Electromechanical heterogeneity in the heart : A key to long QT syndrome?

In the healthy heart, physiological heterogeneities in structure and in electrical and mechanical activity are crucial for normal, efficient excitation and pumping. Alterations of heterogeneity have been linked to arrhythmogenesis in various cardiac disorders such as long QT syndrome (LQTS). This inherited arrhythmia disorder is caused by mutations in different ion channel genes and is characterized by (heterogeneously) prolonged cardiac repolarization and increased risk for ventricular tachycardia, syncope and sudden cardiac death. Cardiac electrical and mechanical function are not independent of each other but interact in a bidirectional manner by electromechanical and mechano-electrical coupling. Therefore, changes in either process will affect the other. Recent experimental and clinical evidence suggests that LQTS, which is primarily considered an "electrical" disorder, also exhibits features of disturbed mechanical function and heterogeneity, which in turn appears to correlate with the risk of arrhythmia in the individual patient. In this review, we give a short overview of the current knowledge about physiological and pathological, long QT-related electrical and mechanical heterogeneity in the heart. Also, their respective roles for future risk prediction approaches in LQTS are discussed.

Possible mechanisms for sensorineural hearing loss and deafness in patients with propionic acidemia.

Propionic acidemia is an inborn error of metabolism caused by deficiency of the mitochondrial enzyme propionyl-CoA carboxylase. Sensorineural deafness and severe hearing loss have been described as long-term complications of this disease, however, the mechanism has not yet been elucidated. We have recently shown by patch clamping experiments and Western blots that acute and chronic effects of accumulating metabolites such as propionic acid, propionylcarnitine and methylcitrate on the KvLQT1/KCNE1 channel complex cause long QT syndrome in patients with propionic acidemia by inhibition of K+ flow via this channel. The same KvLQT1/KCNE1 channel complex is expressed in the inner ear and essential for luminal potassium secretion into the endolymphatic space. A disruption of this K+ flow results in sensorineural hearing loss or deafness. It can be assumed that acute and chronic effects of accumulating metabolites on the KvLQT1/KCNE1 channel protein may similarly cause the hearing impairment of patients with propionic acidemia.

Avoiding sports-related sudden cardiac death in children with congenital channelopathy : Recommendations for sports activities.

For the past few years, children affected by an inherited channelopathy have been counseled to avoid (recreational) sports activities and all competitive sports so as to prevent exercise-induced arrhythmia and sudden cardiac death. An increased understanding of the pathophysiological mechanisms, better anti-arrhythmic strategies, and, in particular, more epidemiological data on exercise-induced arrhythmia in active athletes with channelopathies have changed the universal recommendation of "no sports," leading to revised, less strict, and more differentiated guidelines (published by the American Heart Association/American College of Cardiology in 2015). In this review, we outline the disease- and genotype-specific mechanisms of exercise-induced arrhythmia; give an overview of trigger-, symptom-, and genotype-dependent guidance in sports activities for children with long QT syndrome (LQTS), Brugada syndrome (BrS), catecholaminergic polymorphic ventricular tachycardia (CPVT), or short QT syndrome (SQTS); and highlight the novelties in the current guidelines compared with previous versions. While it is still recommended for patients with LQT1 and CPVT (even when asymptomatic) and all symptomatic LQTS patients (independent of genotype) to avoid any competitive and high-intensity sports, other LQTS patients successfully treated with anti-arrhythmic therapies and phenotype-negative genotype-positive patients may be allowed to perform sports at different activity levels - provided they undergo regular, sophisticated evaluations to detect any changes in arrhythmogenic risk.

The potential impact of new generation transgenic methods on creating rabbit models of cardiac diseases.

