Impaired cardiac contractility in mice lacking both the AE3 Cl-/HCO3- exchanger and the NKCC1 Na+-K+-2Cl- cotransporter: effects on Ca2+ handling and protein phosphatases

Abstract

To analyze the cardiac functions of AE3, we disrupted its gene (Slc4a3) in mice. Cl(-)/HCO3(-) exchange coupled with Na+-dependent acid extrusion can mediate pH-neutral Na+ uptake, potentially affecting Ca2+ handling via effects on Na+/Ca2+ exchange. AE3 null mice appeared normal, however, and AE3 ablation had no effect on ischemia-reperfusion injury in isolated hearts or cardiac performance in vivo. The NKCC1 Na+-K+-2Cl(-) cotransporter also mediates Na+ uptake, and loss of NKCC1 alone does not impair contractility. To further stress the AE3-deficient myocardium, we combined the AE3 and NKCC1 knock-outs. Double knock-outs had impaired contraction and relaxation both in vivo and in isolated ventricular myocytes. Ca2+ transients revealed an apparent increase in Ca2+ clearance in double null cells. This was unlikely to result from increased Ca2+ sequestration, since the ratio of phosphorylated phospholamban to total phospholamban was sharply reduced in all three mutant hearts. Instead, Na+/Ca2+ exchanger activity was found to be enhanced in double null cells. Systolic Ca2+ was unaltered, however, suggesting more direct effects on the contractile apparatus of double null myocytes. Expression of the catalytic subunit of protein phosphatase 1 was increased in all mutant hearts. There was also a dramatic reversal, between single null and double null hearts, in the carboxymethylation and localization to the myofibrillar fraction, of the catalytic subunit of protein phosphatase 2A, which corresponded to the loss of normal contractility in double null hearts. These data show that AE3 and NKCC1 affect Ca2+ handling, PLN regulation, and expression and localization of major cardiac phosphatases and that their combined loss impairs cardiac function.

FIGURE 1.

AE3 gene targeting, genotyping, and mRNA analysis. A, targeting strategy. Top, intron-exon organization of the wild-type allele, with filled boxes indicating exons. Middle, targeting construct, with the neomycin resistance gene (NEO) replacing exon 6, the first exon of the heart-specific variant (exon C1), and exon 7. The herpes simplex virus thymidine kinase gene (TK) was included for negative selection. Bottom, structure of the targeted allele. The locations of the wild-type 4.4- and 3.3-kb BamHI fragments and the mutant 7.4-kb BamHI fragment are shown above the corresponding allele. The locations of the diagnostic probes are indicated below the targeted allele. SmaI (S) and BamHI (B) restriction sites used for preparation of the construct and probes are shown in each allele. B, Southern blot analysis of BamHI-digested wild-type (+/+) and targeted (+/–) embryonic stem cell DNA using the outside probe (left) and tail DNA using the inside probe (right). C, PCR genotyping revealed the presence of wild-type, heterozygous, and null mutant offspring (785 bp, wild-type allele; 605 bp, mutant allele). D, RNA (10 μg/lane) from brain and heart of wild-type (+/+) and null mutant (–/–) mice was hybridized with an AE3 cDNA probe corresponding to sequences from exons 8–14.

FIGURE 2.

Effects of AE3 ablation on cardiac function during ischemia and reperfusion. Hearts from WT and AE3^–/– mice of the mixed 129SvJ and Black Swiss background, retrogradely perfused in a Langendorff apparatus, were subjected to a 25-min equilibration period (Eq), followed by a 30-min period of no-flow ischemia (shaded boxes) and a 30-min period of reperfusion. The hearts were paced at 400 beats/min during the initial equilibration; pacing was terminated during ischemia and then reinitiated 3 min after the start of the reperfusion period. Loss of AE3 did not elicit cardioprotective effects during ischemia and reperfusion. No significant differences were observed between wild-type and AE3 null hearts (n = 6 of each genotype), with respect to left ventricular developed pressure (LVDP)(A) or LVEDP (B). Values are means ± S.E.

FIGURE 3.

Cardiovascular performance of wild-type and AE3 null mutant mice. Pressure measurements from the left ventricle and right femoral artery were obtained from anesthetized adult mice of both the mixed and FVB/N backgrounds under control conditions and in response to increasing levels of β-adrenergic stimulation (intravenous infusion of dobutamine). Results shown include heart rate (A), mean arterial pressure (B), systolic left ventricular pressure (C), left ventricular end-diastolic pressure (D), maximum dP/dt (E), and dP/dt₄₀ (F)(dP/dt calculated at an intraventricular pressure of 40 mm Hg during the contractile phase). For each genotype, n = 6 mice of the mixed background and 7 mice of the FVB/N background. Values shown are means ± S.E. No significant differences were observed between WT and AE3^–/– mice. Although there was a difference in the dP/dt₄₀ values between the two mouse strains (data not shown), there was no significant difference between WT and AE3^–/– mice in either strain. HR, heart rate.

