Evaluation of stress status using the stress map for guide dog candidates in the training stage using variations in the serum cortisol with nerve growth factor and magnesium ions.

Most studies on guide dogs for the blind were conducted to investigate the appropriateness of the animals, including in terms of their breeding, constitution, and temperament. However, research to comprehend the stress status of guide dog candidates in response to their training has been unclear. In this study, the levels of serum cortisol, nerve growth factor (NGF), and magnesium ion (Mg2+) levels of guide dog candidates during the three training stages-the elementary, intermediate, and advanced classes-were examined. Dogs were classified based on the contents of the classes and period during the training in which they were subjected. Since the dogs in the elementary class had the lowest serum NGF and Mg2+ levels, they were understood to be under mental stress and to be unfamiliar with their new surroundings. In contrast, the serum NGF and Mg2+ levels were high in the dogs in the advanced class, though they were demonstrated to be mentally stable and acclimated to their environment. Additionally, they were almost free from the stress caused by daily life, since they had the lowest serum cortisol levels. The status of each dog was plotted on a map consisting of 2 axes representing the serum NGF and Mg2+ levels with high or low cortisol levels. Plots could be divided into three domains corresponding to the elementary, intermediate, and advanced classes. Therefore, for working dogs, serum NGF and Mg2+ levels in addition to serum cortisol levels may be important factors to comprehend the type of stress situation that each dog was in.

Analysis of serum magnesium ions in dogs exposed to external stress: A pilot study.

Magnesium ions (Mg2+) are essential for various enzymatic reactions in the body associated with energy production and activation of the muscles and nerves. Mg2+ is also involved in blood pressure regulation, maintenance of body temperature, and glucose metabolism. Although various factors including foods and physical conditions have been reported to change serum Mg2+ status in humans, serum Mg2+ in dogs exposed to external stress has been unclear. In this study, we examined serum levels of Mg2+ in dogs at different conditions using the guide dog candidates for the blind. Serum Mg2+ was decreased in winter and increased in summer. Guide dog candidates in an elementary class of the training showed markedly lower levels of serum Mg2+, compared with that of dogs in an advanced class. When healthy adult dogs were subjected to forced exercise using a treadmill, a significant reduction in serum Mg2+ levels was observed, particularly in winter. These findings suggest that serum levels of Mg2+ may be influenced by weather fluctuation such as air temperature, nervousness in unaccustomed situations, age, and physical stress induced by exercise. The results indicate that Mg2+ supplementation should be considered for working dogs, dogs moving or traveling to a new environment, and dogs during winter.

Effects of candesartan on electrical remodeling in the hearts of inherited dilated cardiomyopathy model mice.

Inherited dilated cardiomyopathy (DCM) is characterized by dilatation and dysfunction of the ventricles, and often results in sudden death or heart failure (HF). Although angiotensin receptor blockers (ARBs) have been used for the treatment of HF, little is known about the effects on postulated electrical remodeling that occurs in inherited DCM. The aim of this study was to examine the effects of candesartan, one of the ARBs, on cardiac function and electrical remodeling in the hearts of inherited DCM model mice (TNNT2 ΔK210). DCM mice were treated with candesartan in drinking water for 2 months from 1 month of age. Control, non-treated DCM mice showed an enlargement of the heart with prolongation of QRS and QT intervals, and died at t1/2 of 70 days. Candesartan dramatically extended the lifespan of DCM mice, suppressed cardiac dilatation, and improved the functional parameters of the myocardium. It also greatly suppressed prolongation of QRS and QT intervals and action potential duration (APD) in the left ventricular myocardium and occurrence of ventricular arrhythmia. Expression analysis revealed that down-regulation of Kv4.2 (Ito channel protein), KChIP2 (auxiliary subunit of Kv4.2), and Kv1.5 (IKur channel protein) in DCM was partially reversed by candesartan administration. Interestingly, non-treated DCM heart had both normal-sized myocytes with moderately decreased Ito and IKur and enlarged cells with greatly reduced K+ currents (Ito, IKur IK1 and Iss). Treatment with candesartan completely abrogated the emergence of the enlarged cells but did not reverse the Ito, and IKur in normal-sized cells in DCM hearts. Our results indicate that candesartan treatment suppresses structural remodeling to prevent severe electrical remodeling in inherited DCM.

