Gene therapy encoding cell cycle factors to treat chronic ischemic heart failure in rats

Riham R E Abouleisa; Xian-Liang Tang; Qinghui Ou; Abou-Bakr M Salama; Amie Woolard; Dana Hammouri; Hania Abdelhafez; Sarah Cayton; Sameeha K Abdulwali; Momo Arai; Israel D Sithu; Daniel J Conklin; Roberto Bolli; Tamer M A Mohamed

doi:10.1093/cvr/cvae002

. 2024 Jan 4;120(2):152–163. doi: 10.1093/cvr/cvae002

Gene therapy encoding cell cycle factors to treat chronic ischemic heart failure in rats

Riham R E Abouleisa ^1,^b, Xian-Liang Tang ^2,^b, Qinghui Ou ³, Abou-Bakr M Salama ^4,⁵, Amie Woolard ⁶, Dana Hammouri ^7,⁸, Hania Abdelhafez ^9,¹⁰, Sarah Cayton ¹¹, Sameeha K Abdulwali ^12,¹³, Momo Arai ^14,¹⁵, Israel D Sithu ^16,¹⁷, Daniel J Conklin ¹⁸, Roberto Bolli ¹⁹, Tamer M A Mohamed ^20,^21,^22,^23,^24,^25,^26,^✉,^c

¹ Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

² Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

³ Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

⁴ Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

⁵ Department of Cardiovascular Medicine, Faculty of Medicine, Zagazig University, 872 Shaibet an Nakareyah, Zagazig, Al-Sharqia Governorate 7120001, Egypt

⁶ Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

⁷ Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

⁸ Department of Pharmacology and Toxicology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

⁹ Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

¹⁰ Department of Bioengineering, Speed School of Engineering, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

¹¹ Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

¹² Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

¹³ College of Medicine, Alfaisal University, Interconnection of Al Takhassousi،Al Zahrawi Street, Riyadh 11533, Saudi Arabia

¹⁴ Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

¹⁵ College of Medicine, Alfaisal University, Interconnection of Al Takhassousi،Al Zahrawi Street, Riyadh 11533, Saudi Arabia

¹⁶ Department of Medicine, Center for Cardiometabolic Science, Envirome Institute, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

¹⁷ Department of Physiology, School of Medicine, University of Louisville, Louisville, 580 South Preston Street, KY 40202, USA

¹⁸ Department of Medicine, Center for Cardiometabolic Science, Envirome Institute, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

¹⁹ Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

²⁰ Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

²¹ Department of Pharmacology and Toxicology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

²² Department of Bioengineering, Speed School of Engineering, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

²³ Department of Medicine, Center for Cardiometabolic Science, Envirome Institute, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA

²⁴ Department of Biochemistry Faculty of Pharmacy, Zagazig University, 872 Shaibet an Nakareyah, Zagazig, Zagazig, Al-Sharqia Governorate 7120001, Egypt

²⁵ Institute of Cardiovascular Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK

²⁶ Surgery Department, Baylor College of Medicine, 6519 Fannin Street, Houston, TX, 77030, USA

^✉

Corresponding author. Tel: 8323559926, E-mail: tamer.mohamed@bcm.edu

Riham R.E. Abouleisa and Xian-Liang Tang share a specific author position.

Conflict of interest: T.M.A.M. holds equities in Tenaya Therapeutics. The other authors report no disclosures.

PMCID: PMC10936750 PMID: 38175760

Abstract

Aims

Gene therapies to induce cardiomyocyte (CM) cell cycle re-entry have shown a potential to treat subacute ischaemic heart failure (IHF) but have not been tested in the more relevant setting of chronic IHF. Our group recently showed that polycistronic non-integrating lentivirus encoding Cdk1/CyclinB1 and Cdk4/CyclinD1 (TNNT2-4Fpolycistronic-NIL) is effective in inducing CM cell cycle re-entry and ameliorating subacute IHF models and preventing the subsequent IHF-induced congestions in the liver, kidneys, and lungs in rats and pigs. Here, we aim to test the long-term efficacy of TNNT2-4Fpolycistronic-NIL in a rat model of chronic IHF, a setting that differs pathophysiologically from subacute IHF and has greater clinical relevance.

Methods and results

Rats were subjected to a 2-h coronary occlusion followed by reperfusion; 4 weeks later, rats were injected intramyocardially with either TNNT2-4Fpolycistronic-NIL or LacZ-NIL. Four months post-viral injection, TNNT2-4Fpolycistronic-NIL–treated rats showed a significant reduction in scar size and a significant improvement in left ventricular (LV) systolic cardiac function but not in the LV dilatation associated with chronic IHF. A mitosis reporter system developed in our lab showed significant induction of CM mitotic activity in TNNT2-4Fpolycistronic-NIL–treated rats.

Conclusion

This study demonstrates, for the first time, that TNNT2-4Fpolycistronic-NIL gene therapy induces CM cell cycle re-entry in chronic IHF and improves LV function, and that this salubrious effect is sustained for at least 4 months. Given the high prevalence of chronic IHF, these results have significant clinical implications for developing a novel treatment for this deadly disease.

Keywords: Cell cycle, Heart failure, Cardiomyocyte

1. Introduction

Heart failure (HF) is an increasingly common syndrome with a high morbidity and mortality rate. Twenty-six million people worldwide suffer from HF. Besides its human toll, this growing disease contributes to increased healthcare costs worldwide.^1,2 The most frequent form of HF, accounting for approximately half of HF cases, is ischaemic heart failure (IHF), caused by myocardial ischaemia and death of cardiomyocytes (CMs).³ IHF generally cannot be cured due to the limited regeneration capacity of CMs.⁴ The lost CMs are replaced by fibrous tissue, which is accompanied by an adverse structural and functional remodelling that worsens systolic function and causes progressive dilatation over time, resulting in chronic IHF.^5,6

Over the past decades, several studies have shown the potential of interventions that induce the cell cycle in CMs to treat acute/subacute HF.^7,8 However, to our knowledge, no study has demonstrated the ability of an intervention to induce CM cell cycle re-entry and thereby improve systolic function in models of chronic IHF. Chronic IHF is more clinically relevant because, in many IHF patients, IHF develops gradually over the years as a result of recurring ischaemic insults without a major myocardial infarction (MI); furthermore, in those patients who do experience a large MI, the development of chronic IHF is often not clear until several weeks later, due to remodelling and spontaneous functional recovery of viable myocardium.^9,10

Our previous studies showed that ectopic expression of four cell cycle genes in combination (Cdk1/CyclinB1 and Cdk4/CyclinD1; known as ‘4F’) using adenoviral vectors induces cell cycle re-entry in 15–20% of CMs in vitro and in vivo.¹¹ 4F-induced CM cell cycle re-entry improved left ventricular (LV) function in a model of subacute IHF in mice.¹¹ To preclude the possibility of uncontrolled cell cycle re-entry and oncogenesis in other tissues in the clinical setting, we developed TNNT2-4Fpolycistronic-NIL where all the four cell cycle factors were cloned into one polycistronic lentivirus backbone with each factor driven by a cardiac-specific troponin-T (TNNT2) promoter in a non-integrating lentivirus (NIL) as the delivery method. NIL is known for its high infection efficiency and transient expression of the encoded protein, which is limited to 2–3 days, followed by a significant decline in expression.^12–15 Our previous study showed that NIL encoding TNNT2 promoter drives the 4F expression (TNNT2-4Fpolycistronic-NIL) specifically in CMs with 80–100% infection efficiency. TNNT2-4Fpolycistronic-NIL induced cell cycle in 10–15% of the CMs within 4 days post-infection; however, the cell cycle activity was abolished by Day 10 post-infection, indicating the transient nature of the cell cycle induction in the CMs.¹⁶ We showed that TNNT2-4Fpolycistronic-NIL improved LV function in subacute IHF models (rats and pigs treated 7 days post-infarction) and prevented the consequent IHF-induced congestion in other organs, including the lungs, kidneys, and liver.^16,17 In the current study, we tested the effects of TNNT2-4Fpolycistronic-NIL on CM cell cycle re-entry, cardiac function, and dilatation in a chronic IHF model in rats. This study is the first to test the impact of induced CM cell cycle re-entry on chronic IHF.

