Therapeutic efficacy of AAV-mediated restoration of PKP2 in arrhythmogenic cardiomyopathy

Kyriakopoulou, Eirini; Versteeg, Danielle; de Ruiter, Hesther; Perini, Ilaria; Seibertz, Fitzwilliam; Döring, Yannic; Zentilin, Lorena; Tsui, Hoyee; van Kampen, Sebastiaan J.; Tiburcy, Malte; Meyer, Tim; Voigt, Niels; Tintelen, van J. Peter; Zimmermann, Wolfram H.; Giacca, Mauro; van Rooij, Eva

doi:10.1038/s44161-023-00378-9

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Published: 07 December 2023

Therapeutic efficacy of AAV-mediated restoration of PKP2 in arrhythmogenic cardiomyopathy

Nature Cardiovascular Research volume 2, pages 1262–1276 (2023)Cite this article

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Abstract

Arrhythmogenic cardiomyopathy is a severe cardiac disorder characterized by lethal arrhythmias and sudden cardiac death, with currently no effective treatment. Plakophilin 2 (PKP2) is the most frequently affected gene. Here we show that adeno-associated virus (AAV)-mediated delivery of PKP2 in PKP2^{c.2013delC/WT} induced pluripotent stem cell-derived cardiomyocytes restored not only cardiac PKP2 levels but also the levels of other junctional proteins, found to be decreased in response to the mutation. PKP2 restoration improved sodium conduction, indicating rescue of the arrhythmic substrate in PKP2 mutant induced pluripotent stem cell-derived cardiomyocytes. Additionally, it enhanced contractile function and normalized contraction kinetics in PKP2 mutant engineered human myocardium. Recovery of desmosomal integrity and cardiac function was corroborated in vivo, by treating heterozygous Pkp2^c.1755delA knock-in mice. Long-term treatment with AAV9–PKP2 prevented cardiac dysfunction in 12-month-old Pkp2^{c.1755delA/WT} mice, without affecting wild-type mice. These findings encourage clinical exploration of PKP2 gene therapy for patients with PKP2 haploinsufficiency.

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Main

Arrhythmogenic cardiomyopathy (ACM) is a progressive genetic cardiac disorder with a prevalence ranging from 1:2,000 to 1:5,000 (ref. ¹). Early diagnosis is often hindered by phenotypic complexity and variable disease penetrance². At the clinically concealed phase of the disease, patients often present asymptomatic or with mild electrocardiogram abnormalities, while possessing high risk of sudden cardiac death³. As disease progresses, structural remodeling characterized by fibro-fatty tissue infiltration within the myocardium becomes evident, ultimately leading to life-threatening ventricular arrhythmias and heart failure, a condition that often requires heart transplantation^3,4.

Approximately 50% of patients with ACM carry genetic mutations in the desmosomal genes: plakophilin 2 (PKP2), plakoglobin (JUP), desmoplakin (DSP), desmocollin (DSC2) and desmoglein (DSG2)⁵. Desmosomes are robust multiprotein structures localized within the intercalated discs (IDs), where they facilitate mechanical coupling of the adjacent cardiomyocytes (CMs)⁶. Despite the classical notion that desmosomes function individually, a close connection and interaction of desmosomes with more ID components such as ion channels, gap junctions and adherens junctions has been described, together forming the area composita or connexome^6,7,8,9.

PKP2 is the most commonly affected gene in patients with ACM¹⁰. Specifically, mutations in PKP2 have been strongly associated with the onset and development of arrhythmogenic right ventricular cardiomyopathy, a distinctive subtype of ACM marked by its pronounced impact on the right ventricle. While there is growing evidence revealing contributions from both the biventricular and left ventricular regions to the ACM phenotype, it is worth noting that arrhythmogenic right ventricular cardiomyopathy continues to be the predominant subtype within the ACM spectrum, primarily inherited through an autosomal dominant pattern¹¹. The vast majority of genetic alterations affecting PKP2 are truncating variants¹². These mutant transcripts are often degraded by nonsense-mediated messenger RNA decay, causing PKP2 haploinsufficiency, which is an important pathogenic ACM driver^12,13,14. The fundamental role of PKP2 in ACM disease pathogenesis and progression has been highlighted by several studies^{9,14,15,16,17,18,19,20,21,22}. A study on a cardiomyocyte-specific, inducible deletion of Pkp2 in mice revealed PKP2 as a crucial regulator of calcium cycling and cardiac rhythm¹⁵. These findings were further corroborated on a heterozygous Pkp2 knockout mouse model exposed to environmental stress stimuli²². Another study demonstrated endogenous correlation of PKP2 transcript abundance with the abundance of transcripts encoding inflammatory/immune response factors, the presence of which is thought to mediate ACM progression¹⁷. Interestingly, a study aiming to explore the pathogenic mechanisms leading to ventricular dilation and decreased systolic function associated with ACM, revealed that truncating PKP2 mutations impair CM contractility by disrupting sarcomere stability and localization¹⁴. Of relevance, it was recently shown that cardiac levels of PKP2 directly correlate to protein levels of other desmosomal and adherens junction proteins in patients²⁰. These data suggest that PKP2 fulfills a key anchoring role for the stabilization and function of other desmosomal and ID proteins. PKP2 loss as a consequence of truncating variants induces desmosomal instability and the eventual degradation of the area composita-related proteins, whereby downstream disease processes become activated.

Despite an improvement in our knowledge about the molecular triggers underlying ACM, targeted and effective therapeutic interventions for this disease remain lacking. While current treatment options are more focused on treating disease symptoms²³, targeting the primary cause of disease consequently leading to ACM would have curative potential.

Here we show that adeno-associated virus (AAV)-mediated PKP2 restoration results in the re-formation of the desmosomal complex and consequently an improvement in contractile function in PKP2^{c.2013delC/WT} induced pluripotent stem (iPS) cell-derived CMs, PKP2^{c.2013delC/WT} engineered human myocardium (EHM) and Pkp2 mutant knock-in mice. Molecular restoration of desmosomal and non-desmosomal protein components within the ID was observed following exogenous administration of Pkp2, which successfully prevented the functional decline induced by PKP2 haploinsufficiency. Of particular importance, the overexpression of PKP2 in healthy cells and mice did not elicit alterations in desmosomal protein levels, nor did it induce a decline in cardiac function. These findings suggest that restoration of PKP2 levels in patients with ACM harboring a pathogenic PKP2 mutation could lead to a therapeutic benefit.

