Analysis Of Calmodulin Dependent Kinase II (CaMKII)-delta and -gamma subunit expression between three different human smooth muscle tissues

Physiology 2014 (London, UK) (2014) Proc Physiol Soc 31, PCB194

Poster Communications: Analysis Of Calmodulin Dependent Kinase II (CaMKII)-delta and -gamma subunit expression between three different human smooth muscle tissues

J. Lartey1, J. Taggart1, S. C. Robson1, M. J. Taggart1

1. Institute of Cellular Medicine, University of Newcastle, Newcastle upon Tyne, United Kingdom.

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Calmodulin-dependent protein kinases (CaMK) have been suggested to be important mediators of smooth muscle function via putative actions on several signaling modalities including myofilament activation, sarcoplasmic reticular Ca2+ homeostasis and gene transcription[1]. Recently, we have presented data to suggest that tissue-specific differences exist in the responsiveness of human smooth muscles – myometrium, myometrial arteries and placental arteries – to inhibitors of CaMKIIs[2] . CaMKII expression in smooth muscle tissues is thought to be predominantly due to two of four possible isoforms – CaMKII delta (CaMKIID) and gamma (CaMKIIG). Furthermore, there are currently five CaMKIID and six CaMKIIG isovariants listed in the human NCBI reference sequence database. Here, we examine if there are variations in the CaMKIID and CaMKIIG isovariants that may explain the above tissue-specific responses to CaMKII inhibition. We used quantitative PCR to measure CaMKIID and CaMKIIG isovariant expression in myometrial strips (MYO) and myometrial (MA) and placental arteries (PA) obtained, following written informed consent, from normal pregnant women undergoing elective Caesarean section at term. Microdissected tissues were subjected to RNA isolation (Qiagen RNeasy fibrous kit #74707) and reverse transcription with AffinityScript cDNA synthesis kit #600559. Using PRIMERBLAST (NCBI) and Primer3, we were able to design isovariant-specific primers to human CaMKIID isovariant 1(NM_172127), isovariant 2 (NM_172128), and isovariant 3 (NM_001221) and to CaMKIIG isovariant 1 (NM_172171) and CaMKIIG isovariant 2 (NM_172169) gene sequences. Primer probes for the remaining isovariants failed due to high GC content, high Tm differences or undetectable transcript expression. Human smooth muscle RNA (Clontech, #636547) was used as an internal calibrator. CaMKIID isovariant 1 expression (fold-change relative to calibrator RNA) was less in MA (1.33±0.15) compared to PA (3.74±0.59) and MYO (3.08±0.35 p<0.05, n=10, mean±SEM, One way-ANOVA, Tukeys Post test). CaMKIIG isovariant 1 expression was higher in MYO (4.73±0.71) compared to MA (2.14±0.30) and PA (2.26±0.28, <0.05, n=10). The expression of the other CaMKIID and CaMKIIG isovariants were similar to that outlined for the sequences above. Previously, we observed that the extent of relaxation to CaMKII inhibition by KN-93 followed the order of MA>PA>MYO [2]. Our results show that this effect of CaMKII inhibition was not comparable to tissue-specific expression profiles of the individual CaMKIID and CaMKIIG isovariants. Instead, the tissue-specificity of action of CaMKII inhibition is likely to reflect alterations in protein expression and/or activity.



Where applicable, experiments conform with Society ethical requirements.

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