Targeted Ablation of the Phospholamban Gene Is Associated With a Marked Decrease in Sensitivity in Aortic Smooth Muscle
Abstract Phospholamban (PLB) is a protein associated with the Ca2+-ATPase of the sarcoplasmic reticulum (SR) in cardiac, slow-twitch skeletal, and smooth muscle. PLB inhibits the SR Ca2+-ATPase in cardiac muscle; this inhibition is relieved on phosphorylation. The role of PLB in smooth muscle contractility is less clear. To elucidate the role of PLB in vascular smooth muscle contractility in vivo, we used a model in which the PLB gene was targeted in murine embryonic stem cells, generating mice deficient in PLB (PLB−). The PLB− mice exhibited no gross developmental abnormalities, but marked changes in aortic contractility were observed. The time course of force development with phenylephrine stimulation was faster in the PLB− aorta, suggesting changes in SR Ca2+ release. No differences were observed for KCl contractures between tissue types for either maximum forces observed or time course of force production; relaxation was faster in 7 of 11 arteries, but this trend did not attain statistical significance. The cumulative concentration–isometric force relations for the PLB− aorta were to the right of the wild-type for both KCl and phenylephrine stimulation, indicating a less sensitive tissue. To investigate whether the observed changes were related to SR function, we inhibited the SR Ca2+-ATPase with cyclopiazonic acid (CPA). CPA treatment resulted in a leftward shift of the concentration–isometric force relations for both aorta types, as expected after removal of a major Ca2+ uptake system. Most interestingly, the differences between PLB and wild-type aorta were abolished by SR inhibition. Our results suggest that PLB is a regulator of the SR Ca2+ pump in mouse aorta and plays a regulatory role in both KCl-induced and receptor-mediated contractility in vascular smooth muscle.
Phospholamban has been shown to be a regulator of the Ca2+-ATPase in cardiac SR and an important determinant in the inotropic response of the heart to β-adrenergic stimulation. Dephosphorylated PLB is an inhibitor of the Ca2+-ATPase in cardiac SR, and phosphorylation relieves this inhibition.1 The exact mechanism of the effect of PLB on the SR Ca2+-ATPase is not known but may involve a direct interaction between the two proteins,2 3 followed by conformational changes in the SR Ca2+-ATPase.4 5 6 Phosphorylation of PLB is associated with an increase in the affinity of the cardiac SR Ca2+ pump for Ca2+.7
Although PLB has clearly been shown to be an important regulator of cardiac contractility, its role in smooth muscle function is less clear. Phosphorylation of PLB by cAMP- or cGMP-dependent protein kinase has been shown to increase Ca2+ uptake in vesicles prepared from bovine pulmonary artery8 and aorta9 and in cultured smooth muscle cells from rat aorta.10 Also, in rat aorta, nitric oxide–induced relaxation via the cGMP-dependent protein kinase pathway was associated with phosphorylation of PLB.11 Although these studies suggest a possible role for PLB in vascular smooth muscle Ca2+ homeostasis, the importance of PLB modulation of the SR Ca2+ pump in regulation of smooth muscle function in vivo is not known.
Recently, a targeted mouse was developed12 in which the gene for PLB was ablated (PLB−), providing a unique model for studying the role of PLB in muscle function. We tested the functional consequences for aortic contractility on the basis of our hypothesis that Ca2+ uptake is more effective in the PLB− aorta because of a deinhibited SR Ca2+ pump. In this study, we measured both contraction and relaxation kinetics as well as steady-state parameters for depolarization and receptor-mediated stimulation. Our results indicate that aortic contractility is significantly altered in PLB− mice. However, after inhibition of the SR Ca2+ pump, the differences were abolished. Our results suggest that PLB modulation of the SR Ca2+ pump may play a major role in modulation of vascular smooth muscle contractility.
Materials and Methods
Generation of PLB− Mice
The murine PLB gene was targeted in murine embryonic stem cells,12 and mice homozygous for the targeted PLB allele were generated. Wild-type mice were either siblings or, after the colony was bred to homozygosity, age-matched mice of similar background (C3H/HCN×C57Bl/6J). Genotypes of all animals were determined by PCR analysis of tail DNA biopsies. The genotypes were also routinely verified by Southern blot analysis of cardiac DNA. Moreover, no PLB mRNA was detected in either heart or aorta from PLB− mice by RT-PCR.
