Prothymosin α Gene Transfer Modulates Myocardial Remodeling after Ischemia-Reperfusion Injury

Abstract

Background

Prothymosin α (ProT), a polypeptide, attenuates inflammation and inhibits transforming growth factor (TGF)-β signaling in pulmonary tissues. We investigated the potential role of ProT in myocardial ischemia-reperfusion (MyoIR) injury using ProT cDNA transfer.

Methods

Serum ProT levels were investigated in cardiogenic shock patients with MyoIR (n = 9). In addition, the myocardium of Sprague-Dawley rats (n = 52) was subjected to 25 min of ischemia followed by an injection of adenoviral vectors (2 × 10⁹ plaque-forming units) carrying ProT or the luciferase gene, 10 min before reperfusion. Echocardiography, serum ProT, and biochemical analyses of organ functions were performed before euthanasia, 14 days after treatment. Immunohistochemistry and immunoblotting of the myocardial tissue were also performed.

Results

Serum ProT levels were transiently elevated in the rats and patients early after MyoIR, which was reduced to baseline levels in control rats and patients. ProT gene transfer persistently mobilized ProT serum levels, reduced dilatation, attenuated fibrotic changes, and preserved the left ventricular ejection fraction after MyoIR. Tissue thrombospondin-1 level was abundant, and matrix metalloproteinase-2, collagen I, and collagen IV levels were decreased in the treatment group. While TGF-β protein level remained stable, ProT transduction mobilized Smad7, which counteracted TGF-β. ProT reduced tissue microRNA-223 expression, inhibited the associated interleukin-1β, and preserved RAS p21 protein activator 1 protein abundance.

Conclusions

An increase in transient serum ProT levels could be a protective response in the acute stage of MyoIR. ProT gene transfer further preserved ventricular morphology and function through anti-inflammatory and anti-fibrotic effects in the subacute stage after injury.

INTRODUCTION

In the modern era of prevalent primary percutaneous coronary intervention (PCI), coronary artery disease remains the major cause of heart failure,^1,2 and over 60% of patients with heart failure have coronary artery disease.³ Moreover, these patients have been reported to have a 20% incidence of developing new cardiovascular events within 6 months after the initial coronary intervention.³ Reperfusion of the myocardium after a coronary intervention for an ischemic episode can trigger a second wave of myocardial injury. The activation of oxidative stress and inflammatory responses induced by reperfusion injury contributes to the occurrence of transient myocardial dysfunction.⁴ The generation of fibroblasts in the subsequent myocardial-repair (subacute) stage can result in cardiac remodeling.^1,5 In addition, the presence of heart failure secondary to myocardial ischemia-reperfusion (MyoIR) is a crucial factor determining long-term survival.¹ Mechanical circulatory support such as extracorporeal membrane oxygenation (ECMO) remains palliative rather than therapeutic for patients experiencing cardiogenic shock after the development of myocardial infarction.⁶ This necessitates investigations for novel therapies.

Prothymosin α (ProT) is a highly conserved mammalian polypeptide containing 109-113 amino acids. ProT is involved in the elicitation of anti-inflammatory reactions, anti-apoptotic responses, immune modulation, cardiac endothelial cell migration, and angiogenesis.^7,8 Our previous study showed that ProT in pulmonary tissue was involved in inhibiting the canonical transforming growth factor (TGF)-β signaling pathway through the post-translational regulation of acetylated-Smad7, an inhibitory Smad in TGF-β signaling.⁷ TGF-β and its downstream mediators Smad2/3 contribute to the pathological process of cardiac fibrosis induced by MyoIR.⁹ However, the role of ProT in MyoIR has not been fully explored. In this study, we investigated serum ProT protein levels among patients who had MyoIR. We further hypothesized that ProT gene transfer might reverse myocardial remodeling after MyoIR. A rat model was used to test this hypothesis and validate the preliminary clinical findings. Collectively, this study aimed to provide a novel target gene for potential treatment against MyoIR.

METHODS

Human serum collection

The study protocol was approved by the ethics committee of National Cheng Kung University (NCKU) Hospital (approval number: B-ER-107-380). We included patients aged 20-70 years who were admitted for PCI due to acute coronary syndrome from August 1, 2020, to December 31, 2020. A total of 9 patients were included in the study. ECMO support was necessary for all patients within half an hour from PCI owing to extensive myocardial injury. Detailed information on patient inclusion and clinical management is provided in the extended methods section in the Data Supplement and Supplementary Table 1. Informed consent was obtained from each study subject or the subject’s legal surrogates before blood sampling, as appropriate. Blood samples were obtained on ECMO day 1 (D1, the day of MyoIR) and D3 after ECMO removal (when the myocardium had recovered from reperfusion injury). Serum from male gender-matched healthy volunteers was obtained as a negative control. The serum was stored at -80 °C and processed for further analysis of ProT protein concentrations.

