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Mutations in genes encoding sarcomeric proteins are the most common cause of inherited cardiomyopathies. However, cardiac disease caused by mutations in non-sarcomeric proteins exhibit remarkably similar phenotypes, suggesting that common modes of pathogenesis might exist. It is of particular interest whether non-sarcomeric mutations result in impaired contractile function akin to sarcomeric diseases. Accordingly, this thesis describes the effect of cardiomyopathy-causing mutations to an energy sensing protein (AMPK γ2) and a small heat shock protein (αB-crystallin) on cardiac myofilament biomechanics and Ca²⁺ handling. Mutations in the regulatory γ2 subunit of AMP-activated kinase (AMPK), encoded by the PRKAG2 gene, cause a cardiomyopathy characterized by ventricular hypertrophy, electrophysiological abnormalities, and glycogen accumulation. Data from animal models in which the mutant transgene is overexpressed suggest that the electrophysiological aspects might be caused by excess glycogen, but that this is insufficient to account for all facets of the phenotype. A novel knock-in mouse expressing R299Q AMPK γ2 (homologous to the human R302Q mutation) was generated to model PRKAG2 cardiomyopathy more accurately. Assessment of the cardiac phenotype at two months to determine the early events in disease pathogenesis revealed systolic and diastolic dysfunction in mutant animals, with no excess glycogen detected. Analysis of acetyl-CoA carboxylase phosphorylation at S79 suggested increased basal AMPK activity in mutant animals. Increased sarcomeric Ca^2+ sensitivity of contractile activation was observed in demembranated cardiac trabeculae from mutant animals, associated with decreased phosphorylation of cardiac troponin I at S23/S24 and increased phosphorylation at S150. Treatment with protein kinase A normalized this difference in Ca^2+ sensitivity, suggesting that reduced PKA phosphorylation is the primary abnormality in mutant tissue. Cardiomyocytes from mutant animals exhibited slowed contractile and Ca^2+ reuptake rates, associated with reduced phosphorylation of phospholamban and myosin binding protein C, as well as an increased response to isoproterenol. The lack of glycogen accumulation in these animals, combined with abnormal contractile and Ca²⁺ handling properties, reveals a more nuanced interpretation of the mechanism of PRKAG2 cardiomyopathy development. Further, the newly identified interaction between energy- (AMPK) and stress- (PKA) signalling networks may be of importance in numerous additional disease pathways. This thesis clarifies the role of αB-crystallin, and its cardiomyopathy-causing mutant (R157H), in regulating cardiac muscle stiffness. Specifically, mass spectrometry data revealed that αB-crystallin forms large oligomers and binds to titin Ig domains. Nuclear magnetic resonance confirmed this binding and suggested that αB-crystallin's C-terminal is responsible for titin binding. A viscoelastic model was developed to match stress relaxation in cardiac muscle fibres. Measurements using this model indicated that αB-crystallin significantly increases myocardial stiffness and that the R157H mutation weakens this effect. Further, a 9-AA peptide from αB-crystallin's C-terminus was shown to bind titin and increase overall muscle stiffness. These results reveal a novel method of cardiac muscle stiffness regulation that might contribute to the pathogenesis of cardiomyopathy.

Retinal imaging with a fundus camera or scanning laser ophthalmoscope (SLO) allows a fast, non-invasive view to the body’s microvasculature. Evidence suggests that features associated with retinal blood vessels (for example, narrowing of arteries and increased vascular tortuosity) measured near to the optic disc are early biomarkers of disease such as diabetes, hypertension (HT) and cardiovascular disease. Ultra-widefield (UWF) ophthalmic imaging allows unique views of peripheral locations as well as the posterior pole, potentially facilitating a more comprehensive study of the state or health of the microvasculature than is afforded by conventional retinal imaging. It is envisaged that this will reveal new candidate biomarkers derivable from the retina which could help identify the presence of disease or improve risk stratification for serious illness. In this thesis, 532 individuals were recruited from a trial evaluating the added benefit of using computed tomography (CT) imaging in the diagnosis of coronary heart disease (CHD) to measure atherosclerotic plaque in the arteries of the heart muscle tissue. The trial participants were deeply phenotyped which allowed access to additional information including: presence and severity of CHD, hypertensive status, presence of diabetes, age, gender, and smoking status (all risk factors commonly associated with CHD). After CT imaging patients were invited for undilated UWF ophthalmic imaging using an Optos P200C SLO. To accurately measure features of blood vessels indicative of microvascular health or disease in these images required the development of a novel semi-automatic computerised technique to segment and analyse the retinal vasculature. This involved implementation of a supervised vessel segmentation algorithm utilising multi-scale matched filters, a neural network classifier and hysteresis thresholding. A true positive rate (TPR) of 0.702 (and standard deviation of 0.059), false positive rate (FPR) of 0.011 (0.006) and accuracy (Acc) of 0.965 (0.006) was achieved by the algorithm. This compared to TPR of 0.674 (0.062), FPR of 0.017 (0.004), and Acc of 0.957 (0.006) for state-of-the-art fundus camera vessel segmentation applied to UWF SLO. After segmentation and prior to the measurements of retinal vessel parameters, the distortions introduced by the instrument mapping the 3D retinal surface onto a 2D image plane were also accounted for utilising an established technique. This is especially important for measuring in the periphery of UWF images and has not previously been reported for biomarker discovery. Measurements from UWF SLO were compared between those participants with CHD (where a reduction in arterial width was hypothesised based on existing research with fundus cameras into cardiovascular disease), and those without to investigate whether a difference between the two groups existed. After appropriate statistical correction for confounding variables (i.e. age, gender, and hypertensive status) the results did not show a statistically significant difference for presence of CHD or for risk stratification. However, the analysis techniques that were developed in this thesis do allow a rapid investigation of retinal vascular parameters in UWF SLO. This has application to a number of other diseases, such as HT, where a more pronounced change to the appearance of vessels is anticipated, and to different areas of the peripheral retina not previously measurable with standard imaging techniques and existing algorithms.

