Abstract
Gene therapy-based biological pacemakers have been proposed as an alternative to their hardware-based counterparts. In this context, short-term ectopic expression of the T-box transcription factor 18 (TBX18) in the ventricle has been reported to generate potent short-term pacemaker function in various animal models. Here, we investigated the impact of adeno-associated virus (AAV)-mediated long-term expression of TBX18, and compared the outcomes to those of the pacemaker ion channel Hcn2. Our findings revealed that CMV-driven ectopic TBX18 expression in mouse hearts led to severe cardiac fibrosis. At lower, non-fibrogenic levels, TBX18 maintained its transcriptional function but failed to induce pacemaker phenotypes. TBX18-expressing cells showed suppressed expression of key working myocardial genes, but the pacemaker gene program was not induced. Electrophysiological studies showed abnormal automaticity in TBX18-expressing cells, combined with prolonged repolarization and various current changes. However, no hyperpolarization-activated funny current was detected. In a complete AV-block rat model, AAV-mediated Hcn2 expression induced robust ectopic pacemaker activity in the presence of isoproterenol, whereas TBX18 expression neither generated such activity nor augmented Hcn2-mediated pacing. In conclusion, at functionally non-fibrogenic levels, TBX18 is neither sufficient nor necessary to induce pacemaker activity. In contrast, Hcn2 generates reliable pacing, making it a more viable candidate for biological pacemaker development.
Authors
Jianan Wang, Mathilde R. Rivaud, Mischa Klerk, Arie R. Boender, Ruud N. Visser, Rinske Sparrius, Hee Young Lee, Karel van Duijvenboden, Huiling Zhou, Yuting Yang, Emiel J.M. Kramer, Kyung Ho Park, Larry C. Park, Silke Schrödel, Christian Thirion, Eric Ehrke-Schulz, Anja Ehrhardt, Osne F. Kirzner, Klaus Neef, Hanno L. Tan, Arie O. Verkerk, Vincent M. Christoffels, Gerard J.J. Boink
Abstract
Enhanced TGFβ signaling caused by mutations in Fibrillin-1 (FBN1) in patients with Marfan syndrome (MFS) leads to myxomatous degeneration of the mitral valve (MDMV). MDMV can result in mitral valve prolapse, severe regurgitation, and sudden cardiac death. However, it remains unknown whether lymphatic vessel (LV) dysfunction contributes to MDMV development in MFS. Here, we show that lymphangiogenesis in murine mitral valves (MVs) begins postnatally. However, this process is inhibited in a mouse MFS model, Fbn1 mutant (Fbn1C1039G/+) mice, accompanied by disrupted lymphatic cell-cell junctions, impaired lymphatic drainage, and an abnormally widespread distribution of MHCII+ infiltrating macrophages. Treatment of Fbn1 mutant mice with VEGF-C156S, a selective VEGFR3 agonist, stimulates the ERK and Akt pathways, increases LV density in MVs, and ameliorates MDMV. Fbn1 mutant MVs display disorganized valvular endothelial cells (VECs) and decreased expression of the anti-inflammatory modulator Zfp36 (zinc finger protein 36) in VECs and immune cells. Treatment with FTY720 (Fingolimod), a ZFP36 activator and S1P antagonist, rescues MDMV phenotypes in Fbn1 mutant mice by reducing immune cell infiltration and restoring lymphatic cell junctions and drainage. These findings suggest that the Fbn1 mutation causes LV hypoplasia and defective lymphatic drainage in MVs, driven in part by pro-inflammatory VECs, leading to MFS-related MDMV.
