Early postnatal changes in sarcoplasmic reticulum calcium transport function in spontaneously hypertensive rats
Nicholas Freestone, .1 Jaipaul Singh, 1 Ernst-Georg K r a u s e 2 and Roland Vetter 2 tDepartment of Applied Biology, University of Central Lancashire, Preston, PR1 2HE, UK; 2Max Delbriick Center for Molecular Medicine, Robert Rdssle Strafle 10, D-13122 Berlin-Buch, Germany
Abstract
This comparative study investigates the relationship between sarcoplasmic reticulum (SR) calcium(Ca2+)-ATPase transport activity and phospholamban (PLB) phosphorylation in whole cardiac homogenates of spontaneously hypertensive rats (SHR) and their parent, normotensive Wistar Kyoto (WKY) strain during early postnatal development at days 1, 3, 6, 12 and at day 40 to ascertain any difference in SR Ca 2+ handling before the onset of hypertension. At day 1, the rate ofhomogenate oxalate- supported Ca 2÷ uptake was significantly higher in SHR than in WKY (0.25 + 0.02 vs 0.12 + 0.01 nmoles CaWing wet ventricu- lar weight/min, respectively; p < 0.001). This interstrain difference disappeared with further developmental increase in SR Ca 2+ transport. Western Blot analysis and a semiquantitative ELISA did not reveal any difference in the amount ofimmunore- active PLB (per mg of total tissue protein) between strains at any of the ages studied. In addition, levels of phosphorylated PLB formed in vitro in the presence ofradiolabelled ATP and catalytic (C) subunit of protein kinase A did not differ between SHR and WKY at days 1, 3, 6 and 12. At day 40, C subunit-catalyzed formation of 3zP-PLB was reduced by 66% (p < 0.001) in SHR when compared to age-matched WKY. In the early postnatal period between day 1 and 12 SR Ca2+-transport values were linearly related to the respective 32p-PLB levels of both SHR and WKY rats. The results indicate that cardiac SR of SHR can sequester Ca 2+ at a much higher rate immediately after birth compared to WKY rats. The disappearance of this interstrain difference with further development suggests that some endogenous neuroendocrine or nutritional factor(s) from the hyper- tensive mother may exert an influence upon the developing heart in utero resulting in a transiently advanced maturation of the SR Ca 2+ transport function in SHR pups at the time of birth. (Mol Cell Biochem 163/164: 57~56, 1996)
The sarcoplasmic reticulum (SR) in cardiac muscle regulates the relaxation of the muscle and acts as a source of Ca 2+ for myofilament activation during the excitation-contraction coupling process [1]. Several previous studies have shown that this process undergoes developmental changes during fetal and postnatal heart growth and matches the increased functional demands during development. For example, a progressive postnatal increase in the rate of cardiac relaxa-
tion in rodents such as rabbit and rat [2, 3] is paralleled by an increased Ca 2+ transporting activity of the cardiac SR [3- 7]. Recent evidence points to a developmentally regulated el- evation in the expression of the SR Ca 2+ ATPase isoform SERCA2a in the myocardium as an important contributing mechanism to these changes [ 5 4 , 8, 9]. In addition, altera- tions in membrane lipids [10-11] as well as levels and state of phosphorylation of the CaZ+-ATPase modulatory protein phospholamban could be other contributing factors [ 12-15]. Phospholamban, a homopentameric SR protein has been
*Present address: Laboratory ofNeural and Secretory Signalling, Department ofNeurobiology, Babraham Institute, Babraham, Cambridge, CB2 4AT, UK Address for offprints: R. Vetter, Max Delbnick Center for Molecular Medicine, Robert ROssle Stra~3e 10, D-I3122 Berlin-Buch, Germany
shown to regulate the rate of SR Ca 2÷ transport through changes in the affinity of the Ca2+-ATPase for Ca 2.. The Ca 2+ transporting activity of this enzyme is suppressed while phospholamban is dephosphorylated. This inhibition is re- lieved upon phosphorylation of this protein by cyclic AMP (cAMP)-dependent protein kinase (at serine 16) which in intact tissue occurs in response to [3-adrenergic stimulation [ 12-14]. Phospholamban can also become phosphorylated at a distinct amino acid residue by Ca2+-dependent protein kinases [13, 15].
The spontaneously hypertensive rat (SHR) is a frequently used genetic animal model of clinical hypertension. With respect to the heart muscle, this strain of rats manifests a progression from a stable form of hypertrophy with normal cardiac function to chronic heart failure with impaired heart function which corresponds well to the clinical course of patients with hypertension [16]. It is of particular interest that differences in cardiac development and blood pressure be- tween SHR and their normotensive counterparts are evident before the onset of hypertensive/hemodynamic factors [ 17, 18]. Thus, SHR exhibits cardiomyocyte hyperplasia and car- diomegaly at birth as well as higher blood pressure compared to WKY [ 17, 18]. There are also differences in cardiac con- tractile properties and cellular Ca 2÷ handling in the mature SHR if compared to WKY rats [ 19-21 ]. The report of Clubb et al. [17] points to interstrain differences in the function of cellular organelles involved in excitation-contraction cou- pling before hypertensive hemodynamic factors become important. As the SR plays a central role in cardiac excita- tion-contraction coupling, we initiated a comparative study on the Ca 2+ transport function of this intracellular organelle in the early postnatal period of rats of the WKY and SHR strains. Furthermore, the question was addressed as to whether the amount and status of phosphorylation of phos- pholamban at the protein kinase A site differed between both strains in the early postnatal period.
