Alterations of mechanical parameters in chemically skinned preparations of rat myocardium as a function of isoenzyme pattern of myosin

Summary Myofibrillar ATPase activity, maximum unloaded shortening velocity, and isometric tension development were evaluated in left ventricular preparations of 5-week-old rats with a high endogeneous level of thyroid hormones and hypothyroid rats after 4-week treatment with propylthiouracil (PTU). The range of possible alterations of the above functional parameters was defined in relation to myosin isoenzyme distribution. The mechanical behaviour of the ventricular preparations was investigated in native myocardium as well as in the glycerinated state.The essential result of the present study is that alterations of myofibrillar ATPase activity and mechanical v_max, evaluated in glycerinated preparations, are limited to a well-defined range of similar magnitude for both functional parameters: 32–40% of maximum values (obtained from rat myocardium with homogeneous myosin V₁). Isometric tension was only insignificantly decreased in glycerinated preparations of the PTU-treated group.The alteration in the apparent maximum shortening velocity of native myocardium (v₀) was of the same magnitude as changes in v_max of chemically skinned preparations. Physical training revealed a shift in the direction of V₁-type myosin with increased ATPase activity and shortening velocity; aging and pressure overload showed an opposite effect. The documented mechanical alterations do not contradict an adaptational interpretation of the myosin isoenzyme redistribution in pressure-induced hypertrophy.Supported by the Deutsche Forschungsgemeinschaft.     

Regulation of pulmonary arterial myosin phosphatase activity in neonatal circulatory transition and in hypoxic pulmonary hypertension: a role for CPI-17

Neonatal circulatory transition is dependent upon tightly regulated pulmonary circuit relaxation. Persistent pulmonary hypertension of the newborn (PPHN) is characterized by pulmonary arterial myocyte relaxation failure. We examined the effect of short course (72 hour) in vivo normobaric hypoxia in newborn swine on smooth muscle contractile enzyme activity and regulatory phosphoprotein abundance, in tissue homogenates of 2nd to 4th generation pulmonary arteries. Myosin light chain kinase (MLCK) and phosphatase (MLCP) protein contents were unchanged in hypoxic pulmonary arteries compared to controls. MLCP activity increased in normoxic animals from birth to day 3. This was ablated by hypoxia; phosphatase activity, measured as in vitro myosin light chain dephosphorylation, was decreased significantly (P < 0.005) in the hypoxic group. Inhibitory site phosphorylations of MLCP myosin binding subunit at threonines 696 and 850 were similar in both hypoxic and normoxic subjects, suggesting that downregulation of MLCP in hypoxia does not involve this pathway. However, content of regulatory protein CPI-17 (protein kinase C-related phosphatase inhibitor) increased from birth in hypoxic subjects (P < 0.05); active (phosphorylated) CPI-17 protein abundance declined after birth in normals, but increased in hypoxic arteries (P < 0.05). This corresponded with the decrease in phosphatase activity. We speculate that CPI-17 may play a role in myosin phosphatase upregulation during neonatal circulatory transition, and in hypoxic inhibition of pulmonary phosphatase activity in PPHN.     

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 V_max 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 V_max values in micromoles of Pi per mg per min were: right ventricle 2.10; left ventricle, 2.84. For Ca²⁺ activated myosin the V_max 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.     

Biochemical characterization of ventricular myosin from spontaneously hypertensive turkeys

Several stimuli are able to alter the synthesis of cardiac myosin isoenzymes. Particularly in the rat a shift toward a low-ATPase isomyosin is generally observed during development and in cardiac hypertrophy due to pressure overload. On the contrary in spontaneously hypertensive turkeys both ageing and the increasing degree of cardiac hypertrophy are accompanied by a different behaviour of ventricular myosin. In fact in a previous study we have shown that the Ca2+-activated ATPase activity of ventricular myosin increases about three folds from young normotensive to old hypertensive animals. Accordingly the peptide pattern obtained after chymotryptic digestion of myosin showed that some peptides, which are not evident or barely discernible in young animals, are present in the adult ones. In this study we compare the ventricular myosin from young normotensive and adult hypertensive turkeys with atrial myosin. The results obtained suggest that in the ventricles of hypertensive turkeys the synthesis of an isomyosin with biochemical properties close to those of atrial myosin occurs.     

