Woo Jung Cho

Titin is a target of matrix metalloproteinase-2: implications in myocardial ischemia/reperfusion injury.

Background— Titin is the largest mammalian (≈3000 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. # Clinical Perspective {#article-title-54}Background— Titin is the largest mammalian (≈3000 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.

Inhibition of matrix metalloproteinase activity in vivo protects against vascular hyporeactivity in endotoxemia

Persistent arterial hypotension is a hallmark of sepsis and is believed to be caused, at least in part, by excess nitric oxide (NO). NO can combine with superoxide to produce peroxynitrite, which activates matrix metalloproteinases (MMPs). Whether MMP inhibition in vivo protects against vascular hyporeactivity induced by endotoxemia is unknown. Male Sprague-Dawley rats were administered either bacterial lipopolysaccharide (LPS, 4 mg/kg ip) or vehicle (pyrogen-free water). Later (30 min), animals received the MMP inhibitor doxycycline (4 mg/kg ip) or vehicle (pyrogen-free water). After LPS injection (6 h), animals were killed, and aortas were excised. Aortic rings were mounted in organ baths, and contractile responses to phenylephrine or KCl were measured. Aortas and plasma were examined for MMP activity by gelatin zymography. Aortic MMP and inducible nitric oxide synthase (iNOS) were examined by immunoblot and/or immunohistochemistry. Doxycycline prevented the LPS-induced development of ex vivo vascular hyporeactivity to phenylephrine and KCl. iNOS protein was significantly upregulated in aortic homogenates from endotoxemic rats; doxycycline did not alter its level. MMP-9 activity was undetectable in aortic homogenates from LPS-treated rats but significantly upregulated in the plasma; this was attenuated by doxycycline. Plasma MMP-2 activities were unchanged by LPS. Specific MMP-2 activity was increased in aortas from LPS-treated rats. This study demonstrates the in vivo protective effect of the MMP inhibitor doxycycline against the development of vascular hyporeactivity in endotoxemic rats.

Calcium extrusion by plasma membrane calcium pump is impaired in caveolin-1 knockout mouse small intestine

Plasma membrane calcium ATPase (PMCA) is an important calcium extrusion mechanism in smooth muscle cells. PMCA4 is the predominant isoform operating in conditions of high intracellular calcium during contraction. PMCA appears to be localized in lipid rafts and caveolae. In this study we examined the effects of the PMCA4-selective inhibitor caloxin 1c2 (5 microM) in intestine of caveolin-1 knockout mice and in bovine tracheal smooth muscle after caveolae disruption on PMCA4 function. Small intestinal tissues from control mice treated with caloxin 1c2 showed a higher contractile response of the longitudinal smooth muscle to Carbachol (10 microM) when compared to control tissues treated with a similar concentration of a control peptide. This effect of caloxin 1c2 was not found in tissues from caveolin-1 knockout mice. Immunohistochemistry and Western blotting of membrane fractions showed that PMCA was co-localized with caveolin-1 in smooth muscle plasma membrane in control tissues. One of the PMCA4 splice variant bands was missing in the lipid raft-enriched fraction prepared from caveolin-1 knockout tissue. In bovine tracheal smooth muscle tissue, caveolae disruption by cholesterol depletion led to the diminution of caveolin-1 and PMCA4b immunoreactivities, previously co-localized in the smooth muscle plasma membrane, and to the loss of the increase in Carbachol-induced contraction by caloxin 1c2. Our results suggest that the calcium removal function of PMCA4 in smooth muscle cells is dependent on its presence in intact caveolae. We suggest that this is due to the close spatial arrangement that allows calcium extrusion from a privileged cytosolic space between caveolae and sarcoplasmic reticulum.

Matrix metalloproteinase-2, caveolins, focal adhesion kinase and c-Kit in cells of the mouse myocardium

Matrix metalloproteinase‐2 (MMP‐2) may play roles at intracellular and extracellular sites of the heart in ischaemia/reperfusion injury. Caveolins (Cav‐1, ‐2 and ‐3) are lipid raft proteins which play roles in cell sig‐nalling. This study examined, using immunohistochemistry and two photon confocal microscopy, if MMP‐2 and caveolins co‐localize at the plasma membrane of cardiac cells: cardiomyocytes (CM), fibroblasts (FB) and capillary endothelial cells (CEC) in the left ventricle (LV) of the Cav‐1+/+ and Cav‐1−/− mouse heart. In Cav‐1+/+ mouse LV MMP‐2 and Cav‐1 co‐localized at CM plasma membranes, and at multiple locations in FB and CEC. MMP‐2 co‐localized with Cav‐2 only at CEC. MMP‐2 co‐localized with Cav‐3 at CM plasma membranes and Z‐lines, and partially at FB and CEC. In Cav‐1−/− LV Cav‐1 and MMP‐2 were absent or reduced everywhere. Cav‐2 appeared at CEC despite the absence of Cav‐1. Cav‐3 appeared at CM plasma membranes and Z‐lines, FB and CEC. Also, FAK in FB and c‐Kit in interstitial Cajal‐like cells (ICLC) were completely absent. By transmission electron microscopy in Cav‐1+/+, regular size caveolae (Cav) were at CEC, irregular size Cav were at CM and a few were at FB. In Cav‐1−/− there were few Cav at CM and FB and some at CEC. To conclude, MMP‐2 is closely associated with caveolins at FB and CEC as well as at CM. Also, MMP‐2 is closely associated with FAK at FB and c‐Kit at ICLC. Thus, Cav‐1 expression is not necessary for Cav‐2 expression. Cav‐3 or Cav‐3 with Cav‐2 has the capability to make Cav.

