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Am J Physiol Heart Circ Physiol 294: H2391-H2399, 2008. First published March 7, 2008; doi:10.1152/ajpheart.00011.2008
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Contractile regulation by overexpressed ETA requires intact T tubules in adult rat ventricular myocytes

Ka Young Chung, Misuk Kang, and Jeffery W. Walker

Molecular and Cellular Pharmacology Program and Department of Physiology, University of Wisconsin, Madison, Wisconsin

Submitted 4 January 2008 ; accepted in final form 6 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Endothelin (ET)-1 regulates the contractility and growth of the heart by binding G protein-coupled receptors of the ET type A receptor (ETA)/ET type B (ETB) receptor family. ETA, the predominant ET-1 receptor subtype in myocardium, is thought to localize preferentially within cardiac T tubules, but the consequences of mislocalization are not fully understood. Here we examined the effects of the overexpression of ETA in conjunction with T-tubule loss in cultured adult rat ventricular myocytes. In adult myocytes cultured for 3 to 4 days, the normally robust positive inotropic effect (PIE) of ET-1 was lost in parallel with T-tubule degeneration and a decline in ETA protein levels. In these T tubule-compromised myocytes, an overexpression of ETA using an adenoviral vector did not rescue the responsiveness to ET-1, despite the robust expression in the surface sarcolemma. The inclusion of the actin polymerization inhibitor cytochalasin D (CD) during culture prevented gross morphological changes including a loss of T tubules and a rounding of intercalated discs, but CD alone did not rescue the responsiveness to ET-1 or prevent ETA downregulation. The rescue of a normal PIE in 3- to 4-day cultured myocytes required both an increased expression of ETA and intact T tubules (preserved with CD). Therefore, the activation of ETA localized in T tubules was associated with a strong PIE, whereas the activation of ETA in surface sarcolemma was not. The results provide insight into the pathological cardiac conditions in which ETA is upregulated and T-tubule morphology is altered.

cytochalasin D; inotropism; endothelin type A receptor; heart failure

THE POTENT vasoconstrictor peptide endothelin (ET)-1 is produced not only by vascular endothelial cells (46) but also by endocardial endothelial cells and cardiac myocytes (14). In the heart, ET-1 produces inotropic, chronotropic, and growth-promoting actions (30, 38). Among these physiological effects of ET-1, the regulation of contractility has received considerable attention, with a positive inotropic effect (PIE) reported in rat, mouse, rabbit, ferret, and human but not in guinea pig or dog myocardium (18, 22, 43–45). Biological functions of ET-1 are mediated by two G protein-coupled receptor (GPCR) subtypes, ET type A receptor (ETA) and ET type B receptor (ETB), and both have been identified in various tissues including ventricular myocytes (38). In normal ventricular myocardium, ~60–85% of ET-1 receptors are ETA (8, 20, 27, 32), and the contractile regulation by ET-1 appears to be mediated by ETA (9, 19).

ET-1 has also been implicated in the pathophysiology of various cardiovascular disease states including hypertension, hypertrophy, and heart failure. Numerous studies have demonstrated increased plasma and tissue levels of ET-1 and an increased expression of ETA in diseased human myocardium and in animal models of heart failure (1, 11, 20, 28, 31, 33, 39, 49). Furthermore, ETA selective or nonselective antagonists have shown beneficial effects in some animal studies and in clinical trials although other studies have failed to find benefits (6, 28, 34).

Cardiac T tubules are invaginations of the surface membrane occurring in the vicinity of Z-lines, which facilitate the propagation of external signals into the inner myocyte cross section (5). T tubules display a unique composition of membrane lipids and Ca2+, Na+, and K+ transport proteins along with numerous signaling molecules. Key sarcoplasmic reticulum proteins such as ryanodine receptors and sarco(endo)plasmic reticulum Ca2+-ATPase pumps are also concentrated at Z-lines adjacent to T tubules (5). Previous studies reported that ETA is mainly localized at T tubules in adult ventricular myocytes (3, 37), but the physiological and pathophysiological significance of the T-tubular localization of ETA is not fully understood. In pathological conditions such as failing myocardium, the structure of T tubules appears to change both in humans and animal models. The majority of studies have described a reduced or disorganized T tubule structure in heart failure although an increase of T tubule density has also been reported (2, 12, 17, 24, 41).

