Biology subjects

George, S. A.

Publications and source records attributed to George, S. A..

9 recordsLinked to original sources

bioRxiv2026

Exercise prevents cardiac electrical remodeling in doxorubicin-treated female mice but does not provide cardioprotection in males.

BackgroundDoxorubicin (DOX) causes sex-specific cardiotoxicity. Metabolic impairment is a well-established cardiotoxic effect of DOX treatment that can contribute to other detrimental effects such as increased reactive oxygen species, reduced ATP, inflammation etc. We hypothesized that preserving cardiac metabolism by exercise can attenuate DOX cardiotoxicity. MethodsMale and female C57BL/6J mice at 15 weeks of age were randomly assigned to one of four groups, 1) Control (sedentary), 2) EX (exercised, treadmill running), 3) DOX (doxorubicin at 5 mg/kg/week for 6 weeks), and 4) EXDOX (exercise + doxorubicin). Echocardiography was performed every other week during the 6-week protocol to measure cardiac mechanical function. At the end of the protocol, optical mapping and seahorse analysis were performed to measure electrophysiology and metabolism, respectively. RNA sequencing, cytokine array assay and transmission electron microscopy were also performed to determine sex-specific mechanisms of DOX cardiotoxicity. ResultsDOX reduced stroke volume and left ventricular diameter in males only and exercise did not prevent these effects of DOX. In female mice, DOX prolonged action potential duration (APD) and slowed conduction velocity (CV), and importantly, exercise prevented DOX-induced CV slowing. Exercise-induced cardioprotection against DOX in female mice was associated with preservation of aerobic metabolism and attenuation of inflammation which modulated ion channel gene expression. Specifically, Cacna1c was increased in both DOX and EXDOX females, but not in males and correlated with APD prolongation. Interestingly, despite CV slowing, Gja1 and Scn5a were increased. However, increased Kcnj8 along with metabolic impairment could cause membrane hyperpolarization and underlie CV slowing. ConclusionsDOX cardiotoxicity is sex specific. Mechanical dysfunction is more prevalent in DOX-treated males while arrhythmogenic electrical remodeling is more prevalent in DOX-treated females. Exercise therapy during DOX did not prevent DOX induced mechanical dysfunction in male hearts but attenuated electrical remodeling in females by preserving metabolism and attenuating inflammation.

physiology↗

bioRxiv2025

Seahorse Metabolic Analysis for Human and Mouse Cardiac Organotypic Slices

The mammalian heart relies on high rates of mitochondrial oxidative phosphorylation to meet its energy demand, with fatty acids serving as the primary fuel source in healthy adult hearts. While metabolic flexibility, the ability to switch between metabolic fuel substrates, is known to change during development and cardiac diseases, standardized methods for assessing substrate usage in intact, living cardiac tissue remain limited. Here, we present a protocol that adapts the Seahorse Mito Fuel Flex Test for use in living organotypic cardiac slices. This method enables the quantification of fuel dependency and capacity for fatty acids (FA), glucose (GLC), and glutamine (GLN) by sequentially inhibiting their respective mitochondrial oxidative phosphorylation pathways with the inhibitors etomoxir, UK5099, and BPTES. First, we validated the protocol by comparing results from organotypic cardiac slices to the standard published protocol using isolated adult mouse primary cardiomyocytes. Next, we demonstrated the sensitivity of this assay by modulating metabolism with AICAR, an AMPK activator, at varying concentrations, to demonstrate improved metabolism and then metabolic suppression at higher toxic doses. Finally, we applied this protocol to organotypic cardiac slices from different chambers of human donor hearts. This protocol provides a high-throughput, physiologically relevant platform for investigating cardiac metabolism, applicable across species and adaptable to other tissue types. It enables the study of metabolic remodeling in development and disease while overcoming the limitations of traditional cell-based assays by preserving native tissue architecture, physiology, and multicellular heterogeneity.

bioengineering↗

bioRxiv2025

Quadruple Parametric Optical Mapping to Measure Cardiac Metabolism-Excitation-Contraction Coupling

