O'Neal Copeland

Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle.

A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n=21). Total phosphorylation was 2.7 ± 0.07 mol Pi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n=19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the three sites was not random, but indicated positive and negative interactions between the three sites. Phosphorylation at Ser-282 was not proportional to the number of sites available. The 2P band contained 302 but not 273; the 3P band contained 273 but not 302.

Myosin binding protein C phosphorylation in normal, hypertrophic and failing human heart muscle

Phosphorylation of myosin binding protein C (MyBP-C) was investigated in intraventricular septum samples taken from patients with hypertrophic cardiomyopathy undergoing surgical septal myectomy. These samples were compared with donor heart muscle, as a well-characterised control tissue, and with end-stage failing heart muscle. MyBP-C was partly purified from myofibrils using a modification of the phosphate-EDTA extraction of Hartzell and Glass. MyBP-C was separated by SDS-PAGE and stained for phosphoproteins using Pro-Q Diamond followed by total protein staining using Coomassie Blue. Relative phosphorylation level was determined from the ratio of Pro-Q Diamond to Coomassie Blue staining of MyBP-C bands as measured by densitometry. We compared 9 myectomy samples and 9 failing heart samples with 9 donor samples. MyBP-C phosphorylation in pathological muscle was lower than in donor (myectomy 40+/-2% of donor, P<0.0001; failing 45+/-3% of donor, P<0.0001). 6 myectomy samples were identified with MYBPC3 mutations, one with MYH7 mutation and two remained unknown, but there was no correlation between MYBPC3 mutation and MyBP-C phosphorylation level. In order to determine the number of phosphorylated sites in human cardiac MyBP-C samples, we phosphorylated the recombinant MyBP-C fragment, C0-C2 (1-453) with PKA using (gamma32)P-ATP up to 3.5 mol Pi/mol C0-C2. This measurement of phosphorylation was used to calibrate measurements of phosphorylation in SDS-PAGE using Pro-Q Diamond stain. The level of phosphorylation in donor heart MyBP-C was calculated to be 4.6+/-0.6 mol Pi/mol and 2.0+/-0.3 mol Pi/mol in myectomy samples. We conclude that MyBP-C is a highly phosphorylated protein in vivo and that diminished MyBP-C phosphorylation is a feature of both end-stage heart failure and hypertrophic cardiomyopathy.

The effect of mutations in alpha tropomyosin (E40K and E54K), that cause familial dilated cardiomyopathy, on the regulatory mechanism of cardiac muscle thin filaments

E40K and E54K mutations in α-tropomyosin cause inherited dilated cardiomyopathy. Previously we showed, using Ala-Ser α-tropomyosin (AS-α-Tm) expressed in Escherichia coli, that both mutations decrease Ca2+ sensitivity. E40K also reduces Vmax of actin-Tm-activated S-1 ATPase by 18%. We investigated cooperative allosteric regulation by native Tm, AS-α-Tm, and the two dilated cardiomyopathy-causing mutants. AS-α-Tm has a lower cooperative unit size (6.5) than native α-tropomyosin (10.0). The E40K mutation reduced the size of the cooperative unit to 3.7, whereas E54K increased it to 8.0. For the equilibrium between On and Off states, the KT value was the same for all actin-Tm species; however, the KT value of actin-Tm-troponin at pCa 5 was 50% less for AS-α-Tm E40K than for AS-α-Tm and AS-α-Tm E54K. Kb, the “closed” to “blocked” equilibrium constant, was the same for all tropomyosin species. The E40K mutation reduced the affinity of tropomyosin for actin by 1.74-fold, but only when in the On state (in the presence of S-1). In contrast the E54K mutation reduced affinity by 3.5-fold only in the Off state. Differential scanning calorimetry measurements of AS-α-Tm showed that domain 3, assigned to the N terminus of tropomyosin, was strongly destabilized by both mutations. Additionally with AS-α-Tm E54K, we observed a unique new domain at 55 °C accounting for 25% of enthalpy indicating stabilization of part of the tropomyosin. The disease-causing mechanism of the E40K mutation may be accounted for by destabilization of the On state of the thin filaments; however, the E54K mutation has a more complex effect on tropomyosin structure and function.

Molecular mechanism of the E99K mutation in cardiac actin (ACTC Gene) that causes apical hypertrophy in man and mouse.

