Cyclical stretch induces structural changes in atrial myocytes

Abstract

Atrial fibrillation (AF) often occurs in the presence of an underlying disease. These underlying diseases cause atrial remodelling, which make the atria more susceptible to AF. Stretch is an important mediator in the remodelling process. The aim of this study was to develop an atrial cell culture model mimicking remodelling due to atrial pressure overload. Neonatal rat atrial cardiomyocytes (NRAM) were cultured and subjected to cyclical stretch on elastic membranes. Stretching with 1 Hz and 15% elongation for 30 min. resulted in increased expression of immediate early genes and phosphorylation of Erk and p38. A 24-hr stretch period resulted in hypertrophy-related changes including increased cell diameter, reinduction of the foetal gene program and cell death. No evidence of apoptosis was observed. Expression of atrial natriuretic peptide, brain natriuretic peptide and growth differentiation factor-15 was increased, and calcineurin signalling was activated. Expression of several potassium channels was decreased, suggesting electrical remodelling. Atrial stretch-induced change in skeletal α-actin expression was inhibited by pravastatin, but not by eplerenone or losartan. Stretch of NRAM results in elevation of stress markers, changes related to hypertrophy and dedifferentiation, electrical remodelling and cell death. This model can contribute to investigating the mechanisms involved in the remodelling process caused by stretch and to the testing of pharmaceutical agents.

Fig. 1

Stretching for 30 min. induces immediate early genes expression and phosphorylation of Erk and p38. (A) Expression of c-fos, c-jun, c-myc and egr-1 upon stretching (n = 7). (B) Representative blots showing phosphorylated and total Erk and p38 levels after 30 min. of stretch. (C) Relative levels of phosphorylated Erk (n = 8). (D) Relative levels of phosphoryated p38 (n = 7). Data are expressed relative to control. *P < 0.05, **P < 0.01, compared with unstretched control. Egr-1: early growth response protein 1; Rplp0: ribosomal protein, large, P0; C: control; Str: stretch.

Fig. 2

Stretching causes changes associated with hypertrophy and dedifferentiation. (A) Atrial myocytes were stained for α-actinin with specific antibodies (green) and actin was stained with Texas Red-phalloidin. Nuclei are stained blue with DAPI. (B) Relative cell diameter after 24 hrs of stretching (n = 11). (C) Relative protein levels of troponin I and troponin T (n = 18 and 19, respectively). (D) Representative blots showing levels of troponin I and troponin T after stretching for 24 hrs. (E) β/α-MHC ratio (n = 7–19). (F) Skeletal α-actin expression (n = 8–19). Data are expressed relative to control. *P < 0.05, **P < 0.01, ***P < 0.001 compared with unstretched control. C: control; Str: stretch; β/α-MHC: β/α-myosin heavy chain; ACTA1: skeletal α-actin; Rplp0; ribosomal protein, large, P0.

Fig. 3

Stretching increases mRNA expression of stress specific markers and induces release of ANP. (A) ANP release into the medium (n = 3–9). (B) ANP expression (n = 8–21). (C) BNP expression (n = 8–20). (D) GDF15 expression (n = 8–19). Data are expressed relative to control. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with unstretched control. ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide; GDF15: growth differentiation factor-15; Rplp0: ribosomal protein, large, P0.

Fig. 4

Stretching for 24 hrs induces cell death, but not via apoptosis or autophagy. (A) The amount of TUNEL-positive cells after 24 hrs of stretching (n = 11). (B) The relative amount of cells in the medium after stretching (n = 9–10). (C) The amount of LDH in the medium (n = 6). (D) Representative blots showing levels of LC3-II after 24 hrs of stretching and levels of phosphorylated and total Akt after 30 min. of stretching. (E) Relative protein levels of LC3-II (n = 10). (F) Relative levels of phosphorylated Akt (n = 8). Data are expressed relative to control. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with unstretched control. PC: positive control, staurosporine (500 nM 24 hrs); LC3: microtubule-associated protein light chain 3.

Fig. 5

Changes in gene expression related to electrical remodelling. Effect of 24 hrs stretch on gene expression of Scn5a, LTCC, Kcnd3, Kcnq1, Kcnj2, Kcnj3, Kcnn1 and NCX1 (n = 9–11). Data are expressed relative to control. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with unstretched control. Scn5a, sodium channel, voltage-gated, type V, alpha subunit; LTCC, α1c subunit of L-type Ca²⁺-channel; Kcnd3, potassium voltage-gated channel, Shal-related subfamily, member 3; Kcnq1, potassium voltage-gated channel, KQT-like subfamily, member 1; Kcnj2, potassium inwardly rectifying channel, subfamily J, member 2; Kcnj3, inwardly rectifying channel, subfamily J, member 3; Kcnn1, potassium intermediate/small conductance calcium-activated channel, subfamily N, member 1; Ncx1, solute carrier family 8 (sodium/calcium exchanger).

Fig. 6

Mechanisms involved in the stretching response. Effect of 24 hrs of stretching on (A) Rcan1 expression (n = 19). (B) The effect of 1 μmol/l KN93 and 1 μmol/l cyclosporin A on the stretch-induced increase in skeletal α-actin expression (n = 7–18). (C) Expression levels of CaMKIIδ (n = 11), and expression of angiotensin II receptor type 1a (n = 8). (D) The effect of 1 μmol/l losartan and 10 μmol/l eplerenone on the stretch-induced increase in expression of skeletal α-actin (n = 5–18). (E) The effect of 10 μmol/l pravastatin on the expression of skeletal α-actin (n = 7). (F) Expression levels of Rac1, eNOS and iNOS (n = 9). (G) Overview of stretch-activated mechanisms. Data in A, C and G are expressed relative to control, in B, D, E and F data are expressed relative to unstretched control with 0.1% DMSO. *P < 0.05, **P < 0.01 and ***P < 0.001 compared with unstretched control and in Figure C, D, E and F compared with stretch with DMSO. Rcan1: regulator of calcineurin 1; Rplp0: ribosomal protein, large, P0; ACTA1: skeletal α-actin; GDF15: growth differentiation factor-15; DMSO: dimethyl sulfoxide; CsA: cyclosporin A; Epl: eplerenone; Los: losartan; Prava: pravastatin; CaMKII: Ca²⁺/calmodulin-dependent protein kinases IIδ; AIIR type 1a: angiotensin II receptor type 1a; Rac1: ras-related C3 botulinum substrate 1; eNOS: endothelial nitric oxide synthase; ANP: atrial natriuretic peptide; BNP: brain natriuretic peptide; β/α-MHC: β/α-myosin heavy chain; Egr-1: early growth response protein 1.