The role of stretch, tachycardia and sodium‐calcium exchanger in induction of early cardiac remodelling

Figures

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  FIGURE 1. Morphological characteristics of neonatal rat ventricular myocytes (MC) and non‐myocytes (NMC) upon mechanical stretch (ST) and tachycardia (TC). A, Induction of MC hypertrophy and B, elongation presented as length‐to‐width ratio after 72 h of ST. C and D, Frequency‐dependent changes in MC size and elongation after 48 h of tachycardia. F and G, ST induces NMC hypertrophy after 72 h without effects on cell elongation (length‐to‐width ratio). H, I, Increase in NMC cell size and elongation after TC (5 Hz). E, J, Immunofluorescent staining of representative fields from bio‐flex and 35 mm dishes. * P < .05 vs control # P < .05 for pairwise comparison. Average cell count: n = 700‐1000 per group

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  FIGURE 2. Gene expression changes induced by stretch (ST) and tachycardia (TC) in neonatal rat ventricular myocytes (MC) and non‐myocytes (NMC). A, Up‐regulation of remodelling marker genes after 24 h of ST in MC. B, Up‐regulation after 24 h of ST in NMC. C, D, Rate‐dependent gene expression changes after 3 h of TC in MC (C) and NMC (D). Dashed line represents the baseline expression of the corresponding control. * P < .05 vs control, # P < .05 for pairwise comparison. n = 4‐13 cell cultures

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  FIGURE 3. Excitation‐transcription (ET) coupling in stretch (ST) and tachycardia (TC). The experiments were performed in 4 groups: stretch/tachycardia + vehicle, control + vehicle, stretch/tachycardia + inhibitor and control + inhibitor. The treatments were normalized to their corresponding control group (group 1 to group 2; group 3 to group 4). A, Marker gene expression upon inhibition of sarcolemmal and intracellular signalling targets in stretched myocytes. B, RCAN1 gene expression in the absence and presence of the NCX inhibitor KB‐R7934 after 3 h of TC stimulation. The dashed line represents the baseline expression of the corresponding controls. * P < .05 # P < .05 (stretch + inhibitor vs control + inhibitor). Numbers in bars represent the number of technical replicates

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  FIGURE 4. Differential NCX protein expression between stretch (ST) and tachycardia (TC). A, Immunofluorescent staining of representative fields from bio‐flex and 35 mm dishes, scale bar = 20 µm. B, Increased NCX expression after 24 h of stretch is independent of the CaMKII signalling pathway. C, Tachycardia (8 Hz, 3 h) does not alter NCX expression. * P < .05 vs control/ vs ST, # P < .05. Average cell count n = 500‐600 per group

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  FIGURE 5. Effect of stretch (ST) and tachycardia (TC) on CaMKII phosphorylation at T286. A, Immunofluorescent staining of representative fields from bio‐flex and 35 mm dishes, scale bar = 20 µm. B, Unchanged phosphorylation of CaMKII after 24 h of ST. C, Increased phosphorylation of CaMKII upon 3 h of tachycardia (8 Hz). *P < .05 vs control, # P < .05. Average cell count n = 1000‐1500 per group

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  FIGURE 6. Microarray gene expression in neonatal rat ventricular myocytes (MC). The hybridization was performed in 6 groups: stretch (ST), control for ST, tachycardia (TC), control for TC, ST combined with NCX inhibition (NCXI) and TC combined with NCXI. A and D, Genes regulated either via stretch (ST) or tachycardia (TC) stimulation or by both triggers. B and E, Genes regulated by the NCX inhibitor KB‐R7934 (NCXI) in combination with ST or TC. C, Venn diagrams that display group interactions were generated from the microarray dataset thereby considering P < .05 and fold change cut‐off from at least 1.2‐fold up‐ or −1.2 down‐regulated (0.83 2‐ΔΔCT ratio). TC/ST + KB‐R7934 (NCXI) was presented normalized to control, B, statistical significance confirmed comparing ST/TC vs ST/TC + KB‐R7934 (NCXI). n = 3 biological replicates per group