AJP - Heart Add DOIs to your references at manuscript stage!
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 291: H507-H516, 2006. First published March 31, 2006; doi:10.1152/ajpheart.00862.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/H507    most recent
00862.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Larsen, K.-O.
Right arrow Articles by Christensen, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Larsen, K.-O.
Right arrow Articles by Christensen, G.

CALL FOR PAPERS
Cytoskeletal Networks and the Regulation of Cardiac Contractility

Alveolar hypoxia induces left ventricular diastolic dysfunction and reduces phosphorylation of phospholamban in mice

Karl-Otto Larsen,1,2,3 Ivar Sjaastad,1,3,4 Aud Svindland,5 Kurt A. Krobert,6 Ole Henning Skjønsberg,2 and Geir Christensen1,3

1Institute for Experimental Medical Research, Ullevål University Hospital, University of Oslo, 2Department of Pulmonary Medicine, Ullevål University Hospital, Faculty Division, University of Oslo, 3Center for Heart Failure Research, University of Oslo, 4Department of Cardiology, Ullevål University Hospital, 5Department of Pathology, Aker University Hospital, and 6Department of Pharmacology, University of Oslo, Oslo, Norway

Submitted 12 August 2005 ; accepted in final form 26 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic obstructive pulmonary disease (COPD) may lead to pulmonary hypertension (PH) and reduced function of the right ventricle (RV). However, COPD patients may also develop left ventricular (LV) diastolic dysfunction. We hypothesized that alveolar hypoxia induces LV diastolic dysfunction and changes in proteins governing Ca2+ removal from cytosol during diastole. Mice exposed to 10% oxygen for 1, 2, or 4 wk were compared with controls. Cardiac hemodynamics were assessed with Doppler echocardiography and a microtransducer catheter under general anesthesia. The pulmonary artery blood flow acceleration time was shorter and RV pressure was higher after 4 wk of hypoxia compared with controls (both P < 0.05). In the RV and LV, 4 wk of hypoxia induced a prolongation of the time constant of isovolumic pressure decay (51% RV, 43% LV) and a reduction in the maximum rate of decline in pressure compared with control (42% RV, 42% LV, all P < 0.05), indicating impaired relaxation and diastolic dysfunction. Alveolar hypoxia induced a 38%, 47%, and 27% reduction in Ser16-phosphorylated phospholamban (PLB) in the RV after 1, 2, and 4 wk of hypoxia, respectively, and at the same time points, Ser16-phosphorylated PLB in the LV was downregulated by 32%, 34%, and 25% (all P < 0.05). The amounts of PLB and sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) were not changed. In conclusion, chronic alveolar hypoxia induces hypophosphorylation of PLB at Ser16, which might be a mechanism for impaired relaxation and diastolic dysfunction in both the RV and LV.

diastole; calcium; echocardiography

PATIENTS suffering from lung diseases with respiratory failure, of whom patients with chronic obstructive pulmonary disease (COPD) are the largest group, can develop pulmonary hypertension (PH) and right ventricular (RV) dysfunction (1). The RV dysfunction has been attributed to increased RV afterload due to PH (6). However, COPD patients with respiratory failure may also have left ventricular (LV) diastolic dysfunction (2). The LV diastolic dysfunction has been explained by a leftward displacement of the interventricular septum caused by RV pressure overload (29). However, in a dog model with pulmonary emphysema, LV diastolic dysfunction was present without bowing of the septum to the left (12), suggesting that intrinsic mechanisms in the LV myocardium participate in LV diastolic dysfunction. The molecular mechanisms leading to dysfunction of the LV in chronic alveolar hypoxia are not known.

Ca2+ plays a pivotal role in regulating both the relaxation and the contraction phase of the cardiac cycle. The rate of relaxation is primarily controlled by uptake of Ca2+ into the sarcoplasmic reticulum (SR) through the activity of the SR Ca2+ ATPase (SERCA), but the Na+/Ca2+ exchanger (NCX) at the sarcolemma also removes Ca2+ in diastole (3). Phospholamban (PLB) regulates SERCA2a activity, and phosphorylation at the Ser16 residue by protein kinase A (PKA) or at the Thr17 site by Ca2+/calmodulin-dependent protein kinase II (CaMKII) relieves the Ca2+ pump inhibition, enhancing relaxation rates and contractility (31). Catecholamines initiate the adrenergic signaling pathways by binding to and activating beta-adrenergic receptors, leading to stimulation of adenylyl cyclase activity and activation of PKA. How chronic alveolar hypoxia and PH affect LV diastolic function and proteins involved in removal of Ca2+ from cytosol in the LV is not known.

We hypothesized that chronic alveolar hypoxia induces changes in proteins governing removal of cytosolic Ca2+ in diastole in both the pressure-loaded RV and the normally loaded LV. To examine this hypothesis, mice were exposed to normobaric hypoxia for up to 4 wk, and proteins involved in removal of Ca2+ from cytosol during diastole were measured in the RV and the LV, as well as in the LV free wall and interventricular septum. We also obtained hemodynamic parameters by RV and LV catheterization and Doppler echocardiography.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and was approved by the Norwegian National Animal Research Committee. The animals were housed with a 12:12-h day-night cycle at 21°C, and food and water were available ad libitum.

