The null mutation of cardiac Na+-Ca2+ exchanger (NCX1) gene in mice caused death of embryo in utero at embryonic day (ED) 9.0-9.5 and this embryonic lethality appears resulted from abnormal heart development. In the present study, we investigated whether transgenic re-expression of NCX1 in mutant cardiac myocytes could rescue these lethal defects. Transgenic mice expressing the canine NCX1 in a cardiac specific manner were bred into the NCX1 knock-out background but did not prevent the fetal lethality associated with the NCX1 null allele. However, the NCX1 knock-out embryos with an NCX1 transgene survived with heart beatings until ED 10.5 which was one day longer than the survival of the NCX1 knock-out embryos (ED 9.5). At ED 10.5, however, the partially rescued NCX1 embryos might have succumbed to the lack of an organized vasculature in the yolk sacs. The placental labyrinth layer was reduced in size and largely avascular. The transgenic re-expression of NCX1 rescued heart beatings and survived longer, but was still insufficient for the mice to be completely rescued. Importantly, NCX1 was observed to express in the yolk sac and the placenta of wild type mice. The results suggest that defects in extra-embryonic compartments are causal to the lethality, and that NCX1 may play an important role in establishing vascularization in extra-embryonic tissues.
Animals ; Embryo/*metabolism/pathology ; Embryo Loss ; Female ; Gene Deletion ; *Gene Expression ; Genetic Complementation Test ; Mice ; Mice, Knockout ; Mice, Transgenic ; Myocytes, Cardiac/metabolism ; Phenotype ; Placenta/metabolism/pathology ; Sodium-Calcium Exchanger/*genetics/*metabolism ; Survival Rate ; Yolk Sac/embryology/metabolism/pathology

BACKGROUNDChronic heart failure (CHF) is associated with calcium transients and calcium handling proteins. Angiotensin converting enzyme (ACE) inhibitor has been demonstrated to have beneficial effect on CHF. Yet studies addressed to the relationship between ACE inhibitor and calcium transients in CHF are rare. The aim of this study was to investigate the influence of ACE inhibitor (perindopril) on the contractility and calcium transients and calcium handling proteins in ventricular myocytes from rats with experimental heart failure.

METHODSMale Wistar rats were randomized to heart failure group treated with perindopril [CHF-T, 3 mg.kg(-1).d(-1)], heart failure group without treatment (CHF-C) and sham-operated group (PS). Heart failure was induced by abdominal aortic constriction. All groups were further followed up for 12 weeks. Left ventricular myocytes were then isolated. Single cell shortening fraction and [Ca(2+)]i were simultaneously measured by laser scanning confocal microscope under the field stimulation (1.0 Hz). Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were performed to evaluate the changes of mRNA and protein of Na(+)-Ca(2+) exchanger (NCX1), sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) and phospholamban (PLB).

RESULTSThe fraction of cell shortening (FS%) and [Ca(2+)]imax (nmol/L) were significantly reduced in group CHF-C compared with group PS (FS%: 7.51 +/- 1.15 vs 13.21 +/- 1.49; [Ca(2+)]i max: 330.85 +/- 50.05 vs 498.16 +/- 14.07; both P < 0.01), and restored at least partially in CHF-T group. In CHF-C group, the left ventricular mRNA of NCX1 and PLB were significantly upregulated in comparing with PS group (RNCX1/beta-Actin: 0.51 +/- 0.12 vs 0.19 +/- 0.06, P < 0.01; RPLB/beta-Actin: 0.26 +/- 0.12 vs 0.20 +/- 0.08, P < 0.05), while SERCA2 mRNA was downregulated (0.48 +/- 0.10 vs 0.80 +/- 0.11, P < 0.01). The mRNA levels of NCX1 and SERCA2 in CHF-T group were between the CHF-C and PS group, and the differences of the latter two groups were significant (all P < 0.05). In CHF-C and CHF-T groups, the protein expression of NCX1 were 1.141 +/- 0.047 and 1.074 +/- 0.081 times of that in PS group respectively (both P < 0.05), and SERCA2 protein levels were 0.803 +/- 0.100 and 0.893 +/- 0.084 times of that in PS group respectively (both P < 0.05). The protein expression of NCX1 and SERCA2 in the CHF-C and CHF-T groups is significantly different (both P < 0.05).

CONCLUSIONACE inhibitor could improve cardiac function of failing heart through directly enhancing the contractility of single cardiomyocyte, and these effects are probably mediated by its roles in preventing the deleterious changes of calcium transients and calcium handling proteins in CHF.

Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Calcium ; metabolism ; Calcium-Binding Proteins ; genetics ; Calcium-Transporting ATPases ; genetics ; Heart Failure ; drug therapy ; metabolism ; Heart Ventricles ; drug effects ; Male ; Myocytes, Cardiac ; drug effects ; metabolism ; Perindopril ; pharmacology ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; Sodium-Calcium Exchanger ; genetics

Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1 076-1 096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1 076-1 096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).
Amino Acid Sequence ; Animals ; Antibodies, Blocking ; metabolism ; pharmacology ; Calcium Channel Blockers ; pharmacology ; Calcium Channels, L-Type ; genetics ; immunology ; metabolism ; Guinea Pigs ; Membrane Potentials ; Molecular Sequence Data ; Myocytes, Cardiac ; drug effects ; metabolism ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Wistar ; Sodium-Calcium Exchanger ; antagonists & inhibitors ; genetics ; immunology