INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

ALTHOUGH THE PRINCIPAL pathophysiological feature of diabetes mellitus (DM) is called diabetic microangiopathy, the presence of myocardial dysfunction "diabetic cardiomyopathy" independent of coronary artery disease has been well documented in streptozotocin-induced DM (29, 32). The recently established Otsuka Long-Evans Tokushima Fatty (OLETF) rat model of DM (9) manifests stable clinical and pathological features that resemble human type 2 DM. Mizushige et al. (13) recently elucidated the causal link between microangiopathy and the pathogenesis of diabetic cardiomyopathy in OLETF rats. They found that early diastolic transmitral inflow exhibited prolonged deceleration time and low peak velocity in the prediabetic stage (13). Abnormal relaxation is usually one of the earliest manifestations of cardiac dysfunction, and diastolic impairment is seen before systolic impairment (16), but the pathogenesis of diabetic cardiomyopathy is not well understood. Specifically, the coupling of mechanical work and energetics of the heart in diabetic cardiomyopathy is not yet understood.

We have recently reported a linear relationship between myocardial oxygen consumption per beat (VO₂) and the systolic pressure-volume area (PVA: a total mechanical energy per beat) in the rat left ventricle in the blood-perfused whole heart preparation, similar to that observed in canine hearts (24), from a curved end-systolic pressure (ESP)-volume (V) relationship (ESPVR) (3, 4, 23). The VO₂ intercept of the linear VO₂-PVA relationship is mainly composed of VO₂ for Ca²⁺ handling in the excitation-contraction coupling, which is primarily consumed by sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2) and basal metabolism (3, 4), as in canine hearts (14, 20). We have recently shown (27, 28) that a new "oxygen (O₂) cost of left ventricular contractility" index can evaluate changes in VO₂ for total Ca²⁺ handling in the excitation-contraction coupling versus changes in left ventricular contractility in terms of the coupling of mechanical work and energetics. This cost is not dependent on a heart rate between 240 and 300 beats/min (18).

We hypothesized that diastolic dysfunction may manifest itself in early diabetic cardiomyopathy due to dysfunction of SERCA2. If so, it is predicted that VO₂ for total Ca²⁺ handling in the excitation-contraction coupling may decrease, and the O₂ cost of left ventricular contractility may increase due to resultant activation of the Na⁺/Ca²⁺ exchanger and Na⁺-K⁺-ATPase (8, 12). To prove this hypothesis, we investigated left ventricular systolic and diastolic functions linked with intracellular Ca²⁺ handling in terms of the coupling of mechanical work and energetics in spontaneously DM, OLETF rats. The use of an appropriate animal model may lead to a better understanding of the processes that cause diabetic cardiomyopathy.

	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).

Animals. Five-week-old male OLETF rats and control male Long-Evans Tokushima Otsuka (LETO) rats were obtained from Tokushima Research Institute (Tokushima, Japan). A previously reported study (7) shows that OLETF rats drinking 30% sucrose solution for 8 wk decreased the conduction velocity of motor nerves and thus impaired autonomic nerve function. Starting at 25 wk of age, OLETF rats were allowed free access to tap water containing 30% sucrose for 8 wk (up to 32 wk) to cause severe DM with neuropathy. Blood was obtained from a midline incision site and tested with a glucose sensor from Sanwa Chemical (Nagoya, Japan).

Surgical preparation. Experiments were performed on 20 excised, cross-circulated rat heart preparations (6 L-I OLETF, 3 L-I LETO, 6 L-II OLETF, and 5 L-II LETO rat hearts) as reported previously (3, 4, 27). In each experiment, two retired breeder male crj:Wistar rats weighing 603 ± 42 g (32-40 wk of age, 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 administered heparin (1,000 units iv). The beating heart was excised from OLETF and LETO rats without interruption of coronary perfusion and supported by cross circulation with the metabolic supporter rat as previously reported in detail (3). The excised heart was maintained at 37°C.

