Interaction of Cardiovascular Nonmodifiable Risk Factors, Comorbidities and Comedications With Ischemia/Reperfusion Injury and Cardioprotection by Pharmacological Treatments and Ischemic Conditioning

Risc cardiovascular; Isquèmia/reperfusióCardiovascular risk; Ischemia/reperfusionRiesgo cardiovascular; Isquemia/reperfusiónPreconditioning, postconditioning, and remote conditioning of the myocardium enhance the ability of the heart to withstand a prolonged ischemia/reperfusion insult and the potential to provide novel therapeutic paradigms for cardioprotection. While many signaling pathways leading to endogenous cardioprotection have been elucidated in experimental studies over the past 30 years, no cardioprotective drug is on the market yet for that indication. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic preclinical evaluation of promising cardioprotective therapies prior to their clinical evaluation, since ischemic heart disease in humans is a complex disorder caused by or associated with cardiovascular risk factors and comorbidities. These risk factors and comorbidities induce fundamental alterations in cellular signaling cascades that affect the development of ischemia/reperfusion injury and responses to cardioprotective interventions. Moreover, some of the medications used to treat these comorbidities may impact on cardioprotection by again modifying cellular signaling pathways. The aim of this article is to review the recent evidence that cardiovascular risk factors as well as comorbidities and their medications may modify the response to cardioprotective interventions. We emphasize the critical need for taking into account the presence of cardiovascular risk factors as well as comorbidities and their concomitant medications when designing preclinical studies for the identification and validation of cardioprotective drug targets and clinical studies. This will hopefully maximize the success rate of developing rational approaches to effective cardioprotective therapies for the majority of patients with multiple comorbidities. Significance Statement Ischemic heart disease is a major cause of mortality; however, there are still no cardioprotective drugs on the market. Most studies on cardioprotection have been undertaken in animal models of ischemia/reperfusion in the absence of comorbidities; however, ischemic heart disease develops with other systemic disorders (e.g., hypertension, hyperlipidemia, diabetes, atherosclerosis). Here we focus on the preclinical and clinical evidence showing how these comorbidities and their routine medications affect ischemia/reperfusion injury and interfere with cardioprotective strategies.P.F. was supported by the National Research, Development and Innovation Office of Hungary (Research Excellence Program–TKP, National Heart Program NVKP 16-1-2016-0017) and by the Higher Education Institutional Excellence Program of the Ministry of Human Capacities in Hungary, within the framework of the Therapeutic Development thematic program of Semmelweis University. D.D. is supported by grants from National Institutes of Health National Heart, Lung, and Blood Institute [R01-HL136389, R01-HL131517, R01-HL089598, and R01-HL163277], the German Research Foundation [DFG, Do 769/4-1], the European Union (large-scale integrative project MAESTRIA, no. 965286). G.H. is supported by the German Research Foundation [SFB 1116 B8]. D.H. is supported by the Duke–NUS Signature Research Programme funded by the Ministry of Health, Singapore Ministry of Health’s National Medical Research Council under its Clinician Scientist–Senior Investigator scheme [NMRC/CSA-SI/0011/2017], Centre Grant [CGAug16M006], and Collaborative Centre Grant scheme [NMRC/CGAug16C006]. I.A. is supported from Boehringer-Ingelheim for the investigation of the effects of empagliflozin on the myocardium and from the European Union (ERDF) and Greek national funds through the Operational Program “Competitiveness, Entrepreneurship and Innovation,” under the call “RESEARCH – CREATE – INNOVATE” (project code: 5048539). S.M.D. acknowledges the support of the British Heart Foundation [PG/19/51/34493 and PG/16/85/32471]. S.L. is supported by the South African National Research Foundation and received COST Seed funding from the Department of Science and Innovation in South Africa. M.R-M. is supported by the Instituto de Salud Carlos III of the Spanish Ministry of Health [FIS-PI19-01196] and a grant from the Spanish Society of Cardiology [SEC/FEC-INV-BAS 217003]. C.J.Z. is supported by a grant from European Foundation for the Study of Diabetes (EFSD), a research grant from Boehringer-Ingelheim and an institutional grant from Amsterdam UMC Cardiovascular Research. R.S. is supported by Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) [Project number 268555672—SFB 1213, Project B05]

