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Genetic manipulation of calcium-handling proteins in cardiac myocytes. I. Experimental studies

Departments of ¹Biomedical Engineering and ²Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan

Submitted 6 May 2004 ; accepted in final form 13 August 2004

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Two genetic experimental approaches, de novo expression of parvalbumin (Parv) and overexpression of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a), have been shown to increase relaxation rates in myocardial tissue. However, the relative effect of Parv and SERCA2a on systolic function and on -adrenergic responsiveness at varied pacing rates is unknown. We used gene transfer in isolated rat adult cardiac myocytes to gain a fuller understanding of Parv/SERCA2a function. As demonstrated previously, when Parv is expressed in elevated concentration (>0.1 mM), the transduced myocytes showed a reduction in sarcomere-shortening amplitude: 129 ± 17, 81 ± 8, and 149 ± 14 nm for control, Parv, and SERCA2a, respectively. At physiological temperature, shortening amplitude responses of Parv and SERCA2a myocytes to the -adrenergic agonist isoproterenol (Iso) were not statistically different from that of control myocytes. However, in SERCA2a myocytes, in which baseline was slightly elevated and the Iso-stimulated value was slightly lower, the increase in shortening was slightly less than in Parv or control myocytes: 108 ± 14, 169 ± 39, and 34 ± 12% for control, Parv, and SERCA2a, respectively. In another test set, Parv myocytes had the strongest early postrest potentiation among all groups studied (rest time = 2–10 s), and SERCA2a myocytes were the least sensitive to variations in stimulation rhythm. To replicate the deficient Ca²⁺ removal observed in heart failure, we used 150 nM thapsigargin. Under these conditions, control myocytes exhibited slowed relaxation, whereas Parv myocytes retained their rapid kinetics, showing that Parv is still able to control relaxation, even when SERCA2a function is impaired.

parvalbumin; sarco(endo)plasmic reticulum calcium-adenosinetriphosphatase 2a; diastolic dysfunction; gene transfer; thapsigargin; postrest potentiation

Recently, two strategies featuring genetic manipulation have been proposed to restore Ca²⁺-handling function in myocytes from the failing heart (30): 1) restoration of the SERCA2a-to-PLB and/or SERCA2a-to-NCX ratio or 2) introduction of parvalbumin (Parv), a specialized Ca²⁺ buffer (10). The SERCA2a-to-PLB ratio has been manipulated by overexpression of SERCA2a (20) and expression of antisense PLB (6) and in PLB-knockout mice (27). These approaches have restored Ca²⁺-handling function in a number of animal models of heart failure (13, 14, 17, 29, 37, 39). However, in recent studies, increasing the SERCA2a-to-PLB ratio did not rescue the failing heart in all animal models (38, 41). Paradoxically, in human studies, individuals expressing a nonfunctional mutated form of PLB (infinite SERCA2a-to-PLB ratio) have a high risk of developing cardiomyopathy early in adult life (8, 19, 40).

Another strategy has employed the delayed Ca²⁺-buffering properties of Parv to increase the relaxation properties in cardiac muscle in vitro (11, 43) and in vivo (42). The mechanism by which Parv increases relaxation performance in cardiac myocytes has been described elsewhere (10). Briefly, Parv is an 11-kDa protein naturally found in fast-twitch skeletal muscles and in the brain, but not in the heart (15). Parv functions as a delayed Ca²⁺ buffer and has been shown to be associated with increased relaxation rates in skeletal muscle (23). The Parv molecule has two binding sites with high affinity for Ca²⁺ (10⁸ M^–1) and moderate affinity for Mg²⁺ (10⁴ M^–1) (34). Because in myocytes the level of free Mg²⁺ is several orders of magnitude larger than the level of intracellular free Ca²⁺ at rest, a large portion of Parv is bound to Mg²⁺ at rest. When the Ca²⁺ level transiently rises in the myocyte, Mg²⁺ dissociates from Parv, and Ca²⁺ subsequently binds (10). Over an optimal Parv concentration range, the delayed Ca²⁺-buffering capacity of Parv is evident during the initial phase of Ca²⁺ decay (increasing diastolic function), with no or little impact during the rising phase of the Ca²⁺ transient, thus preserving systolic function (11). As was the case for the first strategy (modification of the SERCA2a-to-PLB ratio), Parv also improved relaxation kinetics in animal models of diastolic dysfunction (9, 42, 43).

