Tipifarnib Reduces Extracellular Vesicles and Protects From Heart Failure

Abstract

Background: Heart failure (HF) is one of the leading causes of mortality worldwide. Extracellular vesicles, including small extracellular vesicles or exosomes, and their molecular cargo are known to modulate cell-to-cell communication during multiple cardiac diseases. However, the role of systemic extracellular vesicle biogenesis inhibition in HF models is not well documented and remains unclear.

Methods: We investigated the role of circulating exosomes during cardiac dysfunction and remodeling in a mouse transverse aortic constriction (TAC) model of HF. Importantly, we investigate the efficacy of tipifarnib, a recently identified exosome biogenesis inhibitor that targets the critical proteins (Rab27a [Ras associated binding protein 27a], nSMase2 [neutral sphingomyelinase 2], and Alix [ALG-2-interacting protein X]) involved in exosome biogenesis for this mouse model of HF. In this study, 10-week-old male mice underwent TAC surgery were randomly assigned to groups with and without tipifarnib treatment (10 mg/kg 3 times/wk) and monitored for 8 weeks, and a comprehensive assessment was conducted through performed echocardiographic, histological, and biochemical studies.

Results: TAC significantly elevated circulating plasma exosomes and markedly increased cardiac left ventricular dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, injection of plasma exosomes from TAC mice induced left ventricular dysfunction and cardiomyocyte hypertrophy in uninjured mice without TAC. On the contrary, treatment of tipifarnib in TAC mice reduced circulating exosomes to baseline and remarkably improved left ventricular functions, hypertrophy, and fibrosis. Tipifarnib treatment also drastically altered the miRNA profile of circulating post-TAC exosomes, including miR 331-5p, which was highly downregulated both in TAC circulating exosomes and in TAC cardiac tissue. Mechanistically, miR 331-5p is crucial for inhibiting the fibroblast-to-myofibroblast transition by targeting HOXC8, a critical regulator of fibrosis. Tipifarnib treatment in TAC mice upregulated the expression of miR 331-5p that acts as a potent repressor for one of the fibrotic mechanisms mediated by HOXC8.

Conclusions: Our study underscores the pathological role of exosomes in HF and fibrosis in response to pressure overload. Tipifarnib-mediated inhibition of exosome biogenesis and cargo sorting may serve as a viable strategy to prevent progressive cardiac remodeling in HF.

Keywords: cell communication; fibrosis; heart failure; ventricular remodeling.

Figure 1:. Tipifarnib is effective in reducing exosome biogenesis and secretion in TAC mice.

(A) Schematic presentation on the timeline of the Tipifarnib study in 9–10-week-old C57BL/6J male TAC mice (n=10/group). (B) Nanoparticle tracking analysis (NTA) for exosome concentration in serum of sham, TAC and Tipifarnib treated TAC (TAC+Tipifarnib) mice (n=10/group). (C) Representative image of the western blotting analyses of protein extracted from mouse hearts to study the effect of Tipifarnib treatment on the expression of proteins involved in exosome biogenesis. GAPDH served as the loading control. (D) Mean ± SEM of western blotting experiment (n=6/group). Data are expressed as Mean ± SEM. Mean of the different groups was compared using one-way ANOVA followed by Tukey post-tests with 95% confidence interval.

Figure 2:. Treatment with Tipifarnib improves cardiac function

(A) M-Mode 2D-echocardiographic measurements at baseline/0 weeks, 4 weeks and 8 weeks to determine Left Ventricular (LV) functions i.e., ejection fraction (EF), fractional shortening (FS), LV mass, LV internal dimension in diastole (LVID;d), LV internal dimension in systole (LVIDd) and stroke volume (SV) (n=10/group). Data are expressed as Mean ± SD. Mean of three groups was compared using one-way ANOVA, followed by Tukey post-tests with 95% confidence interval. ^a1Sham vs TAC; ^b1TAC vs TAC+Tipifarnib and ^c1Sham vs TAC+Tipifarnib for 4 weeks and ^a2Sham vs TAC; ^b2TAC vs TAC+Tipifarnib and ^c2Sham vs TAC+Tipifarnib for 8 weeks. (B) Representative echocardiographic images (M-mode) from sham, TAC and Tipifarnib treated TAC mouse heart at 8 weeks. (C) Post TAC, heart-to-body weight ratio (HW/BW) and Heart weight (HW) to Tibia length (TL) (gm/cm) ratio at 8 weeks for sham, TAC and TAC+Tipifarnib. Data are expressed as Mean ± SEM. Mean of the different groups was compared using one-way ANOVA followed by Tukey post-tests. with 95% confidence interval. (D) Images of sham, TAC and TAC+Tipifarnib mouse hearts post 8 weeks. (Scale, in cm.)

