Original article
Stretch-induced regulation of angiotensinogen gene expression in cardiac myocytes and fibroblasts: Opposing roles of JNK1/2 and p38α MAP kinases

https://doi.org/10.1016/j.yjmcc.2008.09.121Get rights and content

Abstract

The cardiac renin–angiotensin system (RAS) has been implicated in mediating myocyte hypertrophy, remodeling, and fibroblast proliferation in the hemodynamically overloaded heart. However, the intracellular signaling mechanisms responsible for regulation of angiotensinogen (Ao), a substrate of the RAS system, are largely unknown. Here we report the identification of JNK1/2 as a negative, and p38α as a major positive regulator of Ao gene expression. Isolated neonatal rat ventricular myocytes (NRVM) and fibroblasts (NRFB) plated on deformable membranes coated with collagen IV, were exposed to 20% equiaxial static-stretch (0–24 h). Mechanical stretch initially depressed Ao gene expression (4 h), whereas after 8 h, Ao gene expression increased in a time-dependent manner. Blockade of JNK1/2 with SP600125 increased basal Ao gene expression in NRVM (10.52 ± 1.98 fold, P < 0.001) and NRFB (13.32 ± 2.07 fold, P < 0.001). Adenovirus-mediated expression of wild-type JNK1 significantly inhibited, whereas expression of dominant-negative JNK1 and JNK2 increased basal and stretch-mediated (24 h) Ao gene expression, showing both JNK1 and JNK2 to be negative regulators of Ao gene expression in NRVM and NRFB. Blockade of p38α/β by SB202190 or p38α by SB203580 significantly inhibited stretch-induced (24 h) Ao gene expression, whereas expression of wild-type p38α increased stretch-induced Ao gene expression in both NRVM (8.41 ± 1.50 fold, P < 0.001) and NRFB (3.39 ± 0.74 fold, P < 0.001). Conversely, expression of dominant-negative p38α significantly inhibited stretch response. Moreover, expression of constitutively active MKK6b (E) significantly stimulated Ao gene expression in the absence of stretch, indicating that p38 activation alone is sufficient to induce Ao gene expression. Taken together p38α was demonstrated to be a positive regulator, whereas JNK1/2 was found to be a negative regulator of Ao gene expression. Prolonged stretch diminished JNK1/2 activation, which was accompanied by a reciprocal increase in p38 activation and Ao gene expression. This suggests that a balance in JNK1/2 and p38α activation determines the level of Ao gene expression in myocardial cells.

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.

References (44)

  • RousselE et al.

    Early responses of the left ventricle to pressure overload in Wistar rats

    Life sciences

    (2008)
  • WangY et al.

    Cardiac hypertrophy induced by mitogen-activated protein kinase kinase 7, a specific activator for c-Jun NH2-terminal kinase in ventricular muscle cells

    J. Biol. Chem.

    (1998)
  • SanghiS et al.

    Activation of protein kinase A by atrial natriuretic peptide in neonatal rat cardiac fibroblasts: role in regulation of the local renin–angiotensin system

    Regul. Pept.

    (2005)
  • XuJ et al.

    Role of cardiac overexpression of ANG II in the regulation of cardiac function and remodeling postmyocardial infarction

    Am. J. Physiol.

    (2007)
  • KumarR et al.

    The intracellular renin–angiotensin system: implications in cardiovascular remodeling

    Curr. Opin. Nephrol. Hypertens.

    (2008)
  • SerneriGG et al.

    Cardiac angiotensin II formation in the clinical course of heart failure and its relationship with left ventricular function

    Circ. Res.

    (2001)
  • WollertKC et al.

    The renin–angiotensin system and experimental heart failure

    Cardiovasc. Res.

    (1999)
  • JordeUP

    Suppression of the renin–angiotensin–aldosterone system in chronic heart failure: choice of agents and clinical impact

    Cardiol. Rev.

    (2006)
  • DostalDE et al.

    The cardiac renin–angiotensin system: conceptual, or a regulator of cardiac function?

    Circ. Res.

    (1999)
  • ZhangX et al.

    Identification and activation of autocrine renin–angiotensin system in adult ventricular myocytes

    Am. J. Physiol.

    (1995)
  • BakerKM et al.

    Cardiac actions of angiotensin II: role of an intracardiac renin–angiotensin system

    Annu. Rev. Physiol.

    (1992)
  • DostalDE et al.

    Intracardiac detection of angiotensinogen and renin: a localized renin–angiotensin system in neonatal rat heart

    Am. J. Physiol.

    (1992)
  • Cited by (32)

    • P38α MAPK inhibits stretch-induced JNK activation in cardiac myocytes through MKP-1

      2016, International Journal of Cardiology
      Citation Excerpt :

      Although the heart is able to dynamically adapt to physiologic increases in mechanical load, chronic overload of the myocardium results in the activation of maladaptive processes, which adversely affect both the structure and function of the heart. Mechanical stress induces activation of the cardiac renin-angiotensin system [4,5], which results in increased local production Ang II [6–8] and stimulation of myocardial remodeling and hypertrophy. We have reported that the stress-activated protein kinase family members c-jun NH2-terminal kinase (JNK) and p38, have opposing roles on stretch-induced angiotensinogen (Aogen) expression in neonatal rat ventricular myocytes (NRVM) [5].

    • Interleukin-10 inhibits chronic angiotensin II-induced pathological autophagy

      2015, Journal of Molecular and Cellular Cardiology
      Citation Excerpt :

      As JNK1/2 is known to be a critical regulator of autophagy, next we asked whether IL-10 alters Ang II-induced JNK1/2 activity. In alignment with our previous study [26], chronic long-term (48 h) Ang II treatment significantly inhibited JNK1/2 phosphorylation. Interestingly, Ang II-induced JNK1/2 inhibition was modestly rescued by IL-10; however, this affect was statistically insignificant (Suppl. Figure S2).

    View all citing articles on Scopus
    View full text