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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 4, 2004 as doi:10.1096/fj.03-1231fje. |
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* Department of Medicine, University of California, San Diego, La Jolla, California, USA; and
Department of Biology, San Diego State University, San Diego, California, USA
2 Correspondence: Department of Medicine, University of California, San Diego, 9500 Gilman Dr., Basic Sciences Building / 5063, La Jolla, CA 92093-0618, USA. E-mail: wdillman{at}ucsd.edu
SPECIFIC AIMS
Phospholamban (PLN) plays a crucial role in physiological regulation of cardiac contractility. By inhibiting the calcium pump of the sarcoplasmic reticulum (SERCA), phospholamban reduces diastolic calcium uptake into the sarcoplasmic reticulum (SR) and hence the amount of SR calcium available for the next contraction. Inhibition of SERCA can be relieved by cyclic AMP-mediated phosphorylation of phospholamban. Complete ablation of phosholamban (PLN(–/–) mice) greatly enhances contractility of the heart, not only under normal physiological conditions, but also under pathological conditions where contractility is normally depressed. While ablation of phospholamban represents the most extreme example of modification of SERCA-phospholamban interaction, it is impractical as a therapy in adults and has recently been implicated as a cause of heart failure in humans. Even phospholamban mutations which do not directly influence interaction of SERCA and phospholamban may have unforeseen consequences, indirectly contributing to development of heart failure.
Avoiding mutations in phospholamban altogether, we constructed an antibody specifically targeted to the cytoplasmic portion of phospholamban. Feasibility of using antibodies directed against phospholamban for improving SERCA function has been demonstrated previously, but only in vitro using isolated sarcoplasmic reticulum vesicles or in digitonin-permeablized cardiac myocytes. Our study describes development of a recombinant single chain antibody of avian origin (PLN-Ab) which specifically targets phospholamban, thus increasing the activity of SERCA, and which can be expressed in vivo using adenoviral vectors.
PRINCIPAL FINDINGS
1. Cloning and in vitro testing of PLN-Ab
PLN-Ab was created using RNA from bone marrow and spleen from chickens immunized with a synthetic peptide encompassing the cytosolic sequence of human phospholamban (aa3-19) including phosphorylation sites Ser 16 and Thr 17. Genes encoding variable regions of the heavy and light chain of chicken IgY were cloned into a phagemid expression vector (pComb3H), resulting in a fusion of single chain antibody product to filamentous phage coat protein III which could be used for selection by phage display. The resulting bacteriophages were screened for affinity to phospholamban by multiple rounds of panning using polystyrol tubes coated with BSA cross-linked phospholamban peptide. Selected DNA was subcloned into a bacterial expression plasmid (pARA) to produce PLN-Ab protein with a C terminus hemagglutinin (HA)-tag for protein detection.
When used in a Western blot, PLN-Ab was as effective in recognizing monomeric and pentameric forms of phospholamban as the commercially available anti-phospholamban antibody 2D12. Similarly, PLN-Ab had the same capacity to activate SR calcium uptake in isolated vesicles (more than 3-fold over control, P<0.05) as 2D12 anti-phospholamban antibody. Similar action of PLN-Ab and 2D12 on SR calcium uptake indicates that avian antibody behaves similarly to mammalian- (mouse) derived antibody 2D12 when applied in vitro.
To investigate how PLN-Ab interacts with phospholamban we used a novel protein construct based on fluorescence resonance energy transfer (FRET) to determine changes in conformation of the cytosolic portion of phospholamban. The construct (CcPlnY) contains the cytoplasmic domain of phospholamban, which was cloned between cyan (CFP) and yellow (YFP) variants of fluorescent proteins. When expressed in neonatal cardiac myocytes, the ratio of yellow to cyan fluorescence decreases in presence of isoproterenol, indicating a conformational change in the cytoplasmic portion of phospholamban which would be expected with protein phosphorylation (Fig. 1
). When examining the ratio of yellow to cyan fluorescence of CcPlnY in vitro, PLN-Ab also decreased the ratio, indicating conformational changes similar to those observed in intact neonatal cardiac myocytes (Fig. 1)
. This in vitro action of PLN-Ab was similar to that mediated by 2D12 and the catalytic subunit of protein kinase A, which leads to phosphorylation of phospholamban in vivo. A nonspecific antibody used as control did not affect the fluorescence ratio, indicating that only those antibodies specifically directed against phospholamban can alter its conformation in a manner similar to that of protein phosphorylation.
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2. Effects of PLN-Ab expression on function in cardiac myocytes
To investigate effects on intracellular calcium handling, PLN-Ab was co-expressed with phospholamban in neonatal rat myocytes by electroporating cells with plasmids expressing the two proteins. Co-expression with phospholamban was required because this protein is only minimally expressed in neonatal rat myocytes. Calcium transients were recorded using indo-1. Normalized indo-1 transients from myocytes expressing PLN-Ab demonstrated an acceleration of calcium transients with a reduction in the half time (t1/2) of diastolic calcium removal by 40%. To test whether such a result could also be observed in adult myocytes, PLN-Ab was cloned into an adenoviral vector. The effect of PLN-Ab on function in adult myocytes was determined by performing in vivo gene therapy using adenovirus expressing PLN-Ab or empty vector (control) mixed with green fluorescent protein (CMV driven GFP) expressing adenovirus (10:1 pfu/mL ratio) followed after 4 days by myocyte isolation. Indo-1 calcium transients and video edge detection were measured in freshly isolated myocytes expressing GFP and coexpressing either PLN-Ab or the empty (control) vector. Cardiac myocytes isolated from adult mice infected in vivo demonstrated a similar acceleration in the decline of the indo-1 calcium transient when signals were normalized with a significant reduction (P>0.05) in the half time (t1/2) of calcium removal. When myocyte shortening was measured using edge detection, both the extent of fractional shortening and rate of relaxation were seen to increase significantly (P<0.05) in PLN-Ab treated mice.
