Research ArticleHeart Disease

Cardiac AAV9-S100A1 Gene Therapy Rescues Post-Ischemic Heart Failure in a Preclinical Large Animal Model

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Science Translational Medicine  20 Jul 2011:
Vol. 3, Issue 92, pp. 92ra64
DOI: 10.1126/scitranslmed.3002097


As a prerequisite for clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated virus (AAV)–S100A1 gene therapy in a preclinical large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and animals, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium (Ca2+) handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered, by retrograde coronary venous delivery, AAV serotype 9 (AAV9)–S100A1 to the left ventricular, non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed these functional and structural changes by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca2+ cycling, sarcoplasmic reticulum calcium handling, and energy homeostasis. Transgene expression was restricted to cardiac tissue, and extracardiac organ function was uncompromised. This translational study shows the preclinical feasibility of long-term therapeutic effectiveness of and a favorable safety profile for cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our results present a strong rationale for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure.


Heart failure (HF) is the common endpoint of a variety of cardiac diseases, including myocardial infarction (MI) and genetic cardiomyopathies, and is a major cause of morbidity and mortality worldwide (1). Loss of myocardial tissue triggers a sequence of molecular, cellular, and physiological responses, leading to ventricular remodeling and the inability of the left ventricle to maintain an output of blood sufficient for the metabolic requirements of the tissues of the body. Patients with HF show a characteristic maladaptive neurohumoral response including an activation of the renin-angiotensin-aldosterone system and an increased catecholamine concentration in the blood, both causing a temporary increase in cardiac output but long-term detrimental effects such as water and sodium retention and adverse remodeling of cardiac tissue (2). The current standard of care for patients with HF is either symptomatic therapy (for example, by use of diuretics) or treatment that targets the above-mentioned detrimental vicious cycle [with β-adrenergic receptor (β-AR) blockers, angiotensin-converting enzyme (ACE) inhibitors, or aldosterone antagonists]. These approaches provide a moderate increase in survival, limited improvement in cardiac ejection fraction (EF), and reversal of pathological myocardial remodeling (27). Despite these benefits of conventional drugs, the disease progresses relentlessly and more than 50% of patients with end-stage HF die within 5 years (2, 8). This situation reflects a lack of therapies that target the underlying causes of the disease (1, 2, 8, 9).

Coordinated regulation of Ca2+ cycling in the cardiomyocyte is required during each cycle of cardiac relaxation and contraction. Cytosolic Ca2+ is sequestered in the sarcoplasmic reticulum (SR) lumen by cardiac SR Ca2+-ATPase (SERCA2a), permitting muscle relaxation. Subsequently the stored Ca2+ is released through the ryanodine receptor (RyR2) to activate myofilament contraction. Abnormal cardiomyocyte Ca2+ handling is a key factor in HF pathogenesis (10). Among recently discovered molecules controlling this process, the Ca2+ sensor protein S100A1 has emerged as an attractive target for genetically targeted HF therapy (11). In cardiomyocytes, S100A1 regulates the calcium-controlled network of SR, sarcomeric, and mitochondrial function through modulation of RyR2, SERCA2, titin, and mitochondrial F1-ATPase activity (1118). As a result, cardiomyocytes and hearts with increased S100A1 expression show increased systolic and diastolic performance, a result of improved Ca2+ transient amplitudes resulting from augmented SR Ca2+ load and subsequent systolic Ca2+ release together with decreased diastolic SR Ca2+ leak and enhanced Ca2+ resequestration (11, 13, 15, 17). Concurrently, S100A1 increases mitochondrial high-energy phosphate production and thus coordinates the energy supply with the increased adenosine 5′-triphosphate (ATP) demand by the enhanced cardiomyocyte Ca2+ turnover (11, 12, 15).

