CCND2 Modified mRNA Activates Cell Cycle of Cardiomyocytes in Hearts With Myocardial Infarction in Mice and Pigs
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Abstract
BACKGROUND:
Experiments in mammalian models of cardiac injury suggest that the cardiomyocyte-specific overexpression of CCND2 (cyclin D2, in humans) improves recovery from myocardial infarction (MI). The primary objective of this investigation was to demonstrate that our specific modified mRNA translation system (SMRTs) can induce CCND2 expression in cardiomyocytes and replicate the benefits observed in other studies of cardiomyocyte-specific CCND2 overexpression for myocardial repair.
METHODS:
The CCND2-cardiomyocyte-specific modified mRNA translation system (cardiomyocyte SMRTs) consists of 2 modRNA constructs: one codes for CCND2 and contains a binding site for L7Ae, and the other codes for L7Ae and contains recognition elements for the cardiomyocyte-specific microRNAs miR-1 and miR-208. Thus, L7Ae suppresses CCND2 translation in noncardiomyocytes but is itself suppressed by endogenous miR-1 and -208 in cardiomyocytes, thereby facilitating cardiomyocyte-specific CCND2 expression. Experiments were conducted in both mouse and pig models of MI, and control assessments were performed in animals treated with an SMRTs coding for the cardiomyocyte-specific expression of luciferase or green fluorescent protein (GFP), in animals treated with L7Ae modRNA alone or with the delivery vehicle, and in Sham-operated animals.
RESULTS:
CCND2 was abundantly expressed in cultured, postmitotic cardiomyocytes 2 days after transfection with the CCND2-cardiomyocyte SMRTs, and the increase was accompanied by the upregulation of markers for cell-cycle activation and proliferation (eg, Ki67 and Aurora B kinase). When the GFP-cardiomyocyte SMRTs were intramyocardially injected into infarcted mouse hearts, the GFP signal was observed in cardiomyocytes but no other cell type. In both MI models, cardiomyocyte proliferation (on day 7 and day 3 after treatment administration in mice and pigs, respectively) was significantly greater, left-ventricular ejection fractions (days 7 and 28 in mice, days 10 and 28 in pigs) were significantly higher, and infarcts (day 28 in both species) were significantly smaller in animals treated with the CCND2-cardiomyocyte SMRTs than in any other group that underwent MI induction.
CONCLUSIONS:
Intramyocardial injections of the CCND2-cardiomyocyte SMRTs promoted cardiomyocyte proliferation, reduced infarct size, and improved cardiac performance in small and large mammalian hearts with MI.
Novelty and Significance
What is Known?
The proliferative capacity of adult mammalian cardiomyocytes (CMs) is too limited to repopulate the scarred region of infarcted hearts.
Overexpression of the cell-cycle regulator cyclin D2 (CCND2 in humans) promoted CM proliferation and improved recovery from myocardial infarction (MI) in previous preclinical rodent or pigs studies, but excessive or long-term cell-cycle activation could lead to safety concerns.
A novel Cardiomyocyte-Specific Modified mRNA Translation system (CM SMRTs) has been developed to transiently upregulate the expression of target molecules in CMs.
What New Information Does This Article Contribute?
The CCND2-CM SMRTs significantly, but only transiently (<28 days), upregulated CCND2 expression in CMs with exceptional cell-type specificity.
When the CCND2-CM SMRTs was used to drive CCND2 expression in CMs of infarcted pig and mouse hearts, the treatment significantly increased CM proliferation, but for only a few weeks; and significantly improved recovery from acute myocardial infarction (AMI) without increasing the risk of arrhythmia.
Treatment with the CCND2-CM SMRTs is unlikely to be associated with long-term safety concerns because the target molecule is only transiently expressed.
Using small and large mammalian models of AMI, the current study demonstrates that the transient and exclusive overexpression of CCND2 in CMs via the novel CCND2-CM SMRTs results in significantly increase of CM proliferation, which in turn, accompanied by the prominent reduction of infarct size, improved LV contractile function with no long-term safety concerns.
In This Issue, see p 447
Meet the First Author, see p 448
Editorial, see p 505
Patients with severe acute myocardial infarction (MI) often progress to end-stage congestive heart failure, which is one of the most significant problems in public health. From a cellular perspective, heart failure is caused by the loss of the contractile unit of the left ventricle: cardiomyocytes. Mammalian cardiomyocytes exit the cell cycle shortly after birth,1–4 but the results from our studies with neonatal pigs indicated that when MI was induced on postnatal day 1 (P1), cardiomyocytes re-entered the cell cycle and proliferated, leading to the complete restoration of cardiac function with little evidence of scarring by P30.3,5–8 Anecdotal evidence in a pediatric patient suggests that the regenerative capacity of newborn infant hearts may be similar,9 but less than 1% of cardiomyocytes in adult human hearts are replaced each year,10 and studies in adult mice suggest that cardiomyocyte proliferation increases only marginally in response to cardiac injury.11 Thus, the meager proliferative capacity of adult cardiomyocytes cannot repair the damage caused by MI.
