Myocyte Contractility Research Articles

PDE1 Inhibition Modulates Cav1.2 Channel to Stimulate Cardiomyocyte Contraction.

[Figure: see text].

Abstract P386: Supplementation Of Tyrode With Fatty Acids And 3-hydroxybutyrate Improves Adult Cardiomyocytes Contractility Of Failing Human Hearts.

Due to its high energy consumption and limited ability to store ATP, the heart is highly dependent of exogenous metabolic substrates. Prior in vivo studies have reported that the development of heart failure is accompanied by a transition from the normal preferential metabolism of free fatty acids (FFA) to increases in glucose utilization and even ketone bodies, which normally provide a modest contribution to energy balance. However, the functional significance of the upregulated ketone metabolism in the failing heart is poorly understood. Recognizing that nearly all prior studies examining isolated cardiomyocyte physiology have used glucose as the sole metabolic substrate, we initiated studies to examine the impact of alternative metabolic substrates on contractility in isolated human cardiomyocytes. To understand the role of substrate alteration cardiomyocyte functionalities, we employed freshly isolated adult human left ventricular cardiomyocytes from 11 non-failing hearts (NF) obtained from organ donors and 13 failing hearts (HF) obtained from transplant recipients. Cardiomyocytes were resuspended in a conventional 5mM Glucose Tyrode solution with alternative substrates (Glucose, FFA, R-3-OHB or Mix (Glucose + FFA + 3-OHB)). Myocytes were field stimulated at 1 Hz and sarcomere length, fractional shortening, contraction velocity and relaxation velocity were measured using a video-based sarcomere length detection system (IonOptix Corp). Studies using isolated cardiac myocyte contractility as readout confirm that myocytes from NF human hearts are omnivorous: high levels of myocyte fractional shortening (FS) can be achieved under unstressed conditions (1 Hz, unloaded) with any substrate (FS Glucose : 0.1315±0.012; FS FFA : 0.1428±0.0127; FS 3OHB : 0.1343±0.014; FS MIX : 0.15467±0.02). In the failing heart, glucose alone is insufficient to produce normal unstressed myocyte fractional shortening (FS Glucose : 0.088±0.009***, p<0.001 compare to NF). However, in failing myocytes, supplementation of physiological levels of glucose with FFA or ketones each enhances myocyte contractility and rates of shortening and re-lengthening (FS FFA : 0.109±0.0127; FS 3OHB : 0.107±0.012; FS MIX : 0.112±0.016). These results suggest that future comparisons of NF vs. HF human myocyte contractility should include conditions with a physiological mix of metabolic substrates.

Effects of omecamtiv mecarbil on calcium-transients and contractility in a translational canine myocyte model

Omecamtiv mecarbil (OM) is a selective cardiac myosin activator (myotrope), currently in Phase 3 clinical investigation as a novel treatment for heart failure with reduced ejection fraction. OM increases cardiac contractility by enhancing interaction between myosin and actin in a calcium-independent fashion. This study aims to characterize the mechanism of action by evaluating its simultaneous effect on myocyte contractility and calcium-transients (CTs) in healthy canine ventricular myocytes. Left ventricular myocytes were isolated from canines and loaded with Fura-2 AM. With an IonOptix system, contractility parameters including amplitude and duration of sarcomere shortening, contraction and relaxation velocity, and resting sarcomere length were measured. CT parameters including amplitude at systole and diastole, velocity at systole and diastole, and duration at 50% from peak were simultaneously measured. OM was tested at 0.03, 0.1, 0.3, 1, and 3 µmol\L concentrations to simulate therapeutic human plasma exposure levels. OM and isoproterenol (ISO) demonstrated differential effects on CTs and myocyte contractility. OM increased contractility mainly by prolonging duration of contraction while ISO increased contractility mainly by augmenting the amplitude of contraction. ISO increased the amplitude and velocity of CT, shortened duration of CT concurrent with increasing myocyte contraction, while OM did not change the amplitude, velocity, and duration of CT up to 1 µmol\L. Decreases in relaxation velocity and increases in duration were present only at 3 µmol\L. In this translational myocyte model study, therapeutically relevant concentrations of OM increased contractility but did not alter intracellular CTs, a mechanism of action distinct from traditional calcitropes.

Secondhand Smoke Exposure Impairs Ion Channel Function and Contractility of Mesenteric Arteries.

