An Investigation into the Role of Collagen VI in the Myocardium

Abstract

The role of collagen VI in the myocardium is still largely unknown. Mostly being linked to muscular dystrophies such as Bethlem muscular dystrophy and Ullrich congenital muscular dystrophy, a lack of collagen VI deposition leads to severe matrix deficiencies in skeletal muscle and causes overall degradation of muscle fibres. Research within the myocardium in human heart failure has shown an increase in deposition of collagen VI along remodelled t-tubules which are structurally essential for calcium-induced calcium release. Furthermore, recent research from the Crossman lab demonstrates a reduced ejection fraction in the hearts of collagen VI alpha 1 (Col6α1) knockout rats. Moreover, isolated cardiac myocytes from the knockouts demonstrate increased calcium transients and a propensity for arrhythmia. Loss of ejection fraction and disturbed calcium handling are prominent features of heart failure. This led to the proposition that collagen VI may be a target for heart failure therapies, however the lack of knowledge of how this molecule functions in the myocardium motivated this thesis. It was hypothesized the absence of collagen would lead to reduced force production of the myocardium as indicated by reduced ejection fraction in knock outs and that this was caused by either a loss of mechanical linkage to the extracellular matrix and/or disturbed calcium handling. Moreover, these hypothesized changes likely result from a disconnection between the extracellular matrix and the dystrophin-glycoprotein complex that is essential for normal muscle function. Free running trabeculae were dissected from the hearts of male collagen VI knockout rats and wild type rats. These trabeculae were then superfused in an oxygenated buffer and attached to a force transducer to measure stress. Trabeculae were loaded with Fura-2 AM indicator to measure calcium dynamics. Stress and calcium dynamics were measured at four stimulation frequencies (0.2Hz, 2Hz, 2.6Hz and 5.6Hz), in five extracellular calcium concentrations (1mM, 1.5mM, 2mM, 2.5mM and 3mM) and response to beta-adrenergic stimulation by addition of isoproterenol. The aim of these experiments was to determine if there were any differences in force production and/or intracellular calcium content of knockout trabeculae in comparison to wild type trabeculae. Furthermore, super-resolution microscopy was used to determine if the protein biglycan can form a link between collagen VI and the dystrophin-glycoprotein complex in cardiac myocytes. Knockout trabeculae displayed a statistically significant biphasic change in active stress in response to stimulation frequency. A similar but non-significant pattern was observed in wild type trabeculae likely due to a low sample number. Knockout trabeculae showed statistically significant increases of active stress in response to increasing extracellular calcium and a similar but non-significant change was observed in wild type trabeculae. Trabeculae from both groups demonstrated increases in active stress in response to isoproterenol, although not significant likely due to large variance. Unexpectedly, there was no increase in calcium transient in parallel with increases in active stress. Although it is possible that non-calcium dependant mechanisms could be involved, this does not correlate with published data. Subsequently it was determined that the sensitivity of the calcium detection in the microscope used was poor compared to published studies and likely impacted the ability to detect changes. Active stress was increased up to a third in the knockouts but was not statistically significant. However, variance in both active stress and intracellular calcium in knockout trabeculae resulted in the study being statistically underpowered. An increased variance in active stress within the myocardium could lead to dyssynchronous contraction and explain the reduced ejection fraction in the knockout heart. Supporting this dyssynchronous hypothesis, two of the six knockout trabeculae demonstrated spontaneous arrhythmic activity compared to none of the wild type trabeculae. Interestingly, there was a statistically significant decrease in passive stress suggesting that collagen VI contributes to the stiffness of the myocardium. This suggests a possible mechanism of desynchrony contraction driven by calcium overload. The absence of collagen VI would likely increase myocyte stretch and increased calcium storage via stretch activated channels. Preliminary super-resolution imaging demonstrated biglycan was located at the myocyte surface and t-tubules and was also within molecular bind distance of collagen VI in the wild type rat heart supporting the hypothesis that collagen VI forms a mechanical connection between the myocyte and the extracellular matrix. The findings of the thesis provide some support for the hypothesis that there is disorganisation in the knockout cardiomyocytes resulting in varied active stress and calcium dynamics. Moreover, similar changes in muscle function, stress, calcium and arrhythmias have been found in animal studies on Duchenne muscular dystrophy that have mutations in dystrophin. However, due to low statistical power and technical issues with the experiments, further research on collagen VI in the myocardium is needed in order to validate this hypothesis.