Dong-Feng Yeih

PKCɛ mediates serine phosphorylation of connexin43 induced by lysophosphatidylcholine in neonatal rat cardiomyocytes.

Lysophosphatidylcholine (LPC) is a potent pro-arrhythmic derivative of the membrane phosphotidylcholine, which is accumulated in heart tissues during cardiac ischemia. However, the cellular mechanism underlying LPC-induced cardiomyocyte damage remains to be elucidated. This study focuses on the effects of LPC on cardiomyocyte gap junction. At 30μM, LPC decreased the spontaneous contraction rates of cardiomyocytes, and caused arrhythmic contraction without affecting cell viability. Connexin43 (Cx43) was seen as large plaques at cell junctions in control cells, whereas upon LPC treatment, the intensity of Cx43 staining was decreased in a concentration-sensitive manner and Cx43 staining appeared as tiny dots at cell junctions with a corresponding increase in cytoplasmic punctate staining. This distributional change of Cx43 was accompanied by an impairment of the gap junction intercellular communication (GJIC). Further, LPC treatment induced protein kinase C (PKC) activation, and PKC-dependent Cx43 phosphorylation at serine (Ser) 368. Pre-treatment with a specific PKCɛ inhibitor, eV1-2, prevented the LPC-induced Cx43 phosphorylation at Ser368 and the loss of Cx43 from gap junctions, both of which may disturb GJIC functions. Furthermore, siRNA knockdown of PKCɛ in H9c2 cells prevented LPC-induced serine phosphorylation of Cx43, confirming the role of PKCɛ in Cx43 serine phosphorylation. Double labeling immunofluorescence showed that LPC increased the colocalization of Cx43 with ubiquitin, and pretreatment with MG132 effectively prevented LPC-induced gap junction disassembly. LPC increased the ubiquitination of Cx43, which was blocked by eV1-2 pretreatment, suggesting that LPC accelerated the intracellular degradation of Cx43 via the ubiquitin-proteasomal pathway. It can be concluded that LPC destroyed the structure and function of gap junctions via PKCɛ-mediated serine phosphorylation of Cx43. PKCɛ inhibitors might therefore be effective in prevention of LPC-related diseases.

Enhanced activity and subcellular redistribution of myocardial hexokinase after acute myocardial infarction

BACKGROUND Hexokinase (HK) is known to possess both anti-oxidant and anti-apoptotic properties. This study investigated the behavior of myocardial HK in response to myocardial infarction (MI). METHODS Adult male Wistar rats with various degrees of MI after coronary ligation were examined 4 weeks after operation and were divided dichotomously into small and large MI groups. The activity and subcellular distribution of HK in the non-infarcted myocardium were determined. In parallel, myocardial oxidative stress determined using aconitase activity and malondialdehyde content, and left ventricular function using echocardiography were studied. RESULTS In the mitochondria and the cytosol, HK activity was enhanced after MI and paralleled the increases in oxidative stress and left ventricular end-diastolic dimension (LVEDD). The enhancement in HK activity varied between subcellular compartments and resulted in an increase in the ratio of cytosol to whole-cell HK activity, which was proportional to oxidative stress and LVEDD. CONCLUSIONS The activities of HK in all subcellular fractions are enhanced in response to MI. However, enhanced proportion of cytosolic HK relative to whole-cell HK activity is associated with higher oxidative stress and LVEDD, suggesting that altered myocardial HK activity and subcellular redistribution might be involved in the pathogenesis of postinfarct heart failure.

The detrended fluctuation analysis of acute-phase heart-rate variability in acute coronary syndromes - a pilot study.

Heart-rate variability (HRV) analyzed by detrended fluctuation analysis (DFA), which resulted in a short-term fractal exponent - alpha, has prognostic implication in chronic phase of heart failure and survivors of myocardial infarction. We adopted DFA to study the acute-phase HRV in acute coronary syndromes (ACS) by recruiting 30 age- and co-morbidity-matched adults in acute-stress simulation in comparison with 33 consecutive ACS patients. The 30 volunteers got stepwise elevated alpha with increased intensity of exercise (0.95+/-0.050 to 1.07+/-0.084 to 1.20+/-0.083, p<0.05). The alpha of 33 ACS patients correlated with the complexity of post-MI course (1.004+/-0.0080 in non-complicated vs. 1.216+/-0.058 in complicated p<0.05). No significance existed in categories of age, ACS type, numbers of diseased vessel, infarct-related artery or Killip classification. The increased alpha value of DFA may imply unresolved cardiac stress which demands further attention.

