Ultrastructural morphometric analysis of cultured neonatal and adult rat ventricular cardiac muscle cells

Heart muscle tissue has previously been reported to have the highest content of preproenkephalin (ppEnk) mRNA of any tissue in the adult rat. We have determined that it is present in the ventricular cardiac muscle cells of the heart and is developmentally regulated. The expression of ppEnk mRNA was observed to be low throughout the first 2 weeks of postnatal development and decreases substantially during week 3. Expression was again low by week 4, but by adulthood (approx. 3 months), it reached a maximum. ppEnk mRNA was actively expressed in primary cardiac muscle cell cultures prepared from both neonatal and adult rats. Its steady-state content in cell cultures was observed to be increased by cyclic AMP and 3-isobutyl-1-methylxanthine. The phorbol ester phorbol 12-myristate 13-acetate elicited a transient effect (i.e. an increase was observed at 4 h and a return to control values by 24 h). We speculate that enkephalin may play a multi-functional role in the differentiation of neonatal cardiac muscle cells and in the terminally differentiated adult heart cell. We demonstrate that the primary culture systems employed in this study will be useful models with which to explore both transcriptional and translational regulation of ppEnk mRNA in the heart.

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Gap junction formation and regulation in cultured adult rat and guinea pig cardiac muscle cells

Intercellular Communication through Gap Junctions - Progress in Cell Research ◽

10.1016/b978-0-444-81929-1.50033-2 ◽

1995 ◽

pp. 151-154

Author(s):

Y. Shibata ◽

A. Miyahara ◽

T. Okayama ◽

A. Kuraoka ◽

H. Iida

Keyword(s):

Guinea Pig ◽

Cardiac Muscle ◽

Gap Junction ◽

Muscle Cells ◽

Cardiac Muscle Cells ◽

Adult Rat ◽

Junction Formation

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Translation of heart preproenkephalin mRNA and secretion of enkephalin peptides from cultured cardiac myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1992.263.5.h1560 ◽

1992 ◽

Vol 263 (5) ◽

pp. H1560-H1566 ◽

Cited By ~ 11

Author(s):

J. P. Springhorn ◽

W. C. Claycomb

Keyword(s):

Cardiac Muscle ◽

Muscle Cells ◽

Neonatal Rat ◽

Ribonuclease Protection Assay ◽

Cardiac Muscle Cells ◽

Adult Rat ◽

Rabbit Reticulocyte Lysate System ◽

Ribonuclease Protection ◽

Carboxy Terminal

Rat ventricular cardiac muscle has previously been shown to contain exceptionally high levels of preproenkephalin mRNA (ppEnk mRNA). We have recently determined that the level of ppEnk mRNA is developmentally and hormonally regulated in rat ventricular cardiac muscle tissue and in cultured myocytes (J. P. Springhorn and W. C. Claycomb. Biochem. J. 258: 73-77, 1989). We demonstrate in the current study that heart ppEnk mRNA is structurally identical at the 5' end to brain ppEnk mRNA using a ribonuclease protection assay and that heart ppEnk mRNA can be translated in vitro using a rabbit reticulocyte lysate system. In vitro synthesized preproenkephalin peptides were immunoprecipitated with a polyclonal antibody directed to the carboxy-terminal seven amino acids of preproenkephalin. We have also established by radioimmunoassay that enkephalin-containing peptides are secreted from cultured neonatal and adult rat ventricular cardiac muscle cells. This secretion is linear with respect to time and can be stimulated by phorbol 12-myristate 13-acetate (PMA) and adenosine 3',5'-cyclic monophosphate (cAMP). It was determined by column chromatography that cAMP induced neonatal rat ventricular cardiac muscle cells to secrete Met5-enkephalin-Arg6-Phe7, whereas PMA plus 3-isobutyl-1-methylxanthine induced adult rat ventricular cardiac muscle cells to secrete Met5-enkephalin. These studies establish that ventricular heart muscle ppEnk mRNA can be translated and that enkephalin peptides are secreted from ventricular cardiac muscle cells.

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Inhibition of p66ShcA redox activity in cardiac muscle cells attenuates hyperglycemia-induced oxidative stress and apoptosis

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00225.2008 ◽

2009 ◽

Vol 296 (2) ◽

pp. H380-H388 ◽

Cited By ~ 25

Author(s):

Ashwani Malhotra ◽

Himanshu Vashistha ◽

Virendra S. Yadav ◽

Michael G. Dube ◽

Satya P. Kalra ◽

...

Keyword(s):

Oxidative Stress ◽

Cytochrome C ◽

Cardiac Muscle ◽

Ventricular Myocytes ◽

Muscle Cells ◽

Molecular Switch ◽

Left Ventricular ◽

Cardiac Muscle Cells ◽

Rat Ventricular Myocytes ◽

Adult Rat

Apoptotic myocyte cell death, diastolic dysfunction, and progressive deterioration in left ventricular pump function characterize the clinical course of diabetic cardiomyopathy. A key question concerns the mechanism(s) by which hyperglycemia (HG) transmits danger signals in cardiac muscle cells. The growth factor adapter protein p66ShcA is a genetic determinant of longevity, which controls mitochondrial metabolism and cellular responses to oxidative stress. Here we demonstrate that interventions which attenuate or prevent HG-induced phosphorylation at critical position 36 Ser residue (phospho-Ser36) inhibit the redox function of p66ShcA and promote the survival phenotype. Adult rat ventricular myocytes obtained by enzymatic dissociation were transduced with mutant-36 p66ShcA (mu-36) dominant-negative expression vector and plated in serum-free media containing 5 or 25 mM glucose. At HG, adult rat ventricular myocytes exhibit a marked increase in reactive oxygen species production, upregulation of phospho-Ser36, collapse of mitochondrial transmembrane potential, and increased formation of p66ShcA/cytochrome- c complexes. These indexes of oxidative stress were accompanied by a 40% increase in apoptosis and the upregulation of cleaved caspase-3 and the apoptosis-related proteins p53 and Bax. To test whether p66ShcA functions as a redox-sensitive molecular switch in vivo, we examined the hearts of male Akita diabetic nonobese (C57BL/6J) mice. Western blot analysis detected the upregulation of phospho-Ser36, the translocation of p66ShcA to mitochondria, and the formation of p66ShcA/cytochrome- c complexes. Conversely, the correction of HG by recombinant adeno-associated viral delivery of leptin reversed these alterations. We conclude that p66ShcA is a molecular switch whose redox function is turned on by phospho-Ser36 and turned off by interventions that prevent this modification.

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