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Am J Physiol Heart Circ Physiol 283: H631-H641, 2002. First published April 11, 2002; doi:10.1152/ajpheart.00046.2002
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Vol. 283, Issue 2, H631-H641, August 2002

Cardiac dysfunction in terms of left ventricular mechanical work and energetics in hypothyroid rats

Yoshimi Ohga1, Susumu Sakata1, Chikako Takenaka1, Takehisa Abe2, Tsuyoshi Tsuji2, Shigeki Taniguchi2, and Miyako Takaki1

Departments of 1 Physiology II and 2 Surgery III, Nara Medical University, Kashihara, Nara 634-8521, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We hypothesized that cardiac dysfunction in hypothyroidism is mainly caused by the impairment of Ca2+ handling in excitation-contraction coupling. To prove this hypothesis, we investigated left ventricular (LV) mechanical work and energetics without interference of preload and afterload in an excised, blood-perfused whole heart preparation from hypothyroid rats. We found that LV inotropism and lusitropism were significantly depressed, and these depressions were causally related to decreased myocardial oxygen consumption for Ca2+ handling and for basal metabolism. The oxygen costs of LV contractility for Ca2+ and for dobutamine in the hypothyroid rats did not differ from those in age-matched normal rats. The expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) significantly decreased and that of phospholamban significantly increased. The present results revealed that changes in LV energetics associated with decreased mechanical work in hypothyroid rats are mainly caused by the impairment of Ca2+ uptake via SERCA2. We conclude that the impairment of Ca2+ uptake plays an important role in the pathogenesis of cardiac dysfunction in hypothyroidism.

pressure-volume relationship; isomyosin; hypothyroidism; contractility; sarcoplasmic reticulum calcium adenosinetriphosphatase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN CANINE AND HUMAN HEARTS, the left ventricular myocardial myosin isozyme is V3, in contrast to V1, in normal adult rat hearts (14, 23). It is well known that ATPase activity of the V1 myosin isozyme is higher than that of the V3 myosin isozyme and that the shortening velocity of V1-dominant myocardium is faster than that of V3-dominant myocardium (16, 35). In the hypothyroid rat (V3-dominant myocardium), the isometric tension of native trabecular preparations substantially decreased (9) and peak developed tension of left ventricular papillary muscle preparations significantly decreased (16), although the isometric tension of glycerinated trabecular preparations hardly changed. We recently reported (21) that the curvilinearity of the left ventricular end-systolic pressure-volume relationship (ESPVR) decreased in in situ ejecting hearts of hypothyroid rats, indicating a decrease in left ventricular contractility. Nevertheless, it is still unknown whether the depressed contractility in in situ ejecting hearts of hypothyroid rats derives from the V3-dominant myocardium, the impairment of Ca2+ handling in excitation-contraction coupling, or abnormal pre- and/or afterload conditions due to hypothyroidism.

We hypothesized that cardiac dysfunction in hypothyroidism is mainly caused by the impairment of Ca2+ handling in excitation-contraction coupling. To prove this hypothesis, we aimed to investigate cardiac function in terms of the coupling of rat left ventricular mechanical work and energetics without the interference of preload and afterload in excised, cross-circulated whole heart preparations from hypothyroid rats. This study may lead to a better understanding of the processes that cause cardiac dysfunction in hypothyroidism.

As appropriate indexes for assessing rat left ventricular mechanical work and energetics we adopted systolic pressure-volume area [PVAmLVV, total mechanical energy per beat at midrange left ventricular volume (mLVV)], the oxygen cost of PVA (slope) and the myocardial oxygen consumption per beat (VO2) intercept (PVA-independent VO2 composed of VO2 for Ca2+ handling in excitation-contraction coupling and for basal metabolism) of the VO2-PVA linear relation, oxygen cost of equivalent Emax (eEmax; left ventricular contractility index), and the slope of the linear relation between VO2 for Ca2+ handling in excitation-contraction coupling and eEmax, as reported previously (1, 12, 13, 37, 38).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The investigation conformed 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).

Animal preparations. Twenty-four age-matched male crj:Wistar rats (20-21 wk) consisting of two groups [12 normal rats (556 ± 37 g body wt) and 12 hypothyroid rats (452 ± 33 g body wt)] were used. Rats were made hypothyroid by drinking water containing 8-propylthiouracil (PTU, 0.8 mg/ml) for 4-5 wk (16). Decreased T3 and T4 levels in plasma, which were decreased to 34.2 ± 7.1% and 2.6 ± 0% of normal, respectively, confirmed hypothyroidism.

Surgical preparation. Experiments were performed on 24 excised, cross-circulated rat heart preparations from the 12 normal and 12 hypothyroid rats as reported previously (12, 13, 37, 38). In each experiment, two retired breeder male crj:Wistar rats weighing 568 ± 39 g (~32 wk), purchased from Charles River Japan (Yokohama, Japan), were anesthetized with pentobarbital sodium (50 mg/kg ip) and used as blood supplier and metabolic supporter rats, respectively. All rats were heparinized (1,000 units iv). The beating heart was excised without interruption of coronary perfusion and supported by cross circulation with the metabolic supporter rat as previously reported in detail (12). The excised heart was maintained at 37°C.

