Functional Microdomains in Heart’s Pacemaker: A Step Beyond Classical Electrophysiology and Remodeling

Spontaneous beating of the sinoatrial node (SAN), the primary pacemaker of the heart, is initiated, sustained, and regulated by a complex system that integrates ion channels and transporters on the cell membrane surface (often referred to as “membrane clock”) with subcellular calcium handling machinery (by parity of reasoning referred to as an intracellular “Ca2+ clock”). Stable, rhythmic beating of the SAN is ensured by a rigorous synchronization between these two clocks highlighted in the coupled-clock system concept of SAN timekeeping. The emerging results demonstrate that such synchronization of the complex pacemaking machinery at the cellular level depends on tightly regulated spatiotemporal signals which are restricted to precise sub-cellular microdomains and associated with discrete clusters of different ion channels, transporters, and regulatory receptors. It has recently become evident that within the microdomains, various proteins form an interacting network and work together as a part of a macromolecular signaling complex. These protein–protein interactions are tightly controlled and regulated by a variety of neurohormonal signaling pathways and the diversity of cellular responses achieved with a limited pool of second messengers is made possible through the organization of essential signal components in particular microdomains. In this review, we highlight the emerging understanding of the functionality of distinct subcellular microdomains in SAN myocytes and their functional role in the accumulation and neurohormonal regulation of proteins involved in cardiac pacemaking. We also demonstrate how changes in scaffolding proteins may lead to microdomain-targeted remodeling and regulation of pacemaker proteins contributing to SAN dysfunction

Reduced response to IKr blockade and altered hERG1a/1b stoichiometryin human heart failure

Heart failure (HF) claims 250,000 lives per year in the US, and nearly half of these deaths are sudden and presumably due to ventricular tachyarrhythmias. QT interval and action potential (AP) prolongation are hallmark proarrhythmic changes in the failing myocardium, which potentially result from alterations in repolarizing potassium currents. Thus,we aimed to examinewhether decreased expression of the rapid delayed rectifier potassiumcurrent, IKr, contributes to repolarization abnormalities in human HF. Tomap functional IKr expression across the left ventricle (LV), we optically imaged coronary-perfused LV free wall from donor and end-stage failing human hearts. The LV wedge preparation was used to examine transmural AP durations at 80% repolarization (APD80), and treatment with the IKr-blocking drug, E-4031, was utilized to interrogate functional expression. We assessed the percent change in APD80 post-IKr blockade relative to baseline APD80 (ΔAPD80) and found that ΔAPD80s are reduced in failing versus donor hearts in each transmural region, with 0.35-, 0.43-, and 0.41-fold reductions in endo-, mid-, and epicardium, respectively (p = 0.008, 0.037, and 0.022). We then assessed hERG1 isoform gene and protein expression levels using qPCR and Western blot. While we did not observe differences in hERG1a or hERG1b gene expression between donor and failing hearts, we found a shift in the hERG1a:hERG1b isoform stoichiometry at the protein level. Computer simulations were then conducted to assess IKr block under E-4031 influence in failing and nonfailing conditions. Our results confirmed the experimental observations and E-4031-induced relative APD80 prolongationwas greater in normal conditions than in failing conditions, provided that the cellularmodel of HF included a significant downregulation of IKr. In humanHF, the response to IKr blockade is reduced, suggesting decreased functional IKr expression. This attenuated functional response is associated with altered hERG1a:hERG1b protein stoichiometry in the failing human LV, and failing cardiomyoctye simulations support the experimental findings. Thus, of IKr protein and functional expression may be important determinants of repolarization remodeling in the failing human LV.We thank the Translational Cardiovascular Biobank & Repository (TCBR) at Washington University for provision of donor/patient records. The TCBR is supported by the NIH/CTSA (UL1 TR000448), Children's Discovery Institute, and Richard J. Wilkinson Trust. We also thank the laboratory of Dr. Sakiyama-Elbert for the use of the StepOnePlus equipment We appreciate the critical feedback on the manuscript by Dr. Jeanne Nerbonne. This work has been supported by the National Heart, Lung & Blood Institute (NHLBI, R01 HL114395). K. Holzem has been supported by the American Heart Association (12PRE12050315) and the NHLBI (F30 HL114310).Holzem, KM.; Gómez García, JF.; Glukhov, AV.; Madden, EJ.; Koppel, AC.; Ewald, GA.; Trénor Gomis, BA.... (2016). Reduced response to IKr blockade and altered hERG1a/1b stoichiometryin human heart failure. Journal of Molecular and Cellular Cardiology. 96:82-92. https://doi.org/10.1016/j.yjmcc.2015.06.008S82929