Material and methods
Animals
Newborn male and female rats, 1, 3, 6, 12, and 40 days old of the SHR and WKY strains from the animal facility of the Max Delbr~ek Center for Molecular Medicine were used. Mothers and young animals were maintained on standard rat chow with water ad libitum. Animals were killed by cervi- cal dislocation. The investigation conforms with German law on the care and use of laboratory animals and has been ap- proved and registered by the Senatsverwaltung for Gesund- heit of the city of Berlin.
Preparation of homogenates
As purification procedures often employed in isolating SR vesicles tend to result in low and variable yields of material [22, 23], we decided to use whole ventricular homogenates for the determination of Ca 2+ uptake [24]. This approach has the further advantage of facilitating the use of small amounts of tissue. Hearts were quickly excised and immediately im- mersed in ice-cold 250 mM sucrose, 120 mM NaC1, 30 mM KC1 to stop the heart from beating and to remove any blood. The hearts were then trimmed of atrial tissue, blotted and weighed before being frozen and stored in liquid nitrogen. Ventricular homogenates were prepared in a cold room (+4°C) by 6 x 10 sec homogenizations in 16 volumes of 250 mM sucrose buffer, 10 mM histidine, pH 7.4 with a Brink- mann Polytron PT 10-35 (Kinematica, GmbH, Luzern, Swit- zerland) at a setting of 6 with a 15 see pause between each homogenization. The final homogenate was filtered through polyamide gauze (90 gm mesh; NeoLab, Heidelberg, Ger- many) and kept in a tube on ice. A sample of this homoge- nate was used within 10 min for the measurement of oxa- late-supported Ca 2+ uptake. Other samples were immediately frozen in liquid nitrogen and stored at-70°C until use for the quantitation of phospholamban and protein. For phosphor- ylation experiments, two volumes homogenate were mixed with one volume of a phosphoprotein protection buffer con- taining (in mM) 250 sucrose, 10 histidine, 150 KH2PO 4, 50 NaF and 30 EDTA before being frozen [24]. For immuno- chemical experiments, tissue homogenates were treated with 0.6 M KC1 in order to remove contractile proteins as de- scribed elsewhere [24].
Oxalate-supported Ca 2+ uptake
Oxalate-supported C a 2+ uptake into SR vesicles was meas- ured at 37°C in 40 mM imidazole-HC1 buffer (pH 7.0), 100 mM KC1, 5 mM MgCI2, 5 mM tris(hydroxymethyl)-amino- methane (Tris)-ATP, 6 mM phosphocreatine, 10 mM K- oxalate, 10 mM NaN 3, 0.2 mM ethylene glycol-bis-(13- aminoethyl ether)-N,N,N',N'-tetraacedic acid (EGTA), 0.1 mM 45CAC12 [sp act 12 dplrdpmol; 0.21 gM free Ca 2+ concen- tration] and 0.75 mg wet heart tissue per 0.25 ml employing a previous described method [24]. After preincubation of the reaction mixture for 2 min in the absence ofhomogenate, the Ca 2÷ uptake was started by the addition of homogenate. At selected time intervals, samples were taken and filtered by suction through 0.45 jam HAWP Millipore filters (Millipore, Eschbom, Germany). Filters were washed twice with ice-cold 40 mM imidazole (pH 7.0), 100 mM KC1 and 2 mM EGTA. Radioactivity associated with dry filters was determined by liquid scintillation counting. Reaction mixtures contained either 2 ~tM catalytic (C) subunit of adenosine Y-5'-cyclic
monophosphate (cAMP)dependent protein kinase (protein ki- nase A), 5gM Ruthenium Red, 10 gM synthetic protein kinase A inhibitor peptide [PKI(6-22)amide] (GIBCO BRL, Life Tech- nologies GmbH, Eggenstein, Germany) or no such additions. Transport rates were calculated by the linear regression of data points at 0.5, 1.0, 1.5 and 2.0 rain measured in duplicate. The reaction mixtures allowed for Ca 2+ uptake into SR vesicles only, with ATP-dependent Ca 2+ transport into mitochondrial vesicles being inhibited by NaN 3 [23].
Protein kinase A-catalyzed phosphorylation
lamban antibody (Biomol, Hamburg, Germany) that recog- nized both phosphorylated and nonphosphorylated phospho- lamban was used. In Western blots, immunoreactive protein was visualized using an enhanced chemiluminescence analy- sis kit (ECL; Amersham, Little Chalfont, UK). In ELISA, immunoreactive phospholamban was detected with 0.1 ml of a peroxidase substrate mixture containing 10 mg o-phenylen- ediamine, 10 tal 30% H202, and 0.2 ml 1.0 M citric acid (pH 4.7) per 10 ml distilled water. The absorbance of the sample was recorded at 492 nm using an Anthos HT II spectropho- tometer microtiter plate reader (Anthos Labtec Instruments, Salzburg, Austria).
