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10.1161/CIRCULATIONAHA.119.041882
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Altering Sphingolipid Metabolism Attenuates Cell Death and Inflammatory
Response after Myocardial Infarction
Running Title: Hadas et al.; Acid Ceramidase Induce Cardioprotection
Yoav Hadas, et al.
The full author list is available on page 22.
Addresses for Correspondence:
Efrat Eliyahu, PhD
Department of Genetics and Genomic Sciences
1470 Madison Ave, 8th floor, room 202A
New York, NY 10029
Tel: 917-832-2298
Email: efrat.eliyahu@mssm.edu
Lior Zangi, PhD
Department of Cardiology
1470 Madison Ave, 7th floor, room 107
New York, NY 10029
Tel: 646-254-2800
Email lior.zangi@mssm.edu
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Abstract
Background: Sphingolipids have recently emerged as a biomarker of recurrence and mortality
after myocardial infarction (MI). The increased ceramide levels in mammalian heart tissues
during acute MI, as demonstrated by several groups, is associated with higher cell death rates in
the left ventricle (LV) and deteriorated cardiac function. Ceramidase, the only enzyme known to
hydrolyze pro-apoptotic ceramide, generates sphingosine, which is then phosphorylated by
sphingosine kinase (Sphk) to produce the pro-survival molecule sphingosine-1-phosphate (S1P).
We hypothesized that Acid Ceramidase (AC) overexpression would counteract the negative
effects of elevated ceramide and promote cell survival, thereby providing cardioprotection after
MI.
Methods: We performed transcriptomic, sphingolipid and protein analyses to evaluate
sphingolipid metabolism and signaling post MI. We investigated the effect of altering ceramide
metabolism through a loss (chemical inhibitors) or gain (modified mRNA (modRNA)) of AC
function post hypoxia or MI.
Results: We found that several genes involved in de novo ceramide synthesis were upregulated
and that ceramide (C16, C20, C20:1 and C24) levels had significantly increased 24 hours after
MI. AC inhibition post hypoxia or MI resulted in reduced AC activity and increased cell death;
by contrast, enhancing AC activity via AC modRNA treatment increased cell survival post
hypoxia or MI. AC modRNA-treated mice had significantly better heart function, longer survival
and smaller scar size than control mice 28 days post MI. We attributed the improvement in heart
function post MI following AC modRNA delivery to decreased ceramide levels, lower cell death
rates and changes in the composition of the immune cell population in the LV manifested by
lowered abundance of pro-inflammatory detrimental neutrophils.
Conclusions: Our findings suggest that transiently altering sphingolipid metabolism through AC
overexpression is sufficient and necessary to induce cardioprotection post MI, thereby
highlighting the therapeutic potential of AC modRNA in ischemic heart disease.
Key Words: sphingolipid metabolism; Acid Ceramidase; Modified mRNA; myocardial
infarction; Cardioprotection.
Nonstandard Abbreviations and Acronyms
AC- Acid Ceramidase
Sphk- Sphingosine kinase
S1P -sphingosine-1-phosphate
modRNA- modified mRNA
LV- Left ventricle
MI- Myocardial infarction
LAD - Left anterior descending artery
Luc- Luciferase
nrCM- Neonatal rat cardiomyocytes
hiPS- human induce pluripotent stem cells
AAR – Area at risk
LVID- Left ventricular internal diameter
EF- Ejection fraction
FS- Fractional Shortening
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Clinical Perspective
What is new?
• We show that Acid Ceramidase delivery using modified mRNA alters sphingolipid
metabolism by hydrolyzing ceramides in the heart post myocardial infarction.
• This metabolic change significant reduces cell death, alters immune response by limiting
neutrophil infiltration and supports cardiac function.
What are the clinical applications?
• Our results suggest that Acid Ceramidase modified mRNA may serve as a cardiac
protection target post myocardial infarction.
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Introduction
Myocardial infarction is an acute, life-threatening medical condition caused by the blockage of a
coronary artery, leading to ischemia and, eventually, to cell death in the affected heart area.
Cardiac tissue has a low regenerative capacity. MI thus leads to cardiac scarring and cardiac
remodeling that increase the risk of heart failure (HF). The extent of MI and the risk of
developing HF after acute MI are positively correlated in patients.1 Several therapeutic strategies
based on inhibiting controlled cell death, attenuating inflammation and inducing regeneration
have been proposed for protecting MI survivors against HF.2, 3
Lipid levels and composition in patient blood during acute MI have been shown to
predict the risk of complications.4 In particular, high plasma ceramide concentrations are
associated with a higher probability of MI recurrence and death.4, 5 Ceramides are simple
membrane sphingolipids that form the backbone of all complex sphingolipids.6 High cellular
ceramide levels can trigger programmed cell death.7 In recent years, a few research groups have
shown that ceramide levels are high in the heart tissues of rodents and humans during acute MI8-
10 and that blocking de novo ceramide synthesis in rodents can improve heart function post MI.8, 9
Ceramidases hydrolyze ceramide to generate free fatty acids and sphingosine, which is
then phosphorylated by sphingosine kinase (Sphk) to produce sphingosine 1-phosphate (S1P), a
pro-survival lipid mediator with both intra- and extracellular functions.11, 12 More specifically,
acid ceramidase (AC) is encoded by the Asah1 gene and catalyzes ceramide hydrolysis to free
fatty acids and sphingosine, which is then phosphorylated by Sphk (1 and 2) to generate S1P.13
Asah1 gene mutations lead to ceramidase deficiency and cause Farber lipogranulomatosis, a
lysosomal storage disease.13 AC is essential for embryogenesis, and Asah1-/- embryos undergo
apoptotic death at the two-cell stage.12 AC is a lysosomal and secreted enzyme, with optimal in
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vitro activity in acidic conditions,14 and belongs to the N-terminal nucleophile hydrolase family.
