Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor.

G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.

[ANATOMICAL CHARACTERISTICS OF SEPTOMARGINAL TRABECULA OF THE RIGHT VENTRICLE OF THE HUMAN FETAL HEART].

Anatomical structure of right ventricular septomarginal trabecula (SMT) was studied at micro-macroscopical level in 99 preparations of human fetal heart formed without malformations and minor abnormalities, obtained at 17­28 weeks of development. SMT was found to be a constant cardiac structure consisting of the body and two branches: anterior and posterior. Body of SMT is a myocardial vallum, unseparable from the interventricular septum, the long axis of which is always located along the conventional line connecting septal insertion of supraventricular crest and right ventricular apex. Posterior margin of SMT body was distinct in 75% of cases and smoothened in 21.9%. Base of the SMT was solid in 46.3% and split into secondary trabeculae in 52.6% of cases. Narrow and wide forms of SMT body were considered as its extreme anatomical variants. It is suggested to distinguish two anatomical types of SMT: a complete type, in which SMT was represented by a complex consisting of body in the form of muscular vallum and both branches, and an incomplete one, in which one of the branches was absent. Human fetal heart SMT is characterized by a variability, which is manifested by certain combinations of anatomic variants of SMT base form, its posterior margin, and the presence, shape and mutual location of its branches.

Regional expression of sodium pump subunits isoforms and Na+-Ca++ exchanger in the human heart.

Cardiac glycosides exert a positive inotropic effect by inhibiting sodium pump (Na,K-ATPase) activity, decreasing the driving force for Na+-Ca++ exchange, and increasing cellular content and release of Ca++ during depolarization. Since the inotropic response will be a function of the level of expression of sodium pumps, which are alpha(beta) heterodimers, and of Na+-Ca++ exchangers, this study aimed to determine the regional pattern of expression of these transporters in the heart. Immunoblot assays of homogenate from atria, ventricles, and septa of 14 nonfailing human hearts established expression of Na,K-ATPase alpha1, alpha2, alpha3, beta1, and Na+-Ca++ exchangers in all regions. Na,K-ATPase beta2 expression is negligible, indicating that the human cardiac glycoside receptors are alpha1beta1, alpha2beta1, and alpha3beta1. alpha3, beta1, sodium pump activity, and Na+-Ca++ exchanger levels were 30-50% lower in atria compared to ventricles and/or septum; differences between ventricles and septum were insignificant. Functionally, the EC50 of the sodium channel activator BDF 9148 to increase force of contraction was lower in atria than ventricle muscle strips (0.36 vs. 1.54 microM). These results define the distribution of the cardiac glycoside receptor isoforms in the human heart and they demonstrate that atria have fewer sodium pumps, fewer Na+-Ca++ exchangers, and enhanced sensitivity to inotropic stimulation compared to ventricles.

Calcium-dependent proteolysis and its inhibition in the ischemic rat myocardium.

Activity of calcium-dependent neutral proteinase and its specific inhibition was investigated in the acutely ischemic myocardium after ligation of the left descending coronary artery in anaesthetized open chest rats. In experiments where the mean arterial blood pressure could be maintained above 70 mm Hg the hearts were quickly removed 5 to 30 min after ligation and homogenized in ice-cold buffer. The activity of the calcium-dependent neutral proteinase and of its endogenous inhibitor were determined in the 10,000 g supernatant of ventricular and septal tissues. Hearts from normal anaesthetized and sham operated animals left on the respirator for 10, 20 and 30 min, were used as controls. Only traces of proteinase activity could be found in the supernatants obtained from normal controls, while in the sham-operated animals the specific activity of the Ca2+-dependent proteinase increased with time, reaching significantly elevated values after 30 min on the respirator. In the ischemic groups enzyme activity also increased with increasing duration of ischemia and was substantially elevated in the ventricular myocardium 20 min after ligation. The increased calcium-dependent proteinase activity was accompanied by significantly reduced activity of its endogenous inhibitor. The concomitant changes in the activities of the myocardial calcium-dependent neutral protease and its endogenous inhibitor suggest that interaction between the enzyme and its inhibitor play a role in the regulation of intracellular calcium-dependent proteolysis.

