The role of minocycline in ischemia-reperfusion injury: a comprehensive review of an old drug with new implications.

Minocycline is a semi-synthetic tetracycline that inhibits bacterial protein synthesis and hence is used for the treatment of many infectious diseases. Over the years, many other interesting properties of minocycline have been identified and been used to make patents which include anti-inflammatory, anti-apoptotic, matrix metalloproteinase inhibitor and free oxygen radical scavenger activity. Ischemia-reperfusion injury is a concern for almost every clinical specialty and minocycline seems to be an attractive cytoprotective agent that can ameliorate the damage due to these properties. Ischemia-reperfusion injury is a complex process and involves various pathways that lead to cell death. This review focuses on the body of evidence describing various proposed mechanisms of action of minocycline and its current experimental use in various animal models of ischemia-reperfusion injury.

Activation of Src protein tyrosine kinase plays an essential role in urocortin-mediated cardioprotection.

Urocortin is a 40 amino acid peptide of the corticotrophin-releasing factor (CRF) family that is synthesized and released by cardiac myocytes. Endogenous urocortin expression is increased during ischemia/reperfusion (I/R) and addition of exogenous urocortin reduces cell death caused by I/R injury. Studies have also showed that the protective action of urocortin is mediated by the activation of ERK1/2. We discovered that a non-receptor tyrosine kinase, Src, is involved in the urocortin-induced activation of ERK1/2 in mouse atrial HL-1 myocytes. The selective Src family kinase inhibitor, PP2, reduced the urocortin-induced phosphorylation of ERK1/2, and so did the expression of a dominant-negative mutant of Src in transfected HL-1 cells. Inhibition of Src by PP2 also reduced urocortin's protective effects in HL-1 cells after hypoxia/reoxygenation (H/R), as assessed by flow cytometry and caspase-3 activation assay. Titration studies indicated that as little as 10(-8)M urocortin was sufficient to induce Src activation. Maximal phosphorylation/activation of Src and ERK1/2 were both detected after 5 min incubation with urocortin. These effects of urocortin were largely mediated by CRF receptor-1, although a minor contribution of CRF receptor-2 cannot be excluded. Here we report for the first time that short-term treatment with urocortin causes rapid phosphorylation of Src, and that the urocortin-activated Src kinase serves as an upstream modulator of ERK1/2 activation, playing an essential role in urocortin-mediated cardioprotection.

Warm-blood cardioplegic arrest induces selective mitochondrial translocation of protein kinase Cepsilon followed by interaction with 6.1 inwardly rectifying potassium channel subunit in viable myocytes overexpressing urocortin.

OBJECTIVE: This study investigates the cardioprotective role and mechanism of action of urocortin in patients undergoing cardiac surgery, with respect to protein kinase Cepsilon expression, activation, and relocation. BACKGROUND: Cardioplegic arrest and subsequent reperfusion inevitably expose the heart to iatrogenic ischemia/reperfusion injury. We previously reported that iatrogenic ischemia/reperfusion injury caused myocyte induction of urocortin, an endogenous cardioprotective peptide. METHODS: Two sequential biopsies were obtained from the right atrium of 25 patients undergoing coronary artery bypass grafting at the start of grafting (internal control) and 10 minutes after release of the aortic clamp. RESULTS: In hearts exposed to iatrogenic ischemia/reperfusion injury, induction of urocortin was documented at both the mRNA (255% of basic levels; P < .05) and the protein (4-fold increase; P < .01) levels. Iatrogenic ischemia/reperfusion injury also induced a selective increase of protein kinase Cepsilon mRNA (225% of internal control; P < .05) and a 2-fold overexpression of total protein kinase Cepsilon (P < .05), which paralleled a 2.9-fold increase in protein kinase Cepsilon phosphorylation (P < .01). Mitochondrial translocation of activated protein kinase Cepsilon was observed only in postcardioplegic samples, using both subcellular fractionation (P < .05) and immunostaining techniques (P < .05). Enhanced protein kinase Cepsilon/mitochondria colocalization was selectively observed in viable myocytes, showing concurrently positive staining for urocortin (P < .05). Finally, co immunoprecipitation experiments documented an iatrogenic ischemia/reperfusion injury-enhanced physical interaction of phosphorylated protein kinase Cepsilon with the 6.1 inwardly rectifying potassium channel subunit of the K(ATP) channels (P < .05). CONCLUSION: After iatrogenic ischemia/reperfusion injury, urocortin expression in viable cells selectively colocalized with enhanced phosphorylation and mitochondrial relocation of protein kinase Cepsilon, suggesting a cardioprotective role for endogenous urocortin. The physical interaction of activated protein kinase Cepsilon with 6.1 inwardly rectifying potassium channel, enhanced by cardioplegic arrest, may represent a conjectural mechanism of urocortin-mediated cardioprotection.

Oral administration of amino acidic supplements improves protein and energy profiles in skeletal muscle of aged rats: elongation of functional performance and acceleration of mitochondrial recovery in adenosine triphosphate after exhaustive exertion.

Sarcopenia is an inevitable age-related degenerative process chiefly characterized by decreased synthesis of muscle proteins and impaired mitochondrial function, leading to progressive loss of muscle mass. Here, we sought to probe whether long-term administration of oral amino acids (AAs) can increase protein and adenosine triphosphate (ATP) content in the gastrocnemius muscle of aged rats, enhancing functional performance. To this end, 6- and 24-month-old male Fisher 344 rats were divided into 3 groups: group A (6-month-old rats) and group B (24-month-old rats) were used as adult and senescent control group, respectively, while group C (24-month-old rats) was used as senescent treated group and underwent 1-month oral treatment with a mixture of mainly essential AAs. Untreated senescent animals exhibited a 30% reduction in total and fractional protein content, as well as a 50% reduction in ATP content and production, compared with adult control rats (p <0.001). Long-term supplementation with mixed AAs significantly improved protein and high-energy phosphate content, as well as the rate of mitochondrial ATP production, conforming their values to those of adult control animals (p <0.001). The improved availability of protein and high-energy substrates in the gastrocnemius muscle of treated aged rats paralleled a significant enhancement in functional performance assessed by swim test, with dramatic elongation of maximal exertion times compared with untreated senescent rats (p <0.001). In line with these findings, we observed that, after 6 hours of rest following exhaustive swimming, the recovery in mitochondrial ATP content was approximately 70% in adult control rats, approximately 60% in senescent control rats, and normalized in treated rats as compared with animals of the same age unexposed to maximal exertion (p <0.001). In conclusion, nutritional supplementation with oral AAs improved protein and energy profiles in the gastrocnemius of treated rats, enhancing functional performance and accelerating high-energy phosphate recovery after exhaustive exertion.

Cardiac release of urocortin precedes the occurrence of irreversible myocardial damage in the rat heart exposed to ischemia/reperfusion injury.

This study evaluates whether cardiac ischemia induces release of urocortin, before and independently from myocyte cell death. Urocortin levels rose after 5-min ischemia and peaked after 10-min ischemia, when cell death was not detected. However, myocyte apoptosis and/or necrosis occurred following 20- and 30-min ischemia, which paralleled a fall in urocortin levels, suggesting that urocortin expression and release are mainly sustained by metabolically challenged, though still viable myocytes. Hence, since cardiac release of urocortin, unlike that of conventional biomarkers, occurs before and apart from cell death, urocortin levels may be clinically useful in the diagnosis of sublethal myocardial ischemia.