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

Ouabain binding to Na(+)/K(+)-ATPase activates Src/epidermal growth factor receptor (EGFR) to initiate multiple signal pathways that regulate growth. In cardiac myocytes and the intact heart, the early ouabain-induced pathways that cause rapid activations of ERK1/2 also regulate intracellular Ca(2+) concentration ([Ca(2+)](i)) and contractility. The goal of this study was to explore the role of caveolae in these early signaling events. Subunits of Na(+)/K(+)-ATPase were detected by immunoblot analysis in caveolae isolated from cardiac myocytes, cardiac ventricles, kidney cell lines, and kidney outer medulla by established detergent-free procedures. Isolated rat cardiac caveolae contained Src, EGFR, ERK1/2, and 20-30% of cellular contents of alpha(1)- and alpha(2)-isoforms of Na(+)/K(+)-ATPase, along with nearly all of cellular caveolin-3. Immunofluorescence microscopy of adult cardiac myocytes showed the presence of caveolin-3 and alpha-isoforms in peripheral sarcolemma and T tubules and suggested their partial colocalization. Exposure of contracting isolated rat hearts to a positive inotropic dose of ouabain and analysis of isolated cardiac caveolae showed that ouabain caused 1) no change in total caveolar ERK1/2, but a two- to threefold increase in caveolar phosphorylated/activated ERK1/2; 2) no change in caveolar alpha(1)-isoform and caveolin-3; and 3) 50-60% increases in caveolar Src and alpha(2)-isoform. These findings, in conjunction with previous observations, show that components of the pathways that link Na(+)/K(+)-ATPase to ERK1/2 and [Ca(2+)](i) are organized within cardiac caveolae microdomains. They also suggest that ouabain-induced recruitments of Src and alpha(2)-isoform to caveolae are involved in the manifestation of the positive inotropic effect of ouabain.

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase.

[Preparation and component analysis of tea pigments].

In this study, the effects of tea catechins and tea theaflavins on myocardial contraction were examined in isolated rat hearts using a Langendorff-perfusion system. We found that both tea catechins and theaflavins had positive inotropic effects on the myocardium. Of the tested chemicals, epicatechin-3-gallate (ECG) and theaflavin-3,3'-digallate (TF(4)) appear to be the most effective tea catechin and theaflavin, respectively. Further studies of ECG-induced positive inotropy revealed the following insights. First, unlike digitalis drugs, ECG had no effect on intracellular Ca(2+) level in cultured adult cardiac myocytes. Second, it activated PKCepsilon, but not PKCalpha, in the isolated hearts as well as in cultured cells. Neither a phospholipase C (PLC) inhibitor (U73122) nor the antioxidant N-acetyl cysteine (NAC) affected the ECG-induced activation of PKCepsilon. Third, inhibition of PKCepsilon by either chelerythrine chloride (CHE) or PKCepsilon translocation inhibitor peptide (TIP) caused a partial reduction of ECG-induced increases in myocardial contraction. Moreover, NAC was also effective in reducing the effects of ECG on myocardial contraction. Finally, pretreatment of the heart with both CHE and NAC completely abolished ECG-induced inotropic effects on the heart. Together, these findings indicate that ECG can regulate myocardial contractility via a novel PKCepsilon-dependent signaling pathway.

Identification of a PKCepsilon-dependent regulation of myocardial contraction by epicatechin-3-gallate.

A new cerebroside, 1-O-beta-D-glucopyranosyl-(2S,3R,8E)-2-[(2'R)-2-hydroxy- palmitoylamino]-8-octadecene-1,3-diol, along with aralia cerebroside and 1-O-beta-D- glucopyranosyl-(2S,3S,4R,8E)-2-[(2'R)-2-hydroxybehenoylamino]-8-octadecene-1,3,4-triol were isolated from the roots of Serratula chinensis S. Moore. The structure of the new cerebroside was established by spectroscopic and chemical means. Occurrence of cerebrosides in Serratula is reported here for the first time.

Cerebrosides from the roots of Serratula chinensis.

In order to separate the main individual theaflavin monomers from black tea, high-speed countercurrent chromatography (HSCCC) and Sephadex LH-20 column chromatography were applied. The results showed that theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B) and theaflavin-3,3'-digallate (TF3) can be obtained by HSCCC using a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:3:1:6, v/v/v/v), but the TF1 was containing epicatechin-3-gallate (ECG). Similarly, Sephadex LH-20 can also effectively separate TF2A(B) and TF3, but epigallocatechin-3-gallate (EGCG) contaminated TF1, too. Combination of HSCCC and Sephadex LH-20, the preferably purified TF1, TF2A(B) and TF3 were obtained than single separation technique. In addition, ECG and EGCG were also suggested to be able to be comprehensively separated by combination of the two techniques.

Combination of HSCCC and Sephadex LH-20 methods An approach to isolation and purification of the main individual theaflavins from black tea.

High-performance liquid chromatography (HPLC) has been used to quantify levels of free amino acids, catechins, and caffeine in Chinese green tea. Levels of free amino acids and catechins in green tea leaves show obvious variation from spring to summer, which is useful information to identify the production season of commercial green tea. Supervised pattern recognition methods such as the K-nearest neighbor (KNN) method and Bayesian discriminant method (a type of linear discriminant analysis (LDA)) were used to discriminate between the production seasons of Chinese green tea. The optimal accuracy of the KNN method was ≤97.61 and ≤94.80% as validated by resubstitution and cross-validation tests, respectively, and that of LDA was ≤95.22 and ≤93.54%, respectively. Compared with LDA, the KNN method did not require a Gaussian distribution and was more accurate than LDA. The KNN method in combination with chemical analysis is recommended for discrimination of the production seasons of Chinese green tea.

Discrimination of the production season of Chinese green tea by chemical analysis in combination with supervised pattern recognition.

Natriuretic peptides and digitalis-like compounds serve as regulators of homeostasis, including control of volume expansion and blood pressure. The aim of the present study was to explore possible interactions between atrial natriuretic peptide (ANP) and ouabain in the heart. ANP (1 nmol/L) had no effect in papillary muscle preparations from guinea pigs. Ouabain (1 µmol/L) induced positive inotropic effect. The addition of ANP prior to ouabain resulted in a significant decrease in the ouabain-induced positive inotropic effect, manifested as an attenuated increase in twitch maximal upward force slope and resting muscular tension. In addition, ANP caused an increase in Na⁺-K⁺-ATPase activity in heart microsomal preparations. The effect of ouabain on Na⁺-K⁺-ATPase activity was shown in a biphasic manner. Ouabain (0.01-1 nmol/L) had a small but significant increase on pump activity, but higher doses of ouabain inhibited activity. ANP attenuated ouabain-induced Na⁺-K⁺-ATPase activity. Furthermore, ouabain (50 nmol/L) or ANP (10 nmol/L) alone induced Akt activation in cardiomyocytes. However, ANP blocked ouabain-induced Akt activation. These results point to the existence of interactions between ANP and ouabain on Na⁺-K⁺-ATPase signaling and function in the heart, which may be mediated by regulation of Na⁺-K⁺-ATPase activity and (or) signal transduction mechanisms.

Daxiang Li

Department of Biochemistry and Cancer Biology

University of Toledo College of Medicine and Life Sciences

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

Daxiang Li

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

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 Biochemistry and Cancer Biology