Investigation of Rhodnius prolixus head anatomy using microtomography.

In the last years, microtomography has proved to be a powerful technique on insects' studies, allowing a detailed view of the structures' internal with a high resolution. One of the most important advantages about the use of microtomography in these studies is the fact that the dissection is not necessary, which decreases considerably the number of samples used on the insects' research. Some insects are used constantly in studies about morphology, metamorphosis, and reproduction, because they work as a model for others, and Rhodnius prolixus is one of the most studied in this group. This insect is also one of the main insect vectors of Chagas disease that kills around 12,000 people every year in Latin America. Some studies using laboratory microtomography conventional scanners combining with the correct staining methods have proved that it could be a powerful tool in biological research, allowing the visualisation of low-density tissues. The main goal of the present work was to use staining protocols to study Rhodnius prolixus with laboratory microtomography conventional scanners. The experiments were carried out at the imaging lab in the Theoretical Biology Department, University of Vienna, using an Xradia MicroXCT and at the University of Oslo, using a Skyscan 2211.

Trypanosoma cruzi: effects of azadirachtin and ecdysone on the dynamic development in Rhodnius prolixus larvae.

The effects of azadirachtin and ecdysone on the Trypanosoma cruzi population in the Rhodnius prolixus gut were investigated. T. cruzi were rarely found in the gut compartments of azadirachtin-treated larvae. High parasite numbers were observed in the stomach of the control and ecdysone groups until 10 days after treatment and in the small intestine and rectum until 25 days after treatment. High percentages of round forms developed in the stomachs of all groups, whereas azadirachtin blocked the development of protozoan intermediate forms. This effect was counteracted by ecdysone therapy. In the small intestine and rectum, epimastigotes predominated for all groups, but more of their intermediates developed in the control and ecdysone groups. Azadirachtin supported the development of round forms and their intermediates into trypomastigotes. In the rectum, trypomastigotes did not develop in the azadirachtin group and developed much later after ecdysone therapy. The parallel between the effects of azadirachtin and ecdysone on the host and parasite development is discussed on the basis of the present results because ecdysone appears to act directly or indirectly in determining the synchronic development of T. cruzi forms from round to epimastigotes, but not metacyclic trypomastigotes, in the invertebrate vector.

A rhamnose-binding lectin from Rhodnius prolixus and the impact of its silencing on gut bacterial microbiota and Trypanosoma cruzi.

Lectins are ubiquitous proteins involved in the immune defenses of different organisms and mainly responsible for non-self-recognition and agglutination reactions. This work describes molecular and biological characterization of a rhamnose-binding lectin (RBL) from Rhodnius prolixus, which possesses a 21 amino acid signal peptide and a mature protein of 34.6 kDa. The in-silico analysis of the primary and secondary structures of RpLec revealed a lectin domain fully conserved among previous insects studied. The three-dimensional homology model of RpLec was similar to other RBL-lectins. Docking predictions with the monosaccharides showed rhamnose and galactose-binding sites comparable to Latrophilin-1 and N-Acetylgalactosamine-binding in a different site. The effects of RpLec gene silencing on levels of infecting Trypanosoma cruzi Dm 28c and intestinal bacterial populations in the R. prolixus midgut were studied by injecting RpLec dsRNA into the R. prolixus hemocoel. Whereas T. cruzi numbers remained unchanged compared with the controls, numbers of bacteria increased significantly. The silencing also induced the up regulation of the R. prolixus defC (defensin) expression gene. These results with RpLec reveal the potential importance of this little studied molecule in the insect vector immune response and homeostasis of the gut bacterial microbiota.

Immune depression in Rhodnius prolixus by seco-steroids, physalins.

A comparative study of the effects of physalins, seco-steroidal substances of Physalis angulata (Solanaceae), on the immune reactions of R. prolixus was carried out. Ecdysis and mortality were not affected by treatment with physalins B, D, F or G (1-10 microg/ml of blood meal). R. prolixus larvae fed with blood containing physalins and inoculated with 1 microl of Enterobacter cloacae beta12 (5 x 10(3)/insect) exhibited mortality rates three times higher than controls. The insects treated with physalin B, and F (1 microg/ml) and inoculated with E. cloacae beta12 showed significant differences on lysozyme activity in the hemolymph compared to untreated insects. Furthermore, physalin D (1 microg/ml) significantly reduced the antibacterial activity. Concerning cellular immune reactions, all insects treated with physalins (1 microg/ml), exhibited drastic reductions in the quantity of yeast cell-hemocyte binding and subsequent internalization. Insects inoculated with bacteria and treated with physalins B, F and G showed reductions of microaggregate formation but physalin D did not. Physalins B and F also reduced total hemocyte count in the hemolymph. These results suggest that, in different ways, probably due to their different chemical structures, physalin B, D, F and G are immunomodulatory substances for the bloodsucking insect, R. prolixus.