The 32p incorporation into phospholamban by protein kinase A was determined by urea-sodium dodecyl sulfate-polyacryl- amide gel electrophoresis (urea-SDS-PAGE), autoradiogra- phy and scintillation counting as described previously [24]. Homogenates were incubated with [7-32P]ATP for 5 min at 30°C in the presence ofC subunit of protein kinase A and in the absence of Ca 2÷. The reaction mixture of 40 gl contained 40 rnM N-2hydroxyethylpiperazine-N'-2ethanesulfonic acid- Tris (pH 7.4), 10 mM MgC12, 1 mM EGTA, 1 mM dithio- threitol, 20 mM NaF, 40 gg homogenate protein, 2 ~tM C subunit and 100 gM [7-32p]ATP (sp act 400 dprrdpmol). The excess of C subunit at a saturating [7-32p]ATP concentration ensures maximum 32p incorporation into phospholamban at the protein kinase A specific site. For inhibition ofphospho- protein phosphatases 20 mM NaF was used. Thus, only those phosphorylation sites not yet phosphorylated in vivo can be filled up by the in vitro phosphorylation reaction. After 2 rain of preincubation, the reaction was started by addition of ho- mogenate. The reaction was stopped by addition of 2 ml of ice-cold 15% trichloroacetic acid containing 50 mM H3PO 4 and 0.5 mM Na-ATP. After addition of 100 gg bovine serum albumin, the denaturated proteins were centrifuged at 3000 x g for 10 rain. The pellet was then solubilized in 2% SDS, 1% mercaptoethanol, 5 mM EDTA, 50 mM H3POa/Tris (pH 6.8), boiled for 2 min and mixed with half a volume of 50% glycerol, 1% mercaptoethanol, 5 mM EDTA and 50 mM H3PO4/Tris (pH 6.8). Electrophoresis, gel staining, destaining, autoradiography and measurement of radioactivity associated with the low molecular weight form ofphospholamban were performed as described earlier [24]. The amount of 32p in- corporated into phospholamban was expressed as nmoles 32p per g of wet tissue weight.
Western blotting and ELISA
Immunochemical identification of phospholamban in West- ern blots and determination of the relative amount of this protein in KCl-extracted homogenates by ELISA were done as described previously [24]. A monoclonal anti-phospho-
Miscellaneous
Protein was determined by the Lowry method [25] using ovalbumin as a standard. Unless stated otherwise all the chemicals used here were obtained from Sigma Chemicals (Deisenhofen, Germany). All reagents were of the purest form available.
Statistical analys&
Values are presented as mean + S.E.M. unless stated other- wise. Statistical analysis was performed by Student's t-test for unpaired observations or one way analysis of variance fol- lowed by Bonferroni group-to-group comparisons. Statisti- cal significance was assumed at p < 0.05.
Results
Time course changes in ventricular and body weights for WKY and SHR rats
Table 1 shows the time course changes in ventricular weight, body weight, ventricular weight/body weight ratio and ven- tricular proteins in the early postnatal growth period (from day 1-40) in WKY and SHR rats. Initial experiments showed no differences in either body or heart weight of male and female rats, thus all the data were combined. The results show a more than 10-fold increase in ventricular weight and body weight between postnatal days 1-40 in WKY strains. A simi- lar increase in both parameters occurred in the SHR strain over the same time period. By contrast, the relative ventricu- lar weight (expressed as a ratio of ventricular wet weight in milligram to body weight in gram) was lower at day 40 in both populations than at day 1 (see Table 1). These changes were paralleled by an approximate doubling in total ventricu- lar protein content from day 1-40 in each strain indicating
Values are mean + S.E.M. Numbers in parentheses are number of animals evaluated. **(P < 0.001), *(P < 0.05) significantly different from values at postnatal day one. §Significantly different (P < 0.05) from WKY values at that age.
that the synthesis of cardiac proteins is extremely high in this period. It is particularly noteworthy that the ventricles of 1 day old SHR rat were significantly heavier compared to that of WKY. Furthermore, the protein content in ventricular homogenates from the two populations was also significantly different immediately after birth with SHR homogenates containing 47% more protein per gram of wet ventricular weight compared to WKY (Table 1) at day 1.
Homogenate SR Ca 2+ uptake
in SR Ca 2÷ transport activity between strains was observed immediately after birth at day 1 with a greater Ca 2+ uptake rate for SHR (0.25 -+ 0.02 m o l e s CaWmg wet weight/min) compared to WKY (0.12 ± 0.01 umoles CaZ÷/mg wet weight/ min) rats. Approx. 50% higher Ca 2÷ transport values in homogenates of 1 day old SHR were also observed when Ca 2+ transport values were related per mg of ventricular protein. These differences were also observed when Ca 2+ uptake was measured either in the presence of C subunit of protein ki- nase A, protein kinase A inhibitor peptide [PKI(6-22)amide] or Ruthenium Red.
