Journal of Molecular and Cellular Cardiology

BackgroundVentricular assist devices (VADs) are used as treatment for end-stage heart failure in children and adults. We previously demonstrated decreased mitochondrial function and changes in cardiolipin, a mitochondrial phospholipid, in explanted pediatric and adult failing hearts. In this study, we tested the hypothesis that VAD unloading of failing hearts leads to positive changes in myocardial cardiolipin in both pediatric and adult hearts. MethodsVentricular tissue was collected from the same patient at time of VAD implantation and at transplant. Ejection fraction (EF), left ventricular internal diameter at end-diastole (LVIDd) and brain natriuretic peptide (BNP) were assessed pre- and post-VAD. Cardiolipin species from paired VAD core and explants were quantified using liquid chromatography mass spectrometry. Mitochondrial respiration was measured in ventricular tissue pre- and post-VAD in paired pediatric samples using the Oroboros Oxygraph-2k. ResultsVAD support led to increased EF and decreased LVIDd and BNP. The predominant cardiolipin species in cardiac mitochondria, tetralinoleoylcardiolipin, was positively remodeled in pediatric post-VAD myocardium, while adult post-VAD myocardium demonstrated significantly increased total cardiolipin and decreased oxidized cardiolipin but did not demonstrate the tetralinoleoylcardiolipin remodeling seen in pediatric hearts. In pediatric patients, VAD support resulted in significant increases in Complex I+II activity, and a trend toward increases in Complex I activity. ConclusionOur data demonstrate age-related differences in VAD-associated cardiolipin remodeling and suggest that improved mitochondrial function in pediatric VAD-supported hearts could be related to increased tetralinoleoylcardiolipin.

BACKGROUNDMono-ADP ribosylation is a post-translational modification that regulates various cellular physiological processes, including cell cycle progression, genomic stability, transcription, and cellular protein turnover. PARP16 is an endoplasmic reticulum (ER)-localized mono-ADP-ribosyltransferase that has been shown to regulate the unfolded protein response and maintain ER homeostasis under stress conditions. Despite its established role in ER stress signaling, the functional significance of PARP16 in cardiac pathophysiology, particularly in cardiac hypertrophy and heart failure, remains poorly understood. In this study, we aim to investigate the role of PARP16 in cardiac hypertrophy and heart failure using in vitro and mouse model systems. METHODSWe analysed PARP16 expression in human heart failure samples as well as in heart failure-based mouse models. We evaluated gene expression by RT-PCR, immunoblotting, and confocal microscopy to understand the role of PARP16 in heart failure under phenylephrine- or isoproterenol-treated conditions. We also investigated the role of PARP16 in regulating cardiac function in genetically engineered mouse models, including whole-body PARP16 knockout, cardiac-specific PARP16 knockout, inducible cardiac-specific PARP16 knockout, and cardiac-specific PARP16 Transgenic mice. We performed echocardiography to assess cardiac function. We also used an in vitro primary cardiomyocyte system to knock down and overexpress PARP16. We performed RNA sequencing and mass spectrometry, followed by molecular docking, molecular dynamics simulation, immunoprecipitation, and luciferase assay to characterise the molecular mechanism by which PARP16 regulates cardiac function. RESULTSHuman heart failure samples showed reduced PARP16 expression. PARP16 expression was also significantly reduced in models of heart failure, including the hearts of isoproterenol-treated C57B/L6 mice and phenylephrine-treated primary cardiomyocytes. PARP16-deficient NRCMs showed signs of pathological remodelling. Whole-body, cardiac-specific, and inducible cardiac-specific PARP16 KO mice exhibited cardiac remodelling and dysfunction. In contrast, cardiac-specific PARP16-overexpressing mice were protected from iso-induced cardiac hypertrophy. Mechanistically, several hypertrophic signalling pathway genes are dysregulated in PARP16 knockout mouse hearts concomitant with upregulated NFAT1 transcriptional activity and nuclear translocation. PARP16 binds to and catalytically downregulates NFAT activity, thereby maintaining cardiac function. Mass spectrometry analysis showed that PARP16 is involved in ADP-ribosylation of NFAT1 at E398 and T533. Pharmacological inhibition of NFAT activation attenuates structural and functional abnormalities associated with PARP16 deficiency. CONCLUSIONSPARP16 binds to and inhibits NFAT1 activity to regulate cardiac function in mice, and its downregulation may activate NFAT1 signalling, leading to hypertrophy. In this manner, PARP16 plays a critical role in cardiac hypertrophy and failure and may serve as a potential therapeutic target for the treatment of heart failure.

Pathologic cardiac hypertrophy requires increased protein synthesis, but the mechanosensors that link membrane stretch to translational control remain poorly understood. Polycystin-1 (PC1), encoded by PKD1, has been proposed as a cardiac mechanosensor, with its C-terminal tail (PC1-CT) promoting hypertrophy in rodent cardiomyocytes. However, its subcellular localization and downstream signaling remain incompletely defined, especially in human cardiomyocytes. Here, we examined endogenous PC1 C-terminus localization and the effects of adenoviral PC1-CT overexpression in human iPSC-derived ventricular cardiomyocytes (hiPSC-CMs) and adult mouse ventricular myocytes. Immunofluorescence revealed a striking striated pattern for both endogenous PC1 C-terminus (detected with a PC1-CT antibody) and the overexpressed PC1-CT fragment. In hiPSC-CMs, the PC1 C-terminus localized between the -actinin bands. In contrast, in adult cardiomyocytes, the overexpressed protein colocalized with -actinin and desmin, suggesting that PC1-CT sarcomeric distribution depends on cardiomyocyte maturation. We performed RNA-seq to assess transcriptional responses downstream of PC1-CT overexpression in hiPSC-CMs relative to LacZ controls. Gene Set Enrichment Analysis (GSEA) revealed enrichment of gene sets related to ribosome biogenesis, RNA processing, and protein synthesis, while classical hypertrophic markers remained unchanged. Pathway analysis suggested increased PI3K activity. PC1-CT overexpression increased phosphorylation of Akt, ERK, S6K1, and ribosomal protein S6 without altering 4EBP1 phosphorylation, suggesting preferential activation of the mTOR-S6K1-S6 branch. Pharmacological studies showed that pan-PI3K inhibition abolished S6 phosphorylation, whereas MEK blockade did not affect it; pertussis toxin and PI3K{gamma}-selective inhibitors also did not affect S6, suggesting a Gi/o-independent PI3K/Akt signaling driving mTOR-S6K1-S6 activation. Collectively, these data identify a sarcomere-associated pool of PC1-CT that engages PI3K-Akt-mTOR-S6K1-S6 signaling to enhance transcriptional programs related to ribosome biogenesis and protein synthesis, without activating a canonical hypertrophic gene program. These findings reveal a mechanistic link between PC1-CT and cardiomyocyte growth.

