Cytoskeletal Protein 4.1R Affects Repolarization and Regulates Calcium Handling in the Heart
The 4.1 proteins are a family of multifunctional adaptor proteins. They promote the mechanical stability of plasma membranes by interaction with the cytoskeletal proteins spectrin and actin and are required for the cell surface expression of a number of transmembrane proteins. Protein 4.1R is expressed in heart and upregulated in deteriorating human heart failure, but its functional role in myocardium is unknown. To investigate the role of protein 4.1R on myocardial contractility and electrophysiology, we studied 4.1R-deficient (knockout) mice (4.1R KO). ECG analysis revealed reduced heart rate with prolonged Q-T interval in 4.1R KO. No changes in ejection fraction and fractional shortening, assessed by echocardiography, were found. The action potential duration in isolated ventricular myocytes was prolonged in 4.1R KO. Ca2+ transients were larger and slower to decay in 4.1R KO. The sarcoplasmic reticulum Ca2+ content and Ca2+ sparks frequency were increased. The Na+/Ca2+ exchanger current density was reduced in 4.1R KO. The transient inward current inactivation was faster and the persistent Na+ current density was increased in the 4.1R KO group, with possible effects on action potential duration. Although no major morphological changes were noted, 4.1R KO hearts showed reduced expression of NaV1.5α and increased expression of protein 4.1G. Our data indicate an unexpected and novel role for the cytoskeletal protein 4.1R in modulating the functional properties of several cardiac ion transporters with consequences on cardiac electrophysiology and with possible significant roles during normal cardiac function and disease.
The cardiac cytoskeleton is important in conferring stability to the myocardium, in sensing the mechanical stretch, and in coordinating the assembly of cellular structures and intercellular signaling.1 One group of cytoskeletal proteins, the spectrin- and ankyrin-associated system, is involved in the complex interplay between actin, spectrin, and various ion transporters in relation to the regulation of intracellular [Ca2+].2 Beta II spectrin, muscle LIM-only protein, and ankyrin B and G have all been associated with cardiac function and regulation of the electrophysiological properties of the myocardium.3 The 4.1 protein family is also part of the spectrin-associated cytoskeleton. It promotes the interaction between spectrin and F-actin and, thus, membrane stability.2 In mammals, the 4 genes, EPB41, EPB41L1, EPB41L2, and EPB41L3, encode proteins 4.1R, 4.1G, 4.1N, and 4.1B. mRNA transcripts from all 4 genes are found in mouse myocardium.4 All 4.1 proteins have a FERM membrane-binding domain, a spectrin–actin binding (SAB) domain, and a C-terminal domain.2 A FERM-adjacent domain regulates the activities of both FERM and SAB domains.5 The FERM and C-terminal domains bind to membrane proteins,6 whereas the SAB domain binds the spectrin–actin cytoskeleton.7 Multiple ion channels, pumps, and exchangers located in the plasma membrane are believed to directly interact with members of the 4.1 protein family. The cytoskeletal docking complex not only regulates cell shape, confers mechanical stability, and permits communication across the cell membrane but may additionally function to cluster and stabilize ion transporters to regions of the plasma membrane to form microdomains with specialized roles.8
Protein 4.1R has been associated with the progression of heart failure. We have previously reported that EPB41 mRNA expression is increased in patients with deteriorating heart failure undergoing ventricular assist device surgery compared with patients with stable heart failure.9 Given the limited information available on the roles of this protein in the normal and diseased myocardium, whether protein 4.1R takes direct part in the mechanical and electrophysiological changes associated to cardiac disease is unclear. It is known that protein 4.1R, like other members of this family, is compartmentalized within myocytes. It has been identified at subcellular locations that include the Z/I-band, M-line, intercalated disks, and lateral plasma membrane and within the nucleus of mouse myocytes.4 4.1R is required for cell surface expression of PMCA4, a plasma membrane Ca2+ATPase.3 However, the functional role of protein 4.1R in the myocardium is unknown.
