Impaired β-adrenergic responsiveness accentuates dysfunctional excitation-contraction coupling in an ovine model of tachypacing-induced heart failure

Abstract

Reduced inotropic responsiveness is characteristic of heart failure (HF). This study determined the cellular Ca2+ homeostatic and molecular mechanisms causing the blunted β-adrenergic (β-AR) response in HF.We induced HF by tachypacing in sheep; intracellular Ca2+ concentration was measured in voltage-clamped ventricular myocytes. In HF, Ca2+ transient amplitude and peak L-type Ca2+ current (ICa-L) were reduced (to 70 ± 11% and 50 ± 3.7% of control, respectively, P <0.05) whereas sarcoplasmic reticulum (SR) Ca2+ content was unchanged. β-AR stimulation with isoprenaline (ISO) increased Ca2+ transient amplitude, ICa-L and SRCa2+ content in both cell types; however, the response of HF cells was markedly diminished (P <0.05).Western blotting revealed an increase in protein phosphatase levels (PP1, 158 ± 17% and PP2A, 188 ± 34% of control, P <0.05) and reduced phosphorylation of phospholamban in HF (Ser16, 30 ± 10% and Thr17, 41 ± 15% of control, P <0.05). The β-AR receptor kinase GRK-2 was also increased in HF (173 ± 38% of control, P <0.05). In HF, activation of adenylyl cyclase with forskolin rescued the Ca2+ transient, SR Ca2+ content and SR Ca2+ uptake rate to the same levels as control cells in ISO. In conclusion, the reduced responsiveness of the myocardium to β-AR agonists in HF probably arises as a consequence of impaired phosphorylation of key intracellular proteins responsible for regulating the SR Ca2+ content and therefore failure of the systolic Ca2+ transient to increase appropriately during β-AR stimulation.

Figure 1. Altered intracellular Ca²⁺ homeostasis in heart failure

Aa, representative systolic Ca²⁺ transients from control (left) and HF (right); Ab, normalised and superimposed Ca²⁺ transients from control (black) and HF (red); Ac, mean data showing unaltered rate of decay of the systolic Ca²⁺ transient in HF (n = 22–28 cells, 9–14 hearts). B, assessment of SR Ca²⁺ content through application of 20 mmol l⁻¹ caffeine as indicated showing the caffeine-induced rise of [Ca²⁺]_i (upper panel), associated NCX current (lower panel) and mean data for SR Ca²⁺ content (right panel, n = 35–52 cells, 11–17 hearts). Ca, representative I_Ca-L records normalised for cell capacitance obtained during a 100 ms test step from −40 mV to 10 mV; Cb, mean I_Ca-L current–voltage data for control (black circles) and HF (red triangles) cells.

Figure 2. Reduction of I_Ca-L decreases Ca²⁺ transient amplitude

A, time course showing typical response of a single ventricular myocyte to the application of 1 μmol l⁻¹ nicardipine (open horizontal bar). Ca²⁺ transient amplitude (upper) and peak I_Ca-L (lower) are reduced during nicardipine application (open symbols). The line through the nicardipine data is a single exponential fit to the data for clarity. B, mean data for the effects of nicardipine on (i) Ca²⁺ transient amplitude and (ii) peak I_Ca-L. Filled bars represent control (pre-drug) data and hashed bars the steady-state nicardipine effects. *P < 0.05 vs. control, n = 12 cells.

Figure 3. Impaired response to β-AR stimulation in heart failure

A, time course showing positive inotropic effects of 100 nmol l⁻¹ and 1 μmol l⁻¹ ISO in representative control (left) and HF (right) cells (note difference in ordinate ranges). B, summary data showing the effect of 100 nmol l⁻¹ ISO on (i) systolic Ca²⁺ transient amplitude, (ii) SR Ca²⁺ content, and (iii) the SR-dependent rate of Ca²⁺ removal (k_SR, n = 13–33 cells from 6–16 hearts). Ca, representative Western blots for SERCA2a, total phospholamban and calsequestrin; Cb, summary showing protein expression for SERCA2a (SERCA), phospholamban (PLN) and calsequestrin (CSQ): c denotes control and f denotes failing data. Cc, mean data summarising the SERCA2a to phospholamban ratio in control and heart failure tissues (n = 6 control and 7 failing hearts). *P < 0.05 vs. control samples; §P < 0.05 vs. before ISO addition.

Figure 4. Impaired augmentation of I_Ca-L to β-AR stimulation in heart failure

Aa, representative current recordings normalised to cellular capacitance obtained under basal conditions (continuous lines) and in the presence of 100 nmol l⁻¹ ISO (dashed lines) for control (upper) and heart failure cells (lower panel) during 100 ms steps from −40 to 10 mV at 0. Hz (note different ordinate scales); Ab, summary data for I_Ca-L response to ISO under conditions described in a. Ba, voltage protocol (upper panel) to determine current–voltage relationships in control cells (middle panel) and HF cells (lower panel) under basal conditions (filled symbols, n = 22–30 cells, 9–10 hearts) and in the presence of 100 nmol l⁻¹ ISO (open symbols, n = 9–12 cells, 5–7 hearts); Bb, normalised activation plots under basal conditions (filled symbols and continuous lines) and in the presence of 100 nmol l⁻¹ ISO (open symbols, dashed lines) for control (•, ○) and HF (▾, ▿) cells. Ca, voltage protocol to determine the voltage dependence of I_Ca-L inactivation (upper panel) and mean normalised inactivation plots under basal conditions and in the presence of 100 nmol l⁻¹ ISO (symbols as for panel B); Cb, calculated I_Ca-L window currents under basal conditions (con) and in the presence of 100 nmol l⁻¹ ISO for control (black) and HF cells (red, n = 4–15 cells, 3–6 hearts). *P < 0.05 vs. control cells; §P < 0.05 vs. before ISO application.

Figure 5. Altered protein expression in heart failure

A, representative phosphospecific Western blots (left) and summary data (right) for Ser16 (a) and Thr17 phosphorylated phospholamban (b). B, representative Western blots (upper) and summary data (lower panels) for protein phosphatase 1 (PP1, a) and protein phosphatase 2A (PP2A, b). C, representative Western blot (upper) and summary data (lower panel) for G-protein receptor kinase 2 (GRK-2). Da, representative gel image and summary data assessing PKA activity; non-P and phos denote non-phosphorylated and phosphorylated (activated) PKA substrate, respectively. Db, representative Western blot and summary data for CAMKIIδ expression. E, representative Western blots and summary data for total RyR2 (a) and Ser2808-phosphorylated RyR (b). *P < 0.05; §P < 0.005 vs. control hearts. n = 6 control and 7 failing hearts.

Figure 6. Direct activation of adenylyl cyclase restores the systolic Ca²⁺ transient in heart failure

A, time course showing systolic Ca²⁺ transients in the presence of 100 nmol l⁻¹ isoprenaline and effect of 3 μmol l⁻¹ forskolin applied as indicated in a representative control (a) and heart failure (b) cell. B, mean data summarising the effects of isoprenaline (ISO, open bars) and 3 μmol l⁻¹ forskolin (FSK, hashed bars) on Ca²⁺ transient amplitude (a), SR Ca²⁺ content (b) and k_SR (c) in isoprenaline in control (black) and HF cells (red, n = 7–20 cells, 3–10 hearts). *P < 0.05 vs. control cell under same conditions. §P < 0.05 vs. ISO in same cell.