Role of atrial tissue remodeling on rotor dynamics an in vitro study

The objective of this article is to present an in vitro model of atrial cardiac tissue that could serve to study the mechanisms of remodeling related to atrial fibrillation (AF). We analyze the modification on gene expression and modifications on rotor dynamics following tissue remodeling. Atrial murine cells (HL-1 myocytes) were maintained in culture after the spontaneous initiation of AF and analyzed at two time points: 3.1 +/- 1.3 and 9.7 +/- 0.5 days after AF initiation. The degree of electrophysiological remodeling (i.e., relative gene expression of key ion channels) and structural inhomogeneity was compared between early and late cell culture times both in nonfibrillating and fibrillating cell cultures. In addition, the electrophysiological characteristics of in vitro fibrillation [e.g., density of phase singularities (PS/cm2), dominant frequency, and rotor meandering] analyzed by means of optical mapping were compared with the degree of electrophysiological remodeling. Fibrillating cell cultures showed a differential ion channel gene expression associated with atrial tissue remodeling (i.e., decreased SCN5A, CACN1C, KCND3, and GJA1 and increased KCNJ2) not present in nonfibrillating cell cultures. Also, fibrillatory complexity was increased in late- vs. early stage cultures (1.12 +/- 0.14 vs. 0.43 +/- 0.19 PS/cm(2), P < 0.01), which was associated with changes in the electrical reentrant patterns (i.e., decrease in rotor tip meandering and increase in wavefront curvature). HL-1 cells can reproduce AF features such as electrophysiological remodeling and an increased complexity of the electrophysiological behavior associated with the fibrillation time that resembles those occurring in patients with chronic AF.This work was supported in part by grants from the Spanish Ministry of Science and Innovation (PLE2009-0152), the Instituto de Salud Carlos III (Ministry of Economy and Competitiveness, Spain: PI13-01882, PI13-00903, and TEC2013-50391-EXP), and the Red de Investigacion Cardiovacular (RIC) from Instituto de Salud Carlos III (Ministry of Economy and Competitiveness, Spain).Climent, A.; Guillem Sánchez, MS.; Fuentes, L.; Lee, P.; Bollensdorff, C.; Fernandez-Santos, M.; Suarez-Sancho, S.... (2015). Role of atrial tissue remodeling on rotor dynamics an in vitro study. AJP - Heart and Circulatory Physiology. 309(11):H1964-H1973. doi:10.1152/ajpheart.00055.2015SH1964H197330911Allessie, M. (2002). Electrical, contractile and structural remodeling during atrial fibrillation. Cardiovascular Research, 54(2), 230-246. doi:10.1016/s0008-6363(02)00258-4Allessie, M. A., de Groot, N. M. S., Houben, R. P. M., Schotten, U., Boersma, E., Smeets, J. L., & Crijns, H. J. (2010). Electropathological Substrate of Long-Standing Persistent Atrial Fibrillation in Patients With Structural Heart Disease. Circulation: Arrhythmia and Electrophysiology, 3(6), 606-615. doi:10.1161/circep.109.910125Atienza, F., Almendral, J., Jalife, J., Zlochiver, S., Ploutz-Snyder, R., Torrecilla, E. G., … Berenfeld, O. (2009). Real-time dominant frequency mapping and ablation of dominant frequency sites in atrial fibrillation with left-to-right frequency gradients predicts long-term maintenance of sinus rhythm. Heart Rhythm, 6(1), 33-40. doi:10.1016/j.hrthm.2008.10.024Atienza, F., Almendral, J., Ormaetxe, J. M., Moya, Á., Martínez-Alday, J. D., Hernández-Madrid, A., … Jalife, J. (2014). Comparison of Radiofrequency Catheter Ablation of Drivers and Circumferential Pulmonary Vein Isolation in Atrial Fibrillation. Journal of the American College of Cardiology, 64(23), 2455-2467. doi:10.1016/j.jacc.2014.09.053Bikou, O., Thomas, D., Trappe, K., Lugenbiel, P., Kelemen, K., Koch, M., … Bauer, A. (2011). Connexin 43 gene therapy prevents persistent atrial fibrillation in a porcine model. Cardiovascular Research, 92(2), 218-225. doi:10.1093/cvr/cvr209Bollmann, A., Sonne, K., Esperer, H.