In the present study, we employed an adult rat myocyte model of spontaneous T-tubule degeneration in cell culture to examine the importance of T tubules in ETA function. In conjunction with the overexpression of cyan fluorescent protein (CFP)-tagged ETA, this cellular model reinforced earlier findings that a T-tubular localization of ETA is crucial for the contractile regulation in adult ventricular myocytes (37). This model also permitted a comparison of the functional consequences of the activation of ETA in T tubules versus surface sarcolemma (when T tubules are lost). The results shed light on the altered ET-1 signaling in cardiac disease states that is accompanied by the remodeling of myocyte membranes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. All reagents were obtained from Sigma Chemical (St. Louis, MO) unless noted otherwise. Collagenase was from Worthington (Lakewood, NJ). Complete protease inhibitor cocktail was from Roche (Mannheim, Germany). ETA monoclonal antibody was from BD Biosciences (San Jose, CA), and Alexa 488-conjugated secondary antibodies were from Molecular Probes (Eugene, OR).

Isolation of adult rat ventricular myocytes. Animal handling practices used in this study have been reviewed by and received approval from the Animal Care Committee of the University of Wisconsin. Ventricular cardiac myocytes were isolated from adult male Sprague-Dawley rats by enzymatic digestion with collagenase and hyaluronidases, as previously described (13). The myocytes were maintained in 1 mM Ca2+ Ringer solution containing (in mM) 125 NaCl, 5 KCl, 2 NaH2PO4, 5 sodium pyruvate, 1.2 MgSO4, 11 glucose, 0.5 CaCl2, and 25 HEPES (pH 7.4).

Primary culture and adenoviral infection of adult rat ventricular myocytes. Isolated myocytes were resuspended in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum, 50 U/ml penicillin and 50 µg/ml streptomycin and (in mM) 1 CaCl2, 5 taurine, 5 carnitine, and 5 creatine. The cells were plated on laminin-coated coverslips by incubating at 37°C, 5% CO2-95% room air for 2 h. After 2 h incubation, culture medium was replaced with adenovirus-diluted serum-free DMEM. After 1 h incubation, serum-free DMEM was added and changed every day.

Adenovirus construction. A human ETA was fused to the NH2 terminus of CFP in a pShuttle vector driven by cytomegalovirus promoters (Stratagene). ETA-CFP-carrying adenoviruses were generated using an AdEasy adenoviral vector system (Stratagene) according to the manufacturer's instructions. The viruses were amplified and purified by the use of a ViraKit AdenoMini-4 purification kit (Virapur, San Diego, CA).

Western blot analysis. Proteins separated by SDS-PAGE were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). Nonspecific sites were blocked by Blotto containing 150 mM NaCl, 20 mM Tris (pH 7.4), 0.05% (vol/vol) Tween 20, and 5% powdered milk for 1 h at room temperature, and Western blot analysis was carried out with enhanced chemiluminescence detection (GE Healthcare).

Immunofluorescence. The myocytes were skinned with 100 µg/ml saponins in relaxing solution containing (in mM) 100 KCl, 1 MgCl2, 2 EGTA, 4.5 ATP, and 10 imidazole (pH 7.0) and then washed and blocked by 2% bovine serum albumin in relaxing solution. Skinned myocytes were incubated with primary antibody overnight at 4°C. Following an extensive wash with 2% bovine serum albumin in relaxing solution, the myocytes were incubated with Alexa 488-conjugated anti-mouse IgG secondary antibody (diluted 1:200) for 1 h at room temperature. After the myocytes were extensively washed, images were acquired with a Bio-Rad MRC 1024 laser-scanning confocal microscope equipped with an argon/krypton laser controlled by 24-bit LaserSharp software.

Twitch measurements. The myocytes were resuspended in 1 mM Ca2+ Ringer solution. Cell twitches were initiated by electric field stimulation with a SD9 stimulator (Grass Instrument) in a modified PH1 chamber (Warner Instrument) mounted on a Nikon Diaphot inverted microscope. The stimulation protocol was 0.5 Hz, 10-ms duration, and 50 V at room temperature. Individual myocytes were monitored with a model VED 104 video edge detector (Crescent Electronics), and cell shortening was recorded using Felix software (Photon Technology).

