Abnormal cellular Ca2+ handling and excitation-contraction coupling are associated with reduced contractile function and life-threatening arrhythmias in heart failure (HF). One of the mechanisms contributing to this process is aberrant Ca2+ release through ryanodine receptors (RyRs). In HF and myocardial infarction (MI), the loss of sarcoplasmic reticulum Ca2+ through ‘leaky’ RyRs is due to post-translational modifications of these receptors, which lead to alterations in RyR Ca2+ sensitivity and open probability. Cellular remodeling and alterations in microdomain signaling can further augment spontaneous Ca2+ release through ‘leaky’ RyRs, resulting in contractile dysfunction and arrhythmia. We further investigated how local signaling pathways involving reactive oxygen species (ROS), Ca2+/calmodulin-dependent kinase II (CaMKII) and nitric oxide (NO) modulate different subsets of RyRs (i.e. coupled and non-coupled RyRs) in healthy and diseased hearts. We studied RyR organization and function in healthy, myocardial infarction (MI) and human HF cardiomyocytes. Our data show a specific microdomain-dependent modulation of dyadic RyRs in the healthy heart, which is subject to remodeling with disease. In MI and HF, altered microdomain modulation of RyRs enhances spontaneous Ca2+ release from non-dyadic sites, dependent on CaMKII activation and mitochondrial ROS production. This specific modulation may generate a substrate for arrhythmia that appears to be responsive to selective pharmacological inhibition.