Negative Force-frequency Relationship Research Articles

Enhancing the functional maturity of hiPSC-derived cardiomyocytes to assess inotropic compounds

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) present an attractive in vitro platform to model safety and toxicity assessments—notably screening pro-arrhythmic compounds. The utility of the platform is stymied by a hiPSC-CM contractile apparatus and calcium handling mechanism akin to fetal phenotypes, evidenced by a negative force-frequency relationship. As such, hiPSC-CMs are limited in their ability to assess compounds that modulate contraction mediated by ionotropic compounds (Robertson, Tran, & George, 2013). To address this limitation, we utilize Agilent's xCELLigence Real-Time Cell Analyzer ePacer (RTCA ePacer) to enhance hiPSC-CM functional maturity. A continuous, progressive increase of electrical pacing is applied to hiPSC-CMs for up to 15 days. Contraction and viability are recorded by measurement of impedance using the RTCA ePacer. Our data confirms hiPSC-CMs inherently demonstrate a negative impedance amplitude frequency that is reversed after long-term electrical pacing. The data also indicate positive inotropic compounds increase the contractility of paced cardiomyocytes and calcium handling machinery is improved. Increased expression of genes critical to cardiomyocyte maturation further underscores the maturity of paced cells. In summary, our data suggest the application of continuous electrical pacing can functionally mature hiPSC-CMs, enhancing cellular response to positive inotropic compounds and improving calcium handling. SummaryLong-term electrical stimulation of hiPSC-CM leads to functional maturation enabling predictive assessment of inotropic compounds.

The effect of intravenous ferric carboxymaltose on cardiac reverse remodelling following cardiac resynchronization therapy-the IRON-CRT trial.

AimsIron deficiency is common in heart failure with reduced ejection fraction (HFrEF) and negatively affects cardiac function and structure. The study the effect of ferric carboxymaltose (FCM) on cardiac reverse remodelling and contractile status in HFrEF.Methods and resultsSymptomatic HFrEF patients with iron deficiency and a persistently reduced left ventricular ejection fraction (LVEF <45%) at least 6 months after cardiac resynchronization therapy (CRT) implant were prospectively randomized to FCM or standard of care (SOC) in a double-blind manner. The primary endpoint was the change in LVEF from baseline to 3-month follow-up assessed by three-dimensional echocardiography. Secondary endpoints included the change in left ventricular end-systolic (LVESV) and end-diastolic volume (LVEDV) from baseline to 3-month follow-up. Cardiac performance was evaluated by the force–frequency relationship as assessed by the slope change of the cardiac contractility index (CCI = systolic blood pressure/LVESV index) at 70, 90, and 110 beats of biventricular pacing. A total of 75 patients were randomized to FCM (n = 37) or SOC (n = 38). At baseline, both treatment groups were well matched including baseline LVEF (34 ± 7 vs. 33 ± 8, P = 0.411). After 3 months, the change in LVEF was significantly higher in the FMC group [+4.22%, 95% confidence interval (CI) +3.05%; +5.38%] than in the SOC group (−0.23%, 95% CI −1.44%; +0.97%; P < 0.001). Similarly, LVESV (−9.72 mL, 95% CI −13.5 mL; −5.93 mL vs. −1.83 mL, 95% CI −5.7 mL; 2.1 mL; P = 0.001), but not LVEDV (P = 0.748), improved in the FCM vs. the SOC group. At baseline, both treatment groups demonstrated a negative force–frequency relationship, as defined by a decrease in CCI at higher heart rates (negative slope). FCM resulted in an improvement in the CCI slope during incremental biventricular pacing, with a positive force–frequency relationship at 3 months. Functional status and exercise capacity, as measured by the Kansas City Cardiomyopathy Questionnaire and peak oxygen consumption, were improved by FCM.ConclusionsTreatment with FCM in HFrEF patients with iron deficiency and persistently reduced LVEF after CRT results in an improvement of cardiac function measured by LVEF, LVESV, and cardiac force–frequency relationship.

Effects of change in temperature on the cardiac contractility of broad-snouted caiman (Caiman latirostris) during digestion.