Cardiac excitation contraction coupling has been studied by recording transmembrane potential (Vm) and intracellular calcium (Ca2+), simultaneously. Recently, imaging metabolic substrates such as NADH in addition to Vm and Ca2+ has been reported to improve our understanding of cardiac physiology and the interaction between these different aspects of heart function, known as metabolism-excitation-contraction-coupling (MECC). In this study, we developed and validated the quadruple parametric optical mapping technique to simultaneously measure Vm, Ca2+ and two metabolic substrates, NADH and FAD. Langendorff perfused murine hearts of both sexes were subjected to either pharmacological interventions: 0.1 {micro}M nifedipine or 0.1 {micro}M flecainide, or pathological intervention: ischemia/reperfusion and the four facets of cardiac function were measured. Hearts were stained with RH237 and Rhod2-AM dyes to measure Vm and Ca2+, respectively. Two LED light sources (520 and 365 nm) were used to excite the dyes and induce NADH/FAD autofluorescence, respectively, and all four optical signals were simultaneously recorded and analyzed to measure12 parameters of cardiac function. Nifedipine significantly altered Ca2+ related parameters, including increased Ca2+ rise-time and Ca2+ decay, and prolonged Vm-Ca2+ delay. Flecainide significantly affected all three aspects of MECC with increased Vm rise-time and Ca2+ decay, while decreasing conduction velocity (CV) and the redox ratio. Ischemia also altered MECC with increased Vm rise-time and NADH intensity, decreased CV and redox ratio. Using quadruple parametric optical mapping, we demonstrate known cardiac drug response to nifedipine and flecainide and report novel aspects of cardiac function that are modulated by these drugs. These novel drug effects would not have been identified without such a multi-parametric approach to assessing cardiac physiology. Further, we were able to determine chronological changes in MECC during ischemia and reperfusion to better understand the interplay between electrical, mechanical, and metabolic function in the heart.

bioengineering↗

bioRxiv2023

Moderate Endurance Exercise Increases Arrhythmia Susceptibility and modulates Cardiac Structure and Function in a Sexually Dimorphic manner.

BackgroundWhile moderate endurance exercise has been reported to improve cardiovascular health, its effects on cardiac structure and function are not fully characterized, especially with respect to sexual dimorphism. We aimed to assess the effects of moderate endurance exercise on cardiac physiology in male versus female mice. MethodsC57BL/6J mice of both sexes were run on a treadmill for six weeks. ECG and echocardiography were performed every two weeks. After six weeks of exercise, mice were euthanized, and triple parametric optical mapping was performed on Langendorff perfused hearts to assess cardiac electrophysiology. Arrhythmia inducibility was tested by programmed electrical stimulation. Left ventricular (LV) tissue was fixed, and RNA sequencing was performed to determine exercise-induced transcriptional changes. ResultsExercise-induced LV dilatation was observed in female mice alone, as evidenced by increased LV diameter and reduced LV wall thickness. Increased cardiac output was also observed in female exercised mice but not males. Optical mapping revealed further sexual dimorphism in exercise-induced modulation of cardiac electrophysiology. In female mice, exercise prolonged action potential duration and reduced voltage-calcium influx delay. In male mice, exercise reduced the calcium decay constant, suggesting faster calcium reuptake. Exercise increased arrhythmia inducibility in both male and female mice, however, arrhythmia duration was increased only in females. Lastly, exercise-induced transcriptional changes were sex-dependent: females and males exhibited the most significant changes in contractile versus metabolism-related genes, respectively. ConclusionsOur data suggest that moderate endurance exercise can significantly alter multiple aspects of cardiac physiology in a sex-dependent manner. While some of these effects are beneficial, like improved cardiac mechanical function, others are potentially pro-arrhythmic.

physiology↗

bioRxiv2023

Identification of novel oncogenic transcriptional targets of mutant p53 in Esophageal Squamous Cell Carcinoma

Missense mutations in the DNA binding domain of p53 are observed frequently in Esophageal Squamous Cell Carcinoma (ESCC). Recent studies have revealed the potentially oncogenic transcriptional networks regulated by mutant p53 proteins. However, majority of these studies have focused on common hotspot p53 mutations while rarer mutations are poorly characterized. We had previously identified SMARCD1 as an oncogenic transcriptional target of rare non-hotspot p53 mutants detected from squamous cell carcinoma of the oral tongue (SCCOT). We now report the characterization of non-hotspot p53 mutations from ESCC. In-vitro tumorigenic assays performed following ectopic-expression of non-hotspot mutant p53 proteins caused enhancement of oncogenic properties in squamous carcinoma cell lines. Genome-wide transcript profiling of ESCC tumor samples stratified for p53 status, revealed several genes exhibiting elevated transcript levels in tumors harbouring mutant p53. Of these, ARF6, C1QBP and TRIM23 were studied further due to their previously reported pro-oncogenic roles. Reverse transcription quantitative PCR (RT-qPCR) performed on RNA isolated from ESCC tumor samples revealed significant correlation of TP53 transcript levels with those of the three target genes. Ectopic expression of wild type and several mutant p53 forms followed by RT-qPCR, Chromatin affinity-purification and Promoter-luciferase assays indicated the exclusive recruitment of p53 mutants - P190T and P278L, to the target genes leading to activation of expression. Several functional assays following knockdown of the target genes revealed a significant suppression of tumorigenicity in squamous carcinoma cell lines. Rescue experiments confirmed the specificity of the knockdown. The tumorigenic effect of the genes was confirmed in nude mice xenograft assays. This study has therefore identified novel oncogenic targets of rare non-hotspot mutant p53 proteins relevant for ESCC besides validating the functional heterogeneity of the spectrum of tumor specific p53 mutations.

