Mechanism underlying the negative inotropic effect in rat left ventricle in hyperthermia: the role of TRPV1.

We have previously reported that the negative inotropic effects of hyperthermia (42 °C) on left ventricular (LV) mechanoenergetics using the excised, cross-circulated rat heart model. Here, we investigated the role of TRPV1 on LV mechanoenergetics in hyperthermia. We analyzed the LV end-systolic pressure-volume relation (ESPVR) and the linear relation between the myocardial oxygen consumption per beat (VO2) and the systolic pressure-volume area (PVA; a total mechanical energy per beat) during infusion of capsazepine (CPZ) in hyperthermia, or capsaicin (Cap) under 300 bpm pacing. LV ESP decreased in each LV volume and the resultant downward-shift of LV ESPVR was suppressed by CPZ infusion in hyperthermia-hearts. In Cap-treated hearts, LV ESPVR shifted downward from the control ESPVR, similar to hyperthermia-hearts. The slopes of VO2-PVA relationship were unchanged. The VO2 intercepts in hyperthermia-hearts did not decrease because of decreased E-C coupling VO2, and inversely increased basal metabolic VO2, which was suppressed by CPZ, though the VO2 intercepts in Cap-treated hearts significantly decreased. The levels of phosphorylated phospholamban at serine 16 decreased significantly in hyperthermia-hearts, as well as Cap-treated hearts. These results indicate that a Cap-induced decrease in the LV contractility, like in cases of hyperthermia, are due to the down-regulation of the total calcium handling in E-C coupling, suggesting that negative inotropic effect in hyperthermia-heart is, at least in part, mediated through TRPV1 signaling pathway.

Confocal and multiphoton calcium imaging of the enteric nervous system in anesthetized mice.

Most imaging studies of the enteric nervous system (ENS) that regulates the function of the gastrointestinal tract are so far performed using preparations isolated from animals, thus hindering the understanding of the ENS function in vivo. Here we report a method for imaging the ENS cellular network activity in living mice using a new transgenic mouse line that co-expresses G-CaMP6 and mCherry in the ENS combined with the suction-mediated stabilization of intestinal movements. With confocal or two-photon imaging, our method can visualize spontaneous and pharmacologically-evoked ENS network activity in living animals at cellular and subcellular resolutions, demonstrating the potential usefulness for studies of the ENS function in health and disease.

The energy-saving effect of a new myosin activator, omecamtiv mecarbil, on LV mechanoenergetics in rat hearts with blood-perfused isovolumic contraction model.

A novel myosin activator, omecamtiv mecarbil (OM), is a cardiac inotropic agent with a unique new mechanism of action, which is thought to arise from an increase in the transition rate of myosin into the actin-bound force-generating state without increasing calcium (Ca2+) transient. There remains, however, considerable controversy about the effects of OM on cardiac contractility and energy expenditure. In the present study, we investigated the effects of OM on left ventricular (LV) mechanical work and energetics, i.e., mechanoenergetics in rat normal hearts (CTL) and failing hearts induced by chronic administration of isoproterenol (1.2 mg/kg/day) for 4 weeks (ISO-HF). We analyzed the LV end-systolic pressure-volume relation (ESPVR) and the linear relation between the myocardial oxygen consumption per beat (VO2) and systolic pressure-volume area (PVA; a total mechanical energy per beat) in isovolumically contracting rat hearts at 240- or 300-bpm pacing in the absence or presence of OM. OM did not change the ESPVR in CTL and ISO-HF. OM, however, significantly decreased the slope of VO2-PVA relationship in both CTL and ISO-HF, and significantly increased the mean VO2 intercept without changes in basal metabolism in ISO-HF. These results suggested that OM improved the oxygen cost of PVA (contractile efficiency) with the unchanged LV contractility in both CTL and ISO-HF but increased VO2 for Ca2+ handling in excitation-contraction (E-C) coupling in ISO-HF. We concluded that OM improves contractile efficiency in normal and failing hearts but increases O2 consumption of Ca2+ handling in failing hearts in isovolumically contracting rat model.

