V1-Cal hydrogelation enhances its effects on ventricular remodeling reduction and cardiac function improvement post myocardial infarction

Abstract

Myocardial infarction (MI) is a major cause of disability and mortality worldwide. A cell permeable peptide V1-Cal has shown remarkable therapeutic effects on ML However, using V1-Cal to improve long-term cardiac function after MI is presently limited by its short half-life. Herein, we co-assembled V1-Cal with a well-known hydrogelator Nap-Phe-Phe-Tyr (NapFFY) to obtain a new supramolecular hydrogel V1-Cal/NapFFY. We found that the hydrogel could significantly enhance the therapeutic effects of V1-Cal on ventricular remodeling reduction and cardiac function improvement in a myocardial infarction rat model. In vitro experiments indicated that co-assembly of V1-Cal with NapFFY significantly increased mechanic strength of the hydrogel, enabling a sustained release of V1-Cal for more than two weeks. In vivo experiments supported that sustained release of V1-Cal from V1-Cal/NapFFY hydrogel could effectively decrease the expression and activation of TRPV1, reduce apoptosis and the release of inflammatory factors in a MI rat model. In particular, V1-Cal/NapFFY hydrogel significantly decreased infarct size and fibrosis, while improved cardiac function 28 days post MI. We anticipate that V1-Cal/NapFFY hydrogel could be used clinically to treat MI in the near future.

1. Introduction

Myocardial infarction (MI), which is mainly induced by acute, persistent ischemia or hypoxia of cardiac tissues, is a major cause of mortality and disability worldwide. Its occurrence is commonly seen in Europe and the United States, and its incidence rate has been on the rise in China in recent years. MI not only affects the life safety and life quality of patients, but also leads to a huge social burden [1,2]. Pathologically, MI could lead to various pathological changes including oxidative stress, alteration in energy metabolism, apoptosis, release of inflammatory factors, and cardiomyocyte death, which in turn trigger the profibrotic and hypertrophic signaling cascades to develop into ventricular remodeling and even heart failure [3–5]. Taken above factors into consideration, reasonable control of inflammation and apoptosis would be an effective strategy to prevent fibrosis and ventricular remodeling at early stage, while in the meantime promoting myocardial repair.

Up to now, besides traditional surgical revascularization procedures, more and more innovative methods have been developed to treat MI, such as stem cell therapeutics [6], gene therapeutics [7], exosome therapeutics [8], and growth factor therapeutics [9,10]. Nevertheless, due to the unstable performance of stem cells, genes, exosomes, or growth factor, their application in clinical transformation is greatly limited. Luckily, nanotechnology provides new alternative for the loading of therapeutic reagents to increase their stability and maintain their long circulation in living organisms. For examples, nanomaterials based on liposomes [11,12], micelles [13], dendrimers [14], carbon nanotubes [15,16], or supramolecular hydrogels [17–22] were reported to load drugs to improve ventricular remodeling. However, these biomaterials still have some limitations. Liposomes have low toxicity and high drug loading efficiency while their stability is poor and they are easy to be metabolized. Micelles and dendrimers have versatility in synthesis and functionalization, but they have inherent toxicity and low encapsulation efficacy [23]. Carbon nanotubes are also limited in use due to their intrinsic low biosafety [24]. As one new type of soft biomaterials, hydrogels have been widely used in the biomedical field in recent years [25,26]. According to their different components, hydrogels can be classified into polymer hydrogels, supramolecular peptide hydrogels, etc. Among them, supramolecular peptide hydrogels, which are synthesized by peptide-based molecule assembly through non-covalent interactions and allow administration of local injection, show higher biocompatibility and biodegradation than polymer hydrogels [27,28]. Thus, supramolecular peptide hydrogels are commonly used to encapsulate drugs or growth factors to treat related disease. For example, Liang et al. designed a supramolecular peptide hydrogel to promote periodontal bone regeneration by sustained release of two bioactive factors which are co-assembled inside the hydrogel [29].

Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation-gated channel, which could be activated by a series of physical or chemical noxious stimuli such as capsaicin, heat stimulation, acidic environment, or endogenous mediators [30,31]. Previous studies have shown that over-activation of TRPV1 plays an important role in the pathological changes post myocardial ischemia [32–35]. Gross et al. derived an 11-amino acid peptide from C-terminus TRPV1 TRP domain (R701-S711) and conjugated it with TAT_47–57 to yield V1-Cal (YGRKKRRQRRRGSGRATTILDTEKS, Fig. 1a) for its intracellular delivery [35]. They found that V1-Cal could effectively reduce myocardial infarct size [35,36]. Specifically, in isolated and in vivo myocardial ischemia–reperfusion (MIR) rodent models, V1-Cal efficiently reduced MI by limiting MIR injury with 61.2% and 60.7%, respectively [35]. Since V1-Cal has a short half-life for conventional administration, a recent study reported that, by continuous pump delivery of V1-Cal, it reduced the injury of vascular endothelial cells and promoted the angiogenesis post arterial injury in diabetes rodent models [36]. But to the best of our knowledge, using a biomaterial as a carrier for sustained release of V1-Cal to treat MI has not been reported.

Fig. 1.

(a) Chemical structure of NapFFY hydrogelator and the peptide sequence of V1-Cal. (b) Schematic illustration of a supramolecular hydrogel V1-Cal/NapFFY improving cardiac function of a rat MI model by myocardial injection.

Based on above literature research, naturally we think, could we co-assemble V1-Cal with a supramolecular hydrogelator to form a hydrogel for sustained release of V1-Cal to reduce ventricular remodeling, improve cardiac function, and finally treat MI? To achieve this goal, we co-assembled V1-Cal with a well-known hydrogelator Nap-Phe-Phe-Tyr-OH (NapFFY, Fig. 1a), and indeed obtained a new supramolecular hydrogel V1-Cal/NapFFY. The results indicated that, after the V1-Cal/NapFFY hydrogel was injected into the MI area, V1-Cal was slowly released which effectively decreased the expression and activation of TRPV1, reduced apoptosis and the release of inflammatory factors in MI rat model (Fig. 1b). At 28 day post MI, V1-Cal/NapFFY hydrogel significantly decreased infarct size and fibrosis, while improved cardiac function of the rat model.

2. Results and discussion

2.1. Preparations of hydrogels Gel NapFFY) Gel V1-Cal/NapFFY, and Gel V1-Scr/NapFFY

After synthesis and characterizations of NapFFY (Scheme S1, Fig. S1-S3, Table S1), we investigated its hydrogelation condition with (or w/o) V1-Cal according to previous methods [29]. To obtain Gel NapFFY, we firstly dissolved 10 mg NapFFY powder in 1 mL phosphate buffered saline (PBS, 0.01 M, pH 7.4) at 25 °C and adjusted the solution pH value to 8.0, then heated up the solution to 55 °C until it became clear. A transparent hydrogel at 1.0 wt% formed 30 min later after the solution cooling down to room temperature (25 °C) (the inserted image in Fig. 2a). For Gel V1-Cal/NapFFY hydrogel, 10 mg NapFFY hydrogelator and 1 mg V1-Cal were dissolved in 1 mL PBS (0.01 M, pH 7.4) at 25 °C, then the gel was obtained with the same method as above. To prove the vital role of V1-Cal in the treatment of MI, here we scrambled the peptide sequence of V1-Cal to prepare another hydrogel Gel V1-Scr/NapFFY. In detail, we used a scrambled peptide sequence IDKLRTAEIST of above 11-amino acid peptide from C-terminus TRPV1 TRP domain R701-S711 (i.e., RAITILDTEKS) to conjugate with TAT47-57 to yield V1-Scr (YGRKKRRQRRRGSGIDKLRTAEIST). Similarly, Gel V1-Scr/NapFFY was obtained using above method after dissolving 10 mg NapFFY and 1 mg V1-Scr in 1 mL PBS (0.01 M, pH 7.4) at 25 °C. As can be seen from the inserted images in Fig. 2b-c, both Gel V1-Cal/NapFFY and Gel V1-Scr/NapFFY are transparent, suggesting that V1-Cal or V1-Scr co-assembles (but not physically mix) with the hydrogelator NapFFY to form corresponding hydrogel.

Fig. 2.

