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in adolescence or adulthood for the induction of bNAbs awaits the development of an immunization regimen that can safely induce bNAbs. This study at the very least proposes that successful infant vaccination against HIV-1 is plausible by demonstrating that the infant immune system is capable of generating the highly sought-after bNAbs. These findings raise several questions regarding how bNAbs develop in infants. Do evolving virus quasispecies with sequential ‘sweeps’ of viruses drive bNAb development in neonates, as has been described in adults1? Are bNAbs that develop in infants generated against different and perhaps new targets compared to those from adults? Are infant bNAbs polyreactive as in adults17? Finally, it remains unclear whether infant bNAbs are as highly mutated as adult ones, and if so, whether this somatic hypermutation
in response to HIV-1 early in life occurs more rapidly than in adults. Answering these questions to determine whether the infant immune system is uniquely poised to respond to HIV-1 compared to that of adults can further our understanding as to whether infant immunization would be better than immunization of adults in generating bNAbs—a key step in learning how to protect infants from HIV-1 transmission. Certainly, it is exciting to consider that understanding the ontogeny and specificity of bNAbs in the context of both the infant B and helper T cell repertoires may provide new insights into practical vaccination strategies toward induction of plasma neutralization breadth in all vaccinated individuals. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests:
details are available in the online version of the paper (doi:10.1038/nm.3598). 1. Liao, H.X. et al. Nature 496, 469–476 (2013). 2. Moore, P.L. et al. Nat. Med. 18, 1688–1692 (2012). 3. Doria-Rose, N.A. et al. Nature 509, 55–62 (2014). 4. Gruell, H. & Klein, F. Nat. Med. 20, 478–479 (2014). 5. Goo, L., Chohan, V., Nduati, R. & Overbaugh, J. Nat. Med. 20, 655–658 (2014). 6. Hraber, P. et al. AIDS 28,163–169 (2014). 7. Tomaras, G.D. et al. J. Virol. 85, 11502–11519 (2011). 8. Bonsignori, M. et al. J. Virol. 85, 9998–10009 (2011). 9. Walker, L.M. et al. Science 326, 285–289 (2009). 10. Scheid, J.F. et al. Nature 458, 636–640 (2009). 11. Georgiev, I.S. et al. Science 340, 751–756 (2013). 12. Scharf, L. et al. Cell Reports 7, 785–795 (2014). 13. Blattner, C. et al. Immunity 40, 669–680 (2014). 14. Falkowska, E. et al. Immunity 40, 657–668 (2014). 15. Sather, D.N. et al. J. Virol. 83, 757–769 (2009). 16. Klein, F. et al. Cell 153, 126–138 (2013). 17. Mascola, J.R. & Haynes, B.F. Immunol. Rev. 254, 225–244 (2013). 18. Gray, E.S. et al. J. Virol. 85, 4828–4840 (2011).
