Cardiomyocyte Function Research Articles

Discovery of SIRT1-Activating Hydrogen Sulfide Donating Derivatives for Efficient Resistant of Myocardial Ischemic Injury.

Activating SIRT1 or promoting SIRT1 expression are both protective against myocardial ischemia. Combining these approaches would be an effective strategy for treating ischemic heart disease. Herein, we identified lead compounds with SIRT1 activation activity through screening the natural product library, and five series of H2S donating derivatives were designed and synthesized. Among them, compound 17 exerted an effective cardioprotective effect in vitro and in vivo. The addition of H2S scavenger attenuated the protective activity, emphasizing the critical involvement of H2S in the myocardial ischemia process. Interestingly, 17 exhibited stronger direct SIRT1 activative ability and induced higher SIRT1 expression capability compared to the lead. Furthermore, 17 attenuates oxidative stress-induced cardiomyocytes apoptosis by activating the SIRT1-PGC1α signaling pathway. Our study validated the promising potential of activating SIRT1 and promoting SIRT1 expression through H2S to improve cardiomyocytes function, providing novel insights into the protective mechanisms during the progression of ischemic heart disease.

Stem Cell Treatment for Dilated Cardiomyopathy: A Review of Recent Scientific Advances

Cardiomyopathy, a disorder of mechanical and electrical cardiac action, is a significant cause of morbidity and mortality in the United States and is seen in both adult and pediatric populations. Dilated cardiomyopathy, a subtype involving pathological enlargement of the heart, in particular causes significant reduction in quality of life and is a significant risk factor for heart failure development. Traditional treatment for dilated cardiomyopathy largely focuses on symptomatic relief and delayed progression of disease but does not offer a cure or reversal of disease process. In the past few decades, stem cell therapy has become a promising therapeutic because these cells can differentiate into human cardiomyocytes and offer a potential restoration of normal cardiomyocyte function. This review highlights 3 methodological derivations of cardiomyocytes: bone marrow–derived hematopoietic stem cells, mesenchymal stem cells, and adipose-derived stem cells. While most of the current research has focused on hematopoietic stem cells for dilated cardiomyopathy, further research is needed to elucidate which stem cell line is superior and most effective for disease treatment and reversal. This review highlights future directions of this line of research, such as direct comparison between cell types, analysis of the long-term effects of stem cell treatment, increased sample sizes, and potential use of this treatment in the pediatric and adult populations.

Role of protein domains in trafficking and localization of the voltage-gated sodium channel β2 subunit

The voltage-gated sodium (NaV) channel is critical for cardiomyocyte function since it is responsible for action potential initiation and its propagation throughout the cell. It consists of a protein complex made of a pore forming α subunit and associated β subunits, which regulate α subunit function and subcellular localization. We previously showed the implication of N-linked glycosylation and S-acylation of β2 in its polarized trafficking. Here, we present evidence of β2 dimerization. Moreover, we demonstrate the implication of the cytoplasmic tail, extracellular loop, and transmembrane domain on proper β2 folding and export to the cell surface of polarized Madin-Darby canine kidney cells. Substantial alteration, or lack of any of these domains, leads to accumulation of β2 in the endoplasmic reticulum, along with impaired complex N-glycosylation, which is needed for its efficient surface delivery. We also show that these alterations to β2 affected to certain extent NaV1.5 surface localization. Conversely, however, NaV1.5 had little or no influence on β2 trafficking, its localization to the surface, or homodimer formation. Altogether, our data link the architecture of the β2 domains to the establishment of its proper subcellular localization. These findings could provide valuable insights to gain a deeper comprehension of the elusive biology of β subunits in excitable cells, such as neurons and cardiomyocytes.

