Radial and longitudinal diffusion of myoglobin in single living heart and skeletal muscle cells

Abstract

We have used a fluorescence recovery after photobleaching (FRAP) technique to measure radial diffusion of myoglobin and other proteins in single skeletal and cardiac muscle cells. We compare the radial diffusivities, D(r) (i.e., diffusion perpendicular to the long fiber axis), with longitudinal ones, D(l) (i.e., parallel to the long fiber axis), both measured by the same technique, for myoglobin (17 kDa), lactalbumin (14 kDa), and ovalbumin (45 kDa). At 22 degrees C, D(l) for myoglobin is 1.2 x 10(-7) cm(2)/s in soleus fibers and 1.1 x 10(-7) cm(2)/s in cardiomyocytes. D(l) for lactalbumin is similar in both cell types. D(r) for myoglobin is 1.2 x 10(-7) cm(2)/s in soleus fibers and 1.1 x 10(-7) cm(2)/s in cardiomyocytes and, again, similar for lactalbumin. D(l) and D(r) for ovalbumin are 0.5 x 10(-7) cm(2)/s. In the case of myoglobin, both D(l) and D(r) at 37 degrees C are about 80% higher than at 22 degrees C. We conclude that intracellular diffusivity of myoglobin and other proteins (i) is very low in striated muscle cells, approximately 1/10 of the value in dilute protein solution, (ii) is not markedly different in longitudinal and radial direction, and (iii) is identical in heart and skeletal muscle. A Krogh cylinder model calculation holding for steady-state tissue oxygenation predicts that, based on these myoglobin diffusivities, myoglobin-facilitated oxygen diffusion contributes 4% to the overall intracellular oxygen transport of maximally exercising skeletal muscle and less than 2% to that of heart under conditions of high work load.

Figure 1

Scheme of experimental setup. aom, acoustooptic modulator; be, beam expander; d, field diaphragm for laser beam (d1) and fluorescence (d2); dm, dichroic mirror (510 nm); bpf, band pass filter (500–530 nm); pmt, photo multiplier tube; ld-40x, water immersion long-distance objective with ×40 magnification (see text for details).

Figure 2

Microinjection procedure for FRAP measurements in skeletal muscle fibers. To achieve locally a state of evenly distributed fluorophores, microinjections were performed at two sites along the longitudinal fiber axis (abscissa, peaks of curves refer to injection sites). In most of the experiments, the distance between the injection sites was around 400 μm. Measurements of the fluorescence intensity (ordinate) distribution were performed at different times after microinjection (after 3, 10, 15, 20, and 35 min in this example). The scans at 20 min and 35 min reveal a fiber segment 200 μm in length (horizontal bar) with almost constant fluorescence intensity.

Figure 3

Arrangement of bleached areas and measuring fields in muscle cells. Diffusion was recorded in skeletal muscle fibers in longitudinal (A) and in radial (B) direction and in cardiomyocytes in longitudinal (C) and in radial (D) direction.

Figure 4

Experimental FRAP curves recorded for fluorescently labeled Mb and their respective best-fit curves: longitudinal diffusion (A) and radial diffusion (B). Curves were measured in dilute (d) and concentrated (c) aqueous solution within glass capillaries and in soleus muscle fibers (s).

Figure 5

FRAP curves obtained for radial diffusion of Mb in a glass capillary filled with dilute aqueous solution (A) and in a soleus muscle fiber (B). The steep curves were recorded after bleaching for measurements of radial diffusion. The flat curves represent the superimposed effect of longitudinal diffusion. Best-fits for the radial diffusion process are shown as solid lines.