Cardiomyopathy, mitochondria and Barth syndrome: iPSCs reveal a connection Kunil K Raval & Timothy J Kamp Barth syndrome is a rare X-linked genetic disorder caused by mutations in the tafazzin (TAZ) gene that result in dilated cardiomyopathy, skeletal myopathy and neutropenia. Tafazzin has a mitochondrial function, and a new study using cardiomyocytes derived from induced pluripotent stem cells (iPSCs) from humans with Barth syndrome identifies increased mitochondrial reactive oxygen species (ROS) production as a key intermediate causing cardiac contractile dysfunction (pages 616–623). In the early 1980s, Dutch physician Peter Barth reported a family with an extensive history of male infants presenting with dilated cardiomyopathy, neutropenia and skeletal myopathy1, the majority of whom succumbed to combined infection and heart failure within a year after birth. Subsequent studies have demonstrated that Barth syndrome is an X-linked monogenic disease caused by mutations in TAZ 2, encoding an acyltransferase essential in the modification of cardiolipin, a key phospholipid enriched in mitochondrial inner membranes. Thus, mutations in TAZ provide an obvious link to mitochondrial abnormalities, but the precise mechanisms leading to pathology such as cardiomyopathy are not well understood, not Kunil K. Raval and Timothy J. Kamp are at the Department of Medicine, Stem Cell and Regenerative Medicine Center, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, USA. e-mail: [email protected]
least because it is difficult to study the effect of these mutations in vivo in humans. Recent breakthroughs allowing the reprogramming of somatic cells to pluripotency have enabled the generation of patient- and disease-specific human iPSC lines3,4. In this issue of Nature Medicine, Wang et al.5 generated iPSCs from two patients with Barth syndrome harboring distinct mutations in TAZ to identify ROS production as an intermediate in the pathophysiology of the disease (Fig. 1). Wang et al.5 first differentiated iPSCs derived from the two patients with Barth syndrome into cardiomyocytes (termed iPSC-CMs) and demonstrated that they showed the impaired cardiolipin acylation characteristic of Barth syndrome. Mature cardiolipin is known to have a role in the functioning of oxidative phosphorylation by stabilizing the electron transport chain (ETC) super complex and also in trapping protons on either side of the mitochondrial membrane. The authors thus investigated mitochondrial function in the Barth syndrome iPSC-CMs in
nature medicine volume 20 | number 6 | june 2014
the presence of galactose—a condition that favors oxidative phosphorylation. Compared to controls, Barth syndrome iPSC-CMs had a proton leak across the inner membrane and a reduced efficiency of the F1F0 ATP synthase, resulting in increased basal O2 consumption and, ultimately, a depleted ATP pool. The authors confirmed these findings were the result of mutations in the TAZ gene by reintroducing TAZ mRNA modified to be more stable and less toxic (modRNA) into the iPSC-CMs, which resulted in normal function of the mitochondria. In addition Cas9 genome editing introducing mutations into the TAZ gene of control iPSCs produced the disease phenotype in the genetically engineered iPSC-CMs. Mitochondria regulate cardiac differentiation via redox signaling, which affects sarcomere formation6. A feature identified in pathological samples from Barth syndrome cardiac tissue is myofilament disarray. To be able to directly compare the effects of TAZ mutations on sarcomeric structure in 585
npg
© 2014 Nature America, Inc. All rights reserved.
news and views
586
Barth syndrome iPSC-CM ROS
Healthy iPSC-CM
ROS
TAZ modRNA MitoTEMPO Linoleic acid
ROS ROS
ROS
Mitochondrial inner membrane MLCL
H+ H+
e– e– H+
L4-CL
H+
+ H+ H
H+ H+ H+
MLCL
ETC
+ O2
e – e–
e– O2 H2O ROS
ATP synthase ADP ATP
L4-CL
H+
TAZ
TAZ
e– O2
ETC H O 2
ATP synthase ADP ATP
Figure 1 Proposed pathophysiology of Barth syndrome cardiomyopathy revealed by iPSC model. Healthy iPSC-CMs have organized myofilaments and mitochondria with active transacylase tafazzin (TAZ). This results in conversion of monolysocardiolipin (MLCL; immature cardiolipin) to L 4-CL (mature cardiolipin) in the inner mitochondrial membrane and normal electron transport complex (ETC) and ATP synthase function. In contrast, a Barth syndrome iPSC-CM with mutant TAZ lacks L4-CL, leading to ETC complex malfunction resulting in increased reactive oxygen species (ROS) production that contributes to myofilament disarray and contractile dysfunction. modRNA encoding TAZ can rescue the disease phenotype, as can buffering mitochondrial ROS with mitoTEMPO or providing a precursor for L4-CL, linoleic acid.
