Abstract
BackgroundCa2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling.Methodology and Principal FindingsFive week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity.Conclusions/SignificanceGender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases.
Highlights
In cardiac muscle, numerous studies have implicated intracellular calcium (Ca2+) as a critical mediator in several signaling functions including contraction and the activation of gene transcription [1,2,3,4,5,6]
Based on in vitro phosphatase activity determined either by in-gel phosphatase activity assay [29] or using the generic phosphatase substrate pNPP in total left ventricular (LV) homogenates after immunoprecipitation with antibodies raised against calmodulin-dependent protein kinase phosphatase (CaMKP), we found that thoracic aortic constriction (TAC) induced a gender-specific alteration in CaMKP activity that was increased significantly in male TAC mice only (Fig. 5)
The weak CaMKP protein level does not prove a causal relationship, we propose that the presence of pCaMKII in the nuclear fraction from female TAC mice (Fig. 8A) resulted in the cytoplasmic accumulation of phospho-histone deacetylase 4 (HDAC4) (Fig. 6A)
Summary
Numerous studies have implicated intracellular calcium (Ca2+) as a critical mediator in several signaling functions including contraction and the activation of gene transcription [1,2,3,4,5,6]. Several hypertrophic signaling pathways are interconnected and converge to the nucleus to activate various transcription factors [7]. Alterations in Ca2+-handling play a pivotal role in pathological left ventricular (LV) remodeling. The multifunctional CaMKII signaling molecule is considered to play a major role in the twin pathological phenotypes of heart failure and arrhythmia [13,14]. The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. We investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKIIMEF2 signaling
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