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Review
New insights into SERCA2a gene therapy in heart failure: pay attention to the negative effects of B-type natriuretic peptides
  1. Yuting Zhai1,
  2. Yuanyuan Luo2,
  3. Pei Wu1,
  4. Dongye Li1
  1. 1 Institute of Cardiovascular Disease Research, Xuzhou Medical University, Xuzhou, Jiangsu, China
  2. 2 Department of Cardiology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, Jiangsu, China
  1. Correspondence to Professor Dongye Li, Institute of Cardiovascular Disease Research, Xuzhou Medical University, Xuzhou, Jiangsu 221002, China; dongyeli{at}xzhmu.edu.cn

Abstract

Sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) is a target of interest in gene therapy for heart failure with reduced ejection fraction (HFrEF). However, the results of an important clinical study, the Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial, were controversial. Promising results were observed in the CUPID 1 trial, but the results of the CUPID 2 trial were negative. The factors that caused the controversial results remain unclear. Importantly, enrolled patients were required to have a higher plasma level of B-type natriuretic peptide (BNP) in the CUPID 2 trial. Moreover, BNP was shown to inhibit SERCA2a expression. Therefore, it is possible that high BNP levels interact with treatment effects of SERCA2a gene transfer and accordingly lead to negative results of CUPID 2 trial. From this point of view, effects of SERCA2a gene therapy should be explored in heart failure with preserved ejection fraction, which is characterised by lower BNP levels compared with HFrEF. In this review, we summarise the current knowledge of SERCA2a gene therapy for heart failure, analyse potential interaction between BNP levels and therapeutic effects of SERCA2a gene transfer and provide directions for future research to solve the identified problems.

  • heart failure
  • SERCA2a
  • B-type natriuretic peptide
  • gene therapy

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Introduction

Heart failure (HF) is the end-stage of cardiovascular diseases, with high morbidity, hospitalisation and mortality. HF currently affects more than 23 million people worldwide, and its prevalence increases with age.1 2 Despite advancements in the treatment of HF over the past several decades, the survival rate of patients with HF remains poor. After the diagnosis of HF, the 5 and 10-year survival rates are 50% and 10%, respectively.1 The burden of HF is tremendous. The ESC-HF Pilot study revealed that the 1-year hospitalisation rates for hospitalised and ambulatory patients with acute and chronic HF were 43.9% and 31.9%, respectively.3 In the USA, the future costs of HF will have increased markedly by 2030 because of ageing of the population.4 Therefore, the development of a novel therapeutic approach for the management of HF is necessary.

In addition to neurohormone activation, the disequilibrium of Ca2+ homeostasis is an important pathophysiological mechanism of heart failure with reduced ejection fraction (HFrEF). Approaches to restore abnormal Ca2+ handling have been developed for several decades. Sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy is one research focus. Many fundamental studies have shown that cardiac function in HF can be improved by SERCA2a gene transfer.5–12 Based on the promising results of preclinical studies, SERCA2a gene therapy has moved from bench to bedside. The Calcium Upregulation by Percutaneous Administration of Gene Therapy in Cardiac Disease (CUPID) trial began in 2007 and was the first phase 1/2 study to assess the effects of SERCA2a gene transfer in patients with HFrEF.13 Unexpectedly, the results of the clinical trials were controversial.

The CUPID 1 trial was a small pilot study to confirm feasibility and safety of SERCA2a gene therapy for patients with advanced HFrEF.13 The study suggested that SERCA2a gene therapy was tolerated well and that it improved several independent efficacy/biological activity parameters in the patients and that the treatment reduced the recurrent HF event rate compared with the placebo group.14 15 Based on these findings, CUPID 2, a larger phase 2b study, was designed to confirm the effects of SERCA2a gene therapy on clinical outcomes of patients with advanced HFrEF.16 However, unlike the positive findings of the CUPID 1 trial, the CUPID 2 trial failed to improve the recurrent HF hospitalisations among the participants.17 To the best of our knowledge, B-type natriuretic peptide (BNP) plays a role in decreasing SERCA2a expression18–21 and may interact with the treatment effects of SERCA2a gene therapy.