A thin latex balloon (balloon membrane volume 0.08 ml) fitted into the left ventricle was connected to a pressure transducer (Life Kit DX-312; Nihon Kohden, Tokyo, Japan) and a 0.5-ml precision glass syringe with fine scales (minimum scale 0.005 ml). The unstretched balloon volume was below 0.20-0.25 ml. Left ventricular volume was changed and measured by adjusting the intraballoon water volume with the syringe in 0.02- to 0.05-ml steps between 0.08 and 0.28 ml. Systolic unstressed volume (V0) was determined by filling the balloon to the level at which peak isovolumic pressure and hence PVA (see Data analysis) were zero. The sum of intraballoon water volume and balloon material volume (0.08 ml) was used as an initial estimate of V0. This procedure was repeated during different left ventricular volume-loading runs in control (control Vol-run) and during Ca2+ (Ca2+ Vol-run) or dobutamine (dobutamine Vol-run) infusion at the maximum rate. V0 was then specifically determined as the volume-axis intercept of the best-fit ESPVR in each Vol-run. We obtained the best-fit ESPVR and end-diastolic pressure-volume relationship (EDPVR) with the two different exponential functions by means of the least-squares method (Delta-Graph; DeltaPoint, Monterey, CA) on a personal computer (1, 32, 37). Correlation coefficients of the best-fit ESPVRs and EDPVRs were >0.99.

The left ventricular epicardial electrocardiogram was recorded, and the mean heart rate was constantly maintained at 300 beats/min in normal rats and 262 beats/min in hypothyroid rats by electrical pacing of the right atrium (Table 1). Although the EDPVR at 300 beats/min pacing showed an upward shift due to incomplete relaxation, 262 beats/min pacing caused complete relaxation in the hypothyroid rats. In some cases, to analyze a pressure-time curve, the mean pacing rate was maintained at 264 ± 13 beats/min in normal (n = 10) and 264 ± 9 beats/min in hypothyroid (n = 10) rats. The systemic arterial blood pressure of the supporter rat served as coronary perfusion pressure (100-130 mmHg). Arterial pH, PO2, and PCO2 of the supporter rat were maintained within their physiological ranges with supplemental oxygen and sodium bicarbonate.

                              
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  		Table 1.   Variables of left ventricular mechanical work

Oxygen consumption. Myocardial oxygen consumption was obtained as the product of coronary flow and coronary arteriovenous oxygen content difference. The measurements of coronary flow and arteriovenous oxygen content difference were previously reported in detail (12). VO2 was obtained as myocardial oxygen consumption divided by heart rate. As shown previously (1, 12, 13, 37, 38), the VO2-PVA relation was linear in the rat left ventricle. Its slope represents the oxygen cost of PVA. Its VO2 intercept represents PVA-independent VO2 and is mainly composed of VO2 for Ca2+ handling in the excitation-contraction coupling and for basal metabolism (12). The total Ca2+ handling VO2 is mainly consumed for Ca2+ uptake via sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) (13, 36). The right ventricle was kept collapsed by continuous hydrostatic drainage of the coronary venous return, so that the right ventricular PVA and hence PVA-dependent VO2 were assumed to be negligible (12, 37). The right ventricular component of PVA-independent VO2 was calculated by multiplying biventricular PVA-independent VO2 in each contractile state by the ratio of right ventricular weight divided by the sum of right and left ventricular weights. The right ventricular PVA-independent VO2 was subtracted from the total VO2 to yield left ventricular VO2. The left ventricle, including the septum, and the right ventricle were weighed to normalize left ventricular volume. They were 1.07 ± 0.08 and 0.29 ± 0.06 g in normal rats (n = 12) and 0.85 ± 0.10 and 0.21 ± 0.03 g in hypothyroid rats (n = 12), respectively. The left and right ventricular weights were significantly different between the two groups.

Experimental protocol. Left ventricular pressure, VO2, and PVA data during isovolumic contractions were obtained at five to six different volumes (mean volume range: 0.17 ± 0.02 ml/g) (Vol-run) in each normal and hypothyroid rat heart. Left ventricular volume was increased in steps up to an end-diastolic pressure of 10 mmHg in a control Vol-run. The control Vol-run was performed without any inotropic interventions. After the control Vol-run, a Ca2+-induced (Ca2+ Ino-run; n = 6) or a dobutamine-induced different inotropic run (dobutamine Ino-run; n = 6) was performed at mLVV [0.16 ml= water volume infused into the balloon (0.08 ml) + V0 (0.08 ml)] in normal and hypothyroid rats (n = 12 each). Mean values for mLVV and V0 (normalized for 1 g) were 0.16 ± 0.01 and 0.076 ± 0.005 ml/g in normal rats (n = 12) and 0.19 ± 0.02 and 0.094 ± 0.011 ml/g in hypothyroid rats (n = 12). These values were significantly different between the two rat groups because of the significant difference in left ventricular weight (Table 1). The infusion rates of 1% CaCl2 solution and 66 µM dobutamine were increased in steps up to 10-20 ml/h (n = 6) and up to 4-10 ml/h (n = 6) in the two rat groups (n = 12 each). The Ca2+ Vol-run or dobutamine Vol-run was performed during Ca2+ or dobutamine infusion at the maximum rate. The measured blood Ca2+ concentration reached 3.1-8.1 mM. The calculated blood dobutamine concentration reached 1.1-2.4 µM under a coronary flow rate of 3-4 ml/min. In the normal rats, mean V0 was 0.075 ± 0.004 ml/g in control Vol-run and 0.075 ± 0.004 ml/g in Ca2+ Vol-run (n = 6) and 0.076 ± 0.006 ml/g in control Vol-run and 0.076 ± 0.006 ml/g in dobutamine Vol-run (n = 6). There were no significant differences in mean V0 values between control and Ca2+ Vol-runs and between control and dobutamine Vol-runs. In the hypothyroid rats, mean V0 was 0.093 ± 0.011 ml/g in control Vol-run and 0.092 ± 0.010 ml/g in Ca2+ Vol-run (n = 6) and 0.094 ± 0.011 ml/g in control Vol-run and 0.095 ± 0.010 ml/g in dobutamine Vol-run (n = 6). There were no significant differences in mean V0 values between control and Ca2+ Vol-runs and between control and dobutamine Vol-runs. The V0 values of control, Ca2+, and dobutamine Vol-runs in the normal and hypothyroid rats are summarized in Table 2.

