Jianming Hao

Antiproliferative and antifibrotic effects of mimosine on adult cardiac fibroblasts.

Prolyl 4-hydroxylase catalyzes the hydroxylation of collagen pro-alpha chains for the deposition of cardiac collagen. The effect of prolyl 4-hydroxylase on synthesis and degradation of collagen was studied in cultured adult cardiac fibroblasts using mimosine, a prolyl 4-hydroxylase inhibitor. Mimosine inhibited [3H]thymidine incorporation in cultured fibroblasts in a dose-dependent manner (100-600 microM). Immunofluorescence in fibroblasts and biochemical detection of mature type I collagen in culture serum revealed a strong inhibition of synthesis and secretion of mature collagens, respectively, in the presence of 200 microM mimosine. Western blot analysis for procollagen was carried out in cultured fibroblasts, and 200 microM mimosine treatment was associated with increased intracellular accumulation of procollagen from 4.14+/-0.27 to 10. 19+/-0.37 (arbitrary units). Immunofluorescence studies confirmed a marked increase of intracellular procollagens in fibroblasts treated with mimosine, which suggests a loss of coordinated monomeric procollagen synthesis and secretion of triple helical mature collagens. Modest inhibition of collagen type I mRNA abundance was observed in mimosine-treated fibroblasts, whereas no effect was noted for mRNAs of collagen type III, alpha-prolyl 4-hydroxylase or beta-prolyl 4-hydroxylase when compared to untreated control values. Treatment of fibroblasts with 200 microM mimosine was associated with elevation of matrix metalloproteinase (MMP)-9 activity. The cytotoxicity of mimosine treatment was found minimal at the concentrations indicated above. Thus the antifibrotic effects induced by mimosine on cultured adult cardiac fibroblasts was associated with inhibition of prolyl 4-hydroxylase and diminished extracellular secretion of procollagen, despite the reactive elevation of intracellular procollagen synthesis. We suggest that specific inhibition of prolyl 4-hydroxylase may provide a novel therapeutic approach for the modulation of cardiac fibrosis.

Effect of chronic AT1 receptor blockade on cardiac Smad overexpression in hereditary cardiomyopathic hamsters

OBJECTIVE As the pharmacological suppression of angiotensin has been associated with cardioprotective effects in cardiomyopathy, our primary aim was to determine whether the expression of Smad protein components of the cardiac TGF-beta signaling cascade is modulated by chronic AT(1) receptor blockade. Furthermore, we examined the relationship between cardiac Smad protein expression and altered collagen turnover in the cardiomyopathic heart. METHODS Male UM-X7. 1 cardiomyopathic (CMP) Syrian hamsters at early (65 days) and late (200 days) stages of cardiomyopathy were subjected to 4 week losartan (15 mg/kg/day) treatment. Expression of left ventricular (LV) receptor-activated (Smad 2) and common-mediator (Smad 4) Smads from control (F1-beta strain) hamsters, non-treated cardiomyopathic (CMP), and losartan-treated CMP animals was assessed. Collagen turnover, including fibrillar collagen synthesis/accretion and cardiac MMP activity was assessed. RESULTS Elevated mRNA abundance of fibrillar collagens and ANF were present in cardiomyopathic hearts and these trends were normalized in the early stage losartan-treated group. 4-Hydroxyproline and zymographic assays confirmed fibrosis and elevated MMP-1 and -2 activities in CMP hearts. Losartan treatment was associated with a modest reduction of cardiac 4-hydroxyproline concentration, and a significant reduction of both MMP-1 and MMP-2 activities. While TGF-beta(1) mRNAs were elevated in both CMP groups vs. controls, total TGF-beta protein content was not different in CMP vs. controls. In LV preparations containing nuclear extract, elevated Smad 2 and Smad 4 protein expression was noted in cardiomyopathic hearts vs. controls. Losartan treatment of late-stage CMP hamsters was associated with a significant reduction in Smad 2 and a modest reduction of Smad 4 protein expression vs. untreated CMP samples. CONCLUSIONS Altered cardiac Smad expression, present in both early and late stage cardiomyopathy, is positively correlated with the occurrence of cardiac fibrosis and elevated collagen turnover in failing CMP hearts. Four week AT(1) blockade is associated with normalized expression of cardiac Smad 2 proteins, and these changes occur in parallel with some aspects of collagen turnover in failing cardiomyopathic hearts.

