Abstract
Background: Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment for myocarditis is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis with biomarkers and pathological features indistinguishable from other forms of myocarditis. DSP-associated myocarditis can progress to dilated cardiomyopathy with heightened arrhythmia risk. Methods: To model the cardiomyocyte aspects of DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients and gene-edited healthy control hiPSC lines. Homozygous and heterozygous DSP disrupted EHTs were generated to contain 90% hiPSC-CMs and 10% healthy control human cardiac fibroblasts. We measured innate immune activation and function at baseline and in response to Toll-like receptor (TLR) stimulation in EHTs. Results: At baseline, DSP-/- EHTs displayed a transcriptomic signature of immune activation which was mirrored by EHT cytokine release. Importantly, DSP-/- EHTs were hypersensitive to TLR stimulation demonstrating greater contractile function impairment compared to isogenic controls. Compared to homozygous DSP-/- EHTs, heterozygous DSP patient-derived EHTs had less functionally impairment but also displayed heightened sensitivity to TLR stimulation. When subjected to strain, heterozygous DSP EHTs developed greater functional deficit indicating reduced contractile reserve compared to healthy control. Colchicine or NFΚB inhibitors improved baseline force production and strain-induced force deficits in DSP EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Conclusions: Genetic reduction of DSP renders cardiomyocytes susceptible to innate immune activation and strain-dependent contractile deficits. EHTs replicate electrical and contractile phenotypes seen in human myocarditis implicating cytokine release as a key part of the myogenic susceptibility to inflammation. This heightened innate immune activation and sensitivity is a target for clinical intervention.
Authors
Daniel F. Selgrade, Dominic E. Fullenkamp, Ivana A. Chychula, Binjie Li, Lisa Dellefave-Castillo, Adi D. Dubash, Joyce Ohiri, Tanner O. Monroe, Malorie Blancard, Garima Tomar, Cory Holgren, Paul W. Burridge, Alfred L. George Jr., Alexis R. Demonbreun, Megan. Puckelwartz, Sharon A. George, Igor R. Efimov, Kathleen J. Green, Elizabeth M. McNally
Abstract
Cardiomyocyte sarcomeres contain localized ribosomes, but the factors responsible for their localization and the significance of localized translation are unknown. Using proximity labeling, we identified Ribosomal Protein SA (RPSA) as a Z-line protein. In cultured cardiomyocytes, the loss of RPSA led to impaired local protein translation and reduced sarcomere integrity. By employing CAS9 expressing mice along with adeno-associated viruses expressing CRE recombinase and single-guide RNAs targeting Rpsa, we knocked out Rpsa in vivo and observed mis-localization of ribosomes and diminished local translation. These genetic mosaic mice with Rpsa knockout in a subset of cardiomyocytes developed dilated cardiomyopathy, featuring atrophy of RPSA-deficient cardiomyocytes, compensatory hypertrophy of unaffected cardiomyocytes, left ventricular dilation, and impaired contractile function. We demonstrate that RPSA C-terminal domain is sufficient for localization to the Z-lines and that if the microtubule network is disrupted RPSA loses its sarcomeric localization. These findings highlight RPSA as a ribosomal factor essential for ribosome localization to the Z-line, facilitating local translation and sarcomere maintenance.
Authors
Rami Haddad, Omer Sadeh, Tamar Ziv, Itai Erlich, Lilac Haimovich-Caspi, Ariel Shemesh, Jolanda van der Velden, Izhak Kehat
Abstract
One of the features of pathological cardiac hypertrophy is enhanced translation and protein synthesis. Translational inhibition has been shown to be an effective means of treating cardiac hypertrophy, although system-wide side effects are common. Regulators of translation, such as cardiac-specific long non-coding RNAs (lncRNAs), could provide new, more targeted, therapeutic approaches to inhibit cardiac hypertrophy. Therefore, we generated mice lacking a previously identified lncRNA named CARDINAL to examine its cardiac function. We demonstrate that CARDINAL is a cardiac-specific, ribosome associated lncRNA and show that its expression is induced in the heart upon pathological cardiac hypertrophy; its deletion in mice exacerbates stress-induced cardiac hypertrophy and augments protein translation. In contrast, overexpression of CARDINAL attenuates cardiac hypertrophy in vivo and in vitro, and suppresses hypertrophy-induced protein translation. Mechanistically, CARDINAL interacts with developmentally regulated GTP binding protein 1 (DRG1) and blocks its interaction with DRG family regulatory protein 1 (DFRP1); as a result, DRG1 is downregulated, thereby modulating the rate of protein translation in the heart in response to stress. This study provides evidence for the therapeutic potential of targeting cardiac-specific lncRNAs to suppress disease-induced translational changes and to treat cardiac hypertrophy and heart failure.
