Cardiac MyD88 Mediates Inammatory Injury and Adverse Remodeling in Diabetes

Background: Hyperglycemia-associated inammation contributes to adverse remodeling and brosis in diabetic heart. MyD88 is an adapter protein of many Toll-like receptors (TLRs) and is recruited to TLRs to initiate inammatory signalling pathway in endotoxin-activated innate immunity. However, the role of MyD88 in diabetic cardiomyopathy is unknown. Methods: For genetic deciency, cardiomyocyte-specic MyD88 knockout and littermate control mice were induced type 1 diabetes (T1D) by intraperitoneal injection of 50 mg/kg/day streptozotocin for ve days consecutive and then fed for 4 moths. For pharmacological inhibition, MyD88 inhibitor LM8 were administered daily for 8 weeks by oral gavage in T1D and T2D (db/db) mice. The effect of genetic and pharmacological inhibition MyD88 to myocardial injure which were induced by 33 mM glucose in vivo. Results: In this study, we rst found that MyD88 expression was increased in cardiomyocytes of diabetic mouse hearts. Cardiomyocyte-specic MyD88 knockout protected mice against hyperglycemia-induced cardiac inammation, injury, hypertrophy, and brosis in T1D model. In cultured cardiomyocytes, MyD88 inhibition either by siRNA or by small-molecular inhibitor LM8 markedly blocked TLR4-MyD88 complex formation, reduced pro-inammatory MAPKs/NF-κB cascade activation and decreased pro-inammatory cytokine expression under high glucose condition. Moreover, pharmacologic inhibition of MyD88 by LM8 showed signicantly anti-inammatory, anti-hypertrophic and anti-brotic effects in the hearts of both T1D and T2D mice. These benecial effects of MyD88 inhibition were correlated to the reduced activation of TLR4-MyD88-MAPKs/NF-κB signaling pathways in the hearts. Conclusion: Taken together, MyD88 in cardiomyocytes mediates diabetes-induced cardiac inammatory injuries and genetic or pharmacologic inhibition of MyD88 shows signicantly cardioprotective effects, indicating MyD88 as a potential therapeutic target for diabetic cardiomyopathy. differentiation primary-response protein 88; Myh6, Myosin heavy chain 6; MyHC, Myosin heavy chain, cardiac muscle; NF-κB, nuclear factor-κB; PAMPs, pathogen ‐ associated molecular patterns; PBS, phosphate buffered solution; PCR, Quantitative Polymerase Chain SDS-PAGE, gel electrophoresis; type 1 type 2 diabetes; factor α; tetraethyl

