The endoplasmic reticulum (ER) is the largest, multifunctional, membrane-like, cellular organelle, composed of smooth and rough ER and forms an interconnected network of space [1]. ER exerts a pivotal role in three physiological cellular processes: (1) modulation of correct protein secretion, folding and translocation from ER lumen, (2) regulation of intracellular Ca²⁺ uptake, storage and signalling and (3) production of several membrane cellular lipids such as cholesterol, ceramides and/or glycerophospholipids ^[2][3][2,3].

A significant percentage of intracellular proteins are synthesised in ER lumen, wherein its oxidative environment facilitates the formation of disulphide bonds on proteins by different chaperones, foldases and cofactors. Generating disulphide bonds leads to proper secretory and transmembrane protein folding ^[4][5][4,5]. Alteration of ER protein folding capacity may cause an increased proportion of unfolded and misfolded proteins in ER lumen which triggers loss of ER homeostasis and proteostasis and generates a detrimental cellular environment ^[6][7][8][6,7,8]. Several molecular and biophysical mechanisms are triggered to reverse and restore ER homeostasis such as (1) ER-associated degradation (ERAD), which triggers the misfolded protein degradation from ER lumen; (2) Unfolded protein response (UPR) involving the restoration of ER proteostasis by activation of three transduction signalling –IRE1, ATF6 and PERK branch-; and (3) Reticulophagy, the process of ER remodelling by autophagy of membranes and associated proteins (see reviews ^{[9][10][11][12][13][14]}[9,10,11,12,13,14]). Pathophysiological factors occurring in cardiovascular diseases (CVDs) such as metabolic derangement, hypoxia, hypertrophy or inflammation require an increased protein expression, thus enhancing the disruption of the cellular proteostasis ^{[15][16][17][18][19][20][21]}[15,16,17,18,19,20,21]. As a consequence of the increased requirement of protein synthesis, ER homeostasis is ruptured and different subpopulations of cardiac cells suffer an unfolded and misfolded protein accumulation, which in turn, induces ER stress ^{[22][23][24][25]}[22,23,24,25]. Accumulation of deleterious proteins triggers ER stress signalling which exerts a bivalent role both beneficial and/or harmful in cardiovascular function ^{[26][27][28][29][30][31][32][33]}[26,27,28,29,30,31,32,33]. Furthermore, ER homeostasis is closely associated with normal cardiovascular function, and ER stress is considered a cause and a consequence of an extensive variety of CVDs such as ischaemic heart disease, hypertension, heart failure and dilated cardiomyopathy ^{[34][35][36][37]}[34,35,36,37].

2. ERS and UPR Signalling

Since ER is crucial for the correct functioning of the cell, there are ER stress response mechanisms that control the degradation of the unfolded or misfolded proteins aiming to maintain ER homeostasis. The core mechanism of control is the activation of unfolded protein response (UPR). The central function of UPR is the inhibition of protein synthesis and the increase in the folding capacity of the ER. UPR may be activated by three different signal transduction pathways, initiated by three proteins located in ER membrane: inositol requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6). In basal conditions, these molecules are bound to a chaperone named Bip (or GRP78) and remain attached to the ER membrane. However, when misfolded or unfolded proteins are accumulated, they dissociate and trigger three different signalling pathways induced by IRE1, PERK and ATF6 to resolve ER stress (Figure 1) ^[38][39][38,39]. IRE1 is the most conserved factor across evolution involved in the UPR pathway. IRE1 possess an endoribonuclease activity domain responsible for its molecular function and is represented by two isoforms, IRE1α