Cardiomyocyte Contractility Research Articles

Innovative models for cardiotoxicity assessment in human pluripotent stem cell-derived cardiomyocytes that enable clinically relevant treatment regimen

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): This work was supported by the European Research Area Network on Cardiovascular Diseases 807 (2016T092) and Hartstichting (2019T104) Background/Introduction/Purpose Cardiotoxicity continues to be a significant factor in drug withdrawal, partly due to the unpredictable nature of animal models. Moreover, the limitation of cancer treatment arises from the potential risk of cardiotoxicity. Identifying patients at risk of developing heart symptoms after therapy will strongly contribute to improved treatment possibilities. Cardiomyocytes derived from human pluripotent stem cells (hPSCs) represent an inexhaustible cell reservoir and hold the potential to provide personalized solutions to address this issue. However, conventional 2D cultures have several limitations, such as tissue complexity and the difficulty to apply repeated dose treatments. Methods Therefore, we developed an engineered heart tissue (EHT) platform to predict functional aspects of cardiotoxicity. We evaluated the effect of several anti-cancer drugs on cardiomyocyte contractility after repetitive treatment and quantified force of contraction and contraction kinetics. However, these traditional EHTs lack the complexity of diverse cell types. Thus, in a next step, we generated an innovative and user-friendly Heart-on-Chip (HoC) model from hPSC-derived cardiomyocytes, endothelial and smooth muscle cells. Under flow, these miniaturized micro-EHTs (μEHTs) self-organize into a cardiomyocyte core surrounded by an endothelial barrier layer mimicking the cardiomyocyte-endothelial interface as observed in vivo. Results Our versatile drug screening systems permit following individual tissues over time. We therefore treated EHTs with DMSO or 1 µM Doxorubicin for 24 hours and monitored tissue contraction force over a time course of 28 days. Doxorubicin treatment initially caused a drop of force, but demonstrated a recovery of force and kinetics 10 days after treatment. To recapitulate a clinically relevant intermittent treatment regime, a second treatment of 1 µM Doxorubicin was administered 21 days after the first dose and showed a similar decrease in contraction force compared with the first exposure to the drug. μEHTs cultured under flow exhibit improved contractile performance and conduction velocity when compared to static culture conditions. Importantly, the presence of an endothelial barrier delayed the cardiotoxic effect of drugs, indicating that endothelial cells form a barrier mimicking the systemic delivery route of drugs to the heart. Conclusions Our systems are characterized by increased physiological accuracy and enable the study of hPSC-cardiomyocyte recovery and the effects of accumulated dose after multiple dosing, allowing individualized cardiotoxicity evaluation. In the future, we can further increase the complexity by including multi-organ-on-chip approaches, as for example heart-liver-on-chip to revolutionize the screening of therapeutic targets and the prediction of drug efficacy.

Senolysis ameliorates increased susceptibility to ventricular arrhythmias in prediabetes

Abstract Background Obesity and associated prediabetes are major risk factors for cardiovascular disease that promote cellular senescence and predispose to ventricular arrhythmias (VA). In consequence, targeting cellular senescence may represent a feasible therapeutical approach that can decrease VA susceptibility in aged hearts. The aim of the present study was to characterize the impact of senolysis on glucose control, left ventricular function (LVF) and VA susceptibility in a prediabetic mouse model. Methods Ten – 12 week-old C57Bl/6J mice were fed a high-fat diet over a period of 12 weeks to generate diet-induced-obesity (DIO). Afterwards, mice were treated with dasatinib (5 mg/kg) and quercetin (50 mg/kg) (DQ) or vehicle (10 % DMSO) by oral gavage three times in 7 days. Cardiac senescence was assessed by measuring Cdkn1a mRNA expression levels. Glucose control was monitored by glucose tolerance testing. LVF was evaluated by echocardiography and myocardial contractility was assessed by sarcomere length measurements in paced single cardiomyocytes. VA susceptibility testing was performed by programmed ventricle stimulation combined with an epicardial fluorescence imaging of the LV anterior wall in isolated hearts. VA susceptibility was quantified by an established scoring system. Results Cardiac senescence, as assessed by expression of Cdkn1a mRNA (encoding p21), was increased in DIO mice (P=0.0004, Figure 1A), whereas DQ reduced cardiac senescence (P<0.0001, Figure 1A). Glucose control was impaired in DIO mice (P<0.0001) and could be partially restored by DQ (P=0.003). LVF was ameliorated by DQ in DIO mice (P=0.018). In agreement, contractility in single cardiomyocytes was improved (Amplitude of sarcomere contraction: P=0.0003, figure 1B; speed of sarcomere contraction: P=0.046). VA susceptibility was increased in DIO mice (number of inducible VAs: P=0.001, figure 1C; VA score: P=0.0002; VT inducibility: P=0.001) whereas DQ limited VA susceptibility (number of induced VAs: P=0.004, Figure 1C; VA score: P=0.004; VT inducibility: P=0.025) in isolated hearts. Conclusions Our findings demonstrate that senolysis ameliorates glucose tolerance and LVF while reducing VA susceptibility in obesity and prediabetes. In consequence, senolysis may offer a perspective therapeutical approach for obese patients at risk for VAs. Further experiments will address underlying molecular mechanisms to identify potential therapeutical targets.