To study the role of protein 4.1R on myocardial function and regulation, we used protein 4.1R–deficient (knockout) mice (4.1R KO) previously characterized by Shi et al.10 In these animals, we measured echocardiographic and ECG parameters and we analyzed cellular functional properties, including action potentials and Ca2+ homeostasis, by the assessment of the functional activity of several Na+, Ca2+, and K+ transporters. We demonstrated that the lack of protein 4.1R results in defects of repolarization and cytoplasmic [Ca2+] regulation, and we identified the involvement of a number of cardiac ion transporters, suggesting a significant role of protein 4.1R in the regulation of myocardial function.
Materials and Methods
4.1R KO, bred in C57bl/6 background, were kindly provided by Dr Phillipe Gascard (Life Science Division, Ernest Orlando Lawrence Berkeley National Laboratory, Berkeley, Calif).10 Only 6-month-old male mice were used in these experiments, and C57bl/6 male mice from Harlan (UK) were used as controls. Echocardiography was performed using a 15-MHz probe on an Acuson Sequoia 256 system in M-mode. ECG recording was performed in conscious mice using a radiotelemetry system (Data Science International, St Paul, Minn).
Cardiomyocytes were enzymatically dissociated and examined with a ×60 objective. Cell planimetry was performed using ImageJ software (http://rsb.info.nih.gov/ij). For Ca2+ transient experiments, myocytes loaded with indo-1-acetoxymethyl ester (Molecular Probes) were field-stimulated at 37°C. Confocal studies were performed in myocytes loaded with fluo-4 acetoxymethyl ester. To measure Ca2+ transient synchronicity, time-to-peak of Ca2+ transients at each x pixel was measured and the variance calculated and taken as a measure of synchronicity of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). Action potentials were measured in current-clamp mode. Measurement of ion transporter current density was carried out using the whole-cell configuration of the patch-clamping technique. Pipette resistance was 1.5 to 2.5 MΩ.
Electron Microscopy, Immunofluorescence, and Western Blotting
Thin sections for electron microscopy and 0.2-μm frozen sections for immunofluorescence were prepared and analyzed as described previously.11 Heart homogenates were prepared for Western blot analysis as previously described.4 A total of 100 μg of protein was loaded per lane of SDS polyacrylamide gels.
Statistical comparison of data was performed using a 2-way ANOVA or Student’s t test where appropriate. Data are expressed as means±SEM (n) unless otherwise specified. For cellular studies, n is the number of myocytes. In the figures, * indicates P<0.05; **, P<0.01; and ***, P<0.001.
The in vivo assessment of cardiac function in 4.1R KO mice was performed using M-mode echocardiography and ECG recording. Left ventricular ejection fraction and fractional shortening were not significantly different between control (wild type [WT]) and 4.1R KO (left ventricular ejection fraction [%]: WT, 67.7±2.7 ; 4.1R KO, 64.6±2.8 ; fractional shortening [%], WT, 32.8±2 ; 4.1R KO, 30.8±2 ). The ECG analysis (Figure 1 and Table I in the online data supplement, available at http://circres.ahajournals.org) showed a significant reduction in heart rate in 4.1R KO compared with control (WT) (Figure 1B and 1C). This was accompanied by a significantly prolonged Q-T interval (Figure 1D through 1F). At rest, the changes in ECG parameters were not accompanied by increased arrhythmic events, which remained very low in both groups (number of premature ventricular contractures per hour: WT, 1.75±0.81 ; 4.1R KO, 2.16±0.87 ; number of supraventricular arrhythmias per hour [including AV blocks and atrial ectopic beats], WT, 4.65±0.5 ; 4.1R KO, 5±0.5 ).
Heart weight and heart weight-to-body weight ratio were unchanged in 4.1R KO mice (heart weight [g] WT, 0.277±0.01 ; 4.1R KO, 0.245±0.01 ; heart weight-to- body weight ratio (g/kg): WT, 9.04±0.56 ; 4.1R KO, 7.99±0.3 ). Cardiomyocyte size measured with planimetry (supplemental Figure IA through IC), and cell capacitance (supplemental Figure ID) was also unchanged in the 4.1R KO heart.