Cigarette smoke, including secondhand smoke (SHS), has significant detrimental vascular effects, but its effects on myogenic tone of small resistance arteries and the underlying mechanisms are understudied. Although it is apparent that SHS contributes to endothelial dysfunction, much less is known about how this toxicant alters arterial myocyte contraction, leading to alterations in myogenic tone. The study's goal is to determine the effects of SHS on mesenteric arterial myocyte contractility and excitability. C57BL/6J male mice were randomly assigned to either filtered air (FA) or SHS (6 h/d, 5 d/wk) exposed groups for a 4, 8, or 12-weeks period. Third and fourth-order mesenteric arteries and arterial myocytes were acutely isolated and evaluated with pressure myography and patch clamp electrophysiology, respectively. Myogenic tone was found to be elevated in mesenteric arteries from mice exposed to SHS for 12 wk but not for 4 or 8 wk. These results were correlated with an increase in L-type Ca2+ channel activity in mesenteric arterial myocytes after 12 wk of SHS exposure. Moreover, 12 wk SHS exposed arterial myocytes have reduced total potassium channel current density, which correlates with a depolarized membrane potential (Vm). These results suggest that SHS exposure induces alterations in key ionic conductances that modulate arterial myocyte contractility and myogenic tone. Thus, chronic exposure to an environmentally relevant concentration of SHS impairs mesenteric arterial myocyte electrophysiology and myogenic tone, which may contribute to increased blood pressure and risks of developing vascular complications due to passive exposure to cigarette smoke.

Cardioprotective mechanisms of mitochondria-targeted S-nitrosating agent and adenosine triphosphate-sensitive potassium channel opener are mutually exclusive

BackgroundMyocytes exposed to stress exhibit significant swelling and reduced contractility. These consequences are ameliorated by adenosine triphosphate–sensitive potassium (KATP) channel opener diazoxide (DZX) via an unknown mechanism. KATP channel openers also provide cardioprotection in multiple animal models. Nitric oxide donors are similarly cardioprotective, and their combination with KATP activation may provide synergistic benefit. We hypothesized that mitochondria-targeted S-nitrosating agent (MitoSNO) would provide synergistic cardioprotection with DZX. MethodsMyocyte volume and contractility were compared following Tyrode's physiologic solution (20 minutes) and stress (hyperkalemic cardioplegia [CPG] ± DZX; n = 5-20 each; 20 minutes) with or without MitoSNO (n = 5-11 each) at the end of stress, followed by Tyrode's solution (20 minutes). Isolated mouse hearts received CPG ± DZX (n = 8-10 each) before global ischemia (90 minutes) with or without MitoSNO (n = 8 each) at the end of ischemia, followed by reperfusion (30 minutes). Left ventricular (LV) pressures were compared using a linear mixed model to assess the impact of treatment on the outcome, adjusting for baseline and balloon volume. ResultsStress (CPG) was associated with reduced myocyte contractility that was prevented by DZX and MitoSNO individually; however, their combination was associated with loss of cardioprotection. Similarly, DZX and MitoSNO improved LV function after prolonged ischemia compared with CPG alone, and cardioprotection was lost with their combination. ConclusionsMitoSNO and DZX provide cardioprotection that is lost with their combination, suggesting mutually exclusive mechanisms of action. The lack of a synergistic beneficial effect informs the current knowledge of the cardioprotective mechanisms of DZX and will aid planning of future clinical trials.

B-PO05-213 REVERSAL OF HYPOCALCEMIA-INDUCED DILATED CARDIOMYOPATHY TREATED WITH CRT-D

Hypocalcemia is a rare cause of dilated cardiomyopathy (DCM). Pathophysiology involves decreased myocyte contractility and prolongation of QTc interval. CRT-D is a proven therapy in patients with DCM. We present a novel case of hypocalcemic DCM and syncope that was refractory to GDM, including calcium replacement, that responded to CRT. N/A A 52-year-old woman with DCM and chronic hypocalcemia secondary to hypoparathyroidism presented for biventricular ICD placement. Hypocalcemia began after hemithyroidectomy. She was first diagnosed with heart failure after 10 years of hypocalcemia (mean 6.6 +/- .92 mg/dL). ECG was sinus rhythm with left bundle branch block (QRS 146 ms). TTE showed LVEF of 20% and 4-chamber enlargement. Cardiac catheterization did not show coronary artery disease. After 3 months of GDM and correction of calcium, she continued to have NYHA class III symptoms without change in EF. She was hospitalized for syncope concerning for malignant arrhythmia. QTc was 524 ms (JTc = 378 ms). Cardiac MR did not show evidence of fibrosis or scar. She had class 1b indication for CRT due to LVEF <35% and LBBB and underwent successful placement of Biotronik BiV ICD with LV lead placed into posterolateral branch. At 3-month follow-up she had 100% biventricular pacing. Her LVEF increased from 20% to 35%, LVIDd and LVIDs decreased by 15% (6.76 to 5.73 cc) and 24% (6.10 to 4.64 cc). After prolonged correction of calcium, QTc normalized (474 ms). CRT-D therapy has multiple indications in hypocalcemia-induced DCM, including improvement of LV size and function and prevention of fatal arrhythmias related to QTc prolongation.