BackgroundPediatric dilated cardiomyopathy (DCM) is a rare, progressive heart disease with variable outcomes that range from recovery to heart transplantation. To date, there are no prognostic biomarkers for children with DCM. Identifying circulating biomarkers that are associated with clinical outcomes is critical for personalized management. MethodsmiRNAs were identified by RNA-seq, whereas proteins were identified by SomaScan(R). Machine learning methodologies were used to explore the predictive ability of circulating factors identified from serum samples collected at the time of presentation with acute heart failure. ResultsThirty patients experienced poor outcomes (cardiac transplantation, mechanical circulatory support, or death) and 19 patients recovered left ventricular function. Distinct miRNA and protein signatures differentiated outcomes groups. Top candidate proteins (COL2A1, CXCL12, and ADGRF5) and miRNAs (miR-874-3p, miR-335-3p, miR-323a-3p) demonstrated strong discriminatory performance within the study cohort (recovered vs poor outcomes; Area Under the Curve of 0.92). Ingenuity Pathway Analysis implicates cardiac remodeling, fibrosis, and inflammatory signaling as central pathways differentiating patient outcomes. ConclusionsCirculating miRNA and protein signatures at presentation identify a circulating molecular signature associated with divergent clinical trajectories in pediatric DCM. These findings support the potential utility of multi-omic biomarkers for early risk stratification and provide insight into mechanisms underlying divergent outcomes. CLINICAL PERSPECTIVEWhat Is New? O_LICirculating miRNA and protein profiles measured at presentation distinguish children with pediatric DCM who recover from those who progress to advanced heart failure. C_LIO_LIA combined multi-omic biomarker demonstrated strong discriminatory performance in this cohort (AUC 0.92). C_LIO_LIPathway analysis implicates extracellular matrix remodeling, fibrosis, and inflammatory signaling in children with adverse clinical trajectories. C_LI What Are the Clinical Implications? O_LISerum-based molecular biomarkers may enable earlier risk stratification in children presenting with dilated cardiomyopathy. C_LIO_LIMulti-omic integration may improve identification of pediatric patients at risk for transplantation, mechanical circulatory support, or death. C_LIO_LIThese findings support further validation of circulating biomarker panels to guide personalized management in this rare disease. C_LI RESEARCH PERSPECTIVEWhat New Question Does This Study Raise? O_LICan integrated circulating miRNA-protein signatures identify biologically distinct trajectories of recovery versus progression in children with dilated cardiomyopathy? C_LIO_LIDo circulating molecular profiles reflect underlying disease mechanisms that determine divergent clinical outcomes in pediatric DCM? C_LI What Question Should Be Addressed Next? O_LIDo the pathways identified by integrated miRNA-protein analysis (fibrosis, remodeling, and inflammation) play causal roles in determining recovery versus progression? C_LIO_LICan multi-omic biomarkers be incorporated into prospective studies to improve early risk stratification and guide clinical management? C_LI

Severe forms of inflammation-induced acute and chronic myocarditis have a poor prognosis. Promising therapeutic efforts focused on monoclonal antibodies (mAbs) inhibiting inflammation-inducing molecules. However, most mAbs target only one or a limited number of such molecules. Since inflammation involves multiple redundant pathways, we postulated that an mAb inhibiting multiple inflammatory pathways would be a potent therapeutic agent. We initially tested the commercially available anti-natural killer (NK) cell mAb (anti-NK1.1), which binds a receptor expressed on NK cells and depletes them. Since NK cells are key cellular orchestrators of inflammation, by reducing their number, we aimed to inhibit multiple inflammatory pathways. Our initial studies demonstrated that administration of this antibody significantly improved myocardial outcomes in mouse models of acute myocardial infarction and of heart failure. Since NK1.1 is not expressed in human cells, we built on these promising preclinical results by developing a novel mAb targeting CD160 on human NK cells for evaluation as an immunosuppressive therapy. We found that the anti-CD160 mAb depletes both murine and human NK cells. We also found that, while CD160+ cells were largely present in the NK population, they also occurred among CD8+ and {gamma}/{delta} T cell subsets in human cells. Anti-CD160 therapy entirely prevented the deterioration of the myocardial function of mice with autoimmune-induced acute myocarditis. This outcome suggests our novel approach for inhibiting multiple inflammatory pathways may provide a potent strategy for improving outcomes of inflammation-driven myocarditis, as well as of other inflammation-driven diseases. Key PointsO_ST_ABSQuestionC_ST_ABSCan the depletion of CD160+ cells prevent autoimmune-induced myocarditis? FindingsIn this study we found that CD160 is expressed by mouse and human natural killer cells and other subtypes of cytotoxic T cells, and that a monoclonal antibody targeting CD160 depletes NK cells. In a preclinical model of experimental autoimmune myocarditis, administration of the anti-CD160 monoclonal antibody prevented myocardial dysfunction and systemic inflammation. MeaningOur results are compatible with the hypothesis that early autoimmune-induced myocardial dysfunction is promoted by CD160+ cells, which elevate inflammation-induced circulating factors (or factors released by tissue-resident cytotoxic immune cells) that cause myocardial dysfunction in the absence of myocardial necrosis or fibrosis, and further, that targeting CD160+cells with a mAb that depletes NK cells (and probably CD160 expressing cytotoxic T cells) entirely prevents the deterioration of myocardial function in such mice. This outcome suggests our novel approach for inhibiting multiple inflammatory pathways may provide a potent strategy for improving outcomes of inflammation-driven myocarditis, as well as of other inflammation-driven diseases.

