Mouse Myocardium Research Articles

Cardiomyocyte-specific knockout of ADAM17 ameliorates left ventricular remodeling and function in diabetic cardiomyopathy of mice

Angiotensin-converting enzyme 2 (ACE2) has proven beneficial in attenuating diabetic cardiomyopathy (DCM) but has been found to be a substrate of a disintegrin and metalloprotease protein-17 (ADAM17). However, whether ADAM17 plays a role in the pathogenesis and intervention of DCM is obscure. In this study, we created cardiomyocyte-specific knockout of ADAM17 (A17α-MHCKO) mice, and left ventricular dimension, function, pathology and molecular biology were assessed in ADAM17fl/fl control, A17α-MHCKO control, ADAM17fl/fl diabetic and A17α-MHCKO diabetic mice. Both differentiated H9c2 cells and neonatal rat cardiomyocytes (NRCMs) were used to explore the molecular mechanisms underlying the effect of ADAM17 on DCM. The results showed that protein expression and activity of ADAM17 were upregulated whereas the protein expression of ACE2 was downregulated in the myocardium of diabetic mice. Cardiomyocyte-specific knockout of ADAM17 mitigated cardiac fibrosis and cardiomyocyte apoptosis and ameliorated cardiac dysfunction in mice with DCM. Bioinformatic analyses detected a number of genes enriched in metabolic pathways, in particular the AMPK signaling pathway, expressed differentially between the hearts of A17α-MHCKO and ADAM17fl/fl diabetic mice. The mechanism may involve activated AMPK pathway, increased autophagosome formation and improved autophagic flux, which reduced the apoptotic response in cardiomyocytes. In addition, hypoxia-inducible factor-1α (HIF-1α) might act as an upstream mediator of upregulated ADAM17 and ADAM17 might affect AMPK signaling via α1 A-adrenergic receptor (ADRA1A). These results indicated that ADAM17 activity and ACE2 shedding were enhanced in DCM, which was reversed by cardiomyocyte-specific ADAM17 knockout. Thus, inhibition of ADAM17 may provide a promising approach to the treatment of DCM.

Palmatine Alleviates Acute Myocardial Infarction Through Activating pAMPK/Nrf2 Signaling Pathway in Mouse Model

Acute myocardial infarction is one of the major leading causes for heart failure, which can lead to the irreversible loss of cardiomyocytes and impaired cardiac function. Hence, the efficient therapeutic agents are still urgent. Our study aimed to explore the role of a natural isoquinoline alkaloid, palmatine, in an acute myocardial infarction mouse model. In this study, intragastric administrated palmatine significantly enhanced left ventricle ejection fraction and left ventricle end-systolic of infarcted mice heart. Meanwhile, palmatine administration partially recovered myocardial structure and attenuated the cardiac fibrosis and infiltration of inflammatory cells. In addition, the usage of palmatine further enhanced the increased transforming growth factor (TGF)-beta1 level, reduced the elevated tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta level in the myocardium of acute myocardial infarction–induced mice, as well as elevated the reduced superoxide dismutase production and inhibited the increased malondialdehyde secretion in infarcted myocardium of mice. Meanwhile, acute myocardial infarction led the significant upregulation of Bcl-2-associated X and downregulation of B-cell lymphoma-2 in the myocardium, and palmatine administration statistically enabled to recover the expression changes of these two apoptosis-related proteins. Moreover, palmatine administration obviously elevated the expression levels of phosphorylated AMP-activated protein kinase and nuclear factor erythroid 2–related factor 2 in the myocardium of acute myocardial infarction–induced mice. In a word, our study indicated that palmatine could protect infarcted myocardium of mice from apoptosis, inflammation, and oxidative stress. Our results suggested that palmatine might be a novel therapeutic agent for acute myocardial infarction.Graphical

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk.

Heart disease is the leading cause of morbidity and mortality worldwide. Due to their low cost, ease of handling, and abundance of transgenic strains, rodents have become essential models for cardiovascular research. However, spontaneous lethal cardiac arrhythmias that often cause mortality in heart disease patients are rare in rodent models of heart disease. This is primarily due to the species differences in cardiac electrical properties between humanand rodents and poses a challenge to the study of cardiac arrhythmias using rodents. This protocol describes an approach to enable efficient transgene expression in mouse and rat ventricular myocardium using echocardiography-guided intramuscular injections of recombinant virus (adenovirus and adeno-associated virus). This work also outlines a method to enable reliable assessment of cardiac susceptibility to arrhythmias using isolated, Langendorff-perfused mouse and rat hearts with both adrenergic and programmed electrical stimulations. These techniques are critical for studying heart rhythm disorders associated with adverse cardiac remodeling after injuries, such as myocardial infarction.

Downregulation of miR-34c-5p alleviates chronic intermittent hypoxia-induced myocardial damage by targeting sirtuin 1.

