Disease modeling of a mutation in α‐actinin 2 guides clinical therapy in hypertrophic cardiomyopathy

Abstract Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease accompanied by structural and contractile alterations. We identified a rare c.740C>T (p.T247M) mutation in ACTN2, encoding α‐actinin 2 in a HCM patient, who presented with left ventricular hypertrophy, outflow tract obstruction, and atrial fibrillation. We generated patient‐derived human‐induced pluripotent stem cells (hiPSCs) and show that hiPSC‐derived cardiomyocytes and engineered heart tissues recapitulated several hallmarks of HCM, such as hypertrophy, myofibrillar disarray, hypercontractility, impaired relaxation, and higher myofilament Ca2+ sensitivity, and also prolonged action potential duration and enhanced L‐type Ca2+ current. The L‐type Ca2+ channel blocker diltiazem reduced force amplitude, relaxation, and action potential duration to a greater extent in HCM than in isogenic control. We translated our findings to patient care and showed that diltiazem application ameliorated the prolonged QTc interval in HCM‐affected son and sister of the index patient. These data provide evidence for this ACTN2 mutation to be disease‐causing in cardiomyocytes, guiding clinical therapy in this HCM family. This study may serve as a proof‐of‐principle for the use of hiPSC for personalized treatment of cardiomyopathies.

The paper by Prondzynski and colleagues describes a small family with novel ACTN2 variant p.T247M, which is not found in large population databases. It has been suggested in the literature that the level of evidence for ACTN2 being a causative gene for hypertrophic cardiomyopathy is not strong. This work advances the evidence for ACTN2 being, at least for some variants, causing HCM. The authors generate iPSC derived cardiomyocytes from the proband with the ACTN2 p.T247M variant and find the cells are larger and have abnormal force and abnormal action potentials. The authors describe abnormal force/calcium relationships. They suggest that the Ltype calcium channel blocker diltizem may be effective and treat less affected family members (n=2) with this agent and show reduction of the QT interval. The work around the ACTN2 variant and its role in HCM is quite good. This alone adds to the literature.
Major comment 1. The weakness of the work is around the suggestion that diltiazem and reduced QT interval is reliable indicator of outcomes. The presence of cardiomyopathy alone is well known to prolong QT intervals and is not felt to be a primary finding. Furthermore, reducing the QT interval in this setting is not of clear clinical value. All the wording and conclusions around using diltiazem to treat arrhythmias and long QT should be softened since this is not a clinical trial and the number of treated subjects is simply to small and without adequate outcomes to make this assertion. Furthermore, the data linking the L type channel to a-actinin is quite limited. If there is an effect on calcium handling and action potentials, it is likely quite downstream and much more would need to be shown to assert a more proximal relationship.
2. Allelic balance is better assessed using RNAseq rather than a subcloning method that evaluates very few clones.

Minor comments
3. It is worthwhile to emphasize that this is a later onset HCM, compared to some genetic mutations which can present quite early in life in young adults or even children. Is there any clinical history on the parents of 1-2 to know which side of the family it derived? Any early death in either paternal or maternal side? 4. Was the 43 yo affected individual screened for abnormal rhythms, if so how? Is there more data on this patient? MRI findings-details? 5. The clinical descriptions need to be rewritten since there is some unusual and non-native English here.
Referee #3 (Comments on Novelty/Model System for Author): The manuscript by Prondzynski et al. reports on a novel HCM mutation in alpha-Actinin-2, which is associated with hypertrophic cardiomyopathy (HCM). The authors are using an impressive amount of different techniques and experimental approaches to characterize the pathological phenotype present in the index patient using iPS cell derived cardiac myocytes, which are either cultured in 2 dimensional cultures or as EHT tissue model. The authors also control for genetic background effects by rescuing the mutation by CRISPR Cas9-mediated homology repair demonstrating a partial rescue of the phenotype. Given the importance of heart failure as a prevalent disease and the importance to develop novel patient-specific therapeutic approaches, this manuscript details how one can come up with novel insight, which guided the researchers to propose some novel treatment regime, which had some impact on the pathological phenotype. iPS cells, rescuing the genotype homology repair, and careful phenotype analysis are executed to a very high standard in this manuscript.
