Modified mRNA-Mediated CCN5 Gene Transfer Ameliorates Cardiac Dysfunction and Fibrosis without Adverse Structural Remodeling

Abstract

Modified mRNAs (modRNAs) are an emerging delivery method for gene therapy. The success of modRNA-based COVID-19 vaccines has demonstrated that modRNA is a safe and effective therapeutic tool. Moreover, modRNA has the potential to treat various human diseases, including cardiac dysfunction. Acute myocardial infarction (MI) is a major cardiac disorder that currently lacks curative treatment options, and MI is commonly accompanied by fibrosis and impaired cardiac function. Our group previously demonstrated that the matricellular protein CCN5 inhibits cardiac fibrosis (CF) and mitigates cardiac dysfunction. However, it remains unclear whether early intervention of CF under stress conditions is beneficial or more detrimental due to potential adverse effects such as left ventricular (LV) rupture. We hypothesized that CCN5 would alleviate the adverse effects of myocardial infarction (MI) through its anti-fibrotic properties under stress conditions. To induce the rapid expression of CCN5, ModRNA-CCN5 was synthesized and administrated directly into the myocardium in a mouse MI model. To evaluate CCN5 activity, we established two independent experimental schemes: (1) preventive intervention and (2) therapeutic intervention. Functional analyses, including echocardiography and magnetic resonance imaging (MRI), along with molecular assays, demonstrated that modRNA-mediated CCN5 gene transfer significantly attenuated cardiac fibrosis and improved cardiac function in both preventive and therapeutic models, without causing left ventricular rupture or any adverse cardiac remodeling. In conclusion, early intervention in CF by ModRNA-CCN5 gene transfer is an efficient and safe therapeutic modality for treating MI-induced heart failure.

Keywords: CCN5; cardiac fibrosis; heart failure (HF); left ventricular rupture; modified mRNA (modRNA); myocardial infarction (MI).

Figure 1

Preventive intervention of ModRNA-CCN5 attenuates MI-induced cardiac dysfunction. (A) MI was induced in the mouse heart and subsequently, ModRNA-Con or -CCN5 was directly injected into the LV endocardium of the mouse. A week later, echocardiography, histology, and molecular analysis were performed to determine the effects of modified mRNA-CCN5. (B) Representative M-mode echocardiographic images are shown. (C) Fractional shortening (FS), LVIDd, and LVIDs were compared, sham (n = 6) vs. MI + ModRNA-Con (n = 7) vs. MI + ModRNA-CCN5 (n = 9). (D) The results of the MRI analysis are shown. Left ventricular ejection fraction (LVEF), LV mass, and infarct size were compared, sham (n = 3) vs. MI + ModRNA-Con (n = 4) vs. MI + ModRNA-CCN5 (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Preventive CCN5 treatment ameliorates MI-induced cardiac fibrosis without LV rupture. (A) Heart tissues from the preventive intervention were used for all experiments. The red-stained area indicated normal tissue and the blue-stained area indicated fibrotic tissue in Masson’s trichrome staining. WGA staining was performed to measure the cellular size of the heart. The green-stained area indicated a cellular membrane. Scale bar: 50 μm. (B) Fibrotic area was analyzed using a computer-assisted method. ImageJ plug-in software was used to measure the ratio of fibrotic area. (C) Cell surface area (CSA) was analyzed using a computer-assisted method. ImageJ plug-in software was used to measure CSA. (D) Cardiac tissue lysates (n = 6 for sham, n = 7 for ModRNA-Con, and n = 9 for ModRNA-CCN5) were immunoblotted with antibodies against FAP, Fibulin-1, α-SMA, CCN5, GAPDH, and TGF-β1. (E) Synthesized cDNAs were used to analyze the mRNA expression level of α-Sma, Tgf-β1, Fap, and Bnp by qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Therapeutic intervention of ModRNA-CCN5 mitigates MI-induced cardiac dysfunction. (A) MI was induced in the heart. Then, 2 weeks later, ModRNA-Con or -CCN5 was directly injected into the endocardium of the mouse LV (therapeutic intervention). Subsequently, 4 weeks later, echocardiography, MRI, histology, and molecular analysis were performed to determine the effects of modified mRNA-CCN5. (B) Representative M-mode echocardiographic images are shown. (C) Fractional shortening (FS), LVIDd, and LVIDs were compared, sham (n = 7) vs. MI + ModRNA-Con (n = 7) vs. MI + ModRNA-CCN5 (n = 8). (D) MRI was performed in each group of mice (n = 3 for sham, n = 4 for ModRNA-Con, and n = 4 for ModRNA-CCN5) and some critical values are presented. Left ventricular ejection fraction (LVEF), LV mass, and infarct size were compared, sham vs. MI + ModRNA-Con vs. MI + ModRNA-CCN5. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Therapeutic intervention with ModRNA-CCN5 reduces MI-induced cardiac dysfunction and fibrosis. (A) The heart tissues from the therapeutic intervention were used for all experiments. The red-stained area indicated normal tissue and the blue-stained area indicated fibrotic tissue in Masson’s trichrome staining. WGA staining was performed to measure the cellular size of the heart. The green-stained area indicated a cellular membrane. Scale bar: 50 μm. (B) Fibrotic area was analyzed using a computer-assisted method. ImageJ plug-in software was used to measure the ratio of fibrotic area. (C) Cell surface area (CSA) was analyzed using a computer-assisted method. ImageJ plug-in software was used to measure CSA. (D) Cardiac tissue lysates were immunoblotted with antibodies against SERCA2a, NCX1, p-PLB (Ser 16), total-PLB, and GAPDH. (E) Additional lysates were immunoblotted with antibodies against TGF-β, p-Smad2, Smad2/3, ANF, Fibronectin, Periostin, α-SMA, Vimentin, CCN5, and GAPDH. For each experimental group, animals were used as follows: n = 7 for sham, n = 7 for ModRNA-Con, and n = 8 for ModRNA-CCN5, * p < 0.05, ** p < 0.01, n.s.: not significant.