Numerous microRNAs (miRs) are abnormally expressed in response to hypoxia-induced myocardial damage. Herein, miR-34c-5p as a potential pharmaco-target was investigated in a mouse model of chronic intermittent hypoxia (CIH)-induced myocardial damage. A mouse model of myocardial damage was established using CIH with 7% or 21% O2 alternating 60 s for 12 h/day, 21% O2 for 12 h/day. AntagomiR-34c-5p (20 nM/0.1 ml; once a week for 12 weeks) was used as a miR-34c-5p inhibitor in a mouse model with tail-vein injection. In another experiment, mice were administrated with Sirt1 activator SRT1720 (50 mg/kg/day) by intraperitoneal injection. Gene Expression Omnibus database showed a significant upregulation of miR-34c-5p expression in the ischemic myocardium of male mice. In CIH-stimulated mice, miR-34c-5p expression was also significantly increased compared with normal mice. Treatment of antagomiR-34c-5p significantly restrained CIH-triggered myocardial apoptosis. After administration of antagomiR-34c-5p or Sirt1 activator SRT1720, cardiac hypertrophy and oxidative stress were attenuated in CIH-stimulated mice. We also found sirtuin 1 (Sirt1) as a direct target of miR-34c-5p, which was able to mediate Sirt1 protein expression in cardiomyocytes. AntagomiR-34c-5p injection markedly elevated Sirt1 protein expression in CIH-stimulated mice. AntagomiR-34c-5p or Sirt1 activator SRT1720 administration exhibited the antioxidative activity and cardioprotective roles in CIH-stimulated mice.

Prolonged Myocardial Regenerative Capacity in Neonatal Opossum.

Early neonates of both large and small mammals are able to regenerate the myocardium through cardiomyocyte proliferation for only a short period after birth. This myocardial regenerative capacity declines in parallel with withdrawal of cardiomyocytes from the cell cycle in the first few postnatal days. No mammalian species examined to date has been found capable of a meaningful regenerative response to myocardial injury later than 1 week after birth. We examined cardiomyocyte proliferation in neonates of the marsupial opossum (Monodelphis domestica) by immunostaining at various times after birth. The regenerative capacity of the postnatal opossum myocardium was assessed after either apex resection or induction of myocardial infarction at postnatal day 14 or 29, whereas that of the postnatal mouse myocardium was assessed after myocardial infarction at postnatal day 7. Bioinformatics data analysis, immunofluorescence staining, and pharmacological and genetic intervention were applied to determine the role of AMPK (5'-AMP-activated protein kinase) signaling in regulation of the mammalian cardiomyocyte cell cycle. Opossum neonates were found to manifest cardiomyocyte proliferation for at least 2 weeks after birth at a frequency similar to that apparent in early neonatal mice. Moreover, the opossum heart at postnatal day 14 showed substantial regenerative capacity both after apex resection and after myocardial infarction injury, whereas this capacity had diminished by postnatal day 29. Transcriptomic and immunofluorescence analyses indicated that AMPK signaling is activated in postnatal cardiomyocytes of both opossum and mouse. Pharmacological or genetic inhibition of AMPK signaling was sufficient to extend the postnatal window of cardiomyocyte proliferation in both mouse and opossum neonates as well as of cardiac regeneration in neonatal mice. The marsupial opossum maintains cardiomyocyte proliferation and a capacity for myocardial regeneration for at least 2 weeks after birth. As far as we are aware, this is the longest postnatal duration of such a capacity among mammals examined to date. AMPK signaling was implicated as an evolutionarily conserved regulator of mammalian postnatal cardiomyocyte proliferation.

Activation of Autophagic Flux Maintains Mitochondrial Homeostasis during Cardiac Ischemia/Reperfusion Injury.

Reperfusion injury after extended ischemia accounts for approximately 50% of myocardial infarct size, and there is no standard therapy. HDAC inhibition reduces infarct size and enhances cardiomyocyte autophagy and PGC1α-mediated mitochondrial biogenesis when administered at the time of reperfusion. Furthermore, a specific autophagy-inducing peptide, Tat-Beclin 1 (TB), reduces infarct size when administered at the time of reperfusion. However, since SAHA affects multiple pathways in addition to inducing autophagy, whether autophagic flux induced by TB maintains mitochondrial homeostasis during ischemia/reperfusion (I/R) injury is unknown. We tested whether the augmentation of autophagic flux by TB has cardioprotection by preserving mitochondrial homeostasis both in vitro and in vivo. Wild-type mice were randomized into two groups: Tat-Scrambled (TS) peptide as the control and TB as the experimental group. Mice were subjected to I/R surgery (45 min coronary ligation, 24 h reperfusion). Autophagic flux, mitochondrial DNA (mtDNA), mitochondrial morphology, and mitochondrial dynamic genes were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with a simulated I/R injury to verify cardiomyocyte specificity. The essential autophagy gene, ATG7, conditional cardiomyocyte-specific knockout (ATG7 cKO) mice, and isolated adult mouse ventricular myocytes (AMVMs) were used to evaluate the dependency of autophagy in adult cardiomyocytes. In NRVMs subjected to I/R, TB increased autophagic flux, mtDNA content, mitochondrial function, reduced reactive oxygen species (ROS), and mtDNA damage. Similarly, in the infarct border zone of the mouse heart, TB induced autophagy, increased mitochondrial size and mtDNA content, and promoted the expression of PGC1α and mitochondrial dynamic genes. Conversely, loss of ATG7 in AMVMs and in the myocardium of ATG7 cKO mice abolished the beneficial effects of TB on mitochondrial homeostasis. Thus, autophagic flux is a sufficient and essential process to mitigate myocardial reperfusion injury by maintaining mitochondrial homeostasis and partly by inducing PGC1α-mediated mitochondrial biogenesis.