                              
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  		Table 2.   Specifically determined V0

Cardiac arrest was induced by infusing KCl (1 M) into the coronary perfusion tubing at a constant rate in the two rat groups (n = 7 each of 12 rats), which was adjusted to abolish electrical excitation under monitoring ventricular electrocardiograms but not to generate any KCl-induced constriction of coronary vessels. VO2 and PVA data were obtained by minimal volume loading to avoid volume-loading effects on VO2 data.

In every Vol-run, Ino-run, and KCl arrest-run, a steady state, at which left ventricular pressure, coronary arteriovenous O2 content difference, and coronary flow were stable, was reached 3 min after changing left ventricular volume or after KCl-induced arrest or 6 min after changing the infusion rate of Ca2+ or dobutamine. In each steady state, data were sampled at 500 Hz for 2 s simultaneously and the sampling was usually repeated three times at intervals of 0.5-1 min.

Data analysis. We attempted to fit experimentally obtained left ventricular pressure-volume data to the two different exponential equations to obtain ESPVRs and EDPVRs and thus determined PVA by subtracting the area under the EDPVR curve from the area under the ESPVR curve (1, 32, 37). On the basis of our previous proposal (1, 32, 37, 38), we obtained control ESPVR and EDPVR, end-systolic pressure (ESPmLVV) and end-diastolic pressure (EDPmLVV) at mLVV, and PVAmLVV to assess left ventricular mechanical work and energetics in the two groups.

Oxygen cost of left ventricular contractility. We obtained the specific best-fit curves for the observed ESPmLVV and ESP (0 mmHg) at specifically determined V0 with the best-fit ESPVR functions obtained by the control Vol-runs and maximum dobutamine or Ca2+ Vol-run and the specific best-fit curves for the observed EDPmLVV and EDP (0 mmHg) at specifically determined V0 with the best-fit EDPVR function obtained by the control Vol-runs and maximum dobutamine or Ca2+ Vol-run by the least-squares method and calculated PVAmLVV during dobutamine or Ca2+ infusion on a personal computer (1, 32, 37, 38). The parallelism of the VO2-PVA linear relation during dobutamine (1) or Ca2+ (12, 32, 38) infusion has been confirmed in normal rat hearts. In the present study, we confirmed the parallelism of the VO2-PVA linear relation, i.e., there were no significant differences in the slopes of the VO2-PVA linear relations before and during dobutamine infusion (see Fig. 3, B and D) or before and during Ca2+ infusion in each normal and hypothyroid rat heart. The slopes of the VO2-PVA linear relations of the normal and hypothyroid rats in control, Ca2+, and dobutamine Vol-runs are summarized in Table 3. In only one normal heart, the slope showed a significant difference between control and dobutamine Vol-runs. There were no significant differences in the mean slopes of the VO2-PVA linear relations in normal rat hearts (n = 12) between control (1.16 ± 0.08 × 10-2 µl O2 · mmHg-1 · ml-1) and Ca2+ (1.19 ± 0.11 × 10-2 µl O2 · mmHg-1 · ml-1) Vol-runs and between control (1.14 ± 0.12 × 10-2 µl O2 · mmHg-1 · ml-1) and dobutamine (1.00 ± 0.19 × 10-2 µl O2 · mmHg-1 · ml-1) Vol-runs. There were no significant differences in the mean slopes of the VO2-PVA linear relations in the hypothyroid rat hearts (n = 12) between control (1.25 ± 0.18 × 10-2 µl O2 · mmHg-1 ·ml-1) and Ca2+ (1.10 ± 0.17 × 10-2 µl O2 · mmHg-1 · ml-1) Vol-runs and between control (1.34 ± 0.40 × 10-2 µl O2 · mmHg-1 · ml-1) and dobutamine (1.26 ± 0.30 × 10-2 µlO2 · mmHg-1 · ml-1) Vol-runs. On the basis of this parallelism, the lines including all VO2-PVA data obtained during dobutamine or Ca2+ infusion in steps at the mLVV were drawn in parallel to the control VO2-PVA relation line, as described previously (12, 32, 37, 38). The gradually increased VO2-intercept values (PVA-independent VO2 values) of the lines proportional to the enhanced left ventricular contractility induced by dobutamine or Ca2+ were obtained by this procedure.

