Abstract
Myocardial Infarction (MI) is a leading cause of death worldwide.
Metabolic modulation is a promising therapeutic approach to prevent
adverse remodeling after MI. However, whether material‐derived cues can
treat MI through metabolic regulation is mainly unexplored. Herein, a
Cu^2+ loaded casein microgel (CuCMG) aiming to rescue the pathological
intramyocardial metabolism for MI amelioration is developed. Cu^2+ is
an important ion factor involved in metabolic pathways, and
intracardiac copper drain is observed after MI. It is thus speculated
that intramyocardial supplementation of Cu^2+ can rescue myocardial
metabolism. Casein, a milk‐derived protein, is screened out as Cu^2+
carrier through molecular‐docking based on Cu^2+ loading capacity and
accessibility. CuCMGs notably attenuate MI‐induced cardiac dysfunction
and maladaptive remodeling, accompanied by increased angiogenesis. The
results from unbiased transcriptome profiling and oxidative
phosphorylation analyses support the hypothesis that CuCMG prominently
rescued the metabolic homeostasis of myocardium after MI. These
findings enhance the understanding of the design and application of
metabolic‐modulating biomaterials for ischemic cardiomyopathy therapy.
Keywords: casein, copper, hydrogels, metabolic homeostasis, myocardial
infarctions
__________________________________________________________________
Ischemic myocardium is featured with metabolic disequilibrium and
copper depletion. This study raised a material‐based therapeutic
strategy by intramyocardial supplement of Cu^2+ to rescue myocardial
metabolism. The milk‐derived casein microgel is screened out as Cu^2+
carrier through molecular‐docking by loading capacity and
accessibility. The Cu^2+ loaded microgel prominently rescued cardiac
functions by preserving metabolic homeostasis.
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1. Introduction
Myocardial infarction (MI) is one of the major causes of morbidity and
mortality worldwide.^[ [54]^1 , [55]^2 ^] Despite significant progress
in treatment and technology to revascularize the coronary artery that
has been developed in recent years, MI still poses a high global
medical burden due to the following ischemic cardiomyopathy.^[ [56]^3 ,
[57]^4 ^] Heart function is sensitive to energy supply.^[ [58]^5 ^] MI
leads to a sudden cessation of local blood and oxygen supply which
changes the level of oxidative phosphorylation (OXPHOS). The disorder
of myocardial energy metabolism after ischemia ultimately leads to
changes in left ventricular function and structure, increasing the
probability of cardiac remodeling and heart failure.^[ [59]^6 ^]
Strategies that regulate metabolic homeostasis after MI are promising
therapeutic candidates against heart failure. Although no medication
has been applied clinically to modulate cardiac metabolism directly in
clinically acute MI, more and more results have been presented to
rescue heart failure by reversing the detrimental metabolic processes
in ischemic cardiomyopathy. Inhibitors and activators of certain
metabolic pathways have shown cardioprotection effects in various
studies.^[ [60]^7 , [61]^8 , [62]^9 ^] However, drug toxicity remains a
challenge due to the risks of altering systemic metabolic stability.^[
[63]^10 ^] More recently, certain long non‐coding RNA^[ [64]^11 ^] and
circular RNA^[ [65]^12 ^] have been identified to attenuate
mitochondria malfunction after MI. Adeno‐associated virus (AAV), which
carried certain genes to edit metabolic pathways in cardiomyocytes,^[
[66]^13 ^] achieved protective effects on cardiac functions in mice
with left anterior descending (LAD) branch ligation.^[ [67]^14 ^]
However, AAVs have to be administered weeks before the ischemic events
to allow sufficient transfection, which may be less clinically relevant
as patients cannot be treated beforehand.^[ [68]^15 ^]
Bioengineering strategies based on materials have the potential to
overcome the systemic side effects and take effect immediately upon
administration for metabolic regulation.^[ [69]^16 , [70]^17 , [71]^18
^] The delivery of inherent metabolic factors based on bioengineering
cargoes can enhance tissue regeneration with high performance.^[
[72]^19 ^] Gentaro Ikeda et al. directly transferred mitochondria to
infarct heart tissue by injecting mitochondria‐rich extracellular
vesicles (EVs), successfully restored myocardial bioenergy, and
enhanced cardiac function after MI.^[ [73]^20 ^] Pengfei Chen et al.
developed a plant‐derived photosynthetic system based on nanothylakoid
units encapsulated in the cell membrane to improve anabolism in
degenerated chondrocytes.^[ [74]^21 ^] More user‐friendly materials,
such as scaffolds that can continuously release regulatory metabolites,
i.e., citrate^[ [75]^22 ^] and succinate,^[ [76]^23 ^] have been
developed to modulate energy metabolism for bone regeneration. Thus, we
speculate that materials loading with metabolic modulation factors may
exert positive results to enhance cardiac metabolic homeostasis and
reconstruction.
Copper (Cu) is an essential trace element that plays an important role
in metabolic processes.^[ [77]^24 , [78]^25 , [79]^26 ^] They are
cofactors of many key mitochondrial enzymes (i.e., cytochrome c oxidase
(CCO) and superoxide dismutase 1 (SOD1)), all of which play important
roles in maintaining mitochondrial metabolic homeostasis.^[ [80]^27 ^]
Cu depletion has been reported to affect mitochondrial OXPHOS and
impair metastasis in tumors.^[ [81]^28 ^] In the case of MI, Cu level
in the ischemic myocardium decreased significantly after LAD branch
ligation in mice, which did not recover over time within 28 days.^[
[82]^29 ^] Therapeutic methods emphasized in Cu supplementation have
shown promising results. In these researches, the functions of Cu
involved in several mechanisms, such as suppressing the transformation
of fibroblasts into myofibroblasts,^[ [83]^30 ^] increasing the
stability of hypoxia‐inducible factor‐1 (HIF‐1),^[ [84]^31 ^] acting as
a catalyst to enhance the local release of nitride oxide which increase
endothelial cell activity.^[ [85]^32 ^] However, it is unclear whether
supplementing Cu^2+ can rescue myocardial metabolism after infarction.
As the serum level of Cu elevated after MI,^[ [86]^33 ^] oral Cu
supplementation or intravascular injection may induce potential side
effects, i.e., systemic toxicity. Besides, it is hard to control the
dosage of Cu through dietary uptakes. The Cu overload caused by dietary
supplementation induced endoplasmic reticulum stress and mitochondrial
damage, which even led to myocardial cell apoptosis.^[ [87]^34 ^] Local
administration is an effective method to increase therapeutic functions
and decrease systemic side effects. However, the difficulty of heart
delivery requests a highly controllable release of Cu in order to
rescue the long‐term Cu deficiency with a single dose.
Natural proteins have many advantages as drug carriers, such as good
biocompatibility, abundant sources, safety, clear components and
structures for interaction prediction, and usually strong interactions
with metal ions. Protein‐based biomaterials have multiple reaction
sites and can be made into various forms for in vivo tissue
engineering.^[ [88]^35 , [89]^36 ^] Among them, microgels have received
widespread attention as carriers for therapeutic factors due to the
feasibility of minimally invasive delivery and stable retention for
controllable release.^[ [90]^37 , [91]^38 ^] The size, shape, surface
properties, and composition of the microgel can be controlled by the
preparation process, according to the requirements of various
applications.^[ [92]^39 , [93]^40 , [94]^41 ^] Crosslinkers such as
glutaraldehyde and genipin are usually used to prepare protein
microgels without the demand for molecular premodification,^[ [95]^42
^] however, the residual cross–linkers are usually cytotoxic, which may
lead to adverse effects after implantation. Methacrylate‐modified
proteins are also widely used for microgel fabrication because of the
high controllability,^[ [96]^43 ^] however, the side chain modification
may alter protein structure, and lead to an increase in preparing time
and effort. Previously, we used a ruthenium/sodium persulfate (Ru/SPS)
photocross–linking system to form a hydrogel by forming a bi‐tyrosine
bond between tyrosine residues in protein molecules, without the
necessity for pre‐modification.^[ [97]^44 ^] We thus speculate Ru/SPS
system can also be used for microgel formation.