Inhibition of GSDMD Activates Poly(ADP-ribosyl)ation and Promotes Myocardial Ischemia-Reperfusion Injury.

The precise control of cardiomyocyte viability is imperative to combat myocardial ischemia-reperfusion injury (I/R), in which apoptosis and pyroptosis putatively contribute to the process. Recent researches indicated that GSDMD is involved in I/R as an executive protein of pyroptosis. However, its effect on other forms of cell death is unclear. We identified that GSDMD and GSDMD-N levels were significantly upregulated in the I/R myocardium of mice. Knockout of GSDMD conferred the resistance of the hearts to reperfusion injury in the acute phase of I/R but aggravated reperfusion injury in the chronic phase of I/R. Mechanistically, GSDMD deficiency induced the activation of PARylation and the consumption of NAD+ and ATP, leading to cardiomyocyte apoptosis. Moreover, PJ34, a putative PARP-1 inhibitor, reduced the myocardial injury caused by GSDMD deficiency. Our results reveal a novel action modality of GSDMD in the regulation of cardiomyocyte death; inhibition of GSDMD activates PARylation, suggesting the multidirectional role of GSDMD in I/R and providing a new theory for clinical treatment.

Activation of STIM1/Orai1‑mediated SOCE in sepsis‑induced myocardial depression

Unbalanced Ca2+ homeostasis serves an essential role in the occurrence and development of septic myocardial injury. However, the mechanism of Ca2+ homeostasis in septic myocardial depression is poorly understood due to the complexity of Ca2+ transporters in excitable cells. It was therefore hypothesized that cardiac dysfunction, myocardial injury and cardiac apoptosis in septic myocardial depression are associated with elevated intracellular Ca2+ concentrations caused by stromal interaction molecule 1 (STIM1)/Orai calcium release‑activated calcium modulator 1 (Orai1)‑mediated store‑operated Ca2+ entry (SOCE). A septic myocardial depression model was established using the cecal ligation and puncture operation (CLP) in mice and was simulated in H9C2 cells via lipopolysaccharide (LPS) stimulation. Cardiac function, myocardial injury, cardiac apoptosis and the expression levels of Bax, Bcl‑2, STIM1 and Orai1 were quantified invivo at6, 12and24h. Changes in the intracellular Ca2+ concentration, SOCE and the distribution of STIM1 were assessed invitro within 6h. The morphological changes of heart tissue were observed by hematoxylin‑eosin staining. Myocardial cellular apoptosis was determined by TUNEL method. The expression of Bax, Bcl‑2, STIM1 and Orai1 were visualized by western blot. Cytosolic calcium concentration and SOCE were evaluated by confocal microscopy. The results demonstrated that cardiac contractile function was significantly reduced at6h and morphological changes in cardiac tissues, as well as the myocardial apoptosis rate, were markedly increased at6, 12and24h following CLP. mRNA and protein expression levels of Bax/Bcl‑2 were significantly enhanced at6and12h and glycosylation of Orai1 in the myocardium of septic mice was significantly increased at6h following CLP. The intracellular Ca2+ concentration, SOCE, was significantly increased at1‑2h and the clustering and distribution of STIM1 were markedly changed in H9C2 cells at1and2h. These findings suggested that myocardial dysfunction, cardiac injury and myocardial depression may be related to increased intracellular Ca2+ concentration resulting from STIM1/Orai1‑mediated SOCE, which may provide a potential method to alleviate septic myocardial depression.

