During the development of cardiac hypertrophy, enhanced diastolic Ca²⁺ transients have been observed in the nucleus of cardiomyocytes. It has been established that nuclear signaling pathways during hypertrophic stimulation depend on nuclear calcium. It has not yet been conclusively determined whether nuclear Ca²⁺  signals act causally on the development of cardiac hypertrophy or represent a consequence of progressive calcium dysregulation.

Methods: We established a protocol for inducing a hypertrophic response upon phenylephrine (PE, 100µM) stimulation in cultured neonatal rat cardiac myocytes (NRVCM). Using HPLC-purified scAAV6-vectors, we introduced the nuclear calcium-calmodulin inhibitor CaMBP4. We measured the impact of CaMBP4 on the hypertophic response by measuring the changes in cell area. Furthermore, RNAseq was performed on FACS-sorted NRVCMs. Translational activity was then shown by employing a puromycin assay. Transcription factor activity was assessed using the RNAseq data and a regulatory build of the mouse genome.

Results: CaMBP4-transduced (CaMBP4⁺) NRVCM were protected from catecholamine-evoked cardiac hypertrophy, compared to the mCherry-transduced and untransduced controls, respectively. However,
we found that CaMBP4⁺ cells expressed high levels of hypertrophic markers and Mef2 target genes (such as ANF, BNP, Acta2, Xirp1, Xirp2), unveiling an uncoupling of phenotype and gene expression. Gene

set enrichment analysis (GSEA) revealed a cluster of genes involved in the regulation of translation (such as Eif3d, Eif2s1), whose PE-evoked upregulation was prevented by CaMBP4 transduction. We confirmed that the PE-evoked upregulation of translational activity was blunted by expression of CaMBP4. Analysis of transcription factor activity revealed a number of hypertrophy regulatingCa²⁺ -calmodulin responsive transcription factors including Atf3, E2f3, Runx1, E2f2, Klf2, Klf4 (downregulated in PE-stimulated CaMBP4⁺ cells) and several others, which are upregulated in PE-stimulated CaMBP4⁺ cells,

including Mef2a.

Conclusion:  We identified nuclear Ca²⁺ -calmodulin dependent signaling to play a pivotal role in cardiomyocyte hypertrophy evoked by catecholamine stimulation. Whereas classical MEF2 target genes are still upregulated in CaMBP4 expressing cells, we found that Ca²⁺-calmodulin signaling interference with the increase in protein translation. Future studies are needed to identify a Calcium responsive nuclear signaling Element (CaRNE) that integrates Ca²⁺-calmodulin dependent regulation of transcription, protein translation and development of cardiomyocyte hypertrophy.