Characterizations of the three hydrogels, (a-c) The dynamic storage moduli (G’) (black) and the loss moduli (G”) (red) of 1.0 wt% Gel NapFFY, Gel V1-Scr/NapFFY, and Gel V1-Cal/NapFFY (insets: photographs of corresponding hydrogels), (d-f) TEM images of 0.1 wt% Gel NapFFY, Gel V1-Scr/NapFFY, and Gel V1-Cal/NapFFY, respectively. Scale bar: 200 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

2.2. CACs and MGCs of Gel NapFFY, Gel V1-Scr/NapFFY, and Gel V1-Cal/NapFFY

To further determine the gelation ability of NapFFY, V1-Cal/NapFFY and V1-Scr/NapFFY, we investigated their critical aggregation concentrations (CACs) and minimum gelation concentrations (MGCs). Plots of fluorescence emission intensity versus concentration revealed two regimes, indicating critical aggregation concentration (CAC) of 16.6 µM for NapFFY, 16.0 µM for V1-Cal/NapFFY, and 16.4 µM for V1-Scr/NapFFY (Fig. S4), respectively. According to their CAC values, we calculated the co-assembly efficiency of V1-Cal with NapFFY and V1-Scr with NapFFY was 90.8% (calculated by using the method in the Supporting Information). The inverted tube test indicated that the MGCs for Gel NapFFY, Gel V1-Cal/NapFFY, and Gel V1-Scr/NapFFY were measured to be 0.25 ± 0.05 mM (Fig. S5). The similar CAC and MGC values of these three hydrogels indicated that they have similar self-assembly capabilities.

2.3. Rheology, CD spectra, and TEM characterizations of Gel NapFFY, Gel V1-Scr/NapFFY, and Gel V1-Cal/NapFFY

Next we evaluated the viscoelastic properties of the obtained hydrogels (Gel NapFFY, Gel V1-Scr/NapFFY, and Gel V1-Cal/NapFFY) using rheology. Firstly we studied the dynamic strain scanning of the three hydrogels. All the storage modulus (G’) and loss modulus (G”) values of the three hydrogels showed a weak dependence ranging from 0.01% to 1% of their strains (with G’ dominating G”) at 1.0 wt% as shown in Fig. S6 in the Supporting Information, indicating that all these studied samples are hydrogels. We conducted dynamic frequency scanning of these three hydrogels by setting the strain amplitude at 0.1%. Fig. 2a-c showed that, at the strain amplitude of 1.0%, all G’ and G” values of the three hydrogels increased with the increase of frequency from 0.1 to 10 Hz. In addition, their G’ values were several times higher than those of G”, indicating that all these hydrogels can tolerate external forces. Meanwhile, both G’ and G” values of Gel V1-Scr/NapFFY are close to those of Gel V1-Cal/NapFFY, respectively, indicating that Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY have close mechanical strengths. Interestingly, both Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY were elastically stronger than Gel NapFFY, suggesting that co-assembly between the peptide (V1-Scr or V1-Cal) with the hydrogelator NapFFY significantly enhances the mechanical strength of Gel NapFFY.

After rheological tests, circular dichroism (CD) spectra of these three hydrogels were obtained to investigate the molecular packing of their corresponding hydrogelators (Fig S7). CD spectroscopy measurements indicated that Gel NapFFY exhibited a similar spectrum as Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY. In detail, CD spectrum of Gel NapFFY shows a remarkable positive Cotton effect at 202 nm and a clear negative CD absorption at 232 nm, indicating β-sheet secondary structures formed in the hydrogel [37]. A remarkable positive Cotton effect at 202 nm and a clear negative CD absorption at 220 nm were also observed in CD spectrum of Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY. These results indicated that the encapsulation of V1-Cal or V1-Scr almost did not interfere with the molecular arrangements of the hydrogelators in the hydrogels. CD spectra of NapFFY also indicated that, when its concentration was lower than its CAC it did not form any secondary structure, while it formed above β-sheet secondary structure at concentrations higher than its CAC (Fig. S8).

Then we conducted transmission electron microscopy (TEM) observation to investigate the internal networks in these three hydrogels. As shown in Fig. 2d-f, in general, all the hydrogels were composed of long and flexible nanofibers. And very close nanofiber density between Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY was observed. However, the nanofiber densities of Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY were much higher than that of Gel NapFFY, which is consistent with above rheological results that Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY were mechanically stronger than Gel NapFFY. All these results indicated that the co-assembly between the peptide (V1-Scr or V1-Cal) and NapFFY does not affect the microscopic morphology of the formed hydrogels, but obviously enhances the mechanical strength of the related hydrogels. Previous studies indicated that, due to the dynamic motion of cardiac tissue, co-assembled hydrogel with higher mechanical strength is more suitable for its application in myocardial injection [38,39]. Hence, the co-assembled hydrogels Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY here are more suitable to be used for myocardial injection to treat MI than Gel NapFFY.

