Synergistic interactions between Ca2+ entries through L-type Ca2+ channels and Na+-Ca2+ exchanger in normal and failing rat heart

Abstract

We used confocal Ca2+ imaging and the patch-clamp technique to investigate the interplay between Ca2+ entries through L-type Ca2+ channels (LCCs) and reverse-mode Na+-Ca2+ exchange (NCX) in activating Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) in cardiac myocytes from normal and failing rat hearts. In normal myocytes exposed to N(6),2'-O-dibutyryl adenosine-3',5'-cyclic monophosphate (db-cAMP, membrane-permeable form of cAMP), the bell-shaped voltage dependence of cytosolic Ca2+ transients was dramatically broadened due to activation of SR Ca2+ release at high membrane potentials (30-120 mV). This broadening of Ca2+-transient voltage dependence could be prevented by KB-R7943, an inhibitor of the reverse-mode NCX. Trans-sarcolemmal Ca2+ entries were measured fluorometrically in myocytes during depolarizing steps to high membrane potentials. The total Ca2+ entry (deltaF(Tot)) was separated into two Ca2+ entry components, LCC-mediated (deltaF(LCC)) and NCX-mediated (deltaF(NCX)), by exposing the cells to the specific inhibitors of LCCs and reverse-mode NCX, nifedipine and KB-R7943, respectively. In the absence of protein kinase A (PKA) stimulation the amplitude of the Ca2+-inflow signal (deltaF(Tot)) corresponded to the arithmetic sum of the amplitudes of the KB-R7943- and nifedipine-resistant components (deltaF(Tot)=deltaF(LCC)+deltaF(NCX)). PKA activation resulted in significant increases in deltaF(Tot) and deltaF(LCC). Paradoxically, deltaF(Tot) became approximately threefold larger than the sum of the deltaF(NCX) and deltaF(LCC) components. In myocytes from failing hearts, stimulation of PKA failed to induce a shift in Ca2+ release voltage dependence toward more positive membrane potentials. Although the total and NCX-mediated Ca2+ entries were increased again, deltaF(Tot) did not significantly exceed the sum of deltaF(LCC) and deltaF(NCX). We conclude that the LCC and NCX Ca2+-entry pathways interact synergistically to trigger SR Ca2+ release on depolarization to positive membrane potentials in PKA-stimulated cardiac muscle. In heart failure, this new form of Ca2+ release is diminished and may potentially account for the compromised contractile performance and reduced functional reserve in failing hearts.

Figure 1. Effects of db-cAMP on Ca²⁺ currentI_Ca, intracellular Ca²⁺ transients, and SR Ca²⁺ load in myocytes from normal hearts

A, representative recordings of fluorescence intensity (F/F₀) changes and whole-cell currents from a myocyte during depolarizing steps to 0, 30 and 90 mV from a holding potential of −50 mV under reference conditions (top panel), after exposure of the cell to 0.3 mm db-cAMP (middle panel), and after application of 10 μm KB-R7943 (bottom panel). B, voltage dependence of Ca²⁺ current (I_Ca) and intracellular Ca²⁺ transients in myocytes. Data points are means ± s.e.m. (n = 6–17). C, representative recordings of caffeine-induced intracellular Ca²⁺ transients (upper traces) and Na⁺–Ca²⁺ exchange current (I_NCX) (lower traces) in myocytes under reference conditions, after addition db-cAMP, and in the presence of both db-cAMP and KB-R7943.

Figure 2. Effects of db-cAMP on trans-sarcolemmal Ca²⁺ entry in myocytes from normal hearts

A, representative line-scan images and time-dependent profiles of Ca²⁺ changes recorded before (upper panels) and after exposure of the myocyte to db-cAMP (lower panels) during depolarizing steps to 0, 30 and 90 mV from a holding potential of −50 mV. B, voltage dependence of the amplitude of the Ca²⁺-entry signals measured under reference conditions and in the presence of db-cAMP at the end of the depolarization steps. Data points are means ± s.e.m. (n = 6–9).

Figure 3. Pharmacological analysis of trans-sarcolemmal Ca²⁺ entries in myocytes from normal hearts

A, representative recordings of intracellular Ca²⁺ changes during depolarizing steps to 90 mV from a holding potential of −50 mV under different experimental conditions. From top to bottom: reference (corresponding to ΔF_Tot), in the presence of 10 μm KB-R7943 (corresponding to ΔF_LCC), in the presence of 5 μm nifedipine (corresponding to ΔF_NCX), and in the presence of 10 μm KB-R7943 + 5 μm nifedipine (both ΔF_NCX and ΔF_LCC are blocked). B, bar plot summarizing data. Data points are means ± s.e.m. (n = 5–7). Black bars inside the hatched and grey bars for reference conditions denote the arithmetic sum of the peak amplitudes of KB-R7943- and nifedipine-insensitive components in the presence or absence of db-cAMP, respectively.

Figure 4. Effects of db-cAMP on I_Ca, intracellular Ca²⁺ transients and SR Ca²⁺ load in myocytes from hearts with isoproterenol (ISO)-induced HF

A, representative recordings of F/F₀ changes and whole-cell currents from a myocyte during depolarizing steps to 0, 30 and 90 mV from a holding potential of −50 mV under reference conditions (top panel), after exposure of the cell to 0.3 mm db-cAMP (middle panel), and after application of 10 μm KB-R7943 (bottom panel). B, voltage dependence of I_Ca and intracellular Ca²⁺ transients in myocytes. Data points are means ± s.e.m. (n = 4–7). C, representative recordings of caffeine-induced intracellular Ca²⁺ transients (upper traces) and I_NCX (lower traces) in myocytes before (reference) and after addition db-cAMP, and following execution of a loading protocol to increase SR Ca²⁺ content.

Figure 5. Pharmacological analysis of trans-sarcolemmal Ca²⁺ entries in myocytes from hearts with ISO-induced HF

A, representative recordings of intracellular Ca²⁺ changes during depolarizing steps to 90 mV from a holding potential of −50 mV under different experimental conditions. From top to bottom: reference (corresponding to ΔF_Tot), in the presence of 10 μm KB-R7943 (corresponding to ΔF_LCC), in the presence of 5 μm nifedipine (corresponding to ΔF_NCX), and in the presence of 10 μm KB-R7943 + 5 μm nifedipine (both ΔF_NCX and ΔF_LCC are blocked). B, bar plot summarizing the data. Data points are means ± s.e.m. (n = 4–6). Black bars inside the hatched and grey bars for reference conditions denote the arithmetic sum of the peak amplitudes of KB-R7943- and nifedipine-insensitive components in the presence or absence of db-cAMP, respectively.

Figure 6. Effects of db-cAMP on I_Ca, intracellular Ca²⁺ transients and SR Ca²⁺ load in myocytes from hearts with overload-induced HF

A, representative recordings of F/F₀ changes and whole-cell currents from a myocyte during depolarizing steps to 0, 30 and 90 mV from a holding potential of −50 mV under reference conditions (top panel), after exposure of the cell to 0.3 mm db-cAMP (middle panel), and after application of 10 μm KB-R7943 (bottom panel). B, voltage dependence of I_Ca and intracellular Ca²⁺ transients in myocytes. Data points are means ± s.e.m. (n = 4–6). C, representative recordings of caffeine-induced intracellular Ca²⁺ transients (upper traces) and I_NCX (lower traces) in myocytes before (reference) and after addition db-cAMP, and following execution of a loading protocol to increase SR Ca²⁺ content.