Force-frequency Relationship Research Articles

Biventricular contractility during exercise in adults with small, unrepaired atrial septal defects.

Adult patients with a small, unrepaired atrial septal defect (ASD) have higher mortality and increased risk of several comorbidities compared to general population. Further, reduced submaximal and peak exercise capacity was recently demonstrated in these patients. The mechanisms behind these findings remain unexplained and, therefore, biventricular contractility during exercise was assessed by evaluating the force-frequency relationship in the same group of patients. Adults patients with a small, unrepaired ASD and healthy age- and gender-matched controls were examined using echocardiography during supine bicycle exercise. Continuous tissue velocity Doppler was used to evaluate isovolumetric acceleration (IVA) and systolic velocities during an incremental workload protocol. All data were analyzed post hoc in a blinded fashion. We included 30 patients previously diagnosed with a small, unrepaired ASD (mean age 35years, 63% female) and 25 controls (mean age 34years, 64% female). Patients had similar values of IVA and systolic velocities at rest when compared with the healthy controls. Further, no differences in IVA was found at peak heart rate for neither the left ventricle (90±39 vs 129±68cm/s2 , P=0.1547) nor the right ventricle (128±56 vs 154±56cm/s2 , 0.5691). There were no correlations between peak velocities and the lower exercise capacity previously reported in these patients. Adult patients with a small, unrepaired ASD have normal biventricular contractility at rest and during exercise when compared with healthy peers. Consequently, the pathophysiological mechanisms behind the impaired exercise capacity previously demonstrated in these patients remains unknown and will be a target for future work.

Influence of sildenafil on the purinergic components of nerve-mediated and urothelial ATP release from the bladder of normal and spinal cord injured mice.

Background and purposePDE inhibitors such as sildenafil alleviate lower urinary tract symptoms; however, a complete understanding of their action on the bladder remains unclear. We are investigating the effects of sildenafil, on post and preganglionic nerve‐mediated contractions of the mouse bladder, and neuronal and urothelial ATP release.Experimental approachBladders were used from young (12 weeks), aged (24 months), and spinal cord transected (SCT), mice, for in vitro contractility experiments. An arterially perfused in situ whole mouse model was used to record bladder pressure. Nerve‐mediated contractions were generated by electrical field stimulation (EFS) of postganglionic nerve terminals or the pelvic nerve. ATP release during EFS in intact detrusor strips, and during stretch of isolated mucosa strips, was measured using a luciferin‐luciferase assay.Key resultsSildenafil (20 μM) inhibited nerve‐mediated contractions in young mice, with an increase in f 1/2 values in force–frequency relationships, demonstrating a greater effect at low frequencies. Sildenafil reduced the atropine‐resistant, purinergic component of nerve‐mediated contractions, and suppressed neuronal ATP release upon EFS in vitro. Sildenafil reduced the preganglionic pelvic nerve stimulated bladder pressure recordings in situ; comparable to in vitro experiments. Sildenafil reduced stretch‐induced urothelial ATP release. Sildenafil also relaxed nerve‐mediated contractions in aged and SCT mice.Conclusion and implicationsSildenafil has a greater effect on the low‐frequency, purinergic‐mediated contractions and suppresses neuronal ATP release. In addition, sildenafil reduces stretch‐induced urothelial ATP release. These results demonstrate a novel action of sildenafil to selectively inhibit ATP release from nerve terminals innervating detrusor smooth muscle and the urothelium.

The Effect of Vitamin D Supplementation on Skeletal Muscle in the mdx Mouse Model of Duchenne Muscular Dystrophy.

