Endothelial-derived extracellular vesicles from obese/hypertensive adults increase factors associated with hypertrophy and fibrosis in cardiomyocytes

Abstract

Obesity and hypertension, independently and combined, are associated with increased risk of heart failure and heart failure-related morbidity and mortality. Interest in circulating endothelial cell-derived microvesicles (EMVs) has intensified because of their involvement in the development and progression of endothelial dysfunction, atherosclerosis, and cardiomyopathy. The experimental aim of this study was to determine, in vitro, the effects of EMVs isolated from obese/hypertensive adults on key proteins regulating cardiomyocyte hypertrophy [cardiac troponin T (cTnT), α-actinin, nuclear factor-kB (NF-kB)] and fibrosis [transforming growth factor (TGF)-β, collagen1-α1], as well as endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production. EMVs (CD144⁺ microvesicles) were isolated from plasma by flow cytometry in 12 normal weight/normotensive [8 males/4 females; age: 56 ± 5 yr; body mass index (BMI): 23.3 ± 2.0 kg/m²; blood pressure (BP): 117/74 ± 4/5 mmHg] and 12 obese/hypertensive (8 males/4 females; 57 ± 5 yr; 31.7 ± 1.8 kg/m²; 138/83 ± 8/7 mmHg) adults. Human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were cultured and treated with EMVs from either normal weight/normotensive or obese/hypertensive adults for 24 h. Expression of cTnT (64.1 ± 13.9 vs. 29.5 ± 7.8 AU), α-actinin (66.0 ± 14.7 vs. 36.2 ± 10.3 AU), NF-kB (166.3 ± 13.3 vs. 149.5 ± 8.8 AU), phosphorylated-NF-kB (226.1 ± 25.2 vs. 179.1 ± 25.5 AU), and TGF-β (62.1 ± 13.3 vs. 23.5 ± 8.8 AU) were significantly higher and eNOS activation (16.4 ± 4.3 vs. 24.8 ± 3.7 AU) and nitric oxide production (6.8 ± 1.2 vs. 9.6 ± 1.3 µmol/L) were significantly lower in iPSC-CMs treated with EMVs from obese/hypertensive compared with normal weight/normotensive adults. These data indicate that EMVs from obese/hypertensive adults induce a cardiomyocyte phenotype prone to hypertrophy, fibrosis, and reduced nitric oxide production, central factors associated with heart failure risk and development.

NEW & NOTEWORTHY In the present study we determined the effect of endothelial microvesicles (EMVs) isolated from obese/hypertensive adults on mediators of cardiomyocyte hypertrophy [cardiac troponin T (cTnT), α-actinin, nuclear factor-kB (NF-kB)] and fibrosis [transforming growth factor (TGF-β), collagen1-α1] as well as endothelial nitric oxide synthase (eNOS) expression and NO production. EMVs from obese/hypertensive induced significantly higher expression of hypertrophic (cTnT, α-actinin, NF-kB) and fibrotic (TGF-β) proteins as well as significantly lower eNOS activation and NO production in cardiomyocytes than EMVs from normal weight/normotensive adults. EMVs are a potential mediating factor in the increased risk of cardiomyopathy and heart failure with obesity/hypertension.

INTRODUCTION

The prevalence of obesity [body mass index (BMI) ≥30 kg/m²] and hypertension [blood pressure (BP) >130/80 mmHg] continue to rise in the United States (1). Obesity is often a precipitating factor in the development of hypertension and the combined existence of both risk factors markedly increases cardiovascular-related morbidity and mortality (2, 3). Indeed, adults with obesity/hypertension are at higher risk of developing cardiomyopathy and heart failure than the presence of each risk factor alone (4). Cardiac remodeling characterized by pathological cardiomyocyte hypertrophy and fibrosis, resulting in ventricular dysfunction and ultimately failure, are central factors exacerbated by obesity and hypertension (5, 6). However, the underlying mechanisms that intensify these cardiac maladaptations with obesity/hypertension are not well understood.