In many reptiles, digestion has been associated with the selection of higher body temperatures, the so-called post-prandial thermophilic response. This study aimed to investigate the excitation-contraction (E-C) coupling in postprandial broad-snouted caimans (Caiman latirostris) in response to acute warming within a preferred body temperature range of crocodiles. Isometric preparations subjected to a temperature transition from 25°C to 30°C were used to investigate myocardial contractility of postprandial caimans, that is, 48 h after the animals ingested a rodent meal corresponding to 15% of body mass. The caiman heart exhibits a negative force-frequency relationship that is independent of the temperature. At 25°C, cardiac muscle was able to maintain a constant force up to 36 bpm, above which it decreased significantly, reaching minimum values at the highest frequency of 84 bpm. Moreover, E-C coupling is predominantly dependent on transsarcolemmal Ca2+ transport denoted by the lack of significant ryanodine effects on force generation. On the contrary, ventricular strips at 30°C were able to sustain the cardiac contractility at higher pacing frequencies (from 12 to 144 bpm) due to an important role of Na+ /Ca2+ exchanger in Ca2+ cycling, as indicated by the decay of the post-rest contraction, and a significant contribution of the sarcoplasmic reticulum above 72 bpm. Our results demonstrated that the myocardium of postprandial caimans exhibits a significant degree of thermal plasticity of E-C coupling during acute warming. Therefore, myocardial contractility can be maximized when postprandial broad-snouted caimans select higher body temperatures (preferred temperature zone) following feeding.

Optimizing the Direction and Order of the Motion Unveiled the Ability of Conventional Monolayers of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Show Frequency-Dependent Enhancement of Contraction and Relaxation Motion.

Contractility of the human heart increases as its beating rate is elevated, so-called positive force-frequency relationship; however, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been reported to exert a negative force-frequency relationship. We tested the hypothesis that the regulation of motion directions by electrical pacing and/or oxygen supply may improve the electro-mechanical properties of hiPSC-CMs monolayers. To better evaluate the spatial and temporal relationship between electrical excitation and contractile motion, we simultaneously observed the field potential and motion vector of hiPSC-CMs sheets. Under spontaneous contraction, although an electrical excitation originating from a region propagated unidirectionally over the cell sheet, contraction wave started from multiple sites, and relaxation wave was initiated from a geometric center of hiPSC-CMs sheet. During electrical pacing, contraction and relaxation waves were propagated from the stimulated site. Interestingly, the maximum contraction speed was more increased when the hiPSC-CMs sheet was stimulated at an area relaxation initiated under spontaneous condition. Furthermore, motion vector analysis demonstrated that “positive contraction velocity-frequency relationship” in contraction and “frequency-dependent enhancement of relaxation” were produced in the cell sheet by optimizing the direction and order of the contractile motion with pacing at the relaxation-initiating area. A close analysis of motion vectors along with field potential recording demonstrated that relaxation process consists of fast and slow phases, and suggest that intracellular Ca2+ dynamics may be closely related to functions of Ca2+-ATPase pump and Na+-Ca2+ exchangers. Namely, the slow relaxation phase occurred after the second peak of field potential, suggesting that the slow phase may be associated with extrusion of Ca2+ by Na+-Ca2+ exchangers during repolarization. Increase of oxygen concentration from 20 to 95% as well as β-adrenergic stimulation with isoproterenol accelerated the fast relaxation, suggesting that it could depend on Ca2+ uptake via Ca2+-ATPase during the depolarization phase. The ratio of maximum contraction speed to field potential duration was increased by the β-adrenergic stimulation, indicating the elevated contraction efficiency per Ca2+-influx. Thus, these findings revealed potential ability of conventional monolayers of hiPSC-CMs, which will help apply them to translational study filling the gap between physiological as well as pharmacological studies and clinical practice.

The Degree of t-System Remodeling Predicts Negative Force-Frequency Relationship and Prolonged Relaxation Time in Failing Human Myocardium.

The normally positive cardiac force-frequency relationship (FFR) becomes flat or negative in chronic heart failure (HF). Here we explored if remodeling of the cardiomyocyte transverse tubular system (t-system) is associated with alterations in FFR and contractile kinetics in failing human myocardium. Left-ventricular myocardial slices from 13 failing human hearts were mounted into a biomimetic culture setup. Maximum twitch force (F), 90% contraction duration (CD90), time to peak force (TTP) and time to relaxation (TTR) were determined at 37°C and 0.2–2 Hz pacing frequency. F1Hz/F0.5Hz and F2Hz/F0.5Hz served as measures of FFR, intracellular cardiomyocyte t-tubule distance (ΔTT) as measure of t-system remodeling. Protein levels of SERCA2, NCX1, and PLB were quantified by immunoblotting. F1Hz/F0.5Hz (R2 = 0.82) and F2Hz/F0.5Hz (R2 = 0.5) correlated negatively with ΔTT, i.e., samples with severe t-system loss exhibited a negative FFR and reduced myocardial wall tension at high pacing rates. PLB levels also predicted F1Hz/F0.5Hz, but to a lesser degree (R2 = 0.49), whereas NCX1 was not correlated (R2 = 0.02). CD90 correlated positively with ΔTT (R2 = 0.39) and negatively with SERCA2/PLB (R2 = 0.42), indicating that both the t-system and SERCA activity are important for contraction kinetics. Surprisingly, ΔTT was not associated with TTP (R2 = 0) but rather with TTR (R2 = 0.5). This became even more pronounced when interaction with NCX1 expression was added to the model (R2 = 0.79), suggesting that t-system loss impairs myocardial relaxation especially when NCX1 expression is low. The degree of t-system remodeling predicts FFR inversion and contraction slowing in failing human myocardium. Moreover, together with NCX, the t-system may be important for myocardial relaxation.

