Takeshi Yoneda

Cardiac Endothelin-1 Plays a Critical Role in the Functional Deterioration of Left Ventricles During the Transition From Compensatory Hypertrophy to Congestive Heart Failure in Salt-Sensitive Hypertensive Rats

BACKGROUND To investigate whether endogenous ET-1 participates in an adaptive process of left ventricular hypertrophy (LVH) or a maladaptive process from LVH to congestive heart failure (CHF), we used a Dahl salt-sensitive (DS) rat model, in which systemic hypertension caused compensated concentric LVH at the age of 11 weeks followed by marked LV dilatation and global hypokinesis at the age of 17 weeks. METHODS AND RESULTS By specific sandwich enzyme immunoassay, serum and myocardial ET-1 levels at the LVH stage were not elevated compared with age-matched Dahl salt-resistant (DR) rats, despite the marked increase of LV/body weight ratio (LV/BW). However, at the CHF stage, serum and LV ET-1 levels increased by 3. 8-fold and 5.4-fold, respectively. LV ET-1 contents had close relationships with the fractional shortening (r=0.763) and the systolic wall stress (r=0.858) measured by in vivo transthoracic echocardiography. Immunohistochemistry demonstrated that the remarkably increased ET-1 in LV is located mainly in cardiomyocytes. By competitive reverse transcriptase-polymerase chain reaction, LV prepro-ET-1 mRNA levels increased by 4.1-fold in CHF rats. We randomized 11-week-old LVH rats to chronic treatment with the endothelin receptor antagonist bosentan (Bos, 100 mg. kg-1. d-1, n=14), the alpha1-receptor antagonist doxazosin (Dox, 1 mg. kg-1. d-1, n=12), or vehicle (Cont, n=14). Bos treatment did not alter the LV geometry and function at 15 weeks; however, it attenuated the decrease of LV fractional shortening by 51% (P<0.01) without reducing the LV/BW at 17 weeks. Conversely, Dox, which decreased the blood pressure to the same extent as Bos, did not affect the progression of LV dysfunction. Bos (93%; P<0.0001 versus Cont) but not Dox (42%; P=0.8465 versus Cont) ameliorated the survival rate at 17 weeks (Cont; 36%). CONCLUSIONS The accelerated myocardial synthesis of ET-1 contributes directly to LV contractile dysfunction during the transition from LVH to CHF. Unelevated levels of LV ET-1 at the established LVH stage and lack of effects on LV mass by chronic bosentan treatment suggest that myocardial growth is mediated through alternative pathways. These studies indicate that chronic ET antagonism may provide an additional strategy for heart failure therapy in humans.

Excessive activation of matrix metalloproteinases coincides with left ventricular remodeling during transition from hypertrophy to heart failure in hypertensive rats

OBJECTIVES We sought to elucidate how the local activation of matrix metalloproteinases (MMPs) is balanced by that of the endogenous tissue inhibitors of MMP (TIMPs) during left ventricular (LV) remodeling. BACKGROUND Although it is known that the extracellular matrix (ECM) must be altered during LV remodeling, its local regulation has not been fully elucidated. METHODS In Dahl salt-sensitive rats with hypertension, in which a stage of concentric, compensated left ventricular hypertrophy (LVH) at 11 weeks is followed by a distinct stage of congestive heart failure (CHF) with LV enlargement and dysfunction at 17 weeks, we determined protein and messenger ribonucleic acid (mRNA) levels of LV myocardial TIMP-2 and -4 and MMP-2, as well as their concomitant activities. RESULTS No changes were found at the LVH stage. However, during the transition to CHF, TIMP-2 and -4 activities, protein and mRNA levels were all sharply increased. At the same time, the MMP-2 mRNA and protein levels and activities, as determined by gelatin zymography, as well as by an antibody capture assay, showed a substantial increase during the transition to CHF. The net MMP activities were closely related to increases in LV diameter (r = 0.763) and to systolic wall stress (r = 0.858) in vivo. CONCLUSIONS Both TIMPs and MMP-2 remained inactive during hypertrophy, per se; they were activated during the transition to CHF. At this time, the activation of MMP-2 surpassed that of TIMPs, possibly resulting in ECM breakdown and progression of LV enlargement.

Anti-Ischemic Effect of a Novel Cardioprotective Agent, JTV519, Is Mediated Through Specific Activation of δ-Isoform of Protein Kinase C in Rat Ventricular Myocardium

