Protein Turnover in the Failing Heart: An Ever-Changing Landscape

Despite years of research, we still have only limited understanding of the molecular basis of heart failure (HF).1 Our current therapies for HF with reduced ejection fraction (HFrEF), although reasonably successful in reducing morbidity and mortality, target compensatory mechanisms and have only limited effects on cardiomyocytes themselves. Particularly alarming, we have no therapies that improve outcomes in patients with HF with preserved ejection fraction (HFpEF). Each cardiomyocyte is meant to span an individual's entire life, but its constituent proteins and organelles are in constant flux. Every protein within the human heart is regenerated within 30 days, and the majority of amino acids used in new protein synthesis are recycled from prior protein breakdown through cellular degradation pathways (Figure 1).2, 3 This turnover helps to refresh key cellular contractile elements including actin and myosin, to eliminate proteins with normal ‘wear and tear’, and to remove misfolded proteins susceptible to aggregation.3, 4 This requires a steady energy supply and protein quality control systems that identify, target, and break down proteins and organelles in need of repair. The US Food and Drug Administration and many investigators in the HF field have recently urged a shift toward research and drug development efforts that primarily target specific structural abnormalities intrinsic to the failing cardiomyocyte, but doing so requires a molecular understanding of the HF syndrome.1 Studies utilizing transmission electron microscopy to examine cardiomyocytes in failing hearts have shown that a majority of cardiomyocytes are structurally viable.5 Nonetheless, these cardiomyocytes are dysfunctional. Several plausible hypotheses have been proposed to explain this discrepancy. The inability to contract or perform work in structurally viable cardiomyocytes may be attributable to: (i) insufficient energy in the form of ATP in that ATP is sufficient to sustain life but insufficient to allow for normal contraction and relaxation; (ii) existing abnormalities of calcium cycling, particularly in the sarcoplasmic reticulum, that alter the kinetics of calcium uptake and release, leading to dysfunction of contractile elements including actin and myosin; (iii) abnormalities of contractile protein turnover that lead to inappropriate synthesis and protein folding and/or inappropriate degradation causing dysfunctional contraction and relaxation; (iv) mitochondrial dysfunction involving the electron transport chain, formation of reactive oxygen species, errors in substrate utilization, and issues in ion homeostasis; and (v) stunned, hibernating myocardium attributable to chronic ischaemia.6-8 At the least, the first and third hypotheses are driven by abnormalities in protein turnover at the level of contractile elements and organelles, including the mitochondria.6, 8 Thus, understanding the specific role of protein turnover may provide insights into the genesis of the viable but dysfunctional cardiomyocyte and the mechanism of functional myocardial recovery.6, 9 A key cellular system for protein and organelle degradation is the lysosomal autophagy pathway, only recently illuminated by the Nobel Prize-winning work of Dr Yoshinori Ohsumi and his colleagues. Autophagy is defined as the breakdown of cytosolic components including whole organelles within vacuoles and lysosomes, first identified by electron microscopy by Dr C. de Duve in 1963.10 No progress was made in this field until it was discovered that autophagy is up-regulated in yeast during starvation, presumably as a result of a limited energy supply. Since then, much has been learned about the regulation of autophagy through the discovery of unique genes that, when mutated, lead to the accumulation of autophagy intermediates or so-called autophagy-related genes. This system is tightly regulated in order to avoid the indiscriminate destruction of cellular structures. Importantly, human analogues of yeast autophagy-related genes are consistently being discovered and implicated in a variety of diseases.10 The main non-lysosomal pathway of protein degradation is the ubiquitin–proteasome system (UPS). The system utilizes a barrel-shaped protein complex capable of degrading proteins into their individual amino acid constituents.11 The UPS is integral to protein quality control. It helps proteins maintain their intended conformation in hostile environments and targets those that have adapted a toxic structure for degradation.12, 13 The UPS is also involved in removing abnormally folded protein from the endoplasmic reticulum (ER) through targeting and export to the cytosol, a process known as ER-associated degradation. Cells that fail to break down these exported products undergo programmed cell death, known as ER stress.12, 14 Several enzymes are involved in this process, such as the E3 or ubiquitin protein ligase, which has over 1000 variations, leaving open the possibility of the existence of cardiac-specific E3 proteins. Additional pathways for protein degradation exist, including caspase proteases, matrix metalloproteinases (MMPs), and calpain proteases.15 Further, evidence suggests crosstalk amongst several of these pathways, such as inhibition of the UPS that increases the activity of the lysosomal autophagy pathway and vice versa, as well as calpain system activity that influences autophagy activity.16 As more light is shed on the complexity of protein turnover within the heart, it is likely that more examples will become evident. At a minimum, current evidence suggests links between the systems involved in protein and organelle degradation and the development of HF and myopathies in general. As suggested above, mitochondrial dysfunction is hypothesized to play a distinct role in the development of failing cardiomyocytes.6, 8, 17 As mitochondria become damaged, reactive oxygen species are generated that, if exposed to cytosolic proteins and/or organelles, may induce cell death.8, 17 This is particularly relevant in terminally