We generated a transgenic mouse model expressing the apical hypertrophic cardiomyopathy-causing mutation ACTC E99K at 50% of total heart actin and compared it with actin from patients carrying the same mutation. The actin mutation caused a higher Ca2+ sensitivity in reconstituted thin filaments measured by in vitro motility assay (2.3-fold for mice and 1.3-fold for humans) and in skinned papillary muscle. The mutation also abolished the change in Ca2+ sensitivity normally linked to troponin I phosphorylation. MyBP-C and troponin I phosphorylation levels were the same as controls in transgenic mice and human carrier heart samples. ACTC E99K mice exhibited a high death rate between 28 and 45 days (48% females and 22% males). At 21 weeks, the hearts of the male survivors had enlarged atria, increased interstitial fibrosis, and sarcomere disarray. MRI showed hypertrophy, predominantly at the apex of the heart. End-diastolic volume and end-diastolic pressure were increased, and relaxation rates were reduced compared with nontransgenic littermates. End-systolic pressures and volumes were unaltered. ECG abnormalities were present, and the contractile response to β-adrenergic stimulation was much reduced. Older mice (29-week-old females and 38-week-old males) developed dilated cardiomyopathy with increased end-systolic volume and continuing increased end-diastolic pressure and slower contraction and relaxation rates. ECG showed atrial flutter and frequent atrial ectopic beats at rest in some ACTC E99K mice. We propose that the ACTC E99K mutation causes higher myofibrillar Ca2+ sensitivity that is responsible for the sudden cardiac death, apical hypertrophy, and subsequent development of heart failure in humans and mice.

Myofibrillar Ca2+-Sensitivity Is Uncoupled From Troponin I Phosphorylation In Hypertrophic Obstructive Cardiomyopathy Due To Abnormal Troponin T

AIMS We studied the relationship between myofilament Ca(2+) sensitivity and troponin I (TnI) phosphorylation by protein kinase A at serines 22/23 in human heart troponin isolated from donor hearts and from myectomy samples from patients with hypertrophic obstructive cardiomyopathy (HOCM). METHODS AND RESULTS We used a quantitative in vitro motility assay. With donor heart troponin, Ca(2+) sensitivity is two- to three-fold higher when TnI is unphosphorylated. In the myectomy samples from patients with HOCM, the mean level of TnI phosphorylation was low: 0.38 ± 0.19 mol Pi/mol TnI compared with 1.60 ± 0.19 mol Pi/mol TnI in donor hearts, but no difference in myofilament Ca(2+) sensitivity was observed. Thus, troponin regulation of thin filament Ca(2+) sensitivity is abnormal in HOCM hearts. HOCM troponin (0.29 mol Pi/mol TnI) was treated with protein kinase A to increase the level of phosphorylation to 1.56 mol Pi/mol TnI. No difference in EC(50) was found in thin filaments containing high and low TnI phosphorylation levels. This indicates that Ca(2+) sensitivity is uncoupled from TnI phosphorylation in HOCM heart troponin. Coupling could be restored by replacing endogenous troponin T (TnT) with the recombinant TnT T3 isoform. No difference in Ca(2+) sensitivity was observed if TnI was exchanged into HOCM heart troponin or if TnT was exchanged into the highly phosphorylated donor heart troponin. Comparison of donor and HOCM heart troponin by mass spectrometry and with adduct-specific antibodies did not show any differences in TnT isoform expression, phosphorylation or any post-translational modifications. CONCLUSION An abnormality in TnT is responsible for uncoupling myofibrillar Ca(2+) sensitivity from TnI phosphorylation in the septum of HOCM patients.

Quantifying the Release of Biomarkers of Myocardial Necrosis from Cardiac Myocytes and Intact Myocardium

BACKGROUND Myocardial infarction is diagnosed when biomarkers of cardiac necrosis exceed the 99th centile, although guidelines advocate even lower concentrations for early rule-out. We examined how many myocytes and how much myocardium these concentrations represent. We also examined if dietary troponin can confound the rule-out algorithm. METHODS Individual rat cardiac myocytes, rat myocardium, ovine myocardium, or human myocardium were spiked into 400-μL aliquots of human serum. Blood was drawn from a volunteer after ingestion of ovine myocardium. High-sensitivity assays were used to measure cardiac troponin T (cTnT; Roche, Elecsys), cTnI (Abbott, Architect), and cardiac myosin-binding protein C (cMyC; EMD Millipore, Erenna®). RESULTS The cMyC assay could only detect the human protein. For each rat cardiac myocyte added to 400 μL of human serum, cTnT and cTnI increased by 19.0 ng/L (95% CI, 16.8-21.2) and 18.9 ng/L (95% CI, 14.7-23.1), respectively. Under identical conditions cTnT, cTnI, and cMyC increased by 3.9 ng/L (95% CI, 3.6-4.3), 4.3 ng/L (95% CI, 3.8-4.7), and 41.0 ng/L (95% CI, 38.0-44.0) per μg of human myocardium. There was no detectable change in cTnI or cTnT concentration after ingestion of sufficient ovine myocardium to increase cTnT and cTnI to approximately 1 × 108 times their lower limits of quantification. CONCLUSIONS Based on pragmatic assumptions regarding cTn and cMyC release efficiency, circulating species, and volume of distribution, 99th centile concentrations may be exceeded by necrosis of 40 mg of myocardium. This volume is much too small to detect by noninvasive imaging.