Figure 1 shows a representative time-course of oxalate-sup- ported SR Ca 2+ uptake in whole ventricular homogenates of 1 day old SHR and WKY rats under conditions of Ca 2+ trans- port stimulation and inhibition. Addition of either C subunit of protein kinase A or 5 gM of the Ca 2+ release-channel blocker, Ruthenium Red, resulted in an increased Ca 2+ uptake both in WKY and SHR. By contrast, inhibition of endogenous protein kinase A by 10 gM of the protein kinase A inhibitor peptide [PKI(6-22)amide] caused a marked reduction in Ca 2÷ uptake in both strains. These results indicate that the cardiac S R C a 2+ pump activity of newborn WKY and SHR can be modulated by protein kinase A-catalyzed phosphorylation of phospholamban. Similar Ca 2÷ uptake curves were obtained for 3, 6, 12 and 40 day old animals (data not shown). Figure 2 shows the Ca 2+ transport rates in whole ventrieular homo- genates from day 1-40 in both SHR and WKY under con- trol conditions. It demonstrates that the Ca 2+ uptake rate of both strains increased with age and reached a maximum level around day 12 which was not significantly different from the values obtained for 40 day old animals. The peak uptake values at day 12 represent an approx 4-fold increase in Ca 2+ transport from day 1 in SHR and an approx. 7-fold increase in WKY. In fact, the only significant difference (p < 0.001)
Phospholamban phosphorylation
To examine whether the observed early developmental changes and the interstain difference of Ca 2+ transport at day 1 were related to alterations in the control of SR Ca 2+ pump activity by phospholamban, in vitro phosphorylation experi- ments were performed. Figure 3 shows the postnatal changes between day 1 and day 40 in the content ofradiolabelled 32p_ phospholamban that was formed after incubation of cardiac homogenates of SHR and WKY rats in the presence of satu- rating [7-32p]ATP concentration and an excess of exogenous C subunit of protein kinase A. The results show that the in vitro 32p incorporation into phospholamban increased with age in both SHR and WKY rats with maximal values obtained at day 12. There was no significant interstrain difference at days 1, 3, 6, and 12. However, at day 40 32p incorporation into phospholamban was significantly lowered by 66% in SHR (0..90 + 0.16 nmoles 32p/g wet weight) compared to WKY rats (2.71 ± 0.17 m o l e s 32p/g wet weight).
61
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TIME (min) TIME (rain)
Fig 1. Time course of oxalate-supported Ca 2÷ uptake in whole ventricular homogenates of representative 1 day old WKY and SHR rats. Homogenates were incubated in (in mM) 40 imidazole (pH 7.0), 100 KC1, 5 MgCI 2 5 Tris-ATP, 6 phosphocreatine, 10 K-oxalate, 10 NAN3, 0.2 EGTA and 0.1 45CAC12 at 37°C. Each point is the mean of duplicate determinations which were performed under control conditions without any other additions (solid circles) and in the presence of the following additions: 2 gM C subunit of protein kinase A (solid triangles), 5 gM Ruthenium Red (open circles) and 10 gM synthetic protein kinase A inhibitor peptide [PKI(6-22)amide] (open triangles). See also Materials and methods_
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AGE (days)
Fig 2. Postnatal changes in oxalate-supported Ca 2+ uptake rate in whole ventricular homogenates of WKY and SHR rats at postnatal days 1, 3, 6, 12 and 40. Ca 2+ uptake was measured under control conditions as indicated in Fig. 1. Data are means + S.E.M. for 5-9 animals in each group. ***p < 0.001 vs age-matched WKY.
Immunoreactive phospholamban
To prove whether the difference between W K Y and SHR in phospholamban phosphory la t ion at day 40 was due to an alteration in total phospholamban content, the levels o f im- munoreac t ive phospho lamban were de te rmined us ing an ELISA. Figure 4 shows that the abundance o f immunoreac- tive phospholamban did not differ between 40 day old W K Y and SHR. There was also no interstrain difference in other age groups studied (data not shown). This indicates that the reduced level o f 32P-phospholamban that were observed in homogenates o f 40 day-old SHR after max imum protein ki- nase A-dependent phosphorylat ion is not due to an alteration in the total phospholamban protein level but perhaps due to higher endogenous phosphoryla t ion o f phospho lamban in SHR.
Relationship between oxalate-supported Ca2+-uptake and phospholamban phosphorylation
In order to examine whether the postnatal changes in SR C a 2÷
pump activity and phospholamban phosphorylat ion occur in a coordinated manner, Ca 2+ uptake rates were plot ted against the amount o f 32P-phospholamban formed in vitro in the pres- ence o f C subunit. Figure 5 shows the relationship between SR Ca 2+ uptake rate and phospho lamban phosphory la t ion
from day 1-12 post par tum for SHR and W K Y rats. The re- suits show for both strains a linear relationship between SR
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Fig. 3. Protein kinase A-catalyzed in vitro phosphorylation of phospho- [amban in ventricular homogenates of 1, 3, 6, 12 and 40 day old WKY and SHR rats. In vitro phosphorylation of homogenates was performed for 5 min at 30 °C under stringent phosphoprotein protection conditions [24] in the presence of 1 mM EGTA. Phosphorylation was terminated by adding trichloroacetic acid. Heated sodium dodecyl sulfate(SDS)-solubilized sam- ples (40 gg protein) were subjected to urea-SDS-PAGE and autoradiogra- phy. The radioactivity associated with the low molecular weight form of phospholamban was determined by scintillation counting of the respective gel band cut out from dried gels [24]. Phospholamban was identified by immunostaining and the typical molecular weight shift that occurs after heating the solubilized samples. Values are means ± S.E.M. for 5-9 ani- mals in each group. ***p < 0.001 vs 40 day old WKY.
