Balanced changes in Ca buffering by SERCA and troponin contribute to Ca handling during β-adrenergic stimulation in cardiac myocytes

Abstract

Aims: During activation of cardiac myocytes, less than 1% of cytosolic Ca is free; the rest is bound to buffers, largely SERCA, and troponin C. Signalling by phosphorylation, as occurs during β-adrenergic stimulation, changes the Ca-binding affinity of these proteins and may affect the systolic Ca transient. Our aim was to determine the effects of β-adrenergic stimulation on Ca buffering and to differentiate between the roles of SERCA and troponin.

Methods and results: Ca buffering was studied in cardiac myocytes from mice: wild-type (WT), phospholamban-knockout (PLN-KO), and mice expressing slow skeletal troponin I (ssTnI) that is not protein kinase A phosphorylatable. WT cells showed no change in Ca buffering in response to the β-adrenoceptor agonist isoproterenol (ISO). However, ISO decreased Ca buffering in PLN-KO myocytes, presumably unmasking the role of troponin. This effect was confirmed in WT cells in which SERCA activity was blocked with the application of thapsigargin. In contrast, ISO increased Ca buffering in ssTnI cells, presumably revealing the effect of an increase in Ca binding to SERCA.

Conclusions: These data indicate the individual roles played by SERCA and troponin in Ca buffering during β-adrenergic stimulation and that these two buffers effectively counterbalance each other so that Ca buffering remains constant during β-adrenergic stimulation, a factor which may be physiologically important. This study also emphasizes the importance of taking into account Ca buffering, particularly in disease states where Ca binding to myofilaments or SERCA may be altered.

Keywords: Buffering; Calcium; Phospholamban; SERCA; Troponin.

Figure 1

The effects of ISO on Ca signalling in ventricular myocytes from WT and PLN-KO mice. (A) Timecourse of effects on a WT cell. The cell was stimulated with voltage-clamp pulses. Caffeine/BDM (caff) and ISO (100 nM) were applied as shown. (B) Expanded records of the effects of applying caffeine (5 mM, for the period shown by the horizontal bars) to measure SR Ca content. Traces show (from top to bottom): Ca, membrane current, and integrated current. (C) Timecourse of effects on a PLN-KO cell. (D) Expanded records of the effects of applying caffeine/BDM to measure SR Ca content. (E) Amplitude of the systolic Ca transient. (F) Rate constant of decay of the systolic Ca transient. (G) Amplitude of the caffeine-evoked increase of Ca. (H) Calculated SR Ca content. *P < 0.05; **P < 0.01; ***P < 0.001. PLN-KO: n = 8–9 cells/6 animals; WT: n = 12 cells/5 animals.

Figure 2

The effects of ISO on Ca buffering in WT and PLN-KO myocytes. (A) Buffer curves derived from caffeine-evoked currents. In both panels, data from control and ISO (100 nM) are shown with linear regressions. Left-hand panel shows WT and right-hand PLN-KO. (B) Mean data showing the effects of ISO on the buffer slope in WT (left) and PLN-KO (right). (C) The effects of ISO on buffer power. The bars show the ratio of buffer power in ISO/buffer power in control for WT (left) and PLN-KO (right). (D) The calculated increase of cytoplasmic total Ca during systole. Solid bars, control; open, ISO. (E) Quantitative analysis of Ca fluxes. The two left-hand columns are WT and the right-hand PLN-KO myocytes. From top to bottom: [Ca²⁺]_i; current; net change of cell Ca (calculated from Ca influx and efflux); change of total cytoplasmic Ca; change of SR Ca. **P < 0.01; ***P < 0.001. PLN-KO: n = 9–11 cells/6 animals; WT: n = 5–12 cells/4–6 animals.

Figure 3

The effects of thapsigargin on Ca handling and buffering. (A) Timecourse. The cell was stimulated with voltage-clamp pulses in the presence of ISO (100 nM). Caffeine/BDM (caff) or thapsigargin (Thap, 5 µM) was applied as indicated. Stimulation was stopped during these periods. (B) Measurement of SR Ca content and Ca buffering. The three traces show R/R_rest, membrane current, and calculated change of total SR Ca. The left-hand panel was obtained in ISO and the right following 90 s exposure to thapsigargin (5 µM), horizontal bars represent the application of caffeine/BDM. (C) Specimen, normalized Ca transients obtained at the times shown by arrows in A. (D) SR Ca content in control (left) and thapsigargin (right). (E) The bottom graph shows the buffer curves obtained in ISO and thapsigargin with linear regressions. (F) Slope of buffer curve in ISO (left) and thapsigargin (right). *P < 0.05; ***P < 0.001, n = 10 cells/3 animals.

Figure 4

Comparison of the effects of ISO in ssTnI and WT mice. (A) The effects of ISO (100 nM) on systolic Ca in WT (top) and ssTnI ventricular myocytes (bottom). (B) Mean data for the effects of ISO on the amplitude and rate constant of decay (C) of the systolic Ca transient. (D) Measurement of SR Ca content and Ca buffering. Traces show (from top to bottom): R/R_rest, membrane current, and change of total Ca. The horizontal bars represent the application of caffeine/BDM (caff). (E) The effects of ISO on SR Ca content WT (left) and ssTnI (right). (F) Buffer curves from control (Con) and ISO with linear regressions. (G) The effects of ISO on the buffer slopes in WT (left) and ssTnI (right). (H) Mean data for the effects of ISO on the change in Ca_T for WT (left) and ssTnI (right). *P < 0.05; **P < 0.01; ***P < 0.001. WT: n = 8 cells/6 animals; ssTnI: n = 10 cells/5 animals.