Lusitropic Responses Research Articles

Feed Forward Modeling: an efficient approach for mathematical modeling of the force frequency relationship in the rabbit isolated ventricular myocyte

Background and Objective. This study addresses the Force-Frequency relationship, a fundamental characteristic of cardiac muscle influenced by β 1-adrenergic stimulation. This relationship reveals that heart rate (HR) changes at the sinoatrial node lead to alterations in ventricular cell contractility, increasing the force and decreasing relaxation time for higher beat rates. Traditional models lacking this relationship offer an incomplete physiological depiction, impacting the interpretation of in silico experiment results. To improve this, we propose a new mathematical model for ventricular myocytes, named ‘Feed Forward Modeling’ (FFM). Methods. FFM adjusts model parameters like channel conductance and Ca2+ pump affinity according to stimulation frequency, in contrast to fixed parameter values. An empirical sigmoid curve guided the adaptation of each parameter, integrated into a rabbit ventricular cell electromechanical model. Model validation was achieved by comparing simulated data with experimental current–voltage (I-V) curves for L-type Calcium and slow Potassium currents. Results. FFM-enhanced simulations align more closely with physiological behaviors, accurately reflecting inotropic and lusitropic responses. For instance, action potential duration at 90% repolarization (APD90) decreased from 206 ms at 1 Hz to 173 ms at 4 Hz using FFM, contrary to the conventional model, where APD90 increased, limiting high-frequency heartbeats. Peak force also showed an increase with FFM, from 8.5 mN mm−2 at 1 Hz to 11.9 mN mm−2 at 4 Hz, while it barely changed without FFM. Relaxation time at 50% of maximum force (t50) similarly improved, dropping from 114 ms at 1 Hz to 75.9 ms at 4 Hz with FFM, a change not observed without the model. Conclusion. The FFM approach offers computational efficiency, bypassing the need to model all beta-adrenergic pathways, thus facilitating large-scale simulations. The study recommends that frequency change experiments include fractional dosing of isoproterenol to better replicate heart conditions in vivo.

Activation of the cannabinoid type 2 (CB2) receptor improves cardiac contractile performance in fish, Brycon amazonicus

In addition to their well-known classical effects, cannabinoid CB1 and CB2 receptors have also been involvement in both deleterious and protective actions on the heart under various pathological conditions. While the potential therapeutic applications of the endocannabinoid system in the context of cardiovascular function are indeed a viable prospect, significant debate exists within the literature regarding whether CB1, CB2, or a combination of both receptors exert a favorable influence on cardiac function. Hence, the aim of this study was to investigate the effects of CB1 + CB2 or CB2 agonists on cardiac excitation-contraction (E-C) coupling, utilizing fish (Brycon amazonicus) as an experimental model. The CB2 agonist elicited marked positive inotropic and lusitropic responses in isolated ventricular myocardium, induced cyclic adenosine 3′,5′-monophosphate (cAMP) production, and upregulated critical Ca2+ handling proteins, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+/Ca2+ exchanger (NCX). Our current study demonstrated, for the first time, that CB2 receptor activation-induced effects improved the efficiency of Ca2+ cycling, excitation-contraction coupling (E-C coupling), and cardiac performance in under physiological conditions. Hence, CB2 receptors could be considered a potential therapeutic target for modulating cardiac contractile dysfunctions.

C-type natriuretic peptide induces inotropic and lusitropic effects in human 3D-engineered cardiac tissue: Implications for the regulation of cardiac function in humans.

The role of C-type natriuretic peptide (CNP) in the regulation of cardiac function in humans remains to be established as previous investigations have been confined to animal model systems. Here, we used well-characterized engineered cardiac tissues (ECTs) generated from human stem cell-derived cardiomyocytes and fibroblasts to study the acute effects of CNP on contractility. Application of CNP elicited a positive inotropic response as evidenced by increases in maximum twitch amplitude, maximum contraction slope and maximum calcium amplitude. This inotropic response was accompanied by a positive lusitropic response as demonstrated by reductions in time from peak contraction to 90% of relaxation and time from peak calcium transient to 90% of decay that paralleled increases in maximum contraction decay slope and maximum calcium decay slope. To establish translatability, CNP-induced changes in contractility were also assessed in rat ex vivo (isolated heart) and in vivo models. Here, the effects on force kinetics observed in ECTs mirrored those observed in both the ex vivo and in vivo model systems, whereas the increase in maximal force generation with CNP application was only detected in ECTs. In conclusion, CNP induces a positive inotropic and lusitropic response in ECTs, thus supporting an important role for CNP in the regulation of human cardiac function. The high degree of translatability between ECTs, ex vivo and in vivo models further supports a regulatory role for CNP and expands the current understanding of the translational value of human ECTs. NEW FINDINGS: What is the central question of this study? What are the acute responses to C-type natriuretic peptide (CNP) in human-engineered cardiac tissues (ECTs) on cardiac function and how well do they translate to matched concentrations in animal ex vivo and in vivo models? What is the main finding and its importance? Acute stimulation of ECTs with CNP induced positive lusitropic and inotropic effects on cardiac contractility, which closely reflected the changes observed in rat ex vivo and in vivo cardiac models. These findings support an important role for CNP in the regulation of human cardiac function and highlight the translational value of ECTs.

