Functional and Morphological Parameters with Tissue Characterization of Cardiovascular Magnetic Imaging in Clinically Verified "Infarct-like Myocarditis".

PURPOSE: Cardiac magnetic resonance (CMR) has increasingly proved to be a valuable diagnostic tool for evaluating patients with suspected myocarditis. The objective of this study was to evaluate the diagnostic value of functional and morphological parameters including tissue characterization in patients with "infarct-like myocarditis". MATERIALS AND METHODS: 43 patients with clinically verified cases of "infarct-like myocarditis" (median time to MRI scanning after admission for acute symptoms 3 days) and 35 control patients matched by age and sex were included in this retrospective case control study. In this study we used a 1.5 T MRI scanner conducting steady-state-free-precession sequences, T2-weighted imaging, T1-weighted imaging before and after contrast administration and late gadolinium enhancement sequences. According to the recommendations for CMR diagnosis of myocarditis (Lake Louise consensus criteria), a scan was positive for acute myocarditis if 2 of 3 CMR criteria were present. RESULTS: 30 % of the patients with "infarct-like myocarditis" had a reduced left ventricular ejection fraction, 11 % had an increased LV end-diastolic volume index and 35 % had an increased LV mass index. The sensitivity of wall motion abnormalities was 63 % with a regional distribution in 49 %. In 47 % of cases regional wall motion abnormalities were present in the lateral left ventricular segments. Pericardial effusions were discovered in 65 % of cases with a circular appearance in 21 % and focal manifestation in 44 %. The diagnostic sensitivity, specificity, and accuracy of CMR in patients with "infarct-like myocarditis" were 67 %, 100 % and 82 %, respectively. The LGE alone was the most sensitive test parameter with 86 %, providing a specificity of 100 % and accuracy of 92 %. CONCLUSION: Our study results can be applied to the subgroup of patients with "infarct-like myocarditis", where we found that LGE alone was the most sensitive test parameter. In addition to tissue characterization, the functional and morphological analysis of patients with acute myocarditis provides a useful further diagnostic tool. KEY POINTS: •Infarct-like myocarditis can be diagnosed by CMR with high validity and reliability. •LGE allone performed best with a sensitivity of 86 %. •Functional and morphological CMR parameters in addition to tissue characterization are useful tool in the diagnosis of acute myocarditis.

Localization of the angiotensin II receptor subtypes in the human atrium.

Angiotensin II has two major receptor subtypes, designated AT1 and AT2. Both have been detected in the heart of several species, but most of the known functions of angiotensin II seem to be mediated through the AT1 receptor. The major objective of this study was to specify the cell type on which the AT2 receptor is located in the atrium of human heart. Right atrial biopsies from patients with coronary artery disease were tested in membrane binding assays and found to contain high levels of angiotensin II receptor (820 +/- 175 fmol/mg), 82 +/- 2% of which was of the AT2 subtype. Cryostat sections of these biopsies were incubated with 125I-[Sar1,Ile8] angiotensin II in the presence of selective concentrations of the cold ligands losartan and CGP 42112A to detect the subtypes using microscopic autoradiography. High local densities of the AT2 receptor were observed. Comparison of the labelling patterns thus obtained with adjacent sections stained for vimentin, collagen, neurofilaments or acetylcholinesterase revealed that the high densities of AT2 receptor were always associated with fibrous tissue. However, the AT1 receptor was in general evenly distributed over the tissue at low concentrations. Higher local concentrations of this receptor subtype were observed on nervous tissue. The present finding of high densities of the AT2 receptor on fibroblasts at sites of fibrosis may have important clinical implications. Further studies to elucidate the function of this receptor subtype in the heart are therefore essential and the clinical consequences of the use of AT1 antagonists on post-infarction remodelling should be investigated.

Angiotensin II-receptor subtypes in human atria and evidence for alterations in patients with cardiac dysfunction.