Background: The clinical significance of sarcomere variants of uncertain significance (VUS) in hypertrophic cardiomyopathy (HCM) remains unclear, and VUS are currently regarded as clinically non-actionable despite their increasing prevalence. This study aimed to evaluate genotype?phenotype and genotype?outcome associations according to variant pathogenicity in patients with HCM, with a particular focus on the clinical relevance of sarcomere VUS. Methods: This multicenter retrospective cohort study included 438 patients with HCM who underwent next-generation sequencing-based genetic testing at two tertiary hospitals. Patients were classified into three groups: pathogenic or likely pathogenic (P/LP) variants, VUS, and no sarcomere mutations. Clinical characteristics, imaging phenotypes, and outcomes were compared across groups. The primary endpoint was a composite of cardiovascular death, aborted sudden cardiac death, appropriate implantable cardioverter-defibrillator therapy, and heart transplantation. Time-to-event analyses were performed using Kaplan-Meier methods and Cox proportional hazards models with Firth's penalized partial likelihood approach. Results: P/LP variants were identified in 171 patients (39.0%) and sarcomere VUS in 159 patients (36.3%). Patients with VUS demonstrated intermediate clinical and phenotypic features between P/LP carriers and genotype-negative patients. Kaplan?Meier analysis showed a graded difference in event-free survival across variant classifications. While VUS were not independently associated with adverse outcomes when modeled as a categorical variable, increasing pathogenicity from genotype-negative to VUS and P/LP variants was associated with a stepwise increase in risk of the primary endpoint (hazard ratio 2.05, 95% confidence interval 1.11?4.16 p=0.019). Identified VUS were preferentially enriched in Z-disc and giant sarcomere scaffolding proteins. Conclusion: Sarcomere VUS represent intermediate characteristics along a continuum of sarcomere dysfunction, associated with distinct phenotypic features and clinical outcomes compared with both P/LP variants and the absence of sarcomere mutations. These findings suggest that sarcomere VUS may not be entirely clinically neutral and should be interpreted within a broader genetic and structural context in patients with HCM.

BACKGROUNDExcessive trabeculations and myocardial crypts are recurrent features across cardiomyopathies, yet their developmental origins and clinical significance remain poorly defined. To reveal the link between cardiac morphogenesis and disease, we generated humanized mouse models carrying patient-derived MYBPC3 frameshift mutations associated with overlapping hypertrophic cardiomyopathy (HCM) and left ventricular non-compaction (LVNC). METHODSWe applied CRISPR-Cas9 to introduce distinct MYBPC3 frameshift alleles into the mouse genome and performed comprehensive phenotypic and transcriptomic profiling from fetal life through adulthood. RESULTSAdult homozygous Mybpc3 frameshift mutant mice like humans displayed hallmark HCM; however, without LVNC. Fetal and neonatal mutant hearts exhibited markedly enlarged ventricular trabeculae and crypts that progressed postnatally into the observed adult hypertrophy. Transcriptomic analysis revealed stage-specific dysregulation of oxidative metabolism, nonsense-mediated decay (NMD), and cell cycle pathways, peaking at postnatal days 1 and 7, indicating that these stages represent critical time points in disease onset. The persistent NMD signature, also observed in phenotype-negative heterozygotes, suggests a compensatory stress response. Enlarged trabeculae exhibited 2-fold increased trabecular cardiomyocyte proliferation, reversing the normal compact-trabecular proliferative gradient and leading to impaired ventricular compaction in neonates. Hey2CreERT2 lineage tracing demonstrated invasion of Hey2+ compact cardiomyocytes into the trabeculae and ectopic trabecular expression of the Prdm16 transcription factor, indicating defective ventricular wall patterning and maturation. Postnatally, Hey2+-derived cardiomyocytes became restricted to the outer/compact myocardium in mutants, while the inner/trabecular myocardium underwent accelerated hypertrophy concurrent with Prdm16 downregulation. Mice with a Mybpc3 missense variant also exhibited Hey2+ myocardial lineage expansion into trabeculae but no increased proliferation, implicating additional mechanisms beyond Hey2 regulation. Postnatal Prdm16 restoration, via transgenic expression in Mybpc3-null mice effectively attenuated hypertrophy, establishing a causal link between Mybpc3 loss, Prdm16 decline, and pathological remodeling. CONCLUSIONSMybpc3 governs ventricular wall maturation by regulating cardiomyocyte proliferation, patterning, and maturation, partly via Prdm16. Disruption of these developmental programs precedes and drives adult HCM, highlighting a developmental role for sarcomeric proteins, and revealing postnatal Prdm16 modulation as an antihypertrophic therapeutic strategy.

Background and Aims: Acute myocarditis (AM) is a T cell-mediated myocardial disease with clinical manifestations ranging from mild chest pain to cardiogenic shock. Reliable biomarkers to stratify patients and guide therapy are currently lacking. In particular, the extent of the dysregulation of inflammatory pathways, and the impact on myocardial dysfunction, remain elusive. Methods: Serum analyses were performed in prospectively recruited AM patients (n = 103) from two independent cohorts. Multimodal data integration combining profiling of cytokine and chemokine dysregulation with clinical biomarkers was used to define clinical phenotypes with distinct inflammatory signatures. Machine-learning and regression models were applied to determine biomarkers that indicate clinical severity. Results: Immuno-proteomic profiling revealed conserved inflammatory patterns across AM cohorts, dominated by T cell-related cytokines and chemokines. In addition, AM patients showed dysregulation of fibroblast-derived cytokines, including hepatocyte growth factor (HGF), bone morphogenic protein 4 (BMP4) and the BMP4 inhibitors Gremlin-1 (GREM1) and Gremlin-2 (GREM2). Data integration and unsupervised clustering revealed two immuno-clinical phenotypes, linking T cell activation and fibroblast dysregulation to disease severity. Machine learning-based analysis identified CXCL10, GREM2 and LVEF as critical parameters for stratifying disease severity. Conclusions: These findings highlight a systemic T cell activation signature as diagnostic hallmark of AM. In addition, dysregulation of fibroblast-derived tissue cytokines serves as an indicator for distinct immuno-clinical phenotypes in myocardial inflammatory disease. Thus, the clinically relevant link between T cell-driven immune activation, myocardial inflammation and fibroblast-driven remodelling provides a versatile set of parameters to identify severe manifestations of AM.