Left ventricular mechanoenergetics in excised, cross-circulated rat hearts under hypo-, normo-, and hyperthermic conditions.

We investigated the effects of altering cardiac temperature on left ventricular (LV) myocardial mechanical work and energetics using the excised, cross-circulated rat heart model. We analyzed the LV end-systolic pressure-volume relationship (ESPVR) and linear relationship between myocardial oxygen consumption per beat (VO2) and systolic pressure-volume area (PVA; total mechanical energy per beat) in isovolumically contracting rat hearts during hypo- (32 °C), normo- (37 °C), and hyperthermia (42 °C) under a 300-beats per minute pacing. LV ESPVR shifted downward with increasing cardiac temperature. The VO2-PVA relationship was superimposable in these different thermal conditions; however, each data point of VO2-PVA shifted left-downward during increasing cardiac temperature on the superimposable VO2-PVA relationship line. VO2 for Ca2+ handling in excitation-contraction coupling decreased, which was associated with increasing cardiac temperature, during which sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity was suppressed, due to phospholamban phosphorylation inhibition, and instead, O2 consumption for basal metabolism was increased. The O2 cost of LV contractility for Ca2+ also increased with increasing cardiac temperature. Logistic time constants evaluating LV relaxation time were significantly shortened with increasing cardiac temperature related to the acceleration of the detachment in cross-bridge (CB) cycling, indicating increased myosin ATPase activity. The results suggested that increasing cardiac temperature induced a negative inotropic action related to SERCA activity suppression in Ca2+ handling and increased myosin ATPase activity in CB cycling. We concluded that thermal intervention could modulate cardiac inotropism by changing CB cycling, Ca2+ handling, and basal metabolism in rat hearts.

Methods for the Preparation of an Excised, Cross-Circulated Rat Heart.

The Emax-Pressure-Volume Area (PVA)-VO2 framework proposed by Dr. Suga for canine hearts has dramatically advanced the field of cardiac mechanical work and energetics, i.e., mechanoenergetics. He and his collaborators investigated mechanoenergetics in the left ventricle (LV) of excised, cross-circulated canine heart preparations. We instituted the excised cross-circulated rat whole heart preparations and found a curvilinear end-systolic pressure-volume relation (ESPVR) in the rat LV, in contrast to the linear ESPVR in canine, rabbit, and human LVs. Although Emax, the slope of the linear ESPVR, could be used as an index of LV contractility, it was not applicable for evaluating LV contractility in the rat LV. Thus, we proposed a new index of contractility, equivalent Emax (eEmax) in the rat LV. We also found a linear VO2-PVA relationship in the rat LV. Here, we introduce the methods for the preparation of excised, cross-circulated rat whole hearts and the eEmax-PVA-VO2 framework in the rat LV. Using this method, we can obtain accurate LV volume and myocardial O2 consumption in real time for estimating cardiac mechanoenergetics, which is very challenging in in vivo experiments.

Activation of 5-HT4 receptors facilitates neurogenesis from transplanted neural stem cells in the anastomotic ileum.

An orally administered serotonin-4 (5-HT4) receptor agonist, mosapride citrate (MOS), promotes enteric neurogenesis in anastomoses after gut surgery. We performed gut surgery and transplanted 2 × 10(5) neural stem cells (NSCs) from the embryonic central nervous system after marking them with the cell linker, PKH26. We found that neurons differentiated from transplanted NSCs (PKH [+]) and newborn enteric neurons differentiated from mobilized (host) NSCs (YFP [+]) in the deep granulation tissue of the anastomotic ileum. MOS significantly increased the number of PKH (+) and YFP (+) neurons by 2.5-fold (P < 0.005) (n = 4). The distribution patterns of PKH (+) neurons and YFP (+) neurons were similar along the depth of the anastomosis. A 5-HT4 receptor antagonist, SB-207266, abolished these effects of MOS (n = 4). Our results indicate that neurogenesis from transplanted NSCs is potentiated by activation of 5-HT4 receptors. Thus, a combination of drug administration and cell transplantation could be more beneficial than cell transplantation alone in treating Hirschsprung's disease and related disorders.

