Loss of MD1 exacerbates pressure overload-induced left ventricular structural and electrical remodelling

Figures

• 

  Figure 1. MD1 expression in left ventricles of human and mouse failing heart. (A) Representative western blots of MD1 in heart samples from normal donors (n = 7) and DCM patients (n = 8). (B) qRT-PCR analyses of BNP and β-MHC mRNA in LV tissues of normal donors (n = 7) and DCM patients (n = 8). (C) EF values of normal (n = 7) and DCM hearts (n = 8). (D,E) Representative western blots of MD1 and (F) EF/FS values in WT mouse left ventricles at 4 weeks after sham or AB operation (n = 4). *P < 0.05 vs. normal donors or shams.

• 

  Figure 2. Loss of MD1 aggravates pressure overload-induced LV structural remodelling. (A) Representative western blots of MD1 expression in LV tissues from WT and MD1-KO mice (n = 6). (B) Gross hearts and H&E staining performed at 4 weeks after surgery (n = 7–8). (C) Statistical results of LV myocyte cross-sectional areas (n = 200+ cells). (D–F) HW/BW, HW/TL, and LW/TL values of the indicated groups (n = 13–14). (G) mRNA levels of the hypertrophy markers BNP and β-MHC in WT and MD1-KO left ventricles at 4 weeks after surgery, determined using qRT-PCR (n = 5). (H) Echocardiographic results of the indicated groups (n = 13–14). (I) PSR staining of histological sections prepared from LV samples of WT and MD1-KO mice at 4 weeks after surgery (n = 7–8). (J) Fibrotic areas from histological sections quantified using an image-analysis system (n = 26–28 fields). *P < 0.025 vs. WT-Sham, # P < 0.05 vs. WT-AB.

• 

  Figure 3. Deletion of MD1 alters surface ECG parameters and exacerbates pressure overload-induced LV electrical remodelling. (A) Examples of surface ECG recordings and (B) summary of surface ECG parameters from WT and MD1-KO mice at 4 weeks after surgery (n = 7–8). (C) Representative MAP recordings at a PCL of 150 ms and (D) statistical analysis of APD90 from Langendorff-perfused WT and MD1-KO hearts at 4 weeks after surgery (n = 8–9). (E) Representative MAP recordings of APD alternans and (F) statistical analysis of the threshold interval for APD alternans from the indicated groups (n = 7–8). (G) Examples of MAP recordings after burst pacing and (H) summary of VT inducibility rates in Langendorff-perfused WT and MD1-KO hearts at 4 weeks after surgery (n is indicated above the bar graphs). PCL = pacing cycle length; L = longer APD; S = shorter APD; NSR = normal sinus rhythm. *P < 0.03 vs. WT-Sham, § P < 0.01 vs. MD1-KO-Sham, # P < 0.05 vs. WT-AB.

• 

  Figure 4. MD1 regulates TLR4 signalling and CaMKII signalling in response to pressure overload. Western blot analysis of WT and MD1-KO left-ventricle samples at 4 weeks after surgery for (A) RP105, MD2, and TLR4 expression; (B) ox-CaMKII and total CaMKII expression, and phosphorylation of CaMKIIβ + γ + δ at Thr287. Left: representative immunoblots; right: quantitative results. Data were normalised to GAPDH. Numbers of mice per group are shown inside bars. *P < 0.05 vs. WT-Sham, # P < 0.05 vs. WT-AB.

• 

  Figure 5. MD1 modulates intracellular Ca2+ handling in response to pressure overload. (A) Representative recordings (F/F0) of steady-state Ca2+ transients followed by rapid caffeine administration and (B) confocal line-scan images of mouse LV myocytes. Cells were isolated from WT and MD1-KO mice at 4 weeks after surgery. (C) Statistical analysis of systolic Ca2+ transient amplitude and SR Ca2+ content in the indicated groups (5–6 mice/group). (D) Time to peak [Ca2+]i transient amplitude and duration of [Ca2+]i transient decay (4–5 mice/group). RT50 = half-maximal relaxation time. (E) Quantification of CaSpF in the indicated groups (3–4 mice/group). (F) Original immunoblots of major Ca2+ handling proteins (including total and phosphorylated proteins). Statistical analysis of WT and MD1-KO heart samples at 4 weeks after surgery for (G) expression of total SERCA2, total PLN, and NCX1, phosphorylation of SERCA2 at Ser38, and PLN phosphorylation at Thr17 normalised to total PLN; (H) total RyR2 expression, and RyR2 phosphorylation at Ser2808 and Ser2814 normalised to total RyR2. Data were normalised to GAPDH. Numbers within columns indicate the number of LV myocytes (C–E) and heart samples…

• 

  Figure 6. MD1 regulates the expression of K+ and Na+ channels in failing hearts. Immunoblot analysis of WT and MD1-KO left-ventricle samples at 4 weeks after surgery for (A) KCNH2, KCNE1 and KCNQ1 expression; (B) Kv4.2, Kv4.3 and Nav1.5 expression. Left: representative original immunoblots; right: quantitative results. Data were normalized to GAPDH. Numbers of mice per group are shown inside bars. *P < 0.02 vs. WT-Sham, # P < 0.05 vs. WT-AB.