PD-1 inhibitor induces myocarditis by reducing regulatory T cells, activating inflammatory responses, promoting myocardial apoptosis and autophagy

The present study aimed to explore the pathogenesis of autoimmune myocarditis induced by PD-1 inhibitors and their potential therapeutic targets. Mouse models of autoimmune myocarditis induced by PD-1 inhibitor in mouse models of polymyositis were established. The expression level of PD-1 and regulatory T cells (Tregs), CD4, CD8 + T cells, inflammation, apoptosis and autophagy-related factors, including IL-6, TGF-β, AMA-M2, Fas/FasL, LC3 and p62 were detected in peripheral blood, muscle or myocardium of mice in each group, using ELISA, RT-PCR, Western Blot and immunofluorescence. In addition, HE and TUNEL staining and ultrastructural scanning were performed on the myocardium of mice in each group. Results showed that the expression level of PD-1 in the two myositis groups was significantly lower than that in the control group, and the level of PD-1 was lower in the myocarditis group than that in the polymyositis group. In the myocardium, TGF-β, p62, and Tregs proportion showed the same expression level trend as PD-1, while CD8, IL-6, IL-10 and LC3 showed the opposite trend. Levels of Fas/FasL were significantly higher in both myositis groups, but were slightly lower in the myocarditis group, as was AMA-M2. Inflammation, apoptosis, and autophagy were observed in both myositis groups, but were more severe in the myocarditis group. In summary, the decreased expression level of PD-1 leads to decreased Tregs level in the myocardium, aggravated inflammatory response, apoptosis and autophagy, which may be the pathological mechanism of myocarditis induced by PD-1 inhibitors.

Establishment of a novel myocarditis mouse model based on cyclosporine A.

Myocarditis is a myocardial injury that can easily cause adolescent death. Traditional research models of animal invasion with viral components, lipopolysaccharide (LPS) or porcine myocardial myosin, among others, have the shortcomings of potential biological safety hazards and high animal mortality. To explore the construction of a novel myocarditis model with cyclosporine A and the potential genes and pathways associated with it. BALB/c mice were used in this study, and cyclosporin A and LPS were injected into the peritoneal cavity of mice. The successful establishment of the model was assessed by detecting serum myocardial injury markers and inflammatory factors levels, HE, IHC staining, and RT-qPCR methods. Key genes were obtained using the GSE35182 dataset from the GEO database and validated with the RT-qPCR method. We found that a large number of inflammatory cells infiltrated the myocardium of mice in each group of Cyclosporin A constructed model, while the expression of inflammatory factor indicators was increased, and this model has the characteristics of high degree of local inflammation in myocardial tissue, low mortality, and safe and non-toxic treatment. Using GSE35182 data, we selected 18 Hub genes and validated Hub genes in myocardial tissue with RT-qPCR and found that multiple signaling pathways such as Toll-likereceptor signaling pathway(TLRs), Rap1 signal pathway(Rap1), and Chemokine signaling pathway may be involved in the development of myocarditis. Cyclosporin A can construct a new myocarditis model, and TLRs, Chemokines and Rap1 signaling pathways may be the core pathways of myocarditis.

Molecular Mechanism of Sevoflurane Preconditioning Based on Whole-transcriptome Sequencing of Lipopolysaccharide-induced Cardiac Dysfunction in Mice.

Sevoflurane, a widely used inhalation anesthetic, has been shown to be cardioprotective in individuals with sepsis and myocardial dysfunction. However, the exact mechanism has not been completely explained. In this study, we performed whole-transcriptome profile analysis in the myocardium of lipopolysaccharide-induced septic mice after sevoflurane pretreatment. RNA transcriptome sequencing showed that 97 protein coding RNAs (mRNAs), 64 long noncoding RNAs (lncRNAs), and 27 microRNAs (miRNAs) were differentially expressed between the lipopolysaccharide and S_L groups. Functional enrichment analysis revealed that target genes for the differentially expressed mRNAs between the 2 groups participated in protein processing in the endoplasmic reticulum, antigen processing and presentation, and the mitogen-activated protein kinase signaling pathway. The bioinformatics study of differentially expressed mRNAs revealed that 13 key genes including Hsph1, Otud1, Manf, Gbp2b, Stip1, Gbp3, Hspa1b, Aff3, Med12, Kdm4a, Gatad1, Cdkn1a, and Ppp1r16b are related to the heart or inflammation. Furthermore, the competing endogenous RNA network revealed that 3 of the 13 key genes established the lncRNA-miRNA-mRNA network (ENSMUST00000192774 --- mmu-miR-7a-5p --- Hspa1b, TCONS_00188587 --- mmu-miR-204-3p --- Aff3 and ENSMUST00000138273 --- mmu-miR-1954 --- Ppp1r16b) may be associated with cardioprotection in septic mice. In general, the findings identified 11 potential essential genes (Hsph1, Otud1, Manf, Gbp2b, Stip1, Gbp3, Hspa1b, Aff3, Med12, Kdm4a, Gatad1, Cdkn1a, and Ppp1r16b) and mitogen-activated protein kinase signaling pathway involved in sevoflurane-induced cardioprotection in septic mice. In particular, sevoflurane may prevent myocardial injury by regulating the lncRNA-miRNA-mRNA network, including (ENSMUST00000192774-mmu-miR-7a-5p-Hspa1b, TCONS_00188587-mmu-miR-204-3p-Aff3, and ENSMUST00000138273-mmu-miR-1954-Ppp1r16b networks), which may be a novel mechanism of sevoflurane-induced cardioprotection.