Suppression of lusitropy as a disease mechanism in cardiomyopathies.

In cardiac muscle the action of adrenaline on β1 receptors of heart muscle cells is essential to adjust cardiac output to the body's needs. Adrenergic activation leads to enhanced contractility (inotropy), faster heart rate (chronotropy) and faster relaxation (lusitropy), mainly through activation of protein kinase A (PKA). Efficient enhancement of heart output under stress requires all of these responses to work together. Lusitropy is essential for shortening the heartbeat when heart rate increases. It therefore follows that, if the lusitropic response is not present, heart function under stress will be compromised. Current literature suggests that lusitropy is primarily achieved due to PKA phosphorylation of troponin I (TnI) and phospholamban (PLB). It has been well documented that PKA-induced phosphorylation of TnI releases Ca2+ from troponin C faster and increases the rate of cardiac muscle relaxation, while phosphorylation of PLB increases SERCA activity, speeding up Ca2+ removal from the cytoplasm. In this review we consider the current scientific evidences for the connection between suppression of lusitropy and cardiac dysfunction in the context of mutations in phospholamban and thin filament proteins that are associated with cardiomyopathies. We will discuss what advances have been made into understanding the physiological mechanism of lusitropy due to TnI and PLB phosphorylation and its suppression by mutations and we will evaluate the evidence whether lack of lusitropy is sufficient to cause cardiomyopathy, and under what circumstances, and consider the range of pathologies associated with loss of lusitropy. Finally, we will discuss whether suppressed lusitropy due to mutations in thin filament proteins can be therapeutically restored.

Abstract 9515: Cariporide, a Selective Na+-H+ Exchange Inhibitor, Enhances Cardiac Positive Inotropic and Lusitropic Responses to Dobutamine in Heart Failure: Assessment by Pressure-Volume Analysis

Background: Inability of failing hearts to respond to positive inotropic agents such as dobutamine (DOB) represents a major clinical problem. We have shown previously that in addition to intracellular acidosis, various signaling pathways such as endothelin-1 and angiotensin II also activate cardiac Na + -H + exchange (NHE). In heart failure (HF), increased stimulation of NHE decreases myocardial calcium sensitivity. We hypothesized that blocking NHE with cariporide (CAR), a NHE-1-selective inhibitor, may improve β-adrenergic inotropic responsiveness in HF and enhance left ventricle (LV) positive inotropic and lusitropic responses of DOB. Methods: We compared the effect of clinically relevant doses of CAR (3.5 μg/kg, iv) on LV functional performance and LV contractile response to DOB (6 μg/kg/min, iv, 10 min) in 6 instrumented, conscious dogs after pacing-induced HF. LV contractile performance was measured by pressure (P)-volume (V) analysis. Animals received DOB first. After stopping DOB for 30 minutes, both DOB and CAR were infused. Results: After HF, DOB caused a 17% increase in E ES (5.3 ±1.5 vs 4.4±0.8 mmHg/ml), a16% increase in M SW (53.8±4.9 vs 45.3±3.7 mmHg), and an 11% decrease in the time constant of LV relaxation (τ) (40.4±2.6 vs 45.8±2.5 ms). DOB followed by CAR resulted in significantly greater effects with a 42% increase in E ES (6.1±2.3 vs 4.3±1.9 mmHg/ml) and a 37% increase in M SW (61.4±5.9 vs 45.1±3.2 mmHg), and a 23 % decrease in τ (34.1±3.6 vs 44.5±2.4 ms). Compared with DOB alone, DOB plus CAR caused no significant changes in heart rate (126±11 vs 130±12 bpm) and LV end-systolic P (110±8.6 vs 112±6.5 mmHg), but significantly decreased mean left atrial P (27.2±5.0 vs 32.9±5.4 mmHg), and increased stroke volume (32%, 13.3±1.7 vs 10.1±2.7 ml). DOB plus CAR also produced about 28% increased LV mechanical efficiency, measured as the ratio of stroke work to total P-V area (0.59±0.15 vs 0.46±0.17) ( p <0.05). Conclusions: In pacing-induced HF, Na + -H + exchange inhibition by cariporide enhances LV positive inotropic and lusitropic responses to DOB. This suggests that combination therapy with intravenous cariporide and DOB may be appropriate in HF, thus providing a potential method of enhancing β-adrenergic responsiveness of the failing myocardium.