Angiotensin II (AII) has been implicated as an important factor in the pathophysiology of heart diseases. Following the recent identification of two subtypes of the AII receptor in cardiac tissue of animals, we investigated the possible occurrence of these, or similar, subtypes in human atrial tissue. In right-atrial tissue from patients undergoing heart surgery, we determined the AII-receptor profile in receptor binding studies, using [125I]-angiotensin as radioligand and subtypes to identify and quantify AII-receptor subpopulations. In 35 patients (23 requiring coronary bypasses, 10 valvular surgery and two combined coronary and valvular surgery), the left-ventricular ejection fraction was determined in the preoperative phase, and right- and left-atrial pressure during surgery. In membranes of human right atria, AII receptors are present in high density (median: Bmax = 294 fmol.mg-1 protein, range: 111-2073) and two different subtypes can be distinguished. Type-1 receptors (AT1) accounted for 33 +/- 10% of the population whereas type-2 receptors (AT2) made up 67 +/- 10% of the population. There was no correlation between any of the measured cardiac functions and total AII-receptor density or receptor affinity. However, the percentage of AT1 receptors was higher in the atria of patients with normal right-atrial pressure; left-ventricular ejection fraction was positively and right-atrial pressure inversely correlated with the percentage of AT1 receptors (r = 0.740 and -0.901, respectively; P < 0.001, for both). Moreover, the percentage of AT2 receptors was directly correlated with the levels of left-atrial pressure (r = 0.853; P < 0.001). It is concluded that the ratio of AT1 to AT2 receptors correlates well with right-atrial pressure and left-ventricular function. This is a first indication of a possible involvement of AII-receptor subtypes in the pathophysiology of cardiac dysfunctions.

Localization of angiotensin II receptor subtypes in the rabbit heart.

Angiotensin II (Ang II) is an essential component of the renin-angiotensin system and is partially responsible for the maintenance of hypertension. Two major receptor subtypes have been defined for Ang II and have been detected in the heart of various species. Most of the known functions of Ang II are mediated via the AT1 subtype, whereas the function of the AT2 receptor remains ill defined. In this study we aimed to localize both receptor subtypes in the rabbit heart using film and light microscope autoradiography as well as radioligand binding assays on membranes. Total receptor densities in the atrium and nervous tissue were respectively four and nine times greater than in the ventricle. Conductive tissue shows a density between that of atrial and nervous tissue. In the ventricle, approximately 20% of the Ang II receptors were AT2. This receptor subtype was almost totally absent from nervous, conductive and atrial tissue. The limited resolution of the microscope autoradiography method did not allow us to specify the exact cell-type at this stage.

Angiotensin II receptor subtypes and cardiac function.

All the components of the renin-angiotensin system have been identified in the heart including the angiotensin II receptor subtypes AT1 and AT2. In the normal human heart, there is a decreasing receptor density from the right atrium to the left ventricle. In right atrial membranes prepared from pathological hearts, the percentage of AT1 receptor decreases with the severity of cardiac dysfunction whereas that of AT2 receptor increases. Treatment of hypertrophic rats with AT1 receptor antagonists inhibits cardiac hypertrophy and reverses the increase receptor density, indicating involvement of this Ang II receptor subtype. The role of the AT2 receptor is still largely unknown but it may be involved in cell growth and proliferation. The cloning of both AT1 and AT2 receptors as well as the availability of potent and selective antagonists will help us to understand better the functional role of Angiotensin II in cardiovascular disorders.

Evidence for a novel angiotensin II receptor involved in angiogenesis in chick embryo chorioallantoic membrane.