Oxygen consumption. Myocardial oxygen consumption was obtained as the product of coronary flow and coronary arteriovenous O₂ content difference (3, 27). Total coronary blood flow was continuously measured with an electromagnetic flowmeter (MFV-3100, Nihon Kohden; Tokyo, Japan) placed in the middle of the coronary venous drainage tubing from the right ventricle. Left ventricular thebesian flow was negligible. The coronary arteriovenous O₂ content difference was continuously measured by passing all the arterial and venous cross-circulation blood through the cuvettes of a custom-made arteriovenous O₂ content difference analyzer (PWA-200S, Shoe Technica; Chiba, Japan) as previously reported in detail (3, 27). As shown previously (3, 4, 27), the VO₂-PVA relationship was linear in the rat left ventricle. Its slope represents the O₂ cost of PVA, and its VO₂ intercept represents the PVA-independent VO₂. 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 (3, 27). The right ventricular PVA-independent VO₂ (3, 4, 27, 28) was subtracted from the total VO₂ to yield left ventricular VO₂. The left ventricle (including the septum) and the right ventricle were weighed for normalization of LVV. They were 1.06 ± 0.11 and 0.31 ± 0.04 g (n = 20), respectively. There were no significant differences in left ventricular weight among the four rat groups (L-I OLETF, 1.03 ± 0.08; L-I LETO, 1.04 ± 0.05; L-II OLETF, 1.01 ± 0.07; L-II LETO, 1.16 ± 0.12 g).

Experimental protocol. Left ventricular pressure, VO₂, and PVA data during isovolumic contractions were obtained at four to five different LVVs (mean volume range: 0.16 ± 0.03 ml/g; n = 20) in each L-I OLETF and L-I LETO rat heart and L-II OLETF and L-II LETO rat heart. In each L-I OLETF and L-I LETO rat heart, after the control volume run, a Ca²⁺-induced different inotropic run (Ca²⁺ inotropic run) was performed at a midrange LVV (mLVV) [0.16 or 0.18 ml = water volume infused into the balloon (equals 0.08 or 0.10 ml) plus V₀ (equals 0.08 ml)] during intracoronary infusion of 1% CaCl₂ solution. In each L-II OLETF and L-II LETO rat heart, after the control volume run, a first dobutamine-induced different inotropic run and a second Ca²⁺ inotropic run were performed at the same mLVV during infusion of 66 µM dobutamine and 1% CaCl₂ solution, respectively. Between the two inotropic runs, it took 20 min to attain complete clearance of the blood with dobutamine by the kidney of the supporter rat. Mean values for mLVV and V₀ (normalized for 1 g) were 0.15 ± 0.01 and 0.08 ± 0.01 ml/g (n = 20), respectively. These values were not significantly different among the four rat groups (Table 1). The infusion rates of Ca²⁺ and dobutamine were increased in steps up to 10-20 ml/h (n = 20) and up to 4 ml/h (n = 11), respectively. The blood Ca²⁺ concentration reached 3.1-8.1 mmol/l. The calculated blood dobutamine concentration reached 1.1-1.5 µmol/l under a coronary flow rate of 3-4 ml/min. In every volume run and inotropic run, a steady state where left ventricular pressure, coronary arteriovenous O₂ content difference, and coronary flow were stable was reached 3 min after changing LVV or 5 min after changing the infusion rate of Ca²⁺ or dobutamine.

Data analysis. We attempted to fit experimentally obtained left ventricular pressure-volume data to the exponential equations to obtain ESPVRs (see Table 1) and end-diastolic pressure-volume relationships (EDPVRs) and thus determine PVA by the same method as described previously in detail (3, 4, 27, 28).