Mice 10 to 12 weeks old were either anesthetized by injection of 1 mg sodium pentobarbital/g body wt IP with 1.5 U heparin given to prevent aortic thrombi or by CO2 gas inhalation and were killed by cervical dislocation. The aortas were dissected and rinsed in cold bicarbonate-buffered PSS, and loose fat and connective tissue were removed. The endothelium was removed mechanically by sliding the ring on a 30-gauge stainless steel needle. After the experiment, the aorta was gently blotted and weighed, and its dimensions were measured. Aortic wall thickness (t) was estimated from the equation t=blot weight/(1.05×length×circumference), and the cross-sectional area (CSA) for force normalization was taken as CSA=2×t×length.
The PSS contained (mmol/L) NaCl 118, KCl 4.73, MgCl2 1.2, EDTA 0.026, KH2PO4 1.2, CaCl2 2.5, and glucose 5.5, buffered with 25 NaH2CO3; pH, when the PSS was bubbled with 95%O2/5% CO2, was 7.4 at 37°C. Ca2+-free PSS was similar except that CaCl2 was omitted and 0.5 mmol/L EGTA was included.
The ring was threaded with two 100 μm stainless steel wires, and each wire was placed into an angle-shaped holder; each completed mounting formed a double triangle. The fiber and holder were then mounted on a hook that was attached to a Harvard Apparatus differential capacitor force transducer. Resting tension on each aorta was set to 25 mN, the tension calculated for an in vivo aortic pressure of 100 mm Hg and a cross-sectional area of 0.42 mm2, and this passive tension was maintained throughout the experiment. Data were acquired with BioPac instrumentation and analyzed with the accompanying AcqKnowledge software.
Quantitative Western Blots
Mouse aortas from 100 wild-type mice were homogenized in a motor-driven glass/Teflon tissue grinder in a solution containing (mmol/L): imidazole 10 (pH 7.0), sucrose 250, dithiothreitol 1, and PMSF 0.5. The homogenate was centrifuged at 10 000g for 20 minutes. After centrifugation, the pellet was rehomogenized and centrifuged again at 10 000g. The supernatants from the two centrifugations were pooled and titrated to 0.6 mol/L KCl by the dropwise addition of 2.4 mol/L KCl. After incubation at 4°C for 15 minutes, the supernatant was centrifuged at 100 000g for 60 minutes. The pellet was resuspended in homogenizing buffer and centrifuged again at 100 000g. The final pellet was resuspended in (mmol/L) imidazole 10 (pH 7.0), KCl 150, β-mercaptoethanol 3, and sucrose 250. Protein concentration was determined by the Bio-Rad method, with BSA used for the standard curve.
Aortic microsomes (135 μg) were solubilized (1:1) in SDS sample buffer and loaded onto 10% to 20% SDS polyacrylamide gels. Before loading, one sample was boiled for 5 minutes and then immediately loaded onto the gel. After electrophoresis, the gel was stained with Coomassie blue, destained, and then electroblotted onto 0.22-μm nitrocellulose membranes.13 The blots were incubated overnight with 1 μg/mL monoclonal antibody specific for PLB (UBI). The antibody-antigen complex was detected with an alkaline phosphatase–labeled anti-mouse IgG conjugate (Cappel). The color was detected according to the manufacturer’s instructions with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as substrates.
Data are presented as mean±SEM. “n” refers to number of mice. Data for the time courses of development and relaxation (Tables 2⇓ and 3⇓) were averaged, then fitted to double exponential equations (indicated in the captions for Figs 3⇓ and 4⇓) by use of Origin software. Time-course data were also analyzed with a two-way repeated-measures ANOVA with SAS software. Concentration-response curves for each aorta were fitted to a logistic equation with Origin software; the fitting parameters (ED50, Fo, and Hill parameter) for each type of mouse were then averaged (Tables 3⇓ and 4⇓). Student’s t tests for group comparisons between the parameters for wild-type and PLB− mice were performed with Excel software; a Bonferroni correction was used when multiple comparisons were made. Results were considered significant at a value of P<.05.
