Transcriptional regulation profiling reveals PPARA-mediated fatty acid oxidation as a novel therapeutic target in phospholamban R14del cardiomyopathy

doi:10.21203/rs.3.rs-1902254/v1

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Transcriptional regulation profiling reveals PPARA-mediated fatty acid oxidation as a novel therapeutic target in phospholamban R14del cardiomyopathy

https://doi.org/10.21203/rs.3.rs-1902254/v1

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Carriers of the R14del pathogenic variant in the phospholamban (PLN) gene develop severe cardiomyopathy with extracellular adipocyte infiltration and intracellular cardiomyocyte mitochondrial disturbances. However, the basis of this metabolic dysregulation tailoring potential treatment targets is unknown. Here, we present a combined approach of transcriptional regulation analysis in human primary tissue and validation in a unique long-term (160 days) matured human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. We demonstrate a dysregulated PPARA-mediated mitochondrial fatty acid oxidation (FAO) signalling in PLN-R14del hearts and hiPSC-CMs. PLN-R14del hiPSC-CMs also displayed a higher preference for glycolysis over FAO and presented limited flexibility in energy substrate switching leading to enhanced lipid droplet storage. By activating PPARA in PLN-R14del hiPSC-CMs using bezafibrate, we observed an improved mitochondrial structure and calcium handling function, further indicating the importance of FAO in the disease and the potential of PPARA agonists as a novel therapeutic strategy in cardiomyopathies.

Biological sciences/Physiology/Cardiovascular biology/Cardiovascular diseases/Heart failure

Biological sciences/Stem cells/Pluripotent stem cells

Biological sciences/Biotechnology/Regenerative medicine

Biological sciences/Biological techniques/Epigenetics analysis

Biological sciences/Systems biology/Regulatory networks

The R14del (c.40_42delAGA, p.Arg14del) pathogenic variant in the phospholamban (PLN) gene is associated with biventricular cardiomyopathy with a high risk of life-threatening ventricular arrhythmias, often presenting as dilated cardiomyopathy (DCM) or arrhythmogenic cardiomyopathy (ACM).^1,2 It explains a large proportion of Dutch DCM and ACM patients and has also been identified in many other countries.^3,4 PLN is a small phosphoprotein located in the cardiomyocyte sarcoplasmic reticulum and the nuclear membrane and is the major regulator of SERCA2a/ATP2A2 activity and calcium (Ca²⁺) cycling.¹ We and others have shown that mechanisms within the nuclear, endoplasmic/sarcoplasmic reticulum and mitochondrial network are impaired in PLN-R14del cardiomyopathy.^5–7 Despite these efforts, no effective treatment is available for PLN-R14del variant carriers to prevent disease development.

Macroscopically, PLN-R14del hearts show biventricular subepicardial fibrofatty tissue replacement of the myocardium, which is characterised by extensive interstitial fibrosis, adipocyte infiltration, and islands of isolated cardiomyocytes between adipocytes.^8–10 This adipocyte infiltration and fibrosis create an anatomical barrier resulting in the reentry of the electrical impulse and thereby an increased risk of fatal arrhythmias.^11,12 A novel adult zebrafish plna R14del model also displays this adipocyte accumulation and mitochondrial damage in the diseased myocardium,¹³ while the available murine models show mitochondrial impairment in the absence of adipocyte accumulation.^14,15 Mitochondrial dysfunction decreases ATP production, thereby opening the sensitive K⁺ channels on the sarcolemma channels, which reduces cardiomyocyte excitability and impairs electrical conduction in the heart.¹⁶ Besides altered electrical conduction, metabolic changes also affect cardiac ion channel gating, intracellular calcium handling, and fibrosis formation; all well-known aspects of PLN-R14del pathophysiology.¹⁶ However, the majority of studies have focused on calcium regulation by PLN,^14,17 little effort has been put into elucidating the basis for the metabolic aberrations in human PLN-R14del cardiomyopathy.

The presence of myocardial fibrofatty infiltration is accompanied by intracellular cardiomyocyte lipid abnormalities in cardiomyopathies in adults, such as those due to variants in PKP2 and PNPLA2.^18,19 Several forms of childhood cardiomyopathies caused by mutations in the mitochondrial fatty acid oxidation (FAO) pathway genes, such as HADHA, HADHB, CPT2, and ACADVL, also show intracellular cardiomyocyte lipid droplet storage and adipocyte infiltration in the myocardium and other organs.²⁰ A recent study has linked lipid droplet accumulation in PLN-R14del to the endoplasmic reticulum stress.⁶ However, little is known about the transcriptional regulation of this process and what (metabolic) factors influence lipid droplet accumulation.

A limited number of lipid droplets, which store unutilized fatty acids (FAs) by mitochondria for energy production, are present in healthy cardiomyocytes. However, an increased lipid droplet deposition is associated with impaired FAO.²¹ While FAs are the primary energy source in healthy adult cardiomyocytes, diseased cardiomyocytes switch from FAO to glycolysis for energy production, resembling the energy balance of the fetal heart.^22,23 Yet, this switch to fetus-like energy management is insufficient to meet adult energy consumption needs and it will disturb the capacity of FAO-based ATP synthesis, leading to further starvation of the heart and disease progression.²⁴ The impaired lipid metabolism could hereby contribute to the disease progression in PLN-R14del cardiomyocytes and warrant further investigation.

Here, we explored the role of transcriptional regulation on disturbed (lipid) metabolism in PLN-R14del cardiomyopathy. We showed that even after the removal of the dominant fibrofatty replacement in the subepicardial layer, the fingerprint of (lipid) metabolic dysregulation was still present in the remaining human myocardium. We further confirmed the disturbed (lipid) metabolism in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from PLN-R14del carriers. We also identified key transcription factors involved in the affected (lipid) metabolism, which can serve as promising targets for future therapeutic strategies. Furthermore, we examined the preference of energy sources and the metabolic flexibility in switching energy sources between PLN-R14del and wildtype hiPSC-CMs, including the effect of major energy substrate depletion. Finally, we employed two rescue methods, namely CRISPR/Cas9-based gene correction and an FAO-modulating compound (bezafibrate), to investigate the association of the PLN-R14del pathogenic variant with intracellular lipid accumulation, mitochondrial lipid metabolism and the Ca²⁺ handling properties. Collectively, our data on the negative effects of energy substrate dysregulation in PLN-R14del cardiomyopathy provide novel insights into the new therapeutic strategies and clinical practice (i.e. drug repurposing).

