Role of Membrane Microdomains in Cardiac Protection: Strategies for Diabetic Cardiomyopathy

Abstract

Diabetic cardiomyopathy (DCM) is a cardiac disorder characterized by structural and functional impairments independent of coronary artery disease. Membrane microdomains, including lipid rafts and caveolae, play a crucial role in cardiac signaling, insulin receptor trafficking, and ion channel regulation. In diabetes, disrupting these microdomains leads to impaired insulin signaling, oxidative stress, and mitochondrial dysfunction, exacerbating cardiac pathology. This review explores the role of caveolins and lipid rafts in DCM and how their dysfunction contributes to disease progression. We highlight how therapeutics used to manage diabetic patients may impact microdomain integrity. Future research should focus on targeting membrane microdomains for novel treatments.

I. Introduction

The Centers for Disease Control and Prevention reports that diabetes affects more than 38.4 million people in the U.S. (11.6% of the population), with healthcare expenses for treatment reaching $413 billion annually, including both direct medical costs and indirect costs such as disability, work loss, and premature mortality. In addition, there may be as many as 79 million prediabetic individuals. A large epidemiologic study found that a discharge diagnosis of diabetes was given in nearly 40% of cases, and this diagnosis did not discriminate by race or geographical location (1). Further, cardiovascular disease accounted for nearly 50% of all diabetic hospitalizations (1). Such statistics underscore a strong link between diabetes and heart disease and point to a growing epidemic that is approaching a tipping point. Therefore, it is essential to enhance our understanding of the mechanisms underlying diabetic cardiomyopathy (DCM) to improve therapies that could help prevent cardiac dysfunction associated with diabetes.

Currently, about 5.7 million people over the age of 20 years in the US are clinically diagnosed with heart failure (HF) (2, 3), and projections show that this number will increase 46% by 2030 (4). HF is defined as a “complex clinical syndrome that results from any structural or functional impairment of ventricular filling or ejection of blood” (5) and is classified into stages and functions by the ACCF/AHA (6) and the New York Heart Association (7). Several diseases in their final stages are associated with HF. One disease most prone to HF is metabolic syndrome, which includes insulin resistance, type 2 diabetes mellitus (T2DM), and associated cardiovascular disease, with approximately 50 million people suffering from this condition in the US alone (8). The harmonized definition of metabolic syndrome proposed by Alberti et al. in 2009 requires 3 out of 5 risk factors to be present to establish the diagnosis and include, 1) elevated waist circumference, 2) elevated triglycerides, 3) reduced HDL-C, 4) elevated blood pressure, and 5) elevated fasting glucose (9, 10).

Why is the diabetic heart dysfunctional?

DCM is characterized by impaired cardiac function and structure independent of vascular pathology. In animal models, diabetes contributes to both diastolic and systolic dysfunction. Diastolic impairment is marked by delayed relaxation and elevated left ventricular end-diastolic pressure, while systolic dysfunction manifests as alterations in heart rate, systolic blood pressure, and fractional shortening (11). Similar findings are also observed in humans (12). Structural changes occur in the diabetic heart, including fibrosis, myocyte loss, altered mitochondrial structure, and altered cardiac ultrastructure (13–15). T2DM is a frequent comorbidity associated with HF with preserved ejection fraction (HFpEF), found in approximately 30–40% of T2DM patients; often myocardial dysfunction is present even in the absence of significant coronary artery disease or hypertension, and then called “diabetic cardiomyopathy” (16–19). Multiple mechanisms are involved in the development of HFpEF in individuals with T2DM, including impaired cardiac substrate utilization and metabolism, altered insulin signaling leading to protein kinase C activation, cell-to-cell as well as cell-to-matrix connections, nitric oxide production from endothelial cells, mechanosensing, Ca²⁺ signaling, increased cytokine production (TGF-beta activation ), advanced glycated end product deposition, and impaired endothelial-to-cardiomyocyte crosstalk (18).

In all organs, small vessels (i.e., microvasculature and the arteriolar and capillary bed) are responsible for supplying tissues with oxygen and nutrients. Coronary microvascular disease (aka small vessel disease) is caused by the same risk factors that lead to atherosclerosis-induced cardiovascular disease, such as diabetes, obesity, hypertension, lack of physical activity, unhealthy diet, elevated levels of oxidized LDL-C, autoimmune disorders, and smoking (to name a few) (20, 21). Coronary microvascular disease is more prevalent in women and presents with unspecific symptoms such as chronic fatigue and exercise intolerance, and aggressive management of co-morbidities is a treatment goal (22).

Additional molecular mechanisms proposed for diabetes-induced heart disease include altered substrate supply and utilization, altered energy generation with mitochondrial dysfunction, altered ion channel function, myocyte apoptosis, cardiac insulin resistance, and renin-angiotensin system activation (23). Diabetic hearts show reduced responsiveness to protective strategies to minimize ischemia-reperfusion injury, suggesting substantial dysfunction in survival kinase signaling pathways (24). These results suggest that diabetic DCM is associated with multiple cellular changes, some of which may be linked by a shared regulatory mechanism that could serve as a potential therapeutic target. The reduced insulin sensitivity associated with T2DM is primarily based on impaired function or insulin receptor (IR) signaling resulting from hyperglycemia.

II. Membrane Microdomains and Signaling: Caveolae and Caveolins

An important aspect of cardiac signaling is the proper localization of receptors and integrated effectors in the membrane. Not all membrane regions are created equal, as some contain specialized regions with specific lipid and protein compositions that provide distinct structural and functional properties. These regions are called membrane microdomains, and they are critical for numerous cellular processes such as signaling, trafficking, and membrane organization.

Membrane microdomains that are enriched in cholesterol, (glycol)sphingolipids, and saturated long-chain glycolipids are called “lipid rafts”. Microdomains are not only present in the plasma membrane but also found in the endoplasmic reticulum/Golgi, and nuclear, lysosomal, and mitochondrial membranes (25–29). The organization of lipid rafts in vivo is controlled by cholesterol-binding proteins, which include three caveolins, four stomatins, two flotillins, and podocin (only expressed in kidney podocytes) (30, 31). Given the importance of caveolin in dynamically regulating plasma membranes in disease, this review will focus on caveolin-containing microdomains.

Identified in the 1950s by electron microscopy, caveolae appear as morphologically identifiable invaginations of the plasma membrane (32) of most differentiated cell types, including myocytes, endothelial cells, and adipocytes (33). Interestingly, these are the main cell types involved in glucose regulation. Caveolae expand the cell surface area and play a crucial role in macromolecular transport, preserving plasma membrane integrity, facilitating vesicular trafficking, mediating signal transduction, and regulating mechanotransduction (34). In mammals, caveolae comprise three transmembrane proteins: caveolin-1 (Cav-1), caveolin-2 (Cav-2), and caveolin-3 (Cav-3). Caveolins, act as scaffolds to concentrate lipids (cholesterol and glycosphingolipids) (35), signaling proteins (heterotrimeric G-proteins, H-Ras, nitric oxide synthase (NOS), epidermal growth factor receptor, protein kinases, and Src-like kinases) (36), and structural proteins (37) within caveolae. All three caveolin knockout (KO) mice have cardiovascular phenotypes, suggesting caveolins are critical for the regulation and maintenance of signaling events in the cardiovascular system. However, Cav-3 is specifically required for caveolae formation in cardiac, skeletal, and certain smooth muscles (38). Cav-3 deficiency results in muscle degeneration, heart failure, and increased adiposity (39). Caveolins have a crucial role in insulin signaling, localization of certain glucose transporters, and lipidosis in DM (40, 41). Caveolae and caveolins help safeguard the heart against ischemia-reperfusion injury, with insulin signaling – specifically Akt activation and GLUT regulation–playing a crucial role in this protective mechanism (42, 43). Cardiomyocyte-specific overexpression of Cav-3 (Cav-3 OE) protects from pressure-overload-induced heart failure (44).