The autoproteolytic cleavage of AC generates two active subunits: the α subunit with a
molecular weight of ~14 kDa and the β subunit with a molecular weight of ~43 kDa.15 The
autoproteolytic cleavage of the precursor triggers a conformational change that reveals the active
site and activates the enzyme toward sphingolipid metabolism.16 It has been suggested that
interfering with the signal transduction pathways mediated by sphingolipids could prevent cell
death post MI. Recent studies have suggested that S1P could be used as a therapeutic target in
patients with heart failure17 and MI,18 to prolong cardiac cell survival and consequently improve
heart function. While S1P lyase inhibition causes increased cardiac S1P levels and bradycardia in
rats,19 S1P receptor agonist, FTY720, boosts myocardial salvage and enhances heart function in a
porcine model of ischemia/reperfusion injury.20 Inhibiting de novo ceramide synthesis has also
been suggested as a strategy for reducing the pro-apoptotic effect of ceramide post MI.9 Indeed,
inhibiting acid sphingomyelinase, which hydrolyzes spingomyelin to generate ceramide, limits
ceramide accumulation in post-ischemic hearts.10 Moreover, adiponectin seems to exert its anti-
apoptotic effect on CMs through adiponectin receptor-mediated ceramidase activity.21
We investigated using AC and/or Sphk enzymes to inhibit cell death and initiate cell
survival through ceramide hydrolysis and S1P production. Delivering AC or Sphk proteins is
safe and controlled, but their effects are limited by these proteins’ half-lives. Conversely, using
DNA or viruses (DNA or RNA viruses) is not controlled and may elicit an immune response that
could compromise genome integrity. We therefore delivered AC and Sphk via synthetic
modified mRNA (modRNA), a nucleic acid delivery tool, to transiently alter sphingolipid
metabolism. Our group and others have shown that modRNA is a highly efficient system for
delivery to the heart, rapidly yielding transient expression with no signs of innate
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immunoresponse.22-28 More specifically, we have shown that synthesized modRNAs, in which all
uridine residues are replaced with pseudouridine-5'-triphosphate or N1-methylpseudouridine-5'-
triphosphate (1-mψU), result in more efficient translation, with lower immunogenicity and
greater resistance to RNase cleavage than unmodified mRNA in cardiac cells and tissue.27
ModRNA, which has a unique, transient, pulse-like pharmacokinetic profile, is translated within
minutes and the resulting protein remains detectable for ~5-7 days in vitro and ~10 days in
vivo.27 This distinctive kinetic profile makes modRNA approaches ideal for immediately and
transiently altering sphingolipid metabolism in the heart post MI. While insulin-like growth
factor 1 (IGF-1) modRNA delivered immediately after MI has been shown to decrease CM
apoptosis, this treatment’s long-term effects on heart function are remain unclear.22 We recently
showed that delivering IGF-1 modRNA to the heart post MI induces detrimental epicardial
adipose tissue formation by differentiate epicardial derived cells into fat cells 28 days post MI.25
In this study, we investigated sphingolipid level dynamics and the expression of genes
involved in sphingolipid metabolism and signaling pathways post MI. Loss (chemical inhibitors)
or gain (modRNA) of AC function showed that inhibiting AC activity increased cell death and,
by contrast, enhancing AC activity reduced cell death under hypoxia conditions in vitro or during
MI in vivo. Moreover, we show that enhancing AC activity attenuated inflammation post MI by
decreasing detrimental neutrophil levels in the LV, thereby improving heart function and long-
term survival after ischemic injury.
Methods
All methods and materials used in this study are described in detail in the Supplementary
Materials and Methods section. Below please see a brief summary of the most relevant ones.
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Mice
All animal procedures were performed according to protocols approved by the Icahn School of
Medicine at Mount Sinai Institutional Care and Use Committee. CFW male and female mice
were used. For protein expression, mice were injected intramyocardially with 100 g of Luc,
Sphk1 or modRNA in citrate buffer. MI was induced by permanent LAD ligation. When
required, 100 g of modRNA was injected into the border zone immediately after LAD ligation.
For the inhibitor assay, 10mg/kg of ARN14974 was injected IP at the time and 7 hours post LAD
ligation. Heart function was determined using echocardiography and MRI.
ModRNA Synthesis
Clean PCR products generated with plasmid templates (GeneArt, Thermo Fisher Scientific) were
used as the template for mRNA (for a complete list of the open reading frames used in this study,
see Supplementary Table 1). ModRNAs were produced by transcription in vitro with a
customized ribonucleoside blend and DNA PCR products with plasmid templates, followed by
Antarctic Phosphatase treatment and purification. Please see supplemental materials methods
section for more detailes.
nrCM isolation and hPSC differentiation
Ventrical nrCMs were isolated from 3- to 4-day-old Sprague-Dawley rats by multiple digestion
series using 0.1% collagenase II. We obtained hPSCs-derived CMs by using the embryoid bodies
(EBs) formatting method.
In vitro modRNA transfection
Neonatal rat hPSC-derived CMs were transfected with 2.5 g of modRNA per well (on 24 well
plate) encoding nGFP, AC, Sphk1, SphK2 or S1pr2 using RNAiMAX (Life Technologies).
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Sphingolipid composition and AC activity
To determine the sphingolipid composition of mouse hearts, samples were analyzed at the Stony
Brook Lipidomics facility. AC activity in heart tissue lysate was analyzed by HPLC-MS/MS.
Immunofluorescence
Hearts were perfused and fixed with 4% PFA, followed by incubation with 30% sucrose and
mounting in OCT. Transversal 10 m-thick sections were stained overnight with primary
antibodies followed by appropriate secondary antibodies, as listed in Supplementary Table 2,
depending on the experiment. To detect apoptotic cells in the heart tissue, TUNEL assay was
performed according to manufacturer’s protocol. To stain cells, coverslips with previously
seeded cells were fixed with 4%PFA and stained with appropriate antibodies. Images were
quantified using ImageJ software.
Flow cytometry
Infarct zones of AC modRNA- or Luc modRNA-treated hearts were collected at 2, 7 and 14 days
post MI and processed according to a previously described protocol29 with a few modifications.