Rationale and design of the IRON-CRT trial: effect of intravenous ferric carboxymaltose on reverse remodelling following cardiac resynchronization therapy.

AimsIron deficiency is common in heart failure with reduced ejection fraction (HFrEF). In patients with cardiac resynchronization therapy (CRT), it is associated with a diminished reverse remodelling response and poor functional improvement. The latter is partially related to a loss in contractile force at higher heart rates (negative force–frequency relationship).Methods and resultsThe effect of intravenous ferric carboxymaltose on reverse remodelling following cardiac resynchronization therapy (IRON‐CRT) trial is a multicentre, prospective, randomized, double‐blind controlled trial in HFrEF patients who experienced incomplete reverse remodelling (defined as a left ventricular ejection fraction below <45%) at least 6 months after CRT. Additionally, patients need to have iron deficiency defined as a ferritin below 100 μg/L irrespective of transferrin saturation or a ferritin between 100 and 300 μg/L with a transferrin saturation <20%. Patients will be randomized to either intravenous ferric carboxymaltose (dose based according to Summary of Product Characteristics) or intravenous placebo. The primary objective is to evaluate the effect of ferric carboxymaltose on metrics of cardiac reverse remodelling and contractility, measured by the primary endpoint, change in left ventricular ejection fraction assessed by three‐dimensional (3D) echo from baseline to 3 month follow‐up and the secondary endpoints change in left ventricular end‐systolic and end‐diastolic volume. The secondary objective is to determine if ferric carboxymaltose is capable of improving cardiac contractility in vivo, by assessing the force–frequency relationship through incremental biventricular pacing. A total of 100 patients will be randomized in a 1:1 fashion.ConclusionsThe IRON‐CRT trial will determine the effect of ferric carboxymaltose on cardiac reverse remodelling and rate‐dependent cardiac contractility in HFrEF patients.

Impact of heart rate on cross-bridge cycling kinetics in failing and nonfailing human myocardium.

The force-frequency relationship (FFR) is an important regulatory mechanism that increases the force-generating capacity as well as the contraction and relaxation kinetics in human cardiac muscle as the heart rate increases. In human heart failure, the normally positive FFR often becomes flat, or even negative. The rate of cross-bridge cycling, which has been reported to affect cardiac output, could be potentially dysregulated and contribute to blunted or negative FFR in heart failure. We recently developed and herein use a novel method for measuring the rate of tension redevelopment. This method allows us to obtain an index of the rate of cross-bridge cycling in intact contracting cardiac trabeculae at physiological temperature and assess physiological properties of cardiac muscles while preserving posttranslational modifications representative of those that occur in vivo. We observed that trabeculae from failing human hearts indeed exhibit an impaired FFR and a reduced speed of relaxation kinetics. However, stimulation frequencies in the lower spectrum did not majorly affect cross-bridge cycling kinetics in nonfailing and failing trabeculae when assessed at maximal activation. Trabeculae from failing human hearts had slightly slower cross-bridge kinetics at 3 Hz as well as reduced capacity to generate force upon K+ contracture at this frequency. We conclude that cross-bridge kinetics at maximal activation in the prevailing in vivo heart rates are not majorly impacted by frequency and are not majorly impacted by disease.NEW & NOTEWORTHY In this study, we confirm that cardiac relaxation kinetics are impaired in filing human myocardium and that cross-bridge cycling rate at resting heart rates does not contribute to this impaired relaxation. At high heart rates, failing myocardium cross-bridge rates are slower than in nonfailing myocardium.

