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Parvalbumin isoforms differentially accelerate cardiac myocyte relaxation kinetics in an animal model of diastolic dysfunction

¹Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan; and ²Department of Molecular and Cellular Pharmacology, University of Miami, Miami, Florida

Submitted 22 February 2007 ; accepted in final form 31 May 2007

	ABSTRACT

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The cytosolic Ca²⁺/Mg²⁺-binding protein -parvalbumin (-Parv) has been shown to accelerate cardiac relaxation; however, beyond an optimal concentration range, -Parv can also diminish contractility. Mathematical modeling suggests that increasing Parv's Mg²⁺ affinity may lower the effective concentration of Parv ([Parv]) to speed relaxation and, thus, limit Parv-mediated depressed contraction. Naturally occurring /-Parv isoforms show divergence in amino acid primary structure (57% homology) and cation-binding affinities, with -Parv having an estimated 16% greater Mg²⁺ affinity and 200% greater Ca²⁺ affinity than -Parv. We tested the hypothesis that, at the same or lower estimated [Parv], mechanical relaxation rate would be more significantly accelerated by -Parv than by -Parv. Dahl salt-sensitive (DS) rats were used as an experimental model of diastolic dysfunction. Relaxation properties were significantly slowed in adult cardiac myocytes isolated from DS rats compared with controls: time from peak contraction to 50% relaxation was 57 ± 2 vs. 49 ± 2 (SE) ms (P < 0.05), validating this model system. DS cardiac myocytes were subsequently transduced with - or -Parv adenoviral vectors. Upon Parv gene transfer, -Parv caused significantly faster relaxation than -Parv (P < 0.05), even though estimated [-Parv] was 10% of [-Parv]. This comparative analysis showing distinct functional outcomes raises the prospect of utilizing naturally occurring Parv variants to address disease-associated slowed cardiac relaxation.

gene transfer; mechanical relaxation

A recent approach to redress diastolic dysfunction has focused on the introduction of a non-energy-dependent mechanism for accelerating relaxation. Specifically, -parvalbumin (-Parv), a small (11-kDa) Ca²⁺-binding protein not typically expressed in mammalian cardiac myocytes, has been demonstrated to accelerate relaxation in a variety of models of diastolic dysfunction (5–9, 17, 25, 29). The proposed mechanism for -Parv-based acceleration of cardiac relaxation is described in detail elsewhere (6, 7, 9). Briefly, -Parv functions as a delayed Ca²⁺ buffer, where systolic Ca²⁺ binding is limited by the slow off-rate of Mg²⁺ from -Parv that must occur before Ca²⁺ binding. Ideally, this would exclusively occur in early to mid diastole to speed relaxation while preserving contraction amplitude (6). Whereas -Parv has been shown to correct diastolic dysfunction in disease models and aging (5, 17, 25), -Parv expression-dependent depressed cellular contraction is evident. In vitro studies have defined an optimal -Parv expression window of 10–100 µM to cause myocyte relaxation without compromising systolic function. Mathematical modeling predicts that modification of -Parv's metal-binding affinities could be useful in enhancing the functional properties of -Parv in cardiac myocytes (7, 9). Previous studies exclusively used -Parv to redress cardiac diastolic dysfunction (5–9). In nature, two distinct Parv isoforms, - and -Parv, have 43% dissimilarity in amino acid sequence (Supplemental Fig. 1; supplemental data for this article are available at the American Journal of Physiology-Heart and Circulatory Physiology website). Thus it is postulated that the divergence in Parv isoform primary structure, including select amino acid differences in the more conserved E-F hand consensus sequence, could translate to alterations in cation binding in the intact cardiac myocyte. Biochemical assessment of isolated Parv isoforms in solution provides evidence that -Parv binds Mg²⁺ and Ca²⁺ more tightly than -Parv (12; unpublished observations). An increase in Mg²⁺ affinity, through decreased off-rate, in particular, is modeled to further delay the Ca²⁺-buffering function of Parv.