Cav-3 KO mice have various deleterious features, including muscle degeneration (45), insulin resistance (46), and progressive cardiomyopathy (39). Cav-1 knockdown in adipocytes results in loss of insulin receptor signaling due to decreased insulin receptor and glucose transporter (GLUT4) expression (47). In H9C2 cardiomyoblasts, Cav-1 knockdown inhibits IGF signaling (48), as well as insulin signaling coupled to Akt and glucose transport (49). Cav-3, on the other hand, is a positive regulator of insulin signaling (50). Gene transfer of caveolin to the liver improves glucose metabolism in diabetic mice (51). Cav-3 OE in cardiac myocytes increases Akt phosphorylation, which helps protect the heart from ischemic injuries and high blood pressure-induced mechanical stress (42). Resveratrol recruits GLUT4 to caveolae and increases Akt signaling in Type I DM (52, 53). In addition, caveolae contain a host of signaling molecules, and alterations in the regulation of Ca²⁺ storage and influx may limit diabetic cardiomyopathy (54).

Signaling pathways facilitated by membrane microdomains

The Singer-Nicolson fluid-mosaic membrane-type model was proposed in 1972 (55), but the view of plasma membranes has evolved since then (56). In 2012, Kusumi et al. proposed a three-tier model separating the actin cytoskeleton (40–300 nM), from lipid rafts (2–20 nm), and dynamic protein domains (3–20 nm) (26). Plasma membranes constantly change their structure, control biological functions, and are hubs for “outside-in” and “inside-out” signaling functions in endothelial cells, immune cells, fibroblasts, smooth muscle, and striated cardiac myocytes. Structural changes are hard to correlate to protein level changes (i.e., expression of caveolins) as these may increase or decrease with disease; however, biochemical approaches to assess caveolar/lipid raft microdomain content through measurement of cholesterol in a density gradient or use of electron microscopy (36) to determine membrane ultrastructure could be utilized in disease settings given tissue samples are available. Microdomains in all these cell-types are involved in the development of DCM. Plasma membrane proteins such as the large family of G-protein coupled receptors (GPCRs), Src family-protein kinases, and mitogen-activated protein kinases (MAPK) are associated with caveolar membrane invaginations (caveolae) containing Cav-1, -2, and/or -3, or certain membrane proteins and receptors, such as insulin-like growth factor-1 (IGF-1), aquaporin-1, Thy-1, and N-cadherin – a glycosylphosphatidylinositol-anchored membrane protein and glycoprotein – are uniquely associated with non-caveolar lipid microdomains (57).

Membrane lipid raft-specific cell signaling in the context of DCM (58) include 1) the insulin receptor (IR) (59), 2) GPCRs, 3) endothelial nitric oxide synthase (eNOS) and NO (60), 4) calcium compartmentalization within the endoplasmic reticulum (ER), the sarcoplasmic reticulum (SR) in muscle and endothelial cells, as well as within mitochondria (61–66), 5) cellular communication and mechano-sensing (integrins, gap junctions, tight junctions) (67) and 6) oxidative stress/inflammation (68). We will discuss microdomain-related signaling pathways that are relevant in cardiac cell-types affected by T2DM and will review cell-specific pathway alterations.

Insulin Signaling in Membrane Microdomains

Caveolae are enriched in endothelial cells, adipocytes, muscle cells, and fibroblasts, contain caveolins as structural components, and act as hubs for numerous essential receptors involved in various critical functions. One such receptor localized to caveolae is the insulin receptor 1a (Figure 1A), which binds insulin and transports it to target organs such as the heart, skeletal muscle, and brain and is a key regulator of cell proliferation, differentiation, growth processes, and glucose homeostasis (69–73). The cell membrane is impermeable to glucose and relies on glucose transporters. Three classes of eukaryotic sugar transporters have been identified, including glucose transporters (GLUT), sodium-glucose symporters (SGLTs), and sugars will eventually be exported transporters (SWEETs) (74). Since the insulin receptor is enriched in caveolae, insulin-stimulated glucose homeostasis is critically linked to membrane microdomains/lipid rafts.

Figure 1: Caveolae-mediated insulin transport for insulin receptor-stimulated glucose homeostasis.

A schematic representation illustrating the caveolae-enriched insulin receptor complex and the role of caveolae-mediated insulin transport, and PI3K-Akt-dependent GLUT4 vesicle translocation. (A) The insulin receptor complex consists of transmembrane receptors that bind circulating insulin, initiating downstream signaling cascades involving the phosphorylation of the insulin receptor substrate (IRS), the recruitment of phosphoinositide 3-kinase (PI3K), leading to Akt phosphorylation and PI3K-Akt-dependent GLUT4 storage vesicle translocation from the cytosol to the cell membrane. Membrane-bound GLUT4 increases cellular glucose uptake for glucose homeostasis and cellular metabolism. (B) In endothelial cells, particularly within the cardiac microvasculature (Bi) and the blood-brain barrier (Bii), caveolae play a crucial role in insulin transcytosis. This specialized transport mechanism facilitates the regulated movement of insulin from the bloodstream into underlying tissues, ensuring proper metabolic and neurovascular function. Endothelial dysfunction disrupts in this process and has been implicated in cardiovascular disease, neurodegenerative diseases, metabolic disorders, and insulin resistance, highlighting the significance of endothelial insulin transport in systemic health. (C)Transmission electron microscopic scan of murine cardiac tissue showing the proximity of capillaries, composed of an endothelial cell monolayer, and cardiac myocytes (scale 2 μm). (D) Endothelial cell (red box) and cardiomyocyte (blue box) caveolae are shown in a higher magnification of the outlined area from Fig. 1C (scale 500 nm).

