In this study, we have established an inducible, heart specific model of MKK3 induction that permits the examination of acute p38 MAPK activation. By breeding the inducible transgene alleles into the MK2−/− background, we examined the role of MK2 in acute p38 activation in adult heart. Induction of MKK3 led to cardiomyocyte hypertrophy, cardiac remodeling, contractile dysfunction, pathological gene activation, and lethal bradycardic heart failure. Removal of the MK2 gene rescued some aspects of this pathologic response, including partially ameliorated hypertrophy and contractile dysfunction, and prevention of early lethality. Thus, MK2 has an important role in mediating some aspects of p38 induced heart failure. Its contribution to p38 signaling can be mediated by either affecting the total p38 protein level and signaling intensity () or by specifically blocking the MK2 dependent downstream signaling (as demonstrated by Hsp27 phosphorylation in ). From both in vitro and in vivo analysis, we also established a previously uncharacterized role for MK2 in COX-2 protein synthesis regulation. Therefore, our study not only demonstrates a specific contribution of MK2 in p38 induced cardiomyopathy but also reveals a new mechanistic link between p38 and inflammatory gene expression in heart.
The finding that p38 activation was sufficient to induce ventricular myocyte hypertrophy is supported by wall thickness measured in vivo
by echocardiography, normalized ventricular mass and myocyte CSA from histological sections. The finding that p38 induces hypertrophy in adult heart is in accordance with in vitro
myocyte experiments 3
. However, most in vivo
models of p38 activation have not shown myocyte hypertrophy 6, 7
. One potential explanation for this discrepancy lies in the timing and duration of p38 activation. Earlier in vivo
studies used constitutively activated promoters to achieve p38 activation in an unregulated manner at earlier developmental stages 6, 7
. In contrast, our current study used a tamoxifen dependent Cre gene to activate the p38 pathway in adult heart (see Methods). This may eliminate the potential confounding factors involving p38 mediated regulation of cardiomyocyte proliferation and differentiation as implicated in other reports 28
, or secondary compensatory changes. However, we cannot exclude other complicating factors such as contribution of tamoxifen treatment 24
and GFP expression 29
although we have different control groups to support the specificity of the observed phenotype in MKK3bE expressing hearts.
Considering the fact that p38 is usually activated in a transient fashion in response to acute stress stimuli, our model may be more relevant to pathological induction of the p38 pathway in heart. Our data also shows that MK2 had a major role in p38 mediated cardiac hypertrophy. MK2 inactivation partially reduced LV weight, cross-sectional area and LV wall thickness induced by p38 activation. In short, our data suggests that activation of the p38 pathway in adult heart may still have a significant contribution to cardiomyocyte hypertrophy in addition to other aspects of pathological remodeling. Furthermore, MK2 is a significant downstream signaling component in p38 induced hypertrophy.
In addition to hypertrophy, we also showed that p38 MAPK activation is linked to cardiac remodeling and contractile defects as supported by histology, gene expression and functional data, in good agreement with previous findings 6, 8
. MK2 inactivation significantly improved cardiac contractility in MKK3 transgenic hearts based on hemodynamic measurements. However, MK2 did not rescue the observed myocardial stiffness or fibrotic remodeling. One possibility is that MK2 acts directly downstream of p38 MAPK to mediate negative inotropic signaling on the contractile apparatus 8
. However, the underlying molecular mechanism needs to be further investigated.
In this study, we demonstrate that MK2 has a previously uncharacterized role in regulating COX-2 protein expression. Our data suggests that MK2 modulates the rate of COX-2 protein synthesis without affecting its degradation. We also suggest that the MK2 mediated modulation of COX-2 protein synthesis does not involve the COX-2 mRNA 3′UTR. This observation differs from previous reports that implicates p38 and MK2 in COX-2 induction through mRNA stabilization 14, 15, 30
. MK2 mediated regulation of COX-2 protein synthesis is an important addition to the regulatory mechanisms linking stress stimuli to inflammatory responses in heart cells.
COX-2 protein induction has been found in human patients with myocardial infarction or heart failure 31
, and prostaglandins such as PGF2α
are induced in heart failure patients as well 32
. COX-2 inhibition protects the heart from endotoxin and lipoteichoic acid treatment 33, 34
. COX-2 inhibition also protects the heart from ischemia related damage in coronary artery ligated rat and mouse heart 35, 36
. However, a number of other st udies have reported no effect, or the opposite effect of COX-2 inhibition in myocarditis and ischemia (reviewed in 37
). Evidence from our lab suggests that COX-2 expression in heart is sufficient to induce cardiac hypertrophy and fetal gene expression 38
. Clearly, more investigation will be needed to establish the specific function of COX-2 in MK2 mediated regulation of cardiac pathology and to uncover the underlying molecular basis by which MK2 regulates COX-2 protein synthesis.
The MK2 KO mouse appears to tolerate the loss of MK2 protein well without adverse effects 12
. Therefore, MK2 inhibition may serve as an alternative means of preventing cardiac hypertrophy, heart failure and inhibiting inflammatory response. Indeed, specific MK2 inhibitors are already being evaluated for rheumatoid arthritis 39
and their efficacy in treating heart failure remains to be examined. Nevertheless, the in vivo
evidence and molecular data presented in this study suggest that MK2 is a potential target of intervention for cardiomyopathy and inflammation.
NOVELTY AND SIGNIFICANCE
What is known?
- Chronic activation of a stress inducible p38 mitogen activated protein kinase (MAPK) pathway can lead to pathological changes in the heart, including gene expression, contractile dysfunction, extracellular remodeling, and myocyte death. However, its specific contribution to cardiac hypertrophy remains controversial.
- MAPK activated protein kinase-2 (MK2) is a major downstream protein kinase of p38 MAPK; however, its role in p38 induced cardiac pathology is unknown.
- Cyclooxygenase-2 (COX2) is a known downstream target of p38 signaling and has a role in the development of hypertrophy. However, the significance and mechanism of MK2 mediated pathway in COX2 expression in heart is unknown.
What new information does this article contribute?
- Inducible activation of p38 pathway in adult heart led to hypertrophy and pathological remodeling, a phenotype different from the one that resulted from chronic activation of the p38 pathway.
- MK2 inactivation has a significant but selective impact on the pathological effect of p38 activation in the intact heart.
- MK2 is a critical signal molecule for stress-induced COX2 expression in heart and other cells. The underlying mechanism involves protein synthesis instead of transcriptional regulation, mRNA stability, or protein degradation.
Chronic activation of p38 MAPK can lead to pathological changes in gene expression, contractility, extracellular matrix remodeling, and inflammatory response in the heart. However, the specific role of the p38 kinase pathway in cardiac hypertrophy is controversial. MK2 is a well established p38 downstream kinase, yet its contribution to p38 mediated pathological response in heart has not been investigated. In this report, we developed new transgenic mouse models with cardiomyocyte specific and inducible activation of the p38 pathway in the heart in either an MK2 wildtype or an MK2 null background. Through phenotypic and molecular characterizations, we have established that acute p38 activation in the adult mouse heart causes rapid onset of lethal cardiomyopathy associated with cardiomyocyte hypertrophy, different from the phenotype resulting from chronic p38 activation. Our study also demonstrates for the first time the selective contribution of MK2 to p38 induced pathological changes, including a major role in the post-transcriptional regulation of the pro-inflammatory protein COX-2. This study provides a new understanding of the functional significance of a major stress-response signaling pathway in the heart and of the downstream signaling mechanisms.