antioxidant mitoTEMPO dramatically reversed the observed contractile defects. Thus, this study clearly implicates mitochondrialderived ROS as a key intermediate linking TAZ mutations to contractile dysfunction. Translating these small-molecule–based interventions to patient therapies may not be straightforward, but the results stand as an important landmark in the search for better treatments. On the basis of the data obtained by Wang et al.5, one obvious question for future study is the correlation of genotype with phenotype, as there were potentially important differences in the two different patient mutations studied, such as the different pattern of contractile dysfunction observed with the heart-on-chip assay. It would be interesting to know further clinical information regarding the disease features of the patients sampled— they were adult males, which suggests they have a milder phenotype. Furthermore, given that the existing iPSC approaches produce CMs that exhibit features of the embryonic heart such as a predominant dependence on glycolysis, how directly results from embryonic-like CMs translate to disease in the adult heart is not clear. Is the pathology all initiated in the mitochondria, or are there
other functional impacts, such as at the peroxisome where cardiolipin is also present8? Have other substrates or functions of tafazzin been overlooked that may help explain the variable phenotypes that are associated with distinct mutations? Perhaps the greatest impact of this work and model will be on advancing our understanding of mitochondrial and cardiolipin biology that is central to cardiac dysfunction seen in ischemic heart disease and aging mediated in part by ROS. The good news is that we now have a powerful model system to pursue these questions and further investigate therapies. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper (doi:10.1038/nm.3592). 1. Barth, P.G. et al. J. Neurol. Sci. 62, 327–355 (1983). 2. Bione, S. et al. Nat. Genet. 12, 385–389 (1996). 3. Takahashi, K. et al. Cell 131, 861–872 (2007). 4. Yu, J. et al. Science 318, 1917–1920 (2007). 5. Wang, G. et al. Nat. Med. 20, 616–623 (2014). 6. Hom, J.R. et al. Dev. Cell 21, 469–478 (2011). 7. Feinberg, A.W. et al. Science 317, 1366–1370 (2007). 8. Raja, V. & Greenberg, M.L. Chem. Phys. Lipids 179, 49–56 (2014).
volume 20 | number 6 | june 2014 nature medicine
Kim Caesar/Nature Publishing Group
the iPSC-CMs, Wang et al.5 quantified sarcomeric organization in single cells seeded on micropatterned fibronectin rectangles that mimic the dimensions of adult cardio myocytes. Intriguingly, they observed a significant decrease in organization in the sarcomeres in the disease line with a TAZ frameshift mutation, which could be eliminated with the TAZ modRNA, but the sarcomeres had normal structure in the disease line with a missense mutation. Whether this points to distinct pathology for these different mutations or is the result of differences in the differentiation or reprogramming process needs further investigation. To test for contractile deficits known to be part of the pathophysiology of Barth syndrome, the authors used their previously published heart-on-chip technology7. This approach uses fibronectin patterned elastomeric thin films to generate strips of anisotropic cardiac tissue from seeded iPSC-CMs, and contractile force is extrapolated from the extent of deflection of the thin film with each contraction. iPSC-CM contractions from both patients with Barth syndrome were weaker than those from wild-type cell lines, although there were differences in the characteristics of contractile dysfunction. Addition of the TAZ modRNA was able to return contractile force to normal levels, indicating that this deficit is the result of TAZ mutations. Culture of the iPSC-CMs in glucose resulted in increased ATP levels by promotion of glycolysis; however, this did not improve force production in disease constructs, indicating that deficient mitochondrial ATP synthesis is not directly responsible for cardiac contractile deficits; however, the authors found a potential mechanism when testing for therapies. In perhaps the most important part of their study, Wang et al.5 used the iPSC-CM platform to test small-molecule therapeutic interventions. Of the three interventions tested, linoleic acid, an essential unsaturated fatty acid precursor of mature cardiolipin, was most promising, as it largely corrected the mitochondrial metabolic abnormalities and alleviated contractile dysfunction. They hypothesized that this might be because it encouraged an alternative cardiolipin synthesis pathway. However, they also thought that it might be able to scavenge mitochondrial ROS; in fact, they were able to show that it curtailed mitochondrial ROS production, which was found to be elevated in iPSCCMs. In addition, the mitochondrial targeted
npg
© 2014 Nature America, Inc. All rights reserved.
