Background-Titin is the largest mammalian (Ϸ3000 to 4000 kDa) and myofilament protein that acts as a molecular spring in the cardiac sarcomere and determines systolic and diastolic function. Loss of titin in ischemic hearts has been reported, but the mechanism of titin degradation is not well understood. Matrix metalloproteinase-2 (MMP-2) is localized to the cardiac sarcomere and, on activation in ischemia/reperfusion injury, proteolyzes specific myofilament proteins. Here we determine whether titin is an intracellular substrate for MMP-2 and if its degradation during ischemia/reperfusion contributes to cardiac contractile dysfunction. Methods and Results-Immunohistochemistry and confocal microscopy in rat and human hearts showed discrete colocalization between MMP-2 and titin in the Z-disk region of titin and that MMP-2 is localized mainly to titin near the Z disk of the cardiac sarcomere. Both purified titin and titin in skinned cardiomyocytes were proteolyzed when incubated with MMP-2 in a concentration-dependent manner, and this was prevented by MMP inhibitors. Isolated rat hearts subjected to ischemia/reperfusion injury showed cleavage of titin in ventricular extracts by gel electrophoresis, which was confirmed by reduced titin immunostaining in tissue sections. Inhibition of MMP activity with ONO-4817 prevented ischemia/reperfusion-induced titin degradation and improved the recovery of myocardial contractile function. Titin degradation was also reduced in hearts from MMP-2 knockout mice subjected to ischemia/reperfusion in vivo compared with wild-type controls. Conclusion-MMP-2 localizes to titin at the Z-disk region of the cardiac sarcomere and contributes to titin degradation in myocardial ischemia/reperfusion injury. Key Words: contractile dysfunction Ⅲ ischemia Ⅲ matrix metalloproteinase-2 Ⅲ sarcomere Ⅲ titin M atrix metalloproteinase-2 (MMP-2) is a zinc-dependent protease that is best known for its ability to degrade the extracellular matrix in both physiological and pathological conditions. MMP-2 is synthesized as a zymogen by a variety of cells, including cardiac myocytes, and is activated either by proteases 1 (such as by action of or by posttranslational modifications to the full-length enzyme caused by enhanced oxidative stress. For example, peroxynitrite, which is generated in early reperfusion after ischemia, 2 directly activates several MMPs, 3 including MMP-2, 4 via a nonproteolytic mechanism involving the S-glutathiolation of a critical propeptide cysteine in its autoinhibitory domain. Ediotrial see p 2002 Clinical Perspective on p 2047MMPs are best recognized for their role in tissue remodeling by proteolyzing various components of the extracellular matrix in both health and disease, ie, in angiogenesis, embryogenesis, wound healing, 5 atherosclerosis, 6 aortic aneurysm, 7 and myocardial infarction. 8 More recent studies, however, show that MMP-2 is involved in several acute biological processes independently of its actions on extracellular matrix proteins. This includes platelet activati...

The murine jejunum and lower esophageal sphincter (LES) were examined to determine the locations of various signaling molecules and their colocalization with caveolin-1 and one another. Caveolin-1 was present in punctate sites of the plasma membranes (PM) of all smooth muscles and diffusely in all classes of interstitial cells of Cajal (ICC; identified by c-kit immunoreactivity), ICC-myenteric plexus (MP), ICC-deep muscular plexus (DMP), ICC-serosa (ICC-S), and ICC-intramuscularis (IM). In general, all ICC also contained the L-type Ca(2+) (L-Ca(2+)) channel, the PM Ca(2+) pump, and the Na(+)/Ca(2+) exchanger-1 localized with caveolin-1. ICC in various sites also contained Ca(2+)-sequestering molecules such as calreticulin and calsequestrin. Calreticulin was present also in smooth muscle, frequently in the cytosol, whereas calsequestrin was present in skeletal muscle of the esophagus. Gap junction proteins connexin-43 and -40 were present in circular muscle of jejunum but not in longitudinal muscle or in LES. In some cases, these proteins were associated with ICC-DMP. The large-conductance Ca(2+)-activated K(+) channel was present in smooth muscle and skeletal muscle of esophagus and some ICC but was not colocalized with caveolin-1. These findings suggest that all ICC have several Ca(2+)-handling and -sequestering molecules, although the functions of only the L-Ca(2+) channel are currently known. They also suggest that gap junction proteins are located at sites where ultrastructural gap junctions are know to exist in circular muscle of intestine but not in other smooth muscles. These findings also point to the need to evaluate the function of Ca(2+) sequestration in ICC.

