Systolic heart failure has been widely reported to involve reduced contraction magnitude of individual cardiomyocytes. However, accumulating data indicate that contraction is also slowed in this condition, which contributes to a reduced power of the heartbeat [1]. We and others have identified that a slowed rising phase of the Ca2+ transient is a critical underlying mechanism [1-3]. Although Ca2+ entry through Ca2+ channels is generally reported to be unaltered in failing cells, triggered release of Ca2+ from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) is de-synchronized and slowed [2,3]. This results, at least in part, from altered structure of T-tubules, which are invaginations of the surface sarcolemma. T-tubules become lost and/or disorganized during heart failure, which effectively removes some Ca2+ channels from their dyadic junctions with clusters of RyRs [2,3]. Thus, a significant proportion of RyRs are “orphaned” during heart failure, and Ca2+ release is delayed at these sites, as it is dependent on Ca2+ diffusion following release from intact dyads. However, we previously reported that irregular gaps between T-tubules and the formation of orphaned RyRs accounted for only a fraction of the overall slowing of the Ca2+ transient in this condition [2]. Thus, we presently hypothesized that alterations in RyR function also contribute to slowing of Ca2+ release in a mouse model of congestive heart failure (CHF) following myocardial infarction. Myocardial infarction was induced by left coronary artery ligation in anaesthetized (2.5% isoflurane inhalation) 8-10 week old C57BL/6 mice. SHAM-operated mice served as controls. Using echocardiography, animals which had developed CHF at 1 week following surgery were distinguished from non-failing animals by increased left atrial diameter (>2.0 mm) and infarct size >40% of the total left ventricular circumference. CHF was then allowed to progress until 10 weeks post-infarction when animals were sacrificed. Cardiomyocytes were isolated from viable regions of the septum. Field-stimulated Ca2+ transients (fluo-4 AM) rose markedly slower in CHF than SHAM myocytes as indicated by longer time to peak values (CHF=152±12% of SHAM, ncells =22, 23 in SHAM, CHF, P<0.05). The rise time of Ca2+ sparks was also increased in CHF (SHAM=9.6±0.6 ms, CHF=13.2±0.7 ms, nsparks= 89, 254 in SHAM, CHF, P<0.05), due to a sub-population of sparks (≈20%) with markedly slowed kinetics. Importantly, these slow sparks were distinct from prolonged, plateau-like Ca2+ release events, known to result from RyR subconductance states [4]. Regions of the cell associated with slow spontaneous sparks also exhibited slowed Ca2+ release during the action potential. Thus, greater variability in spark kinetics in CHF promoted less uniform Ca2+ release across the cell. As expected, dyssynchronous Ca2+ transients in CHF additionally resulted from T-tubule disorganization, as indicated by fast Fourier transforms. However, simultaneous imaging of T-tubules and Ca2+ indicated that slow sparks were not associated with orphaned RyRs. We instead hypothesized that an altered spatial configuration of RyRs could account for slow sparks. Western blotting revealed reduced RyR expression in CHF and, using an extended version of the sticky cluster model [5], we observed that reducing RyR density in the dyad could slow the rate of rise of the Ca2+ sparks. More dramatic slowing of Ca2+ release was modeled by distributing RyRs into sub-clusters, as there was a time delay inherent in the propagation of Ca2+ between sub-clusters. Adjusting other parameters in the model such as the physical coupling (“stickiness”) of RyRs, junctional SR volume, cytosolic and SR [Ca2+], and RyR triggering threshold did not produce slow sparks. Thus, our results suggest that altered configuration of dyadic RyRs can produce abnormally slow Ca2+ sparks in CHF. In combination with disrupted T-tubule structure, these alterations promote slowed, dyssynchronous Ca2+ transients in this condition.