2.4. Cumulative release of V1-Cal or V1-Scr from Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY in vitro, respectively

After rheology tests and TEM observations of the hydrogels, we studied in vitro release of V1-Cal or V1-Scr from Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY, respectively. Firstly, using HPLC analysis, we obtained the standard calibration curves of V1-Cal and V1-Scr by plotting the relationship between the peptide concentrations and its HPLC peak areas (Fig. S9, Table S2). After preparing Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY hydrogel as described above, we added 1 mL PBS (0.01 M, pH 7.4) to 200 µL Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY, and incubated it at 37 °C. At different times (1, 2, 3, 5, 8, or 14 day), 1 mL of supernatant was collected for HPLC analysis. Then the culture mixture was replenished with 1 mL PBS immediately. As shown in Fig. 3, V1-Cal and V1-Scr were released continuously within 14 days. In vitro release monitoring results showed that the cumulative release amount of V1-Cal and V1-Scr was 27.8 ± 8.4% and 24.4 ± 4.8% at day 3, respectively. The cumulative release amount of V1-Cal or V1-Scr increased with time and approached to 62.1 ± 8.2% or 60.8 ± 6.1% at day 14, respectively. The release rate of the peptide was high in the beginning 5 days and then gradually decreased with time. These experimental results indicated that both V1-Cal and V1-Scr were released from respective Gel V1-Cal/NapFFY and Gel V1-Scr/NapFFY at a sustainable manner, and suitable for in vivo experiments of MI treatment.

Fig. 3.

In vitro cumulative releases of V1-Cal or V1-Scr from Gel V1-Cal/NapFFY or Gel V1-Scr/NapFFY in PBS (0.01 M, pH 7.4) at 37 °C, respectively.

2.5. Cytotoxicity evaluation of V1-Scr, V1-Cal, GelNapFFY, Gel V1-Scr/NapFFY and Gel V1-Cal/NapFFY

Then we conducted the stability study of the peptides in PBS buffer (pH 7.4, 10 mM). HPLC analysis clearly indicated that either V1-Cal or V1-Scr was quite stable in PBS up to 16 days (Fig. S10). In order to evaluate the cytotoxicity of above peptides (V1-Scr or V1-Cal) and hydrogels (Gel NapFFY, Gel V1-Scr/NapFFY, or Gel V1-Cal/NapFFY), we used cell counting kit-8 (CCK-8) to investigate the survival and proliferation of rat embryonic cardiomyocyte (H9C2 cells). First, we seeded 100 µL culture medium into a well of a cell culture plate as a control group (Control). 0.2 mg/mL V1-Scr or 0.2 mg/mL V1-Cal in 100 µL culture medium, 20 µL of Gel NapFFY at 1.0 wt% in 100 µL culture medium, 20 µL of Gel V1-Scr/NapFFY at 1.0 wt% in 100 µL culture medium, and 20 µL of Gel V1-Cal/NapFFY at 1.0 wt% in 100 µL culture medium were seeded as experimental groups into the wells of a cell culture plate. Then each of above wells was added with 100 µL cell dispersion (2000 cells) in culture medium to a final V1-Cal (or V1-Scr) concentration of 0.1 mg/mL. The cell plates were incubated at 37 °C for 24, 48, or 72 h for cytotoxicity study. As shown in Fig. S11, cell numbers of the H9C2 cells in all groups increased with time, indicating that 0.1 mg/mL peptides (V1-Scr or V1-Cal) and 1.0 wt% hydrogels (Gel NapFFY, Gel V1-Scr/NapFFY, or Gel V1-Cal/NapFFY) did not impose toxicity on the cells. Interestingly, we found that the viabilities of H9C2 cells treated with the hydrogels (Gel NapFFY, Gel V1-Scr/NapFFY, or Gel V1-Cal/NapFFY) were higher than those of cells treated with 0.1 mg/mL peptides (V1-Scr or V1-Cal) or non-treated (Control). This suggests that the hydrogels with (or w/o) co-assembled peptides (V1-Scr or V1-Cal) could promote the proliferation of H9C2 cells [27], All these above results indicate that V1-Scr, V1-Cal, Gel NapFFY, Gel V1-Scr/NapFFY, or Gel V1-Cal/NapFFY is highly compatible to H9C2 cells and could be applied for following in vivo experiments.