Since the creation of the first transgenic rabbit thirty years ago, pronuclear microinjection remained the single applied method and resulted in numerous important rabbit models of human diseases, including cardiac deficiencies, albeit with low efficiency. For additive transgenesis a novel transposon mediated method, e.g., the Sleeping Beauty transgenesis, increased the efficiency, and its application to create cardiac disease models is expected in the near future. The targeted genome engineering nuclease family, e.g., the zink finger nuclease (ZFN), the transcription activator-like effector nuclease (TALEN) and the newest, clustered regularly interspaced short palindromic repeats (CRISPR) with the CRISPR associated effector protein (CAS), revolutionized the non-mouse transgenesis. The latest gene-targeting technology, the CRISPR/CAS system, was proven to be efficient in rabbit to create multi-gene knockout models. In the future, the number of tailor-made rabbit models produced with one of the above mentioned methods is expected to exponentially increase and to provide adequate models of heart diseases.

Transgenic rabbit models to investigate the cardiac ion channel disease long QT syndrome.

Long QT syndrome (LQTS) is a rare inherited channelopathy caused mainly by different mutations in genes encoding for cardiac K(+) or Na(+) channels, but can also be caused by commonly used ion-channel-blocking and QT-prolonging drugs, thus affecting a much larger population. To develop novel diagnostic and therapeutic strategies to improve the clinical management of these patients, a thorough understanding of the pathophysiological mechanisms of arrhythmogenesis and potential pharmacological targets is needed. Drug-induced and genetic animal models of various species have been generated and have been instrumental for identifying pro-arrhythmic triggers and important characteristics of the arrhythmogenic substrate in LQTS. However, due to species differences in features of cardiac electrical function, these different models do not entirely recapitulate all aspects of the human disease. In this review, we summarize advantages and shortcomings of different drug-induced and genetically mediated LQTS animal models - focusing on mouse and rabbit models since these represent the most commonly used small animal models for LQTS that can be subjected to genetic manipulation. In particular, we highlight the different aspects of arrhythmogenic mechanisms, pro-arrhythmic triggering factors, anti-arrhythmic agents, and electro-mechanical dysfunction investigated in transgenic LQTS rabbit models and their translational application for the clinical management of LQTS patients in detail. Transgenic LQTS rabbits have been instrumental to increase our understanding of the role of spatial and temporal dispersion of repolarization to provide an arrhythmogenic substrate, genotype-differences in the mechanisms for early afterdepolarization formation and arrhythmia maintenance, mechanisms of hormonal modification of arrhythmogenesis and regional heterogeneities in electro-mechanical dysfunction in LQTS.

Electro-mechanical dysfunction in long QT syndrome: Role for arrhythmogenic risk prediction and modulation by sex and sex hormones.

Long QT syndrome (LQTS) is a congenital arrhythmogenic channelopathy characterized by impaired cardiac repolarization. Increasing evidence supports the notion that LQTS is not purely an "electrical" disease but rather an "electro-mechanical" disease with regionally heterogeneously impaired electrical and mechanical cardiac function. In the first part, this article reviews current knowledge on electro-mechanical (dys)function in LQTS, clinical consequences of the observed electro-mechanical dysfunction, and potential underlying mechanisms. Since several novel imaging techniques - Strain Echocardiography (SE) and Magnetic Resonance Tissue Phase Mapping (TPM) - are applied in clinical and experimental settings to assess the (regional) mechanical function, advantages of these non-invasive techniques and their feasibility in the clinical routine are particularly highlighted. The second part provides novel insights into sex differences and sex hormone effects on electro-mechanical cardiac function in a transgenic LQT2 rabbit model. Here we demonstrate that female LQT2 rabbits exhibit a prolonged time to diastolic peak - as marker for contraction duration and early relaxation - compared to males. Chronic estradiol-treatment enhances these differences in time to diastolic peak even more and additionally increases the risk for ventricular arrhythmia. Importantly, time to diastolic peak is particularly prolonged in rabbits exhibiting ventricular arrhythmia - regardless of hormone treatment - contrasting with a lack of differences in QT duration between symptomatic and asymptomatic LQT2 rabbits. This indicates the potential added value of the assessment of mechanical dysfunction in future risk stratification of LQTS patients.

On the emerging role of rabbit as human disease model and the instrumental role of novel transgenic tools.