#1447 Calcimimetics improve impaired cardiac function and contractility in chronic phosphate loading by activating the CaSR/cAMP signaling cascade

Abstract Background and Aims High phosphate loading stimulate the synthesis of PTH and FGF23 and is associated with increased cardiovascular (CV) mortality. In hemodialysis patients, the administration of calcimimetics led to reductions in PTH and FGF23 and the latter was associated with a lower rate of CV events. We have previously shown that mice with a high dietary phosphate load show elevated PTH and FGF23 levels and develop impaired systolic cardiac function, which could be improved by calcimimetics. Calcimimetics are allosteric activators of the calcium-sensing receptor (CaSR). Phosphate (Pi) was shown to inactivate the parathyroid CaSR at concentrations >1.4 mM Pi. Whether calcimimetics contribute to CV event reduction indirectly via lowering PTH and FGF23 or directly via activation of the cardiac CaSR is unknown. Method Wild-type mice were fed a high phosphate diet (HPD) ± etelcalcetide (Etl) or a normal phosphate diet (NPD), cardiac function was assessed by echocardiography and Millar catheterization, and cardiac tissue was harvested for histology and RNAseq analyses. In addition, isolated adult mouse cardiomyocytes (AMCM) were stimulated ex vivo with PTH, FGF23 or Pi, contractility and calcium handling were measured with the addition of Etl and activity analyses were performed. Results In addition to the positive effect on HPD-induced systolic cardiac dysfunction, Etl improved impaired LV contractility and reduced fibrosis. RNAseq analyses revealed reduced Ca, PKA and cAMP signaling by HPD. Ex vivo stimulation of AMCM with PTH and FGF23 had no functional effects. In contrast, Pi significantly worsened contractility and Ca handling, which was blunted by Etl co-stimulation. Downstream of cardiac CaSR, Pi reduced the formation of cAMP, which led to PKA-dependent dephosphorylation of PLN and subsequently to lower Serca2a activity, which was ultimately responsible for the reduced contractility. Etl led to improved cardiomyocyte contractility via increased cAMP synthesis and increased PLN phosphorylation and Serca2a activity. Conclusion Our data suggest that Etl ameliorates the Pi-induced pathologic cardiac phenotype associated with high phosphate load via direct activation of CaSR/cAMP signaling cascade, resulting in improvement of cardiomyocyte contractility.

Protection by L-arginine against Epinephrine-induced Arrhythmia and Cardiotoxicity

Nitric oxide is important in control of coronary blood flow, cardiomyocyte contractility, cardiac rhythm, and thrombogenicity. We aimed to investigate the effect of the nitric oxide precursor L-arginine on cardiac arrhythmia and myocardial injury induced by epinephrine in male Sprague-Dawley rats. The possible modulation of L-arginine effects by methylene blue (MethyB), a nitric oxide synthase inhibitor was also examined. Cardiac arrhythmia was induced with 10μg/kg epinephrine given intravenously (i.v.). L-arginine (200mg/kg) or L-arginine and MethyB (100mg/kg) were intraperitoneally (i.p.) administered prior to i.v. epinephrine. Other groups received i.p. saline, only L-arginine or only MethyB (100mg/kg). Results showed that compared with the saline control, epinephrine caused a significant bradycardia (188.7±24.4 vs. 405.6±1.19beats/min), increased QRS duration (0.039±0.006 vs. 0.0185±0.0001s), decreased R wave amplitude (0.18±0.01 vs. 0.23±0.011mv), and ventricular extrasystoles. L-arginine alone showed no significant effect on heart rate (380.8±10.0 vs. 405.6±1.19beats/min) but increased QRS duration (0.029±0.0002 vs. 0.0185±0.0001s), and R wave amplitude (0.47±0.01 vs. 0.23±0.011mv). L-arginine given prior to epinephrine prevented bradycardia, shortened the duration of arrhythmia and decreased the number of extrasystoles. The administration of L-arginine and MethyB almost completely suppressed epinephrine-induced ventricular arrhythmias. Epinephrine caused severe degeneration of muscle fibres, widening of intercellular spaces, cellular infiltrations and interstitial haemorrhage. L-arginine markedly attenuated, while L-arginine/MethyB completely prevented these changes. It is concluded that L-arginine protects against cardiac arrhythmias and cardiac tissue damage caused by epinephrine.