Vitamin D (VitD) has shown to be beneficial in reversing muscle weakness and atrophy associated with VitD deficiency. Duchenne muscular dystrophy is characterized by worsening muscle weakness and muscle atrophy, with VitD deficiency commonly observed. This study aimed to investigate the effect of VitD supplementation on dystrophic skeletal muscle. Eight-week old female control (C57BL/10; n = 29) and dystrophic (C57BL/mdx; n = 23) mice were randomly supplemented with one of three VitD enriched diets (1000, 8000 & 20,000 IU/kg chow). Following a four-week feeding period, the extensor digitorum longus (EDL) and soleus muscles contractile and fatigue properties were tested ex vivo, followed by histological analysis. As expected, mdx muscles displayed higher mass yet lower specific forces and a rightward shift in their force frequency relationship consistent with dystrophic pathology. There was a trend for mdx muscle mass to be larger following the 20,000 IU/kg diet, but this did not result in improved force production. Fiber area in the EDL was larger in mdx compared to controls, and there were higher amounts of damage in both muscles, with VitD supplementation having no effect. Four weeks of VitD supplementation did not appear to have any impact upon dystrophic skeletal muscle pathology at this age.

Engineering microenvironment for human cardiac tissue assembly in heart-on-a-chip platform

Organ-on-a-chip systems have the potential to revolutionize drug screening and disease modeling through the use of human stem cell-derived cardiomyocytes. The predictive power of these tissue models critically depends on the functional assembly and maturation of human cells that are used as building blocks for organ-on-a-chip systems. To resemble a more adult-like phenotype on these heart-on-a-chip systems, the surrounding micro-environment of individual cardiomyocyte needs to be controlled. Herein, we investigated the impact of four microenvironmental cues: cell seeding density, types and percentages of non-myocyte populations, the types of hydrogels used for tissue inoculation and the electrical conditioning regimes on the structural and functional assembly of human pluripotent stem cell-derived cardiac tissues. Utilizing a novel, plastic and open-access heart-on-a-chip system that is capable of continuous non-invasive monitoring of tissue contractions, we were able to study how different micro-environmental cues affect the assembly of the cardiomyocytes into a functional cardiac tissue. We have defined conditions that resulted in tissues exhibiting hallmarks of the mature human myocardium, such as positive force-frequency relationship and post-rest potentiation.

The effects of fatigue and oxidation on contractile function of intact muscle fibers and myofibrils isolated from the mouse diaphragm

The goal of this study was to investigate the effects of repetitive stimulation and the oxidant H2O2 on fatigue of diaphragm intact fibers and in myofibrils measured with different Ca2+ concentrations. Intact fibers were isolated from mice diaphragm, and twitch and tetanic contractions (500 ms duration) were performed at different frequencies of stimulation ranging from 15 Hz to 150 Hz to establish a force-frequency relation before and after a fatigue and recovery protocol, without or after a treatment with H2O2. Fatigue was induced with isometric contractions (500 ms, 40 Hz) evoked every 0.8 seconds, with a total of 625 tetani. After the fatigue, the force recovery was followed by invoking tetanic contractions (500 ms, 40 Hz) every 1 min, with a total duration of 30 min. Individual myofibrils were also isolated from the mouse diaphragm and were tested for isometric contractions before and after treatment with H2O2 and NAC. In a second series of experiments, myofibrils were activated at different pCa (pCa = −log10 [Ca2+]), before and after H2O2 treatment. After 15 minutes of H2O2 treatment, the myofibrillar force was decreased to 54 ± 12% of its control, maximal value, and a result that was reversed by NAC treatment. The force was also decreased after myofibrils were treated with H2O2 and activated in pCa ranging between 4.5 and 5.7. These results suggest that fatigue in diaphragm intact fibers and at the myofibrils level is caused partially by oxidation of the contractile proteins that may be responsible for changing the force in various levels of Ca2+ activation.

A Mathematical Model of dP/dt Max for the Evaluation of the Dynamic Control of Heart Contractility in Septic Shock