Results

PKP2 restoration enhances CM function

In an effort to assess the relevance of PKP2 haploinsufficiency for the ACM population, we conducted a comprehensive analysis of the Dutch ACM registry (https://www.acmregistry.nl/). Of over 228 index patients from the registry, 137 (60%) have a (likely) pathogenic variant underlying their ACM phenotype²⁴. Ninety-seven patients carry (likely) pathogenic PKP2 variants. Thirty-four of these have 8 different single nucleotide substitutions in PKP2 introducing a stop codon, originally described as c.235C>T; p.(Arg79*), c.258T>G; p.(Tyr86*), c.397C>T; p.(Gln133*), c.1848C>A; p.(Tyr616*), c.1951C>T; p.(Arg651*), c.2028G>A; p.(Trp676*), c.2203C>T; p.(Arg735*), c.2421C>A p.(Tyr807*)²⁵. These findings highlight the importance of PKP2 mutations, particularly nonsense mutations, in the pathogenesis and advancement of the disease.

To start examining the effects of PKP2 restoration in a human-relevant cell model, we utilized a patient-derived iPS cell line, harboring the pathogenic mutation PKP2 c.2013delC (PKP2^{c.2013delC/WT}) and generated isogenic control cells as a reference²⁰. To determine a suitable dosage, we delivered two different viral loads (v.l.) (v.l.1 = 0.5 × 10³ viral genomes (v.g.) per cell, v.l.2 = 5 × 10³ v.g. per cell) of an AAV6 vector expressing the wild-type human PKP2 gene under the control of a cytomegalovirus (CMV) immediate-early promoter (AAV6–PKP2) (Fig. 1a). Molecular analysis of the transduced iPS-cell-derived CMs revealed a load-dependent response in PKP2 protein levels, while transduction with 5 × 10³ v.g. per cell resulted in complete restoration of the PKP2 protein levels in the mutant CMs (Fig. 1c,d). To assess the effect of PKP2 restoration on desmosomal integrity, we next transduced the PKP2 mutant iPS-cell-derived CMs with either AAV6–PKP2 or an empty AAV6 vector (AAV6-ctr) and performed mRNA and protein analysis at 4 days and 7 days post-infection, respectively (Fig. 1b). As a reference, we also treated the PKP2-corrected iPS-cell-derived CMs with an equal titer of the AAV6-ctr construct. Real-time polymerase chain reaction (PCR) analysis did not show any effects of the exogenously delivered PKP2 on the mRNA expression levels of other desmosomal components, including JUP, DSP, DSG2 and DSC2 (Extended Data Fig. 1a–e). In contrast, western blot analysis revealed that restoration of PKP2 protein levels induced subsequent recovery of the desmosomal proteins, JUP and DSP, in the AAV6–PKP2-treated PKP2^{c.2013delC/WT} iPS-cell-derived CMs, while the DSC2 and DSG2 protein levels remained unaffected (Fig. 1e–j). Performing these experiments in additional iPS-cell-derived CM lines harboring a different pathogenic PKP2 mutation, PKP2 c.1854C>T (PKP2^c.1854C>T/WT) corroborated these findings, showing rescue of desmosomal protein content in response to viral delivery of human (h)PKP2 (Extended Data Fig. 2a–d). Together, these results indicated that restoration of physiological levels of PKP2 protein improved desmosomal assembly in human CMs harboring PKP2 truncating variants.

**Fig. 1: AAV-mediated restoration of PKP2 in *PKP2*^{c.2013delC/WT} mutant iPS-cell-derived CMs results in molecular rescue of desmosomal assembly.**

As severe ventricular arrhythmia is the hallmark of ACM, we then assessed whether our 2D in vitro model recapitulates the arrhythmic substrate caused by ion channel irregularities reported to be present in other relevant studies^26,27. We, therefore, conducted high-throughput automated patch clamp on iPS-cell-derived CMs to evaluate sodium currents (Fig. 2a). Our study included four distinct conditions, with each experiment conducted on two separate differentiations of CMs.

**Fig. 2: AAV-mediated restoration of PKP2 in *PKP2*^{c.2013delC/WT} mutant iPS-cell-derived CMs improves sodium conduction.**

In comparing sodium conduction between the mutant PKP2 CM line and the isogenic control line we were able to demonstrate a significant reduction in sodium conduction in the mutant CMs, thus successfully replicating the arrhythmic substrate characteristic of ACM (Fig. 2b,c). We subsequently sought to assess the potential of the AAV6–PKP2 gene replacement approach to ameliorate this sodium conduction impairment in the mutant CMs. Our data reveal that treatment with AAV6–PKP2 effectively diminished the differences in sodium conduction, restoring it to a level comparable to that of the isogenic control line (Fig. 2d–f).

These findings show that a gene replacement approach improves sodium conduction in mutant CMs, which might suggest it could rescue the arrhythmic phenotype in patients with ACM.

PKP2 restoration enhances contractile function in engineered human myocardium

As PKP2 plays a major role in intercellular binding and interaction, we next generated three-dimensional (3D) EHM composed of 70% iPS-cell-derived CMs combined with 30% human foreskin fibroblasts (HFFs) in a collagen type I-based hydrogel (Fig. 3a and Extended Data Fig. 3a,b). This model provides a more mature CM phenotype compared to 2D cell cultures, while enabling the sequential assessment of contractile properties at various stages of tissue maturation²⁸.

**Fig. 3: *PKP2*^{c.2013delC/WT} mutant EHM exhibit impaired contractile function compared to the isogenic control.**

Interestingly, PKP2^{c.2013delc/WT} tissues exhibited a significant reduction in expression of cardiac markers, including α-actinin-2 (ACTN2) accompanied by an increase in the expression levels of several fibroblast markers such as vimentin (VIM) and decorin (DCN) compared to the isogenic control (Extended Data Fig. 3c). Furthermore, the mutant tissues showed significantly higher levels of the stress-related gene NPPB (Extended Data Fig. 3c). Notably we did not detect the presence of fibrofatty markers in either of the two genotypes. Despite these transcriptional differences, our histological examination using Masson’s trichrome staining did not reveal substantial differences in fibrosis between the two genotypes at the tissue level (Extended Data Fig. 3d,e). Immunofluorescence assays on 6-week-old EHM revealed colocalization of PKP2 with the ID-specific marker N-cadherin (NCAD), however a more organized ID-like structure was more evident in the control tissues compared to the mutant tissues (Extended Data Fig. 3f). These results suggest that both the PKP2 mutant and isogenic control EHM develop a myocardium-like structure, albeit with differences in the organization of CM junctions, which appear to be improved in both lines compared to the 2D cultures.