2. Methods

2.1. Cloning and preparation of integrating and NIL

cDNA ofTNNT2 promoter, Cre recombinase, AurKB promoter, CCNB, CCND, CDK1, and CDK4 were cloned into the pLenti-MCS-SV-puro backbone (Addgene). To produce the Lentivirus particles, 5 × 106 HEK293 cells were transfected using FuGENE HD transfection reagent (Promega) along with 5 µg pMD2.g, 5 µg psPAX2 (integrating lentivirus) or psPAX2-D64V (NIL; Addgene), and 10 µg of the expression pLenti vector encoding the gene of interest for 48 h. The media containing the virus were collected and filtered through a Nalgene syringe filter 0.45 µm. For in vitro experiments, the virus was mixed with a polybrene-transfecting reagent (1 µg/mL; Millipore Cat# 1003). The virus was then used for in vivo injections, the virus solution was centrifuged at 20 000 g at 4°C for 2 h, and the pellet was resuspended in PBS.

2.2. Rat experiments

2.2.1. Study ethical approval

Animal studies were performed following the NIH Guide for the Care and Use of Laboratory Animals, and the University of Louisville animal use guidelines and the protocols were approved by the Institutional Animal Care and Use Committee (IACUC) and were accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.

2.2.2. Rat heart ischaemic/reperfusion surgery

All surgeries were performed as described in Abouleisa et al.,¹⁶ Tang et al.,¹⁸ and Tokita et al.¹⁹ Briefly, female Fischer 344 (F344) rats at the age ranging from 8 to 12 weeks were anaesthetized with ketamine (37 mg/kg) and xylazine (5 mg/kg), intubated, and ventilated with a rodent respirator. Anaesthesia was maintained with 1% isoflurane inhalation, and body temperature was kept at 37°C with a heating pad. All rats underwent a 2-h occlusion of the left anterior descending coronary artery, followed by reperfusion. Four weeks after MI, echocardiography was performed to assess the decline in cardiac functions after ischaemia/reperfusion (I/R). All rats in this study had ejection fraction (EF) drop > 20 points from baseline. Rats were randomized into two groups (LacZ-NIL, TNNT2-4Fpolycistronic-NIL). Rats were re-anaesthetized with ketamine/xylazine, intubated, and ventilated. The chest was reopened to expose the heart. Viral vectors (1 × 10⁸ T.U. per rat heart in 100 µL PBS) were injected into the left ventricle along the infarct border at five sites (20 µL/site) using a 30G needle. The rat surgeon was blinded to whether 4F or LacZ non-integrated lentivirus was administered in each animal.

Cardiac function was assessed by serial echocardiography at baseline (before MI), 4 weeks after MI (before virus injection), and then 4 or 16 weeks after virus injection. Animals were anaesthetized lightly with isoflurane, placed on the imaging table in the supine position, and prepared for imaging using the Vevo 2100 Imaging System (Visual Sonics) equipped with a 25 MHz transducer. Parasternal longitudinal axis images were acquired and analysed by LV trace using the Vevo LAB 3.2.6 to obtain the LV functional parameters, including the end-diastolic and end-systolic area, volume, stroke volume (SV), fractional shortening (FS), and EF. Imaging and calculations were done by an individual who was blinded to the treatment, and the code was broken after all data were acquired. For the long-term follow-up experiment, oxygen saturation (SPO₂) was assessed during the final echocardiography using a Berry Pulse oximeter (Shanghai Berry Electronic Tech). Euthanasia was performed in accordance with the 2013 AVMA Guidelines on Euthanasia. At the end of the studies, the rats were euthanized by exposure to 5% isoflurane followed by a bolus of KCl into the heart, and the heart was excised and perfused for the measurement of infarct size or for the histopathological analysis of regenerated myocytes. During these experiments, three rats died 1 day post-surgery, and no mortality was observed subsequently in all groups for the duration of the experiment.

2.3. Histological analysis

At the end of the experiments (8 or 20 weeks after MI), animals were sacrificed, and their hearts were harvested for histological studies. The frozen hearts were sectioned longitudinally into 400–500 sections of 8 µm thickness (take a section to throw one add two sections per slide), and one slide for every 10 slides (20–25 slides per animal) was stained with standard Masson’s trichrome staining to determine scar size. The stained sections were imaged using the Keyence BZ9000 imaging system (×4 magnification). ImageJ software was used to measure the scar area (blue) and healthy area (red) on longitudinal sections. Individuals assessing scar area were blinded to the treatment applied in each animal.

2.4. Immunohistochemistry

The hearts were cut longitudinally into half and kept in 4% paraformaldehyde for 48 h. The hearts were then washed with PBS, then placed in 10% sucrose solution for 1 h, followed by 20% sucrose solution for 1 h at room temperature, then placed in 30% sucrose solution overnight at 4°C. The hearts were then processed into frozen optimal cutting temperature (OCT) compound blocks and kept at −80°C for 24 h. The heart was sectioned using a cryostat (Leica Inc.) in 8 µm thick sections, placed on slides, and kept at −20°C until staining. To start staining, the OCT was removed from the section by heating it at 95°C for 5 min, then washing it in PBS for 30 min.

The cleaned sections were permeabilized with 0.1% Triton X-100 for 15 min (Millipore Cat# 55163804) and then blocked with 3% bovine serum albumin (BSA) in PBS for 60 min at room temperature (V.W.R. Cat# 0332). The cells or tissue sections were then probed with primary antibody (1:200 in 1% BSA) for 1.5 h, then washed three times with PBS. They were then labelled with secondary fluorescent antibody (1:200 in 1% BSA). Table 1 shows a list of primary and secondary antibodies used in this study. Tissue sections were then washed three times with PBS and stained with 4',6-diamidino-2-phenylindole (DAPI) 1 µg/mL (Biotium Cat# 40043) to stain the nucleolus blue. Imaging was conducted for the whole well using the high-content imaging instrument Keyence BZ9000. The percentage of GFP-positive CMs was quantified using ImageJ software.

Table 1.

List of primary and secondary antibodies used

Specific protein	Primary antibody	Cat #
Cardiac troponin	Rabbit monoclonal anti-cardiac troponin T antibody	Abcam ab209813
DsRed	Mouse monoclonal anti-R.F.P.	Abcam ab150115
Cardiac troponin	Mouse monoclonal cardiac troponin T antibody	Thermo Fisher (MA5-12960)
CDK4	Rabbit monoclonal anti-CDK4	Abcam ab199728
CCNB	Rabbit monoclonal anti-CCNB	Abcam ab32053
CCND	Rabbit monoclonal anti-CCND	Abcam ab134175
CDK1	Mouse monoclonal anti-CDK1	Abcam ab18

Secondary antibody	Cat #
Alexa Flour™ 647 donkey anti-rabbit IgG	Thermo Fisher A31573
Texas Red-X goat anti-mouse IgG	Thermo Fisher T862
Goat anti-mouse IgG (H+L), F.I.T.C.	Thermo Fisher A16079
Texas Red goat anti-rabbit IgG (H+L)	Thermo Fisher T-6391

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2.5. Cell size analysis

The OCT was removed from the section by heating it at 95°C for 5 min, then washing it in PBS for 30 min. Then the cleaned sections were boiled in citrate retrieval solution (2.4 g/L sodium citrate dehydrate, 0.35 g/L citric acid, pH 6) for 8 min. The slides were then cooled in ice for 2 min and then washed with running tap water. The sections were then probed with wheat germ agglutinin (WGA; 5 µg/mL, Thermo Fisher W32464) for 1 h. The sections were then washed with PBS and stained with DAPI (1 µg/mL, Biotium Cat# 40043) to stain the nucleolus blue. Imaging was conducted using the high-content imaging instrument Keyence BZ9000. ImageJ software was used to measure the cross-sectional cell size of around 1100–1200 cells per group at the border zone.

