Midkine Promotes Atherosclerotic Plaque Formation Through Its Pro-Inflammatory, Angiogenic and Anti-Apoptotic Functions in Apolipoprotein E-Knockout Mice.

BACKGROUND: A recent study suggested that midkine (MK), a heparin-binding growth factor, is associated with atherosclerosis progression in patients with artery disease. It has previously been reported that MK plays a critical role in neointima formation in a restenosis model, whereas the role of MK in the development of atherosclerosis has not been investigated. The present study assessed the effect of MK administration on the process of atherosclerotic plaque formation in apolipoprotein E-knockout (ApoE-/-) mice.Methods and Results:Using an osmotic pump, human recombinant MK protein was intraperitoneally administered for 12 weeks in C57BL/6 ApoE-/-(ApoE-/--MK) and ApoE+/+mice fed a high-fat diet. Saline was administered to the control groups of ApoE-/-(ApoE-/--saline) and ApoE+/+mice. The atherosclerotic lesion areas in longitudinal aortic sections were significantly larger in ApoE-/--MK mice than in ApoE-/--saline mice. The aortic mRNA levels of pro-inflammatory and angiogenic factors, and the percentage of macrophages in aortic root lesions, were significantly higher in ApoE-/--MK mice than in ApoE-/--saline mice, whereas the percentage of apoptotic cells was significantly lower in ApoE-/--MK mice than in ApoE-/--saline mice. CONCLUSIONS: The systemic administration of MK in ApoE-/-mice promoted atherosclerotic plaque formation through pro-inflammatory, angiogenic, and anti-apoptotic effects. MK may serve as a potential therapeutic target for the prevention of atherosclerosis under atherogenic conditions.

Exogenous midkine administration prevents cardiac remodeling in pacing-induced congestive heart failure of rabbits.

Midkine (MK), a heparin-binding growth factor, has been shown to prevent cardiac remodeling after ischemic injury through its anti-apoptotic effect. Cell apoptosis is central to the pathophysiology of cardiac remodeling in congestive heart failure (CHF) of ischemic as well as non-ischemic origin. We hypothesized that MK exerts the anti-apoptotic cardioprotective effect in CHF of non-ischemic etiology. MK protein or vehicle (normal saline) was subcutaneously administered in tachycardia-induced CHF rabbits (right ventricular pacing, 350 beats/min, 4 weeks). The vehicle-treated rabbits (n = 19, control) demonstrated severe CHF and high mortality rate, whereas MK (n = 16) demonstrated a well-compensated state and a lower mortality rate. In echocardiography, left ventricular (LV) end-diastolic dimension decreased in MK versus control, whereas LV systolic function increased. In histological analysis (picrosirius red staining), MK decreased collagen deposition area compared with control. TUNEL staining showed that MK prevented cell apoptosis and minimized myocyte loss in the CHF rabbit ventricle, associated with activation of PI3-K/Akt signaling, producing a parallel decrease of Bax/Bcl-2 ratio. MK prevented progression of cardiac remodeling in the CHF rabbit, likely by activation of anti-apoptotic signaling. Exogenous MK application might be a novel therapeutic strategy for CHF due to non-ischemic origin.

Detection of QT prolongation through approximation of the T wave on Gaussian mixture modeling.

BACKGROUND: To establish a simple and accurate method for the automated identification of the end of a T wave, we approximated electrocardiograph (ECG) traces using a Gaussian mixture model in conjunction with a split-and-merge expectation-maximization algorithm. METHODS AND RESULTS: A total of 286 ECG traces of heart beats of 50 healthy men were used as control data and ECGs from 15 subjects recorded before and after 400mg oral moxifloxacin as positive controls. An experienced cardiologist determined the reference points by visual inspection of the original ECGs. The primary estimated point for the end of the T wave was selected as the point 2 ms before the point at which the gradient of the approximated wave was not steeper than the common threshold value. This point was then adjusted by applying modification rules proposed by an experienced cardiologist. The absolute value of the average interval between the resulting final estimated point and the manually selected reference point was 1.8±7.7 ms for the control data. After treatment with moxifloxacin, the average QT interval, corrected by Bazett's formula, showed a 17.2±27.1 ms prolongation with a lower bound of the 95% confidence interval of 4.9 ms. CONCLUSIONS: When the modification rules were applied, the accuracy of QT measurement was improved, and the present system was capable of detecting QT prolongation correctly.

