Akt increases sarcoplasmic reticulum Ca2+ cycling by direct phosphorylation of phospholamban at Thr17.

Cardiomyocytes adapt to physical stress by increasing their size while maintaining cell function. The serine/threonine kinase Akt plays a critical role in this process of adaptation. We previously reported that transgenic overexpression of an active form of Akt (Akt-E40K) in mice results in increased cardiac contractility and cell size, as well as improved sarcoplasmic reticulum (SR) Ca(2+) handling. Because it is not fully elucidated, we decided to study the molecular mechanism by which Akt-E40K overexpression improves SR Ca(2+) handling. To this end, SR Ca(2+) uptake and the phosphorylation status of phospholamban (PLN) were evaluated in heart extracts from wild-type and Akt-E40K mice and mice harboring inducible and cardiac specific knock-out of phosphatidylinositol-dependent kinase-1, the upstream activator of Akt. Moreover, the effect of Akt was assessed in vitro by overexpressing a mutant Akt targeted preferentially to the SR, and by biochemical assays to evaluate potential interaction with PLN. We found that when activated, Akt interacts with and phosphorylates PLN at Thr(17), the Ca(2+)-calmodulin-dependent kinase IIdelta site, whereas silencing Akt signaling, through the knock-out of phosphatidylinositol-dependent kinase-1, resulted in reduced phosphorylation of PLN at Thr(17). Furthermore, overexpression of SR-targeted Akt in cardiomyocytes improved Ca(2+) handling without affecting cell size. Thus, we describe here a new mechanism whereby the preferential translocation of Akt to the SR is responsible for enhancement of contractility without stimulation of hypertrophy.

Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice.

A trial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca2+ handling. Diastolic Ca2+ release from the sarcoplasmic reticulum via "leaky" ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice. Rapid atrial pacing resulted in increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in Ryr2R176Q/+ mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in Ryr2S2814A knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent Ca2+ leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction.

Intracellular calcium leak due to FKBP12.6 deficiency in mice facilitates the inducibility of atrial fibrillation.

BACKGROUND: Although defective Ca(2+) homeostasis may contribute to arrhythmogenesis in atrial fibrillation (AF), the underlying molecular mechanisms remain poorly understood. Studies in patients with AF revealed that impaired diastolic closure of sarcoplasmic reticulum (SR) Ca(2+)-release channels (ryanodine receptors, RyR2) is associated with reduced levels of the RyR2-inhibitory subunit FKBP12.6. OBJECTIVE: The objective of the present study was to test the hypothesis that Ca(2+) leak from the SR through RyR2 increases the propensity for AF in FKBP12.6-deficient (-/-) mice. METHODS: Surface electrocardiogram and intracardiac electrograms were recorded simultaneously in FKBP12.6-/- mice and wild-type (WT) littermates. Right atrial programmed stimulation was performed before and after injection of RyR2 antagonist tetracaine (0.5 mg/kg). Intracellular Ca(2+) transients were recorded in atrial myocytes from FKBP12.6-/- and WT mice. RESULTS: FKBP12.6-/- mice had structurally normal atria and unaltered expression of key Ca(2+)-handling proteins. AF episodes were inducible in 81% of FKBP12.6-/-, but in only 7% of WT mice (P <.05), and were prevented by tetracaine in all FKBP12.6-/- mice. SR Ca(2+) leak in FKBP12.6-/- myocytes was 53% larger than in WT myocytes, and FKBP12.6-/- myocytes showed increased incidence of spontaneous SR Ca(2+) release events, which could be blocked by tetracaine. CONCLUSION: The increased vulnerability to AF in FKBP12.6-/- mice substantiates the notion that defective SR Ca(2+) release caused by abnormal RyR2 and FKBP12.6 interactions may contribute to the initiation or maintenance of atrial fibrillation.

Ryanodine receptors as pharmacological targets for heart disease.

Calcium release from intracellular stores plays an important role in the regulation of muscle contraction and electrical signals that determine the heart rhythm. The ryanodine receptor (RyR) is the major calcium (Ca2+) release channel required for excitation-contraction coupling in the heart. Recent studies have demonstrated that RyR are macromolecular complexes comprising of 4 pore-forming channel subunits, each of which is associated with regulatory subunits. Clinical and experimental studies over the past 5 years have provided compelling evidence that intracellular Ca2+ release channels play a pivotal role in the development of cardiac arrhythmias and heart failure. Changes in the channel regulation and subunit composition are believed to cause diastolic calcium leakage from the sarcoplasmic reticulum, which could trigger arrhythmias and weaken cardiac contractility. Therefore, cardiac RyR have emerged as potential therapeutic targets for the treatment of heart disease. Consequently, there is a strong desire to identify and/or develop novel pharmacological agents that may target these Ca2+ signaling pathways. Pharmacological agents known to modulate RyR in the heart, and their potential application towards the treatment of heart disease are discussed in this review.