The skeletal muscle is the primary driver of glycemic control and plays a crucial role in insulin-stimulated glucose utilization (75). Skeletal and cardiac muscle differ in metabolic substrate utilization. In contrast, skeletal muscle relies on glucose, the primary substrate of the cardiac muscle is free fatty acids, but under ischemic conditions, the heart changes its substrate utilization (76, 77). Insulin binds to the insulin receptor and triggers the insulin signals through the insulin receptor substrate (IRS)-2 via autophosphorylation, leading to a recruitment of phosphoinositide 3-kinase (PI3K), ultimately promoting Akt activation that leads to the translocation of GLUT4 vesicle from to intracellular storage sites to the plasma membrane (Figure 1A). Insulin travels from the pancreas through the vascular tree and binds to insulin receptors on endothelial cells within the capillary bed. After transcellular transport, insulin can bind to insulin receptors at the end organ (e.g., cardiac myocytes, neurons, etc.) (Figure 1B). The monolayer of cardiac endothelial cells is responsible for delivering insulin and other nutrients to cardiac myocytes. Ultrastructural analysis shows that caveolae in endothelial cells and cardiac myocytes are in close proximity (Figure 1C, D). Because endothelial cell dysfunction is a hallmark of impaired glycemic control during T2DM, intense research to understand and restore insulin signaling is currently underway.

Cav-1 null mice fed a high-fat diet developed hyperinsulinemia, and insulin tolerance tests in young Cav-1 null mice on a normal diet showed significantly reduced insulin sensitivity compared to wild-type controls (78). Cav-1 plays an integral role in localizing insulin receptors and some of its downstream effector proteins within caveolae, thereby facilitating their signaling. This is followed by internalization of the IR, which is then brought back to the membrane surface, a mechanism that circumvents clathrin-mediated receptor desensitization (79). Another study explored the expression of Cav-3 in skeletal muscle and its role in regulating insulin signaling, glucose metabolism, and homeostasis (69–73). The authors found that Cav-3 deficiency decreased insulin receptor stability, suggesting it prevents insulin receptor degradation. Haddad et al. reviewed caveolin’s role in insulin secretion, signaling, and the development of insulin resistance (80).

GPCR-related Signaling in Membrane Microdomains

Beta-adrenergic receptors (βARs) are heptahelical GPCRs that are activated by norepinephrine and epinephrine as part of the sympathoadrenal response system in the heart. To fully grasp GPCR signaling, it is essential to account for the receptors’ subcellular distribution and the downstream signaling components they engage with (81–84). The localization of β₁ARs and β₂ARs vary within cardiac myocytes (85, 86): β₁ARs are widely found within the sarcolemma while β₂ARs are predominantly localized to caveolar microdomains (subsets of membrane/lipid rafts). The βAR subtypes present in cardiac myocytes exhibit distinct signaling characteristics. Activating β1AR triggers ionotropic and chronotropic effects by engaging global G stimulatory (Gs) coupling. This, in turn, stimulates adenylyl cyclase (primarily the AC5 and AC6 isoforms in the heart), leading to the activation of protein kinase A (PKA), Epac, L-type calcium channels, and various downstream effectors (87–91). In contrast, β₂AR interact with both stimulatory (G_s) and inhibitory (G_i) pathways. When β2AR are confined to caveolae, they initially produce brief PKA-dependent ionotropic effects near L-type calcium channels. This is followed by a reduction in contraction, driven by the sequential activation of G_s and G_i proteins (91) (G_i proteins are enriched in caveolae (90)). The distinct localization of βAR subtypes influence cell death responses in heart failure: β₁AR stimulation may trigger myocyte apoptosis, whereas β₂AR activation can exert an anti-apoptotic effect via a G_i-, phosphoinositide 3-kinase (PI3K)-, Akt-dependent pathway (92, 93).

β₂AR situated within caveolae are linked to transverse (T)-tubules, where they generate localized cAMP signals in cardiac myocytes (86, 94). T-tubules are enriched in cholesterol and show high Cav-3 protein abundance (95). The deterioration of T-tubule structure is believed to contribute to the advancement of HF, alongside changes in the subcellular localization of βARs and their related signaling elements (96). With the progression of HF, the confinement of β₂ARs within T-tubules can become disrupted, leading to their redistribution onto the myocyte plasma membrane, where β₁AR is the dominant subtype. This relocation enables β₂ARs to produce a widespread cAMP signal like that generated by β₁AR activation, further aggravating myocyte dysfunction (94). It is of note that the altered distribution of the βARs in heart failure can be replicated by chemically disrupting the T-tubule/caveolae microenvironment (94).

Clinical studies and animal models indicate that α1_A-adrenergic receptors (α1_A-AR) play a role in both the growth of cardiomyocytes and pathological hypertrophy. In conditions with an overwhelming increase in pressure, activation of α1_A-AR may provide protective, pro-survival effects thereby helping to prevent the progression of HF (97). A key component of cardiomyocyte function is the presence of K_v4 channels which is a type of voltage-gated potassium channel localized within caveolae-containing membrane lipid rafts in both the heart and brain (98). In a study that investigated how the heart responds to increased physical demands (like those seen in T2D), it was found that the activation of the α1_A-AR led to the phosphorylation of K_v4 channels located within caveolae, which reduced the transient outward potassium current (98, 99). While this reduction resulted in more significant calcium influx and stronger contraction, an excessive decrease in the transient outward potassium current can prolong the action potential and trigger cardiac arrhythmias. It is important to note that there is another population of voltage-gated potassium channels (K_v4.3) in the same cell that are found in non-caveolar membrane microdomains which are not responsive to α1_A-AR activity (99), suggesting that two different population of the same channel complex can exist in the same cell (i.e., caveolar and non-caveolar localized). Therefore, positioning signaling components within caveolae-containing microdomains is essential for proper cardiac function. A study examining the impact of the α1_A-AR agonist A61603 on cardiac myocyte survival found that it activated ERK signaling, protecting the cells from death caused by doxorubicin-induced oxidative stress without affecting blood pressure (100). ERK, a member of the mitogen-activated protein kinase (MAPK) family, requires caveolin expression for its activation. These findings suggest that α1_A-AR agonists can improve cardiac function by regulating signaling pathways dependent on caveolae and caveolins.

It is interesting to note that while α1-ARs are localized to membrane microdomains, there is some evidence to suggest that a large percentage of functional α1-ARs localize to the nuclear membrane of cardiomyocytes (101). Excitingly, the nuclear membrane also consists of membrane microdomains with specific lipid composition which essentially serves the same role as in the membrane – to act as platforms for protein organization and signaling (102). While further investigation is necessary to determine the prevalence of caveolae in these nuclear lipid rafts, there is evidence to suggest the presence of caveolins within these nuclear membrane microdomains, which regulate gene expression (103). For instance, Jeong et al. described the role of Cav-2 in mediating the nuclear response to insulin, demonstrating its localization to the inner nuclear membrane, where it inhibited heterochromatinization and activated transcription factors Elk-1 and STAT3 (104). These observations highlight caveolin’s role in insulin signaling and nuclear gene regulation.

Interaction between microdomains and Ca²⁺ signaling and ion channel regulation

Cardiac excitation-contraction coupling is triggered by Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channel (L-VDCC) during the upstroke of an action potential. The primary L-VDCC in the heart is Ca_v1.2 (105), which is bound to Cav-3 (27). Intracellular calcium compartmentalization, which safeguards against Ca²⁺ overload, is disrupted in T2DM. Consequently, cardioprotective mechanisms are tightly connected to ion channel regulation.