Full details are available in the Supplementary Material online. The study was approved by the local Biobank Research Ethics Committee and performed according to the Declaration of Helsinki.

Human cardiac tissues

Heart samples collected at autopsy or transplantation were obtained from a cohort of PLN-R14del variant positive patients (n = 6). Control hearts (n = 4) were obtained from donors that were not used for heart transplantation. To further elucidate the PLN-R14del-specific changes, hearts from patients with ischemic cardiomyopathy (n = 4) and non-ischemic cardiomyopathy based on pathogenic variants in genes encoding sarcomeric proteins (n = 6) were also included. The dominant fibrofatty replacement in the subepicardial layer, if present, was removed by a scalpel. The remaining compact myocardium was included in the study. An overview of cardiac tissues is presented in Table S1A.

Human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs)

Wildtype control, PLN-R14del patient line, isogenic control (PLN-R14del was corrected by CRISPR/Cas9), homozygous line (knock-in in the PLN-R14del patient line), and carrier line (hiPSC-CMs derived from an asymptomatic PLN-R14del carrier) were generated under approved IRB and SCRO protocols and have been described previously. An overview of hiPSC-CMs is presented in Table S1B.

Multi-omics analyses

Since we and others have shown the importance of histone acetylation changes in heart diseases, including their regulation of gene expression, we performed ChIP-seq for H3K27ac on cardiac tissues as described previously. Differentially acetylated chromatin regions were identified between control and PLN-R14del hearts, followed by enrichment analysis of transcription factor binding motif, gene to peak annotation, and pathway enrichment analyses. Additionally, a set of PLN-R14del-specific histone acetylation changes were identified by further comparison to two non-PLN-R14del-related cardiomyopathy groups. RNA was isolated from cardiac tissues and hiPSC-CMs, followed by polyA selection, library preparation, and sequenced as described previously. Differentially expressed genes were identified using Deseq2 and gene set enrichment analyses were performed. For a detailed description, please refer to the online Supplementary Material.

Functional assays

First, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay was used to examine the general metabolic activity of PLN-R14del and control hiPSC-CMs. Next, long-term cultured (77–159 days old) PLN-R14del and control hiPSC-CMs in three different culture media, including excessive access to FA or glucose, were included. Two main metabolic activities, namely FAO and glycolysis, were examined using a modified Seahorse XFe24 Extracellular Flux Analyzer. Etomoxir (ETO, a specific irreversible inhibitor of carnitine palmitoyltransferase 1) and 2-deoxy-D-glucose (2DG, a competitive glycolytic inhibitor) were applied to hiPSC-CMs separately to block FAO and glycolysis, respectively, and the dependence of hiPSC-CMs on these two pathways was determined by measuring the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). We also studied the the Ca²⁺ handling properties and the mitochondrial lipid metabolism by applying a FAO-modulating compound (bezafibrate) to PLN-R14del and control hiPSC-CMs for 24 hrs, followed by examining Ca²⁺ transient parameters and activities of mitochondrial trifunctional protein. For a detailed description, please refer to the online Supplementary Material.

Multimodal microscopy

In frozen cardiac tissues from PLN-R14del and healthy individuals, Oil Red O staining was performed to detect lipid droplets. Electron microscopy was performed to further investigate intracellular lipid droplets present in these tissues. Candidate genes obtained from the multi-omics dataset, including ATP2A2, TNNI3, and FAO-related proteins (HADHA and PPARA), were examined and confirmed using immunofluorescence staining. Mitochondrial-related proteins (PPARA, ATP5F1A, and HSP60) and lipid droplets were examined and compared between short-term (25 days) and long-term cultured (160 days) hiPSC-CMs from PLN-R14del and healthy individuals using immunofluorescence staining. Nile red staining was also performed to compare lipid accumulation between PLN-R14del, wildtype control, isogenic control, homozygous, and carrier hiPSC-CMs.

Identification of histone acetylation changes in PLN-R14del cardiac tissue

H3K27ac histone acetylation regulates gene transcription and contributes to phenotypic responses in heart diseases.²⁵ Therefore, we performed H3K27ac ChIP-seq to study histone acetylation changes in 6 PLN-R14del versus 4 control hearts (Fig. 1A and Table S1A). We identified 28,149 ± 9,538, and 25,721 ± 8,460 H3K27ac enriched regions within PLN-R14del and control hearts, respectively. We subsequently combined regions that were identified in at least two independent samples into a set of 23,356 regions to assess differentially acetylated regions between control and PLN-R14del groups. In total, 2,107 autosomal regions showed differential H3K27ac levels between PLN-R14del and control hearts (Fig. 1B and Table S2A). Compared to controls, regions with higher H3K27ac levels in PLN-R14del hearts are referred to as hyperacetylated regions (n = 1,149) and regions with lower H3K27ac levels in PLN-R14del hearts are referred to as hypoacetylated regions (n = 958, Fig. 1C and Fig.S1).

Genes annotated to differentially acetylated regions in PLN-R14del cardiac tissues are enriched in metabolic pathways

To identify genes potentially regulated by the differentially acetylated regions, we focused on regions in close vicinity to promoters and annotated genes 5,000 bases up- and downstream from the transcription start site of a gene as used previously (Fig. 1B).^25,26 Out of 968 hyperacetylated regions, 295 genes were identified in close vicinity to 251 hyperacetylated regions, and out of 1,149 hypoacetylated regions, 568 genes were identified in the close vicinity to 462 hypoacetylated regions (Fig. 1C, Table S2B and 2C). To examine which biological processes and pathways are affected, we performed gene set enrichment analysis using genes annotated to differentially acetylated regions. We observed that hyperacetylation-related genes were mostly involved in fibrosis, (cardiovascular) development, and chromatin assembly (Table S2D and Fig.S2), while hypoacetylation-related genes were related to metabolism (Fig. 1D and Table S2E).

