The IKs current constitutes part of the repolarisation reserve in the human myocardium, and whilst it does not play a major role at resting heart rates, it becomes a crucial component of repolarisation in the setting of increased sympathetic tone and high heart rates. The formation of the IKs current requires the KCNQ1 α-subunit and the KCNE1 β-subunit. Mutations in either of these subunits can lead to long QT syndrome types 1 and 5, respectively. Loss-of-function mutations in the IKs channel can reduce the repolarisation reserve and lead to action potential prolongation, predisposing to lethal cardiac arrhythmias such as torsades de pointes and ventricular fibrillation. It is widely recognised that the IKs channel requires the minor membrane phospholipid PIP2 for its function, and previous work in this laboratory found that mutations in a PIP2-binding region in KCNQ1 led to retention of the channel in the endoplasmic reticulum, suggesting that PIP2 may play a role in anterograde trafficking. Here, the rapamycin-inducible dimerisation system was used to manipulate levels of PIP2 and/or PI4P at the plasma membrane or Golgi, and the effect of this on IKs channel trafficking and function was investigated using molecular biology, confocal microscopy and electrophysiology. Despite difficulties with optimising the rapamycin-induced dimerisation system, it was observed that the IKs channel does not require PIP2 for anterograde trafficking, but is heavily reliant on PIP2 for channel opening. In addition, activation of the β1-adrenergic receptor (β 1-AR) led to an increase in the IKs current amplitude. The potential interplay between β1-AR and PIP2 signalling was also explored by depleting PIP2 during β1-AR stimulation. PIP2 depletion was less effective at inhibiting the IKs current during β1-AR stimulation, but this requires further investigation. In conclusion, the results suggest that the IKs channel is reliant on PIP2 for function, but not anterograde trafficking.

Heart failure patients have dysfunction of the cardiac conduction system that contributes to a high burden of arrhythmias including atrial fibrillation and sudden cardiac death. Heart failure has been associated with the inflammatory response, but it is unknown if inflammation is playing a role in the remodelling of the cardiac conduction system in heart failure. Inflammation has been shown to be present in the myocardium from failing hearts and it is known to have detrimental effects on cardiac function, inducing fibrosis, remodelling of ion channels and even arrhythmias. However, the effect of inflammation on the cardiac conduction system has not been investigated. The aims of this study were to determine if there is an increase of pro-inflammatory cytokines and inflammatory cells in the cardiac conduction system in heart failure. In addition, to identify if there is possible inflammation-associated fibrosis and apoptosis in the cardiac conduction system in heart failure. To test these aims, three models of heart failure were used: a rat model of pulmonary arterial hypertension, a rabbit model of congestive heart failure and a rat model of myocardial infarction. In the rat model of pulmonary arterial hypertension there was a bradycardia, a prolongation of the QT interval, and an increase in the atrioventricular and ventricular refractory periods, suggesting electrical remodelling in these animals. The rats with pulmonary arterial hypertension displayed an increase in pro-inflammatory cytokines such as interleukins 1β and TGFβ in the right side of the heart, including the sinoatrial node and right Purkinje fibres of the cardiac conduction system. In addition, in these areas, there was an increase in components of the extracellular matrix, including fibronectin, collagen I and vimentin. Histology revealed regions of non-myocyte nuclei, only in the right ventricle of the rats with pulmonary arterial hypertension. Immunohistochemistry demonstrated patches of CD68 and vimentin expression (markers for macrophages and fibroblasts, respectively) in the right side of the heart in these animals. TUNEL staining also revealed an increase in apoptosis in the right side of the heart. In the rabbit model of congestive heart failure, the region most affected by inflammation was the right atrium, while few changes were measured in the ventricles or cardiac conduction system. Although these results are surprising, it is suggested that the atria could be more sensitive to the physical stretch produced in this model. In the rat model of myocardial infarction, there were regions of non-myocyte nuclei in the border zone. This region also had increases in pro-inflammatory and fibrosis markers. In conclusion, this work has presented the novel finding that there can be inflammation in the cardiac conduction system in heart failure. This could be contributing to the arrhythmias seen in heart failure patients. This could possibly lead the way to anti-inflammatories as a possible novel therapeutic for heart failure patients.