Effects of Nicotine on Chicken Embryo Development: A Review

Background: Studies have shown that 22.3% of the world’s population use tobacco and nicotine exposure during pregnancy remains a concern in embryonic development. Cigarette smoke contains several toxic and carcinogenic chemicals and has been known to cause pregnancy complications including premature births, low birthweights, and stillbirths. Purpose: This review aims to study nicotine exposure in chicken embryo development comprehensively. Methods: PubMed, Centers for Disease Control and Prevention (CDC), and Antpedia sites were used to search for studies using chicken embryos as a model. Studies that reported findings on nicotine's effects on various developmental processes were considered for this review. Conclusion: In total, 55 published articles were included in this review to discuss findings of nicotine-induced alterations during chicken embryo development. Findings have shown that nicotine affects angiogenesis, and embryonic and chorioallantoic membrane (CAM) growth by inhibiting cell proliferation. Nicotine affects brain and cerebellar cortex development by suppressing tumor protein p53 reactions. Nicotine also causes abnormal axial rotation and incomplete formation and closure of neural tubes. The compounds like green tree extract, vitamin C, and folic acid can reduce the effects of nicotine to suppress femur growth, decrease the contractility of cardiomyocytes, and reduce survival rates. These compounds are not protective measures to completely overcome the teratogenic effects of nicotine.

B1AR regulation of the Cav1.2 via the SAP97 complex

Beta adrenergic (BAR) signaling is stimulated by the catecholamines norepinephrine (NE) and epinephrine (EPI) to control cardiac contractility. The beta-1-AR (B1AR) is the major cardiac subtype. B1AR signals canonically through the Gas subunit, leading to increases in cAMP and PKA; however, it can also signal via the Gai subunit, leading to increased NOS1, cGMP, and PKG levels. B1AR-NOS1 signaling leads to increased contractility and calcium cycling in cardiomyocytes. We found a novel complex within cardiomyocytes in which the B1AR, Cav1.2, and NOS1 proteins are held in proximity by the membrane scaffolding protein SAP97. In heart failure, there is a decrease in SAP97 expression and impaired B1AR signaling. Cardiac-specific deletion of SAP97 disrupts this complex. In the SAP97-cKO mice, there are minimal changes in B1AR-Gas; however, B1AR-Gai signaling is decreased, with a loss of B1AR-NOS1 signaling. Furthermore, we see a decrease in whole cell calcium currents and cardiac contractility. In mice lacking the NOS1, we see the same decrease in calcium currents. We hypothesize SAP97 is necessary to maintain B1AR modulation of Cav1.2 in cardiomyocytes. Using electrophysiology and biochemical techniques, we aim to elucidate the role of SAP97 in B1AR modulation of Cav1.2 and how this is impacted by the impairment of B1AR-Gai signaling. T32HL086350 RO1-HL147263. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Chronic central leptin administration attenuates kidney dysfunction and injury in a model of ischemia/reperfusion-induced acute kidney injury

Kidney disease occurs in approximately 15% of the adult population, accounting for 37 million people in the US alone. Lack of blood flow is a major driver of kidney injury and no therapeutic modalities have shown substantial effcacy in treating ischemia-induced acute kidney injury (AKI). We recently demonstrated that activation of the brain leptin-melanocortin system dramatically improves cardiomyocyte mitochondrial function, energy metabolism and contractility, preserving cardiac function and preventing progression of heart failure following myocardial infarction and ischemia/reperfusion injury. In the present study we examined whether chronic intracerebroventricular (ICV) leptin administration protects the kidney against ischemia/reperfusion (I/R)-induced AKI. Twelve-week old male rats were implanted with an ICV cannula into the right lateral ventricle and 8-10 days after surgery, leptin (0.021 mg/hr, n=8) or saline vehicle (0.5 ml/h, n=8) was infused via osmotic minipump connected to the ICV cannula for 12 days. On day 8 of leptin or vehicle infusion, rats were submitted to unilateral ischemia/reperfusion (UIR) by clamping the left pedicle for 30 min. To control for leptin-induced reductions in food intake, the vehicle group was pair-fed (UIR-PF) to match the same amount of food consumed by leptin-treated (UIR-Leptin) rats. On the 12th day of leptin or vehicle infusion (i.e. 4th day after AKI), single-left kidney glomerular filtration rate (GFR) was measured, blood samples were collected to quantify white blood cells, and kidneys were collected for histological assessment of injury. Chronic leptin infusion reduced body weight by 20±1% compared to only 10±1% reduction in UIR-PF. UIR-Leptin rats also showed reduced right and left kidney weights (right: 1040±24 vs. 1281±36 and left: 1127±71 vs. 1707±45 mg, for UIR-Leptin and UIR-PF, respectively). Leptin infusion improved GFR (0.50±0.06 vs. 0.13±0.03 ml/min/g KW) and reduced kidney injury scores. Leptin treatment also increased circulating white blood cells count compared to UIR-PF rats (16.3±1.3 vs. 9.8±0.6 k/mL). Thus, leptin, via its actions on the central nervous system, confers significant protection against major kidney dysfunction and damage in a model of ischemia/reperfusion-induced AKI. NIH grants: (1R0DK1121411 (do Carmo), 1R0HL1630376 (da Silva/do Carmo), P20GM104357 (Hall), P30GM149404 (Hall) and U54GM115428 (Hall)). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Role for Septin GTPases in regulating cytoplasmic calcium handling in Drosophila cardiomyocytes

