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(Circulation Research. 2003;93:230.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Mechanical Load-Dependent Regulation of Gene Expression in Monocrotaline-Induced Right Ventricular Hypertrophy in the Rat

Harald Kögler, Oliver Hartmann, Kirsten Leineweber, Phuc Nguyen van, Peter Schott, Otto-Erich Brodde, Gerd Hasenfuss

From Georg-August-University Göttingen (H.K., O.H., P.N.v., P.S., G.H.), Department of Cardiology and Pneumology, Göttingen, Germany, and the University of Essen (K.L., O.-E.B.), Institute of Pathophysiology, Essen, Germany.

Correspondence to Harald Kögler, MD, Georg-August-Universität Göttingen, Abteilung Kardiologie und Pneumologie, Robert-Koch-Str. 40, D-37075 Göttingen, Germany. E-mail hkogler{at}med.uni-goettingen.de

	Abstract

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Rats treated with monocrotaline (MCT) develop pulmonary hypertension. Their right ventricles (RVs) exhibit severe pressure overload-induced hypertrophy, whereas the left ventricles (LVs) are normally loaded. In contrast, enhanced neuroendocrine stimulation during the transition to heart failure affects both ventricles. We assessed gene expression levels of Ca²⁺-regulating proteins in RVs and LVs of control and MCT rats in transition to heart failure to identify biomechanical load-regulated genes. In MCT RVs, both mRNA and protein levels of the Ca²⁺-ATPase of the sarcoplasmic/endoplasmic reticulum (SERCA2a) were reduced by 36% (P=0.001) and 17% (P=0.016), respectively, compared with control RVs. Phospholamban and ryanodine receptor mRNA levels likewise were reduced (by 27% [P=0.05] and 21% [P=0.011], respectively) in MCT RVs, whereas sarcolemmal Na⁺-Ca²⁺ exchanger expression was not altered. MCT LVs exhibited no significant expression changes compared with control LVs. Isometrically contracting MCT intact RV trabeculae showed enhanced baseline force development. Although control RV preparations exhibited a positive force-frequency relationship, MCT RVs showed a negative force-frequency relationship and blunted postrest potentiation. Contractile function of MCT LV trabeculae was normal. Maximum Ca²⁺-activated tension was enhanced by 64% in permeabilized RV MCT preparations (P=0.013). ß-Myosin heavy chain protein was upregulated in MCT RVs (P<0.001) but unaltered in MCT LVs. Degradation of troponin T was prominent in MCT RVs, a phenomenon not observed in the LV. Enhanced biomechanical load is necessary to induce the gene expression changes associated with the hypertrophic phenotype of the pressure-overloaded RV. Neuroendocrine factors, which equally affect both chambers, are not sufficient to alter the expression of Ca²⁺-cycling proteins.

Key Words: hypertrophy • mechanical load • gene expression • calcium • contractility

	Introduction

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When a heart develops hypertrophy and, subsequently, heart failure (HF), profound changes at the macroscopic and molecular level occur. This remodeling is associated with characteristic gene expression changes, such as the reactivation of a fetal gene expression program, causing the expression of atrial natriuretic factor and B-type natriuretic peptide and the switching of myosin heavy chain (MHC) isoforms from to ß and of actin isoforms from -cardiac to -skeletal actin.¹ Additionally, the expression levels of Ca²⁺-regulating proteins change in a typical way: the ATPase of the sarcoplasmic/endoplasmic reticulum (SERCA2) is downregulated in human end-stage HF² and in several experimental HF models.^3–5 The sarcolemmal Na⁺-Ca²⁺ exchanger (NCX), in contrast, is upregulated.^6–9 Increased biomechanical load and neuroendocrine stimulation have been implicated as potential triggers of these changes, but the relative importance of these stimuli as regulators of gene expression remains to be elucidated for each individual gene.