The whole transcriptome analysis and the circRNA-lncRNA network construction in arsenic trioxide-treated mice myocardium

Background/AimsArsenic trioxide (ATO) is an effective anti-cancer drug. Nonetheless, it possesses cardiotoxic effects which limit its clinical application. The present study aims to elucidate the molecular basis of ATO-induced cardiotoxicity through using whole transcriptome analysis. MethodsThe whole transcriptome in ATO-treated mice myocardium was analyzed using RNA sequencing technique. These results were confirmed by real-time PCR. The lncRNA-mRNA and circRNA-mRNA co-expression networks were constructed. Finally, a circRNA-lncRNA co-regulated competing endogenous RNA (ceRNA) network was constructed. GO and KEGG pathway analyses were performed. The expression levels of Txnip and Spp1 in ATO-treated neonatal mouse cardiomyocytes were validated by real-time PCR. ResultsA total of 113 mRNAs, 159 lncRNAs, 35 miRNAs, and 94 circRNAs were differentially expressed in ATO-treated mice myocardium. A lncRNA-circRNA co-regulation network was constructed. Function annotation revealed that aberrantly expressed genes may be enriched in the ‘Wnt signaling pathway’, ‘Hippo signaling pathway’, ‘Notch signaling pathway’, etc. Finally, the expression levels of Txnip and Spp1 were validated in ATO-treated cardiomyocytes, which was in accordance with the RNA-sequencing results. ConclusionATO altered coding and noncoding RNA profiles in myocardium of mice. The ATO-related lncRNA-circRNA co-regulation network was constructed. Genes in the co-regulation network are likely to play important roles in the cardiotoxicity of ATO. This study provides new insights into the prevention and treatment of ATO-induced cardiotoxicity.

Investigation on the differentially expressed circular RNAs in myocardium of mice with diabetic cardiomyopathy

Objective: To identify the differentially expressed circular RNA (circRNA) in the myocardium of diabetic cardiomyopathy (DCM) mice, and analyze their possible biological functions and related regulatory network. Methods: C57BL/6 mice, aged 8 weeks, and weighing were 21-27 g. Eight mice were selected as the control group and 15 mice were selected as the experimental group. The diabetic mice model was established by intraperitoneal injection of streptozotocin in the experimental group. One week after injection, the fasting blood glucose level of mice was measured, and 12 diabetic mice were included in the final experimental group. All mice were fed for 12 weeks under the same laboratory conditions. The cardiac structure and function were detected by echocardiography. Diabetic mice with the left ventricular ejection fraction less than 60% and the E/A less than 1.6 were selected as DCM group (n=3). Mice in DCM group and control group were then sacrificed under deep anesthesia. RNA was extracted from myocardial tissue. High-throughput RNA sequencing technology was used to sequence and identify the RNA in the myocardial tissue of DCM group and normal control group, and the difference was analyzed by DeSeq2. The analysis results were verified at the tissue level by RT-qPCR, and the differential circRNA were analyzed by GO and KEGG pathway analysis. The differentially expressed circRNA-microRNA(miRNA) interaction was predicted by the miRNA target gene prediction software. Results: A total of 63 differentially expressed circRNAs were found in the myocardium of DCM mice. The results of RT-qPCR showed that the tissue level expression of 8 differentially expressed circRNAs was consistent with the sequencing results, of which 7 were up-regulated and 1 was down-regulated. KEGG pathway analysis showed that the up-regulated circRNAs was mainly related to AMPK signal pathway and intercellular adhesion junction pathway, and the down-regulated circRNA was mainly related to cardiomyopathy. Go analysis showed that the up-regulated circRNA was mainly related to the binding process of ions, proteins, kinases and other factors in terms of molecular function, and was involved in regulating the intracellular structure, especially the composition of organelles in terms of cell components. The functional analysis of molecular function and cell components showed that the up-regulated circRNA were related to the cell component origin, recruitment and tissue, and thus participated in the regulation of cell biological process. The down regulated circRNA was related to catalytic activity in terms of molecular function, protein kinase binding process, transferase and calmodulin activity, and was closely related to the components of contractile fibers and the composition of myofibrils. These differentially expressed circRNAs were also related to biological processes such as lysine peptide modification, sarcomere composition, myofibril assembly, morphological development of myocardial tissue, myocardial hypertrophy and so on. Conclusions: In this study, we detected the novel differentially expressed circRNAs in the myocardium of DCM mice, and bioinformatics analysis confirmed that these circRNAs are related to oxidative stress, fibrosis and death of cardiomyocytes, and finally participate in the pathophysiological process of DCM.

