Cardioprotective Effect of the Mitochondrial Unfolded Protein Response During Chronic Pressure Overload

Background The mitochondrial unfolded protein response (UPRmt) is activated when misfolded proteins accumulate within mitochondria and leads to increased expression of mitochondrial chaperones and proteases to maintain protein quality and mitochondrial function. Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPRmt in the heart is unclear. Objectives The purpose of this study was to: 1) identify conditions that activate the UPRmt in the heart; and 2) study the relationship among the UPRmt, mitochondrial function, and cardiac contractile function. Methods Cultured cardiac myocytes were subjected to different stresses in vitro. Mice were subjected to chronic pressure overload. Tissues and blood biomarkers were studied in patients with aortic stenosis. Results Diverse neurohumoral or mitochondrial stresses transiently induced the UPRmt in cultured cardiomyocytes. The UPRmt was also induced in the hearts of mice subjected to chronic hemodynamic overload. Boosting the UPRmt with nicotinamide riboside (which augments NAD+ pools) in cardiomyocytes in vitro or hearts in vivo significantly mitigated the reductions in mitochondrial oxygen consumption induced by these stresses. In mice subjected to pressure overload, nicotinamide riboside reduced cardiomyocyte death and contractile dysfunction. Myocardial tissue from patients with aortic stenosis also showed evidence of UPRmt activation, which correlated with reduced tissue cardiomyocyte death and fibrosis and lower plasma levels of biomarkers of cardiac damage (high-sensitivity troponin T) and dysfunction (N-terminal pro–B-type natriuretic peptide). Conclusions These results identify the induction of the UPRmt in the mammalian (including human) heart exposed to pathological stresses. Enhancement of the UPRmt ameliorates mitochondrial and contractile dysfunction, suggesting that it may serve an important protective role in the stressed heart.

T he mammalian heart has the highest oxygen consumption rate of any organ, which may transiently increase several-fold during physiological exercise to support the increased cardiac workload. Chronic increases in cardiac workload resulting from hemodynamic overload or neurohumoral activation eventually lead to heart failure. Approximately 26 million patients worldwide are estimated to have heart failure, and the condition imposes substantial morbidity and mortality despite the use of therapies that target neurohumoral activation and cardiac rhythm disturbance (1).
Mitochondria are crucial for cardiac function through oxidative ATP generation to support muscle contraction and relaxation, the metabolism of nucleotides, amino acids and lipids, intracellular calcium buffering, and the regulation of cardiomyocyte survival. Mitochondrial dysfunction is a central feature of heart failure by contributing to energetic dysfunction, oxidative stress, calcium dysregulation, and cardiomyocyte death, and is considered a potential therapeutic target (2). The vast majority of the The mitochondrial unfolded protein response (UPR mt ) is evolutionarily conserved and evoked when the mitochondrial protein folding environment is compromised and there is an accumulation of misfolded proteins (4). The UPR mt is a mitochondrial-tonuclear signal transduction pathway that leads to increased transcription of numerous mitochondrial protective genes, notably a repertoire of molecular chaperones, proteases, and antioxidant enzymes located primarily in the mitochondrial matrix (5).
A P R I L 1 6 , 2 0 1 9 : 1 7 9 5 -8 0 6 The UPR mt in Cardiac Stress showing that enhancement of UPR mt with NR ameliorated the isoproterenol-induced decrease in maximum respiration rate (Max Resp). Representative traces (D) and averaged data (E) showing that the amelioration of isoproterenol-induced decrease in maximum respiration rate by NR is prevented in the setting of Atf5 knockdown.
Smyrnias et al. The UPR mt in Cardiac Stress  Smyrnias et al.
A P R I L 1 6 , 2 0 1 9 : 1 7 9 5 -8 0 6 The UPR mt in Cardiac Stress range to that reported previously in other systems (10,13) (Online Figure 1A). whereas stimulation with tunicamycin, which induces ER stress, robustly increased the levels of these proteins (Online Figure 1). mRNA levels of cytosolic Hsp70 and Hsp90 were also largely unaltered by the UPR mt -inducing stress stimuli (Online Figure 1).   Smyrnias et al.
Atf5 is reported to be a key transcriptional inducer of the mammalian UPR mt (13,17). Indeed, silencing of Atf5 in cardiomyocytes abolished the Iso-induced increase in UPR mt markers (Online Figure 3A). To confirm that the effects of NR are mediated at least in part through activation of the UPR mt , we silenced Atf5 and then examined the effects of NR on UPR mt markers. The silencing of Atf5 substantially inhibited the NR-mediated increase in UPR mt markers (Online Figure 3B) and abolished the NR-mediated prevention of Iso-induced decrease in mitochondrial respiration ( Figures 2D and 2E), strongly supporting the idea that NR is protective by boosting the UPR mt .
We compared a range of parameters of clinical AS severity, hemodynamic state, cardiac structure and function, myocardial histology, and blood biomarkers that were assessed pre-operatively between the 2 subgroups. There was no significant difference between subgroups in age, sex, comorbidities, systemic blood pressure or AS severity, as assessed by the mean transvalvular pressure gradient at echocardiography (Online Table 3 Smyrnias et al.

