Ischaemia-reperfusion injury, sustained when the blood and oxygen supply is removed and then reinstated, is a common cause of cardiac tissue damage and therefore a popular area of scientific research. It is commonly thought that the mitochondria play an important role in mediating ischaemia-reperfusion injury. Particular attention has been focused on the mitochondrial permeability transition pore. This has many times been shown to open only during the reperfusion phase disrupting the mitochondrial transmembrane potential and therefore the synthesis of ATP. This study endeavours to examine the activity of the pore in rat neonatal primary cardiomyocytes during cell death. Models of apoptotic and necrotic cell death are established, as both have been implicated in ischaemia-reperfusion injury. An in vitro model of ischaemia- reperfusion injury is set up and an in vivo model of ischaemia-reperfusion injury is also examined. Many studies have shown that inhibition of cyclophilin D can successfully block the opening of the mitochondrial pore. In this study, the cyclophilin inhibitor cyclosporin A is applied to determine the importance of cyclophilin D as an attenuator of ischaemia-reperfusion injury. The resulting effect on cell viability is analysed by various assays. Changes in the activity of the mitochondrial permeability transition pore are also monitored using a fluorescent mitochondrial transmembrane potential-sensitive dye. A kinetic assay is established to measure the peptidyl prolyl cis-trans-isomerase activity of cyclophilin D, which is believed to have a role in regulation of the pore. Additionally ATP synthesis is measured by a luciferase-based assay and free radical production is monitored using a radical-sensitive dye during cell death. The effect of cyclosporin A on all measured parameters is examined. The results presented in this study show that the importance of the mitochondrial permeability transition pore can vary widely according to the prevailing conditions. However, the data obtained in both isolated cardiomyocytes and cardiac tissue broadly uphold the concept of the major impact of mitochondrial permeability transition pore induction during ischaemia-reperfusion injury and strongly support the hypothesis of a significant role for cyclophilin D in the molecular mechanism of the pore.

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Most familial dilated and hypertrophic cardiomyopathies are caused by mutations in sarcomeric proteins. What remains to be answered is how mutations cause the cardiac phenotype and why mutations in the same protein can cause cardiomyopathies at different ends of the phenotypic spectrum. We collected human myocardium from HCM patients undergoing surgical myectomy, and familial and acquired DCM patients, undergoing cardiac transplantation. Patients were phenotyped before operation and blood taken for genotyping. We used the ProQ Diamond, Western Blot and in vitro motility assays to assess levels of phosphorylation, expression and function respectively of sarcomeric proteins. In HCM patients with myosin binding protein C mutations expression of MyBPC was reduced by 24% in myofilaments arguing strongly for happloinsufficiency as the disease causing mechanism. HCM myosin and MyBPC mutations, also affect cardiac contractility by dominant negative effects. Myosin mutation, Va1606Met, has direct and indirect effects on cardiac contraction. Filament sliding speed was greater and relaxation at pCa9 less complete than with other HCM myosins tested without mutations. The DCM troponin C mutation, Gly159Asp, acts as a poison polypeptide, changing thin filament regulation. Ca2+ -sensitivity of G159D troponin C was independent of the level of troponin phosphorylation. The uncoupling of the relationship between troponin phosphorylation and Ca2+-sensitivity, provides a novel mechanism for initiation of familial DCM. Post-translational modifications in sarcomeric proteins occur independently of genotype. Troponin I is dephosphorylated in DCM and HCM, leading to changes in Ca2+-sensitivity and cross-bridge turnover rate. Also both troponin and myosin from HCM tissue is functionally abnormal and MyBPC phosphorylation is reduced. In acquired heart failure dephosphorylation of Serines 23/24 on troponin I could account for the contractile defect in seen and MyBPC phosphorylation is also decreased. In summary we observed a hypocontractile molecular phenotype in HCM human heart tissue, similar to that seen in heart failure. These findings conflict with the observed clinical phenotype seen in HCM, which is often regarded as a hyperdynamic or hypercontractile state.

The contractility of human heart samples from patients diagnosed with hypertrophic cardiomyopathy were studied using a quantitative in vitro motility assay. The aim of this work was to investigate the molecular phenotype of thin filament proteins in the HCM heart. Three biopsy samples with thin filament mutations were studied alongside samples acquired from a subset of HCM patients classified with hypertrophic obstructive cardiomyopathy. The primary effect of thin filament mutations was investigated by reconstituting Factin with ACTC E99K into thin filament with donor troponin. The E99K actin filaments had a higher Ca2+-sensitivity then filaments composed of donor F-actin (with no mutation) (EC50 E99K/donor 0.78±0.14, p=0.02). A similar higher Ca2+- sensitivity was found when recombinant TnT K273N was incorporated into donor troponin and compared to native donor troponin (EC50 K273N/donor 0.54±0.17, p=0.006). Troponin was also purified from HOCM heart samples. This troponin did not contain a causative mutation but behaved abnormally in the response of thin filament Ca2+- sensitivity to changes in TnI phosphorylation (EC50 PKA-HOCM/HOCM 1.08±0.25, p=0.3) as mean TnI phosphorylation of PKA-HOCM was 1.56 molsPi/molsTnI and HOCM was 0.29 molsPi/molsTnI. Thus, thin filament Ca2+-sensitivity was uncoupled from TnI phosphorylation in thin filaments with HOCM troponin. When the native TnT subunits were replaced with recombinant TnT this coupling was restored (EC50 HOCM rTnT/HOCM 0.63±0.26, p=0.03). It would appear that the result of HCM-causing mutations are two-fold. The primary effect of the HCM-causing mutations is to increase thin filament Ca2+-sensitivity. However, the contraction machinery appears to be the target of secondary modifications, that occur due to the pathology of the disease. Resulting in further changes, such as changes in protein composition and post-translational modification. One major consequence of these modifications may be to uncouple the relatively labile regulation of thin filament Ca2+-sensitivity by TnI phosphorylation.

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