Rhodnius prolixus: from physiology by Wigglesworth to recent studies of immune system modulation by Trypanosoma cruzi and Trypanosoma rangeli.

This review is dedicated to the memory of Professor Sir Vincent B. Wigglesworth (VW) in recognition of his many pioneering contributions to insect physiology which, even today, form the basis of modern-day research in this field. Insects not only make vital contributions to our everyday lives by their roles in pollination, balancing eco-systems and provision of honey and silk products, but they are also outstanding models for studying the pathogenicity of microorganisms and the functioning of innate immunity in humans. In this overview, the immune system of the triatomine bug, Rhodnius prolixus, is considered which is most appropriate to this dedication as this insect species was the favourite subject of VW's research. Herein are described recent developments in knowledge of the functioning of the R. prolixus immune system. Thus, the roles of the cellular defences, such as phagocytosis and nodule formation, as well as the role of eicosanoids, ecdysone, antimicrobial peptides, reactive oxygen and nitrogen radicals, and the gut microbiota in the immune response of R. prolixus are described. The details of many of these were unknown to VW although his work gives indications of his awareness of the importance to R. prolixus of cellular immunity, antibacterial activity, prophenoloxidase and the gut microbiota. This description of R. prolixus immunity forms a backdrop to studies on the interaction of the parasitic flagellates, Trypanosoma cruzi and Trypanosoma rangeli, with the host defences of this important insect vector. These parasites remarkably utilize different strategies to avoid/modulate the triatomine immune response in order to survive in the extremely hostile host environments present in the vector gut and haemocoel. Much recent information has also been gleaned on the remarkable diversity of the immune system in the R. prolixus gut and its interaction with trypanosome parasites. This new data is reviewed and gaps in our knowledge of R. prolixus immunity are identified as subjects for future endeavours. Finally, the publication of the T. cruzi, T. rangeli and R. prolixus genomes, together with the use of modern molecular techniques, should lead to the enhanced identification of the determinants of infection derived from both the vector and the parasites which, in turn, could form targets for new molecular-based control strategies.

Impact of Trypanosoma cruzi on antimicrobial peptide gene expression and activity in the fat body and midgut of Rhodnius prolixus.

BACKGROUND: Rhodnius prolixus is a major vector of Trypanosoma cruzi, the causative agent of Chagas disease in Latin America. In natural habitats, these insects are in contact with a variety of bacteria, fungi, virus and parasites that they acquire from both their environments and the blood of their hosts. Microorganism ingestion may trigger the synthesis of humoral immune factors, including antimicrobial peptides (AMPs). The objective of this study was to compare the expression levels of AMPs (defensins and prolixicin) in the different midgut compartments and the fat body of R. prolixus infected with different T. cruzi strains. The T. cruzi Dm 28c clone (TcI) successfully develops whereas Y strain (TcII) does not complete its life- cycle in R. prolixus. The relative AMP gene expressions were evaluated in the insect midgut and fat body infected on different days with the T. cruzi Dm 28c clone and the Y strain. The influence of the antibacterial activity on the intestinal microbiota was taken into account. METHODS: The presence of T. cruzi in the midgut of R. prolixus was analysed by optical microscope. The relative expression of the antimicrobial peptides encoding genes defensin (defA, defB, defC) and prolixicin (prol) was quantified by RT-qPCR. The antimicrobial activity of the AMPs against Staphylococcus aureus, Escherichia coli and Serratia marcescens were evaluated in vitro using turbidimetric tests with haemolymph, anterior and posterior midgut samples. Midgut bacteria were quantified using colony forming unit (CFU) assays and real time quantitative polymerase chain reaction (RT-qPCR). RESULTS: Our results showed that the infection of R. prolixus by the two different T. cruzi strains exhibited different temporal AMP induction profiles in the anterior and posterior midgut. Insects infected with T. cruzi Dm 28c exhibited an increase in defC and prol transcripts and a simultaneous reduction in the midgut cultivable bacteria population, Serratia marcescens and Rhodococcus rhodnii. In contrast, the T. cruzi Y strain neither induced AMP gene expression in the gut nor reduced the number of colony formation units in the anterior midgut. Beside the induction of a local immune response in the midgut after feeding R. prolixus with T. cruzi, a simultaneous systemic response was also detected in the fat body. CONCLUSIONS: R. prolixus AMP gene expressions and the cultivable midgut bacterial microbiota were modulated in distinct patterns, which depend on the T. cruzi genotype used for infection.