Background Current management of pediatric dilated cardiomyopathy (DCM) in children relies on guideline-directed medical therapy (GDMT) extrapolated from adult heart failure. However, due to small sample size, randomized trials of GDMT agents in children have failed to demonstrate efficacy and mortality benefits seen in adults, suggesting fundamental differences in disease mechanisms. We hypothesized that distinct age-dependent transcriptional programs underlie this therapeutic discordance. Methods We performed comparative transcriptomic profiling using bulk RNA sequencing on explanted left ventricular tissue from pediatric (n=29) and adult (n=35) DCM patients (adult DCM from previously published data) compared with age-matched non-failing controls (n=22 pediatric, 14 adult). We analyzed differential gene expressions, pathway enrichment across disease etiologies, and the regulation of a conserved 430-gene {beta}1-adrenergic receptor gene signaling network ({beta}1-GSN) known to modulate remodeling in adult heart failure. Results Transcriptional signatures were profoundly distinct, with only 7.4% of differentially expressed genes shared between adult and pediatric cohorts. Pediatric DCM was characterized by transcriptional reprogramming and the activation of developmental pathways, including WNT/{beta}-catenin and Notch signaling. Conversely, adult DCM hearts were enriched for pathways associated with metabolic dysfunction, mitochondrial deficits, and inflammation. Crucially, while the {beta}1-GSN was desensitized and extensively remodeled in adults, the pathway remained activated in children, with only 4 of 430 network genes showing antithetical regulation. Conclusion The lack of pathological {beta}-adrenergic remodeling in children could provide a molecular explanation for the lack of clear efficacy of {beta}-blockers in this population. Collectively, these results suggest pediatric DCM represents a biologically distinct disease entity rather than an earlier manifestation of adult heart failure, and future therapeutic strategies must move beyond adult extrapolation to target pediatric-specific pathways.

AimsGenome-wide association studies have identified numerous cardiac transcription factors in association with atrial fibrillation. Amongst these transcription factors, the paired-like homeodomain transcription factor 2 (PITX2) is the strongest genetic risk variant associated with atrial fibrillation. However, the downstream mechanisms of PITX2 are not completely understood. Here, we explore the role of PITX2 in oxidative metabolism and stress as a unifying mechanism of arrhythmogenesis. Methods and resultsTo identify PITX2 mechanisms, we performed transcriptomic analysis in Pitx2c-deficient neonatal rat atrial myocytes. We identify oxidative phosphorylation as the top dysregulated pathway and direct transcriptional targets lie in mitochondrial electron transport chain complexes I and IV. Using the Seahorse Extracellular Flux Analyzer, we identified a functional decrease in oxidative metabolism in Pitx2c-deficient cardiomyocytes. As electron transport chain complexes I and IV may generate reactive oxygen species (ROS) under mitochondrial dysfunction, we quantified mitochondrial specific ROS using MitoSOX and observed an increase in mitochondrial specific ROS in Pitx2c-deficient cardiomyocytes. We additionally assessed spontaneous cardiomyocyte calcium cycling using Fluo-8AM and observed an increased frequency of pro-arrhythmogenic mechanisms including early and delayed afterdepolarizations as inferred through calcium traces. Further, we identified sarcomere disassembly including a potential role of PITX2 in regulating Titin, where Pitx2c-deficient cardiomyocytes display Titin mis-localization within the sarcomeres. To assess whether ROS drives these phenotypes, we treated neonatal rat atrial myocytes with N-acetylcysteine, a potent ROS scavenger, and observed decreased early and delayed afterdepolarizations, as well as restoration of Titin localization. ConclusionPITX2C maintains atrial metabolism and redox balance; the loss of PITX2C results in reduced oxidative metabolism and an elevation in oxidative stress that ramifies cardiomyocyte dysfunction. Treatment with antioxidant restores AF-associated phenotypes including abnormal calcium cycling and sarcomere disassembly in Pitx2c-deficient atrial cardiomyocytes. TRANSLATIONAL PERSPECTIVEGenetic variants close to the PITX2 gene associate most strongly with atrial fibrillation. This study reveals a mechanistic link between multiple AF-associated phenotypes and mitochondrial dysfunction with subsequent accumulation of reactive oxygen species downstream of PITX2. Importantly, metabolic therapies and reducing oxidative stress may present a potential clinical strategy to reverse and prevent functional and structural remodelling related to AF.

