International Journal of Molecular Sciences Review Connexins in the Heart: Regulation, Function and Involvement in Cardiac Disease Antonio Rodríguez-Sinovas 1,2,3,* , Jose Antonio Sánchez 1,2,3, Laura Valls-Lacalle 1,2,3, Marta Consegal 1,2,3 and Ignacio Ferreira-González 1,2,4,* 1 Cardiovascular Diseases Research Group, Department of Cardiology, Vall d’Hebron Institut de Recerca (VHIR), Vall d’Hebron Barcelona Hospital Campus, Vall d’Hebron Hospital Universitari, Passeig Vall d’Hebron 119-129, 08035 Barcelona, Spain; [email protected] (J.A.S.); [email protected] (L.V.-L.); [email protected] (M.C.) 2 Departament de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain 3 Centro de Investigación Biomédica en Red sobre Enfermedades Cardiovasculares (CIBERCV), Instituto de Salud Carlos III, 28029 Madrid, Spain 4 Centro de Investigación Biomédica en Red (CIBER) de Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, 28029 Madrid, Spain * Correspondence: [email protected] (A.R.-S.); [email protected] (I.F.-G.); Tel.: +34-93-4894184 (A.R.-S.) Abstract: Connexins are a family of transmembrane proteins that play a key role in cardiac physiology. Gap junctional channels put into contact the cytoplasms of connected cardiomyocytes, allowing the existence of electrical coupling. However, in addition to this fundamental role, connexins are also involved in cardiomyocyte death and survival. Thus, chemical coupling through gap junctions plays a key role in the spreading of injury between connected cells. Moreover, in addition to their involvement in cell-to-cell communication, mounting evidence indicates that connexins have additional gap junction-independent functions. Opening of unopposed hemichannels, located at Citation: Rodríguez-Sinovas, A.; Sánchez, J.A.; Valls-Lacalle, L.; the lateral surface of cardiomyocytes, may compromise cell homeostasis and may be involved in Consegal, M.; Ferreira-González, I. ischemia/reperfusion injury. In addition, connexins located at non-canonical cell structures, including Connexins in the Heart: Regulation, mitochondria and the nucleus, have been demonstrated to be involved in cardioprotection and in Function and Involvement in Cardiac regulation of cell growth and differentiation. In this review, we will provide, first, an overview on Disease. Int. J. Mol. Sci. 2021, 22, 4413. connexin biology, including their synthesis and degradation, their regulation and their interactions. https://doi.org/10.3390/ijms22094413 Then, we will conduct an in-depth examination of the role of connexins in cardiac pathophysiology, including new findings regarding their involvement in myocardial ischemia/reperfusion injury, Academic Editor: Robert David cardiac fibrosis, gene transcription or signaling regulation. Received: 16 March 2021 Keywords: connexin; Cx43; gap junction; hemichannel; mitochondria; nucleus; heart; cardiomyocyte Accepted: 20 April 2021 Published: 23 April 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- Connexins are a large family of highly homologous transmembrane proteins, com- iations. prising 20 and 21 different isoforms in mice and humans, respectively [1–3]. All these isoforms are named according to their expected molecular weight in kDa and have distinct biophysical properties. However, and despite their electrophysiological differences, all connexins share a similar structure. They are oriented so that the amino- and carboxytermi- nal (CT) tails of the protein are located within the cell cytoplasm, and they include four Copyright: © 2021 by the authors. α Licensee MDPI, Basel, Switzerland. transmembrane-spanning -helix domains linked by two extracellular segments (E1 and This article is an open access article E2) and one cytoplasmic loop (Figure1)[ 4]. Differences between connexin isoforms are distributed under the terms and mostly due to variations in the amino acid sequence at the CT domain, although also, to conditions of the Creative Commons some extent, at the cytoplasmic loop [5]. The CT domain has multiple serine, threonine and Attribution (CC BY) license (https:// tyrosine residues susceptible to phosphorylation, which is a very important phenomenon creativecommons.org/licenses/by/ regulating connexin trafficking, assembly and function [6–9], and is largely responsible 4.0/). for the appearance of multiple bands in Western blots. Connexin 43 (Cx43) is, by far, Int. J. Mol. Sci. 2021, 22, 4413. https://doi.org/10.3390/ijms22094413 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 65 Int. J. Mol. Sci. 2021, 22, 4413 and tyrosine residues susceptible to phosphorylation, which is a very important phe-2 of 64 nomenon regulating connexin trafficking, assembly and function [6–9], and is largely responsible for the appearance of multiple bands in Western blots. Connexin 43 (Cx43) is, bythe far, best-known the best-known and mostand most ubiquitously ubiquitously expressed expressed isoform, isoform, and and it is it encoded is encoded by the by GJA1the GJA1gene, gene, located, located, in in humans, humans, in in chromosome chromosome 6 6 [ 10[10].]