In order to screen suitable Cu carriers, we chose several proteins
commonly used in bioengineering as the candidates and identified them
with theoretical molecular docking and experimental comparison. The Cu
binding ability of seven proteins, including casein, bovine serum
albumin (BSA), human serum albumin (HSA), collagen, lactoglobulin,
sericin, and silk fibroin, were first studied using molecular
stimulation (Figure [98] 1A). We ultimately chose casein (mainly
composed of αS1‐Casein and β‐Casein), which had the highest Cu loading
capacity, as the carrier material (Figure [99]1B; Figure [100]S1,
Supporting Information). Casein is the main protein (≈80%) in milk,
which has extensive resources and great quality control. Casein has
also shown great biocompatibility, low immunogenicity, and a relatively
long degradation period (>35 days) in vivo,^[ [101]^44 ^] which endow
it with long‐term intracorporal drug release. To facilitate
intramyocardial injection, we prepared casein microgels (CMGs) by
Ru/SPS redox system (Figure [102]1C). Cu^2+ loading and release
capacity of casein (Figure [103]1D), and Cu^2+ induced structural and
mechanical changes of CMGs were further studied to reflect the
interaction between Cu and casein. The physiological functions of Cu^2+
loaded CMGs (CuCMGs) on cardiacmyocytes, fibroblasts, and endothelial
cells, which play important roles in myocardial remodeling were studied
in vitro to evaluate the effects of CuCMGs on cell protection in a
pathologic environment. The therapeutic effects and metabolic
modulating functions of CuCMGs were studied in a rat acute MI model. A
whole genome sequencing was proceeded to identify the important
pathways CuCMGs participated in post‐MI treatment. Emphasized metabolic
modulation, we further analyzed the effects of CuCMGs on leveraging the
electron transport chain (ETC) functions of mitochondria in cardiac
tissues. In this study, we created an intramyocardial injection
microgel, CuCMG, which features in maintaining metabolic homeostasis
after MI for cardiac protection, and provided a new insight into
developing cardiac protecting biomaterials (Figure [104]1E).
Figure 1.
Figure 1
[105]Open in a new tab
Schematic illustration of Cu‐loaded milk‐protein derived microgel with
protective effects on cardiac metabolic homeostasis to improve
myocardial infarction. A) Schematic diagram of molecular docking
simulation. B) Schematic diagram of molecular docking between two main
casein subtypes and Cu^2+. C) Preparation process of casein microgels.
D) Illustration showing morphology change of CMG during Cu^2+ capture.
Scale bars = 100 µm. E) A working model summarizing the metabolic
modulating effects of CuCMG in myocardial infarction.
2. Results
2.1. CMG is a Great Carrier of Cu^2+
To compare the Cu binding ability of different protein‐based
biomaterials, molecule docking simulation was performed to sieve
proteins with a high Cu^2+/protein binding ratio. Commonly used
proteins in tissue engineering, including BSA, HSA, collagen,
lactoglobulin, sericin, silk fibroin, and casein, were evaluated in our
study. The results showed that αS1‐Casein and β‐Casein, the most
significant subtypes in bovine casein showed superior Cu^2+ binding
capacity. The Cu^2+ binding amount of αS1‐Casein was 0.64 µmol mg^−1,
and the Cu^2+ binding amount of β‐Casein was 0.61 µmol mg^−1, both of
which were more than twice that of other proteins (Figure [106] 2A). In
order to further clarify the actual Cu^2+ binding ability of casein
hydrogel, 10% casein and 10% BSA were cross–linked by Ru/SPS redox
system, and the adsorption of Cu^2+ was measured experimentally. As the
concentration of Cu^2+ stock solution increased from 1 to 5 mm, the
Cu^2+ binding amount into the hydrogels increased dramatically.
Nevertheless, in all cases, casein hydrogel had significantly higher
Cu^2+ uptake (over 150% higher) than BSA hydrogel (Figure [107]2B). The
results indicated that casein hydrogel could be a great candidate for
Cu^2+ delivery because of its high loading capacity. Cu release of
casein and BSA were also compared with the same Cu^2+ load (different
protein amount). Both casein hydrogels and BSA hydrogels showed slow
and continuous Cu^2+ release, with only 10% Cu^2+ released after 4
weeks (Figure [108]2C). After Cu loading, the degradation rate of
casein in response to enzymes was altered (Figure [109]2D). In the case
of collagenase, untreated casein hydrogel was completely decomposed on
the first day, but only 17.39% ± 5.00% of Cu‐loaded casein degraded
after 14 days. As casein lacks matrix metalloproteinase 2 (MMP2)
cleavage sites, MMP2 showed weak degradability to casein regardless of
Cu loading. The results indicated that casein hydrogel had the
potential to serve as a Cu sustained‐release reservoir regardless of
the physiological environment rich in protease.
Figure 2.
Figure 2
[110]Open in a new tab
Characteristics of casein hydrogel in loading Cu. A) Molecular docking
results of Cu^2+ bound capability of widely used proteins in tissue
engineering. B) Experimental results of Cu adsorption ability of casein
compared to BSA. C) Release curves of Cu^2+ from Casein hydrogel and
BSA hydrogel. D) Degradation of casein hydrogel with or without Cu^2+
in according to MMP2 or collagenase treatment, respectively. E) The
size change of CMG in according to Cu^2+ addition. F) Size distribution
of CMGs and G) CuCMGs. H) Water content of CMGs and CuCMGs. I)
Equilibrium swelling ratio of CMGs and CuCMGs. J) Typical scanning
electron microscope images of freeze‐dried CMG and CuCMG under
different magnifications. K) Cu^2+ release curve of CuCMGs. L)
Schematic of elastic modulus test of CMGs and CuCMGs. M) Typical
Force‐indentation curves for CMG and CuCMG. N) The elastic moduli of
CMGs and CuCMGs. The dashed line indicates the elastic modulus of
ischemic myocardium in rats. Student's t‐test was used for statistical
analyses. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. ns,
no statistical significance.
Casein microgels (CMGs) were prepared by an emulsification
photopolymerization method and used as a Cu^2+ carrier for in vitro and
in vivo experiments. We found that CMGs retained the great Cu^2+
binding ability of casein hydrogel, with 0.32 ± 0.02 µmol mg^−1 Cu^2+
loaded in CMG from a 2 mm Cu^2+ stock solution, which is even higher
than bulk casein hydrogel. Interestingly, we observed an ultrafast
(within 20 s, Figure [111]2E) and dramatic (nearly 50%,
Figure [112]2F,G) size reduction upon Cu^2+ addition of CMGs (Video
[113]S1, Supporting Information). The water content of CMGs also
significantly decreased from 98.48% ± 0.04% to 57.05% ± 1.80%
(Figure [114]2H). Meanwhile, the equilibrium swelling ratio of CMGs
decreased from 64.75 ± 1.55 to 1.33 ± 0.10 (Figure [115]2I). Scanning
electron microscopy (SEM) showed that the surface microstructure of
CuCMG was smoother than CMG as the original porous structure of the
freeze‐dried CuCMGs disappeared (Figure [116]2J). Cross–linking density
calculations revealed significant changes in CMG before and after Cu
binding, with two orders of magnitude increase from 10^−6 to 10^−4 mol
cm^−3. Meanwhile, the circular dichroism (CD) spectrum showed that
after cross–linking with the Ru/SPS system, the peak at 200 nm in the
original casein shifted toward 240 nm, indicating that the
cross–linking process affected the structure of the protein. This
240 nm peak disappeared after CMG binding Cu^2+, suggesting Cu^2+
further altered the protein structure (Figure [117]S2, Supporting
Information). These structural changes were also reflected in the
differential scanning calorimetry (DSC) thermograms, where the
denaturation temperature (T [m]) of CMG decreased relative to casein
and increased after the adsorption of Cu^2+. Alterations in the peak
area further demonstrated structural changes with microgel preparation
and Cu binding. The peak at 50 nm provided additional evidence of these
changes (Figure [118]S3, Supporting Information). Besides, we found
that the conductivity of casein hydrogel increased from 0.050 ± 0.033
to 0.287 ± 0.033 S cm^−1 after loading Cu^2+, which might be due to the
movement of metal ions (Figure [119]S4, Supporting Information), but
the value is still significantly lower than that of normal biological
tissues (30–70 S cm^−1).^[ [120]^45 ^] Being fabricated to microgels,
the CuCMGs showed faster Cu^2+ release than bulk gels due to the higher
specific surface area but still showed a sustained release, with nearly
40% released after 4 weeks (Figure [121]2K). Cu loading also
significantly increases the elastic modulus of CMGs. By recording the
sphere deformation and the repelling force using a cantilever
(Figure [122]2L), the force‐indentation curves of CMGs and CuCMGs were
obtained (Figure [123]2M). The elastic moduli of CMGs were 22.5 ±
7.9 kPa calculated from the force‐indentation curves, while the elastic
moduli of CuCMGs significantly increased ≈20 times to 443.4 ± 25.8 kPa
(Figure [124]2N). Therefore, the above results all indicated that CMG
was an excellent Cu‐loaded material, and after capturing Cu^2+, the
microgels became denser and more rigid.