Oxygen cost of left ventricular contractility. We obtained the specific best-fit curves for the observed ESP_mLVV and ESP (0 mmHg) at V₀ with the ESPVR function in the control volume run by the least squares method, and we calculated PVA_mLVV during dobutamine or Ca²⁺ infusion on a personal computer (18, 27, 28). The parallelism of the VO₂-PVA linear relation during dobutamine (1) or Ca²⁺ infusion (3, 18, 27) has been confirmed in normal Wistar rat hearts. From 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 (3, 18, 27, 28). 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. Our recently proposed index for the left ventricular contractility, equivalent ESP-to-volume ratio at mLVV (eE_max), was calculated from a triangular area equivalent to PVA_mLVV (18, 27, 28). Mean ESV (equals mLVV  V₀) at mean mLVV (0.15 ml/g) (ESV_0.15) was 0.08 ± 0.01 ml/g (n = 20 each). The O₂ cost of left ventricular contractility was the slope of the relationship between PVA-independent VO₂ and eE_max (18, 27, 28).

Logistic time constant. To evaluate the left ventricular relaxation rate or lusitropism, by using our proposed "logistic" time constant (T_L) derived from a logistic model (11) and the conventional "exponential" time constant (T_E), we analyzed left ventricular isovolumic relaxation pressure-time curves at mLVV in OLETF and LETO rats. Both time constants decrease as the heart rate increases and should therefore be compared under the same pacing rate conditions. We compared the time constants of the hearts paced at ~300 beats/min in L-I OLETF versus L-I LETO rats and also compared those of the hearts paced at 240 beats/min in L-II OLETF versus L-II LETO rats.

Western blots for SERCA2. Membrane proteins from the left ventricular myocardium of each heart were isolated as described previously by Yoshida et al. (31). 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. We confirmed that these membrane fractions did not contain any detectable collagens.

Polyacrylamide gel electrophoresis. The left ventricular myocardium from each heart was frozen and stored at 20°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 (10) of Hoh et al. (6).

Mitochondrial respiratory function. A differential centrifugation was used according to the method of Hatefi et al. (5). Immediately after the mitochondrial preparation, oxygen consumption of mitochondria was measured polarographically with an oxygen electrode installed in a closed cell (UC-12, Central; Tokyo, Japan) at 25°C. The incubation medium contained 0.3 M mannitol, 10 mM KH₂PO₄, 2.5 mM MgCl₂, and 0.1 mM EDTA at pH 7.4 in a total of 0.7 ml. A mitochondrial suspension of 0.03 ml (6.27 ± 2.08 mg protein/ml) was first added, and then 0.2 M succinic acid (0.03 ml) was added to the incubation medium as a substrate. One minute later, 10 mM ADP (0.01 ml) was added. The rate of oxygen consumption in state III (state III O₂) and the respiratory control index (RCI) were determined. State III O₂ was calculated from mitochondrial oxygen consumption in nanograms of atom (1 mole O₂ = 2 atoms O) of oxygen consumed per minute per milligram mitochondrial protein during state III respiration. State II O₂ before ADP and state IV O₂ were also calculated. RCI (dimensionless) was taken as the ratio of the rates of oxygen consumption before and after the addition of ADP.

Statistics. Comparison of paired and unpaired individual values was performed by paired and unpaired t-test, respectively. Multiple comparisons were performed by analysis of variance (ANOVA) with post hoc Bonferroni's test or Fisher's protected least-significant difference method. 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