Reduction of P62/SQSTM1 on Calcium Handling in Stressed Myocytes

Introduction Cytosolic protein, p62/SQSTM1, regulates the protein turnover rate, and it interacts with various stress signaling proteins in cells. Accumulations of p62/SQSTM1 have been reported in hearts with cardiomyopathy. We recently reported that p62/SQSTM1 protein deletions reduced beta-adrenergic response in myocytes and improved chronic ischemia-induced Calcium (Ca) defect. Here, we studied acute and chronic stress-induced Ca handling in myocytes genetically suppressed p62/SQSTM1 protein. Methods Myocytes (n=3 per group) isolated from Wild-type (WT), and p62/SQSTM1+/- (p62 HET) at Base, 5 days, and 10 days after Isoproterenol (ISO; beta-agonist) injections (150 mg/kg). We measured myocytes contractility (Fraction shortening of sarcomeres), systolic Ca release (CaT), SR Ca contents (CaffT), and Fractional Ca release, (CaT/CaffT) in field-stimulated single myocytes. Myocytes loaded with fluorescent Ca indicators (Fura-2AM; cytosolic Ca loading). Heart tissues in each group were processed for western blot (n=3 per group). Results The baseline myocytes contractility and Ca regulation were not different among the groups. P62 HET myocytes significantly increased contractility and fractional Ca release (the function of SR, F/F0; WT base, 0.58±0.07, WT ISO, 0.84±0.01, p62 HET ISO, 0.79±0.04) after acute ISO stimulation (1 uM, p<0.05), similar to WT myocytes. Interestingly, p62 HET myocytes preserved SR Ca contents, whereas WT myocytes increased SR Ca contents. Chronic ischemia-induced p62 HET myocytes improved fractional Ca release; however, both systolic Ca release (F/F0; WT base, 0.24±0.03, WT ISO 10days, 0.29±0.06, p62 HET ISO 10 days, 0.17±0.02) and SR Ca contents (F/F0; WT base, 0.43±0.05, WT ISO, 0.49±0.10, p62 HET ISO, 0.33±0.04) were proportionally reduced (p<0.05). Conclusion Taken together, suppression of p62/SQSTM1 protein restores stress-induced Ca dysregulation in myocytes. p62/SQSTM1 protein may directly contribute to systolic Ca release via beta-adrenergic signaling.

Micro RNA Expression of Heart Failure Patients and Responses to Left Ventricular Assistant Device Support

Purpose Micro RNAs are key regulators of various biological processes including the pathogenesis of heart failure (HF). In this study we compared miRNA profiles of heart failure (HF), left ventricular assist devices (LVAD) and non-failing (NF) hearts patients. Our goal was to identify epigenetic mechanisms involved in HF and responses to LVAD therapy. Methods RNAs were isolated from myocardial tissues of HF patients without mechanical support at the time of transplant (n=6) and after LVAD (n=6) placement in non-ischemic dilated cardiomyopathy patients along with NF controls (n = 6). NanoString analysis was used to compare miRNA expression among in HF, LVAD, and control patients. NanoString miRNA raw count data were preprocessed and normalized using nSolver 4.0 Analysis Software. Ingenuity Pathway Analysis (IPA) was carried out on differentially expressed micro RNA to understand the biological pathways involved. Results Our sample population was 84% male with an average age of 57 and 82% Caucasian, 16% African American and 2% Asian. Forty four miRNAs were differentially expressed in HF patients than NF control and 79 miRNAs were differentially expressed in HF patients when compared to post-LVAD HF patients. IPA analysis revealed that heart enlargement was significantly altered in HF compared to NF control with p value of 2.58E-04. Three miRNA were upregulated (mir590-5p, mir146a-5p and mir-761-5p) and three were downregulated by more than two folds (mir-1, mir-23a-3p and mir497-5). Heart enlargement was also significantly different in pre vs post with p value of 2.57E-02. Five miRNAs were involved with four upregulated (mir-590-5p, mir146a-5p, mir-761 and mir 140-3p) while only one was downregulated (mir-497-5p). Conclusion End stage heart failure is characterized by cardiac remodeling, arrhythmias, fibrosis and a decline in myocyte contractility. Our results identify a large number of miRNAs that are associated with end stage heart failure and cardiac enlargement in addition to the miRNAs associated with remodeling on LVAD therapy. Micro RNA profiles can identify molecular mechanisms of disease and serve as potential biomarkers or therapeutic targets.

Rosuvastatin Reduces L-Type Ca2+ Current and Alters Contractile Function in Cardiac Myocytes via Modulation of β-Adrenergic Receptor Signaling.

