Remodeling of the peripheral cardiac conduction system in response to pressure overload.

How chronic pressure overload affects the Purkinje fibers of the ventricular peripheral conduction system (PCS) is not known. Here, we used a connexin (Cx)40 knockout/enhanced green fluorescent protein knockin transgenic mouse model to specifically label the PCS. We hypothesized that the subendocardially located PCS would remodel after chronic pressure overload and therefore analyzed cell size, markers of hypertrophy, and PCS-specific Cx and ion channel expression patterns. Left ventricular hypertrophy with preserved systolic function was induced by 30 days of surgical transaortic constriction. After transaortic constriction, we observed that PCS cardiomyocytes hypertrophied by 23% (P < 0.05) and that microdissected PCS tissue exhibited upregulated markers of hypertrophy. PCS cardiomyocytes showed a 98% increase in the number of Cx40-positive gap junction particles, with an associated twofold increase in gene expression (P < 0.05). We also identified a 50% reduction in Cx43 gap junction particles located at the interface between PCS cardiomyocytes and the working cardiomyocyte. In addition, we measured a fourfold increase of an ion channel, hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, throughout the PCS (P < 0.05). As a direct consequence of PCS remodeling, we found that pressure-overloaded hearts exhibited marked changes in ventricular activation patterns during normal sinus rhythm. These novel findings characterize PCS cardiomyocyte remodeling after chronic pressure overload. We identified significant hypertrophic growth accompanied by modified expression of Cx40, Cx43, and HCN4 within PCS cardiomyocytes. We found that a functional outcome of these changes is a failure of the PCS to activate the ventricular myocardium normally. Our findings provide a proof of concept that pressure overload induces specific cellular changes, not just within the working myocardium but also within the specialized PCS.

Differentiation of cardiac Purkinje fibers requires precise spatiotemporal regulation of Nkx2-5 expression.

Nkx2-5 gene mutations cause cardiac abnormalities, including deficits of function in the atrioventricular conduction system (AVCS). In the chick, Nkx2-5 is elevated in Purkinje fiber AVCS cells relative to working cardiomyocytes. Here, we show that Nkx2-5 expression rises to a peak as Purkinje fibers progressively differentiate. To disrupt this pattern, we overexpressed Nkx2-5 from embryonic day 10, as Purkinje fibers are recruited within developing chick hearts. Overexpression of Nkx2-5 caused inhibition of slow tonic myosin heavy chain protein (sMHC), a late Purkinje fiber marker but did not affect Cx40 levels. Working cardiomyocytes overexpressing Nkx2-5 in these hearts ectopically up-regulated Cx40 but not sMHC. Isolated embryonic cardiomyocytes overexpressing Nkx2-5 also displayed increased Cx40 and suppressed sMHC. By contrast, overexpression of a human NKX2-5 mutant did not effect these markers in vivo or in vitro, suggesting one possible mechanism for clinical phenotypes. We conclude that a prerequisite for normal Purkinje fiber maturation is precise regulation of Nkx2-5 levels.

Transcriptional regulation of cardiac conduction system development: 2004 FASEB cardiac conduction system minimeeting, Washington, DC.

The development of the complex network of specialized cells that form the atrioventricular conduction system (AVCS) during cardiac morphogenesis occurs by progressive recruitment within a multipotent cardiomyogenic lineage. Understanding the molecular control of this developmental process has been the focus of recent research. Transcription factors representative of multiple subfamilies have been identified and include members of zinc-finger subfamilies (GATA4, GATA6 HF-1b), skeletal muscle transcription factors (MyoD), T-box genes (Tbx5), and also homeodomain transcription factors (Msx2 and Nkx2.5). Mutations in some of these transcription factors cause congenital heart disease and are associated with cardiac abnormalities, including deficits within the AVCS. Mouse models that closely phenocopy known human heart disease provide powerful tools for the study of molecular effectors of AVCS development. Indeed, investigations of the Nkx2.5 haploinsufficient mouse have shown that peripheral Purkinje fibers are significantly underrepresented. This piece of data corroborates our previous work showing in chick, mouse, and humans that Nkx2.5 is elevated in the differentiating AVCS relative to adjacent working ventricular myocardial tissues. Using the chick embryo as a model, we show that this elevation of Nkx2.5 is transient in the network of conduction cells comprising the peripheral Purkinje fiber system. Functional studies using defective adenoviral constructs, which disrupt the normal variation in level of this gene, result in perturbations of Purkinje fiber phenotype. Thus, the precise spatiotemporal regulation of Nkx2.5 levels during development may be required for the progressive emergence of gene expression patterns specific to differentiated Purkinje fiber cells.

Differential regulation of the cardiac sodium calcium exchanger promoter in adult and neonatal cardiomyocytes by Nkx2.5 and serum response factor.

Nkx2.5 and serum response factor (SRF) are critically important transcription factors in cardiac morphogenesis. They are also widely expressed in adult cardiomyocytes, but there is little data to indicate their possible role in adult cardiac cells. In this paper we demonstrate that the interaction of Nkx2.5 and SRF in cardiac-specific gene regulation is different between neonatal and adult cardiomyocytes. Our experimental model utilizes transient transfection and adenovirus mediated gene transfer of the proximal promoter fragment of the cardiac isoform of the sodium-calcium exchanger gene (NCX1). This promoter construct (NCX184) contains a single Nkx2.5-response element (NKE) and a single serum response element (CArG). In rat neonatal cardiomyocytes NCX184 activity is substantially induced with Nkx2.5 or SRF and additively with both. Mutagenesis of these NKE and CArG elements demonstrated the specificity of the interactions, which was confirmed with gel retardation analysis of cardiac ventricular tissue. In contrast, in adult cardiomyocytes, co-infection of Nkx2.5 and SRF adenovirus vectors showed Nkx2.5 induction but SRF did not have additive effects on NCX1 promoter regulation. As opposed to NCX1, the proximal atrial natriuretic factor (ANF) promoter was regulated identically in response to SRF and Nkx2.5 in both adult and neonatal cardiomyocytes. These results show that Nkx2.5-SRF interactions are capable of producing different transcriptional responses in adult versus neonatal cardiomyocytes, implying important differences in NCX1 promoter tertiary complex formation dependent on developmental stage.