Aims Heart failure is a major complication in cancer treatment due to the cardiotoxic effects of anticancer drugs, especially from the anthracyclines such as doxorubicin (DXR). DXR enhances oxidative stress and stimulates matrix metalloproteinase-2 (MMP-2) in cardiomyocytes. We investigated whether MMP inhibitors protect against DXR cardiotoxicity given the role of MMP-2 in proteolyzing sarcomeric proteins in the heart and remodelling the extracellular matrix. Methods and results Eight-week-old male C57BL/6J mice were treated with DXR weekly with or without MMP inhibitors doxycycline or ONO-4817 by daily oral gavage for 4 weeks. Echocardiography was used to determine cardiac function and left ventricular remodelling before and after treatment. MMP inhibitors ameliorated DXR-induced systolic and diastolic dysfunction by reducing the loss in left ventricular ejection fraction, fractional shortening, and E′/A′. MMP inhibitors attenuated adverse left ventricular remodelling, reduced cardiomyocyte dropout, and prevented myocardial fibrosis. DXR increased myocardial MMP-2 activity in part also by upregulating N-terminal truncated MMP-2. Immunogold transmission electron microscopy showed that DXR elevated MMP-2 levels within the sarcomere and mitochondria which were associated with myofilament lysis, mitochondrial degeneration, and T-tubule distention. DXR-induced myofilament lysis was associated with increased titin proteolysis in the heart which was prevented by ONO-4817. DXR also increased the level and activity of MMP-2 in human embryonic stem cell-derived cardiomyocytes, which was reduced by ONO-4817. Conclusions MMP-2 activation is an early event in DXR cardiotoxicity and contributes to myofilament lysis by proteolyzing cardiac titin. Two orally available MMP inhibitors ameliorated DXR cardiotoxicity by attenuating intracellular and extracellular matrix remodelling, suggesting their use may be a potential prophylactic strategy to prevent heart injury during chemotherapy.

Confocal microscopic images were obtained from the immunohistochemical sections of jejeunum to determine the localization/colocalization between caveolin-1, caveolin-2 and caveolin-3 in intestinal smooth muscle cells (SMCs) and interstitial cells of Cajal (ICC) of Cav1(+/+) and Cav1(-/-) mouse. Intestinal regions were segmented [inner circular muscle (icm), outer circular muscle (ocm), myenteric plexus region (mp), and longitudinal muscle (lm)] by LSM 5 and analyzed by ImageJ to show Pearson's correlation (r (p)) and overlap coefficient (r) of colocalization. In the intestine of Cav1(+/+), caveolin-1 (cav1) was colocalized with caveolin-2 (cav2) and caveolin-3 (cav3). Cav2 also was well colocalized with cav3. In the intestine of Cav1(-/-), cav1 and cav2 were absent in all images, but reduced cav3 was expressed in ocm. Caveolae were present in cell types with cav1 in Cav1(+/+), and present with cav3 in ocm of Cav1(-/-). C-kit occurred in deep muscular plexus (ICC-DMP) and myenteric plexus (ICC-MP), in both Cav1(+/+) and Cav1(-/-), and colocalized with cav1 and cav2 in the intestine of Cav1(+/+). Cav3 was absent/present at low immunoreactivity in ICC-DMP and ICC-MP of the intestines of Cav1(+/+) and Cav1(-/-). To conclude, cav1 is necessary for the expression of cav2 in SMC and ICC of intestine and facilitates, but is not necessary for the expression of cav3.