In the present study, we used the monocrotaline (MCT) model of right ventricular (RV) hypertrophy in the rat to address this issue. A single injection of the plant alkaloid MCT causes obliterating vasculitis of lung arterioles,¹⁰ leading to pulmonary hypertension. The chronically elevated RV afterload causes myocardial hypertrophy, and some of the animals develop HF.¹¹ Hemodynamically, this animal model is characterized by unaltered mean arterial blood pressure, indicating normal left ventricular (LV) function, in the presence of enhanced RV systolic pressure.¹¹ Because the LV is normally loaded, changes in gene expression that occur in RV but not LV myocardium are assumed to be induced by biomechanical load. In contrast, both ventricles are exposed to enhanced neuroendocrine stimulation during the final transition to HF. Thus, expression changes of genes primarily regulated by neuroendocrine mechanisms are expected to occur in both ventricles to a similar degree. Using this approach, Leineweber et al¹² demonstrated that increased load in the absence of neuroendocrine activation is not sufficient to induce the ß₁-adrenergic receptor (ß₁-AR) downregulation typically observed in HF but that exposure to these two combined stimuli is necessary to downregulate ß₁-ARs.

In the present study, we report that enhanced biomechanical load is necessary for the downregulation of SERCA2, phospholamban (PLN), and the ryanodine receptor (RyR2) as well as for the upregulation of ß-MHC, whereas NCX does not appear to be load-dependently expressed. The observed changes in gene expression levels are shown to be functionally relevant. A preliminary report has recently appeared.¹³

	Materials and Methods

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Animal Model
Six-week-old male Wistar rats¹² received a single subcutaneous injection of MCT (MCT group, 50 mg/kg body wt) or an equal volume of solvent (control group, 1 mL/kg body wt). With an unrestricted food supply, MCT animals consume less than control animals and gain significantly less weight. To prevent MCT-induced alterations of gene expression and contractile function from being modulated by differences in the nutritional state between groups, MCT rats had an unlimited food supply, whereas the amount of food given to control animals was restricted to the quantity consumed by MCT rats on the previous day.^12,14 All animal procedures were approved by the government committee for animal studies and carried out according to German laws regarding the care and use of laboratory animals.

Rat Intact Muscle Strip Preparation
Rats were euthanized on days 20 to 24 after MCT injection by halothane insufflation, and hearts were rapidly excised and retrogradely perfused with a modified Krebs-Henseleit buffer solution containing (in mmol/L) Na⁺ 140.5, K⁺ 5.1, Mg²⁺ 1.2, Ca²⁺ 0.25, Cl^- 124.9, SO₄^2- 1.2, PO₄^3- 2.0, HCO₃^- 20, glucose 10, and butanedione monoxime 20, equilibrated with carbogen (95% O₂/5% CO₂), pH 7.4. Intact trabeculae or papillary muscles were isolated from the RV or LV wall and mounted isometrically in a superfusion bath between a force transducer (Scientific Instruments) and a hook connected to a micromanipulator for length adjustment. Muscle diameters were similar in all groups directly compared with each other (widthxthickness, in µm): for intact preparations, MCT RVs 305±29x224±17, control RVs 323±38x200±18, MCT LVs 200±29x175±20, and control LVs 238±34x213±38; for skinned fibers (circular cross sections), MCT RVs 159±7 and control RVs 171±12. This excludes the possibility that differences in muscle strip geometry had an impact on contractile function. Preparations were superfused with butanedione monoxime-free Krebs-Henseleit solution, and contractions were elicited using electrical field stimulation (baseline 2 Hz, amplitude 3 to 5 V; stimulator, Scientific Instruments). Intact muscle experiments were carried out at 37°C and 1.25 mmol/L [Ca²⁺]_o.

Characterization of Contractile Phenotype
Several functional tests were performed to assess muscle strip contractility. The force-frequency relationship (FFR) was examined at a range of 1 to 7 Hz. Postrest behavior was examined using rest intervals of 1 to 60 seconds. The tension developed on the first twitch after rest was divided by the mean developed tension of the last 10 beats before rest, whereby values >1 indicate rest potentiation, and values <1 indicate rest decay. The response to ß-AR stimulation was tested using (+/-)-isoproterenol (Sigma), taking into consideration that only the (-)-enantiomer is pharmacologically active. A more detailed description of functional tests is provided in an expanded Materials and Methods section in the online data supplement (available at http://www.circresaha.org).