The laboratory rabbit (Oryctolagus cuniculus) is widely used as a model for human diseases, because of its size, which permits non-lethal monitoring of physiological changes and similar disease characteristics. Novel transgenic tools such as, the zinc finger nuclease method and the sleeping beauty transposon mediated or BAC transgenesis were recently adapted to the laboratory rabbit and opened new opportunities in precise tissue and developmental stage specific gene expression/silencing, coupled with increased transgenic efficiencies. Many facets of human development and diseases cannot be investigated in rodents. This is especially true for early prenatal development, its long-lasting effects on health and complex disorders, and some economically important diseases such as atherosclerosis or cardiovascular diseases. The first transgenic rabbits models of arrhythmogenesis mimic human cardiac diseases much better than transgenic mice and hereby underline the importance of non-mouse models. Another emerging field is epigenetic reprogramming and pathogenic mechanisms in diabetic pregnancy, where rabbit models are indispensable. Beyond that rabbit is used for decades as major source of polyclonal antibodies and recently in monoclonal antibody production. Alteration of its genome to increase the efficiency and value of the antibodies by humanization of the immunoglobulin genes, or by increasing the expression of a special receptor (Fc receptor) that augments humoral immune response is a current demand.

Enhanced complement resistance in drug-selected P-glycoprotein expressing multi-drug-resistant ovarian carcinoma cells.

Multi-drug resistance (MDR) is a major obstacle in cancer chemotherapy. There are contrasting data on a possible correlation between the level of expression of the drug transporter P-glycoprotein (P-gp) and susceptibility to complement-dependent cytotoxicity (CDC). We therefore investigated the sensitivity of human ovarian carcinoma cells and their P-gp expressing MDR variants to complement. Chemoselected P-gp expressing MDR cells showed increased resistance to CDC associated with overexpression of membrane-bound complement regulatory proteins (mCRP) and increased release of the soluble inhibitors C1 inhibitor and factor I. MDR1 gene transfection alone did not alter the susceptibility of P-gp expressing A2780-MDR and SKOV3-MDR cells to CDC. However, subsequent vincristine treatment conferred an even higher resistance to complement to these cells, again associated with increased expression of mCRP. Blocking the function of P-gp with verapamil, cyclosporine A or the anti-P-gp-antibody MRK16 had no impact on their complement resistance, whereas blocking of mCRP enhanced their susceptibility to complement. These results suggest that enhanced resistance of chemoselected MDR ovarian carcinoma cells to CDC is not conferred by P-gp, but is due at least partly to overexpression of mCRP, probably induced by treatment with the chemotherapeutic agents.

Three new Sarcocystis species, Sarcocystis giraffae, S. klaseriensis, and S. camelopardalis (Protozoa: Sarcocystidae) from the giraffe (Giraffa camelopardalis) in South Africa.

Three new Sarcocystis species recovered from muscle fibers of the skeletal musculature of a giraffe in South Africa are described based on light and electron microscopy. Sarcocystis giraffae n. sp. formed slim macrocysts with a parasite-induced connective tissue encapsulation of the host muscle fiber in which the plasma membrane of the latter remained unaltered. The sarcocyst wall represented a new ultrastructural type that is characterized by fingerlike villar protrusions with a hairlike projection at the tip, containing microtubules penetrating the ground substance. Sarcocystis klaseriensis n. sp. formed small filiform microcysts with kinked finger-shaped villar protrusions of the wall, containing scattered microtubules or filaments, and represented another new ultrastructural type. Sarcocystis camelopardalis n. sp. formed small filiform microcysts with straplike villar protrusions of the wall, containing chainlike osmiophilic structures, and represented again a new ultrastructural type.

Sarcocystis kirmsei in the brain of a hill mynah (Gracula religiosa).

Cysts of Sarcocystis kirmsei were found in the brain of an exotic bird. The morphological appearance of this protozoon and the changes in the brain tissue are described. This is the second report of S. kirmsei in the brain of a hill mynah, and the third report of this parasite in the avian brain. The affiliation of S. kirmsei to the genus Sarcocystis was confirmed. On the basis of our findings, the genus Frenkelia, which comprises two species affecting the brain of small mammals, becomes synonymous with Sarcocystis.