FIGURE 4.

Cardiovascular performance of wild-type and AE3/NKCC1 double null mutant mice. Experiments were performed as in Fig. 3, using mice of the mixed 129SvJ and Black Swiss background. Heart rate (A), mean arterial pressure (B), left ventricular systolic pressure (C), and left ventricular end diastolic pressure (D), were determined for wild-type and double null mice (n = 7 for each genotype) under basal conditions and in response to increasing β-adrenergic stimulation. Values are means ± S.E. *, significant group effect between wild-type and double null mice for MAP (p < 0.007) and systolic LVP (p < 0.01). No significant differences were observed for heart rate (HR)(p = 0.53) or LVEDP (p = 0.85).

FIGURE 5.

Dobutamine dose-response relationships for maximum dP/dt, minimum dP/dt, and dP/dt₄₀ in wild-type and AE3/NKCC1 double null mutant mice. Data from the same experiments shown in Fig. 4 were used to calculate maximum dP/dt (A), minimum dP/dt (B), and dP/dt at 40 mm Hg (C) for wild-type and double null mice (n = 7 for each genotype) under basal conditions and in response to increasing β-adrenergic stimulation. Values are means ± S.E. *, significant group effect between wild-type and double null mice for maximum dP/dt (p < 0.004), minimum dP/dt (p < 0.005), and dP/dt₄₀ (p < 0.03) was observed.

FIGURE 6.

Contractile function and mechanics in adult cardiomyocytes isolated from AE3/NKCC1 double null, AE3 null, and NKCC1 null hearts. Ventricular myocytes isolated from adult hearts were paced at 0.5 Hz, and cell shortening was measured. A, significant reductions were observed in fractional shortening relative to diastolic length (p = 0.004), the maximal rates of myocyte shortening (+dL/dt, p < 0.0001), and myocyte relengthening (–dL/dt, p < 0.001) in AE3/NKCC1 double null myocytes when compared with wild-type controls, consistent with impaired contractility in vivo (Fig. 5). B, cardiomyocytes from single AE3 and NKCC1 null mutants did not exhibit impaired mechanics, consistent with normal contractile parameters in AE3 null (Fig. 3) and NKCC1 null (20) mice.

FIGURE 7.

Expression of sarcoplasmic reticulum-associated Ca²⁺-handling proteins in AE3/NKCC1 double null hearts. Proteins from whole tissue homogenates of hearts from WT and AE3/NKCC1 DKO mice were resolved by polyacrylamide gel electrophoresis, and immunoblot (A–C) and densitometric (D) analyses were performed using antibodies specific for PLN (A), PLN phosphorylated on Ser¹⁶ (PS16) and Thr¹⁷ (PT17)(B), and SERCA2a (C). When compared with WT levels, PLN (#, p = 0.014) expression was significantly increased in double null hearts. SERCA2a levels and the absolute levels of PS16 and PT17 were not significantly different from wild-type controls. Statistical analysis was carried out using the paired t test.

FIGURE 8.

Expression of sarcoplasmic reticulum-associated Ca²⁺-handling proteins in AE3 and NKCC1 single null hearts. Proteins from whole tissue homogenates of AE3 KO and WT hearts (A) and from NKCC1 KO and WT hearts (B) were resolved by polyacrylamide gel electrophoresis, and immunoblot analyses were performed using antibodies specific for SERCA2a (top of A and B), PLN (middle of A and B), and PLN phosphorylated on Ser¹⁶ (PS16)(bottom of A and B). Compared with respective WT levels, PLN was increased in AE3 (†, p < 0.03) and NKCC1 (p = 0.1) null hearts. Statistical analysis was carried out using the paired t test.

FIGURE 9.

Phosphorylation of phospholamban in AE3 null, NKCC1 null, and AE3/NKCC1 double null hearts. PLN protein expression and levels of PLN phosphorylated on Ser¹⁶ (PS16) were determined using specific antibodies. PS16 levels, when normalized to PLN expression, were significantly reduced in AE3 (*, p = 0.01), NKCC1 (*, p = 0.03), and AE3/NKCC1 (*, p < 0.001) null hearts.

FIGURE 10.

