6.4.2. PLN as a Target for DCM Therapy

As the major regulator of Serca2a, PLN has also been studied for its potential role to mitigate the calcium cycling deficits contributing to DCM. Early studies found PLN overexpression or deficiency decreased or increased calcium cycling and Serca2a calcium sensitivity, respectively, and β-adrenergic signaling was important in determining the level of PLN inhibition of Serca2a [181,182]. Increasing the ratio of PLN to Serca2a in a transgenic mouse model with 2-fold overexpression of PLN decreased peak height of contraction as well as slowed relaxation rate. This was accompanied by similar decreases in calcium transient peak height and decay rate. Depressed *in vivo* function measured by echocardiography was also present, and this was alleviated by isoproterenol treatment [181]. Contrary to PLN overexpression, PLN deficiency increases calcium cycling and cardiac function. Working heart preparations from PLN-deficient mice exhibited both increased systolic and diastolic function, which was not further enhanced by beta-adrenergic stimulation [182]. Increased calcium cycling and subsequent increases in contraction and relaxation function with PLN depletion are dose dependent, with PLN (+/-) mice exhibiting cardiac function at levels between WT and PLN (-/-) animals [183].

These early studies solidifying the inhibitory role of PLN on calcium cycling led to the hypothesis that PLN inhibition may ameliorate the depressed calcium cycling characteristic of DCM. This hypothesis has been tested in multiple models of DCM. In the MLP-deficient mouse [92], PLN deficiency decreased dilation and ultrastructural abnormalities and increased heart function measured by *in vivo* hemodynamics and echocardiography. These improvements could be explained by increased calcium cycling measured in isolated cardiac myocytes [184]. A pseudo-phosphorylated mutant of PLN (S16E) mimics the conformational change occurring in PLN after PKA phosphorylation at serine-16, decreasing interaction with Serca2a. *In vivo* rAAV gene transfer of S16E-PLN improved multiple measures of heart function in a post-myocardial infarction rat model, including dilation, heart size, ejection fraction and E/A ratio measured by echocardiography at 2 and 6 months post-MI. Additionally, multiple hemodynamic measurements were improved compared to infarcted rats without AAV treatment [185]. Similar improvements in heart function were observed in a large animal model of heart failure with S16E-PLN gene transfer. Sheep undergoing four weeks of pacing stress, followed by *in vivo* percutaneous cardiac recirculation-mediated gene delivery of S16E-PLN recovered hemodynamic function after two weeks, whereas control animals continued to have worsening heart failure [186]. Another approach to inhibition of PLN is to inhibit Phosphatase-1 (PP1), which dephosphorylates PLN and causes it to bind and inhibit Serca2a. Using AAV-9 gene delivery of a short-hairpin RNA against PP1beta with a B-type natriuretic peptide (BNP)-promoter created a heart-failure inducible expression system which was tested in the MLP-deficient mouse. This system increased PLN phosphorylation, which was accompanied by decreased cardiac remodeling and improved fractional shortening and hemodynamic function three months after gene transfer [187].

#### 6.4.3. Serca2a and PLN in Muscular Dystrophy-Associated Cardiomyopathy

Although most research has focused on the role of Serca2a/PLN in DCM in general, a number of studies have looked specifically at the cardiomyopathy occurring in models of muscular dystrophy. Serca2a gene expression is decreased in mice with DMD cardiomyopathy [188], suggesting the Serca2a/PLN complex may also be an important therapeutic target for DCM occurring in DMD.

Two studies examined the effect of Serca1 overexpression in skeletal muscle, and one study looked at Serca2a overexpression in the heart of different mouse models of muscular dystrophy. Crossing Serca1 transgenic mice with *mdx*, *mdx:utr*-/- , and *Sgcd*-/- mice, resulting in a 1.5–4-fold overexpression of Serca1, decreased CK release [189,190], reduced Evan's Blue Dye (EBD) uptake [189] and decreased fibrosis [189], suggesting decreased muscle damage. This was attributed to the ability of Serca1 overexpression to improve calcium handling in isolated myocytes [189]. The result of Serca1

overexpression led to restored treadmill running capability [189] and decreased percent torque loss after eccentric contraction-induced injury [190]. Serca2a overexpression in the aged *mdx* heart was examined using AAV-9 gene delivery. In this study, Serca2a was unable to reverse fibrosis, but did result in some improvement in electrocardiographic abnormalities [162].