The transcription factor binding motifs (TFBMs) overrepresented in hypoacetylated regions are enriched in metabolic pathways

To identify possible upstream acting transcription factors (TFs), which regulate genes involved in the pathogenesis of the disease, we studied the overrepresentation of TFBMs in differentially acetylated regions. By using the DNA sequences of all differentially acetylated regions in PLN-R14del versus control hearts (Fig. 1B), we detected enrichment in 202 TFBMs and annotated them to 200 TF-encoding genes (Table S3A). Consistently, several of the most enriched biological processes annotated to TFs pointed towards altered metabolism, such as adipogenesis and mitochondrial structure (Table S3B and Fig.S3). Notably, PPARA, a major regulator of cardiomyocyte lipid metabolism, particularly FAO, was also annotated from enriched motifs together with other interacting TFs (Fig. 1E). Therefore, we further investigated the localization of PPARA in cardiac tissues by immunofluorescence staining and observed a significant decrease in the nuclear PPARA signal of PLN-R14del cardiomyocytes versus the controls, whereas the PPARA signal in non-myocyte cells remained comparable between PLN-R14del and control hearts (Fig. 1F).

Hypoacetylated regions associated with metabolic pathways specific for PLN-R14del cardiomyopathy as compared to other cardiomyopathies

Besides non-failing control hearts, we also compared PLN-R14del hearts with other cardiomyopathies, including ischemic cardiomyopathy (n = 4) and non-ischemic dilated cardiomyopathy (sarcomeric group, n = 6, Fig. 1G). K-mean clustering analysis revealed four PLN-R14del-specific clusters when compared to other cardiomyopathy groups and the controls (Fig. 1H, Fig.S4, and Table S4A,). Genes located in the vicinity of these PLN-R14del-specific clusters were again highly enriched in metabolic signalling (Table S4B and 4C, and Fig.S5). Examples of metabolic genes, including HADHA/HADHB, SLC25A20, PDK2, and CPT1B, which were annotated in PLN-R14del-specific hypoacetylated clusters were shown in Fig. 1I. Combined, we detected differentially acetylated regions that distinguish PLN-R14del from other types of cardiomyopathies and they annotated metabolic-related genes were profoundly affected.

Enriched metabolic pathways by differentially expressed genes in PLN-R14del hearts

Besides differentially acetylated regions, we obtained 1,668 up- and 1,873 downregulated genes in PLN-R14del versus control hearts using RNA-seq (Fig. 2A and Fig. 2B, Table S5A). In line with the well-established suppression of SERCA2A/ATP2A2 at the protein level,⁶ we showed its suppression at the mRNA level for the first time. Additionally, metabolic genes, such as HADHA and HAHDB, which were annotated from PLN-R14del-specific hypoacetylated clusters, also showed significantly lower mRNA levels in PLN-R14del versus control hearts (Fig. 2C). We further demonstrated decreased HADHA and SERCA2A/ATP2A2 protein levels in PLN-R14del versus the control heart by immunofluorescence staining (Fig. 2D). Consistent with enriched biological processes and pathways by annotated genes from differentially acetylated regions, fibrosis, (cardiovascular) development, and chromatin assembly were enriched by upregulated genes (Table S5B) and metabolism (oxidative phosphorylation, ATP metabolic process, metabolic pathways, etc.) were enriched by downregulated genes (Fig. 2E and Table S5C).

Notably, among 200 TFs annotated from differentially acetylated regions, 39 TFs showed significantly altered mRNA levels in PLN-R14del versus control hearts, including 26 up- and 13 down-regulated TF-coding genes. Enriched protein-protein-interaction network by 39 TF-coding genes again suggests (negatively) affected metabolism (Fig. 2F). Thus, we identified a panel of upstream TFs and downstream targets in metabolic processes, which were disrupted in PLN-R14del cardiomyopathy.

Differentially expressed genes in PLN-R14del hiPSC-CMs suggest altered lipid metabolic pathways

Monolayers of hiPSC-CMs have been used to study the molecular mechanisms underlying several major cardiomyopathies, including ion-related, structural, and metabolic cardiomyopathies. However, the physiological immaturity of hiPSC-CMs severely limits their utility as a prediction model for adolescent genetic myopathies. To improve the cardiac immaturity limitation, we cultured hiPSC-CMs for 160 days in a maturation media designed to provide oxidative substrates adapted to the metabolic needs²⁷ (Table S6). These long-term cultured hiPSC-CMs showed well-developed structural and mitochondrial organization as stained by the sarcomeric and mitochondrial marker (Fig.S7A). Hereafter, we studied the transcriptome profiles in PLN-R14del and healthy control hiPSC-CMs (Fig. 3A and Table S1B).

First, we examined markers of cytoskeletal components (ACTN1, TNNT2, MYH7, and MYL2), ion channels (KCNA5 and KCNJ4), and mitochondrial components (ATP5F1A and HSP60), and lipid metabolism (PPARA, ACAT1, FABP3, and Nile red staining). In general, we observed an increase in mRNA levels of most markers from short- and long-term (25 and 160 days, respectively) cultured PLN-R14del and healthy control hiPSC-CMs and confirmed these observations at the protein level by immunofluorescence staining (Fig. 3B and Fig. 3C). Therefore, we extended the culture time of hiPSC-CMs in the following experiments, which showed an overall improved maturation status but a more distinguishable metabolism-phenotype between PLN-R14del and control hiPSC-CMs.