Animal model. A total of 185 8-wk-old male C57Black/6j mice were either placed in a tightly sealed chamber under normobaric hypoxia with an oxygen fraction of 10% (hypoxia group) for 1, 2, or 4 wk or housed under normoxic conditions (oxygen fraction of 21%) (control group) (22). The carbon dioxide concentration was kept <0.4% inside the chamber. Cardiac pressure measurements and Doppler echocardiography were performed on mice exposed to 1, 2, or 4 wk of hypoxia and corresponding controls under general anesthesia with inhalation of isoflurane (34). For Western blots, histological and morphometrical analysis, mice anesthetized with isoflurane were euthanized by dislocation of the neck, and the heart and lungs were rapidly excised and weighed. Blood samples for hematocrit measurements were taken by direct puncture of the heart while the mice were under general anesthesia before they were euthanized. Blood for plasma norepinephrine (NE) and epinephrine (Epi) assessments was drawn quickly from the inferior vena cava after a rapid induction of general anesthesia. Samples were collected on ice into glutathione-EGTA tubes, centrifuged at 4°C at 3,000 g for 10 min, and frozen at –80°C. Catecholamine concentrations were measured by high-performance liquid chromatography, as previously described (28).

Doppler echocardiography. Echocardiography was performed with a VIVID 7 echocardiograph (GE Vingmed Ultrasound, Horten, Norway) with the use of a 13-MHz linear array transducer (GE) designed for examination of small rodents. The examination was performed on mice exposed to 1, 2, or 4 wk of hypoxia and respective controls as terminal experiments, and it was in principle performed as previously described (9). RV two-dimensional (2-D) images were obtained in the short-axis view at the level that gave a transverse section of the LV at the mitral end of the left posterior papillary muscle. The endocardial contour of the RV free wall was difficult to discriminate in the control animals, and thus the transverse diameter of the RV including the free wall was considered more accurate than separately measuring the cavity and free wall diameters. The pulmonary artery (PA) blood flow was measured at the PA root. The PA acceleration time (PAAT) was measured from the start to the peak of the flow signal. We have previously evaluated intra- and interscorer reliability of Doppler, 2-D, and M-mode measurements in mice (9). However, the reliability of RV diameter and the PAAT measurement has not previously been evaluated. To assess interscorer reliability for these two parameters, the data records were analyzed by two persons who were blinded for the intervention carried out and the score of the other analyzer. Also, one scorer analyzed the measurements twice on separate days to evaluate intrascorer reliability. Both intra- and interscorer reliabilities were found acceptable with a mean difference <10%. No statistically significant differences were found (Table 1).


View this table:
[in this window]
[in a new window]
  		Table 1. Reproducibility of echocardiographic measurements

 
Cardiac pressure measurements. Pressure measurements were conducted in the RV after 4 wk of hypoxia and in the LV after 1, 2, or 4 wk of hypoxia and in respective controls as terminal experiments at each time point. The RV and LV systolic pressures (RVSP and LVSP), the end-diastolic pressures (RVEDP and LVEDP), and the maximum rates of decline in pressure (RV dP/dtmin and LV dP/dtmin) were registered, and the time constant of isovolumic relaxation, {tau}, was calculated by assuming a zero asymptote (15). The catheterizations were performed with a 1.4-Fr Millar microtipped transducer catheter (model SPR-671, Millar Instruments, Houston, TX) inserted through the right internal jugular vein into the RV or through the right carotid artery into the LV (34). Data from 10 consecutive beats were recorded in DASYLab version 5.1 software (Datalog, National Instruments, Mönchengladbach, Germany) and analyzed by using a custom-made program designed in a commercially available software package (MATLAB, The MathWorks, Nattick, MA).

Histology and morphometric analysis. For histological and morphometric examinations, eight hearts from mice subjected to 4 wk of hypoxia and eight hearts from the control group were fixed in 4% paraformaldehyde and embedded in paraffin. The hearts were sectioned transversely from apex to the left atrium and stained with hematoxylin and eosin, Van Gieson, or Masson trichrome. The four midsections were selected from each heart to perform morphometrical assessments of both the RV and the LV. The morphometric analysis was conducted with a Leica DMRA2 microscope (Leica Microsystems, Wetzlar, Germany) connected to a Leica DC camera, and the actual areas of the hearts were circumscribed manually and calculated by an imaging program (Leica QWin Image processing and analysis system), as previously described (34). The average of the four midsections was calculated for both the RV and LV cavity dimensions by measuring the circumference of the cavities and for the area of the myocardium in the RV, septum, and the LV free wall. Immunohistochemical examination with affinity-purified antibodies (rabbit) against collagen type I (1:100 dilution) and III (1:1,200 dilution) (Rockland, Gilbertsville, PA) was performed as previously described (10).

Radioligand binding and adenylyl cyclase assay. RV and LV from mice exposed to 4 wk of hypoxia and controls were placed into a microcentrifuge tube containing ice-cold 50 mM Tris·HCl, pH 7.5, at 20°C, 1 mM EDTA, homogenized with an Ultra-Turrax homogenizer by using a single 30-s burst, and centrifuged at 27,000 g for 20 min at 4°C. The resulting crude membrane pellet was resuspended in 1 ml of ice-cold 50 mM Tris·HCl, pH 7.5, at 20°C, 1 mM EDTA, by using a Dounce glass-glass homogenizer, 10 strokes with the tight-fitting pestle; aliquoted; and used the same day or snap-frozen in liquid N2 and stored at –70°C until use for assays. Frozen membranes were rehomogenized with a Dounce glass-glass homogenizer before use in all the assays. Affinity (pKd) and receptor density (Bmax) were determined from equilibrium binding analysis of (–)-3-[125I]iodocyanopindolol ([125I]CYP) (Amersham Biosciences, Buckinghamshire, UK) binding to each membrane preparation. Membranes were incubated with increasing concentrations of [125I]CYP in the absence (total binding) or presence (nonspecific binding) of 10 µM propranolol for 1 h at 32°C. Adenylyl cyclase activities were measured in the basal situation by determining conversion of [{alpha}-32P]ATP (Amersham Biosciences) to [32P]cAMP and after stimulation with isoproterenol (Sigma-Aldrich, St. Louis, MO) or forskolin (Calbiochem, San Diego, CA), as previously described (21).