2.6. Apoptosis, inflammatory factors, and TRPV1 expression analyses in MI rats

After in vitro characterizations and cytotoxicity evaluation of V1-Cal, Gel NapFFY, Gel V1-Scr/NapFFY, and Gel V1-Cal/NapFFY, we investigated their effects on ventricular remodeling reduction and cardiac function improvement post MI in rats. Eight-week-old male Sprague Dawley rats were divided into six groups randomly (Sham, MI, NapFFY, V1-Scr/NapFFY, V1-Cal, and V1-Cal/NapFFY) (n = 3 for each group). Rats in the Sham group only underwent routine operations of anesthesia, endotracheal intubation, mechanical ventilation, thoracotomy, threading around the left anterior descending coronary artery, injection of PBS in the ischemic risk area, but no ligation of the left anterior descending coronary artery. For rats in the MI, NapFFY, V1-Scr/NapFFY, V1-Cal, V1-Cal/NapFFY groups, their left anterior descending coronary arteries were ligated, and then 100 µL PBS, Gel NapFFY at 1.0 wt%, Gel V1-Scr/NapFFY at 1.0 wt%, V1-Cal at 1 mg/mL, or Gel V1-Cal/NapFFY at 1.0 wt % was respectively injected to 5 points along the infarct areas (20 µL for each point) of one rat. Then we conducted following experiments according to a defined time schedule (Fig 4a).

Fig. 4.

Levels of inflammatory factors, apoptosis, and TRPV1 protein in rats of the six groups (Sham, MI, Gel NapFFY, Gel V1-Scr/NapFFY, V1-Cal, and Gel V1-Cal/NapFFY). (a) Treatment timeline for in vivo experiments, (b) Representative images of TUNEL positive apoptotic cells in the border region of the infarct area in six groups of rats at day 3 post MI. Scale bar: 50 µm. (c) Quantitative analysis of percentage of apoptotic cells in b; n = 3 for each group, (d) Quantitative analysis results of expression levels of inflammatory factors (TNF-alpha, IL-6, and IL-17A) in six groups of rats post MI; n = 6 for each group, (e) Protein expression of TRPV1 and p-TRPV1 in the border region of the infarct area in six groups of rats at day 3 post MI. (f) Quantitative analysis of TRPV1 and p-TRPV1 protein expression in e; n = 3 for each group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Apoptosis occurs in the early post-infarction period, from several hours to one week, which directly affects long-term myocardial remodeling and cardiac function. Moreover, antiapoptotic signaling could promote cardiomyocyte survival [40,41]. Therefore, we detected the apoptotic cells in the border region of the infarct area using terminal-deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) staining on the third day post MI. As shown in Fig. 4b, both V1-Cal and V1-Cal/NapFFY significantly alleviated cell apoptosis in the border area, with the latter more effective than the former. Quantitatively, the percentage of apoptotic cells was 3.8 ± 0.5% in MI group, but reduced to 2.1 ± 0.3% in V1-Cal group, or 0.8 ± 0.2% in V1-Cal/NapFFY group (Fig. 4c). Collectively, V1-Cal/NapFFY exhibited a stronger protective effect against apoptosis than V1-Cal. However, there was no significant difference in the percentage of cell apoptosis in NapFFY and V1-Scr/NapFFY groups compared to that in MI group. These results suggested that the V1-Cal/NapFFY hydrogel is able to effectively protect ischemic myocardium against apoptosis, which might be beneficial in ventricular remodeling reduction and cardiac function improvement of the rat post MI.