Cav-3 OE protects from pathological cardiac hypertrophy (44). During ischemia-reperfusion (I/R) injury, membrane microdomains are crucial in limiting calcium overload, a primary contributor to cellular damage. Gong et al. demonstrated that Cav-3 protects diabetic hearts against acute myocardial infarction and reperfusion injury (106). This likely occurs via the clustering of Ca_v1.2 channels within caveolae, which allows for their regulation by local kinases, such as protein kinase A (PKA) and protein kinase C (PKC). These kinases modulate the activity of Ca_v1.2 channels in response to neurohormonal signals (107). This tight regulation helps to prevent excessive calcium influx during reperfusion, reducing the risk of Ca²⁺-induced apoptosis and necrosis.

Furthermore, evidence suggests that caveolae stabilize the function of potassium channels, such as the ATP-sensitive K⁺ (K_ATP) channels, during metabolic stress. Mitochondrial and sarcolemmal K_ATP channels play a differential role during ischemia-reperfusion injury (108, 109). Activation of K_ATP channels is cardioprotective during ischemia by shortening the action potential and reducing calcium entry into the cell, thus conserving energy and preventing calcium overload (110). Cav-3 interacts with and regulates K_ATP channel activity, highlighting the role of membrane microdomains in modulating ion channel function during cardioprotective responses.

eNOS-related Signaling in Membrane Microdomains

In the cardiovascular system, endothelial nitric oxide synthase (eNOS) is the enzyme responsible for producing nitric oxide (NO). eNOS is localized to caveolae in endothelial cells and cardiac myocytes (111). On one hand, Cav-1 interacts with the eNOS domain, and this direct interaction sequesters and primarily inhibits basal eNOS activity, preventing its binding to calmodulin, thereby diminishing eNOS’s ability to produce NO (112). The same interaction may evoke agonist-induced stimulation of eNOS, allowing an increase of eNOS when needed, because of the enriched localization in caveolae, this activation response can be significant, and may result in an eNOS burst. This phenomenon is termed the “eNOS Cav-1 paradox” of contradictory ways Cav-1 inhibits and activates eNOS (113, 114). Additionally, eNOS and Cav are co-regulated because eNOS-derived NO promotes caveolae-mediated endocytosis, thereby maximizing the paracrine effects of NO (Figure 2) (115). High levels of NO are cytotoxic; therefore, tight regulation of eNOS activation exists besides the well-known calcium-triggered calmodulin-binding of eNOS.

Figure 2: Caveolae-associated eNOS signaling pathway mechanism in health and diabetes. Health Scenario.

Caveolae behaves as a physical and biological signaling hub in the cardiomyocyte controlling NO-mediated signaling cascades through Cav-3 (in cardiomyocyte) as eNOS repressor. During the muscle fiber stretching, potential conformational changes in Cav3 transmembrane domain (TMD) and caveolin scaffolding domain (CSD) results in the release of eNOS. In addition, the transient increase in intracellular Ca²⁺ concentration (partially mediated by Ca_v1.2, also controlled by Cav-3) activates the Ca²⁺-Calmodulin complex, an allosteric competitor of Cav-3-eNOS complex, promoting eNOS dislocation and its subsequent activation. NO inhibits mitochondrial complex IV leading to a reduction in O₂ consumption, oxidative phosphorylation, and ATP synthesis. In addition, NO inhibits Cav1.2 channel and attenuates b₁-Adrenergic receptor-mediated Cav1.2 activation, reducing [Ca²⁺]_I, resulting in a final effect of sarcomere relaxation. Diabetes Scenario 1. Diabetes impairs Cav-3 caveolar localization, altering caveolae ultrastructure, stability, and cholesterol and Cav3 content, impairing cardiomyocyte Cav-3-dependent signaling, like Ca_v1.2 inward Ca²⁺ currents modulation. Caveolae cholesterol content reduction increases molecular freedom degrees, triggering Cav-3 conformational changes that release eNOS, leaving it in a constant active state that will produce a persistent sarcomere relaxed state and poor contractility force, compromising the cardiac function. Diabetes Scenario 2. Diabetes results in Cav-3 localization shift from caveolae to non-caveolar cardiomyocyte membrane. Non-caveolar Cav-3 localization presents significant biophysical differences compared with caveolae, together with a different signaling molecules and receptors landscape. This condition may favor Cav-3-eNOS complexes, inhibiting NO signaling in the diabetic cardiomyocyte. In the absence of NO relaxation signal and impaired Cav-3-Integrin b1D mechanotransduction system, the sarcomere contraction force opposes the cardiomyocyte stretching, producing fiber damage (113, 114, 262–265).

Caveolae play a significant role in regulating NO signaling, where physiological levels of NO induce smooth muscle relaxation (vasodilation), low levels of NO lead to smooth muscle contraction (vasoconstriction), and high levels of NO induce inflammation and cell death (116). The localization of eNOS within caveolae ensures that NO production is tightly regulated and needed in response to cardiac stress due to mechanical stretch, neurohormonal signaling, ischemia, or metabolic changes.

Pulmonary artery smooth muscle cells isolated from patients with idiopathic pulmonary arterial hypertension who underwent lung/heart transplantation showed increased Cav-1 protein expression and caveolae formation (117). In contrast, Cav-1 deficient mice that lack Cav-1 and Cav-2 expression develop dilated cardiomyopathy, pulmonary hypertension, right ventricular hypertrophy, and vasculopathies (118). It was also shown that Cav-1 regulates the production of bioactive NO, thereby regulating blood pressure variability in mice (119). In mice with Type 1 and Type 2 Diabetes, protein expression of Cav-1 was increased, and dose-dependent acetylcholine-induced vascular relaxation attenuated (120).

High levels of LDL cholesterol increased the inhibitory caveolin-eNOS complex formation in endothelial cells leading to reduced basal NO production (121). An independent study by Blair et al. showed that oxidized LDL, but not native LDL, or HDL, depleted cholesterol from caveolae from cholesterol, induced redistribution of eNOS, and caveolin from caveolae to internal membrane sites, and inhibited the ability of acetylcholine to activate the enzyme efficiently (122). Lipoproteins (e.g., oxidized LDL versus HDL) have potent effects on eNOS function in caveolae via actions in both membrane cholesterol homeostasis and enzyme activation (123–125).

Studies show that individuals with T2DM present lower NO production levels than healthy individuals (126–128). A recent review by Bahadoran et al. explores NO resistance in T2DM, highlighting the diminished vasodilatory response of NO in both macrovascular and microvascular systems (129). This impairment arises due to the suppression of NO activity, reduced sensitivity of soluble guanylate cyclase (SGC), and/or dysfunction within the cGMP-protein kinase G (PKG) signaling pathway. It is also postulated that NO production is pivotal during compromised exercise performance in subjects with T2DM (130). The exact mechanism of how T2DM (and/or obesity) affects caveolae and insulin resistance is still unclear.

Cell adhesions and membrane microdomains in the heart

In the continuously contacting heart, endothelial cells, fibroblasts, and myocytes are mechanically challenged. Thus, to survive in this environment cardiac cells rely on stable cell-to-matrix and cell-to-cell adhesions (Figure 3). Caveolin-containing lipid rafts are found at both adhesion sites.