Investigations in mouse,12–14 rat,15 and pig16–18 MI models have targeted many of the pathways that regulate the cell cycle activity of cardiomyocytes in an attempt to promote cardiomyocyte proliferation and improve recovery from myocardial injury. For example, CCND2 (cyclin D2, in humans) controls the G1-to-S phase transition in cardiomyocytes, and cardiomyocyte-targeted cyclin D2 overexpression has been associated with improvements in infarct size and cardiac performance.19–21 Furthermore, when human-induced pluripotent stem cells (hiPSCs) were engineered to overexpress CCND2 (CCND2-OEhiPSC-cardiomyocytes) from the cardiomyocyte-specific MHC (myosin heavy chain) promoter, and the CCND2-OEhiPSC-cardiomyocytes were transplanted into infarcted mouse hearts, the small number of hiPSC-cardiomyocytes that were engrafted at the site of administration proliferated and repopulated the myocardial scar, thereby reducing infarct size and improving cardiac performance.22 However, the methods used to manipulate the expression of regulatory molecules are often accompanied by safety concerns that impede their translation to clinical use. Viral-based therapies, especially those that can be integrated into the genome of the host cell (eg, adeno-associated virus), may promote excessive and enduring expression of the gene of interest, which could result in cardiac hypertrophy, uncontrolled cardiomyocyte proliferation, and an increased risk of arrhythmia18; and while nonintegrating lentiviral vectors are expressed only transiently in vivo,16 they may have off-target side-effects, so their safety in humans remains largely unknown.
modRNA technology provides an alternative strategy for transiently inducing gene expression23 that is efficient, titratable, and minimally immunogenic with bell-shaped pharmacokinetics and no risk of genomic integration.24,25 Researchers have already used modRNA to identify novel targets for improving myocardial recovery after MI in mice26–30 and pigs,31 and the safety profile of modRNA was thoroughly studied during the development of mRNA-based vaccines against SARS-CoV-2. Recently, we have developed a cardiomyocyte-specific modified mRNA translation system (cardiomyocyte SMRTs) that can be used to upregulate the expression of a gene of interest exclusively in cardiomyocytes.26,32 For the experiments described in this report, we used the cardiomyocyte SMRTs to transiently overexpress CCND2 in cardiomyocytes and then conducted experiments in both mouse and pig MI models to investigate whether this novel strategy could activate the cell cycle of cardiomyocytes and improve recovery from LV injury.
METHODS
Data Availability
A detailed description of all materials and methods can be found in the Supplemental Methods and the Major Resources Table in the Supplemental Material. The data that support the findings of this study are available upon reasonable request.
Ethics Statement
The animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Alabama at Birmingham.
RESULTS
CCND2-Cardiomyocyte SMRTs Drives Cardiomyocyte-Specific CCND2 Expression
The cardiomyocyte SMRTs (Figure 1A; Figure S1A)32 is composed of 2 distinct modRNA constructs. One codes for the gene of interest and contains a kink-turn motif, which functions as a binding site for the archaeal ribosomal protein L7Ae, while the other codes for L7Ae and contains recognition elements for the cardiomyocyte-specific microRNAs miR-1 and miR-208.33,34 Thus, L7Ae suppresses translation of the gene of interest in noncardiomyocytes but is recognized and cleaved by endogenous miR-1 and -208 in cardiomyocytes, thereby facilitating cardiomyocyte-specific expression of the modRNA transcript.
Figure 1. CCND2 (cyclin D2)-cardiomyocye (CM)-specific modified mRNA translation system (SMRTs) promoted proliferation in cultured postmitotic cardiomyocytes. A, The CM SMRTs consists of 2 modRNA constructs as illustrated. B and C, Cocultures of postmitotic human-induced pluripotent stem cell–derived cardiomyocyte (hiPSC-CM) and (B) endothelial cells (ECs) or (C) fibroblasts (FBs) were transfected with green fluorescent protein (GFP)-modRNA or the GFP-CM SMRTs; then, the cultured cells were immunofluorescently stained for the expression GFP and markers for CMs (cTnT [cardiac troponin T] and αSA [α sarcomeric actin]), ECs (vWF [von Willebrand factor]), and FBs (vimentin). Nuclei were counterstained with DAPI. Scale bar=50 µm. Yellow arrows indicate (B) ECs or (C) FBs. D through H, hiPSC-CMs were transfected with the Luc-CM SMRTs or the CCND2-CM SMRTs, stained for cTnT expression and for (D) CCND2 expression, (E) the expression of Ki67 (a proliferation marker), (F) Bromodeoxyuridine incorporation (a marker for cell-cycle S-phase), (G) phosphorylated histone H3 (PH3; a marker for the cell-cycle G2/M phase transition), or (H) the expression of AuB (aurora kinase B; a karyokinesis/cytokinesis marker). Nuclei were counterstained with DAPI, and the percentage of hiPSC-CMs that were positive for CCND2, Ki67, BrdU, or PH3, and for G2/prophase-, anaphase-, or abscission-like AuB expression, was determined via immunofluorescence staining. Scale bar=50 µm. I, Time-lapse images were obtained of hiPSC-CMs that underwent cell division (yellow arrows) after treatment with the CCND2-CM SMRTs. J, hiPSC-CM cell counts were quantified for 7 days after treatment with the Luc-CM or CCND2-CM SMRTs. Scale bar=50 µm. n=4 biological replication for D through H and J. All data are presented as mean±SEM. P values from unpaired t test (D through H), or Mann-Whitney U test (H auroraB [G2/prophase] positive CM percentage), or ordinary 2-way ANOVA with Šídák multiple comparisons (J).
modRNAs for L7Ae, Luc (luciferase), CCND2, green fluorescent protein (GFP), nuclear GFP (nGFP [nuclear green fluorescent protein]), and CCND2-nGFP were transcribed in vitro35,36 and validated with a bioanalyzer (Figure S2). Subsequent experiments with GFP-modRNA indicated that 3 μg was the optimal dose for efficient modRNA translation in hiPSC-cardiomyocytes (Figure S3A) and that the GFP signal peaked 2 days after transfection before declining to 50% and 20% of maximum on days 5 and 7, respectively (Figure S3B). Transfection with the optimized dose did not promote hiPSC-cardiomyocyte apoptosis (Figure S3C), and the abundance of CCND2 protein in CCND2-cardiomyocyte SMRTs–treated hiPSC-cardiomyocytes peaked from 1 to 3 days after treatment, when it exceeded the abundance in the CCND2-OEhiPSC-cardiomyocytes used in our previous studies22,37,38 by ≈3-fold, before declining to nearly undetectable levels by day 14 (Figure S4). The specificty of the cardiomyocyte SMRTs was validated by transfecting cocultures of hiPSC-cardiomyocytes and endothelial cells or hiPSC-cardiomyocytes and fibroblasts with 3 μg GFP-modRNA or with the GFP-cardiomyocyte SMRTs (3 μg GFP-modRNA and 1.5 μg L7Ae-modRNA): robust GFP immunofluorescence was observed in all cell types after transfection with GFP-modRNA and in hiPSC-cardiomyocytes that had been transfected with the GFP-cardiomyocyte SMRTs but not in GFP-cardiomyocyte SMRTs–transfected endothelial cells or fibroblasts (Figure 1B and 1C).
