Defining decreased protein succinylation of failing human cardiac myofibrils in ischemic cardiomyopathy
Introduction
Heart failure (HF) is a leading cause of mortality, affecting >6 million people in the United States and over 22 million people worldwide [1,2]. The incidence of HF in the United States is expected to rise by 46% by 2030 [3]. Survival rates for patients over 45 years old with HF have not improved since 1998, while many cancer survival rates have doubled over the last four decades [4,5]. The most important risk factor for HF is ischemic heart disease, which typically leads to ischemic cardiomyopathy (ICM) and HF [6]. Ischemic heart disease is the leading cause of death worldwide, accounting for approximately 18 million, or 31%, of all deaths annually [6]. ICM is the most common cause of HF, affecting >2.5 million people in the United States with an annual incidence of 40,000 cases per year and an annual mortality rate of 200,000 [6]. Therefore, revealing underlying biochemical mechanisms contributing to the onset and progression of HF remains a key step toward improving HF treatment and mortality. Known molecular abnormalities associated with HF include post-translational modification (PTM) of sarcomeric proteins, acidosis, reversion of protein isoforms to their fetal phenotype, changes in membrane protein expression, fibrosis, and mitochondrial impairments in oxidative phosphorylation, the TCA cycle, and fatty acid oxidation [7].
Protein PTMs have been shown to play an integral role in a wide variety of human diseases, including HF [8]. However, it is unknown whether these PTMs are a byproduct of HF or a contributing factor to the development of HF. PTMs can regulate protein function through their influence on protein activity, stability, and cellular localization [9,10]. PTMs are a critical event that can impair the pumping ability of failing hearts and can be caused by fluctuations in energy production and utilization [7,11]. They can also impact cardiac contractility by affecting both contractile proteins and calcium cycling [7]. It is believed that many membrane proteins contributing to excitation-contraction coupling are subject to modification, yet little to no information exists regarding the acylation status of these proteins in HF [[11], [12], [13], [14]].
Research on PTMs in HF has primarily focused on phosphorylation of proteins involved in cardiac muscle contraction, which have been shown to significantly alter both protein and cardiac function [[15], [16], [17], [18]]. In addition, evidence is building for the impact of SUMOylation and oxidation of cardiac proteins in HF [19,20]. However, few studies have explored the significance of lysine acylation in HF. Lysine residues are prone to a multitude of acylation events, including acetylation, malonylation, ubiquitination, carbonylation, and succinylation [9,[21], [22], [23], [24], [25], [26], [27], [28]]. However, because the heart is the organ with the highest concentration of succinyl CoA in the human body, succinylation is thought to function as a key regulatory PTM in the heart [10]. Mitochondrial protein succinylation involves the nonenzymatic, reversible modification of the N-ε-amine of lysine residues with a covalently bound succinyl group provided by succinyl CoA [29,30]. The dependence upon succinyl-CoA makes mitochondrial protein succinylation a general footprint of metabolic status [10,12,[31], [32], [33], [34]]. Lysine acylation is regulated, in part, by the activity of sirtuins, which remove acyl groups from lysine residues [35]. Sirtuins are a highly conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent deacylases with homology to the yeast Sir2 protein [35]. Sirtuin 5 (SIRT5) is the predominant regulator of lysine succinylation, malonylation, and glutarylation within the cytosol and mitochondria [36,37]. SIRT5 is known to play a role in various pathologies, including diabetes, cancer, neurodegeneration, fatty liver disease, and cardiovascular disease [32,[38], [39], [40], [41], [42], [43], [44], [45]]. In the heart, SIRT5 has been reported to regulate the TCA cycle, fatty acid metabolism, oxidative phosphorylation, pyruvate metabolism, ketogenesis, branched chain amino acid metabolism, and ATP synthesis [10,12]. Protein succinylation is known to alter protein activity; including increasing the activity of α-ketoglutarate dehydrogenase (α-KGDH), pyruvate dehydrogenase (PDH), fumarase, and succinate dehydrogenase (SDH) and decreasing the activity of 3-hydroxy-3-methylglutaryl-CoA synthetase (HMGC2) [12,[46], [47], [48]]. Initial studies have assessed the cardiac acetylome in whole heart tissue from animals under basal conditions and in models of diabetes, fasting, and obesity [13,49,50]. While it is known that acetylation and succinylation compete for occupancy on lysine residues of proteins, few studies have investigated the cardiac succinylome [[51], [52], [53]]. The heart has the highest concentration of cellular succinyl-CoA of any organ in the body, which implies that cardiomyocytes are highly dependent on the TCA cycle and branched chain amino acid (BCAA) catabolism for energy production [10,[54], [55], [56], [57]].