Different Responses to Drug Safety Screening Targets between Human Neonatal and Infantile Heart Tissue and Cardiac Bodies Derived from Human-Induced Pluripotent Stem Cells.

Aims Induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) have become a promising tool in cardiovascular safety pharmacology. Immaturity of iPS-CMs remains an ongoing concern. We compared electrophysiological and contractile features of cardiac bodies (hiPS-CBs) derived from human-induced pluripotent stem cells and human neonatal and infantile myocardial slices relevant for drug screening. Methods and Results Myocardial tissue slices were prepared from biopsies obtained from patients undergoing surgery for hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot (TOF). Electrophysiological features and response to Ik,r blockade as well as contractile properties were investigated using microelectrodes and isometric force measurements and were compared to hiPS-CBs. Both native myocardial tissue slices as well as hiPS-CBs showed action potential prolongation after Ik,r blockade, but early afterdepolarisations could be observed in native myocardial tissue slices only. The force-frequency relationship (FFR) varied at lower frequencies and was negative throughout at higher frequencies in hiPS-CBs. In contrast, native myocardial tissue slices exhibited positive, negative, and biphasic FFRs. In contrast to native myocardial tissue slices, hiPS-CBs failed to show an inotropic response to ß-adrenergic stimulation. Although all groups showed ß-adrenergic induced positive lusitropy, the effect was more pronounced in myocardial tissue slices. Conclusion hiPS-CBs were able to reproduce AP prolongation after Ik,r blockade, but to a lesser extent compared to human neonatal and infantile myocardial tissue slices. Early afterdepolarisations could not be induced in hiPS-CBs. Contractile force was differently regulated by β-adrenergic stimulation in hiPS-CBs and the native myocardium. If used for cardiotoxicity screening, caution is warranted as hiPS-CBs might be less sensitive to pharmacologic targets compared to the native myocardium of neonates and infants.

Force-frequency relationship and early relaxation kinetics are preserved upon sarcoplasmic blockade in human myocardium.

In this study, we investigated the quantitative and qualitative role of the sarcoplasmic reticulum (SR) in the regulation of the force‐frequency relationship (FFR). We blocked the function of SR with cyclopiazonic acid (CPA) and ryanodine and measured twitch kinetics and developed force at various stimulation frequencies in nonfailing and failing intact human right ventricular trabeculae. We found that developed forces are only slightly reduced upon SR blockade, while the positive FFR in nonfailing trabeculae and negative FFR in failing trabeculae were both preserved. The contraction kinetics (dF/dt, dF/dt/F, and time to peak), however, were significantly slower at all frequencies tested. Kinetics of first 50% of relaxation (RT50) was not affected by SR blockade. Kinetics of entire relaxation process (RT90) was overall slower at low frequencies, but not at high frequencies. From our findings, we conclude that the SR is not essential for FFR, and its role in regulation of FFR lies mostly in contraction kinetics. Unlike small rodents, human myocardium contractile function is near‐normal in absence of a functional SR with little changes in contractile force, and with preservation with the main regulation of FFR.

An Augmented Negative Force-Frequency Relationship and Slowed Mechanical Restitution Are Associated With Increased Susceptibility to Drug-Induced Torsade de Pointes Arrhythmias in the Chronic Atrioventricular Block Dog.

Introduction: In the chronic AV-block (CAVB) dog model, structural, contractile, and electrical remodeling occur, which predispose the heart to dofetilide-induced Torsade de Pointes (TdP) arrhythmias. Previous studies found a relation between electrical remodeling and inducibility of TdP, while structural remodeling is not a prerequisite for arrhythmogenesis. In this study, we prospectively assessed the relation between in vivo markers of contractile remodeling and TdP inducibility.Methods: In 18 anesthetized dogs, the maximal first derivative of left ventricular pressure (LV dP/dtmax) was assessed at acute AV-block (AAVB) and after 2 weeks of chronic AV-block (CAVB2). Using pacing protocols, three markers of contractile remodeling, i.e., force-frequency relationship (FFR), mechanical restitution (MR), and post-extrasystolic potentiation (PESP) were determined. Infusion of dofetilide (0.025 mg/kg in 5 min) was used to test for TdP inducibility.Results: After infusion of dofetilide, 1/18 dogs and 12/18 were susceptible to TdP-arrhythmias at AAVB and CAVB2, respectively (p = 0.001). The inducible dogs at CAVB2 showed augmented contractility at a CL of 1200 ms (2354 ± 168 mmHg/s in inducible dogs versus 1091 ± 59 mmHg/s in non-inducible dogs, p < 0.001) with a negative FFR, while the non-inducible dogs retained their positive FFR. The time constant (TC) of the MR curve was significantly higher in the inducible dogs (158 ± 7 ms versus 97 ± 8 ms, p < 0.0001). Furthermore, a linear correlation was found between a weighted score of the number and severity of arrhythmias and contractile parameters, i.e., contractility at CL of 1200 ms (r = 0.73, p = 0.002), the slope of the FFR (r = -0.58, p = 0.01) and the TC of MR (r = 0.66, p = 0.003). Thus, more severe arrhythmias were seen in dogs with the most pronounced contractile remodeling.Conclusion: Contractile remodeling is concomitantly observed with susceptibility to dofetilide-induced TdP-arrhythmias. The inducible dogs show augmented contractile remodeling compared to non-inducible dogs, as seen by a negative FFR, higher maximal response of MR and PESP and slowed MR kinetics. These altered contractility parameters could reflect disrupted Ca2+ handling and Ca2+-overload, which predispose the heart to delayed- and early afterdepolarizations that could trigger TdP-arrhythmias.