In this study, we investigated the effect of modifications in the SERCA2a-to-PLB ratio via SERCA2a overexpression and expression of Parv on cardiac myocyte relaxation performance. The overall objective was to gain mechanistic insights into Ca²⁺-handling properties and to understand the consequences of SERCA2a and Parv gene transfer in important aspects of cardiac myocyte mechanical function. Accordingly, we used an experimental approach of gene transfer of Parv or SERCA2a in isolated rat adult cardiac myocytes (present study) combined with a theoretical analysis using mathematical modeling (12).

Our aims in this experimental study were threefold. Our first aim was to determine whether the increase in relaxation rate caused by Parv-delayed Ca²⁺-buffering action could be maintained when the SERCA2a removal mechanism was impaired, as observed in human heart failure. As a mimetic of altered SERCA2a function in heart failure, we used a submaximal dose of thapsigargin (TG, 150 nM), a chemical compound that specifically inhibits SERCA2a (25, 45). Under these conditions, we tested the hypothesis that although TG in control myocytes would cause a significant slowing of relaxation, the rapid relaxation kinetics observed in Parv-transduced myocytes would be unaltered by TG.

Our second aim was to determine the effects of Parv expression and SERCA2a overexpression in the presence of -adrenergic stimulation. In normal myocardium, -adrenergic stimulation markedly enhances heart pump performance. In heart failure, -adrenergic stimulation responsiveness is reduced (2). It is therefore of interest to investigate whether genetic manipulations in Ca²⁺-handling proteins would affect the -adrenergic response in myocytes.

Our final aim focused on postrest potentiation (PRP) tests in Parv- and SERCA2a-transduced myocytes. PRP is defined as the contractile response, relative to a steady state, obtained after a resting period of given duration (2). The nature of the PRP response is believed to be mainly dictated by the ability of the SR to reload Ca²⁺ and by the refractory properties of the myocytes (2). Although frequency response has been tested in Parv-transduced (11) and SERCA2a-overexpressing (37) myocytes, PRP offers additional insights into the interplay between modified Ca²⁺ removal and SR Ca²⁺-reloading properties.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Adult cardiac myocyte isolation, gene transfer, and primary culture. Cardiac myocytes were isolated from adult female Sprague-Dawley rats (200 g), and gene transfer was performed as described previously (11, 44). Briefly, myocytes were plated and incubated for 2 h with no virus (control) or adenovirus containing the Lac-Z gene [200–500 multiplicity of infection (MOI)], the human -Parv gene (500 MOI), or the human SERCA2a gene (200 MOI). These doses have been shown to achieve maximal (95–100%) efficiency of gene transfer (11, 43). The myocytes used for functional studies were transferred to stimulating chambers 18 h after gene transfer. The myocytes were then subjected to electrical stimulation (0.5-Hz frequency) sufficient to elicit contraction in 10–20% of the myocytes and were kept in M199+ solution (Sigma) supplemented with 10 mM glutathione, 26.2 mM sodium bicarbonate, 0.02% bovine serum albumin, and 50 U/ml penicillin-streptomycin, with pH adjusted to 7.4, as described previously (11).

The procedures used in this study were in agreement with the guidelines of the Internal Review Board of 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. The University of Michigan is accredited by the American Association of Accreditation of Laboratory Animal Health Care, 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).

Western blots. Each sample (40,000 myocytes) was collected from two coverslips 3 days after gene transfer and stored at –80°C in SDS-PAGE buffer (11). The following primary antibodies were used for Western blotting: 5c5 (Sigma; 1:5,000 dilution) for actin, Parv19 (Sigma; 1:1,000 dilution) for Parv, MA3-922 (ABR; 1:1,000 dilution) for PLB, and MA3-919 (ABR; 1:1,000 dilution) for exogenous SERCA2 (MA3-919 does not recognize endogenous rat SERCA2a). Goat anti-mouse conjugated to horseradish peroxidase secondary antibody (catalog no. A-9917, Sigma; 1:1,000 dilution) was used as secondary antibody. Antibody C-20 (Santa Cruz Biotechnology; 1:100) combined with donkey anti-goat Ig-horseradish peroxidase (Santa Cruz Biotechnology; 1:500 dilution) was used to detect simultaneously the endogenous (rat) and exogenous (human via gene transfer) SERCA2a by enhanced chemiluminescence. In all cases, actin was used to normalize for loading (11).

To estimate Parv concentration, the transduced cardiac myocyte samples were compared, after loading normalization, with samples collected from rat superior vastus lateralis (SVL) muscles. A Parv concentration of 0.4 mM was used for the SVL-positive control samples (18).