However, it is unclear whether MyD88 is necessary for hyperglycemia-induced pro-in ammatory cascades in diabetes-related cardiac injury.
Given that MyD88 is an important adapter protein in TLRs' pro-in ammatory signaling pathways, we speculate that MyD88 may be a potential target for the treatment of diabetic cardiomyopathy. Recently, we identi ed a series of new small-molecule MyD88 inhibitors. Among them, compound LM8 signi cantly reduced lipopolysaccharide (LPS)-induced in ammatory cytokine expression in macrophages via directly binding to MyD88 and blocking MyD88-TLR4 complex formation [18]. In this study, we tested the role of MyD88 in diabetic cardiomyopathy using a cardiac speci c MyD88 knockout mouse model. The protective roles of MyD88 inhibition in hyperglycemia-induced pro-in ammatory cascades and cardiac injury were further con rmed with MyD88 siRNA and LM8 both in vivo and in vitro. Our data demonstrate an essential role of cardiomyocyte MyD88 in cardiac in ammation of diabetic mice. Pharmacologic inhibitors of MyD88, e.g., LM8, could be potential therapeutic agents for diabetic cardiomyopathy.
All animal care and experimental procedures were approved by the Wenzhou Medical University Animal Policy and Welfare Committee.
(1) Type 1 diabetes (T1D) was induced by intraperitoneal injection of 50 mg/kg/day streptozotocin (STZ, from Sigma-Aldrich, dissolved in citrate buffer, pH 4.5) for ve days consecutive. Control group received the same volume of citrate buffer. After seven days, fasting blood-glucose levels were measured using glucometer (B. BRAUN, Germany). Mice with fasting glucose levels > 12 mM for three consecutive days were considered diabetic, which were maintained at diabetic status for 16 weeks to induced diabetic cardiomyopathy. Body weight and fasting blood glucose levels were measured weekly for 16 weeks. For LM8 treatment in T1D mice, mice were randomized into non-diabetic controls (Ctrl, n = 7), STZ-induced diabetic mice (STZ, n = 7), diabetic mice treated 5 mg/kg LM8 (STZ + LM8-5), diabetic mice treated 10 mg/kg LM8 (STZ + LM8-10). LM8 (5 and 10 mg/kg) was administered as oral gavage every two days from 9th week to 16th week. The diabetic group and control group received the same volume of 1% CMC-Na solution every two days.
(2) Seven-week-old male db/db mice were used as Type 2 diabetes (T2D) model, with littermates db/m mice as controls. Mice were maintained at diabetic status for 8 weeks to induced diabetic cardiomyopathy. For LM8 treatment in T2D mice, mice were randomized into db/m controls (db/m, n = 7), db/db diabetic mice (db/db, n = 7), diabetic mice treated 5 mg/kg LM8 (db/db + LM8-5), diabetic mice treated 10 mg/kg LM8 (db/db + LM8-10). LM8 (5 and 10 mg/kg) was administered as oral gavage every two days from 5th week to 8th week. The diabetic group and control group received the same volume of 1% CMC-Na solution every two days. At 7th week in the experiment, intravenous glucose tolerance tests (IGTT) were performed by intraperitoneal injection of glucose (1 g/kg) and subsequent measurement of the blood-glucose levels per 15min using glucometer. were injected with citrate buffer. Body-weights and fasting glucose levels were measured in all mice weekly for 16 weeks.
At the end of treatment, mice were killed under sodium pentobarbital anesthesia (i.p. injection of 0.2 mL sodium pentobarbital at 100 mg·mL − 1 ). The blood and hearts were collected for subsequent analyses.
Biotin-based pull-down assay Biotinylated LM8 (Bio-LM8) was synthesized and structurally characterized with a purity of 98.1%. Firstly, 1µM Bio-LM8 was added to 200µl pre-blocked streptavidin-agarose beads and incubated for 1h at room temperature. Biotin and LM8 alone were used as a control. Secondly, lysates prepared from 293T cells that overexpress MyD88 or mouse heart tissues were added to the pre-loading Bio-LM8 streptavidinagarose beads and then incubated for 4 h at room temperature. Lastly, 50µl 1 SDS loading buffer were added to the bead precipitates to fully elute protein then loaded on SDS-PAGE for immunoblot analysis. Total lysates were used as an input control.

Immunohistochemistry
The depara nized and rehydrated sections were treated with boiling 0.01M sodium citrate buffer (pH6.0) to restore antigen and then incubated 3% H 2 O 2 for 30 min to block endogenous peroxidase activity. The sections were next blocked with 1% BSA in PBS for 30 min, incubated at 4°C overnight with the TNF-α primary antibody (Santa, cat.no. SC52746 1:200 dilution) followed by incubation for 1 h with HRPconjugated secondary antibodies (1:500 dilution) and then immunoreactivity was detected by diaminobenzidine (DAB) following the manufacturer's protocol. The stained sections were viewed under a light microscope (Nikon, Japan).

Real-time quantitative PCR
Total RNAs were extracted from cells or tissues using TRIZOL (Invitrogen). Reverse transcription and quantitative PCR (qPCR) were carried out using PrimeScript™ RT reagent Kit and SYBR premix taqII (Takara). Bio-Rad CFX96 real time system (Bio-Rad Tech., Shanghai, China) was used for qPCR analysis using standard protocols. The primer sequences of target genes are listed in the supplementary Table S1 (Invitrogen). The amount of each gene was normalized to the amount of β-actin. For genomic PCR, genomic DNA were extracted from the toe of the mice (3 weeks old) using protease K digestion and saturated NaCl sedimentation and then PCR was performed. The primer sequences of target DNA fragment are listed in Table S2 (Invitrogen). Following electrophoresis, PCR products were visualized on 2% agarose gel containing 0.5 µg/mL SYBR.

