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The carboxyl-terminal ahnak domain induces actin bundling and stabilizes muscle contraction¹

HANNELORE HAASE^*,2, INES PAGEL, YANA KHALINA^*,, UDO ZACHARZOWSKY, VERONIKA PERSON, GUDRUN LUTSCH^*, DARIA PETZHOLD^*, MONIKA KOTT^*, JUTTA SCHAPER and INGO MORANO^*,

^* Max Delbrück Center for Molecular Medicine, Berlin, Germany;
Franz Volhard Clinic, Humboldt University (Charité), Berlin, Germany;
Institute of Theoretical and Experimental Biophysics, Academy of Sciences of Russia, Pushchino, Russia;
Max Planck Inst. for Physiological and Clinical Research, Bad Nauheim, Germany; and
Johannes Müller Institute for Physiology, Humboldt University (Charité), Berlin, Germany

²Correspondence: Max Delbrück Center for Molecular Medicine, 13092 Berlin, Germany, E-mail: haase{at}mdc-berlin.de

SPECIFIC AIMS

Ahnak is encoded by an intronless gene located on human chromosome 11q12. It has been implicated in fundamental cellular processes as diverse as cell differentiation, signal transduction, and regulated exocytosis. However, no information is available about the existence of ahnak isoforms that serve different cell type-specific functions. In cardiomyocytes we identified ahnak as a 700 kDa protein that localized to the inner side of the sarcolemma membrane and interacted with the ß subunit of the L-type Ca²⁺ channel and with F-actin via its carboxyl-terminal domain (ahnak-C2) of 382 amino acids. The aim of the present study was to elucidate a potential functional role of ahnak-C2 on actin filaments. We searched for putative ahnak isoforms by subdomain-specific antibodies.

PRINCIPAL FINDINGS

1. Identification of a cardiac-specific, carboxyl-terminal ahnak fragment
A domain-specific anti-ahnak antibody was generated against the carboxyl-terminal amino acid residues (5611–5624) of human ahnak. This antibody, designated as Tail-Ab, differed in immunorecognition pattern from the established ahnak antibody, KIS-Ab. KIS-Ab reacted strongly with the 700 kDa ahnak protein in rat heart and skeletal muscle whereas Tail-Ab barely reacted with the full-length protein in both muscle types. Moreover, the Tail-Ab detected a putative carboxyl-terminal ahnak fragment in cardiac but not in the skeletal muscle. Immunocytochemistry of rat heart revealed Tail-Ab labeling as regularly spaced intracellular striations in longitudinal sections of cardiomyocytes (Fig. 1 A). This labeling pattern was different from that of the KIS-Ab, which showed plasma membrane labeling in sections of the same tissue block (Fig. 1B ). Clear plasma membrane labeling and no intracellular staining were obtained by Tail-Ab (Fig. 1C ) and KIS-Ab (Fig. 1D ) in rat skeletal muscle under the same experimental conditions. The ahnak antibodies revealed comparable subcellular location in skeletal muscle in which the carboxyl-terminal ahnak fragment was not observed.

View larger version (126K): [in this window] [in a new window]   Figure 1. Immunofluorescence micrographs demonstrating the subcellular location of the Tail-Ab and KIS-Ab epitopes in rat heart and skeletal muscle. A, B) Longitudinal sections of rat left ventricular myocytes labeled with the Tail-Ab in regular cross-striations (A) and with the KIS-Ab at the plasma membrane (B). C, D) Longitudinal section of rat skeletal muscle myocytes (M. gastrocnemius) labeled with Tail-Ab (C) and KIS-Ab (D) at the plasma membrane. Primary antibodies are visualized with Cy3-labeled secondary antibodies (red), nuclei are stained with DAPI (blue).