Supplementary Table 1. Characteristics of patients with myocardial ischemia-reperfusion injury after percutaneous coronary intervention.

Variables	Patients (n = 9)
Age (years)	53.73 (50.31-66.93)
Sex (male: female)	9:0
Body weight (kg)	75.0 (52.2-102.0)
Body height (cm)	170.0 (160.0-180.0)
STEMI: NSTEMI	7:2
OHCA	5
IHCA	3
Coronary artery lesion vessels
Left main	1
Left anterior descending	8
Left circumflex	5
Right coronary artery	7
Percutaneous coronary intervention	7
Percutaneous balloon angioplasty	2
Comorbidities
Chronic kidney disease	2
End-stage renal disease	1
Diabetes mellitus	4
Hypertension	1
Ventricular arrhythmia	6
Timing of coronary intervention
< 30 minutes before ECMO implantation	1
< 30 minutes after ECMO implantation	8
ECMO associated comorbidities
Seizures	1
Hemodialysis	3
Sepsis	1
Paroxysmal Afib	3
ECMO support duration (days)	8.0 (4.0-13.0)
Hospital stays (days)	22.0 (18.0-39.0)
APACHE II	23.0 (17.0-29.0)
APACHE IV	84.0 (54.0-102.0)

Continuous data are presented as median (ranges).

Afib, atrial fibrillation; APACHE, “Acute Physiology and Chronic Health Evaluation” score system; CPCR, cardiopulmonary circulatory resuscitation; ECMO, extracorporeal membrane oxygenation; FFP, fresh frozen plasma; IHCA, in-hospital circulatory arrest; NSTEMI, non-ST segment elevation myocardial infarction; OHCA, out-of-hospital circulatory arrest; PRBC, packed red blood cells; STEMI, ST segment elevation myocardial infarction.

Animal model

All procedures were performed in accordance with the guidelines of the Animal Care and Use Committee of NCKU, Taiwan. Age-matched young male Sprague-Dawley rats (weight: 200-250 g) were housed in the Laboratory Animal Center of NCKU with 13 h light and 11 h dark cycles. They were fed a standard chow diet and provided water ad libitum. The detailed procedures for MyoIR are described in the Data Supplement.

Construction of recombinant adenoviral vectors

The detailed method has been described previously.¹⁰ Briefly, to generate the recombinant adenovirus, termed AdProT, the adenoviral transfer vector pAd5L-ProT and the adenoviral type 5 genomic vector were co-transfected into human embryonic kidney 293 cells using the calcium phosphate precipitation method. Recombinant adenovirus encoding firefly luciferase, termed AdLuc, was used as the control virus.^11-13

Treatment protocol

The rats were randomly allocated into MyoIR + AdProT or MyoIR + AdLuc groups after receiving a single dose of AdProT or AdLuc [2 × 10⁹ plaque-forming units] in 50 μL of phosphate-buffered saline (PBS), respectively.^10,11 The treatment was given by injection 15 min after temporary ligation of the left anterior descending coronary artery (LAD), and was randomly performed in the territory of the myocardial area distal to the LAD ligation site (Supplementary Figure 1A). The LAD was then released 10 min after the injection. A total of 25 min of ischemia was applied in this model.^14,15 The sham group received a single injection of PBS after thoracotomy without LAD ligation. The rats were euthanized after imaging on D14 post-treatment.

Supplementary Figure 1.

Diagram of the myocardial ischemia-reperfusion (MyoIR) study protocol. (A) Protocol of temporary ligation of left anterior descending (LAD) coronary artery for a total myocardial ischemic time of 25 minutes. (B) At 15 minutes after temporary LAD ligation, the rats were randomly allocated into two groups, i.e., MyoIR + AdLuc group, treated with adenoviral vectors carrying the luciferase gene, and MyoIR + AdProT group, treated with adenoviral vectors carrying the prothymosin α gene, on the day of ischemia-reperfusion surgery. The surgeon was blinded to treatment allocations. Direct myocardial injection of the adenoviral vectors (2 × 10⁹ plaque-forming unit, PFU) in 50 μL phosphate-buffered saline (PBS) was performed at a site distal to the ligation (temporary) of the LAD coronary artery. Another group of rats, the sham group, comprised normal rats that received a single injection of PBS after thoracotomy. On day 14 post-treatment, the rats were euthanized with pentobarbital sodium (250 mg/kg intraperitoneally). (C) Of 74 animals, 52 received ischemia-reperfusion injury with an overall surgical mortality rate of 11.5% (n = 6). Two rats died before the day of sacrifice. A total of 66 animals (n = 22 for Sham, MyoIR + AdLuc, and MyoIR + AdProT, respectively) were included in the study. AdLuc, transduction with an adenoviral vector carrying luciferase gene; AdProT, transduction with an adenoviral vector carrying prothymosin gene.

