Ian M. C. Dixon

Excessive Tumor Necrosis Factor Activation After Infarction Contributes to Susceptibility of Myocardial Rupture and Left Ventricular Dysfunction

Background—We investigated the potential contributions of tumor necrosis factor-&agr; (TNF-&agr;) on the incidence of acute myocardial rupture and subsequent chronic cardiac dysfunction after myocardial infarction (MI) in TNF knockout (TNF−/−) mice compared with C57/BL wild-type (WT) mice. Methods and Results—Animals were randomized to left anterior descending ligation or sham operation and killed on days 3, 7, 14, and 28. We monitored cardiac rupture rate, cardiac function, inflammatory response, collagen degradation, and net collagen formation. We found the following: (1) within 1 week after MI, 53.3% (n=120) of WT mice died of cardiac rupture, in contrast to 2.5% (n=80) of TNF−/− mice; (2) inflammatory cell infiltration and cytokine expression were significantly higher in the infarct zone in WT than TNF−/− mice on day 3; (3) matrix metalloproteinase-9 and -2 activity in the infarcted myocardium was significantly higher in WT than in TNF−/− mice on day 3; (4) on day 28 after MI compared with sham, there was a significant decrease in LV developed pressure (74%) and ±dP/dtmax (68.3%/65.3%) in WT mice but a less significant decrease in ±dP/dtmax (25.8%/28.8%) in TNF−/− mice; (5) cardiac collagen volume fraction was lower in WT than in TNF−/− mice on days 3 and 7 but higher on day 28 compared with TNF−/− mice; and (6) a reduction in myocyte apoptosis in TNF−/− mice occurred on day 28 compared with WT mice. Conclusions—Elevated local TNF-&agr; in the infarcted myocardium contributes to acute myocardial rupture and chronic left ventricle dysfunction by inducing exuberant local inflammatory response, matrix and collagen degradation, increased matrix metalloproteinase activity, and apoptosis.

Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: Expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts

In fibrosing hearts, myofibroblasts are associated with cardiac extracellular matrix remodeling. Expression of key genes in the transition of cardiac fibroblast to myofibroblast phenotype in post‐myocardial infarction heart and in vitro has not been well addressed. Contractile, focal adhesion‐associated, receptor proteins, fibroblast growth factor‐2 (FGF‐2) expression, and motility were compared to assess phenotype in adult and neonatal rat cardiac fibroblasts and myofibroblasts. Neonatal and adult fibroblasts undergo phenotypic transition to myofibroblastic cells, marked by increased α‐smooth muscle actin (αSMA), smooth muscle myosin heavy chain (SMemb), extra domain‐A (ED‐A) fibronectin, paxillin, tensin, FGF‐2, and TβRII receptor. Elevated ED‐A fibronectin confirmed fibroblast to supermature myofibroblastic phenotype transition. Presence of myofibroblasts in vivo was noted in sections of healed infarct scar after myocardial infarction, and their expression is similar to that in culture. Thus, cultured neonatal and adult cardiac fibroblasts transition to myofibroblasts in vitro and share expression profiles of cardiac myofibroblasts in vivo. Reduced motility with in vitro passage reflects enhanced production of focal adhesions. Developmental Dynamics 239:1573–1584, 2010.

Expression of Gqα and PLC-β in Scar and Border Tissue in Heart Failure Due to Myocardial Infarction

Background—Large transmural myocardial infarction (MI) leads to maladaptive cardiac remodeling and places patients at increased risk of congestive heart failure. Angiotensin II, endothelin, and α1-adrenergic receptor agonists are implicated in the development of cardiac hypertrophy, interstitial fibrosis, and heart failure after MI. Because these agonists are coupled to and activate Gqα protein in the heart, the aim of the present study was to investigate Gqα expression and function in cardiac remodeling and heart failure after MI. Methods and Results—MI was produced in rats by ligation of the left coronary artery, and Gqα protein concentration, localization, and mRNA abundance were noted in surviving left ventricle remote from the infarct and in border and scar tissues from 8-week post-MI hearts with moderate heart failure. Immunohistochemical staining localized elevated Gqα expression in the scar and border tissues. Western analysis confirmed significant upregulation of Gqα proteins in these regions ver...