2.6. Plasma collection

The blood was drawn from the right ventricle and collected on EDTA in a microcentrifuge tube coated with EDTA. The blood was centrifuged at 4600 rpm at 4°C for 10 min. The plasma was collected and stored at −80°C till the process.

2.7. Toxicity tests

The plasma levels of LDH, total cholesterol, HDL cholesterol, triglycerides, ALT, AST, albumin (ALB), and creatinine were performed using Ace Axcel® Clinical Chemistry System (Alfa Wassermann, West Caldwell, NJ). LDL cholesterol was calculated using Friedewald calculation [LDL cholesterol (mg/dL) = total cholesterol − HDL − (triglycerides/5)].

2.8. Apoptosis (TUNEL) assay

TUNEL assay was done using Click-iT™ Plus TUNEL Assay kit (Invitrogen Cat# C10619). Slides of 8 µm thick heart sections were prepared as described above. To start the assay, OCT was removed by heating at 95°C for 5 min and then washing in PBS for 30 min. Slides were immersed in 4% paraformaldehyde (Electron Microscopy Sciences Cat# 15713-S) for 15 min at 37°C, followed by washing twice in PBS for 5 min each. Tissues were then incubated for 15 min with proteinase K solution as a permeabilization reagent followed by a wash in PBS for two times, 5 min each. The tissues were immersed in 4% paraformaldehyde for 15 min at 37°C followed by washing twice in PBS for 5 min each, then rinsed in deionized water. Afterward, TdT reaction and the visualization reaction were performed as recommended by the manufacturer protocol. The samples were blocked with 3% BSA, immunostaining protocol for troponin staining followed as described above, and nuclei stained blue with DAPI. For positive control, DNA strand breaks were induced by incubating permeabilized cells with one unit of DNase I (DNase Max® kit—Qiagen Cat#15200-50) for 30 min at 37°C.

2.9. Statistical analyses

For all assays, power analyses were performed to choose the group sizes, which will provide >80% power to detect a 10% absolute change in the parameter with 5% type I error rate. The Kolmogorov–Smirnov (K–S) test for normality was conducted; all data sets showed normal distribution. Then, differences between the two groups were examined for statistical significance with unpaired t-tests. However, to compare data consisting of more than two groups, we performed one- or two-way analysis of variance (ANOVA) tests followed by Tukey’s multiple comparisons to get the corrected P-value. A value of P < 0.05 was regarded as significant, and error bars indicate standard deviation (SD). The person who performed the analysis was blinded to the experimental groups.

3. Results

3.1. TNNT2-4Fpolycistronic-NIL improves LV systolic cardiac function but does not reverse dilatation

Our previous studies^16,17 showed that TNNT2-4Fpolycistronic-NIL administration 1 week after MI induced transient expression of the 4F and significant induction in CM cell cycle re-entry, which led to improved LV systolic function and prevented LV dilatation and congestion in other organs in rat and pig models of subacute IHF. In this study, we assessed the effect of TNNT2-4Fpolycistronic-NIL administration 4 weeks after MI, when acute myocardial inflammation has subsided, scar formation is largely complete, and most LV dilatation has occurred (chronic IHF) in rats. To validate that TNNT2-4Fpolycistronic-NIL drives transient expression of the 4F in vivo, TNNT2-4Fpolycistronic-NIL or LacZ-NIL control virus was injected intramyocardially at five different sites, and the rats were sacrificed 1 and 2 weeks post-injection. Immunostaining confirmed the expression of 4F in the rat hearts 1 week post-injection and the disappearance of protein expression 2 weeks post-injection. These data confirmed the transient expression of 4F using TNNT2-4Fpolycistronic-NIL (see Supplementary material online, Figure S1). To assess the effect of TNNT2-4Fpolycistronic-NIL on chronic IHF, Fischer rats were subjected to a 2-h coronary occlusion followed by reperfusion; 4 weeks later, echocardiography was performed to assess the alterations in LV function and dimensions. Then, either TNNT2-4Fpolycistronic-NIL or LacZ-NIL (control) was injected into the peri-infarct region of the heart. Four weeks later, echocardiography was performed to assess LV function and dimensions, and then rats were euthanized, and hearts and blood were collected for analysis (Figure 1A). All surgical procedures and echocardiographic and pathologic analyses were performed by investigators blinded to treatment. Four weeks after I/R (before viral injection), both experimental groups exhibited a similar decline in the LV function and similar LV dilatation associated with IHF (see Supplementary material online, Excel Sheet and Figure S2), indicating that the magnitude of the damage caused by I/R was similar. Four weeks after viral injection, all TNNT2-4Fpolycistronic-NIL–treated rats had a significant improvement in LV systolic function as assessed by EF, FS, cardiac output (CO), and SV, while all LacZ-NIL–treated rats showed a significant further decline in LV systolic function (Figure 1B; Supplementary material online, Figure S2). Consistent with the improvement in LV function, histological analyses revealed a ∼30% reduction in scar size in hearts treated with TNNT2-4Fpolycistronic-NIL compared with LacZ-NIL–treated hearts (Figure 2A). Although TNNT2-4Fpolycistronic-NIL–treated rats exhibited improved LV systolic function, the TNNT2-4Fpolycistronic-NIL intervention, however, did not reverse the LV dilatation associated with chronic IHF. TNNT2-4Fpolycistronic-NIL-treated rats showed a significant decrease in the LV area during systole (LVAs) and end-systolic volume (ESV), yet there was no significant improvement in the LV area during diastole (LVAd) or end-diastolic volume (EDV) compared with the LacZ-NIL–treated hearts (Figure 2B and C).

TNNT2-4Fpolycistronic-NIL treatment of chronic IHF rats improves systolic cardiac function within 4 weeks. (A) Schematic diagram of the experimental design. (B) Quantification of the changes in EF (delta EF), FS (delta FS%), SV (delta SV), and CO (delta CO) as assessed by echocardiography between before and after treatment for each individual rat [n = 6–7 rats per group; *P < 0.05, **P < 0.01, ****P < 0.0001 compared to LacZ-NIL (control group); error bars indicate SD, unpaired t-test].

TNNT2-4Fpolycistronic-NIL treatment reduces scar size but does not reverse the cardiac dilatation associated with chronic IHF. (A) Representative images of rat hearts stained with Masson’s trichrome stain [left panel; healthy myocardium stains (red) and fibrotic tissue stains (blue)] at the end of the experiment (scale bar = 2 mm). The right panel shows scar size quantification as a percentage of total LV tissue (n = 6–7 rats per group; *P < 0.05 compared to the LacZ-NIL group; error bars indicate SD, unpaired t-test). Quantification of the change in the LVAs (delta LVAs) and LVAd (delta LVAd, B) and ESV (delta ESV) and EDV (delta EDV, C) between before and after treatment for each individual rat compared to the LacZ-NIL–treated hearts as assessed by echocardiography (n = 6–7 rats per group; **P < 0.05 compared to LacZ-NIL; error bars indicate SD, unpaired t-test). (D) Quantification of LW/BW ratio (n = 6–7 rats per group; *P < 0.05, ***P < 0.001 compared to sham-operated rats; error bars indicate SD, one way ANOVA).

Interestingly, lung congestion associated with chronic IHF was not alleviated in TNNT2-4Fpolycistronic-NIL–treated rats, as indicated by the insignificant change in the lung weight to body weight (LW/BW) ratio compared with LacZ-NIL–treated rats (Figure 2D). The echocardiographic data are presented in Figures 1 and 2 as the changes (delta = cardiac function at the end of experiment—function 4 weeks post-MI for each rat) to show the individual improvement of function after the treatment. All echocardiographic raw data are presented in Supplementary material online, Excel Sheet and Figure S2.