Reduced S100A1 expression in the myocardium accompanies HF in humans and various animal models, and numerous studies have established a clear association between depleted S100A1 in cardiomyocytes and reduced contractile function, corroborating the pathophysiological significance of this protein (15, 1925). Repairing this molecular defect in small animal HF models by cardiac-targeted S100A1 gene transfer reverses malfunction of the SR and mitochondria and restores cardiac function in the long term (15, 2022). Despite this therapeutic profile in rodent models, translation of this cardiac molecular therapeutic approach to the clinic requires large animal HF models, which more closely approximate human physiology, function, and anatomy (26). This is necessary because a mouse heart operates close to its theoretical maximum and beats about 10 times faster than the human heart, indicating that there are fundamental differences in the cardiac inotropic reserve. Human and mouse have mechanistic differences in the regulation of cardiomyocyte Ca2+ cycling: Ca2+ removal from the cytosol in mice relies almost exclusively on SERCA2a (92% of total), whereas in humans Na+/Ca2+ exchanger (NCX) activity accounts for about one-third of the Ca2+ removal, with SERCA2a being largely responsible for the remainder (10, 27). The basic myocardial sarcomeric proteins are also different, with α-myosin heavy chain predominating in adult mice but β-myosin heavy chain predominating in humans (28). Moreover, technical barriers and safety standards for myocardial gene delivery differ significantly between rodent models and clinically relevant large animal models. The target volume of the myocardium necessary to achieve therapeutic effects is more than a hundred times larger in human compared to rats, requiring highly efficient gene delivery. Moreover, in the clinic, cardiac gene transfer will need to be administered percutaneously with certified, approved material. Thus, as a prerequisite to clinical application, we determined long-term therapeutic efficacy, safety, and feasibility of adeno-associated virus serotype 9 (AAV9)–S100A1 gene therapy in a post-ischemic pig model of HF that recapitulates key clinical features of human HF (26, 29) and explored mechanistic features of its action.


MI of pig hearts serves as a preclinical HF model

Percutaneous, catheter-based intermittent balloon occlusion of the proximal left circumflex coronary artery (LCX) resulted in a reproducible perfusion defect of the lateral left ventricular (LV) wall (30 ± 4%, n = 32) with a transmural infarction (Fig. 1, A to E). Infarcted pigs demonstrated systolic LV dysfunction after 2 weeks with transition to failure (EF = 39 ± 4%, n = 18) and remodeling over a follow-up period of 14 weeks (Fig. 1, F to I).

Fig. 1

Porcine HF model. (A) Radioscopic image showing catheter-based left circumflex coronary artery (LCX) occlusion. 1, inflated balloon; 2, guided wire; 3, left anterior descending coronary artery (LAD). (B and C) Perfusion echocardiography showing normal (B) and defective (C) lateral LV wall perfusion corresponding to an open (B) or occluded LCX (C). (B and C, inset) Electrocardiogram of respective heart. Note the concomitant ST-segment elevation in (C). (D) Representative infarcted pig heart 2 weeks after LCX occlusion (black arrow, infarcted area). (E) Triphenyltetrazolium chloride–stained, mid-ventricular section demonstrating scar formation 14 weeks after MI. (F to I) Decreased LV function as assessed by echocardiography [ejection fraction (EF)] and LV catheterization (+dp/dt), as well as LV dilation [end-diastolic diameter (EDD)] and LV hypertrophy [heart weight/body weight (HW/BW)] compared to sham-operated pigs (n = 13) 14 weeks after MI. *P < 0.05, HF (n = 23) versus sham. Data are presented as means ± SEM. Sham pigs received a cardiac catheterization procedure without occlusion of the LCX.

Retrograde coronary venous AAV9-S100A1 gene delivery enhances S100A1 expression levels in failing hearts

Two weeks after MI, pigs (n = 32) were randomized to saline (n = 14), control virus (AAV9-Luc, n = 9), and S100A1 gene-based treatment (n = 9). The AAV9-S100A1 vector carrying the human S100A1 complementary DNA (cDNA) under the control of a cardiomyocyte-specific promoter (CMV-MLC) was delivered at a dosage of 1.5 × 1013 total vector particles (tvp) per animal (~5 × 1011 tvp/kg), targeting non-infarcted anterior and septal LV myocardium (Fig. 2, A and B), whereas AAV9-CMV-MLC2-Luc and saline were given to the control groups. Fourteen weeks after MI, myocardial S100A1 protein expression was decreased in control treatment groups compared to sham-operated, non-infarcted pigs (Fig. 2C). S100A1 gene therapy restored expression of the S100A1 protein in targeted non-infarcted LV myocardium (Fig. 2D). Assessment of S100A1 protein abundance in extracardiac tissues including brain, lung, skeletal muscle, and liver 3 months after gene delivery showed unchanged S100A1 protein expression in both control and AAV9-S100A1 groups (fig. S1). The selective presence of luciferase activity in the anterior LV wall verifies the feasibility of cardiac-restricted and targeted gene delivery (Fig. 2E) by our minimally invasive, catheter-based delivery technique combined with a vector system for cardiomyocyte-specific gene expression.