Calcium handling maturation and adaptation to increased substrate stiffness in human iPSC-derived cardiomyocytes: The impact of full-length dystrophin deficiency.

Cardiomyocytes differentiated from human induced Pluripotent Stem Cells (hiPSC- CMs) are a unique source for modelling inherited cardiomyopathies. In particular, the possibility of observing maturation processes in a simple culture dish opens novel perspectives in the study of early-disease defects caused by genetic mutations before the onset of clinical manifestations. For instance, calcium handling abnormalities are considered as a leading cause of cardiomyocyte dysfunction in several genetic-based dilated cardiomyopathies, including rare types such as Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy. To better define the maturation of calcium handling we simultaneously measured action potential and calcium transients (Ca-Ts) using fluorescent indicators at specific time points. We combined micropatterned substrates with long-term cultures to improve maturation of hiPSC-CMs (60, 75 or 90 days post-differentiation). Control-(hiPSC)-CMs displayed increased maturation over time (90 vs 60 days), with longer action potential duration (APD), increased Ca-T amplitude, faster Ca-T rise (time to peak) and Ca-T decay (RT50). The progressively increased contribution of the SR to Ca release (estimated by post-rest potentiation or Caffeine-induced Ca-Ts) appeared as the main determinant of the progressive rise of Ca-T amplitude during maturation. As an example of severe cardiomyopathy with early onset, we compared hiPSC-CMs generated from a DMD patient (DMD-ΔExon50) and a CRISPR-Cas9 genome edited cell line isogenic to the healthy control with deletion of a G base at position 263 of the DMD gene (c.263delG-CMs). In DMD-hiPSC-CMs, changes of Ca-Ts during maturation were less pronounced: indeed, DMD cells at 90 days showed reduced Ca-T amplitude and faster Ca-T rise and RT50, as compared with control hiPSC-CMs. Caffeine-Ca-T was reduced in amplitude and had a slower time course, suggesting lower SR calcium content and NCX function in DMD vs control cells. Nonetheless, the inotropic and lusitropic responses to forskolin were preserved. CRISPR-induced c.263delG-CM line recapitulated the same developmental calcium handling alterations observed in DMD-CMs. We then tested the effects of micropatterned substrates with higher stiffness. In control hiPSC-CMs, higher stiffness leads to higher amplitude of Ca-T with faster decay kinetics. In hiPSC-CMs lacking full-length dystrophin, however, stiffer substrates did not modify Ca-Ts but only led to higher SR Ca content. These findings highlighted the inability of dystrophin-deficient cardiomyocytes to adjust their calcium homeostasis in response to increases of extracellular matrix stiffness, which suggests a mechanism occurring during the physiological and pathological development (i.e. fibrosis).

Physiological and Pathophysiological Effects of C-Type Natriuretic Peptide on the Heart.