3.2. Improved LV systolic function after TNNT2-4Fpolycistronic-NIL administration is maintained for 4 months

The improvement in LV function at 4 weeks after TNNT2-4Fpolycistronic-NIL administration provides evidence of short-term efficacy. To assess the long-term efficacy and determine if TNNT2-4Fpolycistronic-NIL–induced CM cell cycle re-entry would result in sustained improvement of LV systolic function, rats were subjected to a 2-h coronary occlusion followed by reperfusion (I/R). Four weeks later, echocardiography was performed (see Supplementary material online, Excel Sheet and Figure S3), and TNNT2-4Fpolycistronic-NIL or LacZ-NIL in combination with a mitosis double reporter integrating lentivirus¹⁶ was injected into the peri-infarct region of the heart. Sixteen weeks later, echocardiography was performed to assess cardiac function, and then rats were euthanized, and heart and blood were collected and analysed (Figure 3A).

TNNT2-4Fpolycistronic-NIL–induced CM cell cycle re-entry is maintained for 16 weeks post-treatment. (A) Schematic diagram of the experimental design. (B) Schematic diagram of the mitosis double reporter system. (C) The left panel shows representative images at the site of injection 16 weeks post-injection in rat myocardium. LacZ (top panel) or TNNT2-4Fpolycistronic-NIL (bottom panel) shows the expression of the mitosis double reporter system DsRed (red) and GFP (green) and their co-localization with CMs (TNNT2, grey). The right panel shows quantification of the percentage of GFP-positive CMs of the labelled CMs (n = 6–11 rats per group; *P < 0.05, ***P < 0.001 vs. LacZ-NIL group; error bars indicate SD, one-way ANOVA). (D) Representative images of rat hearts at the border zone (right panel) stained against WGA (red) and nuclear DAPI (blue ). The right panel shows quantification of the cross-sectional area of CMs at the border zone (n = 1100–1200 cells from each group; ****P < 0.0001 compared to the LacZ-NIL group; error bars indicate SD, unpaired t-test). (E) Representative images of rat hearts stained with Masson’s trichrome stain [left panel; healthy myocardium stains (red) and fibrotic tissue stains (blue)] at the end of the experiment (scale bar = 2 mm). The right panel shows scar size quantification as a percentage of total LV tissue (n = 10–11 rats per group, **P < 0.01 compared to the LacZ-NIL; error bars indicate SD, unpaired t-test).

The mitosis double reporter system was developed to detect mitotic events in vivo, as previously reported.¹⁶ In this reporter system, we cloned a Lox-DsRed-Stop-Lox-GFP construct under the CAG promoter in lentivirus; in another lentiviral construct, we cloned the Cre encoding protein sequence under the influence of the AurKB promoter (AurKB-Cre). All infected cells will become DsRed positive; when mitosis occurs, Cre recombinase will be expressed and will cleave the DsRed-Stop sequence, turning these CMs permanently into GFP-positive CMs. Thus, the presence of GFP-positive cells will be an indicator of mitotic events. The data from the AurKB reporter must be interpreted cautiously as mitotic rather than cytokinesis events. As we described before, there is a 30% overestimation of cytokinesis events reported by this reporter.¹⁶ This reporter system shows both the number of infected cells (total red- and green-labelled cells) and the number of mitotic events (green-labelled cells; Figure 3B). Therefore, the quotient of green CMs and green plus red CMs provides a quantification of mitotic CMs.¹⁶ Compared with 0.6% in LacZ-NIL–treated hearts, TNNT2-4Fpolycistronic-NIL–treated hearts showed that ∼12% of the labelled CMs were green either 4 weeks or 4 months post-injection, which is an indication of that mitotic events occur only in the beginning of the treatment and sustained for the 4-month period of the experiment (Figure 3C). These data were in line with our previous findings that TNNT2-4Fpolycistronic-NIL induced CM proliferation in 10–15% of the infected CMs.¹⁶ TNNT2-4Fpolycistronic-NIL–treated hearts showed a significant reduction in the CM cell size at the border zone of the infarcted area (Figure 3D). Furthermore, and consistent with the results in the short-term study, Masson’s trichrome staining showed a 30% reduction in scar size in TNNT2-4Fpolycistronic-NIL–treated hearts compared with LacZ-NIL hearts (Figure 3E). Furthermore, we assessed the scar area and the viable myocardium area using Masson’s trichrome staining; TNNT2-4F-NIL treatment significantly increases the area of the viable myocardium with no significant impact on the scar area compared with the LacZ-NIL treatment (see Supplementary material online, Figure S4A and B). In addition, we investigated apoptosis at the end of the experiment. TNNT2-4Fpolyscistronic-NIL–treated hearts did not show any significant decrease in CM or non-CM apoptosis compared to LacZ-NIL–treated hearts (see Supplementary material online, Figure S4C–E). These data indicate that inducing CM proliferation reduces the scar percentage by generating more viable myocardium but not through scar resorption.

Functionally, all the rats treated with TNNT2-4Fpolycistronic-NIL had improved cardiac function (i.e. EF%, FS%, CO, and SV), while rats treated with LacZ-NIL had a decline in cardiac function over the period of 16 weeks (Figure 4A). As was the case at 4 weeks, the ESV and LVAs were improved in TNNT2-4Fpolycistronic-NIL–treated rats but EDV and LVAd did not differ from LacZ-NIL–treated rats (Figure 4B), nor was there a significant difference in the LW/BW ratio (Figure 5A). TNNT2-4Fpolycistronic-NIL–induced CM cell cycle re-entry did not increase the heart weight (HW) as indicated by no significant change in the HW per BW (HW/BW) ratio nor LV mass compared with LacZ-NIL–treated hearts (Figure 5A). Echocardiographic measurements of the LV dimensions, interventricular septum (IVS), LV posterior wall (PW) thickness, and LV inner dimension (LVID) during systole (s) or diastole (d) were not altered in TNNT2-4Fpolycistronic-NIL–treated hearts compared with LacZ-NIL–treated hearts (Figure 5B). These data indicated that TNNT2-4Fpolycistronic-NIL treatment induced cell cycle re-entry in this rat model of chronic IHF, and that the improvement in LV systolic function was sustained for 16 weeks without a significant effect on LV wall thickness or cardiac dilatation. The echocardiographic data are presented in Figure 4 as the changes (delta = cardiac function at the end of experiment—cardiac function 4 weeks post MI for each rat) to show the individual improvement of function after the treatment. All echocardiographic raw data are presented in the Supplementary material online, Excel Sheet and Figure S4.

TNNT2-4Fpolycistronic-NIL treatment in chronic IHF rats maintained the improvement in the systolic cardiac function and did not reverse the dilatation associated with chronic IHF, 16 weeks post-treatment. (A) Quantification of the change (delta) in EF, FS, SV, and CO and (B) LVAs, LVAd, ESV, and EDV between before and after treatment for each individual rat as assessed by echocardiography (n = 10–11 rats per group; *P < 0.05, **P < 0.01, ****P < 0.0001 compared to LacZ-NIL; error bars indicate SD, unpaired t-test).

TNNT2-4Fpolycistronic-NIL treatment did not alter the HW or the LV wall thickness. (A) Bar graph of the LW/BW, HW/BW, and echocardiographic measurements to the LV mass 20 weeks post-ischaemia (n = 10–11 rats per group; error bars indicate SD, unpaired t-test). (B) Bar graph of the echocardiographic measurements to the LV wall thickness (IVS), left ventricle PW, and LVID during systole (s) or diastole (d) 20 weeks post-IR (n = 10–11 rats per group; error bars indicate SD, two-way ANOVA).

To ensure that there are no detrimental effects of the LacZ-NIL virus on cardiac function, we compared the cardiac function of the LacZ-NIL–treated group with PBS-treated group. There was no significant difference in the cardiac function parameters between PBS-treated rats and LacZ-NIL–treated rats subjected to the same experimental condition after 20 weeks post I/R (see Supplementary material online, Figure S5 and Excel Sheet).

