Published online before print April 1, 2004, doi: 10.1161/01.RES.0000126923.46786.FD
© 2004 American Heart Association, Inc.
Cellular Biology |
Parvalbumin Corrects Slowed Relaxation in Adult Cardiac Myocytes Expressing Hypertrophic Cardiomyopathy-Linked 
-Tropomyosin Mutations
From the Departments of Biomedical Engineering (P.C.), Molecular and Integrative Physiology (C.N.B., E.G.F., J.M.M.), and Internal Medicine (S.M.D.), University of Michigan, Ann Arbor, Mich.
Correspondence to Joseph M. Metzger, Department of Physiology, 7730 Medical Science II, University of Michigan, 1301 E Catherine St, Ann Arbor, MI 48109-0622. E-mail metzgerj{at}umich.edu
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Abstract |
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Hypertrophic cardiomyopathy mutations A63V and E180G in
-tropomyosin (-Tm) have been shown to cause slow cardiac muscle relaxation. In this study, we used two complementary genetic strategies, gene transfer in isolated rat myocytes and transgenesis in mice, to ascertain whether parvalbumin (Parv), a myoplasmic calcium buffer, could correct the diastolic dysfunction caused by these mutations. Sarcomere shortening measurements in rat cardiac myocytes expressing the -Tm A63V mutant revealed a slower time to 50% relengthening (T50R: 44.2±1.4 ms in A63V, 36.8±1.0 ms in controls; n=96 to 108; P<0.001) when compared with controls. Dual gene transfer of -Tm A63V and Parv caused a marked decrease in T50R (29.8±1.0 ms). However, this increase in relaxation rate was accompanied with a decrease in shortening amplitude (114.6±4.4 nm in A63+Parv, 137.8±5.3 nm in controls). Using an asynchronous gene transfer strategy, Parv expression was reduced (from 
0.12 to 0.016 mmol/L), slow relaxation redressed, and shortening amplitude maintained (T50R=33.9±1.6 ms, sarcomere shortening amplitude=132.2±7.0 nm in A63V+PVdelayed; n=56). Transgenic mice expressing the E180G -Tm mutation and mice expressing Parv in the heart were crossed. In isolated adult myocytes, the -Tm mutation alone (E180G+/PV–) had slower sarcomere relengthening kinetics than the controls (T90R: 199±7 ms in E180G+/PV–, 130±4 ms in E180G–/PV–; n=71 to 72), but when coexpressed with Parv, cellular relaxation was faster (T90R: 36±4 ms in E180G+/PV+). Collectively, these findings show that slow relaxation caused by -Tm mutants can be corrected by modifying calcium handling with Parv.
Key Words: tropomyosin • parvalbumin • hypertrophic cardiomyopathy • diastolic dysfunction • gene transfer
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Introduction |
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Hypertrophic cardiomyopathy (HCM) is an inherited disease characterized by an increase in ventricular wall thickness, impairment in relaxation properties (diastolic dysfunction), and an increased risk of sudden death.1,2 Familial HCM has been linked to autosomal-dominant mutations in 10 different sarcomeric proteins including actin, ß-myosin heavy chain, myosin light chains, myosin binding protein C, titin, troponin I and T, and
-tropomyosin (-Tm).1–5 In particular, at least eight HCM causing mutations in -TM have been reported thus far (E62Q, A63V, K70T, V95A, D175N, E180G/V, and L185R).6–9
-Tm consists of two -helical polypeptide chains for a total molecular weight of 67 kDa.10 In cardiac myocytes, -Tm associates in a coiled-coil structure around actin and lays in the groove where the actin-myosin binding sites reside, thus preventing cross-bridge formation.10,11 When calcium binds to troponin C, -Tm moves away from the actin-myosin binding sites, allowing force development. HCM-linked -Tm mutations A63V and E180G have been shown to increase the myofilament sensitivity to calcium, as evident by a leftward shift in the force-pCa relationship.12–14 One physiologically relevant outcome of increased calcium sensitivity is to slow relaxation properties, both in vitro15 and in vivo.13 The diastolic dysfunction caused by these mutants is hypothesized to be a key element that initiates the signaling cascade leading to HCM.16
Gene transfer of parvalbumin (Parv), a fast skeletal calcium buffer not present in the heart, has been shown to accelerate relaxation kinetics in isolated cardiac myocytes and at the organ level.17–19 The mechanisms by which Parv increases relaxation rates have been described elsewhere.20 Recently, Parv has been shown to correct diastolic dysfunction caused by a defect in calcium handling in a hypothyroid animal model,17,19 and in a chemical model by partial inhibition of Serca2a by thapsigargin.21 Because myofilaments play a key role in relaxation,22,23 it is not certain whether gain-in-function through Parv expression would be sufficient to redress slow relaxation performance due to a myofilament defect at the level of the intact single myocyte.
