Table 1.
The abbreviations used for non-failing hearts are as follows: F, female; M, male; W, white; EF, left ventricular ejection fraction measured prior to explant; CVA, cerebrovascular accident; GSW, gunshot wound. Drug therapy acute indicates treatment in the emergency room or intensive care unit prior to brain death: Drugs: DOB, dobutamine (n = 2); DOP, dopamine (n = 4); EPI, epinephrine (n = 1); NE, norepinephrine (n = 4); THY, thyroxine (n = 4), other, nimodipine, phenylephrine, vasopressin, atropine (n = 1 or 3). Drug therapy chronic, indicates drugs (phenobarbital, coumadin, spironolactone, synthroid) taken by patients prior to admission, as reported by family members (n = 1). The abbreviations used for failing hearts are as follows: F, female; M, male; W, white; B, black; NatAm, Native American; A, asian; O, other; EF, left ventricular ejection fraction measured prior to explant; DCM, dilated cardiomyopathy (pre-transplant diagnosis); ICM, ischemic cardiomyopathy; VCM, valvular cardiomyopathy. Drug therapy lists as follows: AM, amiodarone (n = 13); CAPT, captopril (n = 4); CARV, carvedilol (n = 8); DIG, digoxin (n = 10); DOB, dobutamine (n = 6); EN, enalapril (n = 1); LIS, lisinopril (n = 7); MET, metoprolol (n = 7); MIL, milrinone (n = 10); dofetilide, losartan, mexiletine, fosinopril, trazodone, colchicine (n = 1 or 2).
Table 2.
Primers used.
Fig 1.
Upregulation of CTGF, MAS and collagen in human heart failure.
(A) Bar graphs showing real-time PCR analysis of fold increase (2‒ΔΔCt) of MAS receptor mRNA (left) and CTGF mRNA (right) expression in tissue from failed human heart (HF) compared with non-failing (NF) samples (**p<0.01; ***p<0.001). Expression was normalized to GAPDH. (B) Correlation plot between normalized expression of CTGF and MAS mRNA showing strong interaction between them (p value–0.174). (C) Western-blot showing significant upregulation of CTGF in HF tissue samples compared to NF samples (right); GAPDH was used as loading control. The western blot image shown is representative of all the experiments done under similar experimental conditions and the data from multiple experiments quantitated and cumulative data were presented as bar graphs (left) (***p<0.001). (D) Masson’s Trichrome staining of cryo-sections (4μm) of human heart left ventricular wall tissue showing increased collagen deposition (stained blue) in inter-cellular spaces (arrows) of the HF samples compared to NF samples (upper panels); magnification ×20. Immuno-histochemical staining of same set of tissue sections with CTGF antibody, showing more deposition of CTGF (intense brown) in HF sections then NF samples (lower panels); magnification ×20. (E) Bar graphs showing real-time PCR analysis of different sub-types of collagen expression (Col 1A1, Col 1A2, Col 3A1 and Col 4A2) [represented as fold increase (2‒ΔΔCt)] in left ventricular heart tissue from failing (HF) as well as non-failing (NF) samples (***p<0.001). Expression was normalized to GAPDH. All the bar graphs are presented with error bar of ±SD.
Fig 2.
Agonist activated MAS receptor induces CTGF through ERK1/2 and regulates collagen expression in HEK293 cells stably expressing MAS.
(A) Real-time PCR analysis shows significant upregulation of CTGF expression in response to MAS receptor agonist (AR234960; 10μM); while MAS inverse-agonist (AR244555; 10μM) along with agonist suppresses the expression of CTGF below the basal level. (B) Western-blot showing significant upregulation of CTGF in MAS agonist (AR234960) activated samples whereas CTGF expression decreases in presence of inverse-agonist (AR244555). MAS activated by AR234960 induces phosphorylation of ERK1/2, MAS inhibition by the inverse-agonist (AR244555) reduces ERK1/2 activation. MAS expressing HEK293 cells also show significant down-regulation of CTGF in presence of MEK1 inhibitor (PD98059). CTGF and p-ERK1/2 expression were normalized by GAPDH and ERK1/2 respectively. The western blot image shown is a representative of all the experiments done under similar experimental conditions and data from multiple experiments quantitated and cumulative data were presented as bar graph, (*p<0.05; **p<0.01). (C) Bar graphs showing real-time PCR analysis of different collagen sub-types (Col1A1, Col1A2 and Col4A1) [represented as fold increase (2‒ΔΔCt)] in HEK293-MAS cell line. Activated MAS induces collagen synthesis while repression of MAS receptor by its inverse agonist (AR244555) shows significant down-regulation of the same collagen sub-types (*p<0.05; **p<0.01). RT-qPCR was normalized by GAPDH.
