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Am J Physiol Heart Circ Physiol 274: H385-H396, 1998;
0363-6135/98 $5.00
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Vol. 274, Issue 2, H385-H396, February 1998

Troponin I phosphorylation and myofilament calcium sensitivity during decompensated cardiac hypertrophy

Bradley K. McConnell1,2, Christine Schomisch Moravec1,2,3, and Meredith Bond1,2

1 Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland 44106; and 2 Department of Molecular Cardiology, Lerner Research Institute, 3 Center for Anesthesiology Research, Cleveland Clinic Foundation, Cleveland, Ohio 44195

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have measured myocyte cell shortening, troponin-I (Tn-I) phosphorylation, Ca2+ dependence of actomyosin adenosinetriphosphatase (ATPase) activity, adenosine 3',5'-cyclic monophosphate (cAMP) levels, and myofibrillar isoform expression in the spontaneously hypertensive rat (SHR) during decompensated cardiac hypertrophy (76 wk old) and in age-matched Wistar-Kyoto rat (WKY) controls. The decreased inotropic response to beta -adrenergic stimulation previously observed in myocytes from 26-wk-old SHR was further reduced at 76 wk of age. In response to beta -adrenergic stimulation, Tn-I phosphorylation was greater in the 76-wk-old SHR than in the WKY, although cAMP-dependent protein kinase A (PKA)-dependent Tn-I phosphorylation in the SHR did not increase with progression from compensated (26 wk) to decompensated (76 wk) hypertrophy. We also observed a dissociation between the increased PKA-dependent Tn-I phosphorylation and decreased cAMP levels in the 76-wk-old SHR versus WKY during beta -adrenergic stimulation. Baseline Tn-I phosphorylation was significantly reduced in 76-wk-old SHR versus WKY and was associated with decreased basal cAMP levels and increased Ca2+ sensitivity of actomyosin ATPase activity. The change in myofilament Ca2+ sensitivity during beta -adrenergic stimulation in the 76-wk-old SHR (0.65 pCa units) was over twofold greater than in the 76-wk-old WKY (0.30 pCa units). We also determined whether embryonic troponin T isoforms were reexpressed in decompensated hypertrophy and observed significant reexpression of the embryonic cardiac troponin T isoforms in the 76-wk-old SHR. The significant decrease in Ca2+ sensitivity with beta -adrenergic stimulation in 76-wk-old SHR may contribute to the severely impaired inotropic response during decompensated hypertrophy in the SHR.

spontaneously hypertensive rat; inotropic response; adenosine 3',5'-cyclic monophosphate-dependent protein kinase; beta -adrenergic stimulation; actomyosin adenosinetriphosphatase activity

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

SYMPATHETIC STIMULATION of the heart via activation of cardiac muscle beta -adrenergic receptors is an important mechanism to increase cardiac output in response to physiological stress (20). Similar to other animal models of cardiac hypertrophy and failure (34), as well as the failing human heart (6, 7), the spontaneously hypertensive rat (SHR) (5, 32) is characterized by a decreased inotropic response to beta -adrenergic stimulation. We have previously shown during compensatory cardiac hypertrophy in the 26-wk-old SHR, where baseline contractile function is normal, that this decreased inotropic response is associated with 1) increased adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA)-dependent phosphorylation of troponin I (Tn-I) compared with 26-wk-old Wistar-Kyoto rats (WKY), and 2) decreased Ca2+ sensitivity of actomyosin adenosinetriphosphatase (ATPase) activity (28). Additionally, we found no differences in baseline Tn-I phosphorylation or in baseline Ca2+ sensitivity of actomyosin ATPase activity (28).

In this study, we investigate the biochemical alterations that occur during the progression to decompensated cardiac hypertrophy in the 76-wk-old SHR, where the heart is functionally impaired (24). Decompensated hypertrophy in the SHR is characterized by impaired baseline contractile function (24) and an even more severe depression of the inotropic response to beta -adrenergic stimulation than during the early period of compensatory hypertrophy (5). We hypothesized that this further decrease in beta -adrenergic responsiveness during decompensated hypertrophy in 76-wk-old SHR hearts could be due to an additional increase in PKA-dependent Tn-I phosphorylation compared with compensatory hypertrophy in 26-wk-old SHR hearts.

Several alterations in the beta -adrenergic pathway have been reported in the SHR heart, including downregulation of beta -adrenergic receptors (6, 25) and increased guanine nucleotide regulatory protein (Gialpha ) expression, resulting in decreased adenylyl cyclase activity (2, 6). After beta -adrenergic stimulation of SHR hearts, total cardiac cAMP levels have been reported to be decreased (42) or unchanged (18). This latter report (18) would suggest that upstream changes in the beta -adrenergic pathway do not necessarily lead to altered regulation of the pathway at more distal sites. There may also be compartmentalization of cAMP or PKA in cardiac muscle cells (36) such that phosphorylation of a particular PKA substrate is not predicted by changes in total cellular cAMP content (28, 37).

During the period of compensatory hypertrophy, previous evidence from our lab indicates that the amount of Ca2+ available to activate the myofilaments during beta -adrenergic stimulation in the SHR is not decreased compared with the WKY (32). Furthermore, after progression to decompensated cardiac hypertrophy, the size of the sarcoplasmic reticulum Ca2+ store (24) and the amplitude of the cytoplasmic Ca2+ transient, as measured by aequorin (5), are not reduced during beta -adrenergic stimulation. We therefore proposed that decreased Ca2+ availability for activation of contraction would not explain the depressed inotropic response to beta -adrenergic stimulation in SHR hearts and put forward the alternative hypothesis that myofilament Ca2+ sensitivity may be decreased (28). In support of this hypothesis, in our previous study with isolated myocytes from hearts of 26-wk-old SHR and WKY, we showed that the increase in Tn-I phosphorylation after stimulation of the beta -adrenergic pathway is significantly greater in the SHR than the WKY (28).

The Ca2+ affinity of troponin C (Tn-C) is decreased after phosphorylation of two adjacent NH2-terminal serines of Tn-I (Ser-23 and Ser-24 in the rat heart) (29). As a result, Ca2+ sensitivity of actomyosin ATPase activity (19, 30) and Ca2+ sensitivity of force production is decreased (12). Consistent with these observations, we showed that the greater PKA-dependent Tn-I phosphorylation in the SHR than in the WKY at 26 wk of age is associated with a significant rightward shift in the Ca2+ dependence of actomyosin ATPase activity in the SHR, indicating decreased myofilament Ca2+ sensitivity after beta -adrenergic stimulation, compared with the WKY. In contrast, under unstimulated conditions, there was no difference in Tn-I phosphorylation and no difference in Ca2+ dependence of actomyosin ATPase activity between SHR and WKY myocytes at 26 wk of age.