Myofilament Ca²⁺ Responsiveness
Thin RV trabeculae were permeabilized overnight (minimum 10 hours) at 4°C in relaxation solution containing 1% (vol/vol) Triton X-100. Smaller bundles were dissected from these trabeculae, mounted isometrically using T clips, and stretched to the length at which passive tension just began to increase. This corresponded to a sarcomere length of 1.9 µm (laser light diffraction), without a difference between groups. Measurements were carried out at 15°C. The compositions of relaxation and activation solution are provided in an expanded Materials and Methods section in the online data supplement. Intermediate [Ca²⁺] levels were obtained by mixing appropriate amounts of relaxation and activation solutions. The free [Ca²⁺] was calculated by the computer program WinMAXC¹⁵ and using the stability constants provided by Martell and Smith.¹⁶

Plasma NA Levels
Blood drawn from the ophthalmic venous plexus of anesthetized rats before heart excision was collected in a potassium-EDTA S-Monovette (Sarstedt), and glutathione was added at a final concentration of 1 µmol/mL plasma. Samples were centrifuged at 600g for 5 minutes at 4°C, and plasma was removed, snap-frozen in liquid nitrogen, and stored at -80°C. Plasma noradrenaline (NA) was assessed by high-pressure liquid chromatography with fluorometric detection (for details, see Schäfers et al¹⁷).

Protein Expression
RV and LV MCT and control myocardium was snap-frozen in liquid nitrogen immediately after dissection of the trabeculae for force measurements and stored at -80°C. Samples were thawed on ice in 50 µL of homogenization buffer (see online data supplement for details), homogenized, and sonicated at 4°C. Protein concentrations of the suspensions were determined according to the method of Lowry et al.¹⁸ Samples of 40 µg were denatured in electrophoresis buffer (see online data supplement for details) at 95°C and subjected to SDS-PAGE. MHC isoform expression was analyzed by densitometry on a Coomassie-stained 5% SDS-acrylamide gel. Initial current was set at 15 mA and then increased to 25 mA after the bromophenol blue front line reached the separating gel. Western blotting was carried out according to standard protocols, using antibodies against calsequestrin (CS, polyclonal, Affinity Bioreagents), SERCA2a (monoclonal, Affinity Bioreagents), NCX1 (monoclonal, Santa Cruz Biotech), PLN (monoclonal, Upstate Biotechnology), and troponin T (TnT, monoclonal, Sigma). CS served as an internal standard to normalize protein levels.

Quantification of mRNA Expression
DNA-free total RNA was extracted using a Qiagen RNeasy kit and an RNase-Free DNase Set. First-strand cDNA synthesis was performed with the reverse transcriptase SuperScript II and random primers according to the supplier’s instructions (Invitrogen). Polymerase chain reaction (PCR) was performed with a real-time PCR LightCycler (Roche) in a final volume of 20 µL in glass capillaries (see online data supplement for details). After initial denaturation at 95°C for 30 seconds, the samples underwent 45 cycles of 94°C for 0 seconds (recooling immediately after peak), 60°C for 5 seconds, and 72°C for 10 seconds. Emission at 520 nm was measured every cycle at 87°C for SERCA2a, at 83°C for CS and PLN, at 82°C for NCX, and at 85°C for RyR. For details on the primer pairs used, see the online data supplement.

Data Analysis and Statistics
Force was converted to tension by normalizing to the cross-sectional area of each preparation. To characterize the tension-[Ca²⁺] relationship in skinned fiber preparations, tension data and the respective free [Ca²⁺] were fitted to a Hill equation as follows: F_X-F_min=(F_max-F_min)x[Ca_X]ⁿ/[(Ca₅₀)ⁿ+[Ca_X]ⁿ], where F_X is actual tension, F_min is resting tension, F_max is maximal Ca²⁺-activated tension, [Ca_X] is the actual [Ca²⁺], Ca₅₀ is the [Ca²⁺] required for the development of half-maximal tension, and n is the Hill coefficient. Data are expressed as mean±SEM. Statistical analysis used a repeated-measures ANOVA, followed by the Bonferroni correction for multiple comparisons, or (for gene-expression analyses) an unpaired Student t test. Two-sided values of P<0.05 were considered statistically significant.

