Post-translational modification patterns on β-myosin heavy chain are altered in ischemic and nonischemic human hearts
- Department of Biomedical Sciences, College of Medicine, The Florida State University, United States
- Department of Bioengineering, College of Medicine, University of Washington, United States
- Department of Nutrition and Integrative Physiology, The Florida State University, United States
- Translational Science Laboratory, College of Medicine, The Florida State University, United States
- Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, United States
- Department of Surgery, College of Medicine, The Ohio State University, United States
- Department of Biological Science, The Florida State University, United States
Figures
Figure 1 with 1 supplement
Illustrative schematic of the integrative approaches used to identify novel post-translational modifications (PTMs) on human β-myosin heavy chain (β-MHC) and investigate their roles in cardiac muscle regulation.
De-identified human heart samples were obtained from nonfailing, ischemic heart failure, and nonischemic heart failure patients. The presence of PTMs on human β-MHC was confirmed by liquid …
Figure 1—figure supplement 1
Coomassie-stained gel of homogenized human heart tissues.
The red box indicates the band corresponding to β-myosin heavy chain (β-MHC) in the cardiac human samples analyzed here at the predicted molecular weight of ~223 kDa. The lanes are BioRad Dual …
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Figure 1—figure supplement 1—source data 1
Source data for Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/74919/elife-74919-fig1-figsupp1-data1-v2.pdf
Figure 2
Detailed mass spectrometry (MS)/MS spectra of acetylated human β-myosin heavy chain (β-MHC) peptide sequences.
MS/MS spectra of trypsin-digested, acetylated β-MHC peptide sequences 24–35 (K34-Ac, m/z 496.61), 55–67 (K58-Ac, m/z 705.84), 414–434 (K429-Ac, m/z 774.41), 942–952 (K951-Ac, m/z 483.57), and …
Figure 3
Detailed mass spectrometry (MS/MS) spectra of phosphorylated human β-myosin heavy chain (β-MHC) peptide sequences.
MS/MS specta of trypsin-digested, phosphorylated β-MHC peptide sequences 207–234 (S210-P, m/z 1007.49) and 208–234 (T215-P, m/z 964.80). b-ions and y-ions indicate N-terminal and C-terminal ions, …
Figure 4 with 2 supplements
Structural models of post-translational modifications (PTMs) on β-myosin heavy chain (β-MHC).
X-ray crystal structures of the β-MHC. In (A) a post-rigor X-ray structure was used to model K58-Ac, K213-Ac, T215-P PTMs. The four motor subdomains – the N-terminal domain (yellow), upper 50 kDa …
Figure 4—figure supplement 1
Detailed liquid chromatography-mass spectrometry (MS) spectrum of the common internal peptide sequence (IRP).
MS/MS spectrum of trypsin-digested β-myosin heavy chain (β-MHC) peptide sequence (1504–1521, m/z 974.49). b-ions and y-ions indicate N-terminal and C-terminal ions, respectively.
Figure 4—figure supplement 2
Key functional regions of cardiac β-myosin motor domain and locations of the identified post-translational modifications (PTMs).
The locations of PTMs and functional regions are annotated on a schematic of the β-myosin heavy chain (β-MHC) sequence. PTM sites are denoted with pink lines, and significant structural elements are …
Figure 5 with 1 supplement
Calculations of post-translational modification (PTM) occupancy of acetylated residues on β-myosin heavy chain (β-MHC).
Peak areas of all modified peptide sequences (MOD) were normalized to the peak area of a common internal reference peptide sequence (IRP, 1504–1521). (A) Peptide 1 sequence is shown with the site of …
Figure 5—figure supplement 1
Mass spectrometry (MS/MS) spectrum of the doubly modified peptide sequence.
The K213-Ac/T215-P-modified sequence (207–234, m/z 766.36) presented an isotope dot product (idotp) value below 0.5, which indicates that the modified peptide was detectable but not quantifiable. …
Figure 6
Calculations of post-translational modification (PTM) occupancy of phosphorylated residues on β-myosin heavy chain (β-MHC).
Peak areas of all modified peptide sequences (MOD) were normalized to the peak area of a common internal reference peptide sequence (IRP, 1504–1521). (A) Peptide 1 sequence is shown with …
Figure 7
K951-Ac increased flexibility of the myosin tail.
Structural changes in the S2 fragment caused by K951-Ac are shown in representative snapshots from molecular dynamics (MD) simulations. In the snapshots, the ribbon of residue 951 is colored red, …
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Figure 7—source data 1
Source data for Figure 7.
