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A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy. Johannesburg, South Africa 2017. / Almost half of all cases of heart failure have a preserved ejection fraction. However, therapy targeting the mechanisms of this disorder has not improved outcomes. Left ventricular (LV) diastolic dysfunction is a characteristic feature of heart failure with a preserved ejection fraction. A more sound understanding of the mechanisms responsible for LV diastolic dysfunction produced by risk factors may lead to better approaches to preventing this syndrome. Although obesity is thought to be a major risk factor for LV diastolic dysfunction, this does not occur in all obese individuals. In the present thesis I have demonstrated in 737 randomly recruited participants from a community sample of African ancestry, that the relationship between insulin resistance (homeostasis model) and LV diastolic function, as assessed from trans-mitral velocity (E/A) and tissue Doppler imaging of the lateral and septal walls of the LV (e’ and E/e’), is markedly altered by the presence of a more concentrically remodelled LV (as indexed by LV relative wall thickness [RWT]). Importantly, insulin resistance was only associated with LV diastolic function or dysfunction in those with an RWT above a threshold value. In contrast no interactive effects on LV diastolic function between either blood pressure or age and RWT were noted. These data therefore suggest that obesity will only translate into LV diastolic dysfunction if it is associated with insulin resistance and a concentrically remodeled LV. Although hypertension is thought to play an important role in contributing to LV diastolic dysfunction, the pulsatile hemodynamic change primarily responsible for this effect is uncertain. In 524 randomly selected individuals from a community sample I have demonstrated that independent of confounders including left ventricular mass and RWT, aortic backward wave pressure effects (as determined using wave separation analysis), antedate the impact of aortic stiffness (indexed by aortic pulse wave velocity) or the factors determined by aortic stiffness (the time of backward wave return or forward wave pressures) on LV filling pressures (E/e’). These data therefore suggest that to adequately prevent LV diastolic dysfunction, targeting aortic backward wave pressures may be required. As conventional risk factors account for only a portion of the inter-individual variations in LV diastolic function, it is thought that the genetic factors may play a iv significant role. In 694 randomly recruited participants of African ancestry belonging to nuclear families, I demonstrated that independent of conventional risk factors, heritability accounts for approximately 50% of the variation in LV RWT, an important LV structural determinant of LV diastolic function. Moreover, in 442 randomly recruited individuals of African ancestry belonging to nuclear families, I also demonstrated that heritability accounts for approximately 50% of the variation in the index of LV filling pressures, E/e’, independent of LV mass or RWT remodeling and aortic function. These data provide strong evidence that genetic factors responsible for LV diastolic dysfunction and the structural determinants thereof should be sought. In conclusion, the results provided in the present thesis have advanced our knowledge of possible pathophysiological mechanisms that play a role in the development of LV diastolic dysfunction and hence possibly heart failure with a preserved ejection fraction. / MT2017

Hypertrophic cardiomyopathy (HCM) is a heterogeneous, often familial disease, characterized by cardiac hypertrophy, predominantly affecting the interventricular septum. To date, no study has systematically analysed the genetic and phenotypic aspects of the disease in a Swedish population. The aim of this thesis was to identify the genotypes causing HCM in northern Sweden, to characterize the disease phenotypes and correlate these findings. Forty-six patients were recruited for the genetic studies (21 women), 11 familial and 35 sporadic cases. Eight sarcomeric protein genes were screened for mutations. A total of 11 different disease causing mutations were found in four genes. Six of the mutations were previously not described. A novel mutation (a 33 base pair deletion) in the troponin I gene was found in one HCM family. Despite the severe genetic defect, the associated phenotype displayed only mild cardiac hypertrophy and few symptoms. Most mutations (64%) were identified in the myosin binding protein C gene, a gene considered to have a low penetrance. Mutations were identified in 10 of 11 familial HCM cases, but only in three of the 35 sporadic cases. It was found that cardiac amyloidosis can sometimes present itself as HCM. Three HCM patients (7%) carried the ATTR Val30Met mutation, also found in Swedish patients with familial amyloid polyneuropathy (FAP). The patients had no symptoms of polyneuropathy, but cardiac amyloidosis as the cause of hypertrophy was verified by myocardial biopsy in an index case. Amyloid heart disease should therefore be considered as a differential diagnosis in patients with HCM. By studying heart rate variability (HRV), it was found that young patients with HCM had signs of autonomic dysfunction, expressed as a reduced HRV. Treatment with beta-blockade attenuated these