2.2. CuCMGs Protect Cells in Cardiac Tissues under Pathological Environments
The rescue of mitochondrial function in injured cardiomyocytes is
critical to repair infarcted myocardium.^[ [125]^46 ^] To determine the
role of Cu^2+ in preserving mitochondrial functions in pathological
environments in an ischemic heart, we tested whether CuCMG could
protect the mitochondria of H9C2 cells (rat cardiomyocytes) from
oxygen‐glucose deprivation (OGD) injury. The safe concentration range
of Cu^2+ was first determined by live/dead staining and CCK8 assay
(Figures [126]S5 and [127]S6, Supporting Information). A timeline of
the in vitro experimental design is presented in Figure [128] 3A. Only
when the Cu^2+ concentration was lower than 20 µm, the cell viability
was not affected in both normoxia and OGD conditions. Thus, a Cu^2+
concentration of 20 µm was chosen for subsequent cell experiments, and
the CuCMGs concentration was adjusted to 2 mg mL^−1 accordingly so that
the Cu^2+ concentration released within 24 h was also ≈20 µm.
Mitochondria proceed OXPHOS through membrane potential gradients
generated by electron transfer chains (ETCs), thereby driving adenosine
triphosphate (ATP) synthesis. Therefore, mitochondrial membrane
potential (MMP) is one of the important indicators for evaluating
normal mitochondrial function.^[ [129]^47 ^] JC‐1 molecules form
aggregates and show red fluorescence in healthy mitochondria, and as
the MMP decreases, JC‐1 becomes monomers and shows green fluorescence.
The MMP was evaluated in OGD‐treated H9C2 cells by JC‐1 staining.
Figure [130]3B showed that both dissociative Cu^2+ and CuCMGs treatment
effectively improved the MMP in OGD‐treated H9C2 cells, as evidenced by
the shift in fluorescence from green to red. The quantitative analysis
further confirmed these results, that the proportion of aggregates with
red fluorescence increased from 14.08% ± 3.03% in the control group to
27.10% ± 2.00% in the CuCMG group (Figure [131]3C). In addition, to
explore whether CuCMGs treatment could increase intracellular ATP
content in OGD‐treated H9C2 cells, we measured the intracellular ATP
levels of different groups by chemiluminescence method. CuCMGs
significantly rescued the ATP content of H9C2 cells impaired by OGD
treatment (Figure [132]3D). In summary, CuCMGs maintained mitochondrial
function in OGD conditions.
Figure 3.
Figure 3
[133]Open in a new tab
The effects of CuCMG on cardiomyocytes, endothelial cells, and
fibroblasts. A) Schematic diagram of OGD model building, dosing, and
detection times. B) JC‐1 staining of H9C2 cells from Blank, Control,
CMG, Cu, and CuCMG groups. n = 4. Scale bars = 100 µm. C) Statistical
analysis of the JC‐1 staining. D) The ATP levels of H9C2 cells in
different groups after OGD treatment. E) qRT‐PCR analysis of the
expression levels of HIF1A, VEGFA, and FGF2 in HUVECs treated with CMG
and CuCMG separately. F) qRT‐PCR analysis of the expression levels of
Postn, Col1a1, and Ccn2 in pre‐induced MCFs treated with CMG and CuCMG
separately. one‐way ANOVA was used for statistical analyses. *p < 0.05,
**p < 0.01, ***p < 0.001 and ****p < 0.0001. ns, no statistical
significance.
Cu^2+ has been recognized as an effective angiogenesis stimulator,
which affects many processes including endothelial cell proliferation,
migration, and angiogenesis.^[ [134]^48 , [135]^49 ^] In addition,
studies have shown that Cu^2+ could reduce cardiac fibrosis after MI by
inhibiting the transformation of fibroblasts into myofibroblasts.^[
[136]^30 ^] In our experiment, we also found that Cu^2+ treatment
within a certain concentration range could effectively promote the tube
formation of human umbilical vein endothelial cells (HUVECs) and the
production of vascular endothelial growth factor (VEGF) (Figures
[137]S7 and [138]S8, Supporting Information). Also, we evaluated the
function of CuCMGs on HUVECs and found that CuCMG promoted tube
formation by 76.46% with the expression of vascular‐related genes
(HIF1A, VEGFA, and FGF2) in HUVECs promoted (Figure [139]3E; Figure
[140]S9, Supporting Information). Moreover, when evaluating the
function of myocardial fibroblasts (MCFs) activated by transforming
growth factor‐β1 (TGF‐β1), we found that the expression of
fibrosis‐related genes (Postn, Col1a1, and Ccn2) in MCFs were inhibited
(Figure [141]3F).
2.3. CuCMGs Protect the Heart from Ischemic Injury
Due to the decrease in local Cu^2+ content after MI, we hypothesized
that the introduced CuCMGs could serve as a Cu^2+ reservoir to maintain
local Cu^2+ levels and protect myocardial function in the ischemic
heart. Rat MI model proceeded to verify this hypothesis, the schematic
diagram is shown in Figure [142] 4A. We first detected the Cu^2+
supplementation ability of CuCMGs in the myocardium, quantitative
results showed that the amount of Cu^2+ in the ischemic heart decreased
after MI, and the treatment of CuCMGs elevated the local Cu^2+ content
for at least 7 days, while the increase in Cu^2+ content caused by
direct Cu^2+ injection regressed to the level of the MI at Day 7
(Figure [143]4B). To explore the retention of microgels, CY5 labeled
CuCMGs in the heart were traced with a multimodal animal live imaging
system at different time points after MI. ≈50% fluorescence intensity
remained on Day 7, indicating the retention of microgels
(Figure [144]4C,D). Cu^2+ level in serum was also traced for evaluating
the systemic toxicity of CuCMGs after intramyocardial injection, serum
Cu^2+ level in rats gradually increased after MI, intramyocardial
injection of CuCMGs did not significantly change the serum Cu^2+ level
(Figure [145]S10, Supporting Information). Besides, no significant
microstructural changes were observed in the important organs (livers,
spleens, kidneys, and lungs) with the introduction of CuCMGs, further
indicating their good biological safety (Figure [146]S11, supporting
information). Heart function measured by echocardiograms showed no
significant differences in cardiac function among different groups on
Days 3 and 7 after MI (Figures [147]S12 and [148]S13, Supporting
Information). On day 28 after MI, CuCMGs treatment significantly
improved the left ventricular ejection fraction (LVEF) compared to the
MI group, with LVEF values increasing from 44.44% ± 1.70% to 60.93% ±
4.22%. Meanwhile, CuCMGs treatment also increased the left ventricular
fractional shortening (LVFS) from 19.16% ± 0.89% in the MI group to
28.67% ± 2.56% (Figure [149]4E,F). These data indicated the therapeutic
benefit of CuCMGs in improving heart function after MI injury. Masson's
trichrome staining analysis showed that, except for the sham surgery
group, all rats showed myocardial fibrosis on Day 28 after MI, while
the scar area in the CuCMG treatment group was smaller than that in MI
and MI+CMG groups, with the scar area decreased by nearly 50%
(Figure [150]4G,H). In addition, the group treated solely with Cu^2+
also showed a smaller scar area compared to the MI and MI+CMG groups,
even though the scar area in this group was still significantly larger
than that in the CuCMG group. The angiogenesis after MI on Day 28 was
also examined by CD31 (vascular endothelial cell marker)
immunofluorescence staining. More CD31^+ positive area was observed in
the border zone of the ischemic hearts of the MI+CuCMG group as
compared with those in the MI and MI+CMG groups, with CD31^+ area
increasing from 0.68% ± 0.10% in the MI group to 1.16% ± 0.25%
(Figure [151]4I), which indicated better prognosis after blood supply
occlusion.