We defined SERCA2a overexpression as the ratio of transduced SERCA2a to the endogenous level of SERCA2a. To quantify SERCA2a overexpression, we used two different methods. First, we used the antibody C-20, which detects the endogenous rat SERCA2a and the genetically transduced human SERCA2a. To calculate the amount of overexpression of human SERCA2a, we subtracted the amount of endogenous SERCA2a expressed in rat control myocytes from the total amount of SERCA2a expressed in the transduced myocytes and then normalized to the level of endogenous SERCA2a (see Eq. 1).

Second, we used the antibody MA3-919, which detects the transduced human SERCA2a, but not the endogenous rat SERCA2a. We were able to quantify the overexpression, because 1) this antibody also detects rabbit SERCA2a, and 2) the Ca²⁺ uptake rate in rat myocytes is 2.5 times that in rabbit myocytes and SERCA2a affinity for Ca²⁺ is the same in both species (25). Assuming that the difference in function is due to a difference in the number of pumps, we derived the following equation: SERCA2a_{rat(endogenous)} = 2.5 * SERCA2a_{rabbit(endogenous)}. Therefore, with this value, we can use the endogenous SERCA2a expression in rabbit myocytes, as detected with Western blot analysis, to quantify SERCA2a overexpression in our transduced rat myocytes (see Eq. 2). Both methods rely on the two following assumptions: 1) the antibody affinity for SERCA2a is the same across the different species used, and 2) the amount of endogenous SERCA2a in transduced myocytes remains the same as in the control myocytes

(1)

(2)

Sarcomere shortening and Ca²⁺ transients. All experiments were performed at 37 ± 1°C (unless specified otherwise) in M199+ solution. Coverslips were mounted on a custom microscope stage. Unloaded sarcomere shortening and Ca²⁺ transients were measured simultaneously using the fluorescence and contractility system from Ionoptix (Milton, MA). Shortening data were sampled at 240 Hz, and Ca²⁺ fluorescence was sampled at 1,000 Hz. The fluorescence ratio (ratio of excitation at 360 nm to excitation at 380 nm) was measured with fura 2-AM. For each myocyte, 8–10 contractions were averaged and analyzed using the Ionoptix software. The fluorescence signal was filtered using a Savitzky-Golay filter (width = 11, order = 2). Ca²⁺ fluorescence signals were normalized to a maximal value (1.0) after subtraction of background. For sarcomere length (SL) shortening measurements, time from stimulus to peak (T_p), times from peak to one-fourth, one-half, and three-fourths relengthening (T_{p r}, T_{p r}, and T_{p r}), amplitude (ampl), and relengthening maximal velocity (–dl/dt) were measured. For PRP experiments, time from stimulus to one-half relengthening (T_{s r}) was also evaluated. For Ca²⁺ fluorescence measurements, the following parameters were measured: time from stimulus to peak (T_{p Ca}) and times from stimulus to one-fourth, one-half, and three-fourths decay (T_{s d}, T_{s d}, and T_{s d}).

Three independent sets of experimental data were collected: TG, Iso, and PRP. For the first data set (TG), the stimulation-and-measurement protocol was optimized from results obtained in pilot studies (unpublished data), in which we found that 150 nM TG produced a nearly maximal inhibition effect, the drug effect was stable with 7–15 min of incubation, and continuous pacing empties the SR content, annihilating contractions. The protocol is as follows: myocytes were loaded for 5 min with Ca²⁺ fluorescence indicator [5 µM fura 2-AM with 0.01% Pluronic F-127 (both from Molecular Probes)], and after washout, an additional 5 min were allowed for deesterification. The myocytes were then stimulated for 1 min at 0.2 Hz before the baseline measurement was made (also at 0.2 Hz). Then the myocytes were treated with 150 nM TG in the quiescent state (no stimulation) for 8 min. At the end of the TG incubation period, the myocytes were again stimulated for 1 min at 0.2 Hz before measurements were made. TG (Sigma) was diluted to 500 µM in DMSO as stock solution. The stock solution was diluted in M199+ solution. Measurements were made at two external Ca²⁺ concentrations: 1.8 and 5.0 mM.

The same protocol was used for the second set of data (Iso), except for the external Ca²⁺ concentration (fixed at 1.8 mM), dose (10 nM Iso), and incubation time (1–2 min). The dose was selected to produce a strong inotropic effect with a minimal number of fibrillating myocytes, and the incubation time was the minimal amount of time required for the myocytes to stabilize for temperature. In addition to measurements at 37°C, measurements at room temperature (22 ± 2°C) were performed to emphasize the Iso effect on relaxation kinetics.

