**5. Contraction of VSMCs cultured in honeycombs**

VSMCs cultured on plates express the main components of the contractile apparatus and cytoskeleton (including β-non-muscle actin filaments), but VSMCs cultured on plates do not contract under normal culture conditions [23]. The size of honeycombs containing VSMCs is significantly reduced over time [5], which could be due to the contraction of the VSMCs. A key protein for the contraction of VSMCs cultured in honeycombs is filamin. Filamin A has a molecular weight of 280 kDa and a molecular length of approximately 160 nm [24]. Filamin dimers link actin filaments into orthogonal networks or parallel bundles, and an *in vitro* study showed that the manner in which actin filaments are organized depends on the ratio of filamin to actin [25]. Filamin mainly co-localizes with the β-non-muscle actin filaments in gizzard SMCs [26, 27]. When ODC-VSMCs are used, the function of filamin in VSMCs becomes clear. Filamin expression is dramatically decreased in ODC-VSMCs compared to VSMCs (Figure 6). β-Non-muscle actin filaments in ODC-VSMCs cultured on plates are thinner than those of VSMCs, although β-actin expression, as determined by Western blot analysis, is almost the same in ODC-VSMCs as in VSMCs (Figure 6). These results suggest that the 280-kDa form of filamin is responsible for the bundling of β-non-muscle actin filaments in VSMCs.

It is well known that filamin is degraded by µ- and m-calpain [28, 29]. When VSMCs are cultured in honeycombs, 280-kDa filamin is degraded to 180-kDa filamin by calpains [5]. It is expected that degraded filamin cannot induce the bundling of actin filaments, resulting in the relative structural weakness of β-non-muscle actin filaments in VSMCs cultured in honey‐ combs. Observation of the intracellular distribution of filamin and β-actin by immunofluor‐ escence microscopy shows that filamin is situated along fibers and co-localizes with β-actin in subconfluent VSMCs cultured on plates (Figure 7A and B). In honeycombs, filamin staining is detected evenly in the cytoplasm and is not stained as fibers in most cells (Figure 7C and D). It can be assumed that degraded filamin, which does not bind to β-non-muscle actin filaments, is present in the cytoplasm of VSMCs cultured in honeycombs.

The expression levels of proteins involved in both the contractile apparatus (α-actin, myosin heavy chain, and tropomyosin) and cytoskeleton (β-actin and α-actinin) in VSMCs cultured in honeycombs are almost identical to those in VSMCs cultured on plates [5]. However, VSMCs on plates express the 280-kDa form of filamin, whereas VSMCs cultured in honeycombs express both the 280- and 180-kDa forms of filamin. α-Actin and β-actin co-localize in sub‐ confluent VSMCs cultured on plates and in VSMCs cultured in honeycombs, suggesting that the contractile apparatus is aligned with β-non-muscle actin filaments [5].

These data from VSMCs cultured in honeycombs could explain why contraction, which is a result of shortening of the contractile apparatus, is not observed in VSMCs cultured on plates. VSMCs attach to rigid plastic plates via focal adhesions, and the resulting cytoskeletal tension, which is maintained by β-non-muscle actin filaments, inhibits shortening of the contractile apparatus. In honeycombs, VSMCs can contract because filamin degradation reduces cytos‐ keletal tension and allows shortening of the contractile apparatus [5].

Three-Dimensional "Honeycomb" Culture System that Helps to Maintain the Contractile Phenotype of… http://dx.doi.org/10.5772/60960 451

**5. Contraction of VSMCs cultured in honeycombs**

450 Muscle Cell and Tissue

VSMCs cultured on plates express the main components of the contractile apparatus and cytoskeleton (including β-non-muscle actin filaments), but VSMCs cultured on plates do not contract under normal culture conditions [23]. The size of honeycombs containing VSMCs is significantly reduced over time [5], which could be due to the contraction of the VSMCs. A key protein for the contraction of VSMCs cultured in honeycombs is filamin. Filamin A has a molecular weight of 280 kDa and a molecular length of approximately 160 nm [24]. Filamin dimers link actin filaments into orthogonal networks or parallel bundles, and an *in vitro* study showed that the manner in which actin filaments are organized depends on the ratio of filamin to actin [25]. Filamin mainly co-localizes with the β-non-muscle actin filaments in gizzard SMCs [26, 27]. When ODC-VSMCs are used, the function of filamin in VSMCs becomes clear. Filamin expression is dramatically decreased in ODC-VSMCs compared to VSMCs (Figure 6). β-Non-muscle actin filaments in ODC-VSMCs cultured on plates are thinner than those of VSMCs, although β-actin expression, as determined by Western blot analysis, is almost the same in ODC-VSMCs as in VSMCs (Figure 6). These results suggest that the 280-kDa form of

filamin is responsible for the bundling of β-non-muscle actin filaments in VSMCs.

is present in the cytoplasm of VSMCs cultured in honeycombs.

the contractile apparatus is aligned with β-non-muscle actin filaments [5].

keletal tension and allows shortening of the contractile apparatus [5].