Sarcocystis dubeyella n. sp. and Sarcocystis phacochoeri n. sp. (Protozoa: Sarcocystidae) from the warthog (Phacochoerus aethiopicus) in South Africa.

Sarcocystis dubeyella n. sp. and S. phacochoeri n. sp. from muscle fibers of the skeletal musculature of two warthogs in South Africa are described by light and and electron microscopy. Sarcocystis dubeyella sarcocysts are macroscopic (up to 12 mm long and 1 mm wide), with a parasite-induced encapsulation of the host muscle fiber in which the plasma membrane of the latter remained unaltered. The sarcocyst wall is characterized by evenly arranged, irregularly semicircular or rectangular villar protrusions (5.0 x 2.8-11.0 microns) with indented margins and no specific content. Sarcocystis phacochoeri formed filiform microcysts (up to 4 mm long and 0.13 mm wide). Its cyst wall is provided with tightly packed, molarlike villar protrusions (1.6-3.3 x 1.7-3.3 microns), with smooth margins, hollow on one side, and with longitudinal condensations of the fine granular matrix at various locations in the interior.

Characterization of bovine Sarcocystis species by analysis of their 18S ribosomal DNA sequences.

Two Sarcocystis hirsuta isolates from 2 cattle (Bos taurus), 2 Sarcocystis hominis-like isolates from 2 cattle, 1 each Sarcocystis hominis-like isolate from a zoo-born dwarf zebu (B. taurus) and from a zoo-born bison (Bison bison), and 1 each Sarcocystis cruzi isolate from the dwarf zebu and the bison have been characterized by comparing the directly sequenced polymerase chain reaction products of their 18S rRNA genes. Sequences of 2 different isolates from the same species always showed a very high, nearly complete identity to each other both within all or only the conserved overlapping nucleotides. Thus, the 18S rDNA sequences of both S. hirsuta isolates proved to belong to a single species. The same is true for all 3 S. cruzi sequences. The 4 S. hominis-like isolates could not be placed within a single species using these criteria. They formed 2 separate groups (1 cattle isolate and the bison isolate, and the other cattle isolate and the dwarf zebu isolate). The results suggest that zoo-born large mammals do serve as intermediate hosts for Sarcocystis species known from indigenous herbivores, and that there might be another species found in cattle that is morphologically very similar to S. hominis.

Antelopes (Bovidae) kept in European zoological gardens as intermediate hosts of Sarcocystis species.

Four different forms of sarcocysts from the zoo-kept antelopes Addax nasomaculatus. Antilope cervicapra, Taurotragus oryx and Boselaphus tragocamelus (Bovidae) were investigated by light and transmission electron microscopy, in special consideration of the cyst wall. The sarcocysts found in Addax (born in a zoo) were not distinguishable from Sarcocystis medusiformis of Australasian sheep by their morphology and would be the first indication for the occurrence of this species in Europe. Sarcocysts from Antilope (born in a zoo) resembled the tenella/capracanis type of sheep/goats and were, therefore, designated as Sarcocystis sp. (? cf. capracanis) in this paper. Sarcocysts from Taurotragus were similar to a zoonotic species of cattle and hence provisionally designated as S. sp. (? cf. hominis). A sarcocyst form with hair-like villar protrusions of the cyst wall was found in Taurotragus. Boselaphus and Antilope and compared with a common species of cattle: S (? cf. cruzi).

On the diagnostics of Sarcocystis in cattle: sarcocysts of a species unusual for Bos taurus in a dwarf zebu.

Sarcocysts from the musculature of a dwarf zebu born in a German zoo are described by light and transmission electron microscopy. In addition to the three named bovine Sarcocystis species (Sarcocystis cruzi, S. hominis, and S. hirsuta) a fourth species was found which was not previously reported from cattle. It mostly resembles Sarcocystis gracilis Rátz, 1909 from roe deer.

The Mongolian gazelle (Procapra gutturosa, Bovidae) as an intermediate host of three Sarcocystis species in Mongolia.

