Abstract 4369151: Localized Calcium Depletion at The Spindle Poles Is Essential For M-Phase Progression In Cardiomyocytes

Introduction: Cardiomyocyte proliferation is fundamental to heart development and regeneration. Although calcium release from the sarcoplasmic reticulum (SR) is well-known to drive cardiomyocyte contraction, how calcium dynamics are modified during mitosis is not well understood. In cardiomyocytes, RyR2 releases calcium from the SR to trigger contraction, while SERCA2a pumps it back to enable relaxation. We will investigate how the distribution and activity of RyR2 and SERCA2a are altered in proliferating cardiomyocytes. Research Question: How is calcium signaling regulated during M-phase to support cardiomyocyte division? Methods: We used neonatal rat ventricular cardiomyocytes to study calcium signaling during mitosis. Live-cell imaging with SiR-tubulin identified spindle microtubules and M-phase progression. We examined natural and exogenous CDK1/cyclin B1(MOI: 200)-induced M-phase and used antibodies to label SERCA2a, RyR2, and spindle poles. Intracellular calcium was visualized using Rhod-2 AM and confocal microscopy. Drugs were applied to modulate calcium, microtubule integrity, and dynein activity: thapsigargin (6 µM, SERCA2a inhibition), nocodazole (20 µM, microtubule disruption), and ciliobrevin D (120 µM, dynein 1 inhibition), respectively. A cardiomyocyte-specific knockout of Dync1h1 (Dynein Cytoplasmic 1 Heavy Chain 1) in αMHC-Cre;Dync1h1 Flox mice was used to study dynein-dependent SERCA2a transport. We analyzed total cardiomyocyte counts and M-phase activity in the Dync1h1 knockout mice. Results: SERCA2a accumulated at spindle poles during M-phase, reducing local calcium levels without altering overall SR calcium content. Inhibiting SERCA2a with thapsigargin increased calcium at the spindle poles and prevented cardiomyocytes in early M-phase from progressing through metaphase. This treatment resulted in failed cell division, without inducing cell death. However, once cardiomyocytes had passed through metaphase, this treatment no longer inhibited their division. SERCA2a localization required both intact microtubules and dynein 1 activity. Dync1h1 knockout impaired the SERCA2a accumulation, suppressed cardiomyocyte proliferation, and reduced cardiomyocyte numbers in mice. Conclusions: Spatial calcium regulation during M-phase, mediated by SERCA2a accumulation at spindle poles, is critical for mitotic progression in cardiomyocytes. Our findings suggest a strategy to enhance cardiomyocyte proliferation by targeting SERCA2a in research.