Innervating sympathetic neurons regulate heart size and the timing of cardiomyocyte cell cycle withdrawal

Abstract

Sympathetic drive to the heart is a key modulator of cardiac function and interactions between heart tissue and innervating sympathetic fibres are established early in development. Significant innervation takes place during postnatal heart development, a period when cardiomyocytes undergo a rapid transition from proliferative to hypertrophic growth. The question of whether these innervating sympathetic fibres play a role in regulating the modes of cardiomyocyte growth was investigated using 6-hydroxydopamine (6-OHDA) to abolish early sympathetic innervation of the heart. Postnatal chemical sympathectomy resulted in rats with smaller hearts, indicating that heart growth is regulated by innervating sympathetic fibres during the postnatal period. In vitro experiments showed that sympathetic interactions resulted in delays in markers of cardiomyocyte maturation, suggesting that changes in the timing of the transition from hyperplastic to hypertrophic growth of cardiomyocytes could underlie changes in heart size in the sympathectomized animals. There was also an increase in the expression of Meis1, which has been linked to cardiomyocyte cell cycle withdrawal, suggesting that sympathetic signalling suppresses cell cycle withdrawal. This signalling involves β-adrenergic activation, which was necessary for sympathetic regulation of cardiomyocyte proliferation and hypertrophy. The effect of β-adrenergic signalling on cardiomyocyte hypertrophy underwent a developmental transition. While young postnatal cardiomyocytes responded to isoproterenol (isoprenaline) with a decrease in cell size, mature cardiomyocytes showed an increase in cell size in response to the drug. Together, these results suggest that early sympathetic effects on proliferation modulate a key transition between proliferative and hypertrophic growth of the heart and contribute to the sympathetic regulation of adult heart size.

Figure 1. Decreased heart size following 6‐OHDA‐induced neonatal chemical sympathectomy

A, hearts from 1‐week‐old rats injected with 6‐OHDA at P0 were visibly smaller than hearts from sham‐injected animals. B, a time course of heart weight in animals injected with a saline vehicle control (black) or 6‐OHDA (grey) shows a reduction in heart weight in chemically sympathectomized animals compared to age‐matched vehicle controls. C, there was no difference in average body weight of rat pups that received either an injection of saline (black) or 6‐OHDA (grey). D, the difference in heart weight alone resulted in a significant decrease in the heart weight to body weight ratio (heart weight:body weight) in the lesioned animals. (P2 n = 13; P7 n = 14; 8 week (Wk) n = 7; *P < 0.05 vs. age‐matched control; ^# P < 0.05 vs.P2 within condition.)

Figure 2. Neonatal injection with 6‐OHDA depletes sympathetic innervation of the heart

A, representative composite image of a section from a P2 heart. Hearts were sectioned longitudinally and innervation within matched images of the anterior epicardium of the left ventricle was analysed (box) using ImageJ (scale bar = 500 μm). B, representative images of matched sections stained with tyrosine hydroxylase (TH). TH staining was quantified to determine the innervation density in sham (left) and lesioned (right) hearts at P2 (top), P7 (middle) and 8 weeks (bottom). Sympathetic innervation was not detectable in lesioned animals at P2 and P7 and significantly reduced at 8 weeks. (Scale bar = 10 μm.) C, bar plot showing quantification of left ventricular sympathetic nerve density for sham (black) or 6‐OHDA (grey) injected animals demonstrating that injection with 6‐OHDA significantly decreases sympathetic innervation in the heart. (n = 3; nd, not detectable; *P < 0.05 vs. age‐matched sham; ^# P < 0.05 vs. P2 and P7 within condition.)

Figure 3. Sympathetic innervation of cardiomyocytes in culture inhibits cardiomyocyte maturation

A, representative image showing a proliferating cardiomyocyte double‐labelled with α‐actinin and phospho‐histone H3 (PH3; scale bar = 10 μm). B, bar plot showing a higher rate of proliferation for cardiomyoctes co‐cultured with sympathetic neurons for 5 days (black) than for cardiomyocytes grown in isolation (grey; n = 4). C, representative image showing a binucleated cardiomyocyte identified by α‐actinin, staining of cell membranes with wheat germ agglutinin (WGA), and DAPI nuclear staining (scale bar = 10 μm). D, bar plot showing co‐cultures (black) had a decreased number of binucleated cardiomyocytes compared to myocyte‐only cultures (grey; n = 3). E, representative image showing a cardiomyocyte, visualized with α‐actinin staining, outlined with ImageJ, neurons visualized with peripherin, and DAPI nuclear staining (scale bar = 10 μm). F, cardiomyocytes co‐cultured with sympathetic neurons for 4 days (black) were smaller than cardiomyocytes that were cultured for the same period in isolation (grey; n = 3; *P < 0.05 vs. co‐culture.)

