Mitochondrial dysfunction elicits a mitochondrial stress response (MSR) through mitochondrial-nuclear communication and activates ATF4, a master transcriptional regulator of the cellular stress response1. Knowledge of consequences of MSR-triggered ATF4 activation in mitochondrial cardiomyopathy is limited, yet critical for therapeutic approaches.
Mitochondrial phosphatase Ptpmt1 cKO mice (PKO)2 provide a model to investigate in vivo mechanisms of MSR in cardiomyocytes, displaying key features of fetal mitochondrial cardiomyopathy, while evidencing lethality between E16.5–18.52. All mouse protocols were approved by the Institutional Animal Care and Use Committee. Molecular analyses confirmed that loss of PTPMT1 in cardiomyocytes resulted in upregulation of ATF4 and its target genes at E11.5 (Figure A–D), similar to other MSR models. The most well recognized upstream regulator of ATF4 is eIF2α phosphorylation, which increases ATF4 translation. Atf4 is also a direct target of transcriptional repression by HIF1α in cardiomyocytes. Hif1α cKO hearts display increased Atf4 mRNA and protein. mTOR is another upstream regulator of ATF4. We examined these upstream regulators of ATF4 in PKO hearts, and found that phosphorylation of eIF2α was significantly increased in PKO versus control hearts at E11.5, while HIF1α and mTOR phosphorylation were not altered (Figure E), suggesting that eIF2α phosphorylation induced expression of ATF4.
Phosphorylation of eIF2α at serine 51 integrates signals from diverse cellular stress responses (Figure F)1. We generated a “phosphorylation-resistant” eIF2α mutant allele1, in which the serine 51 phosphorylation site was mutated to alanine (eIF2αS51A), and crossed this allele into PKO mice to generate PKO/eIF2αS51A double mutant mice (dMut). Absence of eIF2α phosphorylation in eIF2αS51A and dMut hearts was validated (Figure G). ATF4 protein and downstream targets were significantly decreased to baseline levels in dMut hearts (Figure G–I), confirming that eIF2α phosphorylation was essential for MSR-triggered ATF4 activation in PKO hearts.
Four eIF2α kinases are activated by distinct forms of stress (Figure F)1. RNA-seq analysis from PKOs indicated increased expression of ATF4 targets, but no increase in XBP1- or ATF6 (Figure J), the latter negating the likelihood that a PERK-mediated stress response was involved1. Next, we generated null alleles for GCN2 or HRI, and generated PKO/GCN2 and PKO/HRI double knockout mice (dKO). Loss of HRI abolished activation of eIF2α-ATF4 signaling in PKOs, while eIF2α-ATF4 remained activated in PKO/GCN2 dKO hearts (Figure K–O). Thus, MSR induced eIF2α-ATF4 signaling was dependent on HRI, while other eIF2α kinases were not involved. Our results are consistent with recent in vitro findings in HeLa cells3,4, providing the first demonstration of the in vivo relevance of this pathway in mitochondrial cardiomyopathy. It is not clear whether activation of HRI depends on the amount of heme in cardiomyocytes.
Whether the eIF2α pathway was adaptive or maladaptive remained to be addressed. Although global suppression of protein synthesis by eIF2α phosphorylation conserves energy, and increasing ATF4 translation allows cells to survive periods of stress, persistent ATF4 and suppression of protein synthesis may also be detrimental. Homozygous eIF2αS51A mutants die within 18 hours after birth1. However, constitutive activation of eIF2α by deleting both eIF2α phosphatases Ppp1r15a and Ppp1r15b results in embryonic lethality1. Thus far, in vivo physiological consequences of eIF2α phosphorylation in response to stress have not been addressed. We analyzed morphology and survival of PKO/eIF2αS51A dMut mice. PKO/eIF2αS51A dMut mice died at E13.5, while PKO mice survived at this stage with abnormal heart morphology, demonstrating that blocking eIF2α phosphorylation negatively impacted survival (Figure P).