effects. Abnormal autonomic function might be a substrate for lethal arrhythmias, most often encountered in younger patients with HCM. The results suggest a possible protective effect of beta-blockade, remaining to be studied further. Ventricular function is frequently abnormal in HCM. In particular, diastolic dysfunction has been demonstrated. The recently described myocardial performance index allows the assessment of cardiac function by combining systolic and diastolic performance. We found that patients with hypertrophic cardiomyopathy had evidence of global and regional right ventricular dysfunction, besides left ventricular dysfunction. Hypertrophic cardiomyopathy is traditionally considered to be a disease of the left ventricle. The results show that hypertrophic cardiomyopathy should more be regarded as a biventricular disease. In conclusion, the myosin binding protein C gene is the most common gene causing familial HCM in northern Sweden. This disease gene is considered to be associated with a mild, late-onset disease with ≈50% penetrance at 30 years of age. The low disease penetrance emphasizes the importance of adequate family screening when evaluating patients with HCM, since the familial nature of the disease might easily be overlooked. These particular disease features in northern Sweden contrast to most previous reports, which indicate another disease gene as the most frequent in HCM, associated with a much higher penetrance. Amyloid heart disease, requiring different treatment than HCM, should be kept in mind as a differential diagnosis in the management of patients with HCM. Key words: Hypertrophic cardiomyopathy, genetics, autonomic nervous system, familial amyloid polyneuropathy, echocardiography.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成6年3月25日 服部宇氏の博士論文として提出された

Congestive heart failure represents one of the leading causes of death in industrialized countries. A subset of heart failure situations are linked to genetic mutations, many of which reside in sarcomeric proteins. With the prevalence of mutations as approximately 1:500, the location of a mutation plays a direct role in the severity and lethality of the resulting disease state. The point mutation Arg403Glu (R403Q) located on the myosin heavy chain molecule is no exception with a resulting development of left ventricular hypertrophy, myocyte disarray, and increased cellular fibrosis. Given the severity of this mutation in humans, a mouse model was developed to recapitulate these phenotypic characteristics. An additional confounding factor often overlooked when studying the effects of cardiac disease is the role of biological sex. We, therefore, tested the hypothesis that R403Q mice will display altered crossbridge kinetics, specifically an increase in functioning at the expense of energy efficiency, and that biological sex will impact the cardiac response to the R403Q mutation in terms of both crossbridge functioning and post-translational modifications. To do this, we used both male and female R403Q transgenic mice and quantified myofilament mechanical properties including Ca²⁺ sensitivity, crossbridge cycling kinetics, and tension cost of contraction. In addition, phosphorylation patterning was quantified for one of the central, sarcomeric regulatory proteins, cardiac troponin I. We used 2 month animals which do not display overt pathology in the form of hypertrophy in order to minimize downstream, confounding effects. We were able to find that disease and biological sex played an important role in defining these parameters and suggests females are able to better compensate to the presence of altered sarcomeric interactions. Future investigations should focus on altered kinase activity, possibly driven by interactions with estrogen, in order to better define how females can retain cardiac functioning given a disease condition.

Introduction Keloids are formed by the excessive production of scar tissue, which extends beyond the margins of the original injury, often resulting in lesions of grotesque dimensions. Keloids present a major dilemma to surgeons because of the high recurrence rate with recurrent growth often larger than the original keloid. The high recurrence rate and the poor response of keloids to therapy present a great challenge to surgeons. The numerous therapeutic regimens demonstrate that to date there is no single therapy that is absolutely successful. Therefore, it is necessary to comprehensively establish the pathology of keloids and to determine the aetiopathogenesis of the lesion in order to eventually provide unfailing specific effective treatment and to better understand the mechanisms regulating fibrosis in various fibroproliferative diseases. Aim To evaluate the pathology and aetiopathogenesis of keloid formation. Methods The research protocol for the study was approved by the Nelson R Mandela Faculty of Medicine Ethics Committee. Informed consent was obtained before the biopsies were taken. Keloid and non-lesional skin biopsies were obtained from thirty two patients who had multiple lesions in various locations, bringing the total number of keloids and apparently normal skin biopsies processed and examined to fifty eight. The biopsied specimens were processed for paraffin wax embedment and routine haematoxylin and eosin, differential and immunocytochemical staining. Sections were scrupulously examined using the Olympus BH-2 microscope; features