Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms

Abstract

Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca(2+) concentration ([Ca(2+)](i)) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca(2+) handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (I(Ca,L)) density (-0.964 +/- 0.172 vs. -0.745 +/- 0.128 pA/pF at +10 mV) and Na(+)/Ca(2+) exchanger (NCX) currents (2.1 +/- 0.8 vs. 2.3 +/- 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca(2+)](i) was depressed (at +10 mV, 0.72 +/- 0.07 and 0.56 +/- 0.04 microM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca(2+) spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca(2+) in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the alpha(1C)-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). alpha(1C) protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca(2+) uptake and release synergistically contribute to the depressed [Ca(2+)](i) and the altered AP profile phenotype.

Figure 1

Stimulation frequency dependent differences in the action potential (AP) duration (APD) and membrane resting potential of ventricular myocytes isolated from control and failing ventricles (N_c=12, n_c=18; N_f=5, n_f=11). (A). APD at 50% repolarization (APD₅₀). (B). APD at 90% repolarization (APD₉₀). There is an overall prolongation of AP duration of myocytes isolated from failing compared to control hearts. In this and all subsequent figures circles represent data from control and squares from failing hearts. Mean ± SE, *p<0.05 vs. control.

Figure 2

Records of representative intracellular Ca²⁺ transients (Indo-1 fluorescent ratios 405/485 nm) from control and failing hearts (A), elicited by voltage steps to −40, −20, 0, 20 and 40 mV from a holding voltage of −80 mV. Summary Ca²⁺ transient data are shown in panel B (* p<0.05 failing vs. control). Peak I-V relations for the L-type Ca²⁺ currents are shown in panel C.

Figure 3

Local SR-Ca²⁺ release events (Ca²⁺-sparks) in rabbit ventricular myocytes from control and failing animals. (A) 3D representation of spatiotemporal variations of [Ca²⁺]_i along a 25µm line scan during ∼ 9 seconds. Each frame represents a series of 500 successive scans (3ms/scan; 1.5s/frame); the number of scans was translated into horizontal time scale (horizontal arrow). Ca²⁺ sparks were sampled during the resting period (Frames 1, 2 and 3) following the stimulated twitch (Frame 0). Variations of the ratio of fluorescence intensities F/Fo reflected the local variations of [Ca²⁺]_i as indicated by the pseudo-color bar (see text). (B). Representative sparks in cells isolated from control (left) and failing (right) hearts, respectively. (C) Distribution of the amplitudes of the Ca²⁺ sparks as reflected in the ratio between peak and background. The Ca²⁺ sparks from the failing animals are smaller than the Ca²⁺ sparks from the control animals (p<0.0001, Mann-Whitney test of medians). (D) Comparison of the frequency distribution of spark amplitudes in myocytes from control and failing hearts showed that the two distributions were not statistically different (total number of observations was 474 and 289 in myocytes from control and failing hearts respectively).

Figure 3

Local SR-Ca²⁺ release events (Ca²⁺-sparks) in rabbit ventricular myocytes from control and failing animals. (A) 3D representation of spatiotemporal variations of [Ca²⁺]_i along a 25µm line scan during ∼ 9 seconds. Each frame represents a series of 500 successive scans (3ms/scan; 1.5s/frame); the number of scans was translated into horizontal time scale (horizontal arrow). Ca²⁺ sparks were sampled during the resting period (Frames 1, 2 and 3) following the stimulated twitch (Frame 0). Variations of the ratio of fluorescence intensities F/Fo reflected the local variations of [Ca²⁺]_i as indicated by the pseudo-color bar (see text). (B). Representative sparks in cells isolated from control (left) and failing (right) hearts, respectively. (C) Distribution of the amplitudes of the Ca²⁺ sparks as reflected in the ratio between peak and background. The Ca²⁺ sparks from the failing animals are smaller than the Ca²⁺ sparks from the control animals (p<0.0001, Mann-Whitney test of medians). (D) Comparison of the frequency distribution of spark amplitudes in myocytes from control and failing hearts showed that the two distributions were not statistically different (total number of observations was 474 and 289 in myocytes from control and failing hearts respectively).