Fig. 2

Cardiac-targeted AAV9-S100A1 gene therapy. (A) Radioscopic image showing the retroperfusion catheter (1) to deliver AAVs and the guide wires in the ACV (2) and in the left LAD (3). (B) Location of the non-infarcted posterior LV wall, which is a nontargeted area (4), and the anterior wall, which is a targeted area (5) for gene delivery. (C and D) Representative Western blots showing decreased myocardial S100A1 expression 14 weeks after MI in HF-Luc [48 ± 19%, P < 0.05, HF-Luc (n = 9) versus sham (n = 13)] and reconstituted S100A1 protein expression after AAV9-S100A1 gene delivery [320 ± 030% higher, P < 0.05, HF-S100A1 (n = 9) versus HF-Luc (n = 9)] (D). Equal loading for each tissue was confirmed by calsequestrin (CSQ) staining, a Ca2+ binding protein with unaltered expression in HF. (E) Analysis of luciferase activity of targeted (anterior) and nontargeted (posterior) LV wall in HF-Luc myocardium. *P < 0.05 versus posterior LV wall and liver (n = 9 for HF-Luc myocardial and liver samples). Data are presented as means ± SEM.

S100A1 therapy rescues cardiac function and reverses remodeling in failing pig myocardium in vivo

Twelve weeks after gene therapy, AAV9-S100A1–treated HF animals showed significant improvement in both systolic and diastolic LV performance compared to HF control groups (Fig. 3, A to D). In line with the improvement of global cardiac function, the elevated heart rate seen in HF control groups was normalized by S100A1 therapy (Fig. 3E). Analysis of LV remodeling revealed profound anti-hypertrophic effects after S100A1 gene therapy as reflected by significantly lower adjusted heart weight and smaller end-diastolic LV diameter, as well as reversed LV expression of brain natriuretic peptide (BNP) (Fig. 3, F to H) compared to the HF control groups. Increased cardiac contractile function in S100A1-treated HF pigs was evident with maximum stimulation of the β-AR with dobutamine, indicating preserved β-AR signaling, and thus an increased inotropic cardiac reserve, compared to HF control groups. Sham-treated pigs showed enhanced inotropic reserve compared to all HF groups, including HF pigs treated with S100A1 (Table 1).

Fig. 3

AAV9-S100A1 gene therapy rescues cardiac function and reverses myocardial remodeling in failing myocardium. (A to E) Twelve weeks after AAV9-S100A1 treatment, failing hearts with reconstituted S100A1 expression (n = 9) exhibit restored LV function compared to HF-saline (n = 14) or HF-Luc (n = 9). (F to H) S100A1 gene therapy attenuated LV remodeling, which was corroborated by reversed fetal gene activation. P < 0.05, sham versus HF-saline and HF-Luc; *P < 0.05, HF-S100A1 versus HF-saline and HF-Luc; **P < 0.05, sham versus all other groups. For (H), brain natriuretic peptide (BNP) mRNA was assessed in the LV anterior wall of five animals in each group and normalized to sham. EF, ejection fraction; EDP, end-diastolic pressure; HW/BW, heart weight/body weight; EDD, end-diastolic diameter. Data are presented as means ± SEM. n.s., not significant.

Table 1

Preserved cardiac inotropic reserve and blood biomarkers 14 weeks after AAV9-S100A1 and control AAV9 vector treatment (n = 13 for sham, n = 9 each for HF-Luc and HF-S100A1, and n = 14 for HF-saline). Leukocyte numbers of 9 to 15.7/nl reflect normal values in pigs (48). LVEDP, left ventricular end-diastolic pressure; LVESP, left ventricular end-systolic pressure.

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S100A1 gene therapy restores cardiomyocyte and SR calcium handling in failing myocardium

Isolated cardiomyocytes and SR vesicles from HF control groups, 14 weeks after MI, exhibited decreased systolic Ca2+ transient amplitudes and diastolic Ca2+ overload, as well as diminished SR Ca2+ uptake and enhanced Ca2+ leak (Fig. 4, A to F). In contrast, cardiomyocytes from S100A1-treated myocardium showed significantly higher Ca2+ transient amplitudes and lower diastolic Ca2+ levels than did control HF cardiomyocytes (Fig. 4, A to D). Assessment of Ca2+ fluxes in isolated LV SR vesicles from AAV9-S100A1–treated, myocardium yielded significantly reduced Ca2+ leak and enhanced Ca2+ uptake compared to HF controls (Fig. 4, E and F). Immunoprecipitation of both RyR2 and SERCA2 from S100A1-treated and control failing myocardium showed significantly enhanced S100A1/RyR2 and S100A1/SERCA2 binding ratios in the treated group (Fig. 5, A to F). Consistently, SR vesicles from AAV9-S100A1–treated myocardium exhibited significantly lower [3H]ryanodine binding at 150 nM free Ca2+ concentrations ([Ca2+]) than did myocardium from the control groups, indicating improved diastolic RyR2 closure in the presence of S100A1. Addition of human recombinant S100A1 protein (500 nM) partially reversed abnormal [3H]ryanodine binding in SR vesicles from HF control groups (Fig. 5, G and H) (20).