Short-term NO synthase inhibition and the Na+-binding properties of cardiac Na,K-ATPase.

It is known that hypertension is accompanied by increased [Na+]i. The functional properties of Na,K-ATPase, which transports the Na+ out and K+ into myocardial cells during the relaxation phase, were investigated in the left ventricle (LV), septum (SV) and the right ventricle (RV) of anesthetized dogs with moderate acute blood pressure elevation elicited by short-term (4-hour) NO synthase inhibition. The NO-insufficiency was induced by administration of an L-arginine analogue, the N(G)-nitro-L-arginine methyl ester (L-NAME). Concerning the function of Na,K-ATPase under the conditions of lowered NO synthesis, we focused our attention to the binding of Na+ to the enzyme molecule. Activation of the enzyme by increasing Na+ concentrations revealed significant changes in both the maximal velocity (Vmax) and the affinity for Na+ (K(Na)) in all investigated heart sections. The Vmax increased by 27% in LV, by 87% in SV and by 58% in RV. The K(Na) value increased by 86% in LV, by 105% in SV and by 93% in RV, indicating an apparent decrease in the sensitivity of the Na+-binding site in the Na,K-ATPase molecule. This apparently decreased pump affinity for Na+ together with the increase of Vmax suggest that, during the short-term inhibition of NO synthesis, the Na,K-ATPase is capable of extruding the excessive Na+ from the myocardial cells more effectively at higher [Na+]i, as compared to the Na,K-ATPase of control animals.

[Glycogen phosphorylase activity in infarct-affected and nonaffected parts of the human myocardium after death]. / Aktivnost' glikogenfosforilazy v porazhennykh i ne porazhennykh infarktom uchastkakh miokarda cheloveka posle smerti.

Distinct differences in activity of glycogen phosphorylase were not observed in various parts of an intact human myocardium. This enzymatic activity vas considerably decreased in the left ventricle of the myocardium impaired by infarction; with prolongation of the infarction period the enzymatic activity decreased further. Isozymes of glycogen phosphorylase I, II and III were liberated from ischemic myocardium according to their quantitative ratio in the heart. The glycogen phosphorylase activity, which was observed in blood serum of patients with acute myocardial infarction, was apparently liberated into blood due to the myocardium impairment. Evaluation of the enzymatic activity in blood serum of the patients might serve as an index of the necrosis degree.

Rho kinases regulate endothelial invasion and migration during valvuloseptal endocardial cushion tissue formation.

Rho-associated kinase (ROCK) is a downstream effector of small Rho-GTPases, and phosphorylates several substrates to regulate cell functions, including actin cytoskeletal reorganization and cellular motility. Endothelial-mesenchymal transformation (EMT) is a critical event in the formation of valves and septa during cardiogenesis. It has been reported that ROCK plays an important role in the regulation of endocardial cell differentiation and migration during mouse cardiogenesis (Zhao and Rivkees [2004] Dev. Biol. 275:183-191). Immunohistochemistry showed that, during chick cardiogenesis, ROCK1 and -2 were expressed in the transforming and migrating endothelial/mesenchymal cells in the outflow tract (OT) and atrioventricular (AV) canal regions from which valvuloseptal endocardial cushion tissue would later develop. Treatment with Y27632, a specific ROCK inhibitor, of cultured AV explants or AV endothelial monolayers of stage 14-minus heart (preactivated stage for EMT) on three-dimensional collagen gel perturbed the seeding of mesenchymal cells into the gel lattice. In these experiments, Y27632 did not suppress the expression of an early transformation marker, smooth muscle alpha-actin. Moreover, Y27632 inhibited the mesenchymal invasion in stage 14-18 AV explants, in which endothelial cells had committed to undergo EMT. ML-9, a myosin light chain kinase inhibitor, also inhibited the mesenchymal invasion in cultured AV explants. These results suggest that ROCKs have a critical role in the mesenchymal cell invasion/migration that occurs at the late onset of EMT.