Ecdysis period of Rhodnius prolixus head investigated using phase contrast synchrotron microtomography.

Microtomography using synchrotron sources is a useful tool in biological imaging research since the phase coherence of synchrotron beams can be exploited to obtain images with high contrast resolution. This work is part of a series of works using phase contrast synchrotron microtomography in the study of Rhodnius prolixus head, the insect vector of Chagas' disease, responsible for about 12,000 deaths per year. The control of insect vector is the most efficient method to prevent this disease and studies have shown that the use of triflumuron, a chitin synthesis inhibitor, disrupted chitin synthesis during larval development and it's an alternative method against insect pests. The aim of this work was to investigate the biological effects of treatments with triflumuron in the ecdysis period (the moulting of the R. prolixus cuticle) using the new imaging beamline IMX at LNLS (Brazilian Synchrotron Light Laboratory). Nymphs of R. prolixus were taken from the Laboratory of Biochemistry and Physiology of Insects, Oswaldo Cruz Foundation, Brazil. Doses of 0.05mg of triflumuron were applied directly to the abdomen on half of the insects immediately after feeding. The insects were sacrificed 25days after feeding (intermoulting period) and fixed with glutaraldehyde. The results obtained using phase contrast synchrotron microtomography in R. prolixus showed amazing images of the effects of triflumuron on insects in the ecdysis period, and the formation of the new cuticle on those which were not treated with triflumuron. Both formation and malformation of this insect's cuticle have never been seen before with this technique.

Surgical treatment of annuloaortic ectasia with composite grafts including homologous dura mater valves.

We describe 3 patients in whom a composite dura mater valve-Dacron tube graft was used for replacement of the entire ascending aorta and aortic valve. Two patients had Marfan's syndrome and 1, a chronic aortic dissection, type II. All had severe aortic regurgitation. There were no early or late deaths in a follow-up period of 2, 7, and 12 months, respectively. All 3 patients were asymptomatic when last seen. Two underwent postoperative aortography that disclosed well-functioning valves and good filling of the coronary arteries. It is concluded that annuloaortic ectasia is best treated by the technique of total replacement of the ascending aorta and aortic valve by a composite valve-tube graft, and that the homologous dura mater valve, for its characteristics of central flow, lack of need for anticoagulants, and durability, represents a reasonable alternative for use in this situation.

Effects of lignoids on a hematophagous bug, Rhodnius prolixus: feeding, ecdysis and diuresis.

The effects of lignoids on feeding, ecdysis and diuresis in fourth-instar larvae of Rhodnius prolixus (Hemiptera) were investigated. Up to 100 microg/ml burchellin, podophyllotoxin, pinoresinol, sesamin, licarin A, or nordihydroguaiaretic acid (NDGA) in the diet did not induce antifeedant effects. Pinoresinol and NDGA significantly inhibited ecdysis. In experiments in vivo, burchellin and podophyllotoxin reduced the production of urine after feeding. 5-Hydroxytryptamine (5-HT), a diuretic hormone, partially counteracted this effect of burchellin. In experiments in vitro, using isolated Malpighian tubules, (i) burchellin reduced diuretic hormone levels in the hemolymph but not the amount of diuretic hormone stored in the thoracic ganglionic masses (including axons), (ii) burchellin decreased the volume of urine secreted by isolated Malpighian tubules, and (iii) 5-HT could not overcome the effect of burchellin upon the Malpighian tubules. We conclude that burchellin interfered with the release, but not with the production of diuretic hormone by the thoracic ganglionic mass or induced an antidiuretic hormone and directly affected the Malpighian tubules.