-D., Toepffer, I., & Klein, H. U. (2002). Patients with Persistent Atrial Fibrillation Taking Oral Verapamil Exhibit a Lower Atrial Frequency on the ECG. Annals of Noninvasive Electrocardiology, 7(2), 92-97. doi:10.1111/j.1542-474x.2002.tb00148.xBRUNDEL, B. (2004). Calpain inhibition prevents pacing-induced cellular remodeling in a HL-1 myocyte model for atrial fibrillation. Cardiovascular Research, 62(3), 521-528. doi:10.1016/j.cardiores.2004.02.007Calkins, H., Kuck, K. H., Cappato, R., Brugada, J., Camm, A. J., Chen, S.-A., … Wilber, D. (2012). 2012 HRS/EHRA/ECAS Expert Consensus Statement on Catheter and Surgical Ablation of Atrial Fibrillation: Recommendations for Patient Selection, Procedural Techniques, Patient Management and Follow-up, Definitions, Endpoints, and Research Trial Design. Heart Rhythm, 9(4), 632-696.e21. doi:10.1016/j.hrthm.2011.12.016Claycomb, W. C., Lanson, N. A., Stallworth, B. S., Egeland, D. B., Delcarpio, J. B., Bahinski, A., & Izzo, N. J. (1998). HL-1 cells: A cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proceedings of the National Academy of Sciences, 95(6), 2979-2984. doi:10.1073/pnas.95.6.2979Filgueiras-Rama, D., Price, N. F., Martins, R. P., Yamazaki, M., Avula, U. M. R., Kaur, K., … Berenfeld, O. (2012). Long-Term Frequency Gradients During Persistent Atrial Fibrillation in Sheep Are Associated With Stable Sources in the Left Atrium. Circulation: Arrhythmia and Electrophysiology, 5(6), 1160-1167. doi:10.1161/circep.111.969519Haïssaguerre, M., Jaïs, P., Shah, D. C., Takahashi, A., Hocini, M., Quiniou, G., … Clémenty, J. (1998). Spontaneous Initiation of Atrial Fibrillation by Ectopic Beats Originating in the Pulmonary Veins. New England Journal of Medicine, 339(10), 659-666. doi:10.1056/nejm199809033391003Haralick, R. M., Shanmugam, K., & Dinstein, I. (1973). Textural Features for Image Classification. IEEE Transactions on Systems, Man, and Cybernetics, SMC-3(6), 610-621. doi:10.1109/tsmc.1973.4309314Jalife, J. (2010). Deja vu in the theories of atrial fibrillation dynamics. Cardiovascular Research, 89(4), 766-775. doi:10.1093/cvr/cvq364Koivumäki, J. T., Seemann, G., Maleckar, M. M., & Tavi, P. (2014). In Silico Screening of the Key Cellular Remodeling Targets in Chronic Atrial Fibrillation. PLoS Computational Biology, 10(5), e1003620. doi:10.1371/journal.pcbi.1003620Lee, P., Klos, M., Bollensdorff, C., Hou, L., Ewart, P., Kamp, T. J., … Herron, T. J. (2012). Simultaneous Voltage and Calcium Mapping of Genetically Purified Human Induced Pluripotent Stem Cell–Derived Cardiac Myocyte Monolayers. Circulation Research, 110(12), 1556-1563. doi:10.1161/circresaha.111.262535Lieu, D. K., Fu, J.-D., Chiamvimonvat, N., Tung, K. C., McNerney, G. P., Huser, T., … Li, R. A. (2013). Mechanism-Based Facilitated Maturation of Human Pluripotent Stem Cell–Derived Cardiomyocytes. Circulation: Arrhythmia and Electrophysiology, 6(1), 191-201. doi:10.1161/circep.111.973420Liu, X., Shi, H., Tan, H., Wang, X., Zhou, L., & Gu, J. (2009). Decreased Connexin 43 and Increased Fibrosis in Atrial Regions Susceptible to Complex Fractionated Atrial Electrograms. Cardiology, 114(1), 22-29. doi:10.1159/000210398Mansour, M., Mandapati, R., Berenfeld, O., Chen, J., Samie, F. H., & Jalife, J. (2001). Left-to-Right Gradient of Atrial Frequencies During Acute Atrial Fibrillation in the Isolated Sheep Heart. Circulation, 103(21), 2631-2636. doi:10.1161/01.cir.103.21.2631Martins, R. P., Kaur, K., Hwang, E., Ramirez, R. J., Willis, B. C., Filgueiras-Rama, D., … Jalife, J. (2014). Dominant Frequency Increase Rate Predicts Transition from Paroxysmal to Long-Term Persistent Atrial Fibrillation. Circulation, 129(14), 1472-1482. doi:10.1161/circulationaha.113.004742McDowell, K. S., Vadakkumpadan, F., Blake, R., Blauer, J., Plank, G., MacLeod, R. S., & Trayanova, N. A. (2013). Mechanistic Inquiry into the Role of Tissue Remodeling in Fibrotic Lesions in Human Atrial Fibrillation. Biophysical Journal, 104(12), 2764-2773. doi:10.1016/j.bpj.2013.05.025Narayan, S. M., Krummen, D. E., Shivkumar, K., Clopton, P., Rappel, W.