[Succinate dehydrogenase activity in various parts of the rat heart in systole and diastole]. / Aktivnost' suktsinatdegidrogenazy v raznykh otdelakh serdtsa krys v sistole i diastole.

After fixation of the rat heart at the state of systole and diastole, succinate dehydrogenase (SDH) activity has been revealed. In distributions of the muscle fibers according to their optic density, statistic characteristics connected with the first four moments have been taken into consideration. SDH activity is different in five cardiac parts. Individual changeability is more pronounced in the all cardiac parts at diastole. In comparison to the internal one at systole, it is lower in the all parts, and at diastole--in the atria only. The ratio of the internal and individual changeability is in such a state, that it should be taken into account in all statistical calculations, connected with SDH activity determination in cardiomyocytes.

[Changes in the activity of antioxidant enzymes in ischemia and subsequent reperfusion of the myocardium]. / Izmenenie aktivnosti "antioksidantnykh" fermentov pri ishemii i posleduiushchei reperfuzii miokarda.

Transitory coronary failure of the myocardium was accompanied by a considerable reduction in the activity of superoxide dismutase, glutathione peroxidase and glutathione transferase, with the activity of the enzymes under study being not different in the ischemized and distant from ischemia zones of the myocardium. Reperfusion did influence the activity of superoxide dismutase and glutathione peroxidase after 10 and 40 minutes of ischemia, whereas following 120 minutes of ischemia the activity of superoxide dismutase ascended after 10 and 40 minutes of reperfusion while the activity of glutathione peroxidase remained unchanged.

[Effect of indomethacin and nonachlazine on myocardial and cerebral tyrosine hydroxylase activity in rats]. / Vliianie indometatsina i nonakhlazina na aktivnost' tirozingidroksilazy v miokarde i golovnom mozge krys.

Nonachlazin (6 mg/kg) and indomethacin (10 mg/kg) intraperitoneally have a unidirectional effect on the activity of tyrosin hydroxylase, reducing it in the hypothalamus and in the brain stem and increasing it in the heart septum. In the brain, the effects of nonachlazin and indomethacin are not summarized whereas in the heart their effects are summarized. Unlike indomethacin, nonachlazin exerts a direct effect on the rate of tyrosin hydroxylase reaction but has no effect on the activity of cyclooxygenase and on synthetase of prostaglandins. It is concluded that prostaglandins produce a modulating action on the activity of brain and heart tyrosine hydroxylase and participate in the mechanism of action of nonachlazin.

Regional choline acetyltransferase activity in the guinea pig heart.

Choline acetyltransferase is the enzyme that catalyzes the biosynthesis of acetylcholine, the neurotransmitter of the pre- and postganglionic parasympathetic system. To assess the extent of parasympathetic innervation, enzyme activity was measured in specialized and contractile regions throughout the guinea pig heart. Enzyme activity in the right atrial appendage was 137 nmol g(-1) hr(-1). Activity was greatest in the region of the sinoatrial node (187 nmol g(-1) hr(-1). Also, enzyme activity was high in the regions of the atrioventricular node (153 nmol g(-1) hr(-1), the proximal conduction bundles (133 nmol g(-1) hr(-1), and the base of the anterior papillary muscle of the right ventricle (179 nmol g(-1) hr(-1), which contains the moderator band and Purkinje fibers. In contrast, the enzyme activity in the inferior interventricular septum and the free walls of the right and left ventricles, which are more predominantly contractile tissue, was 67 +/- 6,108 +/- 14, and 56 +/- 11 nmol g(-1) hr(-1), respectively. This activity is significantly lower than in the right atrial appendage. These results suggest that the density of parasympathetic innervation is similar in all the components of the conduction system, from the sinoatrial node to Purkinje tissues. Furthermore, the parasympathetic innervation of regions specialized for conduction is up to four times more dense than that of contractile regions.

