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Tetracycline-inducible CaM kinase II silences hypertrophy-sensitive gene expression in rat neonate cardiomyocytes
T G Valencia1, L D Roberts, H Zeng
1Laboratory of Cardiac and Vascular Molecular Genetics, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas, 76107, USA.
Abstract:
Recent work from this laboratory both in rat primary cardiomyocytes and in ventricular tissue of transgenic mouse models of induced hypertrophy has identified two Ca(2+)/calmodulin-dependent nuclear signaling cascades. The first involves the phosphatase calcineurin (CaN). The second is the CaM kinase kinase cascade which involves CaM kinase I and CaM kinase IV. Each of these signaling cascades strongly up-regulate transcription of hypertrophy-sensitive genes in the rat ventricular cardiomyocyte. We have documented that over-expression of an active form of CaM kinase II silenced transcriptional induction of hypertrophy-sensitive genes. The purpose of this study was to generate an inducible CaM kinase II expression system and correlate its expression with the silencing of hypertrophic-sensitive reporters. A truncated form of CaM KII, CaM KII (1-290) was subcloned downstream and proximal to a promoter under transcriptional control (induction) of the tetracycline-regulated transcription factor, tet-TransActivator (tTA). Hypertrophy-sensitive reporter activity in primary cardiomyocytes was silenced when tet-inducible CaM KII was co-expressed with plasmids harboring active forms of CaN, CaM KI or CaM KIV. For instance, induced CaM KII expression silenced CaN, CaM kinase I, or CaM kinase IV driven ANF reporter activity 4.9-, 2.9-, and 6.9-fold below their maximal values, respectively. Myocyte exposure to doxycycline (DOX) blocked tTA-driven CaM KII expression and restored CaN/CaM KI or CaN/CaM KIV driven reporter activation. This study demonstrates, for the first time, that active CaM KII silences Ca(2+)-sensitive nuclear signaling cascades for transcriptional up-regulation of cardiomyocyte hypertrophy.
Insights
Active CaM kinase II (calcium/calmodulin-dependent protein kinase II) silences gene transcription in cardiomyocytes. This finding reveals a novel mechanism regulating cardiac hypertrophy signaling pathways.
Area of Science:
- Cardiovascular Biology
- Molecular Signaling
- Gene Regulation
Background:
- Two Ca(2+)/calmodulin-dependent nuclear signaling cascades, calcineurin (CaN) and CaM kinase kinase (CaM KI, CaM KIV), up-regulate hypertrophy-sensitive genes in cardiomyocytes.
- Overexpression of active CaM kinase II (CaMKII) was previously shown to silence this transcriptional induction.
Purpose of the Study:
- To generate an inducible CaM kinase II expression system.
- To correlate CaMKII expression with the silencing of hypertrophic-sensitive reporters in cardiomyocytes.
Main Methods:
- A truncated form of CaMKII (CaMKII 1-290) was cloned into a tetracycline-regulated expression system (tet-inducible tTA).
- Primary cardiomyocytes were co-transfected with the inducible CaMKII system and reporter plasmids driven by active CaN, CaM KI, or CaM KIV.
- The effect of doxycycline (DOX) on CaMKII expression and reporter activity was assessed.
Main Results:
- Induced CaMKII expression significantly silenced reporter activity driven by CaN, CaM KI, and CaM KIV.
- Specifically, CaMKII silenced CaN, CaM KI, and CaM KIV driven ANF reporter activity by 4.9-, 2.9-, and 6.9-fold, respectively.
- Doxycycline treatment blocked CaMKII expression and restored reporter activation, confirming the specificity of CaMKII's silencing effect.
Conclusions:
- Active CaM kinase II actively silences Ca(2+)-sensitive nuclear signaling cascades.
- This study provides the first evidence that CaMKII acts as a repressor of transcriptional up-regulation in cardiomyocyte hypertrophy.
- These findings elucidate a novel regulatory mechanism in cardiac hypertrophy.
