17,18 To mimic these circumstances, we injected epinephrine (2 mg/kg) into the peritoneum of S105N Rad TG mice (n=8) and WT mice (n=8). Under the epinephrine loading, nonsustained ventricular tachycardia was induced in 3 of the TG mice (Figure 7D), and consecutive ventricular extrasystoles were more frequently observed (7 TG mice), although no ventricular arrhythmias were observed in WT mice (see supplemental Table II).
Because the increase in ICa,L should facilitate nodal conduction, the abnormal nodal function observed in TG mice seemed anomalous. Rad is also known to suppress the Rho signaling pathway by direct interaction with its substrate, ROCK.3 Furthermore, disruption of Rho signaling results in progressive atrioventricular conduction defects, probably attributable to a dramatic decrease of connexin40.19,20 We examined the atrial expression of connexin40 in mouse hearts by Western blot analysis. As expected, connexin40 expression was significantly lower in S105N Rad TG mice than WT mice (Figure 7E).
The major finding of this study was that dominant negative suppression of endogenous Rad in the heart resulted in an increase in ICa,L, via upregulation of L-type Ca2+ channel expression at the plasma membrane. Using a transgenic approach for cardiac-specific inhibition of Rad by expressing the dominant negative form of Rad (S105N), the present study provides important new insights into the physiological function of Rad in regulating cardiac electrophysiology. Although mouse echocardiography showed no significant changes in cardiac size or function in transgenic animals at 12 weeks of age (supplemental Figure I), the phenotype of the transgenic animals comprised a prolonged QT interval, nodal dysfunction, and extrasystoles. These ECG phenotypes were accompanied by prolonged APDs and enhanced ICa,L, which were attributable to the upregulation of L-type Ca2+ channels in T tubules. Under baseline conditions, TG mice displayed ventricular extrasystoles but no ventricular tachycardia. Furthermore, epinephrine administration exacerbated the ventricular arrhythmias in TG mice but did not induce arrhythmias in WT mice. These data indicate that the Rad signaling pathway plays an important role in cardiac antiarrhythmia via the strong suppression of ICa,L.
We did not predict any nodal dysfunction in TG mice because an increase of ICa,L would be expected to facilitate nodal conduction. The induction of nodal dysfunction was probably attributable, at least in part, to the reduction of connexin40 expression in TG mice. Previous studies have shown that Rad binds directly to ROCK, resulting in the inhibition of Rho/ROCK signaling pathways,3,6 which regulates the connexin40 expression in mouse hearts.19 The GST pull-down assays revealed that S105N Rad, as well as WT Rad, could physically interact with ROCK (data not shown). This implies that overexpression of S105N Rad in TG mice may have led to a decrease of connexin40 expression via suppression of the Rho signaling pathways. Furthermore, this inhibitory effect of Rad on Rho pathways was likely to have been especially strong in the heart because Rad binds to ROCK2, which is highly expressed in the heart, whereas the other isoform (ROCK1) is preferentially expressed in noncardiac tissues such as lung, liver, spleen, kidney, and testis.21 This distinct tissue-specific expression of ROCK isoforms might explain the opposite effects of Gem and Rad on nodal conduction because Gem predominantly binds to ROCK1.3 However, further studies are needed to test this hypothesis.
One major cause of mortality in patients with diabetes mellitus is diabetic cardiomyopathy, which occurs independently from diabetes-mediated vascular complications. Pereira et al22 reported that the systolic dysfunction of type 2 diabetic mice is partly attributable to a reduction of ICa,L, implicating Rad-mediated Ca2+ channel regulation as a possible factor in diabetic cardiomyopathy. Furthermore, diabetic cardiomyopathy is characterized by electrical remodeling, metabolic remodeling with malignant biochemical processes, and anatomical remodeling with progressive loss of cardiomyocytes.23 The abnormal prolongation of QT interval is the most prominent electrical remodeling that occurs in diabetic hearts. QT prolongation is a significant predictor of mortality in diabetes patients because it is associated with an increased risk of sudden cardiac death caused by lethal ventricular arrhythmias.24 One of the mechanisms for QT prolongation in diabetes mellitus is depression of multiple ion currents including the transient outward current, ICa,L, and the delayed rectifier K+ current.22,25 Given that Rad mRNA is upregulated in type 2 diabetes patients2 and Rad protein expression in diabetic mouse heart is upregulated relative to WT mice (data not shown), Rad-mediated regulation of ICa,L might be involved in the electrophysiological remodeling in diabetic cardiomyopathy. Dominant negative suppression of Rad led to QT prolongation and induction of arrhythmias even in the nondiabetic mice used in this study. Considering these experimental and clinical data, it is plausible that upregulation of Rad in diabetic patients might function as a negative regulator to counteract QT prolongation by compensating for the decreased outward K+ currents with downregulation of ICa,L. If so, the preservation of Rad function might be a potential strategy for the prevention of lethal ventricular arrhythmias in diabetic cardiomyopathy.
Our data clearly demonstrated that Rad regulated the trafficking of Ca2+ channel α subunit in both heterologous systems and cardiomyocytes. However, the precise mechanisms of RGK protein-mediated modulation of L-type Ca2+ channels remain to be clarified. Consistent with our data, Beguin et al9 showed that inhibition of ICa,L by another RGK protein, Gem, is attributable to the decreased expression of α subunits in the plasma membrane. However, in our study, the complete inhibition of ICa,L 24 hours after transduction of WT Rad in guinea pig cells could not be explained solely by the suppression of α subunit trafficking to the plasma membrane because our Western blot data still detected a small amount of Ca2+ channel expression in the T tubules. These channels may have remained because the turnover of L-type Ca2+ channels in the plasma membrane is 36 to 48 hours26; thus a 24 hour culture period might not be sufficient for the complete degradation of preexisting channels. Therefore, other mechanisms for suppression of ICa,L by Rad, unrelated to trafficking, are also likely to be involved. One possibility is the direct inhibition of L-type Ca2+ channels by association of Rad with channel subunits. Rem2 has recently been shown to almost completely suppress ICa,L without altering channel expression at the plasma membrane,27,28 which supports this hypothesis. The possibility remains that Rad may regulate the expression of other RGK proteins, which in turn alter L-type Ca2+ channel function. However, the expression level of Rem protein did not change with overexpression of Rad (data not shown), which leads us to conclude that Rem is not associated with the Rad-mediated regulation of the L-type Ca2+ channel. Further studies are required to identify the multiple mechanisms involved in regulation of the L-type Ca2+ channel by Rad.
In summary, dominant negative suppression of Rad in the heart induced QT prolongation and ventricular arrhythmias, caused by the augmentation of ICa,L. The finding that Rad regulates L-type Ca2+ channel function in the heart suggests that the Rad-associated signaling pathway may play a role in arrhythmogenesis in diverse cardiac diseases.
We thank Dr Shunichiro Miyoshi and Dr Yoko Hagiwara for useful technical advice.
Sources of Funding
This study was supported by research grants from the Ministry of Education, Science and Culture, Japan (to M.M., T.A., and K.F.); from Keio Gijuku Academic Developmental Funds (to M.M.); from the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (to K.F.); and from health sciences research grants from the Ministry of Health, Labour and Welfare, Japan (H18-Research on Human Genome-002 to S.O.).
Original received December 9, 2006; revision received April 10, 2007; accepted May 15, 2007.
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