Therefore, this review will provide an explanation for the failure of the CUPID 2 trial by analysing the negative effects of BNP on SERCA2a expression. In addition, as BNP levels are lower in heart failure with preserved ejection fraction (HFpEF) than HFrEF and evidence suggests that impaired SERCA2a function is responsible for diastolic dysfunction, SERCA2a gene therapy can be evaluated in HFpEF in future studies.

SERCA2a is a target of interest for treating HFrEF

SERCA2a is an important Ca2+ handling protein. Impaired SERCA2a function contributes to Ca2+ mishandling, which is a vital pathophysiological mechanism in HFrEF. Restoring SERCA2a function is a valid method for normalising Ca2+ handling and cardiac function.

The relationship between SERCA2a and cardiac function

Ca2+ is an essential ion involved in cardiac excitation-contraction coupling, which is the process of myocardial electrical excitation to cardiac contraction.22 Ca2+ transport is regulated by the sarcoplasmic reticulum (SR), an internal membrane system in cardiomyocytes. SR proteins coordinate with each other and function as a seamless Ca2+ transport system (figure 1). After depolarisation of the sarcolemma, SR Ca2+ release through ryanodine receptors (RyRs) is triggered by a small amount of Ca2+ inflow through the voltage-gated L-type Ca2+ channel. The so-called ‘Ca2+-induced Ca2+-release’ leads to a 10-fold increase in the free intracellular Ca2+ concentration ([Ca2+]i) and provides sufficient Ca2+ for binding to myofilament troponin C and ultimately induces cardiac contraction.23 Cardiac relaxation is induced by a quick decline in [Ca2+]i due to SERCA2a, sarcolemmal Na+/Ca2+ exchange (NCX), mitochondrial Ca2+ uniport and sarcolemmal Ca2+-ATPase.22

Figure 1

The Ca2+ transport system in cardiomyocytes. A small amount of Ca2+ enters the cytosol through the LTCC, resulting in Ca2+ release from the SR to the cytosol through the RyR. Free Ca2+ binds to myofilament troponin C and induces cardiac contraction. Cardiac relaxation is induced by the removal of Ca2+ in the cytosol, which occurs in four ways: (A) SERCA2a, (B) NCX, (C) PMCA, (D) mitochondrial Ca2+ uniport. LTCC, L-type Ca2+ channel; NCX, Na+ /Ca2+ exchange; PMCA, plasma membrane Ca2+-ATPase; RyR, ryanodine receptor; SERCA2a, sarcoplasmic/endoplasmic reticulum calcium ATPase 2a; SR, sarcoplasmic reticulum.

Abnormal function of Ca2+ handling proteins can lead to cytosolic Ca2+ overload and SR Ca2+ store depletion, which are hallmarks of cardiac dysfunction. The main defects of Ca2+ handling involve SR Ca2+ leak through RyRs, decreased SERCA2a function and increased NCX activity. The mechanism of SR Ca2+ leak includes elevation of diastolic Ca2+, reduction of SR Ca2+ storage, trigger of arrhythmias and increase of energy expenditure.24 Moreover, NCX also exerts important function on cardiac contractility.25 Decreased activity of NCX can raise SR Ca2+ storage and accordingly result in increasing Ca2+ transient and cardiac contractility. In end-stage HFrEF, NCX expression and function were reported to be increased, which could be an adaptive change to decreased SERCA2a function.26 However, the sustained activation of NCX would increase Ca2+ removal from cytosol, reduce SR Ca2+ storage and decrease cardiac contractility. Apart from NCX, SERCA2a, another protein responsible for Ca2+ removal, is a key regulator of relaxation rates and contractility of the heart.

SERCA2a, which is encoded by the SERCA2 gene, is an isoform that is mainly expressed in the myocardium and in slow-twitch skeletal muscle tissue. It is located in the SR membrane, accounting for >50% of SR proteins, and has the ability to transport Ca2+ from the cytoplasm to the SR. In ventricles of higher mammalian species and humans, nearly 70% of the activating Ca2+ involved in Ca2+ cycling is transported from the cytoplasm to the SR through SERCA2a and 25%–28% is extruded from the sarcolemma (mainly via NCX). Notably, the proportion of Ca2+ transported by SERCA2a is much higher in rodents, which is greater than 90%.27 Therefore, SERCA2a plays a key role in Ca2+ handling. If a malfunction occurs in SERCA2a, the subsequent Ca2+ overload in the cytosol and Ca2+ exhaustion in the SR will impair cardiac contraction and relaxation.

