bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Mo040: Enhanced neovascularisation does not account for preservation of cardiac function by 11βHSD1 inhibition in a pig model of myocardial infarction
Authors: Al Disi Sara , Ascione Raimondo , Johnson Tom , Sammut Eva , Bruno Vito Domenico , Khan Shazia , Singh Trisha , Baz-Lopez Daniel , James Carol-Ann , Walker Brian , Mills Nicholas , Webster Scott , Freeman Adrian , Whittaker Andrew , Andrew Ruth , Gray Gillian
Published: October 2024

While acute post-myocardial infarction (MI) survival has improved thanks to early intervention, the myocardium still sustains damage that increases the risk of heart failure. Adrenal glucocorticoids (GCs) protect cardiomyocytes immediately after MI, but inhibition of GC regeneration within the heart by 11β-hydroxysteroid dehydrogenase 1 (11βHSD1) during infarct repair prevents subsequent deterioration of ventricular structure and function. In the 11βHSD1-knockout mouse this outcome is associated with enhancement of peri-infarct neovascularisation and prevention of infarct expansion. Here, we tested the hypothesis that pharmacological 11βHSD1 inhibition after MI can preserve function in a translational pig model and investigated the role of neovascularisation. MI was induced in female minipigs via temporary coronary occlusion. A standard clinical therapy (SCT, n =9) group received statin, anti-platelet, βadrenoreceptor antagonist and ACE inhibitor immediatley after MI, and a 11βHSD1i group (n=11) received SCT +11βHSD1 inhibitor (50mg/kg) from 48h after MI until the end of the study. Prior to 11βHSD1i treatment, short-axis cine and late gadolinium-enhanced MRI showed no difference in either infarct mass (4.2±0.4g SCT vs 4.0±0.6g 11βHSD1i) or ejection fraction (EF; 60.6±2.9% SCT vs 56.6±1.8% 11βHSD1i). However, by 28 days after MI, while EF was reduced in SCT pigs to 48±4%, it was maintained in pigs given additional 11βHSD1i treatment (57.9±1.8%,
p
<0.05 vs SCT). Infarct mass did not differ between the two treatments, suggesting that inhibition of infarct expansion did not account for the improvement in function, this was confirmed by a similar area of infarct collagen in histological analysis. Angiogenesis, represented by an increase in CD31
+ve
capillaries, was not enhanced in the infarct or the border zone (BZ) after 11βHSD1i treatment, relative to SCT. While vessel maturation (αSMA
+ve
vessels) was improved in the infarct compared to the remote myocardium, there was no difference between the two groups. Proteomic pathway analysis of the BZ indicated extracellular matrix organisation as the main pathway regulated by 11βHSD1i compared to SCT. In conclusion, 11βHSD1i after MI successfully preserves cardiac function and structure but the mechanism is independent of scar size reduction or enhanced neovascularisation. Instead, the beneficial effects of 11βHSD1i are likely to result from favourable regulation of collagen processing during scar formation.
Abstract Mo043: Advanced waveform analysis of the electrocardiogram signals using complementary signal processing techniques to investigate the response to a QTc prolonging drug vs control including food effect.
Authors: Serna Pascual Miquel , Nandi Manasi , Taubel Jorg , Lorch Ulrike , Djumanov Dilshat , Sadsad Tyrone-Joshua , Mejia Elisa , Rickard James
Published: October 2024

Background:
Thorough QT studies using Electrocardiogram data, are a well-established method for testing the pro-arrhythmic propensity of drugs performed during drug development. ECG analysis is currently reduced to simplified biomarkers, such as rate, intervals and amplitudes. However, ECG waveforms are complex and generally sampled with high fidelity 125-1000 Hz. Our novel mathematical method, Symmetric Projection Attractor Reconstruction analyses the morphology and variability of such waveforms, using every data point to generate a new, faithful 2D waveform representation termed an attractor. Moxifloxacin know QT prolonging medicine and has been shown to cause a mean increase of the QTc interval of 10–14 mS after a single dose of 400 mg. Separately, studies have shown that meals can alter QT interval as well as altering the pharmacokinetic profile of moxifloxacin itself with gender and ethnicity impacting individual responses. In-depth knowledge of these physiological factors could support safer medicines development and clinical use.

Purpose:
This project investigated the feasibility of SPAR analysis of ECG signals to detect drug induced changes which a higher degree of sensitivity, to support safety evaluation during medicines development

Methods:
High resolution triplicate 12 lead ECG time-series data from a Phase one study of healthy human volunteers cross over of placebo, moxifloxacin or moxifloxacin + food was retrospectively analysed using SPAR at 0,1 and 6 hours. MATLAB bespoke software tool was used to perform SPAR analyses to transform ECG time-series into corresponding attractors. Corresponding single point measurements were previously obtained as part of the original Phase one study.

Results:
Initial evaluation of attractors revealed high inter-individual variation, consistent with previous SPAR ECG analysis. Nevertheless, we identified changes in subjects receiving Moxifloxacin +/- food vs placebo control. These changes were not as evident in previously obtained QT intervals.