Identification of metabolic pathways underlying FGF1 and CHIR99021-mediated cardioprotection

SummaryAcute myocardial infarction is a leading cause of death worldwide. We have previously identified two cardioprotective molecules — FGF1 and CHIR99021— that confer cardioprotection in mouse and pig models of acute myocardial infarction. Here, we aimed to determine if improved myocardial metabolism contributes to this cardioprotection. Nanofibers loaded with FGF1 and CHIR99021 were intramyocardially injected to ischemic myocardium of adult mice immediately following surgically induced myocardial infarction. Animals were euthanized 3 and 7 days later. Our data suggested that FGF1/CHIR99021 nanofibers enhanced the heart’s capacity to utilize glycolysis as an energy source and reduced the accumulation of branched-chain amino acids in ischemic myocardium. The impact of FGF1/CHIR99021 on metabolism was more obvious in the first three days post myocardial infarction. Taken together, these findings suggest that FGF1/CHIR99021 protects the heart against ischemic injury via improving myocardial metabolism which may be exploited for treatment of acute myocardial infarction in humans.

Resveratrol mediates the miR-149/HMGB1 axis and regulates the ferroptosis pathway to protect myocardium in endotoxemia mice.

Endotoxemia is a common complication often used to model the acute inflammatory response associated with endotoxemia. Resveratrol has been shown to exert a wide range of therapeutic effects due to its anti-inflammatory and antioxidant properties. This study explored the effect of resveratrol on endotoxemia. Lipopolysaccharide (LPS)-induced endotoxemia mouse model and endotoxemia myocardial injury cell model were established and treated with resveratrol. Cardiomyocyte activity, lactate dehydrogenase (LDH) content in cell supernatant, glutathione (GSH) consumption, lipid reactive oxygen species (ROS) production, and iron accumulation were detected. Cardiac function indexes [left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), ejection fraction (EF)%, and fractional shortening (FS)%] were measured using echocardiography. The creatine kinase muscle/brain isoenzyme (CK-MB) and CK levels in the serum were detected using an automatic biochemical analyzer. The downstream target of miR-149 was predicted, and the binding relationship between miR-149 and high mobility group box 1 (HMGB1) was verified using a dual-luciferase assay. miR-149 and HMGB1 expressions were detected using RT-qPCR and Western blot. After resveratrol treatment, cardiomyocyte viability and GSH were increased, and LDH secretion, lipid ROS production, lipid peroxidation, and iron accumulation were decreased, and cardiac function and cardiomyocyte injury were improved. Resveratrol improved LPS-induced endotoxemia cardiomyocyte injury by upregulating miR-149 and inhibiting ferroptosis. Resveratrol inhibited HMGB1 expression by upregulating miR-149. HMGB1 upregulation reversed the inhibitory effect of miR-149 on LPS-induced ferroptosis in cardiomyocytes. Resveratrol upregulated miR-149 and downregulated HMGB1 to inhibit ferroptosis and improve myocardial injury in mice with LPS-induced endotoxemia. Collectively, resveratrol upregulated miR-149, downregulated HMGB1, and inhibited the ferroptosis pathway, thus improving cardiomyocyte injury in LPS-induced endotoxemia.NEW & NOTEWORTHY Sepsis is an unusual systemic reaction. Resveratrol is involved in sepsis treatment. This study explored the mechanism of resveratrol in sepsis by regulating the miR-149/HMGB1 axis.

Stress relaxation behavior of left ventricular myocardium in mice

BackgroundMyocardial tissues are known to exhibit viscoelastic behavior, i.e., the myocardial wall stress is gradually decaying when stretched at a constant level, referred to as stress relaxation behavior. The study of this behavior becomes important in structural heart diseases, where both passive and active relaxation behaviors of the myocardium may alter. As myocardial tissues display a complex, 3D micro‐architecture, the anisotropy of stress relaxation remains understudied. Our objective in this study was to investigate the anisotropy of (passive) stress relaxation behavior of the left ventricular (LV) myocardium in healthy small animals.MethodsWe used biaxial stress relaxation tests in wild‐type murine specimens (n=2). Full‐thickness myocardium LV free wall (LVFW) specimens were isolated from the mouse hearts (Fig. 1a), with the slab edges being aligned with the longitudinal (apex‐to‐outflow‐tract), circumferential, and radial directions of the LV. The specimens were mounted in a biaxial mechanical testing machine along the circumferential and longitudinal directions. The specimens were stretched in three different loading protocols with circumferential‐to‐longitudinal strain ratios of 30:30, 15:30, and 30:15%. The samples were loaded to the peak strain at a rate of 0.01 s‐1 and held at this stretch for 10 minutes to allow the relaxation. The time‐varying stress was recorded. We used an exponential decay fit to estimate the stress relaxation time constants in both directions.ResultsStress relaxation time constants showed noticeable differences between multiple loading ratios with equibiaxial loading (30:30) having the largest relaxation time constant followed by the circumferential stress in the 30:15 ratio (Fig. 1b). The larger strains led to larger stress relaxation constants (Fig. 1b‐c) indicating a strain‐dependent viscoelastic behavior. The quasi‐static equibiaxial loading of the same specimens resulted in a nearly isotropic behavior (not shown here), exhibiting insignificant contrast between the stresses in the circumferential and longitudinal directions. Interestingly, the contrast in the relaxation behavior between the circumferential and longitudinal directions were similarly insignificant (Fig. 1b). Non‐equibiaxial loadings (15:30 and 13:50) showed significantly faster relaxation for the direction with smaller strains (Fig. 1b).ConclusionsMyocardial tissue viscoelasticity is an important behavior contributing to both diastolic and systolic cardiac function, which remains understudied, especially in case of structural heart diseases associated with impaired relaxation in the LV. Our study suggests that stress relaxation behavior is prominent in the LV myocardium and may exhibit different relaxation behavior depending on strain magnitudes and strain ratios along different directions. The level of anisotropy in stress relaxation was found to be similar to the level of anisotropy in the tissue quasi‐static behavior. These results serve to motivate further studies to fully characterize the viscoelastic behavior of LV myocardium and its contribution to the LV relaxation and filling in murine models of LV diseases.