- https://cdn.elifesciences.org/articles/74919/elife-74919-fig7-data1-v2.xlsx
Figure 8
K58-Ac altered the solvent-exposed surface of myosin’s SH3-like domain.
(A) Representative structural changes in the SH3 domain associated with K58-Ac are shown in the endpoint structures of one unmodified (‘K58’) and two modified (‘K58-Ac’) simulations. K58-Ac formed …
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Figure 8—source data 1
Source data for Figure 8.
- https://cdn.elifesciences.org/articles/74919/elife-74919-fig8-data1-v2.xlsx
Figure 9
K58-Ac/T215-P altered loop 1 structure and dynamics.
Molecular dynamics (MD) snapshots in (A) show representative structures from the unmodified (K213/T215) and modified (K213-Ac/T215-P) simulations. In the unmodified simulations, the loop makes …
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Figure 9—source data 1
Source data for Figure 9 (contacts).
- https://cdn.elifesciences.org/articles/74919/elife-74919-fig9-data1-v2.xlsx
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Figure 9—source data 2
Source data for Figure 9 (distances).
- https://cdn.elifesciences.org/articles/74919/elife-74919-fig9-data2-v2.xlsx
Tables
Table 1
Liquid chromatography-mass spectrometry (LC-MS/MS) analysis of trypsin-digested human β-myosin heavy chain (β-MHC) sequences containing acetylated residues.
*An isotope dot product (idotp) value below 0.5 denotes a detectable but not quantifiable peptide sequence. C = carbamidomethyl. Acetylated residues are highlighted in red while phosphorylated …
| Protein | Peptide sequence | Protease | Modified residues | Modification | Calculated molecular mmass | Observed m/z | Observed molecular mass | Error (ppm) | Charge | . idotp |
|---|---|---|---|---|---|---|---|---|---|---|
| β-MHC | 23(R)LEAQTRPFDLKK(D)36 | Trypsin | K34 | Acetylation (+42) | 1487.82 | 496.61 | 1486.82 | 1.3 | 3+ | 0.99 |
| 54(R)EGGKVTAETEYGK(T)68 | Trypsin | K58 | Acetylation (+42) | 1410.67 | 705.84 | 1409.67 | 3.2 | 2+ | 0.91 | |
| 206(K)DQSPGKGTLEDQIIQANPALEAFGNAK(T)235 | Trypsin | K213/T215 | Acetylation (+42) Phosphorylation (+80) | 3061.48 | 766.37 | 3061.48 | 0.0 | 4+ | 0.48* | |
| 413(K)GQNVQQVIYATGALAKAVYER(M)435 | Trypsin | K429 | Acetylation (+42) | 2321.22 | 774.41 | 2320.21 | –3.6 | 3+ | 0.93 | |
| 941(R)KLEDECSELKR(D)953 | Trypsin | K951 | Carbamidomethyl (+57) Acetylation (+42) | 1448.70 | 483.57 | 1447.69 | 0.57 | 3+ | 0.70 | |
| 1194(K)KHADSVAELGEQIDNLQR(V)1213 | Trypsin | K1195 | Acetylation (+42) | 2065.03 | 689.01 | 2064.01 | –7.4 | 3+ | 0.95 |
Table 2
Liquid chromatography-mass spectrometry (LC-MS/MS) analysis of trypsin-digested human β-myosin heavy chain (β-MHC) sequences containing phosphorylated residues.
*An isotope dot product (idotp) value below 0.5 denotes a detectable but not quantifiable peptide sequence. C = carbamidomethyl. Acetylated residues are highlighted in red while phosphorylated …
| Protein | Peptide sequence | Protease | Modified residues | Modification | Calculated molecular mass | Observed m/z | Observed molecular mass | Error (ppm) | Charge | idotp |
|---|---|---|---|---|---|---|---|---|---|---|
| β-MHC | 206(K)KDQSPGKGTLEDQIIQANPALEAFGNAK(T)235 | Trypsin | S210 | Phosphorylation (+80) | 3020.47 | 1007.49 | 3019.46 | –0.96 | 3+ | 0.99 |
| 206(K)KDQSPGKGTLEDQIIQANPALEAFGNAK(T)235 | Trypsin | K213/T215 | Acetylation (+42) Phosphorylation (+80) | 3061.48 | 766.37 | 3061.48 | 0.0 | 4+ | 0.48* | |
| 207(K)DQSPGKGTLEDQIIQANPALEAFGNAK(T)235 | Trypsin | T215 | Phosphorylation (+80) | 2892.37 | 964.80 | 2891.37 | –0.79 | 3+ | 0.99 |
Table 3
Inventory of β-myosin heavy chain (β-MHC) post-translational modification (PTM) simulations.