In the 1990s, many experiments were conducted to observe changes in SERCA2a expression and function in animal models and human myocardium with HFrEF and found decreased SERCA2a expression or activity in HFrEF.28–30 However, the changes in SERCA2a mRNA, protein and activity were not always consistent.31 32 This discrepancy may be due to different experimental methods, different synthesis and degradation rates of mRNA and protein, or the complex regulatory mechanisms of SERCA2a relative to transcriptional, post-transcriptional and post-translational modifications and protein-protein interactions.

The enzymatic activity of SERCA2a has been found to be regulated by its binding partners such as phospholamban (PLN), sarcolipin, histidine-rich calcium binding protein and calreticulin.33 34 PLN is a major regulator of SERCA2a function and is directly involved in the development of HF. Dephosphorylated PLN binds to SERCA2a and inhibits its affinity for Ca2+, hence reducing Ca2+ influx into the SR. Phosphorylation of PLN by calcium–calmodulin-dependent protein kinases (CAMKII) or protein kinase A relieves its inhibitory effects on SERCA2a function.33 In HF, the ratio of PLN and SERCA2a is increased and PLN is less phosphorylated, leading to more inhibitory effects of PLN.35 SERCA2a activity can also be regulated by S100A1, which is a small protein highly expressed in the human heart. S100A1 influences Ca2+ cycling through interacting with SERCA2a and PLN, RyR, as well as mitochondrial F1-ATPase activity.36 S100A1 can increase SERCA2a activity and thereby enhancing cardiac contractility.37 In failing rat hearts, restoration of the reduced S100A1 level has been proven to increase SR Ca2+ uptake, decrease SR Ca2+ leak, improve cardiac energetic metabolism and normalise myocardial contractile function.38 In addition to the above binding partners, there are several post-translational modifications which include SUMOylation, nitration, phosphorylation, glycosylation, glutathionylation, acetylation and O-GlcNAcylation.39 40 Among these modifications, SERCA2a SUMOylation is a research area attractive to investigators. SUMOylation of SERCA2a increased activity and stability of SERCA2a. In failing hearts of murine, SERCA2a SUMOylation was decreased and normalisation of SUMOylation helped maintain SERCA2a protein level and improve cardiac function.41 42 Furthermore, miRNAs have been reported to modulate cardiac function through regulating SERCA2a function. For example, in animal and human failing hearts, miRNA-25 increased and played a role in reducing SERCA2a expression. Inhibition of miRNA-25 improved cardiac function and survival of HFrEF mice.43 The current laboratory evidence demonstrates that targeting regulators of SERCA2a may represent potential approaches for treating HFrEF. However, this review focuses on the role of increasing SERCA2a expression in treatment for HFrEF, which has moved to clinical trials.

Restoring SERCA2a expression and activity via drugs or the gene transfer method has been confirmed to be beneficial in many experiments in vitro and in vivo. For example, in our previous work, we demonstrated that SERCA2a upregulation by luteolin exerts cardioprotective effects in animal models of ischaemia/reperfusion injury and HFrEF.44–47 Furthermore, SERCA2a gene therapy for HFrEF has been developed for more than 20 years in animal models and has recently been applied in humans.

SERCA2a gene therapy for HFrEF: from bench to bedside

As early as the 1990s, researchers found that SERCA2a gene transfer could increase SERCA2a expression and activity and subsequently improve cardiac function in failing human cardiomyocytes5 and animal models of  HF.6 7 As a result, SERCA2a gene therapy for HFrEF became a topic of interest. During the following 20 years, SERCA2a gene transfer was further shown to reverse left ventricular remodelling and improve survival in both small and large animal models of HFrEF.8 9 In addition, unlike other inotropic agents which cause detrimental effects, including increasing energetic demand, triggering arrhythmias and worsening survival, SERCA2a gene transfer was shown to improve cardiac energetics10 and normalise Ca2+ handling, thus exerting antiarrhythmic effects.11 12 By the way, Ito et al 48 showed that transgenic SERCA2a overexpression could prevent progression from hypertrophy to HFrEF in a mouse model of ascending aortic stenosis, indicating a preventive effect on HFrEF by SERCA2a gene therapy.