PLN inhibition has also been tested in the context of muscular-dystrophy associated cardiomyopathy. The BIO14.6 hamster is a model of limb-girdle muscular dystrophy and exhibits a progressive cardiomyopathy phenotype beginning by about 5 weeks of age [191]. Recombinant AAV gene delivery of S16E-PLN in 5-6 week old BIO14.6 hamsters improved calcium cycling in isolated SR vesicles. This resulted in increased fractional shortening and improved maximum and minimum LV dP/dt at both 5 and 28 weeks post-gene transfer compared to BIO14.6 hamsters without AAV treatment [163]. Another study in the BIO14.6 hamster model used adenoviral gene delivery of an antibody targeted against PLN. Just over 50% of myocytes were infected with the virus, which led to short-term improvement in both echocardiographic and hemodynamic markers of systolic and diastolic function. This was accompanied by improved contractility and calcium handling in isolated myocytes, and increased SR calcium reuptake in whole heart homogenates [164]. Additionally, inhibition of PP1 via gene delivery of Inhibitor-2 in the BIO14.6 hamster resulted in beneficial effects on cardiac dimensions and fractional shortening, as well as improved hemodynamic measurements, decreased fibrosis, and improved survival [165].

Similar to PLN, sarcolipin (SLN) is an inhibitor of Serca. SLN expression is increased in heart muscle of *mdx:utr*-/- mice, dystrophic dogs and human patients with DMD. Deletion of either one or both alleles of SLN in *mdx:utr*-/- mice extended the lifespan of these animals, as well as improved heart function (ejection fraction and fractional shortening). Improved function was attributed to decreased left ventricular internal diameter in diastole (LVIDd) and decreased fibrotic and necrotic tissue. Although calcium handling was not measured in heart tissue in this study, skeletal muscle calcium reuptake was increased as a result of reduced SLN expression [192].

As a result of the extensive pre-clinical literature showing beneficial effects of Serca2a overexpression or PLN inhibition on calcium handling and heart failure outcomes in multiple models of heart failure, including muscular dystrophy-associated cardiomyopathy, our laboratory hypothesized that PLN ablation would improve calcium handling and subsequently improve cardiac function in the *mdx* mouse. Isolated cardiac myocytes from these mice did indeed show enhanced contractility and faster relaxation, which was accompanied by increased calcium transient peak height and decay rate. However, *in vivo* echocardiography revealed severe dilation and decreased systolic and diastolic function. Histological analysis revealed significantly more fibrotic development and EBD uptake, indicating sarcolemma integrity was more severely compromised with PLN ablation [19]. It was concluded that, although PLN ablation improved calcium cycling in isolated myocytes, which could potentially decrease the risk of calcium overload, increased contractile function likely placed additional stress on an already compromised sarcolemma. This likely led to even more extensive membrane damage than occurs with dystrophin deficiency alone [19] (Figure 4).

The results of this study are in contrast to others discussed above [162–165]. One potential contributor to these differences is the level of PLN inhibition. In the context of muscular dystrophy models, one study overexpressed Serca2a [162], and others have inhibited PLN to varying degrees through delivery of a pseudophosphorylated PLN [163], an antibody against PLN [164], or inhibitor-2 [165]. Although complete ablation of PLN improved cardiomyopathy in a DCM model [184], this was not the case in *mdx* mice, highlighting key differences mechanistically between the pathophysiology of muscular dystrophy-associated DCM and other causes of DCM. PLN knockout mice have significantly increased calcium cycling leading to increased contractile function [182,183], which likely increased sarcolemmal stress. Additionally, PLN knockout mice are not responsive to β-adrenergic signaling [182] and therefore have very little cardiac reserve. PLN knockout mice also have an increased ATP utilization and oxygen consumption [193], potentially increasing oxidative stress and potentiating membrane damage in the context of muscular dystrophy, which exhibits

decreased endogenous reducing capability [66]. Finally, gene deletion or transgenic expression of genes may have unknown compensatory effects on development which cannot be controlled for. Whether small increases in Serca2a expression or partial inhibition of PLN in *mdx* mice would yield different results needs to be the focus of future studies.

**Figure 4.** Effects of increased calcium cycling on cardiac myocytes with dystrophin deficiency. Loss of dystrophin destabilizes the sarcolemma and leads to calcium mishandling and overload. Increasing calcium cycling via modulation of Serca2a/PLN function increases calcium uptake into the SR, which could decrease cytosolic calcium concentration. However, increased calcium cycling also increases contractility, which could subsequently cause increased membrane damage and exacerbate calcium overload. In the context of dystrophin deficiency, phospholamban (PLN) ablation led to increased membrane damage and worsened cardiomyopathy [19].
