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Am J Physiol Heart Circ Physiol 302: H1524–H1532, 2012. First published January 27, 2012; doi:10.1152/ajpheart.00955.2010. The onset of left ventricular diastolic dysfunction in SHR rats is not related to hypertrophy or hypertension S. Dupont,1,2 J. Maizel,1,2 R. Mentaverri,1,2 J.-M. Chillon,1,2 I. Six,1,2 P. Giummelly,3 M. Brazier,1,2 G. Choukroun,1,2 C. Tribouilloy,1,2 Z. A. Massy,1,2 and M. Slama1,2 1 INSERM U 1088; 2Jules Verne University of Picardy and Amiens University Medical Center, Amiens; and 3Cardiovascular Pharmacology Laboratory (EA 3452), Nancy, France Submitted 23 September 2010; accepted in final form 4 January 2012 diastole; ultrasonic; calcium; spontaneously hypertensive rats LEFT VENTRICULAR (LV) DIASTOLIC dysfunction is responsible for more than one half of all hospitalizations for congestive heart failure or acute pulmonary oedema, which has been shown to be the consequence of multiple clinical factors, mostly hypertension (15). LV diastolic dysfunction, particularly relaxation abnormalities, is usually recognized to be closely associated with the development of left ventricular hypertrophy (LVH) in hypertensive humans and spontaneously hypertensive rats (SHR), a model of human genetic hypertension (35). Nevertheless, in a previous study, we demonstrated LV relaxation Address for reprint requests and other correspondence: M. Slama, INSERM U 1088, Univ. of Picardy and Amiens Univ. Medical Center, Dept. of Nephrology-Medical Intensive Care Unit, Ave. René Laennec, F-80054 Amiens, France (e-mail: [email protected]). H1524 impairment in young SHR without LVH (37), subsequently confirmed by Aeschbacher et al (1). Di Bello et al. (13) have also demonstrated early LV diastolic dysfunction before the onset of hypertension and LVH. In view of these data, LV diastolic dysfunction may be considered to be an early event in the cascade of processes leading to the development of hypertension and LVH. However, to date, no clear information is available concerning the kinetics of development of diastolic dysfunction. Noteworthy, LVH is associated with structural and functional abnormalities of cardiomyocytes. Under physiological conditions, following the increase in intracytosolic Ca2⫹ concentration ([Ca2⫹]i) during contraction, myocyte relaxation occurs when Ca2⫹ is transported back into the sarcoplasmic reticulum (SR) by the SR Ca2⫹ ATPase [sarco(endo)plasmic Ca2⫹-ATPase isoform (SERCA)] and, to a lesser extent, when Ca2⫹ is pumped out of the cytosol by the Na⫹/Ca2⫹ exchange (NCX) and sarcolemmal Ca2⫹ ATPase. SERCA is regulated by its inhibitor phospholamban (PLB). Phosphorylation of PLB at Ser16 (p16-PLB) by protein kinase A and/or Thr17 by Ca2⫹/calmodulin-dependent kinase II reduces its inhibitory effects on SERCA, resulting in an increased affinity of SERCA for Ca2⫹, increasing SR Ca2⫹ uptake, whereas dephosphorylation of PLB by SRassociated phosphatase (PP1 and PP2a) restores the inhibitory effect of PLB on SERCA. The present study mainly concerned p16-PLB, since it is considered to be the major regulator of PLB activity during -adrenergic stimulation (10). Under pathological conditions, several studies have reported decreased expression of SERCA 2a, the predominant isoform in the heart (43), in failing human myocardium (2, 34) and in animal models of pressure overload-induced hypertrophy, especially in SHR (3). This abnormality of SERCA 2a would lead to abnormalities in Ca2⫹ handling, especially significant alteration of [Ca2⫹]i (4, 5), which appears to be involved in hypertrophic cell growth (28). Chen-izu et al. (9) recently showed that alterations in Ca2⫹ signaling already occur at the onset of hypertension and precede the development of hypertrophy. We therefore hypothesized that LV diastolic dysfunction and abnormal [Ca2⫹]i in genetic hypertension may precede not only LVH but also the onset of hypertension. We decided 1) to analyze LV myocardial function and particularly diastolic function (by invasive and noninvasive methods) in 3-weekold SHR rats before the onset of hypertension and in 5-week-old SHR rats after the onset of hypertension and 2) to investigate variations in [Ca2⫹]i as well as SERCA 2a RNA and protein expression level in these animals. 0363-6135/12 Copyright © 2012 the American Physiological Society http://www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 5, 2017 Dupont S, Maizel J, Mentaverri R, Chillon JM, Six I, Giummelly P, Brazier M, Choukroun G, Tribouilloy C, Massy ZA, Slama M. The onset of left ventricular diastolic dysfunction in SHR rats is not related to hypertrophy or hypertension. Am J Physiol Heart Circ Physiol 302: H1524 –H1532, 2012. First published January 27, 2012; doi:10.1152/ajpheart.00955.2010.