Phosphodiesterase type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins.

Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye domain containing (POPDC) proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that POPDC proteins exist in complexes with members of the PDE4 family in cardiac myocytes. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 modulates cycle length of spontaneous Ca2+ transients firing in intact mouse sinoatrial nodes

Activation of RyR2 by class I kinase inhibitors

Kinase inhibitors are a common treatment for cancer. Class I kinase inhibitors that target the ATP-binding pocket are particularly prevalent. Many of these compounds are cardiotoxic and can cause arrhythmias. Spontaneous release of Ca2+ via cardiac ryanodine receptors (RyR2), through a process termed store overload-induced Ca2+ release (SOICR), is a common mechanism underlying arrhythmia. We explored whether class I kinase inhibitors could modify the activity of RyR2 and trigger SOICR to determine if this contributes to the cardiotoxic nature of these compounds.Centro de Investigaciones Cardiovasculare

The Mitochondrial Ca(2+) Uniporter: Structure, Function, and Pharmacology.

Mitochondrial Ca(2+) uptake is crucial for an array of cellular functions while an imbalance can elicit cell death. In this chapter, we briefly reviewed the various modes of mitochondrial Ca(2+) uptake and our current understanding of mitochondrial Ca(2+) homeostasis in regards to cell physiology and pathophysiology. Further, this chapter focuses on the molecular identities, intracellular regulators as well as the pharmacology of mitochondrial Ca(2+) uniporter complex

53 * altered CaMKII and ROS microdomains favor sparks in orphaned RyR after myocardial infarction

Purpose: In ventricular myocytes of rodents, ryanodine receptors (RyRs) are typically organized at Z-lines where the sarcoplasmic reticulum forms dyads with T-tubules (TTs). In large mammals, TT density is lower and not all RyRs are coupled to the TTs. Recently, we have shown that non-coupled RyRs lack a CaMKII and ROS-dependent microdomain modulation during rate adaptation. Here we examine how this microdomain modulation may be altered in ischemic cardiomyopathy where the fraction of non-coupled RyRs is increased. Methods: Using an established pig model of chronic ischemia and myocardial infarction (MI, N=7), we studied myocytes adjacent to the MI and compared these to myocytes from SHAM pigs (N=5) using whole-cell voltage clamp with Fluo-4 as a [Ca2+]i indicator and confocal line scan imaging. Spontaneous Ca2+ sparks were recorded during a 15s period following stimulation and assigned to different subcellular regions categorized as coupled or non-coupled RyR using a specific algorithm. All data were obtained in at least 3 different animals. Results: In myocytes from SHAM, we confirmed the specific modulation of coupled, but not of non-coupled, RyR as seen in control hearts, i.e. an increase of spark frequency at 2 Hz stimulation that is dependent on CaMKII and NOX2-generated ROS (#sparks/100 μm/s in SHAM: 0.8 ± 0.1 at 0.5 Hz to 2.3 ± 0.2 at 2 Hz, vs. 1.5 ± 0.2 with AIP at 2 Hz, and 1.1 ± 0.2 with gp91 ds-tat peptide at 2 Hz, p-value < 0.05 vs. control, n=7-14 in each group). In MI myocytes this modulation of coupled RyR was absent (#sparks/100 μm/s at 2 Hz not different from at 0.5 Hz and no effect of AIP and gp-91 ds-tat peptide). The fraction of non-coupled RyR was larger in MI (orphaned RyR) and their spark frequency at 2 Hz was significantly higher compared to SHAM. In contrast to SHAM, the response of these orphaned RyR was sensitive to CaMKII inhibition (AIP) (at 2 Hz #sparks/100 μm/s was 5.3 ± 0.5 at baseline vs. 3.1 ± 0.6 with AIP, p-value < 0.05, n=8-10 in each group). A significant reduction in spark frequency was observed in these orphaned RyRs after global ROS scavenging (NAC) (2 Hz #sparks/100 μm/s was 5.2 ± 0.5 at baseline vs. 2.9 ± 0.6 with NAC, p-value < 0.05, n=9-10 in each group) and after mitochondrial ROS inhibition using mitoTEMPO (at 2 Hz #sparks/100 μm/s was 2.9 ± 0.4 at baseline vs. 1.1 ± 0.3 with mitoTEMPO, p-value < 0.001, n=8 in each group) while NOX2 inhibition had no effect (gp91 ds-tat peptide, n=10–14). Conclusion: After MI there is a novel RyR microdomain organization favoring sparks in orphaned RyR, possibly related to mitochondrial ROS production