Multistep ion channel remodeling and lethal arrhythmia precede heart failure in a mouse model of inherited dilated cardiomyopathy.

BACKGROUND: Patients with inherited dilated cardiomyopathy (DCM) frequently die with severe heart failure (HF) or die suddenly with arrhythmias, although these symptoms are not always observed at birth. It remains unclear how and when HF and arrhythmogenic changes develop in these DCM mutation carriers. In order to address this issue, properties of the myocardium and underlying gene expressions were studied using a knock-in mouse model of human inherited DCM caused by a deletion mutation ΔK210 in cardiac troponinT. METHODOLOGY/PRINCIPAL FINDINGS: By 1 month, DCM mice had already enlarged hearts, but showed no symptoms of HF and a much lower mortality than at 2 months or later. At around 2 months, some would die suddenly with no clear symptoms of HF, whereas at 3 months, many of the survivors showed evident symptoms of HF. In isolated left ventricular myocardium (LV) from 2 month-mice, spontaneous activity frequently occurred and action potential duration (APD) was prolonged. Transient outward (I(to)) and ultrarapid delayed rectifier K(+) (I(Kur)) currents were significantly reduced in DCM myocytes. Correspondingly, down-regulation of Kv4.2, Kv1.5 and KChIP2 was evident in mRNA and protein levels. In LVs at 3-months, more frequent spontaneous activity, greater prolongation of APD and further down-regulation in above K(+) channels were observed. At 1 month, in contrast, infrequent spontaneous activity and down-regulation of Kv4.2, but not Kv1.5 or KChIP2, were observed. CONCLUSIONS/SIGNIFICANCE: Our results suggest that at least three steps of electrical remodeling occur in the hearts of DCM model mice, and that the combined down-regulation of Kv4.2, Kv1.5 and KChIP2 prior to the onset of HF may play an important role in the premature sudden death in this DCM model. DCM mice at 1 month or before, on the contrary, are associated with low risk of death in spite of inborn disorder and enlarged heart.

Pathophysiological remodeling of mouse cardiac myocytes expressing dominant negative mutant of neuron restrictive silencing factor.

BACKGROUND: It has been previously reported that the transgenic mouse expressing the dominant negative mutant of the neuron restrictive silencing factor (dnNRSF) in the heart died from lethal arrhythmia, so the present study aimed to clarify the electrophysiological alteration of the ventricular myocyte isolated from the dnNRSF mouse. METHODS AND RESULTS: The action potential (AP) and membrane currents were recorded using the whole-cell patch-clamp method. Intracellular Ca(2+) was measured with Indo-1AM. The AP of dnNRSF myocytes exhibited reduction of resting membrane potential, prolongation of AP duration, and frequent early afterdepolarization (EAD). The EAD was completely inhibited by SEA0400, a specific blocker of the Na(+)-Ca(2+) exchanger (NCX). The most notable alteration of membrane current was a reduction in the inward rectifier K(+) current (I(K1)) density. In addition to re-expression of fetal type cardiac ion channels, a Na(+)-permeable, late inward current was observed in a small population of dnNRSF myocytes. The diastolic intracellular Ca(2+) concentration was also raised in dnNRSF myocytes, and spontaneous Ca(2+) oscillation was induced by ß-adrenergic stimulation. CONCLUSIONS: In dnNRSF myocytes, the "repolarization reserve" of the AP was significantly reduced by specific alterations in membrane currents. Under these conditions, the amplitude of EAD generated by the inward NCX current might be enlarged, thereby increasing the cells' vulnerability to ventricular arrhythmia.