3.3. TNNT2-4Fpolycistronic-NIL treatment has a partial effect on systemic liver and kidney functionality biomarkers in chronic IHF

Since chronic IHF is associated with systemic impairment in the function of other organs such as the liver and kidney, we examined the effect of the improved LV systolic function following TNNT2-4Fpolycistronic-NIL treatment on systemic functional biomarkers of the liver and kidneys. Plasma biomarkers of liver and kidney dysfunction were measured at baseline (before MI), 4 weeks post I/R (before virus injection), and at the end of the experiment (20 weeks). At the 20-week timepoint, LacZ-NIL–treated rats showed a significant decrease in total protein (TP) and ALB and a significant increase in creatinine levels in the plasma compared with their baseline level, which are systemic biomarkers for liver and kidney functionality, while TNNT2-4Fpolycistronic-NIL–treated rats showed no significant change in these parameters compared to their baseline (Figure 6). There was no significant change in the non-specific cell damage marker (LDH) and liver function markers (AST, ALT) 4 weeks or 20 weeks post-infarction in the LacZ-NIL or TNNT2-4Fpolycistronic-NIL treatment groups.

TNNT2-4Fpolycistronic-NIL treatment resulted in a partial beneficiary effect on systemic liver and kidney functionality biomarkers associated with chronic IHF. Quantitative analysis of the plasma levels of TP, ALB, creatinine, lactate dehydrogenase (LDH), ALT, and AST at the baseline, 4 weeks post-ischaemia (before treatment), and 20 weeks post-ischaemia (16 weeks post-treatment; n = 10–11 rats; *P < 0.05, **P < 0.01; error bars indicate SD, two-way ANOVA).

These data indicate that the TNNT2-4Fpolycistronic-NIL-induced improvement in LV function in chronic IHF partially improves the systemic liver and kidney functionality biomarkers.

4. Discussion

We and others have demonstrated the beneficial effect of induced CM cell cycle re-entry on cardiac function in acute and subacute IHF models.^7,20–23 This study is the first to test the impact of the induction of CM cell cycle re-entry on a model of chronic IHF. We used a well-established rat model of chronic IHF characterized by LV systolic dysfunction and dilatation.^24,25 Our results demonstrate that TNNT2-4Fpolycistoronic-NIL-induced CM cell cycle re-entry improves LV systolic function, likely due to the generation of new CMs, and that this beneficial effect is sustained over the course of 4 months. However, there was no significant improvement in the chronic IHF–induced cardiac dilatation and the subsequent congestions in the lungs. This could be due to the fact that once dilatation and congestion occur, it is irreversible.

Despite significant improvements in the treatment of IHF, the prognosis of this syndrome remains bleak.²⁶ This is because current therapies improve symptoms but do not address the underlying cause of IHF, i.e. the loss of CMs.²⁷ Therefore, the disease continues to progress, leading inexorably to worsening LV function and, ultimately, death. A therapy aiming to regenerate the CMs would potentially increase the force generation by the cardiac muscle and improve and reverse the clinical outcome of this deadly syndrome. However, such therapy could be enhanced by a combination of therapies that could increase the vascularization of the scar. We and others have identified different cell cycle regulators that induce stable cytokinesis in adult post-mitotic cells, such as Cyclin A2,^20,28–30 mir199,^21,31 YAP-5SA,^32–35 or the combination of CDK1, CDK4, cyclin B1, and cyclin D1 (collectively known as 4F).^11,36 However, all of the previous animal studies focused on interventions administered within the acute and subacute phase of the MI (within 1–2 weeks post-ischaemia), a time when LV function and dimensions are unstable, and a major cascade of myocardial and systemic pathophysiological events are occurring, with rapid changes from 1 day to the next.^37,38 This acute/subacute phase is very different from the chronic phase of MI (chronic IHF), when a stable scar has formed, LV function and dimensions are less variable, and acute inflammation has subsided.³⁹ Whether cell cycle induction in CMs is effective in this setting of chronic IHF remained unknown. However, from a clinical standpoint, chronic IHF is the most relevant setting. First, it would be difficult to treat patients in the first few days after MI with intramyocardial injection of cell cycle regulators for a variety of reasons, including the instability of LV function, the uncertainty as to the eventual extent of LV dysfunction, and the spontaneous improvement in LV function that occurs commonly after an acute MI.^37,38 Furthermore, many patients have silent MIs or develop chronic IHF over the years without an acute precipitating event such as a large MI. In this patient population, therapies given in the acute/subacute phase of MI are not possible. Our present results demonstrate, for the first time, that administration of cell cycle regulators at 4 weeks after MI (in the setting of chronic IHF) is effective in inducing CM cell cycle re-entry and improving LV function, with some beneficial impact on the liver and kidneys’ functionality biomarkers. However, inducing CM proliferation in the chronic IHF model did not reverse the dilatation or adverse remodelling.

Translational applications of gene therapies that induce CM cell cycle re-entry require transient gene expression, which should be targeted only to the CMs to avoid any oncogenic potential. Here, we used the TNNT2-4Fpolycistoronic-NIL,¹⁶ which provides a transient and CM-specific expression of the 4F. During the past decade, there have been major advances in gene therapy delivery approaches for transient gene expression using either ModRNA⁴⁰ or NIL.¹⁴ Although the modified RNA approach is a promising virus-free delivery system for transient expression, its poor delivery and specificity for CMs limit its applicability for this purpose. Therefore, we used NIL as a tool for transient expression with high infection efficiency, as reviewed in Milone and O'Doherty.⁴¹ In our previous study,¹⁶ this TNNT2-4Fpolycistoronic-NIL induced robust, transient expression of the 4F only in CMs and induced CM cell cycle re-entry in vitro and in vivo. TNNT2-4Fpolycistoronic-NIL also significantly improved LV function in rat and pig models of subacute HF when injected 1 week post-ischaemia.¹⁶

In this study, we demonstrated that TNNT2-4Fpolycistronic-NIL induced only 12% of the infected CMs to undergo mitosis when injected 4 weeks post-ischaemia compared to around 25% when injected 1 week post-ischaemia as we previously reported.¹⁶ This lower level of mitosis may be due to the already established remodelling; that is, the scar may hinder the ability of CMs to proliferate. TNNT2-4Fpolycistronic-NIL–induced CM cell cycle re-entry was sufficient to improve systolic LV function; however, there was no change in the LV dilatation, which could be due to it being too late to reverse such dilatation. Interestingly, despite TNNT2-4Fpolycistronic-NIL–induced CM cell cycle re-entry, there was no gross hypertrophic phenotype. This could be due to the transient nature of the CM cell cycle re-entry, which is not enough to induce hypertrophy, and/or to the fact that, as a result of improved systolic function, any increase in LV mass due to cell cycle re-entry was offset by a reduction in compensatory post-MI LV hypertrophy.

Chronic IHF is usually accompanied by congestion in other tissues, such as the lungs, liver, and kidneys, due to increased systemic venous pressure and reduced plasma ALB levels. The TNNT2-4Fpolycistronic-NIL–induced improvement in LV function was not able to reverse lung congestion; however, it was able to reduce, at least partially, some liver and kidney functionality serum biomarkers, as evidenced by the preservation of the TP, ALB, and creatinine levels similar to their basal levels before ischaemia.

The current guideline-based therapeutic regimens for HF, including beta-adrenergic receptor blockers, angiotensin-converting enzyme inhibitors/angiotensin receptor blockers/neprilysin inhibitors, and mineralocorticoid receptor antagonists, are known to slow the adverse remodelling of IHF.^42,43 Therefore, future work would test the effect of the TNNT2-4Fpolycistoronic-NIL–induced CM cell cycle re-entry in treating chronic IHF patients combined with other guideline-based therapeutic regimens. This combinatory treatment may not only improve LV function in chronic IHF but also reverse adverse remodelling. Another possible therapeutic approach is to combine the TNNT2-4Fpolycistoronic-NIL with other direct cardiac reprogramming⁴⁴ to target the fibroblast pool and convert them to CMs.