Authors
Can Tan, Ziyou Ren, Shreya Kurup, Xianpeng Liu, Zhi-Dong Ge, Shodai Suzuki, Pritika Jakka, Cheryl Tang, M. Luisa Iruela-Arispe, Tsutomu Kume
Abstract
Cardiac macrophages are broadly studied as two subtypes, tissue resident C-X3-C motif chemokine receptor 1 positive (CX3CR1+) that are also C-C motif chemokine receptor 2 negative (CCR2–), and monocyte derived CCR2+. Previous systemic loss of function approaches suggested unique roles for each subtype in the heart with CCR2+ being inflammatory and CX3CR1+ being pro-healing. Here we employed a cardiac-specific gain of function approach to selectively enhance either macrophage subtype. A robust increase in basal CCR2+ macrophages in the heart by targeted C-C motif chemokine ligand 2 (Ccl2) expression did not induce inflammation, cause fibroblast activation, or impair cardiac function. However, increased CCR2+ macrophages reciprocally diminished self-renewing tissue resident macrophages and worsened cardiac fibrosis due to pressure overload stimulation. Conversely, augmented expression of colony-stimulating factor-1 (Csf1) in the heart promoted selective expansion of resident CX3CR1+ macrophages, which exerted no pathophysiological consequences at steady-state. However, pressure overload in these mice with expanded CX3CR1+ macrophages showed a CCR2+ macrophage-dependent inflammation leading to exacerbated cardiac dysfunction, simultaneously still protecting from adverse remodeling and cardiac fibrosis. In conclusion, cardiac-specific selective enrichment of macrophage subtypes shows their intricate interplay and unique functional roles in regulating myocardial inflammation and fibrosis during hypertrophy and at homeostasis.
Authors
Rajesh K. Kasam, Ronald J. Vagnozzi, Yasuhide Kuwabara, Anne Katrine Z. Johansen, N. Scott Blair, Vikram Prasad, Suh-Chin J. Lin, Akanksha Rajput, Michelle Nieman, Jeffery D. Molkentin
Abstract
Dilated cardiomyopathy (DCM) is a genetically heterogeneous disorder, characterized by ventricular dilatation and impaired systolic function, leading to heart failure and sudden cardiac death. Despite advances in genomic technologies, the genetic cause of DCM remains unidentified in more than half of the cases. Here, we performed an integrative analysis of genomic and transcriptomic data from patient-derived cardiac tissue to identify causative variants in genetically undiagnosed DCM. This approach enabled us to identify a homozygous splice-site variant (c.243+6T>A) in the sarcoglycan gene SGCB, which results in exon 2 skipping. This variant was significantly enriched in patients with DCM compared with the general population, with consistent genotype–phenotype correlations observed across multiple families. Protein-level analysis of cardiac tissue from homozygous individuals revealed loss of β-sarcoglycan, the protein product of SGCB, and destabilization of the sarcoglycan complex. Although SGCB has been previously associated with limb-girdle muscular dystrophy, these homozygous individuals showed no biochemical or clinical signs of skeletal muscle involvement, indicating an absence of myopathy. Compared with variant-negative patients with DCM, homozygous individuals also had a higher risk of early-onset adverse cardiac events. Together, these findings identify c.243+6T>A in SGCB as a cause of isolated DCM associated with unfavorable clinical outcomes.
Authors
Fangfang Li, Haruki Shinomiya, Yuki Kuramoto, Koshiro Kanaoka, Yuji Sakahashi, Yasuki Ishihara, Hidetaka Kioka, Seiko Ide, Yumi Yamaguchi-Kabata, Shu Tadaka, Ikuko N. Motoike, Kengo Kinoshita, Kinuko Ohneda, Hidetoshi Sakurai, Takahiro Okumura, Yohei Miyashita, Kota Jojima, Hisakazu Kato, Ken Matsuoka, Kazuya Tanabe, Shunsuke Nishimura, Seiji Takashima, Yoshihiro Asano, Yasushi Sakata
Abstract
BACKGROUND. Right ventricular failure (RVF) is a major determinant of mortality in pulmonary arterial hypertension (PAH), and hepatic dysfunction predicts adverse outcomes. However, the cell-specific effects of PAH/RVF on the human liver remain poorly defined. METHODS. We performed single-nucleus RNA sequencing of autopsy-derived liver tissue from 5 PAH patients and 4 non-PAH controls and compared these findings with publicly available single-nucleus RNA sequencing datasets from non-alcoholic steatohepatitis (NASH) and Fontan-associated liver disease (FALD). Transcriptomic analyses were integrated with histologic assessment, mitochondrial-enriched proteomics, and correlated with clinical markers of PAH/RVF severity. RESULTS. PAH livers showed cell-specific metabolic, inflammatory, and fibrotic remodeling distinct from NASH and FALD. PAH hepatocytes exhibited a hypoxia-adapted, Warburg-like metabolic phenotype with reduced fatty acid metabolism, gluconeogenesis, cytochrome P450 activity, and ketone metabolism. PAH endothelial cells demonstrated increased glycolytic pathway activity and disrupted adhesion/barrier signaling. PAH hepatic stellate cells displayed HIF-1 and PI3K-Akt pathway activation, and increased IL6 expression, which resulted in central vein fibrotic remodeling. PAH macrophages showed complement activation with reduced JAK-STAT signaling. Finally, HSC HIF-1 activity correlated with clinical markers of PAH/RVF severity. CONCLUSION. PAH induces a distinct metabolic and inflammatory hepatopathy characterized by hepatocyte metabolic reprogramming, HSC activation, and macrophage complement signaling. These findings support PAH-associated hepatopathy as a disease-specific end-organ phenotype linked to RVF severity.