Septic shock (SS) patients often show elevated heart rate (HR) despite resuscitation, and this condition is considered an early manifestation of myocardial dysfunction due to an impairment of autonomic nervous system (ANS). We aimed at proposing a mathematical model to assess the autonomic control of ventricular contractility (VC) and HR to track changes in heart functionality during an experimental animal model of SS and resuscitation. SS was induced in six adult swine by polymicrobial peritonitis. We analyzed the beat-to-beat variability of the maximum positive time derivative of left ventricular pressure (dP/dt max), heart period (HP), and aortic blood pressure (ABP). We identified the transfer functions relating fluctuations in ABP and HP to dP/dt max to characterize the static and dynamic properties of the arterial baroreflex and the force-frequency relation mechanisms, respectively. Standard indices of autonomic dysfunction have also been considered as HR variability (HRV) and baroreflex sensitivity (BRS). During baseline, the baroreflex is predominant in controlling VC with a gain value of -5.8 (-7.5,-3) s-1, compared to -1.2 (-1.9,-0.5) mmHg/s ms-1 of the force-frequency autoregulation. During shock, both mechanisms increase their extent in VC control (higher gains and slightly faster dynamics for the baroreflex). After resuscitation, the physiological control of VC is not restored and all the animals still exhibit high HR and reduced HRV and BRS. A condition of cardiovascular inefficiency is persistent after resuscitation and this could be due to autonomic dysfunction. The ANS in SS is crucial to restore homeostasis. Our model could be used to evaluate the efficacy of treatments on VC and related control mechanisms.

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.

Left and Right Ventricular Systolic and Diastolic Functional Reserves Are Impaired in Anthracycline-Treated Long-Term Survivors of Childhood Cancers

The aim of this study was to test the hypothesis that left ventricular (LV) and right ventricular (RV) functional reserves are altered in anthracycline-treated long-term survivors of childhood cancers. One hundred three survivors (55% men) aged 25.0±5.8years at 15.2±5.8years after chemotherapy and 61 healthy control subjects (52% men) were studied. Tissue Doppler-derived mitral and tricuspid systolic (s) and early diastolic (e) velocities and LV myocardial acceleration during isovolumic contraction (IVA) were determined at rest and during bicycle exercise. The slope of the LV force-frequency relationship was derived from changes in IVA with heart rate during exercise (ΔIVA/Δ[heart rate]). LV and RV functional reserves were further assessed by the systolic functional reserve index (Δs×[1-1/s at baseline]) and diastolic functional reserve index (Δe×[1-1/e at baseline]). At baseline, mitral annular tissue Doppler indices were similar between survivors and control subjects (P>.05 for all), while tricuspid s and e velocities were significantly lower in survivors (P<.05 for both). The force-frequency relationship slope (P<.001), LV systolic functional reserve index (P<.001), and RV systolic functional reserve index (P=.001) were significantly lower in survivors than control subjects. For diastolic functional reserve, LV but not RV diastolic functional reserve index was significantly lower in survivors (P<.001). Multivariate analysis revealed survivor status (β=-0.39, P<.001) and baseline LV IVA (β=0.15, P<.044) as significant determinants of the LV force-frequency relationship. LV and RV functional reserves during exercise are impaired in anthracycline-treated long-term survivors of childhood cancer.

Ventricular force-frequency relationships during biventricular or multisite pacing in congenital heart disease.

Traditional indices to evaluate biventricular (BiV) pacing are load dependent, fail to assess dynamic changes, and may not be appropriate in patients with congenital heart disease (CHD). We therefore measured the force-frequency relationship (FFR) using tissue Doppler-derived isovolumic acceleration (IVA) to assess the dynamic adaption of the myocardium and its variability with different ventricular pacing strategies. This was a prospective pilot study of pediatric and young adult CHD patients with biventricular or multisite pacing systems. Color-coded myocardial velocities were recorded at the base of the systemic ventricular free wall. IVA was calculated at resting heart rate and with incremental pacing. FFR curves were obtained by plotting IVA against heart rate for different ventricular pacing strategies. Ten patients were included (mean: 22±7years). The FFR identified a best and worst ventricular pacing strategy for each patient, based on the AUC at baseline, submaximal, and peak heart rates (P<.001). However, there was no single best ventricular pacing strategy that was optimal for all patients. Additionally, the best ventricular pacing strategy often differed within the same patient at different heart rates. This novel assessment demonstrates a wide variability in optimal ventricular pacing strategy. These inherent differences may play a role in the unpredictable clinical response to BiV pacing in CHD, and emphasizes an individualized approach. Furthermore, the optimal ventricular pacing varies with heart rate within individuals, suggesting that rate-responsive ventricular pacing modulation may be required to optimize ventricular performance.

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.

Etiology-dependent impairment of relaxation kinetics in right ