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Myofilament Ca2+ sensitivity in intact versus skinned rat ventricular muscle
W D Gao1, P H Backx, M Azan-Backx
1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Circulation Research
|March 1, 1994
Summary
Skinning rat trabeculae alters myofilament calcium sensitivity, even with magnesium adjustments. This study quantifies changes in force-calcium relations after skinning, revealing persistent differences from intact muscle.
Area of Science:
- Cardiovascular Physiology
- Muscle Contraction Mechanisms
- Biochemistry
Background:
- Understanding myofilament calcium sensitivity is crucial for cardiac function.
- Skinning procedures are used to study isolated contractile proteins but may alter cellular properties.
- Previous studies have not fully elucidated the impact of skinning on calcium-gated force generation in cardiac muscle.
Purpose of the Study:
- To compare the calcium (Ca2+) sensitivity of myofilaments in intact versus skinned rat trabeculae.
- To investigate the influence of magnesium (Mg2+) concentration on the force-calcium relationship in skinned cardiac muscle.
- To determine if skinning alters the cooperative binding of Ca2+ to myofilaments.
Main Methods:
- Rat right ventricular trabeculae were loaded with fura-2 salt for intracellular calcium ([Ca2+]i) determination.
- Steady-state activation was achieved using ryanodine and electrical stimulation.
- Muscles were skinned using Triton X-100 and subsequently activated with solutions of varying [Ca2+].
- Force-calcium relationships were analyzed for cooperativity (Hill coefficient) and half-maximal activation (K1/2).
Main Results:
- Intact trabeculae exhibited high cooperativity (Hill coefficient, 4.87) and low K1/2 (0.62 µmol/L).
- After skinning, cooperativity decreased (Hill coefficient, 2.72) and K1/2 shifted rightward (2.2 µmol/L) with 1.2 mmol/L free Mg2+.
- Adjusting Mg2+ to 0.5 mmol/L partially restored Ca2+ sensitivity (K1/2 = 0.93 µmol/L, Hill coefficient = 3.75) but did not fully replicate intact muscle conditions.
Conclusions:
- Skinning significantly alters the Ca2+ sensitivity and cooperativity of cardiac myofilaments.
- While Mg2+ influences the force-calcium relationship in skinned preparations, it does not fully account for the changes induced by skinning.
- These findings highlight the need for careful consideration of skinning artifacts when interpreting studies on isolated cardiac muscle contractility.