Effect of AT1 receptor blockade on cardiac collagen remodeling after myocardial infarction

OBJECTIVE Previous work has shown that cardiac fibrosis occurs after myocardial infarction (MI) in non-infarcted ventricular tissue and that this event is associated with abnormal cardiac function. Our aim was to investigate the effect of AT1 receptor blockade on cardiac collagen remodeling in post-MI rat heart remote from the infarct site by addressing collagen mRNA abundance, posttranslational hydroxylation of collagen monomers, and mature collagen deposition. Prolyl 4-hydroxylase (PH) mediates hydroxylation of procollagen alpha-chains in the endoplasmic reticulum of cardiac fibroblasts and thus regulates the downstream formation and secretion of helical procollagen molecules. METHODS The effects of losartan (15 mg/kg/day) on collagen deposition and mRNA abundance were monitored in viable left and right ventricles in sham-operated (control) and experimental groups in the presence or absence of losartan. Immunoreactive PH concentration in viable tissues as well as cardiac function in control and experimental groups was determined by ELISA. RESULTS Immunohistochemical staining and 4-hydroxyproline assays confirmed that losartan treatment attenuates fibrosis in experimental hearts. Northern analysis revealed that losartan treatment of 1, 2, or 4 week experimental groups had no effect on collagen mRNA abundance compared to untreated post-MI rats. On the other hand, immunoreactive PH concentration was significantly decreased in the post-MI group treated with losartan. Determination of cardiac mass and cardiac function revealed that losartan treatment was associated with attenuated cardiac hypertrophy and improved left ventricular (LV) function in experimental animals. CONCLUSIONS AT1 blockade is associated with a significant decrease in cardiac fibrosis in treated post-MI rats, and this trend is positively correlated to a significant decrease in immunoreactive PH compared to untreated experimental animals. The expression of cardiac PH may be regulated by angiotensin via AT1 receptor activation, and the suppression of PH with losartan treatment may be an important mechanism for modulation of collagen deposition in the post-MI rat heart.

Alterations in heart membrane calcium transport during the development of ischemia-reperfusion injury.

Global ischemia in guinea-pig hearts for 60 to 90 min depressed microsomal and mitochondrial Ca2+ uptake activities. Reperfusion of the 60 min ischemic hearts resulted in incomplete recovery of contractile function and calcium uptake activities of both mitochondrial and microsomal fractions. On the other hand, reperfusion of the 90 min ischemic hearts further depressed the microsomal Ca2+ uptake activity. Coronary occlusion for 90 min in dog hearts was found to decrease microsomal Ca2+-pump and sarcolemmal Na+-K+ ATPase activities. Reperfusion of these regional ischemic hearts further depressed the microsomal Ca2+ uptake and Ca2+-stimulated ATPase as well as sarcolemmal Na+-K+ ATPase activities whereas mitochondrial Ca2+ uptake was increased. Perfusion of rat hearts for 60 min with hypoxic medium resulted in depression of the sarcolemmal Na+-dependent Ca2+ uptake and ATP-dependent Ca2+ uptake activities. Reperfusion of these hypoxic hearts failed to recover the sarcolemmal Na+-Ca2+ exchange and Ca2+-pump activities. These results demonstrate that membrane defects with respect to Ca2+ transport processes in ischemic/hypoxic hearts may be associated with irreversible injury.