Neuronal, cardiac, and skeletal muscle electrical signaling is achieved through the highly regulated activity of several types of voltage-gated ion channels to produce an action potential (AP). Voltage-gated potassium (Kv) channels are responsible for repolarization of the AP. Kv channels are uniquely and heavily glycosylated proteins. Previous reports indicate glycosylation modulates gating of some Kv channel isoforms; often, terminal sialic acid residues alter Kv channel gating. Here, we questioned whether alterations in glycosylation impact Kv channel gating, thus altering APs and cardiac excitability. ST3Gal-IV, a sialyltransferase expressed at uniform levels throughout the heart, adds sialic acids to N- and O-glycans through alpha 2-3 linkages. Electrocardiograms (ECGs) suggest that cardiac conduction/rhythm are altered in ST3Gal-IV(-/-) animals, which show an increased incidence of arrhythmic beats. AP waveform parameters and two components of IK, the transient outward, Ito, and the slowly inactivating, IK,slow, were compared in neonatal control versus ST3Gal-IV(-/-) and glycosidase treated atrial and ventricular myocytes. Action potential durations (APDs) measured from ST3Gal-IV(-/-) and glycosidase treated atrial myocytes were lengthened significantly (~25-150%) compared to control; however, ventricular APDs were unaffected by changes in glycosylation. Consistently, atrial Ito and IK,slow activation were shifted to more depolarized potentials (by ~9-17 mV) in ST3Gal-IV(-/-) and glycosidase treated myocytes, while ventricular K+ currents were unaltered. Those channels responsible for producing Ito and IK,slow were examined under conditions of full and reduced glycosylation. Sialylation and N-glycosylation uniquely and differently impact gating of two mammalian Shaker family Kv channel isoforms, Kv1.4 and Kv1.5; Kv1.4 gating was unaffected by changes in channel glycosylation, while N-linked sialic acids, acting through electrostatic mechanisms, fully account for glycan effects on Kv1.5 gating. In addition, sialic acids modulate the gating of three Kv channel isoforms that are not N-glycosylated, Kv2.1, Kv4.2, and Kv4.3, through apparent electrostatic mechanisms. Click chemistry was utilized to confirm that these three isoforms are O-glycosylated and sialylated; thus, O-linked sialylation modulates gating of Kv2.1, Kv4.2, and Kv4.3. This study suggests that regulated or aberrant glycosylation alters the gating of channels producing IK in a chamber-specific manner, thus altering the rate of cardiac repolarization and potentially leading to arrhythmias.

Despite decades of research and thousands of studies on cardiac electrophysiology, cardiovascular disease remains among the leading causes of death in the United States today. Despite substantially beneficial advances, we have largely shifted cardiovascular disease from an acute to a chronic issue. It is therefore clear that our current understanding of the heart's functions remain inadequate and we must search for untapped therapeutic approaches to eliminate these deadly and costly ailments once and for all. This thesis will focus on the electrophysiology of the heart, specifically the mechanisms of cell-to-cell conduction. Canonically, the understood mechanism of cardiac conduction is through gap junctions (GJ) following a cable-like conduction model. While both experimentally and mathematically, this understanding of conduction has explained cardiac electrical behavior, it is also incomplete, as evidenced by recent conflicting modeling and experimental data. The overall goal of this thesis is to explore a structure modulating an ephaptic, or electric field, cellular coupling mechanism: the GJ-adjacent perinexus, with three specific aims. First, I identified the perinexus – a recently-established structure in rodent myocardium – in human atrial tissue. I also observed a significant tendency for open-heart surgery patients with pre-operative atrial fibrillation to have wider perinexi, indicating a possibly targetable mechanism of atrial fibrillation, one of the costliest, and most poorly-understood cardiac diseases. Next, I developed a high-throughput, high-resolution method for quantifying the perinexus. Finally, I sought to reconcile a major controversy in the field: whether cardiac edema could either be beneficial or harmful to cardiac conduction. Using a Langendorff perfusion model, I added osmotic agents of various sizes to guinea pig hearts and measured electrical and structural parameters. My findings suggest that while cardiac conduction is multifaceted and influenced by several parameters, the strongest correlation is an inverse relationship between conduction velocity and the width of the perinexus. This study is the first to osmotically expand and narrow the perinexus and show an inverse correlation with conduction. Importantly, my conduction data cannot be explained by factors consistent with a cable-like conduction mechanism, indicating once again that the perinexus could be a therapeutic target for a myriad of cardiac conduction diseases. / Doctor of Philosophy / The ways