A piece of the human heart: variance of protein phosphorylation in left ventricular samples from end-stage primary cardiomyopathy patients

Abstract

Cardiomyocyte contraction is regulated by phosphorylation of sarcomeric proteins. Throughout the heart regional and transmural differences may exist in protein phosphorylation. In addition, phosphorylation of sarcomeric proteins is altered in cardiac disease. Heterogeneity in protein phosphorylation may be larger in hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) as it may be caused by multiple mutations in genes encoding different sarcomeric proteins. Moreover, HCM is characterized by asymmetric remodelling of the heart. In the present study we assessed if local differences in sarcomeric protein phosphorylation are more evident in primary HCM or DCM than in non-failing donors. Thereto, phosphorylation of the two main target proteins of the beta-adrenergic receptor pathway, troponin I (cTnI) and myosin binding protein C (cMyBP-C) was analysed in different parts in the free left ventricular wall of end-stage failing HCM and DCM patients and donors obtained during transplant surgery. Intra-patient variability in protein phosphorylation within tissue samples of approximately 2 g wet weight was comparable between donor, HCM and DCM samples and could partly be attributed to the precision of the technique. Thus, our data indicate that within the precision of the measurements small, biopsy-sized cardiac tissue samples are representative for the region of the free left ventricular wall from which they were obtained.

Fig. 1

A representative gradient gel stained with ProQ Diamond and SYPRO Ruby, demonstrating the phosphorylation status of cMyBP-C and cTnI. MWM indicates molecular weight marker

Fig. 2

Intra-heart variability in protein phosphorylation. a–c cMyBP-C phosphorylation. d–f cTnI phosphorylation. The coefficient of variation is indicated above the bars for each heart

Fig. 3

The coefficient of variation, a measure of how much the individual values of each heart differed from the mean, was on average not significantly different between donor, HCM and DCM for the phosphorylation status of cMyBP-C (a) or cTnI (b) (closed symbols). Within the analysis of cMyBP-C phosphorylation, exclusion of outliers (HCM 1 and DCM 3) lowered the mean coefficient of variation and the SEM in HCM, while the average coefficient of variation of the DCM group was slightly increased (as indicated by the arrow, open symbols). After exclusion of the outliers there was still no significant difference in coefficient of variation among the groups. Groups were compared by one-way ANOVA, P < 0.05 was considered significant