—Left ventricular (LV) diastolic dysfunction, particularly relaxation abnormalities, are known to be associated with the development of LV hypertrophy (LVH). Preliminary human and animal studies suggested that early LV diastolic dysfunction may be revealed independently of LVH. However, whether LV diastolic dysfunction is compromised before the onset of hypertension and LVH remains unknown. We therefore evaluated LV diastolic function in spontaneously hypertensive rats (SHR) at different ages and tested whether LV diastolic dysfunction is associated with abnormal intracellular calcium homeostasis. LV systolic and diastolic functions were evaluated by invasive and echocardiographic methods in 3-week-old (without hypertension) and 5-week-old (with hypertension) SHR and Wistar-Kyoto control rats. Basal intracytoplasmic calcium and sarcoplasmic reticulum (SR) Ca2⫹ contents were measured in cardiomyocytes using fura-2 AM. Sarco(endo)plasmic Ca2⫹-ATPase isoform 2a (SERCA 2a) and phospholamban (PLB) expressions were quantified by Western blot and quantitative RT-PCR techniques. LV relaxation dysfunction was observed in 3-week-old SHR rats before onset of hypertension and LVH. An increase in basal intracytoplasmic Ca2⫹ and a decrease in SR Ca2⫹ release were demonstrated in SHR. Decreased expression of SERCA 2a and Ser16 PLB (p16-PLB) protein levels was also observed in SHR rats, whereas mRNA expression was not decreased. For the first time, we have shown that LV myocardial dysfunction precedes hypertension in 3-week-old SHR rats. This LV myocardial dysfunction was associated with high diastolic [Ca2⫹]i possibly due to decreased SERCA 2a and p16-PLB protein levels. Diastolic dysfunction may be a potential predictive marker of arterial hypertension in genetic hypertension syndromes. H1525 EARLY DIASTOLIC DYSFUNCTION IN SHR RATS MATERIALS AND METHODS Table 1. Invasive hemodynamic parameters in anesthetized WKY and SHR 3 Wk n SAP, mmHg DAP, mmHg MAP, mmHg PP, mmHg HR, beats/min , ms 5 Wk WKY SHR WKY SHR 12 69 ⫾ 4 52 ⫾ 2 58 ⫾ 3 17 ⫾ 3 329 ⫾ 11 9.3 ⫾ 0.4 10 75 ⫾ 3 53 ⫾ 3 60 ⫾ 2 22 ⫾ 2 288 ⫾ 8* 10.7 ⫾ 0.4* 6 86 ⫾ 5† 67 ⫾ 6† 74 ⫾ 5† 19 ⫾ 2 386 ⫾ 12† 8.1 ⫾ 0.2† 8 105 ⫾ 2*† 76 ⫾ 3† 86 ⫾ 2*† 29 ⫾ 2* 332 ⫾ 6*† 9.0 ⫾ 0.2*† Values are means ⫾ SE. WKY, Wistar-Kyoto rats; SHR, spontaneously hypertensive rats; SAP, systolic arterial pressure; DAP, diastolic arterial pressure; MAP, mean arterial pressure; PP, pulse pressure; HR, heart rate; and , time constant of isovolumic LV relaxation. *P ⬍ 0.05 vs. age-matched WKY; †P ⬍ 0.05 vs. the same strain at 3 wk of age. 3 Wk n SAP, mmHg DAP, mmHg MAP, mmHg PP, mmHg HR, beats/min 5 Wk WKY SHR WKY SHR 5 106 ⫾ 4 70 ⫾ 5 82 ⫾ 3 37 ⫾ 7 332 ⫾ 17 5 95 ⫾ 3 63 ⫾ 4 73 ⫾ 3 32 ⫾ 5 344 ⫾ 12 5 101 ⫾ 3 80 ⫾ 3 87 ⫾ 3 21 ⫾ 1 308 ⫾ 15 5 129 ⫾ 4*† 84 ⫾ 5† 99 ⫾ 4* 45 ⫾ 3*† 312 ⫾ 20 Values are means ⫾ SE. *P ⬍ 0.05 vs. age-matched WKY; †P ⬍ 0.05 vs. the same strain at 3 wk of age. to 2% isoflurane. Transthoracic echocardiographic measurements were performed in the left lateral decubitus position with a commercially available echocardiography system (Phillips IE33 with a 15MHz transducer, France) using two-dimensional and Doppler imaging, as described previously (36). Echocardiographic evaluation was carried out in blind fashion. The transthoracic echocardiographic transducer was placed to obtain short- and long-axis, four-chamber (ventricles and atria) and five-chamber (ventricles, atria, and aorta) apical cardiac views. M-mode LV imaging was obtained in the cardiac long-axis and LV end-diastolic diameter (LVEDD), LV end-systolic diameter (LVESD), and posterior and septal diastolic wall thickness (PW and IVS) were measured according to the American Society of Echocardiography guidelines (33). LV mass (LVM) was calculated as follows: LVM ⫽ 1.04 ⫻ [(LVEDD ⫹ PW ⫹ IVS)3 ⫺ LVEDD3]. Shortening fraction (SF) was calculated according to the following equation: SF ⫽ (LVEDD ⫺ LVESD)/LVEDD. From a five-chamber apical view, aortic flow was recorded using pulsed Doppler imaging. LV ejection time (ET) was measured as the time from the beginning to the end of the aortic flow wave. Velocity of circumferential fiber shortening was also calculated as VCF ⫽ SF/ET. Mitral flow was recorded at the tip of the mitral valve from an apical view using Doppler imaging. Isovolumic relaxation time (IVRT) was measured as the interval between the aortic closure click and the start of mitral flow. The Tei index was calculated on the same apical view as follows (39): Tei index ⫽ (DD ⫺ MD)/ET where DD is diastole duration and Table 3. Echocardiographic parameters in SHR and WKY rats 3 Wk n BW, g HR, beat/min LVEDD, mm LVESD, mm PW, mm IVS, mm LVM, g LVM/BW mg/g ESAo, % VTIao, cm VTImi, cm VE, cm/s ETao, ms 5 Wk WKY SHR WKY SHR 31 59 ⫾ 2 375 ⫾ 1 4.5 ⫾ 0.1 2.2 ⫾ 0.1 1.3 ⫾ 0.1 1.1 ⫾ 0.1 0.25 ⫾ 0.01 4.2 ⫾ 0.1 8.3 ⫾ 0.8 4.0 ⫾ 0.1 3.3 ⫾ 0.1 78.3 ⫾ 1.4 76.2 ⫾ 0.8 31 51 ⫾ 1* 372 ⫾ 2 4.3 ⫾ 0.1 2.5 ⫾ 0.1 1.2 ⫾ 0.1 1.1 ⫾ 0.1 0.23 ⫾ 0.01 4.5 ⫾ 0.1 9.7 ⫾ 1.3 4.0 ⫾ 0.1 2.9 ⫾ 0.1* 76.0 ⫾ 1.5 77.5 ⫾ 1.0 27 145 ⫾ 5† 383 ⫾ 2 6.2 ⫾ 0.1† 3.1 ⫾ 0.1† 1.4 ⫾ 0.1 1.2 ⫾ 0.1 0.48 ⫾ 0.01† 3.3 ⫾ 0.1† 9.5 ⫾ 1.3 4.8 ⫾ 0.2† 4.2 ⫾ 0.1† 93.5 ⫾ 2.2† 73.7 ⫾ 0.9† 27 111 ⫾ 2*† 378 ⫾ 2 6.0 ⫾ 0.1† 3.4 ⫾ 0.1*† 1.2 ⫾ 0.1* 1.1 ⫾ 0.1 0.40 ⫾ 0.01† 3.3 ⫾ 0.2† 10.9 ⫾ 0.8 4.4 ⫾ 0.1*† 3.8 ⫾ 0.1*† 91.5 ⫾ 1.9† 72.9 ⫾ 1.1† Values are means ⫾ SE. BW, body weight; LVEDD, left ventricular (LV) end-diastolic diameter; LVESD, LV end-systolic diameter; PW, posterior wall end-diastolic diameter; IVS, interventricular septum end-diastolic diameter; LVM, LV mass; LVM/BW and ESAo, systolic expansion rate of aorta; VTIao and VTImi, aortic and mitral velocity time integral; VE, early wave velocity; ETao, aortic ejection time. *P ⬍ 0.05 vs. age-matched WKY; †P ⬍ 0.05 vs. the same strain at 3 wk of age. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00955.2010 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 5, 2017 Materials. All chemicals were obtained from Sigma-Aldrich. Primers were obtained from Invitrogen (France). Primary mouse antiSERCA 2a and rabbit anti-Ser16 phospho-phospholamban (p16-PLB) antibodies were purchased from Affinity Bioreagent. Rabbit anticalnexin, goat anti-PLB, secondary anti-rabbit, anti-goat, and antimouse peroxidase IgG antibodies were purchased from Santa Cruz. Pentobarbital sodium was obtained from Ceva Santé, France. Animals. Three-week-old and 5-week-old male SHR and WistarKyoto (WKY) rats were purchased from Janvier Breeding Laboratories (Le Genest-Saint-Isle, France). They were maintained in temperature- and humidity-controlled rooms with a 12-h:12-h light/dark cycle and were given standard chow. All rats were handled in accordance with French legislation, and the protocol was approved by an institutional animal care committee. Blood and LV pressure measurements. At 3 and 5 weeks of age, respectively, five WKY, five SHR, five WKY, and five SHR rats were anesthetized with ketamine-zylazine solution. A polyethylene catheter was introduced into the right carotid artery and tunneled subcutaneously to exit behind the head. The surgical procedure was performed in the morning. Animals awoke 1 h after the procedure, and the catheter was then connected to a pressure transducer to record arterial blood pressure in conscious animals after resting for 8 h. Systolic arterial pressure (SAP), diastolic arterial pressure, mean arterial blood pressure (MAP), pulse pressures (PP), and heart rate (HR) were measured by the data acquisition system (IOX v2.4.2.6; EMKA technologies, Paris, France) during a stable period. In parallel, at 3 and 5 weeks of age, respectively, these same parameters were measured in 12 WKY, 10 SHR, 6 WKY, and 8 SHR anesthetized animals. The catheter was then carefully advanced into the left ventricle through the aortic valve. Time constants of isovolumic LV relaxation () were calculated automatically from the LV pressure curve (IOX v2.4.2.6; EMKA technologies) during a stable period. Pulse wave velocity measurements. Rats were anesthetized with ketamine-xylazine solution. A polyethylene catheter was introduced into the right carotid artery via the aortic arch. Another catheter was inserted into the abdominal aorta via the left femoral artery. When blood pressure and heart rate were stable, the two pressure waves were recorded simultaneously for at least 3 min. The propagation time for the pulse wave moving from the aortic arch to the abdominal aorta was measured by the time interval between the upstroke (foot) of each pressure wave front. After euthanasia, the distance between the two catheters was determined with a cotton thread. The pulse wave velocity (PWV) was obtained by dividing this distance by the time interval between the two pressure wave fronts. Echocardiography. Thirty-one SHR and WKY rats (3 week old) and 27 SHR and WKY rats (5 week old) were anesthetized with 1% Table 2. Arterial blood pressure in conscious SHR and WKY rats H1526 EARLY DIASTOLIC DYSFUNCTION IN SHR RATS Ca2⫹-free buffer supplemented with fetal bovine serum (10%), and filtered through a nylon mesh (100 m). Cells were finally resuspended successively in buffers containing increasing extracellular Ca2⫹ concentrations to reach a final Ca2⫹ concentration of 1.2 mM. An average of 70 – 80% of rod-shaped cardiomyocytes was observed on light microscopy. Cells were then plated on laminin-coated 96-well plates at a density of 20,000 cells/well and maintained at 37°C in a humidified atmosphere containing 5% CO2 for least 1 h, before intracellular calcium measurements. Measurement of [Ca2⫹]i. Fresh cardiomyocytes were incubated with 5 M fura-2 AM for 30 min, as described previously (45). Excitation wavelengths were 340 and 380 nm (calcium bound and unbound to fura-2 AM, respectively). Fluorescence emission was monitored at 510 nm by Envision 2103 Multilabel Reader (Perkin Elmer). In view of the recognized uncertainties inherent to the measurement of absolute [Ca2⫹]i, the results are expressed as the (340/380 nm) fluorescence ratio throughout this study, which provides an index of [Ca2⫹]i. To assess the sarcoplasmic reticulum (SR) Ca2⫹ release, various ATP concentrations (0.5, 5, 50 M) were used in the presence of 1 mM Ca2⫹. Values are expressed as variations of the 340-to-380 nm ratio calculated as: 340-to-380 nm ratio after stimulation with ATP normalized to 340-to-380 nm ratio observed at steady state (i.e., before stimulation of cardiomyocytes). The maximum (Rmax) and minimum (Rmin) fluorescence intensities were compared in both WKY and SHR rats, at 3 and 5 weeks of age. Rmax was assessed after addition of Triton X-100 and 5 mM Ca2⫹, and Rmin was determined after addition of 25 mM EGTA. Fig. 1. A–D: Doppler echocardiography systolic and diastolic indexes in 3-week-old and 5-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). IVRT, isovolumic relaxation time; SF, shortening fraction; VCF, velocity circumferential fiber shortening. Three-week-old WKY and SHR (n ⫽ 31) 5-week-old WKY and SHR (n ⫽ 27) are shown. *P ⬍ 0.05 vs. agematched WKY. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00955.2010 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 5, 2017 MD is mitral duration. The systolic expansion rate of the aorta (ESAo) was calculated as described previously (24). HR must be identical to compare echocardiographic parameters between SHR and WKY rats, particularly diastolic function (IVRT), which is highly dependent on HR. Aortic and left ventricle collagen and desmosine contents. Twenty rats (5 rats in each of the 4 groups: 3- and 5-week-old SHR and WKY animals) were assigned for the analyses of aortic composition in collagen and desmosine. Samples of frozen aorta and left ventricle were removed, weighed, and hydrolyzed in 6 M hydrochloric acid for 24 h at 105°C. Protein content was determined with the dinitrofluorobenzene method, taking 92 kDa as the molecular weight of an amino acid. Collagen content was determined with the chloramine T/paradimethylaminobenzaldehyde reaction as [(hydroxyproline content ⫻ 7.46/protein content) ⫻ 100] (25). Elastin and the elastin-specific, cross-linking amino acids desmosine and isodesmosine were assayed by capillary zone electrophoresis and UV detection (16). Isolation of cardiomyocytes. Following echocardiographic measurements, animals were euthanized by pentobarbital sodium. The heart was removed, and cardiomyocytes were isolated from the LV wall, as described previously (32). Briefly, the hearts were rapidly excised, mounted on a Langendorff apparatus, and retrogradely perfused for 5 min with Ca2⫹-free buffer containing (in mM) 120.4 NaCl, 5.4 KCl, 0.6 KH2PO4, 0.6 Na2PO4, 1.2 MgSO4, 10 HEPES, 4.6 NaHCO3, 30 taurine, 10 2,3 butanedione monoxime, and 5.5 glucose at pH 7.2. The hearts were then perfused with a Ca2⫹-free buffer containing 1 mg/ml collagenase II (Worthington) until they started to become flaccid. The LV were removed, minced into small pieces in EARLY DIASTOLIC DYSFUNCTION IN SHR RATS Table 4. Collagen, desmosine, and isodesmosine contents in the aorta and the left ventricle in SHR and WKY rats 3 Wk n Aorta ww, mg Desmosine, g/mg aorta ww Isodesmosime, g/mg aorta ww Collagen, g/mg aorta ww LV ww, mg Collagen, g/mg LV ww 5 Wk WKY SHR WKY SHR 5 6.9 ⫾ 0.2 5 6.5 ⫾ 0.2 5 6.9 ⫾ 0.4 5 6.6 ⫾ 0.3 0.7 ⫾ 0.1 0.5 ⫾ 0.1 0.6 ⫾ 0.1 0.6 ⫾ 0.1 0.8 ⫾ 0.1 0.7 ⫾ 0.1 0.7 ⫾ 0.1 0.8 ⫾ 0.1 59.1 ⫾ 2.6 9.1 ⫾ 0.4 65.7 ⫾ 3.4 9.4 ⫾ 0.5 79.2 ⫾ 5.1† 9.3 ⫾ 0.3 83.1 ⫾ 5.7† 9.6 ⫾ 0.5 3.6 ⫾ 0.3 4.0 ⫾ 0.3 3.5 ⫾ 0.2 3.7 ⫾ 0.2 Quantitative RT-PCR. Total RNA was extracted from left ventricles using Tri-reagent, and total RNA (1 g) was transcribed into complementary DNA using the high capacity cDNA reverse transcription kit according to the manufacturer’s protocol (Applied Biosystems). The following primers were used: SERCA 2a (forward, TGA ATC TGA CCC AGT GGC TGA; reverse, ACT CCA GTA TTG CAG GCT CCA), SERCA 2b (forward, CCT TTG CCA CTC ATT TTC CAG A; reverse, GCA CAC TCT TTA CCA GGC TCC A), PLB (forward, TGA GCT CCC AGA CTT CAC ACA; reverse, TTG ACA GCA GGC AGC CAA A), NCX 1 (forward, TCA TTG GAG ATC TGG CTT CCC; reverse, TGG ACG CAT CTG CAT ACT GGT), and -actin (forward, CAT TGC TGA CAG GAT GCA; reverse, AGA GCC ACC AAT CCA CAC AGA). SYBR Green chemistry was used to perform quantitative determinations of the mRNAs encoding for SERCA 2a, SERCA 2b, PLB, NCX 1, and a housekeeping gene, -actin. cDNAs were amplified using an ABI PRISM 7900 sequence detection system (Applied Biosystems). -Actin was used to normalize differences in RNA isolation, RNA degradation, and the efficiency of the reverse transcription reaction. Changes in mRNA expression were determined using the comparative threshold method (23). RNA expression was also expressed in relation to the levels observed in WKY rats. Preparation of SR-enriched microsomes. After rats were euthanized, the hearts were removed and left ventricles were isolated to obtain SR-enriched microsomes, as described previously (19). The protein concentration was determined by Bradford assay (Bio-Rad). Aliquots were frozen at ⫺80°C until Western blot and SERCA 2a activity analysis. Western blot. Proteins of interest were quantified using Western blot analysis. Proteins from SR-enriched microsomes were denatured in Laemmli buffer containing 125 mM Tris·HCl, 2% SDS, 1% -mercaptoethanol, 10% glycerol, and 0.005% bromophenol blue (pH 6.8). Fifty micrograms of proteins per sample were separated on 8% (SERCA 2a, calnexin) or 15% (PLB, Ser16 PLB, or p16-PLB) acrylamide/bisacrylamide SDS-PAGE under constant current. Proteins were then transferred to polyvinylidene difluoride (0.45 m membrane; Millipore) in a transfer buffer, at 14 V overnight at 4°C. Ponceau red staining technique was used to confirm the amount of protein loaded on the membrane. Membranes were then blocked with 5% non-fat milk in Tris-buffered saline 0.1% Tween 20 for 1 to 2 h at room temperature and