In the myocardium, endothelial cells outnumber cardiomyocytes 3:1 and play a critical role in regulating cardiomyocyte function via mechanical and paracrine-mediated mechanisms (7, 8). Given the influence of endothelial cells on cardiomyocyte function, clinical interest in the interaction between endothelial cell-derived microvesicles (EMVs) and cardiomyocytes has intensified. EMVs are a nucleoid membrane bound vesicles, 0.1–1 µm in diameter, that are released both luminally and abluminally in response to a myriad of stimuli that trigger endothelial cell activation or apoptosis (9). EMVs have been shown to predict the presence of vascular disease, recurrent hospitalization, and all-cause mortality in patients with heart failure (10, 11). However, the causative nature of this association with heart failure is unknown. Endothelial cell dysfunction is considered to play a central etiological role in heart failure (12). A significant and often pathological consequence of endothelial cell dysfunction is the release of EMVs that, in turn, can affect the function of target cells through surface interaction and receptor activation, cellular fusion, incorporation, and the delivery of intravesicular cargo (13). EMV production has been shown to be independently elevated with obesity and hypertension and is thought to be mechanistically involved in the myriad of cardiovascular conditions associated with these major risk factors (14–16). It is unknown whether EMVs from adults with obesity/hypertension detrimentally affect cardiomyocytes; if so, EMVs may represent a mediating factor underlying the increased risk of cardiomyopathy and heart failure with the obese/hypertensive state.

Accordingly, the aim of this study was to determine the effect of EMVs isolated from obese/hypertensive adults on mediators of cardiomyocyte hypertrophy and fibrosis. We hypothesized that EMVs from obese/hypertensive adults would negatively affect proteins that regulate cardiomyocyte hypertrophy and fibrosis as well as nitric oxide (NO) production compared with EMVs from normal weight/normotensive adults. To address this aim, human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were cultured and treated with EMVs isolated from either normal weight/normotensive or obese/hypertensive adults to assess intracellular expression of hypertrophy [cardiac troponin T (cTnT), α-actinin, nuclear factor-kB (NF-kB)] and fibrosis [transforming growth factor (TGF)-β, collagen1-α1] proteins as well as endothelial nitric oxide synthase (eNOS) expression and NO production.

METHODS

Subjects

Twenty-four middle-aged and older adults (46–71 yr) participated in the study: 12 normal weight/normotensive (8 males/4 females; BMI, 19.1–25.0 kg/m²; BP, 108–120/67–81 mmHg) and 12 obese/hypertensive (8 males/4 females; BMI, 30.0–34.3 kg/m²; BP, 130–151/70–93 mmHg). All subjects were sedentary, nonsmokers, nonmedicated, and free of overt cardiometabolic disease as assessed by medical history, resting and exercise electrocardiogram, and fasting blood chemistries. The women in the study were ≥1 yr postmenopausal and were either not taking or had discontinued taking hormone replacement therapy for ≥1 yr before starting the study. All subjects had the research study and its potential risks and benefits explained fully before providing written informed consent. This study was approved by the Institutional Review Board of the University of Colorado Boulder.

Blood Pressure

Resting blood pressure measurements were performed in the sitting position on at least two separate days at least 1 wk apart. Subjects were instructed not to ingest caffeine-containing beverages before all BP measurements. The recordings were made under quiet, comfortable ambient (∼24°C) laboratory conditions. To avoid the possibility of investigator bias, measurements were made with a semiautomated device (Dinamap, Critikon, FL) that uses an oscillometric technique over the brachial artery. Recordings were made in triplicate in the upright sitting position. All measurements conformed to American Heart Association guidelines as established by the Council for High Blood Pressure Research (17).

Body Composition and Metabolic Measures

Body mass was measured to the nearest 0.1 kg using a medical beam balance (Detecto, Webb City, MO), and body fat percentage was determined by dual X-ray absorptiometry (Lunar Radiation, Madison, WI). Body mass index (BMI) was calculated as weight divided by height (kg/m²). Minimum waist circumference was measured according to published guidelines (18). Fasting plasma lipid and lipoprotein, glucose, and insulin concentrations were determined using standard techniques by the clinical laboratory affiliated with the Clinical and Translational Research Center at the University of Colorado Boulder. Insulin resistance was estimated using the homeostasis model assessment (HOMA-IR) derived from fasting glucose and insulin concentrations (19).