The perinatal rat: body weight, hematocrit and regional changes in heart weight and lactate dehydrogenase isozyme composition and activity.

Body weight (BW), hematocrit (Hct), heart weight (HW) and cardiac lactate dehydrogenase (LDH) activity and isozyme pattern were studied in the perinatal rat. BW increased linearly, from 5 days before birth till 10 days after birth, while Hct increased from 30 to 34% within 1 day of birth. HW increased in step with BW. However, the relative contribution to total HW of right ventricle (RV), interventricular septum (S) and atria declined relative to the left ventricle free wall (LVW) beginning 2 to 3 days before birth. RV/LVW declined steadily throughout the study period. LDH specific activity in LVW, RV, S and atria increased greatly prior to birth and less so afterwards, with atria showing the lowest values throughout the study. Total LDH activity in each portion of the heart paralleled the respective regional weight changes. LDH isozyme composition (percent M subunit) declined at the time of birth in LVW, RV and S from 63 to 43%; change in atrial M subunit was smaller. The change in LDH isozyme composition could be accounted for in LVW, RV and S by increasing H subunit activity alone. In atria, both M and H subunit activity increased.

Regional changes in ornithine decarboxylase activity and polyamine levels during thyroxine-induced cardiac hypertrophy.

Ornithine decarboxylase activities (ODC) and polyamine levels were determined in five cardiac regions of the rat heart, following daily administration of 1 mg/kg of thyroxine, in the right and left atria, the right and left ventricles and the septum. The thyroxine stimulated ODC activity in all five regions of the heart. Enzyme activity in the left atrium and the septum peaked a day earlier than in other regions and the decline of ODC activity was slower. Putrescine in control animals was present in all regions except the right atrium, where its content was below detectable levels. Following the administration of thyroxine, the putrescine content of the left atrium, the right ventricle and the septum declined, while spermidine and spermine levels remained unchanged. In direct contrast to the other regions of the heart, thyroxine stimulated an increase in polyamines, as well as in weight which occurred exclusively in the left ventricle. These findings suggest a causal relationship between increased polyamines and hypertrophy.

Isolation and characterization of myosin from subjects with asymmetric septal hypertrophy.

Human cardiac myosin isolated from operatively obtained samples of ventricular septum and left ventricular free wall of subjects with asymmetric septal hypertrophy (ASH) was compared, with respect to structural and enzymatic properties, to myosin isolated from hearts of subjects without heart disease. The following parameters were studied: (1) activation of myosin ATPase activity by K+-EDTA and Ca2+, (2) molecular weight of the heavy and light chains of myosin as determined by electrophoretic migration in polyacrylamide-sodium dodecyl sulfate (SDS) gels and (3) ability to form bipolar aggregates at low ionic strength, as examined by electron microscopy. No difference was present in any of these parameters between human cardiac myosin from subjects with ASH and from subjects without heart disease. Thus, the genetic defect present in subjects with ASH is not expressed in the particular structural and functional characteristics of myosin evaluated in this study.

Intraventricular changes in the beta-adrenoceptor-adenylate cyclase system of the rat heart with the progress of monocrotaline-induced right ventricular hypertrophy.