immunoblotted with the desired primary antibody in 0.5% non-fat milk for 90 min at room temperature (anti-SERCA 2a; 1:5,000) or overnight (anti-p16-PLB, anti-PLB, and anti-calnexin; 1:5,000). After several washings in Tris-buffered saline 0.1% Tween 20, membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies (SERCA 2a; 1:5,000), HRP-conjugated goat anti-rabbit antibodies (p16-PLB and calnexin; 1:5,000), or HRP-conjugated rabbit anti-goat antibodies (PLB; 1:5,000). Each secondary antibody was incubated for 1 h at room temperature before development using the enhanced chemiluminescence detection kit (Amersham). Bands were visualized and quantified using the GeneGenius Bio Imaging System (Syngene, UK). SERCA 2a protein levels were normalized to endogenous calnexin levels. The p16-PLB-to-PLB ratio was also determined. Measurement of SERCA 2a activity. SERCA 2a activity was determined from the rate of inorganic phosphate release, as described previously (18). The Ca2⫹-ATPase activity was calculated as the difference between Ca2⫹, Mg2⫹-ATPase activity (in the presence of calcium) and Mg2⫹-ATPase activity measured in the absence of calcium (3 mM EGTA). Statistical analysis. The results are expressed as the mean ⫾ SE of the indicated number of experiments. All variables were tested for a normal distribution (Kolmogorov-Smirnov test). Statistical analysis was performed using Student’s t-test in the case of a normal distribution and a Mann-Whitney test in the case of an abnormal distribution or number of samples is ⬍10. A value of P ⬍ 0.05 was considered to be significant. RESULTS Arterial and LV pressure. Data on arterial pressure are shown in Tables 1 and 2. Body weight was significantly lower in 3-week-old and 5-week-old SHR than in age-matched WKY rats. No difference in SAP, diastolic arterial pressure, MAP, PP, and HR values were observed between 3-week-old WKY and SHR rats or between anesthetized and conscious animals. At the age of 5 weeks, a higher blood pressure (SAP, MAP, Fig. 2. Measurement of basal intracytosolic calcium levels. Before stimulation with ATP, basal intracellular calcium was measured in cardiomyocytes isolated from 3-week-old and 5-week-old WKY and SHR rats and was expressed as the 340-to-380 nm ratio. In our experiments, the minimum and maximum fluorescence intensities (Rmin and Rmax) were not significantly different between WKY and SHR. Three-week-old SHR and WKY rats (n ⫽ 15) and 5-week-old SHR and WKY rats (n ⫽ 8). *P ⬍ 0.05 vs. age-matched WKY. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00955.2010 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 5, 2017 Values are means ⫾ SE. ww, Wet weight. †P ⬍ 0.05 vs. the same strain at 3 wk of age. H1527 H1528 EARLY DIASTOLIC DYSFUNCTION IN SHR RATS Fig. 3. ATP dose-response. Cardiomyocytes isolated from WKY and SHR rats were stimulated with ATP concentrations ranging from 0.5 to 50 M. ATPinduced SR Ca2⫹ release was measured as: 340-to-380 nm ratio after stimulation with ATP (t ⫽ 10 s) normalized to the steady-state 340-to-380 nm ratio. A: 3-week-old SHR and WKY rats (n ⫽ 15). B: 5-week-old SHR and WKY rats (n ⫽ 8). Data were analyzed by t-test. WKY, *P ⬍ 0.05 vs. age-matched WKY. and PP) was observed in SHR rats compared with WKY control rats in both anesthetized and conscious animals. LV catheterization showed a higher value in both 3-week-old and 5-week-old SHR rats compared with WKY rats (Table 1). PP amplification and PWV. At 3 and 5 weeks, the strains did not differ in terms of PP amplification (expressed either in mmHg or percentage). In contrast, the PWV was significantly higher in the SHR groups (440 ⫾ 40, n ⫽ 6; P ⫽ 0.026 and 500 ⫾ 30 cm/s, n ⫽ 5; P ⫽ 0.015 at 3 and 5 weeks, respectively) than in the WKY groups (320 ⫾ 30, n ⫽ 6 and 380 ⫾ 20 cm/s, n ⫽ 5, respectively) at both time points. Doppler echocardiography findings. M-mode echocardiography data are shown in Table 3. LVEDD and IVS were similar in 3-week-old and 5-week-old SHR compared with agematched WKY rats. Echocardiography also revealed a significant increase of LVESD in 5-week-old SHR compared with WKY control rats, whereas no difference was observed at 3 weeks of age. These parameters were used to calculate LVM. No difference in LVM was observed in 3-week-old and 5-week-old SHR compared with age-matched WKY control rats. This Fig. 4. Quantitative RT-PCR analysis of calcium regulatory gene in the left ventricle from 3-week-old and 5-week-old WKY and SHR. Graphs depicting relative sarco(endo)plasmic Ca2⫹-ATPase isoform (SERCA) 2a and 2b, phospholamban (PLB), and Na⫹/Ca2⫹ exchange (NCX1) expression normalized to -actin in 3-week-old (A) and 5-week-old (B) SHR rats compared with WKY (control or ct). 3-week-old WKY and SHR rats: n ⫽ 10, 5-week-old WKY; and SHR rats: n ⫽ 7. The error bar for WKY refers to SERCA 2a. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00955.2010 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 5, 2017 result was confirmed by weighing left ventricles after euthanasia (WKY: 0.19 ⫾ 0.01 and SHR: 0.18 ⫾ 0.01 g for 3-week-old rats; WKY: 0.38 ⫾ 0.02 and SHR: 0.32 ⫾ 0.03 g for 5-week-old rats) and after normalizing to body weight (WKY: 3.2 ⫾ 0.5 and SHR: 3.4 ⫾ 0.5 mg/g for 3-week-old rats; WKY: 2.6 ⫾ 0.1 and SHR: 2.7 ⫾ 0.1 mg/g for 5-week-old rats). ESAo was similar in 3-week-old and 5-week-old SHR compared with age-matched WKY control rats. Systolic and diastolic functions were then evaluated by Doppler echocardiography. Assessment of diastolic function at constant HR showed that IVRT and Tei index, two parameters of LV relaxation, were significantly increased in both 3-weekold and 5-week-old SHR compared with control WKY rats (Fig. 1, A and B). Conversely, no significant increase in IVRT and Tei index was observed between 3-week-old and 5-weekold rats of either strain. Mitral velocity time integral was also significantly lower in 3-week-old and 5-week-old SHR compared with control WKY rats, whereas aortic velocity time was significantly decreased only in 5-week-old rats (Table 3). Assessment of systolic function revealed a significant decrease in SF and VCF in both 3-week-old and 5-week-old SHR compared with control WKY rats (Fig. 1, C and D). In contrast, no significant increase in SF and VCF was observed between 3-week-old and 5-week-old rats of either strain. Aortic and left ventricle collagen and desmosine contents. At both 3 and 5 weeks of age, the SHR and control WKY EARLY DIASTOLIC DYSFUNCTION IN SHR RATS increase of the 340-to-380 nm ratio in both SHR and WKY cardiomyocytes. ATP-induced release of SR Ca2⫹ was significantly lower in 3-week-old SHR than in age-matched control WKY rats, at ATP concentrations of 5 and 50 M (Fig. 3A). At 5 weeks of age, similar data were obtained with a significant difference between the two strains starting at an ATP concentration of 0.5 M (Fig. 3B). mRNA expression by quantitative RT-PCR. Gene expression was determined to further assess the mechanisms underlying the alterations in calcium cycling observed in left ventricles isolated from 3- and 5-week-old rats. No significant difference in mRNA expression, normalized to -actin encoding for SERCA 2a and SERCA 2b, PLB, and NCX 1, was observed between 3-week-old and 5-week-old SHR and their control WKY rats (Fig. 4, A and B). Similar results were obtained when hypoxanthine-guanine phosphoribosyltransferase was used as housekeeping gene instead of -actin (data not shown). Calcium regulatory protein abundance. The abundance of key calcium regulatory proteins, particularly SERCA 2a, PLB, and p16-PLB, was measured in SR-enriched microsomes isolated from left ventricles of 3-week-old and 5-week-old rats. As shown in Fig. 5, A and C, a decrease of SERCA 2a protein expression was observed in both 3-week-old and 5-week-old SHR rats compared with age-matched control WKY rats. SERCA 2a/calnexin, assessed by the GeneGenius Bio Imaging System, was significantly lower in both 3-week-old and 5-week-old SHR compared with WKY control rats (Fig. 5, B Fig. 5. Western blot analysis of SERCA 2a in the left ventricle from 3-week-old and 5-week-old WKY and SHR. A and B: representative Western blot using anti-SERCA 2a antibodies in 3-week-old rats (WKY, n ⫽ 11; SHR, n ⫽ 13) and 5-week-old rats (WKY, n ⫽ 9; SHR: n ⫽ 7), respectively. Calnexin protein expression was used as internal control. C and D: graphs depicting quantification of SERCA 2a normalized to calnexin. *P ⬍ 0.05 vs. age-matched WKY. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00955.2010 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 5, 2017 groups did not differ in terms of the aortic desmosine, isodesmosine, and collagen contents. However, the collagen contents were higher at 5 weeks than at 3 weeks. Left ventricle collagen contents were similar in SHR and age-matched WKY control rats at both 3 and 5 weeks of age (Table 4). Variation of [Ca2⫹]i in SHR and WKY rats. Calcium is the main mediator in cardiac excitation-contraction coupling and relaxation and abnormalities of calcium currents may therefore explain the disorders observed. We therefore investigated [Ca2⫹]i in cardiomyocytes isolated from the left ventricles of SHR and WKY rats. No significant difference in Rmin and Rmax values was observed between cardiomyocytes isolated from 3-week-old and 5-week-old SHR and WKY rats (data not shown). However, basal [Ca2⫹]i expressed as the 340-to-380 nm ratio was higher in cardiomyocytes isolated from SHR compared with age-matched WKY rats at steady state (before stimulation) and independently of age (3 weeks or 5 weeks old) (Fig. 2). A less marked, but nonsignificant, difference in 340-to-380 nm ratio was observed between cardiomyocytes isolated from 5-week-old rats of the two strains, suggesting that an adaptive mechanism affecting intracellular calcium homeostasis becomes effective between the ages of 3 weeks and 5 weeks in SHR rats. ATP, which triggers release of Ca2⫹ from the SR via activation of metabotropic (P2Y) and ionotropic (P2X) receptors, was used to evaluate SR Ca2⫹ load. ATP, introduced at concentrations of 0.1 to 50 M, induced a dose-dependent H1529 H1530 EARLY DIASTOLIC DYSFUNCTION IN SHR RATS and D). Although no difference in PLB was observed between SHR and WKY rats, p16-PLB and p16-PLB/PLB were significantly lower in 3-week-old SHR than in age-matched