The impact of diabetes on tight junctions in cardiomyopathy dysfunction

Abstract

Diabetic cardiomyopathy is a condition characterized by ventricular dysfunction in diabetic patients that is not caused by other cardiac ailments. It is associated with factors such as left ventricular hypertrophy, metabolic disturbances, and oxidative stress. Tight junctions, which form a barrier between cells, play a role in the vascular complications of diabetes. Proteins such as claudins and occludens are important for the structure and function of tight junctions. Zona occludens (ZO) proteins are also involved in tight junctions and their expression may be affected by diabetes. The review discusses the impact of diabetes on the tight junctions and the role of ZO proteins in diabetic cardiovascular dysfunction.

Introduction

Diabetic cardiomyopathy (DCM) [1] Strictly speaking, diabetic cardiomyopathy refers to myocardial disorders that develop in diabetics but are not triggered by hypertension, coronary artery disease (CAD), or any other type of cardiac ailments [2]. This definition implies that there are diabetic patient groups whose cardiac structural and hemodynamic variations are unrelated to other main triggers such as CAD and hypertension [3]. Among the most prominent factors in the onset and advancement of diabetic cardiomyopathy, left ventricular hypertrophy, metabolic disturbances, extracellular matrix alterations, small vessel disease, cardiac autonomic neuropathy, insulin resistance, oxidative stress, and apoptosis can be named. [1]. However, there are discrepancies in the prevalence of diastolic dysfunction among patients with asymptomatic type 2 diabetes (T2D). Depending on the diagnostic methodologies used, the reported prevalence percentages range from 15 to 78% (47–50%) [2].

Pathophysiology of diabetic cardiomyopathy

Insulin resistance can be considered as a major factor for the progression of cardiovascular disease (CVD). Furthermore, heart failure can promote insulin resistance and is linked with greater risk of type 2 diabetesprogression. Insulin resistance causes early LV diastolic anomalies in hypertension irrelevant to the impact of high blood pressure, obesity and LV hypertrophy [1].

In a healthy subject, almost equal quantities of the energy necessary for myocardial function are provided by glucose metabolism and free fatty acids. Meanwhile, in diabetics, myocardial glucose consumption is drastically reduced, and energy production shifts to beta-oxidation of free fatty acids. Reduced glucose transporter proteins, including glucose transporter-1 & 4, cause the diabetic myocardium to consume less glucose. Furthermore, free fatty acids block pyruvate dehydrogenase, reducing myocardial energy generation and promoting apoptosis by accumulating glycolytic intermediates and ceramide [4, 5]. Aside from the impact of free fatty acids on glucose metabolism and oxidative phosphorylation, the synthesis of adenosine triphosphate from free fatty acid metabolism requires a substantial amount of oxygen. Cytotoxic byproducts (so-called lipotoxicity) of free fatty acid metabolism might affect myocyte calcium management, decreasing cardiac function [6].

Diabetes is known to lead to a variety of metabolic issues that can result in DCM. Hyperglycemia, dyslipidemia, increased free fatty acid (FFA) release, and insulin resistance are just a few of the factors that contribute to this. To better understand this concept, it’s important to note that hyperglycemia in diabetes is caused by impaired glucose uptake and excessive hepatic glucose synthesis. Prolonged hyperglycemia leads to metabolic and molecular changes in cardiac cells, resulting in the production of various mediators and cellular damage. Among these alterations, there is a higher flow of hexosamine and polyol pathways, permanent glycation of proteins, and activation of the enzyme protein kinase C (PKC). Because of these metabolic alterations, mitochondria produce reactive oxygen species (ROS), the ability of the primary antioxidant enzyme glutathione reductase (GR) diminishes, and advanced glycation end-products (AGEs) are formed. DNA injury and accelerated death of cardiomyocytes are an aftereffect of the disturbance of oxidative equilibrium, which is caused by increased oxidative stress [7].Elevated glucose levels block gap junctional intercellular communication (GJIC) in cultured vascular cells such as endothelial cells and smooth muscle cells [8]. Studies reveal that redundant phosphorylation of connexin-43, the key functional unit of the gap junction in vascular cells, is regulated by PKC. Furthermore, it was demonstrate that PKC-dependent increased phosphorylation of connexin-43 in diabetic rats causes ventricular transmission dysfunction in the heart. These findings imply that PKC-induced GJIC impairment could result in a wide spectrum of cardiovascular homeostasis issues and contribute to diabetes-related cardiovascular dysfunction. New insights into the involvement of the gap junction in the pathophysiology of diabetic vascular complications could be gained [9].