Figure 3: Schematic visualization of cell-to-matrix and cell-to-cell junctions in cardiac myocytes:

Dynamic crosstalk between the connective tissue interlacing cardiac myocytes and endothelial cells exists. Connective tissue between cardiomyocytes ensures proper cell anchorage. In cardiac myocytes cell-to-matrix adhesions, and cell-to-cell connections enable intricate signaling mechanisms that regulate cardiac contractility. Caveolae are critical for the localization of integrin, therefore organizing a multiprotein complex containing talin, vinculin, and α-actinin which link the sarcolemma to the actin cytoskeleton and the sarcomere. Integrin-mediated cell adhesions influence cardiac myocyte function by regulating contractility and mechanical stiffness through outside-in and inside-out signaling. F-Actin, a key component of the myocyte cytoskeleton, plays a role in mechanotransduction and cellular signaling, while desmin, an intermediate filament protein in cardiac muscle, supports mitochondrial positioning and structural integrity. These cytoskeletal elements help maintain cellular architecture and facilitate efficient force transmission between myocytes, the connective tissue, and endothelial cells. This bidirectional communication is essential for maintaining cardiac efficiency and for adapting to physiological and pathological conditions, including ischemia, pressure-overload, and metabolic challenges. Mitochondria, essential for the high ATP demand in cardiac myocytes, are tightly regulated by endothelial-derived signals to maintain energy balance and prevent oxidative stress. A dysfunctional endothelial-to-matrix-to-cardiomyocyte communication can lead to mitochondrial dysregulation, contributing to metabolic inefficiency and cardiac dysfunction. Physiological cardiomyocyte-to-cardiomyocyte adhesion sites contain gap junctions, adheres junctions, and desmosomes to ensure the mechanical anchorage between cells and electrical connectivity. Understanding how these adhesion sites are altered during diabetes provides valuable insights into the molecular mechanisms leading to the development of diabetic cardiomyopathy to discover novel therapeutic targets.

Focal adhesions are the most abundant cell-to-matrix junctions that connect the extracellular matrix (ECM) through integrins, talin, vinculin, and alpha-actinin to the actin cytoskeleton. Integrins are a large family of transmembrane proteins that form heterodimers, consisting of both α and β subunits. These integrins function in cell-to-ECM and cell-to-cell junctions and connect the ECM to the cytoskeleton to participate in inside-out and outside-in mechanosignaling (131, 132). Integrins are mechanosensors that convert mechanical stimuli to intracellular signaling cascades (133).

Lack of β1 integrin, talin, or vinculin expression in all murine tissues (global knockout) results in early embryonic lethality (134–137). Mice with cardiomyocyte-specific knockout of β1-integrin show disturbed glucose metabolism, cardiac fibrosis, and heart failure (138). Cardiomyocyte-specific deletion of talin1 and talin2 leads to dilated cardiomyopathy (139). Global hemizygous vinculin knockout mice were intolerant to cardiac pressure-overload-induced hypertrophy. Cardiac-specific vinculin knockout mice presented arrhythmias, ultrastructural disturbed cell-to-cell contact sites, and rapid heart failure development, leading to premature death at an early age (140). Mutations in the vinculin/metavinculin gene are found in families with inherited cardiomyopathy (141). These animal studies suggest that integrin-containing cell-to-matrix junctions are crucial anchorage points in cardiac myocytes.

Multiple mutations in the Cav3 gene were identified in human disease, spanning from distal skeletal myopathy (weakness), to rippling (hypercontractile) muscle disease, and cardiac arrhythmias (Long-QT-syndromes) (142–147). Cav-3 OE mice were protected from cardiac ischemia and pressure-overload-induced cardiac hypertrophy (42, 44). Integrin-containing focal adhesions and caveolin-containing caveolae participate in mechanotransduction (Figure 4) (148). Israeli-Rosenberg et al. showed that β1-integrin and Cav-3 colocalize in cardiac myocytes, and acute mechanical stress by pressure overload increased complex formation between active β1-integrin and Cav-3 in vivo (149). Romanelli et al. recently showed that diabetic cardiomyocytes increased stiffness and that these changes were due to intracellular alterations of the spatial arrangement of proteins involved in cell-to-cell and cell-to-matrix communication (150).

Figure 4: Cardiomyocyte integrin β1D-Cav-3 mechanotransduction in health and Diabetes.

Integrin β1D-Cav-3 mechanosensing during health. Cav-3 modulates Integrin b1D function and together transduce mechanical signals from the cardiomyocyte cellular membrane (anchored through laminin to the extracellular matrix, ECM) to the sarcomere. Intermediary proteins like Pax, Kindlin2, Integrin-linked Kinase (ILK), Talin, Vcl, and b-parvin, Integrin β1D-Cav3 complex transmits the force to the sarcomere through α-actin and F-actin, synchronizing the mechanical stress of the fiber with the sarcomere contraction/relaxation. In addition, the Integrin b1D-Cav3 complex promote the activation of growth, hypertrophy, survival, and proliferation signaling pathways. Integrin β1D-Cav-3 mechanosensing during diabetes. The impairment of Cav-3 expression and membrane redistribution (from caveolar to non-caveolar membrane) during diabetes impairs its modulatory effect over Integrin β1D, compromising the appropriate mechanotransduction that can lead to cardiomyocyte damage. Since Integrin β1D-Cav-3 activates cell survival and proliferative pathways, the disruption of Cav-3 as a signaling hub during diabetes can result in cardiomyocyte damage, lost, and replacement with fibrous tissue, reducing the heart muscle efficiency during diabetic cardiomyopathy (149, 266–270).

Endothelial cells exposed to shear stress showed that caveolae play a critical role in differentially activating extracellular signal-regulated kinase (ERK) but not c-Jun NH₂-terminal kinase (JNK) (151). Shear stress-induced β1-integrin translocation to caveolae and caveolin was found to participate in β1- integrin-mediated signaling (152). It was further shown that integrin-linked kinase (ILK) controls microtubule dynamics required for membrane targeting of proteins and the formation of plasma membrane caveolae (153). It was shown that integrins prevent ERK1/2, PI3K, and Rac-dependent signaling by preventing Cav-1-dependent endocytosis and caveolar internalization (154). Caveolae were shown to be important mechanotransduction sites leading to the activation of ERK1/2 MAP kinases (155). The role of caveolins and integrin in endothelial cell dysfunction is multifaceted and was recently reviewed by Aman et al. (156).

In fibroblasts, caveolae play a crucial role in integrin activation and recycling (157). Yeh et al. highlight the critical roles of Cav-1 and β1 integrin-mediated mechanotransduction, suggesting that Cav-1-induced lipid raft expression may be the rate-limiting step as its expression and phosphorylation is what regulates protein stability of beta1 integrin-mediated focal adhesions (158).

