It is well established that SERCA2a activity is directly modulated by a 52-amino acid endogenous inhibitor, PLN. The inhibitory effect of PLN on SERCA2a activity was first revealed using genetically-altered animal models. Cardiac-specific overexpression of PLN inhibited SR Ca²⁺ uptake and reduced systolic Ca²⁺ levels, contractile parameters, and basal systolic function in mice [27]. In contrast, PLN knockout (KO) mice exhibited enhanced Ca²⁺ cycling and myocardial contractility with no gross developmental abnormalities. The elevated contractile parameters were associated with increased affinity of SERCA2a for Ca²⁺ [28]. Furthermore, it was found that the hyperdynamic cardiac function in PLN-KO mice persisted through the aging process. The persistence of hyperdynamic cardiac function over the long term did not induce any pathological or adverse functional consequences, suggesting that PLN may constitute an important target for treatment in heart disease [29].

In addition, comparative analyses with wild type, heterozygous and homozygous PLN-KO mice revealed that the relative PLN levels correlated well with the affinity of SERCA2a for Ca²⁺ and with the rates of relaxation and contraction in cardiomyocytes, intact hearts and in vivo [27, 28, 30, 31]. These findings suggested that the PLN level may impact SR function and cardiac contractility. Indeed, gene transfer approaches to increase the levels of PLN relative to SERCA2a in isolated cardiomyocytes have been reported to significantly alter intracellular calcium handling by prolonging the relaxation phase of the calcium transient, decreasing calcium release, and increasing end-diastolic calcium concentration [32]. Accordingly, antisense strategies to inhibit PLN indicate improved sarcoplasmic reticulum function, calcium mobilization, as well as significantly improved cell shortening in cardiomyocytes isolated from failing human hearts [32, 33]. Furthermore, overexpression of a dominant negative mutant of PLN has been reported to enhance SERCA2a activity [34]. Taken together, these studies suggest that PLN may have a key role in the maintenance of SERCA2a activity and that the PLN/SERCA2a ratio is a critical determinant of basal contractility in cardiomyocytes [35].

The prominent role of PLN in regulation of Ca²⁺-cycling and excitation-contraction coupling was further supported by the identification of naturally occurring genetic variations in the human PLN gene, which appear to associate with heart failure. There have been four identified naturally occurring mutations in the coding region of the human PLN gene [36–39]. The human V49G point mutation was associated with super-inhibition of SERCA2a and heart failure induction in mouse models [37], while the human R9C-PLN mutation resulted in early death from dilated cardiomyopathy (DCM) in human carriers [36]. The underlying mechanisms included decreases in PLN phosphorylation and chronic inhibition of SERCA2a [36]. The T116G point mutation resulted in a stop codon (L39stop) and homozygosity was associated with DCM at a young age, while the heterozygous individuals exhibited hypertrophy without diminished contractile performance [38]. Another human mutation deleted the arginine 14 amino acid in PLN and heterozygous carriers developed heart failure by mid-age [39]. The R14del-PLN acted as a super-inhibitor of SERCA and cardiac pathology along with early death was observed in mouse models expressing this mutation in the heart [39]. Thus, there are human PLN mutations that are associated with predisposition to dilated cardiomyopathy.

The HS-1 associated protein X-1 (HAX1) was identified as a new PLN binding protein, using a human cDNA library and the yeast-two-hybrid screening approach [40]. Extensive protein studies led to the identification of the minimal binding domains of HAX1 (residues 203–245) and PLN (residues 16–22), indicating a direct physical interaction (Fig. 12.1b). Phosphorylation of PLN or elevation of the concentration of Ca²⁺ leads to dissociation of HAX1 from PLN, similar to findings on the PLN/SERCA2a interaction, thus indicating a physiological/pathophysiological significance of this association in cardiac muscle. Although HAX1 localizes to mitochondria, in the presence of PLN it becomes redistributed and co-localizes with PLN at the endoplasmic reticulum [40]. Analysis of the anti-apoptotic function of HAX1 revealed that the presence of PLN enhanced the HAX1 protective effects from hypoxia/reoxygenation induced cell death [40]. These findings suggest a potential link between the SR Ca²⁺ handling and cell survival, mediated by the PLN/HAX1 interaction.

