Treatment with dimethylthiourea prevents left ventricular remodeling and failure after experimental myocardial infarction in mice: role of oxidative stress.

Oxidative stress might play an important role in the progression of left ventricular (LV) remodeling and failure that occur after myocardial infarction (MI). We determined whether reactive oxygen species (ROS) are increased in the LV remodeling and failure in experimental MI with the use of electron spin resonance spectroscopy and whether the long-term administration of dimethylthiourea (DMTU), hydroxyl radical (.OH) scavenger, could attenuate these changes. We studied 3 groups of mice: sham-operated (sham), MI, and MI animals that received DMTU (MI+DMTU). Drugs were administered to the animals daily via intraperitoneal injection for 4 weeks.OH was increased in the noninfarcted myocardium from MI animals, which was abolished in MI+DMTU. Fractional shortening was depressed by 65%, LV chamber diameter was increased by 53%, and the thickness of noninfarcted myocardium was increased by 37% in MI. MI+DMTU animals had significantly better LV contractile function and smaller increases in LV chamber size and hypertrophy than MI animals. Changes in myocyte cross-sectional area determined with LV mid-free wall specimens were concordant with the wall thickness data. Collagen volume fraction of the noninfarcted myocardium showed significant increases in the MI, which were also attenuated with DMTU. Myocardial matrix metalloproteinase-2 activity, measured with gelatin zymography, was increased with MI after 7 and 28 days, which was attenuated in MI+DMTU. Thus, the attenuation of increased myocardial ROS and metalloproteinase activity with DMTU may contribute, at least in part, to its beneficial effects on LV remodeling and failure. Therapies designed to interfere with oxidative stress might be beneficial to prevent myocardial failure.

Direct evidence for increased hydroxyl radicals originating from superoxide in the failing myocardium.

Experimental and clinical studies have suggested an increased production of reactive oxygen species (ROS) in the failing myocardium. The present study aimed to obtain direct evidence for increased ROS and to determine the contribution of superoxide anion (*O(2)(-)), H(2)O(2), and hydroxy radical (*OH) in failing myocardial tissue. Heart failure was produced in adult mongrel dogs by rapid ventricular pacing at 240 bpm for 4 weeks. To assess the production of ROS directly, freeze-clamped myocardial tissue homogenates were reacted with the nitroxide radical, 4-hydroxy-2,2,6, 6,-tetramethyl-piperidine-N-oxyl, and its spin signals were detected by electron spin resonance spectroscopy. The rate of electron spin resonance signal decay, proportional to *OH level, was significantly increased in heart failure, which was inhibited by the addition of dimethylthiourea (*OH scavenger) into the reaction mixture. Increased *OH in the failing heart was abolished to the same extent in the presence of desferrioxamine (iron chelator), catalase (H(2)O(2) scavenger), and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron; LaMotte) (*O(2)(-) scavenger), indicating that *OH originated from H(2)O(2) and *O(2)(-). Further, *O(2)(-) produced in normal myocardium in the presence of antimycin A (mitochondrial complex III inhibitor) could reproduce the increase of H(2)O(2) and *OH seen in the failing tissue. There was a significant positive relation between myocardial ROS level and left ventricular contractile dysfunction. In conclusion, in the failing myocardium, *OH was produced as a reactive product of *O(2)(-) and H(2)O(2), which might play an important role in left ventricular failure.

Amiodarone protects cardiac myocytes against oxidative injury by its free radical scavenging action.