For the final data set (PRP), the myocytes were loaded with fura 2-AM as described above. The experimental protocol (see Fig. 3A) follows this sequence: conditioning train of 10 pulses (interpulse duration = 1 s), 2 min of rest, another train of 10 prepulses (interpulse duration = 1 s), a variable rest period of duration T, and a train of 10 postpulses (interpulse duration = 1 s). The ratio of postpulse 1 to the average of the last three prepulses (prepulses 8–10) was used to characterize PRP.

View larger version (35K): [in this window] [in a new window]   Fig. 3. Postrest potentiation (PRP) measurements. A: representative PRP sarcomere-shortening traces. Protocol timeline is shown below traces. Duration of rest interval (T) was as follows: 2, 3, 5, 10, 25, and 120 s. Typical "normal" responses for control (n = 9), Parv (n = 8), and SERCA2a (n = 8) myocytes are shown, as well as atypical "abnormal" SERCA2a myocyte response (n = 2). B: summary of sarcomere-shortening (SS) amplitude PRP data. PRP was measured as postpulse 1 data over the average of prepulses 8, 9, and 10. Parv myocytes had the most potentiation, control and "normal" SERCA2a myocytes had a similar trend, but to a lesser extent, and "abnormal" (SE not shown; n = 2) SERCA2a had a negative potentiation at long rest period. C: summary of the time from stimulus to one-half relengthening (Ts r) PRP data. Kinetics data followed a trend similar to that of amplitude data. Values are means ± SE.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Gene transfer. Gene transfer to isolated rat adult cardiac myocytes in primary culture has been shown to be a highly efficient and synchronized means of acute genetic engineering (11, 28). Typically, >95% of the adult cardiac myocytes express the delivered gene product (44). In this study, all experiments were conducted 3 days after gene transfer to yield a robust expression of Parv (11) and SERCA2a (Fig. 1). Figure 1 shows representative Western blots, and, as expected, Parv was present only in the Parv-transduced myocytes. The level of expression was 34.5 ± 11.4% (n = 11) of the Parv level in SVL muscle samples. With the assumption of 0.4 mM Parv in SVL (18), this leads to an estimated 0.138 ± 0.046 mM in the transduced cardiac samples. Two different antibodies were used to detect SERCA2a. The first (anti-SERCA2a#1 in Fig. 1, antibody C-20) detected endogenous SERCA2a and human SERCA2a obtained through gene transfer. Although a small increase in total SERCA2a expression could be seen in some Western blot results, it did not reach statistical significance. However, with use of the second antibody (anti-SERCA2a#2, antibody MA3-919), it was possible to uniquely detect the human SERCA2a expressed by gene transfer. As described in METHODS, we used nontransduced rabbit cardiac myocytes to quantify the level of exogenous SERCA2a in rat myocytes. On average, the transduced rat myocytes expressed 1.15 ± 0.18 times the level of endogenous SERCA2a found in rabbit cardiac myocytes (when normalized to actin). Knowing that the amount of SERCA2a expression in rabbit is 40% of that in rat myocytes (25), we can indirectly obtain an overexpression factor of 46 ± 7% (Eq. 2). The reason for the discrepancy between these two methods is unclear. Because the second method clearly indicates that the overexpressed SERCA2a is present only in the transduced myocytes and that a strong phenotype is present in these myocytes (see Figs. 2–8), it is possible that a difference in affinity for the endogenous rat SERCA2a and exogenous human SERCA2a by antibody C-20 could help explain our findings. The two methods used for quantifying SERCA2a overexpression give values of 15% and 46%. Because antibody MA3-919 clearly shows overexpression of human SERCA2a and given some uncertainties regarding the exact avidity of antibody C-20 between human and rat SERCA2a, we interpret the reason for the accelerated relaxation kinetics of SERCA2a myocytes to be due to 15–46% overexpression of SERCA2a. No significant changes were detected in the PLB level among the different groups of myocytes.