Referee #3 (Remarks for Author): The length of the manuscript is apropriate and I think that overall the paper is extremely wellwritten, and the discussion is focused on the most important aspects. 1. What I am struggling with is the intermediate phenotype of the rescued iPS cell line. If the ACTN2 mutant would be the only disease-causing mutation present in this patient would you not expect a more complete rescue. Several measured parameters as for example cell area, which reflects something like a prohypertrophic state is clearly intermediate to the mutant but also clearly higher than in WT. The same holds true for force, relaxation etc. 2. I am not a geneticist, so it is hard for me to decide whether maybe another coupled pathogenic allele could be present, which acts in combination with ACTN2 to cause the phenotype. You have tested a couple of candidates but clearly a transcriptome analysis and a comparison of affected and normal family members would maybe give some information what else is causing this intermediate pathology after ACTN2 rescue. 3. How is the mutation in ACTN2 mechanistically linked to a gain of function of the L-type calcium channel. Can you enlighten the reader how this is mechanistically linked? What is known to cause a similar gain of function? 4. Although diltiazem was able to lower QT time, it is still far away from normalizing the QT time. This suggest that maybe other elements controlling calcium transients are probably also aberrantly modulated in cells transcribing the mutant allele. Response to Referee #1 We would like to thank the reviewer for his/her careful work on our manuscript and his/her nice, positive comments. We have carefully considered your comments and modified the discussion accordingly.
Please note that all changes in the manuscript are marked with yellow.
This is a very solid, right, and convincing study that shows clearly that information gleaned from hiPSc model system can lead to uncovering new therapeutic approaches for patients with a defined monogenic cardiac disease, namely HCM. Other studies have supported the utility of hIPS models to understand disease, and to model key features of the phenotype, but, to my knowledge, none have taken this in a strictly personalized manner to show that there could be potential therapeutic benefit using surrogate indicators in the same patient. In many ways, this is an elegant "case study" at a molecular level, which is its strength and also its weakness. On the one hand, it shows the way forward for more personalized therapy using hiPSc model systems, and at the same time it is only indicative of a single mutation in a single family when it is clear the disease represents many personal mutations scattered across many sarcomeric genes. The question arises as to the generalizability of the finding. This point should be addressed in the discussion more completely, Also, there are personalized therapies for a distinct class of HCM due to mutations in myosin itself that are being tested by Myokardia. Would these be expected to work in this patient or on the hiPSc model system? If not would lack of effect be also predictive? This would speak to the specificity of the response. If the authors believe that the diltiazem effect would apply to other forms of HCM, they could support this by examining one of the many other hiPSc lines that have been generated. Finally, the use of the novel 3-D muscle tissue system of Eschenhagen is a strength of this paper. It would be interest to highlight how the findings in this study may be different from other previous HCM iPSc models reported by Joe Wu and others.
Response: We thank the reviewer for his/her positive comments. We agree that this is a "case study" evaluating a single HCM mutation out of more than 1000 identified in HCM. However, and specifically, the level of evidence for ACTN2 being a causative gene in HCM is not strong as compared to MYBPC3 and MYH7 genes (Walsh R et al., Genet Med 2017). Furthermore, the interesting fact that the prolonged QTc did not find attention during clinical routine but was revealed after the work in vitro underlines the necessity for more personalized medicine. Therefore, using isogenic control and different assays in vitro we validated its causal role in HCM and could change the treatment of the patient. We modified the discussion to better stress the "case study" and the fact that other less evident genes could be validated by this approach ( Therefore, it could also work for the ACTN2 mutation, which was associated with higher force in EHTs. However, this compound would not be expected to ameliorate the larger LTCC current detected in HCM-cardiomyocytes, which we believe is the primary cause for the prolonged QTc interval observed in the family members carrying the ACTN2 mutation. Thus, the iPSC-approach revealed a mechanism offering a more specific therapeutic intervention (with diltiazem). Heart J). These data are in line with our findings (relaxation), but did not reveal the underlying mechanism. The data are now discussed (page 10, lines 306-308).
Finally, we also believe that the EHT model was crucial for modeling contractile HCMphenotypes and for evaluating diltiazem as a treatment option in this study. We are currently not aware of any hiPSC 3D model that has been used to investigate disease-specific phenotypes for HCM except the EHT model ( Response to Referee #2 We would like to thank the reviewer for his/her comments and critiques on our manuscript. We have softened some of our wordings and performed an additional experiment to address your point. Please note that all changes in the manuscript are marked with yellow. The paper by Prondzynski and colleagues describes a small family with novel ACTN2 variant p.T247M, which is not found in large population databases. It has been suggested in the literature that the level of evidence for ACTN2 being a causative gene for hypertrophic cardiomyopathy is not strong. This work advances the evidence for ACTN2 being, at least for some variants, causing HCM. The authors generate iPSC derived cardiomyocytes from the proband with the ACTN2 p.T247M variant and find the cells are larger and have abnormal force and abnormal action potentials. The authors describe abnormal force/calcium relationships. They suggest that the L-type calcium channel blocker diltiazem may be effective and treat less affected family members (n=2) with this agent and show reduction of the QT interval. The work around the ACTN2 variant and its role in HCM is quite good. This alone adds to the literature.