BS-514-02 TRANSIENT BIOLOGICAL PACING BASED ON AAV6-HCN2/SKM1 GENE TRANSFER IN PIGS WITH COMPLETE HEART BLOCK

In an effort to develop hardware-free pacemakers, we and others have engaged into the development of gene therapy-based biological pacemakers by means of ion channel overexpression. Here we report our progress with regard to adeno associated virus (AAV) vectors-mediated delivery of HCN2/SkM1 to generate long-term biological pacing. To study the long-term efficacy of biological pacing based on AAV-mediated gene transfer of HCN2/SkM1. We tested in vitro AAV-mediated overexpression of HCN2 and SkM1 on transduced neonatal rat ventricular myocytes (NRVMs). In vivo gene transfer was further validated after injecting 1E11 GC of AAV6-cTNT-HCN2 or AAV6-CMV-SkM1 into the left ventricle free wall of adult mice. Functional biological pacemaker studies were performed in pigs with radiofrequency ablation-induced complete AV block (CAVB). Four weeks after the ablation, we proceeded with three groups: non-injected, saline, and AAV6-HCN2/SkM1 (5E12 GC per vector). All animals were then observed for another four weeks to evaluate in vivo biological pacemaker performance. AAV6-HCN2 and AAV6-SkM1 showed both robust expression in vitro (NRVMs) and in vivo (mice) (Figure 1B and 1C). We thus proceeded with in vivo testing in CAVB pigs. Within the first two weeks of gene delivery, maximal beating rate increased in the HCN2/SkM1-transduced animals (Figure 2B) and mean heart rate modestly trended up after one week after injection, although this trend was not sustained (Figure 2A). This increase in beating rates was accompanied by an abrupt reduction of electronically paced beats in HCN2/SkM1 animals showing dependency on backup pacing of less than 50% - one week after of transgene delivery (Figure 2C). Finally, we evaluated corrected ventricular recovery times after overdrive suppression pacing which indicated enhanced intrinsic pacemaker activity in HCN2/SkM1-injected animals (Figure 2D). AAV-mediated gene transfer results in robust long-term overexpression of HCN2 and SKM1 in mouse myocardium. AAV-HCN2/SkM1 gene transfer in CAVB pigs resulted in transient biological pacemaker activity, potentially stemming from insufficient in vivo transduction and/or immunological interference.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Characterization of Human Cardiac Progenitor Cell Secretome

Heart Disease (HD) remains the greatest cause of death worldwide. Due to the heart’s poor capacity to regenerate after damage, there is a critical need for treatments that can restore cardiac cell integrity and function in HD patients. Preclinical and clinical evidence has highlighted the potential role of Cardiac Progenitor Cells (CPCs) in regulation of cardiac repair after injury, with an emerging role of CPC secreted proteins in this process. We have been studying CPCs isolated from human left ventricular myocardium characterized by their high proliferative capacity in vitro. These cells (human highly proliferative clones, hHiPC) are characterized by high expression of CD105/Endoglin and show varying potential for differentiating into endothelial cells in vitro. When injected into the mouse myocardium after surgically induced myocardial infarction (MI), individual hHiPC clones show variable effects on heart remodeling. For instance, when clone 11 was injected into the infarct zone post‐MI we saw a trend towards improvement in the fractional shortening (%) compared to clone 22 and saline injected controls. We hypothesized that hHiPCs associated with improvement in cardiac remodeling after MI will have a secretome characterized by higher expression of pro‐angiogenic and pro‐proliferative proteins, and these are regulated by the BMP/CD105 pathway. An unbiased and global proteomics approach (LC‐MS/MS) was used in our lab to identify proteins secreted from hHiPCs pre‐treated with the CD105 ligand BMP9 (5 ng/mL) compared to non‐treated controls. Our secretome data identified ~700 proteins in which 57% were characterized from UniProt as having a signal peptide sequence. Several pro‐angiogenic secreted proteins were identified in clone 11 including CCN2, CXCL6, and TGFB2. We found a >2.5‐fold significant increase in the secretion of Sclerostin (SOST) in BMP9 treated hHiPC compared to non‐treated controls. SOST is a known BMP‐target protein and WNT inhibitor. Previous evidence has highlighted the role of WNT‐signaling in heart remodeling after myocardial induced injury, but the role of Sclerostin in this process has yet to be elucidated. To further characterize the relationship between BMP9 and the secretion of SOST we performed a Real‐Time q‐PCR and ELISA assay of cell lysates and conditioned media between BMP9 treated and non‐treated controls and found a dramatic increase in the expression of SOST in BMP9 treated clone 11. We also found an increase (4.5‐fold) in CD105 mRNA expression after BMP9 treatment. Similarly, using LC‐MS/MS we found a significant increase in CD105 expression (2.5‐fold, p‐value = 7.39x10‐6) in the membrane fraction of clone 11 compared to clone 22 indicating its potential role in positive heart remodeling effects. Future studies will be conducted in vivo to determine the role SOST/WNT signaling has in heart remodeling after ischemic injury. Further, the increase in pro‐angiogenic factors after BMP9 treatment may suggest the potential relevance of BMP9 treatment in cardiac progenitor cell secretome mediated repair and we will test this hypothesis in the future.

