Aprendizaje reflexivo en la formación

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Figure 3.33. Derived plot of a caffeine transient from a control (continuous line) and an aortic-constricted (broken line) heart both scaled to 100% to allow comparison . [Ca^^]i

measured using the fluorescent indicator, Fura-2 and plotted as the ratio o f the fluorescence output at 340/380 nm. In the control cell resting [Ca^^Jj was 253 nM and increased to 428 nM and in the aortic-constricted cell resting [Ca^^Jj was 254 nM and increased to 364 nM.

Following exposure to 40 mM KCl for one minute, [Ca^^Ji increased to a plateau and on removal o f KCl the [Ca^^Ji declined rapidly to baseline. The results are shown in table 3.10. The typical effect of exposure to 40 mM KCl is illustrated in plate 3B. The magnitude of the increase in [C3?^]\ during exposure to KCl was significantly higher in the combined control group compared to the aortic constricted hearts (p=0.017) but comparing myocytes from sham-operated and aortic-constricted hearts no significant difference was observed (p=0.54).

After a KCl-induced depolarisation, subsequent application o f 10 mM caffeine produced a transient increase o f [Ca^^], in most myocytes. The results are shown in table 3.11. The increase o f [Ca^^Jj in myocytes isolated from aortic-constricted hearts was significantly lower than in the control hearts (69 ± 51 vs 107 ± 68 nM, (mean ± SD) p<0.01). A significant negative relationship was observed between the increase of

10 mM caffeine 340:380 — 0.45 — 0.40 — 0.30 — 0.25 380 3 40 W M W ^^ 1 min 40 mM KCl

B

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Plate 3. In A the increase in [Ca^^]i in response to 10 mM caffeine in a myocyte from a sham-operated heart is illustrated as the ratio values. Resting [Ca^^]i was 309 nM, increasing to 684 nM on application of caffeine. On removal of caffeine [Ca^'^Ji fell rapidly to a nadir of 278 nM before returning to baseline. In B the increase in [Ca^^], in response to 40mM KCl is illustrated followed by a caffeine response in another cell from a sham-operated heart. Resting [Ca^^Ji was 332 nM, increasing to 1007 nM after KCl and 507 nM after caffeine. On removal of caffeine [Ca^^]j fell to a nadir of 296 nM before returning to baseline.

[Ca^^Ji in response to lOmM caffeine and the heart-to-body weight ratio of the animal from which the cells were derived as shown in figure 3.34 (r=-0.68, p<0.01). The values for all the cells isolated from any particular hearts were grouped together and their mean value ± SD was determined and plotted as a function of the heart-to-body weight ratio of the animal from which they were derived.

Table 3.10. Change of [Ca^'^Ji during exposure to 40 mM KCl in isolated myocytes measured using the fluorescent indicator, Fura-2. Values are shown mean ± SD. * denotes p < 0.05 compared to unoperated and combined control group.

Baseline [Ca^^]j, nM + 40 mM KCl (nM) A [Ca^^li, nM

Unoperated 220 ± 56 626 ± 72 4 0 7 ± 128 (n=39) Sham-operated 222 ± 63 55 9 ± 154 3 3 7 ± 120 (n=22) Unoperated and 221 ± 58 6 0 2 ± 150 3 8 1 ± 129 Sham (n=61) Aortic- 2 4 7 ± 103 565 ±216 3 1 7 ± 142* constricted (n=47)

Table 3.11. Change of [Ca^^]i on application of 10 mM caffeine (after a brief exposure to 40 mM KCl) in isolated myocytes measured using the fluorescent indicator, Fura-2. Values are shown mean ± SD. * denotes p<0.01 compared to unoperated and sham- operated groups and combined control group.

Baseline [Ca^^Ji, nM ± 1 0 mM Caffeine (nM) A [C si^ \ nM

Unoperated 234± 64 334 ±102 102 ± 6 4 (n=39) Sham-operated 249 ± 83 3 6 7 ± 134 117±75 (n=22) Unoperated and 244 ± 80 351 ± 122 107 ± 68 Sham (n=61) Aortic- 2 6 2 ± 109 322 ± 147 69 ±51* constricted (n=46)

A second application of 10 mM caffeine, after a 2 minute quiescent period produced a significantly smaller rise in [Ca^^Ji in all groups (45 ± 395 nM, mean ± SD, n=24, p<0.001 in control group vs 37 ± 29 nM, mean ± SD, n=17, p<0.001 in aortic-constricted group). There was no difference in the magnitude of this response between myocytes from control and aortic-constricted hearts (p>0.05).

200-1

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3.0 3.5 4.0 4.5 5.0 5.5

Heart-to-body weight ratio (g.kg )

Figure 3.34. Relationship between SR calcium content, measured in isolated myocytes as the increase in [Ca^^Jj produced on application o f 10 mM caffeine, and heart-to-body weight ratio. Data from 106 myocytes from 18 hearts. The values for all the cells isolated from any particular heart were grouped together and the mean ± SD plotted as a function o f the heart-to-body weight ratio. Values for control hearts shown as open circles and values for aortic-constricted hearts as filled circles. Between 3 and 13 cells were used for the determination of each data point. Data fitted to a linear relationship, r=-0.68, p<0.01.

3.14.4. Effect o f reduced external Na on SR content

Substitution of 147 mM NaCl Tyrode's with 29 mM NaCl Tyrode's (Chapter 2, section 2.3.2, table 2.2) caused an increase of [Ca^^]i which returned towards baseline on reperfusion with 147 mM NaCl Tyrode's. These changes of [Ca^^], are presumably due

to activation of the Na^-Ca^^-exchanger. The increase o f [Ca^"^], was variable with some cells showing a rapid and marked increase as shown in figure 3.35 and plate 4 and other cells showing a smaller increase. The increase was not significantly different between cells from control and aortic-constricted hearts (p=0.64, table 3.12). Figure 3.36 shows the effect of different external concentrations of Na on [Ca^^Ji.

Table 3.12. Change of [Ca^^Ji during superfusion with reduced external Na solution (29 mM NaCl Tyrode's) in isolated myocytes measured using the fluorescent indicator, Fura- 2. Values are shown mean ± SD. No significant differences between aortic-constricted and any control group observed.

Baseline [Ca^^Ji, nM + 29 mM NaCl (nM) A [Ca^^]i,nM

Unoperated 258 ± 74 848 ± 644 591 ±624 (n=51) Sham-operated 264 ± 82 600 ± 399 336 ±389 (n=13) Unoperated and 259 ±75 798 ±608 540 ±591 Sham (n=64) Aortic-constricted 237 ± 75 842 ± 722 604 ±716 (n=39)

Subsequent application of 10 mM caffeine resulted in an increase of [Ca^^Jj which was significantly lower in myocytes from aortic-constricted hearts (table 3.13). A second application of 10 mM caffeine after a 2 minute quiescent period produced a smaller rise in [Ca^"^]i (61 ± 58, mean ± SD, n=25, p<0.001 in control group vs 38 ± 44, mean ± SD, n=13 in aortic-constricted group, p>0.05). There was no significant difference in the magnitude of this response between the myocytes isolated from control and aortic- constricted hearts (p>0.05).

29 m M N aC l 5 0 m M N aC l