The beneficial effects of SERCA2a gene transfer on failing hearts revealed in fundamental studies provided evidence for the execution of clinical trials. The CUPID trial, which began in 2007, was the first phase 1/2 clinical trial to assess the effects of SERCA2a gene therapy in patients with advanced HFrEF.13 In the study, the SERCA2a gene was transferred to cardiac myocytes via adeno-associated viral vector 1 (AAV1). The CUPID 1 phase 1 study was a small, open-label, sequential dose escalation study to confirm feasibility and safety of AAV1/SERCA2a in nine patients with advanced HFrEF.14 After 6–12 months’ follow-up, AAV1/SERCA2a administration showed no safety concerns and improved efficacy/biological activity parameters in several patients. The CUPID 1 phase 2a study was a randomised, double-blind, placebo-controlled, parallel-group, dose-ranging study that treated 39 patients with either placebo or three doses of AAV1/SERCA2a (6×1011 DNase-resistant particle; 3×1012 DNase-resistant particle; 1×1013 DNase-resistant particle).15 Again, the CUPID 1 phase 2a study revealed no safety concerns and indicated improvement in outcomes of AAV1/SERCA2a administration. The high-dose group showed improvement in efficacy parameters at 6 months and demonstrated reduction in clinical events at 12 months compared with the placebo group. Furthermore, the long-term effect of AAV1/SERCA2a was reported in the CUPID 1 phase 2a trial, with decreases in recurrent and terminal events in the high-dose group at 36 months.49 The encouraging results of CUPID 1 trial led to CUPID 2, which was a phase 2b, double-blind, placebo-controlled, multinational, multicentre, randomised event-driven study that began in 2012.17 In the study, 250 patients were randomly assigned to receive either 1×1013 DNase-resistant particle AAV1/SERCA2a (n=123) or placebo (n=127). After median follow-up of 17.5 months, AAV1/SERCA2a did not improve time to recurrent HF hospitalisations and time to terminal events compared with placebo. Though the results of the CUPID 2 trial were unexpected, we should not jump to the conclusion that SERCA2a is not an appropriate target for the management of HF.

Potential causes of the negative results in the CUPID 2 trial

The investigators assessed myocardial tissues from seven patients who required cardiac transplantation, received mechanical assist device implantation or died in the CUPID 2 trial and found that the viral uptake showed a median of 43 copies per μg DNA, representing the lower end of the threshold for dose–response curves (<500 copies per μg DNA) in pharmacology studies.17 Therefore, the negative results of the CUPID 2 trial may be related to insufficient SERCA2a transduction efficacy. Therefore, other investigators and scientists assumed that the low transduction efficacy could be due to a low proportion of empty capsids, a low total particle dose and an inadequate delivery method, and suggested that future studies should focus on developing gene transduction technology to improve gene delivery methods, develop more efficient vector systems and control the effects of antibodies.50–53

Though deficits in gene transduction technology should not be ignored, we still hypothesise that other unknown factors may have contributed to the insufficient therapeutic efficiency of SERCA2a restoration. Considering that BNP downregulates SERCA2a expression, this may reflect a reason for the negative results of the CUPID 2 trial. However, the inhibitory role of BNP on SERCA2a expression is not taken seriously. We will summarise available data supporting the negative effects of BNP in the following passages.

Evidence for interaction between BNP and treatment effects of SERCA2a gene transfer

BNP has been shown to exert an inhibitory effect on SERCA2a expression and may consequently influence the response of patients to effects of SERCA2a gene therapy. In other words, patients with high BNP levels may not benefit from SERCA2a gene therapy.

The inhibitory effects of BNP on SERCA2a expression

BNP is derived from proBNP, which is synthesised by cardiomyocytes and fibroblasts when the left ventricle experiences stretch. According to the 2017 ACC/AHA/HFSA Heart Failure Focused Update,54 BNP levels are significantly correlated with disease severity and recommended to be used as markers for prevention, diagnosis, risk stratification and prognosis of HF.