Since our data obtained from PLN-R14del hearts indicated a disrupted lipid metabolism, we cultured control and PLN-R14del hiPSC-CMs to further elucidate the metabolic activities in three culture media containing different amounts of glucose and lipids, which are the two main metabolic substrates for cardiomyocytes (Fig. 3A). In total, we identified 952, 1,321, and 2,104 differentially expressed genes in PLN-R14del versus control hiPSC-CMs cultured in the maturation, the glucose-rich, and the lipid-rich medium, respectively (Table S7A-C). Notably, regardless of culture media, pathway enrichment analyses using downregulated genes in PLN-R14del versus control hiPSC-CMs consistently pointed towards metabolic activities, such as oxidative phosphorylation/GO:0006119 (Fig. 3D, Fig.S8A-C, and Table S7D-F). However, it is important to note that the metabolic genes involved in the enriched biological processes (i.e., oxidative phosphorylation), which were shared among three conditions, were not the same (Fig.S8D-F). Similarly, regardless of culture media, pathway enrichment analyses using upregulated genes in PLN-R14del versus control hiPSC-CMs consistently pointed towards fibrosis and (cardiovascular) development, which were in line with the results obtained from the ex vivo human cardiac tissues (Table S7D-F).

Disturbed fatty acid oxidation (FAO) and metabolic flexibility in PLN-R14del hiPSC-CMs

Besides the downregulated transcriptional regulation of lipid metabolism in PLN-R14del cardiomyopathy, a significantly lower cellular metabolic activity/viability was also observed in PLN-R14del versus control hiPSC-CMs (Fig. 4A). This suppression remained when excessive glucose or FAs were given to the cells (Fig.S7B-C). To further elucidate whether the obtained transcriptomic data could predict affected metabolism in PLN-R14del cardiomyopathy, we compared the FAO metabolism, a key metabolic program in cardiomyocytes (Fig. 4B), by evaluating mitochondrial respiration via ETO-inhibited FAO and 2-DG-inhibited glycolysis using the Seahorse analysis (Fig. 4C).

In the maturation medium, which contains both glucose and FAs, we observed a comparable oxygen consumption rate (OCR) between PLN-R14del and control hiPSC-CMs at the baseline level and after ETO-inhibited FAO (Fig. 4E), suggesting a similar FAO-dependence of both groups. However, after blocking glycolysis by 2-DG, we observed an increased OCR in control hiPSC-CMs, whereas the OCR of PLN-R14del hiPSC-CMs continued to decline significantly, suggesting control hiPSC-CMs are less dependent on glycolysis and have more metabolic adaptive characteristics than PLN-R14del hiPSC-CMs.

Similarly, in the glucose-rich medium, OCR was comparable between PLN-R14del and control hiPSC-CMs at the baseline level (Fig. 4F). After blocking glycolysis, a significantly higher OCR was observed in control versus PLN-R14del hiPSC-CMs, once again suggesting better metabolic flexibility and substrate utilisation in control hiPSC-CMs.

Notably, in the lipid-rich medium, the basal OCR was significantly lower in PLN-R14del versus control hiPSC-CMs (Fig. 4G), implying an impaired FAO in the PLN-R14del hiPSC-CMs at the beginning. Although OCRs of both PLN-R14del and control hiPSC-CMs decreased after FAO- and glycolysis-blockages, OCRs remained significantly lower in PLN-R14del hiPSC-CMs than in the controls. These findings suggest that despite the presence of excess lipids in the medium, PLN-R14del hiPSC-CMs are less capable of utilizing their FAO metabolism to produce the required energy, therefore being more glycolysis-dependent.

Higher glucose dependency in PLN-R14del hiPSC-CMs

Glucose metabolism, another major metabolic program in cardiomyocytes (Fig. 4B), was also studied. We measured the extracellular acidification rate (ECAR) to study the activity of the glycolytic pathway in PLN-R14del and wild-type hiPSC-CMs by manipulating the FAO and glucose metabolism using ETO and 2-DG respectively (Fig. 4D).

In the maturation medium, basal ECAR levels were comparable between both groups, but it became significantly lower in PLN-R14del versus control hiPSC-CMs after FAO- and glycolysis-blockages (Fig. 4H), indicating a higher glycolysis dependency of PLN-R14del hiPSC-CMs. A significantly higher glycolytic reserve was also observed in PLN-R14del hiPSC-CMs than in the controls.

In the glucose-rich medium, ECAR levels remained comparable between both groups at the basal level, after blocking FAO, and after blocking glycolysis (Fig. 4I). However, the decline of ECAR after glycolysis-blockage was more profound in PLN-R14del versus control hiPSC-CMs, suggesting a higher glycolysis-dependence and a higher glycolytic reserve of PLN-R14del hiPSC-CMs.

In the lipid-rich medium, basal ECAR levels were comparable between both groups (Fig. 4J). Interestingly, a higher ECAR was shown in PLN-R14del versus control hiPSC-CMs after FAO-blockage, which decreased profoundly after glycolysis-blockage, again, confirming the higher glycolysis-dependency in PLN-R14del hiPSC-CMs. These results imply our in vitro maturation-induced PLN-R14del hiPSC-CM mimics the heart failure-related metabolic alterations consisting of the energy production reduction by mitochondria through oxidative phosphorylation and an increase in (anaerobic) glycolysis.

Intracellular lipid droplet accumulation is a key feature of PLN-R14del cardiomyopathy

Data acquired from the cardiac tissues and/or hiPSC-CMs at DNA, RNA, protein, and functional levels consistently pointed towards altered metabolism in PLN-R14del versus control groups, particularly FAO. Since lipid accumulation is known as the hallmark of impaired FAO, we examined the lipid accumulation in cardiac tissues and hiPSC-CMs.

First, we performed a digital quantification in heart slices assessing the percentage of adipose tissue, which showed an increased adipocyte deposition in PLN-R14del versus control hearts (Fig.S8). Next, we used Nile red to localise intracellular lipid droplets in snap-frozen and paraffin-embedded tissues and observed a more frequent perinuclear accumulation of lipid droplets in PLN-R14del cardiac tissue than in the control (Fig. 5A). Transmission electron microscopy revealed a significantly lower mitochondrial length (aspect ratio) and a significantly higher accumulation of intracellular lipid droplets in PLN-R14del versus control hearts (Fig. 5B and Fig. 5C), suggesting impaired mitochondrial FAO. In line with the hearts, highly accumulated intracellular lipid droplets were observed in PLN-R14del versus control hiPSC-CMs, regardless of different culturing media (Fig. 5D). Combined, these findings suggested intracellular lipid droplet accumulation is a key feature in PLN-R14del cardiomyopathy.