CCND2-Cardiomyocyte SMRTs Activates Cardiomyocyte Proliferation, Promotes Myocardial Regeneration, and Improves Recovery From MI in Mice
CCND2 was abundantly expressed in cultured, postmitotic (60-day old) hiPSC-cardiomyocytes 2 days after transfection with the CCND2-cardiomyocyte SMRTs (Figure 1D), and this increase was accompanied by the upregulation of markers for proliferation (Ki67 expression; Figure 1E), for S-phase (Bromodeoxyuridine incorporation; Figure 1F) and the G2/M phase transition (phosphorylated histone H3 [PH3] abundance; Figure 1G) of the cell cycle, and for karyokinesis/cytokinesis (aurora B kinase39 expression; Figure 1H); time-lapse images (Figure 1I; Video S1) and cell counts (Figure 1J) also indicated that treatment with the CCND2-cardiomyocyte SMRTs increased cell division in hiPSC-cardiomyocytes. Furthermore, when the GFP-cardiomyocyte SMRTs (100 μg GFP-modRNA and 50 μg L7Ae-modRNA) was intramyocardially injected into infarcted mouse hearts, immunofluorescence images of sections obtained 3 days later confirmed that the GFP signal was observed in cardiomyocytes but no other cell types (Figure 2A), and live bioluminescence images indicated that luciferase expression in mice treated with intramyocardial injections of the Luc-cardiomyocyte SMRTs (100 μg Luc-modRNA and 50 μg L7Ae-modRNA) after MI induction peaked 24 hours after administration and was maintained for no more than 240 hours (Figure S5). Thus, we began to investigate whether the CCND2-cardiomyocyte SMRTs could promote cardiomyocyte cell cycle activity and improve recovery from MI by conducting experiments in a mouse MI model (Figure 2B).
Figure 2. Intramyocardial injections of the CCND2 (cyclin D2)-cardiomyocte (CM) specific modified mRNA translation system (SMRTs) promoted CM proliferation and improved measures of cardiac function, infarct size, and hypertrophy in a mouse myocardial infarction (MI) model. A, Mice with surgically induced MI received intramyocardial injections of the green fluorescent protein (GFP)-CM SMRTs (100 μg GFP-modRNA and 50 μg L7Ae-modRNA) and were sacrificed 3 days later; then, sections of heart tissue from the site of administration were stained for cTnT (cardiac troponin T) and GFP expression, and nuclei were counterstained with DAPI. Scale bar=50 µm. B, The experimental protocol in the mouse MI model is shown as a schematic. C through E, Sections of heart tissue from the border zone of the infarct were collected from mice 1 week after MI surgery and stained for the presence of cTnT and (C) Ki67, (D) phosphorylated histone H3 (PH3), or (E) AuB (aurora kinase B). AuB staining remains equidistant (symmetrical AuB) between the 2 daughter nuclei during cell division but moves away from the midpoint (asymmetrical AuB) during a multinucleation event3; daughter nuclei are identified with arrows in E. Nuclei were counterstained with DAPI and then CM proliferation, cell-cycle activity, cytokinesis, and multinucleation were quantified as the percentages of cTnT-positive cells that were also positive for Ki67, PH3, and symmetrical and asymmetrical AuB, respectively. Wheat germ agglutinin (WGA) staining was performed to identify cell membrane. Scale bar=50 µm for C; scale bar=10 µm for D; scale bar=10 µm for E. n=3 animals per group. F and G, Left-ventricular ejection fraction (F) and fractional shortening (G) were calculated from echocardiographic images obtained 1 day before MI induction and 0.5, 2, 4, 7, 14, and 28 days afterward. n=8 animals per group. H, LVs collected from mice at week 4 were cut into sections and stained with Sirius red and fast green to visualize fibrotic (red) and normal (green) tissue; then, infarct size was quantified as the ratio of the length of LV scar arc to the circumference of the LV and presented as a percentage. Scale bar=1 mm. n=7 animals per group for vehicle and CCND2-CM SMRTs groups; n=8 animals per group for L7Ae-modRNA and Luc-CM SMRTs groups. I, Sections from the border zone were stained with WGA (wheat germ agglutinin) to visualize cell borders and for the expression of cTnT to visualize CMs. Nuclei were counterstained with DAPI; then, CM cross-sectional sizes were quantified. Scale bar=20 µm. n=5 animals per group. All data are presented as mean±SEM. P values from 1-way ANOVA with Tukey test (C–E, I), or 2-way ANOVA with Šídák multiple comparisons (F and G). aP<0.05 vs vehicle; bP<0.01 vs vehicle; cP<0.001 vs vehicle; dP<0.001 vs L7Ae-modRNA; eP<0.001 vs Luc-CM SMRTs (F and G).
MI was induced via permanent ligation of the left anterior descending coronary artery and then the animals were unbiased assigned into 4 treatment groups. The CCND2-cardiomyocyte SMRTs group received 100 μg CCND2-modRNA with 50 μg L7Ae-modRNA, the Luc-cardiomyocyte SMRTs group received 100 μg Luc-modRNA with L7Ae-modRNA, the L7Ae-modRNA group received 50 μg L7Ae modRNA, and the vehicle group received an equivalent volume of the delivery vehicle; a fifth group of animals, the Sham group, underwent all surgical procedures for MI induction except left anterior descending artery ligation and recovered without any of the experimental treatments. The survival rate for mice in CCND2-cardiomyocyte SMRTs–treated animals was significantly improved when compared with other MI mice (Figure S6A), and Western blot assessments conducted in myocardial tissues collected 3 days after MI and treatment administration or Sham surgery confirmed that CCND2 protein levels were upregulated in the hearts of animals in the CCND2-cardiomyocyte SMRTs group (Figure S6B).