Rho-associated kinase inhibitors promote the cardiac differentiation of embryonic and induced pluripotent stem cells

BACKGROUND Rho-associated kinase (ROCK) plays an important role in maintaining embryonic stem (ES) cell pluripotency. To determine whether ROCK is involved in ES cell differentiation into cardiac and hematopoietic lineages, we evaluated the effect of ROCK inhibitors, Y-27632 and fasudil on murine ES and induced pluripotent stem (iPS) cell differentiation. METHODS Gene expression levels were determined by real-time PCR, Western blot analysis and immunofluorescent confocal microscopy. Cell transplantation of induced differentiated cells were assessed in vivo in a mouse model (three groups, n=8/group) of acute myocardial infarction (MI). The cell engraftment was examined by immunohistochemical staining and the outcome was analyzed by echocardiography. RESULTS Cells were cultured in hematopoietic differentiation medium in the presence or absence of ROCK inhibitor and colony formation as well as markers of ES, hematopoietic stem cells (HSC) and cells of cardiac lineages were analyzed. ROCK inhibition resulted in a drastic change in colony morphology accompanied by loss of hematopoietic markers (GATA-1, CD41 and β-Major) and expressed markers of cardiac lineages (GATA-4, Isl-1, Tbx-5, Tbx-20, MLC-2a, MLC-2v, α-MHC, cTnI and cTnT) in murine ES and iPS cells. Fasudil-induced cardiac progenitor (Mesp-1 expressing) cells were infused into a murine MI model. They engrafted into the peri-infarct and infarct regions and preserved left ventricular function. CONCLUSIONS These findings provide new insights into the signaling required for ES cell differentiation into hematopoietic as well as cardiac lineages and suggest that ROCK inhibitors are useful in directing iPS cell differentiation into cardiac progenitor cells for cell therapy of cardiovascular diseases.

Dimethylthiourea normalizes velocity-dependent, but not force-dependent, index of ventricular performance in diabetic rats: role of myosin heavy chain isozyme.

Hydroxyl radicals and hydrogen peroxide are involved in the pathogenesis of systolic dysfunction in diabetic rats, but the precise mechanisms and the effect of antioxidant therapy in diabetic subjects have not been elucidated. We aimed to evaluate the effects of dimethylthiourea (DMTU), a potent hydroxyl radical scavenger, on both force-dependent and velocity-dependent indexes of cardiac contractility in streptozotocin (STZ)-induced early and chronic diabetic rats. Seventy-two hours and 8 wk after STZ (55 mg/kg) injection, diabetic rats were randomized to either DMTU (50 mg x kg(-1) x day(-1) ip) or vehicle treatment for 6 and 12 wk, respectively. All rats were then subjected to invasive hemodynamic studies. Maximal systolic elastance (E(max)) and maximum theoretical flow (Q(max)) were assessed by curve-fitting techniques in terms of the elastance-resistance model. Both normalized E(max) (E(maxn)) and afterload-adjusted Q(max) (Q(maxad)) were depressed in diabetic rats, concomitant with altered myosin heavy chain (MHC) isoform composition and its upstream regulators, such as myocyte enhancer factor-2 (MEF-2) and heart autonomic nervous system and neural crest derivatives (HAND). In chronic diabetic rats, DMTU markedly attenuated the impairment in Q(maxad) and normalized the expression of MEF-2 and eHAND and MHC isoform composition but exerted an insignificant benefit on E(maxn). Regarding preventive treatment, DMTU significantly ameliorated both E(maxn) and Q(maxad) in early diabetic rats. In conclusion, our study shows that DMTU has disparate effects on Q(maxad) and E(maxn) in chronic diabetic rats. The advantage of DMTU in chronic diabetic rats might involve normalization of MEF-2 and eHAND, as well as reversal of MHC isoform switch.