Figure 3:. Tipifarnib treatment reduces hypertrophy and fibrosis in cardiac tissue of pressure overload HF mice.

(A) Representative image of the immunofluorescence of myocardial sections stained with Alexa fluor 488-labeled wheat germ agglutinin (WGA) antibody, defining myocyte boundaries. Scale bar = 50 μm). (B) Mean ± SEM of cardiomyocytes≈50 myocytes per mouse in each experimental group; n=10/group. Data are expressed as Mean ± SEM. Mean of sham, TAC and Tipifarnib-treated TAC (TAC+Tipifarnib) mice groups was compared using one-way ANOVA followed by Tukey post-tests with 95% confidence interval. (C) Quantitative Reverse Transcription Polymerase Chain Reaction (qRT PCR) of mice heart tissues for hypertrophy–related genes (ANP, BNP and MYH7) in each experimental group after 8 weeks post-TAC; n=7/group. (D) Representative image for Masson’s trichrome staining in the heart tissues of sham, TAC and Tipifarnib treated TAC (TAC+Tipifarnib) Scale bar = 50 μm. (E) Representative coimmunostaining images for Fibronectin and α-SMA in mice cardiac tissue. Immunofluorescence staining was performed using anti-Fibronectin antibody (green), anti-α-SMA antibody (red) and DAPI (blue). Scale bar = 25 μm. (F) qRT PCR of mice heart tissues for fibrosis-related gene markers (ACTA2, COL1A1, POSTN and TGFB1) in each experimental group; n=7/group. Data are expressed as Mean ± SEM. Mean of the different groups was compared using one-way ANOVA followed by Tukey post-tests with 95% confidence interval. ANP: Natriuretic peptide A, BNP: Natriuretic peptide B, MYH7: myosin heavy chain 7, α-SMA/ACTA2: alpha-smooth muscle actin, POSTN: Periostin, COL1A1: Collagen, Type I, TGFB1: Transforming Growth Factor beta1.

FIGURE 4:. Effect of circulating exosomes in cardiomyocytes (CMs) and cardiac fibroblasts (CFs).

Quantitative reverse transcription polymerase chain reaction (qRT PCR) analysis for the expression of the (A) Fibrosis gene markers (ACTA2, COL1A1 and POSTN) (B) Representative immunostaining images for the expression of α-SMA in mouse primary cardiac fibroblast cells. Immunofluorescence staining was performed using anti- α-SMA antibody (red). DAPI was used to stain cell nuclei (blue). Scale bar = 50 μm. (C) Hypertrophy gene markers (ANP, BNP and MYH7) after treatment of serum exosomes derived from sham, TAC and Tipifarnib-treated TAC (TAC+Tipifarnib) mice; n=3/group. Data are expressed as Mean ± SEM. Mean of the different groups was compared using one -way ANOVA followed by Tukey post-tests with 95% confidence interval. α-SMA/ACTA2: alpha-smooth muscle actin, POSTN: Periostin, COL1A1: Collagen, Type I, ANP: Natriuretic peptide A, BNP: Natriuretic peptide B, MYH7: myosin heavy chain 7.

FIGURE 5:. Effect of circulating TAC exosomes on cardiac function and structure in control mice

(A) Schematic presentation on the timeline of the sham or TAC exos (exosomes) study in C57BL/6J male mice. (B) Heart to Body weight ratio (HW/BW) and Heart weight (HW) to Tibia length (TL) (gm/cm) ratio at 8 weeks for sham, TAC and TAC+Tipifarnib. Data are expressed as Mean ± SEM. Mean of the different groups was compared using one-way ANOVA followed by Tukey post-tests with 95% confidence interval. (C) M-Mode echocardiographic measurements of Left Ventricular (LV) functions i.e., Ejection fraction (EF) and Fractional Shortening (FS) after 8 weeks of exosomes injection in mice; n=5/group. Data are expressed as Mean ± SEM. Mean of the Control, sham exos and TAC exos injected Control mice groups were compared using one-way ANOVA followed by Tukey post-tests with 95% confidence interval. (D) Representative image of the immunofluorescence of thin myocardial sections stained with Alexa fluor 488-labeled wheat germ agglutinin (WGA) antibody, defining myocyte boundaries. Scale bar = 100 μm. (E) Mean ± SEM of cardiomyocytes≈25 myocytes per mouse in each experimental group; n=5/group. Data are expressed as Mean ± SEM. Mean of the Control, sham exos and TAC exos mice groups was compared using one-way ANOVA followed by Tukey post-tests with 95% confidence interval. (F) Representative image of the immunofluorescence of thin myocardial sections of mice receiving an intravenous injection of PKH26-labeled TAC exosomes stained with α-Actinin (green) and DAPI (blue) receiving PKH26-labeled TAC exosomes (red). Scale bar = 50 μm.

