Table 1.
Primer sequence.
Fig 1.
Cardiac hypertrophy induced by ISO stimulation.
(A)Representative M-mode of LVs.(B)Echocardiography analyses of left ventricular wall thicknessat the end of diastole. LVAWd: LV anterior wall thickness at diastole, LVPWd: LV posterior wall thickness at diastole. (C)Measurement of ejection fraction(EF%) and fractional shortening(FS%). (D)Ratio of heart weight to tibia length(HW/TL)and heart weight to body weight(HW/BW). (E)Wheat germ agglutinin staining of transverse sections of hearts. Bars, 100μm. (F)Quantification of the size of cardiomyocytes by measuring transverse cell area. (G)The mRNA expression of ANP. n = 7, **P<0.01, ***P<0.001 ISO vs Vehicle.
Fig 2.
Cardiac fibrosis was induced ISO treatment.
(A) Representative micrographs of picrosirius red-stained sections of the heart. Red parts represent collagen. (B) Quantification of cardiac interstitial collagen content from picrosirius red-stained sections with results expressed as collagen volume fraction. (C) Changes in the expression levels of mRNAs transcribed from collagen I. (D) The mRNA expression of collagen type III using real time PCR. n = 7, *** P<0.001 ISO vs Vehicle.
Fig 3.
The β1-adrenoceptor expression was down-regulated in the remodeling heart.
(A) The β1-AR mRNA expression was quantitated using real time PCR. (B)The expression ofβ1-AR was measured by western-blot.(C)Immunohistochemistry of β1-AR visualized in heart. (D)β1-AR expression was quantified from the immunohistochemistry sections of Fig 3C. Bar is 50μm for all fields. n = 7, * P<0.05 ISO vs Vehicle.
Fig 4.
The expression of β2-adrenoceptor was down-regulated in the remodeling heart.
(A)The mRNA level of β2-AR was measured by real time PCR. (B)The expression of β2-AR was detected by western-blot. (C)Immunofluorescence of β2 adrenoceptor visualized in heart. (D)β2-AR expression was quantified from the immunofluorescence sections of Fig 4C. Bar is 100μm for all fields. n = 7, *P<0.05, **P<0.01 ISO vs Vehicle.
Fig 5.
The expression and activation of CREB were not affected in the cardiac remodeling.
(A)The mRNA expression of CREB measured by real-time PCR. (B)The protein expression of phospho-CREB, CREB measured by western-blot.(C) Quantification of CREB/EIF5 is shown. (D)Quantification of phospho-CREB/CREB is shown. n = 7, * P<0.05 ISO vs Vehicle.
Fig 6.
The expression of HuR was increased in the remodeling heart.
(A)The expression of HuR measured by western-blot. (B)Quantification of HuR/EIF5 is shown.(C)Immunohistochemistry of HuR visualized in cell nuclei. (D)Quantification of HuR expression measured byimmunohistochemistryis shown. Five randomly selected fieldsfrom each section were collected to evaluate HuR expression using Image-Pro Plus. n = 7. ***P<0.001 ISO vs Vehicle. (E)Effect of HuR siRNA on HuR protein level. (F) The β1-AR mRNA expression was measured by q-PCR.With HuR knock down, NRCMs were induced by ISO(10-5M) stimulating for 48h. (G)β2-AR mRNA level was detected with deficiency of HuR induced by ISO in NRCMs. n = 4, * P<0.05,***P<0.001.
Fig 7.
Graphic summary for how the β-ARs were down-regulated in ISO-induced cardiac remodeling.
Sustained β-AR activation by isoproterenol enhanced HuR expression rather than inhibited CREB expression and activation, which decreased the β1-AR expression and partially down-regulated the β2-AR expression in the cardiac remodeling model.