WKY control rats (Fig. 6, A and B). Assessment of the p16-PLB-toPLB ratio did not reveal any significant difference in 5-weekold rats (Fig. 6, C and D). SERCA 2a activity. A nonsignificant trend to a decrease in SHR SERCA 2a activity was observed in both 3-week-old (WKY 885 ⫾ 100; SHR 740 ⫾ 126 pmol Pi/mg ⫻ min n ⫽ 6) and 5-week-old rats (WKY 816 ⫾ 63; SHR 696 ⫾ 95 pmol Pi/mg ⫻ min n ⫽ 6). DISCUSSION Fig. 6. Western blot analysis of PLB and phosphorylation of PLB at Ser16 (p16-PLB) in the left ventricle from 3-week-old and 5-week-old WKY and SHR. A and C: representative Western blot using anti-PLB and anti-p16-PLB antibodies in 3-week-old rats (WKY, n ⫽ 12; SHR, n ⫽ 10) and 5-week-old rats (WKY, n ⫽ 9; SHR, n ⫽ 7), respectively. B and D: graphs depicting quantification of p16-PLB normalized to PLB. *P ⬍ 0.05 vs. age-matched WKY. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00955.2010 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 5, 2017 This study demonstrates, for the first time, that 3-week-old SHR rats develop marked myocardial dysfunction with impaired LV relaxation and alteration of LV systolic function, before the onset of hypertension and LVH. Myocardial dysfunction is associated with decreased SR Ca2⫹ release and increased steady-state intracellular calcium concentrations, at least partly due to a decrease of SERCA 2a protein levels. This deregulation was associated with a decrease in PLB phosphorylation, indicating possible functional inhibition of SERCA 2a, in addition to decreased protein expression. The absence of LVH is confirmed by the absence of difference for either LVM or LVM/body weight despite the body weight difference be- tween the two groups. In fact, young SHR rats were been reported to have a lower body weight than age-matched WKY rats, and this weight difference could be explained by the underlying metabolic disorders observed in SHR (14). LV diastolic dysfunction was assessed by IVRT, Tei index, and in SHR rats. IVRT has been described as a reliable marker of LV relaxation in clinical and experimental studies (38). The Tei index is increased in the presence of systolic or diastolic impairment (30). In the present study, IVRT and Tei index were higher in 3-week-old SHR than in age-matched WKY rats, confirming the presence of early LV diastolic dysfunction. These results are consistent with those of a previous study conducted in our laboratory in older rats with LVH and hypertension, confirming the accuracy of these echocardiographic parameters to detect systolic and diastolic dysfunction compared with invasive methods (36, 37). An alteration of aortic stiffness leading to increased PWV has also been observed in 3-week-old SHR rats before the onset of hypertension. Similar results have been reported in 6-week-old SHR (11, 41). This abnormality of aortic compliance has been attributed to media hypertrophy due to an increased number of smooth muscle cell layers rather than a modification in intrinsic elastic properties, particularly collagen and elastin contents (14). Our study results confirmed prior reports because we did not find any difference in collagen and EARLY DIASTOLIC DYSFUNCTION IN SHR RATS Second, IVRT and myocardial index were used to analyze LV relaxation. It would have been preferable to measure Doppler imaging indexes, but these indexes are still difficult to record in small animals weighing 40 – 60 g. In contrast, IVRT and myocardial indexes have been validated in our laboratory and have been shown to be very accurate to assess LV relaxation. was also measured and confirmed by our Doppler findings. Finally, it would have been more appropriate to use a Millar catheter to measure . A polyethylene catheter was used in this study, since it provides an optimal dynamic response with negligible resonance (42). This study demonstrates for the first time that diastolic dysfunction precedes hypertension. This finding may completely change our understanding of the pathophysiology of hypertension. At least three hypotheses can be proposed: 1) diastolic dysfunction may be the cause of hypertension by decreasing stroke volume and stimulating the sympathetic system resulting in hypertension and LV hypertrophy that accentuates LV diastolic dysfunction; 2) diastolic dysfunction may be a part of the same disease, and two mechanisms may be present related to two chromosomal alterations at the same locus; and 3) arterial abnormalities and cardiac dysfunction share the same alterations. In the light of these results, LV diastolic dysfunction could become a valuable predictive marker for hypertension and/or LVH. In the light of these findings, echocardiography may be useful to predict the onset of hypertension in patients with a family history of hypertension. In conclusion, this study demonstrates that early LV myocardial dysfunction precedes the onset of hypertension in SHR rats. This myocardial dysfunction is associated with abnormal Ca2⫹ homeostasis due to decreased SERCA 2a and p16-PLB protein expression. These findings may facilitate the design of appropriate and effective strategies for the treatment of cardiovascular diseases. ACKNOWLEDGMENTS We thank the French Society of Hypertension and the Picardie Regional Council for valuable support. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: S.D., J.M., R.M., I.S., Z.A.M., and M.S. conception and design of research; S.D., J.M., J.-M.C., and P.G. performed experiments; S.D., J.M., P.G., and M.S. analyzed data; S.D., J.M., R.M., J.-M.C., M.B., Z.A.M., and M.S. interpreted results of experiments; S.D. and J.M. prepared figures; S.D., J.M., and R.M. drafted manuscript; S.D., J.M., G.C., C.T., and Z.A.M. edited and revised manuscript; S.D., J.M., R.M., J.-M.C., I.S., P.G., M.B., G.C., C.T., Z.A.M., and M.S. approved final version of manuscript. REFERENCES 1. Aeschbacher BC, Hutter D, Fuhrer J, Weidmann P, Delacretaz E, Allemann Y. Diastolic dysfunction precedes myocardial hypertrophy in the development of hypertension. Am J Hypertens 14: 106 –113, 2001. 2. Arai M, Alpert NR, MacLennan DH, Barton P, Periasamy M. Alterations in sarcoplasmic reticulum gene expression in human heart failure. A possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ Res 72: 463–469, 1993. 3. Arata Y, Geshi E, Nomizo A, Aoki S, Katagiri T. Alterations in sarcoplasmic reticulum and angiotensin II receptor type 1 gene expression in spontaneously hypertensive rat hearts. Jpn Circ J 63: 367–372, 1999. 4. Beuckelmann DJ, Nabauer M, Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation 85: 1046 –1055, 1992. AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00955.2010 • www.ajpheart.org Downloaded from http://ajpheart.physiology.org/ by 10.220.33.6 on May 5, 2017 elastin contents in the aortas and hearts of our younger rats. However, this observation does not rule out the possibility of a qualitative alteration in the elastin, and so further work in this field is required. At the cellular level, the decrease in SERCA 2a protein expression observed in this study would lead to changes in intracellular calcium homeostasis, particularly a higher [Ca2⫹]i, the main cause of LV diastolic dysfunction. No correlation was performed between SERCA 2a protein expression and diastolic function indexes (TRIV, Tei index). However, a few studies have shown that overexpression of SERCA 2a by adenoviral gene transfer improved systolic and diastolic function in both heart failure and LVH (12, 27). Noteworthy, SERCA 2a mRNA expression was not significantly different between WKY and SHR rats, indicating that a lower SERCA 2a protein expression level in SHR is not due to decreased SERCA 2a transcription. One mechanism that could explain this finding is that SERCA 2a protein degradation is more active in SHR than WKY rats, since the SERCA 2a protein may be misfolded, leading to dislocation from the SR lumen back into the cytoplasm, where it is ubiquitinated and degraded by the action of the 26S proteasome pathway, a process known as ERassociated degradation. Calreticulin, a Ca2⫹-binding molecular chaperone localized in SR, interacts with SERCA 2b, but not with SERCA 2a (7). However, overexpression of calreticulin in rat myocardial H9c2 cells in response to oxidative stress enhanced degradation of SERCA 2a (17). A few published studies have shown early oxidative stress in young SHR rats (8, 29), and oxidative stress generated by reactive oxygen species, is known to alter SERCA 2a function (20, 44). The present study demonstrated a nonsignificant trend toward a decrease in SERCA 2a activity in SHR rats. A significant difference would probably have been observed with a larger number of animals. Alteration of SERCA 2a protein levels, and probably the effects of this protein on cardiomyocyte calcium homeostasis, therefore occurs very early in the development of hypertension. Further studies are required to determine whether this alteration is present at birth in these rats. It would also be interesting to measure reactive oxygen species in cardiomyocytes isolated from 3- and 5-week-old SHR and WKY rats. Decreased PLB phosphorylation in 3-week-old rats could be explained by different mechanisms. Enhanced phosphatase activity (PP1 and/or PP2A) has been demonstrated in dilated cardiomyopathy, adrenergic receptor-induced cardiac hypertrophy, and patients with end-stage heart failure (6, 26, 31). Decreased PLB phosphorylation could also be due to a modification of -adrenergic (-AR) receptor expression and/or activity, as Limas and Limas (21) showed that inotropic and lusitropic responses to -adrenergic stimulation are decreased in 5-week-old SHR rats, leading to a decreased number of -AR. Conversely, in another study, the same authors showed an increase in the number of -AR in LVH (22). On the basis of the results obtained in this study in rats, the adaptive mechanism appears to occur in SHR rats between the ages of 3 and 5 weeks. This study presents several limitations. First, our findings only concern an animal model of genetic hypertension and should be confirmed in other animal models of hypertension such as Dahl salt-sensitive rats. Moreover, extrapolation to human hypertension could be hazardous. In contrast, SHR is considered to be the best model of human hypertension (40). 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