Ablation of Akt2 rescues chronic caloric restriction-provoked myocardial remodeling and dysfunction through a CDK1-mediated regulation of mitophagy

Chronic caloric restriction triggers unfavorable alterations in cardiac function albeit responsible scenarios remain unclear. This work evaluated the possible involvement of Akt2 in caloric restriction-evoked cardiac geometric and functional changes and responsible processes focusing on autophagy and mitophagy. Akt2 knockout and WT mice were subjected to caloric restriction for 30 weeks prior to assessment of myocardial homeostasis. Caloric restriction compromised echocardiographic parameters (decreased LV wall thickness, LVEDD, stroke volume, cardiac output, ejection fraction, fractional shortening, and LV mass), cardiomyocyte contractile and intracellular Ca2+ capacity, myocardial atrophy, interstitial fibrosis and mitochondrial injury associated with elevated blood glucocorticoids, autophagy (LC3B, p62, Atg7, Beclin-1), and mitophagy (Pink1, Parkin, TOM20), dampened cardiac ATP levels, mitochondrial protein PGC1α and UCP2, anti-apoptotic protein Bcl2, intracellular Ca2+ governing components Na+-Ca2+ exchanger, phosphorylation of SERCA2a, mTOR (Ser2481) and ULK1 (Ser757), and upregulated Bax, phospholamban, phosphorylation of Akt2, AMPK, and ULK1 (Ser555), the responses except autophagy markers (Beclin-1, Atg7), phosphorylation of AMPK, mTOR and ULK1 were negated by Akt2 ablation. Levels of CDK1 and DRP1 phosphorylation were overtly upregulated with caloric restriction, the response was reversed by Akt2 knockout. Caloric restriction-evoked changes in cardiac remodeling and cardiomyocyte function were alleviated by glucocorticoid receptor antagonism, Parkin ablation and Mdivi-1. In vitro experiment indicated that serum deprivation or glucocorticoids evoked GFP-LC3B accumulation and cardiomyocyte dysfunction, which was negated by inhibition of Akt2, CDK1 or DRP1, whereas mitophagy induction reversed Akt2 ablation-evoked cardioprotection. These observations favor a protective role of Akt2 ablation in sustained caloric restriction-evoked cardiac pathological changes via correction of glucocorticoid-induced mitophagy defect in a CDK1-DRP1-dependent manner.

Molecular Mechanisms behind Reduced ATP Synthase Function in Senescent Cardiomyocytes

Molecular Mechanisms behind Reduced ATP Synthase Function in Senescent Cardiomyocytes

Deleterious role of free fatty acid and ceramide species in the alterations induced by epicardial fat from diabetic patients on cardiomyocyte function

Deleterious role of free fatty acid and ceramide species in the alterations induced by epicardial fat from diabetic patients on cardiomyocyte function

Mitochondria from the systemic heart of Pacific hagfish (Eptatretus stoutii) are insensitive to one hour of anoxia followed by reoxygenation

Pacific hagfish (Eptatretus stoutii) are an ancient agnathan vertebrate known to be anoxia tolerant. To study their metabolic organization and the role of the mitochondria in anoxia tolerance we developed a novel protocol to measure mitochondrial function in permeabilized cardiomyocytes and how this is affected by one hour of anoxia followed by reoxygenation. When measured at 10 °C the mitochondria had a respiration rate of 2.1 ± 0.1pmol/s/mg WW during OXPHOS with saturating concentrations of glutamate, malate, and succinate. This is comparatively low compared to other ectothermic species. The functional characteristics of the mitochondria were quantified with mitochondrial control ratios. These demonstrated that proton leak contributed to just under 50% of the oxygen flux, with the remainder going towards ATP phosphorylation. Finally, when the preparations were exposed to an anoxia-reoxygenation protocol there was no difference in respiration compared to that of a heart sample from the same animal maintained under normoxia for the same time. When Complex I alone or Complex I and II were stimulated following one hour of anoxia there was no decline in oxygen flux observed. However, if Complex II was activated alone there was a significant decline in respiration. This decrease was however also observed in the mitochondria maintained in normoxia for one hour. In conclusion, Pacific hagfish cardiac mitochondria demonstrated a low rate of oxygen consumption, a loosely coupled electron transfer system, and a resistance to one hour of anoxia.

Novel Approach for Cardioprotection: In Situ Targeting of Metformin via Conductive Hydrogel System.