Confocal imaging and image analysis. Confocal images were obtained with a Bio-Rad Radiance 2100 laser-scanning confocal microscope. Quantitative analysis of T tubules and ETA-CFP expression was performed with National Institutes of Health ImageJ software. To analyze T tubule structures quantitatively, two complementary approaches were used. First, myocyte images were processed using fast Fourier transform (FFT), and the intensity of first harmonic of the transformed image was measured. The intensity of first harmonic was normalized by the whole cell area. Second, the percent area was measured. Before analysis, pixels positive for 4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl}1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS) or ETA-CFP were distinguished from negative pixels by the use of a threshold. The percentage of area was obtained by dividing the integrated intensity (total number of pixels above threshold) of the myocyte interior (excluding surface sarcolemma) by the total cell area. Expression patterns of ETA-CFP in subcellular structures of myocytes were also quantified using two methods. One is the intensity of first harmonic described above. The other is percent total intensity. The percent total intensity was obtained by dividing the integrated intensity in a designated area (percinucleus, cell interior, or sarcolemma) by the total integrated intensity.

Quantification of ETA-CFP overexpression. The radiance confocal microscope was calibrated as described previously (16), by the use of a purified bacterial expressed CFP construct provided by Dr. Dapankar Battacharaya. The concentration of the standard CFP protein was estimated by a bicinchoninic acid protein assay and by Coomassie blue binding on a SDS-PAGE gel using bovine serum albumin as a protein standard. With the use of the same microscope settings for the acquisition of cell images (laser power, pin hole dimensions, black levels, objective, etc.), the mean fluorescence was measured as a function of the concentration CFP standard in the solution. The mean fluorescence expressed within ventricular myocytes was then used to estimate the effective fluorescence concentration by comparison with the CFP standard curve, resulting in values of 680 ± 72 and 930 ± 94 nM at 2 and 3 days of culture, respectively. The endogenous ETA concentration was estimated at 106 receptor sites/30 pl per myocyte (37, 40; unpublished data) or 55 nM, such that the levels of overexpression were 12- and 17-fold at 2 and 3 days of culture, respectively.

Statistical analysis. Data are expressed as means ± SE and analyzed using an unpaired Student's t-test or a one-way ANOVA (where appropriate). Values of P < 0.05 were considered to be significant for both statistical tests.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
To address the localization pattern and physiological function of ETA in myocytes with compromised T tubules, isolated adult rat ventricular myocytes (ARVMs) were cultured for up to 3 to 4 days. It is widely recognized that adult rat myocytes lose their rectangular structure and T tubule integrity upon culture (21). Two different strategies were used to quantify changes in T tubule structure during culture: a standard measure of percent myocyte area occupied by T tubules (after thresholding) and a FFT of images to quantify periodicity. The first of these methods (percent area) was adopted in most previous studies to quantify the amount of plasma membrane located inside of myocytes (2, 12, 13), but this method falls short of quantifying organized T tubules. To address this limitation, our laboratory (36) and others (41) have used the FFT analysis, which transforms a two-dimensional fluorescence cell image into a frequency domain image that quantifies periodicity. For ventricular myocytes, the first harmonic in the frequency domain represents the strongest periodicity (which, based upon the spacing between harmonics, represents sarcomeric or T tubule spacing), and the brightness of the first harmonic quantifies its magnitude in the original image. Both analysis methods were performed on the same confocal fluorescence images and showed that T tubule structures were significantly reduced in 3-day cultured ARVMs (Fig. 1).


Figure 1
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  		Fig. 1. Loss of T tubule upon culture. Adult rat ventricular myocytes (ARVMs) were isolated and cultured up to 4 days. Either fresh isolated or cultured ARVMs were stained with 4-{2-[6-(dioctylamino)-2-naphthalenyl]ethenyl} 1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS) to visualize membrane. T-tubule structures were analyzed as described in MATERIALS AND METHODS. A: representative membrane-stained ARVMs (middle), thresholded images (left), and fast Fourier transformed (FFT) images (right). B: quantification of percent area. C: quantification of intensity of first harmonic. *P < 0.05 compared with fresh isolated ARVMs; n ≥ 10. Scale bar = 10 µm.

 
The inotropic response to ET-1 was examined to investigate the functional consequences of T-tubule loss. After 10 min exposure to 10 nM ET-1, a 44 ± 15% PIE was observed in the fresh isolated ARVM, but this inotropic effect was lost after 3 days in culture (Fig. 2A). In parallel with this loss of contractile regulation and the disruption of T-tubule morphology, there was also a dramatic decrease in endogenous ETA localized to T tubules as assessed by immunostaining (Fig. 2B). The level of total endogenous ETA expression monitored by Western blot analysis also decreased by 68 ± 3% over 3 days in culture (Fig. 2C).


