Cells. 2025 Sep 13;14(18):1434. doi: 10.3390/cells14181434.
ABSTRACT
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 contribution and reduced spontaneous activity, indicative of a mature ECC phenotype. When co-cultured with sympathetic neurons (SN), CMs established anatomically distinct neuro-cardiac junctions. Notably, nicotine stimulation triggered spatially restricted, reversible increases in CM Ca2+ transients, confined to varicosity-contacted cells. Pharmacological analysis revealed subtype-specific roles for β1– and β2-adrenergic receptors, and uncovered evidence of functional crosstalk between them. Our study defines a reproducible culture framework that advances CM maturation and enables the high-resolution interrogation of synaptic-like sympathetic modulation. This approach opens new avenues for mechanistic studies and drug testing in developmentally relevant neuro-cardiac systems.
PMID:41002399 | DOI:10.3390/cells14181434