Ischemia/reperfusion (I/R) injury following myocardial infarction is a major cause of cardiomyocyte death and impaired cardiac function. Although clinical data show that metformin is effective in repairing cardiac I/R injury, its efficacy is hindered by non-specific targeting during administration, a short half-life, frequent dosing, and potential adverse effects on the liver and kidneys. In recent years, injectable hydrogels have shown substantial potential in overcoming drug delivery challenges and treating myocardial infarction. To this end, we developed a natural polymer hydrogel system comprising methacryloylated chitosan and methacryloylated gelatin modified with polyaniline conductive derivatives. In vitro studies demonstrated that the optimized hydrogel exhibited excellent injectability, biocompatibility, biodegradability, suitable mechanical properties, and electrical conductivity. Incorporating metformin into this hydrogel significantly extended the administration cycle, mitigated mitochondrial damage, decreased abnormal ROS production, and enhanced cardiomyocyte function. Animal experiments indicated that the metformin/hydrogel system reduced arrhythmia incidence, infarct size, and improved cardiac mitochondrial and overall cardiac function, promoting myocardial repair in I/R injury. Overall, the metformin-loaded conductive hydrogel system effectively mitigates mitochondrial oxidative damage and improves cardiomyocyte function, thereby offering a theoretical foundation for the potential application of metformin in cardioprotection.

Biological Evaluation of Thermosensitive Hydrogels of Chitosan/Hydrolyzed Collagen/β-GP in an In Vitro Model of Induced Cardiac Ischemia.

Ischemic events can culminate in acute myocardial infarction, which is generated by irreversible cardiac lesions that cannot be restored due to the limited regenerative capacity of the heart. Cardiac cell therapy aims to replace injured or necrotic cells with healthy and functional cells. Tissue engineering and cardiovascular regenerative medicine propose therapeutic alternatives using biomaterials that mimic the native extracellular environment and improve cellular and tissue functionality. This investigation evaluates the effect of thermosensitive hydrogels, and murine fetal ventricular cardiomyocytes encapsulated in thermosensitive hydrogels, on the contractile function of cardiomyocyte regeneration during an ischemic event. Chitosan and hydrolyzed collagen thermosensitive hydrogels were developed, and they were physically and chemically characterized. Likewise, their biocompatibility was evaluated through cytotoxicity assays by MTT, LDH, and their hemolytic capacity. The hydrogels, and cells inside the hydrogels, were used as an intervention for primary cardiomyocytes under hypoxic conditions to determine the restoration of the contractile capacity by measuring intracellular calcium levels and the expressions of binding proteins, such as a-actinin and connexin 43. These results evidence the potential of natural thermosensitive hydrogels to restore the bioelectrical functionality of ischemic cardiomyocytes.

Abstract Mo093: Rescue of Desmin Insufficiency Restores Contractile Function in Cardiomyocytes with MYH7 E848G Dilated Cardiomyopathy Variant

Dilated cardiomyopathy (DCM), characterized by systolic dysfunction, is a significant cause of heart failure. Myosin heavy chain 7 ( MYH7 ) pathogenic variants are common causes of DCM; however, no specific disease-modifying therapy for MYH7 DCM exists. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) expressing the pathogenic MYH7 E848G DCM variant, we confirmed hypocontractility at the single cell level as assessed with traction force microscopy. hiPSC-CMs were cultured on polyacrylamide hydrogels with physiological stiffness in 15x100 µm patterned Matrigel-coated rectangles, each containing one cell. Upon 1 Hz electrical stimulation, contracting cells displaced fluorescent beads embedded within the gel, which were measured to estimate twitch force. MYH7 E848G/+ hiPSC-CMs had reduced maximum twitch force (97 ± 11 nN, n=46 cells) compared to MYH7 +/+ hiPSC-CMs (179 ± 18 nN, n=49 cells), recapitulating the disease phenotype. RNAseq analysis of MYH7 E848G/+ hiPSC-CMs compared to isogenic control MYH7 +/+ hiPSC-CMs found significant downregulation of desmin ( DES) (76.4 ± 8.2% reduction, n=4 bio reps), which encodes a muscle-specific type III intermediate filament. Desmin protein expression in MYH7 E848G/+ hiPSC-CMs was significantly attenuated relative to MYH7 +/+ hiPSC-CMs (90.9 ± 1.8% reduction, n=4 bio reps), matching gene expression data. Gene expression of the transcription factor MEF2C , a known transactivator of DES, was also reduced (40.4 ± 7.0% reduction, n=4 bio reps), suggesting MYH7 E848G/+ suppresses the MEF2C - DES signaling axis. We hypothesized restoring desmin protein levels may improve contractility in MYH7 E848G/+ hiPSC-CMs. To test this, we created an adeno-associated virus (AAV9) packaged with full-length desmin cDNA fused with a T2A self-cleaving peptide and mApple fluorescent reporter. hiPSC-CMs were transfected with this mApple-T2A-DES AAV at 1 MOI and contractility was assessed via traction force microscopy. mApple + MYH7 E848G/+ hiPSC-CMs had increased maximum twitch force (141 ± 11 nN, n=75 cells) relative to mApple - MYH7 E848G/+ hiPSC-CMs (65 ± 20 nN, n=21 cells), approaching the maximum twitch force of mApple + MYH7 +/+ hiPSC-CMs (165 ± 10 nN, n=87 cells). In conclusion, suppression of the MEF2C - DES signaling axis is a potential novel mechanism by which pathogenic MYH7 variants cause DCM. By leveraging this mechanistic insight, we found that rescuing desmin insufficiency can partially restore contractile function.

