CALL FOR PAPERS Sex Steroids and Gender in Cardiovascular-Renal Physiology and Pathophysiology Genomic deletion of estrogen receptors ER and ER does not alter estrogen-mediated inhibition of Ca 2 influx and contraction in murine cardiomyocytes

deletion of estrogen receptors ER (cid:1) and ER (cid:2) does not alter estrogen-mediated inhibition of Ca 2 (cid:3) inﬂux and contraction in murine cardiomyocytes. Am J Physiol Heart Circ Physiol 294: H2421–H2427, 2008. modify contraction of vascular smooth muscle and cardiomyocytes, but sug-gestions that they confer protective effects on the cardiovascular system remain controversial. The negative inotropic effects of estrogens are a consequence of L-type Ca 2 (cid:3) channel inhibition, but the underlying mechanisms remain elusive. We tested the hypothesis that membrane-associated estrogen receptors (ER)- (cid:1) and - (cid:2) are involved. We measured the effect of estrogens on Ca 2 (cid:3) current ( I CaL ) in isolated ventricular cardiomyocytes of wild-type (WT), ER (cid:1) knockout (ER (cid:1) KO), and ER (cid:2) KO mice using the whole cell patch-clamp tech-nique at 37°C. No differences in current densities or inactivation proﬁles of I CaL were found under control conditions in WT, ER (cid:1) KO, and ER (cid:2) KO cardiomyocytes, suggesting that absence of either ER has no effect on functional properties of I CaL . In all groups, application of raloxifene (2 (cid:4) M) or 17 (cid:1) - or 17 (cid:2) -estradiol (50 (cid:4) M) reduced I CaL ( P (cid:5) 0.001). Raloxifene decreased I CaL by 44 (cid:6) 9% (mean (cid:6) SE) in WT ( n (cid:7) 5), 34 (cid:6) 5% in ER (cid:1) KO ( n (cid:7) 5), and 30 (cid:6) 5% in ER (cid:2) KO mice ( n (cid:7) 8). 17 (cid:1) -Estradiol reduced I CaL by 41 (cid:6) 10% in WT ( n (cid:7) 4), 34 (cid:6) 12% in ER (cid:1) KO ( n (cid:7) 7), and 38 (cid:6) 8% in ER (cid:2) KO mice ( n (cid:7) 7). 17 (cid:2) -Estradiol inhibited I CaL by 31 (cid:6) 4% in WT ( n (cid:7) 4), 28 (cid:6) 6% in ER (cid:1) KO ( n (cid:7) 3), and 42 (cid:6) 3% in ER (cid:2) KO mice ( n (cid:7) 5). Decreases in cell shortening occurred in parallel with these ﬁndings. Our results suggest that inhibition of I CaL and the decrease in contraction by estrogens do not depend on ER (cid:1) or ER (cid:2) . of 72°C for 5 min and storage at 4°C. PCR products were analyzed using 2% agarose gel electrophoresis. Wild-type animals revealed bands of 387 (P1/P2) and 815 bp (P3/P4) for ER (cid:1) and a band of 155 bp (P4/P5/P6) for ER (cid:2) ; homozygous knockout animals revealed no band for P1/P2, a band of 255 bp for P3/P4 for ER (cid:1) , and a band of 200 bp (P4/P5/P6) for ER (cid:2) . Heterozygotes showed both bands for each primer combi-nation. Voltage-clamp studies. Membrane currents were measured at 37°C using whole cell procedures with an Axopatch ampliﬁer controlled by pCLAMP 8 software (Axon Instruments, Foster City, CA). Capacity current and series resistance compensation were carried out using analog techniques. Cells were voltage-clamped using low resistance (3–6 M (cid:9) ) borosilicate glass micropipettes. Starting from a holding potential of (cid:10) 80 mV, the voltage protocol consisted of an initial 60-ms step to (cid:10) 40 mV to inactivate the sodium and any T-type Ca 2 (cid:3) current, followed by 200-ms test steps ranging from (cid:10) 60 to (cid:3) 60 mV in 5-mV increments. After the step family, another 100-ms test step to 0 mV was added to monitor changes in the voltage-dependent inactivation proﬁle.