View larger version (57K): [in this window] [in a new window]   Fig. 1. Western blot analysis of Ca2+-handling proteins in rat adult cardiac myocytes. Ctrl, control myocytes; Lac-Z, Parv, and SERCA2a, myocytes transduced with Lac-Z, parvalbumin, and sarco(endo)plasmic reticulum Ca2+-ATPase, respectively; SVL, superior vastus lateralis muscle (positive control for Parv). Parv was detected only in Parv-transduced myocytes and at a level comparable to that in SVL samples. Anti-SERCA2a#1 shows endogenous and overexpressed SERCA2a; anti-SERCA2a#2 shows only SERCA2a produced via gene transfer. No changes were observed in expression of phospholamban (PLB; P, pentamer; M, monomer). Actin was used to control for loading.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Functional analysis of Parv and SERCA2a gene transfer. A: representative normalized sarcomere-shortening traces. Parv and SERCA2a had similar effects on increasing relaxation kinetics. B: representative normalized Ca2+-fura 2-AM fluorescence traces. Initial acceleration of Ca2+ decay is similar in Parv and SERCA2a myocytes; however, only SERCA2a retains the faster decay properties toward the end of the transient. C: summary of sarcomere-shortening measurements. Average resting sarcomere length was similar among all groups: 1.80–1.82 µm. D: summary of Ca2+ fluorescence measurements. Values are means ± SE; n = 10–14. Significantly different from control: +P < 0.05; *P < 0.01; #P < 0.001.

View larger version (36K): [in this window] [in a new window]   Fig. 8. Effect of a submaximal dose (150 nM) of thapsigargin (TG) on sarcomere-shortening and Ca2+ fluorescence measurements. A: representative normalized Ca2+ fluorescence measurements for control, Parv, and SERCA2a myocytes at 5.0 mM external Ca2+. B-E: summary of change from baseline in kinetic parameters for Ca2+ fluorescence and sarcomere-shortening data at 1.8 mM (B and C) and 5.0 mM (D and E) external Ca2+. Values are means ± SE of sample size shown in Table 1. *Significantly different from baseline, P < 0.05 (2-tailed paired t-test). Legend in B applies to B–E.

Figure 3B shows the PRP of the sarcomere-shortening amplitude. The results are plotted as the ratio of postpulse 1 to the prepulse steady state (average of the last 3 prepulses) as a function of the duration of the rest period (logarithmic scale): 100% indicates no potentiation, and >100% indicates a positive potentiation response. Control and typical SERCA2a-overexpressing myocytes show a small increase at a shorter rest period (2–10 s), with values increasing to 178.6 ± 11.6% and 154.0 ± 35.5% at 120 s, respectively. Parv myocytes showed a very high potentiation at 120 s (206.7 ± 20.2%). Part of this potentiation is due to the fact that Parv myocytes started from a lower baseline than controls. However, even at 2 s, Parv myocytes showed significant potentiation (124.5 ± 6.4%). For completeness, SERCA2a myocytes with atypical responses are also shown. These atypical SERCA2a myocytes show a flat and elevated potentiation (at low T, i.e., 2–10 s), followed by a negative potentiation at longer rest intervals (25–120 s). The kinetics, as measured by the time from stimulus to one-half relengthening, followed a trend similar to the amplitude, but to a lesser extent (Fig. 3C).

Another way to analyze these data is to observe how the 10 prepulses vary from the first to the last (Fig. 4). This is equivalent to examining how the myocytes transition to a steady state after a long rest period. All the prepulse trains were obtained after 120 s of rest. For each myocyte, six sets of measurements were obtained and averaged. In Fig. 4A, representative traces show the difference in control myocytes between pulse 1 and pulse 9 for Ca²⁺ fluorescence and sarcomere shortening. In both cases, the amplitude reduction was accompanied, to a lesser extent, by a decrease in T_{s r} or T_{s d}. The summaries of the sarcomere-shortening amplitude and T_{s r} are presented in Fig. 4, B and C, respectively. The amplitude curves for the control and SERCA2a myocytes showed a progressive decrease from pulse 1, with SERCA2a myocytes showing a flatter response. In contrast, Parv-treated myocytes showed an abrupt decline from pulse 1 to pulse 2 and then stabilized for the subsequent pulses with a smaller decline. Similar trends were observed in T_{s r}. When the change in T_{s r} was plotted against the change in amplitude (100% = no change from prepulse 1), a strong and similar correlation was found in all groups (Fig. 4D). The magnitude of change in amplitude was larger than the magnitude of change in kinetics, and SERCA2a myocyte data were clustered in the region of minimal change (80–100% ampl), whereas Parv myocytes were clustered around the other end of the trend lines (40–60% ampl). In summary, the PRP data suggest that although expression of Parv and overexpression of SERCA2a accelerate relaxation similarly, they modify the ability of the myocyte to handle changes in pacing rhythm differently. The quantitative analysis of these PRP data is complex and well suited for mathematical modeling interpretation (12).