Fig. 4

AAV9-S100A1 gene therapy rescues SR Ca2+ handling in failing myocardium. (A) Representative Ca2+ tracings from cardiomyocytes isolated from the anterior LV wall of sham-, HF-saline–, AAV9-Luc (HF-Luc)–, and AAV9-S100A1 (HF-S100A1)–treated pig hearts. (B) Light microscopy of isolated pig cardiomyocytes. Scale bar, 100 μm. (C and D) S100A1-treated cardiomyocytes exhibit greater Ca2+ transient amplitude and decreased diastolic Ca2+ levels compared to the HF control group (n = 30 cells from three different animals in each group). (E and F) Fluo-3–based assessment of Ca2+ leakage and uptake in SR vesicles isolated from LV anterior wall segments reveals improved SR function in AAV9-S100A1–treated failing myocardium compared with HF control groups (n = 5 SR preparations from different animals in each group). P < 0.05, sham versus HF-saline and HF-Luc; *P < 0.05, HF-S100A1 versus HF-saline and HF-Luc; **P < 0.05, HF-S100A1 versus all other groups. Data are presented as means ± SEM.

Fig. 5

AAV9-S100A1 gene therapy reverses abnormal S100A1/SR target protein ratios and improves SR Ca2+ handling in failing myocardium. (A and B) Representative immunoprecipitation (IP) for SERCA2 and RyR2 from sham-, saline-, AAV9-Luc–, and AAV9-S100A1–treated myocardium and corresponding Western blots for coprecipitation of S100A1 protein. (C and D) Immunoprecipitation of SERCA2 and RyR2 from sham-, HF control–, and S100A1-treated myocardium. SR from five different hearts (LV anterior wall) was prepared for each group, and immunoprecipitations were conducted in duplicate from each SR preparation with equal amounts of protein (300 μg). (E and F) S100A1/SERCA2 and S100A1/RyR2 binding ratios in sham-, HF control–, and S100A1-treated groups. Ratios are restored after AAV9-S100A1 treatment. (G) SR Ca2+ leak measured by [3H]ryanodine binding at 150 nm free Ca2+ in sham-, HF control–, and S100A1-treated groups. Values are higher in the S100A1-treated group. (H) Addition of human recombinant S100A1 protein to SR vesicles from HF control groups significantly reverses abnormally high [3H]ryanodine binding. P < 0.05, sham versus HF-saline and HF-Luc; *P < 0.05, HF-S100A1 versus HF-saline and HF-Luc; P < 0.05, HF-saline and HF-Luc + S100A1 protein versus corresponding HF-saline or HF-Luc. n = 5 in each group. Internal control for immunoprecipitation is shown in fig. S3.

Cardiomyocyte-targeted AAV9-S100A1 gene therapy expression reconstitutes energy homeostasis in failing myocardium

Myocardium from HF control groups showed reduced energy homeostasis (Fig. 6, A and B), whereas assessment of bioenergetic surrogate markers including high-energy phosphate and dinucleotide content revealed significantly higher phosphocreatine (PC)/ATP and nicotinamide adenine dinucleotide reduced form/oxidized form (NADH/NAD) ratios in S100A1-treated HF myocardium compared to HF control groups (Fig. 6, A and B).

Fig. 6

AAV9-S100A1 gene therapy restores high-energy phosphate and dinucleotide content in failing myocardium. (A and B) Assessment of the bioenergetic marker PC/ATP (A) and NADH/NAD (B) revealed significantly decreased values in HF control groups compared to sham. AAV9-S100A1 gene therapy restores PC/ATP and NADH/NAD ratios to normal, indicative of reconstituted energy homeostasis and redox state in failing hearts (n = 6 different animals in each group; each measurement was carried out in triplicate). P < 0.05, sham versus HF-saline and HF-Luc; *P < 0.05, HF-S100A1 versus HF-saline and HF-Luc. Data are presented as means ± SEM.

Cardiac-targeted S100A1 gene therapy yields a favorable safety profile

Twelve weeks after AAV9-S100A1 gene therapy, leukocyte, erythrocyte, and platelet counts, as well as hemoglobin concentrations were similar in sham and HF animals treated with saline, AAV9-Luc, or AAV9-S100A1 (Table 1). Accordingly, in all groups, sodium, potassium, and glucose blood concentrations were similar, and pancreatic enzymes, kidney retention parameters, and serum liver enzymes were within the physiological normal range for pigs (Table 1).