We investigated the changes in the membranous beta-adrenoceptor-adenylate cyclase system in the right ventricle, left ventricle and interventricular septum during the progress of monocrotaline-induced right ventricular hypertrophy and failure. The beta-adrenoceptor density was decreased in hypertrophied right ventricle 2 to 4 weeks after treatment. When the rats showed symptoms of right ventricular failure 4 weeks after treatment, the beta-adrenoceptor density was decreased in the interventricular septum. Both basal and forskolin-stimulated adenylate cyclase activities were decreased in the right ventricle at 3 and 4 weeks, and in the interventricular septum at 4 weeks, after treatment, which indicates that the catalytic activity of adenylate cyclase is reduced. Changes in isoproterenol plus Gpp (NH) p- or sodium fluoride-stimulated adenylate cyclase activity were generally similar to those in basal activity. These data indicate that a chamber-specific decrease in beta-adrenoceptor density begins in the early stages of right ventricular hypertrophy, and that beta-adrenoceptor density and adenylate cyclase activity in the interventricular septum are decreased in the advanced stages of heart failure in monocrotaline-treated rats.

Antioxidant-related enzymes in myocardial zones and human pericardial fluid in relation to the cause of death.

The aim of this work was to shed light on hypoxic and ischemic processes in the heart that may lead to irreversible or lethal myocardial injury. We determined malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities in human cardiac tissues from 45 medico-legal autopsies of persons who died from different causes. Samples were taken from three different areas of myocardium: the anterior and posterior walls of the left ventricle, and the interventricular septum. We used light microscopy to examine the heart sections (hematoxylin-eosin and Masson's trichromic stains), and studied the K+(Na+ ratio and pericardial fluid. A decrease in GSH-Px activity was found in cases with severe atherosclerosis of the coronary artery in comparison with the group with slight or moderate atherosclerosis. Postmortem activities of GSH-Px and SOD were significantly different in the three myocardial zones studied. An increase in GSH-Px activity in the interventricular septum was noted in cases of cardiac deaths. Antioxidant-related enzymes such as GSH-Px and SOD can therefore be regarded as new biochemical markers indicative of myocardial hypoxia. The possible applications to the postmortem diagnosis of the cause of death are discussed.

Regional changes in creatine kinase and myocyte size in hypertensive and nonhypertensive cardiac hypertrophy.

Several intracellular enzymes have been shown to have altered total activity or isoenzyme composition in cardiac hypertrophy. This study tests the hypothesis that the accumulation of the fetal-type (BB + MB) creatine kinase (CK) isoenzymes in hypertrophied adult myocardium is related to an increase in blood pressure. Consideration was made for the location, size, and hemodynamic load of the myocytes. By using the two-kidney, one-clip (2K1C) rat model of renal hypertension with and without hydralazine treatment, CK (total and isoenzyme), lactate dehydrogenase, and citrate synthase activities and myocyte size were measured. An increased heart weight/body weight ratio occurred in both untreated 2K1C rats (4.15 +/- 0.09) and hydralazine-treated 2K1C rats (4.12 +/- 0.13) as compared with control rats (3.25 +/- 0.10). Blood pressure was high only in untreated 2K1C rats (196 +/- 9 mm Hg), as compared with hydralazine-treated 2K1C rats (142 +/- 6 mm Hg) and control rats (135 +/- 3 mm Hg). Myocytes were isolated from five ventricular regions: left ventricular epicardial and endocardial free wall, left and right halves of the interventricular septum, and right ventricular free wall. Regional differences in normal and hypertrophied myocardium were demonstrated for morphological and biochemical parameters, with the greatest changes occurring in left ventricular endocardium. The shift in CK isoenzyme expression toward accumulating more BB + MB was greater in "hypertensive hypertrophy" (untreated 2K1C rats) than in "nonhypertensive hypertrophy" (hydralazine-treated 2K1C rats). Calculations incorporating isolated myocyte volume showed that the cellular content of total CK remained the same during the hypertrophic process, accounting for a decrease in the tissue activity. Measurement of lactate dehydrogenase and citrate synthase activities suggests that hypertrophied myocardium has relatively higher glycolytic capacity and that this effect is exacerbated in the presence of high blood pressure. We conclude that increased blood pressure is more closely linked to the fetal CK isoenzyme shift than is hypertrophy alone.