Lignoids in insects: chemical probes for the study of ecdysis, excretion and Trypanosoma cruzi-triatomine interactions.

The effects of phytochemicals (lignans and neolignans) are reviewed in a variety of insect species with special focus on the recent advances on feeding, excretion and Trypanosoma cruzi interactions with Rhodnius prolixus. Burchellin, podophyllotoxin, pinoresinol, sesamin, licarin A, and nordihydroguaiaretic acid (NDGA) added to the diet of Rhodnius prolixus larvae induce antifeedant effects only in doses up to 100 microg/ml of blood meal. Additionally, pinoresinol and NDGA significantly inhibit ecdysis (ED(50)<20 microg/ml). Simultaneous application of ecdysone (1 microg/ml) counteracts ecdysial stasis as induced by NDGA in 5th-instar larvae. Experiments in vivo demonstrate that burchellin and podophyllotoxin (100 microg/ml) diminish excretion post-feeding. Simultaneous treatment with 5-hydroxytryptamine (1 mM, 5-HT), a diuretic hormone, partially reverses this effect of burchellin. Experiments in vitro, using isolated Malpighian tubules of R. prolixus, indicate that burchellin (i) decreases diuretic hormone levels in the hemolymph but not the amount of diuretic hormone stored in the thoracic ganglionic masses (including axons); (ii) reduces the volume of urine secreted by isolated Malpighian tubules; and (iii) 5-HT therapy cannot overcome the effect of burchellin on the Malpighian tubules. In R. prolixus fed on blood containing T. cruzi epimastigotes, the number of parasites in the digestive tract decreases drastically in the presence of burchellin and NDGA (10 microg/ml). When these phytochemicals are applied 20 days after T. cruzi infection, burchellin significantly reduces the gut infection, whereas NDGA does not. However, if the insects are pretreated with both compounds 20 days before subsequent infection with epimastigotes, the parasite infection is almost completely abolished. The same holds true when 5th-instar of R. prolixus are inoculated with 0.5 microg/microl/larva of both neolignans 1 day before infection. Taken together, these findings not only provide a better understanding of the lignoid function in insects, but also offer novel insights into basic physiological processes, which make lignoids interesting candidates for new types of insecticides.

Suppression of the prophenoloxidase system in Rhodnius prolixus orally infected with Trypanosoma rangeli.

Investigations were carried out to compare aspects of the prophenoloxidase (proPO)-activating pathway in Rhodnius prolixus hemolymph in response to oral infection and inoculation of the insects with two developmental forms of Trypanosoma rangeli epimastigotes strain H14. In vivo experiments demonstrated that in control insects fed on uninfected blood, inoculation challenge with short epimastigotes resulted in high phenoloxidase (PO) activity. In contrast, previous feeding on blood containing either short or long epimastigotes was able to suppress the proPO activation induced by thoracic inoculation of the short forms. In vitro assays in the presence of short epimastigotes demonstrated that control hemolymph or hemolymph provided by insects previously fed on blood containing epimastigotes incubated with fat body homogenates from control insects significantly increased the PO activity. However, fat body homogenates from insects previously fed on blood containing epimastigotes, incubated with hemolymph taken from insects fed on control blood or blood infected with epimastigotes, drastically reduced the proPO activation. The proteolytic activity in the fat body homogenates of control insects was significantly higher than in those obtained from fat body extracts of insects previously fed on blood containing epimastigotes. These findings indicate that the reduction of the proteolytic activities in the fat body from insects fed on infected blood no longer allows a significant response of the proPO system against parasite challenge. It also provides a better understanding of T. rangeli infection in the vector and offer novel insights into basic immune processes in their invertebrate hosts.

Characterization of inducible lysozyme activity in the hemolymph of Rhodnius prolixus.

1. The characterization and partial purification of an induced lysozyme activity in the hemolymph of adult Rhodnius prolixus inoculated with Micrococcus lysodeikticus is described. 2. Little or no activity against M. lysodeikticus appeared in the first hours after inoculation, but the activity increased reaching a maximum 4 days later, which was maintained to day 12. 3. The activity was characterized as lysozyme on the basis of the following considerations: 1) pH optimum and thermostability at acidic pH; 2) rate of lysis negatively dependent on ionic strength; 3) binding to SP-Sephadex at pH 5.5; 4) apparent molecular weight of 15 kDal. 4. Crude or semi-purified enzyme preparations showed a high degree of stability during handling, freezing and thawing, and standing at 5 degrees C. 5. Incubation of abdominal fat bodies from treated insects resulted in the release of activity into the medium. 6. The relationship between induced lysozyme activity and its role as an insect defense mechanism is discussed.

