Abstract Body: Introduction
Cardiomyocyte proliferation is fundamental to heart development and regeneration. Although pulsatile Ca²⁺ transients (CaTs) drive cardiomyocyte contraction, how Ca²⁺ dynamics are regulated during the cell cycle remains unclear.

Research Question
How are CaTs modulated across cell cycle phases in cardiomyocytes?

Methods
We studied CaTs using human induced pluripotent stem cell (iPSC)-derived cardiomyocytes, human fetal cardiomyocytes, neonatal mouse cardiomyocytes in intact hearts, and cultured neonatal rat ventricular myocytes (NRVMs). Cell cycle phases were identified by morphology, expressed mCherry-Geminin, and immunofluorescence markers (Ki67, phosphorylated histone H3, Aurora B kinase, mitotic spindle labeling). Ca²⁺ dynamics were monitored using Rhod-2 AM and GCaMP8 with real-time in vitro and ex vivo imaging. CDK1 activity was increased by adenoviral expression of constitutively active CDK1 (Adv-Cdk1AF, Adv-cyclin B1) or inhibited using Ro-3306 with MG132 to prevent M-phase exit. Ca²⁺ signals were analyzed using ImageJ/Fiji.

Results
All cycling iPSC-derived cardiomyocytes (n = 17 cells) and 75% of cycling human fetal cardiomyocytes (n = 41 cells) showed decreased or absent CaTs in culture. In intact mouse hearts, cycling cardiomyocytes (n = 21 cells from 3 mouse hearts) showed a 30% decrease in CaT amplitudes compared with non-cycling cardiomyocytes (n = 39 cells from 3 mouse hearts). In NRVMs from P1 rat pups, CaT amplitudes declined from prometaphase, reached a minimum in metaphase, increased during anaphase, and returned to baseline in daughter cells (cell numbers: Ki67^-: n = 88; non-M-phase Ki67⁺: n = 21; interphase: n = 73; prophase: n = 5; prometa/metaphase: n = 11; cytokinesis: n = 8). Peak cytosolic Ca²⁺ decreased from 321.6±138.67 nM in interphase (n = 52 cells) to 125.7±63.16 nM in M-phase (n = 13 cells). Increasing CDK1 activity promoted M-phase entry and reduced CaTs (Ctrl: n = 42 cells; CDK1AF+Cyclin B1: n = 113 cells), whereas CDK1 inhibition restored CaTs in M-phase (Ctrl[MG132]: n = 30 cells; Ro-3306[MG132]: n = 54 cells).

Conclusion
Cardiomyocytes reduce CaT amplitudes during M-phase, and CDK1 activity is required to sustain this reduction. Modulating Ca²⁺ signaling may enhance cardiomyocyte proliferation.