Angiotensin II acts as a growth factor in the cardiovascular system and has been implicated in angiogenesis. The existence of at least two types of angiotensin II receptors, the AT1 and the AT2 receptors, has been suggested by ligand binding studies. We used three different AT receptor antagonists to study the receptor mediating angiotensin II-induced angiogenesis in the chorioallantoic membrane (CAM) of the chick embryo. Angiotensin II caused pronounced angiogenesis of pre- and postcapillary vessels of 30-40%. This response could only be blocked by adding the peptidergic AT2 antagonist CGP-42112A. The nonpeptidergic AT2 antagonist PD123319 and AT1 antagonist losartan (DuP 753) were not effective. In addition, we used radioligand binding studies with a range of ligands to define the nature of the receptor. Our results show a high density of specific single class AT receptor with a total number of binding sites of 1,190 fmol/mg protein and an affinity constant for angiotensin II of 2.7 nM. The inhibitory concentrations (IC50) for CGP-42112A, PD 123319 and losartan were 724, > 100,000, and 59,000 nM, respectively. Our studies suggest that these binding sites act as receptors for angiotensin II-induced angiogenesis. Both functional and radioligand binding studies suggest that the receptor is different from the classical mammalian AT1 and AT2 receptors.

Receptor-mediated effects of angiotensin II on growth of vascular smooth muscle cells from spontaneously hypertensive rats.

This study examines the effects of angiotensin II on hypertrophy and proliferation of aortic smooth muscle cells from spontaneously hypertensive and Wistar-Kyoto rats and the receptor subtypes mediating these effects. In quiescent confluent cells, angiotensin II induced a dose-dependent increase in thymidine and leucine incorporation without stimulating cell proliferation. In nonconfluent cells, angiotensin II stimulated cell proliferation only in combination with a submaximal concentration of fetal calf serum. These effects were enhanced in cells from spontaneously hypertensive rats compared with Wistar-Kyoto rats. The effects of angiotensin II could be blocked by the AT1 receptor antagonist DuP 753 but not by the AT2 receptor ligand PD 123177. In receptor binding studies with cells derived from both rat strains, AT1-typical binding was observed. These data show that the angiotensin II receptors present in vascular smooth muscle cells in culture from both rat strains are of the AT1 receptor subtype. This receptor subtype appears to mediate vascular smooth muscle cell hypertrophy and proliferation as well as vasoconstriction. Although no difference in the receptor profile was detectable between the two rat strains, the affinity for the ligands to the receptor and the receptor density tended to be greater in cells from spontaneously hypertensive rats than in cells from Wistar-Kyoto rats. These results may partly explain the greater hypotensive response to angiotensin II receptor blockade in spontaneously hypertensive rats than in Wistar-Kyoto rats, although both rat strains have the same plasma concentrations of angiotensin II.

Characterization of nuclear angiotensin-II-binding sites in rat liver and comparison with plasma membrane receptors.

Although the action of angiotensin-II (Ang-II) is believed to be mediated by a transmembrane signal transduction mechanism, accumulating evidence suggests that Ang-II may also have a direct nuclear action. We have characterized the nuclear Ang-II-binding site in purified nuclei preparation from rat liver and compared it to plasma membrane Ang-II receptors. [125I]Ang-II binding to isolated nuclei reached equilibration in 30 min at 25 C, slower than binding to plasma membrane, which reached equilibration within 10 min. Scatchard analysis of [125I]Ang-II binding to isolated nuclei revealed a single class of binding sites (Kd = 1.4 nM; binding capacity = 10 fmol/mg protein or 460 sites/nucleus). In the nuclear preparation, Ang-II and its fragments competed for binding a potency order of Ang-III = Ang-II greater than Ang-II-(1-7) greater than Ang-II-(1-6) greater than Ang-II-(1-5). Losartan potassium (DuP 753), a selective blocker of the Ang-II receptor subtype I, fully inhibits nuclear Ang-II binding with affinity similar to that in plasma membrane. The pH optimum for [125I]Ang-II binding to nuclei was 7.0, while binding to plasma membrane was optimal at pH 8.0. Low concentrations (0.05-0.1 mM) of dithiothreitol increased [125I]Ang-II binding to nuclei, but not to plasma membrane. In the absence of detergent, Ang-II-binding sites appear to consist of soluble protein releasable from nuclei by freezing and thawing, hence distinct in physicochemical properties from the membrane-bound receptor. Size-exclusion HPLC estimated the mol wt of the soluble Ang-II-binding sites to be 66 kilodaltons. These nuclear Ang-II-binding sites have some similarities to but also show notable physicochemical differences from plasma membrane Ang-II receptors, and they may play a role in mediating the intracellular action of Ang-II.

