Trials were performed in sheep and rats to elucidate the pathogenesis of the myocardial lesions in gousiekte. In the first trial the macro- and lightmicroscopical lesions and myofibre morphometrical changes were studied in ten sheep exposed daily to Pachystigma pygmaeum at 10 g/kg live body weight for 23 to 31 days. All the treated animals either died or were euthanased in extremis between 31 and 51 days after the commencement of dosing. In the second trial the myocardial ultrastructural lesions were studied in six sheep dosed with Fadogia homblei at a dosage rate of 10 g/kg per day live body weight for 22 to 23 days. All the treated animals either died or were euthanased in extremis between 34 and 57 days after the commencement of dosing. The main objective of the third trial was to compare the myocardial lesions in rats exposed to pavetamine with lesions recorded in sheep exposed to P. pygmaeum and F. homblei plant material. Seven rats were injected intraperitoneally with pavetamine at a dosage rate of 5 mg/kg on day 0 and three were killed on day 6. The remaining four were injected with a second dose of pavetamine at a dosage rate of 3 mg/kg on day 27 and euthanased on day 42. In the sheep exposed to P. pygmaeum pulmonary oedema and hydropericardium were present in eight, hydrothorax in four and ascites in two cases. In two sheep cardiac dilatation was associated with subendocardial pallor (fibrosis) and transmural myocardial mottling. Myofibre hypertrophy was recorded in all the sheep, myofibre necrosis and replacement fibrosis occurred in seven animals the latter being particularly evident in animals with medium to long latent periods. A mononuclear cellular infiltration that varied from mild to severe was evident in all the cases and endocardial thickening, which is an indication of cardiac dilatation, was present in seven animals. Myofibre atrophy occurred in eight animals and was the most striking lesion in a sheep with a short latent period. “Typical” gousiekte lesions, characterised by myofibre necrosis and atrophy, replacement fibrosis and an associated round cell infiltration in the subendocardial region, were present in eight of the sheep. “Atypical” lesions, characterised by hypertrophy of myofibres with multifocal coagulative necrosis or myofibre atrophy, were recorded in two sheep, both of which had short latent periods. The myofibre diameter and nuclear area in the affected animals differed statistically from those of the controls (larger) and anisocytosis and anisonucleosis were particularly striking in sheep with intermediate to long latent periods. The most striking ultrastructural lesions included breakdown of myofibrils, involving in particular what appeared to be thick (myosin) filaments; selective proliferation of organelles such as mitochondria and sarcoplasmic reticulum in areas previously occupied by myofibrils; excessive folding of the myofibre sarcolemma; and advanced myocardial injury characterised by complete loss of myofibrils with loss of intercellular connections and necrosis of myocardial cells. No lesions were present in the rats exposed to a single dose of pavetamine, although they became anorexic and lost weight. Rats exposed to pavetamine twice became anorexic within two to three days after the first exposure and regained weight within a few days (on about day 7). However, they kept on losing weight after the second exposure and continued to do so until termination of the experiment. As a general rule the myocardial lesions were mild in the rats dosed twice with pavetamine. Transmural multifocal myocardial necrosis, with an associated round cell infiltration and replacement fibrosis, was the most striking light- microscopical lesion. The lesions were comparable with “atypical” lesions in ruminants. Ultrastructural lesions in degenerative/necrotic fibres included karyolysis, swelling of the mitochondria and focal lysis of myofilaments. In rats exposed to pavetamine twice there was statistical evidence of myofibre atrophy. Based on the information emanating from this study and previous research the following deductions are made to explain the pathogenesis of the myocardial lesions: 1. Pavetamine has a prolonged effect on the myocardium owing to inhibition of protein synthesis, and also influences the energy production system, which affects the function of myocytes. The structure of the myocytes is not affected during the early stages of the latent period but eventually myofibre hypertrophy, atrophy, degeneration and necrosis are seen. 2. Replacement fibrosis in the subendocardial region is a sequel to the effect of pavetamine on myofibres and the consequence of ischaemia owing to impaired myocardial perfusion of, particularly, the subendocardial region, as a result of decreased myocardial contraction, increased diastolic pressure, tachycardia and myofibre hypertrophy. 3. Cardiac dilatation is a compensatory mechanism, a result of the myofibre damage inflicted by pavetamine and ischaemia (pathological dilatation). 4. Lesions in animals with gousiekte represent a final common pathway of cellular damage rather than a manifestation of a specific type of heart disease. Animals may die during any stage in the development of the lesions. “Atypical” lesions represent a manifestation of the disease in a progression that terminates with dilated cardiomyopathy if the animal does not die during the early stages. These deductions provide an explanation, for the first time, for the latent period between ingestion of the plant and the onset of illness in gousiekte. They also explain the wide range of lesions seen in experimental cases. It furthermore demonstrate that the “typical” lesions of gousiekte are not pathognomonic, and that the absence of “typical” lesions does not rule out a diagnosis of gousiekte in situations where exposure to the causative plants and the clinical history support such a diagnosis. / Thesis (PhD)--University of Pretoria, 2008. / Paraclinical Sciences / unrestricted