Cell-to-cell contact sites in cardiac myocytes consist of gap junctions (GJ), adherens junctions, and desmosomes. Connexins (Cx) form GJ, electrically couple cardiomyocytes by allowing rapid conductance of action potentials, permit intercellular transport of ions/active molecules, and GJ are altered in heart failure (159). In the Golgi apparatus, the C-terminal tail of Cx43 binds to caveolin, both proteins are transported to the plasma membrane, and at the plasma membrane, caveolin dissociates from Cx43 before GJ assembly (160). During HF, Cx43 re-localizes from cell-to-cell junctions to the lateral cell border, and Cx43 protein levels are reduced (161). Lateralized Cx43 was found to be associated with lipid rafts, and it was suggested that this Cx43 pool will undergo autophagic degradation (162). Lipopolysaccharide treatment of astrocytes inhibited Cx43 GJ communication and induced downregulation of Cav-3 via a TLR4-mediated signaling pathway (163). In cardiac myocytes it was shown that Cav-3 directly interacts with Cx43, implicating that Cav-3 plays a role in the functional regulation of GJ (164). Hyperglycemia-induced PKC-activation reduced Cav-3 protein expression, attenuated Akt/eNOS signaling, and induced diastolic dysfunction (165). Since hyperglycemia, oxidative stress, and inflammation alter membrane microdomains, targeting signaling events at cell-to-matrix and cell-to-cell adhesion sites may offer novel treatment options for the diabetic heart.

III. Impact of Diabetes on Membrane Microdomains

Obesity, Oxidative Stress, and Inflammation

Obesity is a key risk factor for the development of insulin resistance and T2DM. So far, existing policies have failed to address the obesity epidemic, and the forecasted trends will increase disease burden and economic costs (166). Metabolic syndrome includes a group of clinical conditions, such as excess body fat, high blood glucose, high blood pressure, and abnormal cholesterol and triglyceride levels that increase the risk of cardiovascular disease. Abdominal obesity is the most frequently observed parameter leading to metabolic syndrome (167, 168).

Caveolae increase the surface area of cells and participate in multiple important cellular functions, including endocytosis, transcytosis, lipid homeostasis, membrane integrity, mechanical transduction, and signal transduction (169). Despite being hyperphagic and having a normal capacity to absorb dietary fat, Cav-1 knockout mice are resistant to diet-induced obesity and present normal insulin, serum glucose, and cholesterol levels but have severely elevated triglycerides and free fatty acids (170). Cav-1 knockout mice revealed a 90% decrease of detectable insulin receptor levels in adipose tissue; therefore, Cav-1 is considered a molecular chaperon stabilizing the insulin receptor (78).

Catalan et al. showed that Cav-1 mRNA expression is increased in visceral and subcutaneous fat tissues of obese normoglycemic and T2DM patients when compared to lean controls (171). Cav-1 expression levels in adipose tissue were positively correlated to BMI and levels of the inflammatory marks sialic acid, fibrinogen, and monocyte chemoattractant protein (171).

Among the many physiological disruptions caused by obesity, a notable one how adipose tissue responds to overnutrition by mounting an immune response (extensively reviewed by Kawai et al.) (172). Obesity has been shown to cause changes in white adipose tissue, increasing inflamed and dysfunctional adipocytes. This, in turn, leads to higher secretion of pro-inflammatory cytokines that can interfere with the normal functions of other organs, particularly the heart (172). Hyperglycemia, which can result from obesity, promotes protein glycation, resulting in altered intracellular signaling, disruptions in gene expression, and increased release of pro-inflammatory molecules and free radicals (173, 174). Glycation, associated with hyperinsulinemia and dyslipidemia, enhances inflammation by promoting the release of pro-inflammatory cytokines, including IL-6 and TNFα, while suppressing the generation of anti-inflammatory mediators like IL-10 (175, 176). This evidence strongly implicates diabetes as a key driver of chronic inflammation, which plays a direct role in the development of vascular and neurovascular diseases affecting cardiac and neural tissues. Notably, prolonged or intensified systemic inflammation triggers microglial activation in the brain (177). Upon activation, microglia-induced inflammation tends to persist for prolonged durations. Studies indicate that, although peripheral inflammation may resolve rapidly, microgliosis can take weeks or months to diminish completely, potentially causing long-term alterations in the neural environment (178).

Inflammation contributes to heightened oxidative stress by promoting excessive reactive oxygen species (ROS) production while weakening antioxidant defenses, resulting in persistently elevated ROS levels (179). While ROS play a crucial role in regulating cell signaling and communication, chronic production can lead to the disassembly, dysfunction, and fragmentation of proteins, lipids, and DNA (180). The buildup of dysfunctional or significantly altered biomolecules naturally disrupts cellular function. When this occurs in endothelial cells, it leads to impaired function of blood vessels and alters its interaction with many other cell types – in particular, cardiomyocytes.

Chronic inflammation and the progression of oxidative stress are inherently linked to the pathogenesis of diabetes (181). This condition can be alleviated with therapies that restore or stabilize caveolae and Cav-3 expression in cardiomyocytes. Cav-3 has been shown to localize to the mitochondria of cardiomyocytes and may suppress mitochondrial-derived reactive oxygen species (182). Caveolin expression in membrane lipid rafts orchestrates a host of physiological and pathological stimuli, including insulin secretion, insulin signaling, insulin resistance, oxidative stress, and diabetic complications (80). Since obesity increases inflammatory cytokine expression, adipocyte-specific Cav-1 protein expression disturbs eNOS signaling, thereby elevating the risk of arterial hypertension. Chronic inflammation and oxidative stress, either directly or via their impact on the vasculature, play a significant role in diabetes-related complications such as retinopathy, nephropathy, and cardio- and neuropathies. Stabilizing caveolae and caveolins in these tissues could, therefore, provide substantial therapeutic benefits.

Diabetes and Endothelial Dysfunction

Oxidative stress and inflammation present significant challenges to endothelial function. Reactive species modify electron-rich thiol residues, resulting in nonspecific protein alterations. These species can also infiltrate the cytosol and organelles through passive membrane diffusion or active transport. Among the many affected targets, phosphatase and tensin homolog (PTEN) and Cav-1 stand out due to their critical roles in cell signaling regulation. Caveolae are essential for macromolecule transcytosis across the endothelium, a process mediated by Cav-1’s direct interaction with proteins that support endothelial health and function. Notably, a decrease in Cav-1 expression disrupts the regulation of eNOS, leading to its uncoupling and contributing to increased oxidative stress (183). A dysfunctional endothelium leads to an inability to properly maintain systemic blood pressure (184), deficits in angiogenesis (185), compromised wound healing (186), accelerates atherosclerosis (187), and disruption in barrier integrity (in both the heart and brain), which can lead to edema and vascular cuffing (187).

PTEN and Cav-1 are intricately linked, as the loss of PTEN function triggers Cav-1 degradation, and conversely, Cav-1 depletion affects PTEN stability. While the exact mechanism remains under investigation, oxidative stress is suspected to play a key role. The disruption of these essential signaling regulators in endothelial cells is thought to contribute to cardiovascular disease while also impairing their ability to support various tissues, including the neurovascular niche. This niche is particularly crucial for regulating the pace of neural stem and progenitor cell proliferation (188). Thus, disrupting the endothelial niche’s ability to maintain the homeostasis of partner cells may be associated with neuropathologies such as Alzheimer’s disease (AD) (189).

