Ca2+RTIN is the abbreviation of cardiomyocyte RyR2 targeting Intra-nanobody. It is also the name of our project. Our project focus on heart failure and want to make contribution to treatment. Meanwhile, RyR2 is one of the most important calcium channels in cardiomyocytes and we expect to achieve the treatment effect by improving Ca2+ handling. So, we add ‘2+’ after ‘Ca’.

Chronic PKA phosphorylation of RyR2 has been shown to lead to cardiac dysfunction. We designed a targeting device, CaRTIN (Cardiomyocyte RyR2 Targeting Intra-Nanobody), to implement RyR2-specific inhibition of phosphorylation. Here, one of the isolated RyR2 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 to combat post-ischemic heart failure. Adeno-associated viral gene delivery elevated AR185 protein expression in rat heart, and this administration normalized the contractile dysfunction of the failing myocardium in vivo and in vitro. Moreover, CaRTIN therapy to failing cardiomyocytes reduced sarcoplasmic reticulum (SR) Ca²⁺ leak, restoring the diminished intracellular Ca²⁺ transients and Ca²⁺ load and reversed the phosphorylation of RyR2. To achieve controlled intra-nanobody release, a BNP promoter based platform was also accessed. Our results established a role of CaRTIN as a promising therapeutic approach for heart failure.

1 Heart failure

What is heart failure?

The primary function of the heart is to pump blood and promote blood circulation to meet the metabolic needs of different tissues throughout the body.

Heart failure is when the heart is unable to pump sufficiently to maintain blood flow to meet the body's needs. Heart failure is the severe and terminal stage of various heart diseases. Patients with hypertension, coronary heart disease and diabetes are at risk of heart failure.

Figure 1

Clinical status and influence of heart failure

Based on the latest statistics, heart disease has become the leading cause of death worldwide. The global incidence of chronic heart failure, the terminal stage of many heart diseases, is 3 percent among adults and as high as 10 percent over the age of 80. About 60 percent of patients die within five years of being diagnosed, with a five-year survival rate similar to that of malignant tumors. Heart failure also brings substantial economic burden to society. In Europe and North America, the hospitalization of heart failure accounts for 1% to 4% of the hospitalization quantity. The United States spent about $39.3 billion on heart failure in 2010, and total spending on patients with heart failure is expected to increase by 50-100% over the next 10 years. At the same time, as patients with severe heart failure will lose their working capacity, heart failure will also cause a considerable burden to the patient’s family.

Figure 2

Current treatment methods and limitations

At present, the treatments of heart failure mainly focus on drug therapies such as beta blockers, angiotensin-converting enzyme inhibitors, aldosterone inhibitors and so on. These drugs have made tremendous contributions to the control of heart failure and have improved the long-term survival of patients. However, these treatments could only control the disease to a certain extent, the prognosis of heart failure is still dismal. As the disease progresses, patients with heart failure will even enter the terminal stage of the disease.

Facing with such conditions, gene therapy has emerged as a novel and promising approach for treating heart failures in recent years.

Figure 3

Cardiac Muscle Contraction and Ca2+ cycling

Calcium ion plays a significant role in mediating cardiac muscle contraction. When cardiac muscles are excited, changes in cell membrane potential can activate the opening of L-type calcium channels on the cell membrane, and the extracellular Ca2+ ions flow into the cells. The Ca2+ influx then binds to Ryanodine receptor 2 (RyR2) which functions as a calcium channel located on sarcoplasmic reticulum (SR), and cause more Ca2+ to release into cytoplasm from SR through RyR2. The increasing Ca2+ ions then result in the contraction of cardiac muscle via a series of reactions. After each contraction, most of the calcium ions will be restored into SR and the calcium concentration in cytoplasm goes back to a low level.

However, when heart failure happens, the calcium channel RyR2 on SR will be hyperphosphorylated by PKA, thereby leading to calcium leakage from SR and reduction of myocardium contractility.

Figure 4

2 CaRTIN

What is RyR2? What is it made up of?

As we have introduced before, Calcium (Ca²⁺) is the important physiological ligand that activates the channels in cardiac muscle during excitation-contraction (EC) coupling. The heart dysfunction will happen when Ca²⁺ cycle is in a mess, which in the end leads to heart failure. That’s why we have to mention the Ca²⁺ release channels (a kind of ryanodine receptor) on the sarcoplasmic reticulum (SR) of striated muscles. They adjust and control Ca²⁺ between cytoplasm and SR as a biphasic channel such that low cytosolic [Ca²⁺] (mM) activates the channels and high cystolic [Ca²⁺] (mM) inactivates the channels, confirming their crucial role in EC coupling. In cardiac muscle, the Calcium release channels on the SR is named as the type 2 ryanodine receptor (RyR2). It is a tetramer comprised of four 565,000 Dalton RyR2 polypeptides and four 12,000 Dalton FK-506 binding proteins (FKBP12.6).

Figure 5

How does it work?

