Left ventricular function of isoproterenol-induced hypertrophied rat hearts perfused with blood: mechanical work and energetics.

We investigated left ventricular (LV) mechanical work and energetics in the cross-circulated (blood-perfused) isoproterenol [Iso 1.2 mg x kg(-1).day(-1) for 3 days (Iso3) or 7 days (Iso7)]-induced hypertrophied rat heart preparation under isovolumic contraction-relaxation. We evaluated pressure-time curves per beat, end-systolic pressure-volume and end-diastolic pressure-volume relations, and myocardial O(2) consumption per beat (Vo(2))-systolic pressure-volume area (PVA; a total mechanical energy per beat) linear relations at 240 beats/min, because Iso-induced hypertrophied hearts failed to completely relax at 300 beats/min. The LV relaxation rate at 240 beats/min in Iso-induced hypertrophied hearts was significantly slower than that in control hearts [saline 24 microl/day for 3 and 7 days (Sa)] with unchanged contraction rate. The Vo(2)-intercepts (composed of basal metabolism and Ca(2+) cycling energy consumption in excitation-contraction coupling) of Vo(2)-PVA linear relations were unchanged associated with their unchanged slopes in Sa, Iso3, and Iso7 groups. The oxygen costs of LV contractility were also unchanged in all three groups. The amounts of expression of sarcoplasmic reticulum Ca(2+)-ATPase, phospholamban (PLB), phosphorylated-Ser(16) PLB, phospholemman, and Na(+)-K(+)-ATPase are significantly decreased in Iso3 and Iso7 groups, although the amount of expression of NCX1 is unchanged in all three groups. Furthermore, the marked collagen production (types I and III) was observed in Iso3 and Iso7 groups. These results suggested the possibility that lowering the heart rate was beneficial to improve mechanical work and energetics in isoproterenol-induced hypertrophied rat hearts, although LV relaxation rate was slower than in normal hearts.

An isoform of Arabidopsis myosin XI interacts with small GTPases in its C-terminal tail region.

Myosin XI, a class of myosins expressed in plants is believed to be responsible for cytoplasmic streaming and the translocation of organelles and vesicles. To gain further insight into the translocation of organelles and vesicles by myosin XI, an isoform of Arabidopsis myosin XI, MYA2, was chosen and its role in peroxisome targeting was examined. Using the yeast two-hybrid screening method, two small GTPases, AtRabD1 and AtRabC2a, were identified as factors that interact with the C-terminal tail region of MYA2. Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner. Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue. It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a.

N-Methyl-D-aspartate receptors contribute to the maintenance of dopaminergic neurons in rat midbrain slice cultures.

Excitatory neuronal activity produces beneficial influences on neuronal survival under several circumstances. We show that cultivation of rat midbrain slices in the presence of elevated extracellular Mg(2+) resulted in a marked decrease in the number of dopaminergic neurons. The effect was prominent when Mg(2+) was added to the medium during the first week of cultivation. Chronic treatment with antagonists of N-methyl-D-aspartate receptors such as 2-amino-5-phosphonovaleric acid, MK-801 and ifenprodil also resulted in a marked loss of dopaminergic neurons, whereas nicotinic receptor antagonists showed no effect. The effect of MK-801 was abolished by chronic depolarization by elevated extracellular K(+), or by application of forskolin or dibutyryl cyclic AMP. Thus, tonic activation of N-methyl-D-aspartate receptors driven by neuronal activity may play an important role in the maintenance of dopaminergic neurons.

Oxygen costs of left ventricular contractility for dobutamine and Ca(2+) in normal rat hearts and the cost for dobutamine in Ca(2+) overload-induced failing hearts.