in adolescence or adulthood for the induction of bNAbs awaits the development of an immunization regimen that can safely induce bNAbs. This study at the very least proposes that successful infant vaccination against HIV-1 is plausible by demonstrating that the infant immune system is capable of generating the highly sought-after bNAbs. These findings raise several questions regarding how bNAbs develop in infants. Do evolving virus quasispecies with sequential ‘sweeps’ of viruses drive bNAb development in neonates, as has been described in adults1? Are bNAbs that develop in infants generated against different and perhaps new targets compared to those from adults? Are infant bNAbs polyreactive as in adults17? Finally, it remains unclear whether infant bNAbs are as highly mutated as adult ones, and if so, whether this somatic hypermutation
in response to HIV-1 early in life occurs more rapidly than in adults. Answering these questions to determine whether the infant immune system is uniquely poised to respond to HIV-1 compared to that of adults can further our understanding as to whether infant immunization would be better than immunization of adults in generating bNAbs—a key step in learning how to protect infants from HIV-1 transmission. Certainly, it is exciting to consider that understanding the ontogeny and specificity of bNAbs in the context of both the infant B and helper T cell repertoires may provide new insights into practical vaccination strategies toward induction of plasma neutralization breadth in all vaccinated individuals. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests:
details are available in the online version of the paper (doi:10.1038/nm.3598). 1. Liao, H.X. et al. Nature 496, 469–476 (2013). 2. Moore, P.L. et al. Nat. Med. 18, 1688–1692 (2012). 3. Doria-Rose, N.A. et al. Nature 509, 55–62 (2014). 4. Gruell, H. & Klein, F. Nat. Med. 20, 478–479 (2014). 5. Goo, L., Chohan, V., Nduati, R. & Overbaugh, J. Nat. Med. 20, 655–658 (2014). 6. Hraber, P. et al. AIDS 28,163–169 (2014). 7. Tomaras, G.D. et al. J. Virol. 85, 11502–11519 (2011). 8. Bonsignori, M. et al. J. Virol. 85, 9998–10009 (2011). 9. Walker, L.M. et al. Science 326, 285–289 (2009). 10. Scheid, J.F. et al. Nature 458, 636–640 (2009). 11. Georgiev, I.S. et al. Science 340, 751–756 (2013). 12. Scharf, L. et al. Cell Reports 7, 785–795 (2014). 13. Blattner, C. et al. Immunity 40, 669–680 (2014). 14. Falkowska, E. et al. Immunity 40, 657–668 (2014). 15. Sather, D.N. et al. J. Virol. 83, 757–769 (2009). 16. Klein, F. et al. Cell 153, 126–138 (2013). 17. Mascola, J.R. & Haynes, B.F. Immunol. Rev. 254, 225–244 (2013). 18. Gray, E.S. et al. J. Virol. 85, 4828–4840 (2011).