                              
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  		Table 3.   Slopes of VO2-PVA relations

Our recently proposed index for left ventricular contractility, eEmax, was defined as an ESP-to-V ratio of the specific virtual triangular PVAmLVV that is energetically equivalent to the real PVAmLVV (1, 32, 37, 38). eEmax is calculated by 2PVAmLVV/ESV<UP><SUB>mLVV</SUB><SUP>2</SUP></UP>. Mean end-systolic volume (ESV = mLVV - V0) at mean mLVV (0.16 ± 0.01 ml/g; ESV0.16) was 0.076 ± 0.005 ml/g in normal rats (n = 12), and mean ESV at mean mLVV (0.19 ± 0.02 ml/g; ESV0.19) was 0.10 ± 0.01 ml/g in hypothyroid rats (n = 12). Mean values of ESVmLVV were not significantly different between normal and hypothyroid rats. The oxygen cost of left ventricular contractility was the slope of the relationship between PVA-independent VO2 and eEmax, indicating changes in VO2 consumed for Ca2+ handling in excitation-contraction coupling per unit change in the contractility under the constant basal metabolism (1, 32, 37, 38).

Delta eEmax/Ca2+ or Delta eEmax/dobutamine was calculated as the ratio of increases in left ventricular contractility for Ca2+ or dobutamine versus the concentrations of Ca2+ or dobutamine, respectively.

Analyses of one-beat left ventricular pressure-time curve by hybrid logistic function and logistic function. To evaluate the left ventricular systolic and diastolic functions, we analyzed the contraction rate (+dP/dtmax), relaxation rate (-dP/ dtmax), and their ratio [(+dP/dtmax)/(-dP/dtmax)] from a best-fit function to one-beat left ventricular pressure-time curve at mLVV during contraction and relaxation with our proposed "hybrid logistic function" (24) in normal (n = 10) and hypothyroid (n = 10) rats. To evaluate left ventricular end-diastolic relaxation rate or lusitropism, we analyzed the "logistic" time constant (TL) and the conventional "exponential" time constant (TE) from respective best-fit functions to the one-beat left ventricular pressure-time curve at mLVV during relaxation with our proposed logistic function (25) and the monoexponential function in normal (n = 10) and hypothyroid (n = 10) rats. Both time constants are decreased as the heart rate increases, and thus they were compared under the same pacing rate condition of 264 beats/min.

Polyacrylamide gel electrophoresis and Western blots for SERCA2 and phospholamban. Membrane proteins from the left ventricular myocardium of each heart were isolated as described previously by Yoshida and Harada (40). 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 were used for immunoblotting of SERCA2 and phospholamban.

Membrane proteins (20-25 µg/lane) were separated on SDS-polyacrylamide gels (10% for SERCA2 and 15% for phospholamban) in a minigel apparatus (Mini-Protean II, Bio-Rad) 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) or anti-phospholamban antibody (1:2,000 dilution, Upstate Biotechnology). The antigens were detected by the luminescence method (ECL Western blotting detection kit, Amersham) with peroxidase-linked anti-mouse IgG (1:2,000 dilution). After immunoblotting was completed, the film was scanned with a scanner, and the intensity of the bands was calculated by NIH Image analysis.

Polyacrylamide gel electrophoresis for myosin isozymes. The left ventricular myocardium from each heart was frozen and stored at -70°C. Myosin was extracted from 50 mg of the left ventricular myocardium, and three myosin isozymes (V1, V2, and V3) were separated by 3.7% polyacrylamide gel electrophoresis in the presence of pyrophosphate according to a slightly modified procedure (21) of Hoh et al. (14).

Statistics. Analysis of covariance (ANCOVA) was applied to compare the two regression lines of left ventricular VO2 on PVA in each heart in the control Vol-run and dobutamine or Ca2+ Vol-run. Comparisons of paired and unpaired individual values were performed by paired and unpaired t-test, respectively. Multiple comparisons were performed by ANOVA and Bonferroni's post hoc analysis. A value of P < 0.05 was considered statistically significant. All data are expressed as means ± SD.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Body and heart weight. The mean body weight of the hypothyroid rats was significantly (P < 0.005) lower by 18.7% than that of the age-matched control rats. The mean left ventricular weight was also significantly (P < 0.005) lower by 20.6% and the mean right ventricular weight was significantly (P < 0.005) lower by 27.6% than that of the age-matched control rats, but neither the ratio of left ventricular weight to left and right ventricular weights nor the ratio of left ventricular weight to body weight in hypothyroid rats differed from those of the normal rats.