The protein S100A1, found in cardiac myocytes, controls intracellular Ca2+ cycling by increasing SERCA2a activity and regulating the open probability of the RyR2, which causes a reduced diastolic SR Ca2+ leak and increased systolic contractility of the cardiomyocyte in various species including human (11, 30). Concurrently, S100A1 enhances ATP generation via mitochondrial F1-ATPase. These features of this protein, together with the evolution of safe and efficient gene transfer technologies that overcome previous barriers such as the lack of gene delivery efficiency and use of invasive techniques, provide a rationale for the use of S100A1 gene therapy to treat HF (11, 19, 31, 32). Our data show long-term therapeutic effectiveness and a favorable safety profile of AAV9-S100A1 gene therapy in a preclinical large animal (porcine) HF model with a clinically relevant, catheter-based gene delivery approach.

Insight into the molecular and cellular basis of S100A1 cardiovascular biology and therapeutic potential has come from rodent models (11). However, important differences exist between small animals and humans with regard to safety and technical aspects of gene therapy. Anatomically, the required target area for gene transfer is much larger in humans than in rodents, requiring a highly efficient gene delivery technique. Clinical application of the S100A1 transgene should be achieved in a nonsurgical, minimally invasive percutaneous approach with certified catheters. In addition, cardiac characteristics differ such as energy homeostasis, the β12-AR ratio, and key aspects of excitation-contraction coupling (26, 33). The significantly lower heart rate in humans than in rodents accentuates pulsatile ejection of the blood and thus affects cardiac diastolic and systolic contractile properties. At the molecular level, this is reflected by a difference in the predominant sarcomeric protein. β-Myosin heavy chain is predominant in humans and shows lower ATPase activity and a reduced maximum velocity, but a higher tension-time integral compared to the α-isoform (28). The NCX/SERCA2a ratio is larger in humans and large animals compared to rodents, which is a concern for therapeutic strategies targeting cardiomyocyte Ca2+ cycling (10, 27). Ablation of phospholamban, a protein that is also involved in cardiomyocyte Ca2+ cycling through its regulation of SERCA2a activity, has opposite effects in humans and mouse models (34). Therefore, large animal models of HF such as pig, sheep, and dog, which more closely approximate human physiology, function, and anatomy, are a critical link in translating basic concepts into clinical therapies (26).

As shown in human HF, myocardial S100A1 protein expression is diminished in our post-ischemic pig HF model concomitant with progressive deterioration of cardiac function, an enhanced heart rate, and adverse remodeling of the myocardium, as well as impaired PC/ATP and NADH/NAD ratios. To correct reduced S100A1 expression in non-infarcted failing myocardium, we used a minimally invasive, percutaneous gene delivery technique developed by von Degenfeld et al. (32). We used an AAV9 vector driving expression of the human S100A1 cDNA under control of a cardiac promoter to achieve long-term restoration of S100A1 expression in targeted LV regions (21, 35). Control experiments showed the absence of extracardiac expression of the therapeutic transgene, demonstrating both selectivity and feasibility of the expression system and delivery technique. Notably, biodistribution of AAVs (as measured by the amount of human S100A1 DNA found in various porcine tissues) further corroborates the safety of this procedure: Only in the liver and skeletal muscle did we detect modest amounts of human S100A1 DNA; in all other nontarget organs, human S100A1 DNA was not detectable (fig. S2).

Genetically targeted correction of reduced myocardial S100A1 expression, a hallmark of advanced HF, not only prevented progressive deterioration but also resulted in long-term rescue of global cardiac function. In line with other previous studies, the therapeutic effects of S100A1 are a result of its ability to improve both systolic and diastolic SR Ca2+ handling (9, 11, 19). We have previously shown that S100A1 binds to and enhances opening of the RyR2 during systole, thereby augmenting SR Ca2+ release independent of Ca2+ load (9, 11, 14, 20). Prevention of diastolic RyR2 leakage by S100A1, together with increased SERCA2 activity to remove Ca2+ from the cytosol into the SR, causes decreased diastolic cytosolic Ca2+ levels and an increased SR Ca2+ load (9, 11, 15, 17, 20, 36). Accordingly, isolated LV cardiomyocytes from AAV9-S100A1–treated failing pig hearts exhibited restoration of intracellular Ca2+ cycling, reflecting improved function of RyR2 and SERCA2. In particular, our results show improved diastolic RyR2 closure in AAV9-S100A1–treated myocardium and correction of defective systolic RyR2 function by recombinant S100A1 protein in failing control groups.