CRISPR/Cas9-based correction of PLN-R14del attenuated intracellular lipid accumulation

We used CRISPR/Cas9-based gene editing to correct PLN-R14del mutation²⁸ and observed a significant reduction of intracellular lipid droplets in R14del-corrected hiPSC-CMs (isogenic control, Fig. 6A). Interestingly, hiPSC-CMs derived from an asymptomatic PLN-R14del carrier and the homozygous hiPSC-CMs showed a comparable amount of the lipid droplets as in PLN-R14del hiPSC-CMs. These findings suggested a tight relationship between PLN-R14del mutation and impaired lipid metabolism, thereby leading to intracellular lipid droplet accumulation.

PPARA-targeted drug increased Ca²⁺ handling and mitochondrial trifunctional protein levels in PLN-R14del hiPSC-CMs

We also applied a PPARA agonist (bezafibrate) to further investigate PPARA-mediated FAO in PLN-R14del cardiomyocytes. First, we measured the Ca²⁺ transient in control and PLN-R14del hiPSC-CMs with and without bezafibrate treatment. We observed significantly increased rise and decay time in treated versus untreated PLN-R14del hiPSC-CMs (Fig. 6B), whereas the treatment did not affect Ca²⁺ handling in control hiPSC-CMs. Next, we examined HADHA and HADHB levels, encoding the mitochondrial trifunctional protein involved in the FAO pathway, which showed suppressed histone acetylation and transcriptional levels in PLN-R14del versus control hearts (Fig. 1I and Fig. 2C). We confirmed the suppression of HADHA and HADHB in long-term cultured untreated PLN-R14del cardiomyocytes and further showed elevated HADHA and HADHB levels in bezafibrate-treated PLN-R14del cardiomyocytes (Fig. 6C). Whereas, the treatment did not alter HADHA and HADHB levels in control hiPSC-CMs. Combined, these findings suggested disturbed HADHA/HADHB-involved FAO in PLN-R14del hiPSC-CMs, the potential association between PPARA-mediated FAO and Ca²⁺ handling, and the role of PPARA as a promising therapeutic target in PLN-R14del cardiomyopathy.

In this study, we provide new information on changes in chromatin activity and global transcriptional regulation in human myocardium obtained from patients with PLN-R14del cardiomyopathy compared to other types of cardiomyopathy and healthy controls. Multi-omics integration points to the inhibited mitochondrial function and (lipid) metabolism in PLN-R14del hearts based on the changed histone acetylation levels of annotated gene promoters, predicted TFBMs and altered gene expression. These datasets will serve as the basis for upcoming biomarker and novel therapeutic strategies.

We particularly focused on the inhibited PPARA-mediated FAO in PLN-R14del hearts. PPARA is a key TF that regulates cardiac lipid metabolism to enhance FAO.²⁹ It is predominantly located within the nucleus,³⁰ however, external signals and pathways regulate the transport of PPARA from the nucleus to other subcellular compartments and mediate the biological functions of PPARA.³¹ We obtained the enriched TFBM of PPARA in differentially acetylated promoters between PLN-R14del and controls. Although the histone acetylation of PPARA promoter and mRNA level of PPARA remained comparable between PLN-R14del and control hearts, we showed a significant decline of PPARA signal inside cardiomyocyte nuclei in PLN-R14del hearts. Taken together, the loss of nuclear PPARA in PLN-R14del cardiomyocytes suggested suppressed PPARA-mediated biological processes that take place inside nuclei, including substrate inflexibility promoting FFA uptake and lipid accumulation, and ensuing cardiovascular complications.

The promoters of PPARA-regulated downstream targets in lipid metabolism, such as HADHA, HADHB, MLYCD, and PNPLA2,^32,33,34 showed suppressed acetylation levels in PLN-R14del hearts. Furthermore, by comparing PLN-R14del hearts with non-PLN-R14del-related cardiomyopathies, we identified PLN-R14del-specific regions and many annotated genes in these regions also involved in lipid metabolism (e.g. AGPAT2, HADHA, HADHB, MLYCD, PLPP1, and PTGDS). Additionally, several of these metabolic genes also showed decreased mRNA levels in PLN-R14del versus control hearts, including HADHA and HADHB. In line with these omics-based data, we also observed accumulated lipid droplets and abnormal mitochondrial morphology in PLN-R14del hearts. Combined, the impaired PPARA-regulated FAO could play a critical role in PLN-R14del cardiomyopathy.

A recent study also showed that 18–33 days old PLN-R14del engineered heart tissues had impaired energy metabolism reflected at the protein level, including suppressed FAs metabolism and accumulation of lipid droplets.⁶ Here, we further explored and identified affected genes in the metabolic regulation in long-term cultured PLN-R14del and control hiPSC-CMs (> 110 days). We demonstrated for the first time that PLN-R14del hiPSC-CMs displayed a lower FAO profile than the controls at both mRNA and functional levels, and this suppression pattern remained consistent even though PLN-R14del hiPCS-CMs were given excessive amounts of FAs or glucose, indicating the profoundly impaired lipid metabolism. FAO changes serve as an indicator of an early adapted or maladapted metabolic response.³⁵ Normally, the majority of the energy demand of the heart comes from mitochondrial FAO, especially free circulating FAs.³⁶ In contrast, diseased cardiomyocytes suffer from a decreased FAO and an increased intracellular lipid accumulation resulting in lipotoxicity and cell death.^37,38 We also showed lipid accumulation and a decreased cell viability of PLN-R14del hiPSC-CMs, further suggesting the importance of impaired FAO as a key pathological mechanism underling PLN-R14del cardiomyopathy.