Isolated cells were first stained with hematopoietic lineage markers followed by a second
staining with specific cell markers. Neutrophils were defined as CD45+ CD11b+ Ly6G+,
Macrophages as CD45+ CD11b+ Lin- F4/80+ Ly6Clow/int and Monocytes as CD45+ CD11b+ Lin-
F4/80- Ly6Clow / Ly6Chi. Data were acquired using Aurora (Cytek) Spectral Flow Cytometer.
Statistical analysis
All statistical analysis was performed with GraphPad-Prism software. Values are reported as
means ± SD. Two-tailed Student's t-tests (*p < 0.05 considered significant) or one-way ANOVA
with Bonferroni correction (*p < 0.05 considered significant) were used for comparisons among
groups. Log-rank (Mantel-Cox) tests were used to analyze survival.
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Results
Cell death and sphingolipid signaling pathway expression dynamics post MI
We characterized the dynamics of cell death and gene expression during and after acute MI by
ligating the left anterior descending artery (LAD) of mouse hearts to cause infarction and then
harvesting hearts at various time points (Fig. 1A).
To assess cell death, we harvested hearts from both sham-operated and ligated mice 1, 2,
4 and 28 days post MI. We used TUNEL staining to assess DNA fragmentation in cardiac cells
and troponin-I (cardiomyocytes marker) immunostaining to distinguish between cardiomyocytes
(CMs) and non-CMs (Fig. 1B). DNA fragmentation levels were highest 24 hours post MI, with
fragmented DNA detected in 9±2% of total cells in the LV (Fig. 1C), 15±3% of CMs (Fig. 1D)
and 4±0.2% of non-CM cells (Fig. 1E). DNA fragmentation levels were lower 48 hours after MI,
in both CMs and non-CM cells, and fell to basal levels by 28 days post MI (Fig. 1C-E).
An unbiased comparison of the LV transcriptome (Fig. 1F) 24 hours post MI identified a
significant change (P=0.02) in the sphingolipid signaling pathway KEGG PATHWAY
map04071.30 This pathway includes 112 genes, 38 of which had significantly changed expression
after MI (Fig. 1G, Supplementary Table 3 and Supplementary Fig. 1F). In addition, 24 hours post
MI, 12 of the 39 (30%) sphingolipid metabolism genes were significantly upregulated
(Supplementary Fig. 1E, F). None of the genes involved in the sphingomyelin hydrolysis
pathway were upregulated at 4 or 24 hours post MI, but four genes involved in the de novo
ceramide synthesis pathway (Sptlc2, CerS2, 3 and 6) displayed 2.2-, 2.3-, 5.2- and 2.2-fold
upregulation, respectively (Supplementary Table 3).
We next determined ceramide levels in the myocardium 24 hours post MI. The levels of
C16-ceramide (8.75-fold increase), C20-ceramide (16.57-fold increase), C20:1-ceramide (8.38-
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fold increase) and C24-ceramide (8.95 increase) were significantly higher 24 hours post MI than
in the absence of MI (Fig. 1H).
In this study, we focused on sphingolipid metabolism and the role of ceramide
degradation by AC and sphingosine phosphorylation to S1P by sphingosine kinase 1 (Sphk1).
We therefore used a separate mouse cohort to measure the mRNA and protein levels
corresponding to these key ceramide metabolism genes. Consistent with our transcriptome
analysis, relative levels of AC mRNA (Supplementary Fig. 1G and Supplementary Table 3) and
AC precursor levels were unaffected after MI (Fig. 1I&J). However, the active AC-enzyme, as
shown by β subunit, was significantly increased along with activity (Fig. 1K). AC-enzyme α
subunit was also upregulated 24 hours post MI (Supplementary Fig. 1I). Moreover, the Sphk1,
mRNA (Supplementary Fig. 1H) and protein (Fig. 1I&J) levels were notably upregulated 24
hours post MI. We also determined relative mRNA levels for AC and Sphk1 as well as Sphk1
protein levels in nrCMs under normoxia or hypoxia (Supplementary Fig. 1A-D). Though we
observed no significant effect on AC and Sphk1 mRNA levels (Supplementary Fig. 1A-B),
Sphk1 protein levels were moderately but significantly higher in cells subjected to hypoxia
(Supplementary Fig. 1C-D).
Modulating sphingolipid metabolism in the heart during acute MI affects cell death
Ceramide metabolites’ effect on cardiomyocyte (CM) death and heart function were investigated
post MI by altering ceramide metabolism via inhibiting or enhancing ceramide degradation and
S1P generation. We used two previously described approaches to limit ceramide degradation in
neonatal rat cardiomyocytes (nrCM): the pan-ceramidase inhibitor B1331 and the acid
ceramidase-specific inhibitor ARN1497432. We also blocked S1P formation with a pan-
sphingosine kinase inhibitor, SK1-II.33 Then, we assessed these inhibitors’ effects under
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normoxia (21% oxygen) and hypoxia (<2% oxygen) (Fig. 2A). In normoxia, inhibiting
ceramidase activity with 50 µM B13 or 20 µM ARN14974 and inhibiting sphingosine kinase
activity with 30 µM SK1-II for 48 hours had no significant effect on nrCM death rates, as shown
by comparison with control cells treated with the solvent used for the inhibitors, DMSO (Fig.
2B). However, increasing B13 concentration to 100 µM or SK1-II concentration to 60 µM
induced nrCM cell death 48 hours after treatment (Fig. 2B). Next, we assessed the combined
inhibition of ceramidase and sphingosine kinase activities. When used together at low doses (50
µM for B13 and 30µM for SK1-II), these two inhibitors induced significantly higher levels of
nrCM cell death than any other treatment except 100 µM B13, for which the same trend was
observed but to a lesser extent (Fig. 2B). In hypoxic conditions, all inhibitor types, at all
concentrations tested, significantly increased CM cell death levels 24 hours after treatment (Fig.
2C). By contrast, 48 hours after treatment, most cells were apoptotic regardless of the presence
or absence of inhibitors, which no longer significantly affected CM death rates (Fig. 2D).
