Kisspeptin-10 increases collagen content in the myocardium by focal adhesion kinase activity

The aim of the study was to evaluate the role of kisspeptin-10 (KiSS-10) in the regulation of collagen content in cardiac fibroblasts. An attempt was also made to describe the mechanism of the effect of KiSS-10 on collagen metabolism. The studies indicate that kisspeptin-10 significantly increases the content of intracellular collagen in the myocardium. KiSS-10 also elevates the level of phosphorylated focal adhesion kinase (FAK) in human cardiac fibroblasts. The inhibition of FAK negates the stimulatory effect of KiSS-10 on collagen deposition in vitro. These changes correlate with an increase in the level of propeptides of procollagen type I (PICP) and III (PIIICP) in fibroblast culture medium and mouse PIIICP in serum. Moreover, this hormone inhibits the release of metalloproteinases (MMP-1,-2,-9) and elevates the secretion of their tissue inhibitors (TIMP-1,-2,-4). KiSS-10 also enhances the expression of α1 chains of procollagen type I and III in vitro. Thus, KiSS-10 is involved in the regulation of collagen metabolism and cardiac fibrosis. Augmentation of collagen deposition by KiSS-10 is dependent on the protein synthesis elevation, inhibition of MMPs activity (increase of TIMPs release) or decrease of MMPs concentration. The profibrotic activity of KiSS-10 is mediated by FAK and is not dependent on TGF-β1.


Cell culture
The presented study was conducted on an immortalized human cardiac fibroblast cell line (ABM, Richmond, BC, Canada).The cells were obtained from ventricles of human heart of healthy adult.The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (Biowest, Nuaillé, France) supplemented with 10% (v/v) foetal bovine serum (FBS) (Biowest, Nuaillé, France), gentamicin (25 µg/ml) (Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA), amphotericin B (0.25 µg/ml) (Capricorn Scientific, Ebsdorfergrund, Germany), insulin (5 µg/ml) (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and vitamin C (50 µg/ml) (Sigma-Aldrich, Saint Louis, Missouri, USA) on the culture plates coated with collagen (10 µg/cm 2 ) (Sigma-Aldrich, Saint Louis, Missouri, USA) at 37 °C under the humidified atmosphere of 5% CO 2 .The cells were grown to confluence before being trypsynized (Trypsin-EDTA 1X in PBS, Biowest, Nuaillé, France) and passaged.All experiments were carried out on the cells from passage 7 to 13.The human cardiac fibroblasts were incubated in DMEM containing 3% (v/v) bovine serum, insulin, vitamin C, antibiotics in the concentrations given above and 10 −11 -10 −5 mol/L of kisspeptin-10 or FAK inhibitor 14 (FAKi, 10 −6 mol/L) or KiSS-10 (10 −8 mol/L) with FAKi (10 −6 mol/L) for 96 h.The results were compared with control cultures without hormones or inhibitors.The medium was changed every day.On the day 5 of the experiment, the total number of fibroblasts and the number of necrotic (stained with trypan blue) cells were counted in the Burker chamber.Intracellular collagen content, expression of α1 chains of procollagen type I (Col1A1) and III (Col3A1) and level of phosphorylated focal adhesion kinase (phospho-FAK) were measured in the cultures on the last day of the experiment.Before determination of the collagen level, the cells were trypsynized and washed four times to remove extracellular collagen that was used to coat the plates.Also, the culture media were collected on day 5 to analyse the concentrations of propeptides of procollagen type I and III (PICP, PIIICP), MMPs, TIMPs, TGF-β1.

Animals and experimental design
Experimental male BALB/c mice (12 weeks old, n = 32) were obtained from the Animal House of the University of Lodz, Poland.All animals were maintained in a standard housing condition i.e. in groups of 2-5 animals, at constant temperature (20-24 °C) and humidity (45-65%), with 12-h light/12-h dark cycle.They were kept with free access to the standard diet and tap water.Before beginning the experiment, the mice were adapted to new environment for two weeks.The animals were then divided into four groups: 1) control (n = 8); 2) placebo-group receiving subcutaneous injections of aqua pro injectione (200 µL, for four weeks, 1 × day) (n = 8); 3) experimental group receiving subcutaneous injections of KiSS-10 (1 nmol/200 µL, for four weeks, 1 × day) (n = 8); 4) experimental group receiving subcutaneous injections of KiSS-10 (10 nmol/200 µL, for four weeks, 1 × day) (n = 8).This animal model was chosen as preferred for cardiovascular research.Kisspeptin-10 doses were selected on the basis of available data 8,18 .The number of animals in each group was determined according to statistical verification.