The different proteins 4.1 have been localized in close proximity to Ca2+ handling proteins in heart muscle,3 but whether they are implicated in the regulation of intracellular [Ca2+] is unknown. Ca2+ transients were recorded from 4.1R KO and WT myocytes. Indo-1 transient amplitude was increased by approximately 33% (Figure 2B), and Indo-1 decay was delayed by approximately 16% (Figure 2C) in 4.1R KO. There was no difference in the time-to-peak or diastolic levels.
To investigate the causes for these changes in Ca2+ transients, a number of specific studies were performed. Firstly, the role of the SR was investigated as previously described.12 SR Ca2+ content was significantly increased by approximately 28% in the 4.1R KO myocytes (Figure 2D). No difference could be detected in fractional release (4.1R KO, 0.74±0.03 ; WT, 0.7±0.08 ; P>0.05) nor the relative contribution of the SR Ca2+ uptake to Ca2+ removal from the cytoplasm (4.1R KO [%], 92±1.5 (9 mice); WT, 95±1.1 ; P>0.05).
The ability of the unitary ryanodine receptor clusters to spontaneously release Ca2+ during rest (Ca2+ sparks), as an indication of SR Ca2+ release function, was also investigated. Ca2+ spark frequency and duration were increased in the 4.1R KO myocytes (Figure 3A through 3C), without changes in fluorescence peak and width (Figure 3D and 3E). Whether this is the consequence of the increased SR Ca2+ content13 or a direct effect of the protein 4.1R deficiency on the SR Ca2+ release unit is unclear.
Local Ca2+-induced Ca2+ release (CICR) in protein 4.1R KO myocytes was further investigated by measuring the synchronicity of Ca2+ release during the Ca2+ transient (Figure 3F). Despite the observation that the global time-to-peak of the Ca2+ transients was unchanged, the variance of the time-to-peak values measured at each pixel on line scan was significantly increased in 4.1R KO myocytes (Figure 3G).
We have previously described a colocalization of protein 4.1R with the plasmalemmal Ca2+ ATPase (PMCA) in cardiac myocytes.3 Ca2+ extrusion carried out by PMCA in the 4.1R KO and WT myocytes was therefore investigated. Using experimental conditions where SR Ca2+ uptake, Na+/Ca2+ exchanger (NCX) and mitochondrial uptake are inhibited14 no significant difference between the groups could be detected (time constant of decline: 4.1R KO, 8.1±0.6 seconds ; WT: 9.8±0.8 seconds ; P=NS), suggesting that PMCA is not involved in the changes in Ca2+ transients observed in protein 4.1R KO myocytes.
The prolonged Q-T interval observed in the ECG from 4.1R KO mice suggests electrophysiological modifications when protein 4.1R is deficient. Figure 4 shows that action potentials recorded in 4.1R KO mice were significantly prolonged. This prolongation was detected at all frequencies studied (1, 3 and 5 Hz). The resting membrane potential was not changed (4.1R KO, −69±1.2 mV ; WT, −68±1.8 mV ).
Action potential prolongation can result from modifications of most ion transporters function.15 Given the importance of NCX and Na+/K+ ATPase (NKP) function in determining Q-T prolongation in mice with ankyrin B mutations,16 the electrophysiological properties of these 2 ion transporters were studied. The NCX current was significantly reduced in 4.1R KO myocytes compared with WT (Figure 5A and 5B), suggesting a relationship between protein 4.1R and NCX. In contrast, the NKP function was not affected by the deficiency of protein 4.1R (supplemental Figure II).