AimsThe development of a method for isolating mitochondria from a specific cell type within a given tissue, while preserving their structural and functional integrity to the greatest possible extent, remains an ongoing challenge. The aim of this study was to establish a protocol for the isolation of mitochondria from rodent cardiomyocytes, characterized by minimal contamination with other cell types and a high yield of mitochondrial fractions originating from distinct subcellular regions of cardiomyocytes. Methods and resultsIn the present study, cardiomyocytes from guinea pig and rat hearts were isolated using a standard enzymatic digestion protocol in a Langendorff heart perfusion system. Traditionally, the isolation of organelles, including mitochondria, from whole cardiac tissue as well as from cardiomyocytes has relied primarily on mechanical tissue homogenization These conventional approaches involve the localized application of high pressure to cells, which may potentially damage delicate organelles, particularly mitochondria. Moreover, such homogenization preferentially releases mitochondria located in the subsarcolemmal region of cardiomyocytes rather than representing the entire mitochondrial population. In our study, we employed an alternative approach based on the gentle mechanical disruption of cardiomyocytes by passing the cell suspension through selected cell strainers using a cell scraper. This strategy facilitated mild disruption of cellular structures, significantly increasing the yield of mitochondria released from interfibrillar regions while preserving mitochondrial functionality. Moreover, this method decrease probability of sample contamination with mitochondria from other cells, based on cell size differences. The effectiveness of this method was confirmed by transmission electron microscopy, and high-resolution respirometry, which revealed no evidence of outer mitochondrial membrane damage, as indicated by the lack of response to the addition of exogenous cytochrome c to the incubation chamber. Moreover, mitochondrial oxygen consumption increased by 7.39 {+/-} 1.25-fold following the addition of 100 {micro}M ADP, reflecting efficient ADP-stimulated respiration. Furthermore, fluorescence measurements were performed. to assess changes in the mitochondrial inner membrane potential ({Delta}{Psi}). The isolated mitochondria were also suitable for electrophysiological studies using the single-channel patch-clamp technique. Additionally, mitochondria isolated using the protocol developed in our laboratory exhibited a high capacity for transplantation into H9c2 cells. ConclusionIn summary, our mitochondrial isolation method is rapid, efficient, and yields functionally competent mitochondria. These preparations are suitable for a wide range of downstream applications, including patch-clamp electrophysiology, analyses of oxygen consumption under various pharmacological conditions, as well as mitochondrial transplantation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=162 HEIGHT=200 SRC="FIGDIR/small/716092v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@613495org.highwire.dtl.DTLVardef@1c34338org.highwire.dtl.DTLVardef@722900org.highwire.dtl.DTLVardef@e1f7a6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rationale: Heart failure with preserved ejection fraction (HFpEF) is a heterogeneous syndrome with substantial unmet diagnostic and therapeutic needs. Circulating lipid metabolism is increasingly implicated in HFpEF pathophysiology but has not been systematically leveraged for molecular stratification. Objective: To determine whether plasma lipidomics can identify molecular phenogroups of HFpEF associated with distinct clinical characteristics and outcomes. Methods and Results: Untargeted plasma lipidomics was performed in non-HF subjects and HFpEF patients from a primary Belgian cohort and an independent Canadian cohort (n=177 in each cohort). In the Belgian cohort, 235 unique lipids spanning 19 subclasses were annotated, including 96 significantly associated with HFpEF (q<0.02). Unsupervised analyses revealed marked lipidomic heterogeneity, with a distinct HFpEF subgroup separable from non-HF subjects. Hierarchical clustering identified three phenogroups with divergent lipid profiles and clinical features. One phenogroup exhibited severe atrial dysfunction, congestion-related biomarkers, elevated indices of cardiac and liver fibrosis, and markedly reduced survival, a second was characterized by prominent metabolic syndrome features, and a third by preserved renal function. Cross-cohort comparison using a supervised classifier trained on 158 shared lipids confirmed analogous lower-risk phenogroups in the Canadian cohort, while the high-risk phenotype was underrepresented. A signature of 10 lipids across six subclasses, including long-chain acylcarnitines, ether phosphatidylcholines, and oxidized sphingomyelins, discriminated the high-risk group and correlated with markers of disease severity. Conclusion: Our findings demonstrate that HFpEF comprises metabolically distinct patient subgroups across cohorts, revealing specific lipidomic dysfunctions that deepen our understanding of HFpEF heterogeneity and underlying pathophysiology.

Right ventricular failure (RVF) is a serious disease with a high mortality but no effective pharmacologic treatments. We reported RVF was reversed by chronic treatment with an 1A-adrenergic receptor (1A-AR) agonist. Recent studies suggest mitochondrial dysfunction contributes to RVF. Therefore, we investigated if reversal of RVF by chronic 1A-AR agonist treatment involved improved mitochondrial function. A mouse model of RVF caused by pulmonary artery constriction (PAC) for 2 wk was chronically treated for a further 2 wk. with a low dose of the 1A-AR agonist A61603 (10 ng/kg/day) or vehicle (no drug control). RV dysfunction was assessed from the fractional shortening of the RV outflow tract (RVOT FS). RVOT FS for sham controls (46.5 {+/-} 1.3 %, n = 9) was reduced 4 wk after PAC (27.6 {+/-} 1.5 %, n = 13, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (34.5 {+/-} 0.6 %, n = 14, P < 0.001). RV myocardial respiration rate (O2 consumption) for sham controls (776 {+/-} 51 pM/s/mg, n = 9) was reduced 4 wk after PAC (493 {+/-} 28 pM/s/mg, n = 15, P <0.0001), but was higher after PAC plus 2 wk A61603 treatment (634 {+/-} 30 pM/s/mg, n = 11, P <0.05). RV myocardial ATP level for sham controls (3.3 {+/-} 0.1 mM, n = 10) was reduced 4 wk after PAC (1.9 {+/-} 0.1 mM, n = 6, P < 0.0001), but was higher after PAC plus 2 wk A61603 treatment (2.6 {+/-} 0.13 mM, n = 7, P < 0.01). In conclusion, reversal of RVF after chronic A61603 treatment involved reversal of mitochondrial dysfunction. Consistent with our previous studies, this study suggests that the 1A-AR is a therapeutic target to treat RVF. HighlightsRV failure is reported to involve mitochondrial dysfunction which might impair RV contraction by decreasing cardiomyocyte ATP level. Using the pulmonary artery constriction model of RV failure, we found that chronic treatment with an 1A-adrenergic receptor agonist increased RV myocardial respiration rate, increased RV myocardial ATP level, and increased RV function. These findings suggest that the 1A-adrenergic receptor is a therapeutic target for treating RV failure, and that the mechanism involves improved RV cardiomyocyte bioenergetic status.

BackgroundPersistent neurohormonal activation is a key driver of maladaptive remodeling and disease progression in heart failure (HF). Sodium-glucose cotransporter 2 inhibitors (SGLT2is) confer robust renoprotective effects in HF; however, the extent to which these benefits involve modulation of renal neurohormonal activity remains unclear. We hypothesized that SGLT2i-mediated renoprotection in HF is associated with attenuation of excessive renal neurohormonal activation. MethodsMale rats with myocardial infarction-induced HF and sham controls were fed standard chow or chow containing empagliflozin (EMPA, 300 mg/kg) for four weeks, followed by assessment of renal inflammatory and neurohormonal markers. Parallel in vitro studies in THP-1 macrophages and HK-2 proximal tubule cells evaluated the direct effects of EMPA on norepinephrine (NE)-dependent tubular inflammatory signaling. ResultsHF rats displayed higher renal cortical renin gene expression and angiotensin II concentrations, which remained unaffected by EMPA. Conversely, EMPA normalized the elevated urinary NE excretion and renal cortical NE content observed in HF rats. Given the inflammatory role of sympathetic hyperactivity, we assessed renal macrophage polarization. EMPA-treated HF rats showed reduced expression of pro-inflammatory markers (Tnf, Ccr2, Nos2, Il-6) and increased expression of markers associated with a reparative macrophage profile (Arg1, Mrc1, CD163), supported by higher CD206 macrophages in kidney sections. While EMPA did not directly alter THP-1 macrophage activation in vitro, it significantly reduced NE-induced SGLT2 expression and interleukin-6 (IL-6) release by HK-2 human proximal tubule epithelial cells. ConclusionThese findings support a model in which SGLT2 inhibitors confer renoprotection in HF by suppressing renal sympathetic hyperactivity, independently of the intrarenal renin-angiotensin system, thereby disrupting a maladaptive renal neuro-epithelial-immune axis and promoting a reparative macrophage phenotype. CLINICAL PERSPECTIVE Whats new?O_LIThis study identifies a renal neuro-epithelial-immune axis underlying empagliflozin-mediated renoprotection in heart failure. C_LIO_LIEmpagliflozin reduces renal cortical and urinary norepinephrine levels in heart failure without altering intrarenal renin-angiotensin system activity, revealing a distinct neurohumoral target of SGLT2 inhibition. C_LIO_LIThis sympatholytic effect is associated with a shift in renal macrophages toward a reparative (M2) phenotype, without changes in total macrophage abundance. C_LIO_LIEmpagliflozin blocks norepinephrine-induced SGLT2 upregulation, limiting proximal tubular glucose reabsorption and IL-6 production, and linking sympathetic signaling to renal inflammation. C_LI What are the clinical implications?O_LIOur findings provide a mechanistic basis for the additive cardiorenal benefits of SGLT2 inhibitors in heart failure, beyond conventional RAS-directed therapies. C_LIO_LITargeting renal sympathetic-driven inflammation may help preserve kidney function and attenuate the progression of cardiorenal syndrome. C_LIO_LISuppression of a renal neuroinflammatory pathway may help explain the early and sustained benefits of SGLT2 inhibitors across heart failure phenotypes, including nondiabetic patients. C_LI

