Enhancement of Cardiac Store Operated Calcium Entry (SOCE) within Novel Intercalated Disk Microdomains in Arrhythmic Disease

Store-operated Ca2+ entry (SOCE), a major Ca2+ signaling mechanism in non-myocyte cells, has recently emerged as a component of Ca2+ signaling in cardiac myocytes. Though it has been reported to play a role in cardiac arrhythmias and to be upregulated in cardiac disease, little is known about the fundamental properties of cardiac SOCE, its structural underpinnings or effector targets. An even greater question is how SOCE interacts with canonical excitation-contraction coupling (ECC). We undertook a multiscale structural and functional investigation of SOCE in cardiac myocytes from healthy mice (wild type; WT) and from a genetic murine model of arrhythmic disease (catecholaminergic ventricular tachycardia; CPVT). Here we provide the first demonstration of local, transient Ca2+ entry (LoCE) events, which comprise cardiac SOCE. Although infrequent in WT myocytes, LoCEs occurred with greater frequency and amplitude in CPVT myocytes. CPVT myocytes also evidenced characteristic arrhythmogenic spontaneous Ca2+ waves under cholinergic stress, which were effectively prevented by SOCE inhibition. In a surprising finding, we report that both LoCEs and their underlying protein machinery are concentrated at the intercalated disk (ID). Therefore, localization of cardiac SOCE in the ID compartment has important implications for SOCE-mediated signaling, arrhythmogenesis and intercellular mechanical and electrical coupling in health and disease

Chapter 3: Pathophysiology

The hallmark pathophysiologic feature of dilated cardiomyopathy is systolic dysfunction. Several pathogenetic mechanisms appear to be operative. These include increased hemodynamic overload, ventricular remodeling, excessive neurohumoral stimulation, abnormal myocyte calcium cycling, excessive or inadequate proliferation of the extracellular matrix, accelerated apoptosis, and genetic mutations. Although beneficial in the early stages of heart failure, these compensatory mechanisms eventually lead to a vicious cycle of worsening heart failure. Genetic causes account for 30\u201340% of DCM and involve genes that encode a heterogeneous group of molecules that participate in force generation, force transmission, sarcomere integrity, cytoskeletal and nuclear architecture, electrolyte homeostasis, mitochondrial function, and transcription. Additional research will improve our understanding of the complex and longitudinal molecular changes that lead from gene mutation to clinical expressio

Ca2+-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca2+ Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca2+ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca2+. We show that hyperphosphorylation by exogenous PKA increases open probability (Po) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca2+; lowering [Ca2+] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca2+-sensitive mode of gating. We additionally found that cytosolic incubation with Mg2+/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca2+. We show that the endogenous kinase is both Ca2+-dependent and sensitive to inhibitors of PKC. Moreover, the Ca2+-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca2+-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca2+ regulation

Prostacyclin mediates endothelial COX-2-dependent neuroprotective effects during excitotoxic brain injury

Ying An,1,2 Natalya Belevych,1,2 Yufen Wang,1,2 Hao Zhang,1 Jason S Nasse,3 Harvey Herschman,4 Qun Chen,1,2 Andrew Tarr,1,2 Xiaoyu Liu,1,2 Ning Quan1,21Institute for Behavior Medicine Research, 2Department of Oral Biology, College of Dentistry, 3Neuroscience Graduate Studies Program, The Ohio State University, Columbus, OH, USA; 4Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA, USAAbstract: In a previous study, we found that intracerebral administration of excitotoxin (RS)-(tetrazole-5yl) glycine caused increased neural damage in the brain in an endothelial COX-2 deleted mouse line (Tie2Cre COX-2flox/flox). In this study, we investigated whether prostacyclin might mediate this endothelial COX-2-dependent neuroprotection. Administration of excitotoxin into the striatum induced the production of prostacyclin (PGI2) in wild type, but not in endothelial COX-2 deleted mice. Inhibition of PGI2 synthase exacerbated brain lesions induced by the excitotoxin in wild type, but not in endothelial COX-2 deleted mice. Administration of a PGI2 agonist reduced neural damage in both wild type and endothelial COX-2 deleted mice. Increased PGI2 synthase expression was found in infiltrating neutrophils. In an ex vivo assay, PGI2 reduced the excitotoxin-induced calcium influx into neurons, suggesting a cellular mechanism for PGI2 mediated neuroprotection. These results reveal that PGI2 mediates endothelial COX-2 dependent neuroprotection.Keywords: neural injury, prostaglandins, neutrophil, conditional COX-2 deletion, PGI

