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Department of Physiology II, Nara Medical University, Kashihara, Nara 634-8521, Japan
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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1-blocker, metoprolol (24 mg·kg–1·day–1), blocked the formation of cardiac hypertrophy and thus PVAmLVV did not decrease. The reversible changes in PVAmLVV reflect precisely the changes in LV work capability in isoproterenol-induced hypertrophied rat hearts mediated by 1-receptors. These results indicate that the present approach may be an appropriate strategy for evaluating the effects of antihypertrophic and antifibrotic modalities.
left ventricular volumetry; conductance catheter; collagen; systolic pressure-volume area
It is known that chronic infusions of an 
,-stimulant, norepinephrine, and a -stimulant, isoproterenol (Iso), induce cardiac hypertrophy accompanied with enhanced fibrosis among cardiac interstitial cells (6, 22, 31). Although chronic infusion of Iso in the rat results in marked cardiac hypertrophy, the various mechanisms for this remodeling are not fully understood (1, 16, 18, 19, 25, 26). Furthermore, we have previously validated the accuracy of the measurement of LVV in in situ normal rat hearts by comparing the stroke volume (SV) measured with a conductance catheter (SVcc) with that measured by electromagnetic flowmetry (SVem) (11). However, we have not evaluated yet the accuracy of this LV volumetry in hypertrophic hearts.
The aims of the present study were at first to induce hypertrophy by stimulating the 1-receptor and to validate the accuracy of LV volumetry in the hypertrophied heart with the conductance catheter system. We compared SVcc with SVem and confirmed the accuracy of LV volumetry. Finally, we recorded a series of P-V loops, obtained ESPVR by curve fitting, and calculated PVAmLVV. We found that the changes in PVAmLVV sensitively reflected the formation and reversibility of Iso-induced hypertrophy mediated by 1-receptors. The present approach could be an appropriate strategy for evaluating the effects of antihypertrophic and antifibrotic agents or genetic targeting.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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1-blocker (31). Osmotic minipumps were removed from the neck 3 days after the implantation under pentobarbital anesthesia. One hour after the removal of the minipump in the Sa, Iso 1.2, Iso 2.4, and Meto + Iso 1.2 groups and 2 days after the removal of the minipump in the Iso 1.2 group [Iso(–), n = 7], LV function was evaluated (see protocol shown in Fig. 1A).
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-chloralose (60 mg/kg ip). The trachea was intubated, and the rat was ventilated with room air. The chest was opened, and the conductance catheter (1.5 Fr, S-I Medicotech; Osaka, Japan) was introduced into the LV through an apical stab with a purse-string suture (13, 15). The positioning was critical to obtain reliable LVV data. The ascending aorta was dissected free from the pulmonary artery. An electromangnetic flow probe (2.5 mm inner diameter, Nihon-Kohden; Tokyo, Japan) was placed around the ascending aorta. LV conductance volume and electromagnetic aortic flow data were simultaneously obtained by varying the preload with gradual inferior vena cava occlusion. We compared SVem against SVcc. SVem was calculated by beat-to-beat integration of the aortic flow signal, and SVcc was obtained as the minimal conductance volume subtracted from the maximal conductance volume in each beat.
Histological analysis. The LV was fixed with 3.7% paraformaldehyde in PBS, embedded in paraffin, and cut into 6-µm slices, which were stained with hematoxylin-eosin for morphological analysis or with Masson's trichrome staining for the detection of fibrosis. For morphometrical analysis, photographs of six LV sections from the Sa (n = 3), Iso 1.2 (n = 3), Iso 2.4 (n = 3), Meto + Iso 1.2 (n = 3), and Iso(–) (n = 3) groups were taken at x400 magnification, and cross-sectional images of cardiac myocytes were digitized by digital micorsope (FUJIX Digital Camera HC-2500). LV cardiac cell size and collagen volume fraction were determined by counting computerized pixels in digital image of myocyte and collagen area stained by Masson's trichrome stain.