Investigations into the Sarcomeric Protein and Ca2+-Regulation Abnormalities Underlying Hypertrophic Cardiomyopathy in Cats (Felix catus)

Hypertrophic cardiomyopathy (HCM) is the most common single gene inherited cardiomyopathy. In cats (Felix catus) HCM is even more prevalent and affects 16% of the outbred population and up to 26% in pedigree breeds such as Maine Coon and Ragdoll. Homozygous MYBPC3 mutations have been identified in these breeds but the mutations in other cats are unknown. At the clinical and physiological level feline HCM is closely analogous to human HCM but little is known about the primary causative mechanism. Most identified HCM causing mutations are in the genes coding for proteins of the sarcomere. We therefore investigated contractile and regulatory proteins in left ventricular tissue from 25 cats, 18 diagnosed with HCM, including a Ragdoll cat with a homozygous MYBPC3 R820W, and 7 non-HCM cats in comparison with human HCM (from septal myectomy) and donor heart tissue. Myofibrillar protein expression was normal except that we observed 20–44% MyBP-C haploinsufficiency in 5 of the HCM cats. Troponin extracted from 8 HCM and 5 non-HCM cat hearts was incorporated into thin filaments and studied by in vitro motility assay. All HCM cat hearts had a higher (2.06 ± 0.13 fold) Ca2+-sensitivity than non-HCM cats and, in all the HCM cats, Ca2+-sensitivity was not modulated by troponin I phosphorylation. We were able to restore modulation of Ca2+-sensitivity by replacing troponin T with wild-type protein or by adding 100 μM Epigallocatechin 3-gallate (EGCG). These fundamental regulatory characteristics closely mimic those seen in human HCM indicating a common molecular mechanism that is independent of the causative mutation. Thus, the HCM cat is a potentially useful large animal model.

Characterisation of the effects of mutation of the caldesmon sequence 691glu-trp-leu-thr-lys-thr696 to pro-gly-his-tyr-asn-asn on caldesmon-calmodulin interaction

We have investigated the functional properties of a mutant (Cg1) derived from the C‐terminal 99 amino acids of chicken caldesmon, 658–756 (658C) where the sequence 691glu‐trp‐leu‐thr‐lys‐thr696 is changed to pro‐gly‐his‐tyr‐asn‐asn. Cg1 bound Ca2+‐calmodulin with (1/7)th of the affinity as compared to 658C or whole caldesmon. NMR titrations indicate that the contacts of Ca2+‐calmodulin with the Trp‐722 region of the peptide are retained but that those at the mutated site are lost. Most importantly Ca2+‐calmodulin is not able to reverse the Cg1‐induced inhibition. We conclude that the interaction of calmodulin with this caldesmon sequence is crucial for the reversal of caldesmon inhibition of actin‐tropomyosin activation of myosin ATPase. The results are interpreted in terms of multi‐site attachment of actin and Ca2+‐calmodulin to overlapping sequences in caldesmon domain 4b.

A post-MI power struggle: adaptations in cardiac power occur at the sarcomere level alongside MyBP-C and RLC phosphorylation.

Compensation postchronic myocardial infarction (CMI) in rats is characterized in trabeculae as increased force and power production during physiological shortening, which occurs alongside classical hypertrophy. Sarcomeric contractile gain is influenced by mechanisms involving reduced myosin binding protein C (MyBP-C) and raised regulatory light chain (RLC) phosphorylation.

Study of O-Glcnacylation of Contractile Proteins in Cardiac Myofibrils by Enzymatic Labelling

Phosphorylation occurs on serine and threonine residues and plays important roles in the regulation of contractile proteins. In heart failure changes in levels of phosphorylation are reported in a number of cardiac sarcomeric proteins. O-linked-N-acetylglucosamine (O-GlcNAc) modification is another possible posttranslational modification on serine and threonine residues and recent publications reported mapping of O-GlcNAc modification sites in some rat contractile proteins, including myosin heavy chain (MHC), actin, cardiac troponin I and myosin light chains (MLC1 and MLC2). O-GlcNAc modification on normal donor hearts (49yr F and unknown) and hypertrophic obstructive cardiomyopathy myectomy samples (33yr M and 42yr M) were studied. Cardiac myofibrils were isolated and the O-GlcNAc groups labelled using an enzymatic labelling system in the presence of PUGNAc (inhibits O-GlcNAc removal enzyme O-GlcNAcase) and protease inhibitors. This method allows coupling of an azido modified N-acetylgalactosamine (UDP-GalNAz) to O-GlcNAc using the mutant enzyme Y289L β1,4-galactosyltransferase (Y289L GalT). The labelled groups were detected by reacting the azide group with an alkyne bearing the tetramethylrhodamine (TAMRA) fluorescent tag for direct imaging following SDS-PAGE. The gel was post-stained with a total protein stain for analysis with densitometry. The labelling process showed no impact on myofibril protein profiles when native and labelled myofibrils were compared. Preliminary results showed that O-GlcNAcylation profiles vary between samples with a total of 7 proteins identified. Strong TAMRA signals from α-actinin and MLC1 were observed in all the four myofibrils samples. In three of the samples the proteins actin, tropomyosin (Tm) and myosin binding protein-C (MyBP-C) were positively labelled. MHC and desmin O-GlcNAcylation were observed in one of the four subjects. This enzymatic labelling method will be investigated further for possibility of quantification and methods for mapping sites of these modifications with mass spectrometry will be explored.