Activation of 5-HT4 receptors facilitates neurogenesis of injured enteric neurons at an anastomosis in the lower gut.

Two-photon microscopy (2PM) can enable high-resolution deep imaging of thick tissue by exciting a fluorescent dye and protein at anastomotic sites in the mouse small intestine in vivo. We performed gut surgery and transplanted neural stem cells (NSC) from the embryonic central nervous system after marking them with the fluorescent cell linker, PKH26. We found that neurons differentiated from transplanted NSC (PKH [+]) and newborn enteric neurons differentiated from mobilized (host) NSC (YFP [+]) could be localized within the granulation tissue of anastomoses. A 5-HT4-receptor agonist, mosapride citrate (MOS), significantly increased the number of PKH (+) and YFP (+) neurons by 2.5-fold (P<0.005). The distribution patterns of PKH (+) neurons were similar to those of YFP (+) neurons. On the other hand, the 5-HT4-receptor antagonist, SB-207266 abolished these effects of MOS. These results indicate that neurogenesis from transplanted NSC is facilitated by activation of 5-HT4-receptors. Thus, a combination of drug administration and cell transplantation could be more beneficial than exclusive cell transplantation in treating Hirschsprung's disease and related disorders including post rectal cancer surgery. The underlying mechanisms for its action were explored using immunohistochemistry of the longitudinal mouse ileum and rat rectal preparations including an anastomosis. MOS significantly increased the number of new neurons, but not when co-administered with either of a protein tyrosine kinase receptor, c-RET two inhibitors. The c-RET signaling pathway contributes to enteric neurogenesis facilitated by MOS. In the future, we would perform functional studies of new neurons over the thick granulation tissue at anastomoses, using in vivo imaging with 2PM and double transgenic mice expressing a calcium indicator such as GCaMP6 and channelrhodopsin.

Characterizations of the α1-adrenoceptor subtypes mediating contractions of the human internal anal sphincter.

Human internal anal sphincter (IAS) is contracted by α1-adrenoceptor stimulation and thus α1-adrenoceptor agonists may be useful in treating fecal incontinence. This study characterizes the contribution of α1-adrenoceptor subtypes in contraction of human IAS and to investigate the age-related risk of patients with fecal incontinence. IAS and inferior mesenteric artery (IMA), as a predictor of systemic arterial pressure, were obtained from 11 patients. Both muscle strips were assessed by isometric-contraction experiments using phenylephrine, further in IAS, in the presence of various subtype selective α1-adrenoceptor antagonists. Immunohistochemistry and gene expression studies were performed in the same samples. The mean pEC50 values with SEM of phenylephrine in IAS (6.30 ± 0.13) were higher than those of IMA (5.60 ± 0.10). Furthermore, the age-related pEC50 change of IAS was observed between age <70 and ≥70 (6.58 ± 0.13 and 6.07 ± 0.16, respectively (P < 0.05)). In IAS, rightward shift of the concentration-response curves of phenylephrine was observed with three α1-adrenoceptor antagonists. Each pKB value of silodosin, BMY-7378 and prazosin was 9.36 ± 0.53, 7.28 ± 0.20 and 8.89 ± 0.12, respectively. These pKB values and gene expression studies indicated that α1A-adrenoceptor subtypes predominantly contributed to human IAS contraction.

A protein tyrosine kinase receptor, c-RET signaling pathway contributes to the enteric neurogenesis induced by a 5-HT4 receptor agonist at an anastomosis after transection of the gut in rodents.