ESPVRs, EDPVRs, and VO₂-PVA relationships in control and during dobutamine or Ca²+ infusion in OLETF and LETO rats. We simultaneously recorded the electrocardiogram, left ventricular pressure, coronary flow, arteriovenous oxygen content difference, and LVV of each heart from the four rat groups at different pacing rates. Left ventricular pressure increased with the increase in LVV. The best-fit ESPVR in the control volume run was an upward convex curve in each heart of L-I OLETF and L-I LETO and L-II OLETF and L-II LETO rats. Although the EDPVR by pacing at 300 beats/min showed a shift upward due to incomplete relaxation in L-II OLETF rats, the EDPVR at 240 beats/min pacing did not shift upward, and the area under the EDPVR is reasonably considered to be zero. An example of control ESPVR and EDPVR in L-II OLETF rats is shown in Fig. 1A. The ESP at a LVV of 0.17 ml/g (ESP_0.17) gradually increased, and the specific best-fit curves for the observed ESP_0.17 and ESP (0 mmHg) at V₀ were obtained with the ESPVR function in the control volume run (Fig. 1B). PVA at a LVV of 0.17 ml/g (PVA_0.17) was then calculated during the first dobutamine (1-4 ml/h) and second Ca²⁺ infusion (1-20 ml/h) in the same heart (Fig. 1B). Mean best-fit parameters, ESP_mLVV, and PVA_mLVV were not significantly different among the four rat groups (Table 1). The mean ESP at mean mLVV (0.15 ml/g) was 76.7 ± 12.4% of the maximum ESP (ESP_max) (n = 20).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Left ventricular (LV) pressure-volume data. LV pressure and volume data during different volume loading run between 0.08 and 0.24 ml/g in control (A) and during dobutamine (Dob, open circles) and Ca2+ (solid circles) infusions (B) at midrange LV volume (mLVV, equalling 0.17 ml/g) in a long-term II (L-II) Otuska Long-Evans Tokushima Fatty (OLETF) rat heart. ESPVR, curved end-systolic pressure-volume relationship. EDPVR, end-diastolic pressure-volume relationship. ESPmLVV, end-systolic pressure at mLVV. PVAmLVV, systolic pressure-volume area at mLVV (hatched area).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Linear relationships of LV control O2 consumption (VO2)-PVA, composite VO2-PVA, and PVA-independent VO2-LV contractility index, equivalent end-systolic pressure-volume ratio at midrange LV volume (eEmax, equalling 0.17 ml/g) during Dob and Ca2+ inotropism runs. LV control linear relationship (thick line connecting small solid circles) between VO2 and PVA and the composite linear relationships between VO2 and PVA (thin line) during Dob (open circles) and Ca2+ (large solid circles)-induced different inotropism runs (A) and linear relationships between PVA-independent VO2 and eEmax during first Dob and second Ca2+ inotropism-runs (B) in the same heart as in Fig. 1.

1

1

1

1

1

1

View larger version (22K): [in this window] [in a new window]   Fig. 3.   A: PVA-independent VO2 per minute in long-term I (L-I) OLETF (n = 6) and age-matched L-I Long-Evana Tokushima Otsuka (LETO, n = 3) and in L-II OLETF (n = 6) and age-matched (L-II) LETO rat hearts (n = 5). *P < 0.05 (unpaired t-test). B: basal metabolic VO2 per minute in L-II OLETF (n = 2) and age-matched L-II LETO rat hearts (n = 3).

Oxygen cost of eE_max for dobutamine and Ca²+. Figure 4A shows that the mean O₂ cost of eE_max for Ca²⁺ showed no significant difference between L-I OLETF and L-I LETO rats or between L-II OLETF and L-II LETO rats. Figure 4B shows that the mean O₂ cost for dobutamine did not show any difference between L-II OLETF and L-II LETO rats. Furthermore, O₂ cost in L-II OLETF and L-II LETO rats did not differ between Ca²⁺ (Fig. 4A) and dobutamine (Fig. 4B). The mean O₂ costs of eE_max for Ca²⁺ (Fig. 4A,a) and dobutamine (Fig. 4B,b) in Wistar rat hearts (n = 14) were similar and neither of them differed from the costs in any other group of hearts (Fig. 4A,a and B,b, our unpublished data).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Mean O2 costs of LV eEmax for Ca2+ and Dob. Mean O2 costs of LV eEmax for Ca2+ (A) in L-I OLETF (n = 6) and age-matched L-I LETO (n = 3), in L-II OLETF (n = 6) and age-matched L-II LETO rat hearts (n = 5), and those for Dob (B) in L-II OLETF (n = 6) and age-matched L-II LETO rat hearts (n = 5). a and b, mean O2 cost of eEmax for Ca2+ (a) and Dob (b) in Wistar rat hearts (n = 14) (unpublished data). *Significantly different (P < 0.01 ANOVA with post hoc Bonferroni's test).