Proper calcium handling in cardiomyocytes is paramount to maintaining cardiomyocyte contractility and heart function. Data from our lab and others have established that store operated calcium entry (SOCE) is an essential component of cardiomyocyte calcium regulation. SOCE refers to calcium influx that is activated by depletion of endo/sarcoplasmic reticulum (E/SR) calcium stores. The pathway is mediated by STIM proteins, which act as calcium sensors in the E/SR, and ORAI calcium selective influx channels in the plasma membrane. Both upregulation and suppression of SOCE in cardiomyocytes result in cardiomyopathy, demonstrating that proper regulation of cardiomyocyte SOCE is essential. However, the mechanisms responsible for SOCE regulation in cardiomyocytes are not well understood. To this end, septin GTPases have emerged as regulators of SOCE in non-cardiac tissues, with septin 1, 2, or 4 depletion resulting in SOCE suppression and septin 7 depletion resulting in SOCE upregulation. The role of septins in cardiomyocytes is nearly completely unknown. Through intravital imaging analysis of Drosophila heart contractility, we show that cardiomyocyte specific RNAi-based depletion of septins 1, 2, or 4 results in dilated cardiomyopathy nearly identical to that caused by SOCE suppression. Overexpression of SERCA, which pumps Ca2+ into SR stores, reversed the septin 2 phenotype, suggesting a mechanism whereby septin depletion reduces SR Ca2+ store refilling due to suppressed SOCE. In further support of SR Ca2+ store depletion, we found through jRGECO-based Ca2+ imaging of contracting hearts that contractile Ca2+ transients were significantly reduced in septin 2-depleted cardiomyocytes compared to controls. Interestingly, septin 7 suppression resulted in hypertrophic cardiomyopathy like that caused by SOCE upregulation, and micro-computerized tomography (microCT) analysis of heart size further confirmed the hypertrophic phenotype of septin 7 depleted hearts. This septin 7 phenotype was reversed by Orai depletion, supporting a role for SOCE dysregulation in the septin 7 phenotype. These results demonstrate for the first time an essential role for septins in cardiomyocyte physiology involving regulation of Ca2+ homeostasis and cycling. National institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Microtubules distinctly contribute to the performance of the healthy and pressure-overloaded RV

Introduction: Microtubules (MT) are a key contributor to cardiomyocyte contractility and mechanics. Recently, our group reported that acute depolymerization of MT reduces viscosity and elasticity and alters anisotropy of healthy and pressure-overloaded right ventricular (RV) tissues. While RV free wall viscosity and elasticity are distinctly upregulated with chronic pressure overload, how organ function is affected by viscoelastic contribution of the MT remains unknown. Our aim was to investigate the role of MT polymerization in RV function from its effect on RV tissue viscoelasticity. We hypothesized that the MT differentially contributes to healthy and pressure-overloaded RV function. Methods: All procedures and methods were approved by Colorado State University IACUC. Pulmonary hypertension (PH) was induced in 6-week-old male rats via 3-week monocrotaline treatment. Healthy age- and sex-matched rats served as control. Pressure-volume (PV) relationships were obtained by RV catheterization before and after intramyocardial injections of 0.3mM colchicine (COL) to acutely depolymerize the MT network and reduce tissue viscoelasticity. Functional impacts were evaluated by monitoring changes in RV end-systolic pressure (ESP), cardiac output (CO), stroke volume (SV), ejection fraction (EF), dP/dt max and min, cardiac effciency (CE), and the end-systolic pressure-volume relation (ESPVR). Results: As expected, monocrotaline treatment induced marked PH and RV contractile dysfunction, indicated by a significant elevation of RV ESP with reduced CO and ESPVR. In the healthy RV, COL treatment led to marked reductions in all functional parameters, indicating that the MT network is critical to maintain RV performance and contractile function in the healthy heart. In the pressure-overloaded RV, COL treatment similarly reduced SV, CO, EF, and dP/dt max and min, but increased CE and ESPVR, indicating that MT depolymerization improved mechanical effciency and contractility. To rule out effects caused by bolus injections alone, we administered a similar quantity of saline and observed no effects on the PV parameters in healthy RV. Discussion: Our study is the first to investigate the acute effect of MT depolymerization on RV function in healthy and diseased states. We found distinct contributions of MT to the contractile function and mechanical effciency of healthy and pressure-overloaded RVs. These findings reveal a critical influence of tissue viscoelasticity on RV physiology and dysfunction, emphasizing the importance of considering myocardial viscoelastic behavior in future studies investigating cardiac remodeling and heart failure pathophysiology. This work is partially supported by the National Science Foundation (NSF) grant 2244994. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comparison of MYOCYTER and MUSCLEMOTION in the analysis of Induced Pluripotent Stem Cell Derived Cardiomyocyte Contractility