Rosuvastatin is one of the most used statins to lower plasma cholesterol levels. Although previous studies have reported remarkable cardiovascular effects of rosuvastatin (RSV), the mechanisms of these effects are largely unknown. In this study, we investigated the acute effects of RSV on L-type Ca2+ currents and contractile function of ventricular myocytes under basal conditions and during β-adrenergic stimulation. The effects of RSV were investigated in freshly isolated adult rat ventricular myocytes. L-type Ca+2 currents and myocyte contractility were recorded using patch-clamp amplifier and sarcomere length detection system. All experimental recordings were performed at 36 ± 1°C. L-type Ca+2 currents were significantly reduced with the administration of 1μM RSV (~ 24%) and this reduction in Ca2+ currents was observed at almost all potential ranges applied. Suppression of L-type Ca2+ current by RSV was prevented by adenylyl cyclase (AC) and protein kinase A (PKA) inhibitors SQ 22536 and KT5720, respectively. However, inhibition of Rho-associated kinases (ROCKs) by Y-27632 or nitric oxide synthase (NOS) by L-NAME failed to circumvent the inhibitory effect of RSV. Finally, we examined the effect of RSV during β-adrenergic receptor stimulation by isoproterenol and observed that RSV significantly suppresses the β-adrenergic responses in both L-type Ca2+ currents and contraction parameters. In conclusion, RSV modulates the β-adrenergic signaling cascade and thereby mimics the impact of β-adrenergic receptor blockers in adult ventricular myocytes through modulation of the AC-cAMP-PKA pathway.

Effects of aerobic exercise training and açai supplementation on cardiac structure and function in rats submitted to a high-fat diet

This study evaluated the effect of aerobic exercise training (AET) and supplementation with açai on cardiac structure and function in rats submitted to a high-fat diet. Two-month old Fischer male rats were divided into 5 groups: Control (C), High-fat Diet (H), High-fat Diet + Açai (HA), High-fat Diet + AET (HT), High-fat Diet + Açai + AET (HAT). The high-fat diet had 21.8% lard and 1% cholesterol (H and HT), or supplemented with 1% lyophilized açai pulp (HA and HAT). The HT and HAT groups performed AET on a treadmill (5 days/week, 1 h/day, 60% of the maximum running speed) for 8 weeks. Exercise tolerance test were performed, and adiposity index calculated. After euthanasia, the left ventricle (LV) was dissected and processed for histological, single myocyte intracellular calcium ([Ca2+]i) transient and contractility, oxidative stress and gene expression analysis. AET improved running capacity and reduced the adiposity index. Both AET and açai supplementation inhibited the increase in the LV collagen content, the deleterious effects on the [Ca2+]i transient and contractility in cardiomyocytes and the increment in oxidative stress, caused by the consumption of a high-fat diet. Aerobic exercise training and açai supplementation can mitigate damage caused by high-fat diet in cardiac structure and function, though the combination of treatments had no additional effects.

Regulating Quantal Size of Neurotransmitter Release through a GPCR Voltage Sensor

Current models emphasize that membrane voltage (Vm) depolarization-induced Ca2+ influx triggers the fusion of vesicles to the plasma membrane. In sympathetic adrenal chromaffin cells, activation of a variety of G-protein coupled receptors (GPCRs) can inhibit quantal size (QS) through the direct interaction of G-protein Giβγ subunits with exocytosis fusion proteins. Here we report that, independently from Ca2+, Vm (action potential) per se regulates the amount of catecholamine released from each vesicle, the QS. The Vm regulation of QS was through ATP-activated GPCR-P2Y12 receptors. D76 and D127 in P2Y12 were the voltage-sensing sites. Finally, we revealed the relevance of the Vm-dependence of QS for tuning auto-inhibition and target cell functions. Together, membrane voltage per se increases the quantal size of dense-core vesicle release of catecholamine via Vm P2Y12(D76/D127) Giβγ QS myocyte contractility, offering an universal Vm-GPCR-signaling pathway for their functions in the nervous system and other systems containing GPCRs. Supported by grants from NSFC, MOST and the CLS-program.

Ophiopogonin D alleviates diabetic myocardial injuries by regulating mitochondrial dynamics