We previously reported that a serotonin 4 (5-HT4) receptor agonist, mosapride citrate (MOS), increased the number of c-RET-positive cells and levels of c-RET mRNA in gel sponge implanted in the necks of rats. The 5-HT4 receptor is a G protein coupled receptor (GPCR) coupled to G protein Gs-cAMP cascades. We investigated the possibility that 5-HT4 receptor activation induced c-RET activation and/or PKA activation by elevating cAMP levels. Rodents were orally administered MOS by adding it to drinking water for 2 weeks after enteric nerve circuit insult via gut transection and anastomosis, together with the RET inhibitors withaferin A (WA) and RPI-1 or the PKA inhibitor H89. We then examined PGP9.5-positive cells in the newly formed granulation tissue at the anastomotic site. MOS significantly increased the number of new neurons, but not when co-administered with WA or RPI-1. Co-administration of H89 failed to alter MOS-induced increases in neurogenesis. In conclusion, the c-RET signaling pathway contributes to enteric neurogenesis facilitated by MOS, though the contribution of PKA activation seems unlikely.

The 5-hydroxytryptamine 4 Receptor Agonist-induced Actions and Enteric Neurogenesis in the Gut.

We explored a novel effect of 5-hydroxytryptamine 4 receptor (5-HT4R) agonists in vivo to reconstruct the enteric neural circuitry that mediates a fundamental distal gut reflex. The neural circuit insult was performed in guinea pigs and rats by rectal transection and anastomosis. A 5-HT4R-agonist, mosapride citrate (MOS) applied orally and locally at the anastomotic site for 2 weeks promoted the regeneration of the impaired neural circuit or the recovery of the distal gut reflex. MOS generated neurofilament-, 5-HT4R- and 5-bromo-2'-deoxyuridine-positive cells and formed neural network in the granulation tissue at the anastomosis. Possible neural stem cell markers increased during the same time period. These novel actions by MOS were inhibited by specific 5-HT4R-antagonist such as GR113808 (GR) or SB-207266. The activation of enteric neural 5-HT4R promotes reconstruction of an enteric neural circuit that involves possibly neural stem cells. We also succeeded in forming dense enteric neural networks by MOS in a gut differentiated from mouse embryonic stem cells. GR abolished the formation of enteric neural networks. MOS up-regulated the expression of mRNA of 5-HT4R, and GR abolished this upregulation, suggesting MOS differentiated enteric neural networks, mediated via activation of 5-HT4R. In the small intestine in H-line: Thy1 promoter green fluorescent protein (GFP) mice, we obtained clear 3-dimensional imaging of enteric neurons that were newly generated by oral application of MOS after gut transection and anastomosis. All findings indicate that treatment with 5-HT4R-agonists could be a novel therapy for generating new enteric neurons to rescue aganglionic disorders in the whole gut.

Persistent overexpression of SERCA2a affects bladder functions under physiological conditions, but not in bladder outlet obstruction-induced sub-acute pathological conditions.

A functional impairment of the bladder and heart in a decompensated state caused by a pressure overload is accompanied by a decrease in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2). The beneficial effects of SERCA2 overexpression in preserving cardiac functions have been previously reported. The aim of the present study was to investigate the effects of overexpressed SERCA2 on bladder functions under physiological and pathological conditions using partial bladder outlet obstruction (BOO) in SERCA2a transgenic Wistar rats (TG). Bladder cystometry and western blot analysis were performed using the wild-type Wistar rats (WT), TG, and BOO models (WTBOO and TGBOO). Persistent overexpression of SERCA2 induces reduced bladder compliance without hypertrophy in TG. BOO induces reduced bladder compliance and hypertrophy in WT and TG in the sub-acute phase, but persistent overexpression of SERCA2a in TG does not aggravate the bladder compliance and hypertrophy. In conclusion, SERCA2a overexpression affects bladder functions under physiological conditions, but not in BOO-induced sub-acute pathological conditions.