Figure 4.
Figure 4
[152]Open in a new tab
Therapeutic effects of CuCMG in treating myocardial infarction. A)
Schematic diagram of MI model and CuCMG administration in rats. B)
Cardiac Cu^2+ levels at different time points after MI. C) Ex vivo
imaging and D) fluorescence intensity analysis of Cy‐5 labeled CuCMGs
in rat hearts. E) Representative echocardiography obtained from the
middle area of the left ventricular papillary muscle in rats on Day 28
after MI. F) Echocardiography analysis of LVEF, LVFS, left ventricular
end‐diastolic volume (LVEDV) and left ventricular end‐systolic volume
(LVESV) at Day 28 after injection. G) Masson's trichrome staining of
rat hearts in Sham, MI, MI+CMG, MI+Cu, and MI+CuCMG groups (first
panel) and enlarged images of the infarcted zone (middle panel,
enlarged from the black box of the first panel) and border zone (lower
panel, enlarged from the red box of the first panel). H) Quantitative
analysis of scar area. I) Images and quantification of CD31^+ area in
the border zone of infarcted hearts on Day 28 after MI. Scale bars =
100 µm. HPF, area of high‐power field. one‐way ANOVA was used for
statistical analyses. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p <
0.0001. ns, no statistical significance.
We further conducted multiple histological analyses to evaluate CuCMGs’
effect on different cell types at the early stage after MI, which may
affect the late stage cardiac functions. CuCMGs treatment significantly
decreased the ratio of TUNEL^+ apoptotic cells by 75.80% and 66.38%
compared to MI group on Day 3 and Day 7, respectively (Figure [153]
5A). The treatment of CuCMGs significantly increased angiogenesis
examined by CD31 for about twice at different time points
(Figure [154]5B). The CD31^+ area decreased from Day 3 to Day 28, which
is consistent with previous reports as most capillary endothelial cells
in the border zone cease to proliferate in the mid to late stage of
MI.^[ [155]^50 , [156]^51 ^] The polarization of macrophages was
analyzed by CD86 (pro‐inflammatory phenotype marker) and CD206
(anti‐inflammatory phenotype marker) staining (Figure [157]5C,D).
CuCMGs did not reduce the number of CD86^+ cells but significantly
increased the number of CD206^+ cells, indicating the anti‐inflammatory
potential of CuCMGs. The major effector in MI‐induced fibrosis,
periostin (Postn) was stained to evaluate the response of fibroblasts.
From Day 3 to Day 7 after MI, Postn^+ area increased from 16.62% ±
6.98% to 25.15% ± 1.63%. CuCMGs or Cu^2+ treatment ameliorated the
process, with significant differences between the two groups observed
on Day 7 (Figure [158]5E), indicating the beneficial effect of
controlled release of Cu^2+ from microgels. Thus, the results from
different cell types indicated that CuCMG showed positive effects on MI
treatment.
Figure 5.
Figure 5
[159]Open in a new tab
The effects of CuCMG in apoptosis, angiogenesis, inflammation, and
fibrosis in the early stage after myocardial infarction. Typical images
and quantification of A) TUNEL, B) CD31, C) CD86, D) CD206, and E)
Postn staining showing the border zone of infarcted hearts at Day 3 and
Day 7 after MI. Scale bars = 100 µm. HPF, area of high‐power field.
one‐way ANOVA was used for statistical analyses. *p < 0.05, **p < 0.01,
***p < 0.001 and ****p < 0.0001. ns, no statistical significance. # p
<0.05 compared to Day 3.
2.4. CuCMGs Rescue the Transcriptome of Ischemic Hearts
To clarify the comprehensive roles of CuCMGs in mediating cardiac
protection following MI, we performed RNA sequencing (RNA‐seq) on the
ventricular tissues of Sham, MI, MI+CMG, and MI+CuCMG hearts on Day 3.
Firstly, principal component analysis (PCA) confirmed the consistency
between each group of biological repeats (n = 4; Figure [160]S14A,
Supporting Information). Hierarchical clustering analysis showed that
CuCMGs local injection significantly altered the cardiac transcriptome
after MI (Figure [161] 6A). Venn diagram revealed that compared to the
Sham group, MI injury altered the expression of 5877 genes in rats, of
which 33.42% (1964 genes) changes could be fully restored to the level
before MI by local injection of CuCMGs (Figure [162]6B). After that, we
found 3151 differentially expressed genes between the MI+CuCMG group
and the MI group, of which 687 genes were upregulated and 2464 genes
were downregulated (Figure [163]6C). Gene Ontology (GO) biological
process analyses showed that the “fatty acid metabolic process”, “ATP
metabolic process”, “tricarboxylic acid cycle” and “oxidative
phosphorylation” were the most enriched in upregulated genes
(Figure [164]6D). Similar to the above description, Kyoto Encyclopedia
of Genes and Genomes (KEGG) pathway analyses revealed that the altered
genes were largely associated with energy metabolism‐related pathways,
with upregulated genes concentrated in pathways such as “oxidative
phosphorylation”, “carbon metabolism”, and “fatty acid metabolism”
(Figure [165]6E). However, the number of differentially expressed genes
between the MI+CMG group and the MI group was very small (Figure
[166]S14B–D, Supporting Information). This indicated that the gene
expression difference caused by CuCMGs was mainly due to the release of
Cu^2+.
Figure 6.
Figure 6
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Intramyocardial injection of CuCMGs rescued transcriptomes of ischemic
heart. A) Hierarchical clustering of differentially expressed genes in
MI, MI+CuCMG, and Sham hearts as assessed by RNA‐seq. B) Venn diagram
of differentially expressed genes in three groups. C) Volcano plot of
all of the expressed genes from the MI and MI+CuCMG group. (|Log[2]Fold
Change| ≥1, adjusted p‐Value < 0.05). D) GO analysis linked to the
biological process of differentially expressed genes in MI+CuCMG hearts
and MI hearts. E) KEGG pathway enrichment analysis of differentially
expressed genes in MI+CuCMG hearts and MI hearts. F) GSEA analysis of
metabolic‐related enrichment plots compared to MI group. G) Heat maps
of metabolic‐related genes.
These findings suggested that intramyocardial injection of CuCMGs
improved cardiac function after MI probably by mediating cardiac
metabolism‐related pathways. To verify this hypothesis, we compared and
analyzed the transcriptome characteristics between MI hearts treated
with or without CuCMGs, as well as between MI hearts and Sham hearts.
Gene Set Enrichment Analysis (GSEA) showed that CuCMGs significantly
restored MI‐induced down‐regulation of metabolic pathways such as
“oxidative phosphorylation”, “glycolysis”, and “fatty acid metabolism”
as compared to MI group (Figure [168]6F). Further sorting out the gene
expression status of each group, it was found that CuCMGs positively
regulated the expression of many metabolic‐related genes after MI,
including “pyruvate metabolism”, “glycolysis”, “fatty acid
degradation”, “citrate cycle”, “fatty acid metabolism” and “carbon
metabolism” (Figure [169]6G). These results supported the hypothesis
that CuCMGs leverage important roles in cardiac metabolism after MI.
