pH(i) and pH(o) at different depths in perfused myocardium measured by confocal fluorescence microscopy

Barbara J. Muller-Borer, Hua Yang, Sayed A.M. Marzouk, John J. Lemasters, Wayne E. Cascio

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

Abstract

Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhodafluor-1 (SNARF-1) were used to measure either intracellular pH (pH(i)) or extracellular pH (pH(o)) in isolated, arterially perfused rabbit papillary muscles. Single-excitation, dual-emission fluorescent images of the endocardial surface and underlying myocardium to a depth of 300 μm were simultaneously recorded from perfused cylindrical muscles suspended in a controlled atmosphere oriented oblique to the focal plane. Contraction was inhibited by the addition of butanedione monoxime. In separate muscles, pH(o) was measured during continuous perfusion of SNARF-1 free acid. pH(i) measurements were made after the muscle was loaded with SNARF-1/AM and the extracellular space was cleared of residual fluorophore. Initial experiments demonstrated the uniformity of ratiometric measurements as a function of pH, image depth, and fluorophore concentration, thereby establishing the potential feasibility of this method for quantitative intramural pH measurements. In subsequent experiments, the method was validated in isolated, arterially perfused rabbit papillary muscle during normal arterial perfusion and as pH(i) and pH(o) were altered by applying CO2 externally, exchanging HEPES and bicarbonate buffers, and changing phi with NH4Cl washout. We conclude that in situ confocal fluorescent microscopy can measure phi and pH(o) changes at the endocardial surface and deeper endocardial layers in arterially perfused ventricular myocardium. This method has the potential to study pH(i) regulation in perfused myocardium at boundaries where diffusion of gases, metabolites, and peptides are expected to modify processes that regulate pH(i).

Original languageEnglish
Pages (from-to)H1937-H1947
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume275
Issue number6 44-6
Publication statusPublished - Dec 1 1998
Externally publishedYes

Keywords

  • Acidosis
  • Alkalosis
  • Carbon dioxide
  • Carboxyseminaphthorhodafluor-1
  • Ventricular myocardium

ASJC Scopus subject areas

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

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