Ca 2÷ uptake and phospholamban phosphorylat ion for the data of 1, 3, 6 and 12 day old animals. Thus, the developmental increase in SR Ca 2* uptake is matched by a proport ional in- crease in the fraction o f cardiac phospholamban that can be phosphoryla ted in vi tro by protein kinase A. The level o f the nonphosphorylated phospholamban fraction is low at birth and increases steadily with further postnatal development. It is noteworthy that the Ca 2+ transport and phosphoryla t ion values obtained for 40 day old rats o f both strains do not fit into the l inear dependence observed in the early postnatal
period.
0.4 E ( -
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a O
0.2
0.0 / [ wKv
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0.01 0.1 1
PROTEIN (pg/wel l)
Fig. 4. Relative phospholamban content in KCl-washed ventrieular homogenates of WKY and SHR rats at day 40. Relative levels of phospholamban were measured by an enzyme-linked immunosorbent as- say. Product formation of peroxidase reaction in a substrate mixture with ophenylenediamine and H202 was monitored spectrophotometrically at 492 nm. A monoclonal mouse anti-phospholamban that cross-reacts with both nonphosphorylated and phosphorylated rat phospholamban was used. Val- ues are means + S.D. for 9 homogenates in each group. Inset: a representa- tive Western blot of KCl-extracted homogenates of a 40 day old WKY (1) and SHR (2) rat. O, origin of SDS-PAGE; 6.5 kD, electrophoretie mobility of the 6.5 kD protein aprotinin; PLB, low molecular weight form of phospholamban; DF, dye front.
D i s c u s s i o n
The aim of the present study was to define early postnatal changes o f the SR Ca 2+ re-uptake activity in SHR and W K Y rats and to investigate whether the well documented cardi- omegaly o f SHR in the early postnatal per iod [ 17, 18] is as- sociated with alterations in the Ca 2+ transport function o f this intracellular organelle. The comparison was chosen because the well documented association o f changes in cellular Ca 2+ handling and SR Ca 2÷ transport function with progress ive
postnatal cardiac growth in normotensive strains [3, 26-28] and with overload hypertrophy in matured adult heart [19 - 21 ]. During postnatal heart growth, both the transcription o f genes encoding SR proteins and Ca z÷ transport catalyzed by the SR Ca 2+ pump are increased in a characteristic manner. The steady state m R N A levels o f the SR Ca 2+ pump and SR Ca 2+ release channel increase steadily [5, 6, 28, 29], whereas phospholamban m R N A levels appear not to change signifi- cantly [29]. Although an altered m R N A level is an impor-
PHOSPHOLAMBAN PHOSPHORYLATION (nmol 32p / g wet wt)
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~.+
0 0.4
21. v
Fig. 5. Relationship between in vitro phospholamban phosphorylation catalyzed by C subunit of protein kinase A and the rate of oxalate-supported SR Ca 2+ uptake in ventricular homogenates of WKY and SHR rats in the early postnatal period of heart growth. The plotted values (means + S.E.M. for 5-8 ammals in each group) are from Figs. 2 and 5. The dotted lines were obtained by linear regression analysis using the respective single values. The ages of the animals are indicated in the figure; see also Legends to Figs. 2 and 3.
tant determinant of SR protein synthesis, it is not the sole factor regulating net Ca 2+ pump activity. In the present study, the SR Ca 2÷ transport activity was measured as oxalate-sup- ported Ca 2+ uptake in whole tissue homogenates which ex- cludes the risk of purifying non-representative SR popu- lations and other uncertainties associated with the use of purified membrane preparations [22, 23]. Another advantage of this approach is that the SR function of small neonatal hearts can be analyzed as only relatively small amounts of tissue are needed for homogenate preparation. For Ca 2+ up- take experiments, a submicromolar free Ca 2+ concentration was chosen which reveals the inhibitory influences of nonphosphorylated phospholamban on the Ca 2+ pump. Phos- phorylation of phospholamban increases the rate-limiting decomposition rate of the intermediate phosphoenzyme of the C a 2+ pump that forms during each Ca 2+ translocation cycle [ 14]. Phosphorylation of this protein also increases the Ca 2+ sensitivity of the Ca 2+ pump [14]. However, the techniques for determining the effect of phospholamban phosphoryla- tion on Ca 2+ transport rate cannot resolve all individual in- fluences. The main problem resides in the fact that the absolute in vivo status ofphospholamban phosphorylation at the cAMP- and the calmodulin-dependent sites is difficult to evaluate although an indirect phosphorylation technique (so- called 'back phosphorylation') has been successfully applied in previous canine and rat studies [30, 31]. To obtain addi- tional information on the postnatal changes in phospho-
lamban, both the in vitro phosphorylation catalyzed by C subunit of protein kinase A and the immunoreactive protein levels of this Ca 2+ pump modulator were determined. The use of a monoclonal anti-phospholamban antibody permitted the quantitation of relative levels of tissue phospholamban, ir- respective of the status ofphosphorylation of this protein [32].