Tight junction (TJ)

Tight junctions (TJs) are sited among epithelial cells (ECs) at cell-cell contact locations. TJs were first discovered in the middle of the 20th century using thin section electron microscopy as complexes where ECs were tightly bound together [10] (Fig. 1). TJs link epithelial cells’ apical surfaces in a continuous manner [11]. It acts as a paracellular barrier, preventing ions and proteins from crossing tissue borders [12]. Tight junctions between endothelial cells form a vascular barrier that becomes more fragile and permeable in the diabetic state due to endothelial cell abnormalities [13]. Tissue edema and damage are results of TJ dysfunction which by itself takes place as a reply to an array of inflammatory stimuli and likewise amid ischemia. Moreover, occludin, claudin family members, junctional adhesion molecules 1 to 3, cingulin 7H6, spectrin, and linker proteins such as the zonula occludens family members (ZO-1/2/3) are all proteins that construct tight junctions [13, 14].

Fig. 1.

Illustration showing the structure of tight junctions between endothelial cells in the context of diabetic cardiovascular dysfunction. Tight junctions play a crucial role in regulating the flow of small molecules and ions between cells, and their dysfunction can lead to vascular complications in diabetes

Tight junction protein-1 dispersion is altered by hyperglycemia (HG) [15]. Inhibition of caludin-5 and − 11 (integral membrane proteins that are essential components of tight junctions, which are structures that regulate paracellular permeability in epithelial and endothelial cells) was observed in human coronary microvascular endothelial cells (HCMECs) exposed to HG. However, this suppression is reversed by TXL (a protein, plays a role in reversing the suppression of gene expression caused by H3K9ac) through the elevation of H3K9ac (acetylated lysine 9 of histone H3) in their specific gene promoters [13].

Tight junctions control the flow of tiny molecules and ions between cells while also determining the polarity of cell. Tight junctions are multiprotein compounds that are located where the membranes of two cells meet. The two functions of TJ include: (1) a fence function that inhibits the mixture of lipids between the apical and basolateral membranes, and (2) a gate function that between cells it controls the flow of ions and molecules [16]. Adhesions based on cadherin and nectin are required for tight junction creation. Tight junctions are made up of more than 40 proteins which are either transmembrane or actin-binding proteins within the cytoplasm. Occludins, caludins and junctional adhesion molecules (JAMs) are the main components of the transmembrane group. The zonula occludens (ZO) proteins are the main cytoplasmic actin binding proteins in tight junctions. The claudins are the most crucial components which they construct TJs backbone. They control the tight junction’s gatekeeping role by limiting the elements that flow through based upon their size. Overall, there are four transmembrane domains within the claudins, while the C-terminus and N-terminus are located within the cytoplasm [17, 18]. Claudins are vital for the arrangement of the TJ, on the other hand, occludins are expandable for welding of TJ. The prominence of occludins lies in their ability to sustain the cohesion and barrier function of TJ. They extend over the membrane four times and establish two extracellular loops, as well as cytoplasmic C- and N-terminus [17, 18]. ZO-1, ZO-2, and ZO-3 which are zona occludens proteins, are fundamentally comparable with two territories essential in intermediating protein-protein interactions. These are consisted of a SH3 and a PDZ territory which are infused with preserved protein-binding territories which are recognized as unique (U) motifs. Mediating interactions between each domain and specific proteins known to localize to tight and adherens junctions. For instance, while some ZO domains are speculated to bind occludin, ZO-1 PDZ domains bind tight junction proteins such as JAMs and claudins. Junctional adhesion molecules are the concluding transmembrane units of the tight junctions. JAMs are single membrane extending proteins with a cytoplasmic tail, an extracellular territory of two IgG-like folds and a transmembrane territory [19]. Regulation of the paracellular flow of solutes and ions and the apical-basolateral polarity are provided by tight junctions, including occludins, JAMs, claudins [4, 5, 20, 21]. The formation of functional tight junctions within tissue requires the dissociation of the ZO-1 scaffold [22].

