Circular RNA circZFPM2 regulates cardiomyocyte hypertrophy and survival

Abstract

Hypertrophic cardiomyopathy (HCM) constitutes the most common genetic cardiac disorder. However, current pharmacotherapeutics are mainly symptomatic and only partially address underlying molecular mechanisms. Circular RNAs (circRNAs) are a recently discovered class of non-coding RNAs and emerged as specific and powerful regulators of cellular functions. By performing global circRNA-specific next generation sequencing in cardiac tissue of patients with hypertrophic cardiomyopathy compared to healthy donors, we identified circZFPM2 (hsa_circ_0003380). CircZFPM2, which derives from the ZFPM2 gene locus, is a highly conserved regulatory circRNA that is strongly induced in HCM tissue. In vitro loss-of-function experiments were performed in neonatal rat cardiomyocytes, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), and HCM-patient-derived hiPSC-CMs. A knockdown of circZFPM2 was found to induce cardiomyocyte hypertrophy and compromise mitochondrial respiration, leading to an increased production of reactive oxygen species and apoptosis. In contrast, delivery of recombinant circZFPM2, packaged in lipid-nanoparticles or using AAV-based overexpression, rescued cardiomyocyte hypertrophic gene expression and promoted cell survival. Additionally, HCM-derived cardiac organoids exhibited improved contractility upon CM-specific overexpression of circZFPM2. Multi-Omics analysis further promoted our hypothesis, showing beneficial effects of circZFPM2 on cardiac contractility and mitochondrial function. Collectively, our data highlight that circZFPM2 serves as a promising target for the treatment of cardiac hypertrophy including HCM.

Keywords: Cardiac hypertrophy; Circular RNA; Heart organoids; Human induced pluripotent stem cell-derived cardiomyocytes; Hypertrophic cardiomyopathy; Non-coding RNA.

Fig. 1

Identification and validation of conserved circRNAs in HCM identifies circZFPM2. A Schematic representation of the circRNA candidate selection pipeline. IGV, Integrative Genomics Viewer; NGS, Next Generation Sequencing. B Degree of sequence conservation (% nucleotide identity) of circZFPM2 between human, pig, rat and mouse. Blue and grey depict ZFPM2 Exons 2 and 3 forming the circle. Red denotes the backsplicing region. C Sanger sequencing results of conserved backsplice regions of circZFPM2 in human, pig, mouse and rat amplified with divergent primers. D Representative gel images of RT-PCR products and RT-qPCR results of RNase R treated murine cardiac RNA (n = 3 mice). E Relative expression of circZFPM2 and ZFPM2 in NRCMs after actinomycin D treatment (n = 3 wells/group). Data are means ± SD

Fig. 2

CircZFPM2 is a cardiomyocyte-enriched, cytoplasmic circRNA that is regulated in HCM. A Relative circZFPM2 expression in a murine organ panel. Non-parametric one-way ANOVA was performed to calculate significances (n = 5–6 mice). B Relative expression of circZFPM2 and ZFPM2 in a timeline of murine cardiac development. Parametric one-way ANOVA was performed to calculate significances to 0.5 days of each group (n = 4–6 mice/group). C Relative circZFPM2 expression in different human cell lines. Non-parametric one-way ANOVA was performed to calculate significances (n = 3–4 wells/group). hiPSC, human induced pluripotent stem cell; hiPSC-CM, hiPSC-derived cardiomyocyte; HCF, human cardiac fibroblast; HUVEC, human umbilical vein endothelial cell. D CircZFPM2 expression in subcellular fractions of hiPSC-CMs (n = 9 fractionations). E Relative expression of circZFPM2 and hypertrophy marker genes in CMs derived from healthy iPSCs (Pnx-CMs) and from HCM patient-derived hiPSCs. Parametric t-test was performed to calculate significances (n = 5–7 wells/group). HCM, hypertrophic cardiomyopathy. F Relative expression of circZFPM2 and ZFPM2 in HCM patient-derived cardiac tissue compared to healthy controls. Parametric t-test was performed to calculate significances (n = 18 subjects/group). Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 3

