6.4.4. Caution with Serca2a/PLN Therapy for Cardiomyopathy

Although many pre-clinical models of heart failure, including those of muscular dystrophy-associated cardiomyopathy, show significant improvement in cardiac morphology, calcium handling, contractile function, and survival with decreased PLN inhibition of Serca2a, caution should be taken when translating this to human disease. Two mutations leading to loss of PLN function have been identified in humans. A point mutation (T116G) resulting in a premature stop codon and a nonfunctional PLN protein (Leu39-stop) leads to severe DCM requiring transplantation at a young age [194]. Co-expression of Leu39-stop PLN and Serca2a in HEK293 cells revealed this truncated PLN protein was unable to decrease Serca2a affinity for calcium, and gene transfer of Leu39-stop PLN in isolated cardiac myocytes had no effects on calcium cycling or contraction/relaxation kinetics [194]. A C→T missense mutation at nucleotide 25 of the PLN gene encodes an Arg→Cys substitution (R9C) in the cytosolic domain, which also results in DCM and early death [195]. Similar to the Leu39-stop mutation, R9C did not inhibit calcium uptake in HEK293 cells [195]. Further study of this mutation revealed that R9C stabilizes the pentamer conformation of the protein, making it unavailable to inhibit Serca2a [196]. Indeed, acute expression of R9C via adenoviral gene transfer in rabbit cardiac myocytes revealed an increase in contractility and relaxation kinetics, with a concomitant increase in calcium peak height and decay rate [197]. Additionally, R9C transfected cardiac myocytes showed decreased responsiveness to beta-adrenergic stimulation [197]. Finally, patients with specific polymorphisms in alpha2c and beta1 adrenergic receptors, which leads to increased release and sensitivity to norepinephrine, have an odds ratio of 10.11 of developing heart failure compared to patients without these polymorphisms [198]. Chronically increased adrenergic stimulation increases load on the heart and decreases cardiac reserve, both of which also occur with PLN inhibition.

Key differences in cardiac physiology and calcium handling exist between mice and humans, which could account for the divergent outcomes. Approximately 90% of calcium reuptake in mice occurs via Serca2a and only 10% occurs via the sarcolemmal Na+/Ca2<sup>+</sup> exchanger. In contrast, approximately one-third of calcium is exported from the cytosol via the Na+/Ca2<sup>+</sup> exchanger in humans. Additionally, resting heart rate is approximately 10-fold lower in humans compared to mice, and the human heart has a greater variability in heart rate response to physiological stress. Finally, humans have a higher cardiac reserve allowing for increased SR calcium uptake and calcium load during physiological stress. Reducing cardiac reserve in humans by inhibiting PLN or increasing Serca2a may therefore have a much different outcome compared with a similar inhibition in rodents [56,199]. This was demonstrated in the CUPID 2 trial, where Serca2a gene delivery was not successful in meeting beneficial clinical outcomes in heart failure patients [180].

#### *6.5. Calcium Bu*ff*ering*

An alternative and energetically neutral approach to improving calcium reuptake and relaxation rate is to introduce expression of *de novo* calcium buffers into cardiac myocytes. The most well-studied calcium buffer in this context is parvalbumin. Parvalbumin (Parv) is ~12 kDa EF-hand calcium/magnesium binding protein naturally expressed in fast-twitch muscle in order to aid in fast relaxation by buffering calcium away from myofilaments [200,201]. Parvalbumin proteins contain two 12 amino acid EF-hand cation binding loops with binding affinities ranging from KCa<sup>2</sup><sup>+</sup> <sup>=</sup> <sup>10</sup>7−109 <sup>M</sup>−<sup>1</sup> and KMg<sup>2</sup><sup>+</sup> = 103−105 M<sup>−</sup>1, for calcium and magnesium, respectively [202,203]. In a resting myocyte, magnesium concentration is ~1 mM and calcium concentration is 10–100 nM, resulting in magnesium occupancy of the EF-hand loops. During contraction, calcium concentration increases, which causes calcium to displace magnesium in the binding loops. Calcium binding to Parv buffers calcium away from myofilaments to aid in rapid relaxation. As calcium is taken back up into the SR during relaxation, cytosolic calcium concentration falls, resulting in magnesium reoccupying the EF-hand cation binding loops. The ability of Parv to bind both magnesium and calcium is important in its function as a delayed calcium buffer. Calcium concentration must be sufficiently increased to induce magnesium dissociation from the EF-hand binding loops. This delay in calcium binding enables calcium to first bind to myofilaments and facilitate contraction of the myocyte before binding to Parv [203].

