Late plasma exosome microRNA-21-5p depicts magnitude of reverse ventricular remodeling after early surgical repair of primary mitral valve regurgitation

Abstract

Introduction: Primary mitral valve regurgitation (MR) results from degeneration of mitral valve apparatus. Mechanisms leading to incomplete postoperative left ventricular (LV) reverse remodeling (Rev-Rem) despite timely and successful surgical mitral valve repair (MVR) remain unknown. Plasma exosomes (pEXOs) are smallest nanovesicles exerting early postoperative cardioprotection. We hypothesized that late plasma exosomal microRNAs (miRs) contribute to Rev-Rem during the late postoperative period.

Methods: Primary MR patients (n = 19; age, 45-71 years) underwent cardiac magnetic resonance imaging and blood sampling before (T0) and 6 months after (T1) MVR. The postoperative LV Rev-Rem was assessed in terms of a decrease in LV end-diastolic volume and patients were stratified into high (HiR-REM) and low (LoR-REM) LV Rev-Rem subgroups. Isolated pEXOs were quantified by nanoparticle tracking analysis. Exosomal microRNA (miR)-1, -21-5p, -133a, and -208a levels were measured by RT-qPCR. Anti-hypertrophic effects of pEXOs were tested in HL-1 cardiomyocytes cultured with angiotensin II (AngII, 1 μM for 48 h).

Results: Surgery zeroed out volume regurgitation in all patients. Although preoperative pEXOs were similar in both groups, pEXO levels increased after MVR in HiR-REM patients (+0.75-fold, p = 0.016), who showed lower cardiac mass index (-11%, p = 0.032). Postoperative exosomal miR-21-5p values of HiR-REM patients were higher than other groups (p < 0.05). In vitro, T1-pEXOs isolated from LoR-REM patients boosted the AngII-induced cardiomyocyte hypertrophy, but not postoperative exosomes of HiR-REM. This adaptive effect was counteracted by miR-21-5p inhibition.

Summary/conclusion: High levels of miR-21-5p-enriched pEXOs during the late postoperative period depict higher LV Rev-Rem after MVR. miR-21-5p-enriched pEXOs may be helpful to predict and to treat incomplete LV Rev-Rem after successful early surgical MVR.

Keywords: exosomes; heart surgery; miR-21-5p; mitral valve (MV) repair; postoperative; reverse remodeling.

FIGURE 1

Comparison of preoperative and postoperative CMRI results in primary MR patients undergoing MVR surgery. The CMRI was performed on patients undergoing MVR surgery (n = 19) before (T0) and 6 months after (T1) surgery. Individual data are shown for each patient at both time points; statistical comparison between T0 and T1 was performed by paired Student’s t-test or the Wilcoxon test depending on data distribution. *p < 0.05; ^**p < 0.01; ^***p < 0.001; ^****p < 0.0001.

FIGURE 2

Analysis of CMRI parameters change in MR patients with different grade of reverse remodeling after MVR surgery. The CMRI data were collected from patients undergoing MVR surgery (n = 19) before (T0) and 6 months after (T1) surgery. The CMRI acquisitions are shown for a representative Low LV reverse remodeling (LoR-REM) and High LV reverse remodeling patient (HiR-REM). For each CMRI parameter measured, the percentage change between T0 and T1 was computed for each patient as %Δ = (T1 – T0)/T0 × 100. The data are compared between LoR-REM and HiR-REM patients. Individual data are shown; horizontal bars represent group average. Statistical significance of the differences was evaluated by independent samples Student’s t-test (with or without Welch’s correction) or the Mann–Whitney test depending on data distribution. *p < 0.05; **p < 0.01; ****p < 0.0001.

FIGURE 3

pEXOs and exosomal miRNAs in primary MR patients subjected to MVR surgery. The pEXOs were isolated from plasma of MR patients (n = 19) collected before (T0) and 6 months after MVR surgery (T1); pEXOs isolated from healthy controls (n = 8) were used as control. (A,B) The exosomal markers TSG101, CD81, and CD3 were assessed on the isolated particles by Western blot as described in the methods. (A) One representative experiment is shown. (B) Densitometric analysis of each protein target normalized for the corresponding experiment’s Ponceau staining shows no difference in marker expression among extracts (N = 3 patients/group). (C) Isolated pEXOs were analyzed by NTA as described in the methods; representative size distribution curves are shown. (D) The pEXOs concentrations were inferred by the NTA’s size distribution curve and shown as particles/ml of starting plasma. (E–G) Exosomal miRNA-1 (E), miRNA-21-5p (F) and miRNA-133a (G) were assessed by qRT-PCR as described in the methods. Individual data, median value and interquartile range are shown in the graphs. Statistical significance of the differences between matching T0 and T1 data was evaluated by paired Student’s t-test or the Wilcoxon test depending on data distribution. Statistical differences between patients and controls were evaluated by one-way ANOVA (with or without Brown–Forsythe modification for heteroscedastic groups) or the Kruskal–Wallis test, depending on sample distribution; *p < 0.05.