Our in vivo data are corroborated by the S100A1-induced reversion of Ca2+ cycling defects in isolated LV cardiomyocytes and SR vesicles. These changes recapitulate characteristic abnormalities of human failing cardiomyocytes concerning Ca2+ handling such as a reduced Ca2+ transient amplitude, increased cytosolic diastolic Ca2+ and an increased diastolic Ca2+ leak, as well as a reduced PC/ATP ratio, consistent with a previous report by Zhang et al. with a similar HF model (29, 37).

The S100A1-mediated long-term improvement of myocardial function was accompanied by improvements in cardiac remodeling, as reflected by a decreased heart-to-body weight ratio. The significant decrease in both end-diastolic LV pressure and diameter in S100A1-treated HF pigs indicates that diminished diastolic wall stress eventually attenuated hypertrophic paracrine signaling and maladaptive growth. The normalized heart rate of S100A1-treated HF pigs suggests that our treatment abrogated the maladaptive β-AR sympathetic overdrive, a likely result of improved cardiac output (38). This important effect could not be observed in small animal HF models, underscoring the clinical relevance of the pig HF model used here (15, 21, 33). Consistent with previous reports revealing improved β-AR–dependent contractile reserve in S100A1-treated rodent HF models (2022), the S100A1-mediated increase in global contractile function was preserved under stimulation with the β-adrenergic agonist dobutamine in failing pig hearts.

From large clinical trials of current standard HF therapy such as β-blockers, ACE inhibitors, and aldosterone antagonists that aim to limit the progress of the disease, we learned that the EF of patients with severe HF treated with these agents shows only a slight increase, showing that cardiac function cannot be rescued by current conventional drug therapy. This circumstance results in a large percentage of highly symptomatic patients with dyspnea (18). Combining the current standard of care with S100A1 gene therapy could translate into clinical benefit of patients with HF because S100A1 significantly increases cardiac contractile function. Therapeutic effects of S100A1 gene therapy are preserved during β-adrenergic antagonist treatment with metoprolol, offering the possibility of ameliorated cardiac reverse remodeling, inotropic action, and anti-arrhythmic effects (21).

Inotropic therapeutic interventions in HF based on manipulation of adenosine 3′,5′-monophosphate (cAMP)–dependent signaling acutely increase cardiac function but show long-term detrimental consequences on heart rate and energy homeostasis and are therefore controversial (39, 40). S100A1 activity does not depend on cAMP-dependent protein kinase A (PKA) and calmodulin-dependent kinase (CaMK) signaling (13, 14, 20). In contrast, long-term S100A1 gene therapy enhanced the PC/ATP ratio, indicative of restored energy flux in failing cardiomyocytes, and improved the NADH/NAD ratio, suggesting restored mitochondrial function (41, 42).

This study further demonstrates a clinically relevant, favorable therapeutic opportunity for post-ischemic S100A1 gene therapy. Patients suffering from cardiac ischemia may not necessarily require immediate intervention with this therapy but can be treated after the clinical event even when already experiencing cardiac dysfunction. Notably, the body weight–adjusted AAV vector dosage used in our study matches the range used in preclinical AAV vector toxicology studies in pigs, which revealed no signs of toxicity by clinical or histopathological assessment (43). In addition, our study highlights the safety of S100A1 gene therapy because it showed cardiac-restricted expression of the S100A1 transgene without adverse effects on blood or organs such as kidney, pancreas, and liver up to 3 months after treatment.

As a final step before clinical application, our translational study shows long-term therapeutic efficacy together with a favorable safety profile of AAV9-S100A1 gene therapy delivered by a clinically relevant approach in a preclinical HF model that approximates human cardiovascular physiology and anatomy. Clinical application of S100A1-targeted gene therapy will have to be tested in a human HF trial and may potentially complement future treatment of end-stage HF.

Materials and Methods

All animal procedures and experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and approved by the local Animal Care and Use Committee of Baden-Württemberg, Germany.

Animal procedures, echocardiography, and catheter-based cardiac gene therapy

Post-ischemic HF was induced in German farm pigs (Bräunling; mean body weight: 30 ± 4 kg) by temporary (2 hours) occlusion of the proximal LCX with a percutaneous transluminal coronary angioplasty balloon (Boston Scientific). On the day of cardiac gene transfer and at the end of the study (2 and 14 weeks after MI), we performed echocardiography and LV catheter–based hemodynamic measurements with a Sonos 5500 imaging system (Philips) and a 5-French pressure catheter (SPC-350; Millar instruments). To examine LV, we administered inotropic reserve dobutamine (100 μg/kg) via an ear vein. Myocardial perfusion echocardiography was used to verify comparability of area at risk during occlusion of the LCX and was performed similar to described protocols (44, 45). Myocardial contrast imaging was performed in a mid short axis view in a harmonic power Doppler mode (Sonos 5500; Philips) with a broadband transducer (S3) with a mean transmission frequency of 1.2 MHz, a receiving frequency of 3.0 MHz, a mechanical index of 1.2, and a 1:3 systolic trigger rate. A commercially available second-generation contrast agent (SonoVue, Bracco) was infused intravenously at a rate of 1 ml/min during image acquisition. Complete myocardial area and the perfusion defect area were traced to allow calculation of their ratio in the mid short axis view.

Myocardial gene transfer of HF pigs (2 weeks after MI) was achieved by selective retroinfusion as described with some modifications (32). Briefly, a 7-French balloon wedge pressure catheter (Arrow) was placed in the anterior cardiac vein (ACV). While the left anterior descending coronary artery (LAD) was occluded distal of the first diagonal branch, with a 3.5 × 8–mm balloon, three times each for 3 min to stop antegrade blood flow, 1.5 × 1013 tvp of the AAV9 constructs in 50 ml of saline were retrogradely injected in the ACV. Afterward, the integrity of the LAD was checked by angiography.

AAV9 vector production

High-titer vectors were produced with a triple transfection approach of 293T cells in cell stacks (Corning) with polyethylenimine, harvested after 48 hours, and purified by filtration and iodixanol as described before (35).

Western blot analysis and immunoprecipitation

Immunoprecipitation for RyR2 and SERCA2 was carried out with Protein A/G PLUS-Agarose beads (sc-2003, Santa Cruz Biotechnology) as previously described (20). Coprecipitated S100A1 was stained (Acris #SP5355P, 1:1000) and reprobed for RyR2 (ABR, MA3-916, 1:1000) and SERCA2 (Santa Cruz Biotechnology, sc-73022, 1:500) and appropriate secondary antibodies coupled to fluorescent 680 or 800 dyes (1:2000). Western blot was carried out as described with anti-S100A1 antibody (SA 5632, 1:1000, custom-made; detecting the human and the pig isoform of S100A1) (23).

Isolation of adult porcine failing cardiomyocytes

Adult porcine cardiomyocytes were isolated from myocardial tissue (2 × 2 cm) of the anterior basal LV wall 12 weeks after S100A1 gene therapy (14 weeks after MI) with a standard enzymatic digestion procedure based on described methods (46). Cardiomyocytes used for Ca2+ measurements were plated at a density of 20,000 cells/cm2 on laminin-coated glass dishes and cultivated as described (20).

Ca2+ transient analysis of isolated adult failing pig cardiomyocytes

Intracellular Ca2+ transients of Fura 2-AM–loaded (1.0 μM for 20 min at 37°C) adult pig cardiomyocytes were measured 2 hours after myocyte isolation as described (20, 21). Measurements were carried out with an inverse Olympus microscope (IX 70) with an ultraviolet (UV) filter connected to a monochromator (Polychrome II, TILL Photonics) with a biphasic electric pulse at 37°C. Twenty steady-state twitches for each cardiomyocyte at 1 Hz and 2.5 mM [Ca2+]e (extracellular calcium concentration) were averaged and analyzed by TILL Vision software (version 4.01) (20). Myocytes were isolated from three pigs of each group.

Assessment SR Ca2+ handling and [3H]ryanodine binding

Preparation of SR vesicles from LV anterior wall samples and SR Ca2+ uptake and SR Ca2+ leak were assessed with the fluorescent Ca2+ indicator Fluo-3 salt, as described, with a heated spectrophotometer (15). For Ca2+ uptake measurements, SR vesicles (0.3 mg/ml) were incubated in 0.2 ml of reaction solution containing 0.15 mM potassium gluconate, 1 mM MgCl2, 0.2 mM EGTA-Ca2+ buffer (free [Ca2+] 0.3 μM), 10 mM NaN3, and 20 mM Mops (pH 7.0), and 10 μM ruthenium red was added to inhibit RyR2 opening. Free [Ca2+] was calculated with Maxchelator (47). [3H]Ryanodine binding experiments were performed with SR vesicle preparations prepared from LV anterior wall samples from each group as described (20). Membranes were incubated at 37°C with 6 nM [3H]ryanodine (Perkin Elmer) in 300 μl of 20 mM Pipes (pH 7.1), 150 mM KCl, 0.5 mM MgCl2, 15 mM NaCl, 3 mM EGTA, 10 mM caffeine, 1% phosphatase inhibitors (Sigma; inhibitor mix I/II), protease inhibitors (one tablet per 5 ml) (Roche Applied Science; Mini Complete EDTA-free protease inhibitors), and 150 nM free Ca2+. Human recombinant S100A1 protein (500 nM) was generated and purified as described (20) and added to HF control groups 30 min before [3H]ryanodine incubation. Nonspecific binding was determined with 1000-fold excess of unlabeled ryanodine.

