Kir2.1 Interactome Mapping Uncovers PKP4 as a Modulator of the Kir2.1-Regulated Inward Rectifier Potassium Currents

Kir2.1, a strong inward rectifier potassium channel encoded by the KCNJ2 gene, is a key regulator of the resting membrane potential of the cardiomyocyte and plays an important role in controlling ventricular excitation and action potential duration in the human heart. Mutations in KCNJ2 result in inheritable cardiac diseases in humans, e.g. the type-1 Andersen-Tawil syndrome (ATS1). Understanding the molecular mechanisms that govern the regulation of inward rectifier potassium currents by Kir2.1 in both normal and disease contexts should help uncover novel targets for therapeutic intervention in ATS1 and other Kir2.1-associated channelopathies. The information available to date on protein-protein interactions involving Kir2.1 channels remains limited. Additional efforts are necessary to provide a comprehensive map of the Kir2.1 interactome. Here we describe the generation of a comprehensive map of the Kir2.1 interactome using the proximity-labeling approach BioID. Most of the 218 high-confidence Kir2.1 channel interactions we identified are novel and encompass various molecular mechanisms of Kir2.1 function, ranging from intracellular trafficking to cross-talk with the insulin-like growth factor receptor signaling pathway, as well as lysosomal degradation. Our map also explores the variations in the interactome profiles of Kir2.1WT versus Kir2.1Δ314-315, a trafficking deficient ATS1 mutant, thus uncovering molecular mechanisms whose malfunctions may underlie ATS1 disease. Finally, using patch-clamp analysis, we validate the functional relevance of PKP4, one of our top BioID interactors, to the modulation of Kir2.1-controlled inward rectifier potassium currents. Our results validate the power of our BioID approach in identifying functionally relevant Kir2.1 interactors and underline the value of our Kir2.1 interactome as a repository for numerous novel biological hypotheses on Kir2.1 and Kir2.1-associated diseases.This work was supported by the National Institutes of Health (NIH) through the National Heart, Lung, and Blood Institute (NHLBI) grant R01HL122352 awarded to J.J., as well as the National Institute of General Medical Sciences (NIGMS) grant R01GM094231 and the National Cancer Institute (NCI) grant U24CA210967 awarded to A.I.N. R.K. is supported by the NCI support grant P30CA046592 awarded to the University of Michigan Rogel Cancer Center. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.S

Transcriptome and proteome mapping in the sheep atria reveal molecular featurets of atrial fibrillation progression.

Atrial fibrillation (AF) is a progressive cardiac arrhythmia that increases the risk of hospitalization and adverse cardiovascular events. There is a clear demand for more inclusive and large-scale approaches to understand the molecular drivers responsible for AF, as well as the fundamental mechanisms governing the transition from paroxysmal to persistent and permanent forms. In this study, we aimed to create a molecular map of AF and find the distinct molecular programmes underlying cell type-specific atrial remodelling and AF progression. We used a sheep model of long-standing, tachypacing-induced AF, sampled right and left atrial tissue, and isolated cardiomyocytes (CMs) from control, intermediate (transition), and late time points during AF progression, and performed transcriptomic and proteome profiling. We have merged all these layers of information into a meaningful three-component space in which we explored the genes and proteins detected and their common patterns of expression. Our data-driven analysis points at extracellular matrix remodelling, inflammation, ion channel, myofibril structure, mitochondrial complexes, chromatin remodelling, and genes related to neural function, as well as critical regulators of cell proliferation as hallmarks of AF progression. Most important, we prove that these changes occur at early transitional stages of the disease, but not at later stages, and that the left atrium undergoes significantly more profound changes than the right atrium in its expression programme. The pattern of dynamic changes in gene and protein expression replicate the electrical and structural remodelling demonstrated previously in the sheep and in humans, and uncover novel mechanisms potentially relevant for disease treatment. Transcriptomic and proteomic analysis of AF progression in a large animal model shows that significant changes occur at early stages, and that among others involve previously undescribed increase in mitochondria, changes to the chromatin of atrial CMs, and genes related to neural function and cell proliferation.This work was supported by the Spanish government (BFU2017-84914-P to M.M.; FPI Fellowship to A.A.-F.; FPU Fellowship to R.R.), and in part by grants to J.J. from the National Heart, Lung and Blood Institute (R01 grant HL122352 NIH/NHLBI), the Leducq Foundation (Transatlantic Network of Excellence Program on Structural Alterations in the Myocardium and the Substrate for Cardiac Fibrillation), and the University of Michigan Health System–Peking University Health Science Center Joint Institute for Translational and Clinical Research (UMHS-PUHSC; project: Molecular Mechanisms of Fibrosis and the Progression from Paroxysmal to Persistent Atrial Fibrillation). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

