The effect of Ca²⁺-calmodulin-dependent protein kinase II on cardiac excitation-contraction coupling in ferret ventricular myocytes

1. The effect of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) on excitation-contraction coupling (E-C coupling) was studied in intact ferret cardiac myocytes using the selective inhibitor KN-93. KN-93 decreased steady-state (SS) twitch [Ca²⁺](i) (by 51%), resting Ca²⁺ spark frequency (by 88%) and SS sarcoplasmic reticulum (SR) Ca²⁺ content evaluated by caffeine application (by 37.5%). 2. Increasing extracellular Ca²⁺ concentration ([Ca²⁺](o)) to 5 mM in KN-93 restored SR Ca²⁺ load and Ca²⁺ spark frequency towards that in control (2 mM Ca(o)²⁺), but SS twitch [Ca²⁺](i) was still significantly depressed by KN-93. 3. KN-93 decreased Ca²⁺ transient amplitude of SS twitches much more strongly than the amplitude of post-rest (PR) twitches. In the control, the time constant (τ) of [Ca²⁺](i) decline of SS twitches was faster than that for PR twitches. This stimulation-dependent acceleration of [Ca²⁺](i) decline was abolished by KN-93. 4. Voltage-clamp experiments demonstrated that KN-93 significantly inhibited sarcolemmal I-type Ca²⁺ current (I(Ca)) during repetitive pulses by slowing recovery from inactivation. This may explain the preferential action of KN-93 to suppress SS vs. PR twitches. 5. In KN-93, even when both I(Ca) and SR Ca²⁺ load were matched to the control levels by manipulation of conditioning voltage-clamp pulses, contraction and twitch Ca²⁺ transients were still both significantly depressed (to 39 and 49% of control, respectively). 6. Since KN-93 reduced SR Ca²⁺ release channel (RyR) activity during E-C coupling, even for matched SR Ca²⁺ load and trigger I(Ca), we infer that endogenous CaMKII is an important modulator of E-C coupling in intact cardiac myocytes. Effects of KN-93 on I(Ca) and SS twitch [Ca²⁺](i) decline also indicate that endogenous CaMKII may have stimulatory effects on I(Ca) and SR Ca²⁺ uptake.