Logistic time constants for the hearts of OLETF and LETO rats. Left ventricular representative isovolumic relaxation pressure-time curves at mLVV are shown in Fig. 5, A and B. T_L and T_E for these curves were 18.2 and 28.9 ms in the L-II OLETF rat (Fig. 5A), respectively, and 13.5 and 19.1 ms in the L-II LETO rat (Fig. 5B), respectively. Mean T_L and T_E at 240 beats/min pacing in L-II OLETF rats were significantly (P < 0.05 unpaired t-test) longer than the constants in L-II LETO rats, but in L-I OLETF rats, neither mean T_L nor T_E at 300 beats/min pacing was significantly different from the corresponding value in L-I LETO rats (Fig. 5C).

View larger version (38K): [in this window] [in a new window]   Fig. 5.   LV representative isovolumic relaxation pressure-time curves. LV representative isovolumic relaxation pressure-time curves obtained with 240 beats/min pacing at midrange LVV (0.17 ml/g in A and 0.13 ml/g in B). Logistic (TL) and exponential (TE) time constants for these curves were 18.2 and 28.9 ms in an L-II OLETF rat (A) and 13.5 and 19.1 ms in an L-II LETO rat (B). Open circles, observed pressure-time data. Solid curves, best-fit logistic curves. Dotted curves, best-fit exponential curves. C: summarized data of TL and TE in L-I OLETF (n = 6) and L-I LETO (n = 3) and L-II OLETF (n = 6) and L-II LETO rat hearts (n = 5). *P < 0.05 (unpaired t-test).

Western blotting of SERCA2. The protein levels of SERCA2 from two hearts in each of the two groups (L-II OLETF and L-II LETO rats) are shown in Fig. 6A. Although the mean protein level of SERCA2 in L-I OLETF rat hearts was 0.82 ± 0.05 (n = 6) and did not differ significantly from other rat hearts, the mean protein level of SERCA2 in L-II OLETF rat hearts was significantly (ANOVA and Bonfferoni's test) lower than that in L-II LETO rat hearts (Fig. 6).

View larger version (49K): [in this window] [in a new window]   Fig. 6.   Immunoblotting of sarco(endo)plasmic reticulum Ca2+- ATPase (SERCA2). Immunoblotting of cardiac SERCA2 (100 kDa) in L-II OLETF and age-matched L-II LETO rat hearts (n = 2 each) and a Wistar rat heart (A). Protein levels of SERCA2 were markedly low in L-II OLETF rat hearts. B: summarized data of SERCA2 expression. *P < 0.001 vs. L-II LETO rat hearts (unpaired t-test).

Myosin isozyme. Myosin isozyme in the left ventricular myocardium was V₃ in L-II OLETF rats (Fig. 7A) and V₁ in L-II LETO rats (Fig. 7B), whereas it was V₁ in L-I OLETF and L-I LETO rats. In adult Wistar rats (32-40 wk), we have previously confirmed a predominance of V₁ (our unpublished data).

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Myosin isozyme. A: myosin isozyme in LV myocardium is V3 in a L-II OLETF rat. B: myosin isozyme is V1 in an age-matched L-II LETO rat.

Mitochondrial respiratory function. Figure 8 summarizes the data on the ADP-to-O ratio (ADP/O ratio, dimensionless), RCI (dimensionless), and state III O₂ (ng atom · min¹ · mg protein¹) of myocardial mitochondria from L-II OLETF (n = 6) and L-II LETO rats (n = 4). Both the ADP/O ratio and state III O₂ in L-II OLETF rat hearts showed a tendency to decrease but to not differ significantly from those in L-II LETO rats, although RCI in L-II OLETF rat hearts was significantly (P < 0.05) smaller than that in L-II LETO rat hearts. State II O₂ and state IV O₂ in L-II OLETF rat hearts also did not differ from those in L-II LETO rats (state II O₂, 141.0 ± 23.6 vs. 121.7 ± 14.8; state IV O₂, 208.1 ± 4.0 vs. 221.1 ± 26.3 ng atom · min¹ · mg protein¹). In L-I OLETF rats (n = 6), the ADP/O ratio, RCI, and state III O₂ did not differ from those in L-I LETO rat hearts (n = 4) (ADP/O ratio, 1.45 ± 0.51 vs. 1.28 ± 0.19; RCI, 2.82 ± 0.32 vs. 2.82 ± 0.13; state III O₂, 440.8 ± 146.0 vs. 471.4 ± 156.0). The myocardial mitochondrial ADP/O ratio, RCI, and state III O₂ in L-II LETO rats (see Fig. 8) did not differ from those in normal Wistar rats (n = 7; ADP/O ratio, 1.28 ± 0.22; RCI, 3.59 ± 0.36; state III O₂, 425.7 ± 26.5).

