Cardiac expression of the CREM repressor isoform CREM-IbΔC-X in mice leads to arrhythmogenic alterations in ventricular cardiomyocytes.

Chronic ß-adrenergic stimulation is regarded as a pivotal step in the progression of heart failure which is associated with a high risk for arrhythmia. The cAMP-dependent transcription factors cAMP-responsive element binding protein (CREB) and cAMP-responsive element modulator (CREM) mediate transcriptional regulation in response to ß-adrenergic stimulation and CREM repressor isoforms are induced after stimulation of the ß-adrenoceptor. Here, we investigate whether CREM repressors contribute to the arrhythmogenic remodeling in the heart by analyzing arrhythmogenic alterations in ventricular cardiomyocytes (VCMs) from mice with transgenic expression of the CREM repressor isoform CREM-IbΔC-X (TG). Patch clamp analyses, calcium imaging, immunoblotting and real-time quantitative RT-PCR were conducted to study proarrhythmic alterations in TG VCMs vs. wild-type controls. The percentage of VCMs displaying spontaneous supra-threshold transient-like Ca(2+) releases was increased in TG accompanied by an enhanced transduction rate of sub-threshold Ca(2+) waves into these supra-threshold events. As a likely cause we discovered enhanced NCX-mediated Ca(2+) transport and NCX1 protein level in TG. An increase in I NCX and decrease in I to and its accessory channel subunit KChIP2 was associated with action potential prolongation and an increased proportion of TG VCMs showing early afterdepolarizations. Finally, ventricular extrasystoles were augmented in TG mice underlining the in vivo relevance of our findings. Transgenic expression of CREM-IbΔC-X in mouse VCMs leads to distinct arrhythmogenic alterations. Since CREM repressors are inducible by chronic ß-adrenergic stimulation our results suggest that the inhibition of CRE-dependent transcription contributes to the formation of an arrhythmogenic substrate in chronic heart disease.

Gene construction, expression and functional testing of an inotropic peptide from the venom of the black scorpion Hottentotta judaicus.

Anti-insect depressant toxins represent a subfamily of scorpion venom-derived ß-toxins that are polypeptides composed of 61-65 amino acid residues stabilized by four disulfide bridges. These toxins affect the activation of voltage-sensitive sodium channels (NaScTx) and exhibit the preferential ability to induce flaccid paralysis in insect larvae. Here we demonstrate the recombinant expression of the novel cardiac inotropic peptide (Bj-IP) that was classified as an anti-insect depressant ßNaScTx isolated from the venom of Hottentotta judaicus. By using "splicing by overlap extension" (SOE)-PCR, allowing for the first time one step de novo synthesis of long-chain scorpion toxin genes, we generated a codon-optimized DNA fragment of Bj-IP for cloning into the Escherichia coli vector pQE30. Moreover, the gene of interest was fused to a 6xHis coding DNA sequence. Subsequent recombinant expression was performed in E. coli KRX. The purification of the polypeptide was achieved by a combination of NiNTA agarose columns and RP (C(18)) high-performance liquid chromatography. The purified fusion protein was digested with factor Xa resulting in the elution of Bj-IP. The yield of recombinant Bj-IP expression was approximately 4.5 mg per liter of culture. Mass spectrometry confirmed the theoretical total mass of Bj-IP (6608 Da). Tag-free Bj-IP was refolded in guanidine chloride buffer with a glutathione redox system which was supplemented with different additives at 16 °C. Supplementation with 10% glycerol produced Bj-IP folding forms that exhibited reproducible biological activity in mouse cardiomyocytes. Cell contractility was increased by almost 3-fold and decay kinetics were hasten by 47% after administration of Bj-IP. Taken together, here we show the recombinant expression of the functionally active cardiac inotropic peptide Bj-IP, a new ßNaScTx from H. judaicus, for promising pharmacological applications. Furthermore, our data suggest that the use of SOE-PCR may help to facilitate in future the high throughput of cloning and/or modification of scorpion toxin genes.