Apolipoprotein C-1 (apo C-1) enriched HDL has been described as an atherogenic form of HDL associated with an increased risk for cardiovascular disease (CVD). The objective of the present study was to develop a rapid method for the separation, purification, and characterization of Apo C-1 from serum. We isolated and characterize HDL subclasses from individuals with and without angiographically-proven CVD who have elevated and normal-to-low HDL-C levels. Ultracentrifugation was linked with immunoaffinity separations for the specific separation of Apo C-1 enriched HDL from other lipoproteins. A 50 μL sample of serum is diluted in TRIS HCl buffer (pH 7.5) and incubated with CNBr-activated Sepharose (Amersham) containing antibodies to apo C-1 (Academy Bio-medical Company). The apo C-1-depleted serum is removed by centrifugation and all apo C-1-containing lipoproteins are released from the Sepharose beads at pH 2. The apo C-1-depleted sample and the apo C-1-containing sample were ultracentrifuged to obtain a lipoprotein density profile in the absence and presence of apo C-1. Density Lipoprotein Profiling (DLP) gives relevant information of lipoproteins, such as density and subclass characterization, and is a novel approach to purify apo C-1-enriched HDL. An additional advantage of this approach is that lipoprotein-a (Lp(a)), which is often an interfering component in the HDL density region, is eliminated. Results show feasibility that these methods could be used in a clinical setting, was achieved. This measurement may yield a precise and quantitative profile of the distribution of apo C-1 for all lipoprotein particles including HDL.

Simvastatin is one of the statins, which are a class of drugs originally developed to fight cardiovascular disease by lowering cholesterol. However, it is now clear that they have effects independent of cholesterol. For example, statin therapy, like exercise, induces adaptations within the heart that protect it against I/R injury. Patients are frequently advised to undergo a combination treatment of statins and chronic exercise, although little is known about how this combination treatment affects cardioprotective adaptations. Both treatments appear to exert their cardioprotective effects through different mechanisms, therefore it appears plausible that combining the two treatments would provide added cardioprotection than either treatment alone. Purpose: To investigate the effects of a combination treatment of statins and exercise upon parameters of post-ischemic myocardial function and damage. Methods: Female Sprague-Dawley rats (6 months of age) were separated into 4 groups for a period of 4 weeks: Sedentary (S, n=10), sedentary plus 10 mg simvastatin (Zocor®)/kg body wt/ day (SD, n=9), exercise (R, n=9), and exercise plus simvastatin (RD, n=9). R and RD were exercised identically on a treadmill for 5 days/week at an intensity of about 70% VO2max and for a duration that was gradually increased to 60 min/day. Twenty-four hrs following the last exercise bout, isolated perfused working hearts were subjected to 30 min of global ischemia followed by 30 min of normoxic reperfusion. Coronary effluents were used to determine lactate dehydrogenase (LDH) leakage and prostaglandin generation. Results: Cardiac function was similar in all groups prior to ischemia. Post I/R recovery of cardiac function in S was 17.6 [greater than or equal to]6.6% of pre-ischemic cardiac output times systolic pressure. Recovery was significantly higher in SD (37.7[greater than or equal to]7.7%) and R (40.1[greater than or equal to]7.8%) and tended to be highest in RD (49.7[greater than or equal to]7.1%). SD had significantly higher pre-ischemic coronary flow per g heart weight (CF/g) than all other groups. At 10 min post-ischemia, simvastatin treatment significantly increased CF/g compared to S (p<0.05). Exercise had an effect on increasing post-ischemic myocardial efficiency and RD had significantly higher post-ischemic myocardial efficiency vs. S (p<0.05). Compared to S, LDH leakage during reperfusion was dramatically decreased in SD, R and RD by approximately similar amounts. Simvastatin treatment doubled the basal production of protective prostaglandins, whereas exercise did not significantly alter their production and combining both treatments yielded a lower prostaglandin release than simvastatin treatment. SD had s significantly higher basal prostaglandin release than R (p<0.05). Conclusion: Although the combination treatment of simvastatin and exercise did not result in a statistically significant addition in cardioprotection compared to either treatment alone, there was a trend for improved parameters of post-ischemic cardiac function and damage upon combining both treatments compared to each treatment alone. Specifically, the prostaglandin, CF/g and efficiency data suggest that exercise and statininduced cardioprotection against I/R injury appears to occur by different mechanisms and combining the two treatments may provide greater protection than either alone.