Inside the epithelial cell sheets, claudins are situated at the TJs between cells, serving as cell-cell adhesion molecules. In mammals, the claudin family consists of 27 proteins with four-transmembrane domains. It was later discovered that common TJ strands are not solely formed from occludins [11].

Claudins-5, -9, and11 are proteins of tight junction which are primarily found in endothelial cells. Their lack can result in cell barrier disruption, which is believed to be the starting point and pathological premise of diabetic cardiovascular disease. The expression of claudin-5 and-11 genes were reduced by HG within HCMECs. There are more than 20 subtypes in the claudin family, all of which are paramount for the arrangement and objective of tight junctions [11]. Claudin-5, -9, and − 11 are predominantly expressed in the endothelial cells of the heart to assemble tight junctions and regulate microvascular permeability [12–14]. This study confirmed that claudin-5 and − 11 were suppressed by HG, resulting in impaired cell membrane localization. In a prior investigation, it was discovered that claudin-9 was solely inhibited by hypoxia in HCMECs [13].

Occludin, which has four transmembrane domains, forms a rate-limiting transport complex within the intercellular cleft [10]. The two extracellular loops of occludin form a junctional seal, while ZO-1, ZO-2, and ZP-3 connect the actin cytoskeleton to the carboxyl tail of occludin. ZO-1 plays a key role in the assembly and organization of transmembrane proteins. Unlike oxidative lipids, ZO-1 proteins are apparently not regulated by vascular endothelial growth factor (VEGF), shear stress, or oxidative lipids. However, they may be altered by oxidants (22). Even though tight junction without occludin are unique, it’s physiological objective with tight junctions is yet vague.

Sequence similarities have been identified among ZO-1, ZO-2, and ZO-3 [11]. In wild-type diabetic hearts, the expression of phosphorylated PKC-θ was reduced, while ZO-1 expression was increased, leading to decreased T-cell infiltration. ZO proteins, specifically ZO-1 and ZO-2, are crucial components of tight junctions in endothelial cells. ZO-1 expression has been reported to be reduced in failing human hearts.

Hypoxia decreases the expression of ZO-1 by increasing the expression of PKC-θ at the endothelial cell borders in a rat model of global hypoxia and reperfusion injury. The expression of ZO-1 was found to be reduced in diabetic Rag1 KO mice, suggesting that PKC-θ is required for ZO-1 expression, as demonstrated by PI treatment [12, 23–27].

Notably, there was a broader and more scattered expression of tight junction protein-1 (ZO-1) in diabetic subjects compared to control subjects. The expression of ZO-1 was found to be more extensive and dispersed among the epidermal cells in diabetic subjects compared to control subjects, as shown in. The positive field per keratinocyte of ZO-1 was significantly higher in diabetic subjects compared to control subjects, as determined by a quantitative analysis [15]. Zona occludens (ZO) proteins are one of the key components of tight junctions. Recent studies have shown that ZO-1 is reduced in failing human hearts. However, both ZO-1 and ZO-2 are present in tight junctions between endothelial cells [23–25].

Compared to control subjects, diabetic subjects exhibit a significantly broader and more distributed expression of tight junction protein-1, which is associated with tight junction formation. In bEnd.3 cells, mRNA levels of ZO-1 were significantly reduced after treatment with glucose at concentrations of 10mM and 25mM. Similarly, mRNA levels of CLD5 were significantly and dose-dependently decreased upon glucose treatment. The protein levels of CLD5 also showed a dose-dependent reduction following glucose treatment, consistent with the mRNA levels.

Gap junctions and its role in the cardiomyopathy

Gap junctions facilitate the exchange of small metabolites, ions, and second messengers between adjacent cells. They are formed by two protein families, connexins and pannexins. Gap junctional channels connect the cytoplasm of two cells, allowing the transfer of molecules such as glucose, ions (Ca2+, K+), and second messengers (IP3, cAMP, cGMP). While it is still unclear if small interfering RNAs are commonly exchanged in vivo, recent research has demonstrated the possibility of small interfering RNA conduction between adjacent cells through gap junctions [28].

Gap junctions, composed of connexin (Cx) protein subunits, enable direct intercellular communication and electrical coupling between adjacent cells. The human genome contains 21 connexin genes, while mice have 20 connexin genes, with 19 of them being sequence-orthologous sets [29].