Quantitative real-time PCR. A real-time quantitative polymerase chain reaction (PCR) system (ABI 7900HT Fast Real-Time PCR System, PE Biosystems, Foster City, CA) was used to measure the amounts of {alpha}- and beta-myosin heavy chain (MHC) mRNA in RV and LV. Total mRNA was isolated from RV and LV from mice subjected to 4 wk of hypoxia and controls by using SV total RNA isolation system (Promega, Madison, WI). All RNA samples were quality-assessed by Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) and RNA integrity numbers. The RNA samples were reverse transcribed by using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Specific mRNA transcripts were quantified by Taqman GX assays (Applied Biosystems, Foster City, CA) for {alpha}-MHC (assay Mm00440354_m1, efficiency 1.99) and beta-MHC (assay Mm00600555_m1, efficiency 2.02). All samples were tested in triplicate, and average values were used for quantification. The amounts of {alpha}- and beta-MHC were determined from the cycle of threshold values and a standard curve, and they are given as percentage of the average of the control group (7).

Western blot analysis. Immunoblot analysis was performed as previously described (27). For inhibition of endogenous phosphatases, phosphatase inhibitor cocktail (Sigma, St. Louis, MO) was dissolved in homogenization solution. Briefly, proteins were separated by 6%, 10%, or 15% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Schleicher and Schuell, Dassel, Germany). Nonspecific binding was blocked in nonfat dry milk at 4°C overnight before the blots were incubated with primary antibody for 1 h at room temperature and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies: rabbit anti-sheep IgG (Pierce, Rockford, UK), anti-mouse IgG, or anti-rabbit IgG (all from Amersham Pharmacia Biotech, Buckinghamshire, UK). The primary antibodies were anti-PLB-Ser16, anti-PLB-Thr17 (both from Badrilla, Leeds, UK), anti-PLB A1, anti-SERCA2a (both from Affinity BioReagents, Golden, CO), and anti-NCX (33). Immunoreactive proteins were visualized by using ECL Plus detection kit (Amersham Life Sciences). Luminescence was detected by LAS-1000 video detection system and quantified with the Image Gauge program (both from Fujifilm, Stockholm, Sweden). Protein concentrations were determined by the bicinchoninic acid assay (Pierce 23235) by using bovine serum albumin as standard.

Data analysis and statistics. Data are presented as means ± SE or SD. Comparisons between groups were made by using unpaired Student's t-test, Mann-Whitney rank sum test in SigmaStat 3.1.1 (Systat Software, Richmond, CA), or ANOVA and subsequent Newman-Keuls post hoc test in Statistica 6.0 (StatSoft, Tulsa, OK). Differences were considered significant for P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Animal model. The body weight of the mice exposed to 4 wk of hypoxia was 5% lower than in the control group (P < 0.05, Table 2). With regard to the tibia length (TL), which was not measured in all the mice, no difference was observed between the groups (Table 2). Thus all organ weights were related to the TL. In the hypoxia group, the RV weight/TL ratio increased by 25%, 39%, and 36% compared with controls at 1, 2, and 4 wk, respectively (all P < 0.001, Table 2). There were no differences between the hypoxia and the control groups with regard to the LV weight normalized to TL (Table 2) or the septum weight-to-TL ratio after 1, 2, and 4 wk. The lung weight normalized to TL was increased by 36%, 36%, and 32%, respectively, in the hypoxia group at 1, 2, and 4 wk compared with controls (all P < 0.001, Table 2), while no difference was observed in the liver weight-to-TL ratio (77.2 ± 2.8 mg/mm in hypoxia group vs. 70.4 ± 3.7 mg/mm in control group, n = 14 each). Four weeks of hypoxia induced a 32% increase in hematocrit (57 ± 2 in hypoxia group vs. 39 ± 1 in control group, P < 0.001, n = 7 each). There were no significant differences in the plasma levels of NE (1,426 ± 297 pg/ml in hypoxia group vs. 1,276 ± 305 pg/ml in control group, n = 5 and n = 6, respectively) or Epi (1,146 ± 373 pg/ml in hypoxia group vs. 971 ± 178 pg/ml in control group, n = 5 and n = 6, respectively) after 4 wk of hypoxia. No ascites or pleural effusions were observed in the animals.


View this table:
[in this window]
[in a new window]
  		Table 2. Organ weights in the control and hypoxia groups

 
Echocardiographic measurements. The function and diameter of the cardiac chambers were assessed by echocardiography after 1, 2, or 4 wk of hypoxia. The RV diastolic diameter increased by 32% and 25%, respectively, in animals subjected to 2 or 4 wk of hypoxia compared with controls (both P < 0.05, Fig. 1, Table 3). Also, the RV fractional shortening was reduced in the hypoxia compared with the control groups after 1 wk (15 ± 3% vs. 28 ± 4%, P < 0.05), 2 wk (18 ± 3% vs. 30 ± 5%, P < 0.05), and 4 wk of hypoxia (10 ± 4% vs. 21 ± 3%, P < 0.05). LV fractional shortening and left atrial diameter were not significantly changed at any time point (Fig. 1, Table 3).


Figure 1
View larger version (48K):
[in this window]
[in a new window]
  		Fig. 1. M-mode echocardiographic recordings from mice breathing room air (control; left) and mice exposed to 4 wk of hypoxia (right). Top: representative traces from aorta (Ao) and left atrium (LA). RV, right ventricle. Bottom: representative traces from left ventricle (LV). IVS, interventricular septum; LVD, LV diameter; PW, posterior wall.