2.5. CuCMGs Improve Cardiac Metabolic Homeostasis after MI
The results of RNA‐seq analysis revealed that CuCMGs regulated
metabolic processes after MI, which prompted further investigation into
how CuCMGs alleviated cardiac metabolic stress in response to heart
injury and remodeling. Respiratory function is one of the most
fundamental energy metabolism activities in organisms, and mitochondria
are the main site for aerobic respiration in cells, providing energy
for cell metabolism. The function of the respiratory chain represents
the most basic function of mitochondria, and the mitochondrial
respiratory chain enzyme complexes ultimately form ATP through a series
of redox processes, providing energy for the body's tissues.^[ [170]^52
, [171]^53 ^] By evaluating the five enzyme complexes related genes on
the electron transport chain in the hearts of MI, MI+CuCMG, and Sham
groups by RNA‐seq, we found that CuCMGs had a positive regulatory
effect on the expression of mitochondrial respiratory‐related genes
after MI (Figure [172] 7A). To validate these results, we performed
qRT‐PCR analysis on typical signature OXPHOS‐related genes which showed
significant differences in RNA‐seq, including the genes of key enzymes
in TCA cycle, citrate synthase (Cs) and isocitrate dehydrogenase
(Idh2),^[ [173]^54 ^] as well as mitochondrial complex related genes
(Ndufs2, Sdha, Uqcrfs1, Cox4a, and Atp5a1, representing genes from
complex I to V).^[ [174]^55 , [175]^56 ^] Our results confirmed the
upregulation of mitochondrial respiratory‐related pathways after CuCMGs
treatment (Figure [176]7B). Further, we proceeded with a western blot
(WB) to detect the protein expression of the corresponding OXPHOS
indicators mentioned above. The results agreed that intramyocardial
injection of CuCMGs rescued the expression of OXPHOS‐related processes
after MI in both gene and protein levels. (Figure [177]7C,D).
Figure 7.
Figure 7
[178]Open in a new tab
CuCMGs improved cardiac metabolic homeostasis after myocardial
infarction. A) Heat maps of five enzyme complexes related genes on the
electron transport chain in MI, MI+CuCMG, and Sham hearts extracted
from RNA‐seq. B) qRT‐PCR analysis of the expression levels of Cs, Idh2,
Ndufs2, Sdha, Uqcrfs1, Cox4a, and Atp5a1. C) Western blot of
corresponding OXPHOS representative proteins in infarcted myocardium.
D) Quantification of Western blot band intensity. E) Schematic diagram
showing ETC and the represented inhibitors. Rotenone (Rot) is an
inhibitor of mitochondrial complex I. Antimycin A (AA) is an inhibitor
of mitochondrial complex III. Sodium azide (AZD) is an inhibitor of
mitochondrial complex IV. F) Representative respiratory experiment of
mitochondrial OXPHOS ability to interrogate sequentially different
substrates and coupling states using myocardial filaments. G) Analysis
of mitochondrial OXPHOS capacity of complex I (CI), complex II (CII),
and complex IV (CIV) in ETC. Complex respiration rates were corrected
to corresponding residual oxygen consumption (ROX). Student's t‐test
was used for statistical analyses. *p < 0.05, **p < 0.01, ***p < 0.001
and ****p < 0.0001. ns, no statistical significance.
The mitochondrial ETC is located on the inner membrane of mitochondria
and consists of five complexes, each with its specific substrates and
inhibitors (Figure [179]7E). Mitochondrial respiration was evaluated by
measuring the oxygen consumption rate (OCR) of the ischemic myocardial
filament adjacent to the border zone of the ventricle using
high‐resolution respirometry based on substrate‐uncoupler inhibitor
titration (SUIT) protocols.^[ [180]^11 , [181]^57 , [182]^58 ^] A
comprehensive analysis was conducted on the OXPHOS ability of each
complex in mitochondria and was evaluated by monitoring OCR while
sequentially adding corresponding substrates and inhibitors.
(Figure [183]7F). Compared with the MI group, in the presence of
adenosine diphosphate (ADP) and cytochrome C (Cyto C), the respiration
of complex I in the MI+CuCMG hearts significantly increased to 143% by
adding substrates pyruvate, malate, and glutamate (PMG). The
respiration of complex II in the MI+CuCMG group increased to 143% of
the MI group in the presence of substrate succinate by adding ascorbate
(As) and tetramethyl‐phenylenediamine (TMPD) as substrates, the
respiration of complex IV in the MI+CuCMG hearts increased to 129%
compared to the MI hearts (Figure [184]7G). In summary, these results
indicated that CuCMGs improved cardiac OXPHOS, maintained cardiac
metabolic homeostasis, and improved cardiac function after MI.
3. Discussion
Myocardial ischemia is mainly caused by coronary artery thrombosis. Due
to the high incidence rate and mortality, it poses a huge threat to
public health.^[ [185]^59 ^] In the infarcted heart, the respiratory
and OXPHOS functions of myocardial mitochondria, which are key
organelles of Cu metabolism, are significantly impaired, affecting
overall metabolic levels and ultimately damaging systolic function.^[
[186]^60 ^] In this study, we used casein as a carrier to construct
Cu‐loaded microgels (CuCMGs) that could be injected into the
myocardium. Through in vitro cell experiments and in vivo rat MI
models, we investigated the cellular protective and metabolic
regulatory effects of CuCMGs in the pathological environment of
ischemia and hypoxia. We found that CuCMGs indeed have the function of
maintaining metabolic homeostasis after MI to protect cardiac function.
Casein, as a protein derived from food sources, not only has lower
costs compared to other natural sources of proteins, but molecular
simulation docking results also demonstrated superior Cu^2+ loading
capacity. Therefore, we used the Ru/SPS redox system to construct
casein microgels (CMGs) through visible light cross–linking. In the
process of CMG loaded with Cu^2+, we observed a rapid size decrease,
accompanied by significant increase in cross–linking density and
elastic modulus. Combined with the changes in CD spectra and DSC
thermograms, we anticipated structural change of protein in CMG after
Cu^2+ loading. The structural change condensed the microgels and
affected the exposure of related sites, which further affected the
sensitivity to collagenase, resulting in slower degradation. As the
elastic modulus of ischemic myocardium increased significantly compared
to normal myocardium, from 10–20 to ≈50 kPa,^[ [187]^61 ^] soft
microgel may be distorted under compression. The elastic modulus of
CuCMG was significantly higher than that of ischemic myocardial tissue,
indicating that the degree of deformation of CuCMG during cardiac
contraction could be ignored after intramyocardial injection, which was
beneficial in eliminating squeeze‐induced Cu release. Therefore, CuCMG
was a good carrier for achieving precise delivery and sustained release
of Cu^2+ to cardiac tissue.
The dysfunction and apoptosis of cardiomyocytes, loss of vascular, and
activation of fibroblasts play important roles in myocardial remodeling
after ischemia.^[ [188]^10 , [189]^62 ^] We thus studied the function
of CuCMGs in rescuing the mitochondrial function of cardiomyocytes,
promoting endothelial cell angiogenesis, and inhibiting fibrotic
expression of fibroblasts. In vivo experiments using rat MI models also
showed that CuCMGs significantly alleviated the deterioration of
cardiac function after ischemia, which could contribute to
cardiomyocyte protection, angiogenesis improvement, or fibrosis
inhibition. In order to determine the main pathways in which CuCMGs
participated in post‐MI, we performed whole genome sequencing on the
ischemic myocardium of rats. The results of RNA‐seq suggested that
intramyocardial injection of CuCMGs improved cardiac function after MI
mainly by mediating pathways related to cardiac energy metabolism,
particularly affecting pathways related to the tricarboxylic acid (TCA)
cycle, OXPHOS, and fatty acid metabolism. As the center for oxidative
metabolism in eukaryotes, mitochondria are the sites for the TCA cycle
and OXPHOS.^[ [190]^63 ^] In mitochondria, the TCA cycle and OXPHOS are
closely coordinated. The nicotinamide adenine dinucleotide (NADH) and
flavin adenine dinucleotide (FADH2) produced by a TCA cycle are
consumed by Complex I (NADH dehydrogenase) and Complex II (succinate
dehydrogenase, SDH) of ETC, respectively. Complex I and Complex II
transfer electrons through ETC and ultimately produce ATP through
OXPHOS. ETC is necessary for maintaining the TCA cycle, and it closely
links the TCA cycle with OXPHOS.^[ [191]^64 ^] By further analyzing the
results of RNA‐seq and validating key genes with qRT‐PCR, we observed a
significant upregulation of complex‐related gene expression on ETC
compared to the MI group after CuCMGs treatment. These changes have
also been observed at the protein levels, as the expression of Cs (the
enzyme initiates the TCA cycle) and Idh2 (the enzyme generates NADH)
increased significantly. The representative proteins in mitochondrial
complex I to V also showed the same trend as their corresponding genes.