PLB arises from a single gene with no known isoforms. PLB gene ablation was verified by PCR analysis of tail DNA biopsies and Southern blot analysis of cardiac DNA. Moreover, no PLB RNA message was detected by RT-PCR analysis of the PLB− aorta.
To demonstrate the presence of PLB in wild-type mouse aorta, enriched microsomal preparations were prepared from aortic homogenates and subjected to Western blot analysis as described in “Methods.” Fig 1⇓ shows an immunoblot of microsomal protein (100 μg) hybridized with an anti-PLB monoclonal antibody. The characteristic mobility shift of PLB was observed when the membranes were boiled in SDS before electrophoresis (Fig 1⇓).
Aortas from PLB− mice were indistinguishable from wild-type by their gross anatomic parameters (Table 1⇓). To assess the physiological consequences of PLB ablation, mechanical measurements on aorta from age-matched PLB− and wild-type mice were made simultaneously in the same experimental bath to ensure similar treatment. Fig 2⇓ shows representative time courses of isometric force for each aorta type for KCl (50 mmol/L) and PE (30 μmol/L) stimulation. There were no differences in the maximum isometric force/area between the PLB− and wild-type aortas for any of the stimulus conditions studied. In 7 of 11 cases, the PLB− aorta relaxed faster than the wild-type aorta. Fig 3⇓ summarizes graphically the average relaxation time courses by PLB− and wild-type aortas after washout of the 50 mmol/L KCl used to activate the contraction. These time courses were fit to a double exponential equation, and the fitting parameters are given in Table 2⇓. The more rapid phase, with a time constant (τ1) of ≈50 seconds, accounted for 65% of the relaxation in the PLB− and 56% in the wild-type aorta, but this trend did not reach statistical significance.
To further characterize the differences in transient behavior between the PLB− and wild-type aortas, we also analyzed the time course of isometric force development. With KCl stimulation (Figs 2A⇑ and 4A⇓), when the primary source of Ca2+ was extracellular, there were no significant differences in the time course of force production between PLB− and wild-type aorta. However, for receptor-mediated PE stimulation (Figs 2B⇑ and 4B⇓), when SR Ca2+ release is an important factor, the PLB− aorta showed a significantly different time course of force development (Table 3⇓). Force development for both PLB− and wild-type aorta was characterized by two components with τ1 of ≈10 seconds and τ2 of ≈2 minutes. The magnitude of the rapid phase of force development for PLB− aorta (61%) was substantially higher (P=.002) than that observed in the wild-type (46%). Thus, the PLB− aorta contracted more rapidly than wild-type aorta when receptor-mediated stimulation was used.
To isolate the effects of PLB gene ablation on SR Ca2+ release, the response to 10 μmol/L PE was measured 10 minutes after transfer to Ca2+-free PSS. The response to PE was transient, and the peak values were smaller than in the presence of Ca2+ (see Fig 8⇓) for both wild-type and PLB− aortas. Importantly, the peak force obtained for the PLB− aorta (19.4±3.3% of control Fmax) was nearly twice as great as those for the wild-type (9.9±3.0% of control Fmax, n=5). This is consistent with a greater SR Ca2+ release in the PLB− aorta suggested by the experiments in the presence of Ca2+ (Fig 4B⇑, Table 3⇑).
Maintenance of flow is of particular importance to vascular function, and this parameter is governed by vessel diameter. Thus, the effects of PLB− ablation on the relations between stimulus and steady-state isometric force were investigated next. Fig 5⇓ shows a representative experiment of PE concentration versus isometric force relations in PLB− and wild-type aortas. The PLB− aortas were less sensitive than the wild-type; the ED50 was fourfold greater in the PLB− aortas than in the wild-type (Table 4⇓).
To eliminate any effects of Ca2+ influx through L-type Ca2+ channels, we also studied the PE concentration-force relation in the presence of 10 μmol/L nifedipine in a different set of animals. The maximum force was slightly reduced in the presence of nifedipine, to 75.4±5.7% and 73.9±2.8% of control for the wild-type (n=4) and PLB− (n=5) aortas, respectively. Importantly, the PE concentration-force relation of the PLB− aorta remained to the right of (Fig 6⇓) that of the wild-type aorta, as in the absence of nifedipine (Fig 5⇑, Table 3⇑).