Expression of Gi-2α and Gsα in myofibroblasts localized to the infarct scar in heart failure due to myocardial infarction

Objective: Patients surviving large transmural myocardial infarction (MI) are at risk for congestive heart failure with attendant alteration of ventricular geometry and scar remodeling. Altered Gi-2α and Gsα protein expression may be involved in cardiac remodeling associated with heart failure, however their expression in scar tissue remains unclear. Methods: MI was produced in Sprague–Dawley rats by ligation of the left coronary artery. Gi-2α and Gsα protein concentration, localization and mRNA abundance were noted in surviving left ventricle remote to the infarct, in border and in scar tissues from 8 week post-MI hearts with moderate heart failure. Results: We observed a 4.5- and 5.0-fold increase in immunoreactive Gi-2α protein concentration occurs in the border and scar regions vs. control values, respectively, in 8-week post-MI rat hearts. Similarly, immunoreactive Gsα protein concentration was increased 3.4- and 8.2-fold, respectively, in these tissues vs. controls. Double-fluorescence labeling and phenotyping studies revealed that both Gi-2α and Gsα proteins were localized to myofibroblasts in the infarct scar and to viable myocytes bordering the scar. Northern analysis revealed that the Gi-2α/GAPDH ratio was increased in both viable and scar regions (1.24- and 1.85-fold respectively) from experimental hearts when compared to sham-operated control values when compared to noninfarcted left ventricle, the value of this ratio in scar tissue was elevated ∼1.5 fold. The Gsα/GAPDH ratio was significantly increased (1.28-fold) only in the scar region vs. control. Conclusion: Our results indicate a marked increase in the expression of Gi-2α and Gsα from myofibroblasts of the infarct scar as well as remnant myocytes bordering the scar in 8-week post-MI rat hearts. We suggest that these changes may be associated with ongoing remodeling in the infarct scar in chronic post-MI phase of this experimental model.

Experimental Models of MMP Activation: Ventricular Volume Overload

In conclusion, these data indicate that early and sustained activation of cardiac MMP-1 in experimental volume overload may be responsible for loss of cardiac matrix collagen content during ventricular remodeling associated with volume overload. Losartan treatment was associated with normalization of MMP-1 activity and collagen content in volume overloaded hearts.

Activated TGFβ Signaling in the Heart After Myocardial Infarction

We have previously shown that myofibroblasts of the healed 8 week infarct scar overexpress transduction proteins that may be linked to elevated deposition of extracellular matrix components in this tissue. Other work suggests that TGFβ1, may be involved in cardiac fibrosis and myocyte hypertrophy. The significance of the altered TGFβ signaling in heart failure in the chronic phase of post-myocardial infarction (MI), particularly in the ongoing remodeling of the infarct scar, remains unexplored. Patterns of cardiac TGFβ1 and Smad 2, 3, and 4 protein expression were investigated 8 weeks after MI and were compared to relative collagen deposition in border tissues (containing remnant myocytes) and the infarct scar (nonmyocytes dominated by myofibroblasts). Both TGFβ1 mRNA abundance (Northern analysis) and protein levels (ELISA) were significantly increased in the infarct scar versus control values, and this trend was positively correlated to increased collagen type I expression in this tissues. Cardiac Smad 2, 3, and 4 proteins were significantly increased in border and scar tissues versus control values. Immunofluorescent studies indicated that Smad proteins were proximally localized to cellular nuclei in the infarct scar. TβRI (53 kDa) protein expression was significantly reduced in the scar, while the 75kDa and 110kDa isoforms of TβRII were unchanged and significantly increased respectively, in the infarct scar. These results indicate that TGFβ/Smad signaling may be involved in the remodeling of the infarct scar after the completion of wound healing per se, via ongoing stimulation of matrix deposition.