Ethnopharmacological relevanceOphiopogonin D (OP-D) is a steroidal saponin extracted from Ophiopogon japonicus (Thunb.) Ker Gawl. (Liliaceae), that has been traditionally used to treat cough, sputum, and thirst in some Asian countries. Recently, various pharmacological roles of OP-D have been identified, including anti-inflammatory, cardioprotective, and anti-cancer effects. However, whether OP-D can prevent diabetic myocardial injury remains unknown. Aim of the studyIn this study, we aimed to observe the effects of OP-D on the diabetic myocardium. Materials and methodsLeptin receptor-deficient db/db mice were used as an animal model for type 2 diabetes. The effects of OP-D on blood glucose, blood lipids, myocardial ultrastructure, and mitochondrial function in mice were observed after four weeks of intragastric administration. Palmitic acid was used to stimulate cardiomyocytes to establish a myocardial lipotoxicity model. Cell apoptosis, mitochondrial morphology, and function were observed. ResultsBlood glucose and blood lipid levels were significantly increased in db/db mice, accompanied by myocardial mitochondrial injury and dysfunction. OP-D treatment reduced blood lipid levels in db/db mice and relieved mitochondrial injury and dysfunction. OP-D inhibited palmitic acid induced-mitochondrial fission and dysfunction, reduced endogenous apoptosis, and improved cell survival rate in H9C2 cardiomyocytes. Both in vivo and in vitro models showed increased phosphorylation of DRP1 at Ser-616, reduced phosphorylation of DRP1 at Ser-637, and reduced expression of fusion proteins MFN1/2 and OPA1. Meanwhile, immunofluorescence co-localization analysis revealed that palmitic acid stimulated the translocation of DRP1 protein from the cytoplasm to the mitochondria in H9C2 cardiomyocytes. The imbalance of mitochondrial dynamics, protein expression, and translocation of DRP1 were effectively reversed by OP-D treatment. In isolated mice ventricular myocytes, palmitic acid enhanced cytoplasmic Ca2+ levels and suppressed contractility in ventricular myocytes, accompanied by activation of calcineurin, a key regulator of DRP1 dephosphorylation at Ser-637. OP-D reversed the changes caused by palmitic acid. ConclusionsOur findings indicate that OP-D intervention could alleviate lipid accumulation and mitochondrial injury in diabetic mouse hearts and palmitic acid-stimulated cardiomyocytes. The cardioprotective effect of OP-D may be mediated by the regulation of mitochondrial dynamics.

The transmembrane peptide DWORF activates SERCA2a via dual mechanisms

The Ca-ATPase isoform 2a (SERCA2a) pumps cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. Abnormally high cytosolic [Ca2+] is a central factor in heart failure, suggesting that augmentation of SERCA2a Ca2+ transport activity could be a promising therapeutic approach. SERCA2a is inhibited by the protein phospholamban (PLB), and a novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing PLB from binding to SERCA2a. However, establishing DWORF’s precise physiological role requires further investigation. In the present study, we developed cell-based FRET biosensor systems that can report on protein–protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the SERCA2a-binding site, we transiently transfected DWORF into a stable HEK cell line expressing SERCA2a labeled with a FRET donor and PLB labeled with a FRET acceptor. We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Surprisingly, we also found that DWORF also activates SERCA’s enzymatic activity directly in the absence of PLB at subsaturating calcium levels. Using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues P15 and W22 as necessary for functional SERCA2a–DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.

Giant ankyrin-G regulates cardiac function

Cardiovascular disease (CVD) remains the most common cause of adult morbidity and mortality in developed nations. As a result, predisposition for CVD is increasingly important to understand. Ankyrins are intracellular proteins required for the maintenance of membrane domains. Canonical ankyrin-G (AnkG) has been shown to be vital for normal cardiac function, specifically cardiac excitability, via targeting and regulation of the cardiac voltage-gated sodium channel. Noncanonical (giant) AnkG isoforms play a key role in neuronal membrane biogenesis and excitability, with evidence for human neurologic disease when aberrant. However, the role of giant AnkG in cardiovascular tissue has yet to be explored. Here, we identify giant AnkG in the myocardium and identify that it is enriched in 1-week-old mice. Using a new mouse model lacking giant AnkG expression in myocytes, we identify that young mice displayed a dilated cardiomyopathy phenotype with aberrant electrical conduction and enhanced arrhythmogenicity. Structural and electrical dysfunction occurred at 1 week of age, when giant AnkG was highly expressed and did not appreciably change in adulthood until advanced age. At a cellular level, loss of giant AnkG results in delayed and early afterdepolarizations. However, surprisingly, giant AnkG cKO myocytes display normal INa, but abnormal myocyte contractility, suggesting unique roles of the large isoform in the heart. Finally, transcript analysis provided evidence for unique pathways that may contribute to the structural and electrical findings shown in giant AnkG cKO animals. In summary, we identify a critical role for giant AnkG that adds to the diversity of ankyrin function in the heart.

Abstract 12677: Impaired Cardiomyocyte Contractility to Angiotensin-(1-12) in a Humanized Angiotensinogen Model of Hypertension