Figure 2
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  		Fig. 2. Endothelin type A receptor (ETA) system in cultured ARVMs. A: contractile responses to 10 nM ET-1 in fresh isolated vs. 3-day cultured ARVMs. To examine positive inotropic effect (PIE), twitch amplitudes were measured before (0 min) and 10 min after ET-1 treatment and plotted as percent increase over 0 min. *P < 0.05 compared with fresh isolated ARVMs; +P < 0.05 compared with before ET-1 treatment; n ≥ 10. B: expression pattern of ETA in fresh isolated vs. cultured ARVMs stained with a ETA-specific primary antibody. C: overall expression levels of ETA in fresh isolated vs. cultured ARVMs. Cell lysate samples were separated by SDS-PAGE, and Western blot analyses were performed using a ETA-specific antibody. *P < 0.05; n ≥ 4. Scale bar = 10 µm.

 
For comparison, inotropic responses to other stimuli were evaluated in myocytes cultured for 3 days. An increase in the extracellular Ca2+ concentration from 1 to 2 mM produced a 80 ± 32% PIE in fresh isolated myocytes and a 46 ± 17% PIE in 3-day cultured myocytes, but this difference was not statistically significant (Fig. 3). Isoproterenol (10 nM) induced a 151 ± 25% PIE in fresh isolated myocytes and a 91 ± 18% PIE in 3-day cultured myocytes, and this difference was also not statistically significant (Fig. 3). These results suggest that, despite the lack of responsiveness to ET-1, the contractile and inotropic potential is otherwise largely preserved in 3-day cultured myocytes.


Figure 3
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  		Fig. 3. Other inotropic responses in cultured ARVMs. Extracellular Ca2+ concentration ([Ca2+]out) was increased from 1 to 2 mM in fresh isolated or 3-day cultured ARVMs. Alternatively, 10 nM isoproterenol (Iso) was perfused onto fresh or cultured ARVMs. To examine PIE, twitch amplitudes were measured before (0 min) and 3 min after the Ca2+ increase or the treatment with Iso. *P < 0.05 compared with before treatment; n ≥ 8.

 
To address the mechanism of this loss of ET-1 responsiveness, ETA-CFP was overexpressed to compensate for the decrease in ETA. In 2-day cultured ARVMs displaying relatively intact T tubules, ETA-CFP was localized at the perinuclear (PN) region, T tubules, and sarcolemma and gave a periodic pattern with the FFT analysis (Fig. 4, A and B). The T tubule and sarcolemmal (SL) ETA also colocalized with the membrane marker di-8-ANEPPS (Fig. 4A). However, when T tubules were lost in 3- or 4-day cultured cells, ETA-CFP was located primarily at the perinucleus and sarcolemma, and the periodic FFT pattern was lost (Fig. 4, A and B). At 2 days in culture, the subcellular distribution of ETA based on the total CFP fluorescence was 18 ± 2% PN, 52 ± 2% intracellular structures including T tubules (Int), and 31 ± 2% SL (Fig. 4C). By 3 to 4 days in culture, the subcellular distribution changed to 12 ± 2% PN, 30 ± 2% Int, and 60 ± 4% SL (Fig. 4C).


Figure 4
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  		Fig. 4. ETA-cyan fluorescent protein (CFP) expression in cultured ARVMs. ETA-CFP was introduced to isolated ARVMs by adenoviral infection, and expression patterns were analyzed as described in MATERIALS AND METHODS. A: representative images of ETA-CFP-expressing ARVMs. Insets: FFT images. B: quantification of intensity of first harmonic (n ≥ 15). C: quantification of percent total intensity. PN, perinuclear region; Int, intracellular structures including T tubules; SL, surface sarcolemma. *P < 0.05; n ≥ 27. Scale bar = 10 µm.