by which cells in the heart communicate have been studied extensively and are thought to be well-understood. However, despite decades of research, cardiovascular disease is a major problem in the developed world today and we remain unable to develop treatments to truly cure many major cardiac diseases. Because of this lack of clinical success in preventing or treating conditions such as atrial fibrillation, Brugada syndrome and sudden cardiac death, all of which are associated with disruptions in the heart’s electrical communication systems, I have sought to better understand the ways by which cellular communication is achieved. Currently, we think of cardiac tissue to propagate electrical signals as if it was a series of cables, just like the electrical wires over our streets and in our homes. However, we have seen experimental evidence, along with computer simulations, that supports the idea of a second mechanism of cellular electrical conduction. This second mechanism is called ephaptic, or electric field, coupling and relies on changes in charges inside and outside the cell to trigger the action potential – the electrical signal which tells the cell to contract. In order for ephaptic coupling to occur, two main conditions must be met. First, there must be a suitably-sized cleft, or ephapse, between adjacent cells. Models have estimated this space to be between 10-100 nm wide. Second, there must be a large concentration of sodium channels, as sodium ions are primarily used to set off the action potential. The region in which I am most interested is the cardiac perinexus, which is the space immediately adjacent to plaques of connexin proteins which link adjacent cells. The perinexus is both of an appropriate size (we’ve measured it between 10 and 25 nm on average) and rich in sodium channels, making it an ideal candidate to be a cardiac ephapse. In recent years, our lab has shown experimentally that expanding this space can disrupt cardiac conduction and my first study showed that clinically, patients with chronic atrial fibrillation (a-fib) prior to open-heart surgery have wider perinexi than patients without chronic a-fib. No one, however, has been able to demonstrate that narrowing the perinexus would be therapeutic by making it easier for cells to communicate via this ephaptic mechanism. Knowing I would need a better method for measuring the width of huge numbers of perinexi, I then developed a faster, more precise measurement program. Finally, I perfused several osmotic agents – substances which would theoretically draw fluid into or out of various compartments of cardiac tissue – into guinea pig hearts and observed changes to both their electrical behavior and tissue structure. Using my new perinexal measurement program, I found that changing the perinexus was the only factor that could explain the conduction changes I observed with each osmotic agent and that parameters associated with cable theory, such as gap junctional protein expression or interstitial resistance, could not explain conduction changes. Therefore, I have indicated, along with my clinical study, that the cardiac perinexus could be a therapeutic target for preventing, managing, or possibly even curing cardiac conduction diseases.

Thesis (PhD)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: Progressive familial heart block I (PFHBI) is an inherited autosomal dominant cardiac conduction disorder which segregates in a large South African (SA) pedigree, two smaller SA families and a Lebanese family. It specifically affects conduction in the ventricles and is of unknown cause. Clinically, PFHBI is detected on electrocardiogram (ECG) by evidence of bundle-branch disease, i.e., as right bundle branch block, left anterior or posterior hemiblock, or complete heart block with broad QRS complexes. The PFHBI-causative gene was mapped to a lOcM region on chromosome 19ql3.3 using linkage analysis, and the locus was subsequently reduced to 7cM by genetic fine mapping. The present study involved a multi-strategy approach to search for the PFHBI gene. The objectives were the further reduction of the PFHBI locus by genetic fine mapping using published and novel markers, searching for short gene transcripts from publicly available databases and the generation of an integrated map of the locus to which genes were mapped. Prioritised genes were screened for PFHBI-causing mutations and, in addition, the PFHBI locus was searched for the presence of a G protein-encoding gene (PI 15- RhoGEF), a connexin (Cx) gene and any genes containing a CTG repeat expansion motif, since these genes are plausible PFHBI candidate genes. Genotyping and fine genetic mapping using known and novel polymorphic dinucleotide (CA)n and novel tetranucleotide (A3G)n repeat markers across the PFHBI locus were performed. Publicly available databases, such as LLNL (Livermore, USA), and GENEMAP (NCBI) were searched for ESTs which, in turn, were extended using clustering programmes, such as UNIGENE (NCBI) and STACK (SANBI), and the resulting consensus sequences were subsequently BLAST-searched against the protein