 

View this table:
[in this window]
[in a new window]
  		Table 3. Echocardiographic data

 
Doppler measurements. Cardiac hemodynamics was assessed by Doppler measurements after 1, 2, or 4 wk of hypoxia. PAAT was 23% shorter in mice subjected to 4 wk of hypoxia than in control animals (P < 0.05, Fig. 2, Table 3). Also, as early as 1 and 2 wk of hypoxia, a significant reduction in PAAT was observed compared with controls (21% and 30%, respectively, Table 3). There was no significant difference in PAAT between the groups exposed to 1, 2, and 4 wk of hypoxia. Hypoxia did not induce changes in peak PA blood flow (Table 3). On the other hand, peak LV outlet tract flow was reduced in the hypoxia groups compared with control animals by 20%, 31%, and 17% after 1, 2, and 4 wk, respectively (all P < 0.05, Table 3). Under the present experimental conditions, the heart rate was not significantly altered in the hypoxia compared with the control groups (Table 3). Cardiac output was reduced by 28%, 33%, and 29% after 1, 2, and 4 wk of hypoxia, respectively (all P < 0.05, Table 3).


Figure 2
View larger version (53K):
[in this window]
[in a new window]
  		Fig. 2. Doppler tracings in the pulmonary artery root. A: representative tracings in mice breathing room air (control; left) and mice subjected to 4 wk of hypoxia (right). B: magnified section from the Doppler tracings in A. The drawing between the panels illustrates the Doppler signals (control, hatched; hypoxia, open). C: pulmonary artery acceleration time (PAAT) in normoxia (control, filled bar) and after 4 wk of hypoxia (open bar) *P < 0.05.

 
Cardiac pressure measurements. To verify whether alveolar hypoxia induces PH as indicated by the Doppler measurements, the intraventricular pressure was measured in the RV after 4 wk of hypoxia. Hypoxia induced a rise in RVSP compared with control (59 ± 3 mmHg vs. 27 ± 1 mmHg, P < 0.001, Fig. 3). The LVSP and LVEDP were not significantly different (Fig. 3). With regard to ventricular relaxation, 4 wk of hypoxia induced a prolongation of the time constant of isovolumic pressure decay {tau} by 51% in the RV (P < 0.05, Fig. 4A), and RV dP/dtmin was reduced by 42% (P < 0.05, Fig. 4C). In the LV, {tau} was prolonged by 22%, and LV dP/dtmin was reduced by 29% after 2 wk of hypoxia, while 4 wk of hypoxia induced a prolongation of {tau} by 43% and a decrease in LV dP/dtmin by 42% (all P < 0.05, Fig. 4, B and D). The prolongation of {tau} and decrease in LV dP/dtmin did not reach statistical significance after 1 wk of hypoxia (Fig. 4, B and D).


Figure 3
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 3. Cardiac hemodynamic measurements. RV and LV pressure measurements in controls (filled bars) and mice exposed to 4 wk of hypoxia (open bars). RVSP, RV systolic pressure; LVSP, LV systolic pressure; LVEDP, LV end-diastolic pressure {dagger}P < 0.001.

 

Figure 4
View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4. Rates of cardiac relaxation. A: time constant ({tau}) of isovolumic pressure decay measured in the RV after 4 wk of hypoxia. B: {tau} measured in the LV after 1, 2, and 4 wk of hypoxia and in the respective control groups. C: maximum decay of RV pressure (RV dP/dtmin) after 4 wk of hypoxia. D: maximum decay of LV pressure (LV dP/dtmin) after 1, 2, and 4 wk of hypoxia and in the respective control groups. Normoxia, filled bars; alveolar hypoxia, open bars *P < 0.05.

 
Morphometric and histological analysis. The RV hypertrophy and dilatation observed in the hypoxia group with echocardiography was verified by morphometry, which showed that 4 wk of hypoxia increased the RV myocardial area-to-total cardiac area (TCA) ratio by 90 ± 18% of control value, indicating RV hypertrophy (P < 0.05). Hypoxia increased the RV circumference by 86 ± 17% compared with control, indicating RV dilatation (P < 0.05). RV hypertrophy and dilatation could also be visualized by light microscopy (Fig. 5). The septum area-to-TCA ratio and the LV free wall area-to-TCA ratio were not changed in hypoxia compared with control. We observed no edema in the RV or in the LV myocardium in the hypoxia group, as assessed by microscopy (data not shown). Four weeks of hypoxia induced minor subendocardial collagen depositions in the papillary muscles of the LV in Van Gieson-stained sections (Fig. 5), and immunohistochemical examination indicated increased amounts of collagen I and III (data not shown).


Figure 5
View larger version (116K):
[in this window]
[in a new window]
  		Fig. 5. Representative histological cardiac sections from mice breathing room air (control; left) or exposed to 4 wk of alveolar hypoxia (right). Top: RV hypertrophy (filled arrow) and dilatation (open arrow) in hypoxia (hematoxylin and eosin-stained section; magnification, x4). Bottom: collagen deposition (arrow) in hypoxia in papillary muscles from LV (Van Gieson staining; magnification, x100).

 
Radioligand binding and adenylyl cyclase assay. The level of beta-adrenergic receptors was not changed in any of the ventricles after 4 wk of hypoxia (Table 4). Hypoxia induced a reduction in the basal (13.2 ± 0.7 vs. 16.0 ± 0.9 pmol·mg protein–1·min–1) and isoproterenol-stimulated (25.3 ± 2.2 pmol·mg protein–1·min–1 over basal vs. 34.0 ± 2.2 pmol·mg protein–1·min–1 over basal) adenylyl cyclase activity in the LV compared with control (both P < 0.05, Table 4).


