Biology subjects

Fritzmann, G.

Publications and source records attributed to Fritzmann, G..

3 recordsLinked to original sources

bioRxiv2025

Missense variants in the myosin binding domains of MYBPC3 and MYBPHL impair sarcomere incorporation

Approximately 40% of genetic hypertrophic cardiomyopathy cases involve mutations in MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C), a key regulator of sarcomere contractility. The atrial-specific paralog, myosin binding protein-H like (MyBP-HL), has been associated with dilated cardiomyopathy in humans and mice. Both proteins bind to the same binding sites in the thick filament C-zone. In the atria, cMyBP-C and MyBP-HL are found at [~]1:1 ratios, while ventricles only express cMyBP-C, which is found at twice the atrial level, indicating a stoichiometric relationship. In the atria, we hypothesize that missense variants in either gene may cause alterations in thick filament binding affinity and changes in the normal [~]1:1 ratio. Notably, MyBP-HL deletion in atrial myofibrils accelerates relaxation kinetics, suggesting that altered stoichiometry impacts biophysical parameters. We hypothesized that deletion, overexpression, or missense variants in either gene would alter the abundance of the other protein in atrial sarcomeres, affecting sarcomere localization and function. To test this, we engineered two constructs: a mini-C construct comprising thick filament-binding domains of cMyBP-C and a MyBP-HL construct. Selected MYBPC3 and MYBPHL missense variants were introduced and expressed in neonatal rat ventricular cardiomyocytes (NRVMs). Sarcomere localization was assessed by co-localization with endogenous cMyBP-C. MYBPC3 variants were selected across a range of pathogenicity, while MYBPHL variants were based on evolutionary conservation of residues. MYBPHL variants Gly275Ser, Arg285His, and Ala342Thr induced significant sarcomere mislocalization, and MYBPC3 variants Pro1181Ala and Asn1257Lys showed variable effects on sarcomere mislocalization. To assess stoichiometric effects, we developed a T2A/P2A polycistronic construct to co-express mini-C, Td-Tomato, and MyBP-HL. Immunoblotting and mass spectrometry confirmed consistent and reproducible expression. We identified several MYBPC3 and MYBPHL variants that reduced the affinity of their protein for myofilament incorporation. These results suggest that this 2A construct is a useful tool for measuring the effect of myosin binding protein missense variants on sarcomere affinity, with implications for assessing pathogenicity of these variants.

biochemistry↗

bioRxiv2024

The effects of conditional loss of myosin binding protein H-like on cardiac function.

Mutations in the myosin-binding protein H-like (MyBP-HL) gene, MYBPHL, are linked to hereditary dilated cardiomyopathy (DCM), atrial fibrillation, and atrioventricular arrhythmias. MyBP-HL is a sarcomeric protein that is highly expressed in the atria with only scarce, distinct clusters of MyBP-HL positive cells within and surrounding the ventricular conduction system. Constitutive knock-out of MyBP-HL in mice causes atrial dilation, arrhythmia, and DCM. Whether MyBP-HL plays a developmental role, or if knock-down in adulthood will recapitulate a similar phenotype has yet to be examined. Moreover, the significance of the MyBP-HL expressing ventricular cells, or the functional need for differential thick filament regulation is currently unknown. We used a conditional floxed Mybphl mouse to further elucidate the role of MyBP-HL. We crossed this mouse with a ROSA26-Cre(ERT2) LoxP mouse to conditionally knock-down Mybphl after tamoxifen treatment. We also crossed the Mybphl flox mouse with a Contactin-2-Cre mouse that deletes Mybphl solely in the cardiac conduction system from birth. Echocardiography was used to measure contractile function, and conscious telemetry allowed for monitoring of heart rhythm and electrical signal conduction changes. We demonstrate that mice with conditional decrease of MyBP-HL in adulthood develop a hypertrophic phenotype with atrial contractile changes, increased total heart weight to body weight, and increased heart rate variability. Deletion of Mybphl solely within the cardiac conduction system trends toward mild hypercontractility, lower heart rates, and interventricular septal thickening. These data show that MyBP-HL is essential for proper cardiac function, and even minor alteration in protein levels cause a diseased cardiac phenotype.

physiology↗

bioRxiv2024

MYBPHL nonsense mutations have poor sarcomere binding, are degraded, and cause abnormal contraction

Heart function depends on the cardiomyocyte contractile apparatus and proper sarcomere protein expression. Mutations in sarcomere genes cause inherited forms of cardiomyopathy and arrhythmias, including atrial fibrillation (AF). Recently, a novel sarcomere component, myosin binding protein-H like (MyBP-HL) was identified. MyBP-HL is mainly expressed in cardiac atria and shares homology to the last three C-terminal domains of cardiac myosin binding protein-C (cMyBP-C). The MYBPHL R255X mutation has been linked to atrial enlargement, dilated cardiomyopathy, and atrial and ventricular arrhythmias. Similar nonsense mutations in MYBPC3 result in no myofilament incorporation and a rapid degradation of the truncated protein and are highly associated with development of hypertrophic cardiomyopathy. However, the MYBPHL R255X mutation occurs too frequently in the human population to be highly pathogenic. We sought to determine whether all MYBPHL nonsense mutations lead to impaired MyBP-HL sarcomere integration and degradation of the mutant protein, or if the MYBPHL R255X mutation has a different consequence. We mimicked human MYBPHL nonsense mutations in the mouse Mybphl cDNA sequence and tested their sarcomere incorporation in neonatal rat cardiomyocytes. We demonstrated that wild type MyBP-HL overexpression showed the expected C-zone sarcomere incorporation, like cMyBP-C. Nonsense mutations showed defective sarcomere incorporation. We demonstrated that wild type MyBP-HL and MyBP-HL nonsense mutations were degraded by both proteasome and calpain mechanisms. Additionally, we observed changes in contraction kinetics and calcium transients in cells transfected with MyBP-HL nonsense mutations compared to MyBP-HL full length. Together, these data support the hypothesis that MYBPHL nonsense mutations are largely similar. Short summaryPremature stop mutations in myosin binding protein H-like prevent sarcomere incorporation of the translated protein. Overexpression of truncating mutants causes contractile defects in neonatal rat cardiomyocytes. These effects occur regardless of the location of the premature stop along the protein.

physiology↗