Although HRI null mice were viable1 and displayed normal cardiac development and function at baseline, the role of HRI activation in mitochondrial cardiomyopathy remained to be addressed. We found that PKO/HRI dKOs died between E14.5-E16.5, while PKO littermates survived with abnormal cardiac morphology (Figure Q). Thus, although milder than effects of the eIF2α S51A mutant, deletion of HRI was also detrimental to survival. Thus HRI-eIF2α activation was protective for fetal mitochondrial cardiomyopathy
To determine the role of HRI in adult mitochondrial cardiomyopathy, we crossed HRI knockout mice with Tafazzin cKO (TKO) mice. TKO mice display mitochondrial dysfunction at 2 months and dilated cardiomyopathy at 4 months, but survive more than one year with impaired cardiac function5. Western blot and qPCR analysis confirmed that eIF2α-ATF4 signaling was activated in TKO hearts but abolished in TKO/HRI dKO hearts (Figure R–S). TKO/HRI dKO mice died between postnatal day (P) 7–10 with enlarged hearts, compared to TKO mice that survived more than one year5. We also observed increased ventricular weight to body weight ratios in dKOs (Figure T–U). Echocardiographic analysis revealed severe cardiac dysfunction in dKOs (Figure V). Thus, MSR triggered HRI-eIF2α was also protective for adult mitochondrial cardiomyopathy.
Overall we demonstrated that an HRI-eIF2α pathway mediated mitochondrial-nuclear communication and MSR-triggered ATF4 activation in both embryonic and adult heart. Our results uncovered a protective role for MSR triggered HRI-eIF2α-ATF4 signaling in both fetal and adult mitochondrial cardiomyopathy. Therefore, intention to inhibit the HRI-eIF2α-ATF4 pathway in mitochondrial cardiomyopathy could be detrimental, rather than beneficial, for cardiac dysfunction.
The data, analytical methods, and study materials that support the findings of this study will be available to other researchers from the corresponding authors on reasonable request. RNAseq data was deposited to the GEO database (Accession number: GSE201042).
Sources of Funding
XF is supported by NIH grants. SME is supported by NIH grants and the Foundation Leducq (16 CVD 03).
Footnotes
Disclosures
Jun Zhao is currently employed by Guardant Health, US. This work is not related to her employment at Guardant Health.
Reference:
- 1.Costa-Mattioli M and Walter P. The integrated stress response: From mechanism to disease. Science. 2020. Apr 24;368(6489):eaat5314. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Chen Z, Zhu S, Wang H, Wang L, Zhang J, Gu Y, Tan C, Dhanani M, Wever E, Wang X, Xie B, Wang S, Huang L, van Kampen AHC, Liu J, Han Z, Patel HH, Vaz FM, Fang X, Chen J and Ouyang K. PTPMT1 Is Required for Embryonic Cardiac Cardiolipin Biosynthesis to Regulate Mitochondrial Morphogenesis and Heart Development. Circulation. 2021;144:403–406. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Fessler E, Eckl EM, Schmitt S, Mancilla IA, Meyer-Bender MF, Hanf M, Philippou-Massier J, Krebs S, Zischka H and Jae LT. A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol. Nature. 2020;579:433–437. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Guo X, Aviles G, Liu Y, Tian R, Unger BA, Lin YT, Wiita AP, Xu K, Correia MA and Kampmann M. Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway. Nature. 2020;579:427–432. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Zhu S, Chen Z, Zhu M, Shen Y, Leon LJ, Chi L, Spinozzi S, Tan C, Gu Y, Nguyen A, Zhou Y, Feng W, Vaz FM, Wang X, Gustafsson AB, Evans SM, Kunfu O and Fang X. Cardiolipin Remodeling Defects Impair Mitochondrial Architecture and Function in a Murine Model of Barth Syndrome Cardiomyopathy. Circ Heart Fail. 2021;14:e008289. [DOI] [PMC free article] [PubMed] [Google Scholar]