A protein diet initiates oogenesis in Rhodnius prolixus.

Oogenesis in R. prolixus females is induced by feeding blood, blood plasma, a suspension of washed erythrocytes, or 5% solutions of egg albumin, bovine serum albumin or hemoglobin. Unfed females and those fed saline, casein hydrolysate, dextran or sucrose do not initiate oogenesis. The juvenile hormone analogue epoxygeranylgeraniol methyl ester induced oogenesis in unfed females as well as in ethoxyprecocene II-treated insects. The results show that a protein meal was sufficient to initiate oogenesis. They also support the view that oogenesis depends on a relationship between protein ingestion and corpus allatum function and that oogenesis is not controlled by hormones released in response to abdominal stretching.

Demonstration of a proallatotoxin-sensitive period in 4th-instar nymphs of Rhodnius prolixus.

1. Topical application of the proallatotoxin ethoxyprecocene II (60 micrograms/nymph) to 4th-instar nymphs of Rhodnius prolixus at various times before and after a blood meal induced precocious metamorphosis. 2. The highest percentage of adultoids was obtained when proallatotoxin was applied prior to feeding or up to day 3 after the blood meal. 3. The proallatotoxin-sensitive period ended 4 days after feeding just before the maximum peak of ecdysteroid concentration in the hemolymph. 4. The significance of these findings is discussed in terms of the juvenile hormone-sensitive period and the hormonal program which controls molting in this insect.

In vitro cytotoxicity of Rhodnius prolixus hemolytic factor and mellitin towards different trypanosomatids.

Trypanosoma cruzi strain Y and clone Dm28c and other trypanosomatids were exposed to two lytic agents, Rhodnius prolixus hemolytic factor (RHF) and mellitin, in vitro. In both cases, the result was a significant decrease in the number of parasites after a 30-min treatment at 37 degrees C. RHF and mellitin had distinct activities on different strains and species of trypanosomatids. These observations suggest that RHF may be an important factor in selecting resistant strains of trypanosomes for development in the vector's gut.

Biological effects of canatoxin in different insect models: evidence for a proteolytic activation of the toxin by insect cathepsinlike enzymes.

Canatoxin is a toxic protein isolated from the jackbean, Canavalia ensiformis. The toxin injected intraperitoneally is lethal for mice and rats; however, it is inactive if given orally. In this study, Manduca sexta (L.) (Lepidoptera), Schistocerca americana (Drury) (Orthoptera), Drosophila melanogaster (L.) (Diptera), Aedes aegypti (L.) (Diptera), Rhodnius prolixus (Stål) (Hemiptera), and Callosobruchus maculatus (F.) (Coleoptera) were fed on canatoxin-containing diets. No effects were seen in M. sexta, S. americana, D. melanogaster, or A. aegypti. No traces of canatoxin were found in their feces, suggesting that the protein was digested completely by these insects, which characteristically have a trypsin-based digestion. In contrast, canatoxin was lethal for insects displaying cathepsin-based digestion. Thus, for C. maculatus, a diet containing 0.25% wt:wt canatoxin caused complete inhibition of larval growth. When R. prolixus were fed on canatoxin, 2 effects were seen: impairment of water excretion and increased lethality 48-96 h after feeding. The lethal effect of canatoxin in R. prolixus was blocked partially or completely when the digestion of the toxin by R. prolixus midgut enzymes was impaired. The data showed that canatoxin is highly toxic when ingested by some species of insects but not affecting others, probably in correlation with the characteristics of the digestive process of the insect.

Effects of some lignans and neolignans on the development and excretion of Rhodnius prolixus.

