Significance: Mitochondrial calcium uniporter (MCU) is the core channel subunit of the mitochondrial Ca ²⁺ uniporter complex (MCUC) and contributes to the regulation of ATP production. Here, we identify that the adaptive and maladaptive remodeling occurring in rodent and human cardiomyocytes is associated with changes in MCU content, which are inversely correlated with those of its regulator microRNA-1 (miR-1). The present study thus defines the molecular mechanism by which MCU content and, consequently, mitochondrial Ca ²⁺ uptake are directly modified by alteration of miR-1, a key regulator affecting physiological and pathological heart growth. These findings provide evidence for the miR-1/MCU axis as a potential target for the development of novel therapeutic approaches.

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.