 
To gain insight into the absolute levels of expression, we estimated the amount of the CFP-tagged receptor by a comparison with a fluorescent CFP standard (see MATERIALS AND METHODS). The effective receptor fluorescence was equivalent to 680 ± 72 nM CFP at 2 days in culture and 930 ± 94 nM CFP at 3 to 4 days in culture. Therefore, CFP-tagged receptors accumulated in the SL compartment as the time in culture increased from an effective fluorescence of 210 nM (0.31 x 680 nM) at 2 days to 560 nM (0.60 x 930 nM) at 3 to 4 days. Overall, these confocal myocyte images and the subsequent analysis support the idea that ETA-CFP is initially expressed in PN Golgi and in T-tubule structures when they are intact and available. Non-Golgi ETA then localizes predominantly at other membranes such as the SL when T tubules are not present.

Twitch responses of myocytes expressing ETA-CFP were also investigated. Two-day cultured control ARVMs showed 34 ± 7% PIE by ET-1 similar to fresh isolated ARVMs (Fig. 5A). Two-day cultured ARVMs overexpressing ETA-CFP had a larger PIE of 73 ± 22% (Fig. 5A). In contrast, 3-day cultured ARVMs, whether overexpressing ETA-CFP or not, showed virtually no response to ET-1. Therefore, the overexpression of the receptor alone could not rescue the loss of function in 3-day cultured ARVMs (Fig. 5A). This result also suggests that ETA-CFP localized in the SL was not functional, at least in the context of regulating myocyte contractility.


Figure 5
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  		Fig. 5. Twitch responses and effect of cytochalasin D (CD) on the ETA system in ARVMs. A: to examine PIE, twitch amplitudes were measured before (0 min) and 10 min after 10 nM ET-1 treatment and expressed as percent increase over 0 min. *P < 0.05 compared with before ET-1 treatment; +P < 0.05; n ≥ 5. B: expression levels of ETA in fresh isolated and cultured ARVMs with or without CD. Cell lysate samples from either fresh isolated or cultured ARVMs were separated by SDS-PAGE, and Western blot analyses were performed using ETA-specific antibody. *P < 0.05; n ≥ 4.

 
To examine the role of T-tubule degeneration in the loss of function in cultured ARVMs, cytochalasin D (CD) was used. CD has been employed by others (4) to investigate the role of cytoskeletal actin since it binds to the barbed end of actin filaments preventing actin polymerization. The inclusion of CD during culture has also been shown to inhibit changes in myocyte morphology (21), which we were able to reproduce in Fig. 6. The T-tubule quantification parameters described above were used to confirm the preservation of T tubules in CD-treated ARVMs (Fig. 6).


Figure 6
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  		Fig. 6. CD and cellular structures of cultured ARVMs. Isolated ARVMs were incubated with or without CD for up to 4 days. A: representative images of di-8-ANEPPS-stained ARVMs. Insets: FFT images. B: quantification of percent area (n ≥ 12). C: quantification of intensity of first harmonic. *P < 0.05 compared with 1-day cultured ARVMs; +P < 0.05; n ≥ 10. Scale bar = 10 µm.

 
Interestingly, although CD largely prevented the remodeling of cellular structures, it could not prevent the decrease in myocyte ETA protein expression over 3 to 4 days in culture (Fig. 5B). This suggests that CD may be able to protect the cells from structural remodeling but not from all the changes that occur in culture. CD treatment by itself was also not able to rescue the reduced PIE in 3-day cultured ARVMs (Fig. 5A), suggesting that the decline in ETA expression during culture leaves an insufficient amount of receptors to mediate inotropic responses. Therefore, we evaluated the impact on the function of combined CD treatment with ETA-CFP overexpression in cultured ARVMs. ET-1 did induce a 55 ± 16% PIE in ARVMs with T tubules preserved and overexpressed ETA-CFP (Fig. 5A), suggesting that T-tubule integrity is critical for the normal physiological function of ETA.

ETA-CFP introduced by adenoviral infection was localized within T tubules at 2 days (with or without CD) and at 3 to 4 days of culture if CD was included (Fig. 7). The inclusion of CD also influenced the subcellular distribution of ETA-CFP expression. ETA-CFP was localized to 9 ± 1% PN, 53 ± 1% Int, and 40 ± 2% SL when CD was included, and this pattern changed only modestly over 2 to 4 days in culture (Fig. 7). Thus preserving T tubule structure appeared to be critical for the stable accumulation of ETA within the intracellular/T-tubular compartment, and preserving T tubules also blunted a disproportionate accumulation of ETA at the SL. Overall, normal inotropic responses of adult ventricular myocytes to ET-1 required both an adequate expression of functional ETAs and an appropriate localization of those receptors in T tubules (as illustrated in Figs. 5–7).