The aim of the present study was to compare the oxygen (O(2)) cost of left ventricular (LV) contractility (equivalent maximal elastance; eEmax, an index for contractility) for dobutamine (a beta-receptor stimulant) with that for calcium (Ca(2+)) in normal rat hearts and to assess the O(2) cost of LV eEmax for dobutamine in Ca(2+) overload-induced failing rat hearts. The mean O(2) cost of LV eEmax (x10(-4) microl O(2) x beat(-1) x mmHg(-1) x ml x g(-2)) for Ca(2+) was 1.30+/-0.37 in 12 normal hearts, and for dobutamine it was 1.26+/-0.30 in eight different normal hearts. In the same five normal hearts, the mean O(2) cost of LV eEmax for dobutamine was 1.15+/-0.27, and for Ca(2+) it was 0.81+/-0.36. These mean values showed no significant differences between Ca(2+) and dobutamine. In five Ca(2+) overload-induced failing hearts, the mean O(2) cost of LV eEmax for Ca(2+) could not be assessed, but the mean O(2) cost of LV eEmax for dobutamine was 1.04+/-0.40. This mean value for dobutamine did not differ significantly from those (see above 1.26+/-0.30 or 1.15+/-0.27) in the normal hearts. The present results indicate, in terms of the coupling of mechanical work and energetics of the heart, that the total Ca(2+) handling VO(2) in excitation-contraction coupling against unit LV contractility change for dobutamine in the contractile failing hearts does not differ from that in the normal hearts. This suggests that in the Ca(2+) overload-induced contractile failing hearts, there were no changes in the sensitivity of the contractile machinery for Ca(2+), in the Ca(2+)/ATP in the total Ca(2+) handling, and in the ATP/VO(2) in the mitochondria.

Effects of formaldehyde on cardiovascular system in in situ rat hearts.

The aim of the present study was to examine the effects of formaldehyde solution on rat left ventricular function and compare it with those in hypertrophic hearts treated with isoproterenol by pressure-volume measurements with the catheter method. After 20-30 min. of intravenous infusion of 3.7% formaldehyde solution (FA) at 10 µl (3.7 mg)/kg/min, normal and hypertrophic hearts showed significant decreases in left ventricle end-systolic pressure (ESP), heart rate and cardiac output per minute, indicating an acute pumping failure. Hypertrophic hearts showed significantly smaller ESP, stroke volumes and cardiac output than those in normal hearts. Systolic pressure-volume area at midrange left ventricular volume (PVA(mLVV) : a mechanical work capability index) was significantly smaller than that in normal hearts and per cent of mean PVA(mLVV) versus pre-infusion mean value in hypertrophic hearts was significantly decreased compared to normal hearts 30 min. after FA infusion. The marked decrease in pH, base excess and no changes in PaO2 and PaCO2 suggest metabolic acidosis. The correction of metabolic acidosis with 9% NaHCO3 did not influence on the acute pumping failure, indicating that metabolic acidosis did not cause it. Ultrastructural observations revealed marked dilation of the sarcoplasmic reticulum with intact sarcolemmal membranes and no disintegration of muscle myofibrils. Ryanodine receptors and calcium (Ca²âº) pumps (SERCA2A) located in the sarcoplasmic reticulum have major roles in the cytosolic Ca²âº handling. Taken together, acute pumping failure by FA may derive from the impairment of Ca²âº handling in the cardiac excitation-contraction coupling.

Increased O2 consumption in excitation-contraction coupling in hypertrophied rat heart slices related to increased Na+ -Ca2+ exchange activity.