View this table:
[in this window]
[in a new window]
  		Table 4. beta-Adrenergic receptor level and adenylyl cyclase activity in the control and hypoxia groups

 
Quantitative real-time PCR. Four weeks of hypoxia increased the amount of beta-MHC mRNA in the RV by 112 ± 11% compared with control (P < 0.05, n = 6 each), but no significant change in the LV was observed. The levels of {alpha}-MHC mRNA were unchanged in RV and LV.

Calcium handling proteins. The expression levels of proteins involved in removal of Ca2+ from the cytosol in RV and LV myocardium from mice subjected to 1, 2, or 4 wk of hypoxia and in the control groups were analyzed. The amounts of PLB and SERCA2a were not changed in the RV or in the LV after 1, 2, and 4 wk of hypoxia. Total Ser16-phosphorylated PLB in the RV was reduced to 62%, 54%, and 73% of controls after exposure to 1, 2, and 4 wk of hypoxia, respectively (all P < 0.05, Fig. 6A). Specific antibodies allowed quantification of both the pentameric and monomeric forms of Ser16-phosphorylated PLB. Both the pentameric and monomeric forms of Ser16 were reduced at all time points. The reduction in Ser16 phosphorylation was more pronounced in the monomeric than in the pentameric form of PLB, the difference being statistically significant at 1 wk (50% monomeric vs. 82% pentameric, P < 0.001) and 4 wk of hypoxia (61% monomeric vs 83% pentameric, P < 0.05).


Figure 6
View larger version (13K):
[in this window]
[in a new window]
  		Fig. 6. Ser16-phosphorylated phospholamban in cardiac tissue. Immunolabeling density for the control groups (filled bars) was set to 100% and compared with 1, 2, and 4 wk of alveolar hypoxia (open bars). A: RV. B: LV, including septum. *P < 0.05.

 
Hypoxia reduced the amount of total Ser16-phosphorylated PLB in the LV to 68%, 66%, and 75% of control at 1, 2, and 4 wk of hypoxia, respectively (all P < 0.05, Fig. 6B). The monomeric form of Ser16-phosphorylated PLB was significantly reduced by 46%, 38%, and 34% of controls after 1, 2, and 4 wk of hypoxia (all P < 0.05), and the pentameric form was decreased by 11%, 31%, and 16%, respectively, in the hypoxia groups at the same time points (all P < 0.05). In the LV free wall, 4 wk of hypoxia resulted in a reduction in the amount of total Ser16-phosphorylated PLB to 76% of control (P < 0.05, Fig. 7A). The monomeric form was reduced to 65% and the pentameric form to 85% of control (P < 0.05). Ser16-phosphorylated PLB was not significantly changed in the septum (Fig. 7A).


Figure 7
View larger version (8K):
[in this window]
[in a new window]
  		Fig. 7. Calcium handling proteins in septum and LV free wall. Immunolabeling density for the control groups (filled bars) was set to 100% and compared with 4 wk of alveolar hypoxia (open bars). A: total Ser16-phosphorylated phospholamban. B: total Thr17-phosphorylated phospholamban. C: Na+/Ca2+-exchanger protein abundance. *P < 0.05.

 
Hypoxia did not induce any significant changes in the amount of Thr17-phosphorylated PLB in the RV or in the whole LV. After 4 wk of hypoxia, the amount of Thr17-phosphorylated PLB increased to 119% of control in the free wall of the LV (P < 0.05, Fig. 7B). There were no significant changes in Thr17-phosphorylated PLB in the septum (Fig. 7B). In the RV, 1 wk of hypoxia increased the amount of NCX to 119% of control value (P < 0.05), while no significant changes were observed in the RV or in the whole LV after 2 and 4 wk of hypoxia. In the LV free wall, the level of NCX in the hypoxia group was 118% of the control group (P < 0.05) but was not significantly altered in the septum (Fig. 7C).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Alveolar hypoxia induced shorter PAAT, elevated pressure in the RV, and RV hypertrophy and dilatation. In both the RV and LV, prolonged {tau} and reduced dP/dtmin were found in the hypoxia group, indicating impaired relaxation and diastolic dysfunction. Ser16 phosphorylation of PLB was substantially reduced in both the RV and the LV at 1, 2, and 4 wk of hypoxia. The active monomeric form of Ser16-phosphorylated PLB was more reduced than the pentameric form. There were no alterations in the levels of PLB or SERCA2a. The amount of NCX was increased in the RV and the LV free wall in alveolar hypoxia.

Effects of alveolar hypoxia on hemodynamics and cardiac function. Doppler echocardiography demonstrated a shorter PAAT after 1, 2, and 4 wk of hypoxia, being consistent with increased RV afterload due to PH. PAAT has to our knowledge not previously been measured in mice applying Doppler technique. However, in a rat model of progressive PH, PAAT was found to be useful in estimating PH (16). In our study, RV catheterization showed increased RV pressure in mice subjected to alveolar hypoxia, supporting the Doppler finding that indicated PH. Thus it is now possible to detect PH noninvasively also in mice, by applying the Doppler technique. We also assessed, for the first time to our knowledge, the RV diameter and function noninvasively in mice.