-J., & Miller, J. M. (2012). Treatment of Atrial Fibrillation by the Ablation of Localized Sources. Journal of the American College of Cardiology, 60(7), 628-636. doi:10.1016/j.jacc.2012.05.022Noguchi, K., Masumiya, H., Takahashi, K., Kaneko, K., Higuchi, S., Tanaka, H., & Shigenobu, K. (1997). Comparative effects of gallopamil and verapamil on the mechanical and electrophysiological parameters of isolated guinea-pig myocardium. Canadian Journal of Physiology and Pharmacology, 75(12), 1316-1321. doi:10.1139/y97-161Pandit, S. V., Berenfeld, O., Anumonwo, J. M. B., Zaritski, R. M., Kneller, J., Nattel, S., & Jalife, J. (2005). Ionic Determinants of Functional Reentry in a 2-D Model of Human Atrial Cells During Simulated Chronic Atrial Fibrillation. Biophysical Journal, 88(6), 3806-3821. doi:10.1529/biophysj.105.060459Pandit, S. V., & Jalife, J. (2013). Rotors and the Dynamics of Cardiac Fibrillation. Circulation Research, 112(5), 849-862. doi:10.1161/circresaha.111.300158Riccio, M. L., Koller, M. L., & Gilmour, R. F. (1999). Electrical Restitution and Spatiotemporal Organization During Ventricular Fibrillation. Circulation Research, 84(8), 955-963. doi:10.1161/01.res.84.8.955Samie, F. H., Mandapati, R., Gray, R. A., Watanabe, Y., Zuur, C., Beaumont, J., & Jalife, J. (2000). A Mechanism of Transition From Ventricular Fibrillation to Tachycardia. Circulation Research, 86(6), 684-691. doi:10.1161/01.res.86.6.684Samie, F. H., Berenfeld, O., Anumonwo, J., Mironov, S. F., Udassi, S., Beaumont, J., … Jalife, J. (2001). Rectification of the Background Potassium Current. Circulation Research, 89(12), 1216-1223. doi:10.1161/hh2401.100818Smith, A. W., Segar, C. E., Nguyen, P. K., MacEwan, M. R., Efimov, I. R., & Elbert, D. L. (2012). Long-term culture of HL-1 cardiomyocytes in modular poly(ethylene glycol) microsphere-based scaffolds crosslinked in the phase-separated state. Acta Biomaterialia, 8(1), 31-40. doi:10.1016/j.actbio.2011.08.021Tsai, C.-T., Chiang, F.-T., Chen, W.-P., Hwang, J.-J., Tseng, C.-D., Wu, C.-K., … Lin, J.-L. (2011). Angiotensin II induces complex fractionated electrogram in a cultured atrial myocyte monolayer mediated by calcium and sodium-calcium exchanger. Cell Calcium, 49(1), 1-11. doi:10.1016/j.ceca.2010.10.005Tsai, C.-T., Chiang, F.-T., Tseng, C.-D., Yu, C.-C., Wang, Y.-C., Lai, L.-P., … Lin, J.-L. (2011). Mechanical Stretch of Atrial Myocyte Monolayer Decreases Sarcoplasmic Reticulum Calcium Adenosine Triphosphatase Expression and Increases Susceptibility to Repolarization Alternans. Journal of the American College of Cardiology, 58(20), 2106-2115. doi:10.1016/j.jacc.2011.07.039Tuomi, J. M., Tyml, K., & Jones, D. L. (2011). Atrial tachycardia/fibrillation in the connexin 43 G60S mutant (Oculodentodigital dysplasia) mouse. American Journal of Physiology-Heart and Circulatory Physiology, 300(4), H1402-H1411. doi:10.1152/ajpheart.01094.2010White, S. M., Constantin, P. E., & Claycomb, W. C. (2004). Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function. American Journal of Physiology-Heart and Circulatory Physiology, 286(3), H823-H829. doi:10.1152/ajpheart.00986.2003Wijffels, M. C. E. F., Kirchhof, C. J. H. J., Dorland, R., & Allessie, M. A. (1995). Atrial Fibrillation Begets Atrial Fibrillation. Circulation, 92(7), 1954-1968. doi:10.1161/01.cir.92.7.1954Zlochiver, S., Muñoz, V., Vikstrom, K. L., Taffet, S. M., Berenfeld, O., & Jalife, J. (2008). Electrotonic Myofibroblast-to-Myocyte Coupling Increases Propensity to Reentrant Arrhythmias in Two-Dimensional Cardiac Monolayers. Biophysical Journal, 95(9), 4469-4480. doi:10.1529/biophysj.108.13647