T-type Ca2+ channel blockers prevent cardiac cell hypertrophy through an inhibition of calcineurin-NFAT3 activation as well as L-type Ca2+ channel blockers.

T-type Ca2+ channels (TCCs) are involved in cardiac cell growth and proliferation in cultured cardiomyocytes. Underlying molecular mechanisms are not well understood. In this study, we investigated the role of TCCs in signal transduction in cardiac hypertrophy compared with L-type Ca2+ channels (LCCs). Cardiomyocytes dissociated from neonatal mouse ventricles were cultured until stabilization. Cell hypertrophy was induced by reapplication of 1% fatal bovine serum (FBS) following a period (24 h) of FBS depletion. Cell surface area increased from 862+/-73 microm2 to 2153+/-131 microm2 by FBS stimulation in control (250+/-1.8%). T-type Ca2+ current (I(CaT)) was inhibited dose-dependently by kurtoxin (KT) and efonidipine (ED) with IC50 0.07 microM and 3.2 microM, respectively in whole-cell voltage clamp. On the other hand, 1 microM KT which inhibits I(CaT) over 90% did not effect on L-type Ca2+ current (I(CaL)). 10 microM ED had the ability of I(CaL) blockade as well as that of I(CaT) blockade. 3 microM nisoldipine (ND) suppressed I(CaL) by over 80%. The increase in cell surface area following reapplication of FBS as observed in control (250+/-1.8%) was significantly reduced in the presence of 1 microM KT (216+/-1.2%) and virtually abolished in the presence of 10 microM ED (97+/-0.8%) and 3 microM ND (80+/-1.1%). Hypertrophy was associated with an increase in BNP mRNA of 316+/-3.6% in control and this increase was reduced as well in the presence of 1 microM KT (254+/-1.8%) and almost abolished in the presence of 10 microM ED (116+/-1.1%) and 3 muM ND (93+/-0.8%). Immunolabeling showed that translocation of nuclear factor of activated T cells (NFAT3) into the nucleus in response to FBS stimulation was markedly inhibited by either KT or ED as well as ND. Calcineurin phosphatase activity was upregulated 2.2-fold by FBS, but KT, ED and ND decreased this upregulation (1.7-fold, 0.8-fold, and 0.7-fold with KT, ED and ND respectively). These results suggest that blockade of Ca2+ entry into cardiomyocytes via TCCs may block pathophysiological signaling pathways leading to hypertrophy as well as via LCCs. The mechanism may be the inhibition of calcineurin-mediated NFAT3 activation resulting in prevention of its translocation into the nucleus.

Midkine plays a protective role against cardiac ischemia/reperfusion injury through a reduction of apoptotic reaction.