In the heart, intercellular electrical coupling is primarily mediated by gap junctions. Connexin-43 expression is associated with various pathological conditions including hypertrophy, heart failure (HF), myocardial ischemia, and arrhythmias. Impaired gap junctional communication in diabetic cardiomyocytes may contribute to arrhythmias observed in a range of heart failure cases [30]. Contribution of cardiac gap junctions in electrical pairing among heart cells and regulated administration inside the heart is observed [31]. In the pathogenesis of diabetes cell-cell relationship by gap junctions; transmission and connexin hemi-channels are entangled.

The founding components of GJs, connexin (Cx) subunits, play a crucial role in electrical coupling between cardiomyocytes [16, 17]. Cx43, Cx40, and Cx45 inside the mammalian heart cells are the ones which are primarily expressed [17]. Interchanges in the expression of patterns of Cx have been correlated with a diversity of heart pathogenesis and take part in the progression of cardiac arrhythmia [32]. Direct intercellular communication is facilitated by channels composed of connexin (Cx) protein subunits, which are organized by GJs.Direct communication among adjacent cells is permitted by GJs amidst the numerous cell connection moderating protein complexes such as desmosomes, tight junctions, and cell adhesion molecules [29].

Desmosomes and its role in the cardiomyopathy

Desmosomes and adherens junctions (AJs) play a crucial role in connecting adjacent cells by attaching the fiber networks of keratin and actin. While AJs transduce and sense mechanical energies between the actomyosin cytoskeleton and plasma membrane, desmosomes and intermediate filaments (Ifs) provide the mechanical stability necessary for tissue coherence and architecture under mechanical tension. Desmosomes are particularly important for maintaining intercellular coherence, as they establish cell adhesion and contribute to mechanical balance [33].

Desmosomes consist of three protein families: desmogleins (DSGs), desmocollins (DSCs), and desmosomal cadherins. These proteins are anchored to the desmosomal plaque by their cytoplasmic tails, while their extracellular regions form the adhesive interface of the desmosome. The core structure of desmosomes is formed by DSG-DSC heterodimers [33].

In heart tissue, desmosomes are essential for coordinating heart muscle cells and maintaining tissue integrity. Along with gap junctions, which facilitate electrical transmission, desmosomes and adherens junctions are integral components of intercalated discs that connect adjacent heart cells. Adherens junctions are believed to modulate desmosome formation.

Genetic changes in desmosomal elements such as DSG2, DSC2, plakophilin 2, plakoglobin, and desmoplakin can lead to cardiac problems like arrhythmogenic right ventricular cardiomyopathy (ARVC). ARVC is characterized by fibrofatty substitution of cardiomyocytes, resulting from impaired cell adhesion caused by alterations in desmosomes. Clinical manifestations of ARVC include arrhythmias and right bundle branch block. Desmosomal distances between adjacent cell membranes have been measured to be approximately 320–350 Å. Overall, desmosomes play a critical role in maintaining tissue coherence and are particularly important in heart tissue for proper cell coordination and adhesion [34].

Connection of desmo&adher

Adherens junctions and desmosomes share systemic similarities as desmosomes originate from adherens junctions, but they serve distinct functions [35]. Both adherens junctions and desmosomes are calcium-dependent, cadherin-based intercellular junctions [36]. Adherens junctions have two focal configurations: [1] adherens junctions, which involve cell-type specific adhesion molecules from the cadherin superfamily that are linked to the actin cytoskeleton, and [2] desmosomes, which act as anchoring structures for intermediate filaments through desmosomal cadherins [34]. While a pathway is provided for the exchange of small molecules and ions between cells, adherens junctions and desmosomes primarily contribute to mechanical integrity and stability [37].

Adherens junctions and its role in the Cardiomyopathy

Adherens junctions are multi-protein complexes consisting of cadherin, β-catenin, and p120 catenin (p120ctn), and are critical subcellular structures for endothelial cell-cell adhesion [38]. Plakoglobin, also known as β-catenin (82 kDa), is the only desmosomal component that is also present in conventional adherens junctions [34]. Adherens junctions are specialized structures that facilitate mechanical communication between neighboring cells. The extracellular region, where cadherins tightly bind to one another, and the intracellular region, where the cytoplasmic end of cadherin is indirectly linked to the actin cytoskeleton, are two important sites for cell-cell mechanical anchoring [37]. Adherens junctions are considered important intercellular adhesion complexes and are composed of three protein families: transmembrane cadherins, armadillo proteins, and cytoskeletal adaptors [19]. Desmoplakin plays a role in the development of adherens junctions during epithelial sheet formation [39].