Simple SummaryC-type natriuretic peptide (CNP) is the third member of the natriuretic peptide family. Unlike atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), CNP was not previously regarded as an important cardiac modulator. However, recent studies have revealed the physiological and pathophysiological importance of CNP in the heart; in concert with its cognate natriuretic peptide receptor-B (NPR-B), CNP has come to be regarded as the major heart-protective natriuretic peptide in the failed heart. In this review, I introduce the history of research on CNP in the cardiac field.C-type natriuretic peptide (CNP) is the third member of the natriuretic peptide family. Unlike other members, i.e., atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), which are cardiac hormones secreted from the atrium and ventricle of the heart, respectively, CNP is regarded as an autocrine/paracrine regulator with broad expression in the body. Because of its low expression levels compared to ANP and BNP, early studies failed to show its existence and role in the heart. However, recent studies have revealed the physiological and pathophysiological importance of CNP in the heart; in concert with the distribution of its specific natriuretic peptide receptor-B (NPR-B), CNP has come to be regarded as the major heart-protective natriuretic peptide in the failed heart. NPR-B generates intracellular cyclic guanosine 3′,5′-monophosphate (cGMP) upon CNP binding, followed by various molecular effects including the activation of cGMP-dependent protein kinases, which generates diverse cytoprotective actions in cardiomyocytes, as well as in cardiac fibroblasts. CNP exerts negative inotropic and positive lusitropic responses in both normal and failing heart models. Furthermore, osteocrin, the intrinsic and specific ligand for the clearance receptor for natriuretic peptides, can augment the effects of CNP and may supply a novel therapeutic strategy for cardiac protection.

Cardioprotective actions of nitroxyl donor Angeli's salt are preserved in the diabetic heart and vasculature in the face of nitric oxide resistance

Background and PurposeThe risk of fatal cardiovascular events is increased in patients with type 2 diabetes mellitus (T2DM). A major contributor to poor prognosis is impaired nitric oxide (NO•) signalling at the level of tissue responsiveness, termed NO• resistance. This study aimed to determine if T2DM promotes NO• resistance in the heart and vasculature and whether tissue responsiveness to nitroxyl (HNO) is affected.Experimental ApproachAt 8 weeks of age, male Sprague–Dawley rats commenced a high‐fat diet. After 2 weeks, the rats received low‐dose streptozotocin (two intraperitoneal injections, 35 mg·kg−1, over two consecutive days) and continued on the same diet. Twelve weeks later, isolated hearts were Langendorff‐perfused to assess responses to the NO• donor diethylamine NONOate (DEA/NO) and the HNO donor Angeli's salt. Isolated mesenteric arteries were utilised to measure vascular responsiveness to the NO• donors sodium nitroprusside (SNP) and DEA/NO, and the HNO donor Angeli's salt.Key ResultsInotropic, lusitropic and coronary vasodilator responses to DEA/NO were impaired in T2DM hearts, whereas responses to Angeli's salt were preserved or enhanced. Vasorelaxation to Angeli's salt was augmented in T2DM mesenteric arteries, which were hyporesponsive to the relaxant effects of SNP and DEA/NO.Conclusion and ImplicationsThis is the first evidence that inotropic and lusitropic responses are preserved, and NO• resistance in the coronary and mesenteric vasculature is circumvented, by the HNO donor Angeli's salt in T2DM. These findings highlight the cardiovascular therapeutic potential of HNO donors, especially in emergencies such as acute ischaemia or heart failure.

Abstract P405: Cardioprotective Actions Of Nitroxyl Donor Angeli's Salt Are Preserved In The Diabetic Heart And Vasculature In The Face Of Nitric Oxide Resistance

Introduction: The risk of fatal cardiovascular events is increased in patients with type 2 diabetes mellitus (T2DM). A major contributor to poor prognosis is impaired nitric oxide (NO•) signalling at the level of tissue responsiveness, termed NO• resistance. Nitroxyl (HNO) induces positive inotropic and lusitropic effects in healthy and failing hearts. Hypothesis: We hypothesised that in a rodent model, T2DM will promote, and HNO will circumvent, NO• resistance in the myocardium and coronary vasculature. Methods: At 8 weeks of age, male Sprague-Dawley rats commenced a high-fat diet. After two weeks, the rats received low-dose streptozotocin (two intraperitoneal injections, 35 mg/kg, over two consecutive days), and continued the diet. Twelve weeks later, hearts were Langendorff-perfused to assess responses to the NO• donor diethylamine NONOate (DEA/NO) and the HNO donor Angeli’s salt. Results: Inotropic, lusitropic and coronary vasodilator responses to DEA/NO were impaired, and responses to Angeli’s salt were preserved or enhanced, in T2DM hearts compared with non-diabetic hearts. Conclusions: This is the first evidence that inotropic and lusitropic responses are preserved, and NO• resistance in the coronary vasculature is circumvented, by the HNO donor Angeli’s salt in T2DM. These findings highlight the cardiovascular therapeutic potential of HNO donors, especially in cardiac emergencies such as acute ischaemia or heart failure. Figure 1. Dose-response curves and maximal responses to DEA/NO or Angeli's salt in diabetic or non-diabetic hearts. (A-C) LV+dP/dt, (D-F) LV-dP/dt and (G-I) coronary flow rate. Data expressed as change from baseline (denoted by Δ), mean ± SEM. Data analysed by two-way RM ANOVA with Sidak's post-hoc test. *P<0.05 vs. non-diabetic. LV, left ventricular; LV+dP/dt, maximal rate of rise in LV pressure; LV-dP/dt, maximal rate of fall in LV pressure.

