isolation-transfection-long-term-culture-adult-mouse-rat

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...

University of Cincinnati

An innovative approach for cardiac regeneration following injury is to induce endogenous cardiomyocyte (CM) cell cycle re-entry. In the present study, CMs from adult rat hearts were isolated and transfected with cel-miR-67 (control) and rno-miR-210. A significant increase in CM proliferation and mono-nucleation were observed in miR-210 group, in addition to a reduction in CM size, multi-nucleation, and cell death. When compared to control, β-catenin and Bcl-2 were upregulated while APC (adenomatous polyposis coli), p16, and caspase-3 were downregulated in miR-210 group. In silico analysis predicted cell cycle inhibitor, APC, as a direct target of miR-210 in rodents. Moreover, compared to control, a significant increase in CM survival and proliferation were observed with siRNA-mediated inhibition of APC. Furthermore, miR-210 overexpressing C57BL/6 mice (210-TG) were used for short-term ischemia/reperfusion study, revealing smaller cell size, increased mono-nucleation, decreased multi-nucleation, and increased CM proliferation in 210-TG hearts in contrast to wild-type (NTG). Likewise, myocardial infarction (MI) was created in adult mice, echocardiography was performed, and the hearts were harvested for immunohistochemistry and molecular studies. Compared to NTG, 210-TG hearts showed a significant increase in CM proliferation, reduced apoptosis, upregulated angiogenesis, reduced infarct size, and overall improvement in cardiac function following MI. β-catenin, Bcl-2, and VEGF (vascular endothelial growth factor) were upregulated while APC, p16, and caspase-3 were downregulated in 210-TG hearts. Overall, constitutive overexpression of miR-210 rescues heart function following cardiac injury in adult mice via promoting CM proliferation, cell survival, and angiogenesis.

MicroRNA-210-mediated proliferation, survival, and angiogenesis promote cardiac repair post myocardial infarction in rodents.

The objective of this study was to develop an injectable and biocompatible hydrogel that can deliver a cocktail of therapeutic biomolecules (secretome) secreted by human adipose-derived stem cells (hASCs) to the peri-infarct myocardium. Gelatin and Laponite® were combined to formulate a shear-thinning, nanocomposite hydrogel (nSi Gel) as an injectable carrier of secretome (nSi Gel+). The growth factor composition and the pro-angiogenic activity of the secretome were tested in vitro by evaluating the proliferation, migration and tube formation of human umbilical endothelial cells. The therapeutic efficacy of the nSi Gel + system was then investigated in vivo in rats by intramyocardial injection into the peri-infarct region. Subsequently, the inflammatory response, angiogenesis, scar formation, and heart function were assessed. Biocompatibility of the developed nSi Gel was confirmed by quantitative PCR and immunohistochemical tests which showed no significant differences in the level of inflammatory genes, microRNAs, and cell marker expression compared to the untreated control group. In addition, the only group that showed a significant increase in capillary density, reduction in scar area and improved cardiac function was treated with the nSi Gel+. Our in vitro and in vivo findings demonstrate the potential of this new secretome-loaded hydrogel as an alternative strategy to treat myocardial infarction.

Stem cell-inspired secretome-rich injectable hydrogel to repair injured cardiac tissue.

Stem cell-based therapies represent a valid approach to restore cardiac function due to their beneficial effect in reducing scar area formation and promoting angiogenesis. However, their translation into the clinic is limited by the poor differentiation and inability to secrete sufficient therapeutic factors. To address this issue, several strategies such as genetic modification and biophysical preconditioning have been used to enhance the efficacy of stem cells for cardiac tissue repair.

Pre-Conditioning Stem Cells in a Biomimetic Environment for Enhanced Cardiac Tissue Repair:  and  Analysis.

Carbonic anhydrase (CAII) binds to the electrogenic basolateral Na-[Formula: see text] cotransporter (NBCe1) and facilitates [Formula: see text] reabsorption across the proximal tubule. However, whether the inhibition of CAII with acetazolamide (ACTZ) alters NBCe1 activity and interferes with the ammoniagenesis pathway remains elusive. To address this issue, we compared the renal adaptation of rats treated with ACTZ to NHCl loading for up to 2 wk. The results indicated that ACTZ-treated rats exhibited a sustained metabolic acidosis for up to 2 wk, whereas in NHCl-loaded rats, metabolic acidosis was corrected within 2 wk of treatment. [Formula: see text] excretion increased by 10-fold in NHCl-loaded rats but only slightly (1.7-fold) in ACTZ-treated rats during the first week despite a similar degree of acidosis. Immunoblot experiments showed that the protein abundance of glutaminase (4-fold), glutamate dehydrogenase (6-fold), and SN1 (8-fold) increased significantly in NHCl-loaded rats but remained unchanged in ACTZ-treated rats. Na/H exchanger 3 and NBCe1 proteins were upregulated in response to NHCl loading but not ACTZ treatment and were rather sharply downregulated after 2 wk of ACTZ treatment. ACTZ causes renal [Formula: see text] wasting and induces metabolic acidosis but inhibits the upregulation of glutamine transporter and ammoniagenic enzymes and thus suppresses ammonia synthesis and secretion in the proximal tubule, which prevented the correction of acidosis. This effect is likely mediated through the inhibition of the CA-NBCe1 metabolon complex, which results in cell alkalinization. During chronic ACTZ treatment, the downregulation of both NBCe1 and Na/H exchanger 3, along with the inhibition of ammoniagenesis and [Formula: see text] generation, contributes to the maintenance of metabolic acidosis.

Acetazolamide causes renal [Formula: see text] wasting but inhibits ammoniagenesis and prevents the correction of metabolic acidosis by the kidney.

Hypobaric hypoxia poses stress to sojourners traveling to high-altitude. A cascade of physiological changes occurs to cope with or adapt to hypobaric hypoxia. However, an insufficient physiological response to the hypoxic condition resulting from imbalanced vascular homeostasis pathways results in high-altitude pulmonary edema (HAPE). The present study aims to identify the implication of miRNAs associating with HAPE and adaptation. We analyzed the expression of 1,113 miRNAs in HAPE-patients (HAPE-p), HAPE-free controls (HAPE-f), and highland natives (HLs). Based on miRNA profiling and in silico analyses, miR-124-3p emerged relevantly. We observed a significant overexpression of miR-124-3p in HAPE-p. In silico analyses revealed a direct interaction of miR-124-3p with vascular homeostasis and hypoxia-associated genes  (endothelial nitric oxide synthase), , and  (V-Ets avian erythroblastosis virus E2 oncogene homolog 1). Moreover, the transcript and biolevel expression of these genes were significantly decreased in HAPE-p when compared with HAPE-f or HLs. Our in vitro analysis in human umbilical vein endothelial cells demonstrated a significant knockdown of these genes both at transcript and protein levels following miR-124-3p overexpression. Conclusively, our results showed that miR-124-3p might play a plausible role in HAPE pathophysiology by inhibiting the expression of , , and .

Perwez Alam

Department of Pathology and Laboratory Medicine,
College of Medicine,
Department of Pathology and Laboratory Medicine, College of Medicine

University of Cincinnati

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Perwez Alam

Department of Pathology and Laboratory Medicine,College of Medicine,Department of Pathology and Laboratory Medicine, College of Medicine

University of Cincinnati

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Department of Pathology and Laboratory Medicine,
College of Medicine,
Department of Pathology and Laboratory Medicine, College of Medicine