In vivo bioluminescence imaging of luciferase

The rats were anesthetized via isoflurane inhalation (2-3% v/v in oxygen). A solution of the luciferase substrate, VivoGloTM luciferin (150 mg/kg, Promega, USA), was injected intraperitoneally.¹⁶ The detailed procedures are described in the Data Supplement.¹⁶

Echocardiography

Transthoracic echocardiography was performed in the anesthetized rats before euthanasia using an ACUSON X300 Premium Edition ultrasound system (Siemens Ultrasound System, USA) with a 9.2 MHz probe (P9-4 phased array transducer, Siemens Medical Solutions) as previously described.^10,17

Quantitative analysis of serum biochemical parameters

Rat blood samples were collected on D3 in a separate experiment and on D14 before euthanasia. Blood glucose, triglyceride, cholesterol, aspartate transaminase, alanine transaminase, blood urea nitrogen, and creatinine levels were analyzed using colorimetric assays (ADVIA 1800 Chemistry System, Siemens Healthineers, Germany).^10,18 An enzyme-linked immunosorbent assay was used to measure the levels of serum ProT as previously described.¹⁹ N-terminal pro-brain natriuretic peptide (NT-ProBNP; Cloud-Clone Corp., USA) levels were measured according to the manufacturer’s instructions.

Histology, immunohistochemistry, and in situ hybridization of rat heart tissue

The rat heart tissues were transversely resected into five chunks, numbered 1 through 5, from the atrium to the apex, including both the right and left ventricles (LVs). Tissue sections 3-5, which comprised the myocardium of reperfusion injury, were used for histological analysis and immunohistochemistry. Some sections were incubated with rabbit anti-ProT antibody (1:200) at 25 °C for 30 min and counterstained using hematoxylin. In situ hybridization was performed using miR-223-3p (sequence 5′-ggg gta ttt gac aaa ctg aca-3′) probe (BioTnA, Taiwan), as described previously.¹⁰ Quantification was performed using a computerized morphometric system (Nikon Eclipse E200).^10,17

Determination of collagen concentration and immunoblot analysis of rat myocardium

Frozen heart tissues were sliced into small cubes and processed in an extraction solvent for the quantification of collagen type I, as described previously.²⁰ Soluble protein (50 μg) prepared from the remaining rat myocardium was loaded into polyacrylamide gels (9-12%) as previously described.^21,22 Detailed procedures for the immunoblot analysis are described in the Data Supplement.

Statistical analysis

Unless otherwise specified, the animal data are presented as mean ± standard deviation, whereas the patient data are presented as median (range). Detailed statistical methods are described in the Data Supplement. Statistical significance was set at p < 0.05. All statistical analyses were performed using SigmaPlot 14.0 software (Systat Software Inc., USA).

RESULTS

Human serum ProT levels were inversely associated with myocardial function after MyoIR

The serum ProT levels were elevated on D1 of ECMO support during the acute phase of MyoIR (Figure 1A). The ProT levels were significantly decreased on D3 after ECMO removal (p = 0.014) when myocardial function gradually recovered from MyoIR (Figure 1A). Assessment of LV function showed a significant improvement in the ejection fraction (LVEF) on D3 after ECMO removal (p = 0.004, Figure 1B).

Figure 1.

Serum prothymosin α (ProT) levels and left ventricular ejection fraction (LVEF) in patients who underwent extracorporeal membrane oxygenation (ECMO) for acute myocardial infarction. These patients required ECMO support within half an hour from percutaneous coronary intervention owing to decompensated myocardial function. (A) Serum ProT levels on day 1 of ECMO (E1) support and day 3 after ECMO removal (ER3); p = 0.02 using one-way repeated measure analysis of variance, n = 9. Dashed line indicates the mean value of healthy volunteers as a negative control, 1,144 pg/mL, n = 6. (B) LVEF measured using echocardiography on E1 and ER3. p = 0.004 using Friedman Repeated Measures Analysis of Variance on Ranks, n = 9.

Changes in the body weight and serum biochemical parameter levels of the rats

A total of 66 animals (n = 22 in each group) were included in the study analysis (Supplementary Figure 1C). There was a reduction in the body weight on D14 after MyoIR (p = 0.029; Supplementary Table 2), whereas there was a significant improvement in the MyoIR rats receiving ProT gene transfer (p = 0.041). There were no significant changes in the levels of serum biochemical parameters after gene transfer in the MyoIR rats on D14.

Supplementary Table 2. Biochemical analysis of serum from rats subjected to myocardial ischemia-reperfusion (MyoIR) on the day of sacrifice.

