Andrea Ciobotaru

Estrogen Contributes to Gender Differences in Mouse Ventricular Repolarization

Rationale: Fast-transient outward K+ (Ito,f) and ultrarapid delayed rectifier K+ currents (IK,slow, also known as IKur) contribute to mouse cardiac repolarization. Gender studies on these currents have reported conflicting results. Objective: Key missing information in these studies is the estral stage of the animals. We revisited gender-related differences in K+ currents, taking into consideration the females’ estral stage. We hypothesized that changes in estrogen levels during the estral cycle could play a role in determining the densities of K+ currents underlying ventricular repolarization. Methods and Results: Peak total K+ current (IK,total) densities (pA/pF, at +40 mV) were much higher in males (48.6±3.0) versus females at estrus (27.2±2.3) but not at diestrus-2 (39.1±3.4). Underlying this change, Ito,f and IK,slow were lower in females at estrus versus males and diestrus-2 (IK,slow: male 21.9±1.8, estrus 14.6±0.6, diestrus-2 20.3±1.4; Ito,f: male 26.8±1.9, estrus 14.9±1.6, diestrus-2 22.1±2.1). Lower IK,slow in estrus was attributable to only IK,slow1 reduction, without changes in IK,slow2. Estrogen treatment of ovariectomized mice decreased IK,total (46.4±3.0 to 28.4±1.6), Ito,f (26.6±1.6 to 12.8±1.0) and IK,slow (22.2±1.6 to 17.2±1.4). Transcript levels of Kv4.3 and Kv1.5 (underlying Ito,f and IK,slow, respectively) were lower in estrus versus diestrus-2 and male. In ovariectomized mice, estrogen treatment resulted in downregulation of Kv4.3 and Kv1.5 but not Kv4.2, KChIP2, or Kv2.1 transcripts. K+ current reduction in high estrogenic conditions were associated with prolongation of the action potential duration and corrected QT interval. Conclusion: Downregulation of Kv4.3 and Kv1.5 transcripts by estrogen are one mechanism defining gender-related differences in mouse ventricular repolarization.

Hormonal regulation of cardiac KCNE2 gene expression

The KCNE2 gene encodes a single transmembrane domain protein that modulates a variety of K+ channel functions in various tissues. Here we show that cardiac KCNE2 transcript levels are approximately 10-fold upregulated at the end of pregnancy. This upregulation was mimicked by 17-beta estradiol but not by 5alpha-dihydrotestosterone treatments in ovariectomized mice. To investigate the mechanism of KCNE2 transcriptional regulation by estrogen, we experimentally identified KCNE2 transcription start sites, delineated its gene structure and characterized its promoter region. Estrogen treatment stimulated KCNE2 promoter activity in a dose-dependent manner and ICI 182,780 blocked estrogen stimulation. A direct genomic mechanism was demonstrated by (i) the loss of estrogen responsiveness in the presence of a DNA-binding domain mutant estrogen receptor alpha or mutant KCNE2 ERE and (ii) binding of ERalpha to the KCNE2 ERE. These findings show that a genomic mechanism of estrogen action alters KCNE2 expression, which may have important physiological implications.

Kcne2 Expression is Regulated both by Estrogen and Cardiac Stress in the Adult Male Mouse Heart

KCNE2 is a single transmembrane modulatory β subunit that can modulate a variety of K+ channel pore-forming α subunits in heterologous systems; recently we have shown KCNE2 to be an estrogen-responsive gene. KCNE2 is linked to LQTs and fatal arrhythmia. Pathological heart hypertrophy is associated with abnormal electrical activity leading to a considerable propensity to arrhythmias. We hypothesized KCNE2 expression might be modulated by pathological heart hypertrophy and by estrogen. The trans-aortic constriction (TAC) procedure was used to induce pressure overload and eventually heart failure (TAC-HF) in male mice. Once the ejection fraction reached ∼30%, the mice were treated with estrogen for 10 days. Real-time PCR showed that transcript levels of KCNE2 were similar between TAC-HF and control (CTRL), while strikingly upregulated ∼3 fold by estrogen treatment. To gain insight into the KCNE2 cell biology in heart failure and after treatment with estrogen, isolated cardiomyocytes were labeled with anti-KCNE2 antibody. In healthy hearts, KCNE2 was distributed both at the surface membrane as well as in the T-tubules, while in failing hearts KCNE2 completely disappeared from the T-tubules but its surface plasma membrane labeling increased. The disappearance of KCNE2 from the T-tubules in TAC-HF was not due to the disruption of their structure, since their integrity was maintained as evident by a similar α-actinin labeling in control and TAC-HF. E2 treatment of TAC-HF significantly increased overall KCNE2 labeling; KCNE2 was distributed both at the surface membrane as well as in the T-tubules. We speculate higher KCNE2 transcript levels, as well as reappearance of KCNE2 in the T-tubules by estrogen treatment of TAC-HF, would increase the association of KCNE2 with Kv4.3 and/or Kv4.2, therefore potentiating Ito,f currents thus resulting in a better cardiac repolarization.

Intralipid Protects Cardiac Function of Late Pregnant Mice against Ischemia/Reperfusion Injury

Female mouse hearts show better functional recovery after ischemia/reperfusion (I/R) injury compared with males. However, the vulnerability of isolated late pregnant (LP) hearts to I/R injury is unknown. Here we investigated the susceptibility of isolated mouse hearts in LP and postpartum (PP) to I/R injury. Isolated hearts (Langendorff) from female mice in diestrus stage (NP), LP, one day PP (PP1) and 7 day PP (PP7) were subjected to 20 minutes of global normothermic (37°C) ischemia followed by 40 minutes of reperfusion. The heart function was recorded throughout the experiments and infarct size was assessed by triphenyltetrazolium staining at the end of reperfusion. Although the function was similar in all 4 groups before ischemia, the functional recovery of LP hearts at the end of reperfusion was significantly lower compared to NP hearts; the rate pressure product (RPP) was reduced from 12926±1479mmHg∗beats/min in NP to 1614±438mmHg∗beats/min in LP mice. Interestingly, the RPP recovered partially in PP1 to 4716±584mmHg∗beats/min and almost fully back to NP levels one week PP. Consistent with the functional recovery findings, the infarct size was markedly larger in LP (59.7±5.2%) compared with NP (15.2±0.8%). The infarct size was restored partially in PP1 and fully back in PP7. Recently we have observed that Intralipid can protect the male mouse heart against I/R injury. To test whether Intralipid can improve the heart function in LP mice, 1% Intralipid was applied to isolated LP hearts at the onset of reperfusion. Intralipid treatment significantly improved the cardiac function of LP mice (RPP=11565±1599mmHg∗beats/min) and reduced the infarct size (17±1.1%) to similar values as in NP. In conclusion, isolated LP hearts have high vulnerability to I/R injury and postischemic treatment with Intralipid can protect the heart against I/R injury.