Figure 1 shows a representative set of left ventricular pressure-time curves at mLVV in each heart paced by 261 beats/min of a normal (Fig. 1A) and a hypothyroid (Fig. 1B) rat. Left ventricular ESPmLVV was smaller in a hypothyroid rat than in a normal rat. Each left ventricular contraction and relaxation rate was slower in a hypothyroid rat than in a normal rat. The mean contraction (b, +dP/dtmax) and relaxation rate (i, -dP/dtmax) were significantly slower (Fig. 2A), and b/i (+dP/dtmax/-dP/dtmax) was significantly larger in the hypothyroid rats than in the normal rats (2.32 ± 0.38 vs. 1.80 ± 0.21). Mean TL and TE were significantly larger in the hypothyroid rats, indicating that the end-diastolic relaxation rate was slower in the hypothyroid rats (Fig. 2B).


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  		Fig. 1.   Left ventricular (LV) pressure-time curves in a normal (A) and a hypothyroid (B) rat under 261 beats/min pacing at midrange LV volume (mLVV), where 0.08 ml of water was infused into the balloon.



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  		Fig. 2.   A: mean best-fit parameter b, corresponding to contraction rate (+dP/dtmax), and best-fit parameter i, corresponding to relaxation rate (-dP/dtmax), in P(t) = a/{1 + exp[-(4b/a)(t - c)]} - h/{1 + exp[-(4i/h)(t - j)]} to LV pressure (P)-time (t) curves at mLVV in normal and hypothyroid rats. a, c, h, and j are other best-fit parameters. B: mean logistic and exponential time constants in normal and hypothyroid rats. *P < 0.05 vs. values in normal rats.

Figure 3, A and C, shows a representative set of left ventricular curved ESPVRs and EDPVRs and end-systolic pressure data at mLVV (ESPmLVV) of a normal and a hypothyroid rat in control conditions and during dobutamine infusions. We had already confirmed (32) that left ventricular ESPVR and EDPVR did not significantly change in the hearts paced by 240 and 300 beats/min, and therefore we paced the hearts in normal and hypothyroid rats at different rates in the present study (Table 1). The control ESPVR (cESPVR) in the hypothyroid rat (Fig. 3C) was obviously lower than that in the normal rat (Fig. 3A). The control EDPVR (cEDPVR) in the hypothyroid rat was slightly higher than that in the normal rat at the larger LVV, but the end-diastolic pressure (EDP) at mLVV (EDPmLVV) at 300 beats/min pacing in the normal rat and EDPmLVV at 262 beats/min pacing in the hypothyroid rat were almost zero. The mean ESPmLVV was significantly smaller and the mean EDPmLVV significantly larger in the hypothyroid rats than in the normal rats, so that PVAmLVV was significantly smaller in the hypothyroid rats than in the normal rats (Table 1). The mean ESVmLVV was similar in the two groups, so that eEmax (=2PVAmLVV/ESV<UP><SUB>mLVV</SUB><SUP>2</SUP></UP>) was significantly smaller in the hypothyroid rats than in the normal rats (Table 1).


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  		Fig. 3.   LV pressure-volume data and myocardial oxygen consumption per beat (VO2)-systolic pressure-volume area (PVA) data in a normal (A and B) and a hypothyroid (C and D) rat. LV pressure-volume data during different volume loading runs between 0.08 and 0.23 ml/g of a normal rat (A) and between 0.09 and 0.25 ml/g of a hypothyroid rat (C) in control (large filled circles) and under dobutamine (Dob) infusion (small solid circles) and LV pressure-volume (A and C) and VO2-PVA data at mLVV (open circles) during Dob infusion in a normal and a hypothyroid rat (B and D) are shown. ESPVR, curved end-systolic pressure-volume relationships in control [cESPVR; correlation coefficient (R) = 0.999] and under Dob (dESPVR; R = 0.999). EDPVR, end-diastolic pressure-volume relationships in control (cEDPVR; R = 0.991) and under Dob (dEDPVR; R = 0.999). PVA, systolic pressure-volume area at mLVV (hatched area in A). LV control linear relations between VO2 and PVA in control (cVO2-PVA) and under Dob (dVO2-PVA) and the composite linear relations between VO2 and PVA (comp VO2-PVA) during Dob-induced different inotropism runs in a normal (B) and hypothyroid rat (D) are shown. No significant differences in the slopes were found between cVO2-PVA and dVO2-PVA in a normal (B) and hypothyroid (D) rat by ANCOVA. Thus the thinnest VO2-PVA lines parallel to the cVO2-PVA relationship line were drawn to obtain PVA-independent VO2 (B and D).

Left ventricular ESPmLVV gradually increased with an increase in the infusion rate of dobutamine in both normal and hypothyroid rats. cESPVR shifted upward (dESPVR) and cEDPVR shifted downward (dEDPVR) during dobutamine infusion in the hypothyroid rat (Fig. 3C); the effects of dobutamine on ESP and EDP appeared to be rather more potent than those in the normal rat (Fig. 3A), but dESPVR and dEDPVR in the hypothyroid rat corresponded to those in the normal rat.

Figure 3, B and D, shows the demonstrative linear VO2-PVA relations in the control Vol-runs (cVO2-PVA) and composite linear VO2-PVA relations (compVO2-PVA) during the dobutamine Ino-runs in the same set of normal and hypothyroid rats. Because the VO2-PVA relations and compVO2-PVA relations were not significantly different at 240 and 300 beats/min (32), we used different pacing rates in normal and hypothyroid rats.