We assessed loss of function in vivo using an AC-specific inhibitor (ARN14974) that has
been shown to reduce enzyme activity in mouse hearts for more than seven hours after
intraperitoneal injection (Fig. 2E).31 First, we first confirmed that the AC-specific inhibitor could
affect AC in our assay (Fig. 2F). We then administered two IP injections (immediately and seven
hours post MI) of the AC-specific inhibitor; this treatment resulted in twice as many cells
containing fragmented DNA 24 hours post MI (Fig. 2G&H). Importantly, lower doses of AC-
specific inhibitor lead to insignificant changes in cell death 24 hours post MI (Supplementary
Fig. 2A-B). To evaluative AC’s role in mouse survival post MI, we compared mouse survival
following treatment with either AC-specific inhibitor or DMSO control post MI. We show
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significantly reduced survival 72 hours post MI and treatment with AC-specific inhibitor (Fig
2I).
Additionally, to investigate the effect of overexpressing AC or Sphk1, alone or together,
in nrCMs under hypoxic conditions (Fig. 3A), we used modRNAs encoding human AC and
Sphk1. First, we confirmed the modRNAs were translated in nrCMs (Fig. 3B). We then cultured
the transfected nrCMs in a hypoxia chamber to assess the modRNAs’ ability to prevent nrCM
cell death. After 48 hours in hypoxia, 60% of the nrCMs transfected with nGFP (the control)
were apoptotic, whereas cell death rates were 22% and 27% lower, respectively, in cells
transfected with the AC modRNA and the Sphk1 modRNA. One of modRNA’s advantages as an
upregulation tool is that several modRNAs can be transfected together into the same cell.25 The
overexpression of both AC and Sphk1 decreased the number of apoptotic cells by 48% (Fig. 3C).
Similarly, we investigated the effects of AC and Sphk1 overexpression on cell death in the LV
post MI (Fig. 3D). We first used immunofluorescence staining to confirm that the various
modRNAs were translated in the LV 24 hours post MI (Fig. 3E). We then confirmed that AC
activity was significantly higher following AC modRNA delivery (Fig. 3F). Finally, we assessed
DNA fragmentation by performing TUNEL assays 48 hours post MI and after the various
treatments (Fig. 3G&H). AC overexpression in the left ventricle immediately after LAD ligation
produced 54% fewer cells with fragmented DNA than hearts treated with Luc modRNA, 48
hours post MI (Fig. 3G&H). Neither Sphk1 overexpression nor lower doses of AC modRNA
significantly changed DNA fragmentation (Fig. 3H and Supplementary Fig. 3A&B), while the
combined approach, upregulating both AC and Sphk1, had an effect similar to that of AC alone
(59% decrease) (Fig. 3G&H). In addition, we evaluated AC modRNA’s effect in human CMs by
transfecting human-induced pluripotent stem cell-derived CMs (hiPS-CMs) with AC modRNA
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(Supplementary Fig. 4A). The AC modRNA was translated to generate AC protein
(Supplementary Fig. 4B), resulting in lower cell death levels under hypoxia (Supplementary Fig.
4C).
Transient AC overexpression improves heart function and survival rate in mice post MI
Based on the beneficial effects of AC both alone and with Sphk1, we investigated how these
modRNAs affect cardiac function post MI by injecting AC, Sphk1, AC+Sphk1 or Luc
modRNAs directly into the LV and evaluating cardiac function post MI (Fig. 4A). Two days post
MI, no significant difference in either area at risk from the left ventricle (AAR/LV) or %FS
(fractional shortening, expressed as a percentage) was observed between groups (Supplementary
Fig. 5A&B). However, 28 days post MI, the %FS of the LV in mice treated with AC, Sphk1 or
AC+ Sphk1 modRNAs was significantly higher than in the control (Supplementary Fig. 5C).
Left ventricular internal diameter end diastole (LVIDd) did not differ significantly between
treated and control mice 28 days post MI (Fig. 4B). Yet left ventricular internal diameter end
systole (LVIDs) was significantly lower in mice treated with AC or AC+Sphk1 modRNAs than
in control mice (Fig. 4C). Comparing %FS values between 2 and 28 days post MI revealed
significantly improved heart function in mice treated with the Sphk1 modRNA and highly
improved heart function in mice treated with AC or AC + Sphk1 modRNAs (Fig. 4D). To
evaluate scar size after MI and the various treatments, we performed Masson trichrome staining
(Fig. 4E) and found that scars were significantly smaller after treatment with AC, Sphk1 or
AC+Sphk1 modRNAs (Fig. 4F). Further, we found no sign of CM hypertrophy in the LV of
treated mice relative to the control (Supplementary Fig. 5D&E). AC modRNA alone yielded the
most significant beneficial effect on the LV post MI. We therefore used magnetic resonance
imaging (MRI) to compare the percent ejection fraction (%EF) of mice treated with AC
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modRNA with that of mice treated with the Luc control modRNA (Fig. 4G). We observed
significantly higher EF (Fig. 4H), with better-preserved wall thickness, specifically in the areas
into which the AC modRNA was injected (posterior and lateral) relative to the injection-free
areas (anterior and septal) (Fig. 4I & Supplementary Movies 1&2). Finally, we determined that
survival rates three months post MI were significantly higher for mice treated with the AC
modRNA than for mice receiving other treatments or for control mice (Fig. 4J).
Transient AC overexpression reduces ceramide levels and attenuates inflammation post MI
To investigate the molecular basis of improved heart function, we collected hearts from mice
treated with AC or Luc control modRNAs and analyzed the effect of AC overexpression on
protein levels and sphingolipid composition 24 hours post MI; we also analyzed whole-
transcriptome dynamics 4, 24 and 48 hours post MI (Fig. 5A). Strikingly, AC overexpression
decreased the levels of all ceramides investigated – significantly for C20 and C22 – in the LV
post MI (Fig. 5B). We investigated this effect’s possible correlation with apoptosis markers, such
as caspase 3 dimerization,34 and cleavage. We found that caspase 3 dimerization levels were
higher in control mice than in sham-operated mice 24 hours post MI and that AC-treated mice
had lower caspase 3 dimer levels than did control mice (Fig. 5C and Supplementary Fig. 6A).
