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The mitogen-activated protein kinases (MAPKs) play an important role in ischemia/reperfusion (I/R) injury. Previous evidence suggests that p38 MAPK inhibition before ischemia is cardioprotective. However, whether p38 MAPK inhibition during ischemia or reperfusion provides cardioprotection is not well known. We tested the hypothesis that p38 MAPK inhibition at different times during I/R protects the heart from arrhythmias, reduces the infarct size, and attenuates ventricular dysfunction. Adult Wistar rats were subject to a 30-minute left anterior descending coronary artery occlusion, followed by a 120-minute reperfusion. A p38 MAPK inhibitor, SB203580, was given intravenously before left anterior descending coronary artery occlusion, during ischemia, or at the onset of reperfusion. The results showed that SB203580 given either before or during ischemia, but not at the onset of reperfusion, decreased the ventricular tachycardia/ventricular fibrillation (VT/VF) incidence and heat shock protein 27 phosphorylation, and increased connexin 43 phosphorylation. The infarct size and cytochrome c level was decreased in all SB203580-treated rats, without the alteration of the total Bax/Bcl-2 expression. The ventricular function was improved only in SB203580-pretreated rats. These findings suggest that timing of p38 MAPK inhibition with respect to onset of ischemia is an important determinant of therapeutic efficacy.

The use of nonselective pharmacological inhibitors has resulted in controversy regarding the mechanism and consequences of p38 activation during myocardial infarction. Classic p38 inhibitors such as SB203580 rely on a critical "gatekeeper" threonine residue for binding. We addressed these controversies by using mice in which the p38␣ alleles were targeted to cause substitution of the gatekeeper residue and resistance to inhibition. In homozygous drug-resistant compared with wildtype hearts, SB203580 failed to inhibit the activating phosphorylation of p38 or to reduce the infarction caused by myocardial ischemia. However, BIRB796, a p38 inhibitor not reliant on the gatekeeper for binding, similarly reduced p38-activating phosphorylation and infarction in both wild-type and knock-in mice, thereby excluding a nonspecific inhibitor-dependent phenotype resulting from the targeting strategy. Furthermore, the activation during myocardial ischemia involved phosphorylation of both the threonine and tyrosine residues in the activation loop of p38 despite the phosphorylation of the threonine alone being sufficient to create the epitope for dual phosphospecific antibody binding. Finally, SB203580 failed to reduce infarction in heterozygous drug-resistant hearts, suggesting that near complete inhibition of p38␣ kinase activity is necessary to elicit protection. These results indicate that, during myocardial ischemia, p38␣ (i) is the dominant-active p38 isoform, (ii) contributes to infarction, (iii) is responsible for the cardioprotective effect of SB203580, and (iv) is activated by a mechanism consistent with autodiphosphorylation despite this necessitating the phosphorylation of a tyrosine residue by an archetypal serine/threonine kinase.

Previous studies have demonstrated that decreased bone mass results from either the impairment of osteoblastic insulin signaling or obesity. Our previous study revealed that 12-week high-fat-diet (HFD) consumption caused obesity as well as peripheral and brain insulin resistance. However, the osteoblastic insulin resistance induced by HFD has not been elucidated. Therefore, we hypothesized that 12-week HFD rats exhibited not only peripheral insulin resistance but also osteoblastic insulin resistance, which leads to decreased jawbone quality. We found that the jawbones of rats fed a 12-week HFD exhibited increased osteoporosis. The osteoblastic cells isolated from HFD-fed rats exhibited the impairment of osteoblastic insulin signaling as well as reduction of cell proliferation and survival. In conclusion, this study demonstrated that insulin resistance induced by 12-week HFD impaired osteoblastic insulin signaling, osteoblast proliferation, and osteoblast survival and resulted in osteoporosis in the jawbone.

The p38 mitogen-activated protein kinases (p38s) are Ser/Thr kinases that are activated as a result of cellular stresses and various pathological conditions, including myocardial ischemia/reperfusion. p38 activation has been shown to accentuate myocardial injury and impair cardiac function. Inhibition of p38 activation and its activity has been proposed to be cardioprotective by slowing the rate of myocardial damage and improving cardiac function. The growing body of evidence on the use of p38 inhibitors as therapeutic means for responding to heart problems is controversial, since both beneficial as well as a lack of protective effects on the heart have been reported. In this review, the outcomes from studies investigating the effect of p38 inhibitors on the heart in a wide range of study models, including in vitro, ex vivo, and in vivo models, are discussed. The correlations of experimental models with practical clinical usefulness, as well as the need for future studies regarding the use of p38 inhibitors, are also addressed.