cancer biology↗

bioRxiv2022

RyR2 inhibition with dantrolene is antiarrhythmic, antifibrotic, and improves cardiac function in chronic ischemic heart disease

BackgroundVentricular tachycardia (VT) is responsible for sudden death in chronic ischemic heart disease (CIHD) patients. The cardiac ryanodine receptor (RyR2) releases Ca2+ from the sarcoplasmic reticulum (SR) and links electrical excitation to contraction. RyR2 hyperactivity has been widely documented in CIHD and may contribute to VT risk and progressive LV remodeling. ObjectiveTo test the hypothesis that targeting RyR2 hyperactivity plays a mechanistic role in VT inducibility and progressive heart failure in CIHD that can be prevented by the RyR2 inhibitor dantrolene. MethodsCIHD was induced in C57BL/6J mice by left coronary artery ligation. Four weeks later, mice were randomized to either acute or chronic (6 weeks via osmotic mini-pump) treatment with dantrolene or vehicle. VT inducibility was assessed by programmed stimulation in vivo and in isolated hearts. Electrical substrate remodeling was assessed by optical mapping. Ca2+ sparks and spontaneous Ca2+ releases were measured in isolated cardiomyocytes. Cardiac remodeling was assessed by histology and qRT-PCR. Cardiac function and contractility were assessed by echocardiography. ResultsCompared to vehicle, acute dantrolene treatment reduced VT inducibility and improved LV contractility in vivo. Optical mapping in isolated hearts demonstrated reentrant VT prevention by dantrolene, which normalized the shortened refractory period (VERP) and prolonged action potential duration (APD), preventing APD alternans. In single CIHD cardiomyocytes, dantrolene normalized RyR2 hyperactivity and prevented spontaneous SR Ca2+ release. Chronic dantrolene treatment reduced peripheral muscle strength but had no adverse effects on body weight or mortality. Chronic dantrolene not only reduced VT inducibility but also reduced peri-infarct fibrosis and prevented the progression of LV dysfunction in CIHD mice. ConclusionRyR2 hyperactivity plays a mechanistic role for VT risk, infarct remodeling, and contractile dysfunction in CIHD mice. Our data provide proof of concept for the anti-arrhythmic and anti-fibrotic efficacy of dantrolene in CIHD. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIThe mouse CIHD model is a more clinically relevant model in which treatment is started late after infarction, when heart failure is already established. C_LIO_LIAcute and chronic dantrolene treatment suppresses VT inducibility by restoring myocyte APD, terminating APD alternans and normalizing VERP. C_LIO_LIChronic dantrolene treatment prevents pathological remodeling and peri-infarct fibrosis, the substrate for reentry VT. Cardiac function is improved with chronic dantrolene therapy. C_LI Clinical ImplicationsO_LITreatment with dantrolene, which is already approved for clinical use, is a promising therapy in patients with ischemic heart disease, in whom other antiarrhythmic drugs are contraindicated. C_LIO_LIDantrolene inhibition of RyR2 not only suppresses VT but also improves cardiac function in chronic ischemic heart disease. C_LI

pharmacology and toxicology↗

bioRxiv2021

Simultaneous Triple-Parametric Optical Mapping of Transmembrane Potential, Intracellular Calcium and NADH for Cardiac Physiology Assessment

Investigation of the complex relationships and dependencies of multiple cellular processes that govern cardiac physiology and pathophysiology requires simultaneous dynamic assessment of multiple parameters. In this study, we introduce triple-parametric optical mapping to simultaneously image metabolism, electrical excitation, and calcium signaling from the same field of view and demonstrate its application in the field of drug testing and cardiovascular research. We applied this metabolism-excitation-contraction coupling (MECC) methodology to test the effects of blebbistatin, 4-aminopyridine and verapamil on cardiac physiology. While blebbistatin and 4-aminopyridine alter multiple aspects of cardiac function suggesting off-target effects, the effects of verapamil were on-target and it altered only one of ten tested parameters. Triple-parametric optical mapping was also applied during ischemia and reperfusion; and we identified that metabolic changes precede the effects of ischemia on cardiac electrophysiology.