p38γ/δ activation alters cardiac electrical activity and predisposes to ventricular arrhythmia

We gratefully acknowledge L. Sen-Martín, J. Alegre-Cebollada (CNIC, Madrid) and L. Carrier (University Medical Center HamburgEppendorf and DZHK, Hamburg) for the cMyBP3-C KO cardiac tissue; D. Roiz-Valle and C. López-Otín (IUOPA; Universidad de Oviedo, Oviedo) for the LmnaG609G/G609G cardiac tissue; and R. J. Davis for the MKK6 KO mice. We thank G. Giovinazzo and the CNIC Pluripotent Cell Technology Unit (CNIC, Madrid) for the hiPSCs. We thank S. Bartlett and F. Chanut (CNIC, Madrid) for English editing, and R. R. Mondragon (University of Michigan, Ann Arbor) for technical support. We are grateful to R. J. Davis (University of Massachusetts Chan Medical School, Worcester), A. Padmanabhan (University of California, San Francisco) and M. Costa and C. López-Otín (IUOPA; Universidad de Oviedo, Oviedo) for critical reading of the manuscript. We thank the staf at the CNIC Genomics and Bioinformatics Units for technical support and help with data analysis and A. C. Silva for help with figure editing and design. This work was funded by a CNIC Intramural Project Severo Ochoa (Expediente 12- 2016 IGP) to G.S. and J.J. G.S. is supported by the following projects: PMP21/00057 IMPACT-2021, funded by the Instituto de Salud Carlos III (ISCIII), and PDC2021-121147-I00 and PID2019-104399RB-I00, funded by MCIN/AEI/10.13039/501100011033—all funded by the European Union (FEDER/FSE); ‘Una manera de hacer Europa’/‘El FSE invierte en tu futuro’/Next Generation EU and co-funded by the European Union/Plan de Recuperación, Transformación y Resiliencia (PRTR). R.R.B. is a fellow of the FPU Program (FPU17/03847). B.G.T. was a fellow of the FPI Severo Ochoa CNIC Program (SVP‐2013‐067639) and an American Heart Association Postdoctoral Fellow (18POST34080175). The following grants provided additional funding: Instituto de Salud Carlos III, PDC2021-121147-I00 Convocatoria: Proyectos Prueba de Concepto 2021 Ministerio de Ciencia e Innovación and PID2022-138525OB-I00 Ministerio de Ciencia e Innovación; US National Heart, Lung, and Blood Institute (R01 grant HL122352); Fondos FEDER, Madrid, Spain, and Fundación Bancaria ‘La Caixa (project HR19/52160013) to J.J.; American Heart Association Postdoctoral Fellowship 14POST17820005 to D.P.B.; and MICINN PGC2018-097019-B-I00, ISCIII-SGEFI/ERDF (PRB3-IPT17/0019, ProteoRed), the Fundació Marató TV3 (grant 122/C/2015) and ‘la Caixa’ Banking Foundation (project code HR17- 00247) to J.V. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S, funded by MICIN/AEI/10.13039/501100011033).S

Human influenza A virus causes myocardial and cardiac-specific conduction system infections associated with early inflammation and premature death.