CNP regulates cardiac contractility and increases cGMP near both SERCA and TnI: difference from BNP visualized by targeted cGMP biosensors.

AimsGuanylyl cyclase-B (GC-B; natriuretic peptide receptor-B, NPR-B) stimulation by C-type natriuretic peptide (CNP) increases cGMP and causes a lusitropic and negative inotropic response in adult myocardium. These effects are not mimicked by NPR-A (GC-A) stimulation by brain natriuretic peptide (BNP), despite similar cGMP increase. More refined methods are needed to better understand the mechanisms of the differential cGMP signalling and compartmentation. The aim of this work was to measure cGMP near proteins involved in regulating contractility to understand compartmentation of cGMP signalling in adult cardiomyocytes.Methods and resultsWe constructed several fluorescence resonance energy transfer (FRET)-based biosensors for cGMP subcellularly targeted to phospholamban (PLB) and troponin I (TnI). CNP stimulation of adult rat cardiomyocytes increased cGMP near PLB and TnI, whereas BNP stimulation increased cGMP near PLB, but not TnI. The phosphodiesterases PDE2 and PDE3 constrained cGMP in both compartments. Local receptor stimulation aided by scanning ion conductance microscopy (SICM) combined with FRET revealed that CNP stimulation both in the t-tubules and on the cell crest increases cGMP similarly near both TnI and PLB. In ventricular strips, CNP stimulation, but not BNP, induced a lusitropic response, enhanced by inhibition of either PDE2 or PDE3, and a negative inotropic response. In cardiomyocytes from heart failure rats, CNP increased cGMP near PLB and TnI more pronounced than in cells from sham-operated animals.ConclusionThese targeted biosensors demonstrate that CNP, but not BNP, increases cGMP near TnI in addition to PLB, explaining how CNP, but not BNP, is able to induce lusitropic and negative inotropic responses.

Reduced cardiac response to the adrenergic system is a key limiting factor for physical capacity in old age.

Ageing is associated with a progressive reduction in physical capacity reducing quality of life. One key physiological limitation of physical capacity that deteriorates in a progressive age-dependent manner is cardiac reserve. Peak cardiac output falls progressively with advancing age such that in extreme old age there is limited ability to enhance cardiac output beyond basal function as is required to support the increased metabolic needs of physical activity. This loss of dynamic range in cardiac output associates with a progressive reduction in the heart's response to adrenergic stimulation. A combination of decreases in the expression and functioning of beta1 adrenergic receptors partially underlies this change. Changes in end effector proteins also have a role to play in this decline. Alterations in the efficiency of excitation-contraction coupling contribute to the reduced chronotropic, inotropic and lusitropic responses of the aged heart. Moderate to vigorous endurance exercise training however has some potential to counter elements of these changes. Further studies are required to fully elucidate the key pivotal mechanisms involved in the age-related loss of response to adrenergic signalling to allow targeted therapeutic strategies to be developed with the aim of preserving physical capacity in advanced old age.

Abstract 16878: Cardiac Overexpression of RXFP1 via AAV9 Gene Therapy Sensitizes Myocardial Inotropic Response to Serelaxin