In both the RV and LV, 4 wk of hypoxia induced a prolonged {tau} and reduction in dP/dtmin, indicating impaired relaxation and diastolic dysfunction that was present in the LV already after 2 wk. Impaired LV relaxation has not previously been observed in any model with chronic hypoxia or in any small animal model of PH. It is, however, known that patients suffering from COPD with hypoxemia may develop LV diastolic dysfunction (2), in addition to RV hypertrophy and RV diastolic dysfunction (24). Studies of COPD patients with LV diastolic dysfunction demonstrate that hypoxemia is present when this cardiac complication is diagnosed (5, 29). Several mechanisms have been suggested to explain LV diastolic dysfunction in COPD and PH; LV interstitial edema (8), leftward displacement of the septum (29), increased myocardial stiffness (12), and diastolic asynchrony in the apical and lateral walls (4). In our study, light microscopic examination did not reveal any signs of interstitial edema in the hypoxia group, and the collagen depositions in the LV papillary muscles were limited and not likely to be of functional relevance. Echocardiography showed no leftward displacement of the septum, and the LV was not subjected to pressure overload that could explain impaired relaxation, given the unaltered LVSP and LVEDP measured in our study.

Effects of alveolar hypoxia on proteins regulating cardiac relaxation. Our results suggest that the mechanism for LV diastolic dysfunction in chronic alveolar hypoxia is related to alterations in proteins governing removal of cytosolic Ca2+ in diastole. The rate of relaxation is important for LV diastolic function, and it is primarily controlled by the uptake of Ca2+ into the SR, which is governed by the SERCA activity. PLB is a reversibly phosphorylated protein that regulates SERCA activity. In its dephosphorylated state, PLB binds to SERCA2a and inhibits Ca2+ pump activity, whereas phosphorylation of PLB relieves the Ca2+ pump inhibition and increases the relaxation rate and contractility (31). Thus the substantial reduction in Ser16-phosphorylated PLB that was found in the LV myocardium at 1, 2, and 4 wk of hypoxia can reduce the relaxation rate and may at least partly explain the observed LV diastolic dysfunction. PLB exists in an active monomeric form and as a pentamer that functions as an inactive or less active reservoir (19). We observed that the amount of active monomeric Ser16-phosphorylated PLB was more reduced than the pentameric form at 1 and 4 wk of hypoxia, which, taken together with a lower level of total Ser16-phosphorylated PLB, might reduce SERCA2 activity and be a mechanism for LV diastolic dysfunction.

Reduced amounts of Ser16-phosphorylated PLB have to our knowledge not been observed in any model of chronic hypoxia or PH. However, in the RV, altered levels of SERCA2a and PLB have been found in a monocrotaline (MCT) rat model with RV pressure overload. Increased levels of phosphorylated PLB and SERCA2 together with decreased amount of total PLB were found (32). These MCT-treated rats also had a 40% prolongation of the Ca2+ transients in isolated perfused RVs, which is difficult to explain from increased amounts of SERCA2 and phosphorylated PLB. In another study with the MCT rat model, reduced SERCA2a protein and mRNA expression and decreased PLB mRNA were found in the RV, which may explain impaired Ca2+ cycling in the pressure-overloaded RV in that model (20). A reduced level of Ser16 phosphorylation, as observed in our study, has also been found in left-sided heart failure (18, 27) and was related to relaxation abnormalities. Thus both the RV and the LV diastolic dysfunction might be related to reduced Ser16-phosphorylated PLB in alveolar hypoxia.

The link between alveolar hypoxia and reduced Ser16-phosphorylated PLB has not been identified. Theoretically, reduced beta-adrenergic receptor signaling could induce the observed reduction in Ser16 phosphorylation of PLB (23). In our study, there were no significant changes in the plasma level of NE or Epi that might have led to altered beta-adrenergic signaling in the hypoxia group. In a rat model with hypoxia, a decreased number of beta-adrenergic receptors were observed in the LV (17), whereas in another study it was shown that the decrease in beta-adrenergic receptors was more pronounced in the pressure-loaded RV than in the LV in a MCT rat model of PH (30). In our study, we found no significant changes in the beta-adrenergic receptor level either in the RV or the LV. It has also been shown in rat that 3 wk of hypoxia induced a reduction of the basal and isoproterenol-stimulated adenylyl cyclase activity in the RV (17). In contrast to this finding, we found a reduction in basal and isoproterenol-stimulated adenylyl cyclase activity in the LV, indicating that desensitization or uncoupling of beta-adrenergic receptor signaling may reduce Ser16-phosphorylated PLB in the LV. However, there were no significant changes in the beta-adrenergic receptor level and adenylyl cyclase activities in the RV, indicating that other mechanisms might be more important for the hypophosphorylation of PLB. Neither can the reduction of Ser16-phosphorylated PLB in the LV be explained by increased LV afterload or increased RV pressure on the septum. In support of this notion, decreased Ser16-phosphorylated PLB was found in the LV free wall and not in the septum. On the other hand, circulating factors such as cytokines may affect both ventricles and alter cellular signals regulating phosphorylation of PLB. It has been shown that exposure to hypoxia increases the levels of circulating cytokines that can reach the heart and alter beta-adrenergic signaling (26).

The amount of Thr17-phosphorylated PLB was increased in the LV free wall after 4 wk of hypoxia but unaltered in the septum. The inverse regulation of cardiac Thr17- and Ser16-phosphorylated PLB has also been observed in a rat heart failure model with aortic banding, where increased phosphorylation at Thr17 was found to be consistent with increased CaM kinase activity in failing hearts (25).

Interestingly, the NCX was increased in the LV free wall after 4 wk of hypoxia. In failing human myocardium, protein levels of NCX relative to SERCA are increased (14). In the MCT rat model with PH, the NCX mRNA and protein levels in both the LV and RV were unchanged (20). In our study, the reduced Ser16-phosphorylated PLB in the hypoxia group may have induced a decrease in the Ca2+ pump activity in the SR, and an increased level of NCX might be a compensatory mechanism to reduce cytosolic Ca2+ during diastole (13).