and IRE1β. IRE1 is activated by auto-phosphorylation and homodimerisation under the loss of ER homeostasis. Activated IRE1 is delivered to ER membrane and recognises a consensus region in the X-box binding protein 1 (XBP1) mRNA, inducing alternative splicing by cleavage of a 26-nucleotide intron. Such a cleavage results in a functional active protein XBP1 named, XBP1s. XBP1s exerts as a transcription factor triggering expression of several UPR target genes such as ERAD components, ER chaperones, ER-translocation and folding enzymes further reducing ER stress levels. However, maintenance of IRE1α results in increased apoptosis. IRE1α interacts with tumour necrosis factor receptor-associated factor 2 (TRAF2) and adaptor protein tumour necrosis factor (TNF) to form a complex [40]. This complex recruits mitogen-activated protein kinase (MAPK), apoptosis signal-regulating kinase (ASK), and caspase-12 in order to trigger apoptosis ^[41][42][41,42]. Like IRE1, activation of PERK occurs by autophosphorylation of its kinase domain. Activated PERK modulates phosphorylation of eukaryotic translation initiation factor 2 alpha (Eif2α), which in turn, inhibits 80S ribosome assembly and thus protein synthesis, inducing a reduction in the ERS. Furthermore, Eif2α enhances the translation of activating transcription factor 4 (Atf4) mRNA. Atf4 induces the transcription of growth arrest and DNA damage-inducible protein 34 (Gadd34) and c/EBP homologous protein (CHOP) resulting in the activation of several proapoptotic signalling. CHOP induces apoptosis by the induction of several caspases and proapoptotic factors. Curiously, Gadd34 regulates the dephosphorylation of Eif2α when ER stress is solved, and it restores the normal protein translation. Dephosphorylation of Eif2α is required to conduct prosurvival signalling. Activation transcription factor 6 (ATF6) is an ER transmembrane protein belonging to the leucine zipper transcription factor family. ATF6 acts as a core modulator of autophagy and apoptosis in response to increased ER stress [43]. When the ER is stressed, ATF6 is delivered from ER membrane by Bip and transported to the Golgi apparatus, where it is cleaved by two different proteases, site-1 protease (S1P) and site-2 protease (S2P), generating a 50 kDa amino-terminal cytoplasmic fragment and acquiring a transcriptional activation function (ATF6f). ATF6f is capable to enter the nucleus and trigger the expression of ERAD components, GRP78 and XBP1. Furthermore, ATF6 may bind to the endoplasmic reticulum response element (ERSE) and thereby activating CHOP and inducing cell apoptosis in several pathologies [44]. Early activation of UPR—named adaptive UPR—exerts a protective role against several injuries promoting cell survival and improving cellular function. Furthermore, UPR is required for different cellular processes such as differentiation and proliferation, pinpointing an important role in appropriate development and cellular physiology ^{[45][46][47][48]}[45,46,47,48]. For example, activation of three branches of UPR- IRE1, PERK and ATF6- is necessary for the expression of several myogenic genes such as Mef2c or MyoD, the correct formation of myotubes and therefore proper embryonic myogenesis ^{[49][50][51][52]}[49,50,51,52]. In addition, the regeneration of skeletal muscle by activation of satellite cells requires the expression of PERK signalling and downstream genes suggesting a crucial role in the regenerative process [53]. Beneficial and physiological effects observed by adaptive UPR are closely related to the maintenance of calcium homeostasis, mitochondrial function and the regulation of homeostatic levels of free radicals in the cell cytoplasm ^[54][55][56][54,55,56]. However, prolonged stimulation of the UPR signalling pathway—known as maladaptive UPR—has a deleterious effect on cellular