Targeting RyR2 with a phosphorylation site–specific nanobody reverses dysfunction of failing cardiomyocytes in rats

Abstract

Chronic PKA phosphorylation of ryanodine receptor 2 (RyR2) has been shown to increase diastolic sarcoplasmic reticulum (SR) Ca2+ leakage and lead to cardiac dysfunction. We hypothesize that intracellular gene delivery of an RyR2‐targeting phosphorylation site—specific nanobody could preserve the contractility of the failing myocardium. In the present study, we acquired RyR2‐specific nanobodies from a phage display library that were variable domains of Camelidae heavy chain—only antibodies. One of the nanobodies, AR185, inhibited RyR2 phosphorylation in vitro and was chosen for further investigation. We investigated the potential of adeno‐associated virus (AAV)9—mediated cardiac expression of AR185 to combat postischemic heart failure (HF). AAV gene delivery elevated the intracellular expression of the AR185 protein in a rat model of ischemic HF, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of SR calcium leaks, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited the PKA‐mediated phosphorylation of RyR2 in failing cardiomyocytes. Our results provide preclinical experimental evidence that the cardiac expression of RyR2 nanobodies with AAV9 vectors is a promising therapeutic strategy for HF.—Li, T., Shen, Y., Lin, F., Fu, W., Liu, S., Wang, C, Liang, J., Fan, X., Ye, X., Tang, Y., Ding, M., Yang, Y., Lei, C., Hu, S. Targeting RyR2 with a phosphorylation site–specific nanobody reverses dysfunction of failing cardiomyocytes in rats. FASEB J. 33, 7467–7478 (2019). http://www.fasebj.org

Chai-Hu-San-Shen Capsule Ameliorates Ventricular Arrhythmia Through Inhibition of the CaMKII/FKBP12.6/RyR2/Ca2+ Signaling Pathway in Rats with Myocardial Ischemia

“…e SR is the main location of intracellular calcium storage in muscle cells [34], and Ca 2+ release from the SR is mediated by RyR2, a specific Ca 2+ release channel [35][36][37]. Increased Ca 2+ leakage from the SR through the RyR2 channel is considered an important mechanism of arrhythmia [38,39], and RyR2 phosphorylation plays a key role in regulation of SR Ca 2+ release [40][41][42][43][44]. Ryanodine receptor R2 can be phosphorylated by PKA and CaMKII [45][46][47].…”

Diagnostic and Therapeutic Aptamers

“…For instance, regardless of the cause, HF patients in the late stage of the pathology have a common feature: defective Ca 2+ signaling in cardiomyocytes, which results in impaired cardiac muscle contraction and relaxation as well as in development of cardiac arrhythmias and adverse remodelling. 100 Indeed, a hallmark of failing cardiomyocytes is a change in excitatory contraction coupling, including reduced amplitude of Ca 2+ transients and delayed onset and decay kinetics of Ca 2+ transients. All these changes in Ca 2+ handling can be attributed, among others, to the impaired function of L-type calcium channel (LTCC), RyR2, SERCA, phospholamban (PLN), and the Na + -Ca 2+ exchanger.…”

“…All these changes in Ca 2+ handling can be attributed, among others, to the impaired function of L-type calcium channel (LTCC), RyR2, SERCA, phospholamban (PLN), and the Na + -Ca 2+ exchanger. 100 In this context, nanobodies (nAbs), small recombinant antigen-binding fragments derived from the atypical monomeric immunoglobulins present in camelid mammals and cartilaginous fish, have been proposed as diagnostic/therapeutic tools to visualize and/or modulate endogenous targets within living cells. 101 , 102 , 103 Compared with traditional antibodies, nAbs, also called intrabodies, have the advantages of low MW (∼15 kDa vs the conventional antibodies immunoglobulin Gs, ∼150 kDa), high affinity, high stability, low immunogenicity, and strong penetrability.…”

“… 101 , 102 , 103 Although promising, only 1 nanobody-based drug has been approved so far (caplacizumab [Sanofi] for targeting the vWF), whereas to the best of our knowledge, only 2 works describing the preclinical use of nAbs for defective calcium signaling components (RyR2 and PLN specific nanobodies) have been shown. 100 , 104 In fact, the main problem with regard to the use of this technology is the requirement of a safe, efficient, and cell-specific gene delivery method for their effective clinical translation. As a matter of fact, nAbs, like any other antibody, rely on conserved disulfide bonds, which in the reducing milieu of the cytosol strongly impair the formation of disulfide bonds.…”

A Small Virus to Deliver Small Antibodies: New Targeted Therapies Based on AAV Delivery of Nanobodies

“…In the case of RyR2, hyperphosphorylation has been postulated as an important pathologic mechanism for myocardial injury and heart failure development, representing an interesting pharmacological target. Following this rationale, Li et al developed a camel nanobody that could inhibit RyR2 phosphorylation [ 73 ]. In order to deliver this nanobody in vivo, they used an AAV9 vector and tested its functionality in a rat model of ischemic heart failure induced by coronary artery ligation.…”