BNP is much more than a biomarker; it also exerts functions such as natriuresis, diuresis, vasodilation and antitissue remodelling, and counteracts the sympathetic nervous system (SNS) and the renin-angiotensin-aldosterone system. These beneficial effects have elicited increasing attention on the treatment of HF by targeting the natriuretic peptide system.55 However, just as a coin has two sides, BNP is no exception. The Acute Study of Clinical Effectiveness of Nesiritide in Decompensated Heart Failure trial demonstrated that recombinant human BNP nesiritide failed to improve rehospitalisation and death of patients and that it increased rates of hypotension.56 The haemodynamic effects of BNP could be related to a reflex response of SNS. Chan et al 57 reported that BNP caused release of norepinephrine via protein kinase G (PKG) induced inhibition of phosphodiesterasetype-3 (PDE3) mediated cAMP hydrolysis. BNP could also inhibit PDE3 through the activation of NPR-B.58 Thireau et al 18 found that the sympathetic overdrive of BNP was associated with Ca2+ handling alterations which include reduced expression of SERCA2a, S100A1 and RyR2, as well as increased NCX, resulting in reduced Ca2+ transients, impaired Ca2+ reuptake and increased Ca2+ spark frequency. The abnormal sympathetic activation and Ca2+ cycling induced by BNP finally resulted in ventricular remodelling and arrhythmias, which could be prevented by metoprolol. The same team further found that combination with BNP and metoprolol was more effective than metoprolol or BNP alone in improving cardiac function and Ca2+ homeostasis.59 Thus, overactivation of SNS by BNP can interfere with Ca2+ handling protein as SERCA2a (figure 2).

Figure 2

The mechanism of SERCA2a downregulation by BNP. The activation of PKG by BNP via GC, leading to cGMP elevation, inhibits the calcineurin-NFAT signalling pathway, which is involved in SERCA2a upregulation. Calcineurin activates NFAT, allowing its translocation to the nucleus with consequent NFAT-dependent SERCA2a transcription.21 Calcineurin also activates the PI3K-Akt-GSK3β signalling pathway. GSK3β phosphorylates NFAT and promotes its cytoplasmic location, resulting in SERCA2a transcriptional regulation. However, whether BNP inhibits SERCA2a expression through this pathway remains unclear (indicated by ‘?’).62 The inhibition of PDE3 mediated cAMP hydrolysis by BNP via NPRA or NPRB activation increase [Ca2+]i and subsequently causes NE release, which finally inhibits SERCA2a expression.18 59 BNP, B-type natriuretic peptide; cAMP, cyclic AMP; cGMP, cyclic guanosine monophosphate; CN, calcineurin;  [Ca2+]i, intracellular calcium; GC, guanylyl cyclase; GSK3β, glycogen synthase kinase 3β; NE, norepinephrine; NFAT, nuclear factor of activated T cells; NPRA, natriuretic peptide receptor A; NPRB, natriuretic peptide receptor B; PDE3, phosphodiesterasetype-3; PI3K, phosphoinositide-3 kinase; PKA, protein kinase A; PKG, protein kinase G; SERCA2a, sarcoplasmic/endoplasmic reticulum calcium ATPase 2a.

Alteration in the BNP mRNA level is always negatively correlated with changes in the SERCA2a mRNA level in the failing myocardium.60 61 Is the negative correlation of expression levels between BNP and SERCA2a an accidental phenomenon? Kögler et al 19 studied the relationship between BNP and SERCA2a and found the following results: (1) patients with HF who received an left ventricular assist device (LVAD) showed increased SERCA2a mRNA expression only when BNP mRNA expression levels were downregulated; (2) a perfect negative correlation between the mRNA levels of BNP and SERCA2a was observed in stretched rabbit muscle strips and in the myocardium of human failing hearts; (3) exogenous recombinant human BNP abolished preload-induced SERCA2a upregulation in a concentration-dependent manner in rabbit right ventricular muscle strips. Toischer and colleagues20 found that an endogenous increase in BNP mRNA levels from afterload and isoproterenol stimulation prevented preload-dependent SERCA2a upregulation in rabbit muscle strips. They further observed the inability of preload to upregulate SERCA2a expression in failing human myocardium with elevated BNP expression, indicating that it was difficult to restore SERCA2a expression if BNP levels increased. The same group subsequently validated the negative effect of BNP in a mouse model of transversal aortic constriction.21 In their study, the activation of PKG by BNP inhibited the calcineurin-nuclear factor of activated T cell (NFAT) pathway, which is involved in the pressure overload-induced upregulation of SERCA2a. Therefore, they suggested that the underlying mechanism of SERCA2a downregulation via BNP is the inhibition of the calcineurin-NFAT pathway. Moreover, the PI3K-Akt signalling pathway, which can be activated by pressure overload or calcineurin, may also participate in downregulating SERCA2a expression (figure 2).62