Circulating EMV Identification, Enumeration, and Collection

Circulating EMVs were determined from peripheral blood samples as previously described by our laboratory (14). Briefly, venous blood from an antecubital vein was collected in sodium citrate tubes and centrifuged at 1,500 g for 10 min at room temperature; thereafter, plasma was collected and stored at −80°C for batch analysis and microvesicle isolation. To harvest microvesicles from each sample for use in cell experiments, plasma was centrifuged at 13,000 g for 2 min to remove cellular debris and then recentrifuged at 20,500 g for 50 min at 4°C to pellet microvesicles. Pelleted vesicles were then resuspended in PBS media supplemented with 10% FBS. To determine the concentration of the isolated microvesicles, media samples were centrifuged at 13,000 g for 2 min and 100 µL was transferred to a TruCount tube (BD Biosciences, Franklin Lakes, NJ). EMVs were determined by incubating samples with fluorochrome-labeled antibodies (CD144; BioLegend, San Diego, CA) for 20 min at room temperature in a dark room. Samples were then fixed with 2% paraformaldehyde (ChemCruz Biochemicals, Santa Cruz, CA) and diluted with RNasefree PBS (14, 20). Micro-vesicle-size threshold was established using Megamix-Plus SSC calibrator beads (Biocytex, Marseille, France) and only events >0.2 µm and <0.8 µm in size and expressing CD144 label were counted and collected. The concentration of EMVs was determined using the formula: (number of events in region containing EMV/number of events in absolute count bead region) × (total number of beads per test/total volume of sample) (21). All samples were analyzed on a BD FACSAria instrument. The event rate was set to 10,000 events/s up to one-million events (22). These methods were in accordance with International Society for Extracellular Vesicles guidelines at the time of study (23). EMVs were resuspended in sterile PBS buffer media and stored at −80°C for later use in cell experiments.

Cardiomyocyte Cell Culture

iPSC-CMs (Cellular Dynamics International; Madison, WI) were cultured on 0.01% fibronectin (Sigma; St. Louis, MO) under normal culture conditions (37°C and 5% CO₂) using proprietary plating media (M1001, Fujifilm Cellular Dynamics), which was removed after 4 h, after which cardiomyocytes were maintained in proprietary culture media (M1003, Fujifilm Cellular Dynamics, Madison, WI) that was replaced every 48 h for 6 days. On day 7, iPSCs-CMs were treated with EMVs from either a normal weight/normotensive or obese/hypertensive adult (2:1 ratio; EMVs:iPSC-CMs) for 24 h.

Protein Expression

EMV-treated iPSC-CMs were harvested for the quantification of intracellular proteins. Briefly, iPSC-CMs were washed in cold PBS and incubated on ice in cold RIPA lysis buffer containing protease and phosphatase inhibitors (Thermo Fisher Scientific) for 10 min. Cell lysates were then sonicated for 20 s (four 5-s cycles spaced by 90 s between each cycle), incubated on ice for 10 min, and centrifuged at 13,000 g at 4°C for 10 min. Supernatant was collected, and protein concentration was determined using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA). Thereafter, protein expression was determined by capillary electrophoresis immunoassay (Wes, Protein Simple, San Clara, CA; 24). Briefly, 1–4 ng of protein isolate was combined with a proprietary master mix (ProteinSimple) containing buffer, fluorescent molecular weight markers, and 40 mM DTT. Samples were vortexed and incubated at 95°C for 5 min, then combined with blocking solution, rabbit anti-human primary antibodies, horseradish peroxidase-conjugated secondary antibody, chemiluminescent substrate, and separation and stacking matrices for automated electrophoresis (375 V for 25 min) and immunodetection using the Wes system. Protein expression was normalized to total protein in the sample and presented as arbitrary units (AU). Rabbit primary antibodies against cTnT, NF-kB, phosphorylated (activated) NF-kB, α-actinin, TGF-β, collagen1-α1, eNOS, phosphorylated eNOS Ser1177 (Cell Signaling Technologies, Danvers, MA; diluted 1:50, 1:250, 1:250, 1:250, 1:250, 1:250, 1:250, respectively) and total eNOS (Thermo Fisher Scientific, Waltham, MA; diluted 1:50) were used.