Measurement of luciferase activity

Luciferase reporter activities were determined with the luciferase assay kit from Promega in a luminometer (Lumat LB9501; Berthold) as described (31).

Real-time reverse transcription–polymerase chain reaction

Genomic DNA and total RNA from tissue were isolated as described (20, 22). Real-time reverse transcription–polymerase chain reaction (RT-PCR) was performed in duplicates with a 1:100 dilution of the cDNA on a MyIQ real-time PCR detection system (Bio-Rad) with the SYBR Green PCR master mix (Bio-Rad). The following GenBank cDNA sequences were used to design oligonucleotide primers to examine expression of porcine BNP (M23596, forward primer 5′-cccgcagtagcatcttcca-3′, reverse primer 5′-ttgctttgaaggggagcag-3′) and human S100A1 (NM006271.1, forward primer 5′-cgatggagaccctcatcaa-3′, reverse primer 5′-tggaagtccacctccccgtc-3′). For normalization, 18S ribosomal RNA was used (forward primer 5′-tcaagaacgaaagtcggagg-3′, reverse primer 5′-ggacatctaagggcatcac-3′). PCR conditions were 95°C, 3 min, and 40 cycles of 95°C, 10 s; 60.5°C, 45 s. Specificity of PCR products was confirmed by gel electrophoresis.

Measurement of bioenergetic markers

PC and ATP in the LV anterior wall samples were assessed as described (15, 20). NADH and NAD levels were enzymatically quantified with a kit from BioVision (#K337-100) following the manufacturer’s protocol.

Hematology, electrolytes, and clinical chemistry

Analyses were carried out according to the International Federation of Clinical Chemistry primary reference procedures for the measurement of catalytic activity of enzymes at 37°C, as well as standard methods with indirect potentiometry for electrolytes.

Statistical analysis

Data are expressed as means ± SEM. Unpaired Student’s t test was used when appropriate. To compare means among three or more independent groups, we performed one-way analysis of variance (ANOVA) measures for statistical comparisons. A Bonferroni test was applied whenever multiple comparisons were conducted. For statistical analysis, GraphPad Prism software was used and the P values shown are adjusted in function for the number of comparisons. For all tests, a value of P < 0.05 was accepted as statistically significant.

Supplementary Material

Fig. S1. Cardiac-targeted AAV9-S100A1 gene therapy.

Fig. S2. AAV biodistribution.

Fig. S3. Specificity controls for immunoprecipitation experiments.


  • * These authors contributed equally to this work.

References and Notes

  1. Acknowledgments: We thank R. Eudenbach, J. Kleinschmidt, and the German Cancer Research Center vector core production unit for generating high-titer AAV vector stocks. Funding: This work was supported by NIH grants R01HL92130 and R01HL92130-02S1 (P.M.) and P01HL075443, R01HL56205, and R01HL061690 (W.J.K.); Deutsche Forschungsgemeinschaft grants 1654/3-2 (O.J.M.) and 562/1-1 (P.M. and S.T.P.); Bundesministerium für Bildung und Forschung grant 01GU0527 (P.M., O.J.M., P.B., and H.A.K.); and BioFuture grant (R.B.). Author contributions: S.T.P. and P.M. wrote the manuscript. S.T.P., C.S., J.K., P.R., W.J.K., H.A.K., O.J.M., and P.M. designed the experiments. S.T.P., C.S., J.K., R.B., P.B., R.H., S.S., B.L., J.L., C.W., and O.J.M. performed the experiments and analyzed the data. G.Q. conducted biochemical assays. Competing interests: P.M. and H.A.K. have filed U.S. and EU patent applications on the therapeutic use of the S100A1 protein to treat heart failure.
  • Citation: S. T. Pleger, C. Shan, J. Ksienzyk, R. Bekeredjian, P. Boekstegers, R. Hinkel, S. Schinkel, B. Leuchs, J. Ludwig, G. Qiu, C. Weber, P. Raake, W. J. Koch, H. A. Katus, O. J. Müller, P. Most, Cardiac AAV9-S100A1 Gene Therapy Rescues Post-Ischemic Heart Failure in a Preclinical Large Animal Model. Sci. Transl. Med. 3, 92ra64 (2011).

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