biostudies-literatureBorile GFondazione Italiana di Ricerca per la Sclerosi Laterale AmiotroficaMinistero dell'università e della ricerca1434https://www.ebi.ac.uk/biostudies/studies/S-EPMC12468161biostudies-literatureUnknown14(18)Neonatal rodent cardiomyocytes (CMs) are a mainstay of in vitro cardiac research, yet their immature phenotype limits the study of key physiological processes such as excitation-contraction coupling (ECC) and sympathetic modulation. Here, we present a defined low-glucose, serum-free (LGSF) culture protocol that drives the structural and functional maturation of neonatal CMs and supports their integration into functional neuro-cardiac co-cultures. After 15 days in LGSF conditions, CMs exhibit elongated morphology, organized sarcomeres, polarized connexin-43, mitochondrial redistribution, and sarcoplasmic reticulum (SR) development, all closely resembling features of adult cells. These structural hallmarks were paralleled by enhanced Ca²⁺ handling, with increased SR conCellsDefined Composition of Culture Media Promotes Rodent Neonatal Cardiomyocyte Maturation and Enables Functional Neuro-Cardiac Co-Culture.PMC12468161PRIN 2022F3NENHPRIN 20229PX74ASYMP-ALSFaggian GSanchez-Alonso JLCampo ADokshokova LZaglia TBorile GPrando VMongillo MMoro NGorelik JfalseDefined Composition of Culture Media Promotes Rodent Neonatal Cardiomyocyte Maturation and Enables Functional Neuro-Cardiac Co-Culture.Neonatal rodent cardiomyocytes (CMs) are a mainstay of in vitro cardiac research, yet their immature phenotype limits the study of key physiological processes such as excitation-contraction coupling (ECC) and sympathetic modulation. Here, we present a defined low-glucose, serum-free (LGSF) culture protocol that drives the structural and functional maturation of neonatal CMs and supports their integration into functional neuro-cardiac co-cultures. After 15 days in LGSF conditions, CMs exhibit elongated morphology, organized sarcomeres, polarized connexin-43, mitochondrial redistribution, and sarcoplasmic reticulum (SR) development, all closely resembling features of adult cells. These structural hallmarks were paralleled by enhanced Ca²⁺ handling, with increased SR con2025-01-01T00:00:00Z2025 Sep2026-05-01T03:19:30.044Z2026-05-01T03:10:47.449ZS-EPMC124681614100239910.3390/cells14181434