Morphology and Metabolism of Intact Muscle Cells Isolated from Adult Rat Heart

Berry, Michael N.; Friend, Daniel S.; Scheuer, James

doi:10.1161/01.res.26.6.679

Cited by 116 publications

(36 citation statements)

References 18 publications

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“…22 Isolation of myocardial cells from other cell types generally followed the procedure of Berry et al 28 Twenty hamster hearts were required to obtain a sufficient yield of isolated myocardial cells. Separation of myocardial cells from the hyaluronidase and collagenase was ensured by layering the myocardial cell suspension over 20 ml of 0.32 M sucrose in Kreb's calcium-free medium and centrifuging for 2 minutes at 50 g in the IEC-J-6 refrigerated centrifuge as described previously.…”

Section: Isolation Of Myocardial Cells From Cardiomyopathic Heartsmentioning

confidence: 99%

Nuclear proteins in the heart of the cardiomyopathic Syrian hamster. Fractionation of phenol-soluble nonhistone proteins by two-dimensional polyacrylamide gel electrophoresis.

Liew¹,

Sole²

1978

Circulation Research

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SUMMARY Nonhistone nuclear proteins (NHNP) were isolated from the hearts of hamsters in the myolytic, hypertrophic, and failing phases of cardiomyopathy and from paired controls. These proteins were solubilized in phenol and fractionated by polyacrylamide gel electrophoresis. One-dimensional gel electrophoresis, using either isoelectrofocusing or sodium dodecyl sulfate, showed quantitative differences between the dystrophic hearts and the controls. A high resolution of NHNP was achieved by two-dimensional gel electrophoresis, revealing both quantitative and limited qualitative differences between the two groups. Proteins focusing from pH 5.0 to 5.6 with molecular weights of 55,000 (P 5 ) and 100,000 (P,) were strikingly increased in the cardiomyopathy. A protein (P 2 ) with an isoelectric point of 5.1 and some NHNP from dystrophic hearts in the entire region from pH 7.0 to 9.0 with molecular weights ranging from 35,000 (P 7 ) to 68,000 (P 4 ) were markedly reduced or absent. Differences in NHNP could be detected during the myolytic phase of cardiomyopathy but were most striking during the terminal phase of the disease. There were no detectable differences between the profiles of proteins derived from whole heart homogenates of dystrophic hamsters and controls. There were no significant differences at the failing phase between NHNP isolated from a purified preparation of myocardial cells and those isolated from whole heart. Therefore, the differences in NHNP appear to be reflections of alterations in nuclear composition of the dystrophic myocardial cell. Some of these observations may represent changes secondary to heart disease. However, if NHNP interacting with DNA play a major role in genetic expression, some of the manifestations of hamster cardiomyopathy could be due to a different constitution of NHNP in the dystrophic heart.

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Section: Isolation Of Myocardial Cells From Cardiomyopathic Heartsmentioning

confidence: 99%

Nuclear proteins in the heart of the cardiomyopathic Syrian hamster. Fractionation of phenol-soluble nonhistone proteins by two-dimensional polyacrylamide gel electrophoresis.

Liew¹,

Sole²

1978

Circulation Research

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“…Our method 21 for isolating cells is a modification of that described by Berry et al 22 Briefly, this involves Langendorff perfusion of rat hearts at 37°C with a well oxygenated, nominally Ca 2+ -free and Mg 2+ -free buffer solution containing (ITIM): NaCl, 134; KC1, 5; sodium phosphate (pH 7.4), 6; and glucose, 10. After about 5 minutes, collagenase (80 U/ml) and hyaluronidase (82 U/ml) were added to the perfusion fluid and the perfusion was continued at 37°C for an additional 20 minutes.…”

Section: Isolation Of Cellsmentioning

confidence: 99%

alpha-Adrenergic reduction of cyclic adenosine monophosphate concentrations in rat myocardium.