Based on the above results, we further focused on the functional
changes of ETC.
ETC is located on the inner membrane of mitochondria and consists of
five enzyme complexes.^[ [192]^65 ^] In addition to Complex I and
Complex II mentioned earlier, Complex III, known as cytochrome c
reductase, can transfer electrons to cytochrome c. Complex IV, called
Cco, is the last protein complex in ETC. It carries the final reaction
of ETC, transferring electrons to oxygen while expelling protons into
the mitochondrial membrane gap. Protons ultimately pass through Complex
V (mitochondrial ATP synthase) to synthesize ATP.^[ [193]^66 ^] In
respiratory experiments for mitochondrial OXPHOS ability following SUIT
protocol, the addition of an inhibitor of a certain complex blocks the
preceding ETC flow, thus Complex III inhibitor could be used to
evaluate OCR change caused by complex II. Research has found that many
enzyme complexes are mediated by Cu^2+ during their functional
processes. Michael O Isei et al. found that Cu^2+ acted on multiple
sites in ETC and could regulate the flavin sites of Complex I and
Complex II in a concentration‐dependent manner. Cu^2+ also mediated the
external ubiquinone binding sites of Complex III, affecting the
generation of reactive oxygen species (ROS).^[ [194]^67 ^] In addition,
copper sulfate (CuSO[4]) pretreatment counteracted the activity damage
in mitochondrial Complexes I, II, IV, and V caused by
1‐methyl‐4‐phenylpyridinium (MPP^+) in rat striatum.^[ [195]^68 ^] In
the mitochondria of inflammatory macrophages, NADH can be rapidly
consumed to produce NAD^+ in the presence of Cu^2+, and this process is
mainly involved by Complex I.^[ [196]^10 ^] In addition, Complex IV
uses Cu^2+ as a cofactor and has multiple Cu^2+ binding sites.^[
[197]^69 ^] Therefore, we speculated that the Cu^2+ released by CuCMGs
might intervene in the function of the entire ETC by affecting the
structure and function of various complexes, thereby maintaining
metabolic homeostasis after MI.
Present as a great carrier for Cu^2+ delivery, CuCMGs show promising
capability in maintaining overall ETC functions and metabolic
homeostasis after MI. In the future, the weight functions of specific
pathways can be further explored to unveil the key mechanism in
CuCMG‐related metabolism regulation. It may also combine with other
therapeutic strategies such as mechanical support, conductivity
improvement, immunomodulation, etc., to further promote cardiac repair
after MI.
4. Conclusion
In summary, more and more studies have been focused on preserving
metabolic homeostasis after MI therapy. In this research, we developed
a metabolism‐modulating system for ischemic myocardium by simply
loading Cu to CMGs. Casein was screened out from the seven most
commonly used protein materials due to its great binding capacity to Cu
by combining molecule docking and experimental measurement. The Cu
loading significantly diminished the size, and increased
anti‐enzymolysis and mechanical modulus of CMGs, indicating a strong
affinity between casein and Cu. The formed CuCMG has shown long‐acting
Cu release and ensured the stability of MMP and ATP synthesis in
cardiomyocytes under OGD environment. In addition, CuCMGs led to
favorable changes in transcriptome levels related to energy metabolism
and cardiac function. These findings confirm the rationale that
biomaterials targeting metabolic homeostasis possess great potential in
cardiac reconstruction, CuCMGs present as a new therapeutic agent for
cardioprotection by modulating the malfunction metabolism in ischemic
hearts.
5. Experimental Section
Molecular Docking Method (MDM)
AlphaFold2 was used to construct the full‐length structures of the
proteins and served them as the initial structures.^[ [198]^70 ^] Using
Cu^2+ as a ligand, the molecular structure was optimized by the
Molecular Orbital PACkage (MOPAC) program and calculated the PM3 atomic
charge for subsequent molecular docking.^[ [199]^71 , [200]^72 ^]
AutoDock Tools 1.5.6 was used to process the initial protein structures
and ligand structure, generating corresponding pdbqt files for
docking.^[ [201]^73 ^] Docking was performed with AutoDock 4.2.6 using
a 100 × 100 × 100 grid box centered on the protein binding site and 100
docking runs. To relieve unrealistic atomic contacts, energy
optimization was done using Amber14 in two steps: 1000 cycles of
steepest descent, followed by 500 cycles of conjugate gradient.
Synthesis of CMGs and CuCMGs
CMGs were prepared by an emulsification photocross–linking method
described as follows. The aqueous phase was prepared by dissolving SPS
(Sinopharm Chemical Reagent Co., Ltd., Shanghai, China),
tris(2,2‐bipyridyl) dichlororuthenium (II) hexahydrate (Ru) (Xianding,
Shanghai, China) and casein (Fonterra, Shanghai, China) in 0.1 m NaOH
solution to obtain a 10% w/v casein solution with 80 mm SPS and 0.3 mm
photoinitiator Ru. The pH was adjusted to 6.5–7.5 and 0.1% w/v Tween60
(Ruibio, Germany) was added as dispersant and mixed thoroughly. The oil
phase was liquid paraffin (HUSHI, Shanghai, China), containing 0.1% w/v
emulsifier Span 80 (Aladdin, Shanghai, China). The volume ratio of the
aqueous phase to the oil phase was 1:10. The aqueous phase was dropped
into the oil phase, fully stirred for 10 min, and then irradiated with
a white LED lamp for 10 min to ensure complete gelation. The obtained
solid materials were thoroughly cleaned with n‐hexane (Aladdin,
Shanghai, China), acetone (Aladdin, Shanghai, China), and phosphate
buffered solution (PBS) in sequence, and stored in a 4 °C refrigerator
before use.
To prepare CuCMGs, the CMGs were incubated in a 2 mm CuCl[2] solution
for at least 24 h to ensure equilibrium adsorption. Before the
following microgel characterization, the microgels were rinsed with
deionized water to remove potential unchelated ions.
Cu Adsorption Capacity Of Casein Hydrogel
The amount of Cu adsorption was measured by a subtraction method.
Namely, 25 µL (v [0]) of 10% w/v protein pre‐gel solution was taken and
illuminated to form complete gelation. Each hydrogel was placed in 1 mL
CuCl[2] solution for 24 h, and the variation of Cu^2+ concentration in
solution before (c [0]) and after adsorption (c) was detected with a Cu
assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China)
according to manufacturer's instructions and measured with a plate
reader (Tecan, SPARK) at 600 nm wavelength. The amount of adsorbed Cu
(Q) was calculated following:
[MATH: Q=c0−cv0*10% :MATH]
(1)
Cu Release Measurement
The following methods were used to test the release profiles of Cu^2+.
10 mg of microgels was placed in a 1.5 mL centrifuge tube, and then
1 mL (v [1]) of PBS was added and placed at 37 °C. Take samples (v[2] )
and supplement the release medium on days 1, 3, 7, 10, 14, 21, and 28,
respectively. Casein hydrogels and BSA hydrogels loaded with the same
amount of Cu^2+ were also immersed in PBS using the same method as
described above. The contents of Cu^2+ (c [1] to c[7] )in the solution
were detected by a copper assay kit as above. The cumulative release
rate was calculated following:
[MATH: Cumulativereleaserate=cn*v1+(c1+c2+c3
+⋯+cn−1)*v20.44*100% :MATH]
(2)
c[n] represented the Cu concentration at the nth sampling point, and
the total Cu loading amount has been determined to be 0.44 µmol
according to previous calculations. Finally, the corresponding
cumulative release curves were plotted.
Characterization of Microgels
A phase‐contrast optical microscope (Lecia DMIL, Germany) was used to
monitor the shape and particle size of each group of microgels.
