RRD of APD modulation is shared, although with differences in magnitude, by interventions of very different nature. RRD can be interpreted as a consequence of the relationship between I(m) and APD and, as such, is expected in all species having positive APD-CL relationship. This implies that the development of agents prolonging APD with direct rate dependency, or even completely devoid of RRD, may be difficult to achieve.

The BS pattern of p.S216L might result from a decrease in I(Na) density, which masked gating abnormalities that might otherwise result in a LQT phenotype. The p.H558R polymorphism decreased p.S216L expressivity, partly by lessening p.S216L effects and partly through the induction of further gating abnormalities suitable to blunt p.S216L effects during repolarization.

Cardiac progenitor cells (CPCs), migrating from heart tissue, in culture aggregate to form cardiospheres (CSs) in which replication and cardiogenic differentiation occur. However, the frequency of functional differentiation in CSs and the role of cell clustering in supporting it remain to be established. The aim of our study is to quantify differentiation of a muscle-type Ca(2+) release mechanism in CS-derived cells, correlate it with cardiac differentiation markers and test its dependency on CS formation. CPCs migrating from murine cardiac explants were studied prior and after CSs formation (Pre-CS and Post-CS). Inducibility of RyR- and IP3-R-mediated Ca(2+) transients in individual cells was tested by exposure to caffeine and ATP, respectively; expression of cardiac and non-cardiac lineage markers was assessed. Caffeine responsiveness was negligible in Pre-CS cells and increased by 7.5 fold in Post-CS cells (3.6 vs. 26.9%; p < 0.05), and was closely correlated with activation of the cardiac TnI gene promoter. ATP-induced responses, frequent in Pre-CS (86%), were slightly increased in Post-CS cells (94%; p < 0.05). Expression of cardiac-specific Ca(2+)-handling proteins (Cav1.2, NCX1, RyR2, SERCA2a) was either limited to the Post-CS stage, or markedly enhanced. CS beating was infrequent, but its pharmacology was compatible with cardiac excitation-contraction coupling. Expression of non-cardiac lineage was low in general, and similar between Pre- and Post-CS cells. Culture conditions inhibiting CSs formation prevented the increase in caffeine responders. In conclusion, clustering in CSs leads to the induction of a muscle-specific functional response in about 30% of CPCs; this is accompanied by development of a cardiac-specific expression pattern.

Cardiac progenitor cells (CPCs) form three-dimensional structure named cardiospheres (CSs). While CSs are seen as partially committed cardiac precursors, it is unknown whether they already display cardiac-type molecular functions. Whereas IP3-mediated Ca 2+ release is a shared property of many cell types, Ca + release through RyR channels is muscle specific. Aims: To test if cardiac-type Ca 2+ release mechanism is present in CPCs and whether it may develop during the CSs stage. Methods: Cells arose from murine cardiac explants in culture were studied prior to CSs formation (pre-CSs) and after expansion of CSs (post-CSs). Ca 2+ transients were detected in wide optical confocal fields by Fluo4-AM fluorescence. RyR- and IP3-R-mediated Ca 2+ release from intracellular stores was tested by exposures to caffeine (CAF, 10 mM) or ATP (200 μM) respectively in Ca 2+ -free conditions. The expression of the cardiac-specific RyR isoform (RyR2) was tested by immunolabeling and western-blot analysis. Results: In isolated cells CAF-induced response was almost exclusive of post-CSs cells (22.4 % vs. 3.62 %; p<0.05); ATP-induced response was already present in pre-CSs cells and showed only a small increase in post-CSs ones (94.3 % vs 86.1 % p<0.05). The ratio between post-CSs and pre-CSs responses was 6.2 for CAF and 1.1 for ATP. Both CAF and ATP responses were suppressed by the SERCA inhibitor CPA (50 μM), thus confirming intracellular stores as the Ca 2+ source. ATP response only was suppressed by the IP3-R blocker 2APB (10 μM). Ryanodine (20 μM) prevented the response to CAF, but not to ATP. In post-CSs RyR2 protein levels were higher than in pre-CSs and similar to those of adult myocytes. Immunoistochemistry analysis of post-CSs cells results in a distribution of RyR2 expression consistent with immature neonatal cardiomyocytes previously described. Conclusions: At variance with IP3-mediated signaling, RyR mediated Ca 2+ release develops during maturation within the CSs environment, along with expression of the cardiac RyR isoform. Detection of caffeine-induced Ca 2+ responses may be useful in identifying cardio-specific functional maturation in progenitor cell populations.