Each row corresponds to a simulated system and reports the modifications that were made and the extent of molecular dynamics (MD) sampling.
| ID | PDB | Condition | Runs | Length per run(ns) | Net sampling (ns) |
|---|---|---|---|---|---|
| 4DB1 unmodified | 4DB1 | Unmodified | 3 | 500 | 1500 |
| 4DB1 K58-Ac | 4DB1 | K58Ac | 3 | 500 | 1500 |
| 4DB1 K213-Ac/T215-P | 4DB1 | K213Ac, T215P | 3 | 500 | 1500 |
| 2FXM K951 | 2FXM | Unmodified | 3 | 500 | 1500 |
| 2FXM K951-Ac | 2FXM | K951Ac | 3 | 500 | 1500 |
Table 4
Average Cα root mean squared deviation (RMSD) values.
| ID | Run number | Cα RMSD (Å) |
|---|---|---|
| 4DB1 unmodified | 1 | 3.9 ± 0.3 |
| 2 | 3.2 ± 0.3 | |
| 3 | 4.1 ± 0.3 | |
| 4DB1 K58-Ac | 1 | 3.8 ± 0.3 |
| 2 | 3.6 ± 0.2 | |
| 3 | 4.1 ± 0.5 | |
| 4DB1 K213-Ac/T215-P | 1 | 3.5 ± 0.4 |
| 2 | 3.6 ± 0.4 | |
| 3 | 3.6 ± 0.3 | |
| 2FXM unmodified | 1 | 6.9 ± 2.4 |
| 2 | 7.5 ± 2.3 | |
| 3 | 7.2 ± 2.4 | |
| 2FMX K951-Ac | 1 | 10.8 ± 2.8 |
| 2 | 9.2 ± 2.5 | |
| 3 | 10.1 ± 2.0 |
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Table 4—source data 1
Source data for Table 4.
- https://cdn.elifesciences.org/articles/74919/elife-74919-table4-data1-v2.xlsx
Author response table 1
| K34 | WT | ISC HF | NON-ISC HF | IRP | WT | ISC HF | NON-ISC HF | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 171243328 | 37933984 | 123456288 | 37951213568 | 35468374016 | 36720361472 | WT | ISC HF | NON-ISC HF | |||
| 304486400 | 474435776 | 254883488 | 30835193856 | 24838664192 | 28284686336 | 4.5121964 | 10.486356 | 3.36206625 | |||
| 137056288 | 474435776 | 254883488 | 33189758776 | 22171635712 | 32563331072 | 9.8746387 | 19.100696 | 9.011359892 | |||
| 86855160 | 136785104 | 100508704 | 30544549888 | 38021373952 | 37236039680 | 4.1294752 | 3.2725629 | 1.374162978 | |||
| 2.8435567 | 3.5975845 | 2.699231843 | |||||||||
| AVG | 5.33997 | 9.1143 | 4.111705241 | ||||||||
| K58 | WT | ISC HF | NON-ISC HF | IRP | WT | ISC HF | NON-ISC HF | ||||
| 356371104 | 95448224 | 426329504 | 37951213568 | 35468374016 | 36720361472 | WT | ISC HF | NON-ISC HF | |||
| 414622496 | 58387644 | 338617856 | 30835193856 | 24838664192 | 28284686336 | 9.3902426 | 2.6910798 | 11.61016632 | |||
| 87149368 | 56710632 | 8662002 | 33189758976 | 22171635712 | 32563331072 | 13.446405 | 2.3506757 | 0.233004789 | |||
| 80678968 | 142589216 | 71101432 | 30544549888 | 38021373952 | 37236039680 | 2.6257909 | 2.5582069 | 0.266004789 | |||
| 2.641354 | 3.7502384 | 1.909478898 | |||||||||
| AVG | 7.02595 | 2.83755 | 6.439355856 | ||||||||
| S210 | WT | ISC HF | NON-ISC HF | IRP | WT | ISC HF | NON-ISC HF | ||||
| 37987736 | 30292644 | 32893104 | 37951213568 | 35468374016 | 36720361472 | WT | ISC HF | NON-ISC HF | |||
| 149246768 | 29204212 | 38378992 | 30835193856 | 24838664192 | 29284686336 | 1.0009624 | 0.8540748 | 0.895772881 | |||
| 35820280 | 27507396 | 54357632 | 33189758976 | 22171635712 | 32563331072 | 4.8401437 | 1.1757561 | 1.356882362 | |||
| 25008872 | 18092996 | 45057196 | 30544549888 | 38021373952 | 37236039680 | 1.079257 | 1.240657 | 1.669289664 | |||
| 0.8187671 | 0.4758638 | 1.210004265 | |||||||||
| AVG | 1.93478 | 0.93659 | 1.282006889 | ||||||||
| Y215 | WT | ISC HF | NON-ISC HF | IRP | WT | ISC HF | NON-ISC HF | ||||
| 58306360 | 60397624 | 63556984 | 37951213568 | 35468374016 | 36720361472 | WT | ISC HF | NON-ISC HF | |||