. The The gene gene nomenclature nomenclature for for the the two two otherother most most common common human human cardiac connexinconnexin isoforms,isoforms, Cx40 Cx40 and and Cx45, Cx45, is GJA5is GJA5 and and GJC1, GJC1,respectively respectively [2]. [2]. FigureFigure 1. Schematic 1. Schematic view view of a ofsingle a single connexin connexin molecule molecule located located at the at plasma the plasma membrane membrane (upper (upper panel).panel). Lower Lower panels panels depict depict hemichannels hemichannels and and intercellular intercellular or or gap gap junctional junctional channels. channels. Created Created with withBiorender.com Biorender.com (accessed (accessed on on 15 April15 Ap 2021).ril 2021). Modified Modified from from [11 ,[11,12].12]. AllAll connexin connexin isoforms isoforms are are integral integral compon componentsents of ofplasma plasma membranes membranes where where they they formform channels channels around around a central a central pore. pore. These These channels channels are formed are formed by the by oligomerization the oligomeriza- of tionsix ofindividual six individual connexin connexin molecules molecules and andare known are known as hemichannels as hemichannels or orconnexons connexons (Figure(Figure 1)1 [4,13].) [4,13 Hundreds]. Hundreds to tothousands thousands of ofthese these hemichannels hemichannels gather gather in inplaques, plaques, termed termed gapgap junctions, junctions, where where they they dock dock with with opposi opposingng connexons connexons from from adjacent adjacent cells, cells, forming forming intercellularintercellular channels channels (Figure (Figure 1)1)[ [4,13].4,13]. In In cardiomyocytes, gapgap junctions junctions are are mainly mainly located lo- catedat the at cellthe polescell poles (Figure (Figure2), perpendicular 2), perpendicular to the to long the axis long of axis the cell,of the within cell, within the intercalated the in- tercalateddiscs, which discs, are which complex are complex structures structures in which in plasma which membranesplasma membranes of neighboring of neigh- cells boringare in cells close are contact in close and contact that also and include that al adherensso include junctions, adherens hemichannels junctions, hemichannels and ion chan- andnels ion [14 channels,15]. Three [14,15]. cysteine Three residues, cysteine located residues, in each located extracellular in each extracellular loop of each loop connexin of eachmolecule, connexin are molecule, important are in important the docking in process.the docking Disulfide process. bonds Disulfide between bonds these between cysteines theseare cysteines needed to are create needed the β to-sheet create conformation the β-sheet conformation required for the required interaction for the between interaction the two betweenopposing the hemichannelstwo opposing [hemich4,5,16].annels In fact, [4,5,16]. Cx43 lacking In fact, these Cx43 cysteines lacking these is not cysteines able to form is notgap able junctional to form gap intercellular junctional channels intercellular [17]. Connexonschannels [17] can. Connexons be formed bycan a be unique formed connexin by a uniqueisoform connexin or by several isoform connexin or by several isoforms connexin (giving isoforms rise to homomeric (giving rise or heteromericto homomeric connex- or heteromericons, respectively), connexons, although respectively), not all are although compatible not [all16]. are In turn,compatible intercellular [16]. In channels turn, in- can tercellularbe homotypic channels (formed can be by homotypic connexons (formed with the by sameconnexons composition) with the orsame heterotypic composition) (when each connexon has a different composition) [18,19]. Such mixed conformations modify channel electrophysiological properties and alter gap junctional conductance [20,21]. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 65 Int. J. Mol. Sci. 2021, 22, 4413 or heterotypic (when each connexon has a different composition) [18,19]. Such mixed3 of 64 conformations modify channel electrophysiological properties and alter gap junctional conductance [20,21]. Figure 2. (A) Confocal images showing expression of connexin 43 (Cx43, red) in cardiac slices obtained from wild-type Figure 2. (A) Confocal images showing expression of connexin