Matrix Metalloproteinase-2 Proteolysis of Calponin-1 Contributes to Vascular Hypocontractility in Endotoxemic Rats

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 nitrate+nitrite increased >15-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.

Colocalization between caveolin isoforms in the intestinal smooth muscle and interstitial cells of Cajal of the Cav1+/+ and Cav1−/− mouse

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 (rp) 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.

The role of caveolae and caveolin 1 in calcium handling in pacing and contraction of mouse intestine

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.

Smooth muscle NOS, colocalized with caveolin-1, modulates contraction in mouse small intestine

Neuronal nitric oxide synthase (nNOS) in myenteric neurons is activated during peristalsis to produce nitric oxide which relaxes intestinal smooth muscle. A putative nNOS is also found in the membrane of intestinal smooth muscle cells in mouse and dog. In this study we studied the possible functions of this nNOS expressed in mouse small intestinal smooth muscle colocalized with caveolin‐1(Cav‐1). Cav‐1 knockout mice lacked nNOS in smooth muscle and provided control tissues. 60 mM KCl was used to increase intracellular [Ca2+] through L‐type Ca2+ channel opening and stimulate smooth muscle NOS activity in intestinal tissue segments. An additional contractile response to LNNA (100 μM, NOS inhibitor) was observed in KCl‐contracted tissues from control mice and was almost absent in tissues from Cav‐1 knockout mice. Disruption of caveolae with 40 mM methyl‐β cyclodextrin in tissues from control mice led to the loss of Cav‐1 and nNOS immunoreactivity from smooth muscle as shown by immunohistochemistry and a reduction in the response of these tissues to N‐ω‐nitro‐L‐arginine (LNNA). Reconstitution of membrane cholesterol using water soluble cholesterol in the depleted segments restored the immunoreactivity and the response to LNNA added after KCl. Nicardipine (1 μM) blocked the responses to KCl and LNNA confirming the role of L‐type Ca2+ channels. ODQ (1 μM, soluble guanylate cyclase inhibitor) had the same effect as inhibition of NOS following KCl. We conclude that the activation of nNOS, localized in smooth muscle caveolae, by calcium entering through L‐type calcium channels triggers nitric oxide production which modulates muscle contraction by a cGMP‐dependent mechanism.

Effect of ischemia reperfusion injury and epoxyeicosatrienoic acids on caveolin expression in mouse myocardium.

Background: Caveolins (Cav) are structural proteins that insert into the plasma membrane to form caveolae that can bind molecules important in cardiac signal transduction and function. Cytochrome P450 epoxygenases can metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have known cardioprotective effects. Subsequent metabolism of EETs by soluble epoxide hydrolase reduces the protective effect. Aims: (1) To assess the effect of ischemia–reperfusion injury on expression and subcellular localization of caveolins. (2) To study the effect of EETs on caveolins. Methods: Hearts from soluble epoxide hydrolase null (KO) and littermate control (WT) mice were perfused in Langendorff mode and subjected to 20 minutes ischemia followed by 40 minutes reperfusion. Immunohistochemistry, immunoblot, and electron microscopy were performed to study localization of caveolins and changes in ultrastructure. Results: In WT heart, Cav-1 and Cav-3 were present in cardiomyocyte and capillary endothelial cell at baseline. After ischemia, Cav-1 but not Cav-3, disappeared from cardiomyocyte; moreover, caveolae were absent and mitochondrial cristae were damaged. Improved postischemic functional recovery observed in KO or WT hearts treated with 11,12-EET corresponded to higher Cav-1 expression and maintained caveolae structure. In addition, KO mice preserved the Cav-1 signaling after ischemia that lost in WT mice. Conclusions: Taken together, our data suggest that ischemia–reperfusion injury causes loss of Cav-1 and caveolins, and EETs-mediated cardioprotection involves preservation of Cav-1.

Dynamic Alterations to α-Actinin Accompanying Sarcomere Disassembly and Reassembly during Cardiomyocyte Mitosis.

Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk) disassembly precedes that of titin (M-line), suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble) and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1) induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.