Background: The pleiotropic hormone relaxin has been shown to mediate physiologic responses during pregnancy by facilitating increases in vascular compliance and cardiac output. While relaxin signaling has been attributed to increases in atrial inotropy, the hormonal effects on ventricular contractility have not been explored in vivo . In this study, we investigated the dose-response effects of recombinant human relaxin (serelaxin) on ventricular mechanics. Methods and Results: Adult male CD1 mice were anesthetized and mechanically ventilated prior to Millar cardiac catheterization. A pressure-volume catheter was advanced into the left ventricle (LV), and baseline systolic and diastolic pressure/volume (PV) loops were recorded (Pre). Mice were injected with either saline, 10 μg/kg (low dose) or 1 mg/kg (high dose) of serelaxin, and PV loops were obtained after stabilization (Post) . Mice injected with 1 mg/kg demonstrated significantly higher LV contractility ( Fig A ) independent of preload ( Fig B ). These responses were not observed with low dose. In order to further examine the role of RXFP1 in modulating these responses, healthy CD1 were injected with AAV9-RXFP1 or empty vector as control and receptor overexpression was confirmed four weeks later via qPCR ( Fig C ). PV loop analysis showed an increased inotropic response [ΔP Max , Δ(dP/dt) Max ] and improved relaxation kinetics [Δ(dP/dt) Min , Δτ] in RXFP1-overexpressing mice at a lower dose of serelaxin, when compared to empty vector mice ( Fig D ). Conclusions: Our data show a novel, dose-dependent correlation between serelaxin and ventricular inotropic and lusitropic responses in mice. These responses were observed at a lower drug dose in cardiac RXFP1-overexpressing mice. We propose that further investigation of this phenomenon could advance the therapeutic, cardioprotective effects associated with relaxin therapy in different etiologies of heart failure that may exhibit changes in RXFP1 expression.

Nitroxyl: A Novel Strategy to Circumvent Diabetes Associated Impairments in Nitric Oxide Signaling.

Diabetes is associated with an increased mortality risk due to cardiovascular complications. Hyperglycemia-induced oxidative stress underlies these complications, leading to an impairment in endogenous nitric oxide (NO•) generation, together with reductions in NO• bioavailability and NO• responsiveness in the vasculature, platelets and myocardium. The latter impairment of responsiveness to NO•, termed NO• resistance, compromises the ability of traditional NO•-based therapeutics to improve hemodynamic status during diabetes-associated cardiovascular emergencies, such as acute myocardial infarction. Whilst a number of agents can ameliorate (e.g. angiotensin converting enzyme [ACE] inhibitors, perhexiline, statins and insulin) or circumvent (e.g. nitrite and sGC activators) NO• resistance, nitroxyl (HNO) donors offer a novel opportunity to circumvent NO• resistance in diabetes. With a suite of vasoprotective properties and an ability to enhance cardiac inotropic and lusitropic responses, coupled with preserved efficacy in the setting of oxidative stress, HNO donors have intact therapeutic potential in the face of diminished NO• signaling. This review explores the major mechanisms by which hyperglycemia-induced oxidative stress drives NO• resistance, and the therapeutic potential of HNO donors to circumvent this to treat cardiovascular complications in type 2 diabetes mellitus.

Generation of Ventricular-Like HiPSC-Derived Cardiomyocytes and High-Quality Cell Preparations for Calcium Handling Characterization.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a valuable human source for studying the basic science of calcium (Ca2+) handling and signaling pathways as well as high-throughput drug screening and toxicity assays. Herein, we provide a detailed description of the methodologies used to generate high-quality iPSC-CMs that can consistently reproduce molecular and functional characteristics across different cell lines. Additionally, a method is described to reliably assess their functional characterization through the evaluation of Ca2+ handling properties. Low oxygen (O2) conditions, lactate selection, and prolonged time in culture produce high-purity and high-quality ventricular-like cardiomyocytes. Similar to isolated adult rat cardiomyocytes (ARCMs), 3-month-old iPSC-CMs exhibit higher Ca2+ amplitude, faster rate of Ca2+ reuptake (decay-tau), and a positive lusitropic response to β-adrenergic stimulation compared to day 30 iPSC-CMs. The strategy is technically simple, cost-effective, and reproducible. It provides a robust platform to model cardiac disease and for the large-scale drug screening to target Ca2+ handling proteins.

Inotropic and lusitropic effects of incremental doses of dobutamine in dogs with right ventricular apical pacing-induced cardiac dysfunction.