Expression of NCX in double null hearts and determination of NCX-mediated Ca²⁺ efflux in AE3/NKCC1 double null myocytes. A, proteins from whole heart homogenates from WT and DKO mice were resolved by polyacrylamide gel electrophoresis. Immunoblot analysis was performed using a monoclonal antibody against NCX (R3F1). Densitometric analysis revealed no significant alteration in NCX levels (p = 0.2). SR Ca²⁺ content in WT (n = 19 cells/4 mice) and double null (DKO; n = 22 cells/4 mice) ventricular myocytes was measured by the rapid and sustained application of caffeine (10 mm). B, fluorescent changes measured using a dual excitation fluorescence photomultiplier system revealed no significant reduction in total SR Ca²⁺ store levels (amplitude (340/380 nm) was 1.12 ± 0.09 for WT and 0.90 ± 0.07 for DKO). In the same experiment, the rate of decay of the caffeine-induced transient was determined. C, time for 70% recovery of transient (TRC 70%) was significantly reduced in DKO myocytes compared with WT controls (5.6 ± 0.5 in WT versus 4.2 ± 0.27 in DKO; p < 0.5).

FIGURE 11.

Expression of PP1-C in AE3 null, NKCC1 null, and AE3/NKCC1 double null hearts. Proteins from whole heart homogenates from WT and mutant mice were resolved by polyacrylamide gel electrophoresis. Expression of PP1-C was determined in AE3 null (A), NKCC1 null (B), and AE3/NKCC1 double null (C) hearts. Densitometric analyses (D) revealed that PP1-C expression was significantly increased in AE3 null (*, p < 0.02), NKCC1 null (*, p < 0.05), and double null hearts (*, p < 0.02).

FIGURE 12.

Carboxymethylation of PP2A-C in AE3 null, NKCC1 null, and AE3/NKCC1 double null hearts. Proteins from whole heart homogenates from wild-type and mutant mice were resolved by polyacrylamide gel electrophoresis. Immunoblot analyses were performed using monoclonal antibodies specific for unmethylated PP2A-C (clone 1D6 and clone 4B7). The PP2A-C subunits migrate either as single or double bands, varying for the same sample between immunoblots, a pattern that has been previously reported (33). Levels of unmethylated PP2A-C were significantly reduced in AE3 null hearts (A and D,*, p < 0.001) and NKCC1 null hearts (B and D,*, p < 0.01). In contrast, levels of unmethylated PP2A-C were significantly increased in AE3/NKCC1 double null hearts (C and D,*, p < 0.05). Total PP2A-C levels were determined upon cold base treatment of nitrocellulose membranes, which results in complete demethylation of PP2A-C (32). Total PP2A-C levels were slightly reduced in AE3 single null hearts (to 86 ± 1.6% of wild type; bottom of A; p < 0.001) and was unaltered in NKCC1 single null and double null hearts (B and C).

FIGURE 13.

Localization of PP2A-C in the myofibrillar fraction of AE3 null, NKCC1 null, and AE3/NKCC1 double null hearts. The myofibrillar fraction (3000 × g pellet) was generated as described under “Experimental Procedures.” Proteins were resolved by polyacrylamide gel electrophoresis, and levels of total PP2A-C were determined as described in Fig. 12, upon cold base treatment of nitrocellulose membranes. Localization of PP2A-C to the myofibrillar fraction was dramatically reduced in AE3 (A and D; p < 0.0001) and NKCC1 (B and D; p < 0.0001) single null hearts. In contrast, PP2A-C levels in the myofibrillar fraction of AE3/NKCC1 double null hearts were comparable with wild-type levels (C and D; p = 0.16).

FIGURE 14.

Acid-base and electrolyte transporter activities have the potential to alter intracellular Ca²⁺ handling. The diagram shows the exchangers (AE1, AE2, AE3, and PAT1), Na⁺-dependent transporters that extrude H⁺ (Na⁺/H⁺ exchanger) or take up (NBCn1, NBC1, and NBC4 cotransporters), the NKCC1 Na⁺-K⁺-2Cl^– cotransporter, and the NCX that have been identified in heart. It should be noted that NBC4 has been identified in the human heart (45) but may not be present in the rodent heart (46). The grouping of transporters is according to general function and is not meant to indicate a polarized distribution or specific location in the cell. Both NKCC1 by itself and the Na⁺-dependent acid-base transporters, when coupled with exchangers to prevent an increase in pH_i from inhibiting their activities, have the potential to mediate sustained Na⁺ loading in cardiac myocytes. Na⁺ loading can reduce the rate of Ca²⁺ extrusion via inhibition or reversal of the NCX.