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry techniques are well-suited to high-throughput characterization of natural products, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social molecular networking (GNPS, http://gnps.ucsd.edu), an open-access knowledge base for community wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of ‘living data’ through continuous reanalysis of deposited data

Atrial Fibrillation and Fibrosis: Beyond the Cardiomyocyte Centric View

Atrial fibrillation (AF) associated with fibrosis is characterized by the appearance of interstitial myofibroblasts. These cells are responsible for the uncontrolled deposition of the extracellular matrix, which pathologically separate cardiomyocyte bundles. The enhanced fibrosis is thought to contribute to arrhythmias “indirectly” because a collagenous septum is a passive substrate for propagation, resulting in impulse conduction block and/or zigzag conduction. However, the emerging results demonstrate that myofibroblasts in vitro also promote arrhythmogenesis due to direct implications upon cardiomyocyte electrophysiology. This electrical interference may be considered beneficial as it resolves any conduction blocks; however, the passive properties of myofibroblasts might cause a delay in impulse propagation, thus promoting AF due to discontinuous slow conduction. Moreover, low-polarized myofibroblasts reduce, via cell-density dependence, the fast driving inward current for cardiac impulse conduction, therefore resulting in arrhythmogenic uniformly slow propagation. Critically, the subsequent reduction in cardiomyocytes resting membrane potential in vitro significantly increases the likelihood of ectopic activity. Myofibroblast densities and the degree of coupling at cellular border zones also impact upon this likelihood. By considering future in vivo studies, which identify myofibroblasts “per se” as a novel targets for cardiac arrhythmias, this review aims to describe the implications of noncardiomyocyte view in the context of AF

Cellular and Molecular Mechanisms of Functional Hierarchy of Pacemaker Clusters in the Sinoatrial Node: New Insights into Sick Sinus Syndrome

The sinoatrial node (SAN), the primary pacemaker of the heart, consists of a heterogeneous population of specialized cardiac myocytes that can spontaneously produce action potentials, generating the rhythm of the heart and coordinating heart contractions. Spontaneous beating can be observed from very early embryonic stage and under a series of genetic programing, the complex heterogeneous SAN cells are formed with specific biomarker proteins and generate robust automaticity. The SAN is capable to adjust its pacemaking rate in response to environmental and autonomic changes to regulate the heart’s performance and maintain physiological needs of the body. Importantly, the origin of the action potential in the SAN is not static, but rather dynamically changes according to the prevailing conditions. Changes in the heart rate are associated with a shift of the leading pacemaker location within the SAN and accompanied by alterations in P wave morphology and PQ interval on ECG. Pacemaker shift occurs in response to different interventions: neurohormonal modulation, cardiac glycosides, pharmacological agents, mechanical stretch, a change in temperature, and a change in extracellular electrolyte concentrations. It was linked with the presence of distinct anatomically and functionally defined intranodal pacemaker clusters that are responsible for the generation of the heart rhythm at different rates. Recent studies indicate that on the cellular level, different pacemaker clusters rely on a complex interplay between the calcium (referred to local subsarcolemmal Ca2+ releases generated by the sarcoplasmic reticulum via ryanodine receptors) and voltage (referred to sarcolemmal electrogenic proteins) components of so-called “coupled clock pacemaker system” that is used to describe a complex mechanism of SAN pacemaking. In this review, we examine the structural, functional, and molecular evidence for hierarchical pacemaker clustering within the SAN. We also demonstrate the unique molecular signatures of intranodal pacemaker clusters, highlighting their importance for physiological rhythm regulation as well as their role in the development of SAN dysfunction, also known as sick sinus syndrome