Statistical analysis
All experiments were randomized. In-vitro experiments were repeated at least 3 times. Data are presented as Means ± SEMs. The statistical signi cance of differences between groups was obtained by the student's t-test or ANOVA multiple comparisons in GraphPad Pro8.0 (GraphPad, San Diego, CA). We used one-way ANOVA followed by Dunnett's post-hoc test when comparing more than two groups and one-way ANOVA, non-parametric Kruskal-Wallis test followed by Dunn's post-hoc test when comparing multiple independent groups. P < 0.05 was considered statistical signi cance.

Results
MyD88 expression was upregulated in the cardiomyocytes of diabetic mice Firstly, we measured the expression level of MyD88 in T1D mouse hearts. Western blot analysis showed that MyD88 expression was signi cantly increased in the hearts of STZ-induced T1D mice compared to the non-diabetic controls (Fig. 1A). The same changes in TLR2 and TLR4 proteins were observed in hearts of T1D mice (Fig. 1A). Interestingly, immunoprecipitation assay of heart tissue lysates revealed increased MyD88 interaction with TLR2 and TLR4 (Fig. 1B), further indicating an activated state of MyD88 in diabetic hearts. To con rm the source of increased MyD88 expression, we performed immuno uorescence in mouse heart sections using doubly-staining for MyD88 and either cardiomyocytespeci c marker actin or broblast-speci c marker vimentin. [14] Our double-staining immuno uorescence and quanti cation data indicated signi cantly increased expression and co-localization of MyD88 mainly in the cardiomyocytes of diabetic mice (Fig. 1C, 1E). Comparatively, a faint expression level of MyD88 was observed in the cardiac broblasts of STZ-treated mice and control mice (Fig. 1D, 1E). Immuno uorescence data was further validated by measuring MyD88 expression in rat primary cardiomyocytes and primary cardiac broblasts by immunoblotting. The result con rmed a much higher expression in cardiomyocytes in comparison to broblasts (Fig. 1F). We then treated the primary cardiomyocytes with HG at 33mM. As shown in Fig. 1G and supplementary Figure S1A, HG challenge for a long time (24 or 48 h) induced MyD88 expression. However, HG stimulation for 25 or 30 min signi cantly activated MyD88, evidenced by increased MyD88 interaction with TLR2 and TLR4 in primary cardiomyocytes (Fig. 1H). This induction was not seen in 33mM mannitol-challenged H9c2 cells, indicating osmotic stress is not involved in MyD88 activation (supplementary Figure S1B). These data show that diabetes/HG could up-regulate and activate MyD88 in cardiomyocytes, indicating the involvement of MyD88 in diabetic cardiomyopathy.

Cardiomyocyte-speci c MyD88 knockout prevented STZinduced cardiac in ammation and injury
To investigate the role of MyD88 in the hearts of T1D mice, cardiomyocyte-speci c MyD88 knockout mice were generated by crossing Myh6 Cre and MyD88 f/f mice (Supplementary Figure S2A-B). Successful cardiac MyD88 deletion was validated by western blot in the isolated adult mouse cardiomyocytes (Supplementary Figure S2C). As shown in Fig. 2A, cardiac MyD88 deletion did not affect hyperglycemia induced by STZ. Similarly, cardiac MyD88 deletion also had no effect on the body weight of both healthy and T1D mice (Fig. 2B). Interestingly, the heart weight/tibia length (HW/TL) ratio was signi cantly increased in STZ-treated MyD88 f/f mice, and cardiac MyD88 deletion completely prevented this increase (Fig. 2C). Moreover, plasma lactate dehydrogenase (LDH) and creatine kinase isoenzyme-MB (CK-MB), two biochemical indices of myocardial injury, were increased in STZ-treated MyD88 f/f mice, and cardiac MyD88 deletion blunted these increases (Fig. 2D-E). H&E staining was performed on transverse and longitudinal heart sections, which show increased structural abnormalities and increased cardiomyocyte area, indicating cardiac hypertrophy in the hearts of STZ-treated mice in comparison to control mice. However, the increased cardiomyocyte area was completely prevented in STZ-treated MyD88-de cient mice ( Fig. 2F and supplementary Figure S3A). Masson's trichrome and Sirius red staining analysis showed increased collagen deposition in the hearts of STZ-treated MyD88 f/f mice, which was also prevented by cardiac MyD88 deletion (Fig. 2G and Figure S3E). Subsequently, gene expression levels of TNF-α, IL-6, and IL-1β were signi cantly increased in STZ-treated MyD88 f/f mice (Fig. 2K). Importantly, MAPKs activation, IκB-α degradation, and increased expression of proin ammatory cytokines were markedly prevented by cardiac MyD88 deletion (Fig. 2J-K and supplementary Figure S3E).