The goal of our study was to evaluate the origin of the increased O(2) consumption in electrically stimulated left ventricular slices of isoproterenol-induced hypertrophied rat hearts with normal left ventricular pressure. O(2) consumption per minute (mVO(2)) of mechanically unloaded left ventricular slices was measured in the absence and presence of 1-Hz field stimulation. Basal metabolic mVO(2), i.e., mVO(2) without electrical stimulation, was significantly smaller, but mVO(2) for the total Ca(2+) handling in excitation-contraction coupling (E-C coupling mVO(2)), i.e., delta mVO(2) (=mVO(2) with stimulation - mVO(2) without stimulation), was significantly larger in the hypertrophied heart. Furthermore, the fraction of E-C coupling mVO(2) was markedly altered in the hypertrophied heart. Namely, mVO(2) consumed by sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) was depressed by 40%; mVO(2) consumed by the Na(+)/K(+)-ATPase (NKA)-Na(+)/Ca(2+) exchange (NCX) coupling was increased by 100%. The depressed mVO(2) consumption by SERCA2 was supported by lower protein expressions of phosphorylated-Ser(16) phospholamban and SERCA2. The increase in NKA-NCX coupling mVO(2) was supported by marked augmentation of NCX current. However, the increase in NCX current was not due to the increase in NCX1 protein expression, but was attributable to attenuation of the intrinsic inactivation mechanisms. The present results demonstrated that the altered origin of the increased E-C coupling mVO(2) in hypertrophy was derived from decreased SERCA2 activity (1ATP: 2Ca(2+)) and increased NCX activity coupled to NKA activity (1ATP: Ca(2+)). Taken together, we conclude that the energetically less efficient Ca(2+) extrusion pathway evenly contributes to Ca(2+) handling in E-C coupling in the present hypertrophy model.

Rescue of Ca2+ overload-induced left ventricular dysfunction by targeted ablation of phospholamban.

In failing hearts, a deficiency in sarco(endo)plasmic reticulum Ca2+ -ATPase (SERCA)2a results in abnormal Ca2+ handling and diminished contraction. In addition, a decrease in the phosphorylation of phospholamban (PLB) has been reported. Gene transfer of antisense PLB (asPLB) can improve contractile function in the failing human myocardium. Gene transfer of SERCA2a improves survival and the energy potential in failing hearts. The aim of present study was to evaluate whether enhancement of SERCA2a function prevents acute Ca2+ overload-induced left ventricular (LV) dysfunction in rat hearts. We ablated PLB using adenoviral gene transfer of asPLB by a new and less invasive gene delivery method, which involved a percutaneous technique. Experiments were performed on 13 excised cross-circulated rat hearts: 5 rats underwent sham operations, 4 rats underwent gene transfer of the reporter gene beta-galactosidase (Ad.beta-gal), and 4 rats underwent gene transfer of asPLB (Ad.asPLB). After clearance of high Ca2+ infused into the coronary, there was LV contractile dysfunction associated with the decreased myocardial O2 consumption per beat (Vo2) intercept (equal to decreased Vo2 for Ca2+ handling in excitation-contraction coupling) of the Vo2-systolic pressure-volume area (PVA; total mechanical energy per beat) linear relation in the hearts that underwent sham operation and had been infected with Ad.beta-gal. Hearts that had been infected with Ad.asPLB were rescued from LV contractile dysfunction associated with an unchanged Vo2 intercept of the Vo2-PVA linear relation. We conclude that SERCA2a function enhanced by adenoviral gene transfer of asPLB prevents Ca2+ overload-induced LV contractile dysfunction in terms of mechanical work and especially energetics.

Isoproterenol-induced hypertrophied rat hearts: does short-term treatment correspond to long-term treatment?

In consideration of clinical implications, it is often complained that short-term experimental diseased heart models do not mimic long-term diseased hearts that are often clinically encountered. The aim of the present study was (i) to compare the left ventricular function between rat cardiac hypertrophy models treated with isoproterenol for 3 days (Iso 3d) and 7 days (Iso 7d) by pressure-volume measurements with a catheter method, and (ii) to follow up the left ventricular function in the same model treated with Iso up to 16 weeks with a less-invasive echocardiography. An infusion of either Iso (1.2 mg x kg(-1) x day(-1) for 3 days-16 weeks) or vehicle (saline 24 microl x day(-1) for 3 days-16 weeks; Sa group) was performed by subcutaneously implanting an osmotic minipump. There were no significant differences in the systolic pressure-volume area at midrange left ventricular volume (PVA(mLVV): a mechanical work capability index) between Iso 3d and 7d groups, though PVA(mLVV) in both groups was significantly reduced from that in the Sa group. From echocardiography, the left ventricular function of the hypertrophy models at 3 days, 1 week, and 2 weeks was unchanged, but the model at a term longer than 4 weeks resulted in prolonged systolic failure. The results indicated that (i) no marked differences in the left ventricular mechanical work capability were found between the Iso 3d and 7d groups, and that (ii) only a 3-day Iso infusion induced the hypertrophy model similar in shape and function to that induced by a 2-week Iso infusion. We concluded that the 3-day model was sufficient.