Conclusion:
This pilot study illustrates that SPAR is highlighting changes on the ECG in fasted and fed states that are less obvious from QT data alone. Further evaluation of the whole dataset to identify generalisable SPAR features, and their comparison with existing markers is now necessary. The individualised nature of ECGs and corresponding attractors indicates the method may have highest utility in personalised medicine.
Abstract Mo048: Cereblon as a potential biomarker for early detection of diabetic cardiomyopathy
Authors: Cho Sung Woo , Kim Hyoung Kyu , Kim Byung Gyu , Han Jin
Published: October 2024

Type 2 diabetes mellitus (T2DM) patients have a higher risk of heart failure and cause cardiomyopathy without other cardiac disease. There is no specific biomarker for discriminating patients with diabetic cardiomyopathy (DCM). In this study, we investigated the cereblon (CRBN) level in mice and human samples to determine its potential as a biomarker for early detection of diabetic cardiomyopathy (DCM). C57BL/db/db male mice at 6 and 8 weeks of age, as the early DCM model with obesity and T2DM were investigated. We confirmed that mitochondrial function was decreased in the 8 weeks old db/db mice without alterations of cardiac function and structures. Compared to the control mice, CRBN protein level was significantly increased and phosphorylation of AMPK, a negative regulator by CRBN, was significantly decreased in the heart tissue of 8 weeks old db/db mice. Blood CRBN level was also significantly increased in both 6 and 8 weeks old db/db mice compared with control mice. In blood of diabetic patients, CRBN level was significantly correlated with IL-6, IL-1b, TNF-a, IGFBP-7, and adiponectin, which are previously reported biomarkers of DCM. However, CRBN level was not correlated with cardiac troponin I, NT-proBNP, and ST2, which are biomarkers of myocardial injury and heart failure. Furthermore, the CRBN level showed a higher tendency in patients with diastolic dysfunction without systolic dysfunction compared to patients with normal function and systolic dysfunction, although the difference was not statistically significant. In conclusion, cardiac mitochondrial dysfunction occured and CRBN levels of heart and blood increased prior to cardiac functional and structural changes in the early DCM mouse model, and CRBN might serve as a potential biomarker for the early detection of DCM in T2DM patients.
Abstract Mo053: Acacetin's Antiarrhythmic Potential in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes Harboring Hypertrophic and Dilated Cardiomyopathy-Related Mutations
Authors: abdelsayed mena , Mercola Mark , Antzelevitch Charles
Published: October 2024

Background:
Hypertrophic cardiomyopathy (HCM) is often caused by mutations such as p.R943X in the Myosin Binding Protein C3 (MYBPC3), leading to early and delayed afterdepolarizations, myofibrillar disarray, and calcium dysfunction, which may induce lethal arrhythmias. Dilated cardiomyopathy (DCM) can also result from mutations, such as the p.R14del mutation in the phospholamban (PLN) protein, which is crucial for calcium signaling regulation, leading to arrhythmogenesis when provoked. This study explores the arrhythmogenic potential in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the HCM MYBPC3 p.R943X mutation and the DCM PLN p.R14del mutation.

Methods:
We utilized a 384-well optical physiological system to record action potentials (APs) in hiPSC-CMs carrying the MYBPC3 p.R943X mutation and the DCM PLN p.R14del mutation, including their isogenic controls. Action potential metrics were measured under spontaneous and electrically paced conditions. The effects of Acacetin, NS-5806, nifedipine, and GS-967 on the recorded AP types were analyzed to understand the underlying channelopathies and arrhythmogenic potential. Special attention was given to the variability between the HCM MYBPC3 p.R943X and DCM PLN p.R14del cell lines' susceptibility to arrhythmogenesis and their isogenic controls.

Results:
The HCM MYBPC3 p.R943X hiPSC-CMs exhibited a significant increase in early afterdepolarizations, non-sustained and sustained ventricular tachycardia, and AP notches compared to their isogenic control. AP notches and arrhythmogenic events were notably provoked by NS-5806 in both the HCM MYBPC3 p.R943X and DCM PLN p.R14del hiPSC-CMs. However, these were significantly ameliorated (p<0.05) by Acacetin administration (5 µM), highlighting its potential as an effective antiarrhythmic in managing both HCM- and DCM-induced arrhythmias. GS-967 also showed efficacy in reducing early afterdepolarizations (EADs) in the HCM model.

Conclusions:
This study provides novel insights into the arrhythmogenic potential of hiPSC-CMs carrying MYBPC3 p.R943X and PLN p.R14del mutations, underscoring the significant role of Acacetin in attenuating these effects. It paves the way for developing targeted therapies that address the complex molecular mechanisms underlying arrhythmogenesis in cardiomyopathies. The findings suggest that Acacetin could be a promising candidate for the pharmacologic treatment of cardiomyopathies characterized by arrhythmogenic risks.
Abstract Mo055: CHD4 Interacts With TBX5 to Maintain the Gene Regulatory Network of Postnatal Atrial Cardiomyocytes
Authors: Sweat Mason , Shi Wei , Keating Erin , Ponek Anna , Conlon Frank , Pu William
Published: October 2024

Atrial fibrillation (AF) is the most common sustained arrhythmia in humans, leading to increased mortality due to increased incidence of stroke and heart failure. Over 200 genes are associated with AF by GWAS studies, suggesting a nuanced gene regulatory network (GRN) is required to maintain atrial cardiomyocyte (aCM) function and rhythm. Among these genes is the transcription factor TBX5, which we and others have shown is a master regulator of aCM identity that is essential for atrial rhythm homeostasis. We showed that TBX5 is required to maintain the chromatin looping, accessibility, and activity of atrial enhancers. Chromodomain helicase DNA binding protein 4 (CHD4), a chromatin remodeling protein that interacts with TBX5, is canonically thought to repress transcription as a component of the NuRD complex. Here, we investigated the contribution of CHD4 to TBX5 regulation of aCM gene expression.

Methods:
We inactivated CHD4 specifically within atrial cardiomyocytes using Cre recombinase driven by the aCM-specific
Nppa
promoter delivered postnatally with AAV9. Atrial remodeling, transcriptomic changes and AF susceptibility were examined.

