1 Abstract

Chronic PKA phosphorylation of RyR2 has been shown to increased diastolic SR Ca²⁺ leak and lead to cardiac dysfunction. Since the change of phosphorylation level of RyR2 is a biomarker of failing heart, we attempted to verify the hypothesis that intracellular gene delivery of a RyR2 targeting phosphorylation site-specific nanobody could preserve contractility of failing myocardium. In present study, we acquired the RyR2-specific nanobodies from a phage display library which are variable domains of camellidae heavy chain-only antibodies (VHH). One of the monoclonal nanobodies, AR185, inhibiting RyR2 phosphorylation in an in vitro assay was then chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of AR185 against post-ischemic heart failure. Adeno-associated virus gene delivery elevated the intracellular expression AR185 protein in the ischemic heart failure model of rats, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca²⁺ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of sarcoplasmic reticulum (SR) calcium leak, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited PKA phosphorylation of RyR2 in failing cardiomyocytes. Our results provided strong pre-clinical experimental evidence of the cardiac expression of RyR2 nanobody with AAV9 vectors as a promising therapeutic strategy for ischemic heart failure.

2 Introduction

In most countries, heart problems are the leading cause of morbidity and mortality⁽¹⁾. Heart failure is the end stage of most heart diseases, with approximately 38 million heart failure patients worldwide⁽²⁾. Despite the use of drugs, implanted cardiac assist devices and surgical treatments, many patients' conditions will still irreversibly deteriorate, eventually being difficult to control and rescue.⁽³⁾ Therefore, studying the molecular mechanism of cardiovascular pathology， developing new therapeutic strategies and preparations have the great significance for the prevention and treatment of heart failure. No matter what the cause, heart failure patients in the late stage of the disease have a common feature that calcium circulation is abnormal in the cardiomyocytes. A hallmark of failing cardiomyocytes is the change of excitatory contraction coupling, including the reduced amplitude of Ca²⁺ transients, delayed onset and decay kinetics of Ca²⁺ transients. These changes eventually lead to decreased contractility, delayed contraction, and reduced diastole. In addition, a rare spontaneous Ca²⁺ release events (calcium leaks) in the resting period of healthy cardiomyocytes occurs more frequently. All the change of Ca²⁺ handling is attributed to impaired function of RyR2, SERCA2a, Na⁺-Ca²⁺ exchanger (NCX).

Changes in expression and function of these ion channel proteins and their negative effect after MI have been reported in many studies^{(4, 5)}, which directly attenuate the cardiac contractility. Therefore, preventing these detrimental effects of myocardial infarction, as well as the effects of ischemic injury due to the changes in expression or function of calcium-channel proteins, is an optimal treatment strategy for cardio-protection.

The activity of RyR2 function is regulated by various mechanisms, and many factors will affect it. The post-translational regulation abnormality of RyR2 is the main cause of the dysfunction of RyR2 protein. Phosphorylation is a crucial post-translational modification of RyR2 protein.^{(6, 7)} The "calcium leakage" caused by RyR2 hyperphosphorylation is considered to be an important pathological mechanism for myocardial injury and heart failure development.^{(8, 9)} The study found that the expression of catecholamines in the blood of chronic heart failure patients was up-regulated, and intracellular protein kinase A (PKA) levels and activities continued to increase. After PKA is continuously activated, hyperphosphorylation of RyR2 protein causes an increase of the dissociation rate of FKBP12.6 and RyR2, resulting in a change of RyR2 structure, which increases the sensitivity of RyR2 to calcium ions, resulting in a small amount of calcium ions can stimulate the release of RyR2 channels, causing the calcium leakage in the resting stage of cardiomyocytes. The earliest article demonstrating changes in ryr2 phosphorylation in patients with heart failure was published by Marx et al. in 2000⁽¹⁰⁾. Marx et al.⁽¹⁰⁾, Wehrens et al⁽¹¹⁾. and Shan et al.⁽¹²⁾ proposed a hypothesis: sympathetic excitation in heart failure, activation of PKA-mediated RyR2 S2808 Hyperphosphorylation, while inhibiting the binding of FKBP12.6 to RyR2, increases the probability of RyR2 opening.