In this study, we used rat adult cardiac myocytes and a dual gene transfer approach. We sought to induce diastolic dysfunction with gene transfer of the HCM mutant, -TmA63V, and then to attempt to correct the slow relaxation using Parv gene transfer. In complementary studies, we performed a genetic cross between mice expressing a HCM mutant -Tm (E180G), previously shown to display diastolic dysfunction,13 with Parv transgenic mice.24 Overall, we found that facilitating the calcium sequestration process, using Parv, corrected the slowed relaxation caused by the -Tm mutants in both rat and mouse cardiac myocytes.
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Materials and Methods |
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The methods for most of the experiments have been previously published.18 The following is a brief summary of the methodology; for an expanded Materials and Methods section, see the online data supplement at http://circres.ahajournals.org. All experiments were conducted at 37°C. Mice were obtained from Charles River (Wilmington, Mass). Rats were obtained from Harlen (Indianapolis, Ind). The procedures used in this study were in agreement with the guidelines of the Internal Review Board of the University of Michigan and approved by the University of Michigan Committee on the Use and Care of Animals. Veterinary care was provided by the University of Michigan Unit for Laboratory Animal Medicine, and the animal care use program conforms to the standards of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 86-23).
The experimental plan consisted of two parts. In the first series of experiments, cardiac myocytes were isolated from adult female rats and submitted to two different adenovirus gene transfer strategies: synchronous and asynchronous. In the synchronous gene transfer strategy, the myocytes were either nontransduced (Control or Ctrl), or transduced with wild-type -Tm (Tm), A63V -Tm mutant (A63V), Parv (PV), or with both Parv and A63V -Tm mutant (A63V+PV). These recombinant vectors have been described.12,17 It should be noted that all -Tms (WT and A63V) had a C-terminal Flag tag, and that all transductions occurred 1 hour after myocyte isolation. The C-terminal Flag was shown previously not to alter isometric force or sarcomere shortening.12,15 In the asynchronous gene transfer strategy, an additional group of myocytes was generated by transducing the myocytes with A63V -Tm mutant 1 hour after isolation, followed by gene transfer to the same myocytes 1.5 days later with Parv (A63V+PVdel) (see Figure 3A). In all cases, sarcomere shortening measurements were performed 4 days after isolation and samples were then collected for Western blotting.
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The second series of experiments consisted of a genetic complementation approach involving crossing two lines of transgenic mice. One transgenic line expressed the -Tm mutant E180G,13 and the other line expressed Parv in the heart.24 The offspring formed four groups (E180G–/PV–, E180G–/PV+, E180G+/PV–, and E180G+/PV+). Gene and protein expression was identified by PCR and by Western blots. The adult mouse myocytes were isolated, and calcium fluorescence and sarcomere shortening measurements were performed between 3 and 6 hours after isolation using methods detailed previously (see the expanded Materials and Methods).