Abstract Mo085: Direct effect of sodium-glucose cotransporter 2 (SGLT2) inhibitors on cardiomyocyte function as a potential therapy for Phospholamban cardiomyopathy

Introduction: Phospholamban (PLN) cardiomyopathy is a dilated and arrhythmogenic cardiomyopathy caused by a single deletion of arginine in PLN gene (PLN-R14del). Clinically, It presents with heart failure, ventricular arrhythmias and sudden cardiac death, and there is still no effective and/or specific treatment besides the heart transplant. Hypothesis: Sodium-glucose cotransporter 2 (SGLT2) inhibitors have shown cardiovascular outcomes improvement in general heart failure patients in multiple clinical trials. We hypothesize that SGLT2 inhibitors could ameliorate the contractile dysfunction in PLN cardiomyopathy. Goals: Identify whether there is a direct cardiac effect of SGLT2 inhibitors on PLN cardiomyopathy. Approach: We used a human iPSC-derived cardiomyocyte (hiPSC-CMs) model of PLN cardiomyopathy that mimics the impaired contractility of the disease to evaluate the contractile function after 4 days of Empagliflozin treatment by using functional cellular imaging assays. Additionally, we evaluated the effect of Empagliflozin treatment on CaMKII phosphorylation and activation of apoptosis mediators by measuring protein expression. All the experiments were compared to results using a CRISPR-engineered isogenic (healthy) line control hiPSC-CMs. Results: 4 days of Empagliflozin treatment significantly restored contractile function, decreased levels of CaMKII phosphorylation, and decreased apoptotic activity in hiPSC-CMs with PLN cardiomyopathy when compared to their healthy (isogenic) control. Conclusions: Our study provided evidence that SGLT2 inhibitors might serve as a therapeutic strategy to ameliorate contractile dysfunction and suppress arrhythmia in PLN cardiomyopathy.

Abstract We046: Building Mass or Strength: Stage-specific Effects of Insulin and Wnt on Cell Fate Decisions in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Background: Reactivation of the so-called fetal gene programs and pathways have been reported in failing hearts, however, it remains unclear whether this phenomenon is beneficial or detrimental to the remaining functional myocardium. During cardiac development, the orchestration of these embryonic growth pathways, facilitate temporal and stage-specific cell fate decisions such as required for specification and growth of the various myocardial compartments. Previously, we demonstrated that molecular activation of the Wnt pathway resulted in massive expansion of immature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Yet, it is unknown what exact cues guide the cell fate decisions of immature hiPSC-CMs to either proliferate or terminally differentiate into more mature cardiomyocytes. Purpose: This work uncovers the embryonic growth pathways that control the cues for proliferation versus terminal differentiation in functional immature hiPSC-CMs. Methods: We utilised spontaneously beating hiPSC-CMs at day 11 of differentiation and performed a combinatory screen for various dosages of CHIR99021/Wnt and Insulin/Akt pathway. Proliferation was determined by quantification of cell number and the proportion of Ki67 positive hiPSC-CMs. Terminal differentiation was evaluated by presence of more mature sarcomere markers and cell cycle exit. Results: Our combinatory screen for Insulin/Akt and CHIR99021/Wnt demonstrates synergistic effects on proliferation of immature hiPSC-CMs, whereas the absence of these pathway activators leads to rapid cell cycle exit and terminal differentiation of hiPSC-CMs. Detailed characterization reveals that CHIR99021/Wnt also importantly regulates sarcomere homeostasis and architecture in hiPSC-CMs. Moreover, we identify a novel temporal interplay between CHIR99021/Wnt via TCF and Insulin/Akt via FOXO as regulators of cell-fate decision in immature hiPSC-CMs. Conclusions: Molecular targeting of the embryonic growth pathways in acute and chronic forms of heart failure requires a detailed understanding of the specific effects on cardiomyocyte function and sarcomere architecture. This work provides a starting point to develop future strategies to target acute and chronic forms of heart failure via modulation of embryonic growth pathways.