The novel CaMKII inhibitor GS-680 reduces diastolic SR Ca leak and prevents CaMKII-dependent pro-arrhythmic activity

RationaleCa/calmodulin-dependent protein kinase II (CaMKII) was shown to increase diastolic sarcoplasmic reticulum (SR) Ca leak, which can result in delayed afterdepolarizations and triggered arrhythmias. Since increased CaMKII expression and activity has been mechanistically linked to arrhythmias in human heart failure (HF) and atrial fibrillation (AF), specific strategies aimed at CaMKII inhibition may have therapeutic potential. ObjectiveWe tested the antiarrhythmic and inotropic effects of a novel selective and ATP-competitive CaMKII inhibitor (GS-680). Methods and resultsTrabeculae were isolated from right atrial appendage biopsies of patients undergoing cardiac surgery. Premature atrial contractions (PACs) were induced by stimulation with isoproterenol (ISO, 100 nM) at increased [Ca]o (3.5 mM). Interestingly, compared to vehicle, PACs were significantly inhibited by exposure to GS-680 (at 100 and 300 nM). GS-680 also significantly decreased early and delayed afterdepolarizations in isolated human atrial myocytes. Moreover, GS-680 (at 100 or 300 nM) significantly inhibited diastolic SR Ca leak, measured as frequency of spontaneous SR Ca release events (Ca sparks) in isolated human atrial myocytes (Fluo-4 loaded) similar to the well-established peptide CaMKII inhibitor AIP. In accordance, GS-680 significantly reduced CaMKII autophosphorylation (Western blot) but enhanced developed tension after 10 or 30 s pause of electrical stimulation (post-rest behavior). Surprisingly, we found a strong negative inotropic effect of GS-680 in atrial trabeculae at 1 Hz stimulation rate, which was not observed at 4 Hz and abolished by beta-adrenergic stimulation. In contrast, GS-680 did not impair systolic force of isolated ventricular trabeculae from explanted hearts of heart transplant recipients at 1 Hz, blunted the negative force-frequency relationship (1–3 Hz) and significantly increased the Ca transient amplitude. ConclusionThe novel ATP-competitive and selective CaMKII inhibitor GS-680 inhibits pro-arrhythmic activity in human atrium and improves contractility in failing human ventricle, which may have therapeutic implications.

The reduced myofilament responsiveness to calcium contributes to the negative force-frequency relationship in rat cardiomyocytes: role of reactive oxygen species and p-38 map kinase.

The force-frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) on contractile force in response to an elevation of heart rate is present in the normal rat or in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as calcium-calmodulin-dependent protein kinase II (CaMKII) and p-38 MAP kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. The myocytes were field-stimulated via two-platinum electrodes. Sarcomere length was recorded with a video camera. Ca2+ transients and intracellular pHi were recorded by epifluorescence. Increasing frequency from 0.5 to 3Hz decreased cell shortening without changes in pHi. This negative FFR was changed to positive FFR when the myocytes were pre-incubated with the ROS scavenger MPG, the NADPH oxidase blocker apocynin, or by inhibiting mitochondrial ROS production with 5-HD. Similar results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93, or the p-38K inhibitor, SB-202190. Consistently, the levels of phosphorylation of p-38K and the oxidation of CaMKII were significantly higher at 2Hz than at 0.5Hz. Despite the presence of positive inotropic effect during stimulation frequency enhancement, Ca2+ transient amplitudes were reduced in MPG- and SB-202190-treated myocytes. In conclusion, our results indicate that the activation of the intracellular pathway involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca2+.

