Monika Szilard

M cells and transmural heterogeneity of action potential configuration in myocytes from the left ventricular wall of the pig heart

OBJECTIVE Heterogeneity of action potential configuration in the left ventricle (LV), and the contribution of M cells to it, has been observed in the human heart and is important for arrhythmogenesis. Whether the pig heart has similar properties remains a controversial but important issue as the pig heart is currently under study for use in xenotransplantation. METHODS Single myocytes were enzymatically isolated from the epicardium (EPI, ncells = 29), midmyocardium (MID, ncells = 38), and endocardium (ENDO, ncells = 13) of the free LV wall (npigs = 26, 14-22 weeks old, 55-80 kg), and studied at different stimulation rates during whole-cell recording (normal Tyrodes solution, K(+)-aspartate-based pipette solution, 50 microM K5fluo-3 as [Ca2+]i indicator, 37 degrees C). Standard six-lead ECGs were recorded from anesthetized pigs. RESULTS The action potential duration (APD) was not significantly different at 0.25 Hz vs. 2 Hz for the majority of cells in all three layers. However, a subpopulation of cells behaved like M cells and had a very steep frequency response (APD90 at 0.25 Hz 538 +/- 30 ms, vs. 337 +/- 9 ms at 2 Hz, P < 0.05, n = 22). These cells were found predominantly in the MID layer (34% of cells), but also (24%) in EPI. M cells had a more pronounced spike-and-dome configuration, with a significantly larger phase 1 magnitude and plateau voltage. The frequency response of these parameters was different from the other cell types. [Ca2+]i transients tended to be larger in M cells. For the in vivo ECG of anesthetized pigs, the QT time was close to the APD90 of M cells, and J waves were seen in 7/12 recordings. CONCLUSIONS In young adult pigs, M cells can be identified by a steep frequency response of the APD and by a spike-and-dome configuration. These cells are mostly, but not exclusively, found in the midmyocardium, and could contribute to the ECG characteristics. Their properties may however be different from those of other species, including humans.

Repeated stunning precedes myocardial hibernation in progressive multiple coronary artery obstruction

OBJECTIVE The aim of this study was to characterize a regional myocardial flow-function relationship in collateral dependent myocardium produced by multiple coronary artery obstruction. METHODS Ameroid constrictors were placed around the proximal right (RC) and circumflex (CX) coronary arteries and a silicon tubing cuff around the proximal LAD (left anterior descending artery) (luminal stenosis +/- 77%) in 18 dogs. Weekly two-dimensional echocardiography was performed for regional function (anterior [A], inferoposterior [IP], wall thickening [WT]), and fractional shortening (FS). Colored microspheres injected at baseline and before sacrifice, before and after dipyridamole (0.5 mg/kg) injection, determined resting flow (RF) and coronary reserve (CR), respectively. RESULTS Coronary angiography performed at four weeks after surgery confirmed occlusion of RC and CX with collateralization and a tight stenosis of LAD. Initially, an episodic reduction in A and IP WT was observed which became persistent later (AWT: 16 +/- 3%; IPWT: 16 +/- 4%, FS: 20 +/- 4%, p < 0.005 vs. baseline [BS]). With dobutamine a biphasic response (improvement in A and IP WT between 5-15 and dysfunction between 20-30 microg/kg/min) was observed. Seven dogs were sacrificed at eight weeks and showed normal RF but reduced transmural CR (A: 75 +/- 18%; IP: 46 +/- 22% of control). Seven dogs underwent PTCA of the LAD at eight weeks and showed gradual improvement in AWT with normalization at 12 weeks (AWT: 30 +/- 5%, p < 0.001 vs. eight weeks). At sacrifice RF and CR in the A wall were normal but there was reduced subendocardial RF in the IP region (64% of BS). Further, biopsy samples showed normal histological findings and high energy phosphate content in all dogs. Radioligand binding assays using 125I-iodocyanopindolol showed downregulation of beta-adrenergic receptor density in the dysfunctional regions compared with control. CONCLUSIONS In this canine model of viable, collateral dependent and reversibly dysfunctional myocardium, there was early episodic dysfunction followed by persistent dysfunction which was initially associated with normal RF and later with subendocardial hypoperfusion.

In Vivo Effects of Contrast Media on Coronary Thrombolysis

OBJECTIVES The aim of the present study was to evaluate the influence of radiographic contrast media (CM) on alteplase-induced coronary thrombolysis. BACKGROUND Contrast media inhibit fibrinolysis in vitro and interact with endothelial cells, platelets and the coagulation system. The in vivo effects of CM on thrombolysis are not known. METHODS Occlusive coronary artery thrombosis was induced in 4 groups of 10 dogs by the copper coil technique. After 70 min of occlusion the dogs were randomized to intracoronary injection of 2 ml kg(-1) of either saline, a low-osmolar ionic CM (ioxaglate), a low-osmolar nonionic CM (iohexol) or a high-osmolar ionic CM (amidotrizoate). Thrombolysis with alteplase and co-therapy with aspirin and heparin was initiated after 90 min of occlusion. The coronary artery flow was monitored with an electromagnetic flowmeter throughout the experiment. RESULTS Iohexol and amidotrizoate, but not ioxaglate, were associated with longer reperfusion delays (time to optimal reperfusion: 67+/-48 min and 65+/-49 min, respectively, vs. 21+/-11 min after placebo; p < 0.05) and shorter periods of coronary perfusion (optimal perfusion time: 21+/-26 min and 21+/-28 min, respectively, vs. 58+/-40 min after placebo; p < 0.05). No significant differences were observed between groups with regard to activated partial thromboplastin times, circulating thrombin-antithrombin III complex concentrations and fibrinogen. CONCLUSIONS In this animal model administration of iohexol and amidotrizoate before thrombolysis significantly delayed reperfusion. This interaction should be considered in the design of clinical trials of thrombolytic therapy that evaluate coronary artery patency and in patients receiving local infusions of fibrinolytic agents.