A MHC isoform shift with increased beta-MHC has been suggested to be involved in delayed myocardial relaxation (11). In our study, increased beta-MHC was only observed in the pressure-overloaded RV similar to findings in a model with PH induced by MCT (20). Thus a shift in MHC isoforms may play a role in the development of impaired relaxation in the pressure-overloaded RV but not in the normally loaded LV.

In the current study, we have used Doppler echocardiography and cardiac catheterization to characterize a mouse model of alveolar hypoxia. RV hypertrophy, RV dilatation, and RV and LV diastolic dysfunction were found. Reduced Ser16 phosphorylation of PLB in both the RV and LV might be a possible mechanism for the diastolic dysfunction observed in both ventricles. The hypophosphorylation of PLB and increased expression of NCX occur both in the pressure-overloaded RV and the normally loaded LV free wall. These findings indicate that other mechanisms than increased mechanical load might alter the levels of proteins governing removal of cytosolic Ca2+ and thereby be involved in the development of cardiac dysfunction due to alveolar hypoxia.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Anders Jahre's Fund for Promotion of Science, The Research Council of Norway, and The Norwegian Council for Cardiovascular Diseases.


   ACKNOWLEDGMENTS
 
We are grateful to Unni Lie Henriksen, Kristin Arnkværn, Lisbeth Winer, Cathrine Husberg, Torstein Lyberg, Liv Marit Skaug, and Kristin Godang for skillful laboratory work, to Ann Kristin Josefsen and Morten Eriksen for animal care, and to Tævje Andreas Strømme, Roy Trondsen, Paul Fredrik Gjerpe, Gunnar Nicolaysen, Alexandra V. Finsen, and Per Reidar Woldbæk for expert technical help.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K.-O. Larsen, Institute for Experimental Medical Research, Surgical Bldg., 4th floor, Ullevål Univ. Hospital, Kirkeveien 166, N-0407 Oslo, Norway (e-mail: karlottl{at}medisin.uio.no)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Barbera JA, Peinado VI, and Santos S. Pulmonary hypertension in chronic obstructive pulmonary disease. Eur Respir J 21: 892–905, 2003.[Abstract/Free Full Text]
  2. Baum GL, Schwartz A, Llamas R, and Castillo C. Left ventricular function in chronic obstructive lung disease. N Engl J Med 285: 361–365, 1971.[ISI][Medline]
  3. Bers DM. Calcium and cardiac rhythms: physiological and pathophysiological. Circ Res 90: 14–17, 2002.[Free Full Text]
  4. Bhargava V and Sunnerhagen KS. Left ventricular asynchrony in patients with pulmonary hypertension. J Appl Physiol 69: 517–522, 1990.[Abstract/Free Full Text]
  5. Boussuges A, Pinet C, Molenat F, Burnet H, Ambrosi P, Badier M, Sainty JM, and Orehek J. Left atrial and ventricular filling in chronic obstructive pulmonary disease. An echocardiographic and Doppler study. Am J Respir Crit Care Med 162: 670–675, 2000.[Abstract/Free Full Text]
  6. Braunwald E, Zipes DP, and Libby P. Heart Disease: A Textbook of Cardiovascular Medicine (6th ed). Philadelphia, PA: Saunders, 2001.
  7. Danzi S and Klein I. Posttranscriptional regulation of myosin heavy chain expression in the heart by triiodothyronine. Am J Physiol Heart Circ Physiol 288: H455–H460, 2005.[Abstract/Free Full Text]
  8. Davis KL, Mehlhorn U, Laine GA, and Allen SJ. Myocardial edema, left ventricular function, and pulmonary hypertension. J Appl Physiol 78: 132–137, 1995.[Abstract/Free Full Text]
  9. Finsen AV, Christensen G, and Sjaastad I. Echocardiographic parameters discriminating myocardial infarction with pulmonary congestion from myocardial infarction without congestion in the mouse. J Appl Physiol 98: 680–689, 2005.[Abstract/Free Full Text]
  10. Finsen AV, Woldbaek PR, Li J, Wu J, Lyberg T, Tønnessen T, and Christensen G. Increased syndecan expression following myocardial infarction indicates a role in cardiac remodeling. Physiol Genomics 16: 301–308, 2004.[Abstract/Free Full Text]
  11. Fitzsimons DP, Patel JR, and Moss RL. Role of myosin heavy chain composition in kinetics of force development and relaxation in rat myocardium. J Physiol 513: 171–183, 1998.[Abstract/Free Full Text]
  12. Gomez A and Mink S. Increased left ventricular stiffness impairs filling in dogs with pulmonary emphysema in respiratory failure. J Clin Invest 78: 228–240, 1986.[ISI][Medline]
  13. Hasenfuss G and Schillinger W. Is modulation of sodium-calcium exchange a therapeutic option in heart failure? Circ Res 95: 225–227, 2004.[Free Full Text]
  14. Hasenfuss G, Schillinger W, Lehnart SE, Preuss M, Pieske B, Maier LS, Prestle J, Minami K, and Just H. Relationship between Na+-Ca2+-exchanger protein levels and diastolic function of failing human myocardium. Circulation 99: 641–648, 1999.[Abstract/Free Full Text]
  15. Hunter JJ, Tanaka N, Rockman HA, Ross J Jr, and Chien KR. Ventricular expression of a MLC-2v-ras fusion gene induces cardiac hypertrophy and selective diastolic dysfunction in transgenic mice. J Biol Chem 270: 23173–23178, 1995.[Abstract/Free Full Text]