Background: Angiotensin-(1-12) [Ang-(1-12)] is a chymase-dependent source for Angiotensin II (Ang II) inotropic activity that may be impaired in a model of sustained hypertension with high cardiac Ang II content due to insertion of the human angiotensinogen (AGT) gene in the rat genome. Accordingly, we evaluated the effects of Ang-(1-12) and Ang II on myocyte contractility and [Ca 2+ ] i regulation in 9 adult male transgenic rats expressing the human AGT gene [TGR(hAGT)L1623)] and 9 SD controls. Methods: We compared LV myocyte contraction, relaxation and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to Ang-(1-12) (4x10 -6 M) and Ang II (10 -6 M) in freshly isolated LV myocytes. Results: Myocyte contraction (dL/dt max , 109.6 vs 127.9 μm/s), relaxation (dR/dt max , 95.3 vs 107.5 μm/s) and [Ca 2+ ] iT (0.15 vs 0.24) were depressed in TGR(hAGT)L1623 rats compared to SD controls. Moreover, cell contractile and [Ca 2+ ] iT responses following exposure to Ang-(1-12) or Ang II were markedly blunted. In SD myocytes, versus baseline, Ang II or Ang-(1-12) superfusion produced significant increases in dL/dt max [Ang II: 44% vs Ang-(1-12): 34%], dR/dt max (33% vs 26%) and [Ca 2+ ] iT (31% vs 25%). Importantly, the magnitude of the responses to the two agents in TGR(hAGT)L1623 myocytes was significantly reduced. Versus the changes in SD myocytes, Ang-(1-12) caused significantly less increases in dL/dt max (22%), dR/dt max (16%) and [Ca 2+ ] iT (15%) in TGR(hAGT)L1623 myocytes . Ang II also caused similar significantly attenuated increases in dL/dt max (27%), dR/dt max (25%) and [Ca 2+ ] iT (23%). The Ang-(1-12)-induced inotropic effects were completely prevented in the presence of the inhibitory cAMP analog, Rp-cAMPS (10 –4 M, 2 hours) in both SD and TGR(hAGT)L1623 myocytes, but were further augmented only intransgenic rats after incubation of myocytes with the G i inhibitor, pertussis toxin (PTX, 2 μg/ml, 36°C, 5 hours). Conclusions: Ang-(1-12) stimulates LV myocyte contractile function and [Ca 2+ ] iT in both SD and TGR(hAGT)L1623 rats. Furthermore, we now show that the blunted inotropic responses to Ang-(1-12) and Ang II in rats expressing the human AGT gene is mediated through a cAMP-dependent mechanism that is coupled to both stimulatory G and inhibitory PTX-sensitive G proteins.

Abstract 12808: Mechanism of Chronic Ranolazine Caused Regression of Cardiac Dysfunction in a Murine of Diabetic Cardiomyopathy: Beneficial Effects on Cardiomyocyte Contractile Function, [Ca 2+ ] i Regulation and Beta-Adrenergic Modulation

Background: Diabetic cardiomyopathy (DCM) increases the risk of heart failure. As yet, no effective therapeutic strategies exist. Recent evidence indicates that intracellular Na + concentration ([Na + ] i ) is augmented in the myocytes from diabetic hearts, where it causes oxidative stress, augments the sarcoplasmic reticulum Ca 2+ leak and contributes to electrical, structural and functional remodeling. Ranolazine (RAN), inhibiting persistent or late inward Na + current has been proposed to be a therapeutic choice for DCM. However, the role and mechanism of chronic RAN in DCM are unclear. We assessed the hypothesis that RAN improves myocyte function, [Ca 2+ ] i regulation, and β-adrenergic receptor (AR) signaling effectiveness, thus limiting DCM. Methods: We compared LV myocyte function, [Ca 2+ ] i transient ([Ca 2+ ] iT ) and responses to the stimulation of β-AR in 3 groups wild-type (WT) female mice over 10 weeks (W):1) DM (n=8), 10 W after receiving streptozotocin (STZ, 200 mg/kg, ip); 2) DM/RAN (n=6), 6 W after STZ, RAN (10 -5 M/kg/day, mini-pump) was initiated and was given for 4 W; and 3) Sham controls (C) (n=8). Results: Versus control, STZ-treated WT mice had DM with significantly elevated blood glucose levels (410 vs 128mg/dl) followed by LV myocyte dysfunction with decreases in myocyte contractility (dL/dt max ) (75.0 vs 140.1 μm/s), relengthening (dR/dt max ) (62.5 vs 116.6 μm/s) and [Ca 2+ ] iT (0.15 vs 0.22). In DM myocytes, the ability of β-AR agonist, isoproterenol (ISO, 10 -8 M) to increase cell contractility was blunted. Versus control, in DM myocytes, ISO-induced increases in dL/dt max (31% vs 60%), dR/dt max (23% vs 50%) and [Ca 2+ ] iT (15% vs 30%) were significantly reduced. By contrary, versus DM alone, DM/RAN myocytes showed normal basal cell contraction (137.8 μm/s), relaxation (117.2 μm/s) and [Ca 2+ ] iT (0.22) with preserved ISO-stimulated positive inotropic effect. Compared control, in DM/RAN, ISO caused similar increases in dL/dt max (62% vs 60%), dR/dt max (52% vs 50%) and [Ca 2+ ] iT (32% vs 30%). Conclusion: Chronic ranolazine leads to the preservation of myocyte function, [Ca 2+ ] iT and β-AR responsiveness in DCM. Thus, antagonizing myocyte [Na + ] i dysregulation might provide a new therapeutic strategy for DM-related decline in myocardial function.