Nitric Oxide Production

To assess NO production, total nitrite in EMV-treated iPSC-CM media was measured using the Total Nitric Oxide and Nitrate/Nitrite Parameters Assay (R&D Systems, Minneapolis, MN). Intraassay coefficient of variation for the media-based ELISA was <8%.

Statistical Analysis

The distribution of the data was assessed by the Shapiro–Wilk test and the homogeneity of variances by the Levene test. Group differences in subject characteristics and cellular proteins were determined by analysis of variance. There were no significant group × sex interactions, therefore the data were pooled and presented together. Pearson’s correlations were determined between variables of interest. All values in the text and table are expressed as means ± SD, and mean values are denoted in the dot-whisker plots. Statistical significance was set a priori at P < 0.05.

RESULTS

Subject characteristics are presented in the Table 1. Body weight, body mass index, body fat percentage, and blood pressure were significantly higher in the obese/hypertensive compared with normal weight/normotensive group. Although fasting triglyceride and insulin concentrations were slightly, albeit significantly, higher in the adults with obesity/hypertension, there were no significant group differences in other metabolic variables including homeostatic model assessment of insulin resistance (HOMA-IR).

Table 1.

Subject characteristics

Variable	Normal Weight/Normotensive	Obese/Hypertensive
n	12	12
Age, yr	56 ± 5	57 ± 5
Sex, males/females	8/4	8/4
Body mass, kg	72.4 ± 9.9	96.6 ± 15.0*
Body mass index, kg/m2	23.3 ± 2.0	31.7 ± 1.8*
Body fat, %	27.0 ± 9.8	38.9 ± 5.2*
Waist circumference, cm	83.1 ± 9.4	100.7 ± 8.9*
Systolic blood pressure, mmHg	116 ± 4	138 ± 8*
Diastolic blood pressure, mmHg	75 ± 5	83 ± 7*
Total cholesterol, mg/dL	184.8 ± 31.8	197.3 ± 17.6
Low-density lipoprotein, mg/dL	113.4 ± 24.2	116.1 ± 13.9
High-density lipoprotein, mg/dL	57.0 ± 15.7	50.6 ± 18.9
Triglycerides, mg/dL	69.4 ± 21.6	142.2 ± 46.1*
Glucose, mg/dL	90.1 ± 7.3	86.0 ± 9.3
Insulin, µU/mL	6.3 ± 2.6	9.1 ± 2.9*
HOMA-IR	1.5 ± 0.7	1.9 ± 0.7

Values are means ± SD; n, number of participants. BMI, body mass index; BP, blood pressure; HDL, high-density lipoprotein; HOMA-IR, homeostatic model assessment of insulin resistance; LDL, low-density lipoprotein. *P < 0.05.

Circulating EMVs

Figure 1 shows circulating EMV concentrations in the normal weight/normotensive and obese/hypertensive adults. Circulating levels of EMVs were significantly higher (∼145%) in the obese/hypertensive (373 ± 164 EMV/µL) than normal weight/normotensive (153 ± 81 EMV/µL) group.

Figure 1.

Circulating concentration of endothelial cell-derived microvesicles (EMVs) in the normal weight/normotensive and the obese/hypertensive groups. Closed circles, male; open circles, female. Mean value for each group is denoted. *P < 0.05.

