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.

Background: Enhanced diastolic calcium (Ca 2+ ) release via ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum (SR) Ca 2+ leak is caused by increased RyR2 phosphorylation by protein kinase A (PKA) or Ca 2+ /calmodulin-dependent kinase-II (CaMKII) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus sought to determine the role of 'striated muscle preferentially expressed protein kinase' (SPEG), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. Methods: Western blotting was performed with right atrial biopsies from paroxysmal (p)AF patients. SPEG atrial knock-out (aKO) mice were generated using adeno-associated virus 9 (AAV9). In mice, AF inducibility was determined using intracardiac programmed electrical stimulation (PES), and diastolic Ca 2+ leak in atrial cardiomyocytes was assessed using confocal Ca 2+ imaging. Phospho-proteomics studies and western blotting were used to measure RyR2 phosphorylation. In order to test the effects of RyR2-S2367 phosphorylation, knock-in mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated using CRISPR-Cas9. Results: Western blotting revealed decreased SPEG protein levels in atrial biopsies from pAF patients in comparison to patients in sinus rhythm. SPEG aKO mice exhibited increased susceptibility to pacing-induced AF by PES and enhanced Ca 2+ spark frequency in atrial cardiomyocytes with Ca 2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phospho-proteomics in hearts from SPEG cardiomyocyte knock-out mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Additionally, western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in pAF patients. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF as well as aberrant atrial SR Ca 2+ leak. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. Conclusions: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated SR Ca 2+ -release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with pAF. Studies in S2367 knock-in mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.

Rationale: Somatic overexpression in mice using adeno-associated virus (AAV) as gene transfer vectors has become a valuable tool to analyze the roles of specific genes in cardiac diseases. The lack of atrial-specific AAV vector has been a major obstacle for studies into the pathogenesis of atrial diseases. Moreover, gene therapy studies for atrial fibrillation would benefit from atrialspecific vectors. Atrial natriuretic factor (ANF) promoter drives gene expression specifically in atrial cardiomyocytes. Objective: To establish the platform of atrial specific in vivo gene delivery by AAV-ANF. Methods and Results: We constructed AAV vectors based on serotype 9 (AAV9) that are driven by the atrial-specific ANF promoter. Hearts from mice injected with AAV9-ANF-GFP exhibited strong and atrial-specific GFP expression without notable GFP in ventricular tissue.

No abstract

Proper cardiac Ca2+ homeostasis is essential for normal excitation-contraction coupling. Perturbations in cardiac Ca2+ handling through altered kinase activity has been implicated in altered cardiac contractility and arrhythmogenesis. Thus, a better understanding of cardiac Ca2+ handling regulation is vital for a better understanding of various human disease processes. ‘Striated muscle preferentially expressed protein kinase’ (SPEG) is a member of the myosin light chain kinase family that is key for normal cardiac function. Work within the last five years has revealed that SPEG has a crucial role in maintaining normal cardiac Ca2+ handling through maintenance of transverse tubule formation and phosphorylation of junctional membrane complex proteins. Additionally, SPEG has been causally impacted in human genetic diseases such as centronuclear myopathy and dilated cardiomyopathy as well as in common acquired cardiovascular disease such as heart failure and atrial fibrillation. Given the rapidly emerging role of SPEG as a key cardiac Ca2+ regulator, we here present this review in order to summarize recent findings regarding the mechanisms of SPEG regulation of cardiac excitation-contraction coupling in both physiology and human disease. A better understanding of the roles of SPEG will be important for a more complete comprehension of cardiac Ca2+ regulation in physiology and disease.

Aims Abnormal intracellular calcium handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signaling pathway contributes to atrial remodeling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models. Methods and results Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF. NFATc1 and NFATc2 mRNA levels were elevated in chronic AF (cAF) patients compared with those in sinus rhythm (SR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM: NFATc2-/- mice. Programmed electrical stimulation revealed that CREM: NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with WT controls, which was reversed in CREM: NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM: NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on RyR2 in CREM: RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras And EF-Hand Domain-Containing Protein (RASEF) as a direct target of NFATc2 mediated transcription. Conclusion Our findings reveal activation of the NFAT signaling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model. TRANSLATIONAL PERSPECTIVE Atrial fibrillation (AF) is a progressive disease characterized by electrical and structural remodeling which promotes atrial arrhythmias. This study provides evidence for increased ‘nuclear factor of activated T-cell’ (NFAT) signaling in patients with chronic AF. Studies in the CREM transgenic model of progressive AF revealed that the NFATc2 isoform mediates atrial remodeling associated with AF substrate development. Chromatin immunoprecipitation sequencing of atrial biopsies from AF patients identified ‘Ras And EF-Hand Domain-Containing Protein’ (RASEF) as a downstream target of NFATc2-mediated transcription, suggesting that targeting these factors might be beneficial for curtailing AF progression.

In this study, we developed and characterized a transgenic mouse model for studying the cardiac pathogenesis of myotonic dystrophy type 1

There are now over 30 genetic loci associated with atrial fibrillation. A better understanding of how these loci relate to disease pathogenesis may provide insight into novel therapeutic targets and ultimately lead to improved clinical care.