In vivo cooling-induced intracellular Ca2+ elevation and tension in rat skeletal muscle

Abstract

It is an open question as to whether cooling-induced muscle contraction occurs in the in vivo environment. In this investigation, we tested the hypotheses that a rise in intracellular Ca²⁺ concentration ([Ca²⁺]i) and concomitant muscle contraction could be evoked in vivo by reducing muscle temperature and that this phenomenon would be facilitated or inhibited by specific pharmacological interventions designed to impact Ca²⁺-induced Ca²⁺-release (CICR). Progressive temperature reductions were imposed on the spinotrapezius muscle of Wistar rats under isoflurane anesthesia by means of cold fluid immersion. The magnitude, location, and temporal profile of [Ca²⁺]i were estimated using fura-2 loading. Caffeine (1.25-5.0 mM) and procaine (1.6-25.6 mM) loading were applied in separatum to evaluate response plasticity by promoting or inhibiting CICR, respectively. Lowering the temperature of the muscle surface to ~5°C produced active tension and discrete sites with elevated [Ca²⁺]i. This [Ca²⁺]i elevation differed in magnitude from fiber to fiber and also from site to site within a fiber. Caffeine at 1.25 and 5.0 mM reduced the magnitude of cooling necessary to elevate [Ca²⁺]i (i.e., from ~5°C to ~8 and ~16°C, respectively, both p < 0.05) and tension. Conversely, 25.6 mM procaine lowered the temperature at which [Ca²⁺]i elevation and tension were detected to ~2°C (p < 0.05). Herein we demonstrate the spatial and temporal relationship between cooling-induced [Ca²⁺]i elevation and muscle contractile force in vivo and the plasticity of these responses with CICR promotion and inhibition.

Keywords: caffeine; calcium-induced calcium release; rapid cooling contracture; temperature.

FIGURE 1

In vivo cooling‐induced [Ca²⁺]i elevation and contraction in rat spinotrapezius. Representative changes of [Ca²⁺]i and tension across a cooling‐rewarming cycle (i.e., ~20–0–20°C) over 180 s. The temporal profile of fura‐2 ratio responses are shown sequentially and labeled for time in the left side two panel photomicrograph columns (a). The fura‐2 ratio values (scale given) were averaged over a fixed area in 10 muscle fibers each of which is color‐coded and corresponds between the upper right photomicrograph and the lower graph (b). Bars = 100 µm

FIGURE 2

Potentiating effect of caffeine on cooling‐induced [Ca²⁺]i elevation and tension. Representative photomicrographs at 10°C in muscle fibers loaded with fura‐2 are depicted for increasing caffeine concentration from left to right (top panels). The left‐hand graph is a representative example of the overall average fura‐2 ratio (a) or absolute tension increase (d) plotted as a function of temperature change for each caffeine concentration. The center graphs show the fura‐2 ratio (b) and tension (e) for each caffeine concentration at 5, 10, 15, and 20°C. In the graphs at right, the temperature threshold at which high [Ca²⁺]i (c) and tension (f) increase are detected is demonstrated to increase progressively with higher caffeine concentration relative to control (i.e., 0 mM; n = 7 muscles). p < 0.05 was considered statistically significant. Values are means ± SEs. CAF, caffeine group. Bars = 100 µm

FIGURE 3

Inhibitory effect of procaine on cooling‐induced [Ca²⁺]i elevation and tension. Representative photomicrographs at 4°C in muscle fibers loaded with fura‐2 are depicted for increasing procaine concentration (left to right; top panels). The left‐hand graph is a representative example of the overall average fura‐2 ratio (a) or absolute tension increase (b) plotted as a function of temperature change for each procaine concentration. The center graphs show the fura‐2 ratio (c) and tension (d) for each procaine concentration at 2, 4, 6, and 8°C. In the graphs at right, the temperature threshold at which high[Ca²⁺]i (e) and tension (f) increase are detected is demonstrated to decrease progressively with higher procaine concentration relative to control (i.e., 0 mM; n = 6 muscles). p < 0.05 was considered statistically significant. Values are means ± SEs. PRO, procaine group. Bars = 100 µm