Notably, we observed enhanced Ca²⁺ handling properties and elevated mitochondrial trifunctional protein levels (HADHA and HADHB) in PLN-R14del hiPSC-CMs after bezafibrate treatment, a PPARA-targeting agonist. Several FDA/EMA-approved PPARA agonists, including bezafibrate, have shown protective effects on cardiomyopathies by restoring the FA metabolism.^39,40 However, to the best of our knowledge, we showed for the first time, the potential of bezafibrate in re-activating mitochondrial FAO and improving Ca²⁺ transients, which provides a novel strategic path for developing precision medicine for PLN-R14del patients, such as targeting FAO upstream regulators (i.e. PPARA).

Besides FAO suppression, the activation of glycolysis-related genes was shown in murine cardiomyocytes carrying another PLN pathogenic variant (p.Arg9Cys).⁴¹ We also showed PLN-R14del hiPSC-CMs exhibited a preference for glucose utilisation. Increased glucose utilisation, which further inhibits FAO by malonyl coenzyme A-mediated inhibition, has been shown in cardiomyocytes from hypertrophic cardiomyopathies and failing hearts.^33,42 The switch from FAs to glucose utilisation can be due to its faster uptake in the cells and the lower oxygen consumption.⁴³ Like lipotoxicity, increased glucose levels can have deleterious effects on cardiomyocytes by introducing oxidative stress-related cell death and decreasing contractile force.^44,45 Thus, the glucose-dependent energy metabolism is associated with the progression of cardiac dysfunction and could be an early pathological process in PLN-R14del hearts.⁴²

Last but not least, we also observed reduced metabolic flexibility in PLN-R14del hiPSC-CMs. Healthy cardiomyocytes have the flexibility of switching substrates for energy production under different conditions.⁴² This metabolic flexibility is a critical factor for maintaining normal cardiac function and preventing the progression of diseased hearts.⁴⁶ Impaired metabolic flexibility has also been observed in mouse cardiomyocytes with the depletion of Ryr2, which show reduced mRNA levels of key regulators in the FA metabolism (e.g. Ppargc1a, Pparα, Pparγ, and Klf15) as well as the glucose metabolism (e.g. Glut4 and Pck1).⁴⁷ Therefore, restoring balanced metabolic activity is beneficial for PLN-R14del cardiomyopathy.

It is important to note that histone acetylation and transcriptome changes in PLN-R14del versus control hearts are derived from both cardiomyocytes and non-myocyte cell types, such as endothelial cells and fibroblasts that are abundant in the heart.⁴⁸ Non-myocyte cell types in the heart also contribute to the disease progression.⁴⁹ Therefore, the responsible cell type(s) or mechanisms for the adipocyte infiltration by either the activation of the already existing pool of adipocytes or transdifferentiation of (cardiac) cells into adipocytes remain unclear. However, we have previously shown that the majority of the bulk data came from cardiomyocytes when compared to 11 non-myocyte cell types in both inherited and acquired heart disease.^25,26 The impaired mitochondrial FAO indicated by the bulk data was validated in PLN-R14del hiPSC-CMs at transcriptional and functional levels, highlighting the possibility of FAO abnormalities as early pathological signs in PLN-R14del cardiomyocytes. Nevertheless, future studies should also focus on investigating interactions between cardiomyocytes and non-myocyte cells during the cardiac energy rearrangement as well as the impact of lipid disturbances in PLN-R14del cardiomyopathy.

In conclusion, we revealed valuable information on the histone acetylation activities and the transcriptome regulation in PLN-R14del hearts and PLN-R14del hiPSC-CMs when compared with controls. Integrating this data, we demonstrated a disturbed energy metabolism in PLN-R14del and identified upstream TFs regulating impaired FAO. CRISPR-Cas9-based therapy to correct PLN-R14del and bezafibrate treatment to re-active mitochondrial FAO further illustrated the tight relationships between the mutation, imapired FAO, lipid accumulation, and Ca²⁺ handling and sheld light to future therapeutic strategies for PLN-R14del patients.

ACKNOWLEDGMENTS

We are very thankful to the patients and their families for providing valuable cardiac tissue for research purposes. We further thank Marc P. Buijsrogge (in memoriam) for collecting cardiac tissues and Joyce van Kuik, Erica Sierra-de Koning and Petra van der Kraak for technical assistance with tissue processing. We thank Utrecht Sequencing Facility for providing sequencing service and data. Utrecht Sequencing Facility is subsidised by the University Medical Centre Utrecht, Hubrecht Institute, Utrecht University and The Netherlands X-omics Initiative (NWO project 184.034.019). We thank Prof. Joseph C. Wu from Stanford University for providing lines SCVI-111 and SCVI-273. Both first shared authors contributed equally and have the right to put their names forward for CV purposes.

SOURCES OF FUNDING

This work was supported by the Dutch PLN patient organisation Foundation PLN (JYP, RGCM, MH, JMIHG, FWA), Leducq grant (CURE-PLaN no. 18CVD01 to JYP, RGCM, JC, DF, IK, MM, PAD, JPvT,, MH, FWA), the NWO VENI grant (no. 016.176.136 to MH), ZonMW Open Competition grant (CONTRACT no. 09120012010018 to KGH, FWA, MH), National Institute of Health grants R01 LM010098 (MH, FWA) and R01HL152055, P01HL141084 (MM), Dutch Cardiovascular Alliance (DCVA) grant (DOUBLE-DOSE no. 2020B005 to MH, FvS, FWA, JPvT), ERA-CVD grant (SCALE no. 2019T109 to JYP, FvS, MH), Netherlands Foundation for Cardiovascular Excellence (CC), two NWO VIDI grants (no. 91714302 to CC and no. 016096359 to MCV), the ErasmusMC fellowship grant (CC), the RM fellowship grant of the UMC Utrecht (CC), and UCL Hospitals NIHR Biomedical Research Centre grant BRC86A (FWA), Horizon2020 ERC-2016-COG EVICARE (725229), Horizon 2020 BRAV3 (SC1-BHC-07-2019), NWO-TTP program (Harvey 2021/TTW/01038252), and ZonMW-PSIDER (ZonMw file No: 40-46800-98-018) to JS.

DISCLOSURES

None

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There is NO Competing Interest.