Abstract Tu122: Induction of Mitochondrial and Endoplasmic Reticulum Stress in Early Response to High-Fat Diet-Induced Hyperglycemia in Mouse Hearts

Background: Diabetic cardiomyopathy (DCM) is the leading cause of mortality in diabetic patients, accounting for about 80% of the death. Mitochondria and endoplasmic reticulum (ER) play a critical role in cardiomyocyte function, yet their interaction and function in diabetic hearts remains unclear. Hypothesis: We hypothesize that mitochondria and ER stress response are essential in maintaining cardiac function and preventing cardiac remodeling during early diabetic insult in the heart. Methods: Utilizing 6-month-old male and female C57BL/6 mice, we divided them into groups receiving a standard chow diet (N=8) and a 60% high-fat diet (HFD, N=8) for four months. Post-diet, we conducted glucose tolerance tests to verify hyperglycemia. We analyzed heart samples for mitochondrial stress (LonP1, ClpX, ClpP, Afg3l2, spg7, GRP75) and ER stress (XbP1, IRE1a, CHOP, PDI, Hspa5, Dnajb9, Hsp90) markers, along with mitochondrial biogenesis (mtDNA, TFAM, Mt-ND4, and Mt COII levels), and whole heart homogenate oxidative potential by electron paramagnetic resonance (EPR) spin probe (CMH). Between the groups, a p-value < 0.05 was considered significant calculated by one-way ANOVA. Results: HFD significantly increased body weight (p<0.0001) and fasting glucose levels (p<0.0001), confirming hyperglycemia. Among the mitochondrial stress response markers, LonP1 was significantly (p<0.05) elevated in HFD hearts compared to the control. However, the ER stress-associated transcription factors, CHOP and XbP1, were significantly upregulated (p<0.05) in female HFD hearts compared to the control and male groups. Further, EPR analysis showed a significant (p<0.0001) four- and two-fold increase in oxidative potential in the heart homogenate of female and male HFD groups, respectively. However, mitochondrial biogenesis was notably higher, observed only in HFD females compared to other groups. Conclusion: Our study indicates that pre-diabetic hyperglycemia enhances gender-specific mitochondrial biogenesis and triggers mitochondrial and ER stress responses, highlighting the importance of mitochondrial-ER interaction in early DCM and suggesting potential therapeutic avenues.

Abstract Mo055: CHD4 Interacts With TBX5 to Maintain the Gene Regulatory Network of Postnatal Atrial Cardiomyocytes

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 Mo070: Metabolic Syndrome Alters cAMP Homeostasis and Contractile Function of Cardiomyocytes