Variables	Sham (n = 8)	MyoIR + AdLuc (n = 8)	MyoIR + AdProT (n = 8)	p value
Body weight (g)	410.4 ± 45.6	344.0 ± 27.4*	424.1 ± 97.4	0.029
Glucose random (dL/mL)	168.4 ± 18.9	176.8 ± 13.3	167.4 ± 16.2	0.413
Triglyceride (mg/dL)	68.8 ± 28.7	66.2 ± 22.4	56.3 ± 15.8	0.711
AST (U/L)	74.8 ± 21.8	80.6 ± 42.0	95.6 ± 25.2	0.087
ALT (U/L)	52.3 ± 8.9	46.5 ± 10.9	51.5 ± 13.7	0.279
Total cholesterol (mg/dl)	68.8 ± 28.7	50.2 ± 7.9	50.5 ± 3.8	0.421
Cr (mg/dL)	0.4 ± 0.1	0.4 ± 0.1	0.4 ± 0.1	0.769
BUN (mg/dL)	15.5 ± 2.4	14.6 ± 3.5	15.7 ± 3.8	0.620

Data are presented as mean ± SD.

AdLuc, luciferase gene transduction; AdProT, prothymosin α gene transduction; ALT, alanine transaminase; AST, aspartate transaminase; BUN, blood urea nitrogen; Cr, creatinine; SD, standard deviation.

* p = 0.041 vs. MyoIR + AdProT, p = 0.048 vs. Sham.

ProT gene transfer induced ProT protein expression and reduced ventricular wall stress

The serum levels of ProT protein were significantly elevated on D3 after MyoIR Figure 2A. The ProT level decreased to baseline on D14 after the injury. The intramyocardial delivery of AdProT also induced persistently elevated serum ProT levels on D3 and D14 (p = 0.018 and p < 0.001, respectively; Figure 2A). There was no significant mobilization of ProT levels in the myocardial tissues on D14 in the MyoIR + AdLuc group (Supplementary Figure 2A and 2B). Significant ProT expression was detected mostly in the nucleus of cardiomyocytes on D14 after ProT gene transduction (Supplementary Figure 2A). Successful transduction of the luciferase gene was identified using In Vivo Imaging Systems (Supplementary Figure 2C and 2D). Furthermore, MyoIR induced an elevation in the serum levels of NT-proBNP on D3 and D14 (p = 0.02 and 0.009, respectively; Figure 2B). ProT gene delivery resulted in a trend of reduced serum NT-proBNP levels on D3 (p = 0.094, Figure 2B). The level further reduced significantly on D14 (p = 0.02).

Figure 2.

Serum levels of prothymosin α (ProT) and N-terminal pro-brain natriuretic peptide (NT-pro-BNP) in animals after ischemia-reperfusion (MyoIR) injury. (A) Serum ProT protein concentrations on days 3 and 14 after MyoIR with or without ProT gene (AdProT) transfer, n = 12/group. (B) Serum NT-pro-BNP levels on days 3 and 14 after MyoIR; AdLuc, transduction with adenoviral vectors carrying luciferase gene; n.s., not statistically significant (p = 0.094), n = 8/group. The colored bars indicate the median value.

Supplementary Figure 2.

Identification of the prothymosin α (ProT) and luciferase proteins after gene transduction in the myocardium. (A) Representative immunohistochemistry of ProT in the heart tissues harvested on day 14 (D14) after myocardial ischemia-reperfusion injury (MyoIR). Magnification: 400×; scale bar: 100 μm. (B) Quantification of ProT-positive cells per cubic millimeter area in myocardial tissue sections; the upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of non-outliers. Extra dots represent outliers, three slides from each section, five sections for each animal, n = 5 per group. (C) In vivo image for luciferase activity on D3 and (D) D14 after MyoIR. The upper row of (C) and (D) is the reference image superimposed with the in vivo imaging system in the lower row. AdLuc, transduction with an adenoviral vector carrying luciferase gene; AdProT, transduction with an adenoviral vector carrying prothymosin α gene; Naïve, rats that did not receive surgery or any treatment.

ProT gene transfer attenuated adverse remodeling of the myocardium after MyoIR

Reduced function in the LV was observed on D14 after MyoIR (Table 1). AdProT treatment preserved the LVEF and reduced the end-systolic diameter of the LV (p < 0.001 and p = 0.014, respectively). Concomitantly, the right ventricle and main pulmonary artery showed dilated remodeling secondary to LV dysfunction after MyoIR. ProT gene delivery at the LV site also mitigated the adverse remodeling of the right ventricle, while the pulmonary artery remained dilated.

Table 1. Echocardiographic measurement 14 days after myocardial ischemia-reperfusion (MyoIR) injury.

