Background: Hypertrophic cardiomyopathy (HCM) is the most common genetic heart disease. While ≈50% of patients with HCM carry a sarcomere gene mutation (sarcomere mutation-positive, HCM _SMP ), the genetic background is unknown in the other half of the patients (sarcomere mutation-negative, HCM _SMN ). Genotype-specific differences have been reported in cardiac function. Moreover, HCM _SMN patients have later disease onset and a better prognosis than HCM _SMP patients. To define if genotype-specific derailments at the protein level may explain the heterogeneity in disease development, we performed a proteomic analysis in cardiac tissue from a clinically well-phenotyped HCM patient group. Methods: A proteomics screen was performed in cardiac tissue from 39 HCM _SMP patients, 11HCM _SMN patients, and 8 nonfailing controls. Patients with HCM had obstructive cardiomyopathy with left ventricular outflow tract obstruction and diastolic dysfunction. A novel MYBPC3 _2373insG mouse model was used to confirm functional relevance of our proteomic findings. Results: In all HCM patient samples, we found lower levels of metabolic pathway proteins and higher levels of extracellular matrix proteins. Levels of total and detyrosinated α-tubulin were markedly higher in HCM _SMP than in HCM _SMN and controls. Higher tubulin detyrosination was also found in 2 unrelated MYBPC3 mouse models and its inhibition with parthenolide normalized contraction and relaxation time of isolated cardiomyocytes. Conclusions: Our findings indicate that microtubules and especially its detyrosination contribute to the pathomechanism of patients with HCM _SMP . This is of clinical importance since it represents a potential treatment target to improve cardiac function in patients with HCM _SMP , whereas a beneficial effect may be limited in patients with HCM _SMN .

Background: The pathogenesis of MYBPC3 -associated hypertrophic cardiomyopathy (HCM) is still unresolved. In our HCM patient cohort, a large and well-characterized population carrying the MYBPC3 :c772G>A variant (p.Glu258Lys, E258K) provides the unique opportunity to study the basic mechanisms of MYBPC3 -HCM with a comprehensive translational approach. Methods: We collected clinical and genetic data from 93 HCM patients carrying the MYBPC3 :c772G>A variant. Functional perturbations were investigated using different biophysical techniques in left ventricular samples from 4 patients who underwent myectomy for refractory outflow obstruction, compared with samples from non-failing non-hypertrophic surgical patients and healthy donors. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and engineered heart tissues (EHTs) were also investigated. Results: Haplotype analysis revealed MYBPC3 :c772G>A as a founder mutation in Tuscany. In ventricular myocardium, the mutation leads to reduced cMyBP-C (cardiac myosin binding protein-C) expression, supporting haploinsufficiency as the main primary disease mechanism. Mechanical studies in single myofibrils and permeabilized muscle strips highlighted faster cross-bridge cycling, and higher energy cost of tension generation. A novel approach based on tissue clearing and advanced optical microscopy supported the idea that the sarcomere energetics dysfunction is intrinsically related with the reduction in cMyBP-C. Studies in single cardiomyocytes (native and hiPSC-derived), intact trabeculae and hiPSC-EHTs revealed prolonged action potentials, slower Ca ²⁺ transients and preserved twitch duration, suggesting that the slower excitation-contraction coupling counterbalanced the faster sarcomere kinetics. This conclusion was strengthened by in silico simulations. Conclusions: HCM-related MYBPC3 :c772G>A mutation invariably impairs sarcomere energetics and cross-bridge cycling. Compensatory electrophysiological changes (eg, reduced potassium channel expression) appear to preserve twitch contraction parameters, but may expose patients to greater arrhythmic propensity and disease progression. Therapeutic approaches correcting the primary sarcomeric defects may prevent secondary cardiomyocyte remodeling.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is caused by sarcomere gene mutations (genotype-positive HCM) in ≈50% of patients and occurs in the absence of mutations (genotype-negative HCM) in the other half of patients. We explored how alterations in the metabolomic and lipidomic landscape are involved in cardiac remodeling in both patient groups. METHODS: We performed proteomics, metabolomics, and lipidomics on myectomy samples (genotype-positive N=19; genotype-negative N=22; and genotype unknown N=6) from clinically well-phenotyped patients with HCM and on cardiac tissue samples from sex- and age-matched and body mass index–matched nonfailing donors (N=20). These data sets were integrated to comprehensively map changes in lipid-handling and energy metabolism pathways. By linking metabolomic and lipidomic data to variability in clinical data, we explored patient group–specific associations between cardiac and metabolic remodeling. RESULTS: HCM myectomy samples exhibited (1) increased glucose and glycogen metabolism, (2) downregulation of fatty acid oxidation, and (3) reduced ceramide formation and lipid storage. In genotype-negative patients, septal hypertrophy and diastolic dysfunction correlated with lowering of acylcarnitines, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines. In contrast, redox metabolites, amino acids, pentose phosphate pathway intermediates, purines, and pyrimidines were positively associated with septal hypertrophy and diastolic impairment in genotype-positive patients. CONCLUSIONS: We provide novel insights into both general and genotype-specific metabolic changes in HCM. Distinct metabolic alterations underlie cardiac disease progression in genotype-negative and genotype-positive patients with HCM.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder. It is mainly caused by mutations in genes encoding sarcomere proteins. Mutant forms of these highly abundant proteins likely stress the protein quality control (PQC) system of cardiomyocytes. The PQC system, together with a functional microtubule network, maintains proteostasis. We compared left ventricular (LV) tissue of nine donors (controls) with 38 sarcomere mutation-positive (HCM_SMP) and 14 sarcomere mutation-negative (HCM_SMN) patients to define HCM and mutation-specific changes in PQC. Mutations in HCM_SMP result in poison polypeptides or reduced protein levels (haploinsufficiency, HI). The main findings were (1) several key PQC players were more abundant in HCM compared to controls, (2) after correction for sex and age, stabilizing heat shock protein (HSP)B1, and refolding, HSPD1 and HSPA2 were increased in HCM_SMP compared to controls, (3) α-tubulin and acetylated α-tubulin levels were higher in HCM compared to controls, especially in HCM_HI, (4) myosin-binding protein-C (cMyBP-C) levels were inversely correlated with α-tubulin, and (5) α-tubulin levels correlated with acetylated α-tubulin and HSPs. Overall, carrying a mutation affects PQC and α-tubulin acetylation. The haploinsufficiency of cMyBP-C may trigger HSPs and α-tubulin acetylation. Our study indicates that proliferation of the microtubular network may represent a novel pathomechanism in cMyBP-C haploinsufficiency-mediated HCM.

