Differential effects of phospholamban and Ca²⁺/calmodulin- dependent kinase II on [Ca²⁺]_i transients in cardiac myocytes at physiological stimulation frequencies

In cardiac myocytes, the activity of the Ca²⁺/calmodulin- dependent protein kinase II (CaMKII) is hypothesized to regulate Ca²⁺ release from and Ca²⁺ uptake into the sarcoplasmic reticulum via the phosphorylation of the ryanodine receptor 2 and phospholamban (PLN), respectively. We tested the role of CaMKII and PLN on the frequency adaptation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) transients in nearly 500 isolated cardiac myocytes from transgenic mice chronically expressing a specific CaMKII inhibitor, interbred into wild-type or PLN null backgrounds under physiologically relevant pacing conditions (frequencies from 0.2 to 10 Hz and at 37°C). When compared with that of mice lacking PLN only, the combined chronic CaMKII inhibition and PLN ablation decreased the maximum Ca²⁺ release rate by more than 50% at 10 Hz. Although PLN ablation increased the rate of Ca²⁺ uptake at all frequencies, its combination with CaMKII inhibition did not prevent a frequency-dependent reduction of the amplitude and the duration of the [Ca ²⁺]_i transient. High stimulation frequencies in the physiological range diminished the effects of PLN ablation on the decay time constant and on the maximum decay rate of the [Ca²⁺]_i transient, indicating that the PLN-mediated feedback on [Ca²⁺] _i removal is limited by high stimulation frequencies. Taken together, our results suggest that in isolated mouse ventricular cardiac myocytes, the combined chronic CaMKII inhibition and PLN ablation slowed Ca²⁺ release at physiological frequencies: the frequency-dependent decay of the amplitude and shortening of the [Ca²⁺]_i transient occurs independent of chronic CaMKII inhibition and PLN ablation, and the PLN-mediated regulation of Ca²⁺ uptake is diminished at higher stimulation frequencies within the physiological range.