Polyacrylamide gel electrophoresis and Western blots for sarco(endo)plasmic reticulum Ca2+-ATPase. Membrane proteins from the LV myocardium of each heart were isolated as described previously (20, 30). The frozen hearts were homogenized and centrifuged at 1,000 g for 10 min. The supernatants were centrifuged at 100,000 g for 60 min at 4°C. The 100,000-g pellets were cellular membrane fractions and used for immunoblotting of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2.
Membrane proteins (20 µg/lane) were separated on SDS-polyacrylamide gels (10% for SERCA2) in a minigel apparatus (Mini-PROTEAN II, Bio-Rad Laboratories) and transferred to polyvinylidene difluoride membranes. The membranes were blocked (4% Block Ace, Dainippon Pharmaceutical; Osaka, Japan) and then incubated with anti-SERCA2 antibody (1:1,000 dilution, Affinity Bio Reagents). The antigens were detected by the enhanced chemiluminescence method (ECL Western blotting detection kit, Amersham) with peroxidase-linked anti-mouse IgG (1:1,000 dilution). After immunoblotting, the film was scanned with a scanner, and the intensity of the bands was calculated by NIH Image analysis.
Measurements of LV volume and pressure.
One hour or 2 days after the removal of the minipump, we evaluated LV function in hypertrophied rat hearts by simultaneous measurements of LVV and LV pressure (LVP). The rat was anesthetized with ethylcarbamate (0.7 g/kg ip) and -chloralose (60 mg/kg ip). The trachea was intubated, and rat was ventilated with room air. The chest was opened, and a conductance catheter (1.5 Fr) was introduced into the LV through an apical stab, as described above. A 3.0-Fr catheter-tip micromanometer was also inserted through the apex into the LV to obtain reliable LVP data.
The principle of conductance catheter method of measuring LVV has been described in detail (3, 4, 11). When hemodynamics was stable, a series of LV P-V loops was obtained during increasing afterload by a gradual occlusion of the ascending aorta. The occlusion was performed for 1–2 s until end-diastolic volume (EDV) slightly increased. Occlusion was limited so as not to evoke any arrhythmia. This intervention was repeated six times at 10-min intervals. The respirator was stopped during data acquisition to avoid respiratory fluctuation influences on cardiac signals.
In the final part of each experiment, parallel conductance and thus constant offset volume were measured by injecting hypertonic saline (10% NaCl solution, 0.025 ml) into the pulmonary artery to change transiently the resistivity of the blood in the LV (13, 15). The calculated constant offset volume was subtracted from the measured LV conductance volume to obtain LV absolute blood volume, i.e., absolute LVV. LVP and the three individual segmental conductance volume signals were digitized and stored at 12-bit accuracy at a sampling frequency of 500 Hz for later analyses. At the end of each experiment, a lethal dose of pentobarbital sodium was injected into the rat. The LV including the interventricular septum, and the right ventricle (RV) was excised and weighed, respectively.
Data analysis. In the in situ rat LV, a curvilinear ESPVR is obtained by drawing an upper enveloping curve on a series of P-V loops in a similar manner to previously reported methods (13, 15). The LV end-systolic P-V data on the upper left shoulder of all P-V loops were plotted and fitted by the method of least squares using the following equation: LVP = A {1 – exp[–B(LVV – V0)]}, where A and B are fitted parameters and V0 is systolic unstressed volume. We obtained the best-fit ESPVR curve in each heart in the Sa, Iso 1.2, Iso 2.4, Meto + Iso 1.2, and Iso(–) groups. V0 has been previously measured in postmortem isolated rat normal LVs. The V0 values are determined to be 0.02 ± 0.005 ml/g (n = 7)(27). In the present study, the mean V0 value of hearts in the Sa group obtained as volume intercepts of the best-fit ESPVRs was 0.025 ± 0.014 ml/g (n = 7)(see Table 2). Therefore, we judged the V0 values obtained by curve fit are reliable.