homeostasis increasing cellular apoptosis, ROS generation and impaired cell function thus displaying a detrimental role in several pathologies ^{[57][58][59][60][61]}[57,58,59,60,61]. Complementary to the previously described mechanisms, there are also alternative processes that resolve ER stress and support UPR protective function such as ER-associated degradation (ERAD). ERAD is an evolutionarily and anciently conserved mechanism which modulates the degradation of misfolded or unfolded proteins from ER resulting in a subsequent reduction in the ERS. In this process, the misfolded or unfolded proteins accumulated in the ER are translocated to the cytosol where they are ubiquitinated and degraded by the proteasome. ERAD substrates are recognised by different ligases and chaperones depending on whether the misfolded or unfolded domain of the protein is located in the ER lumen, within the ER membrane, or on the cytosolic side of the membrane (ERAD-L, ERAD-M and ERAD-C, respectively). ERAD-L and ERAD-M are driven by Hrd1—RING-finger ligase—a core ubiquitin ligase that forms a protein complex with other ligases such as Hrd3, Usa1 or Der1. Whereas ERAD-C substrates are targeted by Doa10p ligase. Hrd1 protein is formed by six transmembrane domains and a cytoplasmic tail in which a catalytic RING finger is necessary for E3 ligase activity. Curiously, the transmembrane regions of Hrd1 may form a retrotranslocation channel to export ER proteins. The RING finger domain is located in the cytosol to serve at least two distinct purposes. First, Hrd1-dependent autoubiquitination of the RING finger domain gates its own channel function. This finding raises the possibility that deubiquitinases might counter the ubiquitination reaction and control the retrotranslocation event as well. Whether autoubiquitination is a general feature that regulates the channel activity of other E3 ubiquitin ligases dedicated to ERAD is unclear at this point. Second, Hrd1 catalyses ubiquitination of the misfolded substrates once exposed to the cytosol, which in turn are tagged for proteasomal degradation. Recently another E3 ligase gp78 has been described acting downstream, or in parallel, to the Hrd1-ligases complex, enhancing the solubility of the retrotranslocated protein substrates by proper proteasomal degradation. Another important mechanism to resolve ER stress is reticulophagy, a type of macro-autophagy leading to the removal of excess unfolded and misfolded proteins from ER lumen. This process consists of the creation of autophagosomes specifically from ER membranes in order to remove excess deleterious proteins of ER. Several molecular mechanisms of reticulophagy have been described ^[62][63][64][62,63,64]. Increased unfolded and misfolded proteins trigger auto-ubiquitination of the E3 ubiquitin-protein ligase tripartite motif-containing protein 13 (TRIM13) which recruits autophagy adaptor sequestosome 1 (p62). The oligomerisation of both proteins is dependent on the binding of N-Degron to the ZZ domain from p62. TRIM13-p62 protein complex oligomerisation is required to recruit LC3B and other chaperones involved in reticulophagy. LC3B induces specific reticulophagy of ER portions enriched in folding elements and chaperones involving lysosome-associated membrane glycoprotein 1 (LAMP1), RAB7 (in ER- engulfing endolysosomes), charged multivesicular body protein 4B (CHMP4B) and vacuolar protein sorting-associated protein 4A (VPS4A) ^{[45][65][66][67][68]}[45,65,66,67,68]. Furthermore, the PERK-EIF2A pathway is responsible for the activation of the ATG12-ATG16-ATG5 complex which in turn establishes a signature mark into autophagy membranes by converting LC3-I into LC3-II [69]. Like ERAD or UPRs, excessive removal of ER membranes could be translated into the disruption of autophagy and increased apoptosis ^[70][71][70,71].