databases. Using the available data, an integrated physical and genetic map of the PFHBI locus was generated and, as the HGP progressed, a number of novel genes were placed thereon. Subsequently, genes were prioritised on the basis of position, function and expression profile. Genetic fine mapping reduced the PFHBI locus from 7cM to 4cM. The EST approach yielded 38 ESTs, of which 24 ESTs matched proteins, such as activating transcription factor 5 (ATF5), actin-binding protein (KPTN) and zinc finger protein 473 (ZFP473) (May 2003). All the map data generated experimentally and computationally were placed on the PFHBI map. The PI 15-RhoGEF was excluded as a PFHBI candidate gene and although homologous sequences to connexin 37 (Cx37) was located on both chromosome 19 radiation hybrid clones (RHG12 and ORIM-7), it was not identified on the DNA clones spanning the PFHBI locus. No evidence of an expansion of a CTG repeat motif sequence in PFHBI-affected individuals was found. Five highly prioritised candidate genes, namely, 5CZ2-associated X protein (BAX), potassium voltage-gated channel Shaker-related subfamily member 7 (KCNA7’), potassium inwardly-rectifying channel, subfamily J, member 14 (KIR2.4), lin-7 homolog B {LIN-7B) and glycogen synthase 1 (GSYI) were selected for mutation screening. No disease associated mutations were identified in the exonic and flanking intronic regions of these genes. In summary, this study reduced the PFHBI locus substantially and generated a detailed map of the region. A number of attractive candidate genes were excluded from causing PFHBI; however, several plausible candidate genes are still present at this gene-rich locus and remain to be screened. Identifying the PFHBI-causative gene and associated mutation will provide a platform for further studies to understand the pathophysiology, not only of PFHBI, but also of other more commonly occurring conduction disturbances. / AFRIKAANSE OPSOMMING: Progressiewe familiele hartblok I (PFHBI) is ‘n autosomaal dominant oorerflike kardiale geleidingstoomis wat in ‘n groot Suid-Afrikaanse (SA) familie, twee kleiner SA families en ‘n Lebanese familie segregeer. Dit affekteer hoofsaaklik die geleiding in die ventrikels en die oorsaak daarvan is onbekend. Klinies word PFHBI op elektrokardiogram (EKG) geidentifiseer as a bondeltak-siekte, naamlik, as regter bondeltakblok, linker anterior of posterior hemiblok, of volledige hartblok met wye QRS komplekse. Die PFHBI-veroorsakende geen is voorheen deur koppelingsanalise tot ‘n lOcM gebied op chromosoom 19ql3.3 gekarteer, en daaropvolgens is die lokus verklein tot 7cM deur genetiese fyn kartering. Die huidige studie behels ‘n veelvuldige-strategie benadering in die soektog na die PFHBI geen. Die doel van die studie was die verdere verkleining van die PFHBI lokus deur gebruik te maak van beide gepubliseerde en nuwe genetiese merkers, die identifisering van kort geentranskripte (ESTs) uit publieke databanke en die generasie van ‘n geintegreerde kaart van die lokus. Geprioritiseerde gene is geanaliseer vir die PFHBI-veroorsakende mutasie en, daarby, is die PFHBI lokus deursoek vir die teenwoordigheid van ‘n G proteien-enkodeeringsgeen (PIJ5-RhoGEF), ‘n konneksien (Kx) geen en enige gene wat ‘n uitgebreide CTG-herhalingsmotief bevat, aangesien hierdie gene as sterk PFHBI kandidaatgene geag is. Genotipering en fynkartering deur die gebruik van bekende asook nuwe polimorfiese dinukleotied- [(CA)n] en nuwe tertranukleotied- [(A3G)n] herhalingsmerkers wat die PFHBI lokus oorbrug, is uitgevoer. Publieke databanke, soos LLNL (Livermore, USA), en GENEMAP (NCBI) is ondersoek vir ESTs wat vervolgens verleng is deur gebruik te maak van groeperende programme soos UNIGENE (NCBI) en STACK (SANBI) en die gevolglike konsensus volgordes is daama met behulp van BLAST geanaliseer teen die protei'endatabanke. Die bekomde data is vervolgens gebruik om ‘n geintegreerde fisiese en genetiese kaart van die PFHBI lokus te produseer en, soos die mens genoomprojek gevorder het, is nuwe gene daarop geplaas. Daarna is gene geprioritiseer vir mutasie analise gebaseer op posisie, funksie en uitdrukkingsprofiele. Genetiese fynkartering het die PFHBI lokus van 7cM tot 4cM verklein. Die EST benadering het 38 ESTs gei'dentifiseer, waarvan 24 ESTs proteien gelyke gehad het, bv aktiverende transkripsie faktor 5 (ATF5), aktien-verbindingsprotei'en (KPTN) en sink-vingerproteien 473 (ZFP473) (Mei 2003). A1 die karterings data wat eksperimenteel en rekenaar-gewys gegenereer is, is op die PFHBI kaart geposisioneer. Die P115-RhoGEF is uitgeskakel as ‘n PFHBI kandidaatgeen en alhoewel ’n volgorde met homologie aan konneksien37 (Kx37) gevind is op albei chromosoom 19 radiasiehibried klone (RGH12 and ORIM-7), is dit nie gei'dentifiseer in die DNS klone wat die PFHBI lokus oorbrug nie. Geen bewyse van uitbreiding van CTG herhalingsmotiewe is gevind in PFHBIaangetasde persone nie. Vyf hoogs-geprioritiseerde kandidaat gene, naamlik, BCL2-geassosieerde X proteien (BAX), kalium spanningsbeheerde kanaal, subfamilie J, lid 14 (KIR2.4), lin-7 homoloog B (LIN-7b) en glikogeen sintase 1 (GYS1), is geselekteer vir mutasie-analise. Geen siekteveroorsakende mutasie is egter gei'dentifiseer in die eksoniese of die naasliggende introniese gebiede van hierdie gene nie. Ter opsomming, hierdie studie het die PFHBI lokus verklein en het ‘n omvattende kaart van die gebied gegenereer. Verskillende kandidaat gene is uitgesluit as die oorsaak van PFHBI, alhoewel daar nog heelwat goeie kandidaat gene in hierdie geen-ryke lokus is wat geanaliseer behoort te word. Die identifiseering van die PFHBI-veroorsakende mutasie sal ‘n platform bied vir verdere studies om die patofisiologie van nie alleen PFHBI nie, maar ook meer algemene geleidingstoomisse, te verstaan.