The Resistance of Oilseed Rape Microspore-Derived Embryos to Osmotic Stress Is Associated With the Accumulation of Energy Metabolism Proteins, Redox Homeostasis, Higher Abscisic Acid, and Cytokinin Contents

The present study aims to investigate the response of rapeseed microspore-derived embryos (MDE) to osmotic stress at the proteome level. The PEG-induced osmotic stress was studied in the cotyledonary stage of MDE of two genotypes: Cadeli (D) and Viking (V), previously reported to exhibit contrasting leaf proteome responses under drought. Two-dimensional difference gel electrophoresis (2D-DIGE) revealed 156 representative protein spots that have been selected for MALDI-TOF/TOF analysis. Sixty-three proteins have been successfully identified and divided into eight functional groups. Data are available via ProteomeXchange with identifier PXD024552. Eight selected protein accumulation trends were compared with real-time quantitative PCR (RT-qPCR). Biomass accumulation in treated D was significantly higher (3-fold) than in V, which indicates D is resistant to osmotic stress. Cultivar D displayed resistance strategy by the accumulation of proteins in energy metabolism, redox homeostasis, protein destination, and signaling functional groups, high ABA, and active cytokinins (CKs) contents. In contrast, the V protein profile displayed high requirements of energy and nutrients with a significant number of stress-related proteins and cell structure changes accompanied by quick downregulation of active CKs, as well as salicylic and jasmonic acids. Genes that were suitable for gene-targeting showed significantly higher expression in treated samples and were identified as phospholipase D alpha, peroxiredoxin antioxidant, and lactoylglutathione lyase. The MDE proteome profile has been compared with the leaf proteome evaluated in our previous study. Different mechanisms to cope with osmotic stress were revealed between the genotypes studied. This proteomic study is the first step to validate MDE as a suitable model for follow-up research on the characterization of new crossings and can be used for preselection of resistant genotypes