Spontaneous Ca2+ transients in rat pulmonary vein cardiomyocytes are increased in frequency and become more synchronous following electrical stimulation

The pulmonary veins have an external sleeve of cardiomyocytes that are a widely recognised source of ectopic electrical activity that can lead to atrial fibrillation. Although the mechanisms behind this activity are currently unknown, changes in intracellular calcium (Ca2+) signalling are purported to play a role. Therefore, the intracellular Ca2+ concentration was monitored in the pulmonary vein using fluo-4 and epifluorescence microscopy. Electrical field stimulation evoked a synchronous rise in Ca2+ in neighbouring cardiomyocytes; asynchronous spontaneous Ca2+ transients between electrical stimuli were also present. Immediately following termination of electrical field stimulation at 3 Hz or greater, the frequency of the spontaneous Ca2+ transients was increased from 0.45 ± 0.06 Hz under basal conditions to between 0.59 ± 0.05 and 0.65 ± 0.06 Hz (P < 0.001). Increasing the extracellular Ca2+ concentration enhanced this effect, with the frequency of spontaneous Ca2+ transients increasing from 0.45 ± 0.05 Hz to between 0.75 ± 0.06 and 0.94 ± 0.09 Hz after electrical stimulation at 3 to 9 Hz (P < 0.001), and this was accompanied by a significant increase in the velocity of Ca2+ transients that manifested as waves. Moreover, in the presence of high extracellular Ca2+, the spontaneous Ca2+ transients occurred more synchronously in the initial few seconds following electrical stimulation. The ryanodine receptors, which are the source of spontaneous Ca2+ transients in pulmonary vein cardiomyocytes, were found to be arranged in a striated pattern in the cell interior, as well as along the periphery of cell. Furthermore, labelling the sarcolemma with di-4-ANEPPS showed that over 90% of pulmonary vein cardiomyocytes possessed T-tubules. These findings demonstrate that the frequency of spontaneous Ca2+ transients in the rat pulmonary vein are increased following higher rates of electrical stimulation and increasing the extracellular Ca2+ concentration

Electrophysiological heterogeneity in large populations of rabbit ventricular cardiomyocytes

Aims: Cardiac electrophysiological heterogeneity includes: (i) regional differences in action potential (AP) waveform, (ii) AP waveform differences in cells isolated from a single region, (iii) variability of the contribution of individual ion currents in cells with similar AP durations (APDs). The aim of this study is to assess intra-regional AP waveform differences, to quantify the contribution of specific ion channels to the APD via drug responses and to generate a population of mathematical models to investigate the mechanisms underlying heterogeneity in rabbit ventricular cells. Methods and results: APD in ∼50 isolated cells from subregions of the LV free wall of rabbit hearts were measured using a voltage-sensitive dye. When stimulated at 2 Hz, average APD90 value in cells from the basal epicardial region was 254 ± 25 ms (mean ± standard deviation) in 17 hearts with a mean interquartile range (IQR) of 53 ± 17 ms. Endo-epicardial and apical-basal APD90 differences accounted for ∼10% of the IQR value. Highly variable changes in APD occurred after IK(r) or ICa(L) block that included a sub-population of cells (HR) with an exaggerated (hyper) response to IK(r) inhibition. A set of 4471 AP models matching the experimental APD90 distribution was generated from a larger population of models created by random variation of the maximum conductances (Gmax) of 8 key ion channels/exchangers/pumps. This set reproduced the pattern of cell-specific responses to ICa(L) and IK(r) block, including the HR sub-population. The models exhibited a wide range of Gmax values with constrained relationships linking ICa(L) with IK(r), ICl, INCX, and INaK. Conclusion: Modelling the measured range of inter-cell APDs required a larger range of key Gmax values indicating that ventricular tissue has considerable inter-cell variation in channel/pump/exchanger activity. AP morphology is retained by relationships linking specific ionic conductances. These interrelationships are necessary for stable repolarization despite large inter-cell variation of individual conductances and this explains the variable sensitivity to ion channel block