	Results

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Animal Model
MCT rats were euthanized on days 20 to 24 after injection of MCT, when they showed lethargy, accelerated breathing, and ruffled fur. At that time, neither ascites nor pleural effusions were observed in any of the MCT animals. Control rats injected with saline solution on the same day were euthanized accordingly. Although we adjusted the intake of food by the control group to the spontaneously reduced intake by MCT rats, body weight was slightly, albeit significantly, reduced in the MCT group (231.5±1.8 g [MCT] versus 247.8±2.7 g [control], P<0.001; n=10 each). Organ weight data are summarized in Figure 1. Although the LV weight/body weight ratio was similar (Figure 1B), the RV weight/body weight ratio had increased from 0.7±0.035 mg/g in the control group to 1.47±0.06 mg/g in the MCT group (Figure 1A, P<0.001), indicating that MCT treatment caused pronounced myocardial hypertrophy, which was restricted to the RV wall. The lung weight/body weight ratio likewise was more than doubled (Figure 1C, P<0.001) in the MCT group. These organ weight data are fully compliant with earlier reports.^11,12 We assessed plasma NA levels in a separate set of animals on days 21 and 24 (n=9) in MCT and control rats (Figure 1D). Plasma NA was 209±8 pg/mL in the control group and 443±44 pg/mL in the MCT group, indicating that substantial generalized neuroendocrine stimulation was present in the MCT group at the time of euthanasia for dissection of the intact muscle preparations.

View larger version (27K): [in this window] [in a new window]   Figure 1. Organ weights normalized for body weight (mg/g) and plasma NA levels (pg/mL). A, RV weight/body weight is significantly enhanced in MCT rats (*P<0.001, n=10/10). B, LV weight/body weight is unaltered (n=10/10). C, Lung weight/body weight is significantly increased in MCT rats (*P<0.001, n=10/10). D, Plasma NA level is significantly enhanced in MCT rats (*P<0.001, n=9/9).

Contractile Properties of Intact Muscle Strips
We examined isometrically contracting intact muscle strips isolated from the RVs and LVs of both control and MCT rats. Figure 2 summarizes FFR and postrest behavior data. Between 2 and 7 Hz, there was a steady increase in developed tension in control RV preparations (n=8), indicating a positive FFR in this group (Figure 2A). The same test carried out in MCT RV preparations (n=7) revealed two striking differences (Figure 2A). First, developed tension at the baseline stimulation rate of 2 Hz was significantly enhanced in the MCT group, from 3.9±0.9 to 10.6±2.0 mN/mm² (P=0.017). Second, developed tension monotonously decreased with an increasing stimulation rate, indicating a negative FFR. The relationships exhibited a highly significant difference between groups (P<0.001). FFRs were superimposable in control LV preparations (n=6) and control RV preparations (Figures 2A and 2B). In contrast, MCT LV preparations (n=7) were largely different from their RV counterparts: Baseline developed tension was similar to that of control LVs; also, the overall FFR was positive in LV preparations from MCT rats (Figure 2B). Multiple linear regression analysis revealed no significant difference between MCT and control LVs.

View larger version (34K): [in this window] [in a new window]   Figure 2. Contractile function in intact isolated muscle strips. Panels are as follows: A and B, FFR; C and D, postrest behavior; A and C, RVs; and B and D, LVs. A, Control RV preparations show positive FFR, whereas the relationship is negative (#P<0.001) and baseline force development is enhanced (*P=0.017) in MCT RVs (n=8 MCT, n=7 control). B, There were no significant FFR differences in LV preparations (n=7 MCT, n=6 control). C, Substantial postrest potentiation in control RV trabeculae (n=9) is shown, but there was significantly blunted response in MCT RV preparations (P<0.001, n=9). D, There was no significant difference in postrest behavior in LV preparations (n=5 MCT, n=5 control).