FIGURE 4

Analysis of pEXOs and exosomal miRNA in MR patients with different grade of reverse remodeling after MVR surgery. The pEXOs were isolated from plasma of MR patients (n = 19) collected before (T0) and 6 months after MVR surgery (T1); pEXOs isolated from healthy controls (n = 8) were used as control. (A) Isolated pEXOs were quantified by NTA as described in the methods. (B–D) Exosomal miRNA-1 (B), miRNA-21-5p (C) and miRNA-133a (D) were quantified through qRT-PCR. Preoperative (T0) and postoperative (T1) levels were compared between LoR-REM and HiR-REM patients. Individual values are shown in the graphs; bars represent median and interquartile range. Statistical significance of the differences between patients’ groups was evaluated by independent samples Student’s t-test (with or without Welch’s correction) or the Mann–Whitney test depending on data distribution. Statistical differences between patients and controls were evaluated by one-way ANOVA (with or without Brown–Forsythe modification for heteroscedastic groups) or the Kruskal–Wallis test, depending on sample distribution; *p < 0.05.

FIGURE 5

Diagnostic efficacy of pEXOs levels and exosomal miR-1, miR-21-5p, and miR-133a. The pEXOs were isolated from plasma of MR patients (n = 19) collected before (T0) and 6 months after MVR surgery (T1). (A) Isolated pEXOs were quantified by NTA as described in the methods. (B–D) Exosomal miRNA-1 (B), miRNA-21-5p (C) and miRNA-133a (D) were quantified through qRT-PCR as described in the method. Patients were divided into HiR-REM and LoR-REM groups. The diagnostic efficiency of T0, T1, and %Δ levels of pEXOs (particles/uL) (A) and of the microRNAs miR-1 (B), miR-21-5p (C) and miR-133a (D) (2^–ΔΔCT) in distinguishing patients with Low and High postoperative reverse LV remodeling was evaluated by the receiver operating characteristic (ROC) curve analysis as described in the methods. The AUC quantification and statistical significance (p-value) are reported in the table (E).

FIGURE 6

Effect of pEXOs from patients with different grade of reverse ventricular remodeling on cardiomyocytes size. The HL-1 cells were seeded at a concentration of 5,000 cells/cm² and allowed to adhere for 24 h before treatment. To enlarge cardiomyocyte size, HL-1 cells were treated with ± 1-μM Angiotensin II (AngII, Sigma) in complete Claycomb medium for 48 h. A PBS suspension of pEXOs isolated at T0 or T1 from HiR- or LoR-REM group (N = 4 patients/group) was added to a final concentration of 1 × 10⁹ particles/ml, 24 h after the beginning of the treatment, and the treatment was maintained for the remaining 24 h; PBS was employed as a control. The cell monolayer was stained with Phalloidin-Atto 550 as described in the methods. The images were acquired with a fluorescence microscope (Leica; 20× magnification); the cell areas were manually measured with ImageJ software (https://imagej.nih.gov/ij/) and expressed in pixels. Statistical significance of the differences between treatments was evaluated by one-way ANOVA. The symbol “+” represents the significance of the differences against PBS alone (+, p < 0.05; ++, p < 0.01; ++++, p < 0.0001); “#” against PBS + AngII (^####p < 0.0001); and “*” between LoR-REM and HiR-REM pEXO treatment in presence of AngII (****p < 0.0001).

FIGURE 7

Effect of miR-21-5p inhibition on cardiomyocytes size. The HL-1 cells were seeded at a concentration of 5,000 cells/cm² and allowed to adhere for 24 h before treatment. (A) The HL-1 cells were treated with ± 1-μM angiotensin II (AngII, Sigma) in complete antibody-free Claycomb medium for 48 h. Moreover, 24 h after the beginning of the treatment, 30-nM miRNA-21-5p inhibitor/negative control was added to the cells using Lipofectamine^® RNAiMAX Transfection Reagent and transfection was maintained for the following 24 h. (B) To stimulate hypertrophy, HL-1 cells were treated with 1-μM angiotensin II (AngII, Sigma) in complete antibody-free Claycomb medium for 48 h. The pEXOs isolated at T1 from LoR-REM or HiR-REM patients were loaded with miRNA-21-5p inhibitor/negative control through a heat shock-mediated protocol, as described in the experimental procedure, then collected by ultracentrifugation. Isolated pEXOs were added at a final concentration of 1 × 10⁹ particles/ml 24 h after the beginning of the AngII treatment, and treatment by pEXOs was maintained for the remaining 24 h in the presence of AngII. (A,B) At the end of each treatment, the cell monolayer was stained with Phalloidin–Atto 550 as described in the methods. The images were acquired with a fluorescence microscope (Leica; 20× magnification); the cell areas were manually measured with ImageJ software (https://imagej.nih.gov/ij/) and expressed in pixels. Statistical significance of the differences between treatments was evaluated by one-way ANOVA. ^**p < 0.01; ^****p < 0.0001.