Variables	Sham (n = 6)	MyoIR + AdLuc (n = 6)	MyoIR + AdProT (n = 6)	p value
Left ventricle ejection fraction (%)	74.68 ± 5.70	46.54 ± 6.74*	69.87 ± 14.81	< 0.001
Left ventricle end systolic diameter (mm)	4.72 ± 1.18	5.83 ± 1.56#	3.72 ± 0.54	0.014
Stroke volume (mL)	0.72 ± 0.33	0.43 ± 0.17	0.48 ± 0.11	0.159
Right ventricle diameter (mm)	3.48 ± 0.68	5.68 ± 1.74†	3.77 ± 0.80	0.011
Pulmonary artery diameter (mm)	3.04 ± 0.54‡	4.70 ± 0.98	4.95 ± 1.43	0.016

Data are presented as the mean ± SD.

AdLuc, luciferase gene transduction; AdProT, prothymosin α gene transduction.

* p = 0.003 vs. MyoIR + AdProT, and p < 0.001 vs. sham; ^# p = 0.012 vs. MyoIR + AdProT; ^† p = 0.032 vs. MyoIR + AdProT, and p = 0.014 vs. sham; ^‡ p = 0.048 vs. MyoIR + AdLuc, and p = 0.024 vs. MyoIR + AdProT.

Histological analysis revealed a significant increase in myocardial fibrosis and heart size on D14 after MyoIR (Figure 3). AdProT transduction significantly reduced myocardial fibrosis and reversed myocardial remodeling (Figure 3B and 3C).

Figure 3.

Myocardial fibrosis and remodeling after ischemia-reperfusion injury. (A) Representative heart tissue cross-section distal to the coronary artery ligation site using the Masson’s trichrome stain. (B) Statistical analysis of the fibrosis area ratio relative to the corresponding myocardial tissue area. The upper and lower borders of the box represent the upper and lower quartiles, respectively. The middle horizontal line represents the median. Three slides from each section, three sections from each animal, n = 3/group. Magnification: 0.86X; scale bar = 1,000 μm. (C) Tissue concentrations of collagen type I per gram of myocardial tissue. The colored bars indicate the median value, n = 10/group. AdLuc, luciferase gene transduction; AdProT, prothymosin gene transduction; MyoIR, myocardial ischemia reperfusion.

Immunoblot analysis of the heart tissues revealed that the protein abundance of thrombospondin-1, representative of the extracellular matrix, was reduced in the MyoIR rats (p = 0.014, Figure 4A and 4B). ProT gene transfer preserved thrombospondin-1 protein abundance (Figure 4A and 4B). Mobilization of collagen type IV and matrix metalloproteinase (MMP)-2 was observed in the heart tissues of the MyoIR rats. There were significant reductions in collagen type IV and MMP-2 protein levels in the treatment group (both p < 0.001, Figure 4A, 4C, and 4D). There was no significant reduction in the MMP-9 protein abundance on D14 after ProT gene delivery (p = 0.323, Figure 4A, and 4E).

Figure 4.

Immunoblotting of protein markers of the myocardial extracellular matrix. (A) Representative immunoblots of thrombospondin (TSP)-1, collagen type IV, matrix metalloproteinase (MMP)-2, and MMP-9. (B) Quantitative analysis of TSP-1. (C) Collagen type IV. (D) MMP-2, and (E) MMP-9 levels; the colored bars indicate the median value, n = 6/group. AdLuc, transduction with an adenoviral vector carrying luciferase gene; AdProT, transduction with an adenoviral vector carrying prothymosin α gene; Da, Dalton; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MyoIR, myocardial ischemia-reperfusion.

ProT modulated inflammation and the TGF-β canonical signaling pathway after MyoIR

Immunoblot analysis of the heart tissues revealed that the protein levels of TGF-β type I receptor kinase activin receptor-like kinase (ALK)5 and the downstream signaling protein ratio of phosphorylated (p)-Smad2 and p-Smad3 were increased in the MyoIR rats (Figure 5A-C, 5E, and 5F). ProT gene delivery reduced ALK5 (but not TGF-β ) levels and the ratio of p-Smad2 and p-Smad3 protein abundances (Figure 5A-C, 5E, and 5F). ProT gene delivery also significantly mobilized Smad7 protein abundance (Figure 5A and 5D). There was a significant increase in the expression of miR-223, an inflammatory miR,²³ after MyoIR (Supplementary Figure 3A and 3B). The associated protein abundance of interleukin (IL)-1β increased, while that of RAS p21 protein activator (RASA)1 decreased (Supplementary Figure 3C-E). ProT gene delivery significantly suppressed miR223 expression and reduced IL-1β protein abundance in the myocardial tissue. Moreover, the RASA1 expression was enhanced in the treatment group (Supplementary Figure 3C and 3E).

