MMI-0100 Inhibits Cardiac Fibrosis In Myocardial Infarction By Direct Actions On Cardiomyocytes And Fibroblasts By Direct Actions On Cardiomyocytes And Fibroblasts Via MK2 Inhibition

Background and PurposeIschemic heart disease is the most common cause of death in the world; in the United States alone, an estimated 785,000 people will have a myocardial infarction (MI) each year, approximately 1 per minute. The adverse remodeling that occurs after MI contributes to the impaired function and heart failure that commonly develops post-MI. Interventional advances-largely early reperfusion therapies-have improved patient survival, but the adverse remodeling processes that lead to heart failure proceed unabated. The size of the infarcted area, the infarcted wound healing, and chronic left ventricular (LV) remodeling determine the extent of the resulting heart failure. To minimize the extent of heart failure after a large or recurrent MI, therapeutic strategies are needed to limit infarct wound healing in the early phase.Mitogen-activated protein kinases (MAPKs) pathway is a very important biological signal path in the eucryotic cell signaling networks, and it plays a vital role in the regulation of gene expression and the Function of the cytoplasm.5 different types of MAPK signaling pathway have been found so far. The ERK1/2 signalling pathways are in charge of regulating cell growth and differentiation, while JNK and p38 MAPK play an important part in inflammation and cell apoptosis. So far it is know that p38 MAPK is related to the cardiomyocyte death, heart failure after AMI, cardiac fibrosis and myocardial hypertrophy in the fetal process of myocardial infarction. Mitogen activated protein kinase activated protein kinase 2 (MAPKAPK2 or MK2), as an intracellular serine/threonine kinase substrate downstream of p38MAPK that has been implicated in many inflammatory diseases that are complicated by scarring and fibrosis, Recent studies in MK2-/-mice have illustrated that MK2 acts downstream of p38 MAPK and is responsible for p38 MAPK-induced heart failure. Besides, MK2-/-mice are also resistant to ischemia reperfusion injury, indicating a critical role of MK2 in ischemic heart disease experimentally.Recent studies have reported that the cell-permeant peptide MMI-0100 inhibits inflammation and fibrosis (intimal hyperplasia) in a mouse vein graft model, bleomycin-induced pulmonary fibrosis and abdominal adhesions post-surgery So, we decided to verify the hypothesis that the MK2 inhibitor, MMI-0100, protect the heart under myocardial infarction via inhibiting MK2 signaling pathway.Based on these recent findings, the present study tested the hypothesis that MMI-0100 therapy post-myocardial infarction would inhibit the extent of fibrosis in vivo. We demonstrated that MMI-0100 reduced fibrosis that developed after 2 weeks in a standard murine myocardial infarction model induced by permanent ligation of the left anterior descending (LAD) coronary artery. Since cardiomyocyte cell death, fibroblast differentiation to myofibroblasts and the secretion of a variety of extracellular matrix proteins, including collagen (resulting infibrosis) are impacted by MK2, we determined whether MMI-0100 confers cardioprotective benefits by acting on both cell types independently in vitro. We found that MMI-0100 inhibits MK2 activity in both cardiac-derived cells (H9C2 and HL-1)and in primary rat cardiac fibroblasts, inhibiting cardiomyocyte caspase 3/7 activity, while enhancing fibroblast caspase 3/7 activity in vitro. These studies report for the first time that the cell permeant peptide MMI-0100 can inhibit fibrosis associated with myocardial infarction, while illustrating mechanisms by which inhibition of MK2 in turn inhibits cardiomyocyte apoptosis and reduces fibrosis by direct effects on cardiac fibroblasts.Method1. Animal experiments:(1) Twelve week-old male C57BL/6 mice were obtained and myocardial infarction was induced by permanent ligation of the left anterior descending (LAD) coronary artery as described Previously. We divided the mice into 5 groups for the experiment:1. MI group:permanent ligation of the LAD without MMI-0100 injection; 2. MI+treatment group:permanent ligation of the LAD with MMI-0100 injection peritoneally (50μg/kg/day) for 14 days; 3. Sham operation group:a sham operation that included every step except the coronary artery ligation; 4. Normal control:No surgery or MMI-0100 injection; 5. Treatment control:No surgery but with MMI-0100 injection daily for 14 days. Durning the 2 weeks, cardiac functions of the mice (LVEDD; LVESD; LV Vol;d; LV Vol;s; FS; EF%) are recorded at baseline,7, and 14 days.