Approximately one third of the population in the US has metabolic syndrome (MetS), a condition associated with increased risk for coronary artery disease and myocardial infarction. But whether MetS alters properties of the myocardium before the occurrence of ischemic insults remains to be fully elucidated. For this purpose, MetS was induced in C57Bl/6 female mice with a dietary paradigm recapitulating Western-style alimentary habits in humans. Animals on regular chow were used as control (Ctrl). Cardiac function was assessed by echocardiography and myocytes were studied under field stimulation, following enzymatic dissociation. From 3-12 months on the diet, MetS mice had increased body weight, impaired glucose metabolism, augmented left ventricular (LV) mass, but preserved cardiac function, with respect to Ctrl mice. Using ECGs, heart rate variability was attenuated in MetS mice, suggesting that metabolic disorders alter cardiac sympathovagal balance. At the cellular level, LV myocytes from MetS mice had increased volume (+24%), enhanced fractional cell shortening (+42%), and faster kinetics of relaxation (-27%), with respect to Ctrl myocytes. Because cAMP and protein Kinase A (PKA) modulates myocardial contractility upon activation of G-protein coupled receptors, including beta-adrenergic receptors (B-AR), levels of these molecules were assessed by ELISA assay. Consistent with enhanced cell shortening and faster relaxation, levels of cAMP and PKA activity were, respectively, 1.8-fold and 1.9-fold larger in MetS myocytes, in comparison to Ctrl cells. Interestingly, inhibition of PKA with H-89 reduced cell shortening (-30%) and delayed kinetics of relaxation (+50%) in MetS myocytes but had no major effects on Ctrl cells. To establish the contribution of the cAMP/PKA signaling in the functional behavior of the heart with MetS, mice were treated with the B-AR blocker propranolol. Under this condition, MetS mice had reduced ejection fraction and impaired indices of diastolic function, with respect to Ctrl animals. Interestingly, myocytes obtained from Ctrl and MetS mice treated with the B-AR blocker had comparable cell shortening and kinetics of relaxation. Collectively, these results indicate that metabolic syndrome alters the functional properties of the myocytes and heart by affecting cAMP homeostasis. These properties may increase myocardial oxygen demand predisposing the heart to ischemic insults.

Abstract Mo068: The role of FGF13 in modulating gap junction protein Connexin-43

Background: Fibroblast growth factor homologous factors (FHFs) are associated with arrhythmias like Brugada syndrome and atrial fibrillation, but their pathological mechanisms remain poorly understood. FHFs are best characterized as regulators of voltage gated sodium channels (VGSCs), however recent studies suggest broader, non-VGSC-related functions, including regulation of microtubules. Whether and how non-VGSC regulatory functions modulate cardiac rhythm is not known. Hypothesis: FGF13 regulates Cx43 trafficking and expression in ventricular cardiomyocytes via destabilizing microtubules. Aims: Building on an unbiased proteomic screen, we investigated if FGF13, the predominant FHF in rodent heart, affects the expression and trafficking of Connexin-43 (Cx43), the predominant gap junction protein in ventricular myocytes. Methods: To identify additional FGF13-dependent functional roles, we employed proximity labeling proteomics in mouse heart with the TurboID biotin ligase fused to FGF13. We used immunoblots, immunohistochemistry, and immunocytochemistry to further characterize the effects of FGF13 ablation on Cx43 pattern distribution and expression. We also leveraged electrophysiology techniques using gap junction inhibitors to detect any Cx43-dependent functional changes in FGF13-ablated cardiomyocytes. Results: The proteomic data revealed that cell junction proteins associated with the intercalated disc (ID) were among the top FGF13 near neighbors, of which Cx43 was heavily enriched, together with microtubule associated proteins (MAPs) known to be critical for cardiomyocyte function and ion channel trafficking. In ventricular cardiomyocytes from constitutive cardiomyocyte-restricted Fgf13 knockout mice, we found markedly perturbed trafficking of Cx43 and reduced colocalization with the ID marker N-Cadherin. Separately, MAP4 abundance was markedly reduced. The addition of colchicine, a microtubule destabilizing drug, to WT cells altered Cx43 distribution pattern similar to that observed in FGF13-ablated cardiomyocytes. Re-expression of FGF13 restored Cx43 trafficking and ID targeting. Electrophysiology experiments using a Cx43 hemichannel inhibitor, Gap19, showed a perturbation of the action potential duration in knockout cardiomyocytes. Isoproterenol-induced action potential prolongation in knockout myocytes was decreased by the addition of Gap19. Conclusions: FGF13 regulates microtubule-based trafficking and targeting of Cx43.