In this study, we compared for the first time the direct effects of - and -Parv on myocyte performance to test the hypothesis that -Parv would cause faster relaxation than -Parv in cardiac myocytes. Sarcomere shortening and relaxation properties on isolated adult cardiac myocytes were obtained from a hypertensive rat model of diastolic dysfunction and CHF (11). The overall objective was to gain insight into the functional relationship between Parv isoforms and shortening and relaxation kinetics in a model of diastolic dysfunction. To optimize analysis and interpretation, we focused on isolated single cardiac myocytes and used an experimental approach of in vitro gene transfer of - or -Parv. Adult rat cardiac myocytes obtained from Dahl salt-sensitive (DS) rats were fed a high-salt diet to yield a diastolic dysfunction phenotype (11). The DS rat has a renin gene polymorphism and a mutation in the ₁-Na⁺-K⁺-ATPase gene, such that a high-salt diet results in hypertension, with low levels of renin and aldosterone. Recently, Doi and colleagues (11) demonstrated a significantly different progression in hypertension between DS rats fed a high-salt (HS) diet starting at 7 wk of age and those starting the diet at 8 wk of age. Specifically, DS rats fed a high-salt diet starting at 7 wk of age developed hypertension and overt signs of CHF due to diastolic dysfunction with preserved systolic performance.

Our findings of significantly slowed relaxation with preserved shortening amplitude in cardiac myocytes from HS animals validate this as a useful cellular model of diastolic dysfunction with preserved systolic performance. Gene transfer of Parv isoforms demonstrated correction of slowed relaxation, with -Parv causing significantly faster relaxation than -Parv (P < 0.05). These results are the first to indicate that naturally occurring variations in Parv primary structure can be useful in tailoring specific contractile performance outcomes in cardiac myocytes with diastolic dysfunction.

	METHODS

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Animal model and hemodynamics. All surgical and experimental procedures involving animals were reviewed and approved by the University of Michigan Committee on the Use and Care of Animals. The American Association of Accreditation of Laboratory Animal Health Care accredits the University of Michigan, and the animal care use program conforms to the standards of the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23).

Sixteen 4- to 5-wk-old male DS rats (Brookhaven variety) were obtained from Harlan (Indianapolis, IN). All rats were fed standard rat chow (0.4% NaCl) ad libitum during a 1-wk travel-recovery period. Subsequently, rats were randomly assigned to a control group (n = 8), which was continuously fed a standard rodent chow, or a high-salt (HS) group (n = 8), which was switched to a diet consisting of commercially available purified rodent chow (Dyets) containing 8% NaCl beginning at 7 wk of age, following published protocols (11). Systolic blood pressure was measured at alternating weeks with a tail-cuff system (IITC, Woodland Hills, CA) starting at 7 wk of age to document the progression of hypertension.

Echocardiography. Rats were continuously anesthetized with 1.5% inhaled isoflurane-in-oxygen while echocardiograms were performed using a GE Vivid 7 and an S10-MHz phased-array transducer. Systolic and diastolic dimensions and wall thickness were measured in M-mode in the parasternal short-axis view at the level of the papillary muscles. Fractional shortening and ejection fraction were calculated from the M-mode parasternal short-axis view. Diastolic function was assessed by conventional pulsed-wave spectral Doppler analysis of mitral valve inflow patterns [early (E) and late (A) filling waves]. Doppler tissue imaging (DTI) was used to measure the early (Ea) and late (Aa) diastolic tissue velocities of the mitral annulus, the septal annulus, and the posterior wall.

Adult cardiac myocyte isolation, gene transfer, and primary culture. Isolated adult (18- to 20-wk-old) cardiac myocytes were obtained from control and HS-treated DS rats. Cardiac myocyte isolation and gene transfer were performed as previously described (7, 30). Briefly, cardiac myocytes were plated and incubated for 2 h with no virus (control), -Parv (500 multiplicity of infection), or -Parv (200 multiplicity of infection). These doses permit functional testing 3 days after gene transfer with 95–100% gene transfer efficiency. At 18 h after isolation and gene transfer, myocytes used for functional studies were transferred to stimulating chambers containing 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 (7). Myocytes were maintained under field stimulation (0.2 Hz, 7.0 V) using a MyoPacer field stimulator (Ionoptix, Milton, MA). Acutely isolated myocyte width/length dimensions were measured directly via light microscopy.

Recombinant adenoviruses. The -Parv recombinant adenoviral vector was constructed as previously described (30). The -Parv recombinant adenovirus contained the carp -Parv cDNA. Gene expression for each vector was under the control of the cytomegalovirus promoter, and the simian virus 40 provided the polyadenylation signal. High-titer, plaque-purified adenoviral stocks were produced and purified, and viral aliquots were stored at –80°C.