During the examination of postrest behavior, control RV preparations (n=8) showed strong rest potentiation, with a maximum postrest twitch amplitude (3.7±0.5-fold potentiation) found after an interval of 30 seconds (Figure 2C). The postrest behavior of control LV preparations (n=5) was similar, with the exception that a plateau had not yet been reached after 60 seconds of rest (Figure 2D). MCT RV preparations also showed rest potentiation (n=9), which was, however, significantly attenuated and reached a maximum of only 1.8±0.2-fold potentiation (Figure 2C, P=0.006 compared with control RV preparations), whereas MCT LV preparations exhibited a postrest behavior similar to that of control LV preparations (Figure 2D). These data indicate that the ability of the sarcoplasmic reticulum (SR) to store Ca²⁺ during extended periods of rest is impaired in MCT RV myocardium, whereas no evidence for SR dysfunction is found in MCT LV myocardium.

We tested the effects of ß-AR stimulation by characterizing the concentration-response relationship for isoproterenol. Again, under baseline conditions in the absence of inotropic intervention, tension development was significantly enhanced by 115% in MCT RV preparations (n=7) compared with control RV preparations (n=9, P=0.012; Figure 3A). At a saturating isoproterenol concentration of 1 µmol/L, developed tension was similar in both groups, indicating reduced contractile reserve in the MCT RV group. In LV preparations from control and MCT rats, neither baseline developed tension nor contractile reserve after ß-adrenergic stimulation was significantly different (Figure 3B).

View larger version (32K): [in this window] [in a new window]   Figure 3. Isoproterenol concentration-response relationship (A and B) and RT50 as a parameter of diastolic function (C and D). Panels A and C indicate RV data; panels B and D, LV data. A, Tension development at low isoproterenol concentration is enhanced in MCT RVs (#P<0.05, *P<0.01; n=7 MCT, n=9 control). There was no significant difference between groups at saturating isoproterenol concentrations. B, LV trabeculae from MCT (n=7) and control (n=6) hearts behave similarly when stimulated with isoproterenol. C, RT50 is significantly enhanced in MCT (n=8) compared with control (n=7) RV trabeculae, indicating impaired relaxation (P<0.001). D, There was no significant difference in RT50 between MCT (n=7) and control (n=6) LV trabeculae.

To assess diastolic function, we determined during the force-frequency test the time span between peak isometric twitch tension and the moment when developed tension reached 50% of the peak value during relaxation (RT₅₀). In MCT RVs, RT₅₀ was significantly higher than in control preparations over the entire range of stimulation rates (Figure 3C, P<0.001), indicating diastolic dysfunction. In contrast, MCT LV preparations exhibited RT₅₀ values similar to those of control LV preparations. Thus, the impairment of relaxation in MCT rats was restricted to the RV.

Myofilament Ca²⁺ Responsiveness
The results obtained on intact multicellular muscle strips indicate that Ca²⁺ handling is dysfunctional in MCT RV myocardium. Nevertheless, baseline tension development was enhanced in MCT RV preparations. A potential cause for this apparent discrepancy would be an increase in myofilament Ca²⁺ responsiveness, thereby enabling the myocardium to produce adequate force despite reduced activating [Ca²⁺]_i levels. Therefore, we compared the tension-[Ca²⁺] relationships of MCT and control RV myocardium in detergent-skinned trabeculae (Figures 4A and 4B). Maximum Ca²⁺-activated tension was significantly enhanced by 64% in MCT RVs (n=8) compared with control RVs (n=13, P=0.013). Figure 4B presents the same data normalized for resting and maximum Ca²⁺-activated tension. It becomes obvious that there is a slight, but significant, leftward shift of the tension-[Ca²⁺] curve of MCT RV fibers (pCa 0.08, P=0.014), indicating Ca²⁺ sensitization, compared with control RV fibers.

View larger version (15K): [in this window] [in a new window]   Figure 4. Myofilament Ca2+ responsiveness. A, Tension-[Ca2+] relationship. Maximum Ca2+-activated tension was significantly enhanced in skinned fibers obtained from MCT RVs (n=8) compared with control RVs (n=13, *P=0.013). B, Same data set normalized to maximum and minimum tension. The midpoint of the tension-[Ca2+] relationship in MCT preparations is shifted toward lower [Ca2+] by 0.08 pCa units (#P=0.014).