We extended our observations on the action of PLN-Ab following gene therapy to ex vivo perfused hearts and in vivo measurements in a mouse model of diabetes where SR calcium handling is diminished. Under normal conditions, 3 weeks of streptozotocin induced diabetes leads to a significant decrease in the rate of relaxation. While injection of empty adenovirus does not improve function in hearts isolated from diabetic mice, injection of PLN-Ab significantly improved function, increasing the rate of relaxation nearly to the level observed in non-diabetic mice. Rate of contraction (+dP/dt) and peak systolic pressure also showed a similar improvement in diabetic mice injected with PLN-Ab adenovirus. This beneficial effect was not due to changes in the level of SERCA expression, as SERCA levels were similar between diabetic mice injected with PLN-Ab and empty adenovirus. Similarly, the level of phospholamban expression (both pentamer and monomer) did not differ between mice injected with PLN-Ab and empty adenovirus. These results suggest that PLN-Ab mediated its effects through its direct interaction with phospholamban, rather than indirectly altering expression of SERCA or phospholamban to levels favoring increased Ca2+ uptake by the sarcoplasmic reticulum.
A similar improvement in cardiac function could be observed in diabetic mice in vivo, using echo cardiography. Two indices of contractility, fractional shortening and velocity of circumferential fiber shortening, were both significantly (P<0.05) higher in the PLN-Ab group. These results support our ex vivo observations that PLN-Ab gene therapy improves contractile function under pathological conditions where contractile function is depressed.
CONCLUSIONS AND SIGNIFICANCE
Increasing contractility is the goal of a number of therapies aimed at improving cardiac output in a variety of disease states which lead to heart failure. Pharmacological approaches which include ß-adrenergic agonists and phosphodiesterase inhibitors, mediate their contractile effects by increasing intracellular levels of cyclic AMP, leading to activation of protein kinase A followed by phosphorylation of cardiac proteins like L-type calcium channels and phospholamban. Unfortunately, chronic stimulation of the adrenergic signal transduction pathway can be damaging to the heart, resulting in cardiac hypertrophy, heart failure, and subsequently increased mortality as observed in some genetic and pharmacologically induced animal models of adrenergic overstimulation. A more specific approach is to alter activity of SERCA either directly or indirectly through modification of its inhibitory protein phospholamaban. Increasing SERCA activity through transgenic overexpression or adenoviral gene therapy leads to an improvement in function in cardiomyopathies where contractility is depressed. In view of the strong inhibitory effect of phospholamban on SERCA, it has also been targeted to modify SERCA activity. Reduction of phospholamban protein synthesis by adenoviral expression of phospholamban antisense was shown to be partially effective in improving contractility in isolated neonatal and adult cardiac myocytes. Another approach, leading to stronger effects on SR calcium transport and contractility in isolated cardiac myocytes, involves expression of phospholamban mutants which exert a dominant negative effect on wild type phospholamban in isolated cardiac myocytes. In the most extreme case, ablation of phospholamban leads to increased SERCA activity and increased contractile force in both normal and some pathophysiological conditions. Recent studies have shown that naturally occurring mutations in the human genome resulting in ablation of phospholamban leads to development of lethal dilated cardiomyopathy. A recent study involving an arg 
cys missense mutation at residue 9 of human phospholamban also leads to heart failure. Expression of this mutant protein in mice recapitulates the human phenotype of dilated cardiomyopathy; however, the effect appears to be indirectly caused by tight PKA binding to the mutant phospholamban rather than phospholamban’s normal interaction with SERCA. Other mutations to phospholamban directed toward its region of protein–protein interaction with SERCA also leads to heart failure. Given that mutations in phospholamban or its ablation may lead to heart failure, we sought to modify activity of phospholamban by expressing an antibody directed against it rather than mutating the protein itself (Fig. 2
). The concept of using an antibody directed against phospholamban to increase SERCA activity has been investigated previously in isolated SR vesicles and permeablized myocytes. Because these antibodies do not pass the sarcolemma, they are not readily applicable to use in vivo. Use of advanced phage display technologies allowed us to obtain cDNA information for both IgY light and heavy chains fused into a functional single chain antibody (PLN-Ab). Through both in vitro and in vivo studies, we were able to demonstrate the feasibility of creating an antibody directed against phospholamban and which can be delivered to cardiac myocytes via adenoviral gene therapy. Results presented in this study are consistent with PLN-Ab mediating its effects through interaction with phospholamban, increasing SERCA activity. This strategy involves expression of a novel protein and avoids direct modification of phospholamban through mutation.
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FOOTNOTES
1 These authors contributed equally to this study. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1231fje; doi: 10.1096/fj.03-1231fje
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