Immunofluorescence images of sections collected from the border zone of the infarct on Day 7 after MI induction and treatment indicated that the proportion of cardiomyocytes that expressed Ki67 (Figure 2C), PH3 (Figure 2D), symmetrical AuB (aurora kinase B; a marker for cytokinesis), or asymmetrical AuB (a multinucleation marker; Figure 2E) was significantly higher in mice that were treated with the CCND2-cardiomyocyte SMRTs than in the Vehicle-, L7Ae-modRNA-, or Luc-cardiomyocyte SMRTs-treatment groups. However, measures of cell cycle activity and proliferation (Ki67 and PH3) did not differ significantly in cardiomyocytes from the CCND2-cardiomyocyte SMRTs and vehicle groups on days 14 or 28 after injection (Figure S7). Notably, miR-1 and miR-208 have been linked to increases in cardiomyocyte apoptosis,40,41 and the proportion of apoptotic cardiomyocytes on day 3 after MI induction was significantly lower in the hearts from L7Ae-modRNA, Luc-cardiomyocyte SMRTs, and CCND2-cardiomyocyte SMRTs animals than in vehicle-treated hearts (Figure S8), perhaps because miR-1 and miR-208 were sequestered at their respective binding sites in the L7Ae-modRNA construct.
Cardiac function was evaluated 1 day before MI induction and 0.5, 2, 4, 7, 14, and 28 days afterward via echocardiographic assessments (Figure S9A) of left ventricular internal diameter end-systole (Figure S9B), left ventricular end-diastole (Figure S9B), left ventricular ejection fraction (Figure 2F), and left ventricular fractional shortening (Figure 2G). Cardiac functional parameters were equivalent at day 0.5 in all groups that underwent MI induction, but left ventricular ejection fraction and left ventricular fractional shortening progressively increased in the CCND2-cardiomyocyte SMRTs group through day 14, while measurements declined in the vehicle group and remained stable in L7Ae-modRNA and Luc-cardiomyocyte SMRTs animals. Left ventricular internal diameter end-systole and left ventricular end-diastole measurements (Figure S9B) indicated that the CCND2-cardiomyocyte SMRTs also protected against LV chamber dilatation, while heart-weight-to-bodyweight (HW/BW) ratios (Figure S9C), cardiac fibrosis (Figure 2H; Figure S10), and the cross-sectional surface areas of border-zone cardiomyocytes (Figure 2I) were significantly smaller in the CCND2-cardiomyocyte SMRTs group than in all other injury groups. Thus, the CCND2-cardiomyocyte SMRTs was associated with significant increases in cardiomyocyte proliferation and with significant improvements in cardiac function, infarct size, and hypertrophy when evaluated in a mouse MI model.
CCND2-Cardiomyocyte SMRTs Promotes CM Proliferation After MI in Pig Hearts
Our investigation of the potency of the CCND2-cardiomyocyte SMRTs for improving recovery from myocardial injury continued with experiments in a more clinically relevant, large-mammalian model (Figure 3A; Figure S1B and S1C). MI was induced in 45-day-old Yorkshire pigs by occluding the left anterior descending coronary artery for 60 minutes before reperfusion, and then the animals were randomly distributed to treatment with intramyocardial injections of 6 mg CCND2-modRNA and 1.5 mg L7Ae-modRNA (the CCND2-cardiomyocyte SMRTs group), 6 mg nGFP-modRNA and 1.5 mg L7Ae-modRNA (the nGFP-cardiomyocyte SMRTs group), or an equivalent volume of the delivery vehicle (the Vehicle group).31 The cardiomyocyte specificity of the cardiomyocyte SMRTs treatment was confirmed via immunofluorescent images of GFP expression in sections collected from infarcted pig hearts after treatment with CCND2-nGFP-modRNA and L7Ae-modRNA (Figure S11): CCND2 and GFP co-expression was only observed in cardiomyocytes.
Figure 3. Intramyocardial injections of the CCND2 (cyclin D2)-cardiomyocyte (CM) specific modified mRNA translation system (SMRTs) promoted CM cell-cycle activity and proliferation in a pig myocardial infarction (MI) model. A, The protocol for experiments in the pig MI model is shown as a schematic. B through D, Sections of heart tissue from the border zone and remote zone of the infarct were collected from pigs 3 days after MI and stained for the presence of cTnT (cardiac troponin T) and (B) Ki67, (C) phosphorylated histone H3 (PH3), or (D) AuB (aurora kinase B); then, CM proliferation, cell-cycle activity, cytokinesis, and multinucleation were quantified as the percentages of cTnT-positive cells that were also positive for Ki67, PH3, and symmetrical (yellow arrows) and asymmetrical (white arrows) AuB, respectively. Scale bar=50 µm for B and C; scale bar=10 µm for D; n=3 animals per group. All data are presented as mean±SEM. P values from 1-way ANOVA with Tukey test (B–D). NS indicates not significant.