Expression of Ca²⁺-Regulating Proteins
We quantitatively analyzed mRNA and protein expression levels of several target genes involved in the maintenance of Ca²⁺ homeostasis in RV and LV myocardium from control and MCT rats. In MCT RVs compared with control RVs, the SERCA2a/CS mRNA ratio was reduced by 36% (n=8 [MCT] versus n=7 [control], P=0.001), whereas in MCT LVs (n=8), the SERCA2a/CS mRNA ratio was similar to that of the respective control LVs (n=7, Figure 5A). Also, at the protein level, the SERCA2a/CS ratio in MCT RVs (n=6) compared with control RVs (n=6) was significantly reduced (-17%, P=0.016), whereas the SERCA2a/CS ratio was unchanged in the LVs (Figure 5B). A representative immunoblot from RV myocardium is shown in Figure 5C. We next examined RyR mRNA expression and likewise found that the RyR/CS mRNA ratio was reduced in MCT RVs (n=8) compared with control RVs (-28%, n=7; P=0.01), whereas in the LV, the RyR/CS mRNA ratio was not different between groups (n=8 and n=7, respectively; Figure 6A). Also, the PLN/CS mRNA ratio had decreased in MCT RV myocardium (-27%; n=8 and n=7, respectively; P=0.049), whereas in the LV, no significant change in the PLN/CS mRNA ratio was observed (n=8 and n=7, respectively; Figure 6B).

View larger version (19K): [in this window] [in a new window]   Figure 5. Expression levels of SERCA2a, normalized to CS. A, SERCA2a mRNA expression is significantly downregulated in MCT compared with control RV myocardium (*P=0.001; n=8 MCT, n=7 control). There was no significant change in SERCA2a mRNA expression in MCT LV preparations (n=8 MCT, n=7 control). B, There was significant downregulation of SERCA2a protein in MCT RV myocardium (#P=0.016, n=6/6). There was no difference in SERCA2a protein in LV myocardium (n=6/6). C, Representative Western blot from MCT and control (C) RVs shows reduced SERCA2a-immunoreactive bands in MCT RVs.

View larger version (31K): [in this window] [in a new window]   Figure 6. Expression levels of RyR, PLN, and NCX. A, RyR/CS mRNA ratio was significantly reduced in MCT (n=8) compared with control (n=7) RVs (*P<0.05). No change was observed in LV myocardium (n=8/7). B, PLN/CS mRNA was significantly downregulated in MCT (n=8) compared with control (n=7) RVs (*P<0.05), whereas no significant change was observed in the LVs (n=8/7). C, NCX/CS mRNA expression in both ventricles was unaltered in MCT myocardium (n=8 MCT RVs, n=7 control RVs, n=8 MCT LVs, and n=7 control LVs). D, Also, at the protein level, NCX expression was not significantly altered in MCT RVs (n=8 MCT RVs, n=7 control RVs) or LVs (n=8 MCT LVs, n=7 control LVs).

We investigated whether NCX mRNA expression is altered in the hearts of MCT rats. Neither in the RV nor in the LV was there a significant change in NCX/CS mRNA expression (n=8 and n=7, respectively; Figure 6C); also, at the protein level, no significant difference was observed between groups (n=6 each, Figure 6D).

Myofilament Protein Expression Analysis
Because we had demonstrated an enhanced myofilament Ca²⁺ responsiveness in MCT RV myocardium, we examined whether quantitative changes do exist at the myofilament protein level. Figure 7A shows a representative section of a 5% SDS-PAGE with high resolution of higher molecular weight proteins. A faint band running at a slightly higher rate than -MHC was consistently observed in every MCT RV myocardial sample examined, whereas a corresponding band was barely detectable in any of the control RV samples. This band represents ß-MHC. ß-MHC expression constituted a mean fraction of 18% (range 10% to 23%) of the total MHC in MCT RV myocardium (n=7), whereas in control RV myocardium, only 6% of total MHC protein (range 2.5% to 10%, n=7) was present in its ß isoform (Figure 7B, P<0.01). In LV myocardium of both control and MCT rats, no ß-MHC expression was observed (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 7. Myofilament proteins. A, Coomassie-stained SDS-PAGE gel showing MHC isoform composition of MCT and control RVs. B, Fraction of total MHC present as ß-isoform in control (con) and MCT RV myocardium (n=7/7, *P<0.01). C, Representative Western blot with antibodies against cardiac TnT. A prominent lower molecular mass band appears exclusively in MCT RV and is absent in con RVs and MCT or con LVs.