Figure 7
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  		Fig. 7. CD and expression pattern of ETA-CFP in cultured ARVMs. ETA-CFP was introduced to isolated ARVMs by adenoviral infection, and expression pattern was analyzed as described in MATERIALS AND METHODS. A: representative images of ETA-CFP-expressing ARVMs. Insets: FFT images. B: quantification of intensity of first harmonic (n ≥ 14). C: quantification of percent total intensity. *P < 0.05 compared with 2-day cultured ARVMs; +P < 0.05; n ≥ 14. Scale bar = 10 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The main findings of the present study were threefold. First, a culture of ARVMs for 3 to 4 days was accompanied by a loss of T tubules, reduced T-tubule ETA expression, and a loss of responsiveness to ET-1. Second, the introduction of exogenous ETA via adenoviral gene transfer restored ET-1 responsiveness, but only if T tubule integrity was maintained. Third, overexpressed ETA was localized to PN Golgi, T tubules, and surface sarcolemma, but surface SL ETA was not able to mediate an enhancement of myocyte contractility by ET-1.

Several methods were used in this study to analyze T-tubule structure or the T-tubular localization of ETA in ARVMs. One was percent area (in pixels) occupied by di-8-ANEPPS fluorescence after thresholding, since many other studies employed this method to quantify T-tubule integrity (2, 12, 24). This approach measures the percent area of fluorescently labeled structures in the cell interior (not including the nucleus or perinucleus) rather than the fluorescence intensity. To complement this approach, we also quantified intracellular periodic fluorescence staining by measuring the intensity of the first harmonic of FFT images (36, 41). This approach distinguishes intact T-tubular structures from disorganized T tubules or non-T-tubular intracellular structures, since the first harmonic represents a repeating frequency in the image. With these complementary methods, we confirmed that organized T-tubule structure largely vanishes during 3 to 4 days in primary culture of ARVMs as reported in previous studies (23, 26, 48). The consequences of such T tubule loss during culture in the context of ion channel function have been studied extensively (26), but to our knowledge, this is the first study to examine changes of GPCR function in T tubule-compromised cultured myocytes. This report also confirms and extends the observations of Orchard and coworkers (21) who first showed that CD dramatically blunts the morphological remodeling of cultured rat ventricular myocytes.

In the present study, ET-1 failed to induce a PIE in 3-day cultured myocytes with compromised T tubules, and ETA overexpression could not recover the abolished PIE. These results suggest that the T-tubular localization of ETA is critical for its normal function. Robu et al. (37) previously proposed the importance of the T-tubular localization of ETA by using ARVMs detubulated by osmotic shock. This detubulation method is relatively harsh, and the few myocytes that survive may not be representative of the population. Little is also known about the fate of ET receptors in various compartments following the osmotic shock treatment such that receptor internalization, degradation, or uncoupling could play a role in the loss of responsiveness to ET-1. The present study reduces some of this uncertainty by the use of a different detubulation strategy and by localizing exogenously introduced ETA with a CFP tag. This complementary approach clearly showed that ETA introduced by viral gene transfer was expressed in T tubules and in surface sarcolemma, but only when localized to periodic T tubules was it capable of regulating myocyte contractility. SL ETA was virtually nonfunctional in terms of regulating contractility.

A likely explanation for this difference in T-tubular versus SL ETA is that only in T tubules can ETA form a functional complex with appropriate downstream signaling molecules (e.g. G{alpha}q, PLC, and PKC) (35, 37) and end effectors (e.g. L-type Ca2+ channels) (12). A recent immunoprecipitation/proteomics approach has provided evidence consistent with endogenous ETA forming a large complex with these molecules (among others) within the T tubules (7). Moreover, based on the present study, adenoviral-expressed ETA-CFP appears capable of integrating productively into these macromolecular signaling complexes in the T tubules. Apparently, something critical for ETA signaling is absent in the surface sarcolemma (e.g., signaling lipids, proteins, scaffolds, etc.), or the pathway is inhibited or uncoupled in some way. The possibility that the density of L-type Ca2+ channels in surface sarcolemma is too low to regulate contractility is not consistent with the finding that the stimulation of β-receptors by isoproterenol triggers a large PIE even in detubulated ventricular myocytes (Fig. 3) (37). β-Adrenergic receptors are known to colocalize with their downstream signaling molecules throughout the entire sarcolemma including in T tubules and surface sarcolemma (5).