Pharmacological inhibition of MyD88 by LM8 suppresses HG-induced in ammation and hypertrophy in cardiomyocytes
The chemical structure of MyD88 inhibitor LM8 was shown (Fig. 4A). We previously demonstrated that LM8 inhibited MyD88-mediated in ammation by preventing its dimerization in macrophages.[18, 20] A biotinylated LM8 (Bio-LM8, in the supplementary Figure S5A), which showed same anti-in ammatory effects compared to LM8 (supplementary Figure S5B), was used for further target validation in cells and heart tissues. Successful binding of LM8 to MyD88 protein was observed in 293T cells and mouse heart tissues by using biotinylated protein interaction pull-down assays (supplementary Figure S5C-D). We then con rmed that HG induced MyD88 dimerization and MyD88-TLR4 complex formation in H9c2 cells, which were prevented by LM8 treatment (Fig. 4B-C and supplementary Figure S6A-B). Subsequently, western blot assay examined HG-induced phosphorylation of ERK and JNK, as well as IκB degradation and P65 nuclear translocation in H9c2 cells, while this HG-induced activation of MAPKs and NF-κB signaling was signi cantly prevented with LM8 treatment at 2.5, 5 and 10 µM (Fig. 4D and supplementary Figure S6C). LM8 treatment dose-dependently inhibited HG-induced P65 nuclear translocation, which was further con rmed by immuno uorescence staining in H9c2 cells (Fig. 4E-F). As a result, HG-induced expression of in ammatory cytokines, including TNF-α, IL-6, and IL-1β, were signi cantly reduced by LM8 treatment dose-dependently (Fig. 4G).
LM8 prevented cardiac in ammation and injury in mouse T1D model Next, the therapeutic effect of MyD88 inhibition by LM8 was tested in mouse T1D model. The administration doses of LM8 at 5 and 10 mg/kg were chosen according to previous studies.[18, 21] LM8 treatment did not affect blood glucose level and body weight in T1D mice (Fig. 5A-B). Similar to the results of cardiac MyD88 knockout, LM8 treatment at both doses signi cantly decreased HW/TL ratio, serum LDH and CK-MB activity in T1D mice compared to the vehicle treated T1D mice (Fig. 5C-E). H&E staining of transverse and longitudinal heart sections demonstrated improvement of T1D-induced cardiac hypertrophy and structural abnormality with LM8 treatment (Fig. 5F and supplementary Figure  S8A). LM8 treatment also reduced T1D-induced cardiac brosis evidenced by reduced Masson's trichrome and Sirius red staining (Fig. 5G and supplementary Figure S8B Figure S8D). Parallelly, LM8 treatment diminished T1D-induced activation of ERK and JNK and IκB-α degradation in diabetic hearts (Fig. 5J and supplementary Figure S8E). Real-time qPCR assay showed dose-dependent inhibition of LM8 against diabetes-induced production of in ammatory cytokines TNF-α, IL-6, and IL-1β in heart tissues ( Fig. 5K and supplementary Figure S8F-G).
Of note, MyD88-TLR4 complex formation was clearly increased in the hearts of T1D mice, which was markedly blocked by LM8 treatment (Fig. 5L and supplementary Figure S8H). Overall, this data demonstrates that LM8 treatment protects hearts against diabetes-induced cardiac injury by inhibiting MyD88-mediated in ammation rather than reducing hyperglycemia in T1D mice.