Determination of the total collagen level
The collagen content in the cells cultured in 24-well culture plates and the heart samples were measured by Woessner method 19 .The fibroblast cultures were dried in a laboratory oven, while the obtained hearts were minced into pieces, dried in a laboratory oven and homogenized with a mortar.The prepared samples were taken for further analysis.The level of total collagen in samples was assessed based on the amount of hydroxyproline, which was hydrolyzed for 24 h at 100 °C in 6 N HCl (1.2-1.5 mL/culture and 3 mL/10 mg of dry tissue, respectively) (Stanlab, Lublin, Poland).All the hydrolizates were neutralized by adding 5 N NaOH (Stanlab, Lublin, Poland).The products of neutralization were diluted to 5 mL with redistilled water.Following this, 0.2 mL samples were collected and diluted with redistilled water to 1 mL final volume.The oxidation of hydroxyproline to pyrrole was carried out by adding 0.5 mL chloramine T (Chempur, Piekary Slaskie, Poland) and methyl glycol (EMSURE®, Merck, Darmstadt, Germany) in a citrate buffer (pH = 6).Then, the samples were shaken and incubated for 20 min at room temperature.Also, excess chloramine T was removed by adding 0.5 mL of 3.15 mol/L perchloric acid (Loba Chemie, Mumbai, India).After 5 min, the samples were incubated with 0.5 mL of 20% (w/v) p-dimethylaminobenzaldehyde (Sigma-Aldrich, Saint Louis, Missouri, USA) in a 60 °C water bath for 20 min.The optical density was measured by spectrophotometer at a wavelength of 560 nm.

Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted from fibroblast cell culture and from the mouse hearts using Total RNA Mini kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's protocol.RNA purity and quantity was detected on a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA).Then, cDNA was obtained through reverse transcription using the a PrimeScript™ RT reagent Kit (Perfect Real Time) (Takara Bio Inc., Kusatsu, Shiga, Japan) according to the manufacturer's protocol.Samples of cDNA were amplified using Universal Probe Library (UPL) (Roche, Indianapolis, USA) and RealTime Ready Custom Single Assay (Roche, Indianapolis, USA) based on TaqMan probes (for Col1A1, Col3A1, GAPDH, RPLP0, YWHAZ, Rpl13a, Hprt1).The qPCR reaction was carried out using FastStart Essential Probe Master (Roche, Indianapolis, USA) according to the manufacturer's instructions.After activation of DNA polymerase and denaturation of the cDNA for 10 min at 95 °C, 55 cycles of PCR reaction were performed: 95 °C for 10 s, 60 °C for 30 s, 72 °C for 1 s.Then, the reaction mixture was cooled at 40 °C for 30 s. Human ribosomal protein 0 (RPLP0), human tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as a reference gene for analysis of human gene expression of α1 chains of procollagen type I (Col1A1) and III (Col3A1) in human cardiac fibroblasts.Mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh), mouse ribosomal protein L13a (Rpl13a) and mouse hypoxanthine phosphoribosyltransferase 1 (Hprt1) were used as a reference gene for analysis of mouse gene expression of Col1A1 and Col3A1 in the mouse hearts.Human RPLP0 was measured using the primers (forward primer: 5′-GGC ACC ATT GAA ATC CTG AG-3′, reverse primer: 5′-GAA GGG GGA GAT GTT GAG C-3′) with the UPL (Universal Probe Library) probe #36 (Roche, Indianapolis, IN, USA), while human YWHAZ was measured using the primers (forward primer: 5′-AAG TGC AAT GGA GAC CTT GG-3′, reverse primer: 5′-GTT GCC CTA GAT GCA GAA GG-3′) with the UPL probe #2 (Roche, Indianapolis, IN, USA).Human GAPDH, mouse Gapdh, mouse Rpl13a and mouse Hprt1 expression was determined using Real-Time ready Custom Single Assay (Roche, Indianapolis, IN, USA) 20 .Each reaction was conducted in duplicate.The relative gene expression with multiple reference genes was determined and the geometric mean of the concentration ratio was calculated by LightCycler® 96 software (Roche, Indianapolis, USA).

