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

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) .