Figure 5.

Immunoblotting for assessing classical transforming growth factor (TGF)-β signaling protein levels in myocardial tissue on day 14 after ischemia-reperfusion injury. (A) Representative immunoblots of TGF-β, activin receptor-like kinase (ALK) 5, Smad7, Smad2, phosphorylated (p)-Smad2, Smad3, and p-Smad3. (B) Quantitative analysis of TGF-β, (C) ALK5, (D) Smad7, (E) Ratio of p-Smad2 over Smad2, and (F) Ratio of p-Smad3 over Smad3; the colored bars indicate the median value, n = 6/group. AdLuc, transduction with an adenoviral vector carrying luciferase gene; AdProT, transduction with an adenoviral vector carrying prothymosin α gene; Da, Dalton; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MyoIR, myocardial ischemia-reperfusion.

Supplementary Figure 3.

Prothymosin α gene (AdProT) delivery modulated the expression of microRNA (miR)-223-associated signaling protein interleukin (IL)-1β and RAS p21 protein activator (RASA)1 after myocardial ischemia-reperfusion (MyoIR) injury. (A) Representative images of in situ hybridizations of myocardial tissue for miR-223. Magnification: 400×; scale bar: 100 μm. (B) Quantitative results. The upper and lower borders of the box represent the upper and lower quartiles. The middle horizontal line represents the median. The upper and lower whiskers represent the maximum and minimum values of non-outliers. Extra dots represent outliers. Ten visual fields under a microscope with 400× magnification for each tissue sample and three tissue samples from each animal were obtained, and n = 4/group. (C) Representative immunoblotting of IL-1β and RASA1, and (D) Quantification of immunoblotting for IL-1β and (E) RASA1. The colored bars indicate median value; n = 6/group for immunoblotting. AdLuc, transduction with an adenoviral vector carrying luciferase gene.

DISCUSSION

Our animal study revealed that MyoIR resulted in myocardial fibrosis and ventricular remodeling (Figure 6). AdProT transduction reversed the myocardial remodeling in extracellular matrix composition including collagen I, IV, and TSP-1. Ventricular function was thereby preserved at the subacute stage after MyoIR. Cannavo et al.²⁴ reported an increase of more than 1.5-fold in ProT protein abundance in mouse myocardial tissue and serum at D7 after myocardial infarction. They also showed that the epicardial injection of human recombinant ProT protein at the time of surgically induced myocardial infarction without reperfusion restricted the mouse myocardial infarct size 24 h after treatment. The protective effect was associated with the occurrence of an anti-apoptotic mechanism through an Akt-related signaling pathway.²⁴ Although the pathophysiology is different, apoptosis is a dominant process that occurs after MyoIR rather than after myocardial infarction without reperfusion.²⁵ In addition, it would be preferable to consider MyoIR in an myocardial infarction animal model as translational research, as it may resemble clinical situations in the modern era of PCI.¹⁴ Moreover, our patient study showed a significantly elevated serum ProT concentration on D1 after MyoIR. Although further study is required, the clinical findings might indicate the existence of a protective response of ProT to the injury. Taken together, we speculate that the persistent mobilization of ProT expression may be protective against LV remodeling after reperfusion injury.

Figure 6.

Graphical abstract: Gene therapy for myocardial ischemia-reperfusion (MyoIR) injury. MyoIR injury might lead to myocardial fibrosis in the recovery stage, thereby reducing the left ventricle function. Intramyocardial prothymosin α gene delivery resulted in a 67.5% reduction of fibrosis area and a 23.3% improvement in left ventricle ejection fraction after ischemia-reperfusion. The mechanism is associated with inhibition of downstream signaling of transforming growth factor-β and suppression of inflammatory responses. This study shows that prothymosin α is a potential therapeutic gene for patients who experience MyoIR injury. ALK5, activin receptor-like kinase 5; IL-1β, interleukin-1β; LVEF, left ventricle ejection fraction; miR-223, microRNA-223; P, phosphorylated; ProT, prothymosin α gene; RASA1, RAS p21 protein activator 1; TGF-β, transforming growth factor-β.

TGF-β is an important mediator in modulating the extracellular matrix during the repair process after MyoIR.⁹ Binding of TGF-β induces the transphosphorylation of ALK5, which further triggers the phosphorylation of Smad2 and Smad3.^9,26 On the other hand, Smad7 regulates TGF-β signaling by inducing ubiquitination of ALK5 as negative feedback control.^27,28 Furthermore, Yan et al.²⁸ showed that Smad7 induced the degradation of phosphorylated Smad2/3 by enabling direct binding to form heteromeric complexes. There was no significant mobilization of tissue TGF-β protein abundance after MyoIR in our study. However, there were markedly increased protein levels associated with the canonical TGF-β signaling pathway. Treatment with AdProT in this MyoIR model reconfirmed the inhibitory role of ProT protein in TGF-β signaling after injury. Collectively, our results show that ProT gene expression after MyoIR inhibits both inflammatory protein production and signal transduction of the canonical TGF-β pathway, potentially by enhancing the activation of Smad7.