The reduction in NCX function, together with the prolonged action potential, can partially explain the increased SR Ca2+ content and the slower Ca2+ decline observed in protein 4.1R KO myocytes; however, other ion transporters must be responsible for the action potential prolongation observed in 4.1R KO mice. We investigated 3 other ion currents which contribute to the plateau phase of the action potential: the transient outward current Ito for its predominant role in repolarization of the mouse myocardium,17 the L-type Ca2+ current ICa and, the persistent Na+ current IpNa. Despite a similar steady-state current–voltage relationship in the 2 groups, Ito inactivated more rapidly in 4.1R KO myocytes (Figure 5C through 5F). ICa was not affected by the 4.1R deficiency (ICa density at 0 mV: 4.1R KO, −4.2±0.25 pA/pF ; WT, −4.04±0.25 pA/pF ; P=NS). The voltage–current relationship and the voltage-dependent activation and inactivation of ICa were all unchanged (data not shown). IpNa density, calculated as the 30 μmol/L tetrodotoxin–sensitive current (Figure 6) was significantly increased at −20 mV in 4.1R KO myocytes. In addition, the integral of this current between 50 and 300 ms was also significantly increased in the 4.1R KO myocytes. The combined faster inactivation of Ito and increased IpNa can contribute to the prolongation of the action potential.
To investigate the effects of loss of protein 4.1R on heart structure, we used electron microscopy and immunofluorescence (Figure 7). Thin sections of left ventricle did not reveal major structural alterations between WT (Figure 7A) and 4.1R KO (Figure 7B). In particular, the appearances of the sarcomeres, the T-tubules, SR, and mitochondria were not substantially changed. The intercalated disks and transition zones11 were also not substantially affected. Initial immunofluorescence using antibodies to vinculin and β-catenin (not shown) also revealed that the general organization of the plasma membrane (intercalated discs, lateral plasma membrane, and costameres) was not changed.
Immunofluorescence (Figure 7C) revealed the same pattern for 4.1R in WT myocardium as described previously,4 namely staining at intercalated discs and lateral plasma membrane, plus internal striations not separable by light microscopy from α-actinin (ie, Z-disks or T-tubules) (Figure 7D and 7E). Similarly, staining for NaV1.5 and NCX1 was present at intercalated discs, lateral plasma membrane and internal striations (presumably T-tubules) as described by others.16,18
In the 4.1R KO hearts, some residual 4.1R immunoreactivity was detectable, in the same locations as in the WT hearts (Figure 7L). We attribute this to the way the 4.1R KO mice were constructed10: of the 3 initiation codons in 4.1R, only AUG1 and 2 are eliminated. It might be expected that our anti-4.1R antibody recognizes products initiated at AUG3. Figure 7N through 7S reveals no significant disorganization of NCX1 or NaV1.5; in each case, staining is detectable at lateral plasma membranes, intercalated disks, and internal striations.
Finally, we investigated the abundance of proteins that might be relevant to the phenotype we describe above using Western blotting. Figure 8A shows (left) comparative Coomassie blue–stained gels of the homogenates (100 μg protein per lane) and (right) immunoblots probed with an anti-4.1R antibody directed against epitopes in the spectrin–actin binding domain. As expected, the major 80-kDa isoform (translated from AUG2) is completely lost. In the generation of these 4.1R KO mice, the third initiation codon, AUG 3, was not eliminated: an immunoreactive band of lower molecular mass was detected consistent with expression from AUG3. This 40-kDa band was not significantly altered relative to WT tissue.
Protein 4.1G was increased in 4.1R KO myocardium 1.9-fold relative to WT (Figure 8B), but there was no significant change in either 4.1B or 4.1N (Figure 8C and 8D). Figure 8E through 8G show that the expression of the actin-binding proteins α-actinin, spectrin, and tropomyosin (TM) is also unaltered: of these, spectrin and TM also bind 4.1R.19,20
We also tested whether alterations in ion transporter activity were associated with changes in protein expression. Figure 8H shows that the level of the α-subunit of NaV1.5 is reduced by approximately 40% in 4.1R KO myocardium. The expression of NCX1 (Figure 8I) and PMCA2 (Figure 8J) were not significantly changed.
In this study, we show that deficiency of protein 4.1R in mice results in Q-T prolongation without apparent changes in left ventricular function at rest. At myocyte level, deficiency of protein 4.1R is accompanied by prolongation of the action potential duration, increased amplitude, and slower decay of Ca2+ transients. At the subcellular level, the deficiency of protein 4.1R affects local calcium-induced calcium release, reduces the NCX activity, hastens the inactivation of Ito, and increases IpNa. The functional changes are associated with increased expression of protein 4.1G and decrease expression of NaV1.5α subunit.