Patient-specific induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes (CMs) offer tremendous opportunities for cardiac disease modeling and advancements in precision medicine. There is a need for technologies that allow noninvasive and precise measurements of CM function, such as contractility, to increase throughput for drug screening and testing efforts. Hypoplastic Left Heart Syndrome (HLHS) is a rare and complex congenital heart defect characterized by hypoplasia of the left ventricle, proximal aorta, as well as stenosis or atresia of the mitral and/or aortic valves. Previously it was demonstrated that iPSC-CMs from an HLHS patient with a genetic variant in the alpha myosin heavy chain ( MYH6 gene) exhibited an impaired cardiomyocyte phenotype (dysmorphic sarcomere structure, compensatory expression of MYH7, and altered contractility). Gene editing using CRISPR/Cas9 rescued the phenotype, demonstrating the utility of MYH6 iPSC-CMs as a useful disease model for HLHS. Critical parameters of CM function include contraction and relaxation, assessing systolic and diastolic dysfunction, respectively, in iPSC-CMs. The aim of this study is to compare features from two open-source macros for ImageJ: MYOCYTER and MUSCLEMOTION. MYOCYTER improves upon previous plugins available by allowing for users to select parameters. Both methods allow measurement of movement from high-speed movies. MYOCYTER enables the user to select an optimal threshold, size, and cell count to minimize disturbances such as cellular fragments, background noise, and floating bubbles, to maximize precision during analysis. MUSCLEMOTION allows rapid and easy measurement of movement from high- speed videos and can measure and compare relative or absolute parameters. This study will determine whether a systolic or diastolic dysfunction phenotype is observed under different magnification settings and evaluate the effect of dosing with pharmaceutical compounds. Finally, benefits and drawbacks of both macros will be investigated. Children's Research Institute. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cardiomyocyte Adaptation to Exercise: K+ Channels, Contractility and Ischemic Injury.

Cardiovascular disease is a leading cause of morbidity and mortality, and exercise-training (TRN) is known to reduce risk factors and protect the heart from ischemia and reperfusion injury. Though the cardioprotective effects of exercise are well-documented, underlying mechanisms are not well understood. This review highlights recent findings and focuses on cardiac factors with emphasis on K+ channel control of the action potential duration (APD), β-adrenergic and adenosine regulation of cardiomyocyte function, and mitochondrial Ca2+ regulation. TRN-induced prolongation and shortening of the APD at low and high activation rates, respectively, is discussed in the context of a reduced response of the sarcolemma delayed rectifier potassium channel (IK) and increased content and activation of the sarcolemma KATP channel. A proposed mechanism underlying the latter is presented, including the phosphatidylinositol-3kinase/protein kinase B pathway. TRN induced increases in cardiomyocyte contractility and the response to adrenergic agonists are discussed. The TRN-induced protection from reperfusion injury is highlighted by the increased content and activation of the sarcolemma KATP channel and the increased phosphorylated glycogen synthase kinase-3β, which aid in preventing mitochondrial Ca2+ overload and mitochondria-triggered apoptosis. Finally, a brief section is presented on the increased incidences of atrial fibrillation associated with age and in life-long exercisers.

Abstract LB180: Non-cardiotoxic properties of annamycin, a clinically evaluated anthracycline and potent topoisomerase 2β poison