FIGURE 6:. Transcriptomic analysis of serum-derived TAC and Tipifarnib-treated TAC mice exosomes.

(A) Volcano plot analysis of the differential expression miRNAs between Tipifarnib treated TAC (TAC+Tip) mice and TAC mice with a comparison of fold change (log2) and significance (-log10 P-value). Significance is determined as adj-p<0.01. Quantitative reverse transcription polymerase chain reaction (qRT PCR) analysis of miR 331-5p in (B) Serum exosomes and (C) Heart tissue of sham, TAC and Tipifarnib-treated TAC (TAC+Tip) mice after 8 weeks post TAC surgery by (n=6/group). (D) Adult primary mouse cardiac fibroblasts (CFs) were transfected with miR 331-5p antagomir (inhibitor), or miR 331-5p mimic to quantify the differential miR 331-5p transfer. The cycle of threshold values was normalized to RNU6B. Data are expressed as Mean ± SEM. Mean of the different groups was compared using one-way ANOVA followed by Tukey post-tests (for >2 groups) with 95% confidence interval. (E) The qRT PCR analysis of the expression of fibrotic marker genes: ACTA2 and POSTN in CFs upon TGFβ1 treatment (10 ng/ml), transfection of miR 331-5p with or without TGFβ1 (10 ng/ml) treatment. Data are expressed as Mean ± SEM. Mean of the untreated, TGF β1, miR mimic control, miR 331-5p mimic ± TGFβ1 was compared using one-way ANOVA followed by Tukey post-tests with 95% confidence interval. ACTA2: alpha-Smooth Muscle Actin, POSTN: Periostin, TGFβ1: Transforming Growth Factor β1.

FIGURE 7:. miR 331-5p regulates HOXC8 to prevent fibrosis

(A) Bioinformatics algorithms predicting miR 331-5p binding sites in 3′-UTR or coding regions (CDS) across different species for HOXC8 gene. (B) The quantitative reverse transcription polymerase chain reaction (qRT PCR) analysis of the expression of HOXC8 in heart tissue of sham, TAC and Tipifarnib-treated TAC (TAC+Tip) mice at 8 weeks, n=7/group. (C-D) Representative image of the protein expression levels of HOXC8 and GAPDH in HOXC8 in heart tissue of sham, TAC and TAC+Tipifarnib mice at 8 weeks and bar graph is the quantitative analysis of HOXC8 protein levels after normalization with GAPDH, n=5/group. Data are expressed as Mean ± SEM. Mean of the sham, TAC and Tipifarnib-treated TAC (TAC+Tip) mice was compared using one-way ANOVA followed by Tukey post-tests (for >2 groups) with 95% confidence interval. (E) The qRT PCR analysis of the expression of HOXC8 in cardiac fibroblast cells after transfection of miR 331-5p with or without TGFβ1 (10 ng/ml) treatment and after transfection of miR 331-5p antagomir with and without treatment of Tipifarnib-treated TAC exosomes (TAC+Tip). Data are expressed as Mean ± SEM. Mean of the untreated, miR mimic control, miR 331-5p mimic ± TGFβ1, miR antagomir control and miR 331-5p antagomir ± Tipifarnib treated TAC exosomes were compared using one-way ANOVA followed by Tukey post-tests (for >2 groups) with 95% confidence interval. HOXC8: Homeobox C8, TGF β1: Transforming Growth Factor β1.

FIGURE 8:. Silencing HOXC8 in cardiac fibroblast (CF) cells reduced fibroblast activation towards myofibroblast formation.

The quantitative reverse transcription polymerase chain reaction (qRT PCR) analysis of (A) overexpression of HOXC8 in CFs. (B) Fibrotic marker genes ACTA2 and POSTN expression after TGF β1 (10 ng/ml) treatment and overexpression of HOXC8, n=3/group. (C) Knockdown of HOXC8 in CFs. (D) Fibrotic marker genes ACTA2 and POSTN expression after TGF β1 (10 ng/ml) treatment and TGFβ1 in the HOXC8-knockdown cells, n=3/group. Data are expressed as Mean ± SEM. Mean of different groups was compared using one-way ANOVA followed by Tukey post-tests (for >2 groups) with 95% confidence interval. ACTA2: alpha-Smooth muscle actin, HOXC8: Homeobox C8, POSTN: Periostin, TGF β1: Transforming Growth Factor β1.