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The identification and quantification of specific phosphorylation sites within a protein by mass spectrometry has proved challenging when measured from pepfides after protein digestion because each peptide has a unique ionization efficiency that alters with modification, such as phosphorylation, and because phosphorylation can alter cleavage by trypsin, shifting peptide distribution. In addition, some phosphorylated peptides generated by tryptic digest are small and hydrophilic and, thus, are not retained well on commonly used [C.sub.18] columns. We have developed a novel C-terminal peptide [sup.2]H-labeling derivatization strategy and a mass balance approach to quantify phosphorylation. We illustrate the application of our method using electrospray ionization liquid chromatography--mass spectrometry by quantifying phosphorylation of troponin I with protein kinase A and protein kinase C. The method also improves the retention and elution of hydrophilic peptides. The method defines phosphorylation without having to measure the phosphorylated peptides directly or being affected by variable miscleavage. Measurement of phosphorylation is shown to be linear (relative standard error

Male but not female mice carrying a single R403Q missense allele for cardiac [alpha]-myosin heavy chain (M-[alpha].sup.MH[C.sup.R403Q/+] and F-[alpha]MH[C.sup.R403Q/+], respectively) develop significant hypertrophic cardiomyopathy (HCM) compared with male and female wild-type mice (M-[alpha]MH[C.sup.+/+] and F-[alpha]MH[C.sup.+/+], respectively) after Tilde 30 wk of age. We tested the hypothesis that myofilament mechanical performance differs between M-[alpha]MH [C.sup.403Q/+] and F-[alpha]MH[C.sup.R403Q/+ ] at younger ages (10-20 wk) and could account for sex differences in HCM development. The sensitivity of chemically skinned myocardial strips to [Ca.sup.2+] activation (p[Ca.sub.50]) was signifcantly (P < 0.05) enhanced in male mice independent of genotype (M-[alpha]MH[C.sup.R403Q/+] 5.70 [+ or -] 0.06, M-[alpha]MH[C.sup.+/+]: 5.63 [+ or -] + 0.05, F-[alpha]MH [C.sup.R403Q/+]: 5.57 [+ or -] 0.03, F-[alpha] MH[C.sup.+/+]: 5.54 [+ or -] 0.04) by two-way ANOVA, whereas maximum developed tension was significantly enhanced in [alpha]-MH[C.sup.R403Q/+] independent of sex (M-[alpha]MH[C.sup.R403Q/+]: 29.3 [+ or -] 2.3, M-[alpha]MH [C.sup.+/+]: 26.0 [+ or -] 1.4, F-[alpha]MH[C.sup.R403Q/+]: 30.2 [+ or -] 2.1, F-[alpha]MH[C.sup.+/+]: 26.2 [+ or -] 1.2 mN/[mm.sup.2]). The frequency of maximum work generated by sinusoidal length perturbation was significantly higher in [alpha]MH[C.sup.R403Q/+] mice than in sex-matched controls (M-[alpha]MH[CR403Q/+]: 2.26 [+ or -] 0.47, M-[alpha]MH[C.sup.+/+]: 1.29[+ or -] 0.18, F-[alpha]MH [C.sup.R403Q/+]: 3.21 [+ or -] 0.33, F-[alpha]MH[C.sup.+/+]: 2.52 [+ or -] 0.36 Hz). Unloaded shortening velocity was significantly enhanced in [alpha]MH[C.R403Q/+] and in female mice (M-[alpha]MH [C.sup.R403Q/+]: 2.26 [+ or -] 0.47, M-[alpha]MH[C.sup.+/+]: 1.29 [+ or -] 0.18, F-[alpha]MH[C.sup.R403Q/+]: 3.21 [+ or -] 0.33, F-[alpha]MH [C.sup.+/+]: 2.52 [+ or -] 0.36 muscle lengths/s), and normalized mechanical power, calculated from the tension-velocity relationship, was significantly enhanced in [alpha]MH[C.sup.R403Q/+] independent of sex (M[alpha]MH[C.sup.R403Q/+]: 60 [+ or -] 2 [10.sup.-3], M-[alpha]MH [C.sup.+/+]: 37 [+ or -] 3 [10.sup.- 3], F-[alpha]MH[C.sup.R403Q/+]: 57 [+ or -] 3 [10.sup.-3], F-[alpha]MH [C.sup.+/+] 25 [+ or -] 3 [10.sup.-3] muscle lengths/s x normalized tension). We did not find a statistically significant sex x mutation interaction for any measure of myofilament performance. Therefore, sarcomeric incorporation of the R403Q myosin similarly enhanced left ventricular myofilament mechanical performance in both male and female mice. The sex-dependent development of HCM due to the R403Q myosin may then be inhibited by female sex hormones, which may additionally underlie the observed sex differences for p[Ca.sub.50] and unloaded shortening velocity. hypertrophic cardiomyopathy; myosin heavy chain; isometric tension; calcium sensitivity; force-velocity