bioengineering↗

bioRxiv2020

Heart Slice Culture System Reliably Demonstrates Clinical Drug-Related Cardiotoxicity

The limited availability of human heart tissue and its complex cell composition are major limiting factors for reliable testing drug efficacy, toxicity and understanding mechanism. Recently, we developed a functional human and pig heart slice biomimetic culture system that fully preserves the viability and functionality of 300 {micro}m heart slices for 6 days. Here, we tested the reliability of this culture system in delineating the mechanisms of known anti-cancer drugs that cause cardiomyopathy. We tested three anti-cancer drugs (doxorubicin, trastuzumab, and sunitinib) associated with different mechanisms leading to cardiotoxicity at three concentrations and assessed the effect of these drugs on heart slice viability, structure, function and transcriptome. Slices incubated with any of these drugs for 48 h showed significant loss in viability, cardiomyocyte structure and functionality. Mechanistically, RNA sequencing demonstrated a significant downregulation of cardiac genes and upregulation of oxidative response in doxorubicin-treated tissues. Trastuzumab treatment caused major downregulation in cardiac muscle contraction-related genes, consistent with its clinically known direct effect on cardiomyocytes. Interestingly, sunitinib treatment resulted in significant downregulation of angiogenesis-related genes in line with its mechanism of action. Heart slices are not only able to demonstrate the expected toxicity of doxorubicin and trastuzumab similar to hiPS-derived-cardiomyocytes; they are superior in detecting sunitinib cardiotoxicity phenotypes and mechanism in the clinically relevant concentration range, 100 nM - 1 {micro}M. These results indicate that heart slice tissue culture models have the potential to become a reliable platform for testing drug toxicity and mechanism of action.

cell biology↗

bioRxiv2020

p38delta genetic ablation protects female mice from anthracycline cardiotoxicity

BACKGROUNDThe efficacy of an anthracycline antibiotic doxorubicin (DOX) as a chemotherapeutic agent is limited by dose-dependent cardiotoxicity. DOX is associated with activation of intracellular stress signaling pathways including p38 MAPKs. While previous studies have implicated p38 MAPK signaling in DOX-induced cardiac injury, the roles of the individual p38 isoforms, specifically, of the alternative isoforms p38{gamma} and p38{delta}, remain uncharacterized. OBJECTIVESTo determine the potential cardioprotective effects of p38{gamma} and p38{delta} genetic deletion in mice subjected to acute DOX treatment. METHODSMale and female wild-type (WT), p38{gamma}-/-, p38{delta}-/- and p38{gamma}-/-{delta}-/- mice were injected with 30 mg/kg DOX and their survival was tracked for ten days. During this period cardiac function was assessed by echocardiography and electrocardiography and fibrosis by PicroSirius Red staining. Immunoblotting was performed to assess the expression of signaling proteins and markers linked to autophagy. RESULTSSignificantly improved survival was observed in p38{delta}-/- female mice post-DOX relative to WT females, but not in p38{gamma}-/- or p38{gamma}-/-{delta}-/- male or female mice. The improved survival in DOX-treated p38{delta}-/- females was associated with decreased fibrosis, increased cardiac output and LV diameter relative to DOX-treated WT females, and similar to saline-treated controls. Structural and echocardiographic parameters were either unchanged or worsened in all other groups. Increased autophagy, as evidenced by increased LC3-II level, and decreased mTOR activation was also observed in DOX-treated p38{delta}-/- females. CONCLUSIONSp38{delta} plays a crucial role in promoting DOX-induced cardiotoxicity in female mice by inhibiting autophagy. Therefore, p38{delta} targeting could be a potential cardioprotective strategy in anthracycline chemotherapy. NEW AND NOTEWORTHYThis study for the first time identifies the roles of the alternative p38{gamma} and p38{delta} MAPK isoforms in promoting DOX-cardiotoxicity in a sex-specific manner. While p38{gamma} systemic deletion did not affect DOX-cardiotoxicity, p38{delta} systemic deletion was cardioprotective in female but not in male mice. Cardiac structure and function were preserved in DOX-treated p38{delta}-/- females and autophagy was increased.

physiology↗