In Western blots using antibodies directed against cardiac TnT, in addition to the parent protein, a band with an approximate molecular mass of 27 kDa was labeled exclusively in RV samples of MCT rats, whereas this band was undetectable in control RV as well as control and MCT LV myocardium (Figure 7C). No differences between groups were observed in Western blots using anti-troponin I antibodies.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In HF, the myocardium is subjected to a combination of increased biomechanical load and enhanced neuroendocrine stimulation. The relative contributions of load and humoral factors in mediating the gene expression changes typically associated with this condition are unclear. Therefore, we sought to resolve whether or not enhanced biomechanical load is a necessary prerequisite for this molecular remodeling. In the present study, we report that in rats with MCT-induced RV hypertrophy, RV myocardium exhibits a gene expression pattern distinct from that of LV myocardium despite plasma NA levels that were elevated to about the same extent as observed in human patients with New York Heart Association class II-III¹⁹ HF and indicating generalized neuroendocrine stimulation and affecting both ventricles equally. Specifically, we observed significant parallel downregulation of several genes involved in the maintenance of Ca²⁺ homeostasis, SERCA2a, PLN, and RyR in MCT RVs, whereas no alterations in the expression levels of these genes were found in MCT LV myocardium compared with myocardium of saline-treated control rats. This suggests that the enhanced mechanical load on the RV due to pulmonary hypertension is necessary to induce these changes. NCX, on the other hand, was not differentially expressed, indicating that the regulation of NCX expression in this experimental model is not sensitive to mechanical load.

We¹² have recently shown that both the activity and density of the NA transporter are reduced in hypertrophied MCT RV myocardium. It might be argued that this, in principle, could cause local RV synaptic cleft NA levels to increase to levels higher than in the LV despite identical plasma NA levels. This, in turn, would imply that it may not be correct to assume an increased biomechanical load to be the sole factor altered within the RVs of MCT rats. However, the transition to HF in these animals is characterized by a reduction in ß₁-AR expression,¹² such that local downregulation of the NA transporter will to some extent be offset by a reduced sensitivity of the ß₁-adrenergic signal transduction cascade in RV myocardium of MCT rats. Consistent with this notion, hypertrophied hearts of MCT rats exhibit a reduction in basal and stimulated adenylyl cyclase activity that is more pronounced in the RV than the LV,¹¹ suggesting that even slightly increased local levels of NA are not likely to result in enhanced stimulation of the ß₁-AR cascade. Thus, although local effects of increased NA synaptic cleft concentrations cannot be completely excluded, the difference in biomechanical load in this animal model remains the primary factor expected to alter gene expression.

What are the functional consequences of these expression changes? We did not directly assess the Ca²⁺ uptake function of the SR. However, it can be assumed that decreased abundance of SERCA2a, which was confirmed at the protein level, will (1) slow the decline in [Ca²⁺]_i (and thus, in force) during relaxation, (2) cause a net loss of Ca²⁺ from the cell due to alternative transsarcolemmal elimination via NCX, and (3) limit the maximum inotropic response to ß-AR stimulation, which is mainly mediated by enhanced Ca²⁺ uptake into the SR after disinhibition of SERCA2a activity after PLN phosphorylation. Consistent with these assumptions, we demonstrated in isometrically contracting muscle strips isolated from MCT RV (1) increased relaxation time, (2) negative FFR and blunted postrest potentiation, and (3) decreased contractile reserve after treatment with isoproterenol, whereas none of these functional changes were observed in MCT LV preparations. It should be noted that MCT RV myocardium exhibited pronounced fibrosis. However, fibrosis will affect contractile function primarily by reducing the functional cross-sectional area, thereby diminishing the level of developed tension, without altering the relative magnitude of changes induced by interventions such as altered stimulation rate or ß-AR stimulation. Therefore, the observed alterations in contractility reflect intrinsic myocardial properties, and we feel confident to conclude that the biomechanical load-induced downregulation of SERCA2a expression in this model is functionally relevant. This is also supported by a finding²⁰ reported previously and indicating that in myocardium from MCT rats, the diastolic decline in [Ca²⁺]_i is prolonged. Potential consequences of RyR downregulation are more difficult to predict, especially because it has recently become obvious that in HF, RyR function is modulated by posttranslational modification, eg, phosphorylation.²¹ A decrease in RyR density in the junctional SR could reduce the gain of Ca²⁺-induced Ca²⁺ release, but this was not tested in the present study. A downregulation of PLN may be considered as a compensatory response that in the presence of reduced SERCA2a levels will serve to maintain the SERCA2a/PLN stoichiometry.