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Myocardial mitochondrial respiratory function. ADP-to-O ratio (ADP/O) ratio (A), respiratory control index (RCI, B), and oxygen consumption at state III (C) in L-II OLETF (n = 6) and age-matched L-II LETO rat hearts (n = 4). *P < 0.05 (unpaired t-test).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

L-I OLETF rats (40-46 wk) showed no slowing of the left ventricular relaxation rate and had unchanged expression of SERCA2 and V₁ myosin isozyme. It seems likely that left ventricular diastolic dysfunction does not occur, although the left ventricular diastolic dysfunction associated with increased left ventricular collagen content has been observed in OLETF rats at a similar age (13). The discrepancy between our results and those of a previous study (13) may derive from the difference between the methods for evaluating left ventricular diastolic function (i.e., our direct measurements vs. indirect, noninvasive measurements) and the difference between preparations (excised hearts vs. in situ hearts). In in situ hearts, the interferences of preload and afterload with left ventricular functions may result in left ventricular diastolic dysfunction at the prediabetic stage (15-27 wk) as shown by Mizushige et al. (13).

In L-II (62-66 wk) OLETF rats, we found diabetic cardiomyopathy with lusitropic dysfunction and with unchanged coronary flow and coronary flow reserve, but up to 66 wk, no systolic dysfunction was observed at 240 beats/min pacing, as shown by comparison with L-II LETO rats. The left ventricular lusitropic dysfunction in L-II OLETF rats may be related to lower protein levels of SERCA2. The functional defect in SERCA2 also has been observed in streptozotocin-induced DM rats, but this defect has been reported not to be due to changes at the transcriptional or translational level (33). The discrepancy between our results and those in the previous report may be explained by the different DM type and time course (62-66 wk of age in L-II OLETF rats vs. diabetic with streptozotocin for 3-5 wk).

On the other hand, Mizushige et al. (13) suggested that the left ventricular diastolic dysfunction was associated with an increase in cardiac collagen content at the prediabetic stage (15-27 wk). Although the ratio of extracellular matrix (including collagen) to cardiac myocyte content had further increased at 31-47 wk in both OLETF and LETO rats, there was no significant difference between the two groups in this ratio (13), but in the present study, L-II OLETF rats at 62-66 wk showed a significant slowing of the left ventricular relaxation rate compared with that in L-II LETO rats. Therefore, it seems likely that left ventricular diastolic dysfunction in the L-II OLETF rats is not simply related to the increased collagen content but is related to the decreased protein level of SERCA2. The decreased protein level of SERCA2 did not result from the increased collagen content, because the cellular membrane fractions did not contain any collagen.

Curved ESPVR, linear VO₂-PVA relationship, and oxygen cost of PVA. No significant differences were found between L-II OLETF and L-II LETO rats in ESP_mLVV or PVA_mLVV. Each mean slope of the VO₂-PVA relationship (O₂ cost of PVA) in the four rat groups is almost the same as that previously reported (3, 4, 18, 27, 28). In hyperthyroid rabbits, where myosin isozyme is V₁, the increased O₂ cost of PVA is observed (2). In hyperthyroid dogs, where myosin isozyme remains V₃, unaltered O₂ cost of PVA is observed (21). Therefore, it seems likely that there exists a correlation between O₂ cost of PVA and myosin isozyme, but the present results showing unchanged O₂ cost of PVA indicate that the efficiency of chemomechanical energy transduction (the inverse of O₂ cost of PVA) was unchanged among the four rat groups, despite the different myosin isozymes. Mitochondrial oxidative phosphorylation appeared to be unchanged between L-I OLETF and L-I LETO rat hearts and between L-II OLETF and L-II LETO rat hearts. This finding further supports the unchanged efficiency of chemomechanical energy transduction.

