isolation-culture-adult-mouse-cardiomyocytes-for-cell-signaling-vitro

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and <em>in vitro</em> Cardiac Hypertrophy

Technological advances have made genetically modified mice, including transgenic and gene knockout mice, an essential tool in many research fields. Adult ...

University of Toledo College of Medicine and Life Sciences

We have shown that increased production of reactive oxygen species (ROS) was required for ouabain-induced hypertrophy in cultured cardiac myocytes. In the present study we assessed whether long-term exposure of myocytes to nontoxic ROS stress alone is sufficient to induce hypertrophy. A moderate amount of H2O2 was continuously generated in culture media by glucose oxidase. This resulted in a steady increase in intracellular ROS in cultured cardiac myocytes for at least 12 h. Such sustained, but not transient, increase in intracellular ROS at a level comparable to that induced by ouabain was sufficient to stimulate protein synthesis, increase cell size, and change the expression of several hypertrophic marker genes. Like ouabain, glucose oxidase increased intracellular Ca2+ and activated extracellular signal-regulated kinases 1 and 2 (ERK1/2). These effects of glucose oxidase were additive to ouabain-induced cellular changes. Furthermore, glucose oxidase stimulated endocytosis of the plasma membrane Na+/K+-ATPase, resulting in significant inhibition of sodium pump activity. While inhibition of ERK1/2 abolished glucose oxidase-induced increases in protein synthesis, chelating intracellular Ca2+ by BAPTA-AM showed no effect. These results, taken together with our prior observations, suggest that ROS may cross talk with Na+/K+-ATPase, leading to the activation of hypertrophic pathways in cardiac myocytes.

Involvement of Na+/K+-ATPase in hydrogen peroxide-induced hypertrophy in cardiac myocytes.

Our previous studies on cardiac myocytes showed that positive inotropic concentrations of the digitalis drug ouabain activated signaling pathways linked to Na(+)-K(+)-ATPase through Src and epidermal growth factor receptor (EGFR) and led to myocyte hypertrophy. In view of the known involvement of phosphatidylinositol 3-kinase (PI3K)-Akt pathways in cardiac hypertrophy, the aim of the present study was to determine whether these pathways are also linked to cardiac Na(+)-K(+)-ATPase and, if so, to assess their role in ouabain-induced myocyte growth. In a dose- and time-dependent manner, ouabain activated Akt and phosphorylation of its substrates mammalian target of rapamycin and glycogen synthase kinase in neonatal rat cardiac myocytes. Akt activation by ouabain was sensitive to PI3K inhibitors and was also noted in adult myocytes and isolated hearts. Ouabain caused a transient increase of phosphatidylinositol 3,4,5-trisphosphate content of neonatal myocytes, activated class IA, but not class IB, PI3K, and increased coimmunoprecipitation of the alpha-subunit of Na(+)-K(+)-ATPase with the p85 subunit of class IA PI3K. Ouabain-induced activation of ERK1/2 was prevented by Src, EGFR, and MEK inhibitors, but not by PI3K inhibitors. Activation of Akt by ouabain, however, was sensitive to inhibitors of PI3K and Src, but not to inhibitors of EGFR and MEK. Similarly, ouabain-induced myocyte hypertrophy was prevented by PI3K and Src inhibitors, but not by an EGFR inhibitor. These findings 1) establish the linkage of the class IA PI3K-Akt pathway to Na(+)-K(+)-ATPase and the essential role of this linkage to ouabain-induced myocyte hypertrophy and 2) suggest cross talk between these PI3K-Akt pathways and the signaling cascades previously identified to be associated with cardiac Na(+)-K(+)-ATPase.

Association of PI3K-Akt signaling pathway with digitalis-induced hypertrophy of cardiac myocytes.

We have previously shown that the Na/K-ATPase binds and inhibits Src. Here, we report the molecular mechanism of Na/K-ATPase-mediated Src regulation and the generation of a novel peptide Src inhibitor that targets the Na/K-ATPase/Src receptor complex and antagonizes ouabain-induced protein kinase cascades. First, the Na/K-ATPase inhibits Src kinase through the N terminus of the nucleotide-binding domain of the alpha1 subunit. Second, detailed mapping leads to the identification of a 20-amino acid peptide (NaKtide) that inhibits Src (IC50 = 70 nm) in an ATP concentration-independent manner. Moreover, NaKtide does not directly affect the ERK and protein kinase C family of kinases. It inhibits Lyn with a much lower potency (IC50 = 2.5 microm). Third, highly positively charged leader peptide conjugates including HIV-Tat-NaKtide (pNaKtide) readily enter cultured cells. Finally, the following functional studies of pNaKtide demonstrate that this conjugate can specifically target the Na/K-ATPase-interacting pool of Src and act as a potent ouabain antagonist in cultured cells: 1) pNaKtide, unlike PP2, resides in the membranes. Consistently, it affects the basal Src activity much less than that of PP2. 2) pNaKtide is effective in disrupting the formation of the Na/K-ATPase/Src receptor complex in a dose-dependent manner. Consequently, it blocks ouabain-induced activation of Src, ERK, and hypertrophic growth in cardiac myocytes. 3) Unlike PP2, pNaKtide does not affect IGF-induced ERK activation in cardiac myocytes. Taken together, we suggest that pNaKtide may be used as a novel antagonist of ouabain for probing the physiological and pathological significance of the newly appreciated signaling function of Na/K-ATPase and cardiotonic steroids.