Authors
Madelyn J. Blake, Sally E. Prins, Jeffrey C. Blake, Lynn M. Hartweck, Jenna B. Mendelson, Steeve Provencher, Sandra Breuils-Bonnet, Sebastien Bonnet, Kurt W. Prins
Abstract
Authors
Ryan A. Moon, Rafael Sobrano Fais, Minwoo Kim, Neal T. Vogel, Jenna B. Mendelson, Lynn M. Hartweck, John P. Carney, Melissa K. Gardner, Sally E. Prins, Sasha Z. Prisco, Tim Lahm, Kurt W. Prins
Abstract
Cardiomyocytes primarily rely on fatty acid oxidation (FAO), which provides more than 70% of their energy. However, excessive FAO can disrupt cardiac metabolism by increasing oxygen demand and suppressing glucose utilization through the Randle cycle. Although inhibition of FAO has been investigated in heart failure, its overall therapeutic impact remains uncertain. To determine the consequences of enhanced FAO, we generated cardiomyocyte-specific ACC1 and ACC2 double-knockout (ACC dHKO) mice, which exhibit constitutively elevated FAO. ACC dHKO mice developed dilated cardiomyopathy and heart failure. Lipidomic analysis revealed marked depletion of cardiolipin caused by reduced linoleic acid, a direct consequence of excessive FAO. This cardiolipin deficiency impaired mitochondrial electron transport chain (ETC) activity, leading to mitochondrial dysfunction. Pharmacologic inhibition of FAO with etomoxir or oxfenicine restored cardiolipin levels, normalized ETC activity, and prevented cardiac dysfunction in ACC dHKO mice. These findings demonstrate that unrestrained FAO disrupts both lipid and energy homeostasis, culminating in heart failure in this model. Collectively, these results indicate that although FAO is essential for cardiac energy production, therapeutic strategies aimed at stimulating cardiac FAO may be detrimental rather than beneficial in heart failure.
Authors
Chai-Wan Kim, Goncalo Vale, Xiaorong Fu, Jeffrey G. McDonald, Chongshan Dai, Chao Li, Zhao V. Wang, Gaurav Sharma, Chalermchai Khemtong, Craig R. Malloy, Stanislaw Deja, Shawn C. Burgess, Matthew A. Mitsche, Jay D. Horton
Abstract
BACKGROUND. Elevated lipoprotein(a) [Lp(a)] is associated with a higher risk of atherosclerotic cardiovascular disease (ASCVD). Although Lp(a) is a genetically determined risk factor, the plasma proteomic features associated with Lp(a) and whether they provide information about ASCVD risk beyond Lp(a) concentration are not well characterized. OBJECTIVE. We sought to identify plasma proteomic features associated with Lp(a) concentration and to evaluate whether an Lp(a)-associated proteomic signature is associated with ASCVD phenotypes in young, healthy adults. METHODS. In the Coronary Artery Risk Development in Young Adults (CARDIA) study, we measured Year 7 Lp(a) and 184 cardiovascular proteins using the Olink proximity extension assay in 3,920 participants without prior coronary heart disease. Lp(a)-associated proteomic signatures were derived using LASSO regression in a split-sample design and tested for association with coronary artery calcification (CAC), incident CHD, and hs-CRP over 27 years of follow-up. External replication was performed in the UK Biobank (n=37,996). RESULTS. Lp(a) was associated with CAC (OR 1.23 [1.13-1.34]; p<0.0001) and incident CHD (HR 1.23 [1.07-1.41]; p=0.004). Lp(a) correlated with proteomic features reflecting immune activation, coagulation, and vascular dysfunction. A quantitative Lp(a) proteomic score was independently associated with incident CAC (standardized beta = 0.40, p<0.0001) and hs-CRP (standardized beta = 0.11, p = 0.00015) after adjustment for Lp(a) concentration. In the UK Biobank, a recalibrated Lp(a)-associated proteomic score was associated with CRP, incident CHD, and all-cause mortality. CONCLUSIONS. In young adults, Lp(a) is associated with distinct proteomic features that independently predict ASCVD phenotypes beyond Lp(a) concentration, generating hypotheses regarding biological pathways linked to Lp(a)-related cardiovascular risk.