Major comment 1. The weakness of the work is around the suggestion that diltiazem and reduced QT interval is reliable indicator of outcomes. The presence of cardiomyopathy alone is well known to prolong QT intervals and is not felt to be a primary finding. Furthermore, reducing the QT interval in this setting is not of clear clinical value. All the wording and conclusions around using diltiazem to treat arrhythmias and long QT should be softened since this is not a clinical trial and the number of treated subjects is simply to small and without adequate outcomes to make this assertion. Furthermore, the data linking the L type channel to a-actinin is quite limited. If there is an effect on calcium handling and action potentials, it is likely quite downstream and much more would need to be shown to assert a more proximal relationship.
Response: We thank the reviewer for this comment. We agree that cardiomyopathy as such can be associated with prolonged QT intervals and this cannot be excluded as a contributor in the case of this mutation. However, we provide evidence for a more specific effect of the actinin mutation on the LTCC current and believe that the larger diltiazem effect in mutation-carrier EHTs and the new data on the interaction of wild-type and mutated actinin with Cavα1.2 (see below) support this view.
We are fully aware of the fact that shortening of the QT interval in the HCM patients as such is of unknown therapeutic significance. We apologize if we used too strong wording and softened the wording in the abstract, results and discussion, accordingly (page 2, lines 47 and 48; page 7, line 212; page 8, line 236; page 10 lines 330-333).
We also agree that the link between ACTN2 and L-type calcium channel was not fully elucidated. And since reviewer #3 also asked for mechanistic insights, we performed an additional experiment in collaboration with the group of Daniele Catalucci (Milan, Italy). Indeed, it has been shown that αactinin 2 interacts with ion channels and contributes to their modulation, such as the L-type calcium channel (LTCC) complex. In particular, by binding to the IQ segment of the Cavα1.2, pore unit of the LTCC, α-actinin 2 modulates LTCC density and function at the plasma membrane. Therefore, we tested whether the α-actinin 2 mutation affects the binding of α-actinin 2 to Cavα1.2. By a bioluminescence resonance energy transfer (BRET) assays performed in live cardiac myocyte-like HL-1 cell, we observed a similar binding affinity of Cavα1.2 to WT and mutant α-actinin 2 (new Figure S4). However, while this binding affinity of Cavα1.2 to WT α-actinin 2 strongly increased with the co-transfection of Cavα2, the LTCC accessory subunit and chaperone of the LTCC pore unit, it did not in case of mutant α-actinin 2. Thus, the HCM mutation decreased the interaction of α-actinin 2 with the LTCC complex, possibly affecting the activity of the channel. This may explain the electromechanical phenotype of the investigated HCM-affected family, characterized by higher ICa,L density. This experiment has been added in the revised version of the manuscript and discussed (Results, page 6, line 175; page 7, lines 196-210; Discussion, pages 10-11, lines 291-301 and line 347; Methods, page 19, lines 621-628).
2. Allelic balance is better assessed using RNAseq rather than a subcloning method that evaluates very few clones.
Response: Thank you for the suggestion. We performed RNAseq in different samples and confirmed the presence of both wild-type and mutant mRNA in a 50:50 distribution in both HCM-EHTs and in septal myectomy of patient II.4, confirming the allelic balance (see new Figure S2C; Results page 5, line 143; Methods, page 16, lines 509-526).
Minor comments 3. It is worthwhile to emphasize that this is a later onset HCM, compared to some genetic mutations which can present quite early in life in young adults or even children. Is there any clinical history on the parents of 1-2 to know which side of the family it derived? Any early death in either paternal or maternal side?
Response: We agree with the reviewer and modified the clinical characterization accordingly (page 4, lines 121-123). Furthermore, we do not have any information from the parents of individual I.2, which could indicate transmission from either the father or the mother. Response: Thank you for notifying it. We asked an English native speaker to correct the clinical description of the family (page 4, lines 102-123).

Response to Referee #3
We would like to thank the reviewer for his/her careful work on our manuscript, the positive feedback and constructive comments and suggestion to improve the manuscript. We have performed new experiments to address your points. Please note that all changes in the manuscript are marked with yellow.