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Results |
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To demonstrate the feasibility of synchronous (simultaneous) dual-gene transfer in rat cardiac myocytes, we used a dual-labeling immunofluorescence technique. The results are presented in Figure 1A. The simultaneous dual transduction (Parv in green and WT
-Tm-Flag in red) was highly successful (>95% myocytes expressing both). The insets show a diffuse pattern for Parv, typical of soluble cytoplasmic proteins, and a striated pattern for the WT -Tm-Flag, indicative of efficient sarcomeric incorporation, similar to what has been reported with single gene transfer of Tm12 or Parv.18
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In the first set of experiments, five groups were tested (Ctrl, Tm, A63V, PV, and A63V+PV; see Materials and Methods). First, Western blots were used to quantify the protein expression in each group (Figure 1B). The lower band with the anti-Tm antibody indicates the endogenous -Tm, whereas the upper band indicates the -Tm Flag (either WT or A63V). The total level of Tm expression was not significantly different from control myocytes in any of the -Tm-transduced groups, indicating replacement rather than overexpression as shown previously.12 The level of replacement of endogenous -Tm was 27.8±2.0%, 25.7±1.0%, and 23.8±1.2% for Tm Flag, A63V, and A63V+PV, respectively (n=6 to 9). As expected, Parv was expressed only in groups transduced with the Parv vector, and showed an estimated level of expression of 0.154±0.015 and 0.129±0.045 mmol/L (n=2 to 3) for the PV and PV+A63V groups, respectively. In all cases, actin was used to normalize for protein loading.
Figure 2 provides a summary of the sarcomere-shortening experiments. Gene transfer with WT -Tm Flag had no significant effects on the sarcomere shortening amplitude or on the relaxation kinetics when compared with control (Figures 2B through 2D). In contrast, -Tm mutant A63V transduced myocytes exhibited marked slowing of relaxation, especially in the late part of the cycle (T90R). In addition, myocytes expressing A63V showed a slight increase in sarcomere shortening amplitude. When combined with Parv (A63V+PV), the dual-transduced myocytes reversed the slow relaxation, and even showed a significant acceleration of the relaxation performance when compared with controls. However, as seen when Parv is expressed beyond optimal levels, 18 there was a decrease in sarcomere shortening amplitude for both the PV and A63V+PV groups.
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To determine whether Parv could restore relaxation performance while preserving sarcomere shortening amplitude, we optimized the level of Parv by using an asynchronous dual gene transfer strategy. The strategy is detailed in the Methods and illustrated in Figure 3A. To maintain a phenotype characterized by significant diastolic dysfunction, we maintained the incubation time for A63V to 4 days. For Parv, the myoplasmic Parv concentration required to increase relaxation while preserving sarcomere shortening amplitude is 0.05 mmol/L.18 This occurs at 2.5 days after gene transfer.18 Accordingly, myocytes were initially transduced with the A63V vector, and then, 1.5 days later, these same myocytes were transduced with the Parv vector. We then examined the function of these myocytes (termed A63V+PVdel; del for delayed) at 4 days after isolation: this translates to myocytes 4 days after A63V and 2.5 days after Parv gene transfer.
Figure 3B, shows that the level of -Tm replacement was similar for groups A63V+PV and A63V+PVdel, whereas levels of Parv expression were reduced, as expected, in the A63V+PVdel group. Finally, Figures 3C and 3D provide summaries of contractile performance for this optimization strategy. The A63V+PVdel group retained faster relaxation kinetics than the A63V group, and brought the sarcomere shortening amplitude back to the level of control myocytes (131±6 nm, control; 132±6 nm, A63V+PVdel; n=48 to 56). Although the one-way ANOVA test did not reveal significant difference between the sarcomere shortening amplitude of the control and the A63V+PV (nondelayed) myocytes as it did in Figure 2D, a two-tailed unpaired t test showed significance (131±6 nm, control; 110±6 nm, A63V+PV; n=48; P=0.015).