An interesting feature of the contractile phenotype of intact MCT RV trabeculae was the considerably enhanced baseline force development (1.25 mmol/L Ca²⁺, 2-Hz stimulation, and absence of inotropic intervention), which seemingly contradicts the defective Ca²⁺ cycling. Our observation of a substantially increased maximum Ca²⁺-activated tension and slight, but significant, Ca²⁺ sensitization in permeabilized preparations provides a reasonable explanation for this phenomenon, inasmuch as enhanced Ca²⁺ responsiveness allows for the maintenance of adequate force development despite reduced levels of [Ca²⁺]_i. Interestingly, other studies^22,23 investigating the effect of RV pressure overload on myofilament function did not observe enhanced Ca²⁺ responsiveness, in disagreement with our findings. However, in one of the studies using pulmonary artery banding in ferrets,²² the degree of hypertrophy was rather mild, raising the possibility that the biomechanical stimulus was not sufficient to trigger myofilament alterations. On the other hand, the other study, which used pulmonary artery banding in rats,²³ examined myofilament phenotype after long-term pressure overload with overt clinical signs of HF. This makes direct comparison with the present study difficult. We cannot exclude the possibility that in our model of MCT-induced RV hypertrophy, if animals survive for several months to reach a state of end-stage HF, a different myofilament phenotype might also arise.

The MHC isoform composition has been shown to affect the Ca²⁺ responsiveness of the myofilaments.^24,25 Therefore, we probed MCT myocardium for changes in MHC isoform composition and, consistent with findings in other myocardial hypertrophy models in the rat,^26,27 observed significant upregulation of ß-MHC at the protein level. It has been proposed that expression of the slowly cycling ß-MHC by increasing the duty cycle would increase the force-time integral for a given Ca²⁺ saturation of troponin C, thereby causing Ca²⁺ sensitization of the myofilaments,²⁸ and a leftward shift of the pCa-tension relationship of the same order of magnitude observed in the present study has been reported in hypothyroid rats expressing high levels of ß-MHC.²⁴ However, this concept has recently been challenged,²⁴ and an increase in maximum Ca²⁺-activated tension, as reported in the present study, has not been found to be associated with ß-MHC expression.^24,29 Thus, additional alterations of myofilament composition are likely to exist in MCT RV myocardium. Because an isoform shift of TnT has been reported to occur in failing human hearts,³⁰ we examined whether similar changes can be found in MCT-induced RV hypertrophy. Only 1 TnT band could be detected in control RV and control and MCT LV myocardial homogenates, whereas an additional prominent band with a molecular mass of 27 kDa was consistently observed in MCT RV samples. We have recently reported that treatment with reactive oxygen species induces specific proteolysis of TnT in rabbit myocardium.³¹ Whether this band represents a different isoform or a proteolytic fragment is currently under investigation. The potential functional significance of this change, especially with respect to the observed changes in myofilament Ca²⁺ responsiveness, remains to be elucidated.

In summary, we find downregulated SERCA2a, RyR, and PLN expression as well as upregulation of ß-MHC in pressure overload-induced RV hypertrophy in the rat. Neuroendocrine activation alone is not sufficient; enhanced biomechanical load is necessary to induce these changes. MCT-induced pulmonary hypertension along with consecutive RV hypertrophy and failure is an experimental tool ideally suited to investigate the relative roles that mechanical and humoral stimuli play in the regulation of gene expression in the myocardium.

	Acknowledgments

 
This study was supported by Sonderforschungsbereich Transregio 2 of the German Research Foundation (DFG). We are grateful to Elisabeth Barski for expert technical assistance.

	Footnotes

 
Original received March 4, 2003; revision received June 25, 2003; accepted June 25, 2003.

	References

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