Human influenza A virus (hIAV) infection is associated with important cardiovascular complications, although cardiac infection pathophysiology is poorly understood. We aimed to study the ability of hIAV of different pathogenicity to infect the mouse heart, and establish the relationship between the infective capacity and the associated in vivo, cellular and molecular alterations. We evaluated lung and heart viral titres in mice infected with either one of several hIAV strains inoculated intranasally. 3D reconstructions of infected cardiac tissue were used to identify viral proteins inside mouse cardiomyocytes, Purkinje cells, and cardiac vessels. Viral replication was measured in mouse cultured cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were used to confirm infection and study underlying molecular alterations associated with the in vivo electrophysiological phenotype. Pathogenic and attenuated hIAV strains infected and replicated in cardiomyocytes, Purkinje cells, and hiPSC-CMs. The infection was also present in cardiac endothelial cells. Remarkably, lung viral titres did not statistically correlate with viral titres in the mouse heart. The highly pathogenic human recombinant virus PAmut showed faster replication, higher level of inflammatory cytokines in cardiac tissue and higher viral titres in cardiac HL-1 mouse cells and hiPSC-CMs compared with PB2mut-attenuated virus. Correspondingly, cardiac conduction alterations were especially pronounced in PAmut-infected mice, associated with high mortality rates, compared with PB2mut-infected animals. Consistently, connexin43 and NaV1.5 expression decreased acutely in hiPSC-CMs infected with PAmut virus. YEM1L protease also decreased more rapidly and to lower levels in PAmut-infected hiPSC-CMs compared with PB2mut-infected cells, consistent with mitochondrial dysfunction. Human IAV infection did not increase myocardial fibrosis at 4-day post-infection, although PAmut-infected mice showed an early increase in mRNAs expression of lysyl oxidase. Human IAV can infect the heart and cardiac-specific conduction system, which may contribute to cardiac complications and premature death.JV is a PhD fellow of the La Caixa Foundation International Fellowship Programme (La Caixa/CNB). This work was supported by the European Molecular Biology Organizat ion (STF-7649 to AF), the Spanish Ministry of Science, Innovation and Universities (MCIU), (BFU2011-26175 and BFU2014-57797-R to AN), and the network Ciber de Enfermedades Respiratorias (CIBERES) including the Improvement and Mobilit y Programme. The CNIC is a Severo Ochoa Center of Excellence (SEV-2015-0505). CNIC is supported by MCIU and the Pro CNIC Foundation. This study was supported by grants from Fondo Europeo de Desarrollo Regional (CB16/11/00458), grants SAF2015-65607-R and SAF2016-80324-R from MCIU (A.H. and D.F-R.) and fellowship SVP-2014-068595 to J.A.N-A. This study was supported by Frankel Cardiovascular Centre, Michigan Medicine (Grant 332475). JJ is supported in part by the National Heart, Lung, and Blood Institute (R01 Grant HL122352). S.F.N is supported in part by the National Heart, Lung, and Blood Institute grants R21HL138064 and R01HL129136.S

Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154620/1/fsb2fj201700349r.pd

Metodologías colaborativas a través de las tecnologías: hacia una evaluación equitativa.

Presentación de buenas prácticas metodológicas en el área de tecnología educativa, transferidas por el Grupo de investigación consolidado GTEA, Universidad de Málaga (SEJ-462

SERCA Pump Optimizes Ca(2+) Release by a Mechanism Independent of Store Filling in Smooth Muscle Cells

Thapsigargin-sensitive sarco/endoplasmic reticulum Ca(2+) pumps (SERCAs) are involved in maintaining and replenishing agonist-sensitive internal stores. Although it has been assumed that release channels act independently of SERCA pumps, there are data suggesting the opposite. Our aim was to study the relationship between SERCA pumps and the release channels in smooth muscle cells. To this end, we have rapidly blocked SERCA pumps with thapsigargin, to avoid depletion of the internal Ca(2+) stores, and induced Ca(2+) release with either caffeine, to open ryanodine receptors, or acetylcholine, to open inositol 1,4,5-trisphosphate receptors. Blocking SERCA pumps produced smaller and slower agonist-induced [Ca(2+)](i) responses. We determined the Ca(2+) level of the internal stores both indirectly, measuring the frequency of spontaneous transient outward currents, and directly, using Mag-Fura-2, and demonstrated that the inhibition of SERCA pumps did not produce a reduction of the sarco/endoplasmic reticulum Ca(2+) levels to explain the decrease in the agonist-induced Ca(2+) responses. It appears that SERCA pumps are involved in sustaining agonist-induced Ca(2+) release by a mechanism that involves the modulation of Ca(2+) availability in the lumen of the internal stores

TGF-β1, released by myofibroblasts, differentially regulates transcription and function of sodium and potassium channels in adult rat ventricular myocytes.