Unbiased transcriptome analysis revealed that only 30 genes displayed at least 1.5-fold
expression changes (p<0.05) in either direction (Supplementary Fig. 6B and Supplementary
Table 4) four hours after MI. By contrast, the number of genes displaying at least 1.5-fold
expression (p<0.05) in either direction rose to 299 by 24 hours post MI (Supplementary Fig. 6B
and Supplementary Table 5) and to 1675 by 48 hours post MI (Supplementary Fig. 6B and
supplementary Table 6). GO enrichment analysis revealed that AC overexpression altered many
immune-related pathways 24 hours post MI (Supplementary Table 7). Two days post MI, most
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of the genes displaying expression changes were involved in the cell cycle, cell apoptosis and
immune response (Supplementary Fig. 6C). Out of all the genes that were upregulated two days
post MI in mice that received ligation rather than sham, 144 genes had lower expression levels in
AC modRNA-treated hearts than in Luc control modRNA-treated hearts. GO enrichment
analysis with Enrichr35, 36 revealed that among the 144 genes with lower expression levels are
genes involved in lipid metabolism-related genes and cell death. Interestingly, neutrophil
chemotaxis and inflammatory response also show lower expression in the AC modRNA-treated
hearts (Fig. 5D and Supplementary Fig. 6D). The partial transcriptome for sphingolipid
metabolism-related genes was unchanged 4 and 24 hours post MI in AC modRNA-treated hearts
(data not shown). However, 48 hours post MI, we observed significant expression changes for
many of the sphingolipid metabolism-related genes included in RNA sequencing analysis
(Supplementary Fig. 6D). To verify RNAseq data, we selected the Ngp gene encoding the
neutrophilic granule protein, this gene’s expression was downregulated at 4, 24 and 48 hours
post MI in AC modRNA-treated hearts relative to Luc modRNA-treated hearts. We confirmed
changes in this gene’s expression patterns by performing RT-qPCR (please see primers in
Supplemental Table 8) on RNA isolated from the hearts two days post MI; the results obtained
were consistent with our transcriptomic results (Fig. 5E).
The significantly decreased expression of inflammation-related genes in AC modRNA-
treated hearts led us to investigate the composition of the immune cell populations in the infarct
zone of the heart at days 2, 7 and 14 post MI (Fig. 5F). At day 2 post MI, neutrophil infiltration
was reduced significantly in the infarct zone of AC modRNA-treated hearts compared to those
from controls (Fig. 5H-I). No significant differences were found in Ly6Chi and Ly6Clow
monocyte populations (Fig. 5J-K), nor macrophages (Fig. 5L-N). Seven days post MI,
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macrophage numbers increased dramatically whereas neutrophils decreased; both cells remained
near these levels by day 14 post MI. (Supplementary Fig. 6E-F).
Discussion
CM cell death and cardiac inflammation are key to the severity of cardiac dysfunction post MI.
Decreasing CM cell death and inflammation in patients with acute MI or other cardiovascular
diseases is highly desirable.2, 37 There is growing evidence that blood ceramide levels can predict
the extent of cardiovascular disease, complications and mortality.4, 5, 38, 39 Total ceramide levels,
particularly of ceramides with fatty acid chains of 16 or more carbon atoms, are high in the
myocardium in rodent models of MI8, 10 and in human patients with HF.8, 10
We characterized the significant changes to programmed cell death, the expression of
genes involved in sphingolipid metabolism or signaling, sphingolipid levels and AC activity, 24
hours post MI (Fig. 1). We observed significant increases in the levels of C16, C20, C20:1 and
C24 ceramides (Fig. 1H). Further, we found that the heart accommodated the increased ceramide
levels through two different molecular mechanisms. The first involved sharply upregulated
Sphk1 mRNA and protein levels (Fig. 1I&J and Supplementary Fig. 1H), and the second
involved moderately upregulated AC enzymatic activity despite the absence of change in AC
mRNA or protein levels (Fig. 1I-K and Supplementary Fig. 1G&I). These findings suggest that
sphingolipid metabolism, especially Sphk1 and AC enzymatic activity, are important factors in
cardiac tissue response to ischemic conditions.
Moderate concentrations of either ceramidase or sphingosine kinase inhibitors were not
toxic to nrCMs under normal culture conditions. However, incubating nrCM cells with a higher
concentration of either inhibitor separately, or lower concentrations of the two inhibitors
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together, induced cell death. Curtailing these enzymes may have caused ceramide and/or
sphingosine to rise to toxic levels. The significant increase in nrCM cell death observed in
conditions of moderate ceramidase or sphingosine kinase inhibition in hypoxic conditions
suggests that a combination of hypoxia and enzyme inhibition can increase the rate of pro-
apoptotic sphingolipid accumulation and cell death (Fig. 2C). Furthermore, in our in vivo loss-of-
function study, limiting AC activity in mouse hearts led to elevated cardiac cell death 24 hours
post MI. These results suggest that AC activity is a crucial cardioprotective factor in the ischemic
heart (Fig. 2E-H). That inhibiting AC post MI significantly decreases survival indicates AC’s
active role is vital to mouse survival post MI (Fig. 2I).
Ceramide has been shown to induce cell death in various cell types,7 including murine
and human CMs.40, 41 Conversely, the phosphorylation of sphingosine, a ceramide degradation
product, generates a major cell survival and cardioprotective agent, S1P.20,42 Klevsting et al.
suggested that Ceramide accumulation in the myocardium may be partially reliant due to
sphingomyelinase activity, and a heterozygous knockout of Smpd1, which encodes acid
sphingomyelinase, reduced ceramide accumulation in mouse hearts, albeit insufficiently to
improve either heart function or survival 30 days post MI.10 Recent studies in mouse models
have demonstrated constraining de novo ceramide synthesis improves heart function post MI8
and moderately decreases the infarct area and inflammation levels following myocardial I/R
injury.9 Our transcriptomic analysis revealed upregulated genes involved in de novo ceramide
synthesis, including the Sptlc2 and CerS2 3 and 6 genes, with no change in mRNA levels for the
natural sphingomyelinase (Smpd3) and lower transcript levels for other sphingomyelinases
(including Smpd1) 24 hours post MI (Supplementary Table 3). We here use AC for ceramide
hydrolysis43 to prevent ceramide toxicity and promote survival.