Abstract Introduction. Cardiotoxicity is an important side effect limiting the clinical use of all anthracyclines, including doxorubicin (DOX). DOX targets topoisomerase II alpha (Top2α) and beta (Top2β), two enzymes that regulate DNA topology. Top2β is present in cardiomyocytes and has been proposed as a major contributor to DOX-induced cardiotoxicity. Annamycin (ANN), a novel clinically evaluated DOX analog, displays suppressed or no cardiotoxic properties in preclinical in vivo experiments. The objective of this study was to directly assess and compare the potency of DOX and ANN against Top2α and Top2β and determine their impact on physiology of human cardiomyocytes and mice hearts following chronic exposure. Our initial data are clearly aligned with lack of drug-related cardiotoxic events in ANN-treated patients in ongoing clinical trials. Methods. The potency of ANN and DOX to inhibit Top2 was tested in DNA relaxation assays using recombinant Top2α and Top2β with kinetoplast as the DNA substrate. ANN and DOX cytotoxicity was assessed in a panel of cancer cell lines cardiomyocytes (murine and human). In addition, the effects on cardiomyocyte physiology (beating rate, contraction, electric potential,) were assessed in human cardiomyocytes using the xCELLigence RTCA CardioECR. The pathophysiology of the heart after chronic exposure to the drugs was also evaluated in mice models. Cardiotoxicity of liposome formulated ANN (L-ANN) is currently being examined in ongoing clinical studies of subjects with acute myeloid leukemia (NCT05319587) or soft tissue sarcoma with metastases to the lungs (NCT04887298). Results. The nanomolar range potency of ANN in tested cancer cell lines was 2-10 times greater than that of DOX. The DNA relaxation assay demonstrated high potency of ANN against recombinant Top2α and Topβ with IC50 values 0.23±0.06 and 0.22± 0.06 µM, respectively, whereas the IC50 for DOX was 0.65± 0.22 and 0.34± 0.31µM, respectively. Importantly, ANN did not significantly affect electric potential, contractility, and viability of human cardiomyocytes up to highest tested dose of 1,500 nM (72h exposure). In contrast, DOX caused a dramatic change in heart beating rate and decreased in contractility and field potential amplitude. In rat cardiomyocyte culture (H92C), ANN was 14-fold less cytotoxic than DOX (IC50 1,264 nM for ANN vs. 88 nM for DOX). No cardiotoxicity of L-ANN was detected in myocardium of nude mice exposed weekly for 12 weeks to therapeutic doses of L-ANN (4 mg/kg, 40% of LD10). Finally, data from ongoing clinical trials revealed no drug related cardiac events in patients receiving L-ANN (confirmed by an independent review of the Duke’s ECHO core laboratory, USA). Conclusions. Our preclinical studies demonstrate non-cardiotoxic properties of ANN. These data are clearly aligned with lack of drug-related cardiotoxic events in ANN-treated patients in ongoing clinical trials. Interestingly, ANN appeared to be a significantly more potent inhibitor of Top2β than DOX, suggesting that Top2β may not be a universal mediator of cardiotoxicity of anthracyclines. Additional studies are needed to reevaluate the role of Top2β in anthracycline-induced cardiotoxicity. Citation Format: Rafal J. Zielinski, Krzysztof Grela, Roberto Cardenas-Zuniga, Stanislaw Skora, Izabela Fokt, Mihai Gagea, Wolfram Dempke, Waldemar Priebe. Non-cardiotoxic properties of annamycin, a clinically evaluated anthracycline and potent topoisomerase 2β poison [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB180.

Roles of cMyBP-C phosphorylation on cardiac contractile dysfunction in db/db mice

Type 2 diabetes mellitus (T2DM) is a metabolic disease and comorbidity associated with several conditions, including cardiac dysfunction leading to heart failure with preserved ejection fraction (HFpEF), in turn resulting in T2DM-induced cardiomyopathy (T2DM-CM). However, the molecular mechanisms underlying the development of T2DM-CM are poorly understood. It is hypothesized that molecular alterations in myopathic genes induced by diabetes promote the development of HFpEF, whereas cardiac myosin inhibitors can rescue the resultant T2DM-mediated cardiomyopathy. To test this hypothesis, a Leptin receptor-deficient db/db homozygous (Lepr db/db) mouse model was used to define the pathogenesis of T2DM-CM. Echocardiographic studies at 4 and 6 months revealed that Lepr db/db hearts started developing cardiac dysfunction by four months, and left ventricular hypertrophy with diastolic dysfunction was evident at 6 months. RNA-seq data analysis, followed by functional enrichment, revealed the differential regulation of genes related to cardiac dysfunction in Lepr db/db heart tissues. Strikingly, the level of cardiac myosin binding protein-C phosphorylation was significantly increased in Lepr db/db mouse hearts. Finally, using isolated skinned papillary muscles and freshly isolated cardiomyocytes, CAMZYOS® (mavacamten, MYK-461), a prescription heart medicine used for symptomatic obstructive hypertrophic cardiomyopathy treatment, was tested for its ability to rescue T2DM-CM. Compared with controls, MYK-461 significantly reduced force generation in papillary muscle fibers and cardiomyocyte contractility in the db/db group. This line of evidence shows that 1) T2DM-CM is associated with hyperphosphorylation of cardiac myosin binding protein-C and 2) MYK-461 significantly lessened disease progression in vitro, suggesting its promise as a treatment for HFpEF.

Cardiac lipotoxicity and fibrosis underlie impaired contractility in a mouse model of metabolic dysfunction-associated steatotic liver disease.