In the next set of experiments, we studied acutely isolated cardiac myocytes from a cross of two transgenic lines of mice, one expressing -Tm mutant E180G13 and the other expressing Parv in the heart.24 For each transgene, the MHC promoter was used to direct expression to the heart. The resulting offspring were either, nontransgenic (E180G–/PV–), expressing Parv only (E180G–/PV+), expressing E180G only (E180G+/ PV–), or both (E180G+/ PV+) (see Figure 4A). As described previously for E180G mice (line 160), 13 no signs of cardiac hypertrophy were detected in any of the transgenic groups, and survival rate was similar among all the groups. Assessment of genotype was performed using PCR analysis and confirmed by Western blots (Figure 4B). The lower band of the anti-Tm detection represents the E180G mutant, which is migrating more rapidly due to the single mutation in -Tm,13 whereas the upper band represents the wild-type -Tm. The level of replacement of -Tm shown in Figure 4B is comparable to the 60% replacement observed in the founder mouse line.13 Parv mice had an estimated concentration of 0.3 mmol/L in the heart (line 268)24 (Figure 4B).
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Figure 5 shows the results of sarcomere shortening experiments obtained from cardiac myocytes isolated from transgenic mice. The E180G+/PV– group exhibited slower relaxation when compared with controls (E180G–/PV–). This was especially evident in the later part of relaxation (T90R) (Figures 5A and 5B). The Parv group (E180G–/PV+), on the other hand, showed extremely rapid relaxation kinetics and, due to its high Parv concentration, an attenuation in sarcomere shortening amplitude (Figure 5C). In myocytes from E180G+/PV+ mice, both amplitude and relaxation kinetics were almost indistinguishable from the Parv alone group. The only difference was in very late relaxation. For the two PV-positive groups, a small slower second phase of relaxation was present (see arrow Figure 5A). For the E180G–/PV+ group the amplitude of this second phase was 5.02±0.34%, and for the E180G+/PV+ group the amplitude was 7.96±0.29% of the entire amplitude.
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Intracellular calcium fluorescence ratio (360/380 nm) was also measured using Fura-2 (Figure 5D). When comparing the E180G+/PV– myocytes to the E180G–/PV– myocytes, the former showed only a small increase of 6.5% in the time from stimulation to 50% decay (TS50D), whereas showing no significant change in the later part of the decay (TS90D). This suggests that the changes observed in sarcomere shortening experiments are due to a modification of myofilament function, as opposed to a change in calcium handling. Finally, consistent with Parv expression, calcium fluorescence decay was greatly accelerated in myocytes expressing Parv (E180G–/PV+ and E180G+/PV+).
In these transgenic mice, we modulated the functional calcium buffering effect of Parv by increasing the pacing frequency from 0.2 to 2.0 Hz (Figure 6), as described previously.18 At 2.0 Hz, myocytes from group (E180G+/PV+) still retained faster relaxation kinetics than the -Tm mutant group (E180G+/PV–), and the sarcomere shortening amplitude deficit in E180G+/PV+ myocytes relative to E180G+/PV– myocytes went from 65% at 0.2 Hz to 36%.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, results obtained by using two complementary gene-based strategies, gene transfer in isolated myocytes and transgenic mice, showed that delayed myocyte relaxation arising from a defect in the myofilaments could be corrected by the myoplasmic calcium buffer parvalbumin. In addition, we designed a strategy of asynchronous dual-gene transfer to isolated cardiac myocytes that permitted optimization of Parv’s relaxation enhancing effects, while preserving normal myocyte sarcomere shortening amplitude.