In addition to apoptosis, an important healing process after MI is inflammation response. Extended inflammation after MI leads to mass cardiomyocytes death, enhanced matrix degradation, and increased fibrosis. Therefore, the inflammatory cascade after MI is an attractive therapeutic target [42,43]. TNF-alpha and IL-6 have been considered as important inflammatory factors at early stage of myocardial ischemia injury, and are highly expressed in the first 3–4 days post MI [43]. IL-17A, on the other hand, is involved in post-infarction fibrosis, and its expression level was found not changed at early stage, but elevated in 1–2 weeks after infarction, with a peak on day 7 [44,45]. Therefore, in this study, we measured TNF-alpha, IL-6, and IL-17A levels at day 1, 3, and 7 after MI using enzyme-linked immunosorbent assay (ELISA). As shown in Fig 4d, compared with those in Sham group, expression levels of TNF-alpha and IL-6 in rats of all other five groups elevated markedly on day 1 and day 3 after MI, and the levels of IL-17A in rats of other five groups raised to a high level on day 7 post-MI. Compared to those in MI, NapFFY, and V1-Scr/NapFFY groups, expression levels of all these three inflammatory factors in rats of V1-Cal and V1-Cal/NapFFY groups were obviously lower, indicating that the elevations of these inflammatory factors in MI were effectively inhibited by both V1-Cal or Gel V1-Cal/NapFFY treatment. And Gel V1-Cal/NapFFY treatment showed the best inhibitory effect on these inflammatory factors. Interestingly, compared to those in MI group, there was no significant difference in the expression levels of TNF-alpha, IL-6, or IL-17A at all time points in either NapFFY or V1-Scr/NapFFY groups. All these above results together suggested that: 1) neither V1-Scr nor Gel NapFFY has the suppression effect on the elevations of these inflammatory factors in MI; 2) while V1-Cal can ameliorate inflammation post MI, its anti-inflammatory effect was additionally enhanced when V1-Cal was co-assembled in the hydrogel and released in a sustainable manner.

Since V1-Cal possesses a peptide sequence mimicking the conserved TRP box in TRPV1, it could serve as a decoy to block the binding of TRPV1 with other molecules such as 12(S)-HETE and calcineurin, which in turn inhibit TRPV1 activation [36]. To evaluate whether the co-assembled peptides (i.e., V1-Scr and V1-Cal) or hydrogels affect TRPV1 activation, we detected the total and phosphorylated (p)-TRPV1 protein expressions in the border region of the infarct area in rats at day 3 post MI. Compared with those in Sham group, total TRPV1 level and p-TRPV1 level in the border region of rats in MI group increased by 3.6-fold and 3.1-fold, respectively. However, compared with those in MI group, total TRPV1 level and p-TRPV1 level were obviously lower in V1-Cal and V1-Cal/NapFFY groups, indicating that the elevated expressions of total TRPV1 and p-TRPV1 were suppressed by both V1-Cal and Gel V1-Cal/NapFFY. Again, Gel V1-Cal/NapFFY showed the strongest inhibition on both total TRPV1 and p-TRPV1 expressions among the four treated groups (Fig. 4e-f). Compared with those in MI group, there was no significant difference in the expressions of total TRPV1 and p-TRPV1 in either Gel NapFFY or Gel V1-Scr/NapFFY group. And all these above results together suggested that: 1) neither V1-Scr nor Gel NapFFY has the suppression effect on the elevated expression of either total TRPV1 or p-TRPV1 in MI; 2) while V1-Cal can inhibit the elevated expressions of both total TRPV1 and p-TRPV1 post MI, its inhibitory effect was additionally enhanced when V1-Cal was co-assembled in the hydrogel of Gel V1-Cal/NapFFY.