Multiple Potential Molecular Contributors to Atrial Hypocontractility Caused by Atrial Tachycardia Remodeling in Dogs

Background-Atrial fibrillation impairs atrial contractility, inducing atrial stunning that promotes thromboembolic stroke. Action potential (AP)-prolonging drugs are reported to normalize atrial hypocontractility caused by atrial tachycardia remodeling (ATR). Here, we addressed the role of AP duration (APD) changes in ATR-induced hypocontractility. Methods and Results-ATR (7-day tachypacing) decreased APD (perforated patch recording) by approximate to 50%, atrial contractility (echocardiography, cardiomyocyte video edge detection), and [Ca2+](i) transients. ATR AP waveforms suppressed [Ca2+](i) transients and cell shortening of control cardiomyocytes; whereas control AP waveforms improved [Ca2+](i) transients and cell shortening in ATR cells. However, ATR cardiomyocytes clamped with the same control AP waveform had approximate to 60% smaller [Ca2+](i) transients and cell shortening than control cells. We therefore sought additional mechanisms of contractile impairment. Whole-cell voltage clamp revealed reduced I-CaL; I-CaL inhibition superimposed on ATR APs further suppressed [Ca2+](i) transients in control cells. Confocal microscopy indicated ATR-impaired propagation of the Ca2+ release signal to the cell center in association with loss of t-tubular structures. Myofilament function studies in skinned permeabilized cardiomyocytes showed altered Ca2+ sensitivity and force redevelopment in ATR, possibly due to hypophosphorylation of myosin-binding protein C and myosin light-chain protein 2a (immunoblot). Hypophosphorylation was related to multiple phosphorylation system abnormalities where protein kinase A regulatory subunits were downregulated, whereas autophosphorylation and expression of Ca2+-calmodulin-dependent protein kinase II delta and protein phosphatase 1 activity were enhanced. Recovery of [Ca2+](i) transients and cell shortening occurred in parallel after ATR cessation. Conclusions-Shortening of APD contributes to hypocontractility induced by 1-week ATR but accounts for it only partially. Additional contractility-suppressing mechanisms include I-CaL current reduction, impaired subcellular Ca2+ signal transmission, and altered myofilament function associated with abnormal myosin and myosin-associated protein phosphorylation. The complex mechanistic basis of the atrial hypocontractility associated with AF argues for upstream therapeutic targeting rather than interventions directed toward specific downstream pathophysiological derangements. (Circ Arrhythm Electrophysiol. 2010;3:530-541.

Dysfunction in the βII Spectrin-Dependent Cytoskeleton Underlies Human Arrhythmia.

Background: The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked with cardiac pathologies including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction in cardiac electrical activity is not well understood, and often overlooked in the cardiac arrhythmia field. Methods and Results: Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that βII spectrin, an actin-associated molecule, is essential for the post-translational targeting and localization of critical membrane proteins in heart. βII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/βII spectrin interaction leading to severe human arrhythmia phenotypes. Mice lacking cardiac βII spectrin display lethal arrhythmias, aberrant electrical and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, βII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes that include the Na/Ca exchanger, RyR2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in βII spectrin-deficient mice. Conclusions: Our findings identify βII spectrin as critical for normal myocyte electrical activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology

2015 ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death the Task Force for the Management of Patients with Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death of the European Society of Cardiology (ESC) Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC)

N/