Effect of ramipril and losartan on collagen expression in right and left heart after myocardial infarction

Although increased deposition of collagen proteins has been described after myocardial infarction (MI), little is known of timedependent transcriptional alteration of specific cardiac collagen sub-types as well as the degradative mechanisms for cardiac collagens in right and left ventricular myocardium remote to large left ventricular infarction. We sought to study collagen mRNA abundance and the deposition of specific collagen subtypes in noninfarcted left and right rat heart muscle at different times after MI. We also assessed the activity of different myocardial matrix metalloproteinases (MMP) using zymography to gain some information about degradative pathways for collagen. Furthermore, we assessed passive compliance properties of the right ventricle in experimental hearts. Finally we investigated the role of the renin angiotensin system in the collagen gene expression by administration of an angiotensin converting enzyme (ACE) inhibitor (ramipril) and an angiotensin II receptor type I antagonist (losartan) in experimental animals. We observed that the mRNA abundance of types I and III collagen were increased 3 days after myocardial infarction in both viable left and uninfarcted right ventricular tissues, that they peaked at 7–14 days, and were maintained at relatively high levels in the 28 and 56 days experimental groups. Stiffness of the right ventricular myocardium was significantly increased in the 56 days experimental group when compared to that of control values. These findings correlated with increased immunohistochemical staining patterns of different collagen species in the surviving right (and left) cardiac interstitium of 14, 28, and 56 day experimental cardiac groups. The elevation of fibrillarcollagen mRNA abundance in noninfarcted muscle from ventricular chambers was not significantly altered after treatment of experimental animals with ramipril and losartan for up to 14 days. MMP activity was increased in viable left ventricle at 14, 28 and 56 days and at 14 days in the right ventricle in experimental animals when compared to controls. These results indicated that (1) activation of transcription of collagen types I and III gene occurs in acute and chronic MI, and that fibrillar collagen proteins are deposited in the noninfarcted cardiac interstitium after a lag period relative to increased corresponding mRNA abundance; (2) an increase in MMP activity in chronic experimental hearts indicates that increased collagen deposition may be due to an increment in collagen synthesis rather by reduced degradation of collagen, and that MMP activation may be important in remodeling of the noninfarcted cardiac stroma; (3) an increase of right ventricular stiffness was associated with increased deposition of collagen; (4) as losartan treatment is not associated with any normalization of elevated collagen mRNA abundance, the upregulation of collagen gene expression in this model is not mediated by AT, receptor; and (5) the reduction of cardiac fibrosis mediated by ACE inhibition and losartan treatment may reside at the post-translational level in cardiac collagen metabolism.

Cardiotrophin-1: Expression in experimental myocardial infarction and potential role in post-MI wound healing

Cardiotrophin-1 (CT-1), a member of the IL-6 family of cytokines, has been shown to be elevated in the serum of patients with ischemic heart disease and valvular heart disease, and induces cardiomyocyte hypertrophy in vitro. We investigated expression of CT-1 in post-MI rat heart and the effect of CT-1 on cultured primary adult rat cardiac fibroblasts. Elevated CT-1 expression was observed in the infarct zone at 24 h and continued through 2, 4 and 8 weeks post-MI, compared to sham-operated animals. CT-1 induced rapid phosphorylation of Jak1, Jak2, STAT1, STAT3, p42/44 MAPK and Akt in cultured adult cardiac fibroblasts. CT-1 induced cardiac fibroblast protein synthesis and proliferation. Protein and DNA synthesis were dependent on activation of Jak/STAT, MEK1/2, PI3K and Src pathways as evidenced by decreased 3H-leucine and 3H-thymidine incorporation after pretreatment with AG490, PD98059, LY294002 and genistein respectively. Furthermore, CT-1 treatment increased procollagen-1-carboxypropeptide (P1CP) synthesis, a marker of mature collagen synthesis. CT-1 induced cell migration of rat cardiac fibroblasts. Our results suggest that CT-1, as expressed in post-MI heart, may play an important role in infarct scar formation and ongoing remodeling of the scar. CT-1 was able to initiate each of the processes considered important in the formation of infarct scar including cardiac fibroblast migration as well as fibroblast proliferation and collagen synthesis. Further work is required to determine factors that induce CT-1 expression and interplay with other mediators of cardiac infarct wound healing in the setting of acute cardiac ischemia and chronic post-MI heart failure.

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.