Phospholamban cardiomyopathy is an inherited cardiac disorder, caused by mutations in the PLN gene, encoding Phospholamban (PLB). PLB regulates relaxation of cardiomyocytes by inhibiting SERCA, which transports calcium from the cytoplasm to the sarcoplasmic reticulum. Mutations in PLN cause severe arrhythmogenic cardiomyopathies. One common mutation in the Netherlands is R14del (c.40_42delAGA), which is associated with altered calcium transients and formation of PLN-aggregates in cardiomyocytes. It has been assumed that these aggregates affect cardiomyocyte function. In heterozygous patient tissue, as well as a heterozygous R14del mouse model, it can be observed that formation of aggregates does not affect all cardiomyocytes. Mouse models indicate that aggregation increases over time. Underlying triggers for aggregate formation have not been elucidated yet.
We hypothesized that cardiomyocytes with aggregates show higher fractions of R14del mRNA. To test this, we used cryosections of four severely affected PLN R14del patients and isolated cardiomyocytes with and without aggregates. These cardiomyocytes were subjected to RT-qPCR with allele-specific probes to quantify fraction of mutant and wildtype mRNA. Bulk tissue analysis was performed in comparison. Whereas bulk tissue and cardiomyocytes without aggregates show equal fractions of R14del and WT mRNA, we can show increased levels of R14del in cardiomyocytes with aggregates of up to 80%. Currently, we analyze cardiac tissue from heterozygous PLN R14del mice, in order to test whether this finding is also mimicked in the mouse model and to further assess, whether this is already detectable at early – preclinical - time points.
Our findings indicate PLN expression imbalance among individual cardiomyocytes. This might lead to functional heterogeneity among the cardiomyocytes which could contribute to the observed arrhythmias in PLN R14del patients. Thus, reduction of the aggregates may prove an interesting target for therapeutic interventions.