Western blot assessments conducted in organs from pigs sacrificed 3 days after MI induction and treatment confirmed that CCND2 protein levels were upregulated in the hearts of CCND2-cardiomyocyte SMRTs animals, but not in other major organs, and were unchanged in all organs (including the hearts) of animals in the nGFP-cardiomyocyte SMRTs and vehicle groups (Figure S12). Notably, the cardiomyocytes in neonatal pig hearts continue to undergo multinucleation events (ie, mitosis without cytokinesis) for up to 6 months after birth,42 which likely explains why some residual evidence of Ki67 expression, PH3 abundance, and asymmetrical AuB expression was observed in the border zones of nGFP-cardiomyocyte SMRTs- and vehicle-treated animals, as well as in the remote (uninjured) zones of animals in all 3 groups. However, Ki67-positive (5.99-fold increase relative to vehicle, P=4.69×10−5) and PH3-positive (4.17-fold increase relative to vehicle, P=9.37×10−4) cardiomyocytes were significantly more common in samples from the border-zones of hearts from CCND2-cardiomyocyte SMRTs animals than from the nGFP-cardiomyocyte SMRTs and vehicle treatment groups (Figure 3B and 3C). Treatment with the CCND2-cardiomyocyte SMRTs also significantly upregulated AuB expression (both symmetrical and asymmetrical) in border-zone cardiomyocytes but not in cardiomyocytes from the remote zone and whereas asymmetrical AuB was expressed in cardiomyocytes from both zones, symmetrical-AuB-positive cardiomyocytes were essentially absent in the border zones of hearts treated with the nGFP-cardiomyocyte SMRTs or the delivery Vehicle and in the remote zones of hearts from all 3 groups (Figure 3D).
CCND2-Cardiomyocyte SMRTs Reduces Infarct Size and Improves Global and Regional Cardiac Function After AMI in Pigs
Cardiac function was evaluated before MI induction (day 0) and 10 and 28 days afterward via echocardiography and on day 28 via cardiac magnetic resonance imaging (cMRI; Figure 3A). Echocardiographic (Figure 4A) measurements of left ventricular ejection fraction (Figure 4B) and left ventricular fractional shortening (Figure 4C) were equivalent in all 3 treatment groups on day 0 and significantly greater in animals treated with the CCND2-cardiomyocyte SMRTs than in nGFP-SMRTs- or vehicle-treated animals on day 10 and day 28; measurements appeared to increase from day 10 to day 28 in animals from the CCND2-cardiomyocyte SMRTs group while measurements in the vehicle-treatment group declined, but the differences between the two time points were not significant. cMRI (Figure 4D) assessments on day 28 also indicated that left ventricular ejection fraction was significantly greater (1.41-fold increase relative to vehicle, P=3.88×10−3; Figure 4E), as well as stroke volume (Figure 4F), in CCND2-cardiomyocyte SMRTs-treated animals than in either of the other 2 treatment groups and that the increases were primarily attributable to improvements in end-systolic (Figure 4G), rather than end-diastolic (Figure 4H), volume. Furthermore, when cMRI assessments (Figure 4I) were used to calculate radial strain (εRR) and LV wall thickening in accordance with the American Heart Association’s 17-segment model43 (Figure S13), the most significant regional contractility improvements in CCND2-cardiomyocyte SMRTs–treated animals compared to the nGFP-cardiomyocyte SMRTs or vehicle treatment groups occurred in the anterolateral and anterior walls (Figure 4J and 4K). Area under the curve calculations also indicated that both εRR and LV wall thickening were significantly greater in the CCND2-cardiomyocyte SMRTs group on day 28 after MI (Figure 4L and 4M).
Figure 4. Intramyocardial injections of the CCND2 (cyclin D2)-cardiomyocyte (CM) specific modified mRNA translation system (SMRTs) improved global and regional measures of cardiac function in infarcted pig hearts. A through C, Echocardiographic images (A) were obtained for pigs in the CCND2-CM SMRTs, nuclear green fluorescent protein (nGFP)-CM SMRTs, and vehicle groups on day 10 and day 28 after acute myocardial infarction (AMI) induction and used to calculate (B) left-ventricular ejection fraction (LVEF) and (C) left-ventricular fractional shortening (LVFS). D through H, Cardiac magnetic resonance imaging (cMRI) was performed on day 28 (D) and used to calculate (E) LVEF, (F) stroke volume, (G) end-systolic volume, and (H) end-diastolic volume. I through M, Late gadolinium enhancement (LGE)-cMRI was performed on day 28 and used to calculate radial strain (εRR) and LV wall thickening (LVWT). I, Corresponding T1 (left) and LGE (right) images are displayed for a pig in the vehicle group; the infarcted region and the border and remote zones are identified with arrows in the LGE image. Six-segment curves corresponding to (J) LV radial strain (εRR) and (K) LV wall thickening (LVWT) were plotted, and (L and M) the area under the curve (AUC) of both parameters were calculated. n=5 for vehicle group; n=6 for nGFP-CM SMRTs group; n=7 for CCND2-CM SMRTs group. All data are presented as mean±SEM. P values from ordinary 2-way ANOVA with Tukey test (B–C, J–K), or Kruskal-Wallis H test (E), or 1-way ANOVA with Tukey test (F–H, L and M). aP<0.05 vs vehicle; bP<0.01 vs vehicle; cP<0.001 vs vehicle; dP<0.05 vs nGFP-CM SMRTs; eP<0.01 vs nGFP-CM SMRTs; fP<0.001 vs nGFP-CM SMRTs (J and K). A indicates anterior; AL, anterolateral; AS, anteroseptal; I, inferior; IL, inferolateral; IS, inferoseptal; and NS, not significant.
Infarct size was evaluated on day 28 via late gadolinium enhancement cMRI. The gadolinium-retaining region (Figure 5A) was noticeably smaller in CCND2-cardiomyocyte SMRTs–treated hearts than in hearts from animals in the nGFP-cardiomyocyte SMRTs- or vehicle-treatment groups, and calculated values for infarct size (Figure 5B), infarct mass (Figure 5C), and infarct mass percentage (ie, the ratio of the infarct mass to LV weight; Figure 5D) were significantly smaller (by more than 50%, P<0.01) in pigs treated with the CCND2-cardiomyocyte SMRTs than in either of the other 2 groups. The masses of the infarct core (Figure 5F) and the gray zone (Figure 5G; ie, the region of both necrotic and viable myocardium surrounding the infarct core44; Figure 5E) were also significantly smaller in CCND2-cardiomyocyte SMRTs–treated hearts than in Vehicle-treated hearts (by 64.07%, P=4.54×10−4, and 54.23%, P=7.68×10−3, respectively) or hearts from the nGFP-cardiomyocyte SMRTs group, and the results from late gadolinium enhancement-cMRI measurements were consistent with observations in serial sections of the LVs from pigs in each of the 3 treatment groups (Figure 5I and 5J): the fibrotic area was significantly smaller in sections from CCND2-cardiomyocyte SMRTs–treated animals (Figure 5J).