pertinent to the study were photographed with the Olympus DP 10 microscope digital camera system. The stored images were studied, using the Camedia graphics processing programme. Results The results of the study showed that keloids comprise many distinct regions categorized as: the zone of hyalinising collagen bundles, fine fibrous areas, areas of inflammation, zone of dense regular connective tissue, nodular fibrous area and area of angiogenesis. Fibroblastic phenotypes present ranged from spindle, fibrohistiocytic, epitheloid, elongated flattened condensed fibroblastic cells to few wavy, fuzzy, polygonal and atrophic cell types. Immunocytochemically these cells were vimentin-positive and actin- and desmin-negative. Few myofibroblastic phenotypes were also identified and these were vimentin- and alpha smooth muscle actin-positive and desmin-negative. The fibroblastic and myofibroblastic phenotypes were in proliferative or degenerative stages and pathological features exhibited were the presence of vesicular, degenerate or calcified nuclei; nuclear and plasma membrane damage; cytoplasmic and nucleoplasmic clearing; atrophy, pyknosis and swelling. Severe, moderate to mild paravascular inflammation was observed around the microvessels of the sub-papillary plexus and within the keloid. There was compression and occlusion of small blood vessels, coagulation necrosis and dissolution of mural cells of small blood vessels and small peripheral nerves. Also present in keloids were oedematous areas, disorganised and hyalinised connective tissue fibres and increased numbers of degranulated and degranulating mast cells. Elastic fibres in keloids were minimal or absent whereas at the border of keloids there was an increase.Discussion Degenerate, occluded and compressed microvessels were a widespread pathological feature in keloids. This resulted in impaired vascular supply to each of the keloid regions which impacted directly on the pathology of keloids where degeneration and necrosis, manifesting the lack of nutrients and oxygen to tissue, were found throughout the keloid. The vascular supply was impaired because of the chronic inflammatory destruction of the microvessels and the elevated stress within keloids. Factors contributing to increased intrinsic stress were: 1) the lack of elastic fibres in keloids which decreased the elastic limit, leading to effects of excessive deformational force which were compression and stiffening of tissue; 2) the high tension skin covering keloid prone areas had low stretch and a low elastic modulus; 3). protruding hard connective tissue such as bony prominences or cartilage into the dermis of keloid prone skin; 4) contractile forces exerted by wound healing fibroblastic cells; and 5) external forces. Compression and occlusion of blood vessels induced ischaemic and reperfusion tissue injury. During the reperfusion phase blood rich in growth factors returned to tissue stimulating tissue growth. Tissue growth was also promoted by elevated internal stress which stimulated increasing levels of gene expression, collagen synthesis and mitotic activity. All these growth promoting effects resulted in keloid formation. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2013.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 5 uppsatser.

Background-In cardiomyocytes from patients with hypertrophic cardiomyopathy, mechanical dysfunction and arrhythmogenicity are caused by mutation-driven changes in myofilament function combined with excitation-contraction (E-C) coupling abnormalities related to adverse remodeling. Whether myofilament or E-C coupling alterations are more relevant in disease development is unknown. Here, we aim to investigate whether the relative roles of myofilament dysfunction and E-C coupling remodeling in determining the hypertrophic cardiomyopathy phenotype are mutation specific. Methods and Results-Two hypertrophic cardiomyopathy mouse models carrying the R92Q and the E163R TNNT2 mutations were investigated. Echocardiography showed left ventricular hypertrophy, enhanced contractility, and diastolic dysfunction in both models; however, these phenotypes were more pronounced in the R92Q mice. Both E163R and R92Q trabeculae showed prolonged twitch relaxation and increased occurrence of premature beats. In E163R ventricular myofibrils or skinned trabeculae, relaxation following Ca2+ removal was prolonged; resting tension and resting ATPase were higher; and isometric ATPase at maximal Ca2+ activation, the energy cost of tension generation, and myofilament Ca2+ sensitivity were increased compared with that in wildtype mice. No sarcomeric changes were observed in R92Q versus wild-type mice, except for a large increase in myofilament Ca2+ sensitivity. In R92Q myocardium, we found a blunted response to inotropic interventions, slower decay of Ca2+ transients, reduced SERCA function, and increased Ca2+/calmodulin kinase II activity. Contrarily, secondary alterations of E-C coupling and signaling were minimal in E163R myocardium. Conclusions-In E163R models, mutation-driven myofilament abnormalities directly cause myocardial dysfunction. In R92Q, diastolic dysfunction and arrhythmogenicity are mediated by profound cardiomyocytesignaling and E-C coupling changes. Similar hypertrophic cardiomyopathy phenotypes can be generated through different pathways, implying different strategies for a precision medicine approach to treatment.