In conclusion, the current work provides the first evidence that using transient and CM-specific gene therapy to induce CM cell cycle re-entry is effective in reducing scar size and improving LV systolic function over 4 months, although it is not sufficient to reverse the chronic IHF-associated LV dilatation and ventricular remodelling. This new approach has potentially vast implications for the treatment of the large patient population that suffers from chronic IHF.

4.1. Limitations of the study

There is still a major open question whether the generation of new myocardium was associated with the resorption of the scar or not. Our data showed that there is no significant increase in apoptosis following TNNT2-4F-NIL treatment, but there is a significant increase in the viable myocardium. These data indicate that inducing CM proliferation reduces the scar percentage by generating more viable myocardium but not through scar resorption, even though further single-cell RNA-seq assessments of the fibroblasts, resident macrophages, and other inflammatory cell profiles, following TNNT2-4F-NIL, will help ensure no changes in the inflammatory response, which could lead to scar resorption. Another major limitation is that it is not possible to co-label the mitotic CMs with the TNNT4-4F-NIL expressing CMs, as the expression of the 4F disappears within 2 weeks after injection. Furthermore, there is no accurate way to calculate how many new CMs are generated. However, we partially addressed this caveat with the mitotic double reporter¹⁶ to be able, at least, to count the mitotic CMs 4 months post-injection.

Supplementary material

Supplementary material is available at Cardiovascular Research online.

Supplementary Material

cvae002_Supplementary_Data

cvae002_supplementary_data.zip^{(2.2MB, zip)}

Acknowledgements

We would like to acknowledge Wenjian Wu, Halina Ruble, and Heather Stowers for their technical role in animal care and surgeries.

Contributor Information

Riham R E Abouleisa, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA.

Xian-Liang Tang, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA.

Qinghui Ou, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA.

Abou-Bakr M Salama, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA; Department of Cardiovascular Medicine, Faculty of Medicine, Zagazig University, 872 Shaibet an Nakareyah, Zagazig, Al-Sharqia Governorate 7120001, Egypt.

Amie Woolard, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA.

Dana Hammouri, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA; Department of Pharmacology and Toxicology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA.

Hania Abdelhafez, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA; Department of Bioengineering, Speed School of Engineering, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA.

Sarah Cayton, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA.

Sameeha K Abdulwali, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA; College of Medicine, Alfaisal University, Interconnection of Al Takhassousi،Al Zahrawi Street, Riyadh 11533, Saudi Arabia.

Momo Arai, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA; College of Medicine, Alfaisal University, Interconnection of Al Takhassousi،Al Zahrawi Street, Riyadh 11533, Saudi Arabia.

Israel D Sithu, Department of Medicine, Center for Cardiometabolic Science, Envirome Institute, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA; Department of Physiology, School of Medicine, University of Louisville, Louisville, 580 South Preston Street, KY 40202, USA.

Daniel J Conklin, Department of Medicine, Center for Cardiometabolic Science, Envirome Institute, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA.

Roberto Bolli, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA.

Tamer M A Mohamed, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA; Department of Pharmacology and Toxicology, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA; Department of Bioengineering, Speed School of Engineering, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA; Department of Medicine, Center for Cardiometabolic Science, Envirome Institute, University of Louisville, 580 South Preston Street, Louisville, KY 40202, USA; Department of Biochemistry Faculty of Pharmacy, Zagazig University, 872 Shaibet an Nakareyah, Zagazig, Zagazig, Al-Sharqia Governorate 7120001, Egypt; Institute of Cardiovascular Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK; Surgery Department, Baylor College of Medicine, 6519 Fannin Street, Houston, TX, 77030, USA.

Author contributions

R.R.E.A: designing research studies, virus preparation, conducting experiments, analysing data, writing the manuscript, and funding; X.-L.T.: animal surgery, echocardiography, acquiring and analysing data, and reviewing the manuscript; Q.O., A.-B.M.S., A.W., D.H., H.A., S.C., S.K.A., M.A., I.D.S., and D.J.C.: acquiring and analysing data for echocardiography, histology, and serum biomarkers and reviewing the manuscript; R.B.: supervising the animal experiments, reviewing the manuscript, and funding; T.M.A.M.: designing research studies, supervision, writing and reviewing the manuscript, final approval of the version to be published, and funding.

Funding

This work was supported by the National Institutes of Health (NIH) F32HL149140 to R.R.E.A., R01HL147921, P30GM127607, R15HL168688, and R01HL166280 to T.M.A.M., and HL78825 to R.B. and by the American Heart Association grant number 16SDG29950012 (T.M.A.M.).

Data availability

The raw data that support the findings of this study, such as the raw echo data, are available from the corresponding author (T.M.A.M.), upon request.

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36. Ou Q, Jacobson Z, Abouleisa RRE, Tang XL, Hindi SM, Kumar A, Ivey KN, Giridharan G, El-Baz A, Brittian K, Rood B, Lin YH, Watson SA, Perbellini F, McKinsey TA, Hill BG, Jones SP, Terracciano CM, Bolli R, Mohamed TMA. Physiological biomimetic culture system for pig and human heart slices. Circ Res 2019;125:628–642. [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Mechanic OJ, Gavin M, Grossman SA. Acute myocardial infarction. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2022.
38. Saleh M, Ambrose JA. Understanding myocardial infarction. F1000Res 2018;7:F1000. [DOI] [PMC free article] [PubMed] [Google Scholar]
39. Richardson WJ, Clarke SA, Quinn TA, Holmes JW. Physiological implications of myocardial scar structure. Compr Physiol 2015;5:1877–1909. [DOI] [PMC free article] [PubMed] [Google Scholar]
40. Zangi L, Lui KO, von Gise A, Ma Q, Ebina W, Ptaszek LM, Später D, Xu H, Tabebordbar M, Gorbatov R, Sena B, Nahrendorf M, Briscoe DM, Li RA, Wagers AJ, Rossi DJ, Pu WT, Chien KR. Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction. Nat Biotechnol 2013;31:898–907. [DOI] [PMC free article] [PubMed] [Google Scholar]
41. Milone MC, O'Doherty U. Clinical use of lentiviral vectors. Leukemia 2018;32:1529–1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Heidenreich PA, Bozkurt B, Aguilar D, Allen LA, Byun JJ, Colvin MM, Deswal A, Drazner MH, Dunlay SM, Evers LR, Fang JC, Fedson SE, Fonarow GC, Hayek SS, Hernandez AF, Khazanie P, Kittleson MM, Lee CS, Link MS, Milano CA, Nnacheta LC, Sandhu AT, Stevenson LW, Vardeny O, Vest AR, Yancy CW. 2022 AHA/ACC/HFSA guideline for the management of heart failure: a report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 2022;145:e895–e1032. [DOI] [PubMed] [Google Scholar]
43. McDonagh TA, Metra M, Adamo M, Gardner RS, Baumbach A, Böhm M, Burri H, Butler J, Čelutkienė J, Chioncel O, Cleland JGF, Coats AJS, Crespo-Leiro MG, Farmakis D, Gilard M, Heymans S, Hoes AW, Jaarsma T, Jankowska EA, Lainscak M, Lam CSP, Lyon AR, McMurray JJV, Mebazaa A, Mindham R, Muneretto C, Francesco Piepoli M, Price S, Rosano GMC, Ruschitzka F, Kathrine Skibelund A; ESC Scientific Document Group . 2021 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure: developed by the task force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC) with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur Heart J 2021;42:3599–3726. [DOI] [PubMed] [Google Scholar]
44. Mohamed TM, Stone NR, Berry EC, Radzinsky E, Huang Y, Pratt K, Ang YS, Yu P, Wang H, Tang S, Magnitsky S, Ding S, Ivey KN, Srivastava D. Chemical enhancement of in vitro and in vivo direct cardiac reprogramming. Circulation 2017;135:978–995. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

cvae002_Supplementary_Data

cvae002_supplementary_data.zip^{(2.2MB, zip)}

Data Availability Statement

The raw data that support the findings of this study, such as the raw echo data, are available from the corresponding author (T.M.A.M.), upon request.