Cardiomyopathy, mitochondria and Barth syndrome: iPSCs reveal a connection Kunil K Raval & Timothy J Kamp Barth syndrome is a rare X-linked genetic disorder caused by mutations in the tafazzin (TAZ) gene that result in dilated cardiomyopathy, skeletal myopathy and neutropenia. Tafazzin has a mitochondrial function, and a new study using cardiomyocytes derived from induced pluripotent stem cells (iPSCs) from humans with Barth syndrome identifies increased mitochondrial reactive oxygen species (ROS) production as a key intermediate causing cardiac contractile dysfunction (pages 616–623). In the early 1980s, Dutch physician Peter Barth reported a family with an extensive history of male infants presenting with dilated cardiomyopathy, neutropenia and skeletal myopathy1, the majority of whom succumbed to combined infection and heart failure within a year after birth. Subsequent studies have demonstrated that Barth syndrome is an X-linked monogenic disease caused by mutations in TAZ 2, encoding an acyltransferase essential in the modification of cardiolipin, a key phospholipid enriched in mitochondrial inner membranes. Thus, mutations in TAZ provide an obvious link to mitochondrial abnormalities, but the precise mechanisms leading to pathology such as cardiomyopathy are not well understood, not Kunil K. Raval and Timothy J. Kamp are at the Department of Medicine, Stem Cell and Regenerative Medicine Center, School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, USA. e-mail: [email protected]
least because it is difficult to study the effect of these mutations in vivo in humans. Recent breakthroughs allowing the reprogramming of somatic cells to pluripotency have enabled the generation of patient- and disease-specific human iPSC lines3,4. In this issue of Nature Medicine, Wang et al.5 generated iPSCs from two patients with Barth syndrome harboring distinct mutations in TAZ to identify ROS production as an intermediate in the pathophysiology of the disease (Fig. 1). Wang et al.5 first differentiated iPSCs derived from the two patients with Barth syndrome into cardiomyocytes (termed iPSC-CMs) and demonstrated that they showed the impaired cardiolipin acylation characteristic of Barth syndrome. Mature cardiolipin is known to have a role in the functioning of oxidative phosphorylation by stabilizing the electron transport chain (ETC) super complex and also in trapping protons on either side of the mitochondrial membrane. The authors thus investigated mitochondrial function in the Barth syndrome iPSC-CMs in
nature medicine volume 20 | number 6 | june 2014
the presence of galactose—a condition that favors oxidative phosphorylation. Compared to controls, Barth syndrome iPSC-CMs had a proton leak across the inner membrane and a reduced efficiency of the F1F0 ATP synthase, resulting in increased basal O2 consumption and, ultimately, a depleted ATP pool. The authors confirmed these findings were the result of mutations in the TAZ gene by reintroducing TAZ mRNA modified to be more stable and less toxic (modRNA) into the iPSC-CMs, which resulted in normal function of the mitochondria. In addition Cas9 genome editing introducing mutations into the TAZ gene of control iPSCs produced the disease phenotype in the genetically engineered iPSC-CMs. Mitochondria regulate cardiac differentiation via redox signaling, which affects sarcomere formation6. A feature identified in pathological samples from Barth syndrome cardiac tissue is myofilament disarray. To be able to directly compare the effects of TAZ mutations on sarcomeric structure in 585
npg
© 2014 Nature America, Inc. All rights reserved.
news and views
586
Barth syndrome iPSC-CM ROS
Healthy iPSC-CM
ROS
TAZ modRNA MitoTEMPO Linoleic acid
ROS ROS
ROS
Mitochondrial inner membrane MLCL
H+ H+
e– e– H+
L4-CL
H+
+ H+ H
H+ H+ H+
MLCL
ETC
+ O2
e – e–
e– O2 H2O ROS
ATP synthase ADP ATP
L4-CL
H+
TAZ
TAZ
e– O2
ETC H O 2
ATP synthase ADP ATP
Figure 1 Proposed pathophysiology of Barth syndrome cardiomyopathy revealed by iPSC model. Healthy iPSC-CMs have organized myofilaments and mitochondria with active transacylase tafazzin (TAZ). This results in conversion of monolysocardiolipin (MLCL; immature cardiolipin) to L 4-CL (mature cardiolipin) in the inner mitochondrial membrane and normal electron transport complex (ETC) and ATP synthase function. In contrast, a Barth syndrome iPSC-CM with mutant TAZ lacks L4-CL, leading to ETC complex malfunction resulting in increased reactive oxygen species (ROS) production that contributes to myofilament disarray and contractile dysfunction. modRNA encoding TAZ can rescue the disease phenotype, as can buffering mitochondrial ROS with mitoTEMPO or providing a precursor for L4-CL, linoleic acid.