Next, we sought to investigate the desmosomal composition of the mutant and isogenic control EHM. Molecular analysis by western blot confirmed a two-fold decline of PKP2 protein levels in PKP2^{c.2013delC/WT} EHM compared to PKP2^WT/WT EHM, accompanied by a reduction in JUP and DSP protein levels, which is consistent with the molecular phenotype observed in 2D cultures (Fig. 3b,d–f). The desmosomal cadherins DSC2 and DSG2 also showed reduced protein levels in the mutant tissues compared to the control tissues, which for DSC2 was not observed in our 2D model (Figs. 1i and 3c,g–h). This discrepancy might be attributed to the different levels of maturation that the two models present. It is also possible that the mechanical stress that is involved in EHM culture exacerbates the overall phenotype observed, therefore leading to a decline of DSC2 protein. In parallel, video-optical recordings of EHM contraction revealed a significant decline in contractile function (decrease in force of contraction) in PKP2 mutant EHM compared to the PKP2^WT/WT EHM (Fig. 3i–o). Other contractility properties, including beating frequency, contraction velocity and relaxation velocity, did not show significant differences between mutant and isogenic control tissues throughout maturation (Extended Data Fig. 4a–g).

To determine whether PKP2 restoration could improve the functional phenotype observed in mutant tissues, we transduced PKP2^{c.2013delC/WT} and PKP2^WT/WT iPS-cell-derived CMs with either the AAV6–PKP2 or the AAV6-ctr and reconstituted the CMs to EHM 3 days post-transduction (Fig. 4a,b). Our results showed successful PKP2 restoration in the PKP2^{c.2013delC/WT}–AAV6–PKP2 tissues at 6 weeks after EHM formulation, which was paralleled by a strong increase in JUP, DSP and DSG2 protein levels (Fig. 4c–g). Interestingly, DSC2 protein levels did not respond to PKP2 restoration (Extended Data Fig. 5a,b). Apart from the desmosomal proteins, we also assessed the levels of other ID-related factors including NCAD and αCAT. PKP2 restoration was sufficient to restore the levels of these proteins in the mutant tissues, potentially implying that a stronger junction is formed between the adjacent CMs (Fig. 4c,h,i). Importantly, PKP2 overexpression in the PKP2^WT/WT tissues did not lead to a significant increase in PKP2 or other desmosomal protein levels, potentially implying that an excess of PKP2 is being degraded by the cellular housekeeping machinery.

**Fig. 4: AAV-mediated restoration of PKP2 in *PKP2*^{c.2013delC/WT} EHM leads to an increase in desmosomal and junctional protein levels, which further translates into improved contractility.**

At a functional level, AAV6–PKP2 transduction resulted in a progressive improvement in contraction amplitude and normalization of the altered contraction kinetics, that is, an elongation of contraction and relaxation duration towards PKP2^WT/WT levels in the PKP2^{c.2013delC/WT} EHM from day 28, reaching statistical significance on day 42 post-casting (Fig. 4j–o). Noteworthily, PKP2 expression in the AAV6–PKP2-treated isogenic control EHM did not lead to any side effects on the contractile phenotype, further supporting our hypothesis that PKP2 overexpression is not detrimental to healthy CMs.

These data show that AAV-mediated restoration of PKP2 leads to molecular and functional benefits in ACM–EHM models with PKP2 haploinsufficiency.

In vivo PKP2 delivery restores CM junctions

To further increase clinical relevance, we made use of our previously described murine ACM model, harboring the mouse equivalent of the human pathogenic PKP2 c.2013delC variant (Pkp2^{c.1755delA/WT}). In brief, the Pkp2^{c.1755delA/WT} mice exhibit a significant reduction of cardiac desmosomal and adherens junction protein levels compared to Pkp2^WT/WT mice²⁰. To administer Pkp2 in vivo, we generated an AAV9 vector overexpressing the murine wild-type Pkp2 fused to a MYC epitope to enable detection of the exogenously delivered PKP2 (AAV9–PKP2) (Fig. 5a). As a proof of concept, we initially administered an AAV9–PKP2 dose of 2 × 10¹⁴ v.g. kg⁻¹ via intraperitoneal injections (IP) on 5-day-old Pkp2 mutant pups and their wild-type control, and collected tissues 2 weeks after injection (Fig. 5b). Efficiency of transduction was approximately 76% of CMs (Supplementary Fig. 6a,b). Immunohistochemistry on paraffin sections of the murine hearts revealed correct localization of the exogenous PKP2 within the IDs, indicating functionality of the exogenous protein (Fig. 5c). Immunoblot analysis showed successful restoration of PKP2 protein levels in the treated mutant mice, corresponding to a significant recovery of JUP and a partial recovery in DSP and DSG2 (Fig. 5d–i), whereas DSC2 was not responsive to different PKP2 levels (Extended Data Fig. 7a, b). These findings are in line with what has been observed in our EHM models, consistently indicating the re-building of the desmosomal complex upon PKP2 restoration in our preclinical ACM models.

**Fig. 5: AAV-mediated restoration of PKP2 in *Pkp2*^{c.1755delA/WT} pups and adult mice leads to the recovery of desmosomal and non-desmosomal components of the ID.**

Since ACM symptoms often become apparent during early adulthood, we investigated the effect of AAV9–PKP2 administration at a more clinically relevant stage. For this purpose, we intravenously injected Pkp2 mutant and wild-type mice at 2 months of age with a dose of 5 × 10¹³ v.g. kg⁻¹ of either AAV9–PKP2 or AAV9-ctr (efficiency of transduction: 71% CMs in the left ventricle (LV)). Two weeks later we isolated tissues for molecular analysis (Fig. 5j). PKP2 restoration in the mutant mice resulted in elevated levels of the desmosomal proteins, JUP, DSP and DSG2, whereas DSC2 did not show an increase (Fig. 5k,l–o and Extended Data Fig. 7c,d). Importantly, PKP2 restoration also led to a significant recovery of adherens junction proteins, including NCAD and α-CAT, in line with what we observed on the EHM models (Fig. 4c,h,i). These results point to the formation of stronger junctions in the CMs of Pkp2 mutant mice injected with AAV9–PKP2 and further support the therapeutic potential of PKP2 restoration in patients with ACM.