Abstract 13050: Acute Alcohol-Induced Enhanced Cardiac Depression in a Canine Model of Chronic Alcohol Consumption: Adverse Effects on Left Ventricle and Myocyte Contraction, [Ca 2+ ] i Transient and Ca 2+ Current

Background: Acute alcohol ingestion produces transient RAS activation. Alcoholics have an enhanced RAS activation after acute ethanol ingestion. Thus, chronic alcohol users may have enhanced cardiac functional responses to acute alcohol intake. However, it is unclear whether and how chronic alcohol intake alters cardiac response to acute alcohol exposure. We tested the hypothesis that acute alcohol may exacerbate cardiac depression and [Ca 2- ] i dysregulation, thus play important role in development of irreversible cardiomyopathy in alcoholics. Methods: We compared LV and cardiomyocyte response to acute alcohol in 6 chronically-instrumented conscious dogs before and 6 months after the once daily ingestion of alcohol (400 ml, 22% providing 33% of total daily caloric intake). Results: In conscious dogs, 6 months of alcohol significantly decreased LV contractility by 48% measured by the slopes of pressure-volume relations (E ES , 48%, 4.4 vs 8.4 mmHg/ml and M SW , 50.8 vs 98.6 mmHg) and increased the time constant of relaxation (τ, 78%, 47.8 vs 26.9 ms). In the alcoholic animals, when compared with the same animals prior to alcohol, acute alcohol (0.2 g/kg, iv, plasma level 62.3 ± 8 mg/dL) caused a greater decrease in LV contractility (47% vs 23%) and increase in τ (27% vs11%). In isolated myocytes, abrupt exposure to alcohol (250 mM) produced significant decreases in cell contraction, [Ca 2+ ] i transient ([Ca 2+ ] i ) and Ca 2+ current (I Ca,L ) in both normal and alcoholic myocytes. However, compared with the isolated cells obtained from LV biopsied tissues of the same animals prior to alcohol, the acute alcohol-induced decrease in I Ca,L (alcoholic: 50%, 1.3 vs 2.6; normal: 29%, 4.0 vs 5.6 pA/pF) was much greater in the alcoholic myocytes, the resulting reductions in the cell contractility, dL/dtmax (alcoholic: 46%, 36vs67; normal: 24%, 82vs108 μm/s), the percent shortening (49% vs 24%) and relengthening, dR/dtmax were doubled. Conclusion: In chronically alcohol-fed dogs, acute alcohol produces increased direct inhibition in LV and myocyte contractility, relaxation and exacerbates [Ca 2+ ] i hemostasis. The chronic alcohol-induced increased cardiac sensitivity to acute alcohol may play an important role in the functional impairment in alcoholic cardiomyopathy.

Abstract 13045: Chronic Beta3-Adrenergic Receptor Antagonist Therapy Rescues Diabetic Cardiomyopathy in a Mouse Model With Type 2 Diabetes: Insights into Cellular Mechanism

Background: Diabetic cardiomyopathy (DCM) leads to progressive decline in cardiac function, increasing the risk for heart failure. There are no known effective prevention approaches or therapeutic strategies. Recent evidence in diabetes mellitus (DM) human and animal hearts suggests that the up-regulation of β 3 -adrenergic receptor (AR)-mediated inhibitory pathway may be responsible for the progression of DCM. However, its precise role is still being debated. We hypothesize that β 3 -AR antagonists (β 3 -ANT) may rescue the detrimental effects of β 3 -AR activation, improve cardiomyocyte function, and preserve normal β-AR regulation, leading to the regression of DCM. Methods: We compared LV myocyte function, [Ca 2+ ] i transient ([Ca 2+ ] iT ) at baseline and responses to β-AR simulation with isoproterenol (ISO,10 -8 M) in 3 groups wild-type female mice over 14 weeks (W):1) Type 2 DM ( T2 ) (n=9), 14 W fed high-fat diet (HFD), but after HFD for 4 W receiving streptozotocin (STZ, 40 mg/kg/day, i.p. 5 days); 2) T2/β 3 -ANT (n=7), T2 mice at 10 W received L-748,337, a selective β 3 -ANT (10 -7 M/kg/day, mini-pump) for 4 W; and 3) Vehicle controls (C) (n=9). Results: Versus C, T2DM was induced in mice received HFD and low dose STZ with significantly elevated blood glucose levels (T2: 388, T2/β 3 -ANT: 369 vs C: 128 mg/dl). In T2, LV myocyte basal function and [Ca 2+ ] iT regulation were impaired measured as significantly decreased myocyte contractility (dL/dt max ) (76.8 vs 135.2 μm/s), relengthening (dR/dt max ) (62.1 vs 113.8 μ m/s) and [Ca 2+ ] iT (0.16 vs 0.21). Furthermore, versus C, in T2 myocytes, ISO-induced increases in dL/dt max (T2: 40% vs C: 58%), dR/dt max (35% vs 54%) and [Ca 2+ ] iT (19% vs 30%) were significantly reduced. By contrary, versus T2, T2/β 3 -ANT myocytes showed normal basal cell contraction (127.8 μm/s), relaxation (109.4 μm/s) and [Ca 2+ ] iT (0.21) with preserved ISO-stimulated positive inotropic effect. Versus C, in T2/β 3 -ANT, ISO caused similar increases in dL/dt max (57%), dR/dt max (52%) and [Ca 2+ ] iT (30%). Conclusion: Chronic β 3 -ANT leads to the preservation of LV myocyte function, [Ca 2+ ] iT , and β-AR responsiveness in a mouse model of type 2 diabetes. Thus, antagonizing β 3 -AR might provide a new therapeutic strategy for DM-related decline in myocardial function.