Effects of a novel histone deacetylase inhibitor, N-(2-aminophenyl) benzamide, on a reversible hypertrophy induced by isoproterenol in in situ rat hearts.

The aim of the present study was performed to determine whether a novel histone deacetylase (HDAC) inhibitor, N-(2-aminophenyl)-4-{[benzyl(2-hydroxyethyl)amino]methyl} benzamide (K-183), prevents a reversible cardiac hypertrophy induced by isoproterenol and improves left ventricular (LV) dysfunction in rats. Either isoproterenol or vehicle was infused for 3 days by osmotic minipump. One hour prior to the implantation of isoproterenol, K-183 or trichostatin A (TSA) was injected twice a day for 3 days. We recorded continuous LV pressure-volume (P-V) loops of in situ hearts one hour after removal of the osmotic minipump. LV work capability (systolic P-V area at midrange LV volume: PVA(mLVV)) and hemodynamics were evaluated. K-183 per se induced neither cardiac hypertrophy nor collagen production. Although K-183 did not prevent the hypertrophy, where PVA(mLVV) remained decreased, K-183, differently from TSA, significantly attenuated the decrease of cardiac output and the increase of effective arterial elastance in the hypertrophied heart. These results indicate that the novel HDAC inhibitor K-183 has some beneficial effects on hemodynamics, although K-183 has no effects of anti-hypertrophic modalities.

Transcoronary gene transfer of SERCA2a increases coronary blood flow and decreases cardiomyocyte size in a type 2 diabetic rat model.

The Otsuka Long-Evans Tokushima fatty rat is an animal model of Type 2 diabetes mellitus (DM), which is characterized by diastolic dysfunction associated with decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a). The aim of this study was to examine whether gene transfer of SERCA2a can influence coronary blood flow and cardiomyocyte diameter in this model. DM rats were injected with adenovirus carrying SERCA2a (DM+SERCA) or beta-galactosidase gene (DM+betaGal). Coronary blood flow was measured in cross-circulated excised hearts 3 days after infection. Although in all groups coronary blood flow remained unchanged even if left ventricular (LV) volume or intracoronary Ca(2+) infusion was increased, the DM+SERCA group showed a sustained increase in coronary blood flow compared with the other groups. This result suggests that the sustained high coronary blood flow is a specific response in SERCA2a-overexpressed hearts. Although the LV weight-to-body weight ratio (LV/BW) and cardiomyocyte diameter were higher in the DM and DM+betaGal groups than in the non-DM group, in the DM+SERCA group, these measurements were restored to non-DM size. The percentages of collagen area in the three DM groups was significantly higher than results shown in non-DM rats, and there were no significant differences in collagen area percentage among the three DM groups. These results suggest that a lowered LV/BW by SERCA2a overexpression is due mainly to reduced size of cardiomyocytes without any changes in collagen area percentage. In conclusion, in DM failing hearts, SERCA2a gene transfer can increase coronary blood flow and reduce cardiomyocyte size without reduction in collagen production.

Na+/Ca2+ exchange inhibition protects the rat heart from ischemia-reperfusion injury by blocking energy-wasting processes.