Authors
Sascha N. Goonewardena, Shanshan Yao, Tomasz Jurga, Lanyue Zhang, Donald Lloyd-Jones, Dilna Damodaran, Bharat Thyagarajan, David R. Jacobs Jr, Supriya Shore, Eric J. Brandt, Clary Clish, Kahraman Tanriverdi, Jane E. Freedman, Chirag J. Patel, Mark A. Sarzynski, Brian T. Emmer, John T. Wilkins, Ron Do, Vera Bittner, Ravi Shah, Marios K. Georgakis, Robert S. Rosenson, Venkatesh Murthy
Abstract
Pseudoxanthoma Elasticum (PXE) is a rare disease caused by loss of function of the gene Abcc6 and characterized by ectopic calcification of multiple tissues, but the physiological reasons underlying ectopic calcification in PXE remain unclear. In a murine model of Abcc6 deficient PXE where animals develop robust cardiac calcification after heart injury, we show the critical importance of the liver in mediating ectopic cardiac calcification. Tissue-specific deletion of Abcc6 in the liver, but not in the heart was sufficient to cause post-injury cardiac calcification. Metabolomics and gene expression demonstrated deficiencies in nucleotide metabolism, cellular energetics and defects in cellular respiration, underlying ectopic calcification in PXE. Functional abnormalities in cellular respiration in the injured heart were similar in globally or liver-specific Abcc6-deficient animals demonstrating that hepatic Abcc6 expression regulates cellular respiration in the injured heart. We show that ectopic calcification in PXE is primarily dystrophic and treatment with clodronate or etidronate, that prevent the growth of calcium hydroxyapatite mineralization, was sufficient to rescue the phenotype of ectopic cardiac calcification in Abcc6-deficient states. Taken together, these observations highlight the role of the liver in regulating target tissue metabolic and mitochondrial function in causing ectopic calcification in Abcc6-deficient states.
Authors
Yijie Wang, Baiming Sun, Feiyang Ma, Bo Tao, Yiqian Gu, Zhiqiang Zhou, Jason Kim, Linlin Zhang, Zhihao Liu, Johanna ten Hoeve, Linsey Stiles, Lucia Fernandez-del-Rio, Calvin Pan, Orian Shirihai, Shili Xu, Thomas G. Graeber, Tamer Sallam, Matteo Pellegrini, Aldons J. Lusis, Arjun Deb
Abstract
Aortic aneurysms are age-linked aortic dilations that progress silently and carry high rupture mortality. Immune cells are recognized drivers of aneurysm pathogenesis. Clonal hematopoiesis is an age-related expansion of somatically mutated hematopoietic stem cells that reshapes immune function and contributes to diverse age-associated diseases. However, its contribution to aneurysm pathogenesis remains unclear. In this study, targeted ultradeep sequencing of patient specimens revealed a high prevalence of clonal hematopoiesis-associated mutations that correlated with faster aneurysm expansion. Thus, we modeled clonal hematopoiesis by competitively transplanting Tet2-deficient bone marrow into ApoE-knockout mice and induced aneurysms with angiotensin II. Tet2-clonal hematopoiesis mice developed significantly greater aortic dilation than controls. Interestingly, Tet2-deficient macrophages adopted an ACP5-positive, osteoclast-like state and produced more MMP9. Both genetic and pharmacological inhibition of osteoclast-like differentiation suppressed the Tet2-mediated aneurysmal growth in vivo. Thus, Tet2-driven clonal hematopoiesis accelerates aortic aneurysm progression through MMP9-producing osteoclast-like macrophages and therefore represents a tractable therapeutic axis.
Authors
Jun Yonekawa, Yoshimitsu Yura, Junmiao Luo, Katsuhiro Kato, Shuta Ikeda, Yohei Kawai, Tomoki Hattori, Ryotaro Okamoto, Mari Kizuki, Emiri Miura-Yura, Keita Horitani, Kyung-Duk Min, Takuo Emoto, Hiroshi Banno, Mikito Takefuji, Kenneth Walsh, Toyoaki Murohara
Copyright © 2026 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)