Multiscale Determinants of Delayed Afterdepolarization Amplitude in Cardiac Tissue

Spontaneous calcium (Ca) waves in cardiac myocytes underlie delayed afterdepolarizations (DADs) that trigger cardiac arrhythmias. How these subcellular/cellular events overcome source-sink factors in cardiac tissue to generate DADs of sufficient amplitude to trigger action potentials is not fully understood. Here, we evaluate quantitatively how factors at the subcellular scale (number of Ca wave initiation sites), cellular scale (sarcoplasmic reticulum (SR) Ca load), and tissue scale (synchrony of Ca release in populations of myocytes) determine DAD features in cardiac tissue using a combined experimental and computational modeling approach. Isolated patch-clamped rabbit ventricular myocytes loaded with Fluo-4 to image intracellular Ca were rapidly paced during exposure to elevated extracellular Ca (2.7 mmol/L) and isoproterenol (0.25 μmol/L) to induce diastolic Ca waves and subthreshold DADs. As the number of paced beats increased from 1 to 5, SR Ca content (assessed with caffeine pulses) increased, the number of Ca wave initiation sites increased, integrated Ca transients and DADs became larger and shorter in duration, and the latency period to the onset of Ca waves shortened with reduced variance. In silico analysis using a computer model of ventricular tissue incorporating these experimental measurements revealed that whereas all of these factors promoted larger DADs with higher probability of generating triggered activity, the latency period variance and SR Ca load had the greatest influences. Therefore, incorporating quantitative experimental data into tissue level simulations reveals that increased intracellular Ca promotes DAD-mediated triggered activity in tissue predominantly by increasing both the synchrony (decreasing latency variance) of Ca waves in nearby myocytes and SR Ca load, whereas the number of Ca wave initiation sites per myocyte is less important

Dantrolene rescues aberrant N-terminus intersubunit interactions in mutant pro-arrhythmic cardiac ryanodine receptors

Aims The ryanodine receptor (RyR2) is an intracellular Ca2+ release channel essential for cardiac excitation–contraction coupling. Abnormal RyR2 channel function results in the generation of arrhythmias and sudden cardiac death. The present study was undertaken to investigate the mechanistic basis of RyR2 dysfunction in inherited arrhythmogenic cardiac disease. Methods and results We present several lines of complementary evidence, indicating that the arrhythmia-associated L433P mutation disrupts RyR2 N-terminus self-association. A combination of yeast two-hybrid, co-immunoprecipitation, and chemical cross-linking assays collectively demonstrate that a RyR2 N-terminal fragment carrying the L433P mutation displays substantially reduced self-interaction compared with wild type. Moreover, sucrose density gradient centrifugation reveals that the L433P mutation impairs tetramerization of the full-length channel. [3H]Ryanodine-binding assays demonstrate that disrupted N-terminal intersubunit interactions within RyR2L433P confer an altered sensitivity to Ca2+ activation. Calcium imaging of RyR2L433P-expressing cells reveals substantially prolonged Ca2+ transients and reduced Ca2+ store content indicating defective channel closure. Importantly, dantrolene treatment reverses the L433P mutation-induced impairment and restores channel function. Conclusion The N-terminus domain constitutes an important structural determinant for the functional oligomerization of RyR2. Our findings are consistent with defective N-terminus self-association as a molecular mechanism underlying RyR2 channel deregulation in inherited arrhythmogenic cardiac disease. Significantly, the therapeutic action of dantrolene may occur via the restoration of normal RyR2 N-terminal intersubunit interactions