But this assumption is currently controversial. The biggest controversy is whether RyR2 S2808 hyperphosphorylation plays a decisive role in heart failure. Wehrens et al.⁽¹¹⁾ believe that RyR2 S2808 phosphorylation plays a critical role in the experimental myocardial ischemia-induced heart impaired process. They established a heart failure model which induced by myocardial infarction in RyR2 S2808 knockout mice and wild-type mice. Heart function tests were performed 4 weeks after ischemia. The results showed that RyR2 S2808 knockout mice showed significant improvement in cardiac function such as ejection fraction, shortening fraction, maximal rate of left ventricular pressure. At the same time, they also found that RyR2 of RyR2 S2808 knockout mice could not be phosphorylated by PKA, and PKA lost regulation of FKBP12.6, resulting in FKBP12.6 not being able to dissociate from RyR2. However, Zhang et al.⁽¹³⁾ and Houser et al.⁽¹⁴⁾ believe that PKA-mediated hyperphosphorylation of RyR2 S2808 does not alter myocardial contractility and does not improve symptoms of heart failure and arrhythmia. They used a similar experimental protocol and found no improvement in the heart function of the mouse knocked out by RyR2 S2808. However, the biggest problem with Hauser et al. is that the number of samples is too small. Inhibition of RyR2 phosphorylation may be one of the effective treatments of ischemic heart failure.

Here, we hypothesis that targeting RyR2 using anti-phosphorylation agents may improve treatment efficacy. We identified a camel single-domain antibody to RyR2 that have the ability to inhibit PKA dependent S2808 phosphorylation in vitro. To evaluate its potential effect in the treatment of heart failure, an adeno-associated virus (AAV) based intracellular antibody delivery strategy were adopt to achieve cardiac-specific gene-therapy and demonstrated therapeutic effect both in cell-based assays and in vivo models.

3 Results

Generation of Anti-RyR2 nanobodies that specifically inhibits the Phosphorylation of RyR2 S2808

We first obtained and purified RyR2 from rat heart by using GST-fused FKBP12 as the published strategies described. To construct the camel VHH library, blood samples of 30 non-immunized, four-year-old male Bactrian camel were collected. B lymphocyte cDNA encoding VHHs was used to construct a phage display VHH library that consisted of approximately 3×10⁸ individual colonies. VHH gene corresponded to the size of insert of over 98% colonies. For confirming the heterogeneity of the individual clones from the library, we sequenced fifty randomly selected clones, and each clone showed a distinct VHH sequence.

In order to select nanobodies with specific ability to bind RyR2, bio-panning was performed with immobilized RyR2 protein. After the third round of panning, the result showed an obvious enrichment of phage particles that carried RyR2-specific VHH (Fig. 1A). Phage clones exhibited increased binding to RyR2 after the second round of panning. During four rounds of panning there was no phage clone that was found binding to BSA (Fig. 1B). VHH fragments of 300 individual colonies that were randomly chosen were expressed in an ELISA for screening colonies which bound to RyR2. Among these clones, 276 antibody fragments specifically bound to RyR2. One antibody fragment which did not bind to RyR2 was choose as a negative control, termed as VHH-AR117. To obtain antibodies that functionally inhibit of RyR2 phosphorylation, each of the antibody fragments was tested for its effect in an ELSA based RyR2 phosphorylation assay. 4 antibody fragments were potent inhibitors of RyR2 phosphorylation. The complementary determining regions (CDRs) were confirmed by sequence analysis and the result revealed that there was only one unique clone in this panel of antibody fragments, termed as VHH-AR185. To investigating the basis of dephosphorylation of RyR2 by VHH-AR185, the binding affinity of VHH-AR185 to RyR2 was measured by surface plasmon resonance. VHHs were purified for these experiments by expressing and secreting from the E. coli cytosol. As shown in Fig. 1D, the affinity (KD) of VHH-AR185 to RyR2 was estimated to be 1.93 nM. The result of affinity studies likely explained the inhibition of RyR2 phosphorylation due to the extremely slow dissociation rate of VHH-AR185 from RyR2.