Identification and characterization of angiotensin II receptor subtypes in rabbit ventricular myocardium.

CGP 42 112 A and DuP 753 block [125I]-angiotensin II binding in rabbit ventricular myocardial membranes in a clearly biphasic manner, indicating the existence of high- and low-affinity sites for these highly selective agents. Assays using concentrations of either agent large enough to prevent high-affinity binding show that their respective high-affinity sites are distinct, and each corresponds to the low-affinity site of the other. The two receptor subsets, present in nearly equal proportions, are also distinguishable by their different sensitivities to dithiothreitol. These findings afford strong evidence for the existence of two distinct angiotensin II receptors in rabbit myocardium, corresponding to the A and B subtypes recently described in adrenals.

Stereospecific action of diltiazem on the mitochondrial Na-Ca exchange system and on sarcolemmal Ca-channels.

The benzothiazepine diltiazem is a potent Ca-channel blocker, which also inhibits the Na-dependent Ca-efflux from heart mitochondria. In this study, the action of the 4 stereoisomers of diltiazem has been investigated using guinea-pig heart and liver mitochondria. The rate of the Na-dependent Ca-efflux from liver mitochondria has been found to be 10 times smaller than in heart mitochondria. Otherwise, the exchange systems from the two tissues have been found to be pharmacologically indistinguishable. Both the (+)-optical isomers of the cis- and trans-forms of diltiazem inhibit Na-Ca exchange activity with comparable potency (IC50 of 10-20 microM), while the (-)-optical isomers are ineffective (IC50 greater than 200 microM). Radioligand binding experiments have revealed that only one stereoisomer of diltiazem, the (+)-cis form, interacts with high affinity with the Ca-channel receptors of guinea-pig heart sarcolemma preparations (KD = 120 nM). The results have shown that the Ca-channel of plasma membranes and the mitochondrial Na-Ca exchanger have different stereospecific requirements for the binding of diltiazem.

Behavioral effects and general pharmacology of 4-(5-chloro-benzofuranyl-2)-1-methylpiperidine HC1, an antidepressant inhibiting both monoamine oxidase A and 5-hydroxytryptamine uptake.

In psychopharmacological tests in rats and mice, 4-(5-chloro-benzofuranyl-2)-1-methylpiperidine HC1 (CGP 4718 A) was found to exert behavioral effects typical of both monoamine oxidase (MAO)-A and 5-hydroxytryptamine (5-HT) uptake inhibitors (reserpine antagonism, L-5-HTP potentiation, antiaggressive activity in isolated mice). The potential antidepressant activity of the drug was indicated in rats by antagonism of reserpine and its effect in the social-conflict test. CGP 4718 A did not impair motor coordination and had no influence on locomotor activity up to high doses in mice and rats. In monkeys, it increased directed individual activities, including sex-related behaviors and diminished locomotor activity and passivity. Electroencephalographic studies in cats revealed a significant decrease in paradoxical sleep after treatment with CGP 4718 A. In isolated organs, no significant antagonism of norepinephrine, 5-HT, acetylcholine or histamine was found. Cardiovascular studies in cats showed only transient effects on blood pressure and no effect on heart rate. In conscious dogs no cardiovascular effects were found. No potentiation of the pressor effect of tyramine in rats was detectable after repeated doses of up to 300 mg/kg p.o. A weak cardiodepressant effect was seen in isolated guinea pig atria. In conclusion, in animal experiments CGP 4718 A combines an interesting spectrum of antidepressant, activating and antiaggressive properties with a lack of cardiovascular and tyramine-potentiating effects.