Assessment of myocardial viability in a porcine model of chronic coronary artery stenosis with dual dose dobutamine magnetic resonance imaging

In a non-surgical porcine coronary stenosis model resulting in chronic left ventricle dysfunction, we aimed in this study to evaluate the potential of magnetic resonance imaging (MRI) to distinguish dysfunctional but viable from necrotic myocardium by using multiple levels of dobutamine inotropic stimulation during a cine MRI protocol (F.P. van Rugge et al. Circulation 1994; 90: 127–138). We compared our results with histopathology. We were able to demonstrate a biphasic effect at increasing doses of dobutamine in a subgroup of animals with a high-grade coronary stenosis, while in another subgroup the coronary stenosis produced a chronic myocardial infarction, in which no functional recovery could be obtained. In this experimental protocol, dual dose dobutamine MRI proved to be an accurate and reproducible technique to perform viability studies in chronic obstructive coronary artery disease. It permits distinguishing chronic ischemic, but viable myocardium from infarcted tissue. The detection of chronically underperfused but potentially salvageable myocardium is of significant clinical importance since it may aid in determining which patients are eligible for revascularization.

A nonsurgical porcine model of left ventricular dysfunction. Validation of myocardial viability using dobutamine stress echocardiography and positron emission tomography

BACKGROUND: Although several shortterm animal models of stunning and hibernation have been studied extensively, it has been difficult to produce a consistent animal model of chronic hibernation. The aim of the present study was to develop a nonsurgical porcine stent model of coronary stenosis in order to investigate the relationship between chronic dysfunctional myocardium and viability using 2D-echo, dobutamine stress echo (DSE) and positron emission tomography (PET). METHODS AND RESULTS: Focal progressive coronary stenosis was induced by implantation of an oversized stent in the left anterior descending (LAD) and/or circumflex (LCX) coronary artery in a total of 115 pigs, according to various experimental protocols: copper stent in the LAD (group I, n = 5); noncoated stainless steel stent in the LAD combined with balloon overstretch (group II, n = 7); poly(organo)phosphazene-coated stent in the LAD (group III, n = 77); and poly(organo)phosphazene-coated stent in both the LAD and the LCX (group IV, n = 26). Occurrence of left ventricular dysfunction was evaluated weekly by 2D-echo. At the time of left ventricular dysfunction the presence of viable myocardium within the dysfunctional region was investigated with DSE and PET, and confirmed by histology. The degree of coronary artery stenosis was measured by quantitative coronary angiography and morphometry. Severe coronary artery stenosis in the presence of dysfunctional, but viable, myocardium was induced in groups III and IV (47% and 11% of the animals, respectively). CONCLUSIONS: The authors developed a nonsurgical porcine stent model of progressive coronary stenosis using an oversized polymer-coated stent resulting in chronically decreased myocardial function, with residual inotropic reserve and viable myocardium. This condition may arise from repetitive periods of ischemia, or from sustained hypoperfusion, or a combination of these processes eventually leading to myocardial hibernation. (Int J Cardiovasc Intervent 2000; 3: 111-120)

Protein kinase C expression and subcellular distribution in chronic myocardial ischemia. Comparison of two different canine models

We studied protein kinase C (PKC) isozyme expression and activity distribution in two models of chronically ischemic canine myocardium: (1) single vessel obstruction (SVO), produced by tight stenosis of LAD followed by preconditioning and acute ischemia (40 min); (2) three vessel obstruction (3VO), produced by LAD-stenosis and gradual occlusion of right coronary artery and left circumflex. In both models after 8 weeks of chronic ischemia the dogs were either sacrificed or had PTCA of the LAD with a follow up of another 4 weeks. Control dogs were sham operated. PKC activity was measured in subcellular fractions of tissue samples from anterior and posterior regions in the presence of histone and γ-[32P]-ATP. PKC isozymes were detected by Western blotting. All regions perfused by the obstructed coronaries were dysfunctional at 8 weeks when compared to baseline, with improvement of anterior wall function after PTCA of LAD. PKC activity was elevated in the membrane fraction of SVO, but unchanged in the 3VO model. PKCs α, ε, and ζ prevailed in cytosol fraction of the controls (cytosol/membrane ratios were ± 3.34, 1.38 and 4.56 for α, ε and ζ, respectively), consistent with PKC activity distribution, while δ was not detected. There was no significant difference between the groups concerning the relative membrane amount of the isozymes. PKCs α and ε were decreased in the cytosol fraction of both models at 8 weeks (for anterior region, by 56 and 57% in SVO and by 49 and 46% in 3VO, respectively) without there being any differences between anterior and posterior regions, and were low also in the PTCA group. PKC ζ distribution however varied between the two models. The amount of PKC ζ isozyme was downregulated by 45% after 8 weeks of chronic ischemia and returned towards the control values after PTCA in the anterior region of SVO, while it did not change in anterior wall after 8 weeks in 3VO but was significantly decreased (by 47%) in posterior region after PTCA. In conclusion, our results suggest modified PKC signalling in chronically ischemic canine myocardium.