The leading cause of death among patients with metabolic dysfunction-associated steatotic liver disease (MASLD) is cardiovascular disease. A significant percentage of MASLD patients develop heart failure driven by functional and structural alterations in the heart. Previously, we observed cardiac dysfunction in hepatocyte-specific peroxisome proliferator-activated receptor alpha knockout (Ppara HepKO), a mouse model that exhibits hepatic steatosis independent of obesity and insulin resistance. The goal of the present study was to determine mechanisms that underlie hepatic steatosis-induced cardiac dysfunction in Ppara HepKO mice. Experiments were performed in 30-week-old Ppara HepKO and littermate control mice fed regular chow. We observed decreased cardiomyocyte contractility (0.17 ± 0.02 vs. 0.24 ± 0.02 μm, p < 0.05), increased cardiac triglyceride content (0.96 ± 0.13 vs. 0.68 ± 0.06 mM, p < 0.05), collagen type 1 (4.65 ± 0.25 vs. 0.31 ± 0.01 AU, p < 0.001), and collagen type 3 deposition (1.32 ± 0.46 vs. 0.05 ± 0.03 AU, p < 0.05). These changes were associated with increased apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling staining (30.9 ± 4.7 vs. 13.1 ± 0.8%, p < 0.006) and western blots showing increased cleaved caspase-3 (0.27 ± 0.006 vs. 0.08 ± 0.01 AU, p < 0.003) and pro-caspase-3 (5.4 ± 1.5 vs. 0.5 ± 0.3 AU, p < 0.02), B-cell lymphoma protein 2-associated X (0.68 ± 0.07 vs. 0.04 ± 0.04 AU, p < 0.001), and reduced B-cell lymphoma protein 2 (0.29 ± 0.01 vs. 1.47 ± 0.54 AU, p < 0.05). We further observed elevated circulating natriuretic peptides and exercise intolerance in Ppara HepKO mice when compared to controls. Our data demonstrated that lipotoxicity, and fibrosis underlie cardiac dysfunction in MASLD.

Inherited physical capacity: Widening divergence from young to adult to old.

Cardiorespiratory performance segregates into rat strains of inherited low- and high-capacity runners (LCRs and HCRs); during adulthood, this segregation remains stable, but widens in senescence and is followed by segregated function, health, and mortality. However, this segregation has not been investigated prior to adulthood. We, therefore, assessed cardiorespiratory performance and cardiac cell (cardiomyocyte) structure-function in 1- and 4-month-old LCRs and HCRs. Maximal oxygen uptake was 23% less in LCRs at 1-month compared to HCRs at 1-month, and 72% less at 4 months. Cardiomyocyte contractility was 37-56% decreased, and Ca2+ release was 34-62% decreased, in 1- and 4-month LCRs versus HCRs. This occurred because HCRs had improved contractility and Ca2+ release during maturation, whereas LCRs did not. In quiescent cardiomyocytes, LCRs displayed 180% and 297% more Ca2+ sparks and 91% and 38% more Ca2+ waves at 1 and 4 months versus HCRs. Cell sizes were not different between LCRs and HCRs, but LCRs showed reduced transverse-tubules versus HCRs, though no discrepant transverse-tubule generation occurred during maturation. In conclusion, LCRs show reduced scores for aerobic capacity and cardiomyocyte structure-function compared to HCRs and there is a widening divergence between LCRs and HCRs during juvenile to near-adult maturation.

AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

In heart failure, signaling downstream the β2-adrenergic receptor is critical. Sympathetic stimulation of β2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind β2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. cAMP accumulation in real time downstream of the β2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

LncRNA CHKB-DT Downregulation Enhances Dilated Cardiomyopathy Through ALDH2.

Human cardiac long noncoding RNA (lncRNA) profiles in patients with dilated cardiomyopathy (DCM) were previously analyzed, and the long noncoding RNA CHKB (choline kinase beta) divergent transcript (CHKB-DT) levels were found to be mostly downregulated in the heart. In this study, the function of CHKB-DT in DCM was determined. Long noncoding RNA expression levels in the human heart tissues were measured via quantitative reverse transcription-polymerase chain reaction and in situ hybridization assays. A CHKB-DT heterozygous or homozygous knockout mouse model was generated using the clustered regularly interspaced palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, and the adeno-associated virus with a cardiac-specific promoter was used to deliver the RNA in vivo. Sarcomere shortening was performed to assess the primary cardiomyocyte contractility. The Seahorse XF cell mitochondrial stress test was performed to determine the energy metabolism and ATP production. Furthermore, the underlying mechanisms were explored using quantitative proteomics, ribosome profiling, RNA antisense purification assays, mass spectrometry, RNA pull-down, luciferase assay, RNA-fluorescence in situ hybridization, and Western blotting. CHKB-DT levels were remarkably decreased in patients with DCM and mice with transverse aortic constriction-induced heart failure. Heterozygous knockout of CHKB-DT in cardiomyocytes caused cardiac dilation and dysfunction and reduced the contractility of primary cardiomyocytes. Moreover, CHKB-DT heterozygous knockout impaired mitochondrial function and decreased ATP production as well as cardiac energy metabolism. Mechanistically, ALDH2 (aldehyde dehydrogenase 2) was a direct target of CHKB-DT. CHKB-DT physically interacted with the mRNA of ALDH2 and fused in sarcoma (FUS) through the GGUG motif. CHKB-DT knockdown aggravated ALDH2 mRNA degradation and 4-HNE (4-hydroxy-2-nonenal) production, whereas overexpression of CHKB-DT reversed these molecular changes. Furthermore, restoring ALDH2 expression in CHKB-DT+/- mice alleviated cardiac dilation and dysfunction. CHKB-DT is significantly downregulated in DCM. CHKB-DT acts as an energy metabolism-associated long noncoding RNA and represents a promising therapeutic target against DCM.