Vasodilating agents and platelet function: intracellular free calcium concentration, cyclic nucleotides, and shape-change response.

The adenylate-cyclase activator forskolin, the guanylate-cyclase stimulator sodium nitroprusside, the phosphodiesterase inhibitor Ro 15-2041, different Ca-entry blockers, as well as various vasodilators, and the atrial natriuretic peptide were tested for antiplatelet activity. Thrombin, vasopressin, ADP, arachidonic acid, and the dihydropyridine Ca agonist CGP 28392 were used as platelet activators. The physiological and biochemical parameters of platelet function studied included shape-change reaction, intracellular free-Ca modulation, and cyclic nucleotide formation. When inhibition of the shape-change response occurred, it was accompanied by inhibition of the increase in intracellular free Ca. Furthermore, the results suggest a possible intracellular site of action of Ca entry blockers in platelets, and confirm the importance of modulation of cyclic nucleotides in the regulation of platelet function, regardless of the mechanism of platelet activation. Additional antiplatelet activity of antihypertensive agents may have a beneficial effect in reducing the associated risk of thrombo-embolic complications in essential hypertension.

Smoking-induced increases in plasma vasopressin and reduced platelet hormone sensitivity.

Cigarette smoking is a major risk for coronary atherosclerosis, but the mechanism of this is still unclear. The present study demonstrates that smoking produces a variable increase in plasma vasopressin concentration but that sensitivity of platelets to this elevated endogenous vasopressin release is blunted. This suggests that cigarette smoking contributes to atherosclerosis through the vascular effects of the hormones whose release it stimulates rather than by platelet activation. The mechanism for this blunted responsiveness to vasopressin was also investigated in vitro. The rise in intracellular free calcium concentration of platelets was markedly reduced following a second administration of vasopressin, whereas the in vitro shape change response was usually unaltered and could only be reduced with specific procedures for platelet preparation. This suggests that only a small increase of intracellular free calcium is necessary for a complete shape change response induced by vasopressin. The results indicate that the shape change is mediated by an increase in intracellular free calcium which is independent from the phosphoinositol pathway and the calcium is released from intracellular pools other than by those activated by serotonin or thrombin.

In vitro comparative studies of the calcium-entry activators YC-170, CGP 28392, and BAY K 8644.

Recently, the novel dihydropyridine derivates YC-170, CGP 28392, and BAY K 8644 have been reported to act in the opposite way to Ca2+-entry blockers. We have found that these compounds inhibit the binding of [3H]nitrendipine on guinea pig heart membranes (Ki: 6 nM BAY K 8644, 115 nM CGP 28392 and 690 nM YC-170). Like those of nifedipine (Ki 1 nM), the curves had slopes close to unity, and, unlike those of some nondihydropryridine Ca2+ antagonists, were not altered in the presence of diltiazem, indicating a competitive interaction at dihydropyridine-sensitive sites. In isolated guinea pig atria, these agents exerted positively inotropic effects similar in their potency ratio to those observed in the binding experiments (BAY K 8644 1, CGP 28392 1:17, YC-170 1:600). The maximum inotropic effects of BAY K 8644 and CGP 28392, and of YC-170 corresponded respectively to two-thirds and one-third of those induced by isoprenaline or extracellular Ca2+. In the isolated rat mesenteric artery, perfused with a depolarizing solution, vasoconstrictor Ca2+ dose-response curves are shifted to the right by nifedipine. By contrast, BAY K 8644 and CGP 28392 caused a distinct leftward shift of the Ca2+ dose-response curves, at concentrations of 3-300 nM and 30-300 nM, respectively, and YC-170 a marginal shift at concentrations of 200-2000 nM, i.e., similar ranges to their inhibitory effects on [3H]nitrendipine binding. At higher concentrations, all three compounds produced Ca2+-antagonistic effects. These results indicate that the compounds act at dihydropyridine-sensitive sites and exert partial agonistic activities in vascular and myocardial tissue.

