P485Formation of contraction bands of heart muscle cells by spatially high-regional ablation of beta-dystroglycan via multiphoton excitation-evoked CALI

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Purpose: Contraction band necrosis, a key morphological hallmark of ischemia-reperfusion injury, is caused by regional contracture at individual sarcomeres of cardiomyocytes. However, mechanisms underlying formation of the contraction bands remain undetermined. We found by immunohistochemistry that sites of contraction bands in rat ventricular myocytes show low expression of β-dystroglycan (β-DG), a membrane-spanning protein that physically links myofilaments to the extracellular matrix at the costameres. Based on this observation, we hypothesized that regional ablation of β-DG leads to genesis of contraction bands at the ablated sites.

Methods: To address this possibility, we applied multiphoton (MP) excitation-evoked chromophore-assisted laser inactivation (MP-CALI) to neonatal rat ventricular myocytes (approximately 5.0 x 10(5) cells cultured on a 35-mm diameter glass-bottom dish for 2 days) that were transfected with cDNA encoding the protein fused with EGFP (βDG-EGFP), where EGFP fluorescence was homogeneously distributed along the cell membrane. Experiments were performed by using a MP laser-scanning microscope (LSM510 META NLO; Carl Zeiss) with a titanium-sapphire laser (MaiTai; Spectra-Physics; wavelength: 850 nm; pulse width: 100-fs; repetition frequency: 76 MHz; pixel dwell time: 1.61 ms; scan area: 7 mm x 1.4 mm; total scan time: 810 ms) via a 63X objective lens (N.A.=1.4). As the marker of actin filaments in cardiomyocytes, red fluorescent protein (RFP)-linked Lifeact plasmids were co-transfected to the myocytes with βDG-EGFP plasmid.

Results: In all the 12 cells examined, MP-CALI of βDG-EGFP at a localized area of interest resulted in instantaneous bleaching of EGFP, followed by progressive aggregation of actin filaments (Lifeact-RFP positive) at the site of irradiation within 1 minute, resulting in formation of contraction bands at energy power of 1.1 mJ, where myocytes showed no evidence of hypercontracture. Incidence of contraction-band formation was dependent on energy power of excitation with a threshold of 0.85 mJ. On the other hand, MP irradiation of myocytes without βDG-EGFP transfection failed to generate contraction bands. Edaravone (1 mmol/L), a potent scavenger of free radicals, attenuated the formation, indicating mediation of free radicals generated by MP-CALI of βDG-EGFP.

Conclusions: Regional ablation of βDG resulted in contraction-band formation in neonatal cardiomyocytes. Our present observations indicate an essential role of βDG in formation of contraction-band necrosis in the heart.

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