PVA-independent VO₂. All mean VO₂ intercepts of the VO₂-PVA relationship (PVA-independent VO₂) in the four rat groups were similar, and this is in agreement with our previous results (3, 4, 18, 27, 28). The PVA-independent VO₂ corresponds primarily to the VO₂ for total Ca²⁺ handling in the excitation-contraction coupling and that for basal metabolism (3, 4, 27, 30). The significantly decreased PVA-independent VO₂ per minute with unchanged basal metabolism in L-II OLETF rat hearts suggests a decrease in VO₂ for total Ca²⁺ handling in the excitation-contraction coupling per minute. VO₂ for Ca²⁺ handling in the excitation-contraction coupling is primarily consumed by SERCA2 (4, 26). Therefore, the decrease in VO₂ for Ca²⁺ handling indicates a decrease in O₂ consumption by SERCA2 per minute. This is causally related to the decreased pacing rate so as not to cause incomplete left ventricular relaxation. This decrease in the pacing rate seems to be due to dysfunction of SERCA2. In fact, in L-II OLETF rat hearts, depressed expression of SERCA2 was found.

Oxygen cost of left ventricular eE_max. Dobutamine is known to stimulate -adrenergic receptors and thereby to activate protein kinase A (17), resulting in enhanced Ca²⁺ handling in the excitation-contraction coupling, whereas Ca²⁺ directly enhances the Ca²⁺ handling by increasing Ca²⁺ influx. Although the mechanism of enhancing the Ca²⁺ handling is different, the paired O₂ costs of left ventricular contractility (changes in total Ca²⁺ handling VO₂ in the excitation-contraction coupling per beat against unit change in left ventricular contractility) for Ca²⁺ and for dobutamine were similar in each L-II OLETF and L-II LETO rat heart. This result is consistent with that for Wistar rats (1). In non-insulin-dependent diabetic hearts, Schaffer et al. (19) could not find any changes in the myocardial -receptor population but reported reduced responsiveness to a -adrenergic agonist. Nevertheless, the O₂ cost of left ventricular contractility for dobutamine was similar for L-II OLETF and L-II LETO rats. Furthermore, the O₂ cost of left ventricular contractility for Ca²⁺ was similar for L-I OLETF and L-I LETO rats and for L-II OLETF and L-II LETO rats. All these results indicate that changes in the total Ca²⁺ handling VO₂ in the excitation-contraction coupling against unit change in left ventricular contractility induced by Ca²⁺ or dobutamine did not alter on a beat-to-beat basis in the present long-lasting, severe type 2 diabetic rat hearts. Decreasing the pacing rate from 300 to 240 beats/min made it possible to maintain unchanged systolic function and unchanged O₂ cost of left ventricular contractility in L-II OLETF rats.

Left ventricular diastolic dysfunctions in L-II OLETF rat hearts. In L-II OLETF rat excised hearts, at a lower pacing rate (240 beats/min) than normal (300 beats/min), a significant slowing of the left ventricular relaxation rate was observed, but systolic function was unchanged. These results indicate that the present long-lasting, severe type 2 diabetic rat hearts do not show terminal diabetic cardiomyopathy. In in situ hearts, the preload and afterload and hormonal and neuronal influences could interfere with left ventricular function, resulting in left ventricular diastolic dysfunction at the prediabetic stage (15-27 wk) as shown by Mizushige et al. (13). In contrast, in the present cross-circulated (blood-perfused) excised hearts, left ventricular function can be characterized by myocardium per se. Therefore, it seems unlikely that the present long-lasting, severe diabetic rat hearts causes terminal diabetic cardiomyopathy.