As BNP can downregulate endogenous SERCA2a expression, BNP may also exert negative effects on exogenous SERCA2a transgene expression and consequently inhibit the efficacy of SERCA2a gene therapy. Takizawa et al 63 introduced the 5′ upstream region of the SERCA2 gene into adult rat myocardium by gene transfer and then performed abdominal aortic constriction surgery and found that the transcriptional activity of the exogenous SERCA2 gene was lower in the operated group than in the sham-operated control group. This result indicated that transcription of the exogenous SERCA2a gene could be inhibited by factors related to pressure overload. Interestingly, as mentioned before, BNP is one of these factors that increased with pressure overload.

Above findings suggest that BNP may reduce the therapeutic efficacy of SERCA2a gene transfer by inhibiting SERCA2a transgene transcription. In other words, patients with high levels of BNP may be less responsive to SERCA2a gene therapy than patients with low levels of BNP. The new insights provide us with significant opportunities for further studies in relationship of BNP with SERCA2a gene therapy.

Analysis of the relationship between BNP and SERCA2a in the CUPID trial

Compared with the CUPID 1 trial in which the BNP levels were not an entry criterion, a protocol amendment was made to require the participants to have elevated BNP levels (BNP>225 pg/mL or N-terminal proBNP>1200 pg/mL; BNP>275 pg/mL or N-terminal proBNP>1600 pg/mL if atrial fibrillation is present) during screening for the CUPID 2 trial.17 Could the elevated levels of BNP have interacted with the outcomes of SERCA2a gene transfer?

Anand et al 64 explored the interaction between baseline levels of BNP and treatment effects in the Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist Trial study which showed no improvement of spironolactone on the primary outcomes of patients with HFpEF, finding that the greater benefit of spironolactone was seen in patients with low BNP levels compared with patients with high BNP levels. Similar effects of BNP levels on outcomes were shown in a post hoc analysis of the Irbesartan in Heart Failure with Preserved Ejection Fraction trial.65 Therefore, baseline BNP levels may affect treatment effects of drugs, indicating that using elevated levels of BNP as entry criteria should be reconsidered.

In the CUPID 1 phase 1 trial, nine patients were enrolled and treated with AAV1/SERCA2a. By analysing the data in the literature,14 we found that two patients who continued to deteriorate during the study had relatively higher baseline NT-proBNP levels. In contrast, the efficacy parameters of four patients with lower baseline NT-proBNP levels all improved (table 1). These findings supported the BNP hypothesis to some extent. By the way, as the two exacerbated patients showed positive neutralising antibodies (Nabs), the investigators assumed that the failure of improvement in outcomes might be caused by a neutralisation effect of Nabs. However, we cannot draw any conclusions that BNP levels or Nabs interact with treatment effects of AAV1/SERCA2a because the data are too preliminary.

Table 1

Baseline of N-terminal proBNP levels and efficacy parameters of nine patients in CUPID 1 trial

In the CUPID 2 trial, subgroup analysis demonstrated no significant interaction between the effect of AAV1/SERCA2a and baseline BNP levels for the primary endpoint,17 suggesting that BNP levels might not be related with the negative results of the CUPID 2 trial. However, it is a non-prespecified subgroup analysis, leading to a possible selection bias. Moreover, the number of patients is too small for the subgroup analysis. Therefore, we should not jump to a conclusion that BNP levels are not responsible for failure of the CUPID 2 trial. Regarding the strong fundamental evidence for negative effects of BNP on SERCA2a expression and activity, BNP remains a potential factor interacting with efficacy of transfer. Additional exhaustive studies of interaction between BNP levels and treatment effects of SERCA2a gene transfer are necessary.

If our assumption is correct, another question is why the CUPID 1 trial, which also includes patients with high BNP levels, showed positive results. One possibility is that there was difference in BNP levels between the two studies. Another possibility is that the positive results reported in CUPID 1 were due to chance because the CUPID 1 was a small study.

