The importance of the RAS in the pathophysiology of heart failure has been highlighted by the vast number of clinical and experimental investigations [1
]. 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
]. In the current study, we have focused on identifying the pivotal signaling mechanism responsible for mechanical stretch-induced upregulation of Ao gene expression in neonatal rat cardiac myocytes and fibroblasts. Results indicate that a balance in p38α and JNK1/2 activation primarily dictate Ao regulation in both cell types.
Mechanical stretch initially stimulated JNK activation, which was accompanied by a decrease in Ao gene expression in both NRVM and NRFBs. However, with prolonged stretch there was diminished JNK activation, which was accompanied by reciprocal increases in p38 activation and Ao gene expression. Thus, in both cell types, JNK was initially more highly phosphorylated following stretch, whereas p38 activation continues to dominate afterwards. Cardiac ventricular volume-overload studies remain to be performed to determine whether these mechanisms are operational in vivo. However, it is interesting to note that our observations are in agreement with those obtained from mouse and rat models of ventricular pressure overload [31
]. Exposito et al. [31
] have shown that thoracic aortic constriction (TAC)-induced cardiac hypertrophy in the mouse, JNKs are activated as early as 7 h post-TAC, whereas p38 phosphorylation is primarily elevated after 3 days. Recently, it has been shown that in Wistar rats with acute left ventricular pressure overload, transient JNK phosphorylation occurred within the first 24 h and declined [32
]. Taken together, these studies indicate that JNK plays an important role in the early phase of mechanical stretch.
Our results demonstrate that JNK1/2 negatively regulates Ao gene expression. SP600125 inhibits all three isoforms of JNK (JNK1/2/3) and upregulates Ao gene expression several fold. Similar results were obtained in a recent study using transgenic mice, in which cardiac selective deletion of JNK genes revealed that JNK1 preserved cardiac function in the early phase of acute hemodynamic stress [23
]. Pharmacological inhibition of JNK pathway by SP600125 or adenovirus-mediated expression of dominant-negative JNK1/2 increased basal Ao gene expression several fold and conversely expression of wild-type JNK1 significantly inhibited stretch-mediated Ao gene expression, suggesting that JNK1/2 has at least two roles in the regulation of Ao expression in cardiac cells. One role is to preserve basal low-level expression of Ao, whereas the second is to counterbalance the stimulatory effects on Ao expression with increased mechanical load. The data implicating a causative role for JNK signaling in mediating the cardiac growth response has been controversial [15
]. These results are intriguing in light of some acute in vitro
studies, which argue in favor of a pro-hypertrophic role for JNK in cardiac myocytes [18
]. However, a number of recent studies have not only challenged the proposed role of JNK as a pro-hypertrophic signaling effector in cardiac myocytes, but also suggest that JNK serves as a negative regulator of this response [15
]. At present, this discrepancy is poorly understood. While compelling evidence indicates that JNK mediates cardiac protection, the present study is the first to demonstrate the molecular mechanism of action and suggests that JNK activation exerts its protective effect by preserving the low level of local RAS in acute phase of mechanical load. This is further supported by the fact that a local increase in Ao gene expression alone has a significant impact on cardiac pathophysiology [36
]. Over-expression of Ao in the myocardium mediates phenotypic cardiac myocyte remodeling, which leads to age-dependent cardiac dysfunction and failure [36
]. Consistent with over-expression, adenovirus-mediated delivery of Ao antisense attenuates hypertension and cardiac hypertrophy [37
]. Although cardiac myocytes represent the bulk of the myocardial volume, cardiac fibroblasts are the most numerous cell types in the heart and play an essential role in myocardial function. Previously we have demonstrated the expression of Ao in cardiac fibroblasts and its upregulation in different pathological conditions [38
]. Although there is differential expression of mechanoreceptors in cardiac myocytes and fibroblasts [41
], it was interesting to note that similar molecular mechanisms of Ao gene regulation occur at the MAP kinase level in both types of cardiac cells in response to mechanical stretch.
Although the underlying cellular and molecular mechanisms remain to be defined, several studies using pharmacological inhibitors and genetic manipulations suggest a detrimental role for p38α in mediating events associated with cardiac hypertrophy, remodeling and contractile dysfunction[13
]. Results from the present study implicate the role of RAS system upregulation in p38α-mediated myocardial effects. Expression of MKK6b (E) significantly stimulated Ao gene expression, which indicates that activation of p38 by itself is sufficient to stimulate Ao gene expression. Taken together, these data suggest that p38 is required for stimulation of Ao gene expression by mechanical stretch, and activation of p38 alone is sufficient for upregulation of Ao gene expression. Given the detrimental effect of Ao gene expression in the myocardium [36
], we speculate that p38 exerts its detrimental effects at least in part via upregulation of local Ao gene expression. This hypothesis is further supported by several studies which convincingly demonstrate that angiotensin II-induced hypertension, negative inotropy, reactive oxygen species production and organ damage are mediated by p38 [22
]. The downstream effectors responsible for mediating p38α effects on Ao gene expression remain to be determined. In isolated rat kidney cells, cyclic AMP response element binding protein (CREB) has been shown to be an important regulator of Ao gene expression [43
]. Phosphorylated CREB has been shown to enhance Ao gene expression by binding to CRE in the 5’-flanking region of the rat Ao gene [44
]. In the myocardium, CREB has been shown to be activated by mitogen and stress-activated kinase 1 (MSK-1), a downstream target of p38 [20
]. Studies are ongoing in our laboratory to identify the upstream activator/s and the downstream transcription factor/s as molecular targets of p38 that mediate the observed upregulation of Ao gene expression in cardiac myocytes and fibroblasts. Inhibition of JNK1/2 by SP600125 resulted in stretch-induced upregulation of Ao gene expression at 4 h, suggesting that JNK activation is responsible for the initial suppression of Ao gene expression in mechanically stretched NRVM and NRFB.
Although, Results clearly indicate that JNK1/2 suppresses and p38α mediates stretch-induced Ao gene expression in isolated cardiac cells, it remains to be determined whether this mechanism is operational in the hemodynamically overloaded myocardium. Isolated cardiac cells may not completely mimic in vivo conditions since cells were grown as monolayer on a single extracellular matrix. Future in vivo studies employing pharmacological inhibitors and transgenic animal models of p38α and JNK1/2 are therefore warranted.
In summary, we have identified two important regulators of mechanical stretch-induced Ao gene expression in cardiac cells. Our results demonstrate that stress-activated kinases JNK1/2 and p38α have opposing roles in mechanical stretch-induced Ao gene expression; p38 mediates, and JNK suppresses, Ao gene expression. This study also reveals that prolonged stretch causes an imbalance in activation of JNK1/2 and p38α, resulting in upregulation of Ao gene expression in cardiac cells. To our knowledge, this is the first study to demonstrate that a balance between JNK1/2 and p38α is an important regulatory determinant of Ao, an essential precursor of the cardiac RAS. Given the importance of the RAS in mediating cardiac hypertrophy and remodeling, this suggests that both JNK1/2 and p38α may be viable therapeutic targets for the treatment of heart disease.