The sarcocysts of three Sarcocystis species (Sporozoa: Coccidia: Sarcocystidae) found in the Mongolian gazelle are described using light and transmission electron microscopy. The host-parasite interface and the ultrastructure of the cyst wall are represented for the first time in the macrocysts of Sarcocystis mongolica Machul'skii, 1947, localised in connective tissue cells. The cyst wall is attributed to type 1 of the classification by Dubey et al. (1989). Two other species are described for the first time from Procapra gutturosa in Mongolia: S. sp. with hair-like villar protrusions (type 7) and S. danzani n. sp. with villar protrusions possessing a structure hitherto unknown, characterised by a hollow.

The black-backed jackal (Carnivora: Canidae) in Namibia as intermediate host of two Sarcocystis species (Protozoa: Sarcocystidae).

Two structurally different sarcocysts are reported from the black-backed jackal (Canis mesomelas) in Namibia by means of light and transmission electron microscopy for the first time. They cannot be attributed to any of the hitherto described Sarcocystis species from Carnivora, of which the ultrastructure of the cyst wall is known.

Cyst wall ultrastructure of two Sarcocystis spp. from European mouflon (Ovis ammon musimon) in Germany compared with domestic sheep.

Muscle samples from six wild and two captive European mouflons (Ovis ammon musimon) in Germany as well as one domestic sheep from a German zoo were infected with sarcocysts (Sarcocystis: Sarcocystidae, Apicomplexa). Sarcocystis tenella and S. arieticanis were identified by light and electron microscopy. Both species are determined for the first time from wild sheep, and this is the first description of S. arieticanis from wild sheep.

On the morphological diagnostics and host specificity of the Sarcocystis species of some domesticated and wild bovini (cattle, banteng and bison).

Sarcocysts of Sarcocystis cruzi, S. hominis and S. hirsuta were described and compared by means of light (LM) and transmission electron microscopy (TEM) from cattle, bantengs (born in a zoo) and bisons (born in zoo). No morphological differences were observed in the three forms of sarcocysts from each of the three host species. The morphological criteria of the three species were discussed and, partly, newly defined. S. cruzi can easily be distinguished from the other two species. It shows 6-13 micron long hair-like villar protrusions in the freshly extracted sarcocyst, has large compartments often bump-like bulged out (by LM, in the fresh state), and a "thin cyst wall" ( < 1 micron; LM, histology). TEM investigation is not necessary in most cases. S. hominis and S. hirsuta: with a "thick, radially striated" and 2.5-9.0 micron high cyst wall (LM). S. hominis can be most reliably determined by means of TEM: the villar protrusions arise with a broad basis from the cyst surface, are finger-like in the outline and have microfilaments in the core. LM: the villar protrusions are broadly seated on the cyst surface, without optical interruption. Likewise, S. hirsuta can be determined most reliably by TEM (in some cases also in semithin sections): the villar protrusions are club- or bulb-shaped in the outline, with a stalklet at the base and with microfilaments and large osmiophilic granules in the interior. LM (best in the fresh state): the palisade of villar protrusions often shows a bright or dotted horizontal line in the middle and a basal line with dark dots, arranged like a string of pearls.

Sarcocysts (Sarcocystis sp.: Sporozoa) in the European badger, Meles meles.

Sarcocysts from the tongue muscle of a European badger (Meles meles) are reported for the first time and described by light and transmission electron microscopy. Judging from the ultrastructure of the cyst wall, the parasite is similar to the species Sarcocystis gracilis Rátz, 1909 sensu Erber, Boch & Barth (1978) from roe deer and possibly identical with it. This is noteworthy regarding the intermediate host specificity.

The European badger (Carnivora:Mustelidae) as intermediate host of further three Sarcocystis species (Sporozoa).

Three species of sarcocysts are described by light and electron microscopy from the European badger (Meles meles): Sarcocystis hofmanni n. sp. (a species otherwise occurring in roe deer, Capreolus capreolus), S. sp., cf. sebeki (c species usually parasitizing certain murids); and S. melis n. sp. (c species presumably specific for the badger).