In cells lacking desmoplakin and regular intermediate filament attachments, adherens junctions cannot form normally [35]. Poorly managed diabetes and cardiopulmonary bypass can decrease endothelial adherens junction protein activation, expression, and localization [40]. Overall, adherens junctions are important for regulating cell-cell adhesion and maintaining tissue integrity.

Conclusion

Tight junctions play a crucial role in the pathophysiology of diabetic vascular complications. These intercellular junctions, located between endothelial cells, form a barrier that regulates the flow of small molecules and ions between cells. In diabetes, the tight junctions become more fragile and permeable, leading to endothelial cell abnormalities and increased vascular permeability. This dysfunction of tight junctions can contribute to tissue edema, damage, and impaired cardiovascular homeostasis. Proteins such as claudins, occludins, and ZO proteins are important components of tight junctions, and their expression and phosphorylation can be altered by hyperglycemia and other metabolic disturbances associated with diabetes.

The alterations in tight junction proteins discussed in this review have several important clinical implications for the diagnosis, treatment, and prognosis of diabetic cardiovascular dysfunction. For diagnosis, reduced expression and disrupted localization of tight junction proteins like claudin-5, claudin-11, and ZO-1 can serve as potential biomarkers for endothelial dysfunction in diabetic cardiomyopathy. Quantitative assessment of tight junction protein levels and distribution patterns in cardiac or vascular tissue samples may aid in the early detection and diagnosis of diabetic cardiovascular complications. Additionally, monitoring changes in tight junction protein expression could help identify patients at higher risk of developing diabetic cardiomyopathy, allowing for earlier intervention.

For treatment, therapies targeting the restoration of tight junction integrity, such as modulating the expression or function of claudins, occludins, and ZO proteins, may have therapeutic potential for mitigating diabetic cardiovascular dysfunction. Interventions that can reverse the hyperglycemia-induced suppression of tight junction proteins may help improve endothelial barrier function and reduce vascular permeability in diabetes. Furthermore, pharmacological agents that can stabilize tight junctions or prevent their disruption may be explored as adjunctive treatments for diabetic cardiomyopathy.

Regarding prognosis, the degree of tight junction protein dysregulation may correlate with the severity and progression of diabetic cardiovascular disease. Patients with more pronounced alterations in tight junction proteins are likely to have poorer cardiovascular outcomes, such as increased risk of heart failure, arrhythmias, and other complications. Tracking the changes in tight junction protein levels and localization over time could provide valuable prognostic information and guide the management of diabetic patients at risk of cardiovascular complications.

In summary, the review highlights the importance of evaluating the status of tight junction proteins as potential biomarkers for the early detection and diagnosis of diabetic cardiovascular complications. Monitoring the changes in tight junction protein levels and distribution patterns could help identify high-risk patients and guide targeted interventions. Additionally, therapies aimed at restoring tight junction integrity, either by modulating the expression or function of these proteins, may hold promise as novel treatment strategies for mitigating the vascular consequences of diabetes. Future research should further explore the specific signaling pathways and molecular mechanisms by which hyperglycemia, oxidative stress, and other diabetes-related factors disrupt the structure and function of tight junctions in the cardiovascular system. Investigating the potential cross-talk between tight junctions and other intercellular adhesion complexes, such as gap junctions and desmosomes, may also provide valuable insights into the comprehensive pathophysiology of diabetic cardiomyopathy. Ultimately, a better understanding of the role of tight junction proteins in the development and progression of diabetic cardiovascular dysfunction could inform the design of more effective diagnostic tools and targeted therapeutic interventions for this debilitating complication of diabetes.

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Since the manuscript is a review article and does not involve the collection of original data or materials that raise ethical considerations, the availability of data and materials is not applicable in this context.

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Ethical approval

As this manuscript is a review article and does not involve any original research or data collection involving human subjects or animals, ethical approval is not applicable in this case.

Conflict of interest

The authors declare there is no conflict of interest.