Abstract We097: Dual Targeting CXCR4 and CXCR7 Prevents Doxorubicin-Induced Cardiotoxicity by Eliminating Cardiac Inflammation and Preserving Cardiomyocyte Function

Background: Doxorubicin (DOX)-induced cardiotoxicity (DICT) has become a serious health burden for cancer survivors. The detrimental effects of CXCR4-mediated excessive inflammation in myocardium and the protective effects of CXCR7 in cardiomyocytes have been demonstrated. Aims: We tested the protective effects against DICT of a dual-targeting compound TC14012, which has specific antagonistic activity against CXCR4 and potent agonistic activity on CXCR7. Methods: Eight-week-old male C57BL/6J mice were injected with DOX for consecutive 5 weeks, during which mice were treated with TC14012 twice a week. One week after the last dose injection, mice were euthanized after heart function examination. Markers of cardiac inflammation and fibrosis were evaluated. Cardiac RNA-seq was performed to explore the underlying molecular pathways. Key findings: TC14012 supplement significantly prevented DOX-induced cardiac systolic dysfunction, reflected by improved ejection fraction and fraction shortening, along with remarkable attenuation of DOX-induced cardiac perivascular and interstitial collagen deposition and neutrophils and macrophage infiltration. RNA-seq revealed that TC14012 prevention of DICT was accompanied with a remarkable stimulation of signals involving in cardiac contraction and energy reserve metabolic process, particularly glycogen and glucan metabolism that are crucial for meeting the cardiac energy demand in response to DOX induction. These signals could be further enriched into AMPK signaling pathway and validated by upregulation of AMPK phosphorylation. Besides, TC14012 prevention of DICT was accompanied with a significant reduction in cardiac mast cell activation and degranulation. These signals could be further enriched into Rap1 signaling pathway, and TC14012 was validated to inhibit Rap1 activity and mast cell activation. Most importantly, TC14012 treatment did not affect the suppressive effects of DOX on tumor growth in a syngeneic EL4 lymphoma-bearing, immunocompetent mouse model. Conclusions: We preliminary conclude that TC14012 prevents DICT by activating cardiac CXCR7 to reprogram cardiomyocyte metabolism and prevent cardiac damage and dysfunction via activating AMPK, and by antagonizing CXCR4 to inhibit cardiac mast cell infiltration and activation via blunting Rap1 activity but does not affect the anti-tumor activity of DOX.

Abstract Tu142: Pannexin-2 Deficiency Exacerbates Stress-induced Cell Injury in Cardiomyocytes

Pannexins are a family of channel proteins involved in a variety of biological processes, including cell death signaling and immune functions. While Pannexin isoforms 1 and 3 have been widely studied, mainly in the brain, much less is known about the role of isoform 2 (Pannexin2, or Panx2). A recent study showed that deficiency in Panx2 sensitizes pancreatic beta cells to cytokine-induced apoptosis, while another study revealed that Panx2 promotes ultraviolet-induced apoptosis in keratinocytes. The role of Panx2 in the regulation of cardiomyocyte function has not been investigated. In the current study, we explored the subcellular distribution profile of Panx2 expression in cardiomyocytes (AC16 cells and adult cardiomyocytes) and examined the role of Panx2 in regulating cardiomyocyte response to stress. Western blotting analysis revealed palmitoylation of Panx2 in the heart, suggesting that this channel is located intracellularly, instead of in the plasma membrane. Confocal imaging revealed that Panx2 expression is in the proximity of the sarcoplasmic reticulum and mitochondria, confirming its intracellular localization. The stress test results indicated that the knockdown of Panx2 by siRNA exacerbated FCCP-reduced oxidative stress and cell injury in AC16 cells. Similarly, adult cardiomyocytes isolated from Panx2-KO mice were more vulnerable to ischemia-reperfusion injury compared with wild-type cells. Finally, we found that the level of Panx2 expression altered in the cardiac tissue of a mouse model with pressure overload-induced heart failure, implying that Panx2 may be involved in the pathological remodeling of a failing heart.