SERCA2a as a potential target in HFpEF with low BNP levels

In contrast to HFrEF, no evidence-based treatment has been established for HFpEF.66 In fact, despite similar clinical symptoms and prognoses between patients with HFrEF and HFpEF, the HF phenotypes differ significantly from each other with respect to patient characteristics,67 risk factors,68 69 myocardial structure and function,70 71 and pathophysiological mechanism24 26 69 71–76 (table 2).

Table 2

Comparison between HFrEF and HFpEF

The underlying pathophysiology of HFpEF remains poorly understood. Very recently, Tromp et al 77 studied the biomarker profiles in HFpEF and HFrEF, finding that biomarkers interacted with HFpEF were in the domains of inflammation and angiogenesis, while the cardiac stretch markers NT-proBNP and pro-atrial natriuretic peptide interacted with HFrEF. Furthermore, the angiogenesis marker neuropilin and remodelling marker osteopontin were only found to be predicted outcomes in HFpEF. The results indicated that the underlying pathophysiology of HFpEF was associated with inflammation and endothelial function. NT-proBNP levels were reported to be higher in HFrEF. Differences in BNP levels between HFrEF and HFpEF have also been reported in other studies. Data from several studies show that plasma levels of BNP are approximately two to four times higher in patients with HFrEF than in those with HFpEF.65 78–80 Anjan et al 81 studied 159 patients with HFpEF and found that 46 (29%) had normal BNP levels (≤100 pg/mL). Therefore, if SERCA2a gene therapy is only suitable for patients with low BNP levels, then the HFpEF phenotype may be a good choice for SERCA2a gene therapy before a method for preventing the negative effects of BNP on SERCA2a is found. In fact, SERCA2a may play a more important role in HFpEF.

The importance of Ca2+ mishandling in the pathophysiology of HFpEF

The pathophysiological components of HFpEF include cardiac dysfunction (eg, diastolic dysfunction, mild systolic dysfunction, limitations in systolic and chronotropic reserves), vascular impairments (eg, inadequate vasodilatation and endothelial dysfunction) and abnormalities in the periphery (eg, autonomic imbalance and anaemia), which can be caused by ageing, hypertension and metabolic diseases.82 Due to the complex pathophysiology of HFpEF, no effective therapeutic targets have been identified. Nonetheless, existing evidence provides some clues. Among the numerous pathophysiological components, the components involved in the underlying mechanism of diastolic dysfunction are important potential targets because diastolic dysfunction is a key characteristic of HFpEF and is related to worse outcomes in patients with HFpEF.83

Diastolic dysfunction in HFpEF is characterised by two properties: passive stiffness and abnormalities in active relaxation.84 These may be determined by several factors, including changes in the function of myofilament proteins, myocardial fibrosis and alterations of Ca2+ handling proteins.85 However, which abnormality reflects the most meaningful mechanism that could serve as a primary therapeutic target in HFpEF remains unclear. Selby et al 86 used human myocardial tissue samples from patients with left ventricle hypertrophy (LVH) to validate the factors responsible for diastolic dysfunction and found that tachycardia-induced incomplete relaxation of the myocardium was mainly caused by elevated cytosolic Ca2+ levels during diastole. Although the patients had LVH rather than HFpEF, the study indicated that Ca2+ mishandling may play a crucial role in incomplete relaxation and exercise intolerance among patients with HFpEF. However, in contrast to the findings in HFrEF,74 SERCA2a activity was enhanced and the NCX reserve was decreased in the myocardium of patients with LVH. Therefore, Selby et al suggested that the increased diastolic Ca2+ concentration was caused by a decreased NCX reserve rather than changes in SERCA2a function. Though the study of Selby et al stressed the role of decreased Ca2+ extrusion in diastolic dysfunction in LVH, the results may not directly apply to patients with HFpEF.

Many studies have shown SERCA2a upregulation in early diastolic dysfunction, which is considered an adaptive change in cardiac hypertrophy.87 88 Unfortunately, unlike the established decrease in the expression or activity of SERCA2a in HFrEF, studies on SERCA2a in HFpEF are limited due to a lack of appropriate HFpEF animal models and the difficulty of obtaining tissue samples from patients with HFpEF. Whether elevated cytosolic Ca2+ levels are caused by changes in SERCA2a in HFpEF remains to be established. Nonetheless, available data indicate that abnormalities in SERCA2a function play a role in pathophysiology of HFpEF.