Here we show that the Ca 2+ re-uptake activity of the car- diac SR of newborn 1 day-old SHR is elevated compared to age-matched pups of the normotensive WKY strain. This dif- ference could also be observed in the presence of the Ca 2÷ re- lease channel inhibitor Ruthenium Red, C subunit of protein kinase A and after inhibition of endogenous cAMP-depend- ent protein kinase by a specific inhibitory peptide. Thus, in- creased net oxalate-supported Ca 2+ uptake in 1 day-old SHR appears not to be due to interstrain differences in Ca 2+ flux through SR Ca 2+ release channels. Furthermore, a strain-spe- cific alteration of the modulation of the SR Ca 2+ pump by phospholamban is unlikely. This is supported by the finding that the myocardial levels of immunoreactive phospholamban did not differ between SHR and WKY rats in all age groups studied (data not shown). Moreover, the amount of 32p in- corporated into phospholamban in vitro by exogenous C subunit was not significantly different between 1 day-old SHR and WKY rats. This is an indirect indication that the in
vivo phosphorylation status of phospholamban (at the pro- tein kinase A-specific site) does not differ between new- born SHR and WKY rats. In this context, it should be
;~otea that tissue handling and phosphorylation experi- ments were performed under conditions which protect phosphoproteins pre-existing in vivo from dephosphoryla- tion after removal of the hearts [24]. Taken together these findings suggest that the elevated SR Ca 2. transport activ- ity observed in cardiac homogenates of SHR shortly after birth cannot be due to an alteration in the modulation of the Ca R+ pump by phospholamban.
A possible alternative mechanism for increased SR Ca 2÷ transport activity in 1 day old SHR is an elevated expression of the Ca 2÷ pump gene. In fact, an elevated expression of the slow skeletal/cardiac SR Ca 2+ ATPase isoform SERCA2a is, at least partially, responsible for the developmental increase in the SR Ca 2+ transport activity during postnatal heart growth of rodents such as rat and rabbit [5, 6, 8, 28]. This mecha- nism may also contribute to the marked early postnatal in- crease in SR Ca 2+ pumping in SHR and WKY rats. However, mRNA and protein levels of SERCA2a have not been deter- mined in this work. Thus, it remains also an open question whether an elevated Ca 2÷ pump level due to an increased SERCA2a gene expression could be the underlying mecha- nism for the increased SR Ca 2+ transport in 1 day-old SHR. Moreover, influences arising from possible differences in membrane lipids [10, 11] or Ca 2. pump modulation by CaW calmodulin-dependent phosphorylation processes should not dismissed [15, 33, 34].
It is an interesting aspect of this work that the perinatal dif- ferences in SR Ca 2+ transport, heart weight and ventricular protein between SHR and WKY pups disappeared with fur- ther postnatal heart growth. It is well known that the major- ity of cardiac tissue morphogenesis and cardiomyocyte proliferative growth occurs in utero while the cellular, struc- tural and biochemical makeup of the myocardium undergoes marked changes during postnatal hypertrophic growth [17, 35, 36]. Furthermore, the size of the heart correlates with the imposed functional load which is low in utero but is steadily increasing after birth. In addition, variations in neuro-endo- crine influences and locally generated growth factors are important for both overall gene expression that result in more or less proportional enlargement of the heart and individual expression of genes that contribute to a specific protein phe- notype of the heart [37]. Adult spontaneously hypertensive rats exhibit various changes in neuro-endocrine signals (for review see [16]) such as catecholamines, thyroid hormones and peptide growth factors which could be of relevance for the developing fetal heart in utero. Therefore, exposure of the fetus to a specific maternal neuro-endocrine environment could cause strain-specific alterations in overall and also specific gene expression in the fetal SHR heart. However, the exposure of the developing heart to the intrauterine, mother- specific neuro-endocrine environment is limited to the fetal period. Therefore, one might speculate that the elevation of early neonatal SR Ca 2+ transport, cardiac enlargement and the
increase ventricular protein content in SHR is due to some endogenous neuro-endocrine factor(s) derived from the hy- pertensive mother that exert an influence upon the phenotype of the developing heart in utero. Because these maternal factor(s) are missing after birth, the observed phenotypic changes are expected to exist for only a very limited time in the newborn animal. This could be a possible explanation for the phenomenon that the differences between 1 day old SHR and WKY disappear with further postnatal life. It is notewor- thy that various degrees of elevated levels of plasma cate- cholamines and altered thyroid hormone levels have been reported to be associated with genetic hypertension (for re- view see [16]). As these hormones can play atrophic role on heart development and can also influence the metabolic char- acteristics of this organ, they may be considered as candidate hormones for early interstrain differences in cardiac growth and SR Ca 2+ pump function. In fact, thyroid hormone regu- lates the expression of the SERCA2a gene [38] and can also modify the control of SR Ca 2÷ re-uptake by catecholamines due to its modulatory influence on the sympathetic innerva- tion of the heart [39]. Catecholamines, on the other hand, are important signals for the expression of several cardiac genes [37] and are of major importance for the short-term metabolic regulation of the SR Ca 2+ pump by reversible phospholamban phosphorylation [13, 14, 30].
