Cardiac Gq Receptors and Calcineurin Activation Are Not Required for the Hypertrophic Response to Mechanical Left Ventricular Pressure Overload

Figures

• 

  FIGURE 1. The hypertrophic response to angiotensin II (AngII) infusion after 14 days. (A) Representative hearts and developed LVH indexed by the ratios of LVW/BW and LVW/TL in mice treated with AngII infusion versus saline infusion controls (n = 7–9/group); (B) Hemodynamic measurements. All values are mean ± SEM. ***p < 0.001 vs. saline-treated controls; NS: not significant. LVW/BW: LV weight to body weight ratio; LVW/TL: LV weight to tibia length ratio; SP: systolic pressure; dP/dt: first derivative of pressure with respect to time.

• 

  FIGURE 2. The hypertrophic response to TAC for 21 days. (A) Representative hearts (upper) and developed LVH indexed by the ratios of LVW/BW and LVW/TL (lower) in mice subjected to TAC versus sham-operated controls (n = 7–9/group); (B) Hemodynamic measurements. All values are mean ± SEM. **p < 0.01, ***p < 0.001 vs. sham-operated controls; LVW/BW: LV weight to body weight ratio; LVW/TL: LV weight to tibia length ratio; SP: systolic pressure; dP/dt: first derivative of pressure with respect to time.

• 

  FIGURE 3. Markers of the early induction of hypertrophy in response to angiotensin II infusion (AngII) (left panels) or TAC (right panels) after 48 h. (A) Ratios of LVW/BW and LVW/TL, n = 6–8/group. (B) Gene expression of ANP, BNP and α-skeletal actin, n = 3/group. All values are mean ± SEM. ***p < 0.001 vs. saline treated or sham-operated controls, respectively; NS: not significant. LVW/BW: LV weight to body weight ratio; LVW/TL: LV weight to tibia length ratio; ANP: atrial natriuretic peptide; BNP: B-type natriuretic peptide; α-SA: α-skeletal actin.

• 

  FIGURE 4. NFAT-GATA4 signaling in response to angiotensin II infusion after 48 h. (A) Representative Western blots of NFATc4, phosphorylated glycogen synthase kinase-3 beta (GSK3β) and total GSK3β protein expression in the cytoplasm (upper left panel) and nucleus (upper right panel). GATA4 blots are shown for the nucleus. (B) Cytoplasmic and nuclear data are normalized with GAPDH and histone, respectively (bottom panels). All Western blot runs were performed in duplicate or triplicate. Results are presented as the mean value of 3–4 hearts per group ± SEM. *p < 0.05, **p < 0.01, vs. saline treated controls.

• 

  FIGURE 5. NFAT-GATA4 signaling in response to TAC after 48 h. (A) Representative Western blots of NFATc4, phosphorylated glycogen synthase kinase-3 beta (GSK3β) and total GSK3β protein expression in the cytoplasm (upper left panel) and nucleus (upper right panel). GATA4 blots are shown for the nucleus. (B) Cytoplasmic and nuclear data are normalized with GAPDH and histone, respectively (bottom panels). All Western blot runs were performed in duplicate or triplicate. Results are presented as the mean value of 3–4 hearts per group ± SEM. None of the statistical comparisons between TAC mice and sham-operated controls were significant.

• 

  FIGURE 6. CaMKIIδ, HDAC4 and MEF2A signaling in response to angiotensin II after 48 h. (A) Representative Western blots of CaMKIIδ, phosphorylated HDAC4 and total HDAC4 protein expression in the cytoplasm (upper left panel) and in the nucleus (upper right panel). MEF2A blots are shown for the nucleus. (B) Cytoplasmic and nuclear data are normalized with GAPDH and histone, respectively (bottom panels). The HDAC4 cytoplasmic/nuclear (cyto/nuclear) ratio was used as an indicator of HDAC4 nuclear export. All Western blot runs were performed in duplicate or triplicate. Results are presented as the mean value of 3–4 hearts per group ± SEM. None of the statistical comparisons between mice receiving AngII and saline were significant.

• 

  FIGURE 7. CaMKIIδ, HDAC4 and MEF2A signaling in response to TAC after 48 h. (A) Representative Western blots of CaMKIIδ, phosphorylated (p-) HDAC4 and total HDAC4 protein expression in the cytoplasm (upper left panel) and in the nucleus (upper right panel). MEF2A blots are shown for the nucleus. (B) Cytoplasmic and nuclear data are normalized with GAPDH and histone, respectively (bottom panels). The HDAC4 cytoplasmic/nuclear (cyto/nuclear) ratio was used as an indicator of HDAC4 nuclear export. All Western blot runs were performed in duplicate or triplicate. Results are presented as the mean value of 3–4 hearts per group ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. sham-operated controls.

• 

  FIGURE 8. Schematic summary of two distinct signaling pathways involved in the induction of cardiac hypertrophy in response to pressure overload: (1) The calcineurin-NFAT pathway is activated by Gq-coupled receptors in response to neurohumoral stimulation, not only by AngII but possibly also by endothelin and by catecholamines (acting on alpha-adrenergic receptors); (2) Mechanical loading, such as transverse aortic constriction (TAC), does not activate the systemic renin-angiotensin system but induces hypertrophy mediated by CaMKII-HDAC4-MEF2 signaling that does not require Gq-coupled receptor activation, but is presumably activated by calcium entry controlled by mechano-sensitive channels. Differences in the mode of intracellular calcium elevation in response to different stimuli (mechanical versus neuro-humoral) may account for the differential activation of calcineurin versus CaMKII, but CaMKII also directly inhibits activation of calcineurin, as indicated by the broken red inhibitory path (see section “Discussion”).