MicroRNAs, such as miR-223, are small non-coding RNAs that modulate inflammation reactions in the myocardium after MyoIR.²³ Liu et al. demonstrated that miR-223 activated cardiac myofibroblasts after infarction by inhibiting the RASA1-associated signaling pathway in a rat model of myocardial infarction without reperfusion.²⁹ In our study, ProT gene transfer significantly reduced inflammatory mediators of miR-223 and IL-1β. Although we did not investigate the correlation between miR-223 and RASA1, there was a corresponding increase in the abundance of RASA1 protein in the treatment group. Collectively, we propose that intramyocardial ProT gene delivery reduces tissue miR-223 expression and levels of the associated signaling proteins, thereby restoring the histological and functional integrity of the myocardium after MyoIR.

This study had several limitations. To balance a reasonable perioperative survival and area of myocardial injury, our animal model used an ischemic time of 25 min.¹⁴ Hence, potential differences in survival outcomes between the study groups might not have been revealed. Nevertheless, the effects of ProT gene delivery warrant further investigation when the area of myocardial injury is increased by prolonging the ischemic time to over 25 min. In MyoIR animal models, there is a concern regarding the variable extent of myocardial injury after coronary artery ligation.¹⁴ To minimize bias, the surgeon was blinded to the assignment of treatment until the time of injection.^5,14 In addition, the treatment dose was determined from our previous study on right-sided heart failure.^10,11 Therefore, the optimal dose of treatment for maximum myocardial protection remains to be elucidated. From a clinical study perspective, the number of patients was insufficient to explore the role of serum ProT in acute decompensated heart failure after MyoIR. Extrapolation of the study results is also limited by the extreme clinical presentation of these patients after MyoIR.

CONCLUSIONS

In conclusion, the transient elevation of serum ProT levels early after MyoIR as demonstrated in the rat and patient serum studies might be a protective response against myocardial reperfusion injury. Intramyocardial AdProT delivery preserved LV systolic function through anti-inflammatory and anti-fibrotic effects in the subacute stage after MyoIR. These findings imply the potential protective role of ProT against MyoIR, thereby preventing the progression of heart failure. Further investigations are warranted to explore the role of ProT as a potential therapeutic gene for patients who experience MyoIR.

DATA SUPPLEMENT

Detailed Methods

Human serum collection

Patients who fulfilled all of the following three conditions were included in the study: 1. 20-70 years of age; 2. Decompensated heart failure due to acute coronary syndrome which required percutaneous coronary intervention and ECMO support; 3. Survived to wean off ECMO. Patients with acute cerebrovascular events, active malignancy, or pregnancy were excluded. The surgical procedures of ECMO implantation were performed at the National Cheng Kung University Hospital under the guidance of the Cardiovascular Surgery Department, as previously described.^S1 The patients received standard care perioperatively in the intensive surgical unit, as per previously described protocols.^S1,S2 The patient demographic data are summarized in Supplementary Table 1. Diagnosis of acute coronary syndrome was confirmed as indicated in the clinical guide.^S3 Blood samples were obtained on ECMO day 1 and day 3 after removal. The timing of sampling is based on the functional status of the myocardium, cardiogenic shock versus recovery status, rather than an absolute time point. Serum was obtained from 10 mL of peripheral blood after centrifugation at 5,000 rpm for 10 minutes at each sampling time.

Animal model

Age-matched young male Sprague-Dawley rats (weight, 200-250 g) were housed in accordance with the guidelines of the Animal Care and Use Committee of the National Cheng Kung University, Taiwan (IACUC: 108201). The detailed procedures of ischemia-reperfusion injury are described below: the rats were anesthetized via isoflurane inhalation (2%-3% v/v in oxygen) after intubation with a 16-gauge intravenous catheter. Mechanical ventilation was applied with a respiratory rate of 40-45/minute, Vt of 0.25-0.32 mL per breath, FiO₂: 40%, inspiration cycle 40%, volume control mode. An anterolateral thoracotomy was performed to expose the anterior surface of the heart. One suture with 7-0 prolene was made after identifying the left anterior descending (LAD) coronary artery at the level covered by the left atrial tissue.^S4 The prolene suture was tied over a cotton patch to avoid myocardial cutting injury. The ligation tie was released for myocardial reperfusion after 25 minutes of ischemic time.^S5 The sham group of rats received thoracotomy with the prolene suture line passed underneath the LAD. The chest wound was then closed after de-airing of the left thoracic cavity by inflating the left lung. Buprenorphine was administered subcutaneously 30 minutes before surgery, and another dose of buprenorphine was administered to the wound edge immediately before wound closure for perioperative analgesia.