We have now investigated the changes in Tn-I phosphorylation (associated with changes in myofilament Ca2+ sensitivity) and Tn-I and troponin T (Tn-T) isoform expression during the progression to decompensated cardiac hypertrophy (76 wk) in the SHR. Our results indicate that during beta -adrenergic stimulation in myocytes from 76-wk-old SHR, Tn-I phosphorylation is increased compared with 76-wk-old WKY. Under baseline conditions, Tn-I phosphorylation is decreased in myocytes of 76-wk-old SHR compared with myocytes of WKY and is associated with increased Ca2+ sensitivity of actomyosin ATPase activity as well as decreased basal cAMP levels. We also observed significant reexpression of embryonic cardiac Tn-T isoforms in the 76-wk-old SHR.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Male SHR and WKY were purchased from Taconic farms (Germantown, NY) at 12 wk of age. They were housed in the Cleveland Clinic Animal Care Facility from 24 to 76 wk and were killed at 76 wk of age. The week before the rats were killed, blood pressure was measured in unanesthetized SHR and WKY, using the tail-cuff method. Rats were killed by decapitation in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The Cleveland Clinic's Animal Care Facility is accredited by the American Association for the Accreditation of Laboratory Care. The extent of cardiac hypertrophy in the SHR compared with WKY was determined from measurements of heart weight-to-body weight ratio as previously described (24).

Preparation of left ventricular myocytes and measurement of cell shortening. Left ventricular myocytes were prepared from hearts of 76-wk-old SHR and age-matched WKY controls, using a modified Langendorff perfusion apparatus, as previously described (28). Measurement of cell shortening was performed by quantifying the change in cell length, using video-edge detection (28). Cells were placed on a temperature-regulated perfusion chamber, stabilized at 28°C by a Delta T Culture Dish System (Biotechs), and mounted on the stage of an Olympus CK 12 inverted microscope. The myocytes were allowed to partially attach to the bottom of the perfusion chamber for ~3 min in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered saline (HBS) (containing in mM: 118 NaCl, 4.8 KCl, 1.2 MgCl2, 1.25 CaCl2, 11 glucose, 0.68 glutamine, 5 pyruvate, and 25 HEPES, pH 7.35, supplemented with 0.1 mM minimum essential medium, basal medium Eagle vitamin and amino acid solutions) at 28°C and electrically stimulated at 0.2 Hz (SD9 stimulator; Grass Instruments). Once a stable amplitude of myocyte shortening was attained, the myocytes were superfused with HBS + agonist (1 µM isoproterenol, 10 µM norepinephrine + 10 µM prazosin, 10 µM forskolin, or 250 µM chloro-cAMP) at 1.5 ml/min with continued electrical stimulation. Data Sponge software (Bioscience Analysis Software) was used for data acquisition and analysis.

[32P]orthophosphate labeling of isolated myocytes. Phosphorylation of the myofibrils in intact SHR and WKY left ventricular myocytes by [32P]orthophosphate (32Pi) was performed using previously described methods (28). In brief, myocyte suspensions from 76-wk-old SHR or WKY hearts were labeled with 250 µCi 32Pi for 2 h at 22°C under humidified 100% O2. After 32Pi labeling, 2-ml aliquots of the cell suspensions were transferred to test tubes and incubated for 10 min at 37°C with gentle agitation with 1 µM isoproterenol, 250 µM chloro-cAMP, or 100 µM isobutylmethylxanthine (IBMX). Controls were treated with 2 µl dimethyl sulfoxide (solvent for IBMX). After the 10-min incubation periods, the myocytes were immediately washed twice with 5 ml of ice-cold HBS containing protease inhibitors [5 µg/ml antipain, 10 µg/ml leupeptin, 5 µg/ml pepstatin A, 43 µg/ml phenylmethylsulfonyl fluoride, and 5 mM ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)] and phosphatase inhibitors (0.1 µM sodium orthovanadate and 2 nM calyculin A) and pelleted at 100 g for 3 min at 4°C. The pellet was then homogenized in 2 ml of ice-cold "inhibiting buffer" (19) containing (in mM) 50 KH2PO4, 70 NaF, and 5 EDTA, plus 1% Triton X-100 and protease and phosphatase inhibitors (above), and kept on ice for 30 min. The detergent-extracted myofibrils were then pelleted at 5,000 g for 5 min.

PKA-dependent Tn-I phosphorylation. 32Pi-labeled proteins in the crude myofibrillar fraction were separated on 15% polyacrylamide slab gels by one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) as previously described (28). A PhosphorImager (Molecular Dynamics) with the use of the ImageQuant software package was used to quantify the amount of radioactivity incorporated into each 32Pi-labeled band.

The high isoelectric point of Tn-I (pI = 10.4) makes it difficult to obtain clear separation by isoelectric focusing of the phosphorylated and unphosphorylated forms of this protein. Therefore, we did not directly measure the stoichiometry of Tn-I phosphorylation by two-dimensional gel electrophoresis. In previous studies (28), we normalized Tn-I phosphorylation to myosin light chain-2 (MLC-2) phosphorylation, because this is not a substrate of PKA-dependent phosphorylation and does not demonstrate any strain-dependent differences in stoichiometry of MLC-2 phosphorylation during compensatory cardiac hypertrophy. However, during decompensated hypertrophy, baseline MLC-2 phosphorylation may decrease (31). Therefore, an alternative phosphorylated protein, which shows no change in phosphorylation during progression of cardiac hypertrophy, was chosen for normalization of 32Pi incorporation into Tn-I. In this study, we used tropomyosin (Tm) because Tm is not a substrate of PKA-dependent phosphorylation (Table 1) (17). Basal Tm phosphorylation is also constant irrespective of the degree of left ventricular hypertrophy, and there are no strain-dependent differences in the phosphorylation during decompensated hypertrophy in the SHR (Table 1) (10). The amount of 32Pi incorporation into Tn-I was therefore normalized to 32Pi incorporation into Tm from the same myofibrillar fraction on the same lane of the gel. This was performed in unstimulated cells (controls), after stimulation by isoproterenol, and after downstream activation of the beta -adrenergic pathway.