The type-1 phosphatase (PP1) activity is significantly elevated in human and experimental HF. These increases have been suggested to be a contributing factor in the depressed cardiac function and remodeling. Indeed, cardiac overexpression of the catalytic subunit or PP1c in mouse models at similar levels as those observed in human failing hearts resulted in depressed function, remodeling, HF and early death [41]. Thus, it was suggested that inhibition of this enzyme by its endogenous inhibitor 2 and inhibitor 1 may hold promise for targeting the increased PP1 activity in HF. Gene delivery of inhibitor 2 in a rat model prevented HF progression and prolonged survival [42]. In addition, increased activity of inhibitor 1 (I- 1) exhibited therapeutic promise in HF. Inhibitor-1 is activated by protein kinase A (PKA) phosphorylation and this results in potent inhibition of PP1 and increased PLN phosphorylation, leading to enhanced Ca²⁺-cycling (Fig. 12.3). The role of I-1 in the heart was elucidated by genetically altered mouse models. The I-1 knock-out mouse model (KO) presented with increased PP1 activity, had a depressed cardiac function under basal conditions and attenuated β- adrenergic response [41]. These effects were associated with decreased levels of phosphorylation on Serine 16 and Threonine 17 of PLN [41]. Accordingly, overexpression of a truncated (AA: 1–65) and constitutively active (T35D) form of I-1 (I-1c) demonstrated that I-1c could inhibit PP1 activity, increase Ser16 and Thr17 phosphorylation of PLN and attenuate the hypertrophic response, delaying the onset of HF [43]. In addition, cardiac-specific and inducible expression of I-1c in the adult heart revealed that I-1c enhances basal cardiac function, which is associated with increases in PLN phosphorylation levels [44]. Furthermore, under stress conditions (transverse aortic constriction or in vivo ischemia/reperfusion), either conventional or inducible expression of I-1c was associated with increased PLN phosphorylation and increased SR Ca²⁺-transport. This enhanced SR calcium cycling improved the heart’s ability to accommodate the hypertrophic stimulus, delay the progression from hypertrophy to failure and impact cell survival under stress conditions. Thus, targeting I-1 may be beneficial in alleviating the detrimental effects of HF, through specific modulation of the PLN-coupled PP1 activity.

Importantly, long-term inducible expression of constitutively active inhibitor-1, I-1c, in the heart (up to 20 months) preserved increased PLN phosphorylation and did not affect survival rates or cardiac remodeling due to chronic increases in Ca²⁺-cycling and function [45]. Furthermore, long-term expression of I-1c using recombinant adeno-associated virus type 9 gene transfer in rats with HF enhanced contractility and restored remodeling, associated with increased phosphorylation of PLN at Ser16 and Thr17 [45]. Thus, studies in rodent models suggest that chronic inhibition of protein phosphatase 1, through increases in I-1c, does not accelerate age-related cardiomyopathy and gene transfer of this molecule in vivo improves function and halts remodeling. These studies were subsequently extended to a large model of HF using gene transfer of I-1c. Heart failure was induced by myocardial infarction (MI) and 1 month post MI, the animals presented with severe failure, as indicated by decreases in ejection fraction, rates of contraction (+dP/dt) and relaxation (−dP/dt). Intracoronary injection of recombinant adeno-associated viral vector containing I-1c (AAV9.I-1c) prevented further deterioration of cardiac function and led to a decrease of scar size [46]. These preclinical studies indicate that cardiac overexpression of I-1c by gene transfer may improve hemodynamic function and improve cardiac remodelling. Interestingly, a naturally occurring genetic variant in I-1 (G147D), which diminishes the cardiomyocyte response to ß- adrenergic stimulation, has been also identified [47] and this indicates that human I-1 genetic variants may contribute to depressed Ca²⁺-cycling and functional deterioration in HF.

Overall, the studies in small and large animal models indicate that inhibition of the elevated SR PP1 activity in heart disease by active inhibitor-1, I-1c, may constitute a therapeutic strategy to rectify the disturbed Ca²⁺-homeostasis and function in failing hearts. Finally, the relatively small size of I-1c and its role as an inhibitor of PP1 opens the possibility of pharmacologic manipulation in addition to, or in replacement of, gene therapy.

Heat shock proteins (HSPs) are known to enhance cell survival under various stress conditions. In the context of cardiac function, the small heat shock protein HSP20 has emerged as a key mediator of protection against apoptosis, remodeling and ischemia/reperfusion injury. Interestingly, acute increases of HSP20 levels or activity in isolated cardiomyocytes were associated with enhanced contractile parameters and Ca²⁺-transients [48], which were further supported by cardiac specific HSP20 overexpression in transgenic mouse hearts. HSP20 overexpression resulted in significant enhancement of cardiac function in intact animals, and in augmented Ca²⁺-cycling and SR Ca²⁺-load in isolated cardiomyocytes [5]. Accordingly, knockdown of HSP20 by antisense RNA or microRNA-320 resulted in depressed contractility [49]. The enhanced contractility by HSP20 overexpression was associated with specific increases in the phosphorylation of PLN, relieving its inhibitory effect on the apparent Ca²⁺-affinity of SERCA2a. Interestingly, the activity of PP1, a regulator of PLN signaling, was significantly reduced by HSP20-overexpression, suggesting that the HSP20 stimulatory effects are partially mediated through the PP1/PLN interaction. Further studies revealed an association between HSP20, PP1 and PLN (Fig. 12.1b). Specifically, there is a physical interaction between HSP20 and PP1 [50], which could be indicative of the HSP20 role in the regulation of SR Ca²⁺-cycling by targeting the PP1/PLN complex (Fig. 12.3). In addition, Hsp20 protects against ischemia/reperfusion-induced cardiac injury, β-agonist-mediated remodeling, and apoptosis [6, 7], introducing an additional role of HSP20 function in the heart. Thus, the enhanced contractility together with the cardioprotective role of HSP20 implies a potential dual benefit of targeting HSP20 in treating heart disease.