Cultured adult cardiac myocytes represent an important approach to study the failing heart on the cellular level. We have developed a long-term culture of adult rat ventricular myocytes based on serum-free medium and substrates coated by extracellular matrix proteins allowing the sustained adenovirus-mediated expression of the Ca²⁺ sensor ‘inverse pericam’. We used this single-cell model for the current work to investigate the effects of chronic neuroendocrine Gq-coupled stimulation on cellular and subcellular Ca²⁺ signalling. We assessed the loading state of internal Ca²⁺ stores and the activity of transport systems in Fura-2 loaded myocytes following caffeine applications (10mM), while spatially resolved Ca²⁺ imaging (realtime confocal microscopy) was undertaken to characterise the spatio-temporal features of Ca²⁺ sparks and waves. The control cells displayed a 4-fold augmentation of the wave frequency from day 6 onwards. Also on day 6, the spark distribution changed from a cytoplasmic spread into a sub-plasma membrane ring. Interestingly we could measure a 2-fold increase of the size of the spark spread from day 1 onwards. Furthermore, on days in-vitro 3 and 6, we observed decreased amplitudes of caffeine-induced Ca²⁺ transients. The latter ones displayed two distinct decay time constants: a slow one due to Na/Ca exchangers and a rapid one influenced to a large extent by sarcoplasmic reticulum (SR)-Ca²⁺ ATPases (SERCA), the precise role of which in the failing heart is still under controversy. After one week of culture, we could measure a 2-fold increase of the rapid decay time constant. Chronic stimulation with phenylephrine (10-100µM) or endothelin-1 (100nM) led to a rapid (2 days) dedifferentiation of the myocytes into so called myoballs, which formed spreading complexes from day 6 onwards, the spontaneous activity of which was predominantly waves. In the one week old Gq-coupled stimulated cells, the decreased amplitudes of caffeine responses were abolished. On the contrary, the rapid decay time constant was increased by 50% compared to control cells at the same stage. We conclude that chronic neuroendocrine stimulation leads to a remodelling of the cellular Ca²⁺ handling, especially in terms of SR loading and activity of the SERCA pumps, which could be a key target for the hormones tested. Moreover, neuroendocrine stimulation of adult myocytes might thus be an important part of cardiac remodelling in-vivo that can be mimicked and studied in an in-vitro model of adult myocytes.