                              
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  		Table 1.   [3P]orthophosphate incorporation into tropomyosin

cAMP assay. Left ventricular myocytes were prepared from 76-wk-old SHR and WKY hearts, as previously described (28). Myocyte preparations were incubated either in the absence or presence of 5 mM theophylline for 30 min at 37°C. The myocytes were then stimulated for an additional 10 min at 37°C with 1 µM isoproterenol or incubated for the additional 10-min period at 37°C without isoproterenol. The reactions were terminated with 0.1 M HCl, and then the samples were immediately frozen in liquid N2. The samples were then thawed on ice (4°C), after which ice-cold 10% cold trichloroacetic acid plus 55 µM theophylline was added. The samples were centrifuged at 2,500 g 4°C for 20 min. The supernatants were collected and extracted four times with five times the volume of water-saturated ether. The samples were then dried under vacuum. cAMP content was determined using a cAMP 125I double-antibody radioimmunoassay kit (NEK-033, DuPont). Acid precipitates from the samples were dissolved in NaOH, and protein content was determined according to the bicinchoninic acid protein assay reaction kit. Values are expressed as picomoles of cAMP per milligrams of protein.

Actomyosin ATPase activity. Actomyosin ATPase activity was measured as the decrease in NADH absorbance, as previously described (28). The myofibrillar fraction was suspended in N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer containing (in mM) 85 potassium methanesulfonate (KMS), 3 MgCl2, 2 EGTA, 10 NaF, 0.5 dithiothreitol, 0.5 leupeptin, and 30 BES, pH 7.00 plus 3% Tween 20. The protein concentration of the extracted myofibrils was determined by the Lowry method. The reaction mixture consisted of (in mM) 25 BES (pH 7.0), 2.7 MgCl2, 2 EGTA, 10 NaF, 126 KMS, and varying free Ca2+ concentrations to give final free Ca2+ concentrations from pCa 9 to pCa 3. Calcium buffers were prepared according to an iterative computer program (8, 13).

Ca2+-stimulated actomyosin ATPase activity was measured at 22°C as the oxidation of NADH in a reaction coupled to the lactate dehydrogenase and pyruvate kinase reactions. ATPase activity was recorded from the change in absorption at 340 nm over a 5-min period (28). The reaction solutions contained 200 mM phosphoenolpyruvate, 10 mM NADH, 0.5 mg/ml lactate dehydrogenase, and 12.5 mg/ml pyruvate kinase added to 1 ml of the various calcium solutions [containing myofibrillar fractions (0.1-0.5 mg/ml) isolated from either SHR or WKY cardiac myocytes]. Reactions were initiated by addition of 2 mM ATP. The enzyme activity was expressed as the percentage of maximal Ca2+-stimulated actomyosin ATPase activity per milligram protein. Actomyosin ATPase activity measured as a function of Ca2+ concentration (pCa 9 to pCa 3) was calculated by nonlinear regression of a sigmoidal dose-response curve with a variable Hill coefficient. The actomyosin ATPase activity in the absence of Ca2+ was subtracted from values obtained in the presence of Ca2+ and represented <15% of the total ATPase activity. To confirm that the extent of Tn-I phosphorylation did not change during the actomyosin ATPase assay, we measured the extent of Tn-I phosphorylation in myofibrillar fractions in the assay buffer over the time course of the assay. There was no significant change in the extent of Tn-I phosphorylation over the 5-min assay period (data not shown).

In addition, because crude myofibrillar fractions were used for the actomyosin ATPase activity assay, we determined whether there was a contribution to the ATPase activity measured from other ATPases present in the sample. We found no significant difference in ATPase activity measured in the presence or absence of ATPase inhibitors, including (in mM) 2 oligomycin, 2 rotenone, 200 ouabain, and 2 thapsigargin (data not shown). Therefore, we can conclude that mitochondrial ATPase activity and membrane-associated ATPases were lost or inactivated during Triton extraction and did not contribute to the actomyosin ATPase activity measured in this assay.

SDS-PAGE and Western blot analysis of Tn-I and Tn-T. SHR and WKY rats were killed at 26 and 76 wk of age, and the heart from each rat was rapidly removed. The left ventricle was then isolated and immediately frozen in liquid N2. The tissues were homogenized using a Polytron grinder (Brinkmann) and denatured in sample preparation buffer containing 1% SDS, 0.23 M beta -mercaptoethanol, and 0.2 M tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 6.5), heated at 80°C for 10 min, and pelleted at 100 g for 5 min at 4°C. The protein concentration was determined for each sample by the Lowry assay, and 100 µg of protein were loaded on each lane of the gel. The total protein extracts from each rat heart were resolved by one-dimensional SDS-PAGE (14% gels with the resolving gel acrylamide-to-bisacrylamide ratio of 180:1 and stacking gel acrylamide-to-bisacrylamide ratio of 30:1) at constant current (20 mA/gel, 1 h). The proteins were subsequently transferred to nitrocellulose membranes (0.45-µm pore size) using a Bio-Rad semidry electrotransfer apparatus at 5 mA/cm2 for 35 min. The nitrocellulose membranes were blocked in 1% bovine serum albumin (BSA) in Tris-buffered saline (TBS, 150 mM NaCl; 50 mM Tris · HCl, pH 7.5) at 4°C overnight. The blocked membranes were incubated with primary antisera: 1) rabbit anticardiac Tn-I 6C7 monoclonal antibody at 1:4,000 dilution and 2) mouse anticardiac Tn-T CT3 monoclonal antibody at 1:2,000 dilution. Both antibodies were kindly provided by J. P. Jin (Case Western Reserve University, Cleveland, OH). Primary antisera dilutions were suspended in TBS containing 0.1% BSA and incubated at room temperature for 4 h. After three 10-min washes with TBS plus 0.05% Triton X-100 and 0.1% SDS, and two 5-min TBS rinses, both membranes were then incubated with alkaline phosphatase-labeled anti-mouse immunoglobulin G (from Sigma 1:4,000) secondary antisera in TBS containing 0.1% BSA at room temperature for 1 h. After the membranes were washed as described above, the color-detection reaction was initiated using 0.015% 5-bromo-4-chloro-3-indolyl phosphate/0.03% nitro blue tetrazolium substrate to reveal the expression patterns of Tn-I and Tn-T. With the use of NIH Image software, densitometric scans of the cardiac Tn-T (cTn-T) Western blots were performed to determine the relative intensities of the bands representing the different Tn-T isoforms.