ZBTB20 Regulates SERCA2a Activity and Myocardial Contractility Through Phospholamban.

Intracellular Ca2+ cycling determines myocardial contraction and relaxation in response to physiological demands. SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a) is responsible for the sequestration of cytosolic Ca2+ into intracellular stores during cardiac relaxation, and its activity is reversibly inhibited by PLN (phospholamban). However, the regulatory hierarchy of SERCA2a activity remains unclear. Cardiomyocyte-specific ZBTB20 knockout mice were generated by crossing ZBTB20flox mice with Myh6-Cre mice. Echocardiography, blood pressure measurements, Langendorff perfusion, histological analysis and immunohistochemistry, quantitative reverse transcription-PCR, Western blot analysis, electrophysiological measurements, and chromatin immunoprecipitation assay were performed to clarify the phenotype and elucidate the molecular mechanisms. Specific ablation of ZBTB20 in cardiomyocyte led to a significant increase in basal myocardial contractile parameters both in vivo and in vitro, accompanied by an impairment in cardiac reserve and exercise capacity. Moreover, the cardiomyocytes lacking ZBTB20 showed an increase in sarcoplasmic reticular Ca2+ content and exhibited a remarkable enhancement in both SERCA2a activity and electrically stimulated contraction. Mechanistically, PLN expression was dramatically reduced in cardiomyocytes at the mRNA and protein levels by ZBTB20 deletion or silencing, and PLN overexpression could largely restore the basal contractility in ZBTB20-deficient cardiomyocytes. These data point to ZBTB20 as a fine-tuning modulator of PLN expression and SERCA2a activity, thereby offering new perspective on the regulation of basal contractility in the mammalian heart.

PPARγ Agonist Pioglitazone Prevents Hypoxia-induced Cardiac Dysfunction by Reprogramming Glucose Metabolism.

The heart relies on various defense mechanisms, including metabolic plasticity, to maintain its normal structure and function under high-altitude hypoxia. Pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ), sensitizes insulin, which in turn regulates blood glucose levels. However, its preventive effects against hypoxia-induced cardiac dysfunction at high altitudes have not been reported. In this study, pioglitazone effectively prevented cardiac dysfunction in hypoxic mice for 4 weeks, independent of its effects on insulin sensitivity. In vitro experiments demonstrated that pioglitazone enhanced the contractility of primary cardiomyocytes and reduced the risk of QT interval prolongation under hypoxic conditions. Additionally, pioglitazone promoted cardiac glucose metabolic reprogramming by increasing glycolytic capacity; enhancing glucose oxidation, electron transfer, and oxidative phosphorylation processes; and reducing mitochondrial reactive ROS production, which ultimately maintained mitochondrial membrane potential and ATP production in cardiomyocytes under hypoxic conditions. Notably, as a PPARγ agonist, pioglitazone promoted hypoxia-inducible factor 1α (HIF-1α) expression in hypoxic myocardium. Moreover, KC7F2, a HIF-1α inhibitor, disrupted the reprogramming of cardiac glucose metabolism and reduced cardiac function in pioglitazone-treated mice under hypoxic conditions. In conclusion, pioglitazone effectively prevented high-altitude hypoxia-induced cardiac dysfunction by reprogramming cardiac glucose metabolism.

Noninvasive Quantification of Contractile Dynamics in Cardiac Cells, Spheroids, and Organs-on-a-Chip Using High-Frequency Ultrasound.

Cell-based models that mimic in vivo heart physiology are poised to make significant advances in cardiac disease modeling and drug discovery. In these systems, cardiomyocyte (CM) contractility is an important functional metric, but current measurement methods are inaccurate and low-throughput or require complex setups. To address this need, we developed a standalone noninvasive, label-free ultrasound technique operating at 40-200 MHz to measure the contractile kinetics of cardiac models, ranging from single adult CMs to 3D microtissue constructs in standard cell culture formats. The high temporal resolution of 1000 fps resolved the beat profile of single mouse CMs paced at up to 9 Hz, revealing limitations of lower speed optical based measurements to resolve beat kinetics or characterize aberrant beats. Coupling of ultrasound with traction force microscopy enabled the measurement of the CM longitudinal modulus and facile estimation of adult mouse CM contractile forces of 2.34 ± 1.40 μN, comparable to more complex measurement techniques. Similarly, the beat rate, rhythm, and drug responses of CM spheroid and microtissue models were measured, including in configurations without optical access. In conclusion, ultrasound can be used for the rapid characterization of CM contractile function in a wide range of commonly studied configurations ranging from single cells to 3D tissue constructs using standard well plates and custom microdevices, with applications in cardiac drug discovery and cardiotoxicity evaluation.