Watanabe

Hathaway

Besch

et al. 1977

Circulation Research

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SUMMARY We determined the effect of a-adrenergic receptor stimulation on cyclic adenosine monophosphate (cyclic AMP) concentrations in isolated myocytes derived from adult rat hearts and in isolated perfused rat hearts. Activation of a-adrenergic receptors with either phenylephrine (10~8 M to 10~6 M) or epinephrine (10" M to 10~6 M) plus propranolol (10 ' ! M) resulted in a reduction in cyclic AMP levels in isolated myocytes. The action of phenylephrine was antagonized by phentolamine (10~6 M). Phenylephrine (10 s M) attenuated cyclic AMP generation in response to isoproterenol (10" s M and 10 s M). However, this effect of phenylephrine was not antagonized by phentolamine. Elevation of cyclic AMP concentrations produced by glucagon and by theophylline in isolated myocytes was attenuated by phenylephrine and by epinephrine plus propranolol and the attenuation was antagonized by phentolamine. In isolated perfused rat hearts epinephrine (10 (i M), when given with propranolol, diminished the rate of development of tension and also reduced tissue levels of cyclic AMP. Epinephrine alone, as well as isoproterenol, increased contractility and myocardial cyclic AMP concentrations as expected. These results indicate that catecholamines may increase or decrease cyclic AMP levels in rat myocardium, depending on the intensity of stimulation of receptor types. Increases are mediated by /3-adrenergic receptors, whereas decreases appear to be mediated by a-adrenergic receptors. IN 1967, Robison et al. 1 hypothesized that activation of either a-adrenergic or /3-adrenergic receptors may modify the activity of adenylate cyclase and result in changes in the rate of conversion of ATP to cyclic adenosine monophosphate (cyclic AMP). They further predicted that asympathomimetic agents could induce a decrease in the activity of the enzyme in some tissues. This original hypothesis has since been corroborated by studies which indicate that some a-adrenergic effects are mediated by a reduction in the tissue level of cyclic AMP. 2 " 5 Although there is an abundance of evidence that activation of a-adrenergic receptors can lead to alterations in the electrophysiological and mechanical functions of the heart, 6 " 18 no intracellular mediator or biochemical basis for these effects has been identified. It has been fairly convincingly shown that these a-adrenergic effects are not mediated by an increase in myocardial cyclic AMP concentrations. 1920 The question remains, however, whether any of the a-adrenergic effects on the heart might be mediated by a decrease in tissue cyclic AMP levels. The purpose of the present study was to attempt to demonstrate whether stimulation of myocardial a-adrenergic receptors is associated with a fall in cyclic AMP concentrations. The studies were carried out using isolated adult rat heart cells. To extend the findings to the intact heart, we also performed a limited parallel study of a-adrenergic effects on contractility and cyclic AMP levels of isolated, perfused rat hearts. Methods ISOLATION OF CELLSOur me...

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“…Techniques to isolate individual cardiac myocytes were developed over 40 years ago in an effort to better study physiology of this 'workhorse' cell of the heart. Isolation techniques have been improved to increase yield and quality of cells, with the vital development of coronary perfusion via cannulation of the aorta, first performed in 1970 5 . This article describes cardiomyocyte isolation followed by measurement of contractility and calcium transients using ratiometric fluorescence and cell dimensioning data acquisition software.…”

Section: Introductionmentioning

confidence: 99%

Isolation and Physiological Analysis of Mouse Cardiomyocytes

Roth

Bader

Pfaltzgraff

2014

JoVE

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Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes that accompany or lead to heart failure in a variety of experimental conditions. Video LinkThe video component of this article can be found at

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Morphology and Metabolism of Intact Muscle Cells Isolated from Adult Rat Heart

Cited by 116 publications

References 18 publications

Nuclear proteins in the heart of the cardiomyopathic Syrian hamster. Fractionation of phenol-soluble nonhistone proteins by two-dimensional polyacrylamide gel electrophoresis.

Nuclear proteins in the heart of the cardiomyopathic Syrian hamster. Fractionation of phenol-soluble nonhistone proteins by two-dimensional polyacrylamide gel electrophoresis.

alpha-Adrenergic reduction of cyclic adenosine monophosphate concentrations in rat myocardium.

Isolation and Physiological Analysis of Mouse Cardiomyocytes

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