Materials. Collagenase type II was obtained from Worthington Biochemical (Freehold, NJ). Triton X-100 and Protogel (30% wt /vol acrylamide and 0.8% wt /vol bis-acrylamide stock solutions) were purchased from National Diagnostics. N,N,N,N-Tetramethylethylene-diamine (TEMED), 2-mercaptoethanol, ammonium persulfate, and prestained low-molecular-weight markers were purchased from Bio-Rad. All other chemicals were obtained from Sigma.

Experimental controls and data analysis. Results of all trials for each experimental condition were averaged, and comparisons between SHR and WKY were performed using Student's t-test. Differences between SHR and WKY were considered statistically significant at P < 0.05. Results from each experimental condition were normalized to unstimulated controls, which were taken as 100%. All results are expressed as means ± SE, unless otherwise indicated.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Myocyte cell shortening. In papillary muscle preparations, we previously showed that the inotropic response to beta -adrenergic stimulation was decreased in 26-wk-old SHR (32) and further decreased in 76-wk-old SHR (24). We therefore wanted to determine whether the decreased contractile response in the isolated myocytes from SHR hearts (28) was further reduced during the progression to decompensated cardiac hypertrophy. We measured the change in amplitude of cell shortening in response to isoproterenol stimulation in electrically stimulated myocytes from 76-wk-old SHR and from age-matched WKY. Figure 1A shows typical records of the cell-shortening amplitude in electrically stimulated left ventricular myocytes from 76-wk-old SHR and WKY under baseline conditions and in response to superfusion of 1 µM isoproterenol.


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  		Fig. 1.   Amplitude of cell shortening of electrically stimulated myocytes from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) in response to isoproterenol stimulation. A: representative traces of contractions of single myocytes from 76-wk-old SHR (top trace) and 76-wk-old WKY (bottom trace) in response to superfusion of 1 µM isoproterenol. Cells were electrically stimulated at 0.2 Hz throughout the experiment. Isoproterenol was added to cells after 2 min (arrow) and allowed to continuously superfuse the cells for an additional 10 min. B: average increase in amplitude of cell shortening over baseline of electrically stimulated myocytes from 26- and 76-wk-old SHR and WKY in response to superfusion with 1 µM isoproterenol. Results represent 3-6 myocytes isolated from 5 hearts of each animal strain and age group; n = 32 SHR (26 wk), 21 WKY (26 wk), 25 SHR (76 wk), and 22 WKY (76 wk) cells; * P < 0.0001 vs. WKY (26 wk); ** P < 0.0001 vs. WKY (76 wk) and P < 0.004 vs. SHR (26 wk).

Compared with the response to isoproterenol stimulation in myocytes from 76-wk-old WKY, the increase in amplitude of cell shortening was significantly attenuated (P < 0.0001) in the SHR. Similar responses were observed with 10 µM norepinephrine plus 10 µM prazosin, 10 µM forskolin, and 250 µM chloro-cAMP (data not shown). Compared with our previously reported measurements of cell shortening in 26-wk-old SHR and WKY (28) (Fig. 1B), the increase in cell-shortening amplitude in the WKY after isoproterenol stimulation was similar at both ages, but the percent increase in cell-shortening amplitude was significantly less (P < 0.004) in the 76-wk-old SHR versus 26-wk-old SHR. In our experiments, 2 of 25 SHR and 1 of 22 WKY myocytes showed occasional arrhythmic contractions during the experiment; however, there was no noticeable increase in the frequency of arrhythmic contractions during isoproterenol stimulation.

PKA-dependent phosphorylation of Tn-I in SHR and WKY myocytes. We extended our previous studies of Tn-I phosphorylation in response to stimulation of the beta -adrenergic pathway during compensated cardiac hypertrophy (28) to determine whether the differences in Tn-I phosphorylation between the myocytes of 26-wk-old SHR and WKY were more pronounced in rats at 76 wk of age when there is a further decline in the inotropic response to beta -adrenergic stimulation. We investigated whether Tn-I phosphorylation differed 1) under baseline conditions and 2) after stimulation of the beta -adrenergic pathway in myocytes in 76-wk-old SHR and WKY. In the latter case, we compared Tn-I phosphorylation when the myocytes were stimulated at the beta -adrenergic receptor with isoproterenol or by downstream activation of the pathway by chloro-cAMP or IBMX.

Normalizing baseline 32Pi incorporation into Tn-I to baseline 32Pi incorporation into Tm in myocytes of 76-wk-old SHR and WKY (Fig. 2A), we observed a significant decrease in baseline Tn-I phosphorylation (P < 0.05) in 76-wk-old SHR compared with 76-wk-old WKY. For comparative purposes, we also normalized Tn-I phosphorylation in 26-wk-old SHR and WKY (28) to Tm phosphorylation. Normalization of 32Pi incorporation into Tn-I to 32Pi incorporation into Tm showed that in response to 1 µM isoproterenol (Fig. 2B): 1) Tn-I phosphorylation in 76-wk-old SHR was significantly (P < 0.05) greater than in 76-wk-old WKY; 2) consistent with our previous observations (28), when Tn-I phosphorylation was normalized to MLC-2 phosphorylation, Tn-I phosphorylation in 26-wk-old SHR was also significantly (P < 0.005) greater than in 26-wk-old WKY; and 3) Tn-I phosphorylation at 76 wk of age in the SHR was not significantly different from Tn-I phosphorylation in the 26-wk-old SHR. The slight increase in PKA-dependent Tn-I phosphorylation in the 76-wk-old WKY compared with the 26-wk-old WKY, after activation of the beta -adrenergic pathway, was not statistically significant.