Figure 4. Sympathetic neurons promote cardiomyocyte proliferation via β‐adrenergic signalling

A, when β‐adrenergic signalling was blocked in co‐cultures with propranolol (2 μm) during the entire 5 day culture period there was a decrease in cardiomyocyte proliferation, as measured by the number of PH3+ cardiomyocytes. There was no significant effect of treatment with the β‐adrenergic agonist isoproterenol (10 μm) in myocyte‐only cultures, although proliferation was decreased compared to co‐culture conditions (n = 5). B, β₁ and β₂ signalling was blocked in 5 day co‐cultures with bisoprolol (left) and ICI‐118,551 (right), respectively. When β₂‐adrenergic receptors were blocked with ICI‐118,551, there was a significant decrease in cardiomyocyte proliferation (n = 3). C, when cholinergic signalling was blocked with the cholinergic antagonist scopolamine (10 μm) in co‐cultures, we could not detect an effect on the number of PH3+ cells. Stimulating cholinergic signalling in myocyte‐only cultures with the cholinergic agonist muscarine (2.5 μm) also had no effect. (n = 3; *P < 0.05 vs. co‐culture.)

Figure 5. Sympathetic neurons decrease cardiomyocyte size via β‐adrenergic signalling

A, treatment of co‐cultured cardiomyocytes with propranolol (2 μm) resulted in an increase in average cardiomyocyte cell size, while treatment of myocte‐only cultures with isoproterenol (10 μm) resulted in a decrease in cell size (n = 3). B, β₁‐ and β₂‐receptors in co‐cultures were blocked with bisoprolol (left) and ICI‐118,551 (right), respectively. There was no effect of bisoprolol on cardiomyocyte size, but when β₂‐receptors were blocked with ICI‐118,551 there was a significant increase in cell size (n = 3). C, when cholinergic signalling was blocked with the cholinergic antagonist scopolamine (10 μm) in co‐cultures, there was no effect on size of cardiomyocytes. Stimulating cholinergic signalling in myocyte‐only cultures with the cholinergic agonist muscarine (2.5 μm) also had no statistically significant effect. (n = 3; *P < 0.05 vs. co‐culture control; ^# P < 0.05 vs. myocyte‐only control.)

Figure 6. Sympathetic regulation of cardiomyocyte proliferation and cell growth is transient

A, the area of individual cardiomyocytes was measured for cells cultured in the presence or absence of co‐cultured sympathetic neurons. Cultures were fixed and stained for α‐actinin each day for a 7 day period and cardiomyocyte area was measured. A plot of average cell size over days in vitro shows that cardiomyocytes are significantly smaller at 3 and 4 days when grown with sympathetic neurons. B, a plot of percentage of proliferating cardiomyocytes for cultures grown for 2, 3, 4, 5, 7 and 10 days in vitro shows that neuronal co‐culture delayed cell cycle withdrawal. (n ≥ 3; *P < 0.05 vs. age‐matched co‐culture.)

Figure 7. β‐Adrenergic signalling undergoes a developmental shift from a negative to a positive regulator of cardiomyocyte hypertrophy

A, bar plot comparing the average cell size of 3‐week‐old cardiomyocytes cultured without (left) or with (right) sympathetic neurons. Cultures were either treated with isoproterenol (10 μm; grey) for the last 4 days in vitro or not treated (black). Four days of isoproterenol treatment was sufficient to induce an increase in cardiomyocyte size. B, a plot of average cardiomyocyte size over days in vitro shows that, at 4 and 8 DIV, treatment with isoproterenol (grey line) decreases cardiomyocyte size, compared to the control. As the cardiomyocytes mature in culture, the effect of isoproterenol reverses, with a significant increase in cardiomyocyte size seen at 24 DIV. For each time point, cultures were treated with isoproterenol for the last 4 days of the culture period. (n = 3; *P < 0.05 vs. age‐matched control.)

Figure 8. Meis1, a cell cycle arrest‐associated transcription factor, is developmentally regulated in vivo, and is upregulated following sympathetic lesion

A, bar plot showing that Meis1 mRNA expression transiently increases between P2 and P7 in control animals, returning to P2 levels by 8 weeks. B, bar plot demonstrating that lesion of the sympathetic nervous system at P0 leads to an increase in Meis1 expression at P2 and P7. By 8 weeks, there was no difference in Meis1 expression compared to the sham control. (*P < 0.05 vs. P2; ^# P < 0.05 vs. sham.)

Figure 9. Sympathetic innervation is required for the regulation of cardiomyocyte density in vivo

A, representative image of wheat germ agglutinin (WGA) and α‐actinin staining in ventricular sections of 8‐week‐old rats. WGA delineates the cellular membrane of cardiomyocytes as well as staining non‐myocyte cells in the slice. B, representative images of WGA staining showing cardiomyocytes (asterisk) and non‐cardiomyocyte cells (arrowhead) in sham (left) and lesioned (right) animals. C, bar plot showing that, compared to sham (black), neonatal sympathetic lesions (grey) result in a decrease in cardiomyocyte density in 8‐week‐old animals (n = 3). D, bar plot comparing the density of non‐myocyte cells in sham (black) and lesioned (grey) animals, showing an increase in the density of non‐myocyte cells in lesioned animals (n = 4). E, bar plot showing that the average cardiomyocyte size in sham (black) and lesioned (grey) animals is not significantly different, suggesting that the difference in heart size in lesioned animals cannot be accounted for by decreased cell size. (n = 3; *P < 0.05 vs. sham.)