3. Role of ERS and UPR in Cardiovascular Diseases

ERS and subsequent activation of UPR exhibit both beneficial and deleterious effects in cardiovascular diseases, being thus considered both as a cause and consequence of them. Cardiac pathologies increase the demand and requirements of the ER function since an enhanced proportion of misfolded proteins triggers in many cases the loss of homeostasis of this organelle. Furthermore, ERS exerts a pivotal role in the modulation of both Ca²⁺ homeostasis and mitochondrial function in cardiomyocytes. Prola et al. (2019) have demonstrated that Tunicamycin (TM) treated cardiomyocytes display several changes in their cytoplasm ultrastructure, such as enlarged cytosol, decreased mitochondrial number, increased proportion of mitochondria-associated-membrane (MAM) fraction and expansion and dislocation of the ER near to nucleus and thus away from the sarcomeres. Accordingly, ERS reduced the mitochondrial number and function by downregulating several proteins involved in mitochondrial biogenesis such as PGC1a, TFAM, NRF1 or CS and thus is involved in the reduction of the mitochondrial capability to produce ATP [72]. Initially, adaptive UPR activation is capable of restoring ER and mitochondrial function and thus sustaining cardiac homeostasis. Curiously, the effects of molecular signalling pathways triggered by ERS are different within distinct cardiovascular injuries such as atherosclerosis, myocardial infarction, heart failure, cardiac hypertrophy or ischaemia and reperfusion (I/R) injury among others. For example, in heart failure or hypertrophy cardiac response caused by cardiac pressure overload, the PERK signalling pathway increases autophagy while it reduces ROS levels and apoptosis ratio by upregulation of EIF2A and ATF4, which in turn restores protein-folding capacity ^[31][73][31,73]. A sustained upregulation of the axis EIF2A-ATF4 will produce an increase in the cardiomyocyte apoptosis triggered by CHOP and these processes can influence the progression of cardiac diseases. In addition, PERK restores Ca²⁺ intracellular concentration by modulating Serca2a and Calreticulin, demonstrating its requirement for a proper ER-dependent ion homeostasis [74]. Unlike PERK, ATF6 is involved in the progression of cardiac hypertrophy and heart failure response thus exerting a harmful role. However, a protective role of ATF6 has been described in I/R injury suggesting a dependent and complex function of UPR based on the type of cardiac injury. Effects of UPRs’ downstream pathways have been elucidated using several murine models, which have highlighted the importance of ER stress and dependent molecular mechanisms in cardiac homeostasis and pathology (Figure 2). Curiously, ATF6 deficient mice display a worse cardiac function and recovery from infarction after (I/R) injury and increased damage with respect to controls [26]. Furthermore, ATF6 gain-of-function mice exhibits an alleviated myocardial infarction after I/R injury demonstrating that ATF6 is required to protect the heart from damage and injury caused by myocardial infarction [75]. Like ATF6, Xbp1s deficient mice display a worse recovery from heart failure showing an increased infarct size while in vivo overexpression of this gene is translated into reduced infarct size after I/R injury. Similar to that observed in ATF6 and Xbp1s overexpression mouse models, in vivo gain-of-function of Ire1 results in preserved cardiac function and reduced fibrosis after myocardial infarction ^[27][76][27,76]. Unlike IRE1 or ATF6, PERK deficiency has a beneficial phenotype after heart failure displaying protection against pressure overload myocardial infarction suggesting that while ATF6 and IRE1 exert a protective role against heart failure, PERK and its downstream pathways are detrimental [74]. Furthermore, PERK is a key gene involved in the transcription activation of CHOP, an essential factor to trigger ERS-associated apoptosis. CHOP-deficient mice are resistant to cardiac hypertrophy, increased fibrosis and cardiac dysfunction pinpointing the importance of apoptosis in deleterious processes related to cardiovascular diseases [77]. Furthermore, loss of function of enzymes related to ERAD signalling have been carried out, reflecting the importance of this mechanism in cardiovascular diseases. For example, Hrp1 deficient mice display an exacerbated cardiac dysfunction after myocardial infarction demonstrating that loss of one mechanism either UPR signalling or ERAD components is enough to impede recovery from cardiac injury [78]. Accordingly, different murine models have proved that the three main pathways involved in ER stress signalling—ATF6, IRE1 and PERK—may play crucial roles in the progression of cardiovascular diseases exerting either protective roles such as in the case of ATF6 or IRE1, or deleterious roles, in the case of PERK. In addition, cardiac dysfunction related to Hrp1 double knockout (dKO) mutant mouse pinpoints the importance of ERAD signalling in cardiac homeostasis.