The slopes of the VO2-PVA relations were similar in the normal and hypothyroid rats, but the VO2 intercept of the relation was smaller in the hypothyroid rats (Fig. 3D). The VO2-PVA data points at mLVV during the dobutamine Ino-run shifted rightward and upward from the cVO2-PVA data points. The effect of dobutamine on VO2-PVA data appeared to be rather more potent in the hypothyroid rat (Fig. 3D). However, the VO2-PVA relation in the dobutamine Vol-run (dVO2-PVA) in the hypothyroid rat (Fig. 3D) corresponded to that in the normal rat (Fig. 3B). The slopes did not differ between cVO2-PVA and dVO2-PVA in normal and hypothyroid rats. The effects of Ca2+ infusion on ESP and EDP, ESPVR and EDPVR, and VO2-PVA relations were similar to those of dobutamine (data not shown).

Figure 4, A and B, summarizes the mean slopes and mean VO2 intercepts of the VO2-PVA relations in normal and hypothyroid rats (n = 12 each). The slopes were similar in the two groups, whereas the VO2 intercepts in the hypothyroid rats were significantly (P < 0.05) smaller than those in the normal rats. The VO2 intercept was composed of VO2 for Ca2+ handling in excitation-contraction coupling and for basal metabolism. Figure 4, C and D, summarizes the data for the components of the VO2 intercept in the two groups (n = 7 in each of 12 rats). The mean VO2 for Ca2+ handling in excitation-contraction coupling (Fig. 4C) and for basal metabolism (Fig. 4D) in the hypothyroid rats were significantly smaller than those in the normal rats (decreased by 75.5 ± 5.7% and 50.9 ± 10.4% of the normal rats, respectively).


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  		Fig. 4.   Mean slope (A) and VO2 intercept (B) of the linear VO2-PVA relationship and VO2 consumed in excitation-contraction (E-C) coupling (C) and basal metabolic VO2 (D) in normal (n = 12) and hypothyroid (n = 12) rats. *P < 0.05 vs. values in normal rats.

As the slope of the plot of VO2 for Ca2+ handling in excitation-contraction coupling on eEmax, we obtained the oxygen costs of left ventricular contractility in responses to Ca2+ (n = 6) and dobutamine (n = 6). Surprisingly, the oxygen costs of eEmax for dobutamine and Ca2+ were not significantly different in the hypothyroid rats (n = 12) from those in the normal rats (n = 12) (Fig. 5). Furthermore, the mean ratios of increases in left ventricular contractility in response to the concentrations of Ca2+ (Delta eEmax/Ca2+; 400 ± 170 vs. 450 ± 100 mmHg · ml-1 · g · mM-1) and dobutamine (Delta eEmax/Dob; 660 ± 280 vs. 720 ± 190 mmHg · ml-1 · g · mM-1) did not show significant differences between the normal and hypothyroid rats, respectively. These results indicate that the changes in Ca2+ handling VO2 in excitation-contraction coupling in response to Ca2+ and dobutamine in hypothyroid rats also do not differ from those in normal rats.


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  		Fig. 5.   Mean oxygen costs of LV contractility for Ca2+ and dobutamine in normal (n = 12) and hypothyroid (n = 12) rats.

The Ca2+ handling VO2 in excitation-contraction coupling is mainly consumed by SERCA2 (13, 36). SERCA2 and phospholamban regulate the Ca2+ uptake at the sarcoplasmic reticulum (SR). The protein level of phospholamban was significantly higher and that of SERCA2 was significantly lower in hypothyroid rats (n = 12) than in normal rats (n = 10) (Fig. 6), so that the ratio of phospholamban to SERCA2 was markedly higher in the hypothyroid rats than in the normal rats.


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  		Fig. 6.   Immunoblotting of cardiac sarco(endo)plasmic reticulum ATPase (SERCA2 = 100 kDa) and phospholamban (PLB pentamer = 22 kDa) in normal and hypothyroid rats (n = 2 each; top). Protein levels of PLB were high and those of SERCA2 were low in hypothyroid rats. Bottom: summarized data of PLB and SERCA2 expression and the ratio of PLB to SERCA2 expression in normal (n = 10) and hypothyroid (n = 12) rats. *P < 0.05 vs. values in normal rats (unpaired t-test).

The myosin isozyme in left ventricular myocardium was V3 (95.1 ± 2.0% of total myosin isozyme) in the hypothyroid rats (n = 8), whereas it was V1 (89.5 ± 9% of the total) in the normal rats (n = 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Although the body weight and the left and right ventricular weights of the hypothyroid rats were significantly lighter than those in the age-matched control rat, the ratio of left ventricular weight to body weight was unchanged in the hypothyroid rats. In a previous report, body weight, heart weight, and ratio of left ventricular weight to body weight were significantly decreased in 9- to 10-wk-old rats after a 3-wk period of treatment with the addition of PTU to the drinking water compared with those in the age-matched control rats (16). Furthermore, another report also showed a decreased ratio of heart weight to body weight in 12- to 14-wk-old hypothyroid rats (22).

