Abstract Body: Introduction: Nebulette is a sarcomere protein that connects the thin filament to the Z-disc, assisting in sarcomere assembly and organization. Nebulette knockout mice have normal cardiac function, yet the gene encoding Nebulette, NEBL, is mutated in dilated cardiomyopathy and endocardial fibroelastosis. Recently gene variants near NEBL have been associated with myxomatous mitral valve disease (MMVD) in dogs.
Research Question: We tested the hypothesis that canines with severe cardiac dysfunction due to MMVD have impairment in sarcomere function.
Aims: We aimed to determine the myofilament calcium sensitivity, active tension, and passive tension of permeabilized papillary muscle from canines with and without MMVD.
Methods: Left ventricular papillary tissue was obtained after owners’ consent from domesticated canines with MMVD (n = 8) and without MMVD (n = 5) that had been euthanized for welfare reasons. Frozen papillary muscle (~20 mg) was partially homogenized in 50% glycerol solution and stored at -20°C for five days. Papillary muscle was permeabilized further using a skinning buffer (1% triton, 1% saponin). Multicellular preparations were attached to a motor to control sarcomere length and force-transducer to measure muscle force in various concentrations of activating calcium (-log₁₀[calcium] (pCa) 9.0-4.5). Control vs. MMVD cardiomyocytes were compared using an unpaired t-test, significance at a p-value ≤ 0.05.
Results: MMVD cardiomyocytes had greater myofilament calcium sensitivity compared to age-matched controls (5.44 ± 0.041 vs. 5.36 ± 0.034 respectively, p = 0.003). There were no differences in maximal tension or passive tension between groups.
Conclusion: These data indicate that MMVD papillary cardiomyocytes require less activating calcium to trigger 50% of maximal tension. These results suggest that NEBL mutations may cause cardiac dysfunction in part by modulating thin filament sensitivity to activating calcium. Future studies will explore the effects of these mutations on sarcomere length and uniformity.