Microdomain-specific localization of functional ion channels in cardiomyocytes: an emerging concept of local regulation and remodelling

Cardiac excitation involves the generation of action potential by individual cells and the subsequent conduction of the action potential from cell to cell through intercellular gap junctions. Excitation of the cellular membrane results in opening of the voltage-gated L-type calcium ion (Ca(2+)) channels, thereby allowing a small amount of Ca(2+) to enter the cell, which in turn triggers the release of a much greater amount of Ca(2+) from the sarcoplasmic reticulum, the intracellular Ca(2+) store, and gives rise to the systolic Ca(2+) transient and contraction. These processes are highly regulated by the autonomic nervous system, which ensures the acute and reliable contractile function of the heart and the short-term modulation of this function upon changes in heart rate or workload. It has recently become evident that discrete clusters of different ion channels and regulatory receptors are present in the sarcolemma, where they form an interacting network and work together as a part of a macro-molecular signalling complex which in turn allows the specificity, reliability and accuracy of the autonomic modulation of the excitation-contraction processes by a variety of neurohormonal pathways. Disruption in subcellular targeting of ion channels and associated signalling proteins may contribute to the pathophysiology of a variety of cardiac diseases, including heart failure and certain arrhythmias. Recent methodological advances have made it possible to routinely image the topography of live cardiomyocytes, allowing the study of clustering functional ion channels and receptors as well as their coupling within a specific microdomain. In this review we highlight the emerging understanding of the functionality of distinct subcellular microdomains in cardiac myocytes (e.g. T-tubules, lipid rafts/caveolae, costameres and intercalated discs) and their functional role in the accumulation and regulation of different subcellular populations of sodium, Ca(2+) and potassium ion channels and their contributions to cellular signalling and cardiac pathology

Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes

Distinct subpopulations of L-type calcium channels (LTCCs) with different functional properties exist in cardiomyocytes. Disruption of cellular structure may affect LTCC in a microdomain-specific manner and contribute to the pathophysiology of cardiac diseases, especially in cells lacking organized transverse tubules (T-tubules) such as atrial myocytes (AMs)

Role of Pyk2 in cardiac arrhythmogenesis

Proline-rich tyrosine kinase 2 (Pyk2) is a nonreceptor protein kinase regulated by intracellular Ca(2+), CaMK, and PKC and can be activated by different stress signals involved in heart failure. However, Pyk2 has not been investigated in the human heart, and the functional role of Pyk2 signaling at the whole heart level has not been elucidated. We hypothesize that Ca(2+)-dependent activation of Pyk2 is involved in cardiac electrophysiology. We examined the expression of Pyk2 in nonfailing versus ischemic and nonischemic failing human hearts (n = 6 hearts/group). To investigate Pyk2 function, we optically mapped perfused hearts from wild-type (WT; n = 7) and knockout (Pyk2(-/-); n = 8) mice during autonomic stimulation. Experiments were done in control mice and after 1 wk of transverse aortic constriction. We used the Illumina beadarray approach for transcriptional profiling of WT and Pyk2(-/-) mouse ventricles. Western blot analysis revealed a doubling of Pyk2 activation in nonischemic failing versus nonfailing human hearts. In mouse hearts, we observed a much higher probability of ventricular tachyarrhythmia during ACh perfusion in Pyk2(-/-) versus WT mice. Parasympathetic stimulation resulted in a dose-dependent decrease of atrial action potential duration (APD) in both WT and Pyk2(-/-) mice, whereas in ventricles it induced APD shortening in Pyk2(-/-) mice but not in WT mice. Deficiency of Pyk2 abolished ACh-induced prolongation of atrioventricular delay in Pyk2(-/-) mouse hearts but did not affect heart rate. Lower mRNA and protein levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and higher mRNA levels of Na(+)/Ca(2+) exchanger 1 were detected in Pyk2(-/-) hearts compared with WT hearts. The transverse aortic constriction protocol did not change the phenotype. In conclusion, our results indicate a protective role of Pyk2 with respect to ventricular tachyarrhythmia during parasympathetic stimulation by regulation of gene expression related to Ca(2+) handling. We hypothesize that activation of Pyk2 in the human heart during heart failure may contribute to protection against arrhythmia