In vivo bioluminescence imaging of luciferase

The rats were anesthetized by isoflurane inhalation (2%-3% v/v in oxygen). A solution of the luciferase substrate, VivoGloTM luciferin (150 mg/kg, Promega, Madison, USA), was injected intraperitoneally.^S6 The animals were imaged within 2 minutes of luciferin injection for bioluminescence using an in vivo imaging system (Caliper IVIS Spectrum System, PerkinElmer, Waltham, MA, USA). The total photon emission from the selected and defined areas within the images obtained for each rat was quantified using the Living Image^®4.5.5 software (PerkinElmer). The photon signal was presented as a pseudo-color image representing the light intensity (red being the most intense and blue being the least intense). The images were superimposed on the reference image for orientation.^S6

Histology of rat heart tissue

The tissue samples were fixed in 4% paraformaldehyde, paraffin-embedded, and cut into 5 μm-thick sections. The tissue sections were then stained using hematoxylin and eosin and Masson’s trichrome stains.^S7,S8 The slides were analyzed under a light microscope by observers blinded to the treatment groups.^S9 The fibrosis area, defined as the ratio of the collagen area to the whole tissue section area, was used to quantify myocardial fibrosis. All microscopic images were analyzed using a computerized morphometric system (Nikon Eclipse E200).^S7

Immunoblot analysis

Soluble proteins (50 μg) extracted from the rat myocardium were resolved on polyacrylamide gels (9%-12%) and transferred onto polyvinylidene fluoride membranes, following previously described methods.^S10 The mouse monoclonal anti-TGF-β antibody (1:1000, R&D System, Inc. Minneapolis, MN, USA), anti-Smad2 (1:1000, Cell Signaling Technology, Inc. Danvers, MA, USA), and anti-phosphorylated (anti-pSmad2) (1:1000; Cell Signaling Technology, Inc.), and anti-Smad3 (1:1,000; Cell Signaling Technology, Inc.), and anti-pSmad3 (1:500; Cell Signaling Technology, Inc.), and anti-Smad7 (1:1000; R&D Systems, Inc.), anti-thrombospondin (TSP-1) (1:100, catalog no.: no.MA5-13398; Thermo Fisher Scientific, Waltham, MA, USA), anti-matrix metalloproteinase (MMP-2) (1:1000, EMD Millipore, Temecula, CA, USA), anti-MMP9 (1:1000, Novus Biologicals, Littleton, CO, USA), anti-collagen IV (1:500, EMD Millipore), anti-interleukin (IL)-1β (1:500, GeneTex International Corporation, Irvine, CA, USA), anti-poly (ADP-ribose) polymerase (PARP)-1 (1:1000, Cell Signaling Technology, Inc.), anti-tumor necrosis factor (TNF)-α (1:500; GeneTex International Corporation), anti-RAS p21 protein activator (RASA)1 (1: 1000, Abcam, Cambridge, UK), anti-NACHT, LRR, FIIND, CARD domain, and PYD domain-containing protein (NLRP-3) (1:1000, Novus Biologicals), anti-activin receptor-like kinase (ALK-5) (1:1000), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5000, GeneTex International Corporation) antibodies were used. Bands were visualized using enhanced chemiluminescence and quantified by performing scanning densitometry (ImageJ; 1.48v, National Institutes of Health, Bethesda, MD, USA).^S10

Statistical analysis

Unless otherwise specified, the animal study data are presented as mean ± standard deviation (SD). Patient data are presented as median (range). Intergroup differences were analyzed using one-way analysis of variance (ANOVA), followed by appropriate post hoc tests for multiple comparisons.^S9 The patient data of serum ProT levels and left ventricular ejection fraction (LVEF) values were analyzed using one-way repeated measures ANOVA with post hoc Tukey test. Statistical significance was set at p < 0.05. All statistical analyses were performed using the SigmaPlot 14.0 software (Systat Software Inc., San Jose, CA, USA).

AUTHOR CONTRIBUTIONS

ALS, SYF, and JNR designed the study. PNY and JNR collected the patient serum samples and clinical data. JNR, SYF, CHH, and MSC performed the animal experiments. JNR, CHH, SYF, and MSC collected and analyzed the data. JNR, SYF, WCL, and CFL contributed to the statistical analysis and interpretation of the data. ALS, SYF, and JNR drafted the manuscript. All authors read and approved the final version of the manuscript. ALS and SYF contributed equally to this study.

DECLARATION OF CONFLICT OF INTEREST

All authors declare no conflict of interest.