BACKGROUND: Oxidative stress plays an important role in the pathophysiology of ischemic heart disease and heart failure, and antioxidants might be beneficial in the treatment of these patients. This study was performed to determine the scavenging effects of amiodarone on oxygen free radicals and its protective effects against oxygen radical-mediated injury in cardiac myocytes. METHODS AND RESULTS: The formation of the radical spin adduct with hydroxy radical (.OH) in the presence of H(2)O(2) (10 mmol/L) and Fe(3+)-nitrilotriacetate (20 micromol/L) was monitored by electron paramagnetic resonance spectroscopy combined with a spin trapping agent, 5,5-dimethyl pyrroline-N-oxide (DMPO). Amiodarone decreased the intensity of the DMPO-OH signals in a dose-dependent manner (0.1 to 100 micromol/L), whereas other antiarrhythmia drugs such as disopyramide and atenolol had no such effects. Furthermore, amiodarone (10 micromol/L) protected intact adult canine cardiac myocytes against.OH-mediated myocyte injury, as assessed by the degree of morphological change from rod shape to the irreversible hypercontracture state during the exposure of cells to H(2)O(2) and Fe(3+) in vitro. CONCLUSIONS: Amiodarone can protect cardiac myocytes against oxidative stress-mediated injury by directly scavenging oxygen free radicals. Antioxidant action of amiodarone might potentially contribute to the beneficial effects of this drug in the treatment of patients with ischemic heart disease and congestive heart failure.

Positive inotropic effect of insulin-like growth factor-1 on normal and failing cardiac myocytes.

OBJECTIVE: The acute administration of growth hormone (GH) or insulin-like growth factor-1 (IGF-1) improves cardiac performance, possibly contributing to the beneficial effects of GH therapy on heart failure (HF). GH can induce the production of IGF-1 and thus the actions of GH may be mediated through its IGF-1 induction. However, these effects have not yet been demonstrated in failing hearts and the cellular basis of GH or IGF-1-induced inotropic effects remains unknown. We examined the direct effects of GH and IGF-1 on the contractile function and intracellular calcium ([Ca2+]i) homeostasis in normal and failing myocytes. METHODS: To determine whether GH and IGF-1 have a direct effect on myocardial contractility and whether the GH/IGF-1-induced effect was the results of changes in Ca2+ activation, cell shortening and [Ca2+]i transient were simultaneously measured in the left ventricular myocyte preparations, isolated from normal and rapid pacing-induced HF dogs. RESULTS: Basal shortening of HF myocytes was reduced by 64% (p < 0.01). In normal and HF myocytes, GH (0.4-40 x 10(-3) IU/ml) had no effect on either cell shortening or [Ca2+]i transients. In normal myocytes, IGF-1 exerted a positive inotropic effect in a time- and dose-dependent manner (25-500 ng/ml), associated with a parallel increase of [Ca2+]i transient amplitude. IGF-1 increased the shortening magnitude in normal (121 +/- 5% increase from baseline, p < 0.05) and HF (118 +/- 4% increase from baseline, p < 0.05) myocytes. It also increased [Ca2+]i transient amplitude in normal and HF cells by 124 +/- 4 and 125 +/- 7%, respectively. The percent increase of cell shortening and [Ca2+]i transient amplitude was comparable between normal and HF myocytes. Furthermore, IGF-1 did not shift the trajectory of the relaxation phase in the phase-plane plots of cell length vs. [Ca2+]i, indicating that it did not change myofilament Ca2+ sensitivity. CONCLUSIONS: In both normal and HF conditions, IGF-1 exerted an acute direct positive inotropic effect in adult cardiac myocytes by increasing the availability of [Ca2+]i to the myofilaments, possibly explaining the beneficial effect of GH on HF.

Role of Ca2+ availability to myofilaments and their sensitivity to Ca2+ in myocyte contractile dysfunction in heart failure.

OBJECTIVE: Contractile function is depressed at the isolated myocyte level in heart failure (HF), which could result from the decreased availability of intracellular calcium ([Ca2+]i) to the myofibrils and/or the depressed sensitivity of myofilaments to [Ca2+]i. However, the cellular basis of contractile dysfunction remains unestablished. METHODS: We isolated left ventricular myocytes from dogs with rapid pacing-induced HF. Cell shortening and [Ca2+]i transients were measured by indo-1 fluorescence and the myofilament Ca2+ sensitivity was analyzed by the shortening-[Ca2+]i relation in intact myocytes as well as by the pCa tension relation in skinned cells. RESULTS: Peak cell shortening magnitude was depressed in HF, associated with a parallel decrease of [Ca2+]i transient amplitude. There was a significant positive correlation between these two variables (r = 0.71, P < 0.01). In contrast, myofibrillar sensitivity to Ca2+, determined by both intact and skinned myocytes, was comparable between control and HF. Further, there was no significant difference in Ca2+ sensitivity between control and HF even at shorter (1.8 microns) or longer (2.2 microns) sarcomere length. CONCLUSIONS: Using both intact and skinned cellular preparations, a potential defect in myocyte contractile function in HF was a reduction in Ca2+ availability to the myofilaments, rather than the inherent defects in myofilament sensitivity to Ca2+.