FigureS1.png
Supplementary Figure 1: A) Heatmap representing differentially acetylated regions between PLN-R14del and control group. B) Flowchart representing the detection and annotation of differentially acetylated regions in PLN-R14del patients versus controls. C) Manhattan plot showing the differentially acetylated regions in PLN-R14del versus control hearts. Green: hyperacetylated regions; Red: hypoacetylated regions; Black: non-significant regions.
FigureS2.png
Supplementary Figure 2: A) Four differentially acetylated regions depicted using the UCSC Genome Browser. In each example, the region overlaps with the putative promoter (upstream region, 5’UTR, first exons in region tracks) and shows a significant difference between PLN-R14del patients and controls (dot plots). Arrow beginning indicates gene transcription start site. AcS = acetylation 437 signal. ENCODE = publicly available ENCODE consortium data default display. H3K27ac = layered H3K27ac ChIPseq data in 6 cell types (GM12878 - red, H1-hESC – orange, HSMM – green, HUVEC – light blue, K562 – dark blue, NHEK – purple, NHLF – pink). DHS – DNaseI hypersensitivity clusters in 125 cells, TFs = ChIPseq for 161 TFs. B) Selected examples of enriched protein-protein-interaction networks using protein-coding genes annotated to differentially annotated regions.
FigureS3.png
Supplementary Figure 3: A) Selected examples of protein-protein-interaction networks using annotated transcription factors (TFs) that were predicted to binding to motifs enriched in differentially acetylated regions. B) Selected examples of TFs with both differentially acetylated promoter as well as their corresponding motifs enriched in differentially acetylated regions. Dot plots represent the acetylation signal (AcS) measured for each sample.
FigureS4.png
Supplementary Figure 4:PLN-specificity analysis of differentially acetylated regions (ChIP-seq): K-mean clustering of PLN R14del cardiomyopathy (red), healthy controls (green), ischemic cardiomyopathy (dark blue) and sarcomeric non-ischemic cardiomyopathy (light blue) based on H3K27ac signal was used to partition the regions into 12 different clusters. A) K-mean clusters showing PLN-specific hyper- and hypoacetylation regions when compared with controls and hearts with ischemic or non-ischemic dilated cardiomyopathy in general; B) Examples of 8 PLN-specific regions and genes in the vicinity of these regions (LINC01140, WIPF1, OSR1, IFFO2, LTBP1, GCGR, or CNNM4). No genes have been annotated in the vicinity of region 1689.
FigureS5.png
Supplementary Figure 5:Genes annotated to PLN-specific differentially acetylated regions (ChIP-seq): As an effort to identify differentially acetylated regions in the vicinity of genes involved in lipid metabolism possibly linked to fibrofatty tissue replacement in PLN group, this figure shows selected examples of STRING protein database used to detect interacting proteins annotated to differentially annotated regions as part of the GO term ‘lipid metabolic process’ (GO:0006629). Only the highest confidence interactions are displayed. Disconnected nodes were removed from the network image. Dot plots represent the acetylation signal (AcS) measured for each sample.
FigureS6.png
Supplementary Figure 6: Differentially expressed genes between patients and controls (RNA-seq): A) Top 5 enriched Pathway and GO: Biological process terms by upregulated genes in PLN versus control hearts. Notably, the most upregulated genes are part of fibrosis pathways and extracellular matrix organization. B) Top 5 enriched Pathway and GO: Biological process terms by downregulated genes in PLN versus control hearts. Note the enrichment of metabolic pathways. C) Transcription factor (TF) coding genes, which were predicted to bind to enriched motifs in H3K27ac ChIPseq differentially acetylated regions, with altered mRNA expression levels between PLN and control hearts.
FigureS7.png
Supplementary Figure 7:A) Representative immunofluorescence images showing the welldeveloped mitochondrial organization (stained by Tomm20 in red) and sarcomere (stained by α-actinin in green) in long-termed cultured control and PLN-R14del hiPSC-Cms. Nuclei were stained by DAPI (blue). B) Seahorse XF Cell Mito Stress Test demonstrated a lower oxygen consumption rate (OCR) in PLN-R14del hiPSC-CMs than the controls, suggesting an impaired mitochondrial function in PLN-R14del hiPSC-CMs.
FigureS8.png
Supplementary Figure 8:A) Enrichment analysis using downregulated genes in PLN-R14del versus control hiPSC-CMs cultured in the maturation medium pointed towards altered metabolic-related biological functions. B) Enrichment analysis using downregulated genes in PLN-R14del versus control hiPSC-CMs cultured in the glucose-rich medium pointed towards altered metabolic-related biological functions. C) Enrichment analysis using downregulated genes in PLN-R14del versus control hiPSC-CMs cultured in the lipid-rich medium pointed towards altered metabolic-related biological functions. D) STRING protein-protein-interaction network highlighting oxidative phosphorylation-related protein-coding genes (red) that were downregulated in PLN-R14del hiPSC-CMs cultured in the maturation medium. E) STRING protein-protein-interaction network highlighting oxidative phosphorylation-related protein-coding genes (red) that were downregulated in PLN-R14del hiPSC-CMs cultured in the glucose-rich medium. F) STRING protein-protein-interaction network highlighting oxidative phosphorylation related protein-coding genes (red) that were downregulated in PLN-R14del hiPSC-CMs cultured in the lipid-rich medium.
FigureS9.png
Supplementary Figure 9:A) Histological slides derived from the same tissue region as used for H3K27ac ChIP-seq and RNA-seq stained with Masson’s trichrome (red – cardiomyocytes, blue – fibrosis, white – fatty tissue). LV = left ventricle; Sept = septum. Black line indicates the removal of fibro-fatty tissue replacement prior to ChIPseq. B) Schematic overview showing the mean fatty tissues in control and PLN-R14del hearts using a colour scale. Epicardial fat was excluded from the analysis. LV = left ventricle, RV = right ventricle, Post. = posterior, Ant. = anterior.
FigureS10.jpg
SUPPLEMENTARYMETHODS.docx
TableS1.xlsx
Table S1: A) An overview of cardiac tissues, B) An overview of hiPSC-CM lines.
TableS2.xlsx
Table S2: A) Differentially acetylated regions between PLN-R14del and control hearts (dataset 1), B) genes annotated to hyperacetylated regions in PLN-R14del hearts (dataset 2), C) Genes annotated to hypoacetylated regions in PLN-R14del hearts (dataset 2), D) Gene enrichment analysis of annotated genes from hyperacetylated regions in PLN-R14del versus control hearts, E) Gene enrichment analysis of annotated genes from hypoacetylated regions in PLN-R14del versus control hearts.
TableS3.xlsx
Table S3: A) Enriched transcription factor binding motifs in differentially acetylated regions and TFs coding genes (dataset 3), B) Gene enrichment analysis of transcription factor coding genes.
TableS4.xlsx
Table S4: A) Altered regulatory regions that were specific in PLN-R14del hearts when compared with the controls and hearts with ischemic or non-ischemic dilated cardiomyopathy, B) Annotated genes from the hyper- and the hypoacetylated regions that were PLN-R14del-specific when compared with the other groups, C) Gene enrichment analysis of annotated genes from PLN-R14del-specific regions.
TableS5.xlsx
Table S5: A) Differentially expressed genes between PLN-R14del and control hearts, B) Gene enrichment analysis of upregulated genes in PLN-R14del versus control hearts, C) Gene enrichment analysis of downregulated genes in PLN-R14del versus control hearts.
TableS6.xlsx
Table S6: Media used for culturing hiPSC-CMs and their detailed compositions.
TableS7.xlsx
Table S7: A) Differentially expression genes between PLN-R14del and wild-type hiPSC-CMs cultured in the maturation medium, B) Differentially expression genes between PLN-R14del and wild-type hiPSC-CMs cultured in the glucose-rich medium, C) Differentially expression genes between PLN-R14del and wild-type hiPSC-CMs cultured in the lipid-rich medium, D) Gene enrichment analysis of differentially expressed genes between PLN-R14del and wild-type hiPSC-CMs cultured in the maturation medium, E) Gene enrichment analysis of differentially expressed genes between PLN-R14del and wild-type hiPSC-CMs cultured in the glucose-rich medium, F) Gene enrichment analysis of differentially expressed genes between PLN-R14del and wild-type hiPSC-CMs cultured in the lipid-rich medium.
TableS8.xlsx
Table S8: An overview of antibodies and stainings.
Supplmentaryvideo1CtrlDay12.avi
Supplementary Video 1: Control hiPSC-CMs on day 12.
Supplmentaryvideo2CtrlDay90.avi
Supplementary Video 2: Control hiPSC-CMs on day 90.
Supplmentaryvideo3PLNDay12.avi
Supplementary Video 3: PLN-R14del hiPSC-CMs on day 12.
Supplmentaryvideo4PLNDay90.avi
Supplementary Video 4: PLN-R14del hiPSC-CMs on day 90.