Myocardial relaxation is controlled by the properties of two coupled entities, calcium sequestration and myofilaments. There has been debate regarding the relative roles that calcium removal and myofilaments have on controlling the rate of myocardial relaxation.22,23 Several experiments have been conducted in myocardium in which independent modifications of either calcium handling or myofilament properties had significant impact on relaxation rates. Indeed, the impact on relaxation by modulation of sarcoplasmic reticulum calcium ATPase (Serca2a) function is well established, whether it is to slow relaxation using thapsigargin or phospholamban (PLB) overexpression, or to increase it with Serca2a overexpression or PLB ablation (see reviews25,26). Similarly, myofilament mutations, as the ones presented in this article, have been shown to directly slow relaxation, whereas troponin I phosphorylation can accelerate relaxation.27 However, it was not clear, a priori, whether modifying calcium removal using Parv would be sufficient to correct the slow relaxation caused by mutations in a myofilament protein, -Tm. The results obtained in this study with transgenic mice (Figure 5) and with gene transfer in isolated rat myocytes (Figures 2 and 3
), demonstrate the capability of improved calcium handling by Parv to correct myofilament-mediated slow relaxation.
Biophysical Mechanisms
As demonstrated using either an in vitro motility assay28 or permeabilized myocytes,13 the -Tm mutations (A63V and E180G) have no effect on the total force or on the cooperative nature of the force-pCa relationship. However, a leftward shift in pCa50 (calcium level that gives half of the maximal force) in the order of 0.1 to 0.2 log units has consistently been reported in various experimental settings.12,14 The slower kinetics observed in these -Tm mutants during the relaxation phase either in vitro (Figures 2 and 5) or in vivo13 have been attributed to the increased sensitivity of the myofilaments to calcium. Indeed, for an equivalent calcium transient, myocytes with increased myofilament sensitivity produce a larger force than normal myocytes at any given time and take longer to reach full relaxation.
Other possible effects of the leftward shift in force-pCa relationship would be to produce a larger twitch contraction, and possibly to modify calcium transient decay. The results presented in this study show that sarcomere shortening amplitude is increased with the A63V -Tm mutation in rat transduced cardiac myocytes (Figures 2D and 3D) and in transgenic mice bearing the -Tm E180G mutation (Figure 5C), compatible with the idea of stronger contractions. However, calcium fluorescence measurements in the mouse myocytes show that this increase in calcium sensitivity in the E180G+/PV– mice, when compared with the E180G–/PV– mice, had only a small effect on the TS50D, and no significant influence on the TS90D (Figure 5D). A possible explanation is that troponin C is responsible only for a fraction of the calcium buffering, because many other buffers and calcium sequestration mechanisms also contribute in shaping the calcium transient decay.25 Changes in troponin affinity of the magnitude caused by the -Tm mutations are therefore probably too small to influence the calcium transient properties. Finally, using a mathematical modeling analysis (presented in the online data supplement), we confirmed that a shift in the force-pCa relationship of 0.08 log unit is sufficient to explain the effect of slower sarcomere relengthening, greater sarcomere shortening amplitude, and little effect on the calcium transient.
Parv has been shown to increase the rate of calcium decay in cardiac myocytes18 and the mechanisms involved have been characterized.20 Briefly, Parv is a soluble protein with two calcium/magnesium binding sites. When free intracellular calcium concentration transiently increases, in a large portion of Parv binding sites, bound magnesium is dislodged by calcium, with a delay. This delay causes calcium to be maximally sequestered during the decay phase of the transient, resulting in an early abbreviation of the relaxation period.20 It has been shown that when Parv is expressed at high levels (>0.1 mmol/L), in addition to the acceleration of relaxation there is also an attenuation of the contractile amplitude,18 and that Parv effects are also frequency dependent18 (Figure 6). In the present study, we were able to adjust Parv efficiency (effects on contractile amplitude versus relaxation) by either delaying Parv gene transfer in rat cardiac myocytes, or by using a higher pacing frequency in mouse myocytes. It remains to be determined what level Parv would be optimal in vivo and how it would be possible to maintain that optimal level. Mathematical modeling simulation results reproducing and analyzing the effect of Parv on the -Tm mutations are presented in the online data supplement.