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  		Fig. 2.   Protein kinase A (PKA)-dependent troponin I (Tn-I) phosphorylation in suspensions of left ventricular myocytes from 76-wk-old SHR and WKY in response to isoproterenol stimulation. Myocyte preparations were stimulated for 10 min at 37°C in absence or presence of beta -adrenergic stimulation. A: [32P]orthophosphate (32Pi) incorporation into Tn-I (32Pi-Tn-I) in unstimulated myocytes (arbitrary PhosphorImager units) normalized to 32Pi incorporation into tropomysin (Tm) (32Pi-Tm) (arbitrary units) from same lane of same gel. * P < 0.02 vs. 76-wk-old WKY and 26-wk-old SHR. B: 32Pi incorporation into Tn-I (32Pi-Tn-I) in response to 1 µM isoproterenol, normalized to 32Pi incorporation into Tm (32Pi-Tm) from same lane of same gel. * P < 0.002 vs. 26-wk-old WKY; ** P < 0.02 vs. 76-wk-old WKY. C: 32Pi incorporation into Tn-I (32Pi-Tn-I) in response to 250 µM chloroadenosine 3',5'-cyclic monophosphate (chloro-cAMP), normalized to 32Pi incorporation into Tm (32Pi-Tm) from same lane of same gel. * P < 0.03 vs. 26-wk-old WKY; ** P < 0.02 vs. 76-wk-old WKY. D: 32Pi incorporation into Tn-I (32Pi-Tn-I) in response to 100 µM isobutylmethylxanthine (IBMX), normalized to 32Pi incorporation into Tm (32Pi-Tm) from same lane of same gel. * P < 0.02 vs. 26-wk-old WKY; ** P < 0.02 vs. 76-wk-old WKY. Results represent experiments from 9 SHR (26 wk), 9 WKY (26 wk), 12 SHR (76 wk), and 12 WKY (76 wk).

Regardless of whether the beta -adrenergic pathway was stimulated with the beta -receptor-specific agonist isoproterenol (Fig. 2B) or by downstream activation of the beta -adrenergic pathway with chloro-cAMP (Fig. 2C) or IBMX (Fig. 2D), the absolute increase in Tn-I phosphorylation with stimulation was greater in the 76-wk-old SHR than in the 76-wk-old WKY.

cAMP levels. To investigate a possible mechanism for decreased baseline Tn-I phosphorylation in myocytes from 76-wk-old SHR compared with 76-wk-old WKY, we measured cellular cAMP levels. We found a significant decrease in baseline cAMP levels in the myocytes of 76-wk-old SHR compared with myocytes of 76-wk-old WKY (Fig. 3A). Interestingly, with phosphodiesterase inhibition (theophylline), strain-dependent differences in baseline cAMP levels between the myocytes of 76-wk-old SHR and WKY were no longer observed. The differences in cAMP levels in the presence and absence of theophylline may be due to greater phosphodiesterase activity in the SHR (Fig. 3, A and B). This is indicated by the fact that baseline cAMP levels measured in the presence of theophylline increased 2.0-fold in the 76-wk-old SHR but only 1.2-fold in the 76-wk-old WKY (Fig. 3, A vs. B).


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  		Fig. 3.   Measurement of cAMP levels in suspensions of left ventricular myocytes from 76-wk-old SHR and WKY. Myocyte preparations were incubated in absence or presence of 5 mM theophylline for 30 min at 37°C followed by an additional 10-min stimulation at 37°C in absence or presence of 1 µM isoproterenol. A: baseline stimulation; B: baseline stimulation in presence of theophylline pretreatment; C: isoproterenol stimulation; D: isoproterenol stimulation in presence of theophylline pretreatment. * P < 0.05 vs. 76-wk-old WKY. Results represent 3 separate experiments, each measured in duplicate.

We also measured cAMP levels after beta -adrenergic stimulation in myocytes from 76-wk-old SHR and WKY. In response to isoproterenol stimulation, cAMP levels were significantly less in myocytes from 76-wk-old SHR compared with myocytes from 76-wk-old WKY (Fig. 3C). However, in response to isoproterenol stimulation, cAMP levels measured in the presence of phosphodiesterase inhibition increased in myocytes from both 76-wk-old SHR and WKY (Fig. 3D), but this increase was significantly greater (P < 0.05) in the 76-wk-old SHR than WKY (Fig. 3D). Thus, as a result of phosphodiesterase inhibition, cAMP levels increased 5.3-fold in the 76-wk-old SHR versus 1.9-fold in the WKY (Fig. 3, C vs. D).

Taken together, the cAMP measurements under both baseline and stimulated conditions suggest that phosphodiesterase activity is increased in the SHR compared with WKY myocytes.

Actomyosin ATPase. PKA-dependent phosphorylation of Tn-I decreases the Ca2+ sensitivity of Tn-C (19). We therefore investigated whether the differences in Tn-I phosphorylation after progression from compensated to decompensated cardiac hypertrophy in the SHR, compared with age-matched WKY, results in differences in myofilament Ca2+ sensitivity, as measured by the Ca2+ dependence of actomyosin ATPase activity. Under baseline conditions (unstimulated myocytes), Ca2+ dependence of actomyosin ATPase activity was significantly (P < 0.001) shifted to the left in the 76-wk-old SHR compared with 76-wk-old WKY, indicating increased myofilament Ca2+ sensitivity. This was also observed as a significant decrease in the half-maximal effective concentration (EC50) for Ca2+ (increased pCa units) (Fig. 4, A and B, and Table 2). Figure 4, A and B, and Table 2 also show that after isoproterenol stimulation, the Ca2+ dependence of actomyosin ATPase activity is shifted to the right in both 76-wk-old SHR and WKY, as indicated by significant increases in the EC50 for Ca2+ (decreased pCa units) in both strains.


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  		Fig. 4.   Ca2+-dependent actomyosin adenosinetriphosphatase (ATPase) activity in isolated myofibrillar fractions from left ventricular myocytes of 76-wk-old SHR and WKY. A: 76-wk-old WKY; B: 76-wk-old SHR. open circle , No treatment (Control), black-square, 1 µM isoproterenol. Results are from 6 separate experiments, each measured in duplicate.

                              
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  		Table 2.   Actomyosin adenosinetriphosphatase activity

Unlike our previous results (28), which showed that isoproterenol stimulation confers a significantly greater decrease in Ca2+ dependence of actomyosin ATPase activity in 26-wk-old SHR compared with 26-wk-old WKY, the EC50 for Ca2+ dependence of actomyosin ATPase activity in myofilament preparations from myocytes from 76-wk-old SHR stimulated by isoproterenol (5.47 ± 0.07 pCa units) was not significantly greater (lower pCa) than for the WKY (5.57 ± 0.03). However, at the higher free Ca2+ concentrations that occur during activation of the beta -adrenergic pathway, the SHR curve was significantly shifted to the right compared with the WKY (Fig. 4, A and B), as indicated by a significantly higher (P < 0.05) EC75 (lower pCa) in the SHR (4.94 ± 0.06) than in the WKY (5.12 ± 0.05) after isoproterenol stimulation.

