Background Electrical, structural and Ca2+-handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic-reticulum Ca2+-release events (SCaEs) in longstanding persistent AF, but the occurrence and mechanisms of SCaEs in paroxysmal AF (pAF) are unknown. Method and Results Right-atrial appendages from control sinus-rhythm patients (Ctl) or patients with pAF (last episode median 10-20 days preoperatively) were analyzed with simultaneous measurements of [Ca2+]i (Fluo-3) and membrane-currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, as well as Western blot. Action potential duration, L-type Ca2+-current and Na+/Ca2+-exchange current were unaltered in pAF, indicating absence of AF-induced electrical remodeling. In contrast, there was an increased incidence of delayed afterdepolarizations (DADs) in pAF. Ca2+-transient (CaT)-amplitude and sarcoplasmic-reticulum Ca2+-load (caffeine-induced CaT-amplitude, integrated membrane current) were larger in pAF. CaT-decay was faster in pAF but decay of caffeine-induced CaT was unaltered, suggesting increased Serca2a function. In agreement, phosphorylation (inactivation) of the Serca2a-inhibitor protein phospholamban was increased in pAF. Ryanodine-receptor (RyR2) fractional phosphorylation was unaltered in pAF, whereas RyR2-expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both RyR2 dysregulation and enhanced Serca2a activity promote increased sarcoplasmic-reticulum Ca2+-leak and SCaEs, causing DADs/triggered activity in pAF. Conclusions Increased diastolic sarcoplasmic-reticulum Ca2+-leak and related DADs/triggered activity promote cellular arrhythmogenesis in pAF-patients. Biochemical, functional and modeling studies point to a combination of increased sarcoplasmic-reticulum Ca2+-load related to phospholamban-hyperphosphorylation and RyR2 dysregulation as underlying mechanisms.

Background The progression of atrial fibrillation (AF) from paroxysmal to persistent forms remains a major clinical challenge. Abnormal sarcoplasmic reticulum (SR) Ca2+-leak via the ryanodine receptor (RyR2) has been observed as a source of ectopic activity in various AF models. However, its potential role in progression to long-lasting spontaneous AF (sAF) has never been tested. This study tested the hypothesis that enhanced RyR2-mediated Ca2+-release underlies the development of a substrate for sAF and to understand the underlying mechanisms. Methods and Results CREM-IbΔC-X transgenic (CREM)-mice developed age-dependent progression from spontaneous atrial ectopy to paroxysmal and eventually long-lasting AF. The development of sAF in CREM-mice was preceded by enhanced diastolic Ca2+-release, atrial enlargement and marked conduction abnormalities. Genetic inhibition of CaMKII-mediated RyR2-S2814 phosphorylation in CREM-mice normalized open probability of RyR2-channels and SR Ca2+-release, delayed the development of spontaneous atrial ectopy, fully prevented sAF, suppressed atrial dilation and forestalled atrial conduction-abnormalities. Hyperactive RyR2-channels directly stimulated the Ca2+-dependent hypertrophic pathway NFAT/Rcan1-4, suggesting a role for the NFAT/Rcan1-4 system in the development of a substrate for long-lasting AF in CREM mice. Conclusions RyR2-mediated SR Ca2+-leak directly underlies the development of a substrate for sAF in CREM-mice, the first demonstration of a molecular mechanism underlying AF-progression and sAF substrate development in an experimental model. Our work demonstrates that the role of abnormal diastolic Ca2+ release in AF may not be restricted to the generation of atrial ectopy, but extends to the development of atrial remodeling underlying the AF substrate.

Our findings suggest that JPH2 is necessary for TT maturation during postnatal cardiac development in mice. In particular, JPH2 may be critical in anchoring the invaginating sarcolemma to the sarcoplasmic reticulum, thereby enabling the maturation of the TT network.

Rationale Junctional membrane complexes (JMC) in myocytes are critical microdomains, in which excitation-contraction coupling occurs. Structural and functional disruption of JMCs underlies contractile dysfunction in failing hearts. However, the role of newly identified JMC protein ‘striated muscle preferentially expressed gene’ (SPEG) remains unclear. Objective To determine the role of SPEG in healthy and failing adult hearts. Methods and Results Proteomic analysis of immunoprecipatated JMC-proteins ryanodine receptor type-2 (RyR2) and junctophilin-2 (JPH2) followed by mass spectrometry identified the serine-threonine kinase SPEG as the only novel binding partner for both proteins. Real-time PCR revealed downregulation of SPEG mRNA levels in failing human hearts. A novel cardiac myocyte-specific Speg conditional knockout (MCM-Spegfl/fl) model revealed that adult-onset SPEG-deficiency results in heart failure. Calcium (Ca2+) and transverse-tubule (TT) imaging of ventricular myocytes from MCM-Spegfl/fl mice post heart failure revealed both increased SR Ca2+ spark frequency and disrupted JMC integrity. Additional studies revealed that TT disruption precedes the development of heart failure development in MCM-Spegfl/fl mice. Although total JPH2 levels were unaltered, JPH2 phosphorylation levels were found to be reduced in MCM-Spegfl/fl mice, suggesting that loss of SPEG phosphorylation of JPH2 led to TT disruption, a precursor of heart failure development in SPEG deficient mice. Conclusion The novel JMC protein SPEG is downregulated in human failing hearts. Acute loss of SPEG in mouse hearts causes JPH2 dephosphorylation and TT loss associated with downstream Ca2+ mishandling leading to heart failure. Our study suggests that SPEG could be a novel target for the treatment of heart failure.