(2)Immunohistochemical analysis of the Animal experiment specimens:Mice were cervical dislocated at day 14, hearts were fixed in fresh 4% paraformaldehyde for 24 hours, paraffinembedded, processed, and stained with standard hematoxylin and eosin (H&E) and Masson’s trichrome (MT) to determine the degree of fibrosis; Fluorescent stains of vimentin,a-SMA,TGF-β1 and TUNEL were done to the slides to determine the change of the cell constitution and the degree of cell apoptosis. 2. Cell experiments:(1)Cell culture of primary cardiac fibroblast cells and cardiomyocyte cell lines.Two types of cardiomyocyte, H9C2 and HL-1 cell line, and primary cardiac fibroblast cells are adopted and cultured, as instructed by the published protocols.(2) Induction of hypoxia,determination of experimental time points and cell death.Hypoxia was induced by placing cells in a hypoxia chamber in a mixture of 5% CO2/95% N2 to attain a 1% oxygen concentration. Three experimental groups were tested for each cell type:1) Final [0.5% DMSO]; 2) 20 μM MMI-0100 peptide [in a final 0.5% DMSO]; and 3) 100μM MMI-0100 peptide [in a final 0.5% DMSO] at 3 time points. At the time of performing the experiments, all cultures were approximate-1y 70-90% confluent.3 different time points were adopted for each of the 3 cell lines according to the severity of cell death under hypoxia determined by LDH release:for H9C2 cell line:8hr; 16hr; 24hr; for HL-1 cell line:4hr; 8hr; 12hr; for cardiac fibro-blast cell line:16hr; 32hr; 48hr.(3) Analysis of cell necrosis and apoptosis.Cell death was first determined using an LDH release assay kit; Caspase 3/7 activity was next determined using a commercial Caspase 3/7 activity kit to indicate the degree of cell apoptosis.(4) Western blots to determine the activity of MK2.3 indicators (hnRNPAO, MAPK2 and phosphor-MAPK2) are adopted to analyze the activity of MK2.(5) Analysis of cytokines (TNFa, IL-1β and IL-6)in the used media of cell culture.Quantitative analysis of Cytokines (TNFa, IL-1β and IL-6) are also done in the used media of cell cultures.Results1. Treatment of acute myocardial infarction with MMI-0100 peptide protects cardiac function and attenuates dilation in vivo.Our experiment proved that two weeks after the permanent ligation of the LAD(AMI), ejection fraction decreased 26% and fractional shortening depressed 37% compared to sham-operated control mice. Treatment of AMI with MMI-0100 reduced these loses in EF% and FS% to 12% and 20%, respectively. MMI-0100 similarly attenuated the increase in LV volume and LV end diastolic diameter (LVEDD) characteristic of AMI heart failure. While Treatment of mice with MMI-0100 alone did not have any affect on cardiac function or survival.2. Treatment of acute MI with MMI-0100 treatment decreases cardiac fibrosis by protecting cardiomyocytes and increasing the cardiac flbroblast apoptosis in vivo.(1) In the animal experiments, by Masson’s trichrome staining of the sections, we demonstrated that mice subjected to AMI that then received MMI-0100 exhibited 50% less fibrosis than mice subjected to AMI alone. When investigated at a higher magnification, islands of myocyte sparing within the ischemic region scar were found routinely in hearts where MMI-0100 was given in contrast to the complete fibrosis seen after AMI in all animals uniformly in multiple representative sections. The immunofluorescent staining of the sections shows that the treatment of MMI-0100 decreased the number of fibroblasts, increased the vessel density and decreased the number of TGF-β(+)cells after AMI.Given the mechanistic role of TGF-β in promoting fibrosis in non-infarcted myocardium, it is not surprising the MMI-0100’s anti-TGF- effect (via MK2 inhibition) reduced non-infarcted myocardial fibrosis, resulting in long-term protective effects on cardiac function.(2) In the cell experiments, H9C2 and HL-1 cell lines, which are all derived from cardiomyocytes, have an increase in the activity of caspase3/7 after the hypoxia treatment, but with the treatment of MMI-0100, caspase3/7, which indicates the degree of apoptosis in cells, is inhibited effectively; While for the experiment of cardiac fibroblasts derived from rat heart, MMI-0100 increased the acitivity of caspase3/7. Western blots with the cell lysates show that in H9C2 and HL-1, MMI-0100 inhibited the expression of hnRNPAO, while the expression of MK2 and p-MK2 doesn’t have any obvious change; But in fibroblast, MMI-0100 doesn’t inhibit the hnRNPAO, either do the MK2 or p-MK2.Based on these results, MMI-0100 inhibited the apoptosis of cardiomyocytes under hypoxia, while at the same time increased the death of fibroblasts, which might indicates the mechanism of how MMI-0100 works.ConclusionsTherapeutic inhibition of Mitogen Activated Protein Kinase Activated Protein Kinase II (MK2) activity using rationally designed cell-permeant peptides after acute MI inhibits cardiac fibrosis and retains cardiac function at 2 weeks, by mechanisms that involve inhibiting cardiomyocyte apoptosis, while enhancing primary cardiac fibroblast cell death.