BACKGROUND: Midkine (MK) is a heparin-binding growth factor involved in diverse biological phenomena, eg, neural survival, carcinogenesis, and tissue repair. MK could have a protective action against ischemia/reperfusion (I/R) injury in the heart, because MK was shown to have cytoprotective activity in cultured neurons and tumor cells. We investigated this hypothesis in mice with and without genetic MK deletion. METHODS AND RESULTS: Myocardial injury after I/R was produced by transient occlusion of coronary arteries. In wild-type (Mdk+/+) mice, MK expression was increased after I/R in the periinfarct area. Infarct size/area at risk 24 hours after I/R in MK-deficient (Mdk-/-) mice was larger than in Mdk+/+ mice (55.4+/-9.1% versus 32.1+/-5.3%, P<0.05). Terminal dUTP nick end-labeling-positive myocyte population in the periinfarct area in Mdk-/- mice was higher than in Mdk+/+ mice (6.8+/-0.9% versus 3.2+/-0.6%, P<0.05). Left ventricular fractional shortening 24 hours after I/R in Mdk-/- mice was significantly less than that in Mdk+/+ mice (34.3+/-4.4% versus 50.8+/-2.1%, P<0.05). Supplemental application of MK protein to left ventricle of Mdk-/- mice at the time of I/R resulted in reduction of the infarct size. Application of exogenous MK to cultured cardiomyocytes resulted in increased Bcl-2 expression and decreased apoptosis after hypoxia/reoxygenation. CONCLUSIONS: These results suggest that MK plays a protective role against I/R injury, most likely through a prevention of apoptotic reaction. MK is a potentially important new molecular target for treatment of ischemic heart disease.

A single intracoronary injection of midkine reduces ischemia/reperfusion injury in Swine hearts: a novel therapeutic approach for acute coronary syndrome.

Several growth factors are effective for salvaging myocardium and limiting infarct size in experimental studies with small animals. Their benefit in large animals and feasibility in clinical practice remains to be elucidated. We investigated the cardioprotective effect of midkine (MK) in swine subjected to ischemia/reperfusion (I/R). I/R was created by left anterior descending coronary artery occlusion for 45 min using a percutaneous over-the-wire balloon catheter. MK protein was injected as a bolus through the catheter at the initiation of reperfusion [MK-treated (MKT) group]. Saline was injected in controls (CONT). Infarct size/area at risk (24 h after I/R) in MKT was almost five times smaller than in CONT. Echocardiography in MKT revealed a significantly higher percent wall thickening of the interventricular septum, a higher left ventricular (LV) fractional shortening, and a lower E/e(') (ratio of transmitral to annular flow) compared with CONT. LV catheterization in MKT showed a lower LV end-diastolic pressure, and a higher dP/dt(max) compared with CONT. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling-positive myocytes and CD45-positive cell infiltration in the peri-infarct area were significantly less in MKT than in CONT. Here, we demonstrate that a single intracoronary injection of MK protein in swine hearts at the onset of reperfusion dramatically reduces infarct size and ameliorates systolic/diastolic LV function. This beneficial effect is associated with a reduction of apoptotic and inflammatory reactions. MK application during percutaneous coronary intervention may become a promising adjunctive therapy in acute coronary syndromes.

Midkine gene transfer after myocardial infarction in rats prevents remodelling and ameliorates cardiac dysfunction.

AIM: We have previously reported that therapy with midkine (MK) has a protective effect in mouse models of myocardial infarction (MI) and ischemia/reperfusion. The underlying mechanism was proved to be anti-apoptosis and prevention of left ventricular (LV) remodelling following angiogenesis. Here we investigated the effects of overexpression of MK by adenoviral gene transfer on cardiac function and remodelling in an experimental rat MI model. METHODS AND RESULTS: MI was created in male Wistar rats. Adenoviral vectors encoding mouse MK (AdMK) or beta-galactosidase (AdLacZ; as controls) were injected in myocardium at the onset of MI. One week after injection, in vivo adenoviral gene expression was assessed by western blot and histological analysis. After echocardiographic analysis at 4 weeks and haemodynamic analysis at 6 weeks after MI, AdMK animals had better cardiac function compared with AdLacZ animals. Heart weight (HW) and relative HW of AdMK animals were not different from sham-operated animals after 6 weeks, pointing to a very potent effect in the prevention of ischemic cardiomyopathy. In histological studies at 6 weeks after MI, AdMK animals had less fibrosis in the non-infarcted myocardium and higher vascular density in the border-zone area compared with AdLacZ animals. AdMK animals had strongly upregulated levels of phosphorylated extracellular signal-regulated kinase, Akt, PI 3-kinase, and Bcl-2, whereas the level of Bax was downregulated compared with AdLacZ animals. CONCLUSION: Overexpression of MK prevents LV remodelling and ameliorates LV dysfunction by anti-apoptotic and pro-angiogenic effects. MK gene transfer may provide a new therapeutic modality in ischemic cardiomyopathy and ischemic heart failure.