The kinetic effects of the cardiac myosin point mutations R403Q and R453C, which underlie lethal forms of familial hypertrophic cardiomyopathy (FHC), were assessed using isolated myosin and skinned strips taken from beterozygous (R403Q/+ and R453C/+) male mouse hearts. Compared with wild-type (WT) mice, actin-activated ATPase was increased by 38% in R403Q/+ and reduced by 45% in R453C/+, maximal velocity of regulated thin filament ([V.sub.RTF]) in the in vitro motility assay was increased by 8% in R403Q/+ and was not different in R453C/+, myosin concentration at half-maximal [V.sub.RTF] was reduced by 30% in R403Q/+ and not different in R453C/+, and the characteristic frequency for oscillatory work production (b frequency), determined by sinusoidal analysis in the skinned strip at maximal calcium activation, was 27% lower in R403Q/+ and 18% higher in R453C/+. The calcium sensitivity for isometric tension in the skinned strip was not different in R403Q/+ (p[Ca.sub.50] 5.64 [+ or -] 0.02) and significantly enhanced in R453C/+ (5.82 [+ or -] 0.03) compared with WT (5.58 [+ or -] 0.02). We conclude that isolated myosin and skinned strips of R403Q/+ and R453C/+ myocardium show marked differences in cross-bridge kinetic parameters and in calcium sensitivity of force production that indicate different functional roles associated with the location of each point mutation at the molecular level. myosin heavy chain; ATPase; myosin motility; isometric tension; sinusoidal analysis

Alteration of troponin T (TnT) isoform expression has been reported in human and animal models of myocardial failure. The two adult beef cardiac TnT isoforms (Tn[T.sub.3] and Tn[T.sub.4]) were isolated for comparative functional analysis. Thin filaments were reconstituted containing pure populations of the isoforms. The in vitro motility assay was used to directly compare the effect of the two TnT isoforms on force and unloaded shortening as a function of free calcium. We found no significant differences between the two isoforms in terms of calcium sensitivity, cooperativity, or maximal activation (velocity and force) as assessed in a fully calcium-regulated system. Activation by myosin strong binding was similar for thin filaments containing either of the two TnT isoforms. Whereas maximally activated velocity and cooperativity was depressed at pH 6.5, no difference between thin filaments containing the two isoforms was detected. From the small magnitude of the TnT isoform shifts detected in myocardial failure and the lack of significant mechanical effect detected in the motility assay, variable TnT isoform expression is unlikely to be any functional significance in heart failure. thin filament; myocardial failure; in vitro motility

Abstract It has been reported previously that diabetic cardiomyopathy can be inhibited or reverted with chronic zinc supplementation. In the current study, we hypothesized that total cardiac calcium and zinc content is altered in early onset diabetes mellitus characterized in part as hyperglycemia (HG) and that exposure of zinc ion (Zn2+) to isolated cardiomyocytes would enhance contraction-relaxation function in HG more so than in nonHG controls. To better control for differential cardiac myosin isoform expression as occurs in rodents after β-islet cell necrosis, hypothyroidism was induced in 16 rats resulting in 100% β-myosin heavy chain expression in the heart. β-Islet cell necrosis was induced in half of the rats by streptozocin administration. After 6 wks of HG, both HG and nonHG controls rats demonstrated similar myofilament performance measured as thin filament calcium sensitivity, native thin filament velocity in the myosin motility assay and contractile velocity and power. Extracellular Zn2+ reduced cardiomyocyte contractile function in both groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application. This is a novel finding implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Among the many post-translational modifications examined, only phosphorylation of ryanodine receptor (RyR) at S-2808 was significantly higher in HG compared to nonHG. We did not find in our hypothyroid rats any differentiating effects of HG on myofibrillar protein phosphorylation, lysine acetylation, O-linked N-acetylglucosamine and advanced glycated end-products, which are often implicated as complicating factors in cardiac performance due to HG. Our results suggest that the relaxing effects of Zn2+ on cardiomyocyte function are more pronounced in the HG state due an insulin-dependent effect of enhancing removal of cytosolic Ca2+ via SERCA2a or NCX or by reducing Ca2+ influx via L-type channel or Ca2+ leak through the RyR. Investigations into the effects of Zn2+ on these mechanisms are now underway.