Possible peripheral mechanism for taste disorder in rats administered S-1.

BACKGROUND: Taste disorders are frequently observed in cancer patients undergoing chemotherapy and are serious adverse events which impair the quality of life (QoL) of the cancer patient. Nevertheless, taste disorder mechanisms in cancer patients undergoing chemotherapy have not yet been fully elucidated. The aim of this study was to reveal taste disorder-related peripheral mechanisms using the two-bottle preference test (TBPT) and histological examination of tongues by hematoxylin-eosin staining and immunohistochemistry with protein-gene product 9.5. METHODS: In the TBPT, one bottle was filled with the 0.01 mM quinine hydrochloride (quinine), as a bitter compound, and the other was filled with water. Doses of 50 and 100 mg kg(-1) day(-1) S-1 (tegafur/gimeracil/oteracil potassium) are lethal to Wistar rats. Therefore, doses ranging from 2-20 mg kg(-1) day(-1) were administered to the rats for 3 weeks. The S-1 dose of 2 mg kg(-1) day(-1) corresponds to the clinical dose administered to cancer patients. The part of the tongue containing the circumvallate papillae was excised the following TBPT. RESULTS: The rate of increase in terms of the average preference rate for the quinine vs. all intake (quinine plus water) was significant from the initial S-1 period to the final one, compared with that in control rats, suggesting the possibility of a worsening sensation for the bitter taste. In S-1 rats, the area of taste nerve fibers were significantly decreased and the rate of degeneration of intra-tongue ganglionic nerve cells was significantly increased. These changes were significantly correlated with the rate of increase in average preference rate of the quinine. CONCLUSION: Neuropathy of the gustatory nerve at the periphery may be involved in taste disorders induced by an anticancer drug.

Left ventricular mechanical and energetic changes in long-term isoproterenol-induced hypertrophied hearts of SERCA2a transgenic rats.

Overexpression of cardiac sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) has been suggested as a strategic intervention for cardiac failure. However, its benefit in wild-type (WT) rats with normal SERCA2a levels seems to be small. To investigate whether it would be beneficial in a cardiac failure model with down-regulated SERCA2a levels, we made a cardiac hypertrophy model using isoproterenol infusion (1.2mgkg(-1)day(-1) for 1 or 4weeks; TG-ISO1w and TG-ISO4w, respectively) in SERCA2a transgenic (TG) rats and compared these rats with littermate WT rats that underwent the same treatments (WT-ISO1w and WT-ISO4w). We analyzed the left ventricular (LV) mechanoenergetics in the excised heart using our original cross-circulation system. The downward shift of curvilinear LV end-systolic pressure-volume relations (ESPVRs) observed in WT-ISO4w rats was abolished in TG-ISO4w rats. The slope and VO2 intercept of the VO2 (myocardial oxygen consumption per beat)-PVA (systolic pressure-volume area: total mechanical energy per beat) linear relation did not differ in any of the groups. The most important finding was a significantly smaller O2 cost of LV contractility in the TG-ISO4w group, which means that less O2 is needed to exert the same LV contractility, compared with the other groups. The increased ratio of SERCA2a/phospholamban returned to the level of the WT-control group only in the TG-ISO4w group. Longer-term up-regulation of mitochondrial transcription factor A for genes of mitochondrial enzymes producing ATP was observed in TG rats. In conclusion, longer-term overexpression of SERCA2a will be beneficial in the present cardiac failure model with down-regulated SERCA2a levels.

In vivo imaging of enteric neurogenesis in the deep tissue of mouse small intestine.

