Identification of disease modifiers and novel treatment targets in hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a clinically heterogeneous disease with large differences in disease penetrance and severity between patients. Therefore, we hypothesize that a variety of disease modifiers can contribute and influence the clinical disease course in patients. In this thesis we explored the mutant protein dose and mutation location and the cellular protein quality control (PQC) as potential disease modifiers. We further used -omics techniques to identify differences at the RNA and protein level between different genetic groups to identify novel disease modifiers and potential novel treatment targets. In Chapter 2 we investigated the mutant protein dose and location effect of different TNNT2 mutations on cardiomyocyte function. We observed for two of the mutations, I79N and R94C, that a low dose of mutant protein (<15%) is already sufficient for the maximal increase in myofilament Ca2+-sensitivity, while the effect size is determined by the mutation location. For the R278C mutation, however, we observed increased myofilament Ca2+-sensitivity at low and intermediate dose, whereas a high mutant dose led to decreased myofilament Ca2+-sensitivity. In Chapter 3 we reviewed the current knowledge about the role of the PQC in the pathogenesis of inherited cardiomyopathies. In Chapter 4 we determined the expression levels of several PQC key players in our large HCM patient cohort. Compared to healthy controls, we found increased levels of the stabilizing HSPs HSPB1 and HSPB7 and the ATP-dependent HSPD1 and HSPA2, that have refolding capacity. In part 2 of this thesis we performed omics-techniques to identify novel disease modifiers in an unbiased manner on different cellular levels. In Chapter 5 we performed a proteomics screen of a large number of well-characterized human myectomy samples. One of our main findings was reduced expression of proteins involved in energy metabolism. We also identified increased levels of detyrosinated tubulin as the main difference between mutation-positive and -negative HCM samples. In line with previous studies, we could show in our novel MYBPC32373insG mouse model that detyrosinated tubulin has a negative effect on diastolic function of isolated cardiomyocytes and provides a treatment target to improve cardiac function. In Chapter 7 we defined sex-specific differences in our proteomics data to identify differences at the protein level between male and female mutation-positive HCM samples. Interestingly, we found higher levels of tubulin in females compared to males. As females show more severe diastolic dysfunction, we propose that this is partly due to elevated levels of tubulin. In Chapter 6 we integrated different layers of cellular regulation, namely the histone acetylome, the transcriptome and the proteome, to identify key players that drive pathological changes in HCM patients with a mutation in MYBPC3. We identified 36 upregulated and 17 downregulated protein-coding genes that were changed in all three levels of analysis and may belong to the main disease drivers. For early treatment that may be able to halt or slow disease progression, it is of importance to identify mutation carriers at risk before the onset of symptoms and irreversible cardiac remodeling. Imaging studies have shown reduced cardiac efficiency in early disease stages. As these changes may be reflected in the serum metabolite profile, we performed serum metabolomics on healthy controls, asymptomatic mutation carriers and obstructive HCM patients in Chapter 8 and defined very distinct metabolite profiles between groups. The current treatment of HCM patients is only symptomatic. Omics-studies in well characterized patient cohorts and patient tissues form a basis to unravel genotype-specific pathomechanisms that can ultimately lead to patient-specific treatment approaches. Identification of disease modifiers will further help to improve risk prediction in mutation carriers and novel biomarker panels may aid in determining the proper time point for therapeutic intervention.