Type 3 Diabetes: The link between Insulin Resistance, Deficiency and Alzheimer’s disease

Several risk factors are linked to the development of AD, including insulin resistance and T2DM. Data from the Mayo Clinic Alzheimer’s Disease Registry shows that about 80% of AD patients also have T2DM or another disorder related to impaired glucose metabolism. This correlation suggests a potential mechanistic connection between these two debilitating diseases, although the exact nature of this link remains unclear. As a result, AD is hypothesized to represent an advanced stage of T2D, often referred to as Type 3 Diabetes (190). While not an officially recognized medical diagnosis, this occurs when neurons become unable to respond to insulin, a hormone vital for learning and memory. It has been observed that carrying a mutation in ApoE4 (one of the leading genetic causes of AD) causes impairments in insulin signaling because the insulin receptor (IR) cannot localize in the membrane and instead is stuck in the endosome (191).

Additionally, patients with T2D frequently experience vascular pathologies that restrict blood flow to the brain, which in turn increases the risk for neurodegenerative disorders (i.e., AD). Consequently, impairments in the endothelial niche’s capacity to regulate the homeostasis of associated cells may be connected to neurodegenerative disorders like AD (189). It was shown that Cav-1 improved T2DM-related cognitive decline and described that blocking of excessive mitochondrial fission together with stimulation of defective mitophagy might be the responsible mechanism (192, 193).

IV. Strategies Targeting Membrane Microdomains for Cardiac Protection

Tsuchiya et al. reviewed drug effects on membrane microdomains, including anesthetic agents, beta-blockers, and alpha-agonists (to mention a few) (194). We would like to update drug-microdomain interactions and further discuss the effects of SGLT2 inhibitors, GLP-1 agonists, and statins.

SGLT2 inhibitors for the prevention/treatment of diabetic cardiomyopathy

HFpEF is characterized by normal cardiac contractile function but reduced diastolic function (muscle relaxation) and often precedes HF with reduced contractile function (HFrEF) in T2DM patients. Since SGLT2 inhibitors (SGLT2i aka gliflozins) reduce the cardiovascular mortality and HF hospitalization in T2DM patients, they were recently approved to treat HFpEF and are currently the only HFpEF medication available (besides blood pressure and blood glucose control).

Reduced fibrosis is observed after SGLT2i (195, 196). In renal proximal tubule cells, SGLT1 and SGLT2 localized to lipid rafts via colocalization with Cav-1 (197). Cassis et al. demonstrated that in mice with protein-overload proteinuria, the SGLT2 inhibitor dapagliflozin effectively reduced proteinuria and glomerular damage while also improving podocyte function and preventing their loss. Additionally, the study revealed that mouse podocytes express SGLT2, with its expression increasing under conditions of protein overload. In vitro findings further confirmed that podocytes serve as direct targets of SGLT2 inhibition, offering protection against albumin-induced cytoskeletal remodeling (198). Locatelli et al. showed that empagliflozin normalized podocyte VEGF-A overexpression, reducing its paracrine signaling on endothelial Cav-1 and PV-1, which ultimately preserved glomerular endothelial function and permeability (199). How SGLT2i affects cardiomyocytes is still unclear, and the improved CV outcomes might also be attributed to beneficial SGLT2i effects on non-cardiac muscle cells (e.g., endothelial cells, fibroblasts, and immunomodulating cells). Further studies are needed to investigate SGLT2i’s effects on cardiac microdomains.

GLP-1 agonists for the treatment of obesity and HFpEF

Glucagon-like peptide-1 receptor (GLP-1) agonists were initially developed as anti-diabetic drugs, but due to their profound weight-loss effect they were soon repurposed to primarily treat obesity (200). It has been shown that GLP-1 is primarily located within membrane lipid rafts, regulated by membrane lipids, and directly binding to Cav-1 (201, 202). Recently, it has been shown that GLP-1 harbors a cholesterol-binding site in pancreatic beta-cells (203). The recent double-blind, placebo-controlled SUMMIT trial found that the GLP-1 agonist tirzepatide (Mounjaro^® or Zepbound^™) lowered the risk of death from cardiovascular causes or worsening heart failure by 38% in patients with HFpEF and obesity (204). Cardiac MRI in a subset of patients found a clinically significant reduction in LV mass when compared to a placebo, and the mechanism behind this GLP-1 effect warrants further investigation.

Statin effects on glucose homeostasis

Lipids are crucial building blocks of plasma membranes (205). Besides temperature and fatty acid composition, cholesterol plays a major role in the maintenance of membrane fluidity (>cholesterol = > fluidity = elasticity) which is critical in the formation of membrane microdomains. Caveolin is linked to cholesterol transport and membrane integration (206).

Cardiovascular disease (CVD) increases with aging and is the leading cause of death worldwide. Sex differences exist in the onset of CVD. Men develop the disease earlier than women, but after menopause, women present a worsened CVD outcome (207). In the US, the absolute number of annual CVD deaths among females has exceeded that of males (208). Females with T2DM have an increased risk of CVD when compared to men with T2DM (209). Although approximately 80% of CVD could be prevented by early preventive measures, lifestyle modifications (to address obesity, physical inactivity, unhealthy diet, hypertension, hyperglycemia, hypercholesteremia, poor sleep, and smoking) are often unsuccessful (210). In both sexes, a common strategy to reduce CVD is the reduction of serum levels of low-density lipoprotein cholesterol (LDL-C). Therefore, statin drugs are widely used to reduce LDL-C for primary (211, 212) and secondary CVD prevention (213–216). The American Heart Association (AHA) and the American College of Cardiology (ACC) regularly publish guidelines for the prevention and treatment of CVD, and a risk calculator was created to estimate the 10-year CVD risk (217). Predictors like sex, age, race, systolic and diastolic blood pressure, treatment for hypertension, smoking, diabetes, total cholesterol, HDL cholesterol, or body mass index (instead of lipid values) are included in this 10-year risk calculation (the ”ASCVD Risk Estimator Plus” can be downloaded for free from them AppStore). Diabetic patients have an increased 10-year CVD risk (218, 219), but newer studies show that individuals with T2DM may present profoundly different risk profiles (220). Statins are prescribed to diabetic individuals with a 10-year CVD risk score of 10% or higher, and lifestyle modifications are recommended.

Statins inhibit HMG-CoA reductase, which is the rate-limiting enzyme in the mevalonate pathway, responsible for the production of cholesterol, dolichol (N-linked glycosylation), ubiquinone (CoQ10), Heme A, cytochrome C, and protein isoprenylation (221, 222). Simultaneous inhibition of all mevalonate-originating pathways is responsible for pleiotropic statin effects through anti-inflammatory and anti-proliferative mechanisms (223). Given their impact on cholesterol synthesis, statins have the potential to modulate membrane microdomains. Despite demonstrated CV benefits, the FDA reports that statins can cause serious side effects, including statin-induced skeletal myopathy, memory loss, and increased blood glucose levels (224, 225). Laakso et al. recently reviewed the literature about the increased T2DM risk associated with statin use (226). With relevance to the role of membrane microdomains during diabetic cardiomyopathy we will briefly discuss three potential statin-affected molecular pathways including, 1) glucose uptake to skeletal muscle, 2) insulin secretion and/or signaling, and 3) mitochondrial function.