The absence of a significant difference in the EC50 values for Ca2+ activation of actomyosin ATPase activity in 76-wk-old SHR and 76-wk-old WKY after beta -adrenergic stimulation may be related to age-dependent changes in the WKY rather than changes associated with the developent of cardiac hypertrophy in the SHR. The EC50 for Ca2+ activation of actomyosin ATPase activity was significantly increased (i.e., lower pCa) in 76-wk-old WKY (5.57 ± 0.03) compared with 26-wk-old WKY (5.67 ± 0.04) (28) (P < 0.05). It is interesting to note that Tn-I phosphorylation was slightly, but not significantly, higher after beta -adrenergic stimulation in 76-wk-old WKY compared with 26-wk-old WKY (Fig. 2, B and C). In contrast, there was no significant difference between the EC50 for Ca2+ activation of actomyosin ATPase activity in 76-wk-old SHR (this study; 5.47 ± 0.07) and 26-wk-old SHR (28) (5.51 ± 0.04) in response to beta -adrenergic stimulation.

As a consequence of 1) increased myofilament Ca2+ sensitivity under baseline conditions in the 76-wk-old SHR, and 2) similar decreases in myofilament Ca2+ sensitivity following isoproterenol stimulation in the 76-wk-old SHR and WKY, the change in myofilament Ca2+ sensitivity over baseline during beta -adrenergic stimulation was over twofold greater in the SHR (EC50 change of 0.65 pCa units) compared with the WKY (EC50 change of 0.30 pCa units) (Fig. 4, B vs. A, and Table 2). Similar to our results in 26-wk-old SHR and WKY (28), the Hill coefficient was significantly less in both 76-wk-old SHR and WKY preparations in response to isoproterenol stimulation, compared with unstimulated controls (Table 2).

Tn-I and Tn-T isoforms. Changes in Tn-T isoform composition have been associated with altered myofilament Ca2+ sensitivity (43), in particular, reexpression of embryonic Tn-T isoforms has been previously observed in failing human hearts (3). Because the greater PKA-dependent Tn-I phosphorylation in the 76-wk-old SHR did not confer a significant shift in Ca2+ sensitivity of actomyosin ATPase activity, compared with the 76-wk-old WKY, we investigated whether there were differences in Tn-T isoform expression in the SHR and WKY at 26 and 76 wk of age. On the basis of apparent molecular mass from the Coomassie blue-stained gel (Fig. 5A), the major bands were identified as myosin heavy chain, C-protein, actin, Tn-T, Tm, Tn-I, MLC-1, and MLC-2 from SHR and WKY total heart extracts at 26 and 76 wk of age.


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  		Fig. 5.   Identification of Tn-I and Tn-T isoforms by Western blot analysis from left ventricles of SHR and WKY hearts at 26 and 76 wk of age. A: Coomassie blue-stained gel showing myosin heavy chain (myosin, 205 kDa), C-protein (150 kDa), actin (45 kDa), Tn-T (43 kDa), Tm (35 kDa), Tn-I (30 kDa), and myosin light chain 2 (MLC-2, 20 kDa); B: Western blot of cardiac Tn-I (cTn-I) isoform using rabbit anti-cTn-I 6C7 monoclonal antibody; C: Western blot of Tn-T showing adult (cTn-Ta) and embryonic (cTn-Te) cardiac isoforms using mouse anti-cTn-T CT3 monoclonal antibody. Arrow points to embryonic isoforms in 76-wk-old SHR. Lane 1: 26-wk-old SHR; lane 2: 26-wk-old WKY; lane 3: 76-wk-old SHR; lane 4: 76-wk-old WKY (A-C); D: densitometric scans of cTn-T Western blot. Density of bands is in arbitrary units, normalized to total cTn-T. cTn-T isoforms are labeled as follows: 1, cTn-Te; 2, cTn-Te; 3, cTn-Ta; 4, cTn-Ta.

The identity of the cTn-T isoforms was confirmed by Western blot analysis (Fig. 5C). Quantification of the cTn-T isoforms by normalizing to total cTn-T by densitometry in each lane of the Western blot shows equal expression of adult cTn-T (cTn-Ta) isoforms during the progression of cardiac hypertrophy in the SHR (i.e., between 26 and 76 wk) and no strain-dependent differences (Fig. 5, C and D). However, only in the 76-wk-old SHR, we observed a small but significant reexpression (6.5% of total cTn-T) of embryonic cTn-T (cTn-Te) isoforms (Fig. 5, C and D). Therefore, we report that cTn-Te isoforms are reexpressed in decompensated cardiac hypertrophy of the 76-wk-old SHR.

cTn-I was identified as the major band by Western blot analysis (Fig. 5B) appearing at 30 kDa. Cardiac Tn-I expression did not change during the progression from compensated to decompensated cardiac hypertrophy, and no differences were observed between the SHR and WKY. Also, there were no differences in the expression of other minor bands on the Tn-I Western blot during progression of cardiac hypertrophy from 26 to 76 wk in the SHR and no differences between strains.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously shown that inotropic responsiveness to beta -adrenergic stimulation is significantly reduced in papillary muscles from 26-wk-old SHR (32) and that this response is further impaired with progression to decompensated cardiac hypertrophy (76-wk-old SHR) (24). The isoproterenol-dependent increase in amplitude of myocyte cell shortening is also decreased in myocytes from 26-wk-old SHR hearts compared with WKY (28). In the current study, we show a further decline in the increase in amplitude of cell shortening of myocytes from 76-wk-old SHR hearts to beta -adrenergic stimulation, indicating that the functional changes that occur with disease progression in cardiac muscle preparations are also observed in isolated myocytes.

PKA dependent Tn-I phosphorylation is increased, and myofilament Ca2+ sensitivity decreased after beta -adrenergic stimulation in the 26-wk-old SHR, compared with 26-wk-old WKY (28). We proposed that these changes may contribute to the decreased inotropic response in the SHR (28). We therefore predicted that we would observe a greater increase in PKA-dependent Tn-I phosphorylation after the progression to decompensated cardiac hypertrophy in 76-wk-old SHR, where the response to activation of the beta -adrenergic pathway is further reduced.