Figure 4

Measurement of Ni²⁺-sensitive Na⁺-Ca²⁺ exchanger current density in rabbit ventricular myocytes. (A) Representative current records shown on a slow time scale elicited by the ramp protocol shown (+60 to −120 mV, 90 mV/s, 0.1 Hz, holding at −30 mV). The horizontal bars depict application of 5 mmol/L Ni²⁺. Note the rapid time course of the Ni²⁺-induced block, the reproducibility of the Ni²⁺-insensitive component, and the relative stability of recording without significant rundown. Measurement of Ni²⁺-sensitive Na⁺-Ca²⁺ exchanger current density in rabbit ventricular myocytes isolated from control (B) and failing (C) hearts (top). Representative current traces were elicited by the same ramp protocol used in panel A. Current-voltage relationships before (a) and after (b) application of 5 mmol/L Ni²⁺. (bottom) The difference is the Na⁺-Ca²⁺ exchanger current. (D) There were no differences in the Ni⁺-sensitive current in myocytes isolated from control and failing hearts.

Figure 5

Effect of SR Ca²⁺-ATPase inhibition on Ca²⁺ transients ([Ca²⁺]_i). (A), (B): [Ca²⁺]_i were evoked by 200 msec voltage-clamp steps to +20 mV from a holding potential of −80 mV at a rate of 0.5 Hz in rabbit ventricular myocytes isolated from control (A) and failing (B) ventricular myocardium in the absence and presence of 10 µmol/L thapsigargin. (C) The summarized data (control: N=3 animals, n=13 myocytes; failing: N=3, n=16) demonstrate a substantial increase in the time constant of Ca²⁺ removal from the myocyte (τ_Ca) in myocytes isolated from failing compared to control ventricles under baseline conditions. Blocking the Ca²⁺-ATPase with thapsigargin, however increased the absolute τ_Ca to a comparable value in both groups. (D) There was a slightly but not statistically significant larger increase in τ_Ca in the control group. The relative increase in τ_Ca with thapsigargin was significantly larger in the control compared to failing myocardium (*p<;0.05).

Figure 6

mRNA levels of Ca²⁺ handling proteins in control and failing hearts. (A–D). Scatter plots of normalized α_1c (A), RyR2 (B), SERCA2a (C), PLB (D) mRNA in control and failing hearts measured by kinetic RT-PCR. The steady-state levels of α_1c and RyR2 mRNAs are not altered in the failing compared to the control hearts, while SERCA2a and PLB are significantly decreased in the failing heart. (E) Representative NCX RPA showing the expected bands for the NCX (probe 180 bp, protected fragment 118 bp) and the cardiac Na channel (Nav1.5, probe 170 bp, protected fragment 78 bp) in control and failing hearts. The first lane (P) contains the probes alone and the lane marked t, yeast tRNA and the next six lanes are 10 µg of total RNA from different control and failing ventricles. (F) Scatter plot of normalized NCX mRNA in 10 control and 9 failing hearts, the values plotted are the average of duplicate determinations; the steady-state level of mRNA encoding NCX is not altered in failing compared to control hearts (AU, Arbitrary Units and *p < 0.05).

Figure 7

Protein levels of the major calcium handling proteins in control and failing hearts. Representative Western blots and scatter plots of the α_1c subunit of the Ca channel (A), RyR (B), SERCA2a (C), PLB (D), NCX (E) in control and failing hearts. Scatter plots for each of the Ca²⁺ handling proteins demonstrate a significant down regulation of immunoreactive RyR, SERCA2a, PLB monomer and an up regulation of NCX. There is no change in immunoreactive α_1c (A). (AU, Arbitrary Units and *p < 0.05).