Fibrosis and calcium signalling in the failing heart revelations from super-resolution microscopy

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Poster Session One
Dr David Crossman (1)
1. The University of Auckland
Abstract text

Fibrosis and aberrant Ca2+ handling are hallmarks of heart failure (HF) but are generally considered separate features. However, our work has directly implicated fibrosis in the remodelling of the transverse(t)-tubule system. T-tubules are nanoscale invaginations of the plasma membrane that facilitate a rapid and synchronous contraction. Disruption of t-tubule structure can negatively impact Ca2+ signalling in HF. Using proteomics and super-resolution microscopy we identified increased amount of collagen VI within the t-tubules in the failing human heart. The importance of Collagen VI is to healthy muscle is evident by its mutation causing muscular dystrophy. Recently, circulating levels of ColVI were found to independently associate with poor outcomes in HF patients However, its role in cardiac contraction remains poorly understood. To understand the role of collagen VI in the heart we created a knockout of this gene in the rat (Col6a1-/-). 

 Col6a1-/- and wild type (WT) rat hearts were isolated and enzymatically digested to obtain isolated, living cardiomyocytes. One batch of myocytes were fixed for subsequent immunolabelling and confocal imaging, while a separate group of myocytes were loaded with a ratiometric calcium indicator (fura-2AM, 340/380 ratio). Loaded myocytes were field stimulated for intracellular Ca2+ transient recordings at baseline and in response to pharmacological agents. Application of 20mM caffeine was used as a measure of total Ca2+ from the sarcoplasmic reticulum (SR).

 Col6A1-/- myocytes (n= 15) showed larger systolic Ca2+ transients (1.83 ± 0.16 vs. 1.1 ± 0.07, n=22) and a faster maximum rate of rise in comparison to WT myocytes at 1Hz stimulation frequency (P<0.001). Application of caffeine also showed a higher SR Ca2+ load (p<0.001) in Col6A1-/- myocytes (n=24, 0.80 ± 0.05 vs. 0.59 ± 0.03). Col6A1-/- myocytes displayed an increase in Ca2+ transient amplitude during β-adrenergic stimulation (n=13, P<0.001), with increased susceptibility to impaired Ca2+ handling in 50% of myocytes. These results are remarkedly similar to the disturbance in Ca2+ handling found in the cardiac myocytes of the X chromosome-linked muscular dystrophy (mdx) mouse which has non-functional dystrophin. Using STED microscopy we have identified that collagen VI is located adjacent to biglycan within the t-tubules of rat cardiac myocytes a molecule that is can bind to both the dystrophin glycoprotein complex and collagen VI. Overall, these data indicate that collagen VI may have a role in regulating Ca2+ signalling in cardiac myocytes and is a likely member of the dystrophin-glycoprotein complex.