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

Abstract

Background: The mitochondrial unfolded protein response (UPR^mt) 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 UPR^mt in the heart is unclear.

Objectives: The purpose of this study was to: 1) identify conditions that activate the UPR^mt in the heart; and 2) study the relationship among the UPR^mt, 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 UPR^mt in cultured cardiomyocytes. The UPR^mt was also induced in the hearts of mice subjected to chronic hemodynamic overload. Boosting the UPR^mt 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 UPR^mt 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 UPR^mt in the mammalian (including human) heart exposed to pathological stresses. Enhancement of the UPR^mt ameliorates mitochondrial and contractile dysfunction, suggesting that it may serve an important protective role in the stressed heart.

Keywords: cardiomyocyte; heart; mitochondria; pressure overload; unfolded protein response.

Graphical abstract

Figure 1

Induction of UPR^mt Markers Under Various Stress Conditions in Cardiomyocytes and in an In Vivo Model of Chronic Pressure Overload (A and B) Response of cardiomyocytes to paraquat (100 or 500 μmol/l for 6, 24, and 48 h). (C and D) Response of cardiomyocytes to isoproterenol (1 or 100 μmol/l for 6, 24, and 48 h). (E) Response of cardiomyocytes to G-TPP (10 μmol/l for 4 and 8 h). *p < 0.05 versus respective control for changes in mRNA levels. #p < 0.05 versus 6-h treatment. ˆp < 0.05 versus 24-h treatment. (F) Effect of chronic pressure overload (TAC) on UPR^mt markers. *p < 0.05 versus sham-operated control mice or control conditions in NRVM. Dashed line denotes mRNA levels under control conditions. Data are mean ± SEM, n = 4 to 8/group. Atf5 = cyclic AMP-dependent transcription factor ATF-5; CHOP = CCAT-enhancer-binding protein homologous protein; ClpP = ATP-dependent Clp protease proteolytic subunit; G-TPP = gamitrinib-triphenylphosphonium; Hsp10 = Heat shock 10kDa protein 1; Hsp60 = Heat shock 60kDa protein 1; LonP1 = Lon protease homolog, mitochondrial; mtDNAj = mitochondrial pre-sequence translocase-associated motor complex protein; UPR^mt = mitochondrial unfolded protein response.

Figure 2

Enhancement of UPR^mt Prevents Stress-Induced Decrease in Mitochondrial Maximum Respiration Rate (Max Resp) (A to E) Cardiomyocytes treated with NR (1 mmol/l) demonstrated increased mRNA levels of UPR^mt markers (A). Representative traces (B) and averaged data (C) 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. *p < 0.05 versus untreated control represented by dashed line; #p < 0.05 versus 6-h NR treatment. Data are mean ± SEM, n = 5 to 7/group. Cntrl = control; Iso = isoproterenol; NR = nicotinamide riboside; other abbreviations as in Figure 1.

Figure 3

Enhancement of UPR^mt With NR Is Cardioprotective in Mice Subjected to Chronic Pressure Overload Mice were pre-treated with NR or vehicle for 3 days and subsequently subjected to TAC or sham-constriction. (A) Heart weight/body weight (HW:BW) ratio as a measure of cardiac hypertrophy. (B) Effect of NR on left ventricular ejection fraction (EF). (C) Representative M-mode echocardiography images. (D) Quantification of TUNEL⁺ cardiomyocytes in myocardial sections. (E) Changes in KDEL sequence-containing proteins assessed by immunoblotting. Data are mean ± SEM, n = 4 to 14/group; *p < 0.05. TAC = transverse aortic constriction; other abbreviations as in Figures 1 and 2.

Figure 4

Effect of Boosting the UPR^mt on Myocardial Mitochondrial Respiration in Mice Subjected to TAC or SHAM (A to D) Oxygen consumption rate (OCR) in ventricular muscle for Complex I, Complex II, Complex I + II, and Complex IV. Mice were pre-treated with NR or vehicle and then subjected to TAC or sham. Data are mean ± SEM, n = 4 to 6/group; *p < 0.05. NR = nicotinamide riboside; TAC = transverse aortic constriction.

Figure 5

UPR^mt Markers in Human Myocardium From AS Patients Undergoing Valve Replacement Surgery or Control Subjects (A) mRNA levels of UPR^mt markers in myocardium from aortic stenosis (AS) patients undergoing valve replacement surgery compared with control myocardium. (B) Patients were divided into 2 subgroups based on the mRNA levels of UPR^mt markers relative to the upper limit of normality (2× the SD of control measurements for each respective target). mRNA levels of ClpP, mtDNAj, Atf5, Hsp60, and CHOP are shown. Data in A and B are expressed as median ± 95th percentile, n = 5 to 15/group; *p < 0.05. (C to J) Comparison of clinical and histological characteristics between AS patients with UPR^mt markers in the normal range (subgroup A) and those with elevated UPR^mt markers (subgroup B). Left ventricular ejection fraction (EF) by echocardiography (C); maximal aortic transvalvular pressure gradient (D); serum levels of N-terminal pro–B-type natriuretic peptide (NT-proBNP) (E); serum levels of high-sensitivity troponin T (hsTnT) (F); extent of cardiomyocyte hypertrophy (G); TUNEL⁺-cardiomyocytes (H) and TUNEL⁺-noncardiomyocytes in myocardial sections (I); and myocardial collagen cross-linking (J). Data are mean ± SEM, n = 5 to 15/group. *p < 0.05. Abbreviations as in Figure 1.

Central Illustration

Mitochondrial Unfolded Protein Response Protects the Heart During Disease Stresses Diverse disease stresses induce the mitochondrial unfolded protein response (UPR^mt) in cardiomyocytes, which leads to an increased expression of mitochondrial chaperones and proteases (e.g., LonP1, ClpP, mtDNAj, Hsp10, Hsp 60). These enhance protein quality control and mitochondrial respiration, thereby enhancing contractile function and reducing myocyte cell death and fibrosis. Atf5 = cyclic AMP-dependent transcription factor ATF-5; ClpP = ATP-dependent Clp protease proteolytic subunit; FAD = oxidized flavin adenine dinucleotide; FADH = reduced flavin adenine dinucleotide; H = hydrogen; H₂O = water; Hsp10 = Heat shock 10kDa protein 1; Hsp60 = Heat shock 60kDa protein 1; LonP1 = Lon protease homolog, mitochondrial; mtDNAj = mitochondrial pre-sequence translocase-associated motor complex protein; NAD = oxidized nicotinamide adenine dinucleotide; NADH = reduced nicotinamide adenine dinucleotide; O₂ = oxygen.