CircZFPM2 silencing impairs NRCM function. A Schematic representation of a backsplice site specific siRNA. B Relative expression of circZFPM2 and ZFPM2 in circZFPM2 siRNA and scrambled siRNA (scr. siRNA) treated NRCMs. Non-parametric t-test was performed to calculate significances (n = 3 wells/group) C Relative expression of hypertrophy marker genes in circZFPM2 siRNA and scrambled siRNA (scr. siRNA) treated NRCMs. Parametric t-test was performed to calculate significances (n = 6–9 wells/group). D Representative images and cell size measurement of scrambled siRNA (scr. siRNA), circZFPM2 siRNA and scrambled siRNA plus 100 µM phenylephrine (scr. siRNA + PE) treated NRCMs after Hoechst and cTnT (cardiac troponin T) staining in arbitrary units (AU). Parametric one-way ANOVA was performed to calculate significances to scrambled siRNA control (n ≥ 65 cells/group). E Oxygen consumption rate (OCR) of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated NRCMs, measured by Seahorse XF Mito Stress Test (n = 11–16 wells/group). F Spare respiratory capacity of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated NRCMs, measured by Seahorse XF Mito Stress Test. Parametric t-test was performed to calculate significances (n = 11–15 wells/group). G Production of reactive oxygen species (ROS) of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated NRCMs, measured by the DCFDA/H2DCFDA—Cellular ROS Assay Kit. Data are means ± SEM (n = 6 wells/group). H Percentage of cytotoxicity of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated NRCMs, measured with the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit. Parametric t-test was performed to calculate significances (n = 10 wells/group). I Fold change (FC) of caspase activity of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated NRCMs, measured with the Caspase-Glo 3/7 kit. Parametric t-test was performed to calculate significances (n = 9 wells/group). J Representative images and fold change (FC) of TUNEL positive nuclei of cTnT positive NRCMs after scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treatment. Parametric t-test was performed to calculate significances (n = 6 wells/group). K Percentage of cell viability of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated NRCMs, measured with the Cell Proliferation Reagent WST-1 kit. Parametric t-test was performed to calculate significances (n = 21 wells/group). Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns = not significant. FC, fold change

Fig. 4

CircZFPM2 overexpression in NRCMs rescues cardiomyocyte function. A Schematic representation of the circZFPM2 overexpression cassette in AAV6 expression plasmid. B Relative expression of circZFPM2 and ZFPM2 in HL-1 cells transfected with the AAV6-circZFPM2 overexpression plasmid compared to the control (AAV6-Ctrl) plasmid. Non-parametric t-test was performed to calculate significances (n = 3 wells/group). C Timeline of relative circZFPM2 and ZFPM2 expression in NRCMs transduced with AAV6-circZFPM2 compared to AAV6-Ctrl. (n = 3–4 wells/group). D Relative expression of hypertrophy marker genes in NRCMs transduced with AAV6-circZFPM2 compared to AAV6-Ctrl., with and without hypertrophic stimulus (48 h LIF (leukemia inhibitor factor)). Parametric one-way ANOVA was performed to calculate significances to the respective AAV6-Ctrl. (n = 3–6 wells/group). E Representative images and cell size measurement of NRCMs transduced with AAV6-circZFPM2 compared to AAV6-Ctrl., with and without hypertrophic stimulus (48 h LIF treatment) in arbitrary units (AU). One-way ANOVA was performed to calculate significances (n > 200 CMs from 4 wells/group). F Production of reactive oxygen species (ROS) of NRCMs transduced with AAV6-circZFPM2 compared to AAV6-Ctrl., measured by the DCFDA/H2DCFDA–Cellular ROS Assay Kit. Data are means ± SEM (n = 6 wells/group). G Percentage of cytotoxicity of NRCMs transduced with AAV6-circZFPM2 compared to AAV6-Ctrl., with and without doxorubicin (DOX, 1 µM) treatment, measured with the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit. Parametric t-test was performed to calculate significances (n = 12 wells/group). H Fold change (FC) of caspase activity of NRCMs transduced with AAV6-circZFPM2 compared to AAV6-Ctrl., with and without doxorubicin (DOX, 1 µM) treatment, measured with the Caspase-Glo 3/7 kit. Parametric t-test was performed to calculate significances (n = 12 wells/group). I Percentage of cell viability of NRCMs transduced with AAV6-circZFPM2 compared to AAV6-Ctrl., with and without doxorubicin (DOX, 1 µM) treatment, measured with the Cell Proliferation Reagent WST-1 kit. Parametric t-test was performed to calculate significances (n = 6 wells/group). Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns = not significant