Hypertrophy and Fibrosis

The effect of EMVs on the expression of proteins involved in cardiomyocyte hypertrophy (cTnT, α-actinin, and NF-kB) and fibrosis (TGF-β and collagen-1α) is shown in Figs. 2 and 3, respectively. Expression of cTnT (64.1 ± 14.0 vs. 29.5 ± 7.8 AU), α-actinin (66.0 ± 14.7 vs. 36.2 ± 10.3 AU), NF-kB (166.3 ± 9.1 vs. 149.5 ± 9.6 AU), and phosphorylated-NF-kB (226.1 ± 25.2 vs. 179.1 ± 25.5 AU) were significantly higher in iPSC-CMs treated with EMVs from obese/hypertensive compared with normal weight/normotensive adults (Fig. 2). Expression of TGF-β (62.1 ± 13.3 vs. 23.5 ± 8.8 AU), but not collagen1-α1 (45.2 ± 11.6 vs. 49.6 ± 8.2 AU), was significantly higher in cardiomyocytes treated with EMVs from obese/hypertensive versus normal weight/normotensive adults (Fig. 3). Representative protein histograms of immunodetection by capillary electrophoresis immunoassay for cTNT, α-actinin, NF-kB p65, p-NF-kB p65(Ser536), TGF-β, and collagen1-α1 are shown in Fig. 4.

Figure 2.

Cardiomyocyte expression of cTnT (A), α-actinin (B), NF-kB p65 (C), and p-NF-kB p65 (Ser536; D) in response to treatment with endothelial cell-derived microvesicles (EMVs) from normal weight/normotensive and obese/hypertensive adults. Closed circles, male; open circles, female. AU, arbitrary units. Mean value for each group is denoted. *P < 0.05.

Figure 3.

Cardiomyocyte expression of transforming growth factor-β (TGF-β; A) and collagen1-α1 (B) in response to treatment with endothelial cell-derived microvesicles (EMVs) from normal weight/normotensive and obese/hypertensive adults. Closed circles, male; open circles, female. AU, arbitrary units. Mean value for each group is denoted. *P < 0.05.

Figure 4.

Representative protein histograms of immunodetection by capillary electrophoresis immunoassay for cTNT (A), α-actinin (B), NF-kB p65 (C), phospho (p)-NF-kB p65(Ser536; D), transforming growth factor-β (TGF-β; E), collagen1-α1 (F), endothelial nitric oxide synthase (eNOS; G), and p-eNOS (Ser1177; H).

Nitric Oxide

Figure 5 shows the effect of EMVs on iPSC-CM eNOS expression and NO production. There was no significant difference in total eNOS protein expression in iPSC-CMs treated with EMVs from normal weight/normotensive adults compared with EMVs from obese/hypertensive adults (288.4 ± 75.2 vs. 321.6 ± 51.3 AU). However, expression of phosphorylated eNOS (Ser1177) was significantly lower (∼35%) in iPSC-CMs treated with EMVs from obese/hypertensive adults (16.4 ± 4.3 vs. 25.4 ± 3.7 AU); concordantly NO production was ∼30% lower (6.8 ± 1.2 vs. 9.6 ± 1.3 µmol/L; P < 0.05) in iPSC-CMs treated with EMVs from the obese/hypertensive adults [see representative protein histograms of immunodetection by capillary electrophoresis immunoassay for eNOS and phosphorylated eNOS (Ser1177) in Fig. 4]. NO production was significantly and positively associated with the iPSC-CM expression of phosphorylated eNOS (Ser1177; r = 0.56). In addition, expression of phosphorylated eNOS (Ser1177) was significantly inversely related with EMV-treated iPSC-CM expression of cTnT (r = −0.60), α-actinin (r = −0.40), phosphorylated-NF-kB (r = −0.44), and TGF-β (r = −0.71; Fig. 6).

Figure 5.

Cardiomyocyte expression of endothelial nitric oxide (NO) synthase (eNOS; A) and phospho (p)-eNOS (Ser1177; B), and NO production (C) in response to treatment with endothelial cell-derived microvesicles (EMVs) from normal weight/normotensive and obese/hypertensive adults. Relation between p-eNOS (Ser1177) and NO production (D) in EMV-treated cardiomyocytes. Closed circles, male; open circles, female. AU, arbitrary units. Mean value for each group is denoted. *P < 0.05.

Figure 6.

Relation between expression of p-eNOS (Ser1177) and cTnT (A), α-actinin (B), NF-kB p65 (Ser536; C), and transforming growth factor-β (TGF-β; D) in endothelial cell-derived microvesicle (EMV)-treated cardiomyocytes. Closed circles, male; open circles, female.

