Biology subjects

Barefield, D. Y.

Publications and source records attributed to Barefield, D. Y..

4 recordsLinked to original sources

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↗

bioRxiv2024

Cardiac Localized Polycystin-2 plays a Functional Role in Natriuretic Peptide Production and its Absence Contributes to Hypertension

Cardiovascular complications are the most common cause of mortality in patients with autosomal dominant polycystic kidney disease (ADPKD). Hypertension is seen in 70% of patients by the age of 30 prior to decline in kidney function. The natriuretic peptides (NPs), atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are released by cardiomyocytes in response to membrane stretch, increasing urinary excretion of sodium and water. Mice heterozygous for Pkd2 have attenuated NP responses and we hypothesized that cardiomyocyte-localized polycystin proteins contribute to production of NPs. Cardiomyocyte-specific knock-out models of polycystin-2 (PC2), one of the causative genes of ADPKD, demonstrate diurnal hypertension. These mice have decreased ANP and BNP expression in the left ventricle. Analysis of the pathways involved in production, maturation, and activity of NPs identified decreased transcription of CgB, PCSK6, and NFAT genes in cPC2-KOs. Engineered heart tissue with human iPSCs driven into cardiomyocytes with CRISPR/Cas9 KO of PKD2 failed to produce ANP. These results suggest that PC2 in cardiomyocytes are involved in NP production and lack of cardiac PC2 predisposes to a hypertensive volume expanded phenotype, which may contribute to the development of hypertension in ADPKD.

physiology↗

bioRxiv2020

Enhancer and Promoter Usage in the Normal and Failed Human Heart

The failed heart is characterized by re-expression of a fetal gene program, which contributes to adaptation and maladaptation in heart failure. To define genomewide enhancer and promoter use in heart failure, Cap Analysis of Gene Expression (CAGE-seq) was applied to healthy and failed human left ventricles to define short RNAs associated with both promoters and enhancers. Integration of CAGE-seq data with RNA sequencing identified a combined [~]17,000 promoters and [~]1,500 enhancers active in healthy and failed human left ventricles. Comparing promoter usage between healthy and failed hearts highlighted promoter shifts which altered amino-terminal protein sequences. Comparing enhancer usage between healthy and failed hearts revealed a majority of differentially utilized heart failure enhancers were intronic and primarily localized within the first intron, identifying this position as a common feature associated with tissue-specific gene expression changes in the heart. This dataset defines the dynamic genomic regulatory landscape underlying heart failure and serves as an important resource for understanding genetic contributions to cardiac dysfunction.

genomics↗