The present results indicate that a 4- to 5-wk period of treatment with the addition of PTU to the drinking water leads to impairment of normal growth, lower body weight, lower heart weight, and an unchanged ratio of left ventricular weight to body weight in 20- to 21-wk-old hypothyroid rats compared with those in the age-matched control rats. The discrepancy between the previous reports (16, 22) and ours seems to be due to the difference in age (9-14 vs. 20-21 wk). It seems likely that younger rats are susceptible to an impairment of increase in heart weight.

In the present hypothyroid rats, we found left ventricular systolic and diastolic dysfunctions. It is well known that thyroid hormone affects SERCA2 activity through the expression of both SERCA2 and phospholamban (4, 5, 15, 18-20, 30, 31). We hypothesized that the left ventricular systolic and diastolic dysfunctions in hypothyroid rats are related to the severe impairment of Ca2+ uptake by SERCA2 in excitation-contraction coupling. We actually found higher protein levels of phospholamban and lower protein levels of SERCA2 in the hypothyroid rats. Furthermore, we found a marked decrease in total Ca2+ handling VO2 in excitation-contraction coupling, which is primarily consumed by SERCA2 (13, 36). Therefore, the present results proved our hypothesis that the severe impairment of the SERCA2 activity caused systolic and diastolic dysfunctions in the hypothyroid rats.

Curved ESPVR, linear VO2-PVA relation, and oxygen cost of PVA. Marked differences in ESPmLVV or PVAmLVV were found between normal and hypothyroid rats, whereas the mean slope of the VO2-PVA relation (O2 cost of PVA) in the two groups was almost the same as that previously reported (12, 13, 32, 37, 38). In hyperthyroid rabbits, where the myosin isozyme is V1, an increased O2 cost of PVA is observed (11), although in hyperthyroid dogs, where the myosin isozyme remains V3, an unchanged O2 cost of PVA is observed (34). From these findings, it was predicted that the O2 cost of PVA is related to types of myosin isozyme. The present results, however, indicate that there exists no correlation between O2 cost of PVA and type of myosin isozyme in normal and hypothyroid rats. The unchanged O2 cost of PVA in the hypothyroid rats indicates that the efficiency of chemomechanical energy transduction (the inverse of O2 cost of PVA) remained unchanged despite the different types of myosin isozyme.

PVA-independent VO2. The mean VO2 intercept of the VO2-PVA relation (PVA-independent VO2) in the hypothyroid rats was significantly smaller than that in the normal rats. The PVA-independent VO2 corresponds primarily to the VO2 for total Ca2+ handling in excitation-contraction coupling and for basal metabolism (12, 13, 38, 39). Both the VO2 for total Ca2+ handling in excitation-contraction coupling and that for basal metabolism were significantly smaller in the hypothyroid rats. The decrease in VO2 for total Ca2+ handling reflects a decrease in the uptake activity of SERCA2 on a beat-to-beat basis, as mentioned above.

Thyroid hormone changes the expression of mitochondrially encoded respiratory genes (29). In the hypothyroid state, mitochondrial respiratory function may be depressed by depressed gene expression and further mitochondrial cytochrome oxidase activity is depressed (28). Therefore, it seems likely that hypothyroidism leads to depressed mitochondrial respiration. This speculation is supported by the present results showing noticeably lower basal metabolism in the left ventricle of the hypothyroid rats.

Oxygen cost of left ventricular contractility, eEmax. Dobutamine is known to stimulate beta -adrenergic receptors and thereby to activate protein kinase A (27), resulting in enhanced Ca2+ handling in excitation-contraction coupling, whereas Ca2+ directly enhances Ca2+ handling by increasing Ca2+ influx. Although the mechanism of enhancing Ca2+ handling is different, the O2 costs of left ventricular contractility (changes in total Ca2+ handling VO2 in excitation-contraction coupling against unit change in left ventricular contractility) for Ca2+ and dobutamine were similar in normal and hypothyroid rats. In hypothyroid rat hearts, a decrease in the myocardial beta -receptor population and thus a reduced responsiveness to a beta -adrenergic agonist has been reported (8). In contrast, a greater apparent Ca2+ sensitivity to contractile activation has been also reported in papillary muscle of the hypothyroid rat heart (10). Consequently, the net change between the reduced responsiveness to dobutamine and the greater Ca2+ sensitivity to contractile activation may lead to the present results showing similar O2 costs of left ventricular contractility for dobutamine in normal and hypothyroid rats.

On the other hand, the greater apparent Ca2+ sensitivity to contractile activation in hypothyroid rats (10) would result in a smaller O2 cost of left ventricular contractility for Ca2+. The O2 cost of left ventricular contractility for Ca2+ in the present hypothyroid rats, however, was similar to that in the normal rats. This result suggests that there was no occurrence of greater apparent Ca2+ sensitivity to contractile activation in the hypothyroid rats. Consequently, the above explanation for the unchanged O2 cost of left ventricular contractility for dobutamine also seems to be unlikely.

In the hypothyroid rats, the pacing of the heart at 300 beats/min failed but the pacing at 262 beats/min succeeded. Although this reflects the dysfunction of SERCA2, the decreased pacing rate may lead to unchanged O2 costs of left ventricular contractility for Ca2+ and for dobutamine on a beat-to-beat basis in the hypothyroid rats.