NaKtide, a Na/K-ATPase-derived peptide Src inhibitor, antagonizes ouabain-activated signal transduction in cultured cells.

To explore the haemostatic effects of microbubble-enhanced ultrasound (MEUS) at a very low acoustic intensity on the bleeding liver of rabbits.

Liver haemostasis using microbubble-enhanced ultrasound at a low acoustic intensity.

A six-fold increase in pediatric MRSA infections, prompted us to examine the clinical profile of children with MRSA infections seen at Mercy Children's Hospital, Toledo, Ohio and to characterize the responsible strains.

Higher incidence of perineal community acquired MRSA infections among toddlers.

To construct a population pharmacokinetic (popPK) model for ketamine (Ket), norketamine (norKet), dehydronorketamine (DHNK), hydroxynorketamine (2S,6S;2R,6R)-HNK) and hydroxyketamine (HK) in patients with treatment-resistant bipolar depression.

Simultaneous population pharmacokinetic modelling of ketamine and three major metabolites in patients with treatment-resistant bipolar depression.

Ethanol ablation (EA) is a safe and effective method for treating small liver cancer. However, the ethanol is rapidly washed out by blood perfusion, preventing its accumulation within tumors. Microbubble-enhanced ultrasound (MEUS) is capable of disrupting tumor and liver circulation. We hypothesized that this disruption could be used to enhance EA of normal liver tissue. We treated surgically exposed rabbit liver with a combination of MEUS and EA. The controls were treated with only MEUS or 0.05 mL EA. MEUS treatment was administered with a high-pressure-amplitude, pulsed therapeutic ultrasound device and intra-venous injection of microbubbles. Therapeutic ultrasound was delivered at an acoustic pressure of 4.3 MPa and a duty cycle of 0.22%. Contrast-enhanced ultrasound was performed to estimate liver blood perfusion. Livers were harvested for necrotic volume measurements 48 h after treatment. Contrast-enhanced ultrasound demonstrated that liver perfusion was temporally arrested, with a significant peak intensity decline from -46.9 ± 3.8 to -64.0 ± 3.3 dB, after MEUS treatment. The mean volume ablated in MEUS + EA-treated livers (3.3 ± 2.3 cm(3)) was more than 10 times larger than that in livers treated only with EA (0.3 ± 0.2 cm(3)). The volume of liver ablated by MEUS treatment alone was minor, scattered and immeasurable. These results indicate that MEUS disruption of the liver circulation can greatly promote EA of liver.

Impact of microbubble-enhanced ultrasound on liver ethanol ablation.

Graphene oxide (GO-ene), the two-dimensional carbon lattice decorated by abundant oxygen functionalities, is demonstrated as an efficient green catalyst towards selective hydrolysis of cellulose to glucose. The synergy of its carboxylic/phenolic groups and its layered, soft structure rendered GO-ene superior hydrolytic activity.

Graphene oxide for cellulose hydrolysis: how it works as a highly active catalyst?

Microbubble-enhanced therapeutic ultrasound (MEUS) can block the blood flow in the organs. The aim of this study was to evaluate the hemostatic effect of microbubble-enhanced pulsed, low-intensity ultrasound in a New Zealand White rabbit model of avulsion trauma of the liver. The therapeutic ultrasound (TUS) transducer was operated with the frequency of 1.2 MHz and an acoustic pressure of 3.4 MPa. Microbubble-(MB) enhanced ultrasound (MEUS) (n = 6) was delivered to the distal part of the liver where the avulsion was created. Livers were treated by TUS only (n = 4) or MB only (n = 4) which served as controls. Bleeding rates were measured and contrast enhanced ultrasound (CEUS) was performed to assess the hemostatic effect, and liver hemoperfusion before and after treatment. Generally, bleeding rates decreased more than 10-fold after the treatment with MEUS compared with those of the control group (P<0.05). CEUS showed significant declines in perfusion. The peak intensity value and the area under the curve also decreased after insonation compared with those of the control group (P<0.05). Histological examination showed cloudy and swollen hepatocytes, dilated hepatic sinusoids, perisinusoidal spaces with erythrocyte accumulation in small blood vessels, obvious hemorrhage around portal areas and scattered necrosis in liver tissues within the insonation area of MEUS Group. In addition, necrosis was found in liver tissue 48 h after insonation. We conclude that MEUS might provide an effective hemostatic therapy for serious organ trauma such as liver avulsion injury.

Xiaochen Zhao

Department of Physiology and Pharmacology

University of Toledo College of Medicine and Life Sciences

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Xiaochen Zhao

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physiology and Pharmacology

University of Toledo College of Medicine and Life Sciences

Isolation and Culture of Adult Mouse Cardiomyocytes for Cell Signaling and in vitro Cardiac Hypertrophy

Department of Physiology and Pharmacology