Fig.1 balablablabla

The interaction of VHH-AR185 to RyR2 in the cytoplasm of eukaryotic cells was examined by co-immunoprecipitation experiments. VHH-AR185 and RyR2 were expressed in neonatal cardiomyocytes cells and the lysates of transfected cells were detected. As the result in fig. S1, anti-his antibody was able to efficiently co-precipitate RyR2 from the cells that expressed VHH-AR185-HIS, but could not co-precipitate RyR2 from cells expressing VHH-AR117-HIS. Conversely, anti-RyR2 antibody was able to co-precipitate VHH-AR185-HIS with RyR2, but not VHH-AR185-HIS. This result indicated that the VHH-AR185 could maintain its antigen binding ability in the cytoplasm and fold as a soluble protein.

Fig.s1 balablablabla

To identify the epitopes recognized by AR185, phage clones were isolated by panning the PhD.-7 phage display peptide library with AR185. Three rounds of selection were performed, and, at each round, the library was pre-cleared with a control AR177 nanobody. After the third round of panning, the binding of the isolated phage clones to AR185 was determined by ELISA. Sequence analysis of AR185-positive phage clones identified five and six distinct amino acid sequences, respectively (fig. S2A). Alignment of these sequences revealed the consensus motifs DKLAC, which could be aligned with the (2725) DKLAN (2729) sequence located at P2 Domain of RyR2 (fig. S2B).

Fig.s1 balablablabla

Intrabody AR185 rescues cardiac function and reverses remodeling in failing rat myocardium in vivo

We constructed an AAV9 vector containing a VHH-AR185 expression between the two AAV2 inverted terminal repeats and the vector was pseudotyped with a capsid of AAV serotype 9, termed as AAV9.AR185. VHH-AR117 were also constructed as a negative control, termed as AAV9.AR117. To access cardiac expression of VHH, we used the method of adding “self-cleaving” T2A peptide to co-expressed a GFP reporter downstream of VHHs (Fig. 2A). We used the HEK-293 cells expression of different AAV9 particles in vitro and Transmission electron microscope was used to access the AAV9 particles (Fig. 2B). Next, we evaluated the efficiency of gene expression delivered by AAV in vivo. A dosage of AAV9.AR185 particles was delivered to each rat at 1×10¹² genome containing particles (gcp), whereas AAV9.AR117 vector was given to the control treated group (n = 5) at the same dosage. After four weeks, we removed all organs from the sacrificed rat, weighed, and assayed treated tissue for fluorescence intensity. Efficiency of gene expression and ability of targeting were evaluated by the ratio of fluorescence intensity to mass of tissue under fluorescence microscope (Fig. 2C and D).