Results:
We show that inactivating CHD4 in postnatal aCMs resulted in fibrotic atrial remodeling, spontaneous AF in a subset of mice, and increased AF susceptibility and burden after electrical pacing. Transcriptomic profiling of CHD4 aCM KO atria revealed gene expression changes that were highly correlated to altered gene expression downstream of TBX5 inactivation. Mechanistically, we show TBX5 recruits CHD4 to 33,170 genomic regions that are important for aCM gene regulation, whose expression is concordantly regulated downstream of TBX5 and CHD4. Together, these data reveal that CHD4 is required by TBX5 to promote accessibility of enhancers and activate many TBX5 target genes to maintain atrial rhythm.
Abstract Mo059: Leucine-Rich Repeat-Containing Protein 10 Modulates Human Cardiac L-Type Ca
2+
Channels but not T-Type Ca
2+
Channels
Authors: Siri-Angkul Natthaphat , Woon Marites , Gregorich Zachery , OLORUNDARE OLUFUNKE , Lee Youngsook , Kamp Timothy
Published: October 2024

Background:
Leucine-rich repeat-containing protein 10 (LRRC10) is a novel regulator of the cardiac L-type Ca
2+
channel/current (LTCC/I
Ca,L
). LRRC10 is crucial for cardiac regeneration in multiple species, and LRRC10 variants have been associated with dilated cardiomyopathy in humans. LRRC10 increases I
Ca,L
in HEK-293 cells expressing rabbit Ca
V
1.2 subunit, and knockout of LRRC10 in zebrafish and mouse cardiomyocytes (CM) reduces I
Ca,L
. However, LRRC10-mediated regulation of human cardiac LTCC has not been demonstrated, and the mechanism by which LRRC10 modulates LTCC is not known. Moreover, the effect of LRRC10 on the T-type Ca
2+
channel/current (TTCC/I
Ca,T
), which has been implicated in CM cell cycle regulation, has never been tested.

Methods:
LRRC10
knockout (
LRRC10
–/–
) human induced-pluripotent stem cells (hiPSC) were generated from wild-type (WT) DF19-9-11T hiPSCs using CRISPR-Cas9 techniques.
LRRC10
–/–
and WT hiPSCs were differentiated to CMs using the standard GiWi protocol. Whole-cell I
Ca,L
and I
Ca,T
were examined in
LRRC10
–/–
and WT hiPSC-CMs. Whole-cell I
Ca,L
was also assessed in HEK-293 cells expressing human cardiac LTCC subunits (Ca
V
1.2, β
2
, and α
2
δ) with or without human LRRC10. To identify the LRRC10-interacting site of Ca
V
1.2, GST-pulldown assays were performed using purified GST-fusion constructs of the intracellular domains of rabbit Ca
V
1.2 and lysates from HEK-293 cells expressing human LRRC10. Ca
V
protein sequences were analyzed using the Clustal Omega program.

Results:
Genetic ablation of
LRRC10
in hiPSC-CMs decreased peak I
Ca,L
(WT -29.4±2.2 pA/pF,
LRRC10
–/–
-15.0±1.8 pA/pF, p<0.01) but did not significantly affect I
Ca,T
magnitude (WT -3.9±0.5 pA/pF,
LRRC10
–/–
-4.0±0.7 pA/pF, p>0.05 [NS]). LRRC10 increased peak I
Ca,L
in HEK-293 cells expressing human LTCC (with LRRC10 -156.0±19.8 pA/pF, without LRRC10 -60.2±12.0 pA/pF, p<0.05). LRRC10 was pulled down by the GST-Ca
V
1.2-N-terminus but not by other GST-Ca
V
1.2 constructs. Sequence analyses revealed an invariant exon 2-encoded segment of the N-terminus (NT) that was present in both human and rabbit LTCC Ca
V
1.2 but not in TTCC Ca
V
3.1 and Ca
V
3.2.

Conclusions:
The Ca
2+
influx-potentiating effect of LRRC10 is conserved in human LTCC and is specific to regulate I
Ca,L
, not I
Ca,T
. The invariant Ca
V
1.2 NT segment is a potential LRRC10-interacting site. The
LRRC10
–/–
hiPSC line is a promising tool for future investigations into the roles of LRRC10-LTCC interaction in human cardiac biology and disease.
Abstract Mo061: The major voltage-gated sodium channel accessory subunit in working ventricular myocytes may be β1B, not β1
Authors: Williams Zachary , Neutel Dean , Payne Laura , Jourdan Jane , Gourdie Robert
Published: October 2024

Voltage-gated sodium channels, crucial for the action potential upstroke in cardiomyocytes, consist primarily of a single pore-forming α-subunit and 1 or 2 accessory β-subunits. SCN1B encodes two variants: β1, a transmembrane cell adhesion molecule, and β1B, typically described as a soluble secreted protein. While β1 and β1B share identical sequences in the amino-terminal (NT) extracellular Ig domain, they differ in their carboxy-terminal (CT) sequence. The Ig domain of β1/β1B has been deemed critical in maintaining normal cardiac conduction by various groups. Traditionally, effects on cardiac conduction have been attributed to β1, despite difficulties in detecting it at the protein level in the heart. We hypothesized that β1B, rather than β1, plays a significant role in electrical activity propagation in the heart. Utilizing an NT antibody (detects both variants), a β1-specific CT antibody, and a custom β1B-specific CT antibody, we examined SCN1B gene product profiles in various cardiac cell types, including neonatal rat ventricular myocytes and fibroblasts (NRVMs and NFs), isolated adult mouse cardiomyocytes (MCMs), and tissue slices from isolated adult rat hearts. The β1-specific CT antibody failed to label any bands in Western Blots (WB) of these cells and tissues, whereas the NT antibody detected a 37 kD band in all cell types and showed high levels of localization at cell-cell contacts using immunofluorescence (IF) with NRVMs. Conversely, the CT antibody showed minimal immunolabeling evidence. Moreover, an inhibitor of β1 Regulated Intramembrane Proteolysis failed to generate the expected 19 kD fragment if β1 were present in NRVMs. In adult rat hearts, the NT antibody labeled intercalated discs, while the CT showed minimal signal. Conversely, in WBs of NRVMs using our custom β1B antibody, we observed a 37 kD band consistent with the presence of β1B, and in heart sections, we found labeling of intercalated discs. These findings suggest that the major β subunit in working ventricular myocytes may be β1B, not β1. Further investigation is warranted, as these results could significantly impact our understanding of the roles and functional effects of β1 and β1B on cardiac conduction.
Abstract Mo062: Cpne5 is Necessary for Normal Sinoatrial Node Function
Authors: Dang Katherine , Ren Lu , Atzert Felix , Thai Phung , Mantri Sruthi , Duan Lauren , Beyersdorf Benjamin , Chiamvimonvat Nipavan , Wu Sean , Goodyer William
Published: October 2024

Background:
Cpne5 (Copine 5) is a Ca2+-dependent phospholipid binding protein implicated in membrane trafficking but has no known role within the heart to date. In single-cell RNA-sequencing (scRNA-seq) of the developing mouse heart, we found Cpne5 gene expression to be highly enriched throughout the cardiac conduction system (CCS). Further, variants in CPNE5 were identified in 6 independent GWAS related to heart rate variability in humans. We therefore hypothesize that Cpne5 is necessary for normal CCS function.

