P679Ultrastructural and electron tomography analyses of cardiac muscle: normal muscle compared to presence and absence of myosin binding protein C-phosphorylation

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Abstract

Purpose: Myosin binding protein C is an accessory protein of vertebrate striated muscle thick filaments. Many studies have aimed to elucidate both its structure and function since mutations in the cardiac isoform (cMyBP-C) gene (MYBPC3) are known to be a leading cause of familial hypertrophic cardiomyopathy (HCM) affecting millions of people worldwide. There is strong evidence that phosphorylation of N-terminus M-domain of cMyBP-C is essential for normal cardiac function and that their dephosphorylation is associated with contractile dysfunction and HCM. However, the details of the underlying molecular-level ultrastructure are still poorly understood. The aim of this work is to provide details of the ultrastructure of thick filaments in different states of cMyBP-C phosphorylation.

Methods: Two transgenic mouse strains expressing a constitutive phosphorylation or a non-phosphorylatable cMyBP-C have been compared to a homozygous knockout mouse for cMyBP-C and a wild-type (WT) control. We have carried out detailed transmission electron microscopy (TEM) in intact cardiac muscles, and electron tomography (ET) combined with sub-tomographic averaging.

Results: TEM shows that cardiomyocyte ultrastructure is relatively well maintained under the total deletion of cMyBP-C from the heart and due to the expression of constitutive phosphorylation of cMyBP-C. However, the constitutive expression of non-PKA-phosphorylatable cMyBP-C leads to both sarcomeric disarray and mitochondria disruption. Longitudinal views of the average 3D reconstructions employing ET illustrate the surface density features of thick filaments. In the WT, two projection masses can be identified originating from cMyBP-C. Both the absence of cMyBP-C and the presence of constitutive non-phosphorylatable cMyBP-C seem to be associated with poorer structural preservation of the thick filaments.

Conclusions: This study shows that cMyBP-C constitutive phosphorylation effectively preserves the intracellular arrangement of cardiac myocytes. However, non-PKA-phosphorylatable cMyBP-C leads to a dramatic overall decrease of tissue organization which is much more deleterious than the total deletion of cMyBP-C. This finding provides a structural explanation for cardiac dysfunction in this sample. This study shows that chemically fixed samples can be used for ET to reveal 3D structural information of thick filaments. Results obtained by using this methodology are in good agreement with previous studies using isolated filament approaches. However, in order to obtain higher resolution, advanced methods of sample processing will be required.

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