This study aimed to assess the effects of incremental doses of dobutamine on diastolic function in healthy and rapid ventricular apical pacing (RVAP)-induced cardiac dysfunction anesthetized dogs. Inotropic and lusitropic effects of dobutamine (2, 4, 8, and 12μgkg-1 min-1 ) were assessed through left ventricle (LV) pressure-volume relation and Doppler echocardiography in six female dogs before and after 8weeks of RVAP. Peak rate of LV pressure fall (-dP/dtmin ) improved with doses >4μgkg-1 min-1 in healthy (4,490±970 vs. 3,265±471mmHg/s, p<0.05) and >8μgkg-1 min-1 in RVAP dogs (3,385±1,122 vs. 1,864±849mmHg/s, p<0.05) while the time constant of relaxation (tau) reduced with doses >4μgkg-1 min-1 in both groups (healthy: 24.0±3.7 vs. 28.2±4.9ms; RVAP: 32.6±8.5 vs. 37.5±11.4ms, p<0.05) comparing with baseline. Indices of relaxation (-dP/dtmin and tau) suggested preserved lusitropic response in contrast with markedly reduced indices of contractility in the RVAP group compared with healthy group at same infusion rates. Doppler echocardiography showed significant reduction of elastic recoil in failing hearts. The results of this study demonstrated maximal positive lusitropic effects of dobutamine at a dose of 8μgkg-1 min-1 in ventricular pacing-induced cardiac dysfunction without further impairment of ventricular filling.

Functional abnormalities in induced Pluripotent Stem Cell-derived cardiomyocytes generated from titin-mutated patients with dilated cardiomyopathy.

AimsDilated cardiomyopathy (DCM), a myocardial disorder that can result in progressive heart failure and arrhythmias, is defined by ventricular chamber enlargement and dilatation, and systolic dysfunction. Despite extensive research, the pathological mechanisms of DCM are unclear mainly due to numerous mutations in different gene families resulting in the same outcome—decreased ventricular function. Titin (TTN)—a giant protein, expressed in cardiac and skeletal muscles, is an important part of the sarcomere, and thus TTN mutations are the most common cause of adult DCM. To decipher the basis for the cardiac pathology in titin-mutated patients, we investigated the hypothesis that induced Pluripotent Stem Cell (iPSC)-derived cardiomyocytes (iPSC-CM) generated from patients, recapitulate the disease phenotype. The hypothesis was tested by 3 Aims: (1) Investigate key features of the excitation-contraction-coupling machinery; (2) Investigate the responsiveness to positive inotropic interventions; (3) Investigate the proteome profile of the AuP cardiomyocytes using mass-spectrometry (MS).Methods and resultsiPSC were generated from the patients' skin fibroblasts. The major findings were: (1) Sarcomeric organization analysis in mutated iPSC-CM showed defects in assembly and maintenance of sarcomeric structure. (2) Mutated iPSC-CM exhibited diminished inotropic and lusitropic responses to β-adrenergic stimulation with isoproterenol, increased [Ca2+]out and angiotensin-II. Additionally, mutated iPSC-CM displayed prolonged recovery in response to caffeine. These findings may result from defective or lack of interactions of the sarcomeric components with titin through its kinase domain which is absent in the mutated cells.ConclusionsThese findings show that the mutated cardiomyocytes from DCM patients recapitulate abnormalities of the inherited cardiomyopathies, expressed as blunted inotropic response.

Distinct submembrane localisation compartmentalises cardiac NPR1 and NPR2 signalling to cGMP

Natriuretic peptides (NPs) are important hormones that regulate multiple cellular functions including cardiovascular physiology. In the heart, two natriuretic peptide receptors NPR1 and NPR2 act as membrane guanylyl cyclases to produce 3′,5′-cyclic guanosine monophosphate (cGMP). Although both receptors protect from cardiac hypertrophy, their effects on contractility are markedly different, from little effect (NPR1) to pronounced negative inotropic and positive lusitropic responses (NPR2) with unclear underlying mechanisms. Here we use a scanning ion conductance microscopy (SICM) approach combined with Förster resonance energy transfer (FRET)-based cGMP biosensors to show that whereas NPR2 is uniformly localised on the cardiomyocyte membrane, functional NPR1 receptors are found exclusively in membrane invaginations called transverse (T)-tubules. This leads to far-reaching CNP/NPR2/cGMP signals, whereas ANP/NPR1/cGMP signals are highly confined to T-tubular microdomains by local pools of phosphodiesterase 2. This provides a previously unrecognised molecular basis for clearly distinct functional effects engaged by different cGMP producing membrane receptors.