The manuscript by Prondzynski et al. reports on a novel HCM mutation in alpha-Actinin-2, which is associated with hypertrophic cardiomyopathy (HCM). The authors are using an impressive amount of different techniques and experimental approaches to characterize the pathological phenotype present in the index patient using iPS cell derived cardiac myocytes, which are either cultured in 2 dimensional cultures or as EHT tissue model. The authors also control for genetic background effects by rescuing the mutation by CRISPR Cas9-mediated homology repair demonstrating a partial rescue of the phenotype. Given the importance of heart failure as a prevalent disease and the importance to develop novel patient-specific therapeutic approaches, this manuscript details how one can come up with novel insight, which guided the researchers to propose some novel treatment regime, which had some impact on the pathological phenotype. iPS cells, rescuing the genotype homology repair, and careful phenotype analysis are executed to a very high standard in this manuscript. The length of the manuscript is apropriate and I think that overall the paper is extremely well-written, and the discussion is focused on the most important aspects.  Figure III b). Finally, we have systematically analysed in a blinded manner 36 control cell lines, which showed that the the SD of EHT parameters are 20-35% of the mean (Eschenhagen, personal communication; see results below). All this underlines the need of isogenic control for personalized medicine. We therefore discussed further and added the respective references (page 9, lines 275-286).
Parameters obtained under 1 Hz-pacing in 36 control iPSC-derived engineered heart tissues (Mean ± SD): Force = 0.151 ± 0.053 mN (± 35% of the mean) T180% = 0.143 ± 0.029 sec (± 20% of the mean) T280% = 0.205 ± 0.069 sec (± 34% of the mean) 2. I am not a geneticist, so it is hard for me to decide whether maybe another coupled pathogenic allele could be present, which acts in combination with ACTN2 to cause the phenotype. You have tested a couple of candidates but clearly a transcriptome analysis and a comparison of affected and normal family members would maybe give some information what else is causing this intermediate pathology after ACTN2 rescue.
Response: In the line of the response to your comment 1, we think that the main genetic difference is between the control and isogenic control, but we do not have DNA samples of the control individual to confirm that. You suggest performing a transcriptome analysis of affected and normal family members, but we cannot get cardiac samples from unaffected individuals. Thus, we think it will be difficult to solve this issue with RNAseq with patient's tissue. Further OMIC analyses are planned on several HCM and isogenic control cell lines but will be part of another study. On the other hand, and to respond in part to your question, preliminary RNAseq data obtained on HCM, HCMrep and Ctrl EHTs showed the following correlation of gene expression (counts). This shows a higher variability between HCMrep and Crl EHTs than HCMrep and HCM EHTs, underlying more genetic variability between the two control cell lines than between the HCM and isogenic control lines (n=7 EHTs out of 3-6 batches of cardiomyocyte differentiation).
3. How is the mutation in ACTN2 mechanistically linked to a gain of function of the L-type calcium channel. Can you enlighten the reader how this is mechanistically linked? What is known to cause a similar gain of function?
Response: thank you for your very interesting point, which we also tried to understand. it has been shown that α-actinin 2 interacts with ion channels and contributes to their modulation, such as the L-type calcium channel (LTCC) complex. In particular, by binding to the IQ segment of the Cavα1.2, pore unit of the LTCC, α-actinin 2 modulates LTCC density and function at the plasma membrane. Therefore, we tested whether the α-actinin 2 mutation affects the binding of α-actinin 2 to Cavα1.2. By a bioluminescence resonance energy transfer (BRET) assays performed in live cardiac myocyte-like HL-1 cells we observed a similar binding affinity of Cavα1.2 to WT and mutant α-actinin 2 (new Figure S4). However, while this binding affinity of Cavα1.2 to WT αactinin 2 strongly increased with the co-transfection of Cavα2, the LTCC accessory subunit and chaperone of the LTCC pore unit, it did not in case of mutant α-actinin 2. Thus, the HCM mutation decreased the interaction of α-actinin 2 with the LTCC complex, possibly affecting the activity of the channel. This may explain the electromechanical phenotype of the investigated HCM-affected family, characterized by higher ICa,L density. This experiment has been added in the revised version of the manuscript and discussed ( Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees that were asked to re-assess it. As you will see the reviewers are now globally supportive and I am pleased to inform you that we will be able to accept your manuscript pending minor editorial amendments. ***** Reviewer's comments ***** Referee #2 (Remarks for Author): None Referee #3 (Comments on Novelty/Model System for Author): The use of iPS cell model and to further differentiate them in 3D culture is an advanced model of analysing the pathomechanisms that cause HCM related phenotypes. This is highly innovative and exemplifies state of the art of modelling heart disease with iPS cells.
Referee #3 (Remarks for Author): The authors have addressed my points of concerns and even performed some additional experiments, which demonstrate a biochemical difference between the mutant and wildtype aactinin and provides an functional explanation for the effect of the mutation on LTCC.
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Manuscript Number: EMM-2019-11115
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