Synchronization of chaos in microchip lasers by using incoherent feedback.

We propose a chaos-synchronization scheme using incoherent feedback to the pumping power in two microchip lasers. The population inversion of the slave laser is controlled for synchronization by using the detected signals of the peak heights of chaotic pulse intensities in the two lasers. Matching of the optical frequencies between the two lasers (i.e., injection locking) is not required for synchronization using this method. We numerically demonstrate the incoherent feedback method and investigate synchronization regions against parameter mismatching between the two lasers. Synchronization is maintained within a mismatching of 1% for all laser parameters, which implies that the difficulty in reproducing the synchronized laser pulses is very useful for applications of secure optical communications.

Dual synchronization of chaos in one-way coupled microchip lasers.

We experimentally demonstrate the dual synchronization of chaos in two pairs of Nd:YVO(4) microchip lasers in a one-way coupling configuration over one transmission channel. Dual synchronization is achieved when the optical frequency is matched between the corresponding pairs of lasers by using injection locking. We investigate the influence of optical injection from the two master lasers to one slave laser, and found that the dual synchronization is observed when the injection locking is achieved between either of the master lasers and the slave laser. Under the condition of the injection locking between both of the master lasers and the slave laser, the injection locking is alternately achieved and the accuracy of dual synchronization is degraded. We also confirm these results by numerical calculation.

Dental pulp and periodontal ligament cells support osteoclastic differentiation.

Odontoclasts and cementoclasts are considered to play major roles in the internal resorption of dentin and the external resorption of tooth roots. In this study, we evaluated the osteoclast-inducing ability of human dental pulp and periodontal ligament cells, which are mesenchymal cells in dental tissues. These cells expressed RANKL and OPG mRNA constitutively. As osteoclast precursors, CD14(+) monocytes derived from human peripheral blood were isolated, and incubated together with human dental pulp or periodontal ligament cells. Both cell types spontaneously induced the differentiation of CD14(+) monocytes into osteoclasts without osteotropic factors. These results suggest that dental pulp and periodontal ligament cells are involved in regulating the differentiation and function of osteoclasts.

Adrenoceptor-mediated regulation of myofibrillar Ca2+ sensitivity through the GTP-binding protein-related mechanisms: tension recording in beta-escin-skinned single rat cardiac cells with preserved receptor functions.

To investigate the mechanisms of receptor-mediated regulation of heart muscle contraction, we developed a tension-recording system using beta-escin-skinned single cardiac cells of rats and studied the effects of agonists on myofibrillar Ca2+ sensitivity and Ca2+ release from the sarcoplasmic reticulum (SR). In pCa/tension relations, 1 microM isoproterenol plus 100 microM guanosine 5'-triphosphate (GTP) decreased the myofibrillar Ca2+ sensitivity (pCa50, the [Ca2+] required for half-maximal tension, as an indicator of the sensitivity; from 6.07 to 5.92); this effect was blocked by 1 microM metoprolol or 1 mM guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS). Phenylephrine (10 microM) plus 100 microM GTP increased the Ca2+ sensitivity (pCa50; from 6.12 to 6. 28), and this effect was blocked by 1 microM phentolamine or 1 mM GDPbetaS. After Ca2+ loading into the SR, 10 microM phenylephrine plus 100 microM GTP in a low-ethylene- glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, 0. 1 mM) relaxing solution induced oscillatory contractions that were attenuated by either 1 microM phentolamine or pre-treatment with 10 microM inositol 1,4,5-trisphosphate. Our results demonstrate that beta1-adrenergic stimulation decreases myofibrillar Ca2+ sensitivity and that alpha1-adrenergic stimulation both increases the Ca2+ sensitivity and activates Ca2+ release from the agonist-sensitive SR through GTP-binding protein-related mechanisms.

Dual synchronization of chaos in microchip lasers.