It is well known that filamin is degraded by µ- and m-calpain [28, 29]. When VSMCs are cultured in honeycombs, 280-kDa filamin is degraded to 180-kDa filamin by calpains [5]. It is expected that degraded filamin cannot induce the bundling of actin filaments, resulting in the relative structural weakness of β-non-muscle actin filaments in VSMCs cultured in honey‐ combs. Observation of the intracellular distribution of filamin and β-actin by immunofluor‐ escence microscopy shows that filamin is situated along fibers and co-localizes with β-actin in subconfluent VSMCs cultured on plates (Figure 7A and B). In honeycombs, filamin staining is detected evenly in the cytoplasm and is not stained as fibers in most cells (Figure 7C and D). It can be assumed that degraded filamin, which does not bind to β-non-muscle actin filaments,

The expression levels of proteins involved in both the contractile apparatus (α-actin, myosin heavy chain, and tropomyosin) and cytoskeleton (β-actin and α-actinin) in VSMCs cultured in honeycombs are almost identical to those in VSMCs cultured on plates [5]. However, VSMCs on plates express the 280-kDa form of filamin, whereas VSMCs cultured in honeycombs express both the 280- and 180-kDa forms of filamin. α-Actin and β-actin co-localize in sub‐ confluent VSMCs cultured on plates and in VSMCs cultured in honeycombs, suggesting that

These data from VSMCs cultured in honeycombs could explain why contraction, which is a result of shortening of the contractile apparatus, is not observed in VSMCs cultured on plates. VSMCs attach to rigid plastic plates via focal adhesions, and the resulting cytoskeletal tension, which is maintained by β-non-muscle actin filaments, inhibits shortening of the contractile apparatus. In honeycombs, VSMCs can contract because filamin degradation reduces cytos‐

Figure 6. Expression and localization of filamin and β-actin in ODC-VSMCs. (A) Western blot analysis of filamin and β-actin in VSMCs and ODC-VSMCs. **Figure 6.** Expression and localization of filamin and β-actin in ODC-VSMCs. (A) Western blot analysis of filamin and β-actin in VSMCs and ODC-VSMCs. VSMCs and ODC-VSMCs were cultured on plates to subconfluence. Lane 1, VSMCs; lane 2, molecular marker; and lane 3, ODC-VSMCs. Localization of filamin and β-actin in VSMCs (B, C) and ODC-VSMCs (D, E). VSMCs and ODC-VSMCs were cultured on plates to subconfluence. Green indicates filamin, and red indicates β-actin. Scale bars, 20 µm (B, D); 5 µm (C, E). Data adapted from reference 5.

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VSMCs and ODC-VSMCs were cultured on plates to subconfluence. Lane 1, VSMCs; lane 2, molecular marker; and lane 3, ODC-VSMCs. Localization of

 Figure 7. Localization of filamin and β-actin in VSMCs. (A, B) VSMCs were **Figure 7.** Localization of filamin and β-actin in VSMCs. (A, B) VSMCs were cultured to subconfluence on a plate. (C, D) VSMCs were cultured in honeycombs for 3 days. Green indicates filamin, and red indicates β-actin. Scale bars, 20 µm (A, C); 5 µm (B, D). Data adapted from reference 5.

cultured to subconfluence on a plate. (C, D) VSMCs were cultured in honeycombs for 3 days. Green indicates filamin, and red indicates β-actin. Scale When myocardial cells derived from newborn rats are cultured in honeycombs, the cells attach to the honeycombs and start beating from the first day. The rate of beating increases gradually,

bars, 20 µm (A, C); 5 µm (B, D). Data adapted from reference 5.

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and reaches the highest frequency of 162 beats per minute at day 8 [30]. However, the number of myocardial cells increases significantly. From these data, it is shown that the honeycomb scaffold may induce the functional aspects of cells even though it is unclear whether or not cells cultured in honeycombs proliferate.
