Unequal Allelic Expression of Mutated Cardiac Troponin I from Cell-To-Cell May Induce Contractile Imbalance in Hypertrophic Cardiomyopathy

Hypertrophic Cardiomyopathy (HCM) is mostly caused by inherited, heterozygous mutations in sarcomeric proteins. A common mechanism why different functional effects of different mutations induce similar HCM features is unclear. In previous studies, unequal expression of mutated vs. wildtype mRNA and large variability in force generation between individual cardiomyocytes from HCM-patients with mutations in the myosin heavy chain gene (MYH7) was found. This contractile imbalance of adjacent cardiomyocytes could trigger development of common HCM features like cardiomyocyte disarray and fibrosis. For mutations in MYH7 we provided evidence that the unequal fractions of mutated mRNA from cell to cell most likely are due to burst-like, stochastic transcription, independent for the two alleles. Here we asked, if this hypothesis is unique for MYH7 or if it is a mechanism also in other sarcomeric proteins like cardiac troponin I (cTnI, TNNI3). We analyzed TNNI3 mRNA expression and force generation in a donor and HCM-patients with TNNI3-mutation (c.433C>T, R145W). RNA-fluorescence in situ hybridization experiments revealed that most nuclei contain no active transcription sites (aTS) (95% in donor and 81% in patients), while others show active transcription of one or both alleles. This indicates stochastic and independent burst-like transcription of TNNI3 rather than continuous transcription. Single cardiomyocytes of both, patients and donor, showed highly variable fractions of TNNI3 mutant and wildtype mRNA ranging from essentially only wildtype to essentially only mutant mRNA in allele-specific RT-PCR experiments. Calcium dependent force measurements on cardiomyocytes of the same patient presented substantially larger variability of force compared to donor cardiomyocytes. These results suggest that not only MYH7, but also TNNI3 is transcribed burst-like. This may induce cell-to-cell allelic imbalance and, as a result, contractile imbalance in the myocardium as a trigger for HCM.