2.7. Restoration of cardiac function

Above results indicate that Gel V1-Cal/NapFFY has better inhibitory effect than V1-Cal on apoptosis, inflammation, and TRPV1 activation of MI rats. As we know, occurrences of excessive cell apoptosis and inflammation response during MI could aggravate post-MI ventricular remodeling and cardiac dysfunction [41,46]. Therefore, we investigated whether Gel V1-Cal/NapFFY could improve cardiac function of the MI rats. As shown in Fig. 5a, compared with the rats in Sham group, MI rats showed significant ventricular dysfunction at the end of 4 weeks post MI, as indicated by the wall motion abnormalities from their echocardiography. But the ventricular dysfunction of the MI rats was largely recovered after Gel V1-Cal/NapFFY treatment (Fig. 5a). Echocardiography parameter measurements indicated that, compared with rats in Sham group, left ventricular ejection fraction (EF) and fractional shortening (FS) of MI rats significantly decreased, while their left ventricular internal diameter at end systole (LVIDs) and left ventricular internal diameter at end diastole (LVIDd) increased (Fig. 5b-e). Quantitative analysis showed that, four weeks after Gel V1-Cal/NapFFY treatment, EF value of the MI rats was improved from 48.9 ± 4.1% to 73.6 ± 3.3% (Fig. 5b). And other echocardiography parameters of the MI rats (i.e., FS, LVIDs, and LVIDd) were adjusted to normal to the largest extent after Gel V1-Cal/NapFFY treatment (Fig. 5c-e). In comparison, during rat ventricular dysfunction, while V1-Cal showed less improvement of the echocardiography parameters of the MI rats than Gel V1-Cal/NapFFY, neither Gel NapFFY nor Gel V1-Scr/NapFFY treatment had recovery effect on these parameters (Fig. 5b-e). Immunohistostaining against CD31⁺, a typical mature endothelial marker, were conducted. [47] Compared with those in Sham group, the percentage of CD31⁺ area of rats in MI group increased. Nevertheless, compared with that in MI group, the percentage of CD31⁺ area was obviously higher in V1-Cal or Gel V1-Cal/NapFFY group. Again, among the five treated groups, Gel V1-Cal/NapFFY group possessed the highest percentage of CD31⁺ area (Fig. 5f-g). All these results showed that V1-Cal could promote angiogenesis, which may be one of the mechanisms of V1-Cal improving cardiac function after MI. Additionally, Gel V1-Cal/NapFFY had the best pro-angiogenic effect among these studied materials. Collectively, Gel V1-Cal/NapFFY showed the best effect on cardiac function improvement of the rats post MI.

Fig. 5.

Echocardiography measurements of cardiac functions of the rats at day 28 post MI surgery; n = 8 for each group, (a) Representative echocardiography images of rats in the six groups, (b-e) Quantitative analyses of the EF value (b), FS value (c), LVIDs (d), and LVIDd (e) of the six groups of rats, (f) Immunohistostaining images of CD31⁺ area in sham or MI heart sections of rats after PBS, V1-Cal, Gel NapFFY, Gel V1-Cal/NapFFY, Gel Scr/NapFFY administered, (g) Quantification of percentage of CD31⁺ area in sham or MI heart sections of rats after PBS, V1-Cal, Gel NapFFY, Gel V1-Cal/NapFFY, Gel Scr/NapFFY administered. Results are presented as mean ± SD. n = 3 for each group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

2.8. Effects on ventricular remodeling

Cardiac remodeling is a key contributor to cardiac dysfunction post MI, which is often displayed as the combination of cardiac fibrosis, thinner infarct wall, and scar formation [48,49]. Therefore, we performed Masson’s trichrome staining to evaluate cardiac remodeling of the rats at day 28 post MI. The optical images of Masson’s staining of all 6 rats in each group were shown in Fig. S12. The Masson’s staining revealed marked fibrosis, infarct expansion, as well as wall thinning of the rats in MI group (Fig 6a). In general, massive fibrosis and ventricular remodeling were inhibited by Gel V1-Cal/NapFFY and V1-Cal treatments, but not by Gel NapFFY or Gel V1-Scr/NapFFY treatment (Fig. 6a). Specifically, Gel V1-Cal/NapFFY treatment reduced the infarct size from 41.0 ± 5.0% to 22.0 ± 2.4%, fibrosis area from 20.8 ± 3.3% to 5.9 ± 1.6%, while increased the wall thickness from 708.0 ± 121.2 to 1297.7 ± 185.4 µm of the MI rats (Fig. 6b-d). Again, V1-Cal also showed remodeling reduction effect on the MI rats but was less potent than Gel V1-Cal/NapFFY. Consistently, neither Gel NapFFY nor Gel V1-Scr/NapFFY treatment showed remodeling reduction effect on the MI rats. The development of cardiac fibrosis and remodeling would lead to severe cardiac dysfunction and eventually heart failure [1,41]. Thus, the best reverse effect of Gel V1-Cal/NapFFY on post-MI fibrosis and remodeling is expected to effectively prevent post-infarcted heart failure of the MI rats.

Fig. 6.

