Ultrastructural and Biochemical Localization of N-RAP at the Interface between Myofibrils and Intercalated Disks in the Mouse Heart

Zhang, Jian Q.; Elzey, Brian; Williams, Gail M.; Lu, Shajia; Law, Douglas; Horowits, Robert

doi:10.1021/bi0107445

Cited by 51 publications

(66 citation statements)

References 46 publications

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“…7), where guinea-pig heart homogenates were fractionated with a sucrose gradient. As reported previously [11], N-cadherin, which is a marker of the intercalated disc, was enriched in the F3 fraction. A similar enrichment occurred for the β2 subunit, but not the β1 subunit, whereas Na + ,K + -ATPase α subunits were not consistently detected in the fractions, probably due to the degradation during the procedure.…”

Section: Fractionation Studysupporting

confidence: 52%

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Subunit Composition and Role of Na+,K+-ATPases in Ventricular Myocytes

et al. 2006

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Na + ,K + -ATPases are composed of one α and one β subunit; four α and three β isoforms have been found to date. We elucidated which α and β subunits were present in the ventricular myocytes of rat and guinea-pig and what roles the Na + ,K + -ATPase isozymes play in cardiac contraction. The presence of the α1, α2, and α3 subunits and the β1 and β2 subunits in rat and guinea-pig hearts were confirmed at the protein or mRNA level. Immunocytochemistry showed a patchy presence of α1 in the transverse tubules and surface sarcolemma, whereas α2 was distributed continuously in the transverse tubules alone. The α3 isoform was expressed prominently in the guinea-pig intercalated disc and slightly in the rat. On the other hand, the β1 isoform was located in the transverse tubules and surface sarcolemma, whereas the β2 was mainly located in the intercalated disc. The immunocytochemistry and immunoprecipitation findings indicated that the α1 and α2 form heterodimers with β1 and the α3 with β2 in ventricular myocytes. The application of low concentrations of ouabain enhanced the amplitudes of twitch without a change in resting tension in rat and guinea-pig ventricular stripts, whereas that of high concentrations resulted in a decrease in twitch with an increase in the resting tension. We thus conclude that the α2β1 and α3β2 isozymes are selectively located in the transverse tubules and intercalated disc of the ventricular myocytes, respectively, and the α2β1 is involved in the regulation of the Ca 2+ contents in the SR.Key words: transverse tubules, intercalated disc, localization, Na + pump, subunits. Na + , K + -ATPases are heterodimers comprising α and β subunits [1]. There are four α and three β isoforms, and the expression of the α and β subunits have been reported to be developmentally regulated in a cell-specific manner [2]. Although the expression of α and β subunits in oocytes [3] or Sf-9 cells [1], an insect cell line, revealed every combination of α and β subunits was functional, Na + ,K + -ATPases in cells in situ are thought to exist as a specific combination of α and β subunits [4,5]. Furthermore, single cells have been shown to have multiple α subunits, which are distributed differently in the cell membrane [6]. We elucidated that the α1 subunit, which formed a heterodimer with the β3 subunit, was ubiquitously present in the cell membrane of adrenal chromaffin cells, whereas the α2 subunit, forming a heterodimer with the β2, was located in the cell membrane in the vicinity of Ca 2+ store sites [7]. Our functional and morphological analyses revealed that the α2 subunit was involved in the regulation of the Ca 2+ contents in intracellular Ca 2+ store sites and the α3 subunit was not expressed in chromaffin cells [7]. In astrocytes, the α2 subunit has also been shown to take part in regulating the Ca 2+ contents in Ca 2+ store sites, whereas the α1 subunit has only been assumed to play a house-keeping role [8]. On the other hand, a recent knock-in experiment revealed that the α1 subunit could regulate C...

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Section: Fractionation Studysupporting

confidence: 52%

“…A crude fraction of intercalated disc was obtained with the method described previously [11]. Each 1 g of guinea-pig hearts was homogenized in 30 ml of 10 mM Tris solution (pH, 7.0), and then was centrifuged at 10,000 × g for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Subunit Composition and Role of Na+,K+-ATPases in Ventricular Myocytes

et al. 2006

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“…We are uncertain which isoform is present, nor how the transition is achieved, but we consider it significant that the actinand ␣-actinin-binding protein N-RAP locates at the ID with a split distribution (Luo et al, 1997;Zhang et al, 2001). One possible arrangement for N-RAP is presented in Figure 10.…”

Section: Discussionmentioning

confidence: 99%

The Transitional Junction: A New Functional Subcellular Domain at the Intercalated Disc

Bennett

Maggs

Baines

et al. 2006

MBoC

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We define here a previously unrecognized structural element close to the heart muscle plasma membrane at the intercalated disc where the myofibrils lead into the adherens junction. At this location, the plasma membrane is extensively folded. Immunofluorescence and immunogold electron microscopy reveal a spectrin-rich domain at the apex of the folds. These domains occur at the axial level of what would be the final Z-disc of the terminal sarcomere in the myofibril, although there is no Z-disc-like structure there. However, a sharp transitional boundary lies between the myofibrillar I-band and intercalated disc thin filaments, identifiable by the presence of Z-disc proteins, ␣-actinin, and N-terminal titin. This allows for the usual elastic positioning of the A-band in the final sarcomere, whereas the transduction of the contractile force normally associated with the Z-disc is transferred to the adherens junctions at the plasma membrane. The axial conjunction of the transitional junction with the spectrin-rich domains suggests a mechanism for direct communication between intercalated disc and contractile apparatus. In particular, it provides a means for sarcomeres to be added to the ends of the cells during growth. This is of particular relevance to understanding myocyte elongation in dilated cardiomyopathy.

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“…At the ultrastructural level, N-RAP is localized to the terminal actin bundles that link myofibrils to membrane complexes at the myotendinous junctions in skeletal muscle and at the intercalated disks in heart muscle (Herrera et al, 2000;Zhang et al, 2001). In hearts, these terminal actin bundles are found in the adherens junction region of the intercalated disk (Forbes and Sperelakis, 1985;Severs, 1995), providing for transmission of myofibrillar force to the membrane and to neighboring cells by means of cadherin linkages between cardiomyocytes (Takeichi, 1990;Geiger and Ayalon, 1992;Kemler, 1993;Hertig et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

“…N-RAP is an actin-binding LIM protein that is concentrated at the ends of striated muscle cells (Luo et al, 1997;Zhang et al, 2001;Mohiddin et al, 2003). The N-RAP gene structure and coding sequence are highly conserved between mice and humans.…”

Section: Introductionmentioning

confidence: 99%

N-RAP expression during mouse heart development

Borst

Horowits

2005

Dev. Dyn.

Self Cite

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N-RAP gene expression and N-RAP localization were studied during mouse heart development using semiquantitative reverse transcriptase-polymerase chain reaction and immunofluorescence. N-RAP mRNA was detected at embryonic day (E) 10.5, significantly increased from E10.5 to E16.5, and remained essentially constant from E16.5 until 21 days after birth. In E9.5-10.5 heart tissue, N-RAP protein was primarily associated with developing premyofibril structures containing ␣-actinin,

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Ultrastructural and Biochemical Localization of N-RAP at the Interface between Myofibrils and Intercalated Disks in the Mouse Heart

Cited by 51 publications

References 46 publications

Subunit Composition and Role of Na+,K+-ATPases in Ventricular Myocytes

Subunit Composition and Role of Na+,K+-ATPases in Ventricular Myocytes

The Transitional Junction: A New Functional Subcellular Domain at the Intercalated Disc

N-RAP expression during mouse heart development

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