differentiated cardiomyocytes, in which increased cell death may predispose to organ failure. Autophagy is the primary method by which damaged mitochondria are recycled, a process termed ‘mitophagy’. Although autophagy was initially considered to be a non-selective process, specific protein receptors on damaged mitochondria that play a role in targeting the organelle for recycling have been identified.17 Much remains to be discovered regarding this process, but intuitively it would seem that errors in removing damaged mitochondria would cause pathology within cardiac tissue and contribute to the development of HF.6, 8, 18 A key function of the left ventricle (LV) is the development of negative pressure in diastole, creating a suction-like effect to augment filling.19 Increased LV stiffness is thought to impair this process. LV stiffness can result from both intrinsic abnormalities within the cardiomyocyte itself and abnormalities in the interstitial compartment.20 Compliance of the extracellular matrix is influenced largely by the presence of type 1 collagen and the degree of cross-linking, regulated by MMPs and the tissue inhibitors of MMPs.21 In HF, increased deposition of collagen in the extracellular space, resulting from abnormalities in both collagen synthesis and degradation, gives rise to passive LV stiffness evident by increased resistance to LV filling during atrial contraction. Systemic co-morbidities create inflammatory states and further contribute to the progression of HF through microvascular endothelial dysfunction.20, 22, 23 These concepts apply to both HFrEF and HFpEF phenotypes. Titin, a component of the contractile apparatus, is the biggest contributor to intracellular myocyte stiffness, with the major isoforms N2B and N2BA existing in the cell and providing differing degrees of stiffness. The ratio of the two controls compliance and can be altered by differential splicing in transcription and post-translational modifications, as shown by Methawasin et al., who successfully inhibited the splicing factor RNA binding motif-20 to up-regulate compliant titins to decrease diastolic dysfunction and increase exercise tolerance.24 In a separate study, Screen et al. implicated the unfolded protein response, alterations in autophagy, ER stress, and increased amounts of ubiquinated proteins in the molecular mechanism that results in myocyte loss in tibial muscular dystrophy, an autosomal dominant myopathy related to genetic mutations in titin, begging the question of whether targeting these molecular mechanisms might improve myopathy in this disease.25 Impairment in the UPS has also been implicated in endothelial dysfunction that interferes with sarcomere structure and function in mouse models.26 Further, several groups have suggested that an imbalance in degradation to ubiquinated protein conjugates in failing cardiomyocytes plays a significant role in programmed cell death and eventual contractile dysfunction.27 Likewise, some studies have pointed to altered proteasome subunit composition within the cardiac proteasome as a potential therapeutic target.12 Other studies have suggested that proteasome inhibition may actually slow the progression of HF in pressure-overloaded patients.28, 29 Moreover, pathological calpain protease activation in the setting of calcium influx in cardiomyocytes leads to the degradation of the cytoskeleton, negatively influencing contraction.15 The calpain system has been implicated in worsening HF through a variety of mechanisms including the destruction of titin, and modification of the regulation of this family of proteases has been attempted to improve outcomes.15 Given that protein turnover is essential to all living cells in the human body, it is plausible that protein turnover can be implicated in the pathogenesis of HF and dysfunctional but viable cardiomyocytes. However, despite our improving knowledge of the processes that regulate protein turnover in normal and failing hearts, many questions remain unanswered. Can altering protein turnover improve outcomes in patients with HF? Can components of the protein degradation system be utilized as targets to reverse key cellular dysregulation, such as abnormal expression of titin isoforms and abnormal deposition of collagen type 1 in the interstitium? Do errors in the UPS contribute to HF in as yet undiscovered ways? Can new information on the workings of autophagy be harnessed to treat the failing myocardium? Might targeting a specific organelle through autophagy such as the mitochondria be of benefit? Can the calpain system be utilized as a weapon against excessive protein synthesis as opposed to its pathological role in cytoskeletal destruction? Can dysfunctional actin and myosin or other components of the contractile apparatus be targeted through these mechanisms? Might improving dysfunction in protein degradation resuscitate the cardiomyocyte, as suggested by Kunkel et al.?15 If a specific protein degradation system is activated or deactivated, how will crosstalk amongst the remaining degradation systems affect protein turnover in the rest of the cardiomyocyte? In many clinical circumstances, we still lack knowledge of the fundamental biological mechanism that explains why the heart fails, which mirrors our prior lack of understanding of the role of plaque rupture and thrombus formation in acute coronary syndrome. Answers to some of these questions may provide insight and are worthy of further investigation. Conflict of interest: S.J.G. is supported by the National Heart Lung and Blood Institute T32 post-doctoral training grant (5T32HL069749-14). M.G. has been a consultant for Abbott Laboratories, Astellas, AstraZeneca, Bayer HealthCare AG, CorThera, Cytokinetics, DebioPharm SA, Errekappa Terapeutici, GlaxoSmithKline, Ikaria, Johnson & Johnson, Medtronic, Merck, Novartis Pharma AG, Otsuka Pharmaceuticals, Palatin Technologies, Pericor Therapeutics, Protein Design Laboratories, Sanofi-Aventis, Sigma Tau, Solvay Pharmaceuticals, Takeda Pharmaceutical and Trevena Therapeutics. All other authors have no conflicts of interest to declare.