Objective:
Determine whether Cpne5 is necessary for the normal development and/or function of the CCS.

Methods:
To assess the necessity of Cpne5, homozygous systemic knockout (KO) mice were generated using CRISPR/Cas9. Cpne5KO and littermate wild-type (WT) control mice were characterized using surface electrocardiogram (ECG), continuous telemetry and echocardiogram. Protein expression of Cpne5 and CCS-related ion channels were assessed by immunofluorescence of tissue sections, isolated adult murine SAN cells and whole mount immunostaining with 3D volumetric imaging (iDISCO+). Further, CRISPR-mediated deletion of CPNE5 was performed in human induced pluripotent stem cells (hiPSC). Differentiated SAN-like cells were phenotyped via calcium transient (CaT) dynamics and microelectrode array.

Results:
Successful deletion of Cpne5 in CRISPR-generated KO mice was confirmed by PCR immunofluorescence. Homozygous Cpne5KO mice were viable, showed normal growth and allele frequencies segregated in a normal Mendelian fashion. No gross anatomic defects of the CCS were observed in Cpne5KO mice and transthoracic echocardiogram studies showed slight reduction in left ventricular fractional shortening but otherwise a structurally normal heart. Notably, while surface electrocardiogram (ECG) demonstrated normal intervals (PR, QRS, QTc), Cpne5KO mice exhibited pronounced sinus arrhythmia, sinus pauses, and increased heart rate variability compared to WT controls on long term telemetry. Additionally, CPNE5KO hiPSC-derived SAN-like cells exhibited irregular CaT as well as significant beat period variability compared to isogenic WT controls.

Conclusion:
Systemic loss-of-function of Cpne5 in mice resulted in sinus node dysfunction, consistent with an hiPSC-derived SAN model and human GWAS data, implicating a novel, cell-intrinsic role for Cpne5 in CCS function and/or development. Additional phenotyping and molecular studies are ongoing to further elucidate the underlying mechanism.
Abstract Mo063: Hacking the ubiquitin code to distinctively modulate ion channel functional expression
Authors: Shanmugam Sri Karthika , Kanner Scott , zou xinle , Choudhury Papiya , Soni Rajesh , Kass Robert , Colecraft Henry
Published: October 2024

Ubiquitin is a powerful modulator of ion channel fate, yet the mechanistic basis and scope of its regulation has remained elusive due to the complexity of the ubiquitin code and an inability to achieve substrate-specific control of the ubiquitome. To circumvent this and to specifically interrogate the role of distinct ubiquitin linkages on the cardiac repolarizing I
Ks
channel KCNQ1 (Q1), we developed a toolkit of engineered deubiquitinases (enDUBs) featuring a GFP/YFP-targeted nanobody fused to catalytic domains of DUBs with preferences for hydrolyzing distinctive polyubiquitin linkage types: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K27/K33; and USP21 - non-specific. Co-expressing each of the enDUBs with YFP-tagged Q1 channels in HEK293 cells led to decreased channel ubiquitination but produced quantitative and qualitative differences in Q1 expression. NanoCezanne and nanoTRABID significantly increased Q1 surface density and ionic currents, whereas nanoOTUD1 and nanoUSP21 yielded more moderate effects. In sharp contrast, nanoOTUD4 downregulated Q1 surface density and currents. The impact on steady-state Q1 surface density was achieved by divergent trafficking mechanisms – nanoTRABID and nanoCezanne increased channel forward trafficking to the surface, while nanoOTUD4 reduced channel forward trafficking. The E3 ligases NEDD4L and ITCH both eliminate Q1 functional expression but with different polyubiquitin signatures as assessed by mass spectrometry. The enDUBs nanoOTUD1, nanoCezanne and nanoUSP21 protected Q1 from NEDD4L; whereas nanoTRABID, nanoOTUD1, nanoCezanne and nanoUSP21 shielded Q1 from ITCH. In adult guinea ventricular cardiomyocytes, the enDUBs displayed context-specific regulatory effects, where nanoOTUD1, nanoCezanne and nanoUSP21 significantly enhanced Q1 surface expression and stability, with nanoTRABID having more moderate effects, while nanoOTUD4 sharply decreased Q1 trafficking to the sarcolemma. Finally, linkage-specific enDUBs demonstrated varying capabilities to rescue distinct trafficking-deficient mutant Q1 channels that cause long QT syndrome and predispose to sudden cardiac death. The results reveal a rich multi-faceted role for linkage-specific ubiquitination in regulating ion channel trafficking with implications to develop targeted therapies for trafficking-deficit ion channelopathies.
Abstract Mo076: Combining Monogenic and Polygenic Analysis Improves Sudden Cardiac Death Risk Prediction
Authors: Monroe Tanner , pesce Lorenzo , Kearns Samuel , Dellefave-Castillo Lisa , Webster Gregory , Puckelwartz Megan , McNally Elizabeth
Published: October 2024

Background:
Nonischemic sudden cardiac death (SCD) is a catastrophic cardiac rhythm failure. Heritable arrhythmias and cardiomyopathies contribute significantly to SCD risk, and genetic markers can be used to identify and reduce risk, improving odds of survival. Clinically, genetic evaluation of known rare risk variants using gene panels often fails to identify a genetic etiology. These gene panels focus on genes affiliated with monogenic inheritance, and current genetic analysis is agnostic to polygenic disease architectures.

