We present a model that provides a unified framework for studying Ca2+ sparks and Ca2+ waves in cardiac cells. The model is novel in combining 1) use of large currents (approximately 20 pA) through the Ca2+ release units (CRUs) of the sarcoplasmic reticulum (SR); 2) stochastic Ca2+ release (or firing) of CRUs; 3) discrete, asymmetric distribution of CRUs along the longitudinal (separation distance of 2 microm) and transverse (separated by 0.4-0.8 microm) directions of the cell; and 4) anisotropic diffusion of Ca2+ and fluorescent indicator to study the evolution of Ca2+ waves from Ca2+ sparks. The model mimics the important features of Ca2+ sparks and Ca2+ waves in terms of the spontaneous spark rate, the Ca2+ wave velocity, and the pattern of wave propagation. Importantly, these features are reproduced when using experimentally measured values for the CRU Ca2+ sensitivity (approximately 15 microM). Stochastic control of CRU firing is important because it imposes constraints on the Ca2+ sensitivity of the CRU. Even with moderate (approximately 5 microM) Ca2+ sensitivity the very high spontaneous spark rate triggers numerous Ca2+ waves. In contrast, a single Ca2+ wave with arbitrarily large velocity can exist in a deterministic model when the CRU Ca2+ sensitivity is sufficiently high. The combination of low CRU Ca2+ sensitivity (approximately 15 microM), high cytosolic Ca2+ buffering capacity, and the spatial separation of CRUs help control the inherent instability of SR Ca2+ release. This allows Ca2+ waves to form and propagate given a sufficiently large initiation region, but prevents a single spark or a small group of sparks from triggering a wave.

The clustering of ryanodine receptors (RyR2) into functional Ca2+ release units is central to current models for cardiac excitation-contraction (E-C) coupling. Using immunolabeling and confocal microscopy, we have analyzed the distribution of RyR2 clusters in rat and ventricular atrial myocytes. The resolution of the three-dimensional structure was improved by a novel transverse sectioning method as well as digital deconvolution. In contrast to earlier reports, the mean RyR2 cluster transverse spacing was measured 1.05 microm in ventricular myocytes and estimated 0.97 microm in atrial myocytes. Intercalated RyR2 clusters were found interspersed between the Z-disks on the cell periphery but absent in the interior, forming double rows flanking the local Z-disks on the surface. The longitudinal spacing between the adjacent rows of RyR2 clusters on the Z-disks was measured to have a mean value of 1.87 microm in ventricular and 1.69 microm in atrial myocytes. The measured RyR2 cluster distribution is compatible with models of Ca2+ wave generation. The size of the typical RyR2 cluster was close to 250 nm, and this suggests that approximately 100 RyR2s might be present in a cluster. The importance of cluster size and three-dimensional spacing for current E-C coupling models is discussed.

Rationale Increased contractility of arterial myocytes and enhanced vascular tone during hyperglycemia and diabetes may arise from impaired large conductance Ca2+-activated K+ (BKCa) channel function. The scaffolding protein AKAP150 is a key regulator of calcineurin (CaN), a phosphatase known to modulate expression of the regulatory BKCa β1 subunit. Whether AKAP150 mediates BKCa channel suppression during hyperglycemia and diabetes is unknown. Objective To test the hypothesis that AKAP150-dependent CaN signaling mediates BKCa β1 downregulation and impaired vascular BKCa channel function during hyperglycemia and diabetes. Methods and Results We found that AKAP150 is an important determinant of BKCa channel remodeling, CaN/NFATc3 activation, and resistance artery constriction in hyperglycemic animals on high fat diet (HFD). Genetic ablation of AKAP150 protected against these alterations, including augmented vasoconstriction. D-glucose-dependent suppression of BKCa channel β1 subunits required Ca2+ influx via voltage-gated L-type Ca2+ channels and mobilization of a CaN/NFATc3 signaling pathway. Remarkably, HFD mice expressing a mutant AKAP150 unable to anchor CaN resisted activation of NFATc3 and downregulation of BKCa β1 subunits, and attenuated HFD-induced elevation in arterial blood pressure. Conclusions Our results support a model whereby subcellular anchoring of CaN by AKAP150 is a key molecular determinant of vascular BKCa channel remodeling, which contributes to vasoconstriction during diabetes.