Fig 3.
MAS receptor present on adult human cardiac fibroblast primary cells induces CTGF and collagen expression in response to its agonist.
(A) Real-time PCR analysis shows significant upregulation of CTGF expression in response to MAS agonist (AR234960; 10μM); while MAS inverse-agonist (AR244555; 10μM) along with agonist (AR234960; 10μM) reduced CTGF expression significantly. CTGF expression decreases when MAS signaling is blocked by MEK1 inhibitor treatment. (B) Western-blot showing significant upregulation of CTGF in HCF cells treated with MAS agonist (AR234960; 10μM); the CTGF expression decreases when treated with inverse-agonist (AR244555; 10μM). MAS agonist (AR234960) activation also induces phosphorylation of ERK1/2 in HCF cells. In MAS agonist (AR234960) treated HCF cells, CTGF expression as well as ERK1/2 activation were significantly down-regulated in presence of MEK1 inhibitor (PD98059). CTGF expression and p-ERK1/2 levels were normalized by GAPDH and total ERK1/2 respectively. The western blot image shown is a representative of all the experiments done under similar experimental conditions and data from multiple experiments quantitated and cumulative data were presented as bar graphs. (C) Bar graphs showing real-time PCR analysis of different collagen sub-types (Col1A2 and Col3A1) [represented as fold increase (2‒ΔΔCt)] in HCF cells. Activated MAS induces collagen synthesis while inhibition of MAS receptor shows significant down-regulation of the same collagen sub-types. Inhibiting MEK1 also reduces expression of the same collagen sub-types. RT-qPCR was normalized by GAPDH. (*p<0.05; **p<0.01).
Fig 4.
CTGF regulates collagen in HEK293-MAS cells treated with MAS agonist (AR234960).
MAS expressing HEK293 cells were transiently transfected with control siRNA and CTGF siRNA followed by treatment with MAS agonist (AR234960). (A) Real-time PCR analysis confirms CTGF down-regulation by CTGF siRNA as compared to control siRNA transfected and followed by MAS agonist treatment. GAPDH was used as loading control. CTGF siRNA is specific and has no off-target effect on MAS expression. (B). Western-blot confirmation of CTGF protein levels. The western blot image shown is a representative of all the experiment done under similar experimental condition and data from multiple experiments quantitated and cumulative data were presented as bar graphs (**p<0.01) (C) Same set of sample was used for RT-qPCR analysis of collagen sub-types. Col 1A1, Col 1A2 and Col 4A1 were significantly down-regulated in MAS induced CTGF siRNA transfected samples compared to MAS induced control siRNA samples. RT-qPCR and western blot were normalized by GAPDH. (*p<0.05; **p<0.01; ***p<0.001).
Fig 5.
CTGF regulates agonist (AR234960) induced collagen expression in HCF cells.
HCF cells were transiently transfected with control siRNA and CTGF siRNA followed by treatment with MAS agonist (AR234960). (A) Real-time PCR analysis confirms CTGF down-regulation by CTGF siRNA as compared to control siRNA transfected and followed by MAS agonist treatment. GAPDH was used as loading control. CTGF siRNA did not affect MAS expression in HCF (S3 Fig). (B). Western-blot confirmation of CTGF protein levels. The western blot image shown is a representative of all the experiments done under similar experimental condition and data from multiple experiments quantitated and cumulative data were presented as bar graphs (left) (**p<0.01) (C) Same set of sample was used for RT-qPCR analysis of collagen sub-types. Col1A2 and Col3A1 were significantly down-regulated in MAS induced CTGF siRNA transfected samples compared to MAS induced control siRNA samples. RT-qPCR and western blot were normalized by GAPDH. (*p<0.05; **p<0.01).