These results indicate a role of protein 4.1R on the function of several ion transporters affecting cardiac electrophysiology.
Cytoskeleton and Ion Transporter Function
There is increasing evidence that ion transporters exist in the context of macromolecular complexes formed not only by the ion channel subunits but also by regulatory kinases and phosphatases, proteins from the extracellular matrix, from trafficking, other ion channels, and the cytoskeleton.21 The importance of the cytoskeleton in the regulation of cardiac ion transporter function and generation of cardiac arrhythmias is becoming progressively established.22 The specific role of the spectrin–ankyrin cytoskeleton is of particular interest for the relationship between some of its components and cardiac arrhythmias in humans.23 Mutations of ankyrins for example have been clearly associated to congenital arrhythmias.16,18 In general, beta II spectrin and ankyrin B and G have all been associated with cardiac dysfunction.3
The functional roles of proteins 4.1 in heart have not been investigated previously. Protein 4.1R in particular is of interest because its gene expression changes during the progression of heart failure in patients.9 Protein 4.1R is localized in the proximity of ion transporters in cardiac cells and, more specifically, is required for cell surface targeting of PMCA4.3 Although PMCA has a minimal role in Ca2+ extrusion in normal adult cardiac myocytes,24 it can be important in Ca2+ regulation when compensation for dysfunction in other Ca2+ regulatory mechanisms is required.25 However, 4.1R deficiency did not appear to affect PMCA-mediated Ca2+ extrusion function. Other aspects of PMCA function, particularly signal transduction pathways, may be altered in the 4.1R KO mice, and more studies are required.26
Defects of Ca2+ Handling in Protein 4.1R KO Myocytes
Protein 4.1R KO myocytes showed increased Ca2+ transients amplitude and slower Ca2+ extrusion. Whereas these changes were significant at myocyte level, they did not influence whole heart basal function. Whether this is attributable to compensatory mechanisms present in this transgenic model is unclear. Also whether these changes may become relevant during exercise, stress, or disease is unknown, and further studies in this direction are warranted.
The increased amplitude of the Ca2+ transients could be ascribed to increased SR Ca2+ content in the absence of changes in the trigger for Ca2+ release ICa. Two possible causes for the increased SR Ca2+ content can be identified from this study: the prolonged action potential27 and the reduced NCX activity. Despite a normal NKP function, an increased cytoplasmic [Na+] as the consequence of the increased IpNa is expected in protein 4.1R KO myocytes.28 The combined effects of reduced intrinsic NCX activity, the prolonged action potential, and the increased cytoplasmic [Na+] can favor SR Ca2+ uptake during diastole, resulting in increased SR Ca2+ content and slower Ca2+ extrusion.
Changes in the microarchitecture of the cardiac myocyte have been implicated in defects of excitation–contraction coupling during hypertrophy and failure, resulting in inefficient CICR.29 The role of the cytoskeleton in this process is unclear. Protein 4.1R KO myocytes showed an increased variance of the local CICR and disruption to the synchronous Ca2+ release. This may suggest disruption of the spatial relationship between L-type Ca2+ channels and ryanodine receptors and incorrect coupling. The increased Ca2+ spark frequency may also suggest alteration in local SR Ca2+ release, as it has been suggested for defects of ankyrins.23 The increased SR Ca2+ content observed in this study, however, can be responsible for increased Ca2+ spark frequency.13 The prolonged repolarization observed in these myocytes can also be responsible for impaired synchronicity of CICR.30 Therefore, a direct relationship between protein 4.1R deficiency and ryanodine receptor activity cannot be concluded from our results. In general, as for other ion transporters, the relationship may be indirect, resulting from disruption of mechanisms involved in regulating the ion transporters, and this point warrants further studies.