Human cardiac preparations were used for further expression and localization studies. In normal human heart, the Tail-Ab reacted strongly with a protein of 72 ± 3 kDa (n=5). This protein was also identified by the ahnak-C2 antibody after epitope splitting. This ahnak fragment copurified with the myofibrillar protein fraction. The subcellular distribution of the 72 kDa carboxyl-terminal ahnak fragment was studied by immunofluorescence microscopy in double labeling experiments with the sarcomeric Z-band- and M-band proteins -actinin and myomesin, respectively. Confocal imaging of longitudinal sections of normal human myocardium revealed labeling of ahnak’s Tail-Ab epitope in regular cross-striations (Fig. 2 A). A similar picture was obtained for -actinin in its typical regular cross-striated pattern at the level of the Z-line (Fig. 2B ). The intercalated disc was also stained by the Tail-Ab (Fig. 2A , arrow). Merged images indicate remarkable colocalization of ahnak’s Tail-Ab epitope and -actinin at both the Z-line and the intercalated disc (Fig. 2C ). The images in the lower panel of Fig. 2 compare the location of ahnak’s Tail-Ab epitope with myomesin. Again, the regular cross-striated pattern was clearly obtained with the Tail-Ab (Fig. 2D ) and, as expected for myomesin, at the M-band of the sarcomere (Fig. 2E ). Merged images of both proteins revealed a clear alternate staining pattern for ahnak’s Tail-Ab epitope and myomesin (Fig. 2F ).

View larger version (35K): [in this window] [in a new window]   Figure 2. Immunofluorescence micrographs demonstrating the subcellular location of the Tail-Ab epitope in the human myocardium. A) Longitudinal section of human myocardium labeled in green for ahnak’s Tail-Ab epitope in regular cross-striation. The intercalated disc is also stained (arrow). B) Same section as in A labeled for -actinin in its typical regular cross-striated pattern at the level of Z-lines. C) The merged picture of panels A and B shows colocalization (orange). D) Longitudinal section of human myocardium stained for ahnak’s Tail-Ab epitope displaying a regular cross-striated pattern. E) Same section as in panel D stained for the M-band protein myomesin. The regular cross-striated pattern is prominent. F) Merged channels of panels D and E show a clear alternate pattern of ahnak’s tail epitope (green) and myomesin (red).

2. Ahnak-C2 binds to skeletal muscle fibers and stabilizes their contractility
We used recombinant ahnak-C2 for functional studies with demembraned skeletal muscle fibers. Isometric force development of demembraned skeletal muscle fibers (M. gastrocnemius) was monitored at submaximal (pCa 5.8) and at maximal (pCa 5.0) Ca²⁺ activation during two subsequent contraction/relaxation cycles. The isometric force development under control conditions at pCa 5.0 was 1.81 ± 0.33 mN (n=8). When isometric force development of the first contraction cycle was compared with that of the second contraction cycle, we observed a significant decrease in force development at submaximal and maximal Ca²⁺ activation. Addition of GST-ahnak-C2 to the fibers attenuated this decline of force during the second contraction/relaxation cycle significantly: isometric force development at submaximal Ca²⁺ activation declined to 43.8 ± 4.8% in the presence of 1 µM GST-ahnak-C2, and force development at maximal Ca²⁺ activation (pCa 5.0) could be maintained at the level of the first contraction/relaxation cycle. During this protocol, skinned skeletal muscle fibers bound ahnak-C2 as assessed by immunoblotting.

3. Ahnak-C2 has actin bundling activity
Actin filaments were incubated with recombinant ahnak-C2 and inspected by electron microscopy. GST-ahnak-C2 induced different stages of actin filament bundling ranging from loose bundles to tight packing into paracrystalline-like structures. The thickness of bundles was up to 250 nm (Fig. 3 A). No actin bundling was observed with unfused GST (Fig. 3B ).

View larger version (108K): [in this window] [in a new window]   Figure 3. Electron micrographs demonstrating actin bundling. A) Negatively stained actin-ahnak-C2 complexes formed at a molar ratio of 1:1 reveal paracrystalline-like structures. B) Negatively stained actin filaments after incubation with GST as control.

CONCLUSIONS AND SIGNIFICANCE

The findings presented in this study substantially contribute to our understanding of the role of ahnak in cardiomyocytes. Our results suggest that ahnak interacts with actin at specific sites: full-length ahnak anchors sarcolemma proteins (e.g., L-type Ca²⁺ channels) to the subsarcolemmal actin-based cytoskeleton whereas the cardiac-specific carboxyl-terminal ahnak fragment accumulates at the level of Z-lines and intercalated discs for interaction with myofibrillar F-actin. Both myofibrillar Z-line structures and intercalated discs play a crucial role in the establishment and maintenance of cardiomyocyte cytoarchitecture and contractile function.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0446fje;

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