Fig. 5

CircZFPM2 silencing impairs hiPSC-CMs function. A Relative expression of circZFPM2 and ZFPM2 in circZFPM2 siRNA and scrambled siRNA (scr. siRNA) treated hiPSC-CMs. Non-parametric t-test was performed to calculate significances (n = 4 wells/group). B Relative expression of hypertrophy marker genes in circZFPM2 siRNA and scrambled siRNA (scr. siRNA) treated hiPSC-CMs. Non-parametric one-way ANOVA was performed to calculate significances to the respective AAV6-Ctrl. (n = 3 wells/group). C Oxygen consumption rate (OCR) of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated hiPSC-CMs, measured by Seahorse XF Mito Stress Test (n = 9–12 wells/group). D ATP production of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated hiPSC-CMs, measured by Seahorse XF Mito Stress Test. Parametric t-test was performed to calculate significances (n = 9–12 wells/group). E Spare respiratory capacity of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated hiPSC-CMs, measured by Seahorse XF Mito Stress Test. Parametric t-test was performed to calculate significances (n = 9–12 wells/group). F Production of reactive oxygen species (ROS) of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated hiPSC-CMs, measured by the DCFDA/H2DCFDA – Cellular ROS Assay Kit. Data are means ± SEM. (n = 6 wells/group). G Percentage of cytotoxicity of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated hiPSC-CMs, measured with the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit. Parametric t-test was performed to calculate significances (n = 18 wells/group). H Fold change (FC) of caspase activity of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated hiPSC-CMs, measured with the Caspase-Glo 3/7 kit. Non-parametric t-test was performed to calculate significances (n = 3 wells/group). Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns = not significant

Fig. 6

In vitro transcribed circZFPM2 rescues function of hiPSC-CMs. A Strategy of in vitro transcription of circRNAs. B Relative expression of hypertrophy marker genes in hiPSC-CMs transfected with the in vitro transcribed (IVT) circZFPM2 compared to the Mock-Ctrl. (only transfection reagent). Parametric t-test was performed to calculate significances to the respective Mock-control (n = 6 wells/group). C Relative expression of hypertrophy marker genes in hiPSC-CMs transfected with the IVT circZFPM2 compared to the Mock-Ctrl. after induction of hypertrophy with endothelin-1 (ET-1, 50 nM). Non-parametric one-way ANOVA was performed to calculate significances to the respective “Mock + ET-1”-control (n = 2–3 wells/group). D Schematic representation of the preventive IVT circZFPM2 treatment; created with BioRender.com. E Production of reactive oxygen species (ROS) of hiPSC-CMs transfected with the IVT circZFPM2 compared to Mock-Ctrl., measured by the DCFDA/H2DCFDA – Cellular ROS Assay Kit. Data are means ± SEM. (n = 6 wells/group). F Percentage of cytotoxicity of doxorubicin (DOX, 1 µM) treated hiPSC-CMs transfected with the IVT circZFPM2 compared to Mock-Ctrl., measured with the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit. Parametric one-way ANOVA was performed to calculate significances to the “Mock + DOX”-group (n = 21–24 wells/group). G Fold change (FC) of caspase activity of doxorubicin (DOX, 1 µM) treated hiPSC-CMs transfected with the IVT circZFPM2 compared to Mock-Ctrl., measured with the Caspase-Glo 3/7 kit. Non-parametric one-way ANOVA was performed to calculate significances to the “Mock + DOX”-group (n = 4 wells/group). Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns = not significant