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Heart failure due to cardiomyopathy or cardiac conduction disease is a major cause of mortality and morbidity in both developed and developing countries. Although defined as separate clinical entities, inherited forms of cardiomyopathies and cardiac conduction disorders have been identified that present with overlapping clinical features and/or have common molecular aetiologies. The objective of the present study was to identify the molecular cause of progressive familial heart block type II (PFHBII), an inherited cardiac conduction disorder that segregates in a South African Caucasian Afrikaner family (Brink and Torrington, 1977). The availability of family data tracing the segregation of PFHBII meant that linkage analysis could be employed to identify the chromosomal location of the disease-causative gene. Human Genome Project (HGP) databases have provided additional resources to facilitate the identification of positional candidate genes. Clinical examinations were performed on individuals of the PFHBII-affected family, and, where available, clinical records of subjects examined in a previous study by Brink and Torrington (1977) were re-assessed. Retrospective data suggested redefining the classification of PFHBII. Subsequently, linkage analysis was used to test described dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM) and cardiac conduction-causative loci on chromosomes 1, 2, 3, 6, 7, 9, 11, 14, 15 and 19 for their involvement in the development of PFHBII. Once a locus was mapped, bioinformatics tools were applied to identify and prioritise positional candidate genes for mutation screening. The retrospective and prospective clinical study redefined PFHBII as a cardiac conduction and DCM-associated disorder and simultaneously allowed more family members to be traced.Fortuitously, candidate loci linkage analysis mapped the PFHBII locus to chromosome 1q32, to a region that overlapped a previously described DCM-associated disorder (CMD1D), by the generation of a maximum pairwise lod score of 3.13 at D1S3753 (theta [θ]=0.0) and a maximum multipoint lod score of 3.7 between D1S3753 and D1S414. However, genetic fine mapping and haplotype analysis placed the PFHBII-causative locus distal to the CMD1D locus, within a 3.9 centimorgan (cM) interval on chromosome 1q32.2-q32.3, telomeric of D1S70 and centromeric of D1S505. Bioinformatics analyses prioritised seven candidate genes for mutation analysis, namely, a gene encoding a potassium channel (KCNH1), an extracellular matrix protein (LAMB3), a protein phosphatase (PPP2R5A), an adapter protein that interacts with a cytoskeletal protein (T3JAM), a putative acyltransferase (KIAA0205) and two genes encoding proteins possibly involved in energy homeostasis (RAMP and VWS59). The PFHBII-causative mutation was not identified, although single sequence variations were identified in four of the seven candidate genes that were screened. Although the molecular aetiology was not established, the present study defined the underlying involvement of DCM in the pathogenesis of PFHBII. The new clinical classification of PFHBII has been published (Fernandez et al., 2004) and should lead to tracing more affected individuals in South Africa or elsewhere. The identification of a novel disease-causative locus may point toward the future identification of a new DCM-associated aetiology, which, in turn, might provide insights towards understanding the associated molecular pathophysiologies of heart failure. / AFRIKAANSE OPSOMMING: Hartversaking as gevolg van kardiomiopatie of kardiale geleidingsiekte is ‘n hoof-oorsaak van mortaliteit and morbiditeit in beide ontwikkelde en ontwikkelende lande. Alhoewel gedefinieer as verskillende kliniese entiteite is oorerflike vorms van kardiomiopatie en kardiale geleidingsstoornisse geïdentifiseer met oorvleuelende kliniese eienskappe en/of molukulêre oorsake. Die doelwit van hierdie studie was om die molukulêre oorsaak van progressiewe familiële hartblok tipe II (PFHBII), ‘n oorerflike kardiale geleidingsstoornis, wat in ‘n Suid-Afrikaanse Kaukasiër familie segregeer (Brink en Torrington, 1977), te identifiseer. Die beskikbaarheid van familie data, beteken dat koppelingsanalise gebruik kan word om die chromosomale posisie van die siekte-veroorsakende geen te identifiseer. Menslike Genoom Projek (MGP) databanke het addisionele hulpbronne beskikbaar gestel om die identifikasie