Joan Wikman-Coffelt

A new cardiac myosin characterized from the canine atria

Abstract Canine atrial myosin light chains were electrophoretically distinct from myosins of canine ventricles on 5–20% polyacrylamide gradient slab gels (SDS), giving molecular weights of 26,000 and 21,000 as compared to 28,000 and 18,500 for ventricular myosin light chains. While atrial myosin heavy chains were immunologically identical with ventricular myosin heavy chains, in contrast, there was 8.0% relative cross-reactivity of atrial myosin light chains with left ventricular myosin light chains by radioimunoassay. According to charge separation on two-dimensional polyacrylamide urea gels, atrial myosin light chains were different from those of ventricular myosins. Variances in ATPase activities between atrial and ventricular myosins were strongly demonstrated. There was a lower K + activated ATPase activity in atrial myosin, however the Ca 2+ activated ATPase activity, at ATP saturation levels, was higher in atrial myosin as compared to ventricular myosins.

Myosin chains of myocardial tissue. I. Purification and immunological properties of myosin heavy chains

Abstract An antiserum specific to dog myocardial myosin has been developed against highly purified myosin heavy chains. The antiserum is specific for the heavy chains of myosin, giving a single precipitin line in an immunodiffusion assay for either the heavy chains of myosin or native myosin, and does not react with any other myocardial proteins. In such assays myosin acts as a single, uniform antigen. Using this antiserum, a radioimmunoassay has been developed to quantitate myosin in a homogenate of myocardial tissue containing free myosin dissociated from other cellular components.

Chronological effects of mild pressure overload on myosin ATPase activity in the canine right ventricle.

Abstract Mild pulmonic stenosis in the dog, performed by pulmonary artery banding, caused an increasing elevation in myosin ATPase activity which peaked in enzymatic Vmax values by 5 weeks after surgery (35% above normal) and then steadily declined; activity fell 11% below normal by 16 weeks after operation. During the postoperative period there was a consistent elevation in right ventricular peak systolic pressure, which remained approximately 60% above that of normal animals. The free wall of the right ventricle reached maximum size in wet weight by 5 weeks after pulmonic banding.

Comparative Purification of Myocardial Myosin and Antigenic Specificity of the Two Light Chains

Abstract Dog myocardial myosin preparations, purified according to the procedures presented here, utilizing either one or two (NH4)2SO4 fractionations, contained no major contaminants which could be detected by disc gel electrophoresis, and exhibited high myosin ATPase activity. The low molecular weight components (light chains) were dissociated from the rest of the molecule by denaturing with urea; the chains were further purified by column chromatography. Procedures were a modification of those used for purification of skeletal muscle myosin light chains. According to immunoanalyses the two myocardial myosin light chains showed antigenic specificity.

A Stimulator-Regulated Rapid-Freeze Clamp for Terminating Metabolic Processes of the Heart During Normal Physiological Working Conditions

A stimulator-regulated rapid-freeze clamp has been developed by which squeezing action is used to forge the physiologically functioning rat heart into a 0.5 mm thick wafer, thereby freezing (-80°C) the entire heart, and thus terminating metabolic processes in 5 ms ±0.5 ms at any defined point in the contraction cycle. The pneumatic cylinders contain large ports and frictionless seals. 80 psi of compressed air drives the anvils at an overall rate of 0.14 in/ms.

Alterations of subunit composition and ATPase activity of myosin in early hypertrophied right ventricles of dogs with mild experimental pulmonic stenosis

Abstract Mild pulmonic stenosis was performed in dogs by banding of the pulmonary artery to evaluate the effect of systolic pressure overload on the enzymatic activity and subunit composition of myosin in early hypertrophied right ventricles. Three weeks following pulmonary constriction, 12 hypertrophied dogs were sacrificed and compared to 12 control animals. The weight of the hypertrophied right ventricles (HRV) relative to body weight was 46% greater than the weight of normal right ventricles (NRV) (P

Effects of severe hemodynamic pressure overload on the properties of canine left ventricular myosin: mechanism by which myosin ATPase activity is lowered during chronic increased hemodynamic stress.