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Transcriptional regulation profiling reveals PPARA-mediated fatty acid oxidation as a novel therapeutic target in phospholamban R14del cardiomyopathy

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Abstract

Figures

Introduction

Methods

Human cardiac tissues

Human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs)

Multi-omics analyses

Functional assays

Multimodal microscopy

Results

Identification of histone acetylation changes in PLN-R14del cardiac tissue

Genes annotated to differentially acetylated regions in PLN-R14del cardiac tissues are enriched in metabolic pathways

The transcription factor binding motifs (TFBMs) overrepresented in hypoacetylated regions are enriched in metabolic pathways

Hypoacetylated regions associated with metabolic pathways specific for PLN-R14del cardiomyopathy as compared to other cardiomyopathies

Enriched metabolic pathways by differentially expressed genes in PLN-R14del hearts

Differentially expressed genes in PLN-R14del hiPSC-CMs suggest altered lipid metabolic pathways

Disturbed fatty acid oxidation (FAO) and metabolic flexibility in PLN-R14del hiPSC-CMs

Higher glucose dependency in PLN-R14del hiPSC-CMs

Intracellular lipid droplet accumulation is a key feature of PLN-R14del cardiomyopathy

CRISPR/Cas9-based correction of PLN-R14del attenuated intracellular lipid accumulation

PPARA-targeted drug increased Ca²⁺ handling and mitochondrial trifunctional protein levels in PLN-R14del hiPSC-CMs

Discussion

Declarations

References

Additional Declarations

Supplementary Files

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Transcriptional regulation profiling reveals PPARA-mediated fatty acid oxidation as a novel therapeutic target in phospholamban R14del cardiomyopathy

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Version 1

Abstract

Figures

Introduction

Methods

Human cardiac tissues

Human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs)

Multi-omics analyses

Functional assays

Multimodal microscopy

Results

Identification of histone acetylation changes in PLN-R14del cardiac tissue

Genes annotated to differentially acetylated regions in PLN-R14del cardiac tissues are enriched in metabolic pathways

The transcription factor binding motifs (TFBMs) overrepresented in hypoacetylated regions are enriched in metabolic pathways

Hypoacetylated regions associated with metabolic pathways specific for PLN-R14del cardiomyopathy as compared to other cardiomyopathies

Enriched metabolic pathways by differentially expressed genes in PLN-R14del hearts

Differentially expressed genes in PLN-R14del hiPSC-CMs suggest altered lipid metabolic pathways

Disturbed fatty acid oxidation (FAO) and metabolic flexibility in PLN-R14del hiPSC-CMs

Higher glucose dependency in PLN-R14del hiPSC-CMs

Intracellular lipid droplet accumulation is a key feature of PLN-R14del cardiomyopathy

CRISPR/Cas9-based correction of PLN-R14del attenuated intracellular lipid accumulation

PPARA-targeted drug increased Ca2+ handling and mitochondrial trifunctional protein levels in PLN-R14del hiPSC-CMs

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

PPARA-targeted drug increased Ca²⁺ handling and mitochondrial trifunctional protein levels in PLN-R14del hiPSC-CMs