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The PVA as a function of LVV was obtained by integrating the above exponential function from the extrapolated V0 along the volume axis: PVA = A(LVV – V0) – A{1 – exp[–B(LVV – V0)]}/B.
We had proposed that, in in situ hearts, PVA at an appropriate LVV on the curvilinear PVA-volume relationship is valuable to evaluate LV mechanoenergetics (13, 15). In the present study, we calculated mLVV that was the value of [V0 + (maximum ESV – minimum ESV) on the ESPVR x 1/2] from each P-V loop. Each example of end-systolic pressure (ESP) and PVA at midrange LVV (ESPmLVV and PVAmLVV) is shown in Fig. 1B, bottom. EDPVR is very close to the volume axis, as shown in Fig. 4, and thus the effect of excluding the EDPVR measure would be small. Effective arterial elastance (Ea) is defined as the ratio of ESPESV to SV of the LV under stable hemodynamics. SV was obtained by the formula (EDV – ESV) (Fig. 1B, top). The ejection fraction (EF) was obtained by the formula [SV/EDV–V0)].
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Mean mLVV, ESPmLVV, and PVAmLVV. Mean mLVVs in the Iso 1.2 (45 ± 10% of Sa) and Iso 2.4 groups (42 ± 11% of Sa) were significantly smaller than that in the Sa group (0.123 ± 0.0322 ml/g, P < 0.05), but there were no significant differences between the Iso 1.2 and Iso 2.4 groups (Fig. 5A). In contrast, mean ESPmLVV was unchanged in all groups (Fig. 5B). Mean PVAmLVV in the Iso 1.2 (51 ± 16% of Sa) and Iso 2.4 groups (47 ± 14% of Sa) was significantly smaller than that in the Sa group (7.16 ± 2.72 mmHg·ml·beat–1·g–1, P < 0.05; Fig. 5C), indicating that the LV systolic function, i.e., mechanical work capability, was impaired in hypertrophied rat hearts. Indeed, cardiac output (CO) in the Iso 1.2 (62 ± 14% of Sa) and Iso 2.4 groups (63 ± 9.8% of Sa) was also significantly smaller than that in the Sa group (37.3 ± 5.7 ml/min, P < 0.05; Fig. 5D). None of mean mLVV, PVAmLVV, and CO values differed between the Iso 1.2 and Iso 2.4 groups. Mean PVAmLVV and CO in the Iso(–) group showed the almost the same levels as those in the Sa group, although the mean mLVV still remained significantly smaller than that in the Sa group (P < 0.05; Fig. 5A, C, and D).
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Effects of the 1-blocker Meto on Iso-induced hypertrophied hearts.
Microscopic examination identified marked inhibition of hypertrophy by Meto in the Meto+Iso 1.2 group, accompanied with inhibition of distinct fibrosis (Fig. 6, A and B). The collagen area in the Meto + Iso 1.2 group was significantly smaller than that in the Iso 1.2 group and was the almost the same level as that in the Sa group (Fig. 6C). The sizes of cardiac cells in the Meto + Iso 1.2 group (106 ± 47% of Sa, n = 90) were significantly smaller than those in the Iso 1.2 group and were the almost the same levels as those in the Sa group. The amount of expression of SERCA2a in the Meto + Iso 1.2 group was significantly larger than in the Iso 1.2 group and showed almost the same level as in the Sa group (Fig. 6D).
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None of the mean EDV, SV, and Ea values in the Iso(–) group differed from those in the Sa group, although mean ESV differed from that in the Sa group. None of the mean ESV, EDV, SV, and Ea values in the Meto + Iso 1.2 group differed from those in the Sa group, indicating changes in mean ESV, EDV, SV, and Ea in the Iso 1.2 group were blocked by simultaneous treatment with Meto.