We also studied the effects of PLB− gene ablation for KCl contractures, in which extracellular Ca2+ is the primary source for contraction, because 10 μmol/L nifedipine blocked this response. Fig 7⇓ shows a representative KCl concentration-versus-force curve; the response of the PLB− aorta was again significantly less sensitive than that of the wild-type (Table 4⇑).
To verify that the differences in contractility observed above were associated with alterations in SR Ca2+ pump function, we designed a protocol to inhibit the SR function. Our rationale was that if all Ca2+ homeostatic mechanisms were identical in the wild-type and PLB− aortas, with the exception of those associated with the SR, then removal of the SR should eliminate any differences in contractility. If this hypothesis were true, then the concentration–isometric force relations should be identical after the SR Ca2+ pump activity was eliminated. Fig 8⇓ shows a typical experimental example of this protocol; the internal Ca2+ stores were unloaded by repeated stimulation with KCl (50 mmol/L) and PE (10 μmol/L) in Ca2+-free PSS. After this protocol, we judged the SR to be emptied, because the tissues could no longer produce force on stimulation in the Ca2+-free PSS. The bath contents were then replaced with fresh Ca2+-free PSS that contained 1 μmol/L CPA, a reversible inhibitor of the SR Ca2+-ATPase.14 After a 20-minute incubation with CPA, Ca2+ was reintroduced into the bath. At this point, the concentration-force relation was again determined. As shown in Fig 9⇓, the KCl–isometric force relations for both wild-type and PLB− aortas were shifted leftward (Table 5⇓), consistent with the loss of a significant Ca2+ uptake mechanism. Most significantly, the differences in ED50 between the wild-type and PLB− aortas were abolished by CPA treatment (Table 5⇓), indicating that the differences in contractility were most likely related to alterations of SR function.
PLB has been shown to be an important regulator of cardiac contractility. However, its role in smooth muscle in vivo is less well defined.11 15 We have shown that aortas from PLB− mice exhibit significant differences in their contractility compared with those from wild-type mice. Our working hypothesis was that PLB− aorta would have a greater SR Ca2+ uptake in the absence of PLB inhibition. If this were the case, one might postulate that PLB− aorta would show a faster time course of relaxation than its wild-type counterpart. This was observed in 7 of 11 cases, but this trend was not statistically significant (Figs 2⇑ and 3⇑, Table 2⇑). This may indicate that other processes, such as dephosphorylation of the myosin regulatory light chain, may also be important in determining the rate of relaxation. Further experimentation, including measurements of [Ca2+]i, is needed to more fully assess the role of PLB in relaxation.
It has also been suggested that an enhanced rate of the SR Ca2+-ATPase in the absence of PLB may, over time, lead to an increased Ca2+ content of the SR.12 A greater SR Ca2+ storage and consequently higher Ca2+ gradient would lead to a greater driving force for Ca2+ translocation when SR Ca2+ channels are open. One would anticipate that this would be important for force development when SR Ca2+ release is the major source of Ca2+ for contraction. Consistent with this hypothesis, the magnitude of the rapid phase of a PE contraction in PLB− aorta was twice as great as that of the control (Fig 4B⇑, Table 3⇑). This is also supported by the twofold greater peak amplitude for the PLB− aorta in response to PE in the absence of Ca2+. In contrast, with voltage-dependent stimulation with KCl, in which Ca2+ influx from extracellular stores plays a major role, similar time courses of force production were observed for both the PLB− and wild-type aorta (Fig 4A⇑, Table 3⇑).