An alteration in the short-term regulation of the SR Ca 2+ pump by catecholamines via phospholamban phosphoryla- tion is the most likely reason for the differences in the phospholamban phosphorylation between matured SHR and WKY rats at day 40. At this age, SHR are still in a 'prehyper- tensive stage.' The reduced ~2p incorporation into phospho- lamban of 40 day-old SHR confirms earlier results reported for older SHR with established hypertension [40]. It suggests an increased cAMP-dependent in vivo phosphorylation of phospholamban in matured SHR. In fact, sympathetic drive is higher in SHR than in WKY [16]. As the activity of the cardiac SR Ca 2÷ pump depends critically on the degree of phospholamban phosphorylation one would anticipate a higher SR Ca 2+ re-uptake activity in the myocardium of ma- tured SHR. If so, this activation should be detectable by in
vitro measurements of Ca 2+ transport. However, the appar- ently increased Ca 2+ uptake values in homogenates of 40 day old SHR were not significantly different from the respective values of WKY. The reason for the discrepancy is not com- pletely understood but could be due to an insufficient phos- phoprotein protection in the Ca 2+ uptake assay or due to other factors that are difficult to control in our assay.
In contrast to matured hearts, sympathetic innervation is poorly developed in the early postnatal period [41 ] and phos- phorylation ofphospholamban in the intact heart is expected to be low [30, 31]. Unde r conditions of low in vitro phospho- rylation, changes in the amount of ~2p phospholamban formed in vitro can be considered as a measure of relative changes
of the tissue level ofphospholamban. Therefore, the observed correlation between the increase in homogenate SR Ca 2+ uptake and the increase in 32p-phospholamban between day 1 and 12 suggests that the early postnatal expression of the SR Ca 2+ pump and phospholamban occurs in a coordinated manner irrespective of the genetic differences between SHR and WKY pups.
Acknowledgments
The work was supported by the Anglo-German Foundation/ British Research Council (UK) and Deutscher Akademischer Austauschdienst (Germany). We are grateful to Christel Kemsies for performing the quantitative phospholamban analysis.
2. Maylie JG: Excitation-contractioncouplinginneonatalandadultmyo- cardium of cat. Am J Physio1242: H834-H843, 1982
3. Nakanishi T, Seguchi M, Takao A: Development of the myocardial contractile system. Experientia 44: 936-944, 1988
4. Chemla D, Lecarpentier Y, Martin JL, Clergue M, Antonetti A, Hatt PY: Relationship between inotropy and relaxation in rat myocardium. Am J PhysioI 250: H1008-H1016, 1986
5. Komuro I, Kurabayashi M, Shibazaki Y, Takaku F, Yazaki Y: Molecu- lar cloning and characterization of a Ca 2+ +Mg-'+-dependent adenosine triphosphatase from rat cardiac sarcoplasmic reticulum. Regulation of its expression by pressure overload and developmental stage. J Clin Invest 83: 1102-1108, 1989
6. Lompre AM, Lambert F, Lakatta EG, Schwartz K: Expression of sar- coplasmic reticulum CaZ+-ATPase and calsequestrin genes in rat heart during ontogenic development and aging. Circ Res 69: 1380-1388, 1991
7. Nayler WG, Fassold E: Calcium accumulating and ATPase activity of cardiac sarcoplasmic reticulum before and after birth. Cardiovasc Res 11: 231-237, 1977
8. Fisher DJ, Tate CA, Phillips S: Developmental regulation of the sar- coplasmic reticulum calcium pump in the rabbit heart. Pediatr Res 31 : 474-479, 1992
9. Vetter R, Kemsies C, Schulze W: Sarcolemmal Na+-Ca 2+ exchange and sarcoplasmic reticulum Ca z+ uptake in several cardiac preparations. Biomed Biochim Acta 46:$375-$381, 1987
10. Katz AM, Nash-Adler P, Watras J, Messineo FC, Takenaka H, Louis CF: Fatty acid effects on calcium influx and effiux in sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Bioehim Biophys Acta 687: 17-26, 1982
11. Martonosi A, Donley J, Halpin RA: Sarcoplasmic reticulum. 3. The role ofphospholipids in the adenosine triphosphatase activity and Ca ++ transport. J Biol Chem 243: 61-70, 1968
12. ColyerJ: Controlofthe calcium pump ofcardiac sarcop[asmicretieu- lure. A specific role for the pentameric structure of phospholamban? Cardiovasc Res 27: I766-1771, 1993
13. Wegener AD, Simmerman HK, Lindemann JP, Jones LR: Phospho-
65
lamban phosphorylation in intact ventricles. Phosphorylation of ser- ine 16 and threonine I7 in response to beta-adrenergic stimulation. J Biol Chem 264:11468-11474, 1989