Mice Deficient in Endothelial Cell‐Selective Adhesion Molecule Develop Vascular Endothelial and Left Ventricle Diastolic Dysfunction

The Endothelial Cell‐Selective Adhesion Molecule (ESAM) mediates inflammatory cell transmigration and also involved in pathological angiogenesis. Here we investigated the role of ESAM in affecting vasodilator and cardiac contractile function. To that end ESAM knockout and wild type (WT) mice were employed. Using transthoracic echocardiography it was found that ESAM knockout mice displayed left ventricle (LV) diastolic dysfunction, as indicated by a significantly reduced E/A ratio (E=early, A=late mitral inflow peak velocities), increased E/e’ ratio, isovolumic relaxation time (IVRT) and E wave deceleration time, with no change in the ejection fraction. The systolic blood pressure, as measured by tail‐cuff plethysmography, was not different between ESAM knockout and WT mice, whereas the heart to body weight ratio is increased in ESAM knockout mice. Using an unbiased automated tracing of the microvasculature we found a decreased vascularization (total vascular length) of the myocardium in ESAM knockout mice. In addition, vasodilator function of the pulmonary artery and aorta was assessed using wire myography. We found that the endothelium‐dependent, acetylcholine‐induced relaxation of the pulmonary artery was significantly reduced in ESAM knockout mice, whereas endothelium‐independent, sodium nitroprusside‐induced relaxation was similar in two groups. Thus, we conclude that ESAM deficiency causes vasodilator dysfunction, impaired angiogenesis underlying the development LV diastolic dysfunction.

SIRT1‐mediated Lysine Crotonylation is Involved in the Regulation of CaMKII Activity in Myocardium

ObjectivesPost‐translational modifications (PTMs) can modify proteins and regulate their functions. Lysine crotonylation is a novel protein PTM that can be regulated by silent mating type information regulation 2 homolog 1 (SIRT1). Ca2+/calmodulin‐dependent protein kinase II (CaMKII) plays a significant role in mediating cellular responses to external signaling molecules. Little is known regarding the effect of SIRT1 on CaMKII crotonylation and the impact of crotonylation on the activity of this enzyme.MethodsCre‐Loxp technology was used to generate conditional cardiac‐specific SIRT1 knockout mice (SIRT1 KO). Protein crotonylation modification was studied by PTMomics. CaMKII activity was determined by ELISA. Western blot and co‐immunoprecipitation (CO‐IP) were performed to examine the expression and crotonylation of CaMKIIResultsPTMomics showed changes in protein crotonylation profiles in the myocardium of cardiac‐specific SIRT1 knockout mice. Crotonylation of CaMKII was significantly increased at lysine 301 and the enhanced CaMKII crotonylation was further confirmed by CO‐IP (2.37±0.25 in SIRT1 KO vs. 0.96±0.10 in wild‐type and 1.04±0.10 in SIRT1fl/fl mice, p<0.01). Cardiac‐specific knockout of SIRT1 did not affect the protein expression of CaMKII whereas significantly increased CaMKII activity (2.75±0.17 m units/μL in SIRT1 KO vs. 2.10±0.13 m units/μL in wild‐type and 1.98±0.17 m units/μL in SIRT1fl/fl mice, p<0.05). CO‐IP showed no physical interaction between SIRT1 and CaMKII.ConclusionsSIRT1 deficiency increases CaMKII activity in myocardium at least partially through enhancing lysine crotonylation of CaMKII, which advances our understanding of the role played by PTM in the regulation of CaMKII.