BACKGROUND A new 1,4-benzothiazepine derivative, JTV519, has a strong protective effect against Ca(2+) overload-induced myocardial injury. We investigated the effect of JTV519 on ischemia/reperfusion injury in isolated rat hearts. METHODS AND RESULTS At 30 minutes of reperfusion after 30-minute global ischemia, the percent recovery of left ventricular developed pressure was improved, and the creatine phosphokinase and lactate dehydrogenase leakage was reduced in a concentration-dependent manner when JTV519 was administered in the coronary perfusate both at 5 minutes before the induction of ischemia and at the time of reperfusion. The myocardial protective effect of JTV519 was completely blocked by pretreatment of the heart with GF109203X, a specific protein kinase C (PKC) inhibitor. In contrast, the effect of JTV519 was not affected by alpha(1)-, A(1)-, and B(2)-receptor blockers that couple with PKC in the cardiomyocyte. Both immunofluorescence images and immunoblots of JTV519-treated left ventricular myocardium and isolated ventricular myocytes demonstrated that this agent induced concentration-dependent translocation of the delta-isoform but not the other isoforms of PKC to the plasma membrane. CONCLUSIONS The mechanism of cardioprotection by JTV519 against ischemia/reperfusion injury involves isozyme-specific PKC activation through a receptor-independent mechanism. This agent may provide a novel pharmacological approach for the treatment of patients with acute coronary diseases via a subcellular mechanism mimicking ischemic preconditioning.

Reinduction of T-Type Calcium Channels by Endothelin-1 in Failing Hearts In Vivo and in Adult Rat Ventricular Myocytes In Vitro

Background—In ventricular myocardium, the T-type Ca2+ current (ICa,T), which is temporarily observed during fetal and neonatal periods, has been shown to reappear in failing/remodeling hearts. However, its pathophysiological regulation has not been elucidated. Methods and Results—We utilized Dahl salt-sensitive (DS) rats with hypertension at the stage of concentric left ventricular (LV) hypertrophy (11 weeks old, LVH) and at the heart failure stage (16 to 18 weeks old, CHF). Some were treated with bosentan (100 mg/kg per day) during the period from LVH to CHF. In LVH, neither the presence of ICa,T (measured in the freshly isolated LV myocytes) nor an increase in &agr;-1G mRNA expression were detected. This condition was associated with increases in tissue angiotensin II (AII) but not with endothelin (ET)-1 peptides. In contrast, in CHF, when the tissue AII remained elevated and ET-1 de novo increased, ICa,T was recorded in most of the cells (−0.87±0.18 pA/pF at −30 mV, P <0.01 versus LVH). This was associated with a significant increase in the &agr;-1G mRNA level. The chronic bosentan treatment eliminated both the elevation of &agr;-1G mRNA level and ICa,T from the cells, whereas it did not affect the cell size and membrane capacitance. In addition, 48-hour exposure to ET-1 but not AII induced ICa,T in normal adult myocytes in culture from Sprague-Dawley rats. Conclusions—ICa,T channels reappear in failing but not in hypertrophied LV cardiomyocytes in a manner depending on the tissue ET-1 activation.

The palatability of corn oil and linoleic acid to mice as measured by short-term two-bottle choice and licking tests

Free fatty acids (FFAs) were reported to be recognized in the oral cavity and possibly involved in fatty foods recognition. To understand the importance of oil recognition in the oral cavity, we investigated the effect of various concentrations of a fatty acid or corn oil on fluid intake as well as mices preferences in a two-bottle choice test and a licking test. Linoleic acid (LA), which is a main component of corn oil, was used as a representative FFA. In the two-bottle choice test between a pair of different concentrations of corn oil, the mice consistently adopted the higher concentration of corn oil. In the licking test for corn oil, the licking rates for the serial concentration of corn oils (0, 1, 5, 10 and 100%) were increased in a concentration-dependent manner. On the other hand, in the two-bottle test for a pair of different concentrations of LA (0, 0.125, 0.25 and 1%), 0.25% and 1% LA were preferred to mineral oil, but 0.25% and 1% LA were preferred equally in mice. In the licking test for LA, the mice showed the largest number of initial lickings for the 1% LA, while the licking rates for the high concentration of LA decreased. These results suggest that mice could discriminate the concentration of corn oil and LA in the oral cavity. We also suggest that pure corn oil is a highly preferable solution, while an optimal concentration of LA according to the preferences of mice is a low-range concentration (0.25-1%).

Endothelin-1 Has a Unique Oxygen-Saving Effect by Increasing Contractile Efficiency in the Isolated Rat Heart

BackgroundThe effect of endothelin (ET)-1 on cardiac energetics is not fully understood. Methods and ResultsIn isolated, coronary-perfused rat hearts, we measured left ventricular contractility index (Emax), pressure-volume area (PVA), and myocardial oxygen consumption (M˙V>o2) before and after administration of ET-1 (1×10−9 mol/L). ET-1 increased Emax by 48±16% (P <0.01) and the total M˙Vo2 by 24±11% (P <0.01). The M˙Vo2-PVA relations were linear both before and after ET-1 (r >0.99). ET-1 shifted M˙Vo2-PVA upward, increasing the M˙Vo2 intercept by 24±13%. At the same time, ET-1 decreased the slope (S), with 1/S (contractile efficiency) being 46±5% before and 56±5% after ET-1 (P <0.01). ET-1–induced increases in Emax and in contractile efficiency were abolished by an ETA receptor blocker (S-0139) but not by an ETB blocker (BQ-788). Although high [Ca2+] perfusion increased Emax and the intercept to the same extent as ET-1, it did not change S. NG-Nitro-l-arginine (an inhibitor of nitric oxide synthase) increased the coronary perfusion pressure as much as ET-1, but S again remained unchanged. Dimethylamyloride (Na+/H+ exchanger inhibitor) partially blocked the positive inotropic effect of ET-1 but not the ET-1–induced increase in the contractile efficiency. ConclusionsAgonistic effects of ET-1 on the ETA receptor economized the chemomechanical conversion efficiency of the left ventricular unit myocardium by a mechanism independent of the Na+/H+ exchanger. This unique oxygen-saving effect of ET-1 may play an adaptive role in the failing myocardium, in which local accumulation of ET-1 is present.