Statistical analysis
The obtained results were calculated using StatSoft Statistica 13.1 PL and expressed as mean and standard deviation (x ± SD).The normality of the data distribution was determined using the Shapiro-Wilk test and www.nature.com/scientificreports/ the homogeneity of variances using Levene's test.Then, comparisons between two groups were assessed by the paired t-test or Mann-Whitney U-test for nonparametric data.Multiple comparisons were performed using the parametric one-way ANOVA with Tukey's post hoc test, or the nonparametric Kruskal-Wallis for non-normally distributed data.Differences were considered as significant at p < 0.05.

Ethical approval
The protocol of the study was approved by the Local Ethics Committee for Animal Testing in Lodz (Licence No. 25/ŁB138/2019).All methods were carried out in accordance with relevant guidelines and regulations.The study is reported in accordance with ARRIVE guidelines.

Results
The influence of kisspeptin-10 on the intracellular collagen content and the expression of Col1A1 and Col3A1 in human cardiac fibroblasts The effect of kisspeptin-10 on intracellular collagen content was found to be dependent on the dose of KiSS-10 used.Treatment of the cells with 10 −11 to 10 −6 mol/L of KiSS-10 caused a significant increase in the collagen content compared to the control.The most stimulating effect (p < 0.0001) was observed under the influence of 10 −8 mol/L of kisspeptin-10 (192.39 ± 82.73 vs. 45.24 ± 20.42 µg/10 5 cells).However, the highest concentration of KiSS-10 (10 −5 mol/L) did not change markedly the collagen content in human cardiac fibroblast in vitro (Fig. 1A).

The impact of KiSS-10 on the release of TGF-β1 in vitro
No significant changes in the secretion of TGF-β1 were observed from human cardiac fibroblasts under the influence of 10 −11 mol/L, 10 −8 mol/L or 10 −5 mol/L KiSS-10 compared to controls (Fig. 5A).

Changes in serum concentration of PICP and PIIICP
The results revealed that treatment of mice with kisspeptin-10 (10 nmol/200 µL) for four weeks markedly elevated the serum concentration of PIIICP (31.12 ± 5.57 ng/mL) compared to the control (20.17 ± 8.31 ng/mL), placebo (19.26 ± 9.48 ng/mL) and mice, which received KiSS-10 at the lower dose of 1 nmol/200 µL (22.08 ± 5.70 ng/mL) (Fig. 8B).However, no significant changes in the serum concentration of PICP were found between all groups in mice in vivo (Fig. 8A).