Figure 5. Intramyocardial injections of the CCND2 (cyclin D2)-cardiomyocyte (CM) specific modified mRNA translation system (SMRTs) reduced infarct size after myocardial infarction (MI) induction in pigs. A, Late gadolinium enhancement (LGE)-cardiac magnetic resonance imaging (cMRI) images were obtained in pigs on day 28 after MI induction; the endocardium is marked with a red line, the epicardium is marked with a blue line, and the infarct area is shaded in yellow. B through D, LGE-cMRI images were used to calculate (B) infarct size, (C) infarct mass, and (D) infarct mass percentage relative to the LV weight; size and mass measurements were presented as a percentage of the entire left ventricular (LV). E, An LGE-cMRI image is displayed showing the region of the infarct core (white zone; shaded in yellow) and the surrounding region of necrotic and viable tissue (gray zone; shaded in purple). F through H, The mass of the (F) infarct core and (G) gray zone, and (H) the ratio of the masses of the core and gray zones were calculated from LGE-cMRI images. I, LVs were collected on day 28 and cut into 5 circular sections from the apex to the base. Scale bar=2 cm. J, Sections of the infarct core and the surrounding border zone from infarcted cardiac rings 2, 3, 4 were stained with Picro-Sirius Red and Fast Green to visualize fibrotic (red) and normal (green) tissue; then, infarct size was quantified as the ratio of the area of the fibrotic region to total area of the tissue and presented as a percentage. Scale bar=1 cm. n=5 for vehicle group; n=6 for nuclear green fluorescent protein (nGFP)-CM SMRTs group; n=7 for CCND2-CM SMRTs group. All data are presented as mean±SEM. P values from 1-way ANOVA with Tukey test (B–F, J), or Kruskal-Wallis H test (G and H). NS indicates not significant.
CCND2-Cardiomyocyte SMRTs Does Not Induce Long-Term Cardiomyocyte Proliferation or Increase the Risk of Arrhythmia After Administration to Infarcted Pig Hearts
Four weeks after MI induction, measurements of HW/BW ratios, left-ventricular-weight/bodyweight ratios (Figure S14), and the cross-sectional surface areas of border-zone and remote-zone cardiomyocytes were significantly smaller in hearts from the CCND2-cardiomyocyte SMRTs group than from Vehicle- and nGFP-cardiomyocyte SMRTs–treated hearts (Figure 6A). Furthermore, the number of cardiomyocytes containing one nucleus, but not two or more, was also significantly higher in CCND2-cardiomyocyte SMRTs–treated hearts than in hearts from the other 2 groups (Figure 6A). Thus, the CCND2-cardiomyocyte SMRTs appeared to protect the entire left ventricle from MI-related hypertrophy and to promote cytokinesis to a greater extent than multinucleation in cardiomyocytes.
Figure 6. Intramyocardial injections of the CCND2 (cyclin D2)-cardiomyocyte (CM) specific modified mRNA translation system (SMRTs) promoted CM proliferation for <28 days and did not increase the risk of arrhythmia in infarcted pig hearts. A, Sections from border zone and remote zone were stained with cTnT (cardiac troponin T) and WGA (wheat germ agglutinin) to visualize CMs and their cell borders. Nuclei were counterstained with DAPI; then, CM cross-sectional size and the percentage of CMs with the indicated number of nuclei were quantified. Scale bar=50 µm. B and C, Sections from the border and remote zones of pig hearts were collected on day 28 and stained for the presence of cTnT and (B) Ki67 or (C) phosphorylated histone H3 (PH3); then, CM proliferation and cell-cycle activity were quantified as the percentages of cTnT (cardiac troponin T)-positive cells that were also positive for Ki67 and PH3, respectively. Scale bar=50 µm. D, Programmed electrical stimulation (PES) was performed in all long-term study pig hearts before sacrifice on day 28 to evaluate the arrythmia inducibility. Hearts were paced at 400 ms with additional stimuli provided at progressively shorter intervals; PES was halted immediately after an episode of ventricular arrhythmia (VA) was induced. Only 1 animal in each group demonstrated sustained VA (≥15 heart beats; including ventricular tachycardia and ventricular fibrillation). The outcome of PES study is shown as percentage. n=5 for vehicle group; n=6 for nuclear green fluorescent protein (nGFP)-CM SMRTs group; n=7 for CCND2-CM SMRTs group. All data are presented as mean±SEM. P values from 1-way ANOVA with Tukey test (A [border zone CM cross-sectional size], B and C), or Kruskal-Wallis H test (A [remote zone CM cross-sectional size]), or 2-way ANOVA with Tukey test (A [CM nucleation]), or χ2 test (D). NS indicates not significant.
One of the primary safety concerns associated with the administration of CCND2-overexpressing hiPSC-cardiomyocytes is that long-term, uncontrolled expression of cell-cycle regulatory molecules could lead to excessive cardiomyocyte proliferation and arrhythmia.18 However, Ki67- and PH3-positive border-zone and remote-zone cardiomyocytes were no more prevalent on day 28 in CCND2-cardiomyocyte SMRTs–treated hearts than in hearts from animals in the nGFP-cardiomyocyte SMRTs- or vehicle-treatment groups (Figure 6B and 6C). Furthermore, when the hearts of pigs were paced via programmed electrical stimulation, incidents of ventricular tachycardia and fibrillation were equally common in animals from all 3 treatment groups (Figure 6D; Figure S15). Thus, intramyocardial CCND2-cardiomyocyte SMRTs administration produced only a transient increase in cardiomyocyte proliferation and was not associated with an elevated risk of arrhythmogenic complications.