Although the mean heart rate was significantly smaller in the Iso 1.2 and Iso 2.4 groups (P < 0.05), we have previously reported the curved ESPVR is independent of heart rate in in situ rat hearts within 250–320 beats/min (15) and in excised rat hearts within 250–300 beats/min (21).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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For the first time, we have validated the accuracy of LVV measurement by the conductance catheter method in hypertrophied rat hearts by comparing SVcc with SVem during inferior vena caval occlusion. Consequently, we obtained a high and linear correlation between SVcc and SVem in a hypertrophied rat heart from the Iso 2.4 group as well as in a heart from the Sa group. Both SVcc and SVem values pooled from individual hearts also showed high and linear correlations, although the correlation coefficient in the Iso 2.4 group was slightly lower than that in the Sa group due to the scattering of the data. Because SVem-calculated data from the electromagnetic aortic flowmetry depends on the accuracy of fit of the flow probe to the ascending aorta, larger variability of the accuracy might be a reason for this scattering. We, therefore, believe in the accuracy of the conductance catheter method even in hypertrophied in situ rat hearts during remodeling.
Although many studies on the mechanisms underlying Iso-induced cardiac hypertrophy have been reported (1, 14, 16, 18, 19, 25, 26), the conclusive mechanisms have not yet been determined.
1- and 2-adrenergic receptors (-ARs), which bind circulating catecholamines, activate adenylate cyclase by coupling through the stimulatory G protein Gs. The resulting increase in intracellular cAMP activates cAMP-dependent protein kinase (PKA). PKA has several pathways in cardiomyocytes that influence contractility in response to activated -AR signaling; these include the L-type Ca2+ channel in the sarcolemma, the ryanodine receptor (RyR2), and phospholamban (PLB) in the sarcoplasmic reticulum. Phosphorylation of PLB leads to the increase of SERCA and consequent accelerated Ca2+ accumulation in the sarcoplasmic reticulum. PKA phosphorylation of the RyR2 is markedly increased in failing human hearts (17), suggesting a link between PKA signaling and negative inotropism associated with long-term -AR signaling.
Prolonged -AR stimulation by Iso chronic infusion for 3 days may result in the uncoupling of -ARs from downstream effectors, possibly through two mechanisms proposed as in chronic heart failure: downregulation of receptor number (1) (5) and desensitization of the receptors (2) (7). However, the 1-blocker Meto completely antagonized all of collagen production, myocyte hypertrophy, and the impairment of LV systolic function and work capability. These results suggested that the 1-AR-mediated signal transduction pathway is primary responsible for all events related with cardiac hypertrophy induced by Iso infusion. Meto prevented the molecular changes related with cardiac hypertrophy by blocking the 1-AR-mediated signal transduction pathway at an early stage.
This is in agreement with a previous report by Morisco et al. (18). Iso increased LV weight-to-BW ratio and atrial natriuretic factor transcription in the adult rat in vivo, which was inhibited by a 1-antagonist but not by a 2-antagonist. These results indicate that hypertrophy is mediated by the 1-subtype (18).
Roles of the renin-angiotensin system on Iso-induced hypertrophy are controversial (16, 19). Cardiac tissue ANG II regulates myocyte growth in Iso-induced LV hypertrophy, and the reduction of ANG II partly explains the prevention of cardiac hypertrophy by the converting enzyme inhibitor (19). In contrast, other investigators asserted that neither the circulatory nor cardiac renin-angiotensin system plays a major role in the cardiac trophic responses to -AR stimulation (16).
The amount of expression of SERCA2a was dose dependently decreased in the Iso 1.2 and Iso 2.4 groups as described previously (23). The 1-antagonist Meto completely antagonized the downregulation of SERCA2a expression in hearts from the Meto + Iso 1.2 group, indicating that the amount of SERCA2a expression was decreased mediated by 1-ARs. We predicted diastolic dysfunction due to the impaired Ca2+ uptake function into the sarcoplasmic reticulum resulting from the downregulation of SERCA2a expression. The resultant upward shift of EDPVR, however, was not observed.
