Cellular Physiology and Biochemistry | |
Integrin Stimulation Favors Uptake of Macromolecules by Cardiomyocytes In Vitro | |
关键词: Cardiomyocytes; Integrins; Dysferlin; Membrane; Wounding; Patch repair; RGD; | |
DOI : 10.1159/000324013 | |
学科分类:分子生物学,细胞生物学和基因 | |
来源: S Karger AG | |
【 摘 要 】
Previously, our research group showed that integrin stimulation induces release of cardiac troponin I from viable neonatal rat ventricular cardiomyocytes (NRCMs), but would it also stimulate uptake of exogenous macromolecules? For this purpose, beating NRCMs were incubated without or with an RGD motif-containing peptide (GRGDS) to stimulate integrins in the presence of Texas Red-conjugated ovalbumin (OTR; 45 kDa) or dextran (DTR; 70 kDa). After incubation periods of 8, 16 and 24 hours endocytosis of red label was quantified by fluorescence microscopy. Uptake of OTR and DTR by NRCMs was intensified by GRGDS treatment (p for trend <0.001 and 0.019, respectively) and increased with duration of incubation (p<0.001 for both). The GRGDS-induced uptake of OTR by NRCMs correlated positively with OTR concentration (p<0.001). Experiments with pharmacological inhibitors of endocytosis indicated that in the absence of GRGDS, NRCMs take up OTR by the clathrin-mediated pathway of endocytosis while the GRGDS-dependent OTR uptake occurs by macropinocytosis. Cultures of NRCMs that were stretched cyclically showed ≈4-fold increased uptake of OTR compared to stationary NRCM cultures. Immunofluorescence microscopy revealed that the dysferlin-positive plasma membrane (PM) areas in beating GRGDS-treated NRCMs were ≈3-fold larger than in contracting NRCMs incubated with vehicle (p<0.001). However, in non-beating NRCMs exposure to GRGDS did not induce larger dysferlin-positive PM areas, nor did it stimulate uptake of OTR. After inhibition of dysferlin expression by short hairpin RNA-mediated RNA interference, OTR uptake by contracting NRCMs could no longer be stimulated via GRGDS treatment. We conclude that in NRCMs, stimulation of integrins by RGD motif-containing peptides or stretch cause uptake of labeled macromolecules. The latter process appears to depend on the contractile behavior of the NRCMs and on the PM repair protein dysferlin, probably because of its role in macropinocytosis.
【 授权许可】
CC BY-NC-ND
【 预 览 】
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