Functional and transcriptomic insights into pathogenesis of R9C phospholamban mutation using human induced pluripotent stem cell-derived cardiomyocytes

Dilated cardiomyopathy (DCM) can be caused by mutations in the cardiac protein phospholamban (PLN). We used CRISPR/Cas9 to insert the R9C PLN mutation at its endogenous locus into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. R9C PLN hiPSC-CMs display a blunted β-agonist response and defective calcium handling. In 3D human engineered cardiac tissues (hECTs), a blunted lusitropic response to β-adrenergic stimulation was observed with R9C PLN. hiPSC-CMs harboring the R9C PLN mutation showed activation of a hypertrophic phenotype, as evidenced by expression of hypertrophic markers and increased cell size and capacitance of cardiomyocytes. RNA-seq suggests that R9C PLN results in an altered metabolic state and profibrotic signaling, which was confirmed by gene expression analysis and picrosirius staining of R9C PLN hECTs. The expression of several miRNAs involved in fibrosis, hypertrophy, and cardiac metabolism were also perturbed in R9C PLN hiPSC-CMs. This study contributes to better understanding of the pathogenic mechanisms of the hereditary R9C PLN mutation in the context of human cardiomyocytes.

A protocol to study ex vivo mouse working heart at human-like heart rate

Genetically modified mice are widely used as experimental models to study human heart function and diseases. However, the fast rate of normal mouse heart at 400–600bpm limits its capacity of assessing kinetic parameters that are important for the physiology and pathophysiology of human heart that beats at a much slower rate (75–180bpm). To extend the value of mouse models, we established a protocol to study ex vivo mouse working hearts at a human-like heart rate. In the presence of 300μM lidocaine to lower pacemaker and conductive activities and prevent arrhythmia, a stable rate of 120–130bpm at 37°C is achieved for ex vivo mouse working hearts. The negative effects of decreased heart rate on force-frequency dependence and lidocaine as a myocardial depressant on intracellular calcium can be compensated by using a higher but still physiological level of calcium (2.75mM) in the perfusion media. Multiple parameters were studied to compare the function at the human-like heart rate with that of ex vivo mouse working hearts at the standard rate of 480bpm. The results showed that the conditions for slower heart rate in the presence of 300μM lidocaine did not have depressing effect on left ventricular pressure development, systolic and diastolic velocities and stroke volume with maintained positive inotropic and lusitropic responses to β-adrenergic stimulation. Compared with that at 480bpm, the human-like heart rate increased ventricular filling and end diastolic volume with enhanced Frank-Starling responses. Coronary perfusion was increased from longer relaxation time and interval between beats whereas cardiac efficiency was significantly improved. Although the intrinsic differences between mouse and human heart remain, this methodology for ex vivo mouse hearts to work at human-like heart rate extends the value of using genetically modified mouse models to study cardiac function and human heart diseases.

Abstract 185: Genome Editing of Isogenic Human Induced Pluripotent Stem Cells Allows for Functional and Transcriptomic Insights Into Hereditary Dilated Cardiomyopathy Caused by Phospholamban Mutations

Dilated cardiomyopathy (DCM) can be caused by genetic mutations in numerous cardiac proteins, including phospholamban (PLN). PLN mutations are quite rare and obtaining patient samples for mechanistic insights can be challenging. We used genome editing with CRISPR/Cas9 to successfully insert R14del and R9C PLN mutations into a human induced pluripotent stem cell (hiPSC) line from an individual with no cardiovascular disease. HiPSC-cardiomyocytes (hiPSC-CMs) with the inserted R14del PLN mutation recapitulate the phenotype observed in patient-derived R14del hiPSC-CMs, characterized by abnormal intracellular calcium cycling and arrhythmogenicity. Insertion of R9C PLN results in hiPSC-CMs displaying an abnormal response to β-agonists, defective calcium handling, and a hypertrophic phenotype. In human engineered cardiac tissues (hECTs) created from hiPSC-CMs in a 3D matrix, R14del results in a progressive worsening of developed force and R9C PLN demonstrates an abnormal lusitropic response following β-adrenergic stimulation. Further, transcriptional profiling using RNAseq suggests a role for lipid metabolism in R14del. DNA methylation studies showed that differentially expressed genes were enriched for lipoprotein metabolism and chylomicron-mediated lipid transport pathways in R14del PLN. This was also confirmed with the observation of lipid deposition in R14del hECTs and human myocardial tissues from explanted hearts of affected patients. Furthermore, small RNAseq identified 2 miRNAs that were differentially regulated in R14del hiPSC-CMs (miR-449c-5p and miR-483-3p). For R9C PLN, RNAseq suggests that the mutation results in profibrotic signaling, activation of autophagy, and an altered metabolic state. Our findings demonstrate that gene editing of hiPSCs can be used to successfully create models and delineate molecular mechanisms of human PLN mutations associated with DCM.