Enhancement of calcium influx in human platelets by CGP 28392, a novel dihydropyridine.

CGP 28392, a novel compound structurally related to the dihydropyridine Ca2+-entry blockers, causes a dose-dependent increase in intracellular free Ca2+ in human platelets, as measured with the Quin-2 Ca2+ indicator, with a semimaximal effective concentration of 2.2 X 10(-7) M. This effect occurs in a concentration range in which CGP 28392 competes for specific [3H]nitrendipine binding in guinea pig heart membranes. It can be inhibited by nitrendipine. The data presented furnish direct evidence of the Ca2+-entry-stimulating properties of CGP 28392 and indicate the presence of dihydropyridine-susceptible structures in human platelets.

Lack of beta-adrenoreceptor hypersensitivity after abrupt withdrawal of long-term therapy with oxprenolol.

The possibility of beta-adrenoreceptor hypersensitivity after abrupt withdrawal of long-term therapy (8-18 months) with the slow-release (SR) formulation of oxprenolol (160-320 mg/day) was assessed in six patients with uncomplicated essential hypertension. The chronotropic dose 25 of isoproterenol (the dose that increases the resting heart rate by 25 beats/min), plasma concentration of catecholamines, triiodothyronin and thyroxin, plasma renin activity and aldosterone, hemoglobin, hematocrit and oxyhemoglobin dissociation were measured on the last day of oxprenolol SR intake and 1, 2, 3, 6 and 13 days after abrupt replacement by identical placebo tablets. The chronotropic dose 25 of isoproterenol (microgram/m2), which was greater than 25.6 in all patients on the last day of oxprenolol SR, fell to 4.83 +/- 2.03 on the second day and to 3.50 on the third day after its abrupt withdrawal and reached a minimal value on the thirteenth day (2.78 +/- 0.30). Throughout the study, plasma concentrations of catecholamines, triiodothyronin and thyroxin and oxyhemoglobin dissociation remained unchanged. Plasma renin activity and plasma aldosterone, which were suppressed during oxprenolol administration, rose significantly during placebo, coinciding with a significant fall in hematocrit and hemoglobin. No major subjective symptoms were reported by the patients. Thus, hypersensitivity of beta-adrenoreceptor-mediated responses was not demonstrated after sudden withdrawal of oxprenolol SR.

N4-Acetylcytidine. A previously unidentified labile component of the small subunit of eukaryotic ribosomes.

The nucleoside content of 18 S rRNA from rat liver is determined under conditions known to prevent the destruction of chemically labile modified nucleosides. Two base-modified nucleosides, not completely identified before, are shown to be N6-methyladenosine and 7-methylguanosine. The results further demonstrate the presence of a hitherto unidentified component of 18 S rRNA whose spectra and chromatographic properties are identical with that of N4-acetylcytidine. In addition, this compound is not detectable in 28 S rRNA nor in 16 S rRNA derived from the small ribosomal subunit of Escherichia coli. However, it appears to be conserved in the small ribosomal subunit of eukaryotes, since it is also present in yeast 17 S rRNA and chicken liver 18 S rRNA.

Chemical basis for brain-specific serine transfer RNAs.

Serine tRNA from rat brain can be resolved into six isoaccepting species. Three of these species show the same chromatographic behavior as the seryl tRNAs from other rat organs, whereas the remaining species appear to be specific for brain. The isoacceptor tRNAs were purified to homogeneity by chromatography on benzoylated DEAE-cellulose followed by reversed-phase chromatography. We found that the additional species of serine tRNA in brain differ from their counterparts derived from other rat organs by a lack of a specific guanosine ribose-methylation in the dihydrouridine loop. In addition, when total liver tRNA was compared with total brain tRNA, the same degree of undermethylation with respect to 2'-O-methylguanosine was found as a general phenomenon.