DISCUSSION

The novel findings of the present study are as follows: 1) circulating concentrations of EMVs are markedly higher in obese/hypertensive adults compared with normal weight/normotensive adults, 2) EMVs from obese/hypertensive adults increase the expression of key intracellular proteins that are involved in cardiomyocyte hypertrophy and fibrosis, and 3) EMVs from obese/hypertensive adults diminish cardiomyocyte eNOS activation and NO production. It is well established that obesity/hypertension is associated with increased risk of cardiomyopathy and heart failure (4, 25). The mechanisms underlying this heightened risk are complex and not completely understood. To the best of our knowledge, this is the first study to determine whether EMVs are a potential mediating factor in the increased risk of cardiomyopathy and heart failure with obesity/hypertension.

EMVs are emerging as both a biomarker and mediator of disease pathogenesis (26). Elevated circulating EMVs have been linked to endothelial dysfunction and the development, progression, and severity of cardiovascular and cerebrovascular disease. We (14, 27) and others (28) have demonstrated that circulating EMVs are higher in adults with obesity and adults with elevated blood pressure, independent of overt disease and other risk factors. Moreover, in hypertensive adults, elevated circulating EMVs have recently been shown to be a systemic biomarker of impaired endothelium-dependent vasodilation (14). In a seminal series of studies, Berezin et al. (10, 29, 30) have provided evidence of not only a pathological link between EMVs and heart failure but also their prognostic value in disease severity and clinical outcome. For example, increased number of circulating EMVs in individuals with symptoms of chronic heart failure was associated with increased 3-yr heart failure-related and all-cause mortality as well as heightened risk of recurrent hospitalization because of chronic heart failure (10). In the present study, circulating EMVs were markedly higher (∼145%) in the obese/hypertensive adults than normal weight/normotensive adults. Although this finding was expected given that both obesity and hypertension are independently associated with elevated levels of circulating EMVs, it established enhanced systemic production of EMVs with obesity/hypertension and the likelihood of chronic high exposure of cardiomyocytes to EMVs in vivo in this high-risk pathological state (31). Considering the constant interaction between endothelial cells and cardiomyocytes mediated by extracellular vesicles, increased production of potentially pathogenic EMVs may have deleterious consequences on cardiomyocytes providing novel mechanistic insight underlying the increased risk of heart failure with obesity/hypertension.

In the setting of heart failure, cardiac hypertrophy is a central characteristic. Cardiac TnT is a prominent protein involved in myofibrillar ATPase activity (32), sensitivity of myofilaments to calcium and, in turn, calcium binding (33, 34) and sarcomere function (35). Over 90% of cTnT is bound to the myofibrillar apparatus, with a small percentage of unbound TnT located in the cytosol that can be released into the circulation in response myocardial stress and injury (36). Overexpression of cTnT can compromise myofilament function and, in turn, negatively affect cardiac contractility. Indeed, elevated expression of cTnT is associated with heart failure-related hypertrophy. In both animal and human studies, cTnT protein levels have been linked with disease pathogenesis and severity (32, 37, 38). Increased expression of cTnT has been reported in hypertrophied failing hearts of guinea pigs (39) and diabetic rats (35) as well as human heart in end-stage failure (38). In the present study, EMVs from obese/hypertensive adults induced markedly higher (∼120%) cTnT expression in cardiomyocytes compared with EMVs from normal weight/normotensive adults. EMV-induced overexpression of cTnT supports the notion of a causative role of EMVs in cardiac hypertrophy (10, 29, 30). Moreover, in addition to enhanced cTnT expression, EMVs from obese/hypertensive adults also increased activation of NF-kB and expression of α-actinin in cardiomyocytes further exacerbating a hypertrophic phenotype. NF-kB is a redox sensitive transcription factor involved in the regulation of a myriad of genes associated with inflammation, cellular apoptosis, septic shock, ischemia-reperfusion injury, and the development and progression of atherosclerosis, myocardial injury, and dilated cardiomyopathy (40, 41). Most recently, NF-kB activation has been implicated as an intracellular mediator of pathological cardiac hypertrophy and heart failure (41, 42). Indeed, elevated myocardial NF-kB activation has been reported in human heart failure (42). In fact, targeting NF-kB activation to inhibit cardiac hypertrophy is an area of therapeutic interest for myocardial protection (40, 41, 43). Regarding α-actinin, increased expression has been linked to cardiac remodeling and is a contributing factor early in the development of heart failure (44). A major component of the Z-disk architecture, α-actinin contributes to the structure and organization of the sarcomere and is involved in cardiomyocyte force bearing and transmission (44, 45). However, overexpression of α-actinin disrupts contractile function and contributes to a pathological hypertrophic adaptation (46). Collectively, given the increased risk of cardiac hypertrophy and impaired systolic function with obesity/hypertension, our novel findings suggest a potential mediating role of EMVs in this pathology.

