BPS2026 – Reductive stress induces sarcoplasmic reticulum calcium mishandling in ventricular myocytes

Maintaining intracellular redox balance through pro-oxidants and antioxidants is critical for normal cell function. Oxidative stress, marked by increased production of pro-oxidants, is well established as a major contributor to cardiac arrhythmias, promoting proarrhythmic disturbances in Ca 2+ homeostasis. This includes impaired regulation of luminal Ca 2+ by the sarco/endoplasmic reticulum (S/ER) Ca 2+ release channel, the ryanodine receptor (RyR2), and the S/ER Ca 2+ -ATPase (SERCA2a). Conversely, reductive stress is characterized by an abnormal increase in antioxidants. Recent evidence links reductive stress to heart failure, hypertrophy, and ischemia-reperfusion injury, conditions all associated with Ca 2+ -dependent arrhythmias. However, molecular mechanisms connecting reductive stress to Ca 2+ mishandling remain entirely unexplored. We hypothesize that, like oxidative stress, reductive stress in the heart can modulate S/ER Ca 2+ homeostasis and the activity of RyR2 and SERCA2a. We adapted a redox biosensor to measure reductive stress in intact, live ventricular myocytes (VMs). Importantly, this revealed a more reduced intra-SR state in VMs overexpressing nuclear factor erythroid 2-related factor 2 (Nrf2), an antioxidant transcription factor. While SERCA2a blocker thapsigargin is commonly used to reduce SR Ca 2+ content and induce S/ER oxidative stress, instead we overexpressed dominant-negative phospholamban (dnPLB) in healthy VMs to increase SERCA2a activity and increase SR Ca 2+ content. Notably, this induced reductive stress, confirmed by qPCR and western blot analyses. Importantly, reductive stress significantly disrupted intracellular Ca 2+ homeostasis, altering Ca 2+ transient amplitude and SR Ca 2+ content, and promoting proarrhythmic spontaneous Ca 2+ waves. Permeabilized VMs experiencing reductive stress exhibited increased RyR2-mediated Ca 2+ sparks due to heightened channel oxidation. Collectively, our data suggest that SR redox status is a crucial regulator of SR Ca 2+ handling. Similar to oxidative S/ER stress, reductive S/ER stress can also disrupt S/ER calcium handling in VMs and warrants further investigation in cardiac disease contexts.