Mechanical Stress Conditioning and Electrical Stimulation Promote Contractility and Force Maturation of Induced Pluripotent Stem Cell-Derived Human Cardiac Tissue.

Tissue engineering enables the generation of functional human cardiac tissue with cells derived in vitro in combination with biocompatible materials. Human-induced pluripotent stem cell-derived cardiomyocytes provide a cell source for cardiac tissue engineering; however, their immaturity limits their potential applications. Here we sought to study the effect of mechanical conditioning and electric pacing on the maturation of human-induced pluripotent stem cell-derived cardiac tissues. Cardiomyocytes derived from human-induced pluripotent stem cells were used to generate collagen-based bioengineered human cardiac tissue. Engineered tissue constructs were subjected to different mechanical stress and electric pacing conditions. The engineered human myocardium exhibits Frank-Starling-type force-length relationships. After 2 weeks of static stress conditioning, the engineered myocardium demonstrated increases in contractility (0.63±0.10 mN/mm2 vs 0.055±0.009 mN/mm2 for no stress), tensile stiffness, construct alignment, and cell size. Stress conditioning also increased SERCA2 (Sarco/Endoplasmic Reticulum Calcium ATPase 2) expression, which correlated with a less negative force-frequency relationship. When electric pacing was combined with static stress conditioning, the tissues showed an additional increase in force production (1.34±0.19 mN/mm2), with no change in construct alignment or cell size, suggesting maturation of excitation-contraction coupling. Supporting this notion, we found expression of RYR2 (Ryanodine Receptor 2) and SERCA2 further increased by combined static stress and electric stimulation. These studies demonstrate that electric pacing and mechanical stimulation promote maturation of the structural, mechanical, and force generation properties of human-induced pluripotent stem cell-derived cardiac tissues.

Revealing calcium fluxes by analyzing inhibition dynamics in action potential clamp

In cardiac excitation-contraction coupling (ECC), calcium enters the cytosol via L-type Ca2+ channels (LTCC) and reverse Na+/Ca2+-exchange (NCXrev), or is released from the sarcoplasmic reticulum (SR) by Ca2+-induced Ca2+-release (CICR). The magnitude of Ca2+ influx via the different pathways varies with the state of the cell and is difficult to assess quantitatively, because changes in Ca2+ influx through one pathway affect the others. In rainbow trout ventricular myocytes, the role of the SR has been uncertain for decades. The aim of this work was therefore two-fold: 1) to develop a method to quantify the Ca2+ influx pathways, and 2) to determine the role of CICR from the SR in trout ventricular myocytes. The novelty of our developed method lies in the mathematical analysis of measured transsarcolemmal Ca2+ currents and their impact on the corresponding Ca2+ transient during gradual inhibition of the currents in action potential (AP) clamp. We tested the developed method using an excitation-contraction model and showed that the method was able to recover calcium fluxes from noisy synthetic data. We applied the approach to trout ventricular myocytes and quantified the relative contributions of different Ca2+ influx pathways in ECC and determined the kinetics of these fluxes. Under baseline conditions, NCXrev is the main transmembrane Ca2+ influx pathway contributing 29 ± 6% (of the Ca2+ influx), LTCC 18 ± 7%, and CICR 53 ± 10% to overall Ca2+ transient. Thus, NCXrev is an important regulator of contractility and probably plays a role in the negative force-frequency relationship of trout ventricular preparations. These results demonstrate that trout and neonatal mammalian cardiomyocytes resemble each other not only in terms of morphology and energetics but ECC as well. In summary, the developed method resolves the major problem how to separate highly interconnected fluxes in AP clamp and allows to study Ca2+ fluxes in cardiomyocytes under conditions close to in vivo.

Pathophysiological mechanism and therapeutic role of S100 proteins in cardiac failure: a systematic review.