The relationship between SERCA2a and HFpEF

Despite a lack of direct evidence, we can deduce impaired SERCA2a function in patients with HFpEF from the following findings. First, studies of animal models or patients with HFpEF-related comorbidities, such as ageing, obesity and diabetes, revealed reduced SERCA2a expression and activity.89–91 Second, inflammatory markers, which are important factors in the initiation and progression of HFpEF,92 could downregulate SERCA2a expression.93 Finally, Tanaka et al 73 observed reduced SERCA2a protein expression in a mouse model of HFpEF induced by aldosterone infusion. Therefore, it is reasonable that SERCA2a expression and activity are compromised in the myocardium of patients with HFpEF. This impairment may play a crucial role in the pathophysiological mechanism of HFpEF, suggesting that SERCA2a may serve as a good target for HFpEF.

Further evidence for the key role of SERCA2a in HFpEF is as follows: (1) cardiomyocyte-specific deletion of SERCA2a in mice (SERCA2a KO) caused diastolic HF94; and (2) SERCA2a gene therapy could reverse ageing-induced diastolic dysfunction7 and endothelial dysfunction,95 which are HFpEF-related risk factors. Therefore, it is possible that impaired SERCA2a function in HFpEF contributes to incomplete relaxation and is associated with other mechanisms, including endothelial dysfunction, inflammation and interstitial fibrosis. So, even without considering the negative effects of BNP, SERCA2a may be a better target in HFpEF than in HFrEF due to its high correlation with the underlying mechanisms of HFpEF.

Conclusions and future perspectives

Despite of controversial results, the CUPID trial has shown that the SERCA2a gene therapy is safe. Therefore, gene therapy is still a promising strategy in the treatment for HF. Insights from the CUPID trial may help overcome obstacles and advance development of gene therapy.

Although there is preliminary evidence supporting suppressive effects of BNP on SERCA2a gene therapy, data with regard to the interaction between BNP levels and treatment effects of SERCA2a gene therapy remain absent. Is there a BNP threshold in managing HF by SERCA2a gene transfer? Is it useful and safe to increase vector doses in patients with high levels of BNP? These problems need to be solved in experimental studies and clinical trials in the future. Moreover, greater insight into the underlying mechanism of inhibitory effects of BNP on SERCA2a transgene expression will help increase SERCA2a gene transfer efficiency by inciting methods to prevent this negative mechanism in the future. In addition, confirming the application range of BNP levels will facilitate the selection of patients with HF in future studies.

It is likely that patients with low BNP levels may benefit most from SERCA2a gene therapy. Since advanced HFrEF usually demonstrates extremely high BNP levels, SERCA2a may be a more appropriate target for the HFpEF phenotype, which shows relatively lower BNP levels compared with the HFrEF phenotype.

To confirm the key role of SERCA2a in active relaxation and the hypothesis that SERCA2a dysfunction may reflect the mechanism of incomplete relaxation in HFpEF, a clinical trial is currently under way to test whether istaroxime, an activator of SERCA2a, can improve diastolic function during exercise in patients with HFpEF (ClinicalTrials.gov identifier: NCT02772068). Other subjects need to be addressed in future studies, including (1) the difference in Ca2+ handling between HFpEF and HFrEF subjects, (2) changes in SERCA2a function and their connection with the pathophysiology of HFpEF, and (3) the effects of SERCA2a gene therapy in HFpEF. Exploring these topics in future experiments will help advance gene therapy for HF.

Unlike monogenic diseases, which is an area where gene therapy is more likely to succeed, HF is a complex and systemic disease. Various factors may lead to unreliable results. This review shows that host factors, genetic basis and phenotypes may interact with treatment effects of gene therapy, indicating that clinical application of gene therapy should be more rigorous for complex disease like HF.

References

Footnotes

  • YZ and YL contributed equally.

  • Contributors YZ and YL contributed equally to this paper. YZ and YL conceived the review, collected literature and wrote the first draft of the manuscript. YZ and PW analysed the relevant literature and created the figures. DL conceived the review and critically revised the manuscript.

  • Funding This work was supported by the National Natural Science Foundation of China (81570326) and the Science and Technology Plan Projects of Xuzhou City (KC16SH099).

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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