DISCUSSION
Despite current intensive treatment regimens, patients with severe MI often progress to end-stage congestive heart failure, which is one of the most significant problems in public health. From a cellular perspective, heart failure is caused by the loss of cardiomyocytes, and the regenerative capacity of adult mammalian hearts is limited, because nearly all cardiomyocytes exit the cell cycle shortly after birth. Cell cycle arrest occurs at the G1/S transition,45 also known as the restriction point (R-point), and the R-point transition is partially governed by the activity of CDK4 (cyclin-dependent kinase 4) and its cofactors, the D-type cyclins.46 The CDK4/cyclin D complex phosphorylates RB (retinoblastoma protein), thereby disrupting RB-E2F binding and facilitating the E2F-mediated transcription of genes that activate DNA synthesis and cell cycle progression.46 This regulatory mechanism is consistent with the results from previous investigations in transgenic mice that overexpressed cyclin D2 from the MHC promoter: cyclin D2 levels increased in cardiomyocyte nuclei and preserved cardiomyocyte cell cycle activity in animals with both healthy and hypertrophic hearts.19 However, cardiomyocyte DNA synthesis was not preserved in the hypertrophic hearts of cyclin-D1- or cyclin-D3-transgenic mice, likely because nuclear localization of the overexpressed cyclins was compromised, and their association with CDK4 was impeded.19 The immunostaining results from our in vitro and in vivo experiments confirmed that CCND2 was abundantly expressed in cardiomyocyte nuclei after treatment with the CCND2-cardiomyocyte SMRTs and, consequently, could facilitate the R-point transition and the onset of DNA synthesis and cell cycle progression, including the G2/M transition and cytokinesis. Increases in cardiomyocyte cell division were also observed both in vitro and in vivo, which subsequently contributed to significant declines in infarct size and significant improvements in cardiac function.
The increase in the proportion of cardiomyocytes that expressed PH3 and AuB in the hearts of animals treated with the CCND2-cardiomyocyte SMRTs after MI matched or was even greater than the increases reported in other studies of CCND2-induced cardiomyocyte proliferation.14,17 However, we recognize that PH3-positive and AuB-positive cardiomyocytes may be undergoing either multinucleation events (ie, karyokinesis) or cell division (cytokinesis), so we refined our analysis by distinguishing between asymmetrical AuB expression, which identifies a karyokinesis event within a single cell, and symmetrical AuB expression, which is a marker for cell division.39,47 Notably, whereas our experiments in mice were conducted in adult animals, the pigs used in this investigation were juveniles, so their cardiomyocytes were predominantly bi- or tetranuclear but still included a sizable number (≈10%) of mononuclear cells42 that likely were the primary source of proliferating cardiomyocytes. Nevertheless, our results indicate that the CCND2-cardiomyocyte SMRTs reactivated cytokinesis (and promoted karyokinesis) in the cardiomyocytes of both species.
Our observation that cardiomyocyte-specific CCND2 overexpression prevented cardiomyocyte hypertrophy differs from the results of several previous studies, which suggest that cardiomyocyte CycD2 (cyclin D2, in rodent) expression increases in response to pressure overload or treatment with angiotensin II,48–50 and that cardiomyocyte-specific ablation of CycD2 inhibits the hypertrophy observed in transgenic mice that overexpress cMyc.51 Importantly, the models used in these investigations are fundamentally different from the models used in our experiments because they were specifically designed to induce cardiac hypertrophy, rather than MI. Furthermore, although the previous reports convincingly demonstrated that CycD2 is required for the hypertrophic growth of cardiomyocytes, they did not demonstrate that hypertrophy can be induced via CycD2 overexpression alone and, consequently, do not conflict with our observation that both cardiomyocyte cross-sectional surface areas and HW/BW ratios were lower in the CCND2-cardiomyocyte SMRTs groups than in vehicle-treated animals. Notably, CCND2-cardiomyocyte SMRTs administration was also associated with a decline in the proportion of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive cardiomyocytes, perhaps because miR-1 and miR-208, which seem to promote cardiomyocyte apoptosis,40,41 were sequestered at their respective binding sites in the L7Ae-modRNA construct. The molecular and cellular basis for the progressive postinfarction LV dilatation and heart failure is the result of the inability of damaged and apoptotic myocytes to be replaced. The acute myocardial infarction (AMI)-induced LV hypertrophy is largely attributable to the loss of functional contractile tissue–associated LV dilatation that is linearly associated with the LV infarct size and increase of LV end-diastolic pressure, which results in the increase of LV wall stresses and hypertrophy.52–54 The CCND2-cardiomyocyte SMRTs alleviate this wall stress burden by promoting the generation of new cardiomyocytes and by improving the survival of preexisting cardiomyocytes, which in turn results in the decrease of LV infarct size, the improvement of LV dilatation and wall stresses, and consequently the decrease of LV hypertrophy.52–54
One of the primary concerns associated with many investigative approaches for promoting cardiomyocyte proliferation is that excessive activation of the cardiomyocyte cell cycle could reduce sarcomere stability and increase the risk of arrhythmia.18 For example, we have previously shown that when CCND2 was upregulated in hiPSC-cardiomyocytes via lentiviral transfection, the CCND2-overexpressing hiPSC-cardiomyocytes not only proliferated when transplanted into infarcted pig hearts but also increased the proliferation of endogenous cardiomyocytes.37 Thus, although the treatment was not associated with an increase in arrhythmia, the use of a lentiviral vector could, at least in theory, lead to permanent increases in CCND2-induced cell-cycle activity and excessive cardiomyocyte proliferation. Notably, when adeno-associated virus–mediated miR-199a delivery was evaluated in a pig MI model, the treatment led to persistent miR-199a expression, and the majority of pigs died from sudden arrhythmia despite improvements in contractility and scar size.18