Midkine prevents ventricular remodeling and improves long-term survival after myocardial infarction.

Cardiac remodeling is thought to be the major cause of chronic heart dysfunction after myocardial infarction (MI). However, molecules involved in this process have not been thoroughly elucidated. In this study we investigated the long-term effects of the growth factor midkine (MK) in cardiac remodeling after MI. MI was produced by ligation of the left coronary artery. MK expression was progressively increased after MI in wild-type mice, and MK-deficient mice showed a higher mortality. Exogenous MK improved survival and ameliorated left ventricular dysfunction and fibrosis not only of MK-deficient mice but also of wild-type mice. Angiogenesis in the peri-infarct zone was also enhanced. These in vivo changes induced by exogenous MK were associated with the activation of phosphatidylinositol 3-kinase (PI3K)/Akt and MAPKs (ERK, p38) and the expression of syndecans in the left ventricular tissue. In vitro experiments using human umbilical vein endothelial cells confirmed the potent angiogenic action of MK via the PI3K/Akt pathway. These results suggest that MK prevents the cardiac remodeling after MI and improves the survival most likely through an enhancement of angiogenesis. MK application could be a new therapeutic strategy for the treatment of ischemic heart failure.

Aldosterone modulates I(f) current through gene expression in cultured neonatal rat ventricular myocytes.

Mineralocorticoid receptor (MR) antagonists decrease the incidence of sudden cardiac death in patients with heart failure, as has been reported in two clinical trials (Randomized Aldactone Evaluation Study and Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study). Aldosterone has been shown to increase the propensity to arrhythmias by changing the expression or function of various ion channels. In this study, we investigate the effect of aldosterone on the expression of hyperpolarization-activated current (I(f)) channels in cultured neonatal rat ventricular myocytes, using the whole cell patch-clamp technique, real-time PCR, and Western blotting. Incubation with 10 nM aldosterone for 17-24 h significantly accelerates the rate of spontaneous beating by increasing diastolic depolarization. I(f) current elicited by hyperpolarization from -50 to -130 mV significantly increases aldosterone by 10 nM (by 1.9-fold). Exposure to aldosterone for 1.5 h increases hyperpolarization-activated cyclic nucleotide-gated (HCN) 2 mRNA by 26.3% and HCN4 mRNA by 47.2%, whereas HCN1 mRNA expression remains unaffected. Aldosterone (24-h incubation) increases the expression of HCN2 protein (by 60.0%) and HCN4 protein (by 84.8%), but not HCN1 protein. MR antagonists (1 microM eplerenone or 0.1 microM spironolactone) abolish the increase of I(f) channel expression (currents, mRNA, and protein levels) by 10 nM aldosterone. In contrast, 1 microM aldosterone downregulated I(f) channel gene expression. Glucocorticoid receptor antagonist (100 nM RU-38486) did not affect the increase of I(f) current by 10 nM aldosterone. These findings suggest that aldosterone in physiological concentrations upregulates I(f) channel gene expression by MR activation in cardiac myocytes and may increase excitability, which may have a potential proarrhythmic bearing under pathophysiological conditions.

Pathophysiological significance of T-type Ca2+ channels: expression of T-type Ca2+ channels in fetal and diseased heart.