Fig.2 balablablabla

To explore the therapeutic potential of VHH, we chose the mode of ischemic heart failure induced by coronary artery ligation for this study. Following the ligation operation, rats were divided into different groups as described in Methods and received control virus (AAV9.AR117), AAV9.AR185 treatment or saline (HF) (n=7-8). The sham-operated animals (Sham) were used as healthy controls. Nine weeks after ligation operation and injection of AAV particles, LV dimensions in the short-axis view was measured by cardiac echo and we also calculated and analyzed the value of ejection fraction and fractional shortening. Our data shows that rats of HF group and AAV9.AR117 group exhibited progressive cardiac dysfunction and LV enlargement, while AAV9.AR185-treated animals showed significant improvement. Moreover, Ejection Fraction and fractional shortening was markedly improved in AAV9.AR185 group compared with HF group and AAV9.AR117 group (Fig. 3A). To determine whether AAV9.AR185 treatment prevented adverse remodeling of the heart after MI, Masson trichrome staining of cardiac sections was performed to measure cardiac fibrosis (Fig. 3B). Whereas there was a significant increase in the development of cardiac fibrosis in Rats of HF group and AAV9.AR117 group after HF, whereas the amount of fibrosis was significant reduced in AAV9.AR185-treated animals. Additionally, HF rat and AAV9.AR117 treated rat had development of a significant increase of heart weight to body weight ratios (HW/BW) after MI compared with sham-operated rat, which is indicative of cardiac remodeling in the context of congestive HF（Fig. 3C）. In contrast, there was no significant increase in HW/BW ratio after MI in AAV9.AR185-treated rat compared with sham-operated rat. Sarcomeres and mitochondria were the most important index for analysis of ultra-structures of cardiomyocytes from left ventricle that were observed by transmission electron microscopy (Fig. 3D). In the AAV9.AR185 treated and Sham groups, myofilaments were neatly arranged, sarcomeres were intact and Z lines were clear. Conversely, in the HF and AAV9.AR117 groups, MI leaded to disordered arrangement of sarcomeres, dissolution of myofilaments, and frequent vacuoles. In both HF and AAV9.AR117 groups, a lot of mitochondria were swollen and even ruptured, and the separated mitochondrial cristae frequently appeared. The mitochondria in Sham group were well shaped, and the cristae of the mitochondria were obvious and tightly packed. The observations of mitochondria were improved in the AAV9.AR185 treated group compared with AAV9.AR117 treated group. Comprehensively considering the alteration of cardiac function and changes in structure of different groups, the TEM images further support that VHH-AR185 had therapeutic effect in treating heart failure.

Fig.3 balablablabla

We next accessed the contractile kinetics of isolated LV cardiomyocytes（Table1）. When cardiomyocytes were field-stimulated at a frequency of 1 Hz, HF and AAV9.AR117 treated myocytes had significantly slower velocities of shortening and relengthening in than AAV9.AR185 treated myocytes. Fractional shortening of myocytes that were isolated from HF and AAV9.AR117 treated animals also decreased, and time to 50% peak shortening (TPS50%) and time to 50% relengthening (TR50%) became longer. AAV9.AR185 treatment protected cardiomyocytes contractility reserve from the impairment induced by MI. However, only the index of TR50% in myocytes from AAV9.AR185 treated animals returned to a level similar to those of sham operated animals.

AR185 gene therapy restores cardiomyocyte and SR calcium handling in failing myocardium

We used laser scanning confocal microscopy recorded the fluorescence intensity to measure the sarcoplasmic reticulum Ca²⁺ content of cardiomyocytes from different groups by incubation in the fluorescent dye Fluo-5N/AM. As shown in Fig. 4A, basal sarcoplasmic reticulum Ca²⁺ contents in HF and AAV9.AR117 treated animals were lower than in AAV9.AR185 treated and sham-operated animals.

Additionally, we measured amplitude of calcium transient by incubation in Fluo-4/AM and caffeine perfusion. The representative colorful images in Fig. 4B and Table 2 show line-scan results of evoked Ca²⁺ transients from Shams, HFs, AAV9.AR117s and AAV9.AR185s. When challenged with 20 mM caffeine, less Ca²⁺ was released from the SR of myocytes from AAV9.AR117 group compared with myocytes from AAV9.AR185 treated rats. The results also showed there was significantly reduction of the amplitude of Ca²⁺ transients in the HFs and AR117s compared to that in AR185s. Therefore, the decrease in Ca²⁺ transient amplitude may be the causative factor of the impairment in SR Ca²⁺ load. Rate of Ca²⁺ rise also was significantly slower in HF and AAV9.AR117 myocytes than in AAV9.AR185 treated myocytes (Fig. 4C). AAV9.AR185 treatment increased peak of amplitude of evoked Ca²⁺ release and rate of Ca²⁺ rise during Ca²⁺ release.

Fig.4 balablablabla

Table 3 shows representative line-scan images of Ca²⁺ release during the resting stage of cardiomyocytes from Sham (A), HF (B), AAV9.AR117 (C), and AAV9.AR185 animals (D). The data showed that the frequency of Ca²⁺ release was significantly higher and Ca²⁺ sparks occurred frequently in the HF and AAV9.AR117 group compared with the AAV9.AR185 group. The duration of Ca²⁺ sparks in HF and AR117 myocytes were similar to those in Sham and AR185 myocytes, but the Ca²⁺ rise rate of sparks was slower, fluorescence intensity of Ca²⁺ sparks was decreased and T50 decay was longer.

