Temperature-sensitive sarcomeric protein post-translational modifications revealed by top-down proteomics

Abstract

Despite advancements in symptom management for heart failure (HF), this devastating clinical syndrome remains the leading cause of death in the developed world. Studies using animal models have greatly advanced our understanding of the molecular mechanisms underlying HF; however, differences in cardiac physiology and the manifestation of HF between animals, particularly rodents, and humans necessitates the direct interrogation of human heart tissue samples. Nevertheless, an ever-present concern when examining human heart tissue samples is the potential for artefactual changes related to temperature changes during tissue shipment or sample processing. Herein, we examined the effects of temperature on the post-translational modifications (PTMs) of sarcomeric proteins, the proteins responsible for muscle contraction, under conditions mimicking those that might occur during tissue shipment or sample processing. Using a powerful top-down proteomics method, we found that sarcomeric protein PTMs were differentially affected by temperature. Specifically, cardiac troponin I and enigma homolog isoform 2 showed robust increases in phosphorylation when tissue was incubated at either 4 °C or 22 °C. The observed increase is likely due to increased cyclic AMP levels and activation of protein kinase A in the tissue. On the contrary, cardiac troponin T and myosin regulatory light chain phosphorylation decreased when tissue was incubated at 4 °C or 22 °C. Furthermore, significant protein degradation was also observed after incubation at 4 °C or 22 °C. Overall, these results indicate that temperature exerts various effects on sarcomeric protein PTMs and careful tissue handling is critical for studies involving human heart samples. Moreover, these findings highlight the power of top-down proteomics for examining the integrity of cardiac tissue samples.

Donor hearts in the Sydney Heart Bank: reliable control but is it ‘normal’ heart?

“…RNA and protein degradation is an obvious way to detect a 'bad' sample. A particularly sensitive test is the level of protein phosphorylation, since, counter-intuitively, if tissue is not frozen soon enough or thaws out later, the level of TnI phosphorylation increases (Cai et al 2018).…”

Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics

“…LV myocardium from nonfailing hearts from brain-dead donors with no history of heart diseases but unsuitable for heart transplant were used as control tissues in this study (Ctrl, n = 16). The donor hearts LV tissues were obtained either from the University of Wisconsin Organ and Tissue Donation ( 67 – 69 ) or from the Sydney Heart Bank. Interventricular septal myocardium from patients with obstructive HCM undergoing septal myectomy surgery ( 30 ) for relief of symptoms was analyzed in this study (HCM, n = 16).…”

“…Sarcomeric proteins were extracted as previously reported, with minor modifications ( 67 ). To minimize artificial protein modifications and oxidation, tissue homogenization was performed at 4 °C.…”

“…LC-MS analysis was carried out using a NanoAcquity ultra-high pressure LC system (Waters) coupled to a high-resolution Impact II quadrupole time-of-flight mass spectrometer (Bruker Daltonics) ( 67 ). Five hundred nanograms of total sarcomeric protein (per sample) were separated using a home-packed PLRP column (PLRP-S, 1,000 Å pore size, 10-µm particle size, 500-µm inner diameter) using an organic gradient of 5 to 95% mobile phase B (mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in 50:50 acetonitrile:ethanol) at a constant flow rate of 14 µL/min.…”

Mapping the Proteoform Landscape of Five Human Tissues

“…Prior reports by the Ge group have established N-terminal trimethylation of RLCV and phosphorylation of swine RLCV, but phosphorylation of human RLCV was unlocalized and observed at <10% stoichiometry. 58,59 The removal of N-terminal methionine and trimethylation was confirmed by tandem HCD fragmentation, and the site of phosphorylation was localized to S15, which is analogous to the site identified on swine RLCV (Figure 5B). On a last analytical note, phosproteoforms of cardiac troponin I (cTnI) 60 were not separated by RPLC but were at baseline by CZE (Figure 5C); proteoform quantitation by both techniques showed <10% coefficient of variation between them.…”