Cardiac fibrosis often accompanies hypertrophy and is a precipitating factor in the development of myocardial dysfunction leading to heart failure (47, 48). TGF-β is a multifunctional regulatory cytokine involved in cardiac development, repair, remodeling, and hypertrophy (49) and is constitutively expressed in cardiomyocytes and cardiac fibroblasts (50). Overexpression of TGF-β however can be detrimental leading to increased synthesis and deposition of extracellular matrix components such as collagens, fibronectin, and proteoglycans resulting in the development of fibrosis (49, 51). Experimental models of hypertrophy and heart failure have demonstrated that overexpression of TGF-β is a central factor in the development of fibrosis (51, 52). In the present study, concordant with adverse changes in hypertrophic-related proteins, EMVs from obese/hypertensive adults markedly increased cardiomyocyte expression of TGF-β. Elevations in TGF-β, coupled with higher hypertrophic factors such as cTnT and NF-kB, are consistent with the phenotypic characteristics associated with the pathogenesis of cardiac fibrosis (52). Although TGF-β expression was higher in cardiomyocytes treated with obesity/hypertension-related EMVs, collagen1-α1 expression was not. Given the link between TGF-β and collagen production (53), this finding was somewhat surprising; however, it is plausible that our 24-h EMV treatment window, although sufficient to promote increased TGF-β expression, was not sufficiently long enough to allow downstream TGF-β-stimulated increases in collagen1-α1 expression. Previous in vitro studies have demonstrated that TGF-β can increase mRNA levels for collagen1-α1 within 24 h, but not de novo synthesis of protein in this time frame (53–55). Unfortunately, we did not assess mRNA levels for collagen1-α1 in the present study.

Nitric oxide is an important regulator of cardiac function with potent antihypertrophic and antifibrosis effects (56, 57). NOS3, the gene that codes for eNOS, is constitutively expressed in cardiomyocytes contributing to the regulation of inotropy, hypertrophy, response to stress, and survival (58, 59). Reduced eNOS expression and NO production in cardiomyocytes have been linked with depressed left ventricular contractile function, hypertrophic cardiomyopathy, and heart failure (56, 60). For example, Piech et al. (61) reported reduced expression of eNOS in canine hearts with hypertrophic cardiomyopathy. In human hearts, reduced myocardial eNOS expression and activity are associated with hypertrophy and heart failure (56). In the present study, EMVs from obese/hypertensive adults significantly reduced cardiomyocyte eNOS activity and NO production compared with EMVs from normal weight/normotensive adults. Phosphorylation is the primary posttranslational modification regulating eNOS enzyme activity (62). The expression of p-eNOS(Ser1177) was ∼35% lower in iPSC-CMs treated with EMVs from obese/hypertensive adults. In addition, we observed significant inverse associations between eNOS activation and cTnT, NF-kB, α-actinin, and TGF-β expression consistent with previous studies demonstrating a regulatory interaction between eNOS and the expression and signaling of these proteins (57, 63). Thus, it is plausible that EMV-induced reduction in eNOS activation in addition to lowering NO bioavailability may contribute to a cardiomyocyte phenotype prone to maladaptive hypertrophy and fibrosis (63). In addition, considering NO produced in cardiomyocytes does not act as a freely diffusible messenger but rather is involved in triggering posttranslational modification of various proteins through nitrosylation, reduced NO bioavailability can disrupt many cellular processes. For example, NO exerts potent antiapoptotic effects via S-nitrosylation inhibition of caspase-3 and caspase-9 key proteins involved in apoptosis signaling. Uncontrolled apoptosis renders cardiomyocytes highly susceptible to pathological hypertrophy (56, 64).

