Bj. Muller-borer et al., pH(i) and pH(o) at different depths in perfused myocardium measured by confocal fluorescence microscopy, AM J P-HEAR, 44(6), 1998, pp. H1937-H1947
Citations number
30
Categorie Soggetti
Cardiovascular & Hematology Research
Journal title
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY
Confocal microscopy and the H+-sensitive fluorophore carboxyseminaphthorhod
afluor-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 endocar
dial surface and underlying myocardium to a depth of 300 mu m were simultan
eously 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 mea
sured 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 demonstrate
d the uniformity of ratiometric measurements as a function of pH(o) image d
epth, and fluorophore concentration, thereby establishing the potential fea
sibility of this method for quantitative intramural pH measurements. In sub
sequent experiments, the method was validated in isolated, arterially perfu
sed rabbit papillary muscle during normal arterial perfusion and as pH(i) a
nd pH(o) were altered by applying COP externally, exchanging HEPES and bica
rbonate buffers, and changing pH(i) with NH4Cl washout, We conclude that in
situ confocal fluorescent microscopy can measure pH(i) and pH(o) changes a
t the endocardial surface and deeper endocardial layers in arterially perfu
sed ventricular myocardium. This method has the potential to study pH(i) re
gulation in perfused myocardium at boundaries where diffusion of gases, met
abolites, and peptides are expected to modify processes that regulate pH(i)
.