The effects of six lignans and neolignans as inhibitors of ecdysis and on the water balance in fourth-instar larvae of Rhodnius prolixus were studied by oral, topical and continuous contact treatments. The main results may be summarised as follows: (i) burchellin, pinoresinol, sesamin, licarin A and nordihydroguaiaretic acid (NDGA) did not cause feeding inhibition at doses of 100 micrograms/ml blood; podophyllotoxin had no antifeedant effect but caused a high moulting inhibition and significant toxicity when applied either orally or topically; (ii) the highest ecdysis inhibitory effects were observed with pinoresinol and NDGA when applied orally at a dose of 100 micrograms/ml (58% and 50% of moulting inhibition, respectively); burchellin inhibited 30% of the moulting at this concentration; (iii) by topical treatment none of the compounds presented any influence on the moulting cycle; and (iv) podophyllotoxin and burchellin significantly reduced the excretion of the insect in 24 h; the other compounds had no effect on excretion. The implications of these findings in relation to the pertinent biological events in R. prolixus are discussed.

Chagas' disease and its insect vector. Effect of azadirachtin A on the interaction of a triatomine host (Rhodnius prolixus) and its parasite (Trypanosoma cruzi).

The ED50 for moulting inhibition by injected azadirachtin A is for fourth instar larvae of all the triatomines, Triatoma vitticepes, T. pseudomaculata, T. maculata, T. brasiliensis, T. lecticularis, T. matogrossensis, T. infestans, Rhodnius prolixus. R. neglectus, R. robustus, Panstrongylus megistus, and P. herrera in the range of 10-25 ng/larva. In Rhodnius prolixus, the survival of T. cruzi was studied after treatment with the drug. If the trypomastigotes were fed in presence of 1.0 microgram azadirachtin A/ml blood, the number of parasites decreased near to the limit of detection within 30 days. If the drug was applied 20 days after T. cruzi infection, it still completely abolished the parasite in the host's gut within the subsequent 20 days. The same holds true if the insect larvae were pretreated with azadirachtin A 20 days before the subsequent infection with T. cruzi. Azadirachtin A. if applied at the dose of 1 microgram/ml, did not affect the hemolytic activity of the crop contents or the proteinase content of the intestine. A parallel between azadirachtin effects on the hormone balance of the host and growth inhibition of the parasite is discussed on the basis of the present results.

[Right ventricular myxoma simulating pulmonary valve stenosis. A case report]. / Mixoma de ventrículo direito simulando estenose valvular pulmonar. Relato de caso.

A 17 year old girl was asymptomatic until 3 months ago, when she noticed palpitations and chest pain. Physical examination revealed a systolic murmur + + +/6 in the pulmonary area. Chest X-ray showed discrete dilatation of the pulmonary trunk and the electrocardiogram a first degree heart block. The diagnosis of myxoma was established after echocardiogram and cardiac catheterization when a mobile cardiac mass was identified in the right ventricular outflow tract. The tumor was removed using extracorporeal circulation and the ventricular access through the right atrium. After a 16 month follow-up the patient is dealing a normal life and there are no signs of recurrence.

Ventricular evoked response in patients with hypertrophic obstructive cardiomyopathy treated with DDD pacing.

OBJECTIVE: To assess the changes in ventricular evoked responses (VER) produced by the decrease in left ventricular outflow tract gradient (LVOTG) in patients with hypertrophic obstructive cardiomyopathy (HOCM) treated with dual-chamber (DDD) pacing. METHODS: A pulse generator Physios CTM (Biotronik, Germany) was implanted in 9 patients with severe drug-refractory HOCM. After implantation, the following conditions were assessed: 1) Baseline evaluation: different AV delay (ranging from 150 ms to 50 ms) were sequentially programmed during 5 to 10 minutes, and the LVOTG (as determined by Doppler echocardiography) and VER recorded; 2) standard evaluation, when the best AV delay (resulting in the lowest LVOTG) programmed at the initial evaluation was maintained so that its effect on VER and LVOTG could be assessed during each chronic pacing evaluation. RESULTS: LVOTG decreased after DDD pacing, with a mean value of 59 +/- 24 mmHg after dual chamber pacemaker, which was significantly less than the gradient before pacing (98 + 22 mmHg). An AV delay > 100 ms produced a significantly lower decrease in VER depolarization duration (VERDD) when compared to an AV delay < or = 100 ms. Linear regression analyses showed a significant correlation between the LVOTG values and the magnitude of VER (r = 0.69; p < 0.05) in the 9 studied patients. CONCLUSION: The telemetry obtained intramyocardial electrogram is a sensitive means to assess left ventricular dynamics in patients with HOCM treated with DDD pacing.