Cardiac injury promotes fibroblasts activation and differentiation into myofibroblasts, which are hypersecretory of multiple cytokines. It is unknown whether any of such cytokines are involved in the electrophysiological remodeling of adult cardiomyocytes. We cultured adult cardiomyocytes for 3 days in cardiac fibroblast conditioned medium (FCM) from adult rats. In whole-cell voltage-clamp experiments, FCM-treated myocytes had 41% more peak inward sodium current (I(Na)) density at -40 mV than myocytes in control medium (p<0.01). In contrast, peak transient outward current (I(to)) was decreased by ∼55% at 60 mV (p<0.001). Protein analysis of FCM demonstrated that the concentration of TGF-β1 was >3 fold greater in FCM than control, which suggested that FCM effects could be mediated by TGF-β1. This was confirmed by pre-treatment with TGF-β1 neutralizing antibody, which abolished the FCM-induced changes in both I(Na) and I(to). In current-clamp experiments TGF-β1 (10 ng/ml) prolonged the action potential duration at 30, 50, and 90 repolarization (p<0.05); at 50 ng/ml it gave rise to early afterdepolarizations. In voltage-clamp experiments, TGF-β1 increased I(Na) density in a dose-dependent manner without affecting voltage dependence of activation or inactivation. I(Na) density was -36.25±2.8 pA/pF in control, -59.17±6.2 pA/pF at 0.1 ng/ml (p<0.01), and -58.22±6.6 pA/pF at 1 ng/ml (p<0.01). In sharp contrast, I(to) density decreased from 22.2±1.2 pA/pF to 12.7±0.98 pA/pF (p<0.001) at 10 ng/ml. At 1 ng/ml TGF-β1 significantly increased SCN5A (Na(V)1.5) (+73%; p<0.01), while reducing KCNIP2 (Kchip2; -77%; p<0.01) and KCND2 (K(V)4.2; -50% p<0.05) mRNA levels. Further, the TGF-β1-induced increase in I(Na) was mediated through activation of the PI3K-AKT pathway via phosphorylation of FOXO1 (a negative regulator of SCN5A). TGF-β1 released by myofibroblasts differentially regulates transcription and function of the main cardiac sodium channel and of the channel responsible for the transient outward current. The results provide new mechanistic insight into the electrical remodeling associated with myocardial injury

hiPSC-CM Monolayer Maturation State Determines Drug Responsiveness in High Throughput Pro-Arrhythmia Screen

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer a novel in vitro platform for pre-clinical cardiotoxicity and pro-arrhythmia screening of drugs in development. To date hiPSC-CMs used for cardiotoxicity testing display an immature, fetal-like cardiomyocyte structural and electrophysiological phenotype which has called into question the applicability of hiPSC-CM findings to the adult heart. The aim of the current work was to determine the effect of cardiomyocyte maturation state on hiPSC-CM drug responsiveness. To this end, here we developed a high content pro-arrhythmia screening platform consisting of either fetal-like or mature hiPSC-CM monolayers. Compounds tested in the screen were selected based on the pro-arrhythmia risk classification (Low risk, Intermediate risk, or High risk) established recently by the FDA and major stakeholders in the Drug Discovery field for the validation of the Comprehensive In vitro Pro-Arrhythmia Assay (CiPA). Here we show that maturation state of hiPSC-CMs determines the absolute pro-arrhythmia risk score calculated for these compounds. Thus, the maturation state of hiPSC-CMs should be considered prior to pro-arrhythmia and cardiotoxicity screening in drug discovery programs.Research reported in this publication was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under Award Number R43ES027703. This study was also supported by the Lefkofsky Family Foundation (T.J.H.), the UM Frankel Cardiovascular Center (T.J.H.), and the State of Michigan Economic Development Fund (U-M Michigan Translational Research and Commercialization for Life Sciences Program [U-M MTRAC], T.J.H. and J.J.)S

Ca2+â independent positive molecular inotropy for failing rabbit and human cardiac muscle by αâ myosin motor gene transfer

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154404/1/fsb2fj09140566.pd