Genetic cardiomyopathies consist of a heterogeneous group of myocardial disorders caused by variants that disrupt key regulators of cardiac structure and function. Variants in PLN, encoding phospholamban (PLN), the main inhibitor of the sarco/endoplasmic reticulum Ca{superscript 2}-ATPase 2a (SERCA2a), have been linked to both dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). Among these, the PLN Arg14del (R14del) variant is the most prevalent. PLN R14del cardiomyopathy is characterized by the accumulation of large perinuclear PLN aggregates in cardiomyocytes of end-stage heart failure tissue. However, the mechanisms driving PLN aggregate formation and their role in disease progression remain unresolved. Using a humanized plna R14del zebrafish model, left ventricular tissue from end-stage PLN R14del cardiomyopathy patients and pharmacological modeling in wild type (WT) cardiac slices, we demonstrate that previously described PLN aggregates represent accumulated sarcoplasmic reticulum (SR)-derived PLN-containing vesicles that form due to impaired SERCA2a activity and increased cytosolic Ca{superscript 2} levels. Furthermore, these SR-derived vesicles often localize adjacent to lysosomes. Interestingly, Ca2+ dysregulation in plna R14del hearts leads to reduced lysosomal function, resulting in SR-derived vesicle accumulation at the microtubule organizing center (MTOC). This perinuclear accumulation induces microtubule aster formation and subsequent cellular disorganization, including sarcomere misalignment and nuclear deformation. Strikingly, reactivation of lysosomal function through fasting reduces SR-derived vesicle accumulation, restores microtubule integrity, and rescues cellular organization in plna R14del zebrafish hearts. Together, these findings identify impaired lysosomal clearance of SR-derived vesicles and the resulting microtubule disorganization as key pathological mechanisms driving PLN R14del cardiomyopathy. Additionally, our results highlight lysosomal reactivation as a promising potential therapeutic strategy to halt or reverse PLN R14del cardiomyopathy progression. Main findingsO_LIPLN aggregates in PLN R14del cardiomyopathy represent SR-derived vesicles formed due to Ca{superscript 2} dysregulation. C_LIO_LIThese SR-derived vesicles often localize perinuclearly at the microtubule organizing center (MTOC), where they are positioned adjacent to lysosomes. C_LIO_LICa2+ dysregulation leads to lysosomal dysfunction which drives vesicle accumulation responsible for microtubule remodeling and pathological cellular rearrangements. C_LIO_LILysosomal reactivation restores vesicle clearance and rescues cardiomyocyte structure. C_LI

Background. Hypertrophic cardiomyopathy (HCM) is a clinically variable disease in terms of onset and progression. Pathogenic MYBPC3 variants account for a substantial proportion of HCM diagnoses. This study sought to identify protein biomarkers associated with HCM severity. Methods. Olink-assayed plasma proteins of 144 MYBPC3 pathogenic variant carriers were tested for associations with HCM severity based on HCM diagnostic criteria (unaffected, mildly, or severely affected). The UK Biobank was used to replicate the identified proteins through considering time to onset of HCM (67 cases), cardiomyopathy (156 cases),and associations with cardiac MRI derived left ventricular maximum wall thickness (6,492 participants). Replicated proteins were further prioritised based on cardiac tissue expression and druggability, and annotated using pathway enrichment and association with onset of: heart failure (HF), dilated cardiomyopathy (DCM), sudden cardiac arrest (SCA), and ventricular arrhythmias (VA). Results. Among pathogenic MYBPC3 variant carriers, we identified 27 proteins associated with HCM severity. We independently replicated 21 proteins in the UK Biobank. Of the five prioritised proteins (NT-proBNP, GDF-15, FGF-23, ADM, and NCAM1), all but NT-proBNP were targeted by drugs with repurposing potential. The replicated proteins additionally associated with the incidence of HF (n=5), DCM (n=4), SCA (n=4), and VA (n=4). Conclusion. This study replicated 21 and prioritised five proteins associated with HCM severity in pathogenic MYBPC3 variant carriers. Replication in unselected HCM suggests the prioritised proteins are associated with HCM independent of genotype, providing important leads for plasma-based markers for diagnoses, disease monitoring, and drug targets.