Enhanced Diagnosis of Coronary Artery Disease in Women by Dobutamine Thallium-201 ST-Segment/Heart Rate Slope and Thallium-201 Myocardial SPECT

BACKGROUND/PURPOSE The diagnosis of coronary artery disease (CAD) in women presents a great challenge because of poor exercise capacity and inadequate heart rate response during stress test. The clinical significance of stress-related ST-segment/heart rate slope (ST/HR slope) value for evaluating CAD in women remains controversial. Therefore, we conducted the present study to assess the diagnostic performance of dobutamine ST/HR slope in women, compared with myocardial perfusion study using thallium-201 single-photon emission computed tomography (Tl-201 SPECT). METHODS A total of 51 female patients with suspected CAD underwent simultaneous 12-lead electrocardiographic recording during 3-minute stages of dobutamine infusion as well as Tl-201 SPECT, and coronary angiography was performed within 2 weeks post Tl-201 SPECT. The sensitivity, specificity, positive predictive value and negative predictive value of dobutamine ST/HR slope and Tl-201 SPECT were assessed, and the results of coronary angiography were used as a gold standard. RESULTS The sensitivity, specificity and accuracy of dobutamine ST/HR slope in detecting CAD were 43%, 83% and 61%, and those of Tl-201 SPECT were 71%, 87% and 78%, respectively. However, using both positive results of Tl-201 SPECT and ST/HR slope for detecting CAD, the diagnostic specificity increased from 87% to 96%. Using both negative results of Tl-201 SPECT and ST/HR slope to exclude CAD, the negative predictive value increased from 71% to 85%. The accuracy of dobutamine ST/HR slope in detecting CAD was not affected by the use of beta-blockers. CONCLUSION Dobutamine ST/HR slope is less sensitive and less accurate than Tl-201 SPECT for detecting CAD in women. However, it adds diagnostic benefit to Tl-201 SPECT with only a little extra calculation.

Physiology-based diagnosis algorithm for arteriovenous fistula stenosis detection

In this paper, a diagnosis algorithm for arteriovenous fistula (AVF) stenosis is developed based on auscultatory features, signal processing, and machine learning. The AVF sound signals are recorded by electronic stethoscopes at pre-defined positions before and after percutaneous transluminal angioplasty (PTA) treatment. Several new signal features of stenosis are identified and quantified, and the physiological explanations for these features are provided. Utilizing support vector machine method, an average of 90% two-fold cross-validation hit-rate can be obtained, with angiography as the gold standard. This offers a non-invasive easy-to-use diagnostic method for medical staff or even patients themselves for early detection of AVF stenosis.

A novel system for continuous peripheral arterial pressure-volume loop measurement

This study develops a system to obtain continuous blood pressure signal and impedance plethysmography (IPG) signal, simultaneously. Based on the principle of impedance measurement, the peripheral vessel volume change can be computed from the IPG signal. Equipped with simultaneous information of pressure and volume, a pressure-volume (PV) loop can be constructed. It is well known that the left ventricular pressure-volume loop contains a number of feature points indicating the performance of cardiac function. Therefore, in this study, the same principle is used to try to discuss the peripheral vessel pressure-volume loop. Ten volunteers were recruited for this study. Subjects went through the cold pressor test by immersing their left foot into ice water. Blood pressure signal and impedance changed were recorded using a custom-made system. The results illustrated that the pressure-volume loop, as it was expected, demonstrated a contraction phenomenon after stimulation in five out of ten subjects. The areas of those pressure-volume loops reduced as much as 70% in some subject. However, loop responses to stressors varied from subject to subject and the slope of the loop did not alter significantly. In conclusion, the proposed system is a potential way to measure and to investigate the compliance and characteristic of peripheral blood vessel.