Assessment of cardiac remodeling of the rats using Masson’s staining at day 28 post MI surgery, (a) Representative Masson’s trichrome staining images of heart sections of the six groups of rats. Scale bars: 200 µm for macro photos and 20 µm for micro photos. Quantification analyses of infarct area (b), the left ventricular (LV) wall thickness of the MI zone (c), and fibrosis area in the border zone (d) in a MI rat model. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

2.9. Evaluation of the sustainable release of V1-Cal from Gel V1-Cal/NapFFY in vivo

Finally, we evaluated the in vivo release of V1-Cal from Gel V1-Cal/NapFFY using isotope labelling method. Firstly, in the presence of chloramine-T and Na¹²⁵I, the tyrosine (Y) in V1-Cal was labeled with ¹²⁵I (Scheme S2). Then we used ¹²⁵I-V1-Cal and NapFFY to prepare hydrogel according to above method. ¹²⁵I labeled hydrogel was injected to 5 points along the infarct areas of one rat, and ¹²⁵I levels in heart, blood, liver, spleen, lung, and kidney were monitored at day 1 and 3 days post operation, so as to calculate the release and metabolism of V1-Cal in vivo. In vivo release monitoring results showed that the residual amount of ¹²⁵I-V1-Cal in the hearts of rats was 50.7 ± 4.7% and 16.8 ± 5.4% at day 1 and day 3, respectively, maintained the highest concentration among all tissue and organs studied (Table S3). The quantitative statistical results of the distribution of ¹²⁵I-V1-Cal in blood and other tissues (liver, spleen, lung, kidney) at day 1 and day 3 day post injection are shown in Fig. S13. The results showed that, except heart, lung had the second high concentration of V1-Cal, followed by blood, kidney, spleen, and liver. Above experimental results proved that V1-Cal was slowly released from Gel V1-Cal/NapFFY at a sustainable manner in vivo. To further evaluate the systemic toxicity of our hydrogels, after treatment, we took the major organs (liver, spleen, lung, and kidney) from the rats for H&E staining. As shown in Fig. S14, compared with the organs in control group, no pathological change was observed in the organs of these five experimental groups. All these results indicated that the hydrogels have high biological safety and are suitable for in vivo applications.

3. Conclusions

In conclusion, in order to more effectively reduce ventricular remodeling and improve cardiac function in a rat MI model, we co-assembled the therapeutic peptide V1-Cal with a well-studied hydrogelator NapFFY to prepare the supramolecular hydrogel Gel V1-Cal/NapFFY for sustained release of the peptide. Rheology and TEM tests of Gel V1-Cal/NapFFY showed that the co-assembly of V1-Cal with NapFFY increased mechanical strength of Gel NapFFY, enabling Gel V1-Cal/NapFFY more suitable for myocardial injection to treat MI. Cumulative release profile of V1-Cal from Gel V1-Cal/NapFFY in vitro indicated that V1-Cal was released from Gel V1-Cal/NapFFY at a sustainable manner for more than two weeks. Cytotoxicity and proliferation results showed that Gel V1-Cal/NapFFY was non-toxic to but stimulated the proliferation of H9C2 cells. In vivo experiments indicated that, among all five experimental groups, Gel V1-Cal/NapFFY had the best effect on the decrease of TRPV1 expression and activation, apoptosis reduction and the release of inflammatory factors in a MI rat model. Moreover, Gel V1-Cal/NapFFY showed the best effect on cardiac function improvement, reverse effect on post-MI fibrosis and remodeling of the rats post MI. We envision that our Gel V1-Cal/NapFFY could be used to reduce ventricular remodeling and improve cardiac function post MI in clinic in the near future.

4. Materials and methods

4.1. Enzyme-linked, immunosorbent assay (ELISA)

On day 1, 3, 7 after MI operation, blood samplings were obtained from tail vein of rats. TNF-alpha, Interleukin (IL)-6, IL-17A levels in each serum sample were detected by using corresponding ELISA kits (ABclonal, China).

4.2. Echocardiography

Transthoracic echocardiography was performed at day 28 after MI with a Visual Sonics (VINNO 6 VET) equipped with a 18-MHz imaging transducer. M–mode tracings were recorded at the papillary muscle level to measure the left ventricular ejection fraction (EF) and fractional shortening (FS), systolic left ventricular internal dimension (LVIDs), diastolic left ventricular internal dimension (LVIDd).

4.3. Masson’s trichrome staining

The rats were sacrificed and their hearts were harvested on day 28 after MI operation (n = 6 in each group). Masson’s trichrome-staining was then performed for histological studies. Below data were obtained using Image J software (NIH): the infarction area, the left ventricular wall thickness, and the fibrosis area in the border zone.