Re-expression of fetal genes has been considered to underlie ionic remodeling in diseased heart. T-type Ca(2+) channels have been reported to be functionally expressed in embryonic hearts. In this review, we summarize developmental changes of T-type Ca(2+) channels in mouse ventricles from 9.5 days postcoitum (dpc) to adulthood, using patch clamp and quantitative PCR. In addition, we introduced T-type Ca(2+) channel expression in hypertrophied ventricles caused by myocardial infarction (MI) and aortic banding (AOB). Substantial T-type Ca(2+) channel current was recorded at both 9.5 and 18 dpc. The currents were inhibited by Ni(2+) at low concentrations. The current was not detectable in the adult stage. Ca(v)3.2 (alpha(1H)) mRNA is expressed dominantly at both 9.5 and 18 dpc. Ca(v)3.1 (alpha(1G)) increases from 9.5 to 18 dpc, but remains at low level compared with Ca(v)3.2. In contrast, Ca(v)3.1 is greater than Ca(v)3.2 at the adult stage. In MI, Ca(v)3.1 mRNA correlates negatively with brain natriuretic peptide (BNP) mRNA, whereas Ca(v)3.2 mRNA correlates positively with BNP mRNA. In AOB, these correlations are weak. We also analyzed the neuron-restrictive silencer factor (NRSF) in these hearts because it is the suppressor of transcription of the fetal cardiac gene program. The negative correlation between NRSF and BNP was stronger in MI than in AOB. Our findings show that Ca(v)3.2 underlies the functional T-type Ca(2+) channel in embryonic heart and suggest that NRSF may regulate Ca(v)3.2 expression in diseased hearts.

Subtype switching of L-Type Ca 2+ channel from Cav1.3 to Cav1.2 in embryonic murine ventricle.

BACKGROUND: Embryonic hearts exhibit spontaneous electrical activity, which depends on Ca2+ influx through L-type Ca2+ channels. In this study the expression of the L-type Ca2+ channel alpha1 subunit gene in the developing mouse heart was investigated. METHODS AND RESULTS: Mouse cardiac ventricles 9.5 days post coitum (dpc), 18 dpc and adult were used. At 9.5 dpc the level of Cav1.3 mRNA was higher than that of Cav1.2 mRNA. With development, Cav1.2 mRNA increased and Cav1.3 mRNA decreased. Analysis of Cav1.3 splicing variants showed that Cav1.3(1b) mRNA was expressed at a higher density than Cav1.3(1a) mRNA. Cav1.3 protein was detected only at 9.5 dpc, whereas Cav1.2 protein was expressed from 9.5 dpc and its expression increased with development. L-type Ca2+ currents were prominent at 9.5 dpc. The Ca2+ current amplitude at 9.5 dpc was comparable to that at 18 dpc, and was larger in adults than at the embryonic stage. L-type Ca2+ current at 9.5 dpc was activated and/or inactivated at more negative membrane potentials than at 18 dpc or adult. L-type Ca2+ channels at 9.5 dpc were less sensitive to inhibition by nisoldipine than at adult. CONCLUSIONS: The Cav1.3 channel is functionally expressed in early embryonic mouse ventricular myocytes and potentially underlies ventricular automaticity.

Combined effects of nifekalant and lidocaine on the spiral-type re-entry in a perfused 2-dimensional layer of rabbit ventricular myocardium.

BACKGROUND: Spiral re-entry plays the principal role in the genesis of ventricular tachycardia and ventricular fibrillation (VT/VF). The specific I(Kr) blocker, nifekakant (NIF) has, often in combination with lidocaine (LID), recently been used in Japan to prevent recurrent VT/VF, but the combined effects of these drugs on spiral re-entry had never been investigated. METHODS AND RESULTS: A ventricular epicardial sheet was obtained from 13 Langendorff-perfused rabbit hearts by means of a cryoprocedure, and epicardial excitations were analyzed with a high-resolution optical mapping system. Nifekakant (0.5 micromol/L) caused significant prolongation of action potential duration (APD) and LID (3 micromol/L) attenuated the APD prolongation without affecting the conduction velocity. VT were induced in 6 hearts by cross-field stimulation, and single- or double-loop spirals circulating around variable functional block lines were visualized during the VT. Nifekakant reduced VT cycle length and caused early termination in association with destabilization of the spiral dynamics (prolongation of functional block line, frequent local conduction block, and extensive meandering). These modifications of spiral-type re-entrant VT by NIF were prevented by addition of LID. CONCLUSIONS: The effects of NIF on the spiral excitations are reversed by LID. This interaction should be taken into account when these drugs are used in combination to treat VT/VF.