Objective-Matrix metalloproteinase (MMP)-2 is activated in aorta during endotoxemia and plays a role in the hypocontractility to vasoconstrictors. Calponin-1 is a regulator of vascular smooth muscle tone with similarities to troponin, a cardiac myocyte protein that is cleaved by MMP-2 in myocardial oxidative stress injuries. We hypothesized that calponin-1 may be proteolyzed by MMP-2 in endotoxemia-induced vascular hypocontractility. Methods and Results-Rats were given a nonlethal dose of bacterial lipopolysaccharide (LPS) or vehicle. Some rats were given the MMP inhibitors ONO-4817 or doxycycline. Six hours later, plasma nitrateϩnitrite increased Ͼ15-fold in LPS-treated rats, an effect unchanged by doxycycline. Both ONO-4817 and doxycycline prevented LPS-induced aortic hypocontractility to phenylephrine. LPS activated MMP-2 in the aorta by S-glutathiolation. Calponin-1 levels decreased by 25% in endotoxemic aortae, which was prevented by doxycycline. Calponin-1 and MMP-2 coimmunoprecipitated and both exhibited uniform cytosolic staining in medial vascular smooth muscle cells. In vitro incubation of calponin-1 with MMP-2 led to calponin-1 degradation and appearance of its cleavage product. Conclusion-Calponin-1 is a target of MMP-2, which contributes to endotoxemia-induced vascular hypocontractility. Key Words: metalloproteinases Ⅲ calponin-1 Ⅲ endotoxemia Ⅲ lipopolysaccharide Ⅲ vascular hypocontractility S epsis remains one of the most common causes of death worldwide. 1 Its cardiovascular manifestations include myocardial dysfunction and severe arterial hypotension caused in part by vascular hyporeactivity to vasoconstrictors. 2 Several mechanisms 3 including lipopolysaccharide (LPS)-and interleukin-1␤-mediated activation of inducible nitric oxide (NO) synthase, excess biosynthesis of NO, 4,5 and peroxynitrite (ONOO Ϫ ) 6,7 in the vascular wall are important mediators of the vascular dysfunction in sepsis. ONOO Ϫ directly activates matrix metalloproteinases (MMPs), 8,9 a group of zinc-dependent endopeptidases best known for their ability to degrade extracellular matrix proteins, causing vascular dysfunction and remodeling in many cardiovascular diseases. 10 -16 MMPs are synthesized as inactive zymogens in several cells, including vascular smooth muscle that express 72 kDa MMP-2 abundantly. 10 It is activated by proteolytic removal of the autoinhibitory propeptide domain 10 or, alternatively, by S-glutathiolation of a critical cysteine in this propeptide, on reaction with ONOO Ϫ and glutathione, resulting in 72 kDa S-glutathiolated MMP-2. 9 However, whether MMP-2 activation occurs by S-glutathiolation in the vasculature is unknown.Studies have implicated MMPs and the beneficial effects of MMP inhibition in experimental models of sepsis. 17-23 MMP-2 plays an important role in LPS-induced vascular hypocontractility in rats. In vivo treatment with the MMP inhibitor doxycycline attenuated LPS-induced increase in vascular MMP-2 activity and protected against the loss of vascular contractile tone. 18 Doxycyclin...

Aims: Myocardial ischemia can result in marked mitochondrial damage leading to cardiac dysfunction, as such identifying novel mechanisms to limit mitochondrial injury is important. This study investigated the hypothesis that inhibiting soluble epoxide hydrolase (sEH), responsible for converting epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids protects mitochondrial from injury caused by myocardial infarction.Methods: sEH null and WT littermate mice were subjected to surgical occlusion of the left anterior descending (LAD) artery or sham operation. A parallel group of WT mice received an sEH inhibitor, trans-4-[4-(3-adamantan-1-y1-ureido)-cyclohexyloxy]-benzoic acid (tAUCB; 10 mg/L) or vehicle in the drinking water 4 days prior and 7 days post-MI. Cardiac function was assessed by echocardiography prior- and 7-days post-surgery. Heart tissues were dissected into infarct, peri-, and non-infarct regions to assess ultrastructure by electron microscopy. Complexes I, II, IV, citrate synthase, PI3K activities, and mitochondrial respiration were assessed in non-infarct regions. Isolated working hearts were used to measure the rates of glucose and palmitate oxidation.Results: Echocardiography revealed that tAUCB treatment or sEH deficiency significantly improved systolic and diastolic function post-MI compared to controls. Reduced infarct expansion and less adverse cardiac remodeling were observed in tAUCB-treated and sEH null groups. EM data demonstrated mitochondrial ultrastructure damage occurred in infarct and peri-infarct regions but not in non-infarct regions. Inhibition of sEH resulted in significant improvements in mitochondrial respiration, ATP content, mitochondrial enzymatic activities and restored insulin sensitivity and PI3K activity.Conclusion: Inhibition or genetic deletion of sEH protects against long-term ischemia by preserving cardiac function and maintaining mitochondrial efficiency.