Previous studies on cardiac succinylation have all involved either whole heart tissue or left ventricular tissue from SIRT5 KO mice under basal conditions, chronic pressure overload, or ischemia-reperfusion injury [10,12,58]. No cardiac succinylation studies have investigated the myofibril-enriched protein fraction. In this study, we measure cardiac contractile and relaxation parameters of the sarcomeric proteins in myofibril-enriched proteins isolated from the left ventricles of explanted failing ICM human hearts and myofibril-enriched proteins from non-failing human hearts. Within this same myofibril-enriched protein fraction, we quantify both the differences in total protein expression and the succinylome for the first time. A recent study by Fisher-Wellman et al. has reported that protein hyperacylation in cardiac mitochondria has a marginal impact on bioenergetics [59]. While the relevance of acylation to ischemic HF pathology remains unknown, our study reveals significant hyposuccinylation in failing ICM myofibril samples collected from patients demonstrating severe pathology. The results contained herein demonstrate that ICM patients with HF present with hyposuccinylation of cardiac proteins involved in key metabolic and contractile processes.
Section snippets
Human samples
Heart samples were obtained from the Adult Cardiac Tissue Bank maintained by the Division of Cardiology at the University of Colorado Anschutz Medical Campus (COMIRB 01–568). All patients with HF had ischemic cardiomyopathy and hearts were collected during orthotopic heart transplantation. Hearts from non-failing donors were hearts that could not be placed for technical reasons such as size or mis-matched blood type. Cardiac tissue was rapidly dissected and flash-frozen in liquid nitrogen at
Patient characteristics
Patient characteristics are summarized in Table S1. Median age of the non-failing donors was 45.5 years and the median age of the patients with ischemic cardiomyopathy was 54.5 years. 50% of ischemic HF patients had ventricular assist devices (VADs) whereas no NF donors had VADs. Medications were more commonly used in ischemic HF patients than in NF donors.
Myofibril mechanical parameters are distinct between myofibrils isolated from hearts of ischemic cardiomyopathy patients and non-failing donor hearts
Several key parameters of sarcomeric mechanical interactions were altered in myofibrils isolated from failing ICM hearts (Fig. 1, Table S2).
Discussion
We investigated a critical and unique patient population composed of patients with heart failure due to ischemic cardiomyopathy and non-failing donors, and uncovered novel and noteworthy changes in PTMs associated with changes in myofibril mechanics. Our myofibril mechanics analysis of ischemic failing and non-failing hearts identified key differences in ischemic HF samples. Myofibrils from ischemic cardiomyopathy hearts have lower resting tension than myofibrils from non-failing controls.
Limitations
When assessing the field of proteomics and clinical samples it is vital to understand the source of the data generated. The process of preparing skinned cardiac myofibrils from left ventricular tissue clearly results in the isolation of both mitochondria and myofibrils, since cardiac mitochondria are dispersed among myofibrils [85]. Two distinct cardiac mitochondrial subpopulations have been identified in the hearts of many mammalian species including human hearts [86,87]. These mitochondrial
Declaration of Competing Interest
The authors have no conflict of interest to declare.
Acknowledgements
This study was supported, in part, by the Skaggs School of Pharmacy and Pharmaceutical Sciences ADR Grant program, University of Colorado Anschutz Medical Campus. T.A.M. received support from the NIH [HL147558, HL116848, HL127240 and DK119594] and American Heart Association [16SFRN31400013]. Y.H.L. was supported by a fellowship from the American Heart Association [16POST30960017]. KCW received support from the NIH (2K12HD057022) and Lorna G. Moore Award (University of Colorado Anschutz Medical
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