The results of the current study show that Tn-I phosphorylation is indeed greater after stimulation of the beta -adrenergic pathway in myocytes from 76-wk-old SHR than in age-matched WKY (Fig. 2, B and C), but that there is no further increase in PKA-dependent Tn-I phosphorylation in myocytes from 76-wk-old than in 26-wk-old SHR. It is therefore possible that the mechanism responsible for the greater PKA-dependent Tn-I phosphorylation in the SHR is maximal at 26 wk, with no further increase with disease progression. The answer to this question awaits elucidation of the mechanism responsible for the differences in Tn-I phosphorylation observed.

In our previous study in 26-wk-old SHR and WKY, we showed that the greater increase in PKA-dependent Tn-I phosphorylation in the SHR than WKY is unrelated to changes in beta -adrenergic receptor density, since the effect could be reproduced by activation of the beta -adrenergic pathway downstream of the receptor, including direct activation of PKA by a cell-permeant cAMP analog (28). We therefore concluded that decreased beta -adrenergic density in the SHR (6, 25) does not contribute to the changes we observed. In the current study, when cells were stimulated either at the beta -adrenergic receptor or at downstream sites (by chloro-cAMP or IBMX), a significantly greater increase in PKA-dependent Tn-I phosphorylation was still observed in the SHR, again indicating that the mechanism for this difference is distal to cAMP production or breakdown.

In this study, we observed a significant decrease in baseline Tn-I phosphorylation in myocytes from 76-wk-old SHR compared with 76-wk-old WKY (Fig. 2A). Because earlier studies showed desensitization of myofilaments to Ca2+ as a result of PKA-dependent Tn-I phosphorylation (19, 30), we predicted that sensitization of the myofilaments to Ca2+ would occur in the 76-wk-old SHR under baseline conditions as a result of the decreased PKA-dependent Tn-I phosphorylation. Our results confirmed this prediction (Fig. 4). Note that increased baseline Ca2+ sensitivity was only observed during decompensated cardiac hypertrophy and not at the earlier stage of compensatory hypertrophy in the SHR (28). Perreault et al. (35) reported an increased Ca2+ sensitivity of force development in 18- to 24-mo-old SHR compared with WKY, but these differences were limited to right ventricular preparations from SHR showing evidence of heart failure. Perez et al. (33) found no significant differences in myofilament Ca2+ sensitivity in SHR compared with WKY, but their study was carried out on younger (24 wk old) animals. Their results are consistent with our observations in 26-wk-old SHR (28).

Our observations of increased myofilament Ca2+ sensitivity in decompensated cardiac hypertrophy in the SHR are consistent with observations by Wolff et al. (44) who used permeabilized myocardial preparations from dilated cardiomyopathic human hearts. Wolff et al. also showed increased Ca2+ sensitivity of force development under baseline conditions with no significant difference in Ca2+ sensitivity after beta -adrenergic stimulation.

Decreased baseline Tn-I phosphorylation in the 76-wk-old SHR could be due to increased basal phosphatase activity, decreased basal kinase activity, and/or decreased cAMP levels. We have shown that a likely explanation for decreased baseline Tn-I phosphorylation in the 76-wk-old SHR is decreased cAMP levels. Increased phosphatase activity (protein phosphatase 1 and/or protein phosphatase 2A) in the 76-wk-old SHR could, potentially, also contribute to the decreased baseline Tn-I phosphorylation; however, this hypothesis remains to be tested.

We also observed a significant decrease in cAMP levels in response to beta -adrenergic stimulation in myocytes from 76-wk-old SHR versus WKY (Fig. 3C). These results are consistent with the findings of Sharma et al. (42) who showed both reduced baseline cAMP levels and reduced isoproterenol-stimulated cAMP levels in SHR versus WKY myocytes. However, the measurements of Sharma et al. (42) were obtained from 14- to 16-wk-old SHR and WKY animals and thus represent cAMP levels during compensatory hypertrophy. In contrast, Hilal-Dandan and Khairallah (18) reported no changes in baseline or isoproterenol-stimulated cAMP formation in 18-wk-old SHR and WKY animals. This would be consistent with our previous observations of no change in baseline Tn-I phosphorylation during compensatory hypertrophy (28). Our measurements of cAMP levels in the presence and absence of theophylline also suggest that decreased cAMP levels in the 76-wk-old SHR may arise, in part, from increased phosphodiesterase activity.

We observed a significant decrease in cAMP levels in response to beta -adrenergic stimulation in myocytes from 76-wk-old SHR compared with WKY (Fig. 3C) but a significantly greater increase in PKA-dependent Tn-I phosphorylation in response to beta -adrenergic pathway stimulation (isoproterenol, Fig. 2B; cAMP, Fig. 2C; and IBMX, Fig. 2D). These results therefore indicate that the changes in PKA-dependent Tn-I phosphorylation in response to beta -adrenergic stimulation are independent of differences in total cellular cAMP levels. The disparity between alterations in Tn-I phosphorylation and cAMP levels between SHR and WKY myocytes in response to beta -adrenergic stimulation could be due to cAMP compartmentation in cardiac muscle cells (36). Compartmentation of cAMP implies that only a small subcellular fraction of the total cellular pool of cAMP is directly involved in the activation of specific PKA-dependent substrates (9, 23). It has been proposed that compartmentalization of cAMP accounts for the lack of correspondence between increased total cellular cAMP in response to different stimuli (e.g., forskolin vs. isoproterenol or pimobendan vs. isoproterenol) and the corresponding contractile response (37). Consistent with these observations, our findings of increased Tn-I phosphorylation and decreased cAMP levels following beta -adrenergic stimulation indicate that there can be a dissociation between total cellular cAMP levels and the downstream activation of PKA-dependent substrate phosphorylation. These results also suggest a role for local regulation of PKA, possibly by an A-kinase anchoring protein (38, 27).

We previously showed that after beta -adrenergic stimulation, the amount of Ca2+ stored in the junctional sarcoplasmic reticulum in 76-wk-old SHR is not decreased, compared with the WKY (24). However, it is possible that under baseline conditions, Ca2+ availability at the myofilaments may be decreased. Increased myofilament Ca2+ sensitivity under baseline conditions in the SHR may be a compensatory mechanism for decreased availability of Ca2+ for activation of contraction. This hypothesis is supported by a recent report (14) showing decreased frequency of Ca2+ sparks, by confocal microscopy, in myocytes from hypertrophied hearts of the hypertensive Dahl salt-sensitive rat and from hearts of SHR selectively bred for congestive heart failure (SH/HF rats). In both models, Ca2+-triggered Ca2+ release from the sarcoplasmic reticulum was decreased (14).