PKP2 restoration improves cardiac function in mice

To assess the consequences of long-term Pkp2 administration on cardiac function in vivo, we injected both wild-type and mutant 2-month-old mice with a single dose (3 × 10¹³ v.g. kg⁻¹) of either AAV9-ctr or AAV9–PKP2 and monitored heart function at 4, 8 and 12 months of age (Fig. 6a). We aged the mice up to 12 months as the mutant mice show progressive cardiac dysfunction with age, showing a significant decrease in the ratio of the early (E) to late (A) ventricular filling velocities (E/A ratio) and a significant increase in isovolumic relaxation time (IVRT) compared to the wild-type mice²⁰. Ten months after a single injection, immunohistochemistry indicated the presence of exogenous PKP2 at the IDs of 12-month-old mouse hearts (Fig. 6b and Extended Data Fig. 8). Western blot analysis of the hearts extracted from AAV9–PKP2-treated mutant mice demonstrated restoration of the PKP2 protein levels and a significant recovery of the desmosomal proteins JUP, DSP and DSG2 including also DSC2 which was not responsive in the more immature models (Fig. 6c–h and Extended Data Fig. 7e,f). Interestingly, in line with our observations in the PKP2^WT/WT EHM, wild-type mice injected with the AAV9–PKP2 virus did not show a significant increase in PKP2 levels and also the protein levels of the assessed desmosomal components remained unaltered. In accordance with our previous study, echocardiographic analysis at baseline, 4 months, 8 months and 12 months of age did not reveal any significant differences in ejection fraction (EF) (Fig. 6i), LV mass, LV end diastolic volume (LVEDV) and LV end systolic volume (LVESV) between mutant and wild type (Extended Data Fig. 9a–e). Long-term exposure to either AAV-ctrl or AAV–PKP2 did also not influence these cardiac functional and morphological measures, further supporting that Pkp2 overexpression does not impair cardiac physiology (Extended Data Fig. 9a–e). Importantly, assessment of E/A ratio and IVRT at the 12-month time point revealed a significant improvement in the AAV9–PKP2-treated mutant mice compared to the mutant mice injected with the AAV9-ctr (Fig. 6j,k). These data suggest that Pkp2 administration in the mutant mice reinstates physiological gene expression profiles, improves electrical and mechanical coupling and eventually restores cardiac function. Morphological evaluation did not demonstrate significant differences in heart weight/body weight and heart weight/tibia length ratio among the different experimental groups (Extended Data Fig. 9e,f). Moreover, protein analysis of tissues that are prone to receive AAV9–PKP2 particles including lung, liver, spleen and kidney showed moderate expression of the exogenous PKP2 within the liver, which could be explained by the high liver tissue tropism of AAVs²⁹ (Extended Data Fig. 6c–e). Overall, these data indicate that a single dose of AAV9–PKP2 is able to rescue the molecular and functional phenotype observed in the Pkp2^{c.1755delA/WT} adult mice without causing overt adverse effects in the heart and other organs.

**Fig. 6: A single dose of AAV9-PKP2 in 8-week-old *Pkp2*^{c.1755delA/WT} mice prevents cardiac dysfunction at 12 months of age.**

Discussion

PKP2 haploinsufficiency is often the underlying cause for ACM¹³. The data presented in this manuscript underscore the therapeutic potential of PKP2 gene replacement therapy by showing both molecular and functional rescue in human-relevant preclinical in vitro and in vivo models of ACM, with no apparent detrimental effects of PKP2 overexpression under healthy conditions. These are important findings that support the exploration of PKP2 gene therapy as a targeted therapeutic treatment option for patients suffering from ACM.

Our data show that PKP2 replacement under conditions of PKP2 haploinsufficiency can restore desmosomal integrity and CM function. These results are in line with a prior study by Inoue et al. that demonstrated AAV2-mediated PKP2 restoration in PKP2-deficient iPS-cell-derived CMs to restore other desmosomal components and enhance contractility³⁰. By treating PKP2^{c.2013delC/WT} EHM with AAV6–PKP2 we were also able to show that the functional improvement is longlasting and progressive since the treated tissues showed constant improvement in contractile function from day 28 until day 42 post casting. Moreover, PKP2 overexpression under healthy conditions did not appear to influence desmosomal integrity or CM function, which would be an important safety parameter for moving this technology into patients.

Next to our in vivo efficacy data, we provide evidence that a single systemic administration of AAV9–PKP2 in heterozygous mice harboring a pathogenic PKP2 variant leads to restoration of desmosomal assembly in the transduced CMs, which is accompanied by the recovery of intercellular junction proteins NCAD and α-CAT. In time, PKP2 gene therapy also prevents diastolic dysfunction in treated mutant mice at 12 months of age, while showing no effect in healthy wild-type littermates. This might be due to the lack of excessive PKP2 levels in both our wild-type in vitro and in vivo models. The lack of overexpression under healthy conditions might suggest the presence of a cellular compensatory mechanism correcting for an overdose of PKP2 protein. Similar observations have been reported for the exogenous expression of sarcomere genes in CMs. It has been demonstrated that viral expression of the hypertrophic cardiomyopathy-associated sarcomere gene myosin binding protein C (MYBPC3), can lead to the replacement of the endogenous protein without overexpression. This is due to UPS-mediated degradation of the excess amount of sarcomere proteins to preserve the stoichiometry of the sarcomere complex^31,32,33. So far it is unclear whether UPS-mediated protein degradation also plays a role in maintaining PKP2 protein at physiological levels in CMs. While here we show therapeutic benefit in a mutant Pkp2 mouse model, another recent study utilized gene therapy to correct the arrhythmic phenotype observed in a mouse model of ACM carrying a homozygous knock-in variant in the Dsg2 gene (Dsg2^−/−)³⁴. The authors were able to rescue the arrhythmic but not the fibrotic phenotype in the mutant mice, by AAV9-mediated administration of the truncated isoform of CX43 (GJA1-20k), which is responsible for correct localization of CX43 at the IDs.

