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ESC heart failure 8(6), 5542-5550 (2021). doi:10.1002/ehf2.13574, Published by Wiley, Chichester

Homer proteins are a family of scaffolding proteins involved in many intracellular signaling pathways, in both excitable and non-excitable cells. These proteins participate in the assembly and regulation of functional signaling complexes, facilitating the cross-talk between surface membrane receptors and channels in the membranes of intracellular compartments (Worley PF. et al., 2007). Homer proteins are constitutively expressed in the brain, where their scaffolding function is important for a variety of neuronal processes, such as intracellular Ca2+ homeostasis, synaptic plasticity associated with learning and memory in the mature brain, and neuronal development of the embryonic brain (Xiao B. et al., 1998; Worley PF. et al., 2007; Foa L. et al., 2009). Among the Homer splice variants, Homer 1a isoform acts as a natural dominant-negative by disassembling signalling complexes mediated by other Homer isoforms. The Homer 1a gene is transcribed as an immediate early gene (IEG), in neuronal cells its expression is low under normal conditions and increases rapidly after neuronal activation (Brakeman PR. et al., 1997). Homers proteins are also expressed in cardiac muscle, but their regulation and function remain still poorly understood. Despite their important role as regulators of multimeric signalling complex in nervous system, few reports have focused on the role of Homers in the heart. It has been reported that mRNA coding for Homer 1a rapidly and transiently increases in neonatal cardiomyocytes upon stimulation with either endothelin-1 (ET1) or other hypertrophic agonists (Kawamoto T. et al., 2006). The Homer 1a protein levels are also up-regulated following AngII-induced hypertrophy in neonatal cardiomyocytes (Guo WG. et al., 2010). Recently, it has been demonstrated that the variant Homer 1b/c positively regulates α1-adrenergic dependent hypertrophy, whereas Homer 1a is able to antagonize such effect (Grubb DR. et al., 2011). This study investigated the role of Homer 1a in the cardiac hypertrophic program. Our working hypothesis is that Homer 1a may be one of the molecular modulators of cardiac hypertrophy. For this purpose, we studied the presence, sub-cellular distribution and function of Homer1a in cardiac muscle. Under resting conditions we found that Homer 1a is constitutively expressed in cardiac muscle of both mouse and rat and in HL-1 cells (a specific cardiac cell line). In addition, using immunofluorescence confocal microscopy of adult rat heart sections, we showed that Homer 1a displays a peculiar localization: it is sarcomeric and peri-nuclear. We also analyzed Homer 1a expression under hypertrophic conditions. For this purpose, we used rat neonatal cardiomyocytes stimulated with the adrenergic agonist norepinephrine (NE). A significant increase in both Homer1a mRNA and protein was found after NE stimulation, whereas Homer 1b/c (a different Homer 1 isoform) expression remained unchanged. In this hypertrophic cellular model, we studied the adrenergic pathways involved in NE-inducted Homer 1a up-regulation by using specific α1- and β- adrenergic receptor blockers (prazosin and propranolol, respectively). The results showed that prazosin - but not propranolol - drastically reduced NE-induced up-regulation of Homer 1a mRNA, demonstrating that the α1-adrenergic pathway is involved. The effect of hypertrophic stimulation on Homer 1a expression was also confirmed in NE-stimulated HL-1 cardiomyocytes. In this cell line we found that 1 hour after NE stimulation Homer 1a content increased by a factor of 2.5. Overall, these results confirm our working hypothesis and demonstrate the involvement of Homer 1a in the α1-adrenergic pathway leading to cardiac hypertrophy. In the second part of the study we analyzed the effects of Homer 1a over-expression monitoring different hypertrophic markers, such as MAPK/ERK1/2 phosphorylation, NFAT nuclear translocation, ANF-promoter activity and increase in cell size. The results showed that during NE stimulation Homer 1a modulated many of them (except for NFAT nuclear translocation that did not appear to be affected by Homer 1a over-expression), whereas under resting conditions Homer 1a over-expression per sè was ineffective. In particular, we found that, in NE-stimulated HL-1 cells, over-expressed Homer 1a significantly reduced phosphorylation levels of ERK1/2 by about 40%, negatively modulating MAPK pathway. As regards the ANF promoter activity, this activity was significantly reduced by about 20% in NE-stimulated Homer 1a over-expressing cells. In order to verify the specificity of the Homer 1a effect on ANF, we performed the same experiment over-expressing Homer 1c and we found that, unlike Homer 1a, Homer 1c did not modulate the activity of ANF promoter in NE-stimulated HL-1 cells. Subsequently, we assessed the effect of Homer 1a over-expression on increase in cell size. The results obtained showed that Homer 1a counteracted the increase in NE-stimulated cell size. Finally, a preliminary analysis, in vivo, of Homer 1a expression was performed in three hypertrophic models, i.e. mice with chronic transverse aortic constriction, transgenic mice over-expressing Gαq and rats treated with monocrotaline. At variance with results observed in cellular models in vitro, in these models Homer 1a expression did not result affected by hypertrophic conditions, at least in the time span under investigation. However, for this approach in vivo, a broad time-course is needed and, therefore, further analyses are required. In summary, our data on Homer 1a presence and sub-cellular localization in cardiac tissue demonstrate that Homer 1a is constitutively expressed and displays a sarcomeric and peri-nuclear distribution. In our cellular models in vitro, Homer 1a up-regulation is an early event of the NE-induced hypertrophy and, as inferred from gain-of function studies, Homer 1a isoform antagonizes initiation and development of NE-induced events leading to α1-adrenergic-dependent hypertrophy. In conclusion, our results in vitro indicate that Homer 1a is inserted into a negative feedback mechanism in which acts as negative molecular modulator, counteracting early steps of hypertrophy. However, further studies are needed to elucidate the mechanisms underlying this process.