Pathological assessment of osimertinib-associated cardiotoxicity in EGFR-mutated non-small cell lung cancer patients

Abstract Background Osimertinib, a third-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), inhibits both EGFR-TKI sensitizing mutations and resistant T790M mutations detected in non-small cell lung cancer (NSCLC) patients. Cardiac adverse events (AEs) induced by osimertinib are infrequent; however, cases of severe associated cardiac dysfunction have been reported and remain poorly understood. Purpose To assess pathogenesis of osimertinib-associated cardiac AEs, we analyzed myocardial specimens of three NSCLC cases with osimertinib-associated cardiac dysfunction. Results Analysis of LVEF prior to and after osimertinb administration in 36 NSCLC patients showed significant decrease of LVEF from 69% to 63%. Within this cohort, right ventricular (RV) biopsy was performed in 2 cases to further understand the pathophysiology of cardiac dysfunction. Case 1 was 78-year-old female with advanced NSCLC harboring an EGFR L858R mutation was treated with osimertinib as second line therapy. After 3 moths of osimetinib treatment, she presented with dyspnea, high NT-proBNP and troponin I, and significantly decreased left ventricular ejection fraction (LVEF) at 28%. RV biopsy showed moderate cardiomyocyte hypertrophy without inflammatory cell infiltration. Case 2 was 52-year-old female with advanced NSCLC harboring L858R mutation. She was treated with osimertinib as first line therapy. After 2 weeks of osimertinib, screening echocardiography revealed a reduction of LVEF from 63% to 41% without cardiac symptom. RV biopsy showed mild cardiomyocyte hypertrophy with infiltration of a few inflammatory cells in interstinum. We further analyzed death case of NSCLC. Case 3 was 63-year-old female with advanced NSCLC harboring EGFR ex. 19 del. and T790M mutations. After 6 months of osimertinib, she suffered from severe respiratory failure and severely reduced LVEF at 27%. She died on the 44th day after admission. Pathological autopsy revealed mild to moderate cardiomyocyte hypertrophy without inflammatory cell infiltration in both ventricles. These pathological findings may indicate neither myocyte injury nor myocarditis was induced by osimertinib in myocardium. Conclusion Although additional data collection of advanced NSCLC patients will be important in understanding the pathophysiology of cardiac AEs with osimertinib, osimertinib-associated cardiotoxicity may result from functional inhibition of myocyte contractility by osimertinib without induction of cell death or inflammation. Funding Acknowledgement Type of funding source: None

Novel mutations of TCTN3/LTBP2 with cellular function changes in congenital heart disease associated with polydactyly.

Congenital heart disease (CHD) associated with polydactyly involves various genes. We aimed to identify variations from genes related to complex CHD with polydactyly and to investigate the cellular functions related to the mutations. Blood was collected from a complex CHD case with polydactyly, and whole exome sequencing (WES) was performed. The CRISPR/Cas9 system was used to generate human pluripotent stem cell with mutations (hPSCs‐Mut) that were differentiated into cardiomyocytes (hPSC‐CMs‐Mut) and analysed by transcriptomics on day 0, 9 and 13. Two heterozygous mutations, LTBP2 (c.2206G>A, p.Asp736Asn, RefSeq NM_000428.2) and TCTN3 (c.1268G>A, p.Gly423Glu, RefSeq NM_015631.5), were identified via WES but no TBX5 mutations were found. The stable cell lines of hPSCs‐LTBP2mu/TCTN3mu were constructed and differentiated into hPSC‐CMs‐LTBP2mu/TCTN3mu. Compared to the wild type, LTBP2 mutation delayed the development of CMs. The TCTN3 mutation consistently presented lower rate and weaker force of the contraction of CMs. For gene expression pattern of persistent up‐regulation, pathways in cardiac development and congenital heart disease were enriched in hPSCs‐CM‐LTBP2mu, compared with hPSCs‐CM‐WT. Thus, the heterozygous mutations in TCTN3 and LTBP2 affect contractility (rate and force) of cardiac myocytes and may affect the development of the heart. These findings provide new insights into the pathogenesis of complex CHD with polydactyly.