The clinical outcome of hypertrophic cardiomyopathy patients is not only determined by the disease-causing mutation but influenced by a variety of disease modifiers. Here, we defined the role of the mutation location and the mutant protein dose of the troponin T mutations I79N, R94C and R278C. We determined myofilament function after troponin exchange in permeabilized single human cardiomyocytes as well as in cardiac patient samples harboring the R278C mutation. Notably, we found that a small dose of mutant protein is sufficient for the maximal effect on myofilament Ca²⁺-sensitivity for the I79N and R94C mutation while the mutation location determines the magnitude of this effect. While incorporation of I79N and R94C increased myofilament Ca²⁺-sensitivity, incorporation of R278C increased Ca²⁺-sensitivity at low and intermediate dose, while it decreased Ca²⁺-sensitivity at high dose. All three cTnT mutants showed reduced thin filament binding affinity, which coincided with a relatively low maximal exchange (50.5 ± 5.2%) of mutant troponin complex in cardiomyocytes. In accordance, 32.2 ± 4.0% mutant R278C was found in two patient samples which showed 50.0 ± 3.7% mutant mRNA. In accordance with studies that showed clinical variability in patients with the exact same mutation, we observed variability on the functional single cell level in patients with the R278C mutation. These differences in myofilament properties could not be explained by differences in the amount of mutant protein. Using troponin exchange in single human cardiomyocytes, we show that TNNT2 mutation-induced changes in myofilament Ca²⁺-sensitivity depend on mutation location, while all mutants show reduced thin filament binding affinity. The specific mutation-effect observed for R278C could not be translated to myofilament function of cardiomyocytes from patients, and is most likely explained by other (post)-translational troponin modifications. Overall, our studies illustrate that mutation location underlies variability in myofilament Ca²⁺-sensitivity, while only the R278C mutation shows a highly dose-dependent effect on myofilament function.

BACKGROUND: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. METHODS: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3 _c.2373insG HCM mouse model. RESULTS: Screening of 157 kinase inhibitors in wild-type (N=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (N=24 HCM and N=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (N=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. CONCLUSIONS: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.