One of the challenges of using imaging techniques as a tool to study cellular physiology has been the inability to resolve structures that are not located near the surface of the preparation. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy (2PM), has overcome this limitation, providing deeper optical penetration (several hundred µm) in ex vivo and in vivo preparations. We have used this approach in the gut to achieve the first in vivo imaging of enteric neurons and nerve fibers in the mucosa, submucosa, submucosal and myenteric plexuses, and circular and longitudinal muscles of the small intestine in H-line: Thy1 promoter GFP mice. Moreover, we obtained clear three-dimensional imaging of enteric neurons that were newly generated after gut transection and reanastomosis. Neurogenesis was promoted by oral application of the 5-HT(4)-receptor agonist, mosapride citrate (MOS). The number of newly generated neurons observed in mice treated with MOS for one week was 421±89 per 864,900 µm(2) (n = 5), which was significantly greater than that observed in preparations treated with MOS plus an antagonist (113±76 per 864,900 µm(2)) or in 4 week vehicle controls (100±34 per 864,900 µm(2)) (n = 4 both). Most neurons were located within 100 µm of the surface. These results confirm that activation of enteric neural 5-HT(4)-receptor by MOS promotes formation of new enteric neurons. We conclude that in vivo 2PM imaging made it possible to perform high-resolution deep imaging of the living mouse whole gut and reveal formation of new enteric neurons promoted by 5-HT(4)-receptor activation.

A new calpain inhibitor protects left ventricular dysfunction induced by mild ischemia-reperfusion in in situ rat hearts.

We have previously indicated that a new soluble calpain inhibitor, SNJ-1945 (SNJ), attenuates cardiac dysfunction after cardioplegia arrest-reperfusion by inhibiting the proteolysis of α-fodrin in in vitro study. Nevertheless, the in vivo study design is indispensable to explore realistic therapeutic approaches for clinical use. The aim of the present in situ study was to investigate whether SNJ attenuated left ventricular (LV) dysfunction (stunning) after mild ischemic-reperfusion (mI-R) in rat hearts. SNJ (60 µmol/l, 5 ml i.p.) was injected 30 min before gradual and partial coronary occlusion at proximal left anterior descending artery. To investigate LV function, we obtained curvilinear end-systolic pressure-volume relationship by increasing afterload 60 min after reperfusion. In the mI-R group, specific LV functional indices at midrange LV volume (mLVV), end-systolic pressure (ESP(mLVV)), and pressure-volume area (PVA(mLVV): a total mechanical energy per beat, linearly related to oxygen consumption) significantly decreased, but SNJ reversed these decreases to time control level. Furthermore, SNJ prevented the α-fodrin degradation and attenuated degradation of Ca(2+) handling proteins after mI-R. Our results indicate that improvements in LV function following mI-R injury are associated with inhibition of the proteolysis of α-fodrin in in situ rat hearts. In conclusion, SNJ should be a promising tool to protect the heart from the stunning.

Increased passive stiffness of cardiomyocytes in the transverse direction and residual actin and myosin cross-bridge formation in hypertrophied rat hearts induced by chronic ß-adrenergic stimulation.

BACKGROUND: Left ventricular (LV) hypertrophy is often present in patients with diastolic heart failure. However, stiffness of hypertrophied cardiomyocytes in the transverse direction has not been fully elucidated. The aim of this study was to assess passive cardiomyocyte stiffness of hypertrophied hearts in the transverse direction and the influence of actin-myosin cross-bridge formation on the stiffness. METHODS AND RESULTS: Wistar rats received a vehicle (control) or isoproterenol (ISO) subcutaneously. After 7 days, compared with the controls, ISO administration had significantly increased heart weight and LV wall thickness and had decreased peak early annular relaxation velocity (e') assessed by echocardiography. Elastic modulus of living cardiomyocytes in the transverse direction assessed by an atomic force microscope was significantly higher in the ISO group than in controls. We added butanedione monoxime (BDM), an inhibitor of actin-myosin interaction, and blebbistatin, a specific myosin II inhibitor, to the medium. BDM and blebbistatin significantly reduced the elastic modulus of cardiomyocytes in the ISO group. X-ray diffraction analysis showed that the reflection intensity ratio (I((1,0))/I((1,1))) at diastole was not different before and after treatment with BDM, which induces complete relaxation, in control hearts, but that I((1,0))/I((1,1)) was significantly increased after BDM treatment in the ISO group, indicating residual cross-bridge formation in hypertrophied hearts. CONCLUSIONS: Passive cardiomyocyte stiffness in the transverse direction is increased in hearts with ISO-induced hypertrophy and this is caused by residual actin-myosin cross-bridge formation.