antioxidant mitoTEMPO dramatically reversed the observed contractile defects. Thus, this study clearly implicates mitochondrialderived ROS as a key intermediate linking TAZ mutations to contractile dysfunction. Translating these small-molecule–based interventions to patient therapies may not be straightforward, but the results stand as an important landmark in the search for better treatments. On the basis of the data obtained by Wang et al.5, one obvious question for future study is the correlation of genotype with phenotype, as there were potentially important differences in the two different patient mutations studied, such as the different pattern of contractile dysfunction observed with the heart-on-chip assay. It would be interesting to know further clinical information regarding the disease features of the patients sampled— they were adult males, which suggests they have a milder phenotype. Furthermore, given that the existing iPSC approaches produce CMs that exhibit features of the embryonic heart such as a predominant dependence on glycolysis, how directly results from embryonic-like CMs translate to disease in the adult heart is not clear. Is the pathology all initiated in the mitochondria, or are there
other functional impacts, such as at the peroxisome where cardiolipin is also present8? Have other substrates or functions of tafazzin been overlooked that may help explain the variable phenotypes that are associated with distinct mutations? Perhaps the greatest impact of this work and model will be on advancing our understanding of mitochondrial and cardiolipin biology that is central to cardiac dysfunction seen in ischemic heart disease and aging mediated in part by ROS. The good news is that we now have a powerful model system to pursue these questions and further investigate therapies. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper (doi:10.1038/nm.3592). 1. Barth, P.G. et al. J. Neurol. Sci. 62, 327–355 (1983). 2. Bione, S. et al. Nat. Genet. 12, 385–389 (1996). 3. Takahashi, K. et al. Cell 131, 861–872 (2007). 4. Yu, J. et al. Science 318, 1917–1920 (2007). 5. Wang, G. et al. Nat. Med. 20, 616–623 (2014). 6. Hom, J.R. et al. Dev. Cell 21, 469–478 (2011). 7. Feinberg, A.W. et al. Science 317, 1366–1370 (2007). 8. Raja, V. & Greenberg, M.L. Chem. Phys. Lipids 179, 49–56 (2014).
volume 20 | number 6 | june 2014 nature medicine
Kim Caesar/Nature Publishing Group
the iPSC-CMs, Wang et al.5 quantified sarcomeric organization in single cells seeded on micropatterned fibronectin rectangles that mimic the dimensions of adult cardio myocytes. Intriguingly, they observed a significant decrease in organization in the sarcomeres in the disease line with a TAZ frameshift mutation, which could be eliminated with the TAZ modRNA, but the sarcomeres had normal structure in the disease line with a missense mutation. Whether this points to distinct pathology for these different mutations or is the result of differences in the differentiation or reprogramming process needs further investigation. To test for contractile deficits known to be part of the pathophysiology of Barth syndrome, the authors used their previously published heart-on-chip technology7. This approach uses fibronectin patterned elastomeric thin films to generate strips of anisotropic cardiac tissue from seeded iPSC-CMs, and contractile force is extrapolated from the extent of deflection of the thin film with each contraction. iPSC-CM contractions from both patients with Barth syndrome were weaker than those from wild-type cell lines, although there were differences in the characteristics of contractile dysfunction. Addition of the TAZ modRNA was able to return contractile force to normal levels, indicating that this deficit is the result of TAZ mutations. Culture of the iPSC-CMs in glucose resulted in increased ATP levels by promotion of glycolysis; however, this did not improve force production in disease constructs, indicating that deficient mitochondrial ATP synthesis is not directly responsible for cardiac contractile deficits; however, the authors found a potential mechanism when testing for therapies. In perhaps the most important part of their study, Wang et al.5 used the iPSC-CM platform to test small-molecule therapeutic interventions. Of the three interventions tested, linoleic acid, an essential unsaturated fatty acid precursor of mature cardiolipin, was most promising, as it largely corrected the mitochondrial metabolic abnormalities and alleviated contractile dysfunction. They hypothesized that this might be because it encouraged an alternative cardiolipin synthesis pathway. However, they also thought that it might be able to scavenge mitochondrial ROS; in fact, they were able to show that it curtailed mitochondrial ROS production, which was found to be elevated in iPSCCMs. In addition, the mitochondrial targeted