In this study, we used murine models of PKP2 haploinsufficiency harboring the mouse equivalent of the known pathogenic mutation PKP2 c.2013delC to mimic the genetic condition of patients. However, there are significant differences between the electrophysiological properties of hearts of human and mice³⁵. For example, mouse hearts have weaker Ca²⁺ currents and stronger K⁺ currents³⁶ resulting in a shorter ventricular action potential duration and a heart rate about ten times higher than in humans. These differences, including the sparsity of detectable T-waves in mouse hearts³⁷, complicate the identification of cardiac defects in our models.

In vitro, we employed EHM as a valuable tool to study cardiac function. Nevertheless, EHM have their own set of limitations when it comes to modeling arrhythmias. Arrhythmias in ACM often coincide with the presence of abnormal collagen deposition and lipid droplets. In our PKP2 mutant tissues, we did not observe either of these characteristics. This observation aligns with expectations since these changes typically take years to develop in patients with ACM, whereas our tissue samples were cultured for only 1 month. Additionally, it is worth noting that only the CMs in our model were patient-specific, while the cardiac fibroblasts were not. There is an ongoing debate in the scientific literature regarding whether arrhythmias are primarily driven by CMs alone or in combination with other cell types^38,39,40. Many studies suggest that factors such as fibrosis, CM death and the infiltration of fibro-fatty tissue create the substrate for triggering arrhythmias⁴¹. However, there is also evidence indicating that arrhythmias in ACM can manifest before structural abnormalities become apparent⁴². Notably, our 2D iPS-cell-derived CM cultures exhibited sodium conduction irregularities, indicating the presence of a proarrhythmic substrate in our models that may require additional triggers for arrhythmia manifestation (Fig. 2b,c).

So far cardiac gene therapy has been challenging due to a relatively low targeting efficiency owing to the complex architecture of the heart, being composed of multiple cell types. Despite the predominantly non-integrative nature of AAV vectors⁴³, multi-year transgene expression after gene transfer has been documented in large animals and humans^44,45,46. The fact that we can detect the MYC epitope signal in murine CMs 10 months after the virus administration might be indicating that the promising ‘one and done’ AAV strategy employed in mice could potentially be feasible in human patients as well. In addition, our results indicate that PKP2 restoration in almost 70% of the CMs of the murine ventricles is sufficient to lead to a functional benefit in the mutant animals (Extended Data Fig. 6a,b). While AAV-based gene therapies have been shown to target the liver²⁹, our data indicated nonsignificant levels of exogenous PKP2 in the lung, spleen and kidneys of mice; however, moderate expression of cardiac PKP2 in the liver was observed (Extended Data Fig. 6d,e). Should it be desirable, liver targeting can be avoided by the use of a cardiac-specific promoter to drive PKP2 expression, the use of a cardiotropic capsid and the addition of a liver detargeting sequence. For example, AAV2i8, AAV2i8G9 and AAV-SASTG chimeras, some AAV serotype 9 variants, or vectors obtained through the screening of peptide display libraries or DNA-shuffled libraries^47,48 all display improved cardio tropism. A cardiotropic capsid (BNP116) obtained as an AAV2/AAV8 chimera⁴⁹ is currently used in a gene therapy clinical trial for heart failure⁵⁰.

ACM is an inherited heart condition characterized by progressive structural changes in the myocardium, which increase the risk of arrhythmias and impair contractile function. In the absence of curative options, current therapeutic interventions for patients suffering from ACM are aimed at controlling disease progression and include antiarrhythmic medications, use of implantable cardioverter defibrillators, catheter ablation and often lifestyle changes⁵¹. Approaches that interfere with the underlying cause of the disease, in this case PKP2 haploinsufficiency, could potentially be more efficacious in halting or reversing the disease course. However, identification of the patient population that would benefit from PKP2 gene therapy is currently challenging. Genetic testing could help to identify patients that could suffer from PKP2 haploinsufficiency⁵². However, this type of testing is usually reserved for individuals with a family history of cardiac disease or for those who have already been diagnosed with a cardiac condition and does not necessarily indicate what is happening at the protein level. Readouts in non-cardiac cells that express desmosomal proteins might provide a non-invasive tool to determine cardiac desmosomal protein content indirectly. Researchers have detected a significant reduction of JUP protein in buccal mucosa cells of patients with ACM compared to healthy counterparts^53,54. Additionally, the PKP2 levels in keratinocytes has been shown to mirror the cardiac level of PKP2¹³. Such a non-invasive, quantitative method to assess cardiac levels of PKP2, combined with a comprehensive evaluation by a healthcare provider, could effectively identify patients that would benefit from PKP2 gene replacement therapy.

So far, six AAV gene therapy products have already been approved for clinical use for non-cardiac indications, with over 1,400 patients having already been treated with onasemnogene⁵⁵, an intravenously administered AAV9 vector expressing the survival of motor neuron 1 (SMN1) protein for spinal muscular atrophy (SMA). After the initial drawback of the AAV1/SERCA2α gene therapy for heart failure, which failed in a large phase IIb clinical trial⁵⁶, gene therapy for the heart is also picking up speed again. AskBio takes advantage of a cardiotropic AAV vector with a chimeric AAV2/AAV8 capsid (BNP116)⁴⁹ to overexpress a constitutively active form of Inhibitor-1c (I-1c) in patients with heart failure (NCT04179643). Earlier this year, Rocket Pharmaceuticals has received regenerative medicine advanced therapy designation for an AAV9-based gene therapy to express the B isoform of the lysosomal associated membrane protein-2 (LAMP-2) to tackle Danon disease after the success of a phase I clinical trial (NCT03882437). On top of that, in May 2023 the same company received an Investigational New Drug (IND) approval for clinical gene therapy for PKP2 for ACM patients, a highly promising development for the field’s future. Tenaya Therapeutics has received fast track designation for a phase 1b clinical trial for MYBPC3 gene replacement therapy for hypertrophic cardiomyopathy (NCT05836259), utilizing an optimized AAV9 vector to package the full-length MYBPC3 gene. In addition, the company received orphan drug designation to its PKP2 gene therapy product candidate TN-401 for treatment in 2022 with preclinical results in preparation and a filing for IND in 2023.