Authors
Xin He, Tinqun Yang, Yao Wei Lu, Gengze Wu, Gang Dai, Qing Ma, Mingming Zhang, Huimin Zhou, Tianxin Long, Youchen Yan, Zhuomin Liang, Chen Liu, William T. Pu, Yugang Dong, Jingsong Ou, Hong Chen, John D. Mably, Jiangui He, Da-Zhi Wang, Zhan-Peng Huang
Abstract
Newborn mammalian cardiomyocytes quickly transition from a fetal to an adult phenotype that utilizes mitochondrial oxidative phosphorylation but loses mitotic capacity. We tested whether forced reversal of adult cardiomyocytes back to a fetal glycolytic phenotype would restore proliferative capacity. We deleted Uqcrfs1 (mitochondrial Rieske Iron-Sulfur protein, RISP) in hearts of adult mice. As RISP protein decreased, heart mitochondrial function declined, and glucose utilization increased. Simultaneously, they underwent hyperplastic remodeling during which cardiomyocyte number doubled without cellular hypertrophy. Cellular energy supply was preserved, AMPK activation was absent, and mTOR activation was evident. In ischemic hearts with RISP deletion, new cardiomyocytes migrated into the infarcted region, suggesting the potential for therapeutic cardiac regeneration. RNA-seq revealed upregulation of genes associated with cardiac development and proliferation. Metabolomic analysis revealed a decrease in alpha-ketoglutarate (required for TET-mediated demethylation) and an increase in S-adenosylmethionine (required for methyltransferase activity). Analysis revealed an increase in methylated CpGs near gene transcriptional start sites. Genes that were both differentially expressed and differentially methylated were linked to upregulated cardiac developmental pathways. We conclude that decreased mitochondrial function and increased glucose utilization can restore mitotic capacity in adult cardiomyocytes resulting in the generation of new heart cells, potentially through the modification of substrates that regulate epigenetic modification of genes required for proliferation.
Authors
Gregory B. Waypa, Kimberly A. Smith, Paul T. Mungai, Vincent J. Dudley, Kathryn A. Helmin, Benjamin D. Singer, Clara Bien Peek, Joseph Bass, Lauren Beussink-Nelson, Sanjiv J. Shah, Gaston Ofman, J. Andrew Wasserstrom, William A. Muller, Alexander V. Misharin, G.R. Scott Budinger, Hiam Abdala-Valencia, Navdeep S. Chandel, Danijela Dokic, Elizabeth T. Bartom, Shuang Zhang, Yuki Tatekoshi, Amir Mahmoodzadeh, Hossein Ardehali, Edward B. Thorp, Paul T. Schumacker
Abstract
Nuclear factor kappa-B (NFκB) is activated in arrhythmogenic cardiomyopathy (ACM) patient-derived iPSC-cardiac myocytes under basal conditions and inhibition of NFκB signaling prevents disease in Dsg2mut/mut mice, a robust mouse model of ACM. Here, we used genetic approaches and single cell RNA sequencing to define the contributions of immune signaling in cardiac myocytes and macrophages in the natural progression of ACM using Dsg2mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2mut/mut mice. NFκB signaling in cardiac myocytes mobilizes macrophages expressing C-C motif chemokine receptor-2 (CCR2+ cells) to affected areas within the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA sequencing and cellular indexing of transcriptomes and epitomes (CITE-seq) studies revealed marked pro-inflammatory changes in gene expression and the cellular landscape in hearts of Dsg2mut/mut mice involving cardiac myocytes, fibroblasts and CCR2+ macrophages. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2mut/mut mice were dependent on CCR2+ macrophage recruitment to the heart. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells and fibroblasts in the pathogenesis of ACM.
Authors
Stephen P. Chelko, Vinay R. Penna, Morgan Engel, Emily A. Shiel, Ann M. Centner, Waleed Farra, Elisa N. Cannon, Maicon Landim-Vieira, Niccole Schaible, Kory Lavine, Jeffrey E. Saffitz
Abstract
Authors
Fotios Spyropoulos, Apabrita Ayan Das, Markus Waldeck-Weiermair, Shambhu Yadav, Arvind K. Pandey, Ruby Guo, Taylor A. Covington, Venkata Thulabandu, Kosmas Kosmas, Benjamin Steinhorn, Mark Perella, Xiaoli Liu, Helen Christou, Thomas Michel
Abstract
The ability to fight or flee from a threat relies upon an acute adrenergic surge that augments cardiac output, which is dependent upon increased cardiac contractility and heart rate. This cardiac response depends on β-adrenergic-initiated reversal of the small RGK G-protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavβ subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal requires phosphorylation of Ser272 and Ser300 within Rad’s polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad’s N-terminal domain (NTD) alone is ineffective. Phosphorylation of Ser272 and Ser300 or the addition of four Asp to the CTD reduces Rad’s association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVβ, even in the absence of CaVα, measured here by FRET. Addition of a post-translationally prenylated CAAX motif to Rad’s C-terminus, which constitutively tethers Rad to the membrane, prevents the physiological and biochemical effects of both phosphorylation and Asp-substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVβ, is sufficient for sympathetic up-regulation of Ca2+ currents.