  16. Jones JE, Mendes L, Rudd MA, Russo G, Loscalzo J, and Zhang YY. Serial noninvasive assessment of progressive pulmonary hypertension in a rat model. Am J Physiol Heart Circ Physiol 283: H364–H371, 2002.[Abstract/Free Full Text]
  17. Kacimi R, Richalet JP, Corsin A, Abousahl I, and Crozatier B. Hypoxia-induced downregulation of beta-adrenergic receptors in rat heart. J Appl Physiol 73: 1377–1382, 1992.[Abstract/Free Full Text]
  18. Kass DA, Bronzwaer JG, and Paulus WJ. What mechanisms underlie diastolic dysfunction in heart failure? Circ Res 94: 1533–1542, 2004.[Abstract/Free Full Text]
  19. Kimura Y, Kurzydlowski K, Tada M, and MacLennan DH. Phospholamban inhibitory function is activated by depolymerization. J Biol Chem 272: 15061–15064, 1997.[Abstract/Free Full Text]
  20. Kogler H, Hartmann O, Leineweber K, Nguyen van P, Schott P, Brodde OE, and Hasenfuss G. Mechanical load-dependent regulation of gene expression in monocrotaline-induced right ventricular hypertrophy in the rat. Circ Res 93: 230–237, 2003.[Abstract/Free Full Text]
  21. Krobert KA, Bach T, Syversveen T, Kvingedal AM, and Levy FO. The cloned human 5-HT7 receptor splice variants: a comparative characterization of their pharmacology, function and distribution. Naunyn Schmiedebergs Arch Pharmacol 363: 620–632, 2001.[CrossRef][ISI][Medline]
  22. Levi M, Moons L, Bouche A, Shapiro SD, Collen D, and Carmeliet P. Deficiency of urokinase-type plasminogen activator-mediated plasmin generation impairs vascular remodeling during hypoxia-induced pulmonary hypertension in mice. Circulation 103: 2014–2020, 2001.[Abstract/Free Full Text]
  23. MacLennan DH and Kranias EG. Phospholamban: a crucial regulator of cardiac contractility. Nat Rev Mol Cell Biol 4: 566–577, 2003.[CrossRef][ISI][Medline]
  24. Marangoni S, Scalvini S, Schena M, Vitacca M, Quadri A, and Levi G. Right ventricular diastolic function in chronic obstructive lung disease. Eur Respir J 5: 438–443, 1992.[Abstract]
  25. Pathak A, del Monte F, Zhao W, Schultz JE, Lorenz JN, Bodi I, Weiser D, Hahn H, Carr AN, Syed F, Mavila N, Jha L, Qian J, Marreez Y, Chen G, McGraw DW, Heist EK, Guerrero JL, DePaoli-Roach AA, Hajjar RJ, and Kranias EG. Enhancement of cardiac function and suppression of heart failure progression by inhibition of protein phosphatase 1. Circ Res 96: 756–766, 2005.[Abstract/Free Full Text]
  26. Prabhu SD. Cytokine-induced modulation of cardiac function. Circ Res 95: 1140–1153, 2004.[Abstract/Free Full Text]
  27. Sande JB, Sjaastad I, Hoen IB, Bøkenes J, Tønnessen T, Holt E, Lunde PK, and Christensen G. Reduced level of serine16 phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity. Cardiovasc Res 53: 382–391, 2002.[Abstract/Free Full Text]
  28. Schafroth U, Godang K, Ueland T, and Bollerslev J. Leptin response to endogenous acute stress is independent of pituitary function. Eur J Endocrinol 145: 295–301, 2001.[Abstract]
  29. Schena M, Clini E, Errera D, and Quadri A. Echo-Doppler evaluation of left ventricular impairment in chronic cor pulmonale. Chest 109: 1446–1451, 1996.[Abstract/Free Full Text]
  30. Seyfarth T, Gerbershagen HP, Giessler C, Leineweber K, Heinroth-Hoffmann I, Ponicke K, and Brodde OE. The cardiac beta-adrenoceptor-G-protein(s)-adenylyl cyclase system in monocrotaline-treated rats. J Mol Cell Cardiol 32: 2315–2326, 2000.[CrossRef][ISI][Medline]
  31. Simmerman HK and Jones LR. Phospholamban: protein structure, mechanism of action, and role in cardiac function. Physiol Rev 78: 921–947, 1998.[Abstract/Free Full Text]
  32. Stessel H and Brunner F. Effect of endothelin antagonism on contractility, intracellular calcium regulation and calcium regulatory protein expression in right ventricular hypertrophy of the rat. Basic Clin Pharmacol Toxicol 94: 37–45, 2004.[ISI][Medline]
  33. Thomas MJ, Sjaastad I, Andersen K, Helm PJ, Wasserstrom JA, Sejersted OM, and Ottersen OP. Localization and function of the Na+/Ca2+-exchanger in normal and detubulated rat cardiomyocytes. J Mol Cell Cardiol 35: 1325–1337, 2003.[CrossRef][ISI][Medline]
  34. Woldbaek PR, Hoen IB, Christensen G, and Tønnessen T. Gene expression of colony-stimulating factors and stem cell factor after myocardial infarction in the mouse. Acta Physiol Scand 175: 173–181, 2002.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
K.-O. Larsen, B. Lygren, I. Sjaastad, K. A. Krobert, K. Arnkvaern, G. Florholmen, A.-K. R. Larsen, F. O. Levy, K. Tasken, O. H. Skjonsberg, et al.
Diastolic dysfunction in alveolar hypoxia: a role for interleukin-18-mediated increase in protein phosphatase 2A
Cardiovasc Res, October 1, 2008; 80(1): 47 - 54.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/2/H507    most recent
00862.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Larsen, K.-O.
Right arrow Articles by Christensen, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Larsen, K.-O.
Right arrow Articles by Christensen, G.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.