Novel technique to load indo-1 free acid into single adult cardiac myocytes to assess cytosolic Ca2+

S. J. Sollott, B. D. Ziman, E. G. Lakatta

Research output: Contribution to journalArticlepeer-review

Abstract

This report introduces a novel method to load indo-1 'free acid' selectively into the cytosol of cardiac myocytes, presumably by diffusion through momentarily permeable gap junction sites during mechanical dissociation after low-Ca2+ collagenase treatment. Calibration of indo-1 fluorescence in these cells has been accomplished after subtracting average autofluorescence (AF) from time-matched non-indo-loaded cells, taking into account apparent changes in cell AF due to indo-1. There is wide variation in the degree of uncertainty of individual intracellular Ca2+ concentration ([Ca2+](i)) determinations among cells, related principally to differences in cellular indo-1 content, to nonlinear aspects of the [Ca2+]-to- fluorescence ratio relationship, and to the uncertainty in the AF subtraction. Consequently, a quantitative estimate of uncertainty also may be employed in formulating weighted estimates of cytosolic [Ca2+](i). The following [Ca2+](i) values in rat ventricular cells (nM; in 1 mM bathing extracellular Ca2+ concentration, 25°C) are given as weighted means ± 95% confidence intervals (unweighted values in parentheses): 138 ± 5 (136 ± 6, n = 44) in quiescent cells, 435 ± 74 (482 ± 76, n = 43) at the [Ca2+](i)- transient peak during 0.5 Hz steady-state stimulation, and 760 ± 124 (1,027. ± 250, n = 42) at the [Ca2+](i)-transient peak, postrest. Moreover, these peak [Ca2+](i) values fall near the steepest portion of the force-Ca2+ curve (from intact cardiac muscle), consistent with sensitive inotropic regulation and maximal contractile reserve.

Original languageEnglish (US)
Pages (from-to)H1941-H1949
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume262
Issue number6 31-6
StatePublished - Jan 1 1992
Externally publishedYes

Keywords

  • cytosolic calcium
  • manganous ion

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

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