View larger version (38K): [in this window] [in a new window]   Fig. 4. Analysis of PRP prepulses. Sets of 10 prepulses were measured after 120 s of rest. For amplitude data, individual prepulses were used, whereas beat 1 and averages of beats 2–4, 5–7, and 8–10 were used for kinetics data. A: representative Ca2+ fluorescence and sarcomere-shortening traces of prepulse 1 (Pre#1) and prepulse 9 (Pre#9) normalized to prepulse 1. B and C: summary of prepulse data for sarcomere-shortening amplitude and Ts r. Values are means ± SE; n = 8–9. D: correlation between changes in kinetics (y-axis) and amplitude (x-axis) sarcomere-shortening prepulse data, with each point normalized to corresponding prepulse 1 value.

View larger version (30K): [in this window] [in a new window]   Fig. 5. Effect of 10 nM isoproterenol (Iso) at 37°C. A: representative shortening traces for control, Parv, and SERCA2a myocytes without (baseline) or with 10 nM Iso. B-D: summary of sarcomere-shortening amplitude, Tp r, and Ca2+ fluorescence Ts d in response to 10 nM Iso. Values are means ± SE; n = 8–11. *Significantly different from baseline, P < 0.05. #Significantly different from control myocytes under the same conditions, P < 0.05. Legend in C also applies to B and D.

View larger version (30K): [in this window] [in a new window]   Fig. 6. Effect of 10 nM Iso at 22°C. A: representative normalized shortening traces for control myocytes showing stronger effect of Iso on relaxation kinetics at 22°C. B-D: summary of sarcomere-shortening amplitude, Tp r, and Ca2+ fluorescence Ts d in response to 10 nM Iso. Values are means ± SE; n = 8–9, except in D, where n = 4 for SERCA2a. *Significantly different from baseline, P < 0.05. #Significantly different from control myocytes under the same conditions, P < 0.05. Legend in C also applies to B and D.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effect of elevated external Ca2+ concentration (from 1.8 to 5.0 mM). A and B: summary of sarcomere-shortening amplitude and Tp r. SERCA2a showed a blunted response to elevated external Ca2+ compared with other myocytes. Values are means ± SE; n = 10–17. Measurements at low and high Ca2+ were made on an independent set of myocytes. *Significantly different from 1.8 mM Ca2+, P < 0.05.

For all conditions taken together, >91% (106 of 116) of the myocytes survived the TG treatment (Table 1). Although all surviving myocytes were used to calculate shortening amplitude and Ca²⁺ fluorescence data, only those that retained 25% of the shortening amplitude of the pre-TG treatment (73%, or 77 of 106) were kept for sarcomere-shortening kinetic analysis, because low signal-to-noise ratio (or even the absence of contraction) makes evaluation of the kinetic parameters difficult (or impossible).

Figure 8A shows representative traces of normalized Ca²⁺ fluorescence measurements with and without TG. The magnitude of change from baseline in the Ca²⁺ fluorescence kinetic measurements at low and elevated external Ca²⁺ is summarized in Fig. 8, B and D, respectively. T_{s d} was increased by 30.2 ± 12.1% and 72.4 ± 20.4% at 1.8 and 5.0 mM external Ca²⁺, respectively, in control myocytes, whereas the increase was 62.4 ± 13.4% and 45.2 ± 8.2%, respectively, in SERCA2a myocytes. On the other hand, Parv myocytes showed an increase of only 9.1 ± 6.1% and 22.2 ± 12.1%, respectively. When a two-tailed paired t-test was conducted, only Parv myocytes did not show a significant increase in T_{s d} and T_{s d} (at 1.8 and 5.0 mM external Ca²⁺). The average attenuation of sarcomere-shortening amplitude was similar in all groups under all conditions and was 40.4–55.5% (Table 1). At low external Ca²⁺, only the late relaxation kinetic parameter T_{p r} showed significant slowing in control myocytes (Fig. 8C). At elevated Ca²⁺, in control myocytes the relative increase from baseline in all measured kinetic parameters (T_{p r}, T_{p r}, and T_{p r}) was statistically significant (Fig. 8E). Again, only Parv myocytes were able to preserve their kinetic properties in the presence of TG in all sarcomere-shortening kinetic parameters. The data suggest that Parv could have a protective role in maintaining relaxation properties under conditions where the main Ca²⁺ sequestration mechanism is partially impaired, as seen in heart failure.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
We have reported a direct comparison between the effects of Parv expression de novo and SERCA2a overexpression in isolated rodent cardiac myocytes under a variety of experimental conditions. Using unloaded sarcomere shortening and Ca²⁺ fluorescence, we found that 1) Parv- and SERCA2a-transduced myocytes have similar effects on shortening kinetics, 2) SERCA2a myocytes better preserve contractile amplitude than Parv myocytes (at high levels of expression), and 3) although the shortening amplitude in the presence of 10 nM Iso is similar in all groups at physiological temperature, the increase in shortening amplitude of Parv myocytes in response to -adrenergic stimulation was comparable to that of control myocytes, whereas the response of SERCA2a myocytes was reduced. In addition, we showed that Parv corrects diastolic dysfunction in a chemical model where only the SR Ca²⁺-ATPase is impaired.

