Overt expression of AP-1 reduces alpha myosin heavy chain expression and contributes to heart failure from chronic volume overload.

Reduced expression of alpha-MHC plays a significant role in cardiac contractile dysfunction from hemodynamic overload. Previously, Pur proteins and YY1 have been shown to play a role in alpha-MHC repression during heart failure induced by pressure overload and by spontaneous hypertension, respectively. This was not observed in volume-overload-induced heart failure, suggesting additional regulatory mechanisms for alpha-MHC repression. The present study was performed to identify volume overload responsive transcription factors involved in alpha-MHC gene regulation. DNA binding activity of several transcription factors was evaluated in a functionally characterized rat model of heart failure induced by aorto-caval shunt. After 10 weeks of shunt, severe LV dilatation and reduced LV function were accompanied by increased expression of ANF and beta-MHC, and decreased expression of alpha-MHC. This was associated with dramatic (10-fold) activation of AP-1 together with increased expression of c-fos and c-jun. AP-1 activation was not observed following 4 weeks of shunt when cardiac function was preserved. In cultured cardiomyocytes, induction of AP-1 by PMA attenuated alpha-MHC mRNA by 60%. Transient transfection assays mapped PMA responsive sequence to -582 to -588 bp of alpha-MHC promoter. Deletion or mutation of these nucleotides had minimal effect on basal promoter activity but played a dominant role in PMA-mediated repression of alpha-MHC promoter activity. Over-expression of c-fos and c-jun in cardiomyocytes inhibited alpha-MHC promoter activity in a concentration dependent manner. Data suggest a repressive role of AP-1 in alpha-MHC expression and its possible involvement in the transition from compensatory hypertrophy to heart failure in chronic volume overload.

Adapter molecule DOC-2 is differentially expressed in pressure and volume overload hypertrophy and inhibits collagen synthesis in cardiac fibroblasts.

DOC-2 (differentially expressed in ovarian carcinoma) is involved in Ras-, beta-integrin-, PKC-, and transforming growth factor-beta-mediated cell signaling. These pathways are implicated in the accumulation of extracellular matrix proteins during progression of hypertrophy to heart failure; however, the role of DOC-2 in cardiac pathophysiology has never been examined. This study was undertaken to 1) analyze DOC-2 expression in primary cultures of cardiac fibroblasts and cardiac myocytes and in the heart following different types of hemodynamic overloads and 2) examine its role in growth factor-mediated ERK activation and collagen production. Pressure overload and volume overload were induced for 10 wk in Sprague-Dawley rats by aortic constriction and by aortocaval shunt, respectively. ANG II (0.3 mg.kg(-1).day(-1)) was infused for 2 wk. Results showed that, compared with myocytes, DOC-2 was found abundantly expressed in cardiac fibroblasts. Treatment of cardiac fibroblasts with ANG II and TPA resulted in increased expression of DOC-2. Overexpression of DOC-2 in cardiac fibroblasts led to inhibition of hypertrophy agonist-stimulated ERK activation and collagen expression. An inverse correlation between collagen and DOC-2 was observed in in vivo models of cardiac hypertrophy; in pressure overload and after ANG II infusion, increased collagen mRNA correlated with reduced DOC-2 levels, whereas in volume overload increased DOC-2 levels were accompanied by unchanged collagen mRNA. These data for the first time describe expression of DOC-2 in the heart and demonstrate its modulation by growth-promoting agents in cultured cardiac fibroblasts and in in vivo models of heart hypertrophy. Results suggest a role of DOC-2 in cardiac remodeling involving collagen expression during chronic hemodynamic overload.

Mitogen-activated protein kinases (p38 and c-Jun NH2-terminal kinase) are differentially regulated during cardiac volume and pressure overload hypertrophy.

Chronic pressure overload (PO) and volume overload (VO) result in morphologically and functionally distinct forms of myocardial hypertrophy. However, the molecular mechanism initiating these two types of hypertrophy is not yet understood. Data obtained from different cell types have indicated that the mitogen-activated protein kinases (MAPKs) comprising c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 play an important role in transmitting signals of stress stimuli to elicit the cellular response. We tested the hypothesis that early induction of MAPKs differs in two types of overload on the heart and associates with distinct expression of hypertrophic marker genes, namely ANF, alpha-myosin heavy chain (alpha-MHC), and beta-MHC. In rats, VO was induced by aortocaval shunt and PO by constriction of the abdominal aorta. The PO animals were further divided into two groups depending on the severity of the constriction, mild (MPO) and severe pressure overload (SPO), having 35 and 85% aortic constriction, respectively. Early changes in MAPK activity (2-120 min and 1 to 2 d) were analyzed by the in vitro kinase assay using kinase-specific antibodies for p38, JNK, and ERK2. The change in expression of hypertrophy marker genes was examined by Northern blot analysis. In VO hypertrophy, the activity of p38 was markedly increased (10-fold), without changing the activity of ERK and JNK. However, during PO hypertrophy, the activity of JNK was significantly increased (two- to sixfold) and depended on the severity of the load. The activity of p38 was not changed in MPO hypertrophy, whereas it was slightly elevated (50%) in hearts with SPO. Similarly, ERK activity was not changed in hearts with MPO, but a transient rise in activity was observed in hearts with SPO. The expression of ANF and beta-MHC genes was elevated in both PO and VO hypertrophy; however, this change was much greater in hearts subjected to PO than VO hypertrophy. Alpha-MHC expression was downregulated in PO but remained unchanged in VO hypertrophy hearts. Thus, these results demonstrate differential activation of MAPKs in two types of cardiac hypertrophy and this, in part, may contribute to differential expression of cardiac muscle gene expression, giving rise to unique cardiac phenotype associated with different hemodynamic overloads.

Revisiting the surgical creation of volume load by aorto-caval shunt in rats.

Cardiac hypertrophy is an early landmark during the clinical course of heart failure, and is an important risk factor for subsequent morbidity and mortality. The hypertrophy response to different types of cardiac overload is distinguished both at the molecular and cellular levels. These changes have been extensively characterized for pressure load hypertrophy; however, similar information for volume load hypertrophy is still needed. This study was undertaken to improve the existing method of producing experimental cardiac volume load. Previous investigators have employed surgical aorto-caval shunt (ACS) as a model for volume load hypertrophy (VO) in rats. The procedure is relatively simple and involves glue to seal the aortic hole after ACS. However, it has several limitations mostly related to the use of glue e.g. poor visualization due to hardening of tissues, imperfect sealing of the puncture site and glue seeping through the aortic hole resulting in shunt occlusion. We have modified the procedure using aortic adventitial suture instead of glue and 18G angiocatheter instead of 16G needle, which eliminated the technical difficulties from the former method. The ACS was visually confirmed at sacrifice, and the VO demonstrated by time-related changes in the heart weight/body weight ratio which increased from 78% at 4 weeks to 87% at 10 weeks and increased liver/body weight ratio by 22% at 10 weeks of post aorto-caval shunt. Cardiac expression of atrial natriuretic peptide (ANF) also demonstrated time-related increase in ANF mRNA (+275% increase at 4 weeks, p < 0.05, and +370% increase at 10 weeks, p < 0.001). This modified technique of aorto-caval shunt offers simpler, reproducible and consistent model for VO hypertrophy in rats.