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Consistent with its anti-apoptotic effects, sphingosine may disassemble mitochondrial
ceramide channels,44, 45 but it has also been implicated in apoptotic or necrotic cell death.46
Benaim et al. suggested that sphingosine might disturb cellular calcium homeostasis by blocking
the activity of the sarcoendoplasmic reticulum Ca2+-ATPase (SERC2a),47 which plays a crucial
role in correct cardiac function.48, 49 Sphingosine kinase catalyzes the phosphorylation of
sphingosine to generate S1P and has cardioprotective properties.50 Accordingly, adenovirus-
mediated Sphk1 overexpression in rat hearts has been shown to protect treated hearts from I/R
injury.51 In this study, we decided to decrease ceramide levels in the ischemic heart by increasing
ceramide hydrolysis via modRNA used as a gene delivery tool to induce transient overexpression
of AC. This strategy not only decreased ceramide levels but also expanded the sphingosine
reservoir, the principal source of raw materials for producing the pro-survival molecule S1P. We
found that AC modRNA increased AC expression in vitro and in vivo (Fig. 3B, E). The resulting
high levels of AC protein were associated with more AC activity, leading to less cardiac cell
death post MI (Fig. 3C and Fig. 3F-H). We examined AC and/or Sphk1 overexpression’s in vitro
and in vivo effects on reduced cell death. Sphk1 overexpression generated via modRNA was
found to prevent ischemic induced cell death in vitro but not in vivo. (Fig. 3C and Fig. 3H). This
may be because Sphk1 mRNA and protein levels are naturally higher post MI in vivo than in in
vitro hypoxia settings. (Supplementary Fig. 1B-D, H).
Interestingly, S1P receptor 2 (S1pr2) or Sphk2 overexpression did not significantly
reduce cardiac cell death either in vitro or in vivo (Supplementary Fig. 7&8). Sphk1 is, thus,
effective for preventing apoptotic death but not redundant with Sphk2 for this function.
Consistent with this conclusion, Xia et al. showed that Sphk1 interacted with TNF receptor-
associated factor 2 (TRAF2) and that this interaction was required for TRAF2’s anti-apoptotic
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activity.52 Our pathway analysis for sphingolipid signal transduction revealed upregulated
TRAF2 post MI (Supplementary Table 3). Guo et al. recently reported a cardioprotective role for
TRAF253. Future studies are required to investigate the interaction between Sphk1 and TRAF2 in
the context of MI. Cell death levels did not change following S1pr2 upregulation, possibly due to
this receptor’s high physiological expression levels in the heart.
We observed a clear therapeutic effect 28 days after AC modRNA delivery in mice with
MI (Fig. 4), including preserved posterior and lateral wall thicknesses, which were most marked
in the area into which the AC modRNA was injected. AC modRNA had no significant effect on
%LVFS two days post MI (Supplementary Fig. 5A), whereas its effect was significant 28 days
post MI (Supplementary Fig. 5B). This suggests that AC modRNA may not only prevent cell
death immediately post MI but also influence other cardiac functions in this context. We also
show a non-significant reduction of LVIDd 30 days post-MI (Fig. 4b) and reduce scar size (Fig.
4e) that implies but not prove a reduced remodeling. Further investigation on the long-term
effects of AC treatment will clarify the effect on heart remodeling. In further sphingolipid
analyses, we found that AC modRNA affected cell death by decreasing ceramide levels post MI,
particularly for ceramides 20 and 22, (Fig. 5B), and by strongly curtailing caspase 3 dimer
formation and cleavage post MI (Fig. 5C and Supplementary Fig. 6A). In addition, many genes
involved in sphingolipid metabolism, inflammation and neutrophil degranulation displayed
altered expression following AC modRNA treatment, two days post MI (Fig. 5D and
Supplementary Fig. 6C&D). High ceramide levels can lead to neutrophil recruitment54 and
activation.55 Analyzing the immune cell population in the infarct area revealed significantly
lower neutrophil infiltration into the infarct zone of AC modRNA-treated hearts two days post
MI (Fig. 5I), consistent with the post MI altered immune response shown by GO enrichment
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analysis (Fig. 5D) and lowered expression of the Ngp gene, which is expressed predominantly in
neutrophils and promyelocytes56 (Fig. 5E) in these hearts.
Inflammation and immune cell infiltration into infarct cardiac tissue are crucial to healing
after MI.57 Completely inhibiting the recruitment of a particular immune cell population, such as
neutrophils or macrophages, is detrimental to the heart post MI,58, 59 but reducing the numbers of
neutrophils infiltrating the myocardium has been shown to shrink infarct size and promote
adaptation to hypoxic stress.60, 61 Administering AC modRNA immediately after MI decreased
detrimental neutrophil levels, thereby providing an additional mechanism for improving outcome
post MI. It remains unclear how AC modRNA alters the immune cell population in the LV post
MI; AC modRNA may influence chemokine expression directly or indirectly.
Additionally, we do not yet know which S1P receptor (1-5) is activated in the heart
during cell death rate reduction. The two most abundant receptors in the heart, S1pr1 and 3,
increase significantly post MI (Supplementary Table 3). By contrast, S1pr2 levels are low four
hours after MI and return to normal by 24 h post MI. The roles of S1pr1 and S1pr3 in
cardioprotection are well established,50 but the role of S1p2 in heart function remains unclear.
In addition, AC modRNA’s effects post MI have yet to be investigated in large animals.