Defects of Repolarization in Protein 4.1R KO Myocytes
Among the different currents implicated in shaping the action potential in murine myocytes, we studied ICa, Ito, and IpNa. ICa was unaffected in all parameters studied. Whereas the Ito density–voltage relationship was unchanged in protein 4.1R KO myocytes, the inactivation of Ito was faster with possible effects on repolarization. Furthermore, the increased depolarizing current IpNa can also be responsible for delayed repolarization.31 The relationship between the Na+ current and Q-T prolongation is well established and seems to be important in both congenital and acquired cardiomyopathies.32
Interactions Between Protein 4.1R and Ion Transporters
Our results indicate the existence of primary functional effects of the lack of protein 4.1R on NCX, Ito, and IpNa. The effects on SR Ca2+ handling and local CICR may be secondary, but this needs to be tested. The exact molecular mechanisms involving protein 4.1R, and capable of regulating these functions, are unclear at this stage, and further studies are required. Protein 4.1R, like other members of this family, is compartmentalized within myocytes. It has been located at several subcellular locations that include the Z/I-band, M-line, intercalated disks, and lateral plasma membrane and within the nucleus of mouse myocytes. Little is known about its precise role at these locations.3 One interesting aspect is that the SAB domain is preserved in cardiac 4.1R but is lacking in brain isoforms. This may reflect a role in maintaining strong interactions between the cytoskeleton and the contractile apparatus in the heart.7 As a consequence, the ability of protein 4.1R to regulate ion transporter function could be 1 of the links between mechanical load and regulation of function.
In patients with deteriorating heart failure, mRNA for 4.1 proteins were differentially overexpressed.9 Given this, it was possible that 1 or more 4.1 protein was upregulated to compensate for the loss of 4.1R in the knockout mice. We noted nearly double the intensity of the 4.1G band in the 4.1R KO mice (Figure 8B). Expression of 4.1G may be linked to that of 4.1R.
Of the ion currents we analyzed, it was striking that IpNa was increased (Figure 6). Because this current is linked to NaV1.5, we analyzed the channel-forming α-subunit by immunoblot (Figure 8H). This was reduced by approximately 40% in the 4.1R KO heart. This result was surprising, because it indicates that 4.1R has a role in modulating the cellular content of the protein and also its activity. It is not clear whether this is a direct or indirect effect. 4.1 proteins bind numerous ion channels (eg, the erythrocyte anion exchanger and both ionotropic and metabotropic glutamate receptors6,33,34). 4.1R is required for the stable cellular accumulation of glycophorin C in erythrocytes, through its direct binding to the glycophorin C cytoplasmic domain. 4.1R-NaV1.5 interaction may be analogous. However, in erythrocytes, glycophorin C is lost,18 unlike NaV1.5 in heart. Because 4.1G is upregulated in 4.1R KO heart (Figure 8B) it might be hypothesized that 4.1G compensates in part for the loss of 4.1R in this tissue.
It is also interesting that NaV1.5 binds ankyrin, and interaction with ankyrin is required for its stable cellular accumulation.18 4.1R and ankyrin have an overlapping spectrum of interactions with membrane proteins: for example, both the erythrocyte anion exchanger and CD44 binds 4.1R and ankyrin.33,35,36 In these cases, there are distinct sites for interaction with each protein.
NCX activity is also altered in the 4.1R KO mice. Again, NCX1 binds ankyrin, and ankyrin interaction is required for its retention in cardiac membranes.16 However, we find no evidence for loss of NCX1 protein from the 4.1R KO hearts (Figure 8I). Whether 4.1R has a regulatory role in NCX1 activity through direct interaction remains an open question.
In conclusion, deficiency of protein 4.1R in mice results in defects of repolarization and Ca2+ regulation in the heart that are, at least in part, explained by changes in transient outward current, Na+ currents, and NCX function. This study has shown that protein 4.1R has an important role in determining the functional properties of several ion transporters, with possible significance during normal cardiac function and disease.
We are grateful to Amanda J. Wilson (King’s College London) for cutting sections of mouse heart.
Sources of Funding
Supported by The Magdi Yacoub Institute and the Medical Research Council.
Original received March 28, 2008; revision received August 18, 2008; accepted September 3, 2008.
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