TG as a chemical model of diastolic dysfunction. TG is derived from the plant Thapsia garganica (45), has a very high affinity for SERCA2a (K_d < 1 nM) (25), and is a very specific inhibitor of SERCA2a. In this study, the intent was to mimic diastolic dysfunction by using a submaximal dose of TG to create a partial inhibition of SERCA2a function, a feature often seen in heart failure (see the introduction).

A number of cellular changes occur during heart failure. Indeed, in addition to the impairment observed in Ca²⁺ removal, myosin heavy chain isoform changes from to (4), several K⁺ currents (e.g., I_to and I_k1) are downregulated (32), and the increase in Ca²⁺ diastolic level activates calmodulin-dependent signaling events, which can lead to hypertrophy (7, 33). Isolation of the effect specific to Ca²⁺ removal deficiency in this context becomes difficult. Even more difficult is the task of understanding the basic effects of the addition or modification of Ca²⁺-handling proteins to this complex pathophysiological system. The use of TG to mimic diastolic dysfunction allows the specific isolation of impaired SR Ca²⁺ uptake to directly study the biophysical effects of adding a delayed Ca²⁺ buffer such as Parv. However, this model is not suitable for use in studying the effect of overexpressing SERCA2a in diastolic dysfunction, because TG would also affect the newly expressed Ca²⁺ pumps.

One consequence of using TG in rodent cardiac myocytes to mimic diastolic dysfunction is the progressive emptying of the SR. This also creates systolic dysfunction. This situation can be corrected by using TG combined with some inotropic strategy. In this study, we used elevated external Ca²⁺ to partially improve the sarcomere-shortening amplitude in the presence of TG. A direct comparison between data without TG at 1.8 mM external Ca²⁺ and data with 150 nM TG at 5.0 mM external Ca²⁺ reveals a strong diastolic dysfunction with almost no diminution in systolic function (Table 1). Other strategies, such as an augmentation of Ca²⁺ cellular entry via an increase in action potential duration or a reduced Ca²⁺ cellular exit via a reduced NCX function, could produce similar results (12).

TG does not affect Parv-mediated rapid relaxation. In the presence of 150 nM TG, control (and Lac-Z-transduced) myocytes exhibited slower sarcomere-relengthening kinetics, especially in the late phase of the cycle. However, when Parv myocytes were treated with the same TG dose, no significant changes were observed (Table 1, Fig. 8). The sarcomere-shortening behavior of these two groups in the presence of TG could be explained better by first analyzing their Ca²⁺ transient properties.

Ca²⁺ fluorescence data showed that all phases of the Ca²⁺ transient decay were slowed in control myocytes when TG was applied. In contrast, Parv-transduced myocytes were able to maintain their already rapid kinetics in the presence of TG, at least in the first 50–60% of the decay. The basis for this result is hypothesized as follows. In rat control myocytes, SERCA2a pumps are responsible for almost all Ca²⁺ sequestration (2) and, therefore, drive the entire shape of the Ca²⁺ transient decay. When these pumps become partially inhibited by TG, the entire decay is slowed. However, with Parv, the early part of the decay is mostly dominated by the delayed buffering of Ca²⁺ by Parv. In this case, inhibition of SERCA2a has little influence on this part of the response. In the late part of the Ca²⁺ fluorescence decay, preservation of the rapid kinetics in Parv myocytes was reduced. In the absence of TG, it has been shown that Parv slowly releases its bound Ca²⁺ in the myoplasm during the late decay phase in myocytes (10). The rate of this reaction is dictated by a combination of the SERCA2a sequestration properties and the Ca²⁺ off-rate from Parv. When TG is added to the Parv-transduced myocytes, the Ca²⁺ off-rate is unaffected but the SERCA2a uptake rate is diminished, resulting in an even slower late phase of Parv-transduced myocytes. However, this late phase appears to be below the threshold required to create mechanical contraction, because it has not been observed in any of the sarcomere-shortening measurements presented in this study.