Left ventricular myosin ATPase activity, expressed as enzymatic Vmax values, was analyzed in dogs subjected to severe left ventricular pressure overload (aortic stenosis). K+ and Ca2+ activated myosin ATPase activities in the left ventricle (LV) were significantly depressed (P < .01) in the experimental animals. For normal K+ activated myosin the Vmax values in micromoles of Pi per mg per min were: right ventricle 2.10; left ventricle, 2.84. For Ca2+ activated myosin the Vmax values were: right ventricle, 0.77; left ventricle 0.97, when assayed at 37°C. Myosin enzymatic activity in the left ventricle progressively declined following severe aortic banding, reaching a value similar to that observed for normal right ventricular myosin; NH4+ activated left ventricular myosin ATPase activity remained unchanged (7.20 ± 0.4 μmol PO4/mg.min). Left ventricular myosin from the hearts subject to severe stress simulated normal right ventricular myosin in ATPase activity, chain proportions and degree of calcium binding, Normal left ventricular myosin contained approximately 10% of the myosin protein concentration in the light chains; myosin from the left ventricles of the hemodynamically overloaded hearts contained 20% of the myosin protein concentration in the light chains (P < .001). With only one of the myosin light chains binding calcium left ventricular myosin from the stressed hypertrophied tissue bound approximately 2 mol Ca2+ mol−1 myosin similar to myosin of the normal right ventricle; normal left ventricular myosin bound approximately 1 mol of Ca2+ mol−1 myosin.

Identification of in vivo phosphorylated myosin subunits

Perrie et al. [ 1 J have shown in vitro phosphorylation of Mlz (DTNB) light chain [2] of rabbit skeletal muscle myosin. Myosin light chain Cz was phosphorylated using kinases isolated from skeletal muscle myosin and [T-~* P] ATP. Using similar conditions these investigators were not able to phosphorylate cardiac myosin. The study presented here makes a comparison between in vivo incorporation of 32P04 into each of the cardiac myosin subunits and moles of bound phosphate present in each. These phosphorylated myosin subunits were identified by both two-dimensional gel electrophoresis and isoelectrofocusing.

Activity and molecular changes in right and left ventricular myosins during right ventricular volume overload

Abstract Left ventricular myosin ATPase activity was analyzed in dogs subject to right ventricular volume overload induced by extirpation of pulmonic and tricuspid valves; K+ and Ca2+ activated myosin ATPase activities in both the right and left ventricles of the stressed animals were never elevated, but were significantly depressed (P Normal right ventricular myosin contained approximately twice the moles of light chains per mole of myosin as compared to left ventricular myosin; after 6 weeks of tricuspid insufficiency (TI), left ventricular myosin contained twice the number of moles of light chains per mole of myosin as compared to that of normal (NLV) left ventricular myosin (TI = 20% myosin protein concentration present in light chains; NLV = 10% myosin protein concentration present in light chains (P

Comparison of mild vs severe pressure overload on the enzymatic activity of myosin in the canine ventricles

Abstract Mild pulmonic stenosis, performed in dogs by banding the pulmonary artery, elevated right ventricular peak systolic pressure to 60% above the control; this resulted in an elevation in right ventricular K+ and Ca2+ activated myosin ATPase activities which peaked (35% above normal) in enzymatic Vmax values by 5 weeks postoperatively. In contrast, severe pulmonic stenosis produced by the inflatable Jacobson cuff, elevating right ventricular peak systolic pressure to 300% above the control, did not produce an increase in myosin enzymatic ATPase Vmax values. The first significant deviation from normal myosin ATPase activity was detected after 5 weeks of severe pulmonic stenosis; at this time right ventricular myosin enzymatic Vmax values were depressed significantly from those of normal values by 12%. ( NH 4 + activated myosin Vmax value remained normal). Likewise mild aortic stenosis, induced by banding the ascending aorta, forcing a transaortic pressure gradient of 25 mm Hg, caused a 15% elevation in left ventricular myosin ATPase activities by 5 weeks after operation; myosin enzymatic Vmax values decreased to normal values by 12 weeks after surgery. Severe aortic stenosis, brought about by creating a transaortic pressure gradient of 55 mm Hg, never caused an elevation in left ventricular myosin enzymatic Vmax values, but caused a 15% decrease in K+ and Ca2+ activated myosin by 5 weeks after operation and a decrease to 28% below normal by 12 weeks of pressure overload.