Hypothesis:
We hypothesized that cumulative rare and common variation scales with SCD risk.

Approach:
We established a diverse cohort of 1042 individuals at low or high SCD risk and classified them as cases (n=505) or controls (n=537). We then performed whole genome sequencing and analyzed variation centered around cardiomyopathy, arrhythmia, and epilepsy panel genes, as well as the regulatory variants that map to these genes. We also performed common variant analysis by computing polygenic scores for cardiac metrics including cardiac morphology, arrhythmia, and EKG intervals. We then created a risk stratification algorithm that incorporated each of those analyses.

Results:
We found a 3.4-fold enrichment for pathogenic or likely pathogenic (PLP) cardiomyopathy/arrhythmia variants (P<1e-11) in the high risk group, with 23% of those individuals carrying at least one PLP variant. Rare high-effect epilepsy panel variants were also enriched in the high risk cohort (P=0.006). We also found a greater than two-fold enrichment of rare regulatory variants in the high risk group (P<1e-7). Polygenic scores for blood pressure, QRS interval, and LV ejection fraction were all differentially distributed between the low and high risk groups (P<0.05). The subsequent combined genomic analysis found those scoring in the highest decile of the total genetic risk scale had 13-fold higher odds of being a high-risk case and 73-fold higher odds compared to the lowest decile. Compared to using only the monogenic or only the polygenic analysis, the combined analysis was more predictive.

Conclusions:
Incorporating noncoding and polygenic analyses into sudden cardiac death genetic assessment improves risk stratification.
Abstract Mo077: Cardiac Troponin I3 Kinase in Viral Myocarditis
Authors: Tjen Kelsey , Farahani Nick , Parrow Lauren , Ricci Jake , Bruno Katelyn , Sucov Henry
Published: October 2024

Myocarditis is a leading cause of heart failure and sudden cardiac death. Often caused by viral infection, myocarditis is defined histopathologically as mononuclear immune infiltration in the heart muscle. While most patients recover with no long-term effects, some deteriorate rapidly. It is unknown what controls this variable susceptibility to severe disease. Without this knowledge, the ability to develop targeted therapies for acute myocarditis is limited. Previous studies utilizing mouse genetics identified a locus that controls susceptibility to viral myocarditis. Cardiomyocyte-specific kinase TNNI3K is one candidate gene within this locus, but its role in myocarditis has not been established. Importantly, millions of people throughout the world carry natural variants of TNNI3K. My overall hypothesis is that TNNI3K confers resistance to viral myocarditis in a kinase-dependent manner. In an initial study, we infected strain background-matched wild-type, Tnni3k knockout, and Tnni3k kinase-dead mice with heart-passaged coxsackievirus B3. After 10 days, during the peak of acute myocarditis, mice missing functional Tnni3k protein were found to have higher viral genomes and greater presence of immune cell markers (CD45+), clearly demonstrating the role of Tnni3k in the response to viral myocarditis. H&E-stained heart sections show large clusters of hematoxylin-stained nuclei, indicating infiltration of ventricular tissue by immune cells, with more of these clusters in Tnni3k knockout hearts. Because kinase-dead mutants are similarly compromised as fully null mice, this indicates that the kinase activity of TNNI3K is critical in the antiviral response. These results are the first demonstration of the
in vivo
activity of a specific gene that modifies viral myocarditis sensitivity. Future work will expand on this study by examining the effects of coxsackievirus B3 infection of Tnni3k mutant mice at early and late time points. Additionally, efforts are currently underway to identify the specific mechanisms by which TNNI3K regulates the immune response to viral infection.
Abstract Mo080: Clinically Variable Penetrant MYH7 G256E Mutation Shows Gene-Dose-Dependent HCM Disease Phenotype on a Transcriptomic, Proteomic, and Functional Level
Authors: Heinrich Paul , Lee Soah , Sailer Carolin , Achter Jonathan , Jung Raina , Lee Carissa , Vander Roest Alison , Roberts Brock , Bernstein Daniel , Lundby Alicia , Wu Sean
Published: October 2024

Introduction:
Hypertrophic cardiomyopathy (HCM) is a monogenic disorder caused by heterozygous (HET) mutations in sarcomere genes such as
MYH7
. Recently, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying HET HCM mutations have shed new insights into the molecular mechanisms of HCM. However, the cause of phenotype variability and the role of allelic imbalance in phenotype penetrance of HET missense mutations such as
MYH7
G256E remain unclear.

Hypothesis:
We hypothesize that hiPSC CMs carrying clinically variable penetrant HCM missense mutations such as
MYH7
G256E show a gene dose-dependent disease phenotype indicating their susceptibility for allelic imbalance as a cause for variable disease phenotype penetrance.

Methods:
We generated HET and homozygous (HOM) hiPSC-lines for variable penetrant HCM mutation
MYH7
G256E. We performed multiplexed single-cell RNA sequencing and quantitative proteomics analyses on wildtype (WT), HET, and HOM iPSC-CMs at day 30 of cardiac differentiation. Additional Digital Droplet PCR studies were performed to investigate the
MYH7
mutant allele expression. The functional contractile phenotype was assessed with Sony SI8000 Cell motion imaging system.