References

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5. Arrigo M, Jessup M, Mullens W, Reza N, Shah AM, Sliwa K, Mebazaa A. Acute heart failure. Nat Rev Dis Primers 2020;6:16. [DOI] [PMC free article] [PubMed] [Google Scholar]
6. Knoebel SB. Chronic ischemic heart disease: from concept to fact. J Am Coll Cardiol 1983;1:20–30. [DOI] [PubMed] [Google Scholar]
7. Salama ABM, Gebreil A, Mohamed TMA, Abouleisa RRE. Induced cardiomyocyte proliferation: a promising approach to cure heart failure. Int J Mol Sci 2021;22:7720. [DOI] [PMC free article] [PubMed] [Google Scholar]
8. Garbern JC, Lee RT. Heart regeneration: 20 years of progress and renewed optimism. Dev Cell 2022;57:424–439. [DOI] [PMC free article] [PubMed] [Google Scholar]
9. Silverdal J, Sjöland H, Bollano E, Pivodic A, Dahlström U, Fu M. Prognostic impact over time of ischaemic heart disease vs. non-ischaemic heart disease in heart failure. ESC Heart Fail 2020;7:264–273. [DOI] [PMC free article] [PubMed] [Google Scholar]
10. Vedin O, Lam CSP, Koh AS, Benson L, Teng THK, Tay WT, Braun OÖ, Savarese G, Dahlström U, Lund LH. Significance of ischemic heart disease in patients with heart failure and preserved, midrange, and reduced ejection fraction: a nationwide cohort study. Circ Heart Fail 2017;10:e003875. [DOI] [PubMed] [Google Scholar]
11. Mohamed TMA, Ang YS, Radzinsky E, Zhou P, Huang Y, Elfenbein A, Foley A, Magnitsky S, Srivastava D. Regulation of cell cycle to stimulate adult cardiomyocyte proliferation and cardiac regeneration. Cell 2018;173:104–116 e112. [DOI] [PMC free article] [PubMed] [Google Scholar]
12. Xu Z, Zhang L, Lu J, Xu P, Liu G, Xie X, Mu W, Wang Y, Liu D. Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells. Sci China Life Sci 2016;59:1024–1033. [DOI] [PubMed] [Google Scholar]
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18. Tang XL, Nakamura S, Li Q, Wysoczynski M, Gumpert AM, Wu WJ, Hunt G, Stowers H, Ou Q, Bolli R. Repeated administrations of cardiac progenitor cells are superior to a single administration of an equivalent cumulative dose. J Am Heart Assoc 2018;7:e007400. [DOI] [PMC free article] [PubMed] [Google Scholar]
19. Tokita Y, Tang XL, Li Q, Wysoczynski M, Hong KU, Nakamura S, Wu WJ, Xie W, Li D, Hunt G, Ou Q, Stowers H, Bolli R. Repeated administrations of cardiac progenitor cells are markedly more effective than a single administration: a new paradigm in cell therapy. Circ Res 2016;119:635–651. [DOI] [PMC free article] [PubMed] [Google Scholar]
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36. Ou Q, Jacobson Z, Abouleisa RRE, Tang XL, Hindi SM, Kumar A, Ivey KN, Giridharan G, El-Baz A, Brittian K, Rood B, Lin YH, Watson SA, Perbellini F, McKinsey TA, Hill BG, Jones SP, Terracciano CM, Bolli R, Mohamed TMA. Physiological biomimetic culture system for pig and human heart slices. Circ Res 2019;125:628–642. [DOI] [PMC free article] [PubMed] [Google Scholar]
37. Mechanic OJ, Gavin M, Grossman SA. Acute myocardial infarction. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2022.
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41. Milone MC, O'Doherty U. Clinical use of lentiviral vectors. Leukemia 2018;32:1529–1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
42. Heidenreich PA, Bozkurt B, Aguilar D, Allen LA, Byun JJ, Colvin MM, Deswal A, Drazner MH, Dunlay SM, Evers LR, Fang JC, Fedson SE, Fonarow GC, Hayek SS, Hernandez AF, Khazanie P, Kittleson MM, Lee CS, Link MS, Milano CA, Nnacheta LC, Sandhu AT, Stevenson LW, Vardeny O, Vest AR, Yancy CW. 2022 AHA/ACC/HFSA guideline for the management of heart failure: a report of the American College of Cardiology/American Heart Association Joint Committee on Clinical Practice Guidelines. Circulation 2022;145:e895–e1032. [DOI] [PubMed] [Google Scholar]
43. McDonagh TA, Metra M, Adamo M, Gardner RS, Baumbach A, Böhm M, Burri H, Butler J, Čelutkienė J, Chioncel O, Cleland JGF, Coats AJS, Crespo-Leiro MG, Farmakis D, Gilard M, Heymans S, Hoes AW, Jaarsma T, Jankowska EA, Lainscak M, Lam CSP, Lyon AR, McMurray JJV, Mebazaa A, Mindham R, Muneretto C, Francesco Piepoli M, Price S, Rosano GMC, Ruschitzka F, Kathrine Skibelund A; ESC Scientific Document Group . 2021 ESC guidelines for the diagnosis and treatment of acute and chronic heart failure: developed by the task force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC) with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur Heart J 2021;42:3599–3726. [DOI] [PubMed] [Google Scholar]
44. Mohamed TM, Stone NR, Berry EC, Radzinsky E, Huang Y, Pratt K, Ang YS, Yu P, Wang H, Tang S, Magnitsky S, Ding S, Ivey KN, Srivastava D. Chemical enhancement of in vitro and in vivo direct cardiac reprogramming. Circulation 2017;135:978–995. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B1] 1. Savarese G, Lund LH. Global public health burden of heart failure. Card Fail Rev 2017;3:7–11. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B2] 2. Groeneveld PW, Medvedeva EL, Walker L, Segal AG, Richardson DM, Epstein AJ. Outcomes of care for ischemic heart disease and chronic heart failure in the Veterans Health Administration. JAMA Cardiol 2018;3:563–571. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B3] 3. Chaudhry MA. Heart failure. Curr Hypertens Rev 2019;15:7. [DOI] [PubMed] [Google Scholar]

[cvae002-B4] 4. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabé-Heider F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H, Jovinge S, Frisén J. Evidence for cardiomyocyte renewal in humans. Science 2009;324:98–102. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B5] 5. Arrigo M, Jessup M, Mullens W, Reza N, Shah AM, Sliwa K, Mebazaa A. Acute heart failure. Nat Rev Dis Primers 2020;6:16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B6] 6. Knoebel SB. Chronic ischemic heart disease: from concept to fact. J Am Coll Cardiol 1983;1:20–30. [DOI] [PubMed] [Google Scholar]

[cvae002-B7] 7. Salama ABM, Gebreil A, Mohamed TMA, Abouleisa RRE. Induced cardiomyocyte proliferation: a promising approach to cure heart failure. Int J Mol Sci 2021;22:7720. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B8] 8. Garbern JC, Lee RT. Heart regeneration: 20 years of progress and renewed optimism. Dev Cell 2022;57:424–439. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B9] 9. Silverdal J, Sjöland H, Bollano E, Pivodic A, Dahlström U, Fu M. Prognostic impact over time of ischaemic heart disease vs. non-ischaemic heart disease in heart failure. ESC Heart Fail 2020;7:264–273. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B10] 10. Vedin O, Lam CSP, Koh AS, Benson L, Teng THK, Tay WT, Braun OÖ, Savarese G, Dahlström U, Lund LH. Significance of ischemic heart disease in patients with heart failure and preserved, midrange, and reduced ejection fraction: a nationwide cohort study. Circ Heart Fail 2017;10:e003875. [DOI] [PubMed] [Google Scholar]