We have achieved dual synchronization of chaos in two pairs of one-way coupled Nd:YVO4 microchip lasers, using only one transmission channel, by experiment and numerical calculation. We observed the individual synchronization of chaos in each pair of two lasers by adjusting the optical frequencies for injection locking between the corresponding pairs. The achievement of dual synchronization is dependent on the injection-locking condition, which is different from the locking condition for a single pair of lasers because of the presence of an additional injection signal from the master laser of the other pair.

Endocardial versus epicardial differences of sarcoplasmic reticulum Ca2+-ATPase gene expression in the canine failing myocardium.

It is unknown whether the transmural heterogeneity of sarcoplasmic reticulum (SR) Ca2+-ATPase gene expression is present within the left ventricular (LV) wall. Moreover, the changes of transmural distribution have not been examined in the failing hearts. We thus quantified steady-state mRNA abundance of SR Ca2+ regulatory proteins by Northern blot analysis in both subendocardial and subepicardial LV layers from normal and rapid pacing-induced heart failure (HF) dog hearts. For normal LV, Ca2+-ATPase mRNA abundance (normalized to glyceraldehyde-3-phosphate dehydrogenase [GAPDH] mRNA) was significantly reduced in the subendocardium, whereas calsequestrin mRNA abundance was comparable between the two layers. For HF LV, Ca2+-ATPase mRNA abundance in the subendocardium was also reduced compared to the subepicardium. However, the endocardium to epicardium ratio was comparable between control and HF (0.62 +/- 0.08 vs. 0.65 +/- 0.07; p = NS). Therefore, the transmural gradient of this gene was constant in both control and HF. Even though the data on the transmural heterogeneity of protein level is not available, the subendocardium contained significantly less Ca2+-ATPase mRNA, which might contribute, at least in part, to the transmural gradients of biochemical and mechanical function.

Effects of ACE inhibition on left ventricular failure and oxidative stress in Dahl salt-sensitive rats.

Dahl salt-sensitive (DS) rats fed high-salt diet exert compensated left ventricular (LV) hypertrophy and eventually develop heart failure. Oxidative stress has been shown to be involved in myocardial remodeling and failure and thus might play an important role in this transition from hypertrophy to failure. We measured the amount of reactive oxygen species (ROS) in the myocardium from DS rats by using electron spin resonance spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (hydroxy-TEMPO) and also examined the effects of chronic angiotensin-converting enzyme (ACE) inhibition on the transition. We divided DS rats (5 weeks old, 150-200 g) into three groups: low-salt (0.3% NaCl) diet for 10 weeks (LS group), high-salt (8% NaCl) diet for 10 weeks (HS-10+V group), and high-salt diet and cilazapril (10 mg/kg body weight per day) started after 5 weeks of high-salt diet and maintained for 5 weeks (HS-10+Cil group). Systolic blood pressure (mm Hg) was significantly elevated in the HS-10+V (229+/-5) and HS-10+Cil (209+/-5) groups compared with the LS group (141+/-2). The amount of myocardial ROS was not changed after 5 weeks of high-salt diet, but significantly increased in HS-10+V rats compared with LS rats, and was abolished in the HS-10+Cil group. HS-10+V rats exerted the clinical signs of heart failure, including increased lung weight and pleural effusion, associated with LV hypertrophy and LV cavity dilatation. In the HS-10+Cil group, signs of heart failure were significantly attenuated despite only a modest reduction in systolic blood pressure (-20 mm Hg). The progression of LV failure after hypertrophy in high-salt-loaded DS hypertensive rats was associated with increased myocardial ROS, and ACE inhibitor could prevent this transition from compensated hypertrophy to failure.

Positive inotropic effects of calcium sensitizers on normal and failing cardiac myocytes.