Results:
We detected a large clone-to-clone and batch-to-batch variability in HET iPSC-CMs on a transcriptomic and proteomic level that obscured the identification of a consistent mutant phenotype in HET
MYH7
G256E vs WT CMs. These phenotypes correlated with a 1:1 bulk ratio of mutant vs WT mRNA and protein expression, while overall
MYH7
gene and protein expression remained comparable. Transcriptomic and proteomic analyses of WT, HET, and HOM CMs show a gene dose-dependent regulation of known hypertrophy response markers such as
NPPB
(mRNA: HET vs WT: +1.76x p .adj=7.0*10
-7
, HOM vs WT: +5.42x p.adj=1.5*10
-48
; protein: HET vs WT: +1.15x, 95% CI [0.86, 1.58]; HOM vs WT: +1.51x, 95% CI [1.12, 2.01]) which functionally correlated with enhanced contractility (contraction velocity in μm/s: WT: 5.84 +/-2.24, HET: 6.04 +/-2.89, HOM: 7.77 +/-3.352, p=0.3*10
-2
ANOVA) and the more frequent occurrence of arrhythmias (WT: 1/46, HET: 0/55, HOM: 5/41).

Conclusions:
Hypertrophic disease phenotype in
MYH7
G256E mutation iPSC-CMs shows a gene dose-dependent penetrance on a transcriptomic, proteomic, and functional level. This indicates susceptibility to allelic imbalance as a potential cause for variable disease penetrance.
Abstract Mo082: Polygenic Assessment for Atrial Fibrillation Liability Predicts Ventricular Arrhythmia Risk
Authors: Puckelwartz Megan , Acciani Daniel , Monroe Tanner , pesce Lorenzo , Kearns Samuel , Dellefave-Castillo Lisa , Webster Gregory , McNally Elizabeth
Published: October 2024

Background:
The risks for atrial and ventricular arrhythmias are increased with cardiomyopathy. Currently, left ventricular dysfunction is the major determinate of implantable cardioverter-defibrillator (ICD) placement despite left ventricular ejection fraction only having an odds ratio of 2.9 for subjects with an LVEF <35% with sensitivity and specificity that suggests many patients are inappropriately receiving or not receiving a device. Additional testable variables would aid in arrhythmia risk stratification. There is growing evidence that atrial dysfunction is both a cause and consequence of ventricular disease. Atrial fibrillation (AFib) is the most common atrial arrhythmia and is highly heritable suggesting a strong genetic basis, and AFib occurs at increased frequency with cardiomyopathies. Genome-wide association studies (GWAS) identify >100 loci that contribute to AFib, and signals from GWAS can be used to develop polygenic risk scores (PRS). Subjects in the top 3% of one AFib PRS were found to have ~2 fold greater risk for AFib (OR 1.7-4.6).

Hypothesis:
The genetic pathways represented in an AFib PRS account for a portion of arrhythmia risk associated with cardiomyopathy and can be exploited to predict ventricular arrhythmia risk.

Approach:
As part of an AHA Arrhythmia and Sudden Death Strategically Funded Research Network, we assembled a cohort with varying risk for sudden cardiac death ranging from low-risk individuals with non-ischemic cardiomyopathy without arrhythmia to higher risk individuals who underwent ICD placement for aborted sudden death and sudden death. Whole genome sequencing was completed 1043 individuals across the arrhythmia risk spectrum.

Results:
We examined an AFib PRS to define ventricular outcomes in the cohort. Of the 440 participants with disease, 81% had cardiomyopathy, 8% channelopathy and 7% idiopathic arrest. We found the AFib PRS stratified subjects without significant ventricular arrhythmia risk from subjects with known ventricular arrhythmia (p=0.03), indicating that the AFib PRS includes components that are important markers of ventricular dysfunction.

Conclusions:
The AFib PRS is a useful tool to stratify ventricular arrhythmia risk.
Abstract Mo091: Analysis of GLA Gene Mutation Using a Female Fabry Disease Patient-derived Induced Pluripotent Stem Cells.
Authors: Saito Yukihiro , Nakamura Kazufumi , Iida Toshihiro , Vo Duy Quan , Yuasa Shinsuke
Published: October 2024

Introduction:
Fabry disease is an inborn error of metabolism caused by mutations in the alpha-galactosidase gene (
GLA
) located on the X chromosome, resulting in intracellular accumulation of globotriaosylceramide and systemic tissue injury. In addition to enzyme replacement therapy, chaperone therapy is effective for many missense mutations. One female patient had c.547G>A mutation, which has been reported as p.G183S, and we checked the indication for migalastat, a pharmacological chaperone. However, migalastat reactivity testing using HEK293 cells transfected with mutant cDNA had not been performed for this mutation.

Aims:
We analyzed the c.547G>A mutation and examined the efficacy of migalastat for this mutation.

Methods:
Peripheral blood mononuclear cells from patients were transfected with reprogramming factors using episomal vectors to generate induced pluripotent stem (iPS) cells. Cardiomyocytes were induced from the iPS cells. The mutation site was located at the 3' end of exon 3, and splicing mutation was suspected. Thus, PCR amplification was performed between exon 2 and exon 5 of the cDNA, and the PCR product was sequenced to confirm the mRNA sequence generated in the patient's cells. Real-time PCR was performed using Taqman probe to amplify sites unaffected by the mutation, and expression levels of normal and mutant
GLA
were compared. iPS cells were treated with migalastat and the effect on α-galactosidase activity was examined.

Results:
Since the patient was female, five iPS cell lines expressing only normal GLA and four iPS cell lines expressing only mutant GLA were established due to X-chromosome inactivation. Mutant cDNA was found to have a deletion of 36 bases, part of exon 3, resulting in a deletion of 12 amino acids. Real-time PCR result showed that the
GLA
expression level of mutant GLA-expressing cells was about half that of normal GLA-expressing cells, which was comparable to the
GLA
expression level of the cells from a healthy subject. Mutant GLA-expressing iPS cells had no α-galactosidase activity, which was not increased by treatment with migalastat.