Overall, the potential of gene therapy in treating genetic diseases represents a paradigm shift in the way we approach these conditions. Although PKP2 gene replacement therapy has demonstrated promise in preclinical studies using our knock-in mice, additional studies in suitable models are necessary to establish appropriate dosing regimens and determine potential safety issues. Heterogeneity in targeting of individual CMs could render the heart more susceptible to arrhythmias. A meticulous assessment of the risk of arrhythmias demands rigorous testing in larger animal models that accurately mimic human cardiac physiology in terms of heart rates, size, and function.

To conclude, our data support the notion that PKP2 gene therapy holds promise for improving the clinical outcomes of patients with ACM with PKP2 haploinsufficiency, and reinforce the promise of gene therapy for tackling heart disease.

Methods

Human iPS cell lines

The human PKP2 c.2013delC and PKP2 c.1849C>T iPS cell lines were provided by H.-S. V. Chen at University of California San Diego⁴⁰ and J. Wu at Stanford Cardiovascular Institute (supported by National Institutes of Health R24 HL117756), respectively.

Cell culture

Human iPS cells were grown on Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix-coated wells (Gibco, A1413302). The cells received fresh Essential 8 Medium (Gibco, A1517001) on a daily basis and were passaged at 80–100% confluency levels. In brief, medium was aspirated and dissociation of the cells was performed with TrypLE Express Enzyme (Gibco, 12605010) for 5 min at 37 °C. After incubation, 4 ml of Essential 8 Medium, supplemented with 2 μM thiazovivin (Sigma-Aldrich, 420220), was added to the dissociated cells and transferred to a 15 ml Falcon tube. Cells were centrifuged for 3 min at 300g. Lastly, cells were seeded at a density of 15,000 cells cm⁻² in Essential 8 Medium, supplemented with 2 μM thiazovivin. Medium was refreshed the next day with plain Essential 8.

CM differentiation

The differentiation protocol started when human iPS cells reached 80–90% of confluency (day 0). Cells were fed with RPMI-1640-Medium-GlutaMAX Supplement-HEPES (Gibco, 72400-021) supplemented with 0.5 mg ml⁻¹ human recombinant albumin (Sigma-Aldrich, A9731), 0.2 mg ml⁻¹ l-ascorbic acid 2-phosphate (Sigma-Aldrich, A8960) and 4 μM CHIR99021 (Sigma-Aldrich, 361559). After 48 h (day 2), medium was replaced by RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with 0.5 mg ml⁻¹ human recombinant albumin (Sigma-Aldrich, A9731), 0.2 mg ml⁻¹ l-ascorbic acid 2-phosphate and 5 μM IWP2 (Sigma-Aldrich, 681671). On day 4 and day 6, cells were refreshed with RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with 0.5 mg ml⁻¹ human recombinant albumin and 0.2 mg ml⁻¹ l-ascorbic acid 2-phosphate. From day 8 onwards, the medium of the cells was refreshed every 3–4 days with RPMI-1640-Medium-GlutaMAX Supplement-HEPES supplemented with B-27 Supplement (50×)-serum free (Gibco, 17504001).

CM purity

To assess the purity, 1 × 10⁶ iiPS-cell-derived CMs at 15 days of age were utilized. The CMs were subjected to centrifugation at 300g for 5 min. The medium was removed, and cells were washed with Dulbecco’s Phosphate-Buffered Saline (dPBS) (Gibco, 14190094). Following another centrifugation at 300g for 5 min, the dPBS was aspirated, and cells were fixed by adding 1 ml of ice-cold 70% ethanol while vortexing. After a subsequent centrifugation at 300g for 4 min, the fixative was removed. Permeabilization was achieved by resuspending the cells in blocking buffer consisting of PBS (pH 7.2–7.4), supplemented with 5% fetal bovine serum, 1% bovine serum albumin (Sigma-Aldrich, A9647-100G) and 0.5% Triton X-100 (Sigma‐Aldrich, 93443). Following a 10-min incubation at 4 °C, permeabilized cells underwent another centrifugation for 4 min at 300g, and the supernatant was aspirated. The cell pellet was resuspended in 100 μl of blocking buffer containing anti-Cardiac Troponin T antibody (Abcam, ab45932; 1:2,000) and incubated at 4 °C for 1 h. Subsequently, 500 μl of blocking solution was added, and cells were centrifuged again for 4 min at 300g. After aspirating the supernatant, cells were resuspended once more in 500 μl of blocking buffer, followed by another centrifugation step for 4 min at 300g. Cells were then resuspended in 100 μl of blocking buffer containing Alexa 488-anti-rabbit antibody (Thermo Fisher Scientific, A-21206; 1:4,000) and incubated for 30 min at room temperature. Next, 500 μl of blocking buffer was added, and cells were centrifuged for 4 min at 300g. The supernatant was discarded, and cells were resuspended again with 500 μl of blocking buffer, followed by another centrifugation for 4 min at 300g. Finally, the supernatant was aspirated, and cells were resuspended in 1 ml of dPBS for analysis by fluorescence-activated cell sorting (BD Biosciences, FACS Calibur).

Automated patch clamp

iPS-cell-derived CM collection proceeded with a PBS wash followed by a Versene wash before dissociation with TrypLE Express (all: Gibco) for 10 min at 37 °C. The cells were resuspended in divalent-free HBSS (Gibco) at 4 °C before measurement. Automated patch clamp experiments were conducted with the SyncroPatch 384 (Nanion Technologies GmbH) device with thin borosilicate glass, single aperture 384-well chips (NPC384T 1× S-type). Application of negative pressure (150–250 mbar) attained whole-cell configuration. I_Na recordings were performed at 0.5 Hz using a voltage step protocol with a holding potential of −80 mV followed by a hyperpolarizing step to −110 mV for 100 ms and a 300-ms test pulse to −20 mV. Pipette solution contained (in mmol l⁻¹): egtazic acid 10, HEPES 10, KCl 10, NaCl 10 and KF 110, pH 7.2 (with KOH). Bath solution contained (in mmol l⁻¹): HEPES 10, NaCl 80, N-methyl-d-glucamine (NMDG) 60, glucose 5, KCl 4, CaCl₂ 2 and MgCl₂ 1, pH 7.4 at 22–24 °C (with KOH). Currents were recorded with an integrated amplifier controlled by PatchControl 384 software and analyzed offline using DataControl 384 software (both: Nanion Technologies GmbH)⁵⁷.