Cav3.2 subunit underlies the functional T-type Ca2+ channel in murine hearts during the embryonic period.

T-type Ca2+ channels are implicated in cardiac automaticity, cell growth, and cardiovascular remodeling. Two voltage-gated Ca2+ subtypes (Ca(v)3.1 and Ca(v)3.2) have been cloned for the pore-forming alpha(1)-subunit of the T-type Ca2+ channel in cardiac muscle, but their differential roles remain to be clarified. The aim of this study was to elucidate the relative contribution of the two subtypes in the normal development of mouse hearts. A whole cell patch clamp was used to record ionic currents from ventricular myocytes isolated from mice of early (E9.5) and late embryonic days (E18) and from adult 10-wk-old mice. Large T-type Ca2+ current (I(Ca,T)) was observed at both E9.5 and E18, displaying similar voltage-dependence and kinetics of activation and inactivation. The current was inhibited by Ni2+ at relatively low concentrations (IC(50) 26-31 microM). I(Ca,T) was undetectable in adult myocytes. Quantitative PCR analysis revealed that Ca(v)3.2 mRNA is the predominant subtype encoding T-type Ca2+ channels at both E9.5 and E18. Ca(v)3.1 mRNA increased from E9.5 to E18, but remained low compared with Ca(v)3.2 mRNA during the whole embryonic period. In the adulthood, in contrast, Ca(v)3.1 mRNA is greater than Ca(v)3.2 mRNA. These results indicate that Ca(v)3.2 underlies the functional T-type Ca2+ channels in the embryonic murine heart, and there is a subtype switching of transcripts from Ca(v)3.2 to Ca(v)3.1 in the perinatal period.

Overexpression of calpastatin by gene transfer prevents troponin I degradation and ameliorates contractile dysfunction in rat hearts subjected to ischemia/reperfusion.

Calpain is a Ca(2+)-activated neutral protease that supposedly plays a key role in myocardial dysfunction following ischemia/reperfusion, by degrading certain proteins involved in the contraction mechanism. It is possible that overexpression of calpastatin, an endogenous calpain inhibitor, lessens contractile dysfunction in the heart after reperfusion by preventing cardiac troponin I (TnI) degradation. This claim is tested by overexpression of human calpastatin (hCS) in rat hearts ex vivo using an adenovirus vector; the hearts were transplanted heterotopically into the abdomens of recipient rats to allow expression of hCS. On the fourth day after surgery, the hearts were excised and perfused in vitro to study their recovery from 30 min of global ischemia, which was followed by 60 min of reperfusion. The peak recovery of the left ventricular developed pressure (LVDP), and the values of its first derivative (max dP/dt, min dP/dt) in the hCS-overexpressed hearts were 88.9 +/- 4.8%, 90.8 +/- 9.2% and 106.4 +/- 9.8%, respectively; these values were all significantly greater than in the control hearts transfected with LacZ alone (51.4 +/- 6.9%, 52.6 +/- 8.1% and 54.7 +/- 6.6%, P < 0.05). In western blot analysis of ventricular myocardial samples (at 60-min reperfusion) using a monoclonal anti-TnI antibody, two bands corresponding to intact TnI (30 kDa) and TnI fragments (27 kDa) were distinguished. The fraction of 27-kDa TnI (percent of total TnI immunoreactivity) in hCS-overexpressed hearts was significantly less than the controls (5.7 +/- 2.7% vs. 18.1 +/- 3.2%, P < 0.05), implying a protective action of hCS against TnI degradation. These results suggest that adenovirus-mediated overexpression of hCS in the heart could be a novel biological means to minimize myocardial stunning by ischemia/reperfusion.