Western blot analysis. Myocytes were collected 3 days after isolation in 4x Laemmli sample buffer, as previously described (7). Briefly, we obtained samples by applying 10 µl of sample buffer to an individual coverslip (20,000 myocytes) and scraping with a pulled capillary pipette. Collected samples were stored in sample buffer at –20°C. For immunodetection, commercially available monoclonal antibodies were used according to the manufacturer's instructions with corresponding dilutions: 5c5 -actin (1:1,000 dilution; catalog no. A2172, Sigma) and Parv 19 (1:1,000 dilution; catalog no. P3088, Sigma). For detection in both cases, goat anti-mouse antibody conjugated to horseradish peroxidase (1:10,000 dilution; catalog no. A9917, Sigma) was used as the secondary antibody. Detection was performed using the ECL Western blotting detection kit (Amersham Biosciences). Samples collected from rat superior vastus lateralis (SVL) muscle were used as a positive control for [Parv]. For all Parv samples, the level of Parv expression was normalized to the level of actin expression. The same normalization was also performed on the positive control (SVL). Finally, the Parv-to-actin ratio of Parv-transduced myocytes was normalized to the Parv-to-actin ratio of SVL samples to give the estimated Parv concentration in myocytes relative to the Parv level in SVL. The estimated Parv concentration in myocytes is based on a value of 0.4 mM, which was reported for [Parv] in rat SVL (14).

Sarcomere shortening. All experiments were performed at 37 ± 1°C in M199+ solution. Unloaded sarcomere shortening data were collected as previously described (8). Briefly, sarcomere-shortening data were collected at 240 Hz using the contractility system of Ionoptix. A standardized search pattern was used to sample 8–10 myocytes responsive to electrical stimulation from each coverslip per treatment group. The myocyte transient analysis software (Ionoptix) was used to determine baseline (BL) sarcomere length, peak shortening, percent shortening [(peak height/BL)(100)], time to maximal peak shortening, and time from peak to 25, 50, and 75% relengthening, as well as shortening velocity (–dl/dt, where l is sarcomere length) normalized to peak height and relaxation velocity (dl/dt) normalized to peak height.

Statistical analysis. All statistical analyses were performed using SigmaStat software. For experiments where myocytes were studied as populations for each treatment, one-way ANOVA was used to determine any significant differences among groups. When significant differences were detected, pair-wise multiple comparisons (Tukey's test) were performed to determine interaction effects. For experiments determining the effects of the control vs. the HS diet in DS rats, unpaired t-tests were used to determine significance (P < 0.05). Values are means ± SE.

	RESULTS

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Previous reports indicated that initiation of a high-salt diet in DS rats at 7 wk of age results in chronic hypertension, which progresses to a model of CHF primarily associated with diastolic dysfunction independent of marked systolic dysfunction by 19 wk of age (11). This rodent model of diastolic dysfunction was selected specifically to determine whether this phenotype is present in isolated cardiac myocytes and to test the effectiveness of Parv isoforms to redress the diastolic dysfunction. The protocol for the study is outlined in Fig. 1. The HS diet was initiated at 7 wk of age and maintained for 3 mo. On alternate weeks, tail-cuff blood pressures were measured and echocardiography was performed. In agreement with previous reports, systolic blood pressure in HS-treated animals increased steadily and significantly (Fig. 1). By week 15, systolic pressure was >80 mmHg higher in HS-treated than in control animals. Heart rate, stroke volume, and cardiac index were not altered (Table 1). Further echocardiographic assessment showed a progressive and significant increase in left ventricular (LV) relative wall thickness in HS-treated DS animals (Fig. 1). LV mass and LV mass-to-body weight ratio were also significantly increased in these animals (Fig. 1). DTI analysis showed that the lateral annular E-to-A ratio was significantly depressed in the HS group at weeks 14–18 relative to controls (Fig. 1). Because DTI lateral annular E-to-A ratio is a marker of diastolic performance, a decrease in this ratio provides strong evidence of organ-level diastolic dysfunction (Fig. 1). The statistically significant differences in ejection fraction (%), fractional shortening, and velocity of circumferential shortening between HS and control animals are indicative of systolic dysfunction (Fig. 1, Tables 2, and 3). Collectively, these organ-level findings indicate diastolic and systolic dysfunction in this model system.

View larger version (40K): [in this window] [in a new window]   Fig. 1. Experimental time line for high-salt (HS) diet induction and in vivo hemodynamic characterization of hypertension, hypertrophy, and diastolic dysfunction in the Dahl salt-sensitive (DS) rat (A) using tail-cuff manometry to record systolic blood pressure during weeks 7, 9, 11, 13, 15, and 17 (B) and echocardiography recorded during weeks 6, 8, 10, 12, 14, 16, and 18 to determine left ventricular (LV) mass (C), relative wall thickness (D), LV mass-to-body weight ratio (E), ejection fraction (F), and lateral annular ratio of early to late filling waves (E-to-A ratio, G). DS rats were fed standard (0.04% NaCl) rodent chow [low-salt (control), n = 8] or purified rodent chow containing 8.0% NaCl (high salt, n = 8). Values are means ± SE. P < 0.05 vs. age-matched low-salt group. P < 0.05 vs. previous week within group.

