Ca 2+ ‘Sparks’ and Waves in Intact Ventricular Muscle Resolved by Confocal Imaging

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Abstract
The [Ca2+]i transient in heart is now thought to involve the recruitment and summation of discrete and independent “units” of Ca2+ release (Ca2+ “sparks”) from the sarcoplasmic reticulum, each of which is controlled locally by single coassociated L-type Ca2+ channels (“local control theory of excitation-contraction coupling”). All prior studies on Ca2+ sparks, however, have been performed in single enzymatically dissociated heart cells under nonphysiological conditions. In order to understand the possible significance of Ca2+ sparks to normal working cardiac muscle, we used confocal microscopy to record Ca2+ sparks, which are spatially averaged [Ca2+]i transients (and Ca2+ waves), in individual cells of intact rat right ventricular trabeculae (composed of 2+ indicator fluo 3 under physiological conditions ([Ca2+]o, 1 mmol/L; temperature, 33±1°C). Twitch force was recorded simultaneously. When stretched to optimal length (sarcomere length, 2.2 μm) and stimulated at 0.2 Hz, the trabeculae generated ≈700 μg of force per cell. Spatially averaged [Ca2+]i transients recorded from individual cells within a trabecula were similar to those recorded previously from single cells. The amplitude distribution of the peak ratio of Ca2+ sparks was bimodal, with maxima at ratios of 1.8±0.3 and 2.7±0.2 (mean±SD), respectively. The amplitude of the peak of Ca2+ sparks was ≈170 nmol/L. Ca2+ sparks occurred at a frequency of 12.0±0.8/s (mean±SEM) in line scans covering 94 sarcomeres. Ca2+ waves occurred randomly at a frequency of 0.57±0.08/s and propagated with a velocity of 29.5±1.7 μm/s. The extent of Ca2+ wave propagation was 3.9±0.3 sarcomere lengths (sarcomere length, 2.2 μm). Ca2+ sparks could be identified along the leading edge of the waves at intervals of 1.30±0.11 sarcomere length. Our observations suggest that (1) Ca2+ sparks, similar to those recorded in single cells, occur in trabeculae under physiological conditions and (2) coupling of Ca2+ spark generation between neighboring sites occurs and may lead to (3) the development of Ca2+ waves, which propagate under physiological conditions at a low velocity over limited distances. The results suggest that concepts of excitation-contraction coupling recently derived from isolated myocytes are applicable to intact cardiac trabeculae.