Au cours de l'arthrose (OA), une différenciation hypertrophique des chondrocytes, une minéralisation du cartilage, une angiogenèse ostéochondrale et un remodelage de l'os sous-chondral sont observés dans l'articulation. L'angiogenèse ostéochondrale pourrait être impliquée dans l'OA mais les mécanismes moléculaires qui la gouvernent restent inconnus. Nous supposons qu'au cours de l'OA, la différenciation hypertrophique des chondrocytes jouerait un rôle clé dans le remodelage de la jonction ostéochondrale et notamment dans l'angiogenèse à travers un déséquilibre entre la production de facteurs angiogéniques et angiostatiques. Un lien entre la différenciation hypertrophique, la vascularisation ostéochondrale et la progression de l'OA a été confirmé dans des cartilages humains. Le potentiel angiogénique des chondrocytes hypertrophiques a été étudié dans un modèle de différenciation hypertrophique de chondrocytes articulaires murins en culture primaire. Les chondrocytes articulaires expriment les marqueurs chondrocytaires (Sox9, Acan, Col2a1), tandis que l'expression des marqueurs de l'hypertrophie (Runx2, OC, Osx) augmente avec la différenciation hypertrophique. Les chondrocytes hypertrophiques sont capables de minéraliser leur matrice. L'expression/production de facteurs angiogéniques (VEGF, bFGF¿) augmentent avec la différenciation hypertrophique alors que celles des facteurs angiostatiques (TSP-1...) diminuent. Une analyse microarray a été réalisée afin d'identifier des cibles innovantes. La différenciation hypertrophique des chondrocytes pourrait participer aux mécanismes physiopathologiques de l'OA en favorisant la vascularisation et la dégradation du cartilage articulaire / Osteochondral angiogenesis is an important step in the remodeling of the cartilage/subchondral bone junction in osteoarthritis (OA). Cellular and molecular stimuli of this angiogenesis are largely unknown. We hypothesize that osteochondral angiogenesis in OA is controlled by hypertrophic chondrocyte differentiation, as it occurs during development and growth (endochondral ossification process). Chondrocyte hypertrophy is detected by osteocalcin immunostaining in human OA knee tissues. OA is evaluated by modified Mankin score and osteochondral angiogenesis by the vascular channels number reaching the articular cartilage. An original model of hypertrophic differentiation of mouse articular chondrocytes in primary culture has been developed in order to study the angiogenic potential of hypertrophic chondrocytes compared to articular ones. Hypertrophic chondrocytes and osteochondral angiogenesis are positively correlated and linked to OA progression. The expression of chondrocyte markers (Sox9, Acan, Col2a1) decreases with hypertrophic differentiation in vitro, whereas Runx2, Osteocalcin and Osterix mRNAs levels significantly increase. Hypertrophic chondrocytes are characterized by strong matrix calcifications. Hypertrophic differentiation stimulates the angiogenic factors expression (VEGF, bFGF…) whereas angiostatic factors (TSP-1, chondromodulin 1…) undergo a decreased expression level. A microarray analysis has been realized in order to identify innovative molecular targets. These results suggest a key role of chondrocyte hypertrophy in osteochondral angiogenesis and thus in the remodeling of the cartilage/subchondral bone junction in OA, leading to cartilage degradation.

Introduction: Hypertrophic cardiomyopathy (HCM) is a heterogeneous disorder of the cardiac sarcomere, resulting in myocyte hypertrophy and disarray, interstitial fibrosis, and cardiac dysfunction. Our aim was to determine whether the amount of fibrosis in HCM correlates with echocardiographic measures of diastolic dysfunction, presence of HCM-susceptibility mutations, or polymorphisms in the renin-angiotensin-aldosterone system (RAAS). Methods: Surgical specimens from patients with obstructive HCM undergoing septal myectomy at the Mayo Clinic (2001-2004) were examined and compared with autopsy-derived tissues from age- and sex-matched normal controls. Digital image analysis was used to quantitate the fibrosis in representative microscopic sections. Genotyping was performed for myofilament-HCM using polymerase chain reaction, high-performance liquid chromatography, and direct DNA sequencing. RAAS polymorphism status was similarly established. Results: The study included 59 HCM cases and 44 controls. Patients with HCM exhibited more fibrosis (mean 17%, range 3-45%) than controls (mean 8%, range 3-17%) (P<.0001). A significant relationship existed between amount of fibrosis and maximum wall thickness (P=.02), left ventricular ejection fraction (P=.02), and peak early/late diastolic mitral annulus velocity (E/A ratio) (P=.002). Although there was no association between amount of fibrosis and myofilament-HCM genotype status or polymorphisms in the RAAS cascade, there was a trend toward more fibrosis in patients with ≥1 C-encoding allele in CYP11B2-encoded aldosterone synthase. Conclusions: Patients with HCM undergoing septal myectomy had significantly more myocardial interstitial fibrosis than controls. The amount of fibrosis in HCM patients correlated with degree of septal hypertrophy and left ventricular systolic and diastolic function. Notably, neither mutations in cardiac myofilament proteins or polymorphisms in RAAS exhibited strong associations with severity of myocardial fibrosis.