Contribution of gustation to the palatability of linoleic acid

We investigated the palatability of a low concentration of linoleic acid (LA) in short-term two-bottle choice tests and licking tests. To examine the contribution of gustation, mice were rendered anosmic with olfactory nerve transection surgery and test solutions were prepared using mineral oil (saturated long-chain hydrocarbon) to minimize textural effects. In the two-bottle choice tests between various pairs of different concentrations of corn oil and LA, both anosmic and the sham-operated mice constantly preferred a higher concentration of corn oil and LA. In the licking tests, the initial licking rate for 1% LA was higher than that for mineral oil in anosmic mice. In accordance with the results of the two-bottle choice test, the initial licking rate for corn oil and LA increased in a concentration-dependent manner in both anosmic and sham-operated mice in the licking test, and reached its peak at 100% corn oil and 1% LA. A preference comparison between 1% LA and 100% corn oil showed that anosmic mice preferred 1% LA over 100% corn oil. These results suggest that mice could recognize dietary fat and fatty acid solutions in the oral cavity without any olfactory or textural cues and the fatty acid recognition on their tongues might provide a pivotal cue to how dietary fat is recognized in the oral cavity.

Preference for dietary fat induced by release of beta-endorphin in rats

AIMS To determine whether beta-endorphin contributes to the ingestion of and preference for dietary oil, we examined the relationship between the dynamics of beta-endorphin, before and after the ingestion of corn oil, and the intake volume of corn oil. MAIN METHODS Rats were offered 5% corn oil for 20 min for 5 consecutive days so they could acquire a preference for corn oil. On day 6, seven groups of rats were presented with the oil for defined time periods, and we measured the beta-endorphin levels in the serum and cerebrospinal fluid (CSF) before and after the presentation of corn oil as well as the consumed volume of corn oil at defined time points. KEY FINDINGS Beta-endorphin levels in serum and CSF were significantly increased 15 min after the ingestion of corn oil, followed by a rapid decrease and maintenance at the basal level throughout the rest of the experimental period. The intake of corn oil was the lowest in the time period of 15-30 min, when the beta-endorphin level reached a peak value. The intake volume of corn oil might be inversely correlated with beta-endorphin levels in serum and CSF. The pretreatment of naloxone, an antagonist of the opioid receptor, decreased the initial licking rate for corn oil and increased the latency for corn oil in the licking test. SIGNIFICANCE The beta-endorphin was rapidly released after oil ingestion, which contributed to the hedonic preference and ingestive behavior for fat.

Mercaptoacetate inhibition of fatty acid β-oxidation attenuates the oral acceptance of fat in BALB/c mice

We investigated the effect of beta-oxidation inhibition on the fat ingestive behavior of BALB/c mice. Intraperitoneal administration to mice of mercaptoacetate, an inhibitor of fatty acid oxidation, significantly suppressed intake of corn oil but not intake of sucrose solution or laboratory chow. To further examine the effect of mercaptoacetate on the acceptability of corn oil in the oral cavity, we examined short-term licking behavior. Mercaptoacetate significantly and specifically decreased the number of licks of corn oil within a 60-s period but did not affect those of a sucrose solution, a monosodium glutamate solution, or mineral oil. In contrast, the administration of 2-deoxyglucose, an inhibitor of glucose metabolism, did not affect the intake or short-term licking counts of any of the tasted solutions. These findings suggest that fat metabolism is involved in the mechanism underlying the oral acceptance of fat as an energy source.

Intragastric infusion of glucose enhances the rewarding effect of sorbitol fatty acid ester ingestion as measured by conditioned place preference in mice.

We investigated substances that induce a rewarding effect during the postingestive process using the conditioned place preference (CPP) test. Although mice showed high affinity for a low-energy fat substitute--sorbitol fatty acid esters and low-concentration linoleic acid solution--they did not exhibit a place preference toward a voluntary intake of fat substitute in the CPP test. However, during a conditioning session of CPP that involved intragastric administration of corn oil immediately before the intake of the fat substitute, the test mice displayed a place preference. Similarly, intragastric administration of glucose, galactose, and dextrin also induced CPP; however, fructose, mannose, and a nonmetabolized carbohydrate did not. These results suggest that administration of corn oil and glucose has the same postingestive effect with regard to inducing CPP and that the structural specificity of carbohydrates influences the postingestive effect.