van posisionele kandidaat gene te vergemaklik. Kliniese ondersoeke is uitgevoer op PFHBII-geaffekteerde familielede, en waar beskikbaar is kliniese rekords van persone, wat in ‘n vorige studie deur Brink en Torrington (1977) geassesseer was, herontleed. Retrospektiewe data-analise het die kliniese herdefinisie van PFHBII voorgestel. Daarna is koppelingsanalise gebruik om dilateerde kardiomiopatie (DKM), hipertrofiese kardiomiopatie (HKM) en kardiale geleidingssiekte-veroorsakende loki op chromosoom 1, 2, 3, 6, 7, 9, 11, 14, 15 en 19 te ondersoek vir hul moontlike bydrae tot die ontwikkeling van PFHBII. Toe die lokus gekarteer was, is bioinformatiese ondersoeke gebruik om posisionele kandidaat gene te identifiseer en prioritiseer vir mutasie analise. Die retrospektiewe en prospektiewe kliniese ondersoek het PFHBII herdefinieer as ‘n geleidingsstoornis en DKM-verbonde siekte, en terselfde tyd het dit gelei tot die opsporingvan nog familielede. Toevallig het kandidaat loki-analise die PFHBII lokus op chromosoom 1q32 gekarteer, na ‘n gebied wat met ‘n voorheen-beskyfde DKM-verbonde stoornis (CMD1D) oorvleuel, met die opwekking van ‘n makisimum paargewyse lod-getal van 3.13 by D1S3753 (theta [θ] = 0.0) en ‘n maksimum multipunt lod-getal van 3.7 tussen D1S3753 en D1S414. Genetiese fynkartering en haplotipe-analise het die PFHBII-veroorsakende lokus afwaards van die CMD1D lokus geplaas, in ‘n 3.9 centimorgan (cM) gebied op chromosoom 1q32.2-q32.3, telomeries van D1S70 en sentromeries van D1S505. Bioinformatiese analise het daarnatoe gelei dat sewe kandidaat gene vir mutasie analise geprioritiseerd is, naamlik, gene wat onderskeidelik ‘n kalium kanaal (KCNH1), ‘n ekstrasellulêre matriksproteïen (LAMB3), ‘n proteïen fosfatase (PPP2R5A), ‘n aansluiter proteïen wat met ‘n sitoskilet proteïen bind (T3JAM), ‘n asieltansferase (KIAA0205) en twee gene moontlik betrokke in energie homeostase (RAMP en VWS59) enkodeer. Die PFHBII-veroorsakende geen is nie geïdentifiseer nie, alhoewel enkele volgorde-wisselings geïdentifiseer is in vier van die sewe geanaliseerde kandidaat gene. Alhowel die molekulêre oorsaak van die siekte nie vasgestel is nie, het die huidige studie die onderliggende betrokkenheid van DKM in die pathogenese van PFHBII gedefinieer. Die nuwe kliniese klassifikasie van PFHBII is gepubiliseer (Fernandez et al., 2004) en sal lei tot die identifisering van nog geaffekteerde persone in Suid Afrika of in ander lande. Die identifikasie van ‘n nuwe siekte-verbonde lokus mag lei tot die toekomstige identifikasie van ‘n nuwe DKM-verbonde genetiese oorsaak wat, opsig self, dalk insig kan gee in die molekulêre patofisiologie van hartversaking.

Utilizou-se 30 corações de gatos sem raça definida, machos e fêmeas, adultos de idades variadas que não portavam nenhuma afecção cardíaca. Os corações foram injetados pela aorta torácica com Neoprene Latex 450, corados com pigmento vermelho. Verificou-se quando do predomínio da vascularização ventricular do tipo esquerda (63,34%) que a região irrigada pelo nó sinoatrial fica predominantemente na dependência do ramo proximal atrial direito (78,9%) ou com menor freqüência pelo ramo proximal atrial esquerdo (21,1%). Quando ocorreu a vascularização ventricular do tipo equilibrada (33,34%), a região ocupada pelo nó sinoatrial ficou na dependência mais frequentemente do ramo proximal atrial direito (80%), ou com menor freqüência a nutrição do nó se deu pelo ramo proximal atrial esquerdo (20%). Em um caso isolado, onde ocorreu a vascularização ventricular do tipo direita (3,34%), a nutrição da área ocupada pelo nó sinoatrial, ficou na dependência exclusiva do ramo intermédio atrial direito. Estes resultados indicam que nesta espécie não existe nenhuma relação entre a irrigação do nó sinoatrial e o tipo de vascularização ventricular. / We analyzed 30 hearts of cats without pedigree, males and females, adults of several ages. They were not carrying any heart problems. The hearts were injected by the thoracic aorta with Neoprene Latex 450 and stained with red pigment. We analyzed the prevalence of ventricular vascularization of the left type (63.34%) the irrigated region by the sinoatrial node was predominantly in the dependency of the Ramus proximalis atrii dextri (78.9%) or with less frequency by Ramus proximalis atrii sinister (21.1%). When occurred the ventricular vascularization of the type balanced (33.34%), the region occupied by the sinoatrial node was in the responsibility more often of the Ramus proximalis atrii dextri (80%) or with less frequency the nutrition of the sinoatrial node occurred by Ramus proximalis atril sinister (20%). In a singlecase, where we observed the ventricular vascularization of the right type (3.34%), the nutrition of the area occupied by the sinoatrial node, was the exclusive dependence of the Ramus intermedius atril dextri. These results showed that this specie there are no relationship between both the sinoatrial node irrigation and the type of ventricular vascularization, therefore, the nutrition of the sinoatrial node does not depend on the type of the ventricles vascularization.

