Relaxin promotes growth and maturation of mouse neonatal cardiomyocytes in vitro: clues for cardiac regeneration

Abstract

The demonstration that the adult heart contains myocardial progenitor cells which can be recruited in an attempt to replace the injured myocardium has sparkled interest towards novel molecules capable of improving the differentiation of these cells. In this context, the peptide hormone relaxin (RLX), recently validated as a cardiovascular hormone, is a promising candidate. This study was designed to test the hypothesis that RLX may promote the growth and maturation of mouse neonatal immature cardiomyocytes in primary culture. The cultures were studied at 2, 12, 24 and 48 hrs after the addition of human recombinant H2 RLX (100 ng/ml), the main circulating form of the hormone, or plain medium by combining molecular biology, morphology and electrophysiology. RLX modulated cell proliferation, promoting it at 2 and 12 hrs and inhibiting it at 24 hrs; RLX also induced the expression of both cardiac-specific transcription factors (GATA-4 and Nkx2-5) and cardiac-specific structural genes (connexin 43, troponin T and HCN4 ion channel) at both the mRNA and protein level. Consistently, RLX induced the appearance of ultrastructural and electrophysiological signs of functionally competent, mature cardiomyocytes. In conclusion, this study provides novel circumstantial evidence that RLX specifically acts on immature cardiomyocytes by promoting their proliferation and maturation. This notion suggests that RLX, for which the heart is both a source and target organ, may be an endogenous regulator of cardiac morphogenesis during pre-natal life and could participate in heart regeneration and repair, both as endogenous myocardium-derived factor and exogenous cardiotropic drug, during adult life.

Fig 1

Cell proliferation analysed at the noted time points from the addition of fresh culture medium, alone (open columns) or with RLX added (striped columns): (A) ³H-thymidine incorporation and b-counting (DPM: disintegrations per minute); (B) histoautoradiography of ³H-thymidine–labelled cardiomyocytes. RLX significantly increases the number of cycling cells at 2 and 12 hrs. On the other hand, the cells’ growth rate decreases at 24 hrs in comparison with the control cultures and remains unchanged at 48 hrs. Significance of differences (one-way ANOVA, n = 3): *P ‘ 0.05; **P ‘ 0.01. Bar (lower right) = 20 mm.

Fig 2

Quantitative real-time PCR analysis showing the effects of RLX on the expression of mRNAs for the noted myocardial markers at the different time points. The columns represent fold changes over the corresponding values of the control cultures, assumed as 1. Significance of differences (Student’s t-test, n = 3): *P ‘ 0.05 versus the controls.

Fig 3

Representative merged differential interference contrast and confocal immunofluorescent micrographs of cardiomyocytes cultured for 2 and 48 hrs in the absence or presence of RLX. Immunoreactivities for the different antigens are shown in pseudo-colours. There is a visible increase in the immunoreactivity for the noted antigens upon a 48-hrs incubation with RLX. GATA-4–positive nuclei are indicated by asterisks. Bar (upper left) = 16 mm. The right panels show the computer-aided densitometry of the immunostaining for the noted myocardial marker proteins. Significance of differences (one-way ANOVA, n = 5): *P ‘ 0.05 and **P ‘ 0.01 versus −RLX 2 hrs; ⁺P ‘ 0.05 and ⁺⁺⁺P ‘ 0.001 versus +RLX 2 hrs; ^#P ‘ 0.05, ^##P ‘ 0.01 and ^###P ‘ 0.01 versus −RLX 48 hrs.

Fig 4

Representative electron micrographs of cardiomyocytes cultured for 48 hrs in the absence (A) or presence of RLX (B, C). (A) An immature control cardiomyocyte showing round mitochondria (inset) and glycogen fields (g). (B) A more mature RLX-treated cardiomyocyte showing cytoplasmic leptomeres (asterisks, lower inset) and round mitochondria with densely stacked cristae and electron-dense matrix granules (upper inset, arrows). (C) Another mature RLX-treated cardiomyocyte showing myofibrils and rod-shaped mitochondria with electron-dense matrix granules (inset, arrows). The asterisks label the leptomeres in an adjacent cell. Bar (left) = 1 mm.

Fig 5

Electrophysiological features of the neonatal cardiomyocytes cultured for 24 and 48 hrs in the absence (a) or presence of RLX (b). (A) Representative spontaneous action potentials recorded in current clamp mode at 24 hrs. (B) I_Na traces and (C) I_Ca traces recorded in voltage-clamp mode at 24 hrs: T indicates transient T-type I_Ca. (D) Boltzmann functions depicting I-V curves for I_Na peak, total I_Ca,tot and T-type Ca²⁺ currents, recorded in the presence of nifedipine at 24 and 48 hrs. The related Boltzmann parameters and the statistical analysis are reported in Table 2. Data are mean values from 26 to 35 different cells.

Fig 6

Electrophysiological features of I_to, I_K1 and I_f currents in the neonatal cardiomyocytes cultured for 24 and 48 hrs in the absence (control) or presence of RLX. I-V plots of: (A) outward I_to, and (B) transient inward rectifier I_K1, evaluated by a voltage ramp protocol. (C) I_f trace currents in cardiomyocytes cultured for 24 hrs in the absence (a) or presence of RLX (b) and the relevant I–V plots at 24 and 48 hrs (c). RLX increases I_K1 size by shifting its voltage dependence towards more positive potentials and enhances the size of I_to and I_f. The related Boltzmann parameters and the statistical analysis are reported in Table 2. Data are mean values from 26 to 35 different cells.