S100 proteins are a family of highly acidic calcium-binding proteins involved in calcium handling in many tissues and organs. Some of these proteins are highly expressed in cardiac tissue, and an impairment of some specific S100 proteins has been related to heart failure. To check this hypothesis, we decided to review the literature since 2008 until May 2015. According to the studies collected, recovering S100A1 levels may enhance contractile/relaxing performance in heart failure, reverse negative force-frequency relationship, improve contractile reserve, reverse diastolic dysfunction and protect against pro-arrhythmic reductions of sarcoplasmic reticulum calcium. The safety profile of gene therapy was also confirmed. Increased S100B protein levels were related to a worse outcome in chronic heart failure. S100A8/A9 complex plasma levels, as well as other inflammatory biomarkers, were significantly higher in chronic heart failure patients. S100A2 seems to increase both contractile and relaxation performance in animal cardiomyocytes. Otherwise, S100A6 cardiac expression seems to have no effects on contractility. S100A4 KO mice showed reduced cardiac interstitial fibrosis. Data collected encourage a potential prospective application in human. These proteins could be exploited as biomarkers in stadiation and prognosis of chronic heart failure, as well as therapeutic target to rescue failing heart. Registration details The study protocol has been registered in PROSPERO ( http://www.crd.york.ac.uk/PROSPERO/ ) under registration number CRD42015027932.

Is rate–pressure product of any use in the isolated rat heart? Assessing cardiac ‘effort’ and oxygen consumption in the Langendorff‐perfused heart

New Findings What is the central question of this study? Rate–pressure product (RPP) is commonly used as an index of cardiac ‘effort’. In canine and human hearts (which have a positive force–frequency relationship), RPP is linearly correlated with oxygen consumption and has therefore been widely adopted as a species‐independent index of cardiac work. However, given that isolated rodent hearts demonstrate a negative force–frequency relationship, its use in this model requires validation. What is the main finding and its importance? Despite its widespread use, RPP is not correlated with oxygen consumption (or cardiac ‘effort’) in the Langendorff‐perfused isolated rat heart. This lack of correlation was also evident when perfusions included a range of metabolic substrates, insulin or β‐adrenoceptor stimulation. Langendorff perfusion of hearts isolated from rats and mice has been used extensively for physiological, pharmacological and biochemical studies. The ability to phenotype these hearts reliably is, therefore, essential. One of the commonly used indices of function is rate–pressure product (RPP); a rather ill‐defined index of ‘work’ or, more correctly, ‘effort’. Rate–pressure product, as originally described in dog or human hearts, was shown to be correlated with myocardial oxygen consumption (MV˙O2). Despite its widespread use, the application of this index to rat or mouse hearts (which, unlike the dog or human, have a negative force–frequency relationship) has not been characterized. The aim of this study was to examine the relationship between RPP and MV˙O2 in Langendorff‐perfused rat hearts. Paced hearts (300–750 beats min−1) were perfused either with Krebs–Henseleit (KH) buffer (11 mm glucose) or with buffer supplemented with metabolic substrates and insulin. The arteriovenous oxygen consumption (MV˙O2) was recorded. Metabolic status was assessed using 31P magnetic resonance spectroscopy and lactate efflux. Experiments were repeated in the presence of isoprenaline and in unpaced hearts where heart rate was increased by cumulative isoprenaline challenge. In KH buffer‐perfused hearts, MV˙O2 increased with increasing heart rate, but given that left ventricular developed pressure decreased with increases in rate, RPP was not correlated with MV˙O2, lactate production or phosphocreatine/ATP ratio. Although the provision of substrates or β‐adrenoceptor stimulation changed the shape of the RPP–MV˙O2 relationship, neither intervention resulted in a positive correlation between RPP and oxygen consumption. Rate–pressure product is therefore an unreliable index of oxygen consumption or ‘cardiac effort’ in the isolated rat heart.

Alternagin-C (ALT-C), a disintegrin-like protein from Rhinocerophis alternatus snake venom promotes positive inotropism and chronotropism in fish heart

Alternagin-C (ALT-C) is a disintegrin-like protein purified from the venom of the snake, Rhinocerophis alternatus. Recent studies showed that ALT-C is able to induce vascular endothelial growth factor (VEGF) expression, endothelial cell proliferation and migration, angiogenesis and to increase myoblast viability. This peptide, therefore, can play a crucial role in tissue regeneration mechanisms. The aim of this study was to evaluate the effects of a single dose of alternagin-C (0.5 mg kg−1, via intra-arterial) on in vitro cardiac function of the freshwater fish traíra, Hoplias malabaricus, after 7 days. ALT-C treatment increased the cardiac performance promoting: 1) significant increases in the contraction force and in the rates of contraction and relaxation with concomitant decreases in the values of time to the peak tension and time to half- and 90% relaxation; 2) improvement in the cardiac pumping capacity and maximal electrical stimulation frequency, shifting the optimum frequency curve upward and to the right; 3) increases in myocardial VEGF levels and expression of key Ca2+-cycling proteins such as SERCA (sarcoplasmic reticulum Ca2+-ATPase), PLB (phospholamban), and NCX (Na+/Ca2+ exchanger); 4) abolishment of the typical negative force–frequency relationship of fish myocardium. In conclusion, this study indicates that ALT-C improves cardiac function, by increasing Ca2+ handling efficiency leading to a positive inotropism and chronotropism. The results suggest that ALT-C may lead to better cardiac output regulation indicating its potential application in therapies for cardiac contractile dysfunction.