The ability of Parv to facilitate fast relaxation in the heart has been tested in numerous cell and animal models of diastolic dysfunction. Adenoviral gene transfer of Parv into isolated cardiac myocytes from hypothyroid rats [204], senescent rats [205], and Dahl salt-sensitive rats [206] hastened relaxation by increasing the rate of calcium decay. *In vivo* studies revealed gene delivery of Ad-Parv improved short-term hemodynamic measurements of relaxation, including -dP/dt and time to 50% and 90% pressure decay in hypothyroid rats [207], and decreased tau, a load independent measure of diastolic dysfunction, in senescent rats [208,209]. One advantage to a calcium buffering approach for improved relaxation over increasing Serca2a activity is the energetic efficiency. Mathematical modeling studies indicate Serca2a overexpression leads to a higher peak and total ATP consumption compared to *de novo* Parv expression, which results in a more even distribution of ATP consumption over the course of the contractile cycle [210]. Additionally, Parv expression in cardiac myocytes preserves beta-adrenergic function, which is blunted with increased Serca2a function [211] or PLN inhibition [182].

One drawback to using wild-type Parv for improved relaxation in the heart is the calcium/magnesium binding affinities are not optimized for the relatively slow contractile cycle of the heart. Although Parv is a delayed calcium buffer, requiring magnesium removal from the EF-hand cation binding sites before calcium can bind, the kinetics are optimized for fast-twitch muscle. This results in WT-Parv binding calcium too early in the contractile cycle of cardiac myocytes and inhibiting maximal contractility [206,212]. This is contraindicated in the context of dilated cardiomyopathy, which is characterized by both decreased contractile function and slowed relaxation. A potential solution to this problem with WT-Parv is to genetically modify the EF-hand cation binding site to have optimal binding affinities for the kinetics of the human heart. It was hypothesized and confirmed with mathematical modeling that increasing magnesium affinity and slowing magnesium off-rate even further than WT-Parv would restrict the buffering of calcium to diastole and prevent premature truncation of contraction [210].

To test this hypothesis in myocytes, two genetically modified parvalbumin proteins have been developed. Both involve substitutions of the highly conserved glutamate at residue 12 of the EF-hand cation binding site, one with glutamine (E101Q) [213] and one with aspartate (E101D) [214]. These substitutions eliminate the 7th coordinating oxygen preferred by calcium, resulting in both an increase in magnesium affinity and a decrease in calcium affinity, further delaying the buffering of calcium compared to WT-Parv. Adenoviral gene transfer of these modified parvalbumins increased relaxation rate and unexpectedly also increased contraction amplitude in isolated myocytes from rat (E101Q and E101D), rabbit (E101Q) and canine (E101Q). Additionally, ParvE101Q improved contractility and relaxation in multiple models of heart failure, including thapsigargin-treated rabbit myocytes, failing myocytes from dogs, and *in vivo* hemodynamic function of inducible Serca2a knock-out mice [213,214].

As a result of its primary role in increasing relaxation, most studies with Parv have been done using models of diastolic dysfunction. This is because, as a calcium buffer, the main contribution of Parv in the context of the failing heart has been thought to be sequestration of calcium to enhance relaxation in diastole. In particular, the decreased contractility characteristic of WT-Parv-treated myocytes would be contraindicated for the already compromised contractility in DCM. Whether or not modified parvalbumins could serve to both improve relaxation and increase contractility in models of DCM is unknown and warrants further investigation, particularly because Parv both preserves beta-adrenergic function and is an energetically neutral approach to improving function in the energetically compromised failing heart [215]. Calcium buffering may potentially have a beneficial role in the cardiomyopathy of DMD. Increased diastolic calcium resulting from calcium influx through SACs, sarcolemmal micro-tears, and increased RyR2 leak could theoretically be mitigated through introduction of a calcium buffering system. The localization of these buffers within the myocyte, as well as the buffering capacity and calcium binding kinetics are all factors that need to be optimized and tested when considering this approach in the context of DMD cardiomyopathy.