Fig. 7

CircZFPM2 silencing impairs cell health of HCM-CMs, while IVT circZFPM2 rescues cardiomyocyte function. A Relative expression of hypertrophy marker genes in circZFPM2 siRNA and scrambled siRNA (scr. siRNA) treated HCM-CMs with and without hypertrophic stimulus (endothelin-1, ET-1). Non-parametric one-way ANOVA was performed to calculate significances to the respective control (n = 3 wells/group). B Fold change (FC) of caspase activity of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated HCM-CMs, measured with the Caspase-Glo 3/7 kit. Parametric t-test was performed to calculate significances (n = 12 wells/group). C Percentage of cytotoxicity of scrambled siRNA (scr. siRNA) and circZFPM2 siRNA treated hiPSC-CMs, with and without doxorubicin (DOX, 1 µM) stress, measured with the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit. Parametric t-test was performed to calculate significances (n = 12 wells/group). D Relative expression of hypertrophy marker genes in HCM-CMs transfected with the in vitro transcribed (IVT) circZFPM2 compared to the Mock-Ctrl. Non-parametric t-test was performed to calculate significances to the respective Mock-control (n = 3 wells/group). E Percentage of cytotoxicity of doxorubicin (DOX, 1 µM) treated HCM-CMs transfected with the IVT circZFPM2 compared to Mock-Ctrl., measured with the CytoTox 96 Non-Radioactive Cytotoxicity Assay kit. Parametric one-way ANOVA was performed to calculate significances to the “Mock + DOX”-group (n = 6 wells/group). F Fold change of caspase activity of doxorubicin (DOX, 1 µM) HCM-CMs transfected with the IVT circZFPM2 compared to Mock-Ctrl., measured with the Caspase-Glo 3/7 kit. Parametric one-way ANOVA was performed to calculate significances to the “Mock + DOX”-group (n = 6 wells/group). Data are means ± SD. *p < 0.05; ***p < 0.001; ****p < 0.0001; ns = not significant

Fig. 8

CircZFPM2 overexpression improves contractility in HCM-derived cardiac organoids. A Representative live-cell images of AAV6-GFP transduced human cardiac organoids (hCOs). B−E Morphological parameters of AAV6-circZFPM2 and AAV6-Ctrl. treated HCM-hCOs, displaying B circularity, C area, D diameter and E perimeter. Non-parametric t-test was performed to calculate significances (n = 123–124 hCOs/group). F Relative expression of circZFPM2 and ZFPM2 in AAV6-circZFPM2 and AAV6-Ctrl. treated HCM-hCOs. Non-parametric t-test was performed to calculate significances (n = 3 wells/group) G−N Contractile measurements of AAV6-circZFPM2 and AAV6-Ctrl. treated HCM-hCOs, displaying G time to peak, H relaxation time, I 90-to-90 transient, J 50-to-50 transient, K 10-to-10 transient, L contraction amplitude and M peak to peak time. Non-parametric t-test was performed to calculate significances (n = 34–39 hCOs/group). N Representative contractility plots of AAV6-circZFPM2 and AAV6-Ctrl. treated HCM-hCOs. Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant

Fig. 9

CircZFPM2 downstream signaling modulates mitochondrial function and cardiac muscle contractility. A Heat map of selected genes from the RNA sequencing dataset of circZFPM2 siRNA and control (scr.) siRNA treated hiPSC-CMs. Blue indicates significantly downregulated genes, red indicates significantly upregulated genes. B Volcano plot of the RNA sequencing dataset of circZFPM2 siRNA and scr. siRNA treated hiPSC-CMs. DEGs are highlighted in red. C KEGG 2021, D GOBP 2023 and E GOCC 2023 analysis of dysregulated gene sets from the RNA sequencing dataset. F Volcano plot of RNA pulldown samples from hiPSC-CM lysates against biotinylated circZFPM2 probe and scr. probe. Significantly enriched proteins with a FC ≥ 2 are highlighted in red. Potential interaction partners with biological relevance are highlighted in blue (FC ≥ 2) and green (FC < 2). FC, fold change