In mouse intestine, caveolae and caveolin-1 (Cav-1) are present in smooth muscle (responsible for executing contractions) and in interstitial cells of Cajal (ICC; responsible for pacing contractions). We found that a number of calcium handling/dependent molecules are associated with caveolae, including L-type Ca2+ channels, Na+-Ca2+ exchanger type 1 (NCX1), plasma membrane Ca2+ pumps and neural nitric oxide synthase (nNOS), and that caveolae are close to the peripheral endo-sarcoplasmic reticulum (ER-SR). Also we found that this assemblage may account for recycling of calcium from caveolar domains to SR through L-type Ca + channels to sustain pacing and contractions. Here we test this hypothesis further comparing pacing and contractions under various conditions in longitudinal muscle of Cav-1 knockout mice (lacking caveolae) and in their genetic controls. We used a procedure in which pacing frequencies (indicative of functioning of ICC) and contraction amplitudes (indicative of functioning of smooth muscle) were studied in calcium-free media with 100 mM ethylene glycol tetra-acetic acid (EGTA). The absence of caveolae in ICC inhibited the ability of ICC to maintain frequencies of contraction in the calcium-free medium by reducing recycling of calcium from caveolar plasma membrane to SR when the calcium stores were initially full. This recycling to ICC involved primarily L-type Ca2+ channels; i.e. pacing frequencies were enhanced by opening and inhibited by closing these channels. However, when these stores were depleted by block of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump or calcium release was activated by carbachol, the absence of Cav-1 or caveolae had little or no effect. The absence of caveolae had little impact on contraction amplitudes, indicative of recycling of calcium to SR in smooth muscle. However, the absence of caveolae slowed the rate of loss of calcium from SR under some conditions in both ICC and smooth muscle, which may reflect the loss of proximity to store operated Ca channels. We found evidence that these channels were associated with Cav-1. These changes were all consistent with the hypothesis that a reduction of the extracellular calcium associated with caveolae in ICC of the myenteric plexus, the state of L-type Ca2+ channels or an increase in the distance between caveolae and SR affected calcium handling.

The rubella virus (RV) capsid is an RNA-binding protein that functions in nucleocapsid assembly at the Golgi complex, the site of virus budding. In addition to its role in virus assembly, pools of capsid associate with mitochondria, a localization that is not consistent with virus assembly. Here we examined the interaction of capsid with mitochondria and showed that this viral protein inhibits the import and processing of mitochondrial precursor proteins in vitro. Moreover, RV-infected cells were found to contain lower intramitochondrial levels of matrix protein p32. In addition to inhibiting the translocation of substrates into mammalian mitochondria, capsid efficiently blocked import into yeast mitochondria, thereby suggesting that it acts by targeting a highly conserved component of the translocation apparatus. Finally, mutation of a cluster of five arginine residues in the amino terminus of capsid, though not interfering with its binding to mitochondria, abrogated its ability to block protein import into mitochondria. This is the first report of a viral protein that affects the import of proteins into mitochondria.Rubella virus (RV) is a human pathogen that causes severe birth defects (reviewed in reference 17). Teratogenicity undoubtedly results from deleterious interactions between virus proteins and host cell proteins, but little is known about this phenomenon. The viral genome encodes two nonstructural proteins (p150 and p90) and three structural proteins, the capsid protein, E2, and E1. The capsid protein is a multifunctional RNA-binding protein and is the focus of our studies. The primary function of the capsid protein is to package the viral genome into nucleocapsids, a process that appears to be regulated by phosphorylation (27,29). Recent evidence suggests that, in addition to their structural roles in virus assembly, capsid proteins may be key determinants in virus-host interactions. For example, the hepatitis C virus capsid may affect disease development by modulating apoptotic and innate immune pathways (5,36,42). Moreover, localization of capsids appears to be an important factor in viral pathogenesis. Specifically, it has been reported that nuclear localization of the Japanese encephalitis virus capsid is necessary for neuroinvasion (38). The RV capsid also localizes to subcellular compartments that have no obvious relationship to the virus budding site (Golgi complex). For example, a pool of capsid colocalizes with the nonstructural protein p150 on virus-induced tubular structures (26). Later it was demonstrated that capsid binds p150 and modulates the transcription of viral RNA (8,(47)(48)(49). In addition to its role as a replicase cofactor, a number of studies indicate that a large pool of capsid localizes to mitochondria (2, 22, 32).Among togaviruses, localization of capsid proteins to mitochondria is unique to RV (31). The significance of this phenomenon is not known, but we hypothesize that the mitochondrial pool of capsid is engaged in functions not directly related to virus budding. We...