We have recently reported that exposure of rat hearts to high Ca(2+) produces a Ca(2+) overload-induced contractile failure in rat hearts, which was associated with proteolysis of alpha-fodrin. We hypothesized that contractile failure after ischemia-reperfusion (I/R) is similar to that after high Ca(2+) infusion. To test this hypothesis, we investigated left ventricular (LV) mechanical work and energetics in the cross-circulated rat hearts, which were subjected to 15 min global ischemia and 60 min reperfusion. Sixty minutes after I/R, mean systolic pressure-volume area (PVA; a total mechanical energy per beat) at midrange LV volume (mLVV) (PVA(mLVV)) was significantly decreased from 5.89 +/- 1.55 to 3.83 +/- 1.16 mmHg.ml.beat(-1).g(-1) (n = 6). Mean myocardial oxygen consumption per beat (Vo(2)) intercept of (Vo(2)-PVA linear relation was significantly decreased from 0.21 +/- 0.05 to 0.15 +/- 0.03 microl O(2).beat(-1).g(-1) without change in its slope. Initial 30-min reperfusion with a Na(+)/Ca(2+) exchanger (NCX) inhibitor KB-R7943 (KBR; 10 micromol/l) significantly reduced the decrease in mean PVA(mLVV) and Vo(2) intercept (n = 6). Although Vo(2) for the Ca(2+) handling was finally decreased, it transiently but significantly increased from the control for 10-15 min after I/R. This increase in Vo(2) for the Ca(2+) handling was completely blocked by KBR, suggesting an inhibition of reverse-mode NCX by KBR. alpha-Fodrin proteolysis, which was significantly increased after I/R, was also significantly reduced by KBR. Our study shows that the contractile failure after I/R is similar to that after high Ca(2+) infusion, although the contribution of reverse-mode NCX to the contractile failure is different. An inhibition of reverse-mode NCX during initial reperfusion protects the heart against reperfusion injury.

Calpain inhibitor-1 protects the rat heart from ischemia-reperfusion injury: analysis by mechanical work and energetics.

We hypothesized that calpain inhibitor-1 protected left ventricular (LV) function from ischemia-reperfusion injury by inhibiting the proteolysis of alpha-fodrin. To test this hypothesis, we investigated the effect of calpain inhibitor-1 on LV mechanical work and energetics in the cross-circulated rat hearts that underwent 15-min global ischemia and 60-min reperfusion (n = 9). After ischemia-reperfusion with calpain inhibitor-1, mean end-systolic pressure at midrange LV volume and systolic pressure-volume area (PVA) at midrange LV volume (total mechanical energy per beat) were hardly changed, although they were significantly (P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. Mean myocardial oxygen consumption per beat (Vo(2)) intercepts (PVA-independent Vo(2); Vo(2) for the total Ca(2+) handling in excitation-contraction coupling and basal metabolism) of Vo(2)-PVA linear relations were also unchanged after ischemia-reperfusion with calpain inhibitor-1, although they were significantly (P < 0.01) decreased after ischemia-reperfusion without calpain inhibitor-1. There were no significant differences in O(2) costs of LV PVA and contractility among the hearts in control (or normal) postischemia-reperfusion and postischemia-reperfusion with calpain inhibitor-1. Western blot analysis of alpha-fodrin and the immunostaining of 150-kDa products of alpha-fodrin confirmed that calpain inhibitor-1 almost completely protected the proteolysis of alpha-fodrin. Our results indicate that calpain inhibitor-1 prevents the heart from ischemia-reperfusion injury associated with the impairment of total Ca(2+) handling by directly inhibiting the proteolysis of alpha-fodrin.

Detrimental effects after dobutamine infusion on rat left ventricular function: mechanical work and energetics.