Discussion
This study examines the role of kisspeptin-10 in the regulation of collagen metabolism in the heart; in addition, unlike previous studies, it also attempts to determine the mechanism behind the effect of kisspeptin-10 on collagen deposition within the heart, which could be related to cardiac fibrosis.Our in vitro findings indicate that the intracellular collagen content in human cardiac fibroblasts is increased under the influence of KiSS-10 (10 −11 -10 −6 mol/L).The most stimulatory effect of KiSS-10 on collagen in fibroblasts was observed after exposure to 10 −8 mol/L of kisspeptin-10.The cells treated with 10 −8 mol/L of KiSS-10 demonstrated 4.25-fold elevation in collagen content compared to non-treated controls.However, the highest concentration of KiSS-10 (10 −5 mol/L) did not affect intracellular collagen level compared to the control (Fig. 1A).Moreover, 10 −8 mol/L of KiSS-10 was found to increase the expression of Col1A1 and Col3A1 in vitro (Fig. 1B,C).By contrast, other studies on Human Aortic Smooth Muscle Cells (HASMCs) have shown that kisspeptin-10 did not significantly alter the protein expression of collagen-1 or collagen-3 21 .
Collagen is synthesized by fibroblasts as pre-procollagen.Cleavage of signal peptide leads to the formation of procollagen molecule.This procollagen is excreted from the cell, releasing the carboxy-terminal or the aminoterminal propeptides, i.e. collagen type I (PICP, PINP) and type III (PIIICP, PIIINP), what results in collagen formation.Thus, these factors can be used as markers of collagen biosynthesis, which are elevated during the  overproduction and deposition of collagen in the heart associated with myocardial fibrosis 22 .Our findings indicate that kisspeptin-10 is involved in the regulation of collagen synthesis by cardiac fibroblasts in vitro; the hormone significantly stimulates PICP and PIIICP release from human cardiac fibroblasts compared to controls when applied at concentrations of 10 −6 and 10 −8 mol/L (Fig. 2).
Cardiac fibroblasts also produce metalloproteinases (MMPs) and their inhibitors: TIMPs.MMPs are the predominant proteases that are involved in the cleavage of collagen.Thus, it is critical to achieve a balance between the function of MMPs and TIMPs to maintain ECM homeostasis.However, changes in the levels of MMPs and TIMPs are observed under pathological conditions such as cardiac fibrosis.These alterations are dependent on the type, stage and severity of the disease 2 .According to our in vitro studies, kisspeptin-10 inhibits the release of MMPs (MMP-1, -2, -9) compared to the control (Fig. 3), thereby reducing collagen degradation.Lee et al. 23 also note that kisspeptin-mimicking peptide inhibits the MMP-1 mRNA expression levels, but increases type I procollagen secretion in UV-induced human dermal fibroblasts.The inhibitory influence of kisspeptin on MMPs secretion has also been noted in cancer cells.Ciaramella et al. 12 observed that treatment with KiSS-10 reduces the activity of MMP-2 and MMP-9 in human malignant mesothelioma cells.However, other studies have shown that kisspeptin-10 significantly enhances the activities of MMP-2 and MMP-9 on HASMCs 21 .According to these studies, KiSS-10 also inhibits the activity of MMPs by stimulation of their tissue inhibitors secretion (TIMP-1,-2,-4) (Fig. 4A,B,D).Other reports also indicate that kisspeptin affects MMPs (MMP-1, -2, -3, -7, -9, -10, -14) directly by downregulating their transcription, as well as indirectly by upregulating TIMP transcription (TIMP-1, -3) in trophoblast cells 24 .
Cardiac fibroblasts are also involved in production of growth factors and other signalling molecules that regulate cellular function and protease activity, thereby modulating ECM biosynthesis and breakdown.Fibroblasts secrete, among others, transforming growth factor β1, which is involved in cardiac fibrosis.It is known that TGF-β1 activates fibroblasts and promotes their transition to myofibroblasts, which produce components of the ECM 6 .However, our findings indicate that the addition of KiSS-10 to the human cardiac fibroblasts cultures does not affect secretion of TGF-β1 (Fig. 5A).Therefore, the profibrotic effect of KiSS-10 within the human cardiac fibroblasts is not mediated by TGF-β1 release.
The signalling associated with FAK activity has been reported to participate in fibrogenesis in inter alia skin 25 , lungs 26 , liver 27 and heart 28 .It is known that FAK integrates growth factor and integrin signals to induce myofibroblast differentiation and promote fibrosis formation 29 .Rajshankar et al. 30 demonstrate that FAK activity contributes to tractional collagen remodeling, but also inhibits collagen degradation via metalloproteinase activity in mouse embryonic fibroblasts.Lagares et al. 26 report that FAK expression and activity are upregulated in fibroblasts from lung fibrosis patients.They also noted that pharmacological inhibition of FAK, as well as its siRNA-mediated silencing, ameliorates bleomycin-induced lung fibrosis and reduces collagen deposition in the lungs of mice in vivo.In contrast, Gałdyszyńska et al. 31 reported that inhibition of FAK using 10 −7 mol/L of FAK kinase inhibitor 14 results in increased collagen content within human cardiac fibroblast culture.Some data suggests that kisspeptin has