Treatment with the CCND2-cardiomyocyte SMRTs promoted cell-cycle activity only transiently, and although we did not thoroughly characterize the duration of cardiomyocyte SMRTs-driven CCND2 expression in vivo, CCND2 expression in cultured cardiomyocytes peaked 1 day after treatment with the CCND2-cardiomyocyte SMRTs and declined to nearly undetectable levels by day 14, which is largely consistent with previous reports indicating that modRNA expression endures for 8 to 12 days in mice.26 Cardiomyocyte proliferation in the CCND2-cardiomyocyte SMRTs group also appeared to return to background levels in just a few weeks, because Ki67- and PH3-positive border-zone cardiomyocytes were no more common in pigs treated with the CCND2- cardiomyocyte SMRTs than in vehicle-treated pigs on day 28. Importantly, no spontaneous arrhythmia-induced lethality was observed in pigs during the 4-week follow-up period and programmed electrical stimulation–induced sustained ventricular arrhythmias were equally common in all 3 groups. Incidents of arrhythmia may also have been limited by the declines in infarct gray zone (ie, the necrotic and viable myocardium surrounding the infarct core) size observed in CCND2-cardiomyocyte SMRTs–treated pigs because a heterogeneous mixture of viable and nonviable myocardium increases the likelihood of electronic circuit reentry.55
The successful use of modRNA technology for the development of vaccines against SARS-CoV-2 demonstrates the feasibility of this platform for administration to patients, despite some mild-to-moderate local or systemic side effects (eg, injection site pain, headache, muscle pain, myalgia, chills, and fever) or extremely rare severe adverse events (eg, myocarditis and severe allergic reactions).56,57 Because circulating modRNA is rapidly degraded by RNase, and the transcripts taken up by cells are expressed only transiently, they are unlikely to be associated with some of the long-term safety concerns that have limited the clinical translation of other delivery methods. Cardiomyocyte specificity is also a key component of strategies for improving myocardial repair via the delivery of cell-cycle regulators to the heart because off-target expression of these molecules in other cell types could promote fibrosis, inflammation, and other complications. Here, we demonstrate that our cardiomyocyte SMRTs platform26,32 can be used to rapidly upregulate CCND2 expression in cardiomyocyte nuclei with exceptional cell-type specificity. However, although the efficiency of modRNA transfection is exceptionally high (≈85% in vivo), it is not perfect, so CCND2 may be expressed by a few noncardiomyocytes that were successfully transfected with CCND2-modRNA but not the L7Ae-modRNA construct.
Our study has demonstrated that the cardiomyocyte SMRTs could effectively drive cardiomyocyte-specific CCND2 expression, and that the treatment was associated with significant increases in cardiomyocyte cell-cycle activity and myocardial recovery. Nevertheless, the method of direct myocardial delivery of cardiomyocyte SMRTs currently employed is not readily applicable in clinical practice. Currently, direct intravascular or intracoronary injection of the cardiomyocyte SMRTs is unlikely to be maximally effective, because mRNA is rapidly degraded by RNase in the circulation,58 and although lipid nanoparticle–encapsulated modRNA is Food and Drug Administration (FDA) approved and sufficiently stable to ensure that the modRNA is taken up by cells, most systemically injected lipid nanoparticles home to the liver.59 In addition, the timing of cardiomyocyte SMRTs administration (ie, immediately after MI induction) and open-chest delivery incompatible with most clinical applications need to be overcome. To realize the full potential of modRNA-based gene therapy for patients, different delivery methods (eg, cardiac-targeted lipid nanoparticles) and time points (eg, weeks or months after MI induction) will need to be developed and tested in future studies. Our results are also subject to the same limitations associated with any preclinical model of human disease.
In conclusion, the results presented in this report demonstrate that the cardiomyocyte SMRTs can rapidly and transiently drive the expression of CCND2 in cardiomyocytes with high cell-type specificity and that intramyocardial injections of the CCND2-cardiomyocyte SMRTs reactivated the cardiomyocyte cell cycle, reduced infarct size, and improved cardiac performance in both small and large mammalian models of myocardial injury. These findings demonstrate, for the first time, that modRNA technology can remuscularize infarcted mammalian hearts.
ARTICLE INFORMATION
Author Contributions
J. Sun, Y. Zhou, and J. Zhang conceived and designed the project. J. Sun, L. Wang, R.C. Matthews, Y.-A. Lu, Y. Wei, and G.P. Walcott acquired and analyzed data. Y. Zhou assisted data interpretation. L. Zangi revised article. J. Sun, L. Wang, Y. Zhou, and J. Zhang wrote and revised the article.
Sources of Funding
This work was supported in part by the following funding sources: NIH RO1s: HL114120, HL 131017, HL 149137, NIH UO1 HL134764, NIH PO1 HL160476 (to J. Zhang); NIH RO1 HL153220 and AHA Transformational Project Award 969529 (to Y. Zhou); AHA 20PRE35210006 (to L. Wang); AHA 23PRE1025367 (to J. Sun).
Supplemental Material
Expanded Materials and Methods
Figures S1–S15
Video S1
References 60–72
| AuB | aurora kinase B |
| CCND2 | cyclin D2 |
| CDK4 | cyclin-dependent kinase 4 |
| cMRI | cardiac magnetic resonance imaging |
| CM SMRTs | cardiomyocyte-specific modified mRNA translation system |
| EC | endothelial cell |
| hiPSC-CM | human-induced pluripotent stem cell–derived cardiomyocyte |
| Luc | luciferase |
| MHC | myosin heavy chain |
| MI | myocardial infarction |
| nGFP | nuclear GFP |
| PH3 | phosphorylated histone H3 |
| RB | retinoblastoma protein |
Disclosures None.
Footnotes
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