One striking conclusion from our study in the 76-wk-old SHR and WKY is that activation of PKA, whether by stimulation of the beta -adrenergic pathway at the level of the receptor or at a distal site, can achieve a greater change in Tn-I phosphorylation (from basal to the stimulated state) in the SHR than the WKY. Thus, although Tn-I phosphorylation is decreased under baseline conditions in 76-wk-old SHR, the activity of the beta -adrenergic pathway is clearly not compromised in these severely dysfunctional hearts, at least with respect to phosphorylation of the PKA substrate Tn-I. This therefore indicates that, despite decreased density of beta -adrenegic receptors (6, 25), there is sufficient reserve in the beta -adrenergic signaling pathway that activity of downstream components of the beta -adrenergic pathway can be upregulated to levels observed in the WKY controls or higher. This point is further emphasized by the fact that, in response to beta -adrenergic stimulation, we observed a greater increase in PKA-dependent Tn-I phosphorylation, despite decreased cAMP levels, in 76-wk-old SHR versus WKY.

The greater than normal increase in PKA-dependent Tn-I phosphorylation in the 76-wk-old old SHR from the basal to the stimulated state results in an over twofold greater decrease in myofilament Ca2+ sensitivity than in the WKY on activation of the beta -adrenergic pathway. Although it is clear that many factors may come into play to contribute to the severely impaired inotropic response to beta -adrenergic stimulation in the 76-wk-old SHR, the abnormally large myofilament Ca2+ desensitization, on activation of the beta -adrenergic pathway, may be a significant factor.

Finally, we investigated whether changes in Tn-I or Tn-T isoform expression occurred in the 76-wk-old SHR. Changes in Tn-T expression have been reported to occur during development of hypertrophy and heart failure in aortic-banded guinea pigs (16). Tn-T isoform changes during cardiac development (26) and in diabetic rat hearts (1) correlate with shifts in Ca2+ sensitivity of force development. In particular, Anderson et al. (3, 4) and Wolff et al. (44) have shown partial reexpression of the embryonic cTn-T isoforms in failing human hearts. Expression of cTn-T isoforms in the adult rat heart results from developmentally regulated alternative RNA splicing of a single gene (22). The generation of multiple cTn-T isoforms involves alternative splicing of two exons encoding the NH2-terminal variable region, including an embryonic isoform-specific exon 4 encoding 10 mainly acidic amino acids (22). Differences between the adult isoforms and between the embryonic isoforms are due to four amino acids in the variable NH2-terminal region (22). As a result, four cTn-T isoforms exist: two major adult isoforms of lower molecular weight (higher mobility) and two minor embryonic isoforms of higher molecular weight (lower mobility).

We observed significant reexpression of cTn-Te isoforms in the 76-wk-old SHR (Fig. 5, C and D). Schiaffino et al. (41) concluded that the additional NH2-terminal acidic amino acid sequence of the cTn-Te isoforms would confer increased rather than decreased myofilament Ca2+ sensitivity. Thus reexpression of cTn-Te could contribute to differences in actomyosin ATPase activity observed by increasing myofilament Ca2+ sensitivity. Also, the disparity between greater Tn-I phosphorylation and normal increased Ca2+ dependence of actomyosin ATPase activity in 76-wk-old SHR in response to beta -adrenergic stimulation, compared with 76-wk-old WKY, could be due to a small increase in myofilament Ca2+ sensitivity resulting from cTn-Te expression in the SHR.

We confirmed the absence of any significant differences in Tn-I isoform composition during the progression from compensatory to decompensated cardiac hypertrophy, and no differences were observed between the SHR and WKY. This is consistent with previous observations showing no reexpression of embryonic or skeletal Tn-I isoforms in the adult heart (39) or during development of cardiac hypertrophy or failure (40).

In summary, this study directly compares myofilament Ca2+ sensitivity of actomyosin ATPase activity to Tn-I phosphorylation by PKA. We also show that a likely mechanism for decreased baseline Tn-I phosphorylation and increased Ca2+ dependence of actomyosin ATPase activity in 76-wk-old SHR, compared with WKY, is decreased baseline cAMP levels. Our findings also suggest that increased phosphodiesterase activity in 76-wk-old SHR, compared with WKY, may play a role in the decreased basal cAMP levels observed in the SHR. However, during activation of the beta -adrenergic pathway, we observed a dissociation between cellular cAMP levels and PKA-dependent Tn-I phosphorylation.

In conclusion, the regulatory mechanisms that operate at a distal site in the beta -adrenergic pathway in the 76-wk-old SHR confer a larger than normal increase in PKA-dependent Tn-I phosphorylation, with an accompanying decrease in myofilament Ca2+ sensitivity that is over twofold normal. This desensitization to beta -adrenergic stimulation may provide a mechanism by which severely compromised hearts are partly protected from chronic overstimulation by elevated levels of circulating catecholamines. Although a severely impaired response to sympathetic stimulation would prevent the SHR heart from responding adequately to demands for increased cardiac output, it may help protect the heart from the potentially deleterious effects of overstimulation by catecholamines, thus minimizing excessive Ca2+ influx and preserving energy supplies.

    ACKNOWLEDGEMENTS

The authors thank Drs. Frank Brozovich and Bin-Xian Zhang for helpful discussions and Dr. J. P. Jin for providing the monoclonal antibodies to Tn-I and Tn-T and for help with the Tn-I and Tn-T Western blots. Also, we thank Steve Schomisch and Mike Trentanelli for help with the cAMP measurements.

    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-56256 (M. Bond), HL-49929 (C. S. Moravec), and T32 HL-07714 (B. K. McConnell) and by Established Investigator Awards from the American Heart Association to M. Bond and C. S. Moravec.

Address for reprint requests: M. Bond, Dept. of Molecular Cardiology, FF10, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195.

Received 9 June 1997; accepted in final form 18 September 1997.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Heart Circ Physiol 274(2):H385-H396
0363-6135/98 $5.00 Copyright © 1998 the American Physiological Society


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