PVAmLVV has been proposed a good mechanoenergetic index to evaluate LV function in in situ normal rat hearts (13, 15, 27), and PVA has been shown to linearly relate to myocardial oxygen consumption per beat in in vitro rat hearts (20, 21). The significant decrease of PVAmLVV denotes the decrease of myocardial oxygen consumption including energy requirements for Ca2+ handling in excitation-contraction coupling and basal metabolism (20, 21, 28). The major component of the energy requirements for Ca2+ handling is utilized by SERCA2a (20, 21, 28), suggesting that the downregulation of SERCA2a expression may decrease the energy requirements for Ca2+ handling in the present hypertrophied rat hearts as in hypothyroid rats (20). On the other hand, myocardial oxygen consumption for basal metabolism per LV WW decreased in LV myocardial slices of the same type of hypertrophied rat hearts (29), where the collagen contents significantly increased. It seems likely that collagen does not consume any energy. Therefore, the total energy demand must decrease in the hypertrophied rats hearts. Our recent unpublished observations have noted the unchanged creatine phosphate-to-ATP ratio in LV myocardial slices of the same type of hypertrophied rat hearts (n = 6), indicating an unchanged energy balance between demand and supply. The suppression of energy production may occur responding to the decreased energy demand, resulting in an unchanged energy balance between demand and supply in the present hypertrophied rat hearts. Alternatively, the decreased energy demand may occur responding to the suppression of energy production, i.e., the downregulation of SERCA2a expression may be compensatory changes against the suppressed energy production.
The present cardiac hypertrophy is reversible, because 2 days after the removal of Iso, all changes in hypertrophy (collagen contents, SERCA2a, mLVV, PVAmLVV, and CO) returned to the almost the same level as those in the Sa group. This reversibility could give us the possibility that treatment with an appropriate agent or gene transfer might cure the cardiac hypertrophy associated with collagen production (fibrosis). For example, a histone deacetylase inhibitor has been reported to prevent hypertrophy induced by an infusion of Iso (14). The identification and further elucidation of antihypertrophic transcriptional pathways will offer novel therapeutic targets for drugs for the treatment of congestive heart failure. We suggested that this Iso-induced hypertrophied rat heart is an appropriate model for exploring novel therapeutic (antihypertrophic and antifibrotic) agents or genes.
Simultaneous measurement of LV P-V signals and thus recording P-V loops revealed that LVP parameters, such as ESPESV, were unchanged, but LVV parameters, such as ESV, EDV, SV, and mLVV, were significantly smaller in the hypertrophied heart than those in the normal heart. These volume changes were due to a decrease in EDV (
0.12 ml/g) rather than a decrease in ESV (0.04 ml/g) (see Table 2). The decreased EDV indicates that the LV lumen size is reduced by cardiac myocyte hypertrophy and collagen production. The increase in Ea was attributable to a decrease in SV and no increase in ESPESV but not due to elevated total peripheral resistance.
LV ESPVR obtained by gradual increase in afterload appeared to be similar between normal and hypertrophied hearts, but PVAmLVV, which depicts mechanical work capability at mLVV (13, 15, 27), was markedly decreased in the hypertrophied heart, indicating that the LV function was impaired in hypertrophied rat hearts, although ESPmLVV did not change.
Taken together with these results, the simultaneous measurement of LV P-V signals and analysis using the framework of ESPVR-PVA, especially PVAmLVV, are substantially needed to evaluate LV function in the heart without changes in LVP such as in the hypertrophied rat heart.
We concluded that the changes in PVAmLVV precisely reflect the changes in LV work capability even in reversible Iso-induced hypertrophied rat hearts. These results indicate that the present approach might be an appropriate strategy for evaluating the effects of antihypertrophic and antifibrotic agents and gene targeting therapy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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-Adrenergic neuroeffector mechanisms in cardiac hypertrophy of renin transgenic rats. Hypertension 24: 653–662, 1994.-adrenergic-receptor density in failing human hearts. N Engl J Med 307: 205–211, 1982.[Abstract]
-Adrenergic cardiac hypertrophy is mediated primarily by the 1-subtype in the rat heart. Mol Cell 33: 561–573, 2001.
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