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Regulation of myocardial slow channels by calmodulin and cyclic nucleotides: Further evidence for the phosphorylation hypothesis
8 If]CREASED DNA SYNTHESIS IN THE HEART DURING ACUTE ALLYLAMINE CARDIOTOXICITY. Paul J. Boor and Nancy Kretschmer. Chemical Pathology Division, Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77550. The cardiotoxin allylamine (3-aminopropene, AA) causes acute myocellular degeneration and necrosis; in a previous ultrastructural study (Lab Invest 47:76-86, 1982) we observed marked mitotic activity in endothelial cells following AA. In the present study, we assessed proliferative activity in the heart: groups of male rats were given either a single dose (100 mg/kg) of AA, or two doses on successive days; all rats were killed 24 h after the 19st dose. Three h before killing, rats were given 0.37 mCu/kg (SA: 6.7 mCi/nmol H--Thymidine) IV; under ether anesthesia rats were killed by cardiac perfusion with formalin, the entire heart was sectioned at 4~m for autoradiography. Multiple adjacent fields were viewed and labeled endothelial and interstitial cell nuclei were counted across the interventricular septum (IVS), right ventricle (RV), and left ventriclar free wall (LV). Endothelia~ nuclear labeling was markedly increased in IVS after 2 doses (9.7 • 2.2 mitoses/nm" vs 1.6 i .2 in control; p < .05), LV and RV showed less pronounced increases. Increased endothelial and interstitial cell labeling correlated with histopathologic lesions although increased labeling after 1 dose was also seen in the absence of lesions, Prominant endothelial cell proliferation and interstitial cell activation occur rapidly in acute AA myocardial damage. (Supported by NIH Grant HL-26189)
MORPHOMETRY OF CELL SURFACE CABLES ~ID }~ASSI~E MECHANICS OF CARDIAC }~OCYTES. S. Bloom, M. Loew, and R. Belliardi. Dept. of Pathology and Dent. of Electrical Engineering and Computer Science, The George Uashington University, Washington, D.C., U.S.A.
Cardiac myocytes appear to be a source of cardiac diastolic stiffness. It is possible that the cell surface cables of cardiac myocvtes are the specific source of this stiffness. One way to determine if this is the case is to compare parameters of cable morphometry in cells at different sarcomere lengths. In order to accomplish such morphometry without subjective bias we have devised a comnuter procedure in which a SEM micrograph is digitized, the cables identified bv the comnuter, and cable abundance, length, width, angular orientation, and branching complexity calculated. The data obtained to date are for cells at short sarcomere length only. They show that cables are about 10 nm wide and branch frequently. While generally parallel to the long axis, their angular orientation is quite heterogeneous. It remains to be seen if either angular orientation or cable diameter varies as a function of cell length. Either relationship would suggest a unique mechanism for cable gover nance of diastolic stiffness.
REGULATION OF MYOCARDIAL SLOW CHANNELS BY CALMODULIN A N D CYLCIC NUCLEOTIDES: FURTHER EVIDENCE FOR THE PHOSPHORYLATION HYPOTHESIS. G. Bkaily and N. Sperelakis. Department of Biophysies, University of Sherbrooke, Quebec Canada and Department of Physiology and Biophysics, University of Cineinnati, Cineinnati, Ohio, U.S.A. Studies from our laboratory have demonstrated that the myocardial slow Ca++ channels are regulated by the cyclic nucleotides (both cAMP and cGMP). However, the involvement of calmodulin in the regulation of the myocardial slow channels is not known. Therefore, in the present study, several potent calmodulin inhibitors, exogenous calmodulin, inhibitor of cyclic AMP-dependent-protein kinase (PrKI) and the catalytic subunit of cAMP-PrK were injected intracellularly into cultured heart cell reaggregates by the liposome method. Injection of calmodulin inhibitors (trifluoperazine, W-7 and calmidazolium) blocked the spontaneous slow APs, depolarized, and caused excitation-contraction uncoupling. Application of hyperpolarizing current to repolarize the cells to high resting potentials did not restore the slow APs, but injection of calmodulin did. Injection of PrKI rapidly blocked the inward slow current (Isi) and depolarized the membrane; hyperpolarizing current pulses did not restore the slow APs, but injection of the catalytic subunit did. The data show that calmodulin is involved in regulation the phosphorylation of the myocardial slow channels. In addition, the present results further support the hypothesis that the myocardial slow channels protein (or an associated regulatory protein) must be phosphorylated in order for the slow channels to be available for voltage activation. This study was supported by NIH HL-31942; Dr. Bkaily had a scholarshipfrom C~
MORPHOMETRY OF CELL SURFACE CABLES ~ID }~ASSI~E MECHANICS OF CARDIAC }~OCYTES. S. Bloom, M. Loew, and R. Belliardi. Dept. of Pathology and Dent. of Electrical Engineering and Computer Science, The George Uashington University, Washington, D.C., U.S.A.
Cardiac myocytes appear to be a source of cardiac diastolic stiffness. It is possible that the cell surface cables of cardiac myocvtes are the specific source of this stiffness. One way to determine if this is the case is to compare parameters of cable morphometry in cells at different sarcomere lengths. In order to accomplish such morphometry without subjective bias we have devised a comnuter procedure in which a SEM micrograph is digitized, the cables identified bv the comnuter, and cable abundance, length, width, angular orientation, and branching complexity calculated. The data obtained to date are for cells at short sarcomere length only. They show that cables are about 10 nm wide and branch frequently. While generally parallel to the long axis, their angular orientation is quite heterogeneous. It remains to be seen if either angular orientation or cable diameter varies as a function of cell length. Either relationship would suggest a unique mechanism for cable gover nance of diastolic stiffness.
REGULATION OF MYOCARDIAL SLOW CHANNELS BY CALMODULIN A N D CYLCIC NUCLEOTIDES: FURTHER EVIDENCE FOR THE PHOSPHORYLATION HYPOTHESIS. G. Bkaily and N. Sperelakis. Department of Biophysies, University of Sherbrooke, Quebec Canada and Department of Physiology and Biophysics, University of Cineinnati, Cineinnati, Ohio, U.S.A. Studies from our laboratory have demonstrated that the myocardial slow Ca++ channels are regulated by the cyclic nucleotides (both cAMP and cGMP). However, the involvement of calmodulin in the regulation of the myocardial slow channels is not known. Therefore, in the present study, several potent calmodulin inhibitors, exogenous calmodulin, inhibitor of cyclic AMP-dependent-protein kinase (PrKI) and the catalytic subunit of cAMP-PrK were injected intracellularly into cultured heart cell reaggregates by the liposome method. Injection of calmodulin inhibitors (trifluoperazine, W-7 and calmidazolium) blocked the spontaneous slow APs, depolarized, and caused excitation-contraction uncoupling. Application of hyperpolarizing current to repolarize the cells to high resting potentials did not restore the slow APs, but injection of calmodulin did. Injection of PrKI rapidly blocked the inward slow current (Isi) and depolarized the membrane; hyperpolarizing current pulses did not restore the slow APs, but injection of the catalytic subunit did. The data show that calmodulin is involved in regulation the phosphorylation of the myocardial slow channels. In addition, the present results further support the hypothesis that the myocardial slow channels protein (or an associated regulatory protein) must be phosphorylated in order for the slow channels to be available for voltage activation. This study was supported by NIH HL-31942; Dr. Bkaily had a scholarshipfrom C~