Scanning electron microscopy (GeminiSEM 300, Germany) was used to
detect the surface morphology of freeze‐dried microgels. Far‐UV CD
spectra were acquired on a spectrometer (Chirascan V100, Applied
Photophysics Ltd, China) at 25 °C using a 0.1 cm path‐length quartz
cuvette. Samples were dispersed in deionized water. Spectra were
recorded from 260 to 180 nm at a scan rate of 50 nm min^−1 with a
response time of 1 sec and bandwidth of 1 nm. Three scans were
accumulated and averaged followed by baseline subtraction of buffer
only signal. Thermograms were obtained using a TA Instruments DSC (Q20
V24.10 Build 122, USA) from −80 to 180 °C at a scan rate of 10 °C
min^−1 with a nitrogen purge gas flow of 50 mL min^−1. An empty pan was
used as a reference to obtain the instrument baseline profile. Sample
data was baseline‐corrected by subtracting the reference scan. The
denaturation temperature (T [m]) was determined from the maximum point
of the endothermic peak. A certain compressive force was applied to the
microgel by using the microscale mechanical testing system
(Microsquisher, CellScale) to compress it by 30 µm at a speed of 2 µm
s^−1. The elastic modulus was calculated from strain–stress curve.
Calculation of Cross–linking Density of CMG and CuCMG
Microgels were accurately weighed (mass m [0]) and added to 25 mL of
deionized water. The vials were capped and allowed to swell at 25 °C
with solvent replacement every 6 hours. Samples were periodically
removed every 30 min initially, then per hour when swelling slowed,
blotted dry, and weighed (mass m [1]) until consecutive weighings
differed by no more than 0.002 g, indicating swelling equilibrium was
reached. Each sample was measured in duplicate. The cross–linking
density (ρ[c] ) was calculated as:
[MATH: ρc=−ln(1−vr)+vr+χvr2
VS(vr1/3−0.5vr) :MATH]
(3)
[MATH: vr=m0/ρ0m0/ρ0+(m1−m0)/ρ1 :MATH]
(4)
Here V[S] was solvent molar volume, χ was Flory–Huggins polymer‐solvent
interaction parameter, ρ [0], and ρ [1] were densities of the microgels
and solvent, and m [0], m [1] were masses of the sample before
swelling, and the swollen samples, respectively.
Water content and Equilibrium Swelling Ratio of Microgels
The wet mass (M[S] ) of microgels in each group was measured. The
microgels were then dried in the freeze‐drying machine for 24 h and
their dry mass (M[D] ) was measured. The water content and equilibrium
swelling ratio were calculated as follows:
[MATH: Watercontent=MS−MDMS*100%
:MATH]
(5)
[MATH: Equilibriumswellingratio=MS−MDMD :MATH]
(6)
Degradation of Casein Hydrogel With or Without Cu^2+
Casein hydrogels with or without Cu treatment were put into 10 nm MMP2
(Solarbio, Beijing, China) or 1 mg mL^−1 collagenase (Gibco, USA)
respectively. The initial dry weight (M [0]) and the dry weight at
different time points (M[n] ) of hydrogels in each group were measured.
The degradation rate was calculated as follows:
[MATH: Degradationrate=M0−MnM0*100%
:MATH]
(7)
Cell Culture
H9C2 cells (rat cardiomyocytes) were purchased from Procell (Wuhan,
China). The cells were cultured in high‐glucose Dulbecco's modification
of Eagle medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine
serum (FBS; Gibco, USA) and 1% penicillin/streptomycin (P/S; Gibco,
USA) in a carbon dioxide incubator (37 °C and 5% CO[2]). The OGD model
was established with low‐glucose DMEM supplemented with 2% FBS, and the
cells were incubated in 95% N[2] and 5% CO[2] for 24 h. Immortalized
and green fluorescent protein (GFP) transfected human umbilical vein
endothelial cells (HUVECs) were purchased from iCell (Shanghai, China)
and cultured in high‐glucose DMEM supplemented with 10% FBS and 1% P/S.
Isolation of myocardial fibroblasts (MCFs) from neonatal mice was
performed following literature using a Neonatal Cardiac Fibroblast
Isolation Kit (Miltenyi, Germany).^[ [202]^74 ^] MCFs were cultured
with high‐glucose DMEM supplemented with 5% FBS and 1% P/S.
JC‐1 Staining Analysis
H9C2 cells were allowed to grow with an initial density of 40 000 cells
mL^−1 in 96‐well plates in complete DMEM for 24 h. After OGD treatment
for 24 h, the original culture medium was replaced with fresh medium
with 2 µm JC‐1 (MedChemExpress, USA) The plates were incubated at 37 °C
for 15–20 min, washed twice with PBS, and observed under fluorescence
microscopy (Leica DMi8, Germany).
ATP Content Detection
H9C2 cells were allowed to grow for 24 h in a 12‐well plate as
previously mentioned. After OGD treatment, the cellular ATP levels were
measured according to the manual of the Enhanced ATP Assay Kit
(Beyotime Biotechnology, Shanghai, China). ATP level was calculated
according to a calibration curve with standard ATP samples. The total
amount of protein in each group was detected using the BCA Protein
Assay Kit (Beyotime Biotechnology, Shanghai, China) for normalization
of ATP contents.
Quantitative Reverse Transcription‑Polymerase Chain Reaction (qRT‑PCR)
Cells were lysed and RNA was extracted using a total RNA isolation kit
(Vazyme Biotech, Nanjing, China). The total RNA of heart tissues was
isolated using TRIzol reagent (Life Technologies). The complementary
DNA (cDNA) was synthesized using a PrimeScript RT reagent kit (Accurate
Biology, Hunan, China). qRT‐PCR signal was detected by the LightCycler
480 II PCR System (Roche, Switzerland) with a SYBR green qPCR SuperMix
(TransGen Biotech, Beijing, China). Gapdh and ACTB were used as the
housekeeping genes, respectively. ddCt was used to analyze gene
expression from qPCR datasets. Primers used for qRT‑PCR in this study
were purchased from Sangon Biotech and listed in Table [203]S1
(Supporting Information).
Myocardial Infarction
All animal experiments have been approved by the Zhejiang Experimental
Animal Center (No. ZJCLA‐IACUC‐20010597). Male SD rats (8 w old,
200–250 g) were purchased from the Zhejiang Academy of Medical
Sciences. The animals were anesthetized with an intraperitoneal
injection of 1% w/v sodium pentobarbital and ventilated. The heart was
exposed through a left‐sided thoracotomy. 6‐0 silk suture was used to
ligate the LAD coronary artery to obtain left ventricular infarction. A
total volume of 60 µL of 100 mg mL^−1 CuCMGs, 100 mg mL^−1 CMGs, or
3 mm CuCl[2] solution was injected into the border zones of infarcted
myocardium for MI+CuCMG group, MI+CMG group, and MI+Cu group,
respectively. Rats only experienced a thoracotomy operation (Sham) and
MI rats received no treatments (MI) were used as controls.
Measurement of Cu^2+ Contents in the Serum and Heart Tissue
The postoperative rats were anesthetized and blood was collected from
the hearts to obtain serum. Serum Cu levels were detected with a Cu
assay kit as mentioned above. Heart samples from the infarct area were
obtained, and heart tissue from the same part of the heart of the Sham
group was taken as controls. Heart tissue samples were freshly frozen,
lyophilized, and digested with nitric acid (HNO[3], Sinopharm, China)
overnight (10 mg dried tissue with 1 mL HNO[3]). After filtration, Cu
contents were determined by using a graphite furnace atomic absorption
spectrophotometer (AAS, ICE3500, Thermo, USA).
Ex vivo Fluorescence Imaging
Microgels labeled with Sulfo‐CY5‐NHS (Yusi Biotechnology Co., Ltd.,
Chongqing, China) were used for intramyocardial injection. The rats
were euthanized to harvest their hearts at Day 0 (immediately after
surgery), Day 3, and Day 7 after MI and imaged with a multimodal animal
live imaging system (AniView100, BioLight, China). The excitation light
wavelength was 605 nm, and the emission light wavelength was 680 nm.
