Fluorescence polarization: a novel indicator of cardiomyocyte contraction q Dror Fixler, a Reuven Tirosh, a Tova Zinman, b Asher Shainberg, b and Motti Deutsch a, * a The Biophysical Interdisciplinary Schottenstein Center for the Research and the Technology of the Cellome, Physics Department, Bar-Ilan University, Ramat-Gan 52900, Israel b Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Received 11 November 2002 Abstract The changes measured in intracellular fluorescein fluorescence polarization (IFFP) are used as a new tool for tracing cytoplasmic effects during contractile cycles of cardiac myocytes (1–2-day-old rat hearts), in addition to the established Ca 2þ monitoring and/or videometric methods of tracking cell-shortening. This novel method was found to be non-intrusive to the contraction cycles. The decay of the transient IFFP signal (from 0:220  0:01 to 0:170  0:013) seems to be closely related to the extended phase of con- tractile activation. This fact was further supported when Ca 2þ exchanger inhibitor was introduced and significantly decreased (90%) the rate of beats of contraction and IFFP, but not the Ca 2þ beat rate changes. This result suggests that the IFFP indicator is probably associated with the physiological activation, rather than with Ca 2þ alterations. The IFFP measure monitors the average of effective changes in the micro-viscosity of the cytoplasm protein matrix, associated with cellular activation. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Fluorescence polarization; Cardiac cells; Contraction rate; Amplitude The ability to trace various physiological events during contraction cycles of single cardiomyocytes is greatly increased with the use of intracellular probes. These measurements might contribute to the funda- mental understanding of cellular stimulation and acti- vation. Thus, during contraction cycles of myocardial tissue, rapid changes within single cells can be measured and correlated, e.g., membrane potential, cytosolic Ca 2þ concentration ([Ca 2þ ] i ), and intracellular pH, compared to shortening and tension generation [4,24]. All these physiological variables show a change during stimula- tion and activation responses in a large number of cell types. Processes linking early events following cell stimulation and activation involve conformational changes of the cytosolic enzymes and/or their regulatory proteins, and dynamic reorganization of their intracel- lular matrix [15]. Such structural changes might be easily monitored via fluorescence polarization (FP) emitted from a fluorescent molecule, hosted by the cellular me- dia. Fluorescence polarization is considered to be one of the first functional cytometric parameters [23] and was initially suggested by Cerceks as an indicator for lym- phocyte activation [23]. The FP is defined as the ratio ðFI II  FI þ Þ= ðFI II þ FI þ Þ, where (II) and (+) correspondingly indi- cate the FIs measured parallel and perpendicular to the excitation field vector. The more the molecule rotational movement is restricted—the higher the FP value is and vice versa. The biophysical aspects of the FP of the cellular probe are based on the fact that the spectro- scopic nature of a fluorescent solute is strongly depen- dent on the physical/chemical characteristics of the hosting media among which are viscosity, dielectric Biochemical and Biophysical Research Communications 300 (2003) 23–28 www.elsevier.com/locate/ybbrc BBRC q Abbreviations: BDM, 2,3-butanedione monoxime; DMSO, di- methyl sulfoxide; FDA, fluorescein diacetate; FI, fluorescence inten- sity; FP, fluorescence polarization; IFFP, intracellular fluorescein fluorescence polarization; PBS, phosphate-buffered saline; R, ratio- metric. * Corresponding author. Fax: +972-3-534-2019. E-mail addresses: fixeled@mail.biu.ac.il (D. Fixler), motti_d@net vision.net.il (M. Deutsch). 0006-291X/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S0006-291X(02)02768-7