However, by using hiPS-CMs in a hypoxia chamber, we demonstrated that AC modRNA
reduced cell death to control levels in human settings, an effect similar to that reported here for
nrCMs (Supplementary Fig. 6).
Overall, our data suggest (as summarized in Fig. 5O) that using AC modRNA to achieve
transient overexpression of AC, an essential enzyme in sphingolipid metabolism and cell
survival,12 lowers ceramide levels, decreases cardiac cell death and attenuates but does not
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eliminate detrimental neutrophil infiltration in the LV. Further, these effects improve cardiac
function, leading to improved survival after MI in mice.
Acknowledgments
The authors acknowledge Jason Kondrat, Sunita DSouza and the Pluripotent Stem Cell Core
Facility at the Icahn School of Medicine at Mount Sinai for their help with this manuscript.
Sources of Funding
This work was funded by a cardiology start-up grant and NIH grant R01 HL142768-01 awarded
to the Zangi laboratory and by the Genetics and Genomic Sciences budget in support of the
Eliyahu laboratory.
Disclosures
E.E., L.Z, A.S.V. and Y.H. are Inventors on Provisional Patent Application (MODRNA
ENCODING SPHINGOLIPID METABOLIZING PROTEINS TO PROMOTE CELL
SURVIVAL) 3710/039P, Filed March 2018, which covers the results in this manuscript.
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Authors
Yoav Hadas, PhD1,2,4; Adam S. Vincek, PhD2; Elias Youssef, MSc1,2,4;
Magdalena M. Żak, PhD1,2,4; Elena Chepurko, MDM1,2,4; Nishat Sultana, PhD1,2,4;
Mohammad Tofael Kabir Sharkar, PhD1,2,4; Ningning Guo, PhD2; Rinat Komargodski, MSc1,2,4;
Ann Anu Kurian, MSc1,2,4; Keerat Kaur, PhD1,2,4; Ajit Magadum, PhD1,2,4;
Anthony Fargnoli, PhD1; Michael G. Katz, MD1; Nadia Hossain, MSc1,2,4;
Ephraim Kenigsberg, PhD2; Nicole C. Dubois, PhD3,4; Eric Schadt, PhD2,5; Roger Hajjar, MD6;
Efrat Eliyahu, PhD2,5†; Lior Zangi, PhD1,2,4†
†Co-corresponding authors
Affiliations
1Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, NY;
2Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New
York, NY; 3Department of Developmental and Regenerative Biology and The Mindich Child
Health and Development Institute, Icahn School of Medicine at Mount Sinai, New York, NY;
4Black Family Stem Cell Institute, Icahn School of Medicine at Mount Sinai, New York, NY;
5Multiscale Biology Institute, Icahn School of Medicine at Mount Sinai, New York, NY;
6Phospholamban Foundation, Amsterdam, Netherlands
Supplemental Materials
Supplementary Materials and Methods
Supplementary Tables 1-8
Supplementary Figures 1-8
Supplementary Movies 1 and 2
References 62-63
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Figure Legends
Figure 1. Characterizing cell death dynamics and sphingolipid metabolism in mouse hearts
after MI. A, Hearts were harvested from sham-operated mice or 1, 2, 4 and 28 days post MI. B,
TUNEL assays were performed to assess DNA fragmentation in hearts harvested from either
sham-operated mice or 1, 2, 4 or 28 days post MI. Troponin-I immunostaining was used to
distinguish between cardiomyocytes and non-cardiomyocytes. Percentage of dead cells in left
ventricle at day 1, 2, 4 and 28 post MI was quantified within all cardiac cells (DAPI+ cells) n=3
(C); CMs only (DAPI+, Troponin I+) n=3 (D) and non-CM cells only (DAPI+, Troponin I-) n=3
(E). F, For RNASeq, protein analysis and mass spectrometry, hearts were harvested from sham-
operated mice or 4 or 24 hours post MI. RNA, proteins and lipids were extracted for sphingolipid
determination. G, Hierarchical clustering dendrogram for the sphingolipid signaling pathway
transcriptome in sham-operated hearts and hearts harvested 4 or 24 hours post MI, n=3, 3, 4,
respectively. H, Sphingolipid levels measured in the LV of sham-operated mice or 24 hours post
MI, n=3. I, Western blot of the AC precursor, AC active subunit β and Sphk1 in the LV of sham-
operated hearts and hearts harvested 4 or 24 hours post MI. J, Quantified protein levels of AC
precursor, AC β subunit and Sphk1, n=4. K, HPLC-MS/MS determination of AC activity in the
LV of sham-operated hearts and hearts harvested 4 or 24 hours post MI, n=3. *, P<0.05, One-
way ANOVA, Tukey's Multiple Comparison Test for (J&K) and Holm-Sidak correction for
multiple comparisons (H). Scale bar = 50μm. The results include two independent experiments
for H-K.
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Figure 2. Inhibiting sphingolipid metabolism increases cell death in cardiac cells in vitro
and after MI. A, Primary cardiomyocytes were isolated from the hearts of two- to three-day-old
rats. Two days after isolation, the cells were treated with ceramidase inhibitors (pan-ceramidase
inhibitor B13 and AC-specific ARN 14974) or sphingosine kinase inhibitor (SK1-II) and
transferred either to normoxia (21% oxygen) for 48 hours or hypoxia (<2% oxygen) for 24 or 48
hours. At the end of the incubation period, the cells were stained with an apoptosis marker
(Annexin 5) and a cell viability marker (DAPI) to assess the effects of inhibitors and oxygen
levels on cell death. Cell death was quantified in cells treated with chemical inhibitors under
normoxia for 48 hours, n=4 (B), or hypoxia for 24 hours, n=4 (C) and 48 hours, n=4 (D). E, AC
inhibitor was injected intraperitoneally at the time of MI and 7 hours post MI. Hearts were
harvested 24 hours post MI. Proteins were extracted for AC activity assays, or tissue was fixed
and stained with TUNEL to assess cell death in the LV. F, HPLC-MS/MS determined AC
activity in LV lysates 24 hours post MI and treatment with various AC inhibitor concentrations,
n=3. G, Representative images of heart sections from mice treated with AC inhibitor or DMSO
(control) obtained 24 hours post MI. H, Quantified cell death levels in the LV after treatment
with AC inhibitor or DMSO control, 24 hours post MI, n=4. I, Short-term post MI survival curve
for mice injected with AC inhibitor or DMSO control (n=8). ****, P<0.0001,***, P<0.001,**,
P<0.01, *, P<0.05, NS, not significant. One-way ANOVA, Tukey's Multiple Comparison Test
(B-D&F), two-tailed Student’s t-test (H), Mantel-Cox log-rank test (I). Scale bar 1mm.