Strategies to Correct Diastolic Dysfunction in -Tropomyosin Mutations
HCM-related mutations in -Tm have been characterized in a number of studies.13–15,28 However, no strategies have been tested to correct the diastolic dysfunction observed in these myocytes. Several strategies could be envisioned. First, because these mutations are dominant negative, it could be possible to overexpress wild-type (WT) -Tm, such that these WT -Tm replace the mutant ones. A more technically challenging approach could consist of producing an antisense sequence that would specifically block the mutant -Tm formation while leaving production of WT -Tm unaffected.
Alternatively, instead of trying to directly modify mutant -Tm expression, another possibility would be to modify the system’s control variable, in this case, the calcium transient. In that sense, the strategy would be to correct the slow relaxation phenotype caused by the -Tm mutations, and by applying another phenotype, rapid calcium sequestration. Although this is an indirect compensation strategy, the end result appears as a rescue of the slow relaxation. In this article, we showed that by controlling the kinetics of decay of the calcium transient, by using a specialized calcium buffer (Parv), it is possible to correct the cellular diastolic dysfunction observed in two of these -Tm mutants (A63V, E180G). Other calcium modifying strategies such as increasing the quantity of Serca2a, or removing PLB inhibition in these calcium pumps may also have beneficial effects.26 Indeed, these strategies have been applied to several models of heart failure with success.29–32 Interestingly, the possibility of controlling reduction in shortening amplitude with Parv might turn out to be a positive point, because negative inotropic agents such as ß-blockers have been shown to have long-term beneficial effect in patients with heart failure or HCM.33,34
Limitations
One of the limitations of this study is that all experimental conditions tended to make calcium removal the rate-limiting step. Physiological temperature, low stimulation rate, unloaded contractions, and rapid myosin isoform (V1 as found in rat), all have been suggested to shift the relaxation dominance toward calcium sequestration properties.22,23 It is unclear whether the results obtained in this study could be extrapolated to the human heart. Indeed, the slower myosin isoform (V3) and the mechanical load present in the human heart in vivo tend to bring the relaxation rate-limiting factor toward myofilament properties.23 In any case, this rate-limiting argument should be interpreted with caution, because calcium sequestration and myofilament force production are both coupled. Moreover, the calcium transient is the stimulus for myofilament force production in this system. In large mammals (eg, rabbit, dog, or human), calcium transient duration at 37°C is 150 to 300 ms,25,29,35 whereas isometric force duration is 300 to 500 ms.25,36 This leaves sufficient margin for an abbreviation of the calcium transient, which would then result in a similar reduction in twitch duration. In fact, in several conditions where the myofilaments are believed to be the rate-limiting step, calcium handling-protein manipulations have been shown to increase mechanical relaxation rates (eg, large mammals in vitro29,37 or in rodents in vivo19,30). Nonetheless, experiments in conditions that more closely resemble the human heart in its physiological environment would be important to address this issue.
Another possible limitation is that all the mutations used in this study influenced only one part of the myofilament function, ie, the myofilament sensitivity to calcium. It would be interesting to see if other myofilament mutations that affect other aspects of the contractile apparatus, such as cross-bridge cycling kinetics, could be corrected using a similar strategy.
In conclusion, we showed that altering the calcium transient using Parv successfully reversed the cellular diastolic dysfunction caused by HCM-linked -Tm mutation A63V (in rat myocytes through adenovirus gene transfer) and E180G (in transgenic mice myocytes). It remains to be determined how this method compares with the other calcium handling/myofilament modification strategies mentioned in this study. More importantly, finding the optimal strategy to prevent HCM progression in the living heart bearing these -Tm mutations in vivo still represents a fundamental challenge.
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Acknowledgments |
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This study was supported by the NIH and the American Heart Association.
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Footnotes |
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Original received December 22, 2003; revision received March 17, 2004; accepted March 19, 2004.
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References |
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