[cvae002-B11] 11. Mohamed TMA, Ang YS, Radzinsky E, Zhou P, Huang Y, Elfenbein A, Foley A, Magnitsky S, Srivastava D. Regulation of cell cycle to stimulate adult cardiomyocyte proliferation and cardiac regeneration. Cell 2018;173:104–116 e112. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B12] 12. Xu Z, Zhang L, Lu J, Xu P, Liu G, Xie X, Mu W, Wang Y, Liu D. Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells. Sci China Life Sci 2016;59:1024–1033. [DOI] [PubMed] [Google Scholar]

[cvae002-B13] 13. Nightingale SJ, Hollis RP, Pepper KA, Petersen D, Yu XJ, Yang C, Bahner I, Kohn DB. Transient gene expression by nonintegrating lentiviral vectors. Mol Ther 2006;13:1121–1132. [DOI] [PubMed] [Google Scholar]

[cvae002-B14] 14. Shaw A, Cornetta K. Design and potential of non-integrating lentiviral vectors. Biomedicines 2014;2:14–35. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B15] 15. Sultana N, Magadum A, Hadas Y, Kondrat J, Singh N, Youssef E, Calderon D, Chepurko E, Dubois N, Hajjar RJ, Zangi L. Optimizing cardiac delivery of modified mRNA. Mol Ther 2017;25:1306–1315. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B16] 16. Abouleisa RRE, Salama ABM, Ou Q, Tang XL, Solanki M, Guo Y, Nong Y, McNally L, Lorkiewicz PK, Kassem KM, Ahern BM, Choudhary K, Thomas R, Huang Y, Juhardeen HR, Siddique A, Ifthikar Z, Hammad SK, Elbaz AS, Ivey KN, Conklin DJ, Satin J, Hill BG, Srivastava D, Bolli R, Mohamed TMA. Transient cell cycle induction in cardiomyocytes to treat subacute ischemic heart failure. Circulation 2022;145:1339–1355. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B17] 17. Salama ABM, Abouleisa RRE, Ou Q, Tang XL, Alhariry N, Hassan S, Gebreil A, Dastagir M, Abdulwali F, Bolli R, Mohamed TMA. Transient gene therapy using cell cycle factors reverses renin-angiotensin-aldosterone system activation in heart failure rat model. Mol Cell Biochem 2023;478:1245–1250. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B18] 18. Tang XL, Nakamura S, Li Q, Wysoczynski M, Gumpert AM, Wu WJ, Hunt G, Stowers H, Ou Q, Bolli R. Repeated administrations of cardiac progenitor cells are superior to a single administration of an equivalent cumulative dose. J Am Heart Assoc 2018;7:e007400. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B19] 19. Tokita Y, Tang XL, Li Q, Wysoczynski M, Hong KU, Nakamura S, Wu WJ, Xie W, Li D, Hunt G, Ou Q, Stowers H, Bolli R. Repeated administrations of cardiac progenitor cells are markedly more effective than a single administration: a new paradigm in cell therapy. Circ Res 2016;119:635–651. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B20] 20. Shapiro SD, Ranjan AK, Kawase Y, Cheng RK, Kara RJ, Bhattacharya R, Guzman-Martinez G, Sanz J, Garcia MJ, Chaudhry HW. Cyclin A2 induces cardiac regeneration after myocardial infarction through cytokinesis of adult cardiomyocytes. Sci Transl Med 2014;6:224ra27. [DOI] [PubMed] [Google Scholar]

[cvae002-B21] 21. Gabisonia K, Prosdocimo G, Aquaro GD, Carlucci L, Zentilin L, Secco I, Ali H, Braga L, Gorgodze N, Bernini F, Burchielli S, Collesi C, Zandonà L, Sinagra G, Piacenti M, Zacchigna S, Bussani R, Recchia FA, Giacca M. MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs. Nature 2019;569:418–422. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B22] 22. Huang W, Feng Y, Liang J, Yu H, Wang C, Wang B, Wang M, Jiang L, Meng W, Cai W, Medvedovic M, Chen J, Paul C, Davidson WS, Sadayappan S, Stambrook PJ, Yu XY, Wang Y. Loss of microRNA-128 promotes cardiomyocyte proliferation and heart regeneration. Nat Commun 2018;9:700. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B23] 23. Xiao S, Liang R, Lucero E, McConnell BK, Chen Z, Chang J, Navran S, Schwartz RJ, Iyer D. STEMIN and YAP5SA synthetic modified mRNAs regenerate and repair infarcted mouse hearts. J Cardiovasc Aging 2022;2:31. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B24] 24. Bolger AP, Coats AJ, Gatzoulis MA. Congenital heart disease: the original heart failure syndrome. Eur Heart J 2003;24:970–976. [DOI] [PubMed] [Google Scholar]

[cvae002-B25] 25. Cabac-Pogorevici I, Muk B, Rustamova Y, Kalogeropoulos A, Tzeis S, Vardas P. Ischaemic cardiomyopathy. Pathophysiological insights, diagnostic management and the roles of revascularisation and device treatment. Gaps and dilemmas in the era of advanced technology. Eur J Heart Fail 2020;22:789–799. [DOI] [PubMed] [Google Scholar]

[cvae002-B26] 26. Virani SS, Alonso A, Aparicio HJ, Benjamin EJ, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Cheng S, Delling FN, Elkind MSV, Evenson KR, Ferguson JF, Gupta DK, Khan SS, Kissela BM, Knutson KL, Lee CD, Lewis TT, Liu J, Loop MS, Lutsey PL, Ma J, Mackey J, Martin SS, Matchar DB, Mussolino ME, Navaneethan SD, Perak AM, Roth GA, Samad Z, Satou GM, Schroeder EB, Shah SH, Shay CM, Stokes A, VanWagner LB, Wang NY, Tsao CW; American Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics Subcommittee . Heart disease and stroke statistics—2021 update: a report from the American Heart Association. Circulation 2021;143:e254–e743 . [DOI] [PubMed] [Google Scholar]

[cvae002-B27] 27. Inamdar AA, Inamdar AC. Heart failure: diagnosis, management and utilization. J Clin Med 2016;5:62. [DOI] [PMC free article] [PubMed] [Google Scholar]

[cvae002-B28] 28. Chaudhry HW, Dashoush NH, Tang H, Zhang L, Wang X, Wu EX, Wolgemuth DJ. Cyclin A2 mediates cardiomyocyte mitosis in the postmitotic myocardium. J Biol Chem 2004;279:35858–35866. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Gene therapy encoding cell cycle factors to treat chronic ischemic heart failure in rats

Riham R E Abouleisa

Xian-Liang Tang

Qinghui Ou

Abou-Bakr M Salama

Amie Woolard

Dana Hammouri

Hania Abdelhafez

Sarah Cayton

Sameeha K Abdulwali

Momo Arai

Israel D Sithu

Daniel J Conklin

Roberto Bolli

Tamer M A Mohamed

Abstract

Aims

Methods and results

Conclusion

1. Introduction

2. Methods

2.1. Cloning and preparation of integrating and NIL

2.2. Rat experiments

2.2.1. Study ethical approval

2.2.2. Rat heart ischaemic/reperfusion surgery

2.3. Histological analysis

2.4. Immunohistochemistry

Table 1.

2.5. Cell size analysis

2.6. Plasma collection

2.7. Toxicity tests

2.8. Apoptosis (TUNEL) assay

2.9. Statistical analyses

3. Results

3.1. TNNT2-4Fpolycistronic-NIL improves LV systolic cardiac function but does not reverse dilatation

Figure 1.

Figure 2.

3.2. Improved LV systolic function after TNNT2-4Fpolycistronic-NIL administration is maintained for 4 months

Figure 3.

Figure 4.

Figure 5.

3.3. TNNT2-4Fpolycistronic-NIL treatment has a partial effect on systemic liver and kidney functionality biomarkers in chronic IHF

Figure 6.

4. Discussion

4.1. Limitations of the study

Supplementary material

Supplementary Material

Acknowledgements

Contributor Information

Author contributions

Funding

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

References

ACTIONS

PERMALINK

RESOURCES

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