Causes of abnormal Ca2+ transients in Guinea pig pathophysiological ventricular muscle revealed by Ca2+ and action potential imaging at cellular level.

BACKGROUND: Abnormal Ca(2+) transients are often observed in heart muscles under a variety of pathophysiological conditions including ventricular tachycardia. To clarify whether these abnormal Ca(2+) transients can be attributed to abnormal action potential generation or abnormal Ca(2+) handling/excitation-contraction (EC) coupling, we developed a procedure to determine Ca(2+) and action potential signals at the cellular level in isolated heart tissues. METHODOLOGY/PRINCIPAL FINDINGS: After loading ventricular papillary muscle with rhod-2 and di-4-ANEPPS, mono-wavelength fluorescence images from rhod-2 and ratiometric images of two wavelengths of emission from di-4-ANEPPS were sequentially obtained. To mimic the ventricular tachycardia, the ventricular muscles were field-stimulated in non-flowing Krebs solution which elicited abnormal Ca(2+) transients. For the failed and alternating Ca(2+) transient generation, there were two types of causes, i.e., failed or abnormal action potential generation and abnormal EC coupling. In cells showing delayed initiation of Ca(2+) transients with field stimulation, action potential onset was delayed and the rate of rise was slower than in healthy cells. Similar delayed onset was also observed in the presence of heptanol, an inhibitor of gap junction channels but having a non-specific channel blocking effect. A Na(+) channel blocker, on the other hand, reduced the rate of rise of the action potentials but did not result in desynchronization of the action potentials. The delayed onset of action potentials can be explained primarily by impaired gap junctions and partly by Na(+) channel inactivation. CONCLUSIONS/SIGNIFICANCE: Our results indicate that there are multiple patterns for the causes of abnormal Ca(2+) signals and that our methods are useful for investigating the physiology and pathophysiology of heart muscle.

Altered KCNQ3 potassium channel function caused by the W309R pore-helix mutation found in human epilepsy.

The second tryptophan (W) residue of the conserved WW motif in the pore helix of many K+ channel subunit is thought to interact with the tyrosine (Y) residues of the selectivity filter. A missense mutation causing the replacement of the corresponding residues with an arginine (W309R) occurs in KCNQ3 subunits forming part of M-channels. In this study, we examined the functional consequences of the W309R mutation in heterogously expressed KCNQ channels. Homomeric KCNQ3W309R channels lacked KCNQ currents. Heteromeric KCNQ2/KCNQ3W309R channels displayed a dominant-negative suppression of current and a significant modification in gating properties when compared with heteromeric KCNQ3/KCNQ2 channels mimicking the M-channels. A three-dimensional homology model in the W309R mutant indicated that the R side chain of pore helices is too far from the Y side chain of the selectivity filter to interact via hydrogen bonds with each other and stabilize the pore structure. Collectively, the present results suggest that the second W residues of pore helices and their chemical interaction with the Y residues of the selectivity filter are essential for normal K+ channel function. This pore-helix mutation, if occurs in the brain M channels, could thus lead to a channel dysfunction sufficient to trigger epileptic hyperexcitability.

A simple technique for isolating healthy heart cells from mouse models.

Single heart cells of mouse models provide powerful tools for heart research. However, their isolation is not easy, and it imposes a significant bottleneck on their use in cellular studies of the heart. Aiming to overcome this problem, this report introduces a novel technique that reproducibly isolates healthy heart cells from mouse models. Using simple devices that ensure easy handling and the rapid aortic cannulation of a small mouse heart, cell isolation was done under physiological conditions without using the "KB" medium or 2,3-butanedione monoxime (BDM). The isolated cells consistently had a healthy appearance and a high viability of 75 +/- 5% (mean +/- SD) in Tyrode solution containing 1.8 mM Ca2+. After 8 h of storage at 37 degrees C, they still had a viability of 45 +/- 12%. The cells showed normal contraction properties when field-stimulated, and they generated normal action potentials and membrane currents under the whole-cell clamp condition. The beta-adrenergic signal transduction of the cells was also normal when it was examined with the isoproterenol enhancement of the L-type Ca2+ current.