To the best of our knowledge, this is the first study to assess the effects of EMVs, or any extracellular vesicle subtype for that matter, on cardiomyocyte characteristics. The mechanisms responsible for obese/hypertensive-EMVs inducing higher expression of cTnT, NF-kB, α-actinin, and TGF-β and lower eNOS activation and NO production than EMVs from normal weight/normotensive adults are not clear. EMV RNA and protein cargo have been shown to play an important role in dictating EMV phenotype and cellular effects. Indeed, the ability of EMVs to transport and transfer their content to recipient cells can dramatically affect gene expression and, in turn, induce wide-ranging phenotypic changes in target cells (65). Several studies have demonstrated differential expression of miRNA cargo in EMVs in healthy and diseased populations and links between EMV microRNA cargo and disease severity (20, 66, 67). Studies are ongoing in our laboratory to determine whether the microRNA cargo signature is different in EMVs from normal weight/normotensive compared with obese/hypertensive adults. EMVs can also disrupt the intracellular microRNA milieu in target cells leading to acute and chronic changes in protein expression and signaling (24). For example, EMV-induced changes in intracellular miR-126 have been shown to compromise eNOS activity and NO production (68). Future studies are needed to determine whether EMVs alter the cardiomyocyte microRNA profile.

There are a few experimental considerations regarding the present study that deserve mention. First, with any cross-sectional human study, there exists the possibility that genetic and/or lifestyle behaviors may have influenced the results of our group comparisons. To minimize the effects of lifestyle behaviors and underlying disease, all subjects were sedentary, nonsmokers, nonmedicated, and free of overt cardiometabolic abnormalities. Second, as noted earlier, we did not elucidate potential mechanisms underlying the prohypertrophic and profibrosis effects of obesity/hypertension-related EMVs on cardiomyocytes. The lack of data on the potential effect of EMVs on cardiomyocytes necessitated these initial novel studies to substantiate, identify, and guide mechanistic targets. Last, the in vitro nature of our study does not allow for definitive conclusions regarding clinical risk or future development of pathological hypertrophy and fibrosis. However, it is important to emphasize that all of the proteins studied are well-established cellular markers and mediators of cardiomyocyte hypertrophy and fibrosis with causative links to clinical risk and the development of cardiomyopathy and heart failure. Moreover, the use of iPSC-CMs have been shown to demonstrate similar functional, morphological, and electrophysiological characteristics and responses to pathogenic stimuli to adult human cardiomyocytes making them a well-used and validated model for studying cardiomyocyte-related pathogenesis in vitro (51, 69–71).

In conclusion, the results of this study demonstrate that circulating concentrations of EMVs are elevated in obese/hypertensive adults indicative of increased EMV production. In addition, EMVs from obese/hypertensive adults increased cardiomyocyte expression of cTnT, NF-kB, α-actinin, and TGF-β as well as lowered eNOS activation and NO production. It is well established that these changes in cardiomyocytes promote a highly prohypertrophic, profibrotic phenotype that is etiologically linked to heart failure risk and development (41, 51, 60). EMVs are critical mediators of cross talk between endothelial cells and cardiomyocytes and represent a novel mechanistic factor underlying the increased risk of heart failure in obese/hypertensive adults.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This study was supported, in part, by National Institutes of Health (NIH) Grants HL077450 and HL107715 and Colorado CTSA Grant UL1 TR002535 from NIH.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

H.K.F. and C.A.D. conceived and designed research; H.K.F., J.W.T., J.J.G., K.P.D., and B.L.S. performed experiments; H.K.F., V.P.G., L.M.B., and J.J.G. analyzed data; H.K.F., V.P.G., and C.A.D. interpreted results of experiments; H.K.F. and J.J.G. prepared figures; H.K.F. and C.A.D. drafted manuscript; V.P.G. and C.A.D. edited and revised manuscript; H.K.F., V.P.G., J.W.T., L.M.B., J.J.G., K.P.D., B.L.S., and C.A.D. approved final version of manuscript.