Calcium sensitizers increase myocardial contractile function without affecting Ca2+ homeostasis, which might be beneficial in the treatment of patients with heart failure. However, it remains uncertain whether Ca sensitizers induce quantitatively similar inotropic responses in control and failing hearts. To compare their effects in normal versus failing hearts at the cellular level, shortening mechanics and intracellular calcium ([Ca2+]i) transient were simultaneously measured in the left ventricular myocytes isolated from normal dogs (n = 8) and dogs with rapid pacing-induced heart failure (n = 7). CGP 48506 and EMD 57033 exerted a positive inotropic effect in a dose (0.1-3 microM)-dependent manner in both normal and heart failure myocytes. The percent increase of cell shortening magnitude was comparable between the two groups. CGP 48506 and EMD 57033 did not affect the diastolic cell length and resting [Ca2+]i level. They did not affect the duration of [Ca2+]i transient dynamics. Thus Ca2+ sensitizers exerted comparable positive inotropic effects without affecting the rest cell length and rest [Ca2+]i in normal and heart failure myocytes.

Alpha1-adrenoceptor-Gq-RhoA signaling is upregulated to increase myofibrillar Ca2+ sensitivity in failing hearts.

Alpha1-adrenergic stimulation, coupled to Gq, has been shown to promote heart failure. However, the role of alpha1-adrenergic signaling in the regulation of myocardial contractility in failing myocardium is still poorly understood. To investigate this, we observed 1) the effect of phenylephrine on myofibrillar Ca2+ sensitivity in alpha-toxin-skinned cardiomyocytes, and 2) protein expression of Gq, RhoA, and myosin light chain phosphorylation using tachypacing-induced canine failing hearts. Phenylephrine significantly increased myofibrillar Ca2+ sensitivity in failing but not in normal cardiomyocytes. Whereas Y-27632 (Rho kinase inhibitor) blocked the phenylephrine-induced Ca2+ sensitization in the failing myocytes, calphostin C (protein kinase C inhibitor) had no effect on Ca2+ sensitization. The protein expression of Galpha(q) and RhoA and the phosphorylation level of regulatory myosin light chain significantly increased in the failing myocardium. Our results suggest that alpha1-adrenoceptor-Gq signaling is upregulated in the failing myocardium to increase the myofibrillar Ca2+ sensitivity mainly through the RhoA-Rho kinase pathway rather than through the protein kinase C pathway.

Mitochondrial electron transport complex I is a potential source of oxygen free radicals in the failing myocardium.

Oxidative stress in the myocardium may play an important role in the pathogenesis of congestive heart failure (HF). However, the cellular sources and mechanisms for the enhanced generation of reactive oxygen species (ROS) in the failing myocardium remain unknown. The amount of thiobarbituric acid reactive substances increased in the canine HF hearts subjected to rapid ventricular pacing for 4 weeks, and immunohistochemical staining of 4-hydroxy-2-nonenal ROS-induced lipid peroxides was detected in cardiac myocytes but not in interstitial cells of HF animals. The generation of superoxide anion was directly assessed in the submitochondrial fractions by use of electron spin resonance spectroscopy with spin trapping agent, 5, 5'-dimethyl-1-pyrroline-N-oxide, in the presence of NADH and succinate as a substrate for NADH-ubiquinone oxidoreductase (complex I) and succinate-ubiquinone oxidoreductase (complex II), respectively. Superoxide production was increased 2.8-fold (P<0.01) in HF, which was due to the functional block of electron transport at complex I. The enzymatic activity of complex I decreased in HF (274+/-13 versus 136+/-9 nmol. min(-1). mg(-1) protein, P<0.01), which may thus have caused the functional uncoupling of the respiratory chain and the deleterious ROS production in HF mitochondria. The present study provided direct evidence for the involvement of ROS in the mitochondrial origin of HF myocytes, which might be responsible for both contractile dysfunction and structural damage to the myocardium.

Role of microtubules in the contractile dysfunction of myocytes from tachycardia-induced dilated cardiomyopathy.