P-1 Remodeling of Myocardial Gap Junctions and its Correlation with Cardiac Contractility in Diabetic Rats

not available at time of printing. Poster Sessions 1: Heart Failure/Myocardial Diseases P-1 Remodeling of Myocardial Gap Junctions and its Correlation with Cardiac Contractility in Diabetic Rats Dong-Feng Yeih1, Pen-Chih Liao1, Ho-Tsung Hsin1, Kuo-Chin Chen1, Ai-Hsian Li1, Yung-Zu Tseng2. 1Department of Cardiology, Far-Eastern Memorial Hospital, Taiwan, 2Department of Cardiology, National Taiwan University, Taiwan Introduction: Gap junctions play an essential role in coupling adjacent cardiomyocytes, ensuring organized propagation of action potential and synchronizing beating in cardiomyocytes. However, it remains unclear that temporal remodeling of myocardial gap junctions and its relation to cardiac contractility in diabetic rats. Methods: Male Wistar rats, weighing 250 to 300 grams, were randomized to injection with either vehicle or streptozotocin (50mg, IP). Invasive hemodynamic studies with simultaneous recordings of left ventricular pressure and aortic flow signals were done at 4, 8 and 12 weeks after injection. Maximal systolic elastance (Emax) and maximum theoretical flow (Qmax) were assessed by curve fitting techniques; protein expression of non-phosphorylated Cx43 (P0) and total amount of Cx43 (T) in the left ventricle by western blotting analysis. Results: Emax was significantly lower in diabetic rats than in controls and Emax was significantly depressed with time in diabetic groups. On the other hand, Qmax was preserved and cardiac output was maintained in diabetic rats. Protein expression of total Cx43 was persistently attenuated in diabetic groups, while P0 was enhanced at 8 and 12 weeks after injection. The ratio of P0 to T was significantly increased with time, which correlated well with the declines in Emax in diabetic rats. Conclusions: Temporal remodeling of myocardial gap junctions, with persistently lower total Cx43 and gradually increased non-phosphorylated Cx43 and the ratio of P0 to T, occurred in the evolution of systolic dysfunction in diabetic rats. P-2 Quantification of Left Ventricular Diastolic Dynamics in Normal and Diseased Myocardium by Speckle-Tracking Echocardiography Kaoru Dohi1, Katsuya Onishi2, Takeshi Takamura1, Emiyo Sugiura1, Hiroshi Nakajima1, Kazuhide Ichikawa1, Masaki Tanabe1, Hiroya Tamada1, Masatoshi Miyahara1, Mashio Nakamura1, Masaaki Ito1. 1Department of Cardiology, Mie University Graduate School of Medicine, Japan, 2Department of Molecular and Laboratory Medicine, Mie University Graduate School of Medicine, Japan Background: The present study aimed to quantify left ventricular (LV) diastolic dynamics and to elucidate their relations to systolic properties in normal and diseased myocardium. Methods: 50 patients with hypertensive LV hypertrophy (LVH: EF 61±8%, QRS 96±16ms), 50 patients with dilated cardiomyopathy (DCM: EF 31±10%, QRS 121±32ms), and 50 normal controls (Control: EF 5±6%, QRS 89±9ms) had echo-study with speckle-tracking strain and strain rate imaging. To quantify LV diastolic relaxation and segmental synchrony, global peak relaxation rate (PRR) during early diastole and standard deviation (SD) of the 18 segmental time-to-PRR (TPRR-SD) were calculated from apical 4-, 2-, and long axis views. To elucidate the relations between diastolic and systolic properties, global peak systolic strain (PSS) and SD of the segmental time-to-peak PSS (TPSS-SD) were also evaluated. Results: LV relaxation and diastolic synchrony were significantly impaired in LVH and was more prominent in DCM (PRR: 0.8±0.3* 1/s in LVH, 0.5±0.2*# 1/s in DCM, and 1.2±0.3 1/s in Control, TPRR-SD: 55±16*ms in LVH, 79±35*# ms in DCM, and 42±13ms in Control, *p < 0.05 vs. Control and #p < 0.05 vs. LVH, respectively). There were strong correlations between PSS and PRR, and TPRR-SD and TPSS-SD (r = 0.83 and r = 0.83, p < 0.05, respectively). Conclusion: Speckle-tracking echocardiography quantified LV diastolic dynamics and exhibited the strong relation to systolic properties.