Tvarkingą širdies darbą užtikrina širdies laidžioji sistema (ŠLS). Ją pažeidus sutrinka širdies darbas. Pažeidimo rizika atsiranda operacijos metu, kadangi ŠLS yra raumeninės kilmės audinys ir vizualiai neatskiriamas nuo aplinkinių audinių. ŠLS pažeidimo galima būtų išvengti, jei būtų žinomas tikslus ŠLS išsidėstymas arba egzistuotų ŠLS vaizdinimo metodika. Deja, bet atskirų ŠLS dalių tikslus išsidėstymas vis dar tikslinamas, o patologijų atvejais apskritai nėra žinomas. ŠLS vaizdinimo metodikos, tinkančios in vivo taikymams, taipogi nėra. Atlikus širdies audinių tyrimus nustoviąja fluorescencine spektroskopija nustatyti charakteringi intensyvumų skirtumai. Remiantis šiais skirtumais sukurta ŠLS atskyrimo metodika, paremta intensyvumų santykių skaičiavimu. Suskaičiuota vertė R = I(330)/I(380) yra skirtinga ŠLS, miokardui (MK) ir jungiamąjam audiniui (JA).Tokia metodika yra nejautri tyrimo sąlygoms ir gali būti naudojama ŠLS nustatymui. Ištyrus širdies audinius laikinės skyros spektroskopija nustatyta, kad ŠLS ir MK fluorescencijos gyvavimo trukmė bei santykinė sudėtis reikšmingai nesiskiria, tuo tarpu JA ir ŠLS tiek fluorescencijos gyvavimo trukmės, tiek santykinė komponentinė sudėtis yra skirtinga. Ištyrus širdies audinius konfokaliniu atspindžio mikroskopu nustatyta, jog dėl skirtingų šviesą atspindinčių komponentų bei skirtingo jų išsidėstymo galima identifikuoti MK, JA, Purkinje ląsteles ir ŠLS pluoštus. Atlikus tyrimus in vivo nustatyta, jog operacijos metu... [toliau žr. visą tekstą] / Rhythmical contraction of the heart is controlled by the cardiac conduction system (CCS). However, this highly important system visually could not be distinguished from the surrounding heart tissues – myocardium (MC) and connective tissue (CT); therefore during surgical procedures CCS could be damaged. The reliable method for CCS identification either in vivo or ex vivo does not exist therefore there is a definite need for developing a CCS imaging method. Fluorescence spectroscopy studies of cardiac tissues revealed, that most distinct spectral differences between CCS and the surrounding tissues were observed in 400 nm – 550 nm region under excitation from 330 nm – 380 nm region. The visualization method, based on the intensity ratios calculated for two excitation wavelengths, has been established. The calculated ratio R = I(330)/I(380) is different for CCS, CT and MC tissues, therefore the method may be used for identification of CCS. Time resolved fluorescence spectroscopy revealed no significant difference in composition and lifetimes between CCS and MC. On the other hand, the lifetimes and the relative spectral composition of CT differed significantly from those of CCS. Reflection confocal microscopy allows visualizing MC, CT, Purkinje cells and CCS bundles because of different reflection properties of tissue components and their specific distribution inside the tissue. The results of in vivo performed procedure revealed, that the distribution of fluorescence intensities... [to full text]

Rhythmical contraction of the heart is controlled by the cardiac conduction system (CCS). However, this highly important system visually could not be distinguished from the surrounding heart tissues – myocardium (MC) and connective tissue (CT); therefore during surgical procedures CCS could be damaged. The reliable method for CCS identification either in vivo or ex vivo does not exist therefore there is a definite need for developing a CCS imaging method. Fluorescence spectroscopy studies of cardiac tissues revealed, that most distinct spectral differences between CCS and the surrounding tissues were observed in 400 nm – 550 nm region under excitation from 330 nm – 380 nm region. The visualization method, based on the intensity ratios calculated for two excitation wavelengths, has been established. The calculated ratio R = I(330)/I(380) is different for CCS, CT and MC tissues, therefore the method may be used for identification of CCS. Time resolved fluorescence spectroscopy revealed no significant difference in composition and lifetimes between CCS and MC. On the other hand, the lifetimes and the relative spectral composition of CT differed significantly from those of CCS. Reflection confocal microscopy