On the basis of this model of diastolic dysfunction, we can speculate that, in addition to correction of diastolic function, as seen previously in some animal models (42, 43), Parv could potentially offer some degree of protection against downregulation of Ca²⁺ pumps in vivo, as seen in human heart failure (1, 16).

Parv and SERCA2a: -adrenergic response and systolic function. Although de novo expression of Parv and overexpression of SERCA2a operate through very distinct mechanisms, both increase relaxation rates to a similar extent during single contractions. However, their response differs in at least two important aspects: responsiveness to -adrenergic stimulation and management of contractile amplitude.

In this study, Parv-transduced myocytes showed a reduction in sarcomere-shortening amplitude compared with control myocytes. In a previous study (11), we found that Parv buffers most of its Ca²⁺ in the early phase of the Ca²⁺ transient decay. However, when Parv is expressed at higher concentrations, it can also affect the rising phase of the Ca²⁺ transient, resulting in reduced Ca²⁺ transient and sarcomere-shortening amplitudes (11). In the same study, we demonstrated that there was an optimal range of Parv concentration for which Parv increases relaxation rates without affecting contractile amplitude (11). This phenomenon occurs at 0.01–0.1 mM Parv (or at 2.5 days after gene transfer) for rat cardiac myocytes in vitro. In the present study, we focused on Parv concentrations above this optimal range (0.14 mM, 3.0 days after gene transfer) to have a greater effect on relaxation kinetics. In comparison, under the conditions used in this study, SERCA2a-transduced myocytes showed only a tendency to increase shortening amplitude, without reaching statistical significance. Other studies have shown no change or an increase in amplitude, depending on the species studied and experimental conditions (6, 20). In particular, the increase in amplitude seems to be more apparent at higher stimulation frequency (6). In this study, our use of a low stimulus frequency (0.2 Hz) might explain this relatively small increase in sarcomere-shortening amplitude in SERCA2a-overexpressing myocytes. Pharmaceutical treatments that have negative inotropic effects on the myocardium, such as -blockers, have been shown to help patients with heart failure (22, 36). It is therefore possible that, in addition to its positive impact on relaxation rate, Parv attenuation effects on contractile function may also be beneficial. Interestingly, not only did Szatkowski et al. (42) not find any reductions in systolic function in the Parv-transduced rat heart in vivo, they reported an increase in pressure development after gene transfer. Clearly, more research is required to determine the optimal inotropy/lusitropy strategy in vivo.

As mentioned above, the -adrenergic response is critical in managing the wide range of pumping capacity of the heart. In this study, we examined the dynamic range of shortening amplitude when the Parv- and SERCA2a-transduced myocytes were treated with 10 nM Iso. Parv-transduced and control myocytes showed a comparable increase in sarcomere-shortening amplitude, whereas in SERCA2a myocytes the amplitude increased only moderately (Table 2). Part of the explanation for this limited increase in SERCA2a-transduced myocytes is the slightly more elevated baseline amplitude than in control myocytes. However, at 10 nM Iso, there was a tendency for SERCA2a myocytes to have lower shortening amplitude than controls, but it did not reach statistical significance. A possible explanation for this phenomenon is that the level of SERCA2a in overexpressing myocytes is increased without any change in PLB content (6, 20), most likely leaving a large portion of the new pumps uncoupled to PLB and, therefore, less sensitive to Iso. A possible limitation of the present study is that measurements were made under unloaded conditions and that mechanical load could affect the baseline properties and the responsiveness to -adrenergic stimulation.

In conclusion, we used TG as a chemical means to mimic cellular diastolic dysfunction and found that Parv myocytes retained their rapid relaxation properties. This study also provides, for the first time, a detailed characterization of rat cardiac myocytes transduced with Parv under a variety of conditions: -adrenergic stimulation, elevated external Ca²⁺, lowered temperature, and PRP tests. A direct comparison with SERCA2a overexpression was performed, highlighting the strengths and possible limitations of each approach; SERCA2a overexpression has a reduced range of modulation by -adrenergic stimulation, whereas Parv de novo expression is more susceptible to decrease in contractile function, at least in vitro.

This study, combined with our mathematical analysis (12), helps clarify the effects of adding new Ca²⁺-handling proteins in normal and diseased cardiac myocytes. It remains to be determined whether the results and conclusions found in vitro in rodent myocytes will translate to the organ level of larger mammals, including humans.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. M. Metzger, Dept. of Physiology, 7730 Medical Science II, Univ. of Michigan, 1301 E. Catherine St., Ann Arbor, MI 48109-0622 (E-mail: metzgerj{at}umich.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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