Figure 3. Acid ceramidase overexpression using modRNA decreases cell death in cardiac
cells in vitro and after MI. A, Primary cardiomyocytes were isolated from the hearts of two- to
three-day-old rats. Two days after isolation, the cells were transfected with modRNAs for AC
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and SphK1. B, 18 hours post transfection in normoxia, CMs were fixed and immunostained to
confirm modRNA translation. C, Cell death levels of CMs transfected with the nGFP modRNA
(control), AC modRNA and SphK1 modRNA after 48 hours under hypoxia, n=4. D, modRNAs
for Luc, AC and Sphk1 were injected into mouse hearts at the time of MI. To evaluate injected
modRNA’s effects on the left ventricle, immunostaining (24 hours post MI), AC activity (24
hours post MI) and TUNEL assays were performed. E, Immunostaining for AC and SphK1 24
hours post modRNA injection. F, HPLC-MS/MS measurement of AC activity in the LV 24
hours post modRNA injection and MI, n=3 . G-H, Quantification and representative images of
cell death levels in heart sections from mice treated with Luc modRNA (control), AC modRNA,
Sphk1 modRNA or combination of AC + Sphk1 modRNAs 48 hours post injection, n=7. ****,
P<0.0001, **, P<0.01, *, P<0.05, NS, not significant. One-way ANOVA, Tukey's Multiple
Comparison Test (C & H), Two-tailed Student’s t-test (F). Scale bars; 10μm (B), 50μm (E) and
1mm (G).
Figure 4. AC modRNA improves heart function and mouse survival post MI. A, modRNAs
for Luc, AC, Sphk1 or a combination of modRNAs for AC and Sphk1 were injected into the LV
immediately after MI. Heart function was assessed by echocardiography and MRI. At day 29
post MI, hearts were collected for histological analysis. B-C, Echochardiography to measure left
ventricular internal dimension end diastole (LVIDd) and left ventricular internal dimension end
systole (LVIDs) 28 days post MI, n=6. D, Echocardiography to determine changes in left
ventricle fractional shortening (FS) between day 2 and day 28 post MI, n=6 . E, Representative
images of Masson’s trichrome staining and F, percentage of left ventricle area occupied by scar
tissue 28 days post MI and modRNA injection, n=6. G-H, Representative images of MRI scans
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and quantification of left ventricle ejection fraction (LVEF) 29 days post MI and injection with
AC or Luc modRNA. I, MRI-based measurement of fractional wall thickening percentage 29
days post MI and injection with AC or Luc modRNA, n=6. J, Long-term survival of mice after
MI and transfection with different modRNAs, n=10. ****, P<0.0001, **, P<0.01, *, P<0.05,
NS, not significant. One-way ANOVA, Bonferroni post-hoc tests (B, C, D, F) and two-tailed
Student’s t-tests (H). The results include two independent experiments. Scale bar = 1mm.
Figure 5. AC modRNA reduces ceramide levels and alters the immune cell composition in
the left ventricle post MI. A, modRNAs for Luc or AC were injected into mouse hearts
immediately after MI. Lipids and proteins were extracted from the hearts 24 hours post MI to
analyze sphingolipids by mass spectrometry and proteins by western blot. For RNA sequencing,
hearts were harvested 4, 24 or 48 hours post MI. B, Sphingolipid levels after Luc or AC
modRNA treatment, assessed 24 hours post MI, n=3. C, Western blot and quantification of AC α
subunits and caspase 3 dimers in sham-operated hearts and in hearts treated with Luc or AC
modRNA, 24 hours post MI, n=3. D, Scatterplot and GO enrichment analysis of all genes
downregulated in AC-treated vs. Luc-treated out of all upregulated genes 2 days post MI, n=4. E,
Relative Ngp gene expression levels 2 days post MI in the LV of mice treated with Luc or AC,
quantified using qPCR, n=4. F, To analyze modRNA treatment’s effect on immune cell content
after MI, hearts were collected at 2, 7 and 14 days post MI and Luc or AC modRNA injection
and analyzed using flow cytometry. G, Gating strategy for immune cell populations in the infarct
zone of modRNA-treated hearts. Neutrophils are defined as CD45+ CD11b+ Ly6G+, macrophages
as CD45+ CD11b+ Lin- F4/80+ Ly6Clow/int and monocytes as CD45+ CD11b+ Lin- F4/80- Ly6Clow /
Ly6Chi. H, Representative plots of neutrophil populations in Luc modRNA or AC modRNA-
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treated hearts 2 days post MI. I-N, Flow cytometric quantification of immune cell subsets in the
infarct zone of Luc modRNA- and AC modRNA-treated hearts 2 days post MI included
following populations: neutrophils, n=16 (I); Ly6Clow monocytes, n=16 (J); Ly6Chi monocytes,
n=16 (K); total macrophages, n=16 (L), MHCII-, n=16 (M) and MHCII+, n=16 (N) macrophage
subsets, n=16. O, Summary of proposed AC modRNA action mechanism during and after acute
MI. ***, P<0.001, *, P<0.05, n=3 (B, C), n=4 (E), n=16-18 (I-N). Holm-Sidak correction for
multiple comparisons (B), one-way ANOVA, Tukey's Multiple Comparison Test (C) and two-
tailed Student’s t-tests (E&I-N). Scale bar 20μm. The results include two and three (FACS)
independent experiments.
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