Gousiekte (“quick” disease) is a plant-induced cardiomyopathy of livestock in South Africa, that is characterized by the sudden death of animals within a period of 3-6 weeks after the initial ingestion of toxic plant material. Six species of three genera of the Rubiaceae family viz. Pachystigma pygmaeum, P. thamnus, and P. latifolium; Pavetta harborii and P. schuman-niana, and Fadogia homblei have been identified as the causative agents of the disease. The toxin responsible for the poisoning, named pavettamine, has been isolated and the structure and absolute configuration established as (2S,4R,8R,10S)-1,11-diamino-6-aza-undecane-2,4,8,10-tetraol, or the enantiomer, by mass spectrometry and NMR spectroscopy. Retrosynthetic analysis of the pavettamine molecule as outlined in the dissertation showed that the secondary amine function could be obtained from the amide functional group in an intermediate such as (2R,4S)-N-[(2′R,4′S)-2,4,5-trihydroxypentan-1′-yl]-2,4,5-trihydroxy-pentanamide A. Disconnection of the amide bond then generated two C5 building blocks viz. an amine B and a carboxylic acid C which through a set of functional group transformations led to a common C5 building block, a pentane-1,2,4,5-tetraol D. The terminal primary hydroxy groups required different protecting groups at all times in order to safe-guard the integrity of the two stereogenic centres. In addition identical protecting groups but different to those used for the primary hydroxy groups, were necessary for the secondary hydroxy groups. Further analysis of the C5 building block D showed that it could be obtained from (2S)-malic acid by functional group transformations, chiral sulfoxide methodology and an appropriate protective group strategy. A suitable protective group strategy was developed and an 11 step synthetic route for the C5 building block established. The successful conversion of this moiety through functional group transformations provided the C5 amine B and C5 carboxylic acid C which were linked to give the target compound, the amide D but with the hydroxy groups protected. The synthetic study presented in the dissertation provides an efficient methodology toward the synthesis of any of the 10 possible stereoisomers of pavettamine. / Dissertation (MSc)--University of Pretoria, 2009. / Chemistry / unrestricted

The aim of this study was to investigate the mode of action of pavetamine on rat cardiomyocytes. Pavetamine is the causative agent of gousiekte (“quick-disease”), a disease of ruminants characterized by acute heart failure following ingestion of certain rubiaceous plants. Two in vitro rat cardiomyocyte models were utilized in this study, namely the rat embryonic cardiac cell line, H9c2, and primary neonatal rat cardiomyocytes. Cytotoxicity of pavetamine was evaluated in H9c2 cells using the MTT and LDH release assays. The eventual cell death of H9c2 cells was due to necrosis, with LDH release into the culture medium after exposure to pavetamine for 72 h. Pavetamine did not induce apoptosis, as the typical features of apoptosis were not observed. Electron microscopy was employed to study ultrastructural alterations caused by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormalities after 48 h exposure of the cells to pavetamine. Abundant secondary lysosomes with electron dense material were present in treated cells. Numerous vacuoles were also present in treated cells, indicative of autophagy. During this exposure time, the nuclei appeared normal, with no chromatin condensation as would be expected for apoptosis. Abnormalities in the morphology of the nuclei were only evident after 72 h exposure. The nuclei became fragmented and plasma membrane blebbing occurred. The mitochondrial membrane potential was investigated with a fluorescent probe, which demonstrated that pavetamine caused significant hyperpolarization of the mitochondrial membrane, in contrast to the depolarization caused by apoptotic inducers. Pavetamine did not cause opening of the mitochondrial permeability transition pore, because cyclosporine A, which is an inhibitor of the mitochondrial permeability transition pore, did not reduce the cytotoxicity of pavetamine significantly. Fluorescent probes were used to investigate subcellular changes induced by pavetamine in H9c2 cells. The mitochondria and sarcoplasmic reticula showed abnormal features compared to the control cells, which is consistent with the electron microscopy studies. The lysosomes of treated cells were more abundant and enlarged. The activity of cytosolic hexosaminidase was nearly three times higher in the treated cells than in the control cells, which suggested increased lysosomal membrane permeability. The activity of acid phosphatase was also increased in comparison to the control cells. In addition, the organization of the cytoskeletal F-actin of treated cells was severely affected by pavetamine. Rat neonatal cardiomyocytes were labelled with antibodies to detect the three major contractile proteins (titin, actin and myosin) and cytoskeletal proteins (F-actin, desmin and β-tubulin). Cells treated with pavetamine had degraded myosin and titin, with altered morphology of sarcomeric actin. Vacuoles appeared in the β-tubulin network, but the appearance of desmin was normal. F-actin was severely disrupted in cardiomyocytes treated with pavetamine and was degraded or even absent in treated cells. Ultrastructurally, the sarcomeres of rat neonatal cardiomyocytes exposed to pavetamine were disorganized and disengaged from the Z-lines, which can also be observed in the hearts of ruminants that have died of gousiekte. It is concluded that the pathological alteration to the major contractile and cytoskeleton proteins caused by pavetamine could explain the cardiac dysfunction that characterizes gousiekte. F-actin is involved in protein synthesis and therefore can play a role in the inhibition of protein synthesis in the myocardium of ruminants suffering from gousiekte. Apart from inhibition of protein synthesis in the heart, there is also increased degradation of cardiac proteins in an animal with gousiekte. The mitochondrial damage will lead to an energy deficiency and possibly to generation of reactive oxygen species. The sarcoplasmic reticula are involved in protein synthesis and any damage to them will affect protein synthesis, folding and post-translational modifications. This will activate the unfolded protein response (UPR) and sarcoplasmic reticula-associated protein degradation (ERAD). If the oxidizing environment of the sarcoplasmic reticula is disturbed, it will activate the ubiquitin-proteasome pathway (UPP) to clear aggregated and misfolded proteins. Lastly, the mitochondria, sarcoplasmic reticula and F-actin are involved in calcium homeostasis. Any damage to these organelles will have a profound influence on calcium flux in the heart and will further contribute to the contractile dysfunction that characterizes gousiekte. / Thesis (PhD)--University of Pretoria, 2010. / Paraclinical Sciences / unrestricted