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Obesity is a major risk factor for atrial fibrillation (AF) the most common serious cardiac arrhythmia, but the molecular mechanisms underlying diet-induced AF remain unclear. In this study, we subjected mice to a chronic high-fat diet and acute sympathetic activation ('two-hit' model) to study the mechanisms by which diet-induced obesity promotes AF. Surface electrocardiography revealed that diet-induced obesity and sympathetic activation synergize during intracardiac tachypacing to induce AF. At the cellular level, diet-induced obesity and acute adrenergic stimulation facilitate the formation of delayed afterdepolarizations in atrial myocytes, implicating altered Ca 2+ dynamics as the underlying cause of AF. We found that diet-induced obesity does not alter the expression of major Ca 2+ -handling proteins in atria, including the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA), a major component of beat-to-beat Ca 2+ cycling in the heart. Paradoxically, obesity reduces phospholamban phosphorylation, suggesting decreased SERCA activity, yet atrial myocytes from obese mice showed a significantly increased Ca 2+ transient amplitude and SERCA-mediated Ca 2+ uptake. Adrenergic stimulation further increases the Ca 2+ transient amplitude but does not affect Ca 2+ reuptake in atrial myocytes from obese mice. Transcriptomics analysis showed that a high-fat diet prompts upregulation of neuronatin, a protein that has been implicated in obesity and is known to stimulate SERCA activity. We propose a mechanism in which obesity primes SERCA for paradoxical activation, and adrenergic stimulation facilitates AF conversion through a Ca 2+ -induced Ca 2+ release gain in atrial myocytes. Overall, this study links obesity, altered Ca 2+ signaling, and AF, and targeting this mechanism may prove effective for treating obesity-induced AF., Competing Interests: Competing Interest Statement: D.P.-B., D.J.T., C.C.-C., G.G.S., A.M.D.R., J.S., D.S.R., J.A., D.R.G., and L.M.E.-F. declare no competing interests. T.J.H is co-founder of Cartox, Inc., and scientific advisor to StemBioSys, Inc.

The R403Q mutation of human cardiac β-myosin heavy chain was the first missense mutation of a sarcomeric protein identified as being causal for hypertrophic cardiomyopathy (HCM), in humans. The direct effect of the R403Q mutant myosin on intracellular calcium homeostasis and contractility is not fully known. Here we have used in vitro gene transfer of the R403Q mutant human β-myosin to study its direct effects on single intact adult cardiac myocyte contractility and calcium homeostasis. In the first experiments, adult cardiac myocytes transduced with the R403Q mutant myosin recombinant viral vectors were compared to myocytes transduced with wild-type human β-myosin (wtMYH7). Efficiency of gene transfer was high in both groups (>98%) and the degree of stoichiometric myofilament incorporation of either the mutant or normal myosin was comparable at ∼40% in quiescent myocytes in primary culture. Sarcomere structure and cellular morphology were unaffected by R403Q myosin expression and myofilament incorporation. Functionally, in electrically paced cardiac myocytes, the R403Q mutant myosin caused a significant increase in intracellular calcium concentration and myocyte hyper-contractility. At the sub-cellular myofilament level, the mutant myosin increased the calcium sensitivity of steady state isometric tension development and increased isometric cross-bridge cycling kinetics. R403Q myocytes became arrhythmic after β-adrenergic stimulation with spontaneous calcium transients and contractions in between electrical stimuli. These results indicate that human R403Q mutant myosin directly alters myofilament function and intracellular calcium cycling. Elevated calcium levels may provide a trigger for the ensuing hypertrophy and susceptibility to arrhythmia that are characteristic of HCM.

Myoregulin (MLN) is a protein that regulates the activity of the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) without affecting its affinity for Ca 2+ . MLN's residue Lys27 is located at a site where other SERCA regulators control Ca 2+ affinity. Therefore, we conducted atomistic simulations and ATPase activity experiments to determine whether replacing Lys27 with asparagine, a conserved residue found in various muscle SERCA regulators, would enable MLN to modulate both the Ca 2+ affinity and catalytic activity of SERCA. Our findings indicate that replacing Lys27 with Asn significantly enhances the inhibitory potency of MLN, but it does not affect SERCA's affinity for Ca 2+ . We suggest that the SERCA site modulating Ca 2+ affinity also acts as a catalytic activity switch. Therefore, this site is a key element contributing to the functional divergence among homologous SERCA regulators. This study paves the way for future investigations to explore how biological function diverges during the evolution of the SERCA regulator family., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)