BackgroundPrimary mitral regurgitation resulting from mitral valve prolapse can lead to life-threatening complications, including arrhythmias, heart failure, and sudden cardiac death. Mitral valve prolapse is classically associated with myxomatous mitral valve degeneration, characterized by leaflet thickening, extracellular matrix disorganization, and progressive structural remodeling. Valvular interstitial cells, the predominant stromal population within the valve, maintain extracellular matrix homeostasis; however, their molecular heterogeneity, and state-specific contributions to disease pathogenesis remain incompletely defined. MethodsUsing a fibrillin-1 deficient mouse model and human tissue specimens we integrated single-cell RNA sequencing with spatial transcriptomic profiling to construct a comprehensive atlas of cellular composition and extracellular matrix organization across normal mitral valves, sporadic mitral valve prolapse, and Marfan syndrome-associated mitral valve prolapse. ResultsAnalyses revealed spatially organized cellular niches and substantial heterogeneity within the valvular interstitial cell population. Across murine and human datasets, we identified a conserved activated valvular interstitial cell population enriched for profibrotic extracellular matrix remodeling programs and preferentially localized to mechanically vulnerable leaflet tip regions. This population exhibited coordinated upregulation of collagen- and matrix-associated genes, metabolic signatures consistent with enhanced mitochondrial activity, and transcriptional features suggesting fibro-inflammatory signaling. ConclusionsWe identified a transcriptionally and spatially distinct activated valvular interstitial cell state conserved across species and disease etiologies that is strongly implicated in fibrotic remodeling during myxomatous mitral valve degeneration and provides a candidate therapeutic target.

Abstract Background: ST-elevation myocardial infarction (STEMI) is reported to be a leading cause of mortality worldwide. While cardiac troponins are the gold standard for myocardial injury detection but creatine kinase-MB (CK-MB) and total creatine phosphokinase (CPK) retain prognostic use in resource-limited settings. Objective: To evaluate the prognostic significance of admission CK-MB and CPK levels in STEMI patients and to assess their association with hematological parameters for integrated risk stratification. Methods: This cross-sectional study enrolled 15 consecutive STEMI patients from the Punjab Institute of Cardiology, Lahore, during January 2024. Comprehensive laboratory analysis including cardiac biomarkers (CK-MB, CPK, troponin-I, LDH), complete blood count, renal function, serum electrolytes, and metabolic parameters, was performed on admission. Pearson correlation and comparative statistical analyses were also conducted to assess the relationships between cardiac biomarkers and hematological indices. Results: The cohort includes 15 patients (mean age 50.1 +/- 12.2 years; 73.3% male). Cardiac biomarker elevation was prevalent: CK-MB was elevated in 12/15 (80%), CPK was elevated in 12/15 (80%), with concordant elevation in 11/15 (73.3%), which indicates extensive myocardial necrosis. Troponin-I showed the highest elevation rate at 13/15 (86.7%). Hematological abnormalities included anemia (60%), WBC elevation (53.3%), and RBC reduction (40%). Random glucose averaged 150.80 +/- 63.55 mg/dL, with 66.7% highlighted the hyperglycemia. Remarkably, electrolyte balance was preserved in all of the patients (0% sodium, potassium, and bicarbonate abnormalities), indicating maintained homeostasis. Pearson correlation analysis revealed a significant correlation between CK-MB and CPK (r = 0.615, p = 0.0126), while correlations between cardiac biomarkers and hematological parameters were weak (p > 0.05). Risk stratification identified 53.3% of patients as high-risk who required intensive management. Conclusions: CK-MB and CPK demonstrate significant concordance and retain prognostic value in STEMI patients, particularly in resource-limited settings where troponin access may be constrained. While troponin-I remains the most sensitive biomarker, combined assessment of conventional cardiac enzymes supports reliable evaluation of myocardial injury. Hematological parameters reflect systemic response but show limited correlation with cardiac biomarkers.

BACKGROUNDMyocarditis is an inflammatory cardiac disease in which Th17-driven immune responses contribute to progression toward dilated cardiomyopathy and heart failure. Current therapies mainly rely on corticosteroids but lack specificity, while the role of miR-721, synthesized by Th17 cells, remains largely unexplored in disease pathogenesis. METHODSWe characterized the presence of mmu-miR-721 and its human homolog hsa-RNA-Chr8:96 in extracellular vesicles (EVs) secreted by Th17 cells from IL-17eGFP mice with experimental autoimmune myocarditis (EAM) and myocarditis patients. MxCre-Ppargfl/fl mice and luciferase reporter assays were used to validate the target genes of miR-721 and hsa-RNA-Chr8:96, respectively. The functional role of miR-721 in EAM was investigated by lentiviral vectors overexpression and inhibition using miRNA sponge molecules. Th17 responses and heart inflammation were assessed and echocardiography was performed after in vivo blockade of mmu-miR-721 in EAM mice. RESULTSBoth mmu-miR-721 and hsa-RNA-Chr8:96 were encapsulated in EVs and secreted by Th17 cells of mice and patients with myocarditis. Overexpression of mmu-miR-721 in draining-lymph node cells from EAM mice inhibited Pparg transcription, leading to increased ROR{gamma}t and IL-17 expression and promoting Th17 differentiation. In contrast, in the absence of Pparg, a target of miR-721, no differences in ROR{gamma}t expression were observed, indicating that miR-721 promotes Th17 responses through repression of Pparg. Human PPARG was validated as a target gene of hsa-RNA-Chr8:96 and its overexpression in peripheral blood leukocytes downregulated PPARG mRNA levels, suggesting similar pathways involved in human pathology. In vivo blockade of mmu-miR-721 increased Pparg expression, reducing ROR{gamma}t and IL-17 activation in T cells and leading to decreased leukocyte infiltration in the heart and improved cardiac function. CONCLUSIONSmiR-721 is released by Th17 cells in EVs and promotes Th17 responses during myocarditis through repression of PPAR{gamma}, identifying this miRNA as both a mechanistic driver of disease and a potential therapeutic target. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/713340v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1a69953org.highwire.dtl.DTLVardef@9c36bdorg.highwire.dtl.DTLVardef@1cdce4dorg.highwire.dtl.DTLVardef@a34715_HPS_FORMAT_FIGEXP M_FIG C_FIG Novelty and significanceO_ST_ABSWhat is known?C_ST_ABSO_LImiR-721 and its human homolog are upregulated in the plasma of mice and humans with myocarditis C_LIO_LITh17 cells synthesize miR-721 C_LIO_LIMmu-miR-721 targets Pparg mRNA C_LI What new information does this article contribute?O_LImiR-721 is sorted into extracellular vesicles in the context of acute myocarditis C_LIO_LImiR-721 enhances Th17 differentiation via the Pparg/Rorc double inhibitory axis. C_LIO_LIHsa-RNA-Chr8:96 targets human PPARG mRNA for degradation, inhibiting its expression C_LIO_LIBlockade of miR-721 dampens acute myocarditis development in vivo C_LI This study reveals a novel miRNA-based therapeutic strategy to inhibit Th17 responses and treat myocarditis. Using the experimental autoimmune myocarditis model, the authors unravel the mechanisms by which mmu-miR-721 can enhance Th17 responses and show how targeting this regulatory molecule could ameliorate the progression of the disease. Remarkably, this regulatory axis is suggested to be present in humans as well, since PPARG gene is validated as a target gene for hsa-RNA-Chr8:96. These findings highlights the potential of miR-721 not only as a diagnostic tool but also as a cell-specific therapeutic target to control Th17 responses in the clinical setting.