In vitro morphological bud formation in organ-like three-dimensional structure from mouse ES cells induced by FGF10 signaling.

Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages in vitro. Using an embryoid body (EB) culture system, we developed a gut-like three-dimensional structure from mouse ES cells (the ES 3-D structure). Genetic studies implicate fibroblast growth factor 10 (FGF10)-FGF receptor 2b (FGFR2b) signaling as a critical regulator of lung bud morphogenesis in the embryonic foregut. The aim of the present study was to form a putative respiratory tract in the ES 3-D structure. By local application of FGF10 protein, we successfully demonstrated in vitro morphological formation of putative primitive respiratory tract-like processes, or buds, in the ES 3-D structure. Such organs that are differentiated from ES cells may provide new insights into tissue engineering and regenerative medicine.

Cardiac mechanoenergetics for understanding isoproterenol-induced rat heart failure.

Considering from clinical implication, it is often complained that short-term experimental diseased heart models do not mimic long-term diseased hearts that one often clinically encountered. The left ventricle (LV) function in rat cardiac hypertrophy models treated with isoproterenol (ISO) up to 16 weeks was followed up with a non-invasive echocardiography. Infusion of either ISO (1.2mgkg(-1)day(-1) for 3 days-16 weeks) or vehicle (saline 24µlday(-1) for 3 days-16 weeks; SA group) was performed by subcutaneously implanting osmotic minipump. LV and right ventricle (RV) weight ratios to body weight (mgg(-1)) in SA, ISO3d, ISO7d and ISO4w were: 1.94±0.10 and 0.54±0.04 (n=7), 2.56±0.10 and 0.66±0.05 (n=7), 2.50±0.25 and 0.64±0.07 (n=10) and 2.40±0.08 and 0.59±0.08 (n=9), respectively. From echocardiography, the LV function of the hypertrophy models at 3 days, 1 and 2 weeks was unchanged but the model at the longer-term than 4 weeks resulted in prolonged systolic failure. These results indicated that only 3-day ISO infusion induced the hypertrophy model similar in shape and function to that induced by 2-week ISO infusion; the 3-day model sufficiently represents the effects of 2-week ISO infusion. In this review, left ventricular (LV) function was compared between rat cardiac hypertrophy models treated with ISO for 3 days (ISO3d) and 7 days (ISO7d) by analyzing LV mechanical work and energetics. The LV mechanical work and energetics was unchanged in SA, ISO3d and ISO7d groups. The LV relaxation rate at 240bpm in ISO3d and ISO7d groups was significantly slower than that in SA group with unchanged contraction rate. The amounts of expression of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a), phospholamban (PLB), phosphorylated-Ser(16) PLB (p-PLB), phospholemman (PLM) and Na(+)-K(+)-ATPase (NKA) are significantly decreased in ISO3d and ISO7d groups. Furthermore, the marked collagen production (types I and III) was observed in ISO3d and ISO7d groups. These results suggested the possibility that physiological LV function is compensated, although molecular changes have been generated even in the short-term hypertrophy model. Although a novel histone deacetylase (HDAC) inhibitor, has some beneficial effects on hemodynamics, it has no effects of anti-hypertrophic modalities in ISO3d model. However, a selective sodium proton exchanger-1 (NHE-1) inhibitor normalized ISO-induced down-regulation of SERCA2a without changes in pPLB/PLB expression in the ISO7d model and ameliorates cardiac Ca(2+) handling impairment and prevents the development of cardiac dysfunction. This result indicated that SERCA2a is a key molecule in the ISO7d model. Slow LV relaxation rate in ISO7d model may be due to down-regulation of SERCA2a. In conclusion, lowering the heart rate make it possible to rescue the impairment of LV mechanical work and energetics in the ISO-induced compensatory hypertrophied rat hearts, providing basic evidence for clinical therapy for patients with some types of cardiac failure.