We have previously reported that continuous infusion of dobutamine into the coronary artery induces positive inotropic effects but induces no detrimental effects in cross-circulated, excised normal rat hearts and even in Ca2+ overload-induced contractile failing rat hearts. However, we hypothesized that some detrimental effects on left ventricular (LV) function are induced after continuous dobutamine infusion and the following clearance of blood dobutamine, as is the case after beta-adrenergic receptor stimulation. To test this hypothesis, we investigated LV mechanical work and energetics in the same type of preparations that underwent continuous dobutamine infusion and clearance of blood dobutamine. We found that both mean end-systolic pressure and systolic pressure-volume area (PVA; a measure of total mechanical energy per beat) at midrange LV volume were significantly (P < 0.01) decreased. The mean myocardial oxygen consumption per beat intercept, which is composed of for the total Ca2+ handling in excitation-contraction coupling and basal metabolism, of the and PVA linear relation was also significantly (P < 0.05) decreased (n=8). The mean slope of the linear relation was unchanged in such hearts. Post-dobutamine basal metabolism was unchanged (n = 5 of the 8 hearts). The moderate proteolysis of a cytoskeleton protein, alpha-fodrin was identified (n = 7 of the 8 hearts with the decreased intercept), after clearance of blood dobutamine. In agreement with our hypothesis, the detrimental effect of the post-beta-adrenergic receptor stimulation was induced by a moderate concentration of dobutamine; we found systolic dysfunction due to the impairment of Ca2+ handling in excitation-contraction coupling in the rat LV and proteolysis of a cytoskeleton protein, alpha-fodrin.

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

We hypothesized that cardiac dysfunction in hypothyroidism is mainly caused by the impairment of Ca(2+) 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 Ca(2+) handling and for basal metabolism. The oxygen costs of LV contractility for Ca(2+) and for dobutamine in the hypothyroid rats did not differ from those in age-matched normal rats. The expression of sarco(endo)plasmic reticulum Ca(2+)-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 Ca(2+) uptake via SERCA2. We conclude that the impairment of Ca(2+) uptake plays an important role in the pathogenesis of cardiac dysfunction in hypothyroidism.

Left ventricular mechanoenergetics after hyperpolarized cardioplegic arrest by nicorandil and after depolarized cardioplegic arrest by KCl.

We hypothesized that there are no differences in left ventricular (LV) mechanoenergetics between after hyperpolarized cardioplegic arrest by nicorandil (nicorandil arrest) and after depolarized one by high potassium chloride (KCl arrest). The aim of the present study was to test this hypothesis using LV curved end-systolic pressure-volume relation (ESPVR) and linear pressure-volume area (PVA)-myocardial oxygen consumption per beat (VO2) relation. All hearts underwent 30 min global ischemia (30 degrees C) after infusion of 5 ml of cardioplegia. Cardioplegia consisted of either 30 mmol/l KCl (7 hearts) or nicorandil (100 micromol/l) in Tyrode solution (6 hearts). After a 30-min blood reperfusion, ESPVR and VO2-PVA relation were assessed again. Mean end-systolic pressure (ESP(mLVV)) and mean PVA at midrange LV volume (PVA(mLVV)) significantly (P < 0.05) decreased to 79.1 +/- 13.4% and 85.4 +/- 17.1% of control after KCl arrest and to 85.3 +/- 14.8% and 86.4 +/- 16.9% of control after nicorandil arrest. There were no significant differences in both decreases of mean ESP(mLVV) and PVA(mLVV) between each arrest. The slopes of VO2-PVA relations were also unchanged after each arrest. There was a significant (P < 0.005) difference in the decreases of mean VO2 intercepts of VO2-PVA relations between post-KCl arrest (73.9 +/- 8.2% of control) and post-nicorandil arrest (99.2 +/- 10.1% of control), however. Proteolysis of alpha-fodrin due to Ca2+ overload was significantly marked after KCl arrest. The present results indicate that the total calcium handling in excitation-contraction coupling is transiently impaired after KCl arrest, whereas it is unchanged after nicorandil arrest. This suggests the possibility that nicorandil is a better cardioplegia than KCl.