BackgroundPathogenic desmin (DES) variants have been implicated in early-onset atrial disease, yet the mechanisms by which desmin dysfunction alters atrial structure and function remain unclear. Desmin anchors the cytoskeleton to the nuclear envelope (NE) through the linker of nucleoskeleton and cytoskeleton (LINC) complex, suggesting that defects in this network may drive atrial cardiomyopathy. MethodsHuman desmin wild-type (WT) and the pathogenic variants p.S13F, p.N342D, and p.R454W were stably expressed in HL-1 atrial cardiomyocytes. Desmin organization, nuclear morphology, LINC-complex integrity (nesprin-3, lamin A/C), and DNA leakage, assessed by cyclic GMP-AMP synthase (cGAS), were analyzed by confocal microscopy. Action potential duration (APD) and calcium transients (CaT) were measured optically. Human myocardium samples from DES variant carriers were analyzed for validation. Data-independent acquisition (DIA) mass spectrometry profiled atrial proteomes from desmin-network (DN) and titin variant carriers and controls. The heat-shock proteins (HSPs) inducer geranylgeranylacetone (GGA) was evaluated for rescue effects. Resultsp.N342D caused severe filament-assembly defects with prominent perinuclear aggregates, whereas p.S13F showed mixed phenotypes with frequent perinuclear aggregates, and p.R454W largely preserved filamentous networks. p.N342D and p.S13F induced nuclear deformation with disrupted nesprin-3 and lamin A/C distribution. In p.N342D and p.S13F, desmin aggregates drove focal lamin A/C accumulation, nuclear envelope (NE) rupture, DNA leakage, and increased cGAS activation. DES variants significantly shortened APD20/90 and reduced CaT amplitude, indicating pro-arrhythmic electrical remodeling. Atrial proteomics revealed a DN-specific signature enriched for cytoskeletal, NE, intermediate filament, and chaperone pathways, consistent with the structural injury observed in vitro. GGA prevented desmin aggregation and nuclear morphology changes, and mitigated APD shortening in p.N342D-expressing cardiomyocytes. Human myocardium from DES variant carriers showed concordant desmin aggregation and polarized lamin A/C distribution. ConclusionsDES variants induce a desmin-dependent atrial cardiomyopathy characterized by cytoskeletal disorganization, disruption of LINC-complex, NE rupture with DNA leakage, and pro-arrhythmic electrophysiological remodeling. These findings provide mechanistic insight into how DN variants promote atrial disease. HSPs induction by GGA partially restores structural and functional integrity, identifying a potential therapeutic approach for desmin-related atrial cardiomyopathy. Clinical perspectiveWhat is new? O_LIPathogenic DES variants induce a previously unrecognized atrial cardiomyopathy characterized by desmin aggregation, and desmin-network (DN) collapse, disruption of the linker of nucleoskeleton and cytoskeleton (LINC) complex, and nuclear envelope rupture with DNA leakage. C_LIO_LIVariants that lead to desmin aggregation (e.g., p.N342D) cause focal lamin A/C polarization, cyclic GMP-AMP synthase (cGAS) activation, and structural injury at the nuclear envelope. C_LIO_LIDES variants produce pro-arrhythmic electrical remodeling, including action potential duration shortening and impaired Ca{superscript 2} handling in HL-1 atrial cardiomyocytes. C_LIO_LIAtrial proteomics from DN variant carriers reveals enrichment of pathways related to cytoskeletal, nuclear envelope, intermediate filament, and chaperone, supporting a desmin-dependent remodeling program. C_LIO_LIThe heat-shock protein inducer geranylgeranylacetone (GGA) prevents desmin aggregation, restores nuclear morphology, and mitigates electrical and Ca{superscript 2} handling remodeling. C_LI What are the clinical implications? O_LIThese findings establish DN dysfunction as a distinct cause of atrial cardiomyopathy, providing a mechanistic basis for the association between pathogenic DES variants and atrial arrhythmias, including atrial fibrillation. C_LIO_LINuclear envelope rupture and cytosolic DNA leakage represent new mechanistic evidence which links cytoskeletal injury and atrial arrhythmogenesis. C_LIO_LIIdentifying structural vulnerability in DES variant carriers fosters awareness of genetic counseling for atrial disease, enabling early detection and risk stratification. C_LIO_LIThe protective effects of GGA suggest that restoring proteostasis may be a therapeutic strategy for desmin-related atrial cardiomyopathy and potentially other genetic atrial diseases. C_LI Novelty and significance statementO_ST_ABSNoveltyC_ST_ABSThis study identifies a desmin-dependent atrial cardiomyopathy driven by cytoskeletal aggregation, LINC-complex disruption, and nuclear envelope rupture with DNA leakage. We show that pathogenic DES variants are associated with pro-arrhythmic molecular remodeling and that human atrial proteomics confirm nuclear envelope and cytoskeletal injury as core features. Importantly, the heat-shock protein-inducer GGA rescues structural, molecular, and electrophysiological defects, revealing a modifiable pathway in desmin-mediated atrial disease. SignificanceThese findings provide the first integrated mechanistic explanation linking DN variants to atrial cardiomyopathy. By uncovering nuclear envelope rupture and cGAS activation as key drivers of atrial cardiomyopathy, this work expands the molecular framework for inherited atrial disease and highlights proteostasis enhancement as a potential therapeutic strategy for patients carrying DES and related cytoskeletal variants. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=166 HEIGHT=200 SRC="FIGDIR/small/26348559v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1fb0bfborg.highwire.dtl.DTLVardef@cfc00borg.highwire.dtl.DTLVardef@1493578org.highwire.dtl.DTLVardef@1556b61_HPS_FORMAT_FIGEXP M_FIG C_FIG