View larger version (37K): [in this window] [in a new window]   Fig. 2. Morphological characteristics of cardiac myocytes isolated from DS rats fed standard (0.04% NaCl) rodent chow (low salt, n = 96) or purified rodent chow containing 8% NaCl (high salt, n = 94). A: representative images of myocytes (x20 magnification) obtained from low- and high-salt rats. Scale bar, 20 µm. After 12 wk of a high-salt diet, myocyte length (B) and myocyte area (C) were significantly increased 20% and 44%, respectively. Values are means ± SE. *P < 0.05 vs. low salt.

View larger version (22K): [in this window] [in a new window]   Fig. 3. In vitro sarcomere shortening characterization of cardiac myocytes isolated from DS rats fed standard (0.04% NaCl) rodent chow (low salt, n = 14) or purified rodent chow containing 8% NaCl (high salt, n = 68). A: representative normalized sarcomere shortening traces at day 3 in primary culture. B and C: summary of baseline sarcomere length (Baseline), peak shortening sarcomere length (Peak), and percent shortening, as well as time to peak shortening and time from peak to 75% relaxation. Values are means ± SE. *P < 0.05 vs. low salt.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Effects of parvalbumin (Parv) gene transfer on sarcomere shortening characterization of cardiac myocytes isolated from DS rats 3 days after gene transfer. Treatments include no virus (high salt, n = 68), -Parv (high salt + -Parv, n = 62), and -Parv (high salt + -Parv, n = 77). A: representative normalized sarcomere shortening traces of high-salt, high-salt + -Parv, and high-salt + -Parv transduced cardiac myocytes. B–D: summary of baseline sarcomere length, peak sarcomere shortening length, and percent shortening relative to average values determined for untreated cardiac myocytes from a DS rat fed a standard (0.04% NaCl) rodent chow (low salt, solid horizontal line). Values are means ± SE. *P < 0.05 vs. high salt. P < 0.05 vs. low salt.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Summary of sarcomere relaxation time 3 days after gene transfer in cardiac myocytes isolated from DS rats fed a purified rodent chow containing 8% NaCl. Treatments include no virus (high salt, n = 68), -Parv (high salt + -Parv, n = 62), and -Parv (high salt + Parv, n = 77). Time from peak to 25% (A), 50% (B), and 75% (C) relaxation is shown relative to average values determined for untreated cardiac myocytes obtained from a DS rat fed standard (0.04% NaCl) rodent chow (low salt, solid horizontal line). Values are means ± SE. *P < 0.05 vs. high salt. P < 0.05 vs. low salt.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Time-dependent dose response of - and -Parv (- and -PV) expression effects in cardiac myocytes isolated from DS rats fed purified rodent chow containing 8% NaCl. A: representative Western blot of protein expression of - and -Parv, as well as internal loading control -sarcomeric actin. Positive controls include superior vastus lateralis (SVL) muscle and purified -Parv protein. B: summary of estimated concentration of - and -Parv in isolated cardiac myocytes relative to Parv expression in the SVL on day 3. Average -Parv expression was significantly lower than -Parv expression in transduced cardiac myocytes: 0.03 vs. 0.12 mM. *P < 0.05. C–E: summary of time-dependent effects of Parv expression on baseline sarcomere length (C), peak sarcomere shortening length (D), and amplitude of the peak height (E). Treatments include no virus (high salt) at day 2 (n = 90) and day 3 (n = 68), -Parv (high salt + -PV) at day 2 (n = 71) and day 3 (n = 62), and -Parv (high salt + -PV) at day 2 (n = 71) and day 3 (n = 77). Values are means ± SE. *P < 0.05 vs. high salt within the same day. P < 0.05 vs. day 2 within the same group.

View larger version (12K): [in this window] [in a new window]   Fig. 7. Time-dependent dose-response relationships for - and -Parv expression effect on sarcomere relaxation times (ms). All data were collected from myocytes of high-salt-treated animals assigned to groups as follows: no virus (control, untreated) at day 2 (n = 90) and day 3 (n = 68), -Parv at day 2 (n = 71) and day 3 (n = 62), and -Parv at day 2 (n = 71) and day 3 (n = 77). A–C: time from peak to 25, 50, and 75% baseline. Values are means ± SE. *P < 0.05, - and -Parv vs. control. #P < 0.01, day 3 vs. day 2 within the same group (dose effect). P < 0.05, -Parv vs. -Parv at day 3.