Statins and glucose uptake by GLUT4 in skeletal muscle

Within the body, skeletal muscle is the major site for glucose uptake and homeostasis. The glucose uptake into skeletal muscle cells is mediated through insulin-stimulated glucose uptake by the glucose transporter GLUT4, which cycles between intracellular storage sites and the plasma membrane where it is localized within caveolae (227, 228). In skeletal muscle cells and cardiomyocytes it was shown that lipophilic statins like atorvastatin or simvastatin, but not the hydrophilic pravastatin, reduced GLUT4 expression and induced insulin resistance, which was attributed to statin-induced inhibition of Akt, GSK3β, and the isoprenoid system (229–233). In human skeletal-muscle primary myotubes, Grunwald et al. showed that statin administration reduced Akt phosphorylation, and that Cav3 (important for GLUT4 and IR function) was internalized from the plasma membrane to peri-nuclear sites (234). During maximal exercise performance, statin users switch to anaerobic metabolism sooner than non-statin users (235).

It was shown that simvastatin reduced cardiorespiratory fitness in overweight patients at risk of metabolic syndrome after a 12-week supervised exercise training program (236). Given the strong correlation between exercise, muscle mass, and glucose homeostasis, primary-prevention statin use in T2DM patients should be carefully considered, and the importance of body weight control and exercise-related lifestyle modification should be emphasized.

Statin effects on insulin secretion and β-cell function

While statins reduce serum cholesterol, they may increase the risk of new onset T2DM or worsen glycemic control (237, 238). Clinical studies showed that all statins, except pravastatin, may increase fasting glucose levels (239). Pre-clinical studies show that inhibited cholesterol synthesis reduces beta cell function through multiple mechanisms including reduced isoprenoid production, inhibited calcium channel function or altered mitochondrial function (240–245).

Statin effects on mitochondrial function

Mitochondrial dysfunction is a hallmark of diabetic cardiomyopathy. Statin-induced side effects have been linked to mitochondrial dysfunction (246–248), with statin-induced myopathy/weakness being the most commonly described. Statins may also affect mitochondrial function in cardiac myocytes (249, 250). Godoy et al. showed that atorvastatin reduced the protein expression of IR-β and decreased mitochondrial oxygen consumption in cultured cardiac myocytes (249). Statins have been linked to new onset T2DM (aka increased fasting glucose levels) after atorvastatin and rosuvastatin but not after pravastatin administration (211, 237, 251–253). The West Scotland Coronary Prevention Study showed that pravastatin therapy reduced the risk of developing T2DM by 30% (239). Pravastatin is the only statin that raises HDL-C levels while lowering LDL-C and triglycerides. Statin users presented lower insulin secretion after oral glucose stimulation, indicating reduced beta-cell function (254). Females are more susceptible to statin adverse events, and observational studies report a high incidence of statin-myopathy (255–258).

Of note, for the first time Zaborowska et al. showed that statins directly target lipid rafts (259). The group used a grazing-incidence X-ray diffraction/polarization modulation infrared reflection absorption spectroscopy/Brewster angle microscopy and investigated the effects of two statins with different lipophilicity (259). Condensed monolayers of lipid rafts were generated using a 1:1 ratio of cholesterol and sphingomyelin, allowing for the evaluation of fluvastatin and cerivastatin. The interaction between statins and individual lipid raft components depended on the drug’s lipophilicity and the organization of the lipid layer. With cerivastatin withdrawn from the market in 2001, rosuvastatin and atorvastatin now represent the most potent HMG-CoA reductase inhibitors. To mitigate potential statin-induced effects on glucose metabolism and muscle function, all T2DM patients should be encouraged to adopt intensive lifestyle modifications to prevent diabetic cardiomyopathy.

Challenges and Future Directions

Limitations in current understanding of membrane microdomains in diabetic cardiomyopathy.

Despite advances in understanding membrane microdomains, significant knowledge gaps remain regarding their role in diabetic cardiomyopathy. One of the primary limitations is the complexity of membrane microdomains, which contain diverse lipid and protein compositions that vary by cell type and metabolic state. The dynamic nature of these microdomains makes it challenging to pinpoint specific changes that occur in diabetic cardiomyopathy and how these alterations contribute to disease progression. Furthermore, the interplay between microdomains and insulin signaling is not fully elucidated. While caveolins and lipid rafts are known to regulate insulin receptor localization and function, it is unclear whether their dysfunction is a cause or consequence of insulin resistance in diabetic hearts. Additionally, studies often rely on in vitro models or genetically modified mice, which may not fully replicate the human diabetic condition, leading to translational challenges when developing targeted therapies.

Need for further research to elucidate specific mechanisms and targets within membrane microdomains.

Future research should aim to dissect the molecular mechanisms by which membrane microdomains influence diabetic cardiomyopathy. Specific areas of interest include the regulation of insulin receptor trafficking within lipid rafts, the impact of hyperglycemia on microdomain stability, and how changes in caveolin expression affect cardiac signaling pathways. Advances in super-resolution microscopy and lipidomics may help provide a clearer picture of these microdomains at a nanoscale level. Further investigation is needed into how microdomain-targeted therapies, such as Cav-3 or cholesterol-modulating agents, can mitigate cardiac dysfunction in diabetic patients. Identifying novel protein-lipid interactions within microdomains may also reveal new drug targets that could improve insulin sensitivity and cardioprotective signaling.

V. Conclusions

Membrane microdomains are crucial in regulating cardiac signaling, metabolism, and stress response. In diabetic cardiomyopathy, disruptions in these microdomains (Figure 5) contribute to impaired insulin signaling, increased oxidative stress, and mitochondrial dysfunction, ultimately leading to heart failure. The loss of caveolae-associated proteins, such as Cav-3, exacerbates cardiac dysfunction by altering calcium homeostasis and reducing cardioprotective signaling pathways. Despite growing recognition of these mechanisms, further research is needed to elucidate the role of microdomains in diabetic heart disease fully and to develop targeted therapeutic strategies. Advances in imaging techniques and biomarker discovery may pave the way for novel approaches, enabling more precise interventions that restore membrane microdomain integrity and improve cardiac outcomes in diabetic patients. Gene therapy approaches to restore caveolin expression in various disease settings (260, 261) may apply to diabetes but require further development and the future consideration of small molecules that may do the same. As our understanding of these cellular structures evolves, novel therapeutic strategies aimed at stabilizing membrane microdomains hold promise for mitigating the progression of diabetic cardiomyopathy and reducing the global burden of diabetes-related cardiovascular disease.

Figure 5: Caveolae-associated cardiomyocyte signaling pathways and its impairments in diabetic cardiomyopathy.

Caveolae behaves as a biophysical and signaling hub whose disruption significantly impairs the cardiomyocyte physiology, ultimately leading to the diabetic cardiomyopathy scenario (72, 270–273).