EHM generation

EHM was generated according to the protocol published by Tiburcy et al.⁵⁸. In brief, patient-derived iPS-cell-derived CMs (purity >90%) were mixed together with HFFs (HFF-1, ATCC, SCRC-1041) at a ratio of 70:30. The cell mixture was resuspended in an appropriate volume of Collagen type I (Collagen Solutions, FS22024) diluted into RPMI 2× (Thermo Fisher Scientific, 51800-035). A total of 185 μl of the cell–collagen mixture was cast in each well of a 48 EHM multi-well plate (myrPlate-TM5; myriamed GmbH). The cast mixture was incubated for approximately 45 min at 37 °C and subsequently EHM medium freshly supplemented with TGFβ1 (Peprotech, AF-100-21C) was added. During the initial 3 days following the casting process, tissue medium was refreshed daily with EHM medium supplemented with TGFβ1. Subsequently, the tissue medium was replaced daily with EHM medium for the entirety of the experimental duration.

Contraction analyses

Contraction measurements were performed using video-optic recordings of EHM mediated pole bending in a myrPlate-TM5 culture format at 37 °C (ref. ⁵⁹). Data were recorded from spontaneously contraction EHM for at least 2 min at 50 fps at the indicated time points in a myrImager prototype (myriamed GmbH). Percent pole bending is reported as a surrogate for force of contraction (F); contraction and relaxation times are recorded from 20% to 80% peak contraction and 20% to 80% relaxation; contraction and relaxation velocities are reported as maximal and mininmal dF/dt.

Quantitative real-time PCR

For iPS-cell-derived CMs, RNA isolation was performed utilizing the RNeasy kit (Qiagen, 74104) as per the manufacturer’s guidelines. Complementary DNA synthesis was conducted using the iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR analysis was carried out using the CFX96 Realtime PCR system (Bio-Rad) and iQ SYBR Green (Bio-Rad) in accordance with the manufacturers’ instructions (Supplementary Table 1).

Mouse line generation

All animal studies carried out in this research adhered to the institutional guidelines and complied with the regulations set forth by the Animal Welfare Committee of the Royal Netherlands Academy of Arts and Sciences. Animal experiments were conducted upon approval by the ‘Animal Welfare Body Utrecht’ (I.v.D.) of the Royal Dutch Academy of Sciences and Arts (K.N.A.W.) and are in compliance with national legislation and institutional guidelines.

Mouse lines were maintained on C57B/6J background. Male and female mice were included in studies performed in pups, whereas studies in adult mice only included males.

Echocardiographic analysis

Isoflurane-anesthetized mice were subjected to transthoracic M-mode echocardiographic recordings while placed on a heat mat. The recordings were conducted using a Visual Sonic Ultrasound System connected to a 30 MHz transducer. For each mouse, three measurements were taken for various cardiac parameters including LVEDV, LVESV, end-diastolic interventricular septal wall thickness, end-systolic interventricular septal wall thickness, IVRT and the early (E) and late (A) ventricular filling velocities. The E and A values were utilized to calculate the E/A ratio. Cardiac function, specifically EF (%), was automatically determined by the software using the averaged values of the aforementioned parameters.

Histology and immunohistochemistry

Cardiac tissue from mice or EHM was collected and briefly washed in ice-cold PBS. After rinsing and weighing, they were fixed in 4% paraformaldehyde for 48 or 24 h, respectively, at room temperature. The fixed tissues were then embedded in paraffin and sliced at 4 μm. The sections were dewaxed and rehydrated. For immunohistochemistry, tissue sections were boiled for 20 min in either ethylenediaminetetraacetic acid (EDTA) buffer or sodium citrate buffer. They were then blocked for 45 min at room temperature using a solution containing 0.1% bovine serum albumin and 0.4% TWEEN20 dissolved in dPBS. The tissue was then incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies for 1 h at room temperature. DAPI diluted in dPBS (1:1,000) was used to stain the sections, and they were subsequently mounted with Mowiol and imaged using a Leica TCS SPE confocal microscope. Leica Application Suite (LAS X, version 3.30 or newer) was used for image acquisition, whereas image processing was performed with Fiji. Supplementary Table 2 presents information about all antibodies used for immunofluorescence assays.

Western blot

iPS-cell-derived CMs were dissociated using TrypLE Select Enzyme (10×) and collected in a 1.5-ml Eppendorf tube. The cells were then centrifuged at 300g for 5 min and resuspended in 1 ml of dPBS, followed by another centrifugation round with the same conditions. The cells were then lysed in RIPA buffer containing cOmplete EDTA-free Protease Inhibitor Cocktail (one tablet per 10 ml of RIPA buffer) and PhosSTOP (one tablet per 10 ml of RIPA buffer). For immunoblotting, 10–15 μg of protein extract was used. Horseradish peroxidase-coupled secondary antibodies were used in combination with the Clarity Western ECL Substrate kit for visualization. Immunoblots were imaged using an Amersham Imager 680RGB device and quantified with ImageQuant TL software v7.1 (GE Healthcare).

Immunoblotting was also performed on protein lysates from snap-frozen mouse tissue explanted from the ventricles. The tissue was lysed in approximately 150 μl of RIPA buffer as described above. Immunoblotting was performed using 20–50 μg of protein. In Supplementary Table 3 there are all details regarding antibodies used.

AAV delivery

AAV vectors (serotype 6 or 9) encoding human PKP2, murine Pkp2 or empty vector were generated in collaboration with the Giacca and Zentilin labs (Trieste, Italy).

We used 5 × 10³ v.g. per cell to infect hiPS-cell-derived CMs with AAV6. AAV9-ctr and AAV9–PKP2 were used for in vivo studies. A total of 3 × 10¹¹ v.g. per animal were injected intraperitoneally into 5-day-old pups using an insulin syringe with a 30-gauge needle. Adult mice were injected intravenously through the tail vein with 5 × 10¹² v.g. per animal (one single injection) using a syringe with a 26-gauge needle.