In the absence of PLB, one might also anticipate steady-state differences in contractility. If eliminating PLB enhances the activity of the SR Ca2+ pump, a lower [Ca2+]i for any given Ca2+ release and/or influx would result. One thus might anticipate a lower steady-state force at the same level of stimulation in PLB− aorta. This would be of particular importance for vascular function, because in contrast to the heart, vascular smooth muscle is characterized by the maintenance of tonic forces for circulatory regulation. We tested this hypothesis by measuring the concentration–steady-state isometric force relations to stimulation by PE and KCl (Figs 5 through 7⇑⇑⇑, Table 4⇑). For PE contractures, concentration-force relations for PLB− aorta lie to the right of the wild-type in both the presence and absence of nifedipine blockade of Ca2+ channels. For these steady states, an increased SR Ca2+ uptake would appear to be the dominant effect of PLB− gene ablation.
KCl contractures are characterized by a continuous influx of extracellular Ca2+ through voltage-dependent Ca2+ channels. Because the SR is of finite capacity, one might anticipate that the role of PLB would be of less significance for these contractures. However, the KCl concentration-force relation for the PLB− aorta also was to the right of the wild-type, indicating a significant decrease in sensitivity. These data suggest that the SR uptake does not saturate, supporting the view of Chen and van Breemen,16 who suggest that the SR may extrude Ca2+ directly to the extracellular space. Ablation of the PLB− gene has a significant effect on concentration-force responses and suggests that PLB, through modulation of the SR Ca2+ pump, plays a major role in regulation of contractile function.
As with any transgenic animal model system, the effects of potential compensatory responses need to be considered. Two types of compensation can be envisioned: (1) alterations in aortic contractility as a response to altered cardiac contractility due to PLB ablation in the heart and/or (2) changes in aortic Ca2+ homeostatic mechanisms in response to PLB ablation in the aorta.
The heart rates, body and heart weights, and blood pressures12 of the PLB-deficient mice were similar to those of the age-matched wild-type mice. Cardiac contractility, although in the physiological range, was altered. However, the aorta were physically similar, as shown in Table 1⇑, and no gross physical changes in the aorta, such as those often associated with pressure-overloaded hearts, were observed. Moreover, no differences in the maximum force-generating capacity were noted. Thus, the altered responses of the PLB− aorta were probably not due to alterations of cardiac contractility in this model.
Compensation for a more aggressive SR Ca2+ uptake at the smooth muscle level might involve changes in Ca2+ homeostatic mechanisms, for example, an increase in plasma membrane Ca2+ channels or a decrease in the plasmalemmal Ca2+ pump activity. If SR function were eliminated, any such compensatory mechanisms would be expected to lead to increased sensitivity in the PLB− aorta relative to the wild-type. To eliminate SR function, we used a strategy of emptying the internal Ca2+ stores and pharmacologically inhibiting the SR Ca2+ pump with CPA (Fig 8⇑). After functional elimination of SR, the sensitivity of both types of aorta increased as expected, because of removal of a major Ca2+ uptake system. Importantly, the differences in KCl concentration-force relations between PLB− and wild-type aortas were abolished (Fig 9⇑, Table 5⇑). Thus, compensation at this level is unlikely.
Our studies have shown major changes in contractile parameters in aorta from mice in which the PLB gene had been ablated. Any compensatory changes, if they existed, were not sufficient to reverse the effects of PLB gene ablation. Moreover, our studies with CPA restrict any potential compensatory changes to the SR locus. Thus, at worst, the importance of PLB in regulation of smooth muscle function would be underestimated by our studies. The simplest conclusion, based on the altered contractility of the PLB− aorta, is that PLB−, through modulation of SR function, can play an important role in modulation of vascular smooth muscle sensitivity. Our studies indicate that such modulation can be significant not only when SR Ca2+ release is the dominant source of Ca2+ for contraction but also under conditions (eg, depolarization) in which Ca2+ influx is significant. The physiological significance of the potential role of PLB to regulation of circulation at the whole-animal level will be assessed in future studies.
Selected Abbreviations and Acronyms
|PCR||=||polymerase chain reaction|
|PSS||=||physiological saline solution|
This study was supported in part by NIH grants HL-26057, HL-22619, and HL-52318 (Dr Kranias); TG-07175 (Dr Lalli); and HL-23240 (Dr Paul) and American Heart Association grant 92007130 (Dr Paul). We thank Craig Weber for skillful technical assistance.
- Received June 14, 1996.
- Accepted December 27, 1996.
- © 1997 American Heart Association, Inc.
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