Microtubules of cardiac myocytes are increased in pressure-overloaded cardiac hypertrophy, which interfere with the actin-myosin crossbridge motion and depress muscle contractility. However, it is unknown whether microtubules are increased in non-hypertrophied, dilated cardiomyopathy and, if so, their increase could contribute to the depressed contractility. We assessed the contractile function of isolated left-ventricular (LV) myocytes and also quantitated tubulin mRNA levels as well as free and polymerized tubulin proteins using the LV myocardium obtained from dogs with rapid pacing (240 beats/min, 4 weeks)-induced dilated failing cardiomyopathy (HF; n = 6) and control dogs (n = 6). Myocyte contractility was significantly depressed in HF compared to control. Northern blot analysis indicated that tubulin mRNA levels (normalized to GAPDH mRNA) in HF dogs were upregulated (0.43 +/- 0.04 v 0.13 +/- 0.02; P < 0.01). In contrast, the amount of total tubulins (633 +/- 52 v 697 +/- 42 micrograms/g wet weight; P = N.S.) and the ratio of polymerized tubulin fraction-to-total tubulin (0.44 +/- 0.02 v 0.44 +/- 0.01; P = N.S.) did not differ between the two groups. Immunohistochemical studies showed no apparent differences in the distribution or density of intracellular microtubule network. Further, the exposure of myocytes to colchicine (1 mumol/l, 30 min), which depolymerizes microtubules, did not promote any improvement of the depressed myocyte contraction. Pacing-induced tachycardia increased myocardial tubulin mRNA, but the amount of total and polymerized tubulins were not increased, indicating that alterations in myocyte microtubules do not contribute to the contractile abnormalities in this model of HF.

Negative inotropic effect of basic fibroblast growth factor on adult rat cardiac myocyte.

BACKGROUND: Basic fibroblast growth factor (bFGF) is highly expressed in the myocardium in some cardiac disorders, such as ischemia-reperfusion and cardiac allograft rejection. However, whether bFGF has any effects on myocardial contraction is unknown. METHODS AND RESULTS: We examined the effects of bFGF on myocardial contractility using isolated adult rat cardiac myocyte preparations. bFGF exerted a direct negative inotropic effect that was concentration and time dependent. The pretreatment of myocytes with a neutralizing anti-bFGF antibody (100 ng/mL) abolished the negative inotropic effects of bFGF (100 ng/mL). Platelet-derived growth factor (12.5 ng/mL) and transforming growth factor-beta (1 ng/mL) did not exert such effects, which indicated that bFGF-induced negative inotropism was considered to be specific for this growth factor. bFGF decreased the peak intracellular Ca2+ transient by 46% during systole. The enhanced production of nitric oxide was unlikely to be responsible for the bFGF-induced negative inotropic effect. CONCLUSIONS: bFGF, primarily a potent growth promoter, produced acute negative inotropic effects in the adult cardiac myocyte that could have resulted from alterations in intracellular Ca2+ homeostasis. The negative inotropic effect of bFGF may contribute to myocardial dysfunction associated with ischemia-reperfusion injury and heart transplant rejection.

Role of SR Ca2+-ATPase in contractile dysfunction of myocytes in tachycardia-induced heart failure.

Sarcoplasmic reticulum (SR) Ca2+-ATPase gene expression is reduced in the failing myocardium. However, the functional relevance of these changes to myocardial contractility is not yet established. We assessed myocardial contractile function by analyzing sarcomere motion of isolated myocytes and also quantified SR Ca2+ regulatory protein gene expression by Northern blot analysis in the same hearts obtained from 10 dogs with pacing-induced heart failure (HF; 240 beats/min, 4 wk) and 7 control dogs. Sarcomere-shortening velocity was depressed in HF myocytes, accompanied by the prolongation of intracellular Ca2+ concentration ([Ca2+]i) transient measured by indo 1 fluorescence ratio. SR Ca2+-ATPase mRNA levels (normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA) were significantly depressed in HF, and calsequestrin mRNA was increased. For control and HF dogs, sarcomere-shortening velocity correlated positively with Ca2+-ATPase mRNA levels (r = 0.73, n = 17, P < 0.01) but not with calsequestrin mRNA. Ca2+-ATPase mRNA levels were correlated with 45Ca2+ uptake rate by SR, which was also reduced in HF. Moreover, the inhibition of SR Ca2+-ATPase with thapsigargin or cyclopiazonic acid reproduced in normal myocytes the abnormalities observed in HF myocytes, such as depressed contractility and the prolonged [Ca2+]i transient duration. A downregulation of Ca2+-ATPase gene expression and a resultant decrease in Ca2+ uptake by SR may be responsible for the contractile dysfunction and the alterations of [Ca2+]i transient in HF.