14. Tada M, Katz AM: Phosphorylation of the sareoplasmic reticulum and sarcolemma. Annu Rev Physiol 44: 401-423, 1982
15. Lindemann JP, Watanabe AM: Phosphorylation ofphospholamban in intact myocardium. Role of Ca2+-calmodulin-dependent mechanisms. J Biol Chem 260:4516-4525, 1985
16. Trippodo NC, Frohlich ED: Similarities of genetic (spontaneous) hy- pertension. Man and rat. Circ Res 48:309-319, 1981
17. Clubb FJ, Jr., Bell PD, Kriseman JD, Bishop SP: Myocardial cell growth and blood pressure development in neonatal spontaneously hyperten- sive rats. Lab Invest 56: 189-197, 1987
18. Gray SD: Spontaneous hypertension in the neonatal rat. A review. Clin Exp Hypertens A 6: 755-781, 1984
19. Bing OHL, Brooks WW, Conrad CH, Sen S, Perreault CL, Morgan JP: Intracellular calcium transients in myocardium from spontaneously hypertensive rats during the transition to heart failure. Circ Res 68: 1390-1400, 1991
20. Brooksby P, Levi A J, Jones JV: Contractile properties of ventricular myocytes isolated from spontaneously hypertensive rat. J Hypertens 10: 521-527, 1992
21. Lammerich A, Gtinther J, Storch E, Pfitzer G, Vetter R: Alterations of cardiac contractile function are related to changes in membrane cal- cium transport in spontaneously hypertensive rats. J Hypertens 13: 1313-1224, 1995
22. Feher JJ, Briggs FN, Hess ML: Characterization of cardiac sarcoplas- mic reticulum from ischemic myocardium: Comparison of isolated sarcoplasmie reticulum with unfractionated homogenates. J Mol Cell Cardiol 12: 427-432, 1980
23. Solaro RJ, Briggs FN: Estimating the functional capabilities ofsareo- plasmic reticulum in cardiac muscle. Calcium binding. Circ Res 34: 531-540, 1974
24. Vetter R, Rupp H: CPT-1 inhibition by etomoxir has a chamber-re- lated action on cardiac sarcoplasmic reticulum and isomyosins. Am J Physio1267: H2091-H2099, 1994
25. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measure- ment with the folin phenol reagent. J Biol Chem 193:265 275, 1951
26. Tanaka H, Shigenobu K: Effect ofryanodine on neonatal and adult rat heart: developmental increase in sarcoplasmic reticulum function. J Mol Cell Cardiol 21: 1305-1313, 1989
27. Vetter R, Will H, K~ittner I, Kemsies C, Will Shahab L: Developmen- tal changes of Ca ÷÷ transport systems in chick heart. Biomed Biochim Acta 45: $219-$222, 1986
28. Vetter R, Studer R, Reinecke H, Kolar F, Ostadalova I, Drexler H: Reciprocal changes in the posmatal expression of the sarcolemmal Na+-Ca2+-exchanger and SERCA2 in rat heart. J Mol Cell Cardiol 27: 1689-1701, 1995
29. Arai M, Otsu K, MacLennan DH, Periasamy M: Regulation of sareo- plasmic reticulum gene expression during cardiac and skeletal muscle development. Am J Physiol 262: C614--C620, 1992
30. Karezewski P, Vetter R, Holtzhauer M, Krause EG: Indirect technique for the estimation of cAMP-dependent and CaZ+/calmodulin-depend - ent phospholamban phosphorylation state in canine heart in vivo.
study on the physiological role of the Ca2÷-calmodulin-dependent pro- tein kinase system. J Mol Cell Cardiol 24: 387-396, 1992
34. Hawkins C, Xu A, Narayanan N: Sarcoplasmic reticulum calcium pump in cardiac and slow twitch skeletal muscle but not fast twitch skeletal muscle undergoes phosphorylation by endogenous and exogenous Ca2+/ calmodulin-dependent protein kinase. Characterization of optimal con- ditions for calcium pump phosphorylation. J Biol Chem 269:31198- 31206, 1994
35. Anversa P, Olivetti G, Loud AV: Morphometric study of early postna- tal development in the left and right ventricular myocardium of the rat. I. Hypertrophy, hyperplasia and binucleation of myocytes. Circ Res 46: 49f~502, 1980
36. Rakusan K: Assessment of cardiac growth In: R. Zak (ed.) Growth of the Heart in Health and Disease. Raven Press, New York, 1984, pp 25-40
37. Gupta MP, Gupta M, Jakovcic S, Zak R: Catecholammes and cardiac growth. Mol Cell Biochem (this volume)
38. Rohrer D, Dillmann WH: Thyroid hormone markedly increases the mRNA coding for sarcoplasmic reticulum Ca2÷-ATPase in the rat heart. J Biol Chem 263: 6941~5944, 1988
39. Beekman RE, van Hardeveld C, Simonides WS: On the mechanism of the reduction by thyroid hormone of beta-adrenergic relaxation rate stimulation in rat heart. Biochem J 259: 229--236, 1989
40. Vetter R, Kott M, Rupp H: Differential influences of carnitine palmitoyltransferase-I inhibition and hyperthyroidism on cardiac growth and sarcoplasmic retieulum phosphorylation. Eur Heart J 15 (Suppl.D): 31-37, 1994
41. Kirby ML: Role of extracardiac factors in heart development. Experientia 44:944-951, 1988