FKBP12s are regulatory subunits that stabilize RyR channel function and facilitate coupled gating between neighboring RyR channels which are packed into dense arrays in specialized regions of the SR that release intracellular stores of Ca²⁺ that trigger muscle contraction. RyRs are ligand-activated channels and One FKBP12 molecule is bound to each RyR subunit, and dissociation of FKBP12 significantly alters the biophysical properties of the channels, resulting in the appearance of subconductance states and increased P0 (resting potential of myocardium) due to an increased sensitivity to Ca²⁺-dependent activation. In addition, dissociation of FKBP12 from RyR channels inhibits coupled gating, resulting in channels that gate stochastically rather than as an ensemble. Coupled gating of arrays of RyR channels is thought to be important for efficient EC coupling that regulates muscle contraction.

The function of S2808

According to Marx’s study, PKA phosphorylation regulates the binding of FKBP12.6 to the channel both in vitro and in vivo. PKA phosphorylation of the cardiac RyR2 dissociates the regulatory subunit FKBP12.6 from the channel. Such situation will occur when catecholamine-induced increases in RyR2 phosphorylation at serine 2808 (S2808). If catecholamine stimulation is sustained (for example, as occurs in heart failure), RyR2 becomes hyperphosphorylated and “leaky”, leading to arrhythmias and other pathology. Since that we aim to protect the S2808 from phosphorylation which can rectify calcium current in order to cure heart failure. However, it is difficult to find a way to lower the phosphorylation level of S2808 using traditional gene editing methods, which frustrates us more concerning the production’s long-term and repeatable effect. That’s why our instructor suggested us considering nanobody.

Figure 6

Nanobody

As introduced in Wikipedia, nanobody, which is also named as single-domain antibody (sdAb), is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. With a molecular weight of only 12–15 kDa, nanobodies are much smaller than common antibodies. The first nanobodies were engineered from heavy-chain antibodies found in camelids. Up till now, they have been shown to be just as specific as a regular antibody and in some cases, they are more robust. As well, they are easily isolated using the same phage panning procedure used for traditional antibodies, allowing them to be cultured in vitro in large concentrations. The smaller size and single domain make these antibodies easier to transform into bacterial cells for bulk production, making them ideal for research purposes. With sufficient documents related to support, we assured that it could be used to block biochemistry course in living cells.

Figure 7

Phage display antibody library

To obtain antibodies with high affinity to RyR2, we adopt the phage display technique, one of the Nobel Prize technology (Chemistry, 2018). Phage display is a laboratory technique for the study of protein–protein interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. In our study, a library of variable domains of camellidae heavy chain-only antibodies (VHH) was constructed. A vast majority of VHH clones were inserted into phagemid and expressed on the surface of the phages. In order to select binders to RyR2, bio-panning was performed with immobilized RyR2 protein. To obtain antibodies that functionally inhibit of RyR2 phosphorylation, antibody fragments isolated in the previous step were tested for its effect in an ELISA based RyR2 phosphorylation assay. Finally, AR185 and a negative control AR117 were obtained for further investigation.

Figure 8

BNP Promoter：Heart Failure Inducible

To ensure biosafety and avoid side effects in normal cells, a heart failure inducible BNP promoter was constructed to the upstream of our therapeutic gene. This promoter derives from the approximal region (-408/+100bp) of the human brain natriuretic peptide (hBNP) promoter, which is reported to have the response to AngⅡ, mechanical strain, and other heart-failure-related factors. According to previous reports, its activity remained low under basal conditions and elevated during heart failure. Based on these qualities, we chose to utilize this promoter as a switch to initiate and terminate gene expression.

Precise guidance to myocardium: recombined adeno-associated virus serotype 9 (rAAV9)

Then we came to the terminal part: transduce the “shield” into the myocardium precisely. According to a paper published in 2006, we picked recombined adeno-associated virus serotype 9 (rAAV9), an ideal virus born with specific affinity to cardiac tissue, which was proved by cell and tissue experiments later in our laboratory. Meanwhile rAAV9 has low biotoxicity, since its stable expression lasts 4 weeks according to the Western blot consequence.

Figure 9

References

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3. Eschenhagen, T. "Is ryanodine receptor phosphorylation key to the fight or flight response and heart failure?." Journal of Clinical Investigation120.12(2010):4197-4203. Ullrich, Nina D., H. H. Valdivia, and E. Niggli. "PKA phosphorylation of cardiac ryanodine receptor modulates SR luminal Ca2+ sensitivity." Journal of Molecular & Cellular Cardiology 53.1(2012):33-42.
4. Marx, S. O., et al. "PKA Phosphorylation Dissociates FKBP12.6 from the Calcium Release Channel (Ryanodine Receptor)." Cell 101.4(2000):365-376.
5. Menzel, S., et al. "Nanobody-Based Biologics for Modulating Purinergic Signaling in Inflammation and Immunity." Frontiers in Pharmacology9(2018):266.
6. Wikipedia. Single-domain antibody. https://en.wikipedia.org/wiki/Single-domain_antibody.
7. Wikipedia. Phage display. https://en.wikipedia.org/wiki/Phage_display.
8. Ma, X., et al. "Therapeutic delivery of cyclin-A2 via recombinant adeno-associated virus serotype 9 restarts the myocardial cell cycle: an in vitro study." Molecular Medicine Reports 11.5(2015):3652-3658.