Different cation sensitivities and binding site domains of Na+-Ca2+-K+ and Na+-Ca2+ exchangers.

We examined inhibitory effects of external multivalent cations Ni(2+), Co(2+), Cd(2+), La(3+), Mg(2+), and Mn(2+) on reverse-mode exchange of the K(+)-dependent Na(+)/Ca(2+) exchanger NCKX2 and the K(+)-independent exchanger NCX1 expressed in CCL-39 cells by measuring the rate of Ca(2+) uptake with radioisotope tracer and electrophysiological techniques. The apparent affinities for block of Ca(2+) uptake by multivalent cations was higher in NCKX2 than NCX1, and the rank order of inhibitory potencies among these cations was different. Additional experiments also showed that external Li(+) stimulated reverse-mode exchange by NCX1, but not NCKX2 in the presence of 5 mM K(+). Thus, both exchangers exhibited differential sensitivities to not only K(+) but also many other external cations. We attempted to locate the putative binding sites within the alpha motifs for multivalent cations by site-directed mutagenesis experiments. The cation affinities of NCKX2 were altered by mutations of amino acid residues in the alpha-1 motif, but not by mutations in the alpha-2 motif. These results contrast with those for NCX1 where mutations in both alpha-1 and alpha-2 motifs have been shown previously to affect cation affinities. Susceptibility tests with sulfhydryl alkylating agents suggested that the alpha-1 and alpha-2 motifs are situated extracellularly and intracellularly, respectively, in both exchangers. A topological model is proposed in which the extracellular-facing alpha-1 motif forms an external cation binding site that includes key residues N203, G207C, and I209 in NCKX2, while both alpha-1 and alpha-2 motifs together form the binding sites in NCX1.

Cell-volume regulation by swelling-activated chloride current in guinea-pig ventricular myocytes.

The cell-volume regulation by swelling-activated Cl- current (I(Cl,swell)) was studied in guinea pig ventricular myocytes, using a microscopic video-image analysis. We have previously shown that in ventricular cells depolarized in high-K+ ([K+]o>45 mM) solution, an activation of the cyclic AMP-dependent Cl- current (I(Cl,cAMP)) leads to cell swelling. We first investigated the mechanism underlying the I(Cl,cAMP)-independent recovery (shrinkage) of the swollen cells. They shrank when the membrane potential (Vm) was made negative to the equilibrium potential of Cl- (ECl) by lowering [K+]o or [Cl-]o in the high-K+ solution. This shrinkage was attenuated by the inhibitors (DIDS, glibenclamide, furosemide) of swelling-activated Cl- current (I(Cl,swell)). These findings suggested an involvement of I(Cl,swell) in the observed isosmotic cell shrinkage. On the other hand, an application of hyposmotic (70% of control) solution to the cells at normal [K+]o (ECl>Vm) induced a cell swelling, and the swollen cells underwent a slight but definite spontaneous cell shrinkage during hyposmotic challenge, indicating the operation of the mechanism of regulatory volume decrease (RVD). This RVD was pronounced at low [Cl-]o, at which ECl was much more positive than Vm. On the contrary, when the hyposmotic solution was applied to the cells at high [K+]o, at which ECl was negative to Vm, the cells swelled vigorously and monotonically without showing RVD, the swelling being much greater than that seen at normal [K+]o. Both the RVD at normal [K+]o and the extra cell swelling at high [K+]o were suppressed by DIDS. These results suggest that I(Cl,swell) activated by cell swelling can shrink or inflate the cardiac cells under hyposmotic as well as isosmotic conditions, depending on Vm and ECl.