In developing rat hearts, hypothyroidism decreases the function of SERCA2 and increases the function of Na+/Ca2+ exchanger (6, 17, 30). Banijamali et al. (3) reported that in normal adult rats, 81% of the Ca2+ released from the SR was recirculated by SERCA2; Ca2+ handling relies largely on SR. If the present hypothyroidism had decreased the function of left ventricular SERCA2 and increased the function of left ventricular Na+/Ca2+ exchanger, the O2 cost of left ventricular contractility might have increased (26). However, the unchanged O2 cost of left ventricular contractility in the present hypothyroid rats indicates that there was no change in the relative functions of SERCA2 and Na+/Ca2+ exchanger. This speculation is supported by a recent study showing no increase in left ventricular Na+/Ca2+ exchanger protein in hypothyroid rats compared with that in normal rats (33). The decreased total Ca2+ handling VO2 in excitation-contraction coupling in the present hypothyroid rats suggests a decrease in the total amount of Ca2+ handled in excitation-contraction coupling with no change in the recirculation fraction of Ca2+ caused by SR.

Left ventricular systolic and diastolic dysfunction in hypothyroid rats. In the excised hearts of the hypothyroid rats, at a pacing rate (262 beats/min) lower than normal (300 beats/min), we found significant slowing of the left ventricular contraction and relaxation rates. In in situ hearts, preload and afterload and hormonal and neuronal influences could interfere with left ventricular function. In contrast, in the present cross-circulated (blood perfused) excised hearts, left ventricular function can be characterized by the myocardium per se. Therefore, true left ventricular systolic and diastolic dysfunction seems to be caused in the present hypothyroid rats.

In the present hypothyroid rats, the left ventricular myosin isozyme V1 was transformed to V3 as suggested by Lompre et al. (23). This transformation might be causally related to significant slowing of the left ventricular contraction and relaxation rates (9, 16). However, in the same type of heart preparations of type II diabetic rats as in the present study, where the myosin isozyme was V3 just as in hypothyroid rats, only diastolic dysfunction was found under the decreased pacing rate without any change in the VO2 intercept of the VO2-PVA relation and O2 cost of PVA (1). These findings indicate that hypothyroid and type II diabetic rats are quite different in left ventricular mechanical work and energetic characterization, although their myosin isozymes were the same V3. Therefore, it seems unlikely that the only transformation of myosin isozyme from V1 into V3 is causally related to the left ventricular systolic and diastolic dysfunctions in the present hypothyroid rats, although in long-term hypothyroidism the possibility for the contribution of the V3-dominant myocardium or abnormal pre- and/or afterload conditions due to the hypothyroidism cannot be excluded.

In agreement with our hypothesis, we found not only systolic and diastolic dysfunctions and a decrease in VO2 for Ca2+ handling in excitation-contraction coupling in physiological studies but also depressed expression of SERCA2 and enhanced expression of phospholamban in the hypothyroid rats, as previously reported (15, 18-20, 30). Furthermore, it was reported that in hypothyroid mice, decreases in SERCA2A gene expression are accompanied by prolonged contraction and relaxation of papillary muscles (5). It seems likely that the dysfunction of SERCA2 leads to a primary diastolic dysfunction and the decreased Ca2+ release resulting from the decrease in Ca2+ stored in SR leads to a secondary systolic dysfunction. On the other hand, the possibility of final impairment of ryanodine receptor function cannot be excluded, because a lower level of expression of ryanodine receptor mRNA has been reported in 16-wk hypothyroid rabbits (2).

Recently, it was reported that in the failing rat heart model made by aortic banding, the ratio of SERCA2A to phospholamban decreased in association with worsening metabolism, i.e., decreased ratio of phosphocreatine to ATP (7). Similar to this failing heart model, in the present hypothyroid rat heart, the ratio of SERCA2A to phospholamban decreased, but this was not associated with worsening metabolism, i.e., unchanged O2 costs of PVA and eEmax. The present study revealed, in terms of the coupling of left ventricular mechanical work and energetics, that left ventricular systolic and diastolic functions in the present hypothyroid rats are impaired by the depressed Ca2+ uptake function by SERCA2. We conclude that the impaired Ca2+ uptake function by SERCA2 plays an important role in the pathogenesis of cardiac systolic and diastolic dysfunctions in hypothyroidism. Overexpression of SERCA2A in the failing rat heart made by aortic banding improved cardiac function associated with an improvement of the phosphocreatine-to-ATP ratio, the energy potential (7). Furthermore, in hypothyroid mice, overexpression of SERCA2A also improves both contraction and relaxation of the papillary muscle (5). Therefore, overexpression of SERCA2A would be a promising strategy to rescue various cardiac dysfunctions including that in the present study.


    ACKNOWLEDGEMENTS

This study was partly supported by Grant-In-Aid for Scientific Research 14770016 from the Ministry of Education, Science, Sports and Culture, Japan.


    FOOTNOTES

Address for reprint requests and other correspondence: M. Takaki, Dept. of Physiology II, Nara Medical Univ., 840 Shijo-cho, Kashihara, Nara 634-8521, Japan (E-mail: mtakaki{at}naramed-u.ac.jp).

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.

April 11, 2002;10.1152/ajpheart.00046.2002

Received 20 January 2002; accepted in final form 10 April 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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