Evaluation of left ventricular mechanical work and energetics of normal hearts in SERCA2a transgenic rats.

Cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) is responsible for most of the Ca(2+) removal during diastole and a larger Ca(2+) handling energy consumer in excitation-contraction (E-C) coupling. To understand the cardiac performance under long-term SERCA2a overexpression conditions, we established SERCA2a transgenic (TG) Wistar rats to analyze cardiac mechanical work and energetics in normal hearts during pacing at 300 beats/min. SERCA2a protein expression was increased in TGI and TGII rats (F2 and F3 of the same father and different mothers). Mean left ventricular (LV) end-systolic pressure (ESP) and systolic pressure-volume area (PVA; a total mechanical energy per beat) at midrange LV volume (mLVV) were significantly larger in TGI rats and were unchanged in TGII rats, compared to those in non-TG [wildtype (WT)] littermates. Mean myocardial oxygen consumption per minute for E-C coupling was significantly increased, and the mean slope of myocardial oxygen consumption per beat (VO(2))-PVA (systolic PVA) linear relation was smaller, but the overall O(2) cost of LV contractility for Ca(2+) is unchanged in all TG rats. Mean Ca(2+) concentration exerting maximal ESP(mLVV) in TGII rats was significantly higher than that in WT rats. The Ca(2+) overloading protocol did not elicit mitochondrial swelling in TGII rats. Tolerance to higher Ca(2+) concentrations may support the possibility for enhanced SERCA2a activity in TGII rats. In conclusion, long-term SERCA2a overexpression enhanced or maintained LV mechanics, improved contractile efficiency under higher energy expenditure for Ca(2+) handling, and improved Ca(2+) tolerance, but it did not change the overall O(2) cost of LV contractility for Ca(2+) in normal hearts of TG rats.

NHE-1 blockade reversed changes in calcium transient in myocardial slices from isoproterenol-induced hypertrophied rat left ventricle.

We previously reported that left ventricular (LV) slices from isoproterenol (ISO)-induced hypertrophied rat hearts showed an increase of energy expenditure due to remodeling of Ca(2+) handling in excitation-contraction coupling, i.e., suppressed SERCA2a activity and enhanced Na(+)/Ca(2+)exchanger-1 (NCX-1) activity. Na(+)/H(+) exchanger-1 (NHE-1) inhibitor (NHEI) has been demonstrated to exert beneficial effects in the development of cardiac remodeling. We hypothesized that a novel NHE-1 selective inhibitor, BIIB723 prevents remodeling of Ca(2+) handling in LV slices of ISO-induced hypertrophied rat hearts mediated by inhibiting NCX-1 activity. The significant shortening in duration of multi-cellular Ca(2+) transient in ISO group was normalized in ISO+BIIB723 group. The significant increase in amplitude of multi-cellular Ca(2+) waves (CaW) generated at high [Ca(2+)](o) of LV slices in ISO group was also normalized in ISO+BIIB723 group. However, the enhanced NCX-1 activity was not antagonized by BIIB723. We recently reported that ISO-induced down-regulation of a Ca(2+) handling protein, SERCA2a, was normalized by BIIB723. Therefore, it seems likely that BIIB723 normalized shortened multi-cellular Ca(2+) transient duration and increased CaW amplitude in LV slices mediated via normalization of SERCA2a activity. Furthermore, the results presented here suggest the multi-cellular Ca(2+) transient duration and CaW amplitude in LV slices might be better indices reflecting SERCA2a activity than SERCA2a protein expression level.