| 214473888 | 43148672 | 60914896 | 30835193856 | 24838664192 | 28284686336 | 1.5363503 | 1.7028586 | 1.730837646 | |||
| 40785388 | 87614824 | 33189758976 | 22171635712 | 32563331072 | 6.9554902 | 1.7371575 | 2.153635196 | ||||
| 1100631.5 | 50358960 | 73574128 | 30544549888 | 38021373952 | 37226039680 | 1.83953 | 2.69059771 | ||||
| 0.0360336 | 1.3244908 | 1.975884886 | |||||||||
| AVG | 2.84262 | 1.65101 | 2.137738859 | ||||||||
| K429 | WT | ISC HF | NON-ISC HF | IRP | WT | ISC HF | NON-ISC HF | ||||
| 38319744 | 66542720 | 34278128 | 37951213568 | 35468374016 | 36720361472 | WT | ISC HF | NON-ISC HF | |||
| 39962528 | 31968566 | 25058588 | 30835193856 | 24828664192 | 28284686336 | 1.0097106 | 1.8761142 | 0.933491029 | |||
| 30577718 | 29568352 | 33888364 | 33189758976 | 22171635712 | 32563331072 | 1.2960038 | 1.2870485 | 0.885941873 | |||
| 26531748 | 32985240 | 30807246 | 30544549888 | 38021373952 | 37236039680 | 0.9212998 | 1.3336117 | 1.040690952 | |||
| 0.8686246 | 0.8675447 | 0.82735023 | |||||||||
| AVG | 1.02391 | 1.34108 | 0.921868521 | ||||||||
| K951 | WT | ISC HF | NON-ISC HF | IRP | WT | ISC HF | NON-ISC HF | ||||
| 554000765 | 156454688 | 274431584 | 37951213568 | 35468374016 | 36720361472 | WT | ISC HF | NON-ISC HF | |||
| 431275680 | 104078096 | 254465008 | 30835193856 | 24838664192 | 28284686336 | 14.597709 | 4.411104 | 7.473555345 | |||
| 594530432 | 143491840 | 150870720 | 33189758976 | 22171635712 | 32563331072 | 13.986475 | 4.1901648 | 8.996564607 | |||
| 334659520 | 210390240 | 44803980 | 3.05446E+11 | 38021373952 | 37236039680 | 17.913069 | 6.4718653 | 4.633147624 | |||
| 10.95644 | 5.5334728 | 1.203242353 | |||||||||
| AVG | 14.3634 | 5.15165 | 5.576627007 | ||||||||
| K1195 | WT | ISC HF | NON-ISC HF | IRP | WT | ISC HF | NON-ISC HF | ||||
| 77233384 | 30782836 | 20483700 | 37951213568 | 35468374016 | 36720361472 | WT | ISC HF | NON-ISC HF | |||
| 90984128 | 70924552 | 59125832 | 30835193856 | 24838664192 | 28284686336 | 2.0350702 | 0.8678953 | 0.557829476 | |||
| 44304144 | 16460342 | 29519324 | 33189758976 | 22171635712 | 32563331072 | 2.9506585 | 2.8554093 | 2.09038316 | |||
| 64847704 | 11629701 | 423826652 | 30544569888 | 38021373952 | 37236039680 | 1.3348739 | 0.7424054 | 0.906520403 | |||
| 2.1230532 | 0.3058727 | 1.138215889 | |||||||||
| AVG | 2.11091 | 1.1929 | 1.173237232 |
Additional files
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Supplementary file 1
Summary of the patients’ demographic features.
Deidentified human heart samples were obtained from nonfailing, ischemic heart failure, and nonischemic heart failure patients.
- https://cdn.elifesciences.org/articles/74919/elife-74919-supp1-v2.docx
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Supplementary file 2
Comparison of location of residues bearing post-translational modifications (PTMs) with known cardiomyopathy variants in MYH7.
List of potential pathogenicity of the variants and their location within nearby PTM regions. Cardiomyopathy-loop (CM-loop), likely pathogenic (LP), pathogenic (P), hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM).
- https://cdn.elifesciences.org/articles/74919/elife-74919-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/74919/elife-74919-transrepform1-v2.pdf