Authors
Arianne Papa, Pedro J. del Rivero Morfin, Bi-Xing Chen, Lin Yang, Alex N. Katchman, Sergey I. Zakharov, Guoxia Liu, Michael S. Bohnen, Vivian Zheng, Moshe Katz, Suraj Subramaniam, Joel A. Hirsch, Sharon Weiss, Nathan Dascal, Arthur Karlin, Geoffrey S. Pitt, Henry M. Colecraft, Manu Ben Johny, Steven O. Marx
Abstract
Patients with chronic inflammatory disorders such as psoriasis have an increased risk of cardiovascular disease and elevated levels of LL37, a cathelicidin host defense peptide that has both antimicrobial and proinflammatory properties. To explore if LL37 could contribute to the risk of heart disease, we examined its effects on lipoprotein metabolism and show that LL37 enhances LDL uptake in macrophages through LDLR, SR-B1 and CD36. This interaction led to increased cytosolic cholesterol in macrophages and changes in expression of lipid metabolism genes consistent with increased cholesterol uptake. Structure-function analysis and synchrotron small angle X-ray scattering show structural determinants of the LL37-LDL complex that underlie its ability to bind its receptors and promote uptake. This function of LDL uptake is unique to cathelicidins from humans and some primates and was not observed with cathelicidins from mice or rabbits. Notably, Apoe-/- mice expressing LL37 develop larger atheroma plaques than control mice and a positive correlation between plasma LL37 and OxPL-apoB levels was observed in human subjects with cardiovascular disease. These findings provide evidence that LDL uptake can be increased via interaction with LL37 and may explain the increased risk of cardiovascular disease associated with the chronic inflammatory disorders.
Authors
Yoshiyuki Nakamura, Nikhil N. Kulkarni, Toshiya Takahashi, Haleh Alimohamadi, Tatsuya Dokoshi, Edward L. Liu, Michael Shia, Tomofumi Numata, Elizabeth W.C. Luo, Adrian F. Gombart, Xiaohong Yang, Patrick Secrest, Philip L.S.M. Gordts, Sotirios Tsimikas, Gerard C.L. Wong, Richard L. Gallo
Abstract
Itaconate has emerged as a critical immunoregulatory metabolite. Here, we examined the therapeutic potential of itaconate in atherosclerosis. We found that both itaconate and the enzyme that synthesizes it, aconitate decarboxylase 1 (Acod1, also known as “immune-responsive gene 1”/IRG1) are upregulated during atherogenesis in mice. Deletion of Acod1 in myeloid cells exacerbated inflammation and atherosclerosis in vivo and resulted in an elevated frequency of a specific subset of M1-polarized proinflammatory macrophages in the atherosclerotic aorta. Importantly, Acod1 levels were inversely correlated with clinical occlusion in atherosclerotic human aorta specimens. Treating mice with the itaconate derivative 4-ocytyl itaconate attenuated inflammation and atherosclerosis induced by high cholesterol. Mechanistically, we found that the antioxidant transcription factor, Nuclear factor erythroid-2 Related Factor 2 (Nrf2) was required for itaconate to suppress macrophage activation induced by oxidized lipids in vitro and to decrease atherosclerotic lesion areas in vivo. Overall, our work shows that itaconate suppresses atherogenesis by inducing Nrf2-dependent inhibition of proinflammatory responses in macrophages. Activation of the itaconate pathway may represent an important approach to treat atherosclerosis.
Authors
Jianrui Song, Yanling Zhang, Ryan A. Frieler, Anthony Andren, Sherri C. Wood, Daniel J. Tyrrell, Peter Sajjakulnukit, Jane C. Deng, Costas A. Lyssiotis, Richard M. Mortensen, Morgan Salmon, Daniel R. Goldstein
Abstract
Heterozygous (HET) truncating mutations in the TTN gene (TTNtv) encoding the giant titin protein are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here we investigated 127 clinically identified DCM human cardiac samples with next-generation sequencing (NGS), high-resolution gel electrophoresis, Western blot analysis and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene-sequence predictions were detected in the majority of the TTNtv+ samples. Full length titin was reduced in TTNtv+ compared to TTNtv- samples. Proteomic analysis of washed myofibrils and Stimulated Emission Depletion (STED) super-resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin is structurally integrated in the sarcomere. Sarcomere length-dependent anti-titin epitope position, shape and intensity analyses pointed at possible structural defects in the I/A junction and the M-band of TTNtv+ sarcomeres, which likely contribute, possibly via faulty mechanosensor function, to the development of manifest DCM.
Authors
Dalma Kellermayer, Hedvig Tordai, Balázs Kiss, György Török, Dániel M. Péter, Alex Ali Sayour, Miklós Pólos, István Hartyánszky, Bálint Szilveszter, Siegfried Labeit, Ambrus Gángó, Gábor Bedics, Csaba Bödör, Tamás Radovits, Bela Merkely, Miklós S.Z. Kellermayer
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)