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T. Ueyama, S. Kawashima, T. Sakoda, Y. Rikitake, T. Ishida, M. Kawai, T. Yamashita, S. Ishida, H. Hotta and M. Yokoyama. Requirement of Activation of the Extracellular Signal-regulated Kinase Cascade in Myocardial Cell Hypertrophy.Journal of Molecular and Cellular Cardiology (2000) 32, 947–960. The signal transduction mechanisms mediating hypertrophic responses in myocardial cells (MCs) remain uncertain. We investigated the role of the extracellular signal-regulated kinase (ERK) cascade in myocardial cell hypertrophy by the strategy of using the adenovirus-mediated overexpression of mitogen-activated protein kinase (MAPK)/ERK kinase (MEK), which is the upstream activator of ERK. We generated recombinant adenoviruses expressing constitutively active MEK1 (MEK1 EE) and dominant negative MEK1 (MEK1 DN). Overexpression of MEK1 EE in MCs activated ERK1/2 and subsequently induced atrial natriuretic peptide (ANP) mRNA expression. In addition, MEK1 EE overexpression resulted in an increase in cell size and sarcomeric reorganization. In contrast, overexpression of MEK1 DN in MCs inhibited endothelin-1 (ET-1)-, phenylephrine (PE)-, leukemia inhibitory factor (LIF)-, isoproterenol (ISP)-, and mechanical stretch-induced ERK activation and ANP mRNA expression. MEK1 DN overexpression inhibited ET-1-, PE-, LIF-, and ISP-induced increases in cell size and sarcomeric reorganization. Consistent with the observed effects on cellular morphology, overexpression of MEK1 EE resulted in an increase in amino acid incorporation, while overexpression of MEK1 DN inhibited ET-1-, PE-, LIF-, ISP-, and mechanical stretch-induced increases in amino acid incorporation. These results indicate that the ERK cascade plays an important role in the signaling pathway leading to the development of myocardial cell hypertrophy.

Eighty isolated rat heart preparations were used to study relationships among creatine kinase (CK) release, the loss of vascular competence (no-reflow), and the distribution of morphological changes across the left ventricular wall which occur during 60 min global ischaemia or anoxia and following subsequent oxygenated reperfusion. Hearts were either fixed with glutaraldehyde for light and electron microscopy or were injected with 1% fluorescein to define the distribution of perfusable vessels. The extent of no-reflow in half of the hearts was reduced experimentally by maintaining the diastolic volume of the left ventricular lumen during ischaemia and anoxia with a water-filled balloon. The amount of CK released during 20 min of reoxygenation or reperfusion was inversely proportional to the extent of the no-reflow area observed just prior to reoxygeneration, and also reflected the transmural extent and the severity of myocardial cell damage. Extensive contraction band necrosis was only observed in reperfused regions of anoxic hearts. In isovolumic hearts reoxygenation caused no-reflow to develop in the ventricular myocardium, and this appeared to be associated with hypercontraction. Thus the no-reflow phenomenon has a profound effect on the transmural distribution of myocardial cell damage and enzyme release which follows post ischaemic reperfusion and post anoxic reoxygenation.

A histocytometric study of 42 left ventricles was carried out. The cases consisted of normal hearts as well as a number of hypertrophic hearts which were progressively heavier in weight. The results of this study were analyzed by computer. We found an increase in myocardial cell size correlated with an increase in the ventricular free wall weight, up to a critical weight of 250 g. Beyond that critical weight, cellular hyperplasia was evident, followed by the further development of hypertrophy, in relation to the increase of ventricular weight. At the “critical weight” the progressive increase of cellular diameter, which is less than the theoretical value, abruptly ends and then decreases considerably. In contrast, this same phenomena is not seen for the cell length. The myocardial cell length shows a constant gradual increase always greater than the theoretical value regardless of ventricular weight. For this reason the cellular hyperplasia specifically presents an increase in cellular length beyond the increase in cellular diameter, which is reduced. A multiplication of myocardial nuclei beyond the critical weight was also observed. The results obtained are of physiopathological significance owing to modifications of the capillary network and the interstitial tissues.

Free radical generation may be the principal pathogenic factor responsible for the initiation of ischemia/reperfusion myocardial cell damage. Circumstantial evidence is in favour of the view that impaired function of the membraneous components of ischemic cells is associated with oxygen radical-induced alterations in proteins and lipids. Thus, lipid antioxidants and free radical scavengers as well as antagonists of protein modification may all be required for therapeutic intervention of this aspect of ischemia/reperfusion injury.

The relation between myocardial cell potassium and water contents after hypoxia at 0°C (cold shock) in Krebs-Ringers-Phosphate solution was studied using 252 myocardial slices from six dogs. Losses of potassium were marked and similar after immediate cold shock, cold shock after 1 h equilibration in oxygenated media at 25°C and immediate cold shock plus 1 mm ouabain. However, myocardial water and total monovalent cations were only slightly increased by cold shock after equilibration compared to the marked increases found after immediate cold shock with and without ouabain (P < 0.01). When edematous slices were warmed to 25°C in oxygenated media, myocardial potassium increased towards control values in the absence of ouabain (P < 0.01) and decreased further when ouabain was present (P < 0.01), but in both cases myocardial water and total monovalent cations decreased towards control values (P < 0.01). This dissociation of NaK exchange pump activity and myocardial cell volume regulation can be only partially explained using the classic “pump-leak” theory of cell volume regulation.