Introduction: CARD9, a scaffolding protein expressed in myeloid cells, regulates inflammatory cytokine secretion. Macrophage and neutrophil related inflammation is an underlying factor in cardiovascular disease and heart failure. Hypertension is a serious risk factor with a high prevalence (34%). Our preliminary results indicated CARD9 knockout is protective against transverse aortic constriction (TAC)-induced hypertrophy and fibrosis, and that it slows the progression of in-vivo cardiac dysfunction.
Hypothesis: CARD9 knockout will reduce TAC-induced contractile dysfunction in isolated cardiomyocytes at 4 weeks post-TAC.
Methods: C57BL/6 wild-type (WT) and CARD9-\- mice were assigned to transverse aortic constriction (TAC; n=6 each) or to control (CONT; n=3, 4 respectively). Systolic and diastolic parameters were assessed 4 weeks post-TAC in isolated cardiomyocytes. Western blots were performed for calcium flux regulators PLN and CAMKII, autophagy markers LC3BII/I and p62, and hypertrophy related proteins SMAD2/3 and p38 MAPK.
Results: Cardiomyocyte shortening velocity (μm/sec) was slower in WT-TAC compared to WT-CONT (-160.0+/-25 CONT vs -86.4+/-3.5 TAC; p<0.05). CARD9-\-TAC shortening velocity was significantly better than WT-TAC (-138.2+/-14.72 CARD9-\- vs. -86.4+/-3.5 WT; p<0.05). Peak Shortening (% cell length) was also reduced by TAC in WT (4.4+/-0.29 TAC vs. 8.5+/-1.3 CONT; p<0.01), but not CARD9-\- (8.2+/-0.52 CONT vs. 7.6+/-0.79 TAC; ns). Relengthening velocity (μm/sec) was slower after TAC for WT (135.8+/-24.9 CONT vs. 53.08+/-4.4 TAC; p<0.01) and time to 90% relaxation (sec) was longer (0.205+/-0.008 CONT vs. 0.297 TAC+/-0.019; p<0.01), but not in CARD9-\- (0.1975+/-0.021 CONT vs. 0.221+/-0.007; ns). TAC decreased phosphorylation of p38 MAPK, CAMKII, and PLN for WT but not for CARD9-\-. CARD9-\- decreased p62 expression in TAC mice (p<0.05) but did not change LC3BII/I or p-SMAD2/3.
Conclusions: CARD9 knockout protects mice from TAC-induced cardiomyocyte systolic and diastolic contractile dysfunction at a critical time-point during pressure overload. Normalized phosphorylation of calcium signaling proteins and increased autophagy may play a role in the protective effect of CARD9 knockout.