a stimulatory effect on FAK in some types of cells.Wu et al. 32 noted that kisspeptin regulates the cell motility of endometrial cancer cells through the phosphorylation of FAK and Src-dependent activation of MMP-2.According to Roseweir et al. 33 , kisspeptin-10 also activates FAK in extravillous trophoblastderived cells and participates in the inhibition of placental trophoblast cell migration.In contrast, it has been demonstrated that kisspeptin agonist decreases the phosphorylation of FAK, while the antagonist increases it in human decidual stromal cells 34 .Our present in vitro experiment found that 10 −8 mol/L KiSS-10 stimulates phosphorylation of FAK at Tyr397 in human cardiac fibroblasts compared to controls (Fig. 5B).Moreover, since kisspeptin-10 increases the level of phospho-FAK in cardiac fibroblasts, the study examined whether this signaling pathway mediates the effect of KiSS-10 on collagen deposition.It was found that the cells treated with both KiSS-10 (10 −8 mol/L) and FAKi (10 −6 mol/L) demonstrated a significant reduction intracellular collagen content compared to the culture with KiSS-10 alone (Fig. 5C).Thus, FAK inhibitor autophosphorylation inhibition of FAK at Y397 35,36 abolishes the stimulatory effect of KiSS-10 on collagen level in human cardiac fibroblasts.These observations clearly confirm that the profibrotic influence of KiSS-10 on collagen content is associated with FAK activity.However, kisspeptin activates other signaling pathways and further studies are needed to determine whether they could be also involved in collagen metabolism within the heart.
In vivo studies with a mouse model confirmed that kisspeptin-10 has an influence on collagen deposition in the myocardium.A significant increase in collagen content was noted in the hearts of mice from the experimental group, which received an injection of kisspeptin-10 (10 nmol/200 µL), compared to the control and placebo (Fig. 6).The obtained results also indicate that a significant increase in collagen content in the hearts of mice is associated with an elevation of the collagen biosynthesis marker PIIICP.Treatment of mice with 10 nmol/200 µL kisspeptin-10 for 4 weeks significantly increased the serum concentration of PIIICP compared to the other groups (Fig. 8B).However, in addition, a significant decrease in the expression of Col1A1 and Col3A1 was observed in the experimental groups, i.e. which received kisspeptin-10, compared to the control group and placebo (Fig. 7).The obtained data suggest that kisspeptin-10 could promote collagen accumulation in the heart in vivo due to the intensification of collagen biosynthesis at post-transcriptional levels.It is known that co-and posttranslational modifications are required for formation of functional collagen.The crucial role plays enzymatic modification of proline and lysine residues under the influence prolyl hydroxylase and lysyl hydroxylase.Also, there are other www.nature.com/scientificreports/enzymes that are involved in collagen biosynthesis and formation of covalent intermolecular cross-linking, that is critical for stabilization of collagen.Lysyl oxidase is a copper-dependent monoamine oxidase that mediates collagen cross-linking in the extracellular matrix.However, it is difficult to confront the results of this study with data from the literature because no other reports on the effect of kisspeptin on activity of enzymes that are involved in collagen synthesis have been published so far 37,38 .This stimulatory effect of kisspeptin on collagen deposition and development of cardiac fibrosis was also noted in rats.Zhang et al. 8 reported that treatment with kisspeptin-10 for seven days leads to myocardial changes and cardiomyocyte damage, including an increase in the numbers of collagen fibers in the cardiac muscle and a certain degree of fibrosis, as well as myocardial contractions or mitochondrial cristae fracture.By contrast, studies on lungs have shown that kisspeptin-13 inhibits bleomycin (BLM)-induced pulmonary fibrosis in mice through decreased collagen/α-smooth muscle actin deposition and suppression of the inflammatory response 39 .Moreover, studies indicate that hepatic KiSS1R deficiency promotes liver fibrosis biomarkers in a high-fat diet (HFD)-induced mouse model.On the contrary, activation of hepatic KISS1R plays a protective role against steatosis and reduces fibrosis of the liver in a dietinduced mouse model of non-alcoholic liver disease 40 .

Conclusion
To conclude, KiSS-10 appears to be involved in the regulation of collagen metabolism in the heart.The augmentation of the collagen deposition observed under the influence of kisspeptin-10 is dependent on an elevation of protein synthesis, inhibition of matrix metalloproteinase activity (increase of TIMPs release) or decrease of MMPs concentration.This effect of KiSS-10 is related to its direct action on human cardiac fibroblasts.In addition, this profibrotic activity is mediated by the activity of FAK.These findings indicate that kisspeptin-10 could be involved in pathological cardiac remodeling, and that modulating KiSS-10 concentrations in the body may serve as a new therapeutic approach that could regulate collagen metabolism in the heart and stabilize the connective tissue matrix. https://doi.org/10.1038/s41598-023-47224-3