It should be noted that ETA is naturally expressed and functional in many cell types that do not contain a T-tubule compartment. ETA even regulates Ca2+ handling and contractility in neonatal myocytes and stem cell-derived myocytes with, at best, poorly developed T tubules (unpublished observations). Finally, ETA expressed heterologously in a variety of cell types appears to couple quite normally to phosphoinositide turnover, Ca2+ mobilization, and the endocytotic machinery, even when fused to fluorescent proteins and/or epitope tags (Ref. 10 and N. Evans and J. W. Walker, unpublished observations). Establishing the precise functional status of surface-expressed ETA in adult ventricular myocytes remains an important question (see below) but will require further investigation.

Potential limitations of the present study may include an unintended selection of myocytes by the requirement for culture or by the added stress of adenoviral infection. In our hands, the adenoviral expression of a green fluorescent protein construct did not detectably affect the viability of myocytes in culture, whereas the adenoviral expression of ETA-CFP may have decreased the viability of the myocyte population by twofold or more. In contrast, the addition of CD into the culture media tended to increase myocyte viability. Moreover, besides structural changes, other physiological features may be altered by culturing ventricular myocytes or by adding CD to the culture media. Control experiments in which we examined PIEs induced by extracellular Ca2+ or isoproterenol did not reveal significant differences between freshly isolated and 3-day cultured myocytes. However, some electrophysiological properties have been shown to be altered in cultured myocytes, and protein expression/turnover can be altered as well (26).

Another issue is whether the overexpression of CFP-tagged receptors accurately mimicked the physiology of ET signaling in myocytes. We estimate that the level of overexpression is 12-fold higher (at 2 days in culture) and 17-fold higher (at 3 days in culture) than normal ET receptor levels in freshly isolated adult ventricular myocytes (see MATERIALS AND METHODS). Moreover, the range of the overexpression was 4- to 17-fold at 2 days and 6- to 33-fold at 3 days in culture, and there was no detectable difference in the twitch behavior of myocytes within a given group over these ranges. The impact of a COOH-terminal CFP tag on ETA function has een found to be minimal in cultured in human embryonic kidney-293 cells (N. Evans and J. W. Walker, unpublished observations). Clearly, assessing the full impact of these potential limitations will require further investigation.

Numerous studies have demonstrated an increased expression of ETA (by ~2-fold) in myocardium in patients with cardiac disease and in animal models of heart failure (1, 11, 20, 28, 31, 33, 39, 49). However, several studies reported that ET-1 showed a reduced or absent PIE in myocardium or isolated myocytes from diseased hearts (15, 25, 29, 31, 42, 47). Similarly, Pieske et al. (31) demonstrated that ETA-mediated PIE is attenuated in end-stage failing human ventricular muscle strips desspite an increase in ETA expression. These studies speculated that the desensitization of ETA and a disturbance of downstream signaling were the underlying mechanisms. The present study suggests that a mislocalization of ETA could also contribute to the attenuated responsiveness of myocytes to ET-1.

Interestingly, T tubules have been observed to be disturbed and reduced in human heart failure or animal heart failure models (2, 5, 12, 24, 41). Therefore, it is highly possible that even though ETA is overexpressed in failing myocardium, the failing heart may be less responsive to ET-1 due to a concomitant disruption or depletion of T tubules. Another intriguing possibility to be explored is that mislocalized ETA regulates (or misregulates) processes other than contractility such as local Ca2+ signaling, mitochondrial function, or gene expression with unintended consequences to a heart in stress. Targeting such mislocalized and potentially renegade ETAs may represent a viable pharmacological strategy in the ongoing fight against heart disease.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by the National Heart, Lung, and Blood Institute Grant RO1-HL-08136 and funding from the University of Wisconsin Cardiovascular Research Center. K. Y. Chung was supported by a predoctoral fellowship from the Midwest Affiliate of the American Heart Association.


   ACKNOWLEDGMENTS
 
We thank Nathan Evans, Raquel Sanchos-Solis, Ryan Schmidt, Jennifer Wachholz, and Lance Rodenkirch for technical expertise and insightful discussions. Present address of M. Kang: Novartis Institute for Biomedical Research, Boston, MA 02139.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. W. Walker, 1300 University Ave., Madison, WI 53706 (e-mail: jwalker{at}physiology.wisc.edu)

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   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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