allows visualizing MC, CT, Purkinje cells and CCS bundles because of different reflection properties of tissue components and their specific distribution inside the tissue. The results of in vivo performed procedure revealed, that the distribution of fluorescence intensities... [to full text] / Tvarkingą širdies darbą užtikrina širdies laidžioji sistema (ŠLS). Ją pažeidus sutrinka širdies darbas. Pažeidimo rizika atsiranda operacijos metu, kadangi ŠLS yra raumeninės kilmės audinys ir vizualiai neatskiriamas nuo aplinkinių audinių. ŠLS pažeidimo galima būtų išvengti, jei būtų žinomas tikslus ŠLS išsidėstymas arba egzistuotų ŠLS vaizdinimo metodika. Deja, bet atskirų ŠLS dalių tikslus išsidėstymas vis dar tikslinamas, o patologijų atvejais apskritai nėra žinomas. ŠLS vaizdinimo metodikos, tinkančios in vivo taikymams, taipogi nėra. Atlikus širdies audinių tyrimus nustoviąja fluorescencine spektroskopija nustatyti charakteringi intensyvumų skirtumai. Remiantis šiais skirtumais sukurta ŠLS atskyrimo metodika, paremta intensyvumų santykių skaičiavimu. Suskaičiuota vertė R = I(330)/I(380) yra skirtinga ŠLS, miokardui (MK) ir jungiamąjam audiniui (JA).Tokia metodika yra nejautri tyrimo sąlygoms ir gali būti naudojama ŠLS nustatymui. Ištyrus širdies audinius laikinės skyros spektroskopija nustatyta, kad ŠLS ir MK fluorescencijos gyvavimo trukmė bei santykinė sudėtis reikšmingai nesiskiria, tuo tarpu JA ir ŠLS tiek fluorescencijos gyvavimo trukmės, tiek santykinė komponentinė sudėtis yra skirtinga. Ištyrus širdies audinius konfokaliniu atspindžio mikroskopu nustatyta, jog dėl skirtingų šviesą atspindinčių komponentų bei skirtingo jų išsidėstymo galima identifikuoti MK, JA, Purkinje ląsteles ir ŠLS pluoštus. Atlikus tyrimus in vivo nustatyta, jog operacijos metu... [toliau žr. visą tekstą]

Utilizou-se 30 corações de gatos sem raça definida, machos e fêmeas, adultos de idades variadas que não portavam nenhuma afecção cardíaca. Os corações foram injetados pela aorta torácica com Neoprene Latex 450, corados com pigmento vermelho. Verificou-se quando do predomínio da vascularização ventricular do tipo esquerda (63,34%) que a região irrigada pelo nó sinoatrial fica predominantemente na dependência do ramo proximal atrial direito (78,9%) ou com menor freqüência pelo ramo proximal atrial esquerdo (21,1%). Quando ocorreu a vascularização ventricular do tipo equilibrada (33,34%), a região ocupada pelo nó sinoatrial ficou na dependência mais frequentemente do ramo proximal atrial direito (80%), ou com menor freqüência a nutrição do nó se deu pelo ramo proximal atrial esquerdo (20%). Em um caso isolado, onde ocorreu a vascularização ventricular do tipo direita (3,34%), a nutrição da área ocupada pelo nó sinoatrial, ficou na dependência exclusiva do ramo intermédio atrial direito. Estes resultados indicam que nesta espécie não existe nenhuma relação entre a irrigação do nó sinoatrial e o tipo de vascularização ventricular. / We analyzed 30 hearts of cats without pedigree, males and females, adults of several ages. They were not carrying any heart problems. The hearts were injected by the thoracic aorta with Neoprene Latex 450 and stained with red pigment. We analyzed the prevalence of ventricular vascularization of the left type (63.34%) the irrigated region by the sinoatrial node was predominantly in the dependency of the Ramus proximalis atrii dextri (78.9%) or with less frequency by Ramus proximalis atrii sinister (21.1%). When occurred the ventricular vascularization of the type balanced (33.34%), the region occupied by the sinoatrial node was in the responsibility more often of the Ramus proximalis atrii dextri (80%) or with less frequency the nutrition of the sinoatrial node occurred by Ramus proximalis atril sinister (20%). In a singlecase, where we observed the ventricular vascularization of the right type (3.34%), the nutrition of the area occupied by the sinoatrial node, was the exclusive dependence of the Ramus intermedius atril dextri. These results showed that this specie there are no relationship between both the sinoatrial node irrigation and the type of ventricular vascularization, therefore, the nutrition of the sinoatrial node does not depend on the type of the ventricles vascularization.