The activation of p38 MAPK by dual phosphorylation aggravates myocardial ischemic injury and depresses cardiac contractile function. SB203580, an ATP-competitive inhibitor of p38 MAPK and other kinases, prevents this dual phosphorylation during ischemia. Studies in non-cardiac tissue have shown receptor-interacting protein 2 (RIP2) lies upstream of p38 MAPK, is SB203580-sensitive and ischemia-responsive, and aggravates ischemic injury. We therefore examined the RIP2-p38 MAPK signaling axis in the heart. Adenovirus-driven expression of wild-type RIP2 in adult rat ventricular myocytes caused robust, SB203580-sensitive dual phosphorylation of p38 MAPK associated with activation of p38 MAPK kinases MKK3, MKK4, and MKK6. The effect of SB203580 was recapitulated by unrelated inhibitors of RIP2 or the downstream MAPK kinase kinase, TAK1. However, overexpression of wild-type, kinasedead, caspase recruitment domain-deleted, or kinase-dead and caspase recruitment domain-deleted forms of RIP2 had no effect on the activating dual phosphorylation of p38 MAPK during simulated ischemia. Similarly, p38 MAPK activation and myocardial infarction size in response to true ischemia did not differ between hearts from wild-type and RIP2 null mice. However, both p38 MAPK activation and the contractile depression caused by the endotoxin component muramyl dipeptide were attenuated by SB203580 and in RIP2 null hearts. Although RIP2 can cause myocardial p38 MAPK dual phosphorylation in the heart under some circumstances, it is not responsible for the SB203580-sensitive pattern of activation during ischemia.There is overwhelming evidence that the activation of p38 MAPK 2 during prolonged myocardial ischemia accelerates injury (see Refs. 1 and 2 for review). Thus, in theory at least, inhibitors of p38 MAPK have therapeutic potential in ischemic heart disease. However, enthusiasm for "blanket" pharmacological inhibition of p38 MAPK is tempered by the facts that it is involved in innumerable biological processes (3), its major isoform is essential for early embryogenesis (4), and hepatic toxicity has curtailed the development of at least three clinical trial programs (5). These drawbacks have increased interest in the mechanisms controlling p38 activation in the hope they will reveal new, less crucial, therapeutic targets (5).The activity of p38 MAPK is controlled by dual phosphorylation of the Thr 180 -Gly 181 -Tyr 182 motif within the activation loop/lip (3). The traditional view is that this dual phosphorylation event is achieved by upstream, dual specificity MAPK kinases or MKKs (3, 5). SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole), a pyridinyl imidazole, is the most widely used pharmacological inhibitor of p38 MAPK. Because it occupies the catalytic site without inhibiting upstream MKKs, it should inhibit the phosphorylation events downstream of p38 MAPK without inhibiting the dual phosphorylation of p38 MAPK itself. Nonetheless, there are numerous examples where SB203580 and other pyridinyl imidazole...

Protease enzymes generated from injured cells and leukocytes are the primary cause of myocardial cell damage following ischemia/reperfusion (I/R). The inhibition of protease enzyme activity via the administration of particular drugs may reduce injury and potentially save patients' lives. The aim of the current study was to investigate the cardioprotective effects of treatment with recombinant human secretory leukocyte protease inhibitor (rhSLPI) on in vitro and ex vivo models of myocardial I/R injury. rhSLPI was applied to isolated adult rat ventricular myocytes (ARVMs) subjected to simulated I/R and to ex vivo murine hearts prior to I/R injury. Cellular injury, cell viability, reactive oxygen species (ROS) levels, and levels of associated proteins were assessed. The results demonstrated that administration of rhSLPI prior to or during sI/R significantly reduced the death and injury of ARVMs and significantly reduced intracellular ROS levels in ARVMs during H2O2 stimulation. In addition, treatment of ARVMs with rhSLPI significantly attenuated p38 mitogen-activated protein kinase (MAPK) activation and increased the activation of Akt. Furthermore, pretreatment of ex vivo murine hearts with rhSLPI prior to I/R significantly decreased infarct size, attenuated p38 MAPK activation and increased Akt phosphorylation. The results of the current study demonstrated that treatment with rhSLPI induced a cardioprotective effect and reduced ARVM injury and death, intracellular ROS levels and infarct size. rhSLPI also attenuated p38 MAPK phosphorylation and activated Akt phosphorylation. These results suggest that rhSLPI may be developed as a novel therapeutic strategy of treating ischemic heart disease.

Context: Cardiac cell death and fatal arrhythmias during myocardial ischemia/reperfusion (I/R) can be reduced by p38 MAPK inhibition. However, the effects of p38 MAPK inhibition on cardiac mitochondria have not been investigated. Objective: We tested the hypothesis that p38 MAPK inhibition at different times during I/R protects cardiac mitochondrial functions. Materials and methods: Adult Wistar rats were subjected to 30 min of left anterior descending coronary artery (LAD) occlusion, followed by 120 min of reperfusion. A 2 mg/kg bolus infusion of p38 MAPK inhibitor, SB203580, was given before or during ischemia, or at reperfusion. Mitochondrial function and ultrastructure were assessed and Western blots were performed. Results: Administration of SB203580 at any time point of I/R significantly attenuated the mitochondrial ultrastructure change, mitochondrial swelling, by increasing the absorbance at 540 nm (I/R control 0.42 ± 0.03; pretreatment 0.58 ± 0.04; during ischemia 0.49 ± 0.02; at reperfusion 0.51 ± 0.02, p50.05), similar to reactive oxygen species (ROS) generation (I/R control 1300 ± 48; pretreatment 1150 ± 30; during ischemia 1000 ± 50; at reperfusion 1050 ± 55, p50.05). Only SB203580 given before or during ischemia attenuated mitochondrial membrane depolarization (I/R control 0.78 ± 0.04; pretreatment 1.02 ± 0.03; during ischemia 1.05 ± 0.12, p50.05). In addition, pre-treatment of SB203580 significantly reduced the phosphorylation of p53, CREB, Bax, cytochrome c, and cleaved caspase 3. Discussion and conclusion: The results from this study showed for the first time that p38 MAPK inhibition protects mitochondria from I/R injury.