The Frank-Starling mechanism involves deceleration of cross-bridge kinetics and is preserved in failing human right ventricular myocardium.

Cross-bridge cycling rate is an important determinant of cardiac output, and its alteration can potentially contribute to reduced output in heart failure patients. Additionally, animal studies suggest that this rate can be regulated by muscle length. The purpose of this study was to investigate cross-bridge cycling rate and its regulation by muscle length under near-physiological conditions in intact right ventricular muscles of nonfailing and failing human hearts. We acquired freshly explanted nonfailing (n = 9) and failing (n = 10) human hearts. All experiments were performed on intact right ventricular cardiac trabeculae (n = 40) at physiological temperature and near the normal heart rate range. The failing myocardium showed the typical heart failure phenotype: a negative force-frequency relationship and β-adrenergic desensitization (P < 0.05), indicating the expected pathological myocardium in the right ventricles. We found that there exists a length-dependent regulation of cross-bridge cycling kinetics in human myocardium. Decreasing muscle length accelerated the rate of cross-bridge reattachment (ktr) in both nonfailing and failing myocardium (P < 0.05) equally; there were no major differences between nonfailing and failing myocardium at each respective length (P > 0.05), indicating that this regulatory mechanism is preserved in heart failure. Length-dependent assessment of twitch kinetics mirrored these findings; normalized dF/dt slowed down with increasing length of the muscle and was virtually identical in diseased tissue. This study shows for the first time that muscle length regulates cross-bridge kinetics in human myocardium under near-physiological conditions and that those kinetics are preserved in the right ventricular tissues of heart failure patients.

Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca2+-flux and sarcoplasmic reticulum Ca2+-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca2+-concentration, trans-sarcolemmal Ca2+-flux via L-type Ca2+-channels and Na+-Ca2+-exchanger, and Ca2+-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca2+-channels by verapamil (1 μM) decreased force of contraction to 22±7% compared to baseline (n=4, p<0.05). Ni2+ was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73±3 ms vs. 101±8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca2+-release decreased force development to 54±3% in ventricular (n=13, p<0.05) and to 52±8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca2+-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169±7% force compared to baseline, n=8, p<0.05) and 10 s (198±9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca2+-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

Cardiac hypertrophy in mice expressing unphosphorylatable phospholemman.

AimsElevation of intracellular Na in the failing myocardium contributes to contractile dysfunction, the negative force–frequency relationship, and arrhythmias. Although phospholemman (PLM) is recognized to form the link between signalling pathways and Na/K pump activity, the possibility that defects in its regulation contribute to elevation of intracellular Na has not been investigated. Our aim was to test the hypothesis that the prevention of PLM phosphorylation in a PLM3SA knock-in mouse (in which PLM has been rendered unphosphorylatable) will exacerbate cardiac hypertrophy and cellular Na overload. Testing this hypothesis should determine whether changes in PLM phosphorylation are simply bystander effects or are causally involved in disease progression.Methods and resultsIn wild-type (WT) mice, aortic constriction resulted in hypophosphorylation of PLM with no change in Na/K pump expression. This under-phosphorylation of PLM occurred at 3 days post-banding and was associated with a progressive decline in Na/K pump current and elevation of [Na]i. Echocardiography, morphometry, and pressure-volume (PV) catheterization confirmed remodelling, dilation, and contractile dysfunction, respectively. In PLM3SA mice, expression of Na/K ATPase was increased and PLM decreased such that net Na/K pump current under quiescent conditions was unchanged (cf. WT myocytes); [Na+]i was increased and forward-mode Na/Ca exchanger was reduced in paced PLM3SA myocytes. Cardiac hypertrophy and Na/K pump inhibition were significantly exacerbated in banded PLM3SA mice compared with banded WT.ConclusionsDecreased phosphorylation of PLM reduces Na/K pump activity and exacerbates Na overload, contractile dysfunction, and adverse remodelling following aortic constriction in mice. This suggests a novel therapeutic target for the treatment of heart failure.