Statistical analysis

Data are presented as mean ± standard error of the mean. Statistical differences between two groups were tested by two-sided unpaired or paired Student’s t-tests. In case of three and more groups, one-way or two-way unrepeated or repeated-measures analysis of variance (ANOVA) with appropriate post-hoc testing was performed. The performed tests are specified in the respective figure legends. Statistical testing was performed with GraphPad Prism 9.5.1.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the findings in this study are available within the paper and associated files. Source data are provided with this paper.

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Acknowledgements

This study was funded by: Dutch CardioVascular Alliance with support of the Dutch Heart Foundation, DCVA2017-18 ARENA-PRIME (E.v.R). Dutch CardioVascular Alliance with support of the Dutch. Heart Foundation, DCVA2015-12 eDETECT (J.P.v.T.). Dutch CardioVascular Alliance. with support of the Dutch Heart Foundation, DCVA2018-30 PREDICT2 (J.P.v.T.). Vici grant from the Dutch Research Council (NWO), project 09150181910020 (E.v.R). European Research Council (ERC) Advanced Grant 787971 ‘CuRE’ (M.G.); British Heart Foundation (BHF) Programme Grant RG/19/11/34633 (M.G.); grants 825670 ‘CardioReGenix’ and 874764 ‘REANIMA’ from the European Commission Horizon 2020 programme (M.G.). German Research Foundation (DFG, VO 1568/3-1, VO 1568/4-1, SFB1002 A13, C04, S01, under Germany’s Excellence Strategy—EXC 2067/1-390729940 to N.V., M.T. and W.H.Z.), by the DZHK (German Center for Cardiovascular Research, N.V., M.T. and W.H.Z, 81 × 4300102, ‘DNAfix’ to N.V.), the German Federal Ministry of Education and Research (IndiHEART; 161L0250A; T.M. and W.H.Z.), and the Fondation Leducq (20CVD04) (E.v.R., M.G. and W.H.Z). We would like to acknowledge the Netherlands ACM/PLN Registry, a nationwide observational cohort study, involving individuals diagnosed with ACM and their relatives who are at risk of the condition. This registry is managed by the Netherlands Heart Institute (NHI) located in Utrecht, the Netherlands. The study strictly adheres to the guidelines outlined in the Code of Conduct and Data Usage in Health Research. We gratefully acknowledge Branimir Berecic and Daria Reher for for their invaluable contributions and dedication in training us on EHM construction and providing support in cell culture, respectively.

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Authors and Affiliations

Hubrecht Institute-KNAW and Utrecht University Medical Center, Utrecht, the Netherlands
Eirini Kyriakopoulou, Danielle Versteeg, Hesther de Ruiter, Ilaria Perini, Hoyee Tsui, Sebastiaan J. van Kampen & Eva van Rooij
Institute of Pharmacology and Toxicology, University Medical Center Gottingen (UMG), Göttingen, Germany
Fitzwilliam Seibertz, Yannic Döring, Malte Tiburcy, Tim Meyer, Niels Voigt & Wolfram H. Zimmermann
German Center for Cardiovascular Research (DZHK), partner site Göttingen, Göttingen, Germany
Fitzwilliam Seibertz, Yannic Döring, Malte Tiburcy, Tim Meyer, Niels Voigt & Wolfram H. Zimmermann
Cluster of Excellence ‘Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells’ (MBExC), University of Göttingen, Göttingen, Germany
Fitzwilliam Seibertz, Niels Voigt & Wolfram H. Zimmermann
Nanion Technologies GmbH, Munich, Germany
Fitzwilliam Seibertz
International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy
Lorena Zentilin & Mauro Giacca
Department of Genetics, University Medical Centre Utrecht, Utrecht, the Netherlands
van J. Peter Tintelen
German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany
Wolfram H. Zimmermann
Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP), Göttingen, Germany
Wolfram H. Zimmermann
British Heart Foundation Centre of Research Excellence, School of Cardiovascular Medicine & Sciences, King’s College London, London, UK
Mauro Giacca
Department of Cardiology, University Medical Center Utrecht, Utrecht, the Netherlands
Eva van Rooij

Authors

Eirini Kyriakopoulou
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Danielle Versteeg
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Hesther de Ruiter
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Ilaria Perini
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Fitzwilliam Seibertz
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Yannic Döring
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Lorena Zentilin
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Hoyee Tsui
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Sebastiaan J. van Kampen
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Malte Tiburcy
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Tim Meyer
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Niels Voigt
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van J. Peter Tintelen
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Wolfram H. Zimmermann
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Mauro Giacca
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Eva van Rooij
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Contributions

Conceptualization: E.v.R. and E.K. Data collection: E.K., D.V., H.R., I.P., F.S. and Y.D. Resources: H.T., S.J.v.K., L.Z. and J.P.v.T. Data analysis: E.K., H.R. and F.S. Funding acquisition: M.T., N.V., W.H.Z., M.G. and E.v.R. Software development: T.M. Writing—original draft: E.K. and E.v.R. Writing—review and editing: all authors

Corresponding author

Correspondence to Eva van Rooij.

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Competing interests

E.v.R. is a consultant for Tenaya Therapeutics and Novo Nordisk and is Chief Scientific Officer of Phlox Therapeutics. M.T., T.M. and W.H.Z. are inventors of patents related to the EHM technology and scientific advisors to myriamed GmbH. W.H.Z. is founder and shareholder of myriamed GmbH. M.G. is founder, consultant, member of the Board and equity holder in Purespring Therapeutics, Forcefield Therapeutics and Heqet Therapeutics. All other authors declare that they have no competing interests.

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Kyriakopoulou, E., Versteeg, D., de Ruiter, H. et al. Therapeutic efficacy of AAV-mediated restoration of PKP2 in arrhythmogenic cardiomyopathy. Nat Cardiovasc Res 2, 1262–1276 (2023). https://doi.org/10.1038/s44161-023-00378-9

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Received: 19 May 2023
Accepted: 27 October 2023
Published: 07 December 2023
Issue Date: December 2023
DOI: https://doi.org/10.1038/s44161-023-00378-9

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