Conclusions:
In patient-derived iPS cells and iPS cell-derived cardiomyocytes, c.547G>A produced p.C172_G183del, not p.G183S, and migalastat was ineffective. Further therapeutic development is needed for patients with this mutation.
Abstract Mo092: Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes as an
In Vitro
Model to Evaluate the Efficacy of a Gene Replacement Therapy for
Plakophilin-2-
Associated Arrhythmogenic Right Ventricular Cardiomyopathy
Authors: Perea Gil Isaac , Aycinena J. Alex , Chandrakumar Rishikesan , Cisne-Thompson Olga , Tsui Jonathan , Zepeda Bernardo , Tefera Anley , Shenwai Reva , Farshidfar Farshad , Jones Chayse , Yang Zhihong Jane , Hoey Timothy , Argast Gretchen
Published: October 2024

Background:
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic cardiac disorder characterized by severe arrhythmias and heart dysfunction. Mutations in desmosome gene
Plakophilin-2
(
PKP2
) account for 40% of ARVC cases, with current palliative therapies failing to address the genetic cause. Herein, we generated an
in vitro
ARVC model using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) harboring a pathogenic
PKP2
variant (c.2146G>C), as a platform to test a
PKP2
-gene replacement approach developed at Tenaya Therapeutics for the treatment of
PKP2
-associated ARVC patients.

Methods:
Three isogenic human iPSC lines (wild-type, heterozygous and homozygous
PKP2
mutant) were differentiated to iPSC-CM; and monolayers and engineered heart tissues (EHTs) were generated. Gene and protein expression were assessed by RNA-Seq/RT-qPCR, and immunocytochemistry, respectively. Contractility, electrophysiology and calcium handling were measured. A proprietary adeno-associated virus gene therapy (AAV9:
PKP2
) was used to transduce human iPSC-CM, and changes in gene and protein expression, and contractile function were evaluated.

Results:
A human iPSC-CM beating monolayer was achieved for all three lines. PKP2 expression was depleted in a genotype-dependent manner, and desmosome structure was disrupted in the mutant lines. Functional assessment of human iPSC-CM monolayers showed impaired contractile properties and abnormal electrophysiological and calcium transients in the mutant lines, such as decreased contraction amplitude and prolonged field and action potential duration. Transcriptional analysis revealed changes in desmosome, gap junctions, sarcomere, ion channels, metabolic and apoptosis gene expression. Characterization of EHTs displayed a deficit in contractility, slower action and field potential kinetics and irregular calcium homeostasis in the mutant lines. The administration of AAV9:
PKP2
to the mutant lines restored ion channel and desmosome gene and protein expression, and contractile function.

Conclusions:
Our
PKP2
human iPSC-CM disease model recapitulated the main hallmarks of ARVC phenotype. Administration of AAV9:
PKP2
restored desmosome protein expression and contractility. This model lays a foundation for the understanding of the underlying molecular pathophysiological mechanisms of
PKP2
-ARVC, and the potential for AAV9:
PKP2
as a one-time dose to correct the genetic cause of disease in individuals with
PKP2
-associated ARVC.
Abstract Mo097: Inhibiting the Sarcomere Improves Diastolic Dysfunction in Patient-Derived hiPSC-CM Models of Pediatric Restrictive Cardiomyopathy
Authors: Staudt David , Serrano Ricardo , Feyen Dries , Mercola Mark
Published: October 2024

Introduction:
Pediatric patients with Restrictive Cardiomyopathy (RCM) develop heart failure due to stiffening of the wall of the cardiac ventricle leading to diastolic dysfunction but preserved cardiac contractility. Treatment options are extremely limited, and the evaluation of new therapeutic strategies is hindered due to a lack of models that recapitulate this ventricular stiffening. Here, we evaluate two novel patient-derived human induced pluripotent stem cell (hiPSC) models of restrictive cardiomyopathy due to mutations in the thin filament, and evaluate separate sarcomere-targeted therapeutic strategies to improve diastolic function.

Hypothesis:
Directly decreasing the thin filament’s response to calcium will improve diastolic function in RCM models with less effect on systolic function than myosin inhibition.

Methods:
hiPSC-cardiomyocytes (hiPSC-CMs) were differentiated from hiPSC lines generated from two patients with pediatric RCM, as well as their isogenic controls. After replating on polyacrylamide gels and staining with fluorescent calcium indicators, diastolic tension, generated tension, and calcium sensitivity were measured at baseline and upon treatment with either a troponin calcium desensitizer (W7) or a myosin inhibitor (mavacamten).

Results:
hiPSC-CMs from both patients showed increased diastolic tension, generated tension, and calcium sensitivity compared to their isogenic controls. Treatment with Mavacamten improved diastolic function but severely compromised systolic function, while treatment with W7 improved diastolic function with less effect on systolic function.

Conclusions:
Directly inhibiting the sarcomere in models of RCM improved measures of diastolic dysfunction, but directly targeting the thin filament in these lines showed less effect on systolic function than myosin inhibition, suggesting cardiac troponin calcium desensitizers may be more beneficial in this severe diastolic disease.
Abstract Mo101: 3-Mercaptopyruvate Sulfurtransferase is a Critical Regulator of Branched-Chain Amino Acid Catabolism in Cardiometabolic HFpEF
Authors: Li Zhen , Doiron Jake , Xia Huijing , LaPenna Kyle , Sharp Thomas , YU Xiaoman , Nagahara Noriyuki , Goodchild Traci , Lefer David
Published: October 2024

Background:
Heart failure with preserved ejection fraction (HFpEF) is among the leading causes of cardiovascular mortality and morbidity. Recent multi-omics data sets underscore severely impaired branched-chain amino acid (BCAA) catabolism in the failing hearts of HFpEF patients. 3-mercaptopyruvate sulfurtransferase (3-MST) is a mitochondrial hydrogen sulfide (H
2
S) synthase that regulates mitochondrial biogenesis, function, and substrate metabolism.

Research Questions and Goals:
In the current study, we aimed to elucidate the role of 3-MST in myocardial BCAA catabolism in cardiometabolic HFpEF.