VHH-AR185 inhibits phosphorylation of RyR2 S2808 in failing hearts

To examine whether AAV9.AR185 treatment results in dephosphorylation of RyR2 and in vivo, cardiomyocyte lysates were further subjected to ELISA analysis, our data shows treatment with AAV9.AR185 significantly reduced the level of pRyR2 (S2808) in the cardiomyocytes compared with HF group and AAV9.AR117 treatment (p=0.0003, Dunnett’s test). Moreover, immunohistochemical analysis of the heart tissues in different treatment group also revealed that an increased accumulation of RyR2 phosphorylation was also observed in the AAV9.AR117 treated group, AAV9.AR185 treatment decreased the level of pRyR2 stain of cells in the myocardium, which indicated that VHH185 has blockage effect of RyR2 phosphorylation. Together, these data demonstrate that AAV9.AR185 treatment leads to inhibition of the RyR2 phosphorylation in vivo. (Fig. 5).

Fig.4 balablablabla

4 Discussion

In this experiment, we blocked the PKA regulated phosphorylation site(S2808) by exogenously expressing a intracellular antibody specifically binding to RyR2 in cardiomyocytes. After AAV.AR185 treatment, the Ca²⁺ handing properties of RyR2 in rat hearts isolated form treatment group were very similar to that from Sham group. When the β-adrenergic signaling pathway was activated in vivo, the PKA-regulated site of RyR2 could not bind to phosphate residues, and the phosphorylation of RyR2-S2808 maintained at a low level. At the organic level, AR185 antibody can improve the contractile function of the failing heart, significantly increase the cardiac ejection fraction of heart failure rats, and protect the heart structure from the effects of poor remodeling (Fig 3). At the cellular level, AR185 antibody can reduce the calcium leakage of cardiomyocytes in heart failure rats, maintain the calcium capacity in the sarcoplasmic reticulum of cardiomyocytes, restore calcium homeostasis, and protect the contractility of cardiomyocytes (Fig 4 and Table 1-3). These data is consistent with the previous transgenic animal experiment that down-regulated phosphorylation level of RyR2 S2808 is associated with improved contractility of cardiomyocytes, indicating that S2808 is one of the most important targets in the pathological state of ischemic heart failure.⁽¹¹⁾

To our knowledge, the evidence for efficiency and safety of using antibodies to treat heart disease was limited. Nanobodies are single domain antibodies consisting of the heavy chain variable domain (VHH) in the camelid family which lacks the light chain. Currently, a variety of nanobodies have entered the clinical research stage.^(15-18) Compared with traditional antibodies, nanobodies have the advantages of low molecular weight, high affinity, high stability, low immunogenicity and strong penetrability.⁽¹⁹⁾ Based on the characteristics of nanobodies and VHH, the use of adeno-associated virus vectors to mediate nanobody treatment of heart failure has great potential. In this study, we successfully expressed AR185 nanobodies which specifically bind to RyR2 in rat cardiomyocytes. Our experimental data demonstrate that intracellular antibody treatment is effective in heart disease rats and does not present a significant safety risk.

There are several limitations in our study. We provide evidence that targeting RyR2 with AR185 can be achieved, but rats with heart failure may not fully recapitulate RyR2 in human disease, the experiment data are acquired from a small number of animals, and limited by the shorter observation time, no rats died of heart failure before sacrifice. Furthermore, the mechanisms responsible for therapeutic improvement of AR185 against RyR2 have not been well characterized yet. Hence, these findings will need further validation.

The therapy strategy of AAV9-mediated intracellular antibody which takes advantage of the high specificity and affinity of biomacromolecules can achieve the purpose of specificity regulation of RyR2 and treatment of heart failure. RyR2 is a very promising therapeutic target protein of treatment for heart failure, and intracellular antibody technology in gene therapy is considered as a promising approach. The targets and techniques are worthy of further validation and exploration by researchers.

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