Original articleStretch-induced regulation of angiotensinogen gene expression in cardiac myocytes and fibroblasts: Opposing roles of JNK1/2 and p38α MAP kinases
Introduction
Several lines of evidence from clinical and experimental studies indicate that the renin–angiotensin–system (RAS) has a key role in mediating ventricular hypertrophy in the pressure- and volume-overloaded heart. Angiotensinogen (Ao), a substrate of the RAS system, has been implicated in the pathogenesis of hypertension and congestive heart failure. Angiotensin II (Ang II), the most biologically active peptide of the RAS affects several aspects of cardiac function including contractility, cell metabolism, cellular growth, differentiation, apoptosis and gene expression [1]. Progression of heart failure is associated with steady increase of Ang II formation regardless of the underlying etiology [2], [3], [4]. It is generally accepted that activation of the RAS plays an important role in cardiac pathophysiology, since inhibition of Ang II production by angiotensin converting enzyme inhibitors or treatment with Ang II type-I receptor (AT1) blockers significantly improves cardiac function, reverses ventricular remodeling and reduces morbidity and mortality in patients with heart failure [5], [6]. We and others have shown that all components of RAS system (renin, Ao, ACE, Ang II, Ang II receptors) are present in the ventricular myocardium [7], [8], [9], [10], [11] and expressed by cardiac myocytes and fibroblasts. Elevated cardiac Ang II alone induced cardiac interstitial fibrosis under basal conditions and exacerbated cardiac remodeling and dysfunction and accelerated development of heart failure in mice with myocardial infarction, without affecting systemic hemodynamics [12]. Although the cardiac RAS is upregulated by increased mechanical load, the signaling pathways responsible remain to be determined.
MAP kinases p38 and JNK, sub-classified as stress-activated protein kinases (SAPKs), are specialized transducers of stress responses. Four genes encode p38 kinases (p38α, p38β, p38γ and p38δ), in which p38α is the major isoform expressed in the heart [13], [14]. The p38 cascade is initiated by MAP kinase kinase kinases (MAPKKK) at the level of the plasma membrane, which in turn promotes activation of the dual-specificity kinases, MKK3 and MKK6, which directly phosphorylate Thr and Tyr residues in the Thr-Gly-Tyr (TGY) motif of p38 kinases [14], [15]. Three JNK genes (JNK1, JNK2 and JNK3) have been identified, each of which gives rise to differentially-spliced isoforms [16]. Only JNK1 and JNK2 are present in the myocardium [15]. The JNK branch is initiated by MKKs at the plasma membrane, which promotes activation of dual-specificity kinases MKK4 and MKK7, which in turn directly phosphorylate Thr and Tyr residues in a Thr-Pro-Tyr (TPY) motif of JNK kinases [15]. While MKK7 is a specific activator of JNKs, MKK4 can also phosphorylate the TGY motif of p38 MAP kinases [17].
p38 and JNK cascades have been implicated in both cardiac protection and injury [14], [18], [19], [20]. Many pharmacological and molecular studies demonstrate that p38 activation enhances myocardial injury, whereas p38 inhibition is cardioprotective [13], [14], [21], [22]. Administration of p38 inhibitor has been shown to prevent left ventricular hypertrophy and dysfunction in hypertensive rats and improve cardiac function and attenuate left ventricular remodeling in rats with myocardial infarction [21]. Ventricular myocyte targeted over-expression of constitutively active MKK3 (MKK3bE) and MKK6 (MKK6bE) in transgenic mice produced a cardiomyopathic phenotype with extensive interstitial fibrosis [13]. However, many recent in-vivo studies suggest that JNK actually serves as negative regulator of mechanical load-induced cardiac growth-related responses [18], [19], [21], [23], [24]. In the present study, the objective was to determine the role of stress-activated kinases JNK and p38 in mediating mechanical load-induced Ao gene expression in neonatal rat ventricular myocytes (NRVM) and fibroblasts (NRFB).
Section snippets
Isolation of cardiac cells and mechanical stretch
Primary cultures of NRVM and NRFB were prepared from 1 to 2-day-old Sprague Dawley rats as previously described [25]. Dispersed cardiac cells were separated using a discontinuous Percoll gradient, containing a density of 1.060 g/L (nonmyocyte layer) and 1.086 g/L (myocyte layer). The NRVM were plated on deformable membranes coated with collagen-IV (1 μg/cm2) on Bioflex plates (Flexcell International Corp, Hillsborough, NC), at a density of 0.75 × 106 cells/well in DMEM/M199 medium and maintained
Mechanical stretch upregulates Ao gene expression
We and others have demonstrated that cardiac myocytes and fibroblasts from neonate and adult rats express Ao mRNA and protein [8], [10], [11], [26]. Ao gene expression is increased in ventricular myocytes from acutely infarcted rat hearts [8] and hypertrophied and failing hearts of rats with spontaneous hypertension [7]. In the present study, NRVM and NRFB were used to determine the role of stress activated kinases JNK and p38 in mediating mechanical load-induced Ao gene expression.
Discussion
The importance of the RAS in the pathophysiology of heart failure has been highlighted by the vast number of clinical and experimental investigations [1], [2], [27], [28], [29]. We have previously reported that myocytes and fibroblasts isolated from neonatal and adult rat hearts express Ao, which is upregulated in cardiac myocytes in various forms of load-induced heart failure, including acute myocardial infarction, genetic hypertension and aortic constriction [8], [10], [30]. In the current
Acknowledgments
This work was supported by a grant from the National Institutes of Health (R01-HL-68838) and Scott and White Hospital.
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