Methods:
3-MST knockout or wild-type (WT) mice were subjected to control or L-NAME and high fat diet - induced HFpEF. Echocardiographic measurements, invasive hemodynamic assessment, and exercise capacity testing were conducted to determine HFpEF severity. Metabolomic analysis was performed to study BCAA catabolism. Additionally, a H
2
S donor, SG1002 was administered to increase H
2
S bioavailability in the setting of 3-MST deficiency and HFpEF.

Results:
We observed progressive deterioration in left ventricular (LV) diastolic function and exercise performance from mice subjected to HFpEF. Genetic ablation of 3-MST further-exacerbated HFpEF severity as evidenced by significantly increased E/e’ ratio, elevated LV end-diastolic pressure, and reduced exercise performance. Metabolomic analysis revealed pathological accumulation of intermediate metabolites of BCAA in the HFpEF hearts, suggesting a major derangement in myocardial BCAA catabolism. 3-MST KO hearts exhibited further BCAA catabolic defects as compared to those from WT. Importantly, H
2
S donor treatment significantly attenuated BCAA catabolic defect and alleviated HFpEF severity in both WT and 3-MST KO HFpEF mice.

Conclusion:
Our results demonstrate that deficiency of 3-MST impairs myocardial BCAA catabolism, which results in exacerbated HFpEF severity in a “two-Hit” mouse model of HFpEF. These data suggest 3-MST is critically involved in physiological substrate metabolism within the mitochondria and exerts beneficial effects in HFpEF. Future experiments are underway to delineate the mechanisms by which 3-MST regulates BCAA catabolism.
Abstract Mo105: End Systolic Pressure and Ejection Timing Correlate with Impaired Relaxation
Authors: Wagner Yoshio , Chung Charles
Published: October 2024

Impaired relaxation is a common feature in most cases of diastolic dysfunction, including heart failure with preserved ejection fraction. Isovolumetric relaxation is typically characterized by τ, an exponential time constant, but may be broken into relaxation and stiffness-like parameters (µ and Ek). Recent in vitro work suggests that strain during late systole (protodiastolic period) modifies relaxation. We hypothesize that late systolic pressure and time indexes correlate with myocardial relaxation.

Thirty-four (34) simultaneous aortic and left ventricular micromanometric (Millar) pressure traces were obtained from a comprehensive database of clinical cardiac catheterization patients. These patients underwent coronary artery disease screening and presented but did not have arterial blockages. Pressure and time parameters were calculated over a five-minute interval.

Relaxation rate (1/τ), and stiffness (Ek) correlated to end systolic parameters in an orthogonal pattern to relaxation (µ). 1/τ showed strong correlations with time from peak pressure to aortic valve divided by ejection time (t54/t52, r = .675, p < .01) and moderate correlation to the pressure decline after peak pressure to aortic valve pressure divided by the pulse pressure (P45/P42, r = .577, p < .01). In parallel Ek showed moderate correlation with t54/t52 (r = .592, p < .01), and a moderate correlation with P45/P42 (r = .470, p < .01), the pressure at aortic valve closure (r = -.420, p < .05), and aortic augmentation index (AIx, r = -.360, p < .05). Finally, µ presented a moderate correlation to P45/P42 (r = .501, p < .05), the pressure at aortic valve closure (r = .404, p < .05), and AIx (r = -.357, p < .05).

As predicted, ventricular relaxation in patients correlates with late systolic hemodynamics. Parameters or combinations of parameters may predict relaxation rates or correlate to in vivo late systolic strain. Prospective studies evaluating end systolic strain and relaxation merit consideration. Overall, these observations are consistent with in vitro studies that suggest that impaired cessation of contraction is a regulator of relaxation.
Abstract Mo107: Age-Related Impairment of Mitochondrial Protein Turnover Exacerbates Pathogenesis of Heart Failure with Preserved Ejection Fraction in Old Mice
Authors: Kobak Kamil , Zarzycka Weronika , King Catherine , Borowik Agnieszka , Peelor Frederick , Kinter Michael , Miller Benjamin , Chiao Ying Ann
Published: October 2024

Introduction:
Age is recognized as a major risk factor for heart failure with preserved ejection fraction (HFpEF), yet the precise role of aging in HFpEF development remains unknown. Disruption of mitochondrial protein turnover affects the quality and quantity of proteins crucial for energy metabolism. Although mitochondrial dysfunction is a key feature of both aging and HFpEF heart, the influence of age-related disturbances in mitochondrial protein turnover on HFpEF pathogenesis remains unclear.

Hypothesis:
Age-related impairment of mitochondrial protein turnover intensifies proteostatic perturbations induced by hypertensive and metabolic stress (2-hit stress) to exacerbate pathogenesis of cardiometabolic HFpEF.

Methods:
Young (3 mo.) and old (19 mo.) C57BL/6J male mice were exposed to high-fat diet and L-NAME to induce HFpEF development. Deuterium oxide (D
2
O) labeling coupled with mass spectrometry was used to investigate bulk and individual mitochondrial protein synthesis. The changes in individual mitochondrial protein abundance and protein degradation pathways were also studied.

Results:
Old mice presented exacerbated HFpEF characteristics compared to young mice, with higher mortality and worsened cardiac and extra-cardiac phenotypes. Regardless of age, mice subjected to 2-hit stress had 24% higher bulk mitochondrial protein synthesis compared to controls. Old control and HFpEF mice showed 17% lower mitochondrial protein synthesis compared to younger counterparts, potentially hindering the replacement of dysfunctional mitochondrial proteins. Aging reduced the synthesis rates of 30 out of 115 proteins synthesized more rapidly with HFpEF, including those involved in mitochondrial metabolism. Despite slower synthesis of numerous mitochondrial proteins with age, the overall abundance of these proteins remained unchanged or elevated compared to young counterparts, indicating impaired protein degradation. We found that aging and 2-hit stress differentially inhibited degradation pathways, including autophagy and the ubiquitin-proteasome system, further impairing protein turnover.

Conclusion:
Aging impairs mitochondrial protein turnover in cardiometabolic HFpEF and contributes to poorer disease outcomes.