	DISCUSSION

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Recent experimental and modeling studies by Coutu et al. (6–9) demonstrated the effectiveness of -Parv in enhancement of relaxation performance in cardiac myocytes from normal and diseased models. Wild-type -Parv has two E-F cation-binding sites, with association binding constants of 0.91 x 10⁸ M^–1 for Ca²⁺ and 2.43 x 10⁴ M^–1 for Mg²⁺ (5–7, 12). We modeled -Parv's cardiac relaxation effect as a temporary Ca²⁺ depot, specifically in early to mid diastole, by binding of Ca²⁺ before resequestration to the sarcoplasmic reticulum (6). One negative consequence of wild-type -Parv, particularly evident at cytosolic [Parv] greater than 0.1 mM, was a diminution in myocyte contractility and partial buffering of systolic Ca²⁺. This indicates that -Parv does buffer some Ca²⁺ in systole and accounts for the diminished contractility, particularly at higher [Parv].

We tested the hypothesis in the present study that a naturally occurring alternative Parv isoform demonstrated to have a primary structure different from that of -Parv may, in fact, have a more favorable outcome in terms of myocyte mechanical performance. Theoretically, a given Parv isoform would be considered beneficial if it minimized systolic Ca²⁺ buffering while still retaining some capacity for Ca²⁺ buffering in the diastolic phase of the cardiac cycle. More specifically, in principle, a Parv isoform with increased Mg²⁺ affinity relative to -Parv is modeled to achieve delayed Ca²⁺ binding during a cardiac twitch, thus potentially preserving contractility while speeding relaxation (7). -Parv has an association constant for Mg²⁺ of 2.83 x 10⁴ M^–1. Relative to -Parv, therefore, -Parv has an apparent 16% increase in Mg²⁺ affinity. If this increase in Mg²⁺ affinity is due to reduced off-rate, modeling studies suggest that it may be relevant, inasmuch as Mg²⁺ must dissociate from Parv before binding Ca²⁺. -Parv also has an apparent 218% increase in Ca²⁺ affinity relative to -Parv and would also be expected to affect buffering. Modeling studies in part support this analysis, in particular in showing sarcomere length amplitude effects (see supplemental Fig. 3). There are, however, limitations to this analysis. 1) The biochemically determined cation-binding constants for Parv isoforms have not been determined under identical experimental conditions. 2) It is unknown whether differences in divalent cation affinities or kinetics obtained in biochemical assays would translate to the same differences in the more complex intracellular milieu of the cardiac myocyte, that is, other mono/divalent ion content, pH, ionic strength, and temperature. 3) Affinity differences could be due to a change in association and/or dissociation kinetics, which we did not determine. These confounding issues notwithstanding, the experimental results in cardiac myocytes presented here provide new mechanical evidence that -Parv demonstrates a more potent dose response than -Parv by causing a greater acceleration of relaxation at lower relative [Parv] within the myocyte. Thus, although there are uncertainties regarding the precise binding characteristics of Parv isoforms, these physiological findings are evidence that even small amounts of -Parv, barely detectable above background on Western blot analysis and likely <0.001 mM, may be more potent than 5- to 10-fold higher [-Parv] in terms of hastening relaxation rates of adult cardiac myocytes. The basis for this enhanced function is not known but could, for example, relate to altered association/dissociation rates. The functional results of the present study, which was purposefully conducted in the most straightforward setting of the isolated single myocyte, could have implications for genetic- or protein-based strategies to introduce Parv into the more complex setting of heart muscle in vivo in an attempt to speed myocardial relaxation performance (29).

In summary, an animal model of CHF was demonstrated at the adult cardiac myocyte level to have slow relaxation and preserved contractility and was used here as a cellular model of isolated diastolic dysfunction. Comparative studies for Parv isoforms demonstrated effective correction of slow relaxation in this model. Genetic titration of [Parv] demonstrated conditions under which relaxation could be accelerated and contractility preserved. It is estimated that -Parv is 10 times more potent than -Parv in terms of accelerating mechanical relaxation. This Parv isoform-dependent differential dose response may be useful in designing therapies directed at diastolic dysfunction in the failing heart. In future studies, it will be interesting and important to employ in vivo gene transfer technologies to determine whether the results obtained here in adult cardiac myocytes will effectively translate to the complexities of the diseased heart in small and large mammals in vivo.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. M. Metzger, Dept. of Molecular and Integrative 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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