LM8 prevented cardiac in ammation and injury in db/db mice
To further validate the protective effect of LM8 on diabetic cardiomyopathy, db/db mice were used to produce T2D mouse model. Seven-week-old db/db and db/m control mice were treated with LM8 at 5 and 10 mg/kg for the last 4 weeks. We also saw that MyD88 was over-expressed in hearts of db/db mice, compared to db/m mice (supplementary Figure S9). LM8-treatment did not affect blood glucose and body weight in db/db mice (Fig. 6A-B). LM8-treatment also did not improve insulin resistance in db/db mice, evidenced by IGTT and ITT assay (supplementary Figure S10A-B). Like the results in T1D mice, increased HW/BW ratio and serum CK-MB and LDH levels in db/db mice were signi cantly inhibited by LM8-treatment ( Fig. 6C-E). H&E staining analysis showed that LM8 attenuated cardiac hypertrophy and pathological changes in db/db mice ( Fig. 6F and supplementary Figure S11A). Sirius red and Masson's trichrome staining showed that LM8 treatment also signi cantly reduced brosis in db/db mice heart sections ( Fig. 6G and supplementary Figure S11B-C). LM8 administration also reduced the expression levels of MyHc, Col-I, and TGF-β in the hearts of db/db mice (Fig. 6H-I and supplementary Figure S11D). Parallelly, LM8 treatment diminished activation of ERK, JNK, and NF-κB ( Fig. 6J and supplementary Figure S11E) and production of in ammatory cytokines TNF-α, IL-6, and IL-1β (Fig. 6K) in the hearts of db/db mice. Similarly, LM8-treatment inhibited MyD88-TLR4 complex formation in the hearts of db/db mice ( Fig. 6L and supplementary Figure S11F). Overall, our data in db/db mice show that LM8 protects the heart by inhibiting cardiac in ammation without affecting hyperglycemia or insulin resistance in T2D mouse models. Taken together, this data demonstrates that LM8 treatment protects diabetic hearts by inhibiting MyD88-mediated in ammation rather than reducing hyperglycemia or improvement of insulin resistance in the T2D mouse model.