Furthermore, patients in subgroup B had significantly lower serum levels of both hs-TnT and N-terminal
pro-B-type natriuretic peptide ( Figure 5).
Taken together, these data demonstrate that the UPR mt is induced in the human heart subjected to chronic pathological pressure overload and that a high level of activation is associated with reduced cardiomyocyte cell death, which may consequently limit pathological fibrosis and overall pathological cardiac stress.

DISCUSSION
Numerous cellular processes depend upon normal mitochondrial function, which is required for cellular metabolism, energy generation, and the regulation of cell death. The mitochondrial proteome may be markedly affected by stress conditions that alter the protein-folding environment (3,4). Wellcharacterized, evolutionarily conserved stress responses are evoked in several cellular compartments in response to stresses that disrupt protein integrity, for example, the ER stress response (18), the nucleolar stress response (19), and the cytosolic heat shock response (20). These responses typically lead to an increased expression of proteins that restore organelle homeostasis. The UPR mt was relatively recently identified and serves an analogous function in the mitochondria. While it has been comprehensively characterized in C. elegans (6,7) and identified in other organisms such as yeast (8)  and/or CHOP (10,13). A similar activation of the cardiac UPR mt was observed after the imposition of in vivo hemodynamic overload in the mouse. The level of increase in mRNA levels of UPR mt markers was in a similar dynamic range to that reported after genetic UPR mt induction in C. elegans (21). There were some variations between stressors in the genes that were up-regulated among Atf5, CHOP, mtDNAj, ClpP, LonP1, Hsp10, and Hsp60, which may reflect differences in the precise regulation of individual genes (22). Atf5, similarly to ATFS-1, which mediates the UPR mt in C. elegans (6), is a bZIP transcription factor that shuttles from the mitochondria to the nucleus during mitochondrial stress and has been reported as 1 of the key mediators of the mammalian UPR mt (10,13).
Indeed, we show that silencing of Atf5 in cardiomyocytes blunts the Iso-induced increase in UPR mt markers. CHOP, on the other hand, is best known to be associated with the ER stress response (18), but several studies (including the current study) found a specific involvement of CHOP in the UPR mt independent of ER stress (5,10,23). We found that the activation of the UPR mt occurred distinct from ER stress or the cytosolic heat shock response as evidenced by the lack of corresponding changes in ER stress markers or cytosolic heat shock proteins (Online Figure 1)  The UPR mt in Cardiac Stress A P R I L 1 6 , 2 0  (25). These data identify for the first time an activation of the UPR mt in the pressure-overloaded human heart and suggest that increased activation of the UPR mt may be beneficial.
Activation of the ER stress response has been demonstrated to be an important pathophysiological mechanism in many human diseases (18) and small molecules that modulate this response are under investigation as potential therapeutic agents (26).
Here, we show that the augmentation of a different stress response, the UPR mt , may also have beneficial effects. It should be noted, however, that prolonged or dysregulated induction of the UPR mt could be detrimental, for example, contributing to an accumulation of defective mitochondria (27)   Smyrnias et al.