Purpose: Cardiac Sympathetic Neurons (cSNs) densely innervate the myocardium and operate physiologically both as short term enhancers of cardiac function, and as constitutive regulator of cardiomyocyte (CM) size, by controlling proteolysis through the β2-AR/FOXO3/MuRF-1 dependent signaling axis (1). The density of cSNs decreases progressively from the epi- to the endocardium. We here tested the hypothesis that such innervation pattern would reflect on the degree of constitutive β2-AR input to the different regions of the heart and thus heterogenously control myocardial proteolysis and cell size.
Methods: IF and morphometric analyses were performed on heart slices from neonatal and adult normal and KO hearts. Heart denervation was induced by 6-OH-DOPA (100mg/kg). Surgical dissection of the subepi- and subendocardium was set up.
Results: CM size is heterogenous throughout the mouse myocardium, with the cells in the outer layers (sEPI hereafter) being significantly larger than the similarly-oriented ones in the innermost (sENDO) layers (sEPI: 8590±2121 vs sENDO 4697±1433, in μm3; sEPI/sENDO CM size: 1.83±0.43). Such size differences, that parallel the ‘EPI-to-ENDO’; gradient in cSN density (cSNs/CM: sEPI 0.45±0.06 vs sENDO 0.15±0.02), are completely ablated upon cSN denervation (sEPI/sENDO CM size: 0.83±0.26), and are absent in both β2-AR-/- and MuRF1-/- mice. Interestingly, chronic treatment with either the β2-AR agonist clenbuterol (3mg/kg), or the β2-AR antagonist ICI118,551 (1mg/kg), which diffuse throughout the myocardium resulting in hypertrophic or atrophic remodeling, respectively, also abolish the sEPI-to-sENDO CM size difference, supporting the notion that SN control over CM morphology is operated regionally. In further support of the causal role of cSNs in controlling myocardial heterogeneity, sEPI/sENDO CMs size differences are not present immediately after birth and start to establish at P7, in parallel with the development of myocardial sympathetic innervation and, consistently, never establish in mice permanently denervated at P1. Furthermore, we demonstrated that proteolysis is differentially regulated in the sEPI and sENDO, as sympathetic denervation upregulated the muscle specific atrogenes MuRF1 and Atrogin-1 significantly more in the former than in the latter regions.
Conclusions: Constitutive activity of the cardiac SNs is required for the establishment and maintenance of the correct trophic CM properties. This is the first demonstration of an otherwise homogenous tissue that is shaped by a superimposed pattern of cSNs.
1. Zaglia et al. Cardiovasc Res, 2013