Discussion
Our results showed that MyD88 expression and activity were increased in the cardiomyocytes of diabetic mice. HG treatment signi cantly increased MyD88-TLR4 interaction, activated MAPKs and NF-κB signaling, and induced proin ammatory and pro brotic responses in cultured cardiomyocytes, and MyD88 inhibition with LM8 or siRNA markedly prevented these HG-induced changes in vitro. Importantly, cardiomyocyte-speci c MyD88 gene knockout signi cantly ameliorated hyperglycemia-induced activation of MAPKs and NF-κB and reduced subsequently in ammatory injury and remodeling in the hearts of T1D mice. These protective roles were further con rmed by the fact that LM8 treatment prevented cardiac in ammation and injury, pathological remodeling and brosis in both STZ-treated mice and db/db mice. As summarized in Fig. 7, these results shed new light on the role of MyD88 in in ammatory diabetic cardiomyopathy and provide a mechanistic basis for diabetes/HG-induced cardiac in ammation. This study also indicates that pharmacologic inhibitors of MyD88 could be potential therapeutic agents for diabetic cardiomyopathy.
Myocardial in ammation and brosis are two key pathophysiological mechanisms that drive cardiac remodeling and dysfunction resulting in heart failure. [22] Epidemiological studies suggest that hyperglycemia, a hallmark of either T1D or T2D, is closely associated with a chronic low-level in ammation that contributes to myocardial damage, leading to diabetic cardiomyopathy. [23] Given the central role of in ammation in the progress of diabetic cardiomyopathy, several anti-in ammatory drugsbased approaches have shown therapeutic effects on diabetic cardiomyopathy. [24] However, there are currently no drugs or therapies available to improve cardiac brosis in diabetic cardiomyopathy. In the current study, MyD88 inhibition signi cantly improved cardiac injury and reduced cardiac in ammation and brosis in T1D and T2D mouse models. It is interesting that neither genetic nor pharmacologic inhibition of MyD88 affected hyperglycemia in mice with T1D or T2D, indicating that the cardiac bene ts observed in T1D and T2D mice were mainly due to the anti-in ammatory effect of MyD88 inhibition.
[28] Following recruitment to TLRs except TLR3, MyD88 interacts with IRAK2/4 (Interleukin 1 Receptor Associated Kinase 2/4) through the death domains and leads to the activation of NF-κB and MAPKs (mainly ERK and JNK pathways) and subsequent expression of pro-in ammatory cytokines. [5,32] Given that several TLRs are involved in the pathogenesis of diabetic cardiomyopathy, inhibition of MyD88, a molecular target downstream to TLRs, may be a more appropriate strategy that antagonism of certain TLR. For instance, TLR4 antagonists could not block in ammation mediated by other TLRs, which may be a main reason for the failure of TLR4 antagonists, eritoran and TAK-242, in clinical e ciency of sepsis therapy [33]. In this study, increased MyD88-TLR2/4 interaction was con rmed in the hearts of diabetes mice, accompanied with activation of MAPKs and NF-κB resulting in increased cardiac in ammatory and brotic responses, and these were prevented by either genetic or pharmacological MyD88 inhibition. In addition, MyD88 is expressed in the heart, liver, spleen, lungs, kidney, thymus, lymph nodes and digestive systems at different developmental stages, indicating its important role. [34] However, the systemic MyD88 knockout mice were viable without any overt phenotype, [7] indicating that MyD88 is dispensable physiologically and strategies to inhibit MyD88 at any stage of life are expected not to show strong side-effects in other cell-type or organ systems. This study validates this deduction by good e ciency and high safety of LM8 in both type 1 and type 2 diabetic mice. These data demonstrate that MyD88 is a promising target to treat diabetic cardiomyopathy.
TLR2 and TLR4 were expressed in cardiomyocytes [17,28,35] and cardiac broblasts[36] in hearts. As TLRs adaptor protein, MyD88 expression is found in the heart, but its expression distribution and function in cardiomyocytes, particularly in the diabetic setting, remain unknown. Our data, for the rst time, demonstrate that MyD88 predominantly expressed in cardiomyocytes in hearts. MyD88 expression and its interaction with TLR2 and TLR4 were increased in cardiomyocytes in the setting of high glucose or diabetes. Cardiomyocyte-speci c MyD88 knockout mice demonstrated that MyD88-de ciency protected cardiomyocytes against hyperglycemia-induced cardiac in ammation and injury via reducing MAPKs and NF-κB activation. Cardiac broblasts are also important in the progression of diabetic cardiomyopathy [37,38]. However, our data showed a strong induction of MyD88 only in the cardiomyocyte by diabetes and then cardiac protective effects following the cardiomyocyte-speci c loss of MyD88, indicating a relatively robust role played by cardiomyocytes than cardiac broblasts. It is also important to note that LM8 was systemically delivered to inhibit MyD88 in every cell-type, but we did not observe an additive protective effect of LM8 in comparison to genetic inhibition of cardiomyocyte MyD88. This also indicates that cardiomyocyte MyD88 plays a key role in diabetic cardiomyopathy in comparison to cardiac broblasts. However, a limitation of this study may be the absence of the evaluation of in ltrated macrophages in heart. Given that in ltrated macrophages contribute to diabetic cardiac in ammation [14], it should be necessary in the future to examine whether MyD88 in macrophages also contributes to cardiac in ammatory injuries in diabetes.
This study indicates that strategies aiming to inhibit MyD88 in cardiomyocytes may be a potential therapeutic approach for diabetic cardiomyopathy. However, there are some limitations of the study. First, only cardiac injury/remodeling was evaluated in the mice, and cardiac function was not assessed that is more important in clinic setting and needs to be further investigated in future studies. Second, only the classic and known MAPKs/NF-κB pathways associated with MyD88 signalling were analysed in this study, therefore, a global pathway analysis, especially after MyD88 inhibition, would be valuable to explore the underlying mechanisms and potential safety issues.

Conclusion
In our study demonstrates that cardiomyocyte MyD88 mediates in ammatory diabetic cardiomyopathy via MyD88-MAPKs/NF-κB pro-in ammatory signalling pathway. Genetic and pharmacologic inhibition of MyD88 showed signi cantly cardioprotective effects on hyperglycemiainduced in ammation and brosis in-vitro and in-vivo, indicating that pharmacologic inhibitors of MyD88 could be potential therapeutic agents for diabetic cardiomyopathy.

Declarations
GAPDH was loading control. (K) qPCR analyses of TNF-α, IL-6 and IL-1β in the heart tissues were shown.
(L) Co-IP analysis determined the interaction between TLR4 and MyD88 in the hearts. n = 7 per group, *P<0.05 vs STZ-treated group. Figure 6