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Human cardiac progenitor cell activation and regeneration mechanisms: exploring a novel myocardial ischemia/reperfusion in vitro model.

ABSTRACT: BACKGROUND:Numerous studies from different labs around the world report human cardiac progenitor cells (hCPCs) as having a role in myocardial repair upon ischemia/reperfusion (I/R) injury, mainly through auto/paracrine signaling. Even though these cell populations are already being investigated in cell transplantation-based clinical trials, the mechanisms underlying their response are still poorly understood. METHODS:To further investigate hCPC regenerative process, we established the first in vitro human heterotypic model of myocardial I/R injury using hCPCs and human-induced pluripotent cell-derived cardiomyocytes (hiPSC-CMs). The co-culture model was established using transwell inserts and evaluated in both ischemia and reperfusion phases regarding secretion of key cytokines, hiPSC-CM viability, and hCPC proliferation. hCPC proteome in response to I/R was further characterized using advanced liquid chromatography mass spectrometry tools. RESULTS:This model recapitulates hallmarks of I/R, namely hiPSC-CM death upon insult, protective effect of hCPCs on hiPSC-CM viability (37.6% higher vs hiPSC-CM mono-culture), and hCPC proliferation (approximately threefold increase vs hCPCs mono-culture), emphasizing the importance of paracrine communication between these two populations. In particular, in co-culture supernatant upon injury, we report higher angiogenic functionality as well as a significant increase in the CXCL6 secretion rate, suggesting an important role of this chemokine in myocardial regeneration. hCPC whole proteome analysis allowed us to propose new pathways in the hCPC-mediated regenerative process, including cell cycle regulation, proliferation through EGF signaling, and reactive oxygen species detoxification. CONCLUSION:This work contributes with new insights into hCPC biology in response to I/R, and the model established constitutes an important tool to study the molecular mechanisms involved in the myocardial regenerative process.

SUBMITTER: Sebastiao MJ

PROVIDER: S-EPMC6407246 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Human cardiac progenitor cell activation and regeneration mechanisms: exploring a novel myocardial ischemia/reperfusion in vitro model.

Sebastião Maria J MJ Serra Margarida M Pereira Rute R Palacios Itziar I Gomes-Alves Patrícia P Alves Paula M PM

Stem cell research & therapy 20190307 1

<h4>Background</h4>Numerous studies from different labs around the world report human cardiac progenitor cells (hCPCs) as having a role in myocardial repair upon ischemia/reperfusion (I/R) injury, mainly through auto/paracrine signaling. Even though these cell populations are already being investigated in cell transplantation-based clinical trials, the mechanisms underlying their response are still poorly understood.<h4>Methods</h4>To further investigate hCPC regenerative process, we established ...[more]

PMID: 30845956

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Project description:BackgroundMyocardial ischemia-reperfusion injury (MIRI) is the most common cause of death worldwide. The NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome plays an important role in the inflammatory response to MIRI. Dexmedetomidine (DEX), a specific agonist of α2-adrenergic receptor, is commonly used for sedation and analgesia in anesthesia and critically ill patients. Several studies have shown that dexmedetomidine has a strong anti-inflammatory effect in many diseases. Here, we investigated whether dexmedetomidine protects against MIRI by inhibiting the activation of the NLRP3 inflammasome in vitro.MethodsWe established an MIRI model in cardiomyocytes (CMs) alone and in coculture with cardiac fibroblasts (CFs) by hypoxia/reoxygenation (H/R) in vitro. The cells were treated with dexmedetomidine with or without MCC950 (a potent selective NLRP3 inhibitor). The beating rate and cell viability of cardiomyocytes, NLRP3 localization, the expression of inflammatory cytokines and NLRP3 inflammasome-related proteins, and the expression of apoptosis-related proteins, including Bcl2 and BAX, were determined.ResultsDexmedetomidine treatment increased the beating rates and viability of cardiomyocytes cocultured with cardiac fibroblasts. The expression of the NLRP3 protein was significantly upregulated in cardiac fibroblasts but not in cardiomyocytes after H/R and was significantly attenuated by dexmedetomidine treatment. Expression of the inflammatory cytokines IL-1β, IL-18 and TNF-α was significantly increased in cardiac fibroblasts after H/R and was attenuated by dexmedetomidine treatment. NLRP3 inflammasome activation induced the increased expression of cleaved caspase1, mature IL-1β and IL-18, while dexmedetomidine suppressed H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts. In addition, dexmedetomidine reduced the expression of Bcl2 and BAX in cocultured cardiomyocytes by suppressing H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts.ConclusionDexmedetomidine treatment can suppress H/R-induced NLRP3 inflammasome activation in cardiac fibroblasts, thereby alleviating MIRI by inhibiting the inflammatory response.

Project description:BackgroundMuscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration.MethodsA clamping model of murine (C57b/6 J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 h (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1 × 106 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration.Results2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI.ConclusionWIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.

Project description:IntroductionPrevious studies reported the beneficial effects of pretreatment with melatonin on the heart during cardiac ischemia/reperfusion (I/R) injury. However, the effects of melatonin given after cardiac ischemia, as well as its comparative temporal effects are unknown. These include pretreatment, during ischemia, and at the onset of reperfusion. Also, the association between melatonin receptors and cardiac arrhythmias, mitochondrial function and dynamics, autophagy, and mitophagy during cardiac I/R have not been investigated.ObjectivesWe tested two major hypotheses in this study. Firstly, the temporal effect of melatonin administration exerts different cardioprotective efficacy during cardiac I/R. Secondly, melatonin provides cardioprotective effects via MT2 activation, leading to improvement in cardiac mitochondrial function and dynamics, reduced excessive mitophagy and autophagy, and decreased cardiac arrhythmias, resulting in improved LV function.MethodsMale rats were subjected to cardiac I/R, and divided into 4 intervention groups: vehicle, pretreatment with melatonin, melatonin given during ischemia, and melatonin given at the onset of reperfusion. In addition, either a non-specific melatonin receptor (MT) blocker or specific MT2 blocker was given to rats.ResultsTreatment with melatonin at all time points alleviated cardiac I/R injury to a similar extent, quantified by reduction in infarct size, arrhythmia score, LV dysfunction, cardiac mitochondrial dysfunction, imbalance of mitochondrial dynamics, excessive mitophagy, and a decreased Bax/Bcl2 ratio. In H9C2 cells, melatonin increased %cell viability by reducing mitochondrial dynamic imbalance and a decrease in Bax protein expression. The cardioprotective effects of melatonin were dependent on MT2 activation.ConclusionMelatonin given before or after ischemia exerted equal levels of cardioprotection on the heart with I/R injury, and its beneficial effects on cardiac arrhythmias, cardiac mitochondrial function and dynamics were dependent upon the activation of MT2.

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Human cardiac progenitor cell activation and regeneration mechanisms: exploring a novel myocardial ischemia/reperfusion in vitro model.

Publications

Human cardiac progenitor cell activation and regeneration mechanisms: exploring a novel myocardial ischemia/reperfusion in vitro model.

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