Measurement of Cardiac Function By Echocardiography
Transthoracic echocardiography (VEVO2100, Visual Sonics, Canada) was
used to analyze cardiac function on Day 3, Day 7, and Day 28 after MI.
Rats were anesthetized by inhaling oxygen‐containing isoflurane
(1–1.5%), and their chest hair was also removed before examination.
B‐mode echocardiography and M‐mode echocardiography were used for the
long‐axis views for measuring cardiac parameters.
Histological Analysis
The rats were euthanized to harvest their hearts. The hearts were
soaked in potassium chloride solution to stop beating and fixed in 4%
paraformaldehyde overnight at room temperature. For paraffin
sectioning, heart samples were embedded in paraffin, and sections with
a thickness of 6 µm were cut for following Masson's trichrome staining
which was proceeded using a Modified Masson's Trichrome Stain Kit
(Solarbio, Beijing, China) and observed under an optical microscopy
(Leica DM3000, Germany). For cryotomy, heart samples were perfused with
30% sucrose at 4 °C overnight before being embedded in optimal cutting
temperature compound (OCT) for frozen section. 6 µm sections were cut
for immunofluorescence staining and TUNEL staining. Apoptotic cells
were detected by TUNEL staining using the one‐step TUNEL Apoptosis
Assay Kit (Beyotime, Jiangsu, China) according to the manufacturer's
protocol. The immunofluorescence staining of cTnT was performed
together with TUNEL staining. After blocking with PBS containing 5% w/v
BSA at room temperature for 1 hour. Then, sections were incubated in a
primary antibody solution Mouse anti‐cTnT (1/400 diluted in 1% BSA,
abcam, USA) overnight at 4 °C. Then, sections were washed with PBS and
incubated with a secondary antibody. For other immunofluorescence
staining, Rabbit anti‐CD31 (1/800 diluted in 1% BSA, Servicebio,
China), Rabbit anti‐CD86 (1/500 diluted in 1% BSA, Peoteintech, USA),
Rabbit anti‐CD206 (1/500 diluted in 1% BSA, Peoteintech, USA), Rabbit
anti‐Postn (1/500 diluted in 1% BSA, R&D Systems, USA) were used as the
primary antibody with Alexa Fluor 555 donkey anti‐rabbit IgG as the
secondary antibody (1/500 diluted in 1% BSA, Abcam, Britain). Finally,
after incubating with DAPI (1 µg mL^−1, Servicebio, China) at room
temperature for 10 min, the sections were observed under a fluorescence
microscope (Leica DM6B, Germany). The area of interest was analyzed by
ImageJ software.
RNA‑seq and Genome‑Wide Transcriptome Analysis
On Day 3 after MI, total RNA from the ventricular tissue of rats in the
Sham, MI, MI+CMG, MI+Cu, and MI+CuCMG groups was collected for RNA seq
(n = 4). RNA‐seq experiments were performed by LC‐Bio Technology
(Hangzhou, China). Total RNA was extracted from fresh ventricular
tissue using Trizol reagent (thermofisher) following the manufacturer's
procedure. Then, mRNA was purified from total RNA using Dynabeads Oligo
(dT) (Thermo Fisher, CA, USA).
A cDNA library constructed by technology from the pooled RNA from
samples was sequenced and run with the Illumina Novaseq TM 6000
sequence platform. The Illumina paired‐end RNA‐seq approach was used to
sequence the transcriptome, generating 150 bp paired‐end reads. Reads
obtained from the sequencing machines included raw reads containing
adapters or low‐quality bases which might affect the following assembly
and analysis. To get high‐quality clean reads, reads were further
filtered by Cutadapt. The sequence quality was verified using FastQC.
The reference genome and gene model notes files were downloaded
directly from the genome website.
Principal component analysis (PCA) was performed with the princomp
function of R in this experience. Genes differential expression
analysis was performed by DESeq2 software between two different groups.
The genes with the parameter of false discovery rate (FDR) below 0.05
and absolute fold change ≥ 2 were considered differentially expressed
genes. Differentially expressed genes were then subjected to enrichment
analysis of GO functions and KEGG pathways. GO terms and KEGG pathways
with corrected p < 0.05 were considered significantly enriched in
differentially expressed genes. We performed gene set enrichment
analysis using software GSEA (v4.1.0) and MSigDB to identify whether a
set of genes showed significant differences in the two groups. The
hierarchical clustering heat map was performed using the OmicStudio
tools.
Western blot (WB) Analysis
Protein lysates were prepared from heart tissues using tissue
extraction reagents (Invitrogen) supplemented with proteinase
inhibitors. The samples were separated by 10% sodium dodecyl
sulfate‐polyacrylamide gel electrophoresis (SDS–PAGE) and transferred
onto polyvinylidene fluoride (PVDF) membranes by electrophoresis. After
blocking in 5% skimmed milk, membranes were incubated with the
indicated primary antibodies overnight at 4 °C and then washed with
phosphate‐buffered saline with 0.05% Tween20 (PBST) buffer before
incubation with horseradish peroxidase (HRP)‐conjugated secondary
antibodies for 2 h at room temperature. Protein bands were visualized
using enhanced chemiluminescence (ECL) Reagent (Invitrogen) with the
Bio‐Rad ChemiDoc imaging system. Quantification of WB was analyzed by
ImageJ software. All the antibody information is listed in Table
[204]S2 (Supporting Information).
Mitochondrial Respiration in Myocardial Filaments
Ten milligrams of myocardial tissue adjacent to the border zone was
dissected from each heart and separated into fiber bundles as fine as
possible by sharp tweezers on ice. Then the fiber bundles were
permeabilized in ice‐cold biological sample solution (BIOPS, composed
of 2.77 mm CaK[2]EGTA, 7.23 mm K[2]EGTA, 5.77 mm Na[2]ATP, 6.56 mm
MgCl[2], 20 mm Taurine, 15 mm Na[2]Phosphocreatine, 20 mm Imidazole,
0.5 mm Dithiothreitol, 50 mm 2‐(4‐Morpholino) ethane sulfonic acid
hydrate, pH = 7.1) containing 50 ug mL^−1 saponin (Sigma‐Aldrich,
Germany) by shaking on a shaking table for 30 min. Permeabilized fibers
were washed for 10 min by agitation in ice‐cold mitochondrial
respiration medium 05 (MiR05, composed of 0.5 mm EGTA, 3 mm MgCl[2],
60 mm K‐lactobionate, 20 mm taurine, 10 mm KH[2]PO[4], 20 mm HEPES,
110 mm sucrose, 1 g L^−1 BSA, pH = 7), weighed, and used for
respirometric measurements immediately. 4 mg of myofibers were placed
into each respiratory measurement chamber containing 2 mL of MiR05, and
their respiratory function was measured at 37 °C (OROBOROS Oxygraph‐2k,
Innsbruck, Austria). Data acquisition and analysis were performed by
using DatLab software (OROBOROS INSTRUMENTS).
Statistics Analysis
Statistical analysis was performed using GraphPad Prism software.
Unless otherwise stated, results are presented as mean value ± standard
error of the means (SEM). Statistical significance between two columns
was assessed by a two‐tailed Student's t‐test; for more than two
columns, a one‐way Analysis of Variance (ANOVA) was used.
Conflict of Interest
The authors declare that they have no conflict of interest.
Author Contributions
X.H., G.T., and B.D. contributed equally to this work. The study was
conceptualized by X. H., T.R, J.W., and X.L. The methodology was
developed by X.H., T.R., G.T., B.D., and X.Z. The investigation
involved X. Hong, B.D., X.Z., Y.G., L.Z., H.L., Q.Z., L.Z., Y.Z., D.R.,
and C.G. Visualization was handled by X. H. and T.R. Supervision was
provided by T.R., J.W., and X.L., X.H., and T.R. were responsible for
writing, while T.R., J.W., and X.L. contributed to writing, review, and
editing.
Supporting information
Supporting Information
[205]ADVS-11-2401527-s002.pdf^ (1.6MB, pdf)
Supplemental Video 1
[206]Download video file^ (22.9MB, MP4)
Acknowledgements