Abstract Tu144: DIAPH1 regulates stress induced senescence in Human cardiomyocytes

Introduction: Stress induced senescence (SIS), defined as the loss of cell physiology/function associated with increased senescence associated secretory phenotypes (SASPs), is a potential risk factor for cardiac dysfunction and failure. Human Diaphanous 1(DIAPH1), a formin molecule is known to regulate actin-cytoskeleton dynamics, trafficking of organelles and signal transduction. Our recent work demonstrated the importance of DIAPH1-MFN2 interactions in regulating actin-cytoskeleton dynamics and the mitochondria-sarcoplasmic reticulum(Mito-SR) distance thereby regulating cardiomyocyte function and pathological responses to ischemia reperfusion injury. We tested the hypothesis that DIAPH1 regulated stress induced senescence in cardiomyocytes under aging and hypoxia/reoxygenation (H/R) conditions. Methods: Human cardiomyocytes (HiPSC-CMs) from two different sources of HiPSCs which are double positive for TNT2 and TNNI3 were used. To understand the role of DIAPH1 in SIS, we have used two different approaches. HiPSC-CMs were either kept for 120days in culture or at day25 were treated with mitomycin(200nM/72hrs). DIAPH1 was silenced using shRNA approach ( shDIAPH1 ). Results: We observed increased DIAPH1 gene expression at 120 days of culture vs day 30. Interestingly, shDIAPH1 HiPSC-CMs, attenuated the number of SA-β-galactosidase positive cells, reduced expression of senescence markers SERPINE, CDKN1A, CDKN2A, TP53 , COL1A2 along with improved mitochondrial function when compared to shScr controls. In experiments where HiPSC-CMs at day 25 were treated with mitomycin to induced senescence, we observed that silencing DIAPH1 attenuated the number of SA-β-galactosidase positive cells, reduced F/G actin ratio and robustly decreased DIAPH1-MFN2 interactions as measured by DUOLINK proximity ligation assay. We probed the RNA sequencing datasets from our recent publication (Yepuri et.al., Nat. Comm. 2023) and observed that senescence markers, SASPs and cytokines including CDKN1A, H2AX, LMNA, IL1A, IL6, SERPINE were significantly down regulated in shDIAPH1 HiPSC-CMs exposed to H/R conditions. Conclusion: Taken together our data demonstrates that DIAPH1 mediates stress induced senescence in cardiomyocytes and is linked to actin-cytoskeleton dynamics and DIAPH1-MFN2 interactions. Importantly, our data indicates a causative role of DIAPH1 in SIS and sets the stage for probing DIAPH1 as a therapeutic target for protection of hearts from stress induced senescence.

Abstract Mo096: High-Throughput Functional Determination of TNNI3 Variant Pathogenicity

Introduction: Mutations in the thin filament protein TNNI3 (cardiac troponin I) can perturb calcium cycling and cause hypertrophic, restrictive, and dilated cardiomyopathies. However, the majority of TNNI3 variants observed in patients have unknown functional significance and uncertain contribution to disease. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful system for studying the effect of genetic variants on human cardiomyocyte function. TNNI3 has been challenging to study in these cells as they predominantly express TNNI1. In this study, we combined a novel scalable in vitro gene replacement method with simultaneous measurement of contractility and calcium cycling to determine the functional effect of TNNI3 variants. Hypothesis: Using our gene replacement platform, we expect that TNNI3 variants causing Hypertrophic Cardiomyopathy (HCM) will increase force generation and calcium sensitivity relative to wild-type, whereas TNNI3 variants causing Dilated Cardiomyopathy (DCM) will decrease force generation and calcium sensitivity relative to wild-type. Methods: We generated double knockout TNNI1 -/- TNNI3 -/- hiPSCs. Cardiomyocytes differentiated from these hiPSCs were transduced with lentivirus carrying either wild-type or mutant TNNI3 during replating onto polyacrylamide gels with fluorescent beads. After a week of incubation, the cells were stained with a fluorescent calcium probe, and contractility, diastolic tension, and calcium sensitivity were measured by our high-throughput physiologic imaging and analysis platform. Results: hiPSC-CMs expressing the HCM-associated variant R192H show increased peak force, diastolic tension, and calcium sensitivity relative to wild-type TNNI3, whereas hiPSC-CMs expressing the DCM-associated variant K36Q show decreased peak force and calcium sensitivity relative to wild-type TNNI3. Conclusion: We validated our TNNI3 in vitro gene replacement and high-throughput physiologic imaging and analysis platform by comparing reference HCM and DCM variants to wild-type TNNI3. Ongoing scaled studies will evaluate the naturally occurring variants in ClinVar to characterize their functional effect on cardiac physiology.