Precision Medicine Approaches to Vascular Disease: JACC Focus Seminar 2/5

In this second of a 5-part Focus Seminar series, we focus on precision medicine in the context of vascular disease. The most common vascular disease worldwide is atherosclerosis, which is the primary cause of coronary artery disease, peripheral vascular disease, and a large proportion of strokes and other disorders. Atherosclerosis is a complex genetic disease that likely involves many hundreds to thousands of single nucleotide polymorphisms, each with a relatively modest effect for causing disease. Conversely, although less prevalent, there are many vascular disorders that typically involve only a single genetic change, but these changes can often have a profound effect that is sufficient to cause disease. These are termed “Mendelian vascular diseases,” which include Marfan and Loeys-Dietz syndromes. Given the very different genetic basis of atherosclerosis versus Mendelian vascular diseases, this article was divided into 2 parts to cover the most promising precision medicine approaches for these disease types.     

Isomyosin redistribution in chronic pressure overload: comparison between peptide mapping and electrophoresis under non-denaturing conditions

Summary Chronic pressure overload induces a redistribution in myosin isoenzymes as demonstrated by Ca⁺⁺-activated ATPase activity, electrophoresis under non-denaturing conditions and immunohistochemistry.We compared, in two groups of renal hypertensive rats and control rats, the isoenzymic patterns obtained by electrophoresis under non-denaturing conditions with those observed after heavy chains digestion with S. Aureus V8 protease.In the hypertensive animals in which a shift towards the slow V₂ and V₃ isomyosins was evident, peptide mapping always gave origin to a band which was not present in the controls.Since we consider this peptide as a marker of the redistribution towards the slow isoforms, peptide mapping according to Cleveland appears to be a simple and useful method to assess differences in isomyosin composition, at least between hypertrophic pressure-overloaded and normal rat ventricles. Moreover, in our experience this technique is simple, the patterns obtained from highly purified substrates are very reproducible and the digestion allows easy and clear comparisons.     

Molecular mimicry in the autoimmune pathogenesis of rheumatic heart disease

Molecular mimicry is a hallmark of the pathogenesis of rheumatic fever where the streptococcal group A carbohydrate epitope, N-acetyl glucosamine, and the α-helical coiled-coil streptococcal M protein structurally mimic cardiac myosin in the human disease, rheumatic carditis, and in animal models immunized with streptococcal M protein and cardiac myosin. Recent studies have unraveled the potential pathogenic mechanisms by which the immune response against the group A streptococcus attacks the rheumatic valve leading to chronic rheumatic heart disease. Both B- and T-cell responses are involved in the process, and evidence for the hypotheses of molecular mimicry and epitope spreading are reviewed.     

逼尿肌相关蛋白在慢性前列腺炎大鼠模型中的表达及意义

目的：采用17β-雌二醇联合去势建立大鼠慢性非细菌性前列腺炎模型,检测膀胱逼尿肌蛋白在慢性前列腺炎大鼠膀胱的表达。方法取2月龄 SD 雄性大鼠60只随机分为6组,每组10只,即：空白对照组、生理盐水组、假手术组、单纯17β-雌二醇组、单纯去势组、17β-雌二醇联合去势组。HE 染色判定大鼠慢性前列腺炎模型建造成功后,采用 SP 免疫组化法、Western blot 检测6组大鼠膀胱组织肌动蛋白(α-actin)、肌球蛋白(MLC 2)及高分子量钙调素结合蛋白(h-cad)表达的差异。结果免疫组化结果显示17β-雌二醇联合去势组中α-actin、MLC 2、h-cad 蛋白表达增多,Western blot 显示α-actin 在各组的表达,与空白对照组相比,生理盐水组、假手术组、单纯17β-雌二醇组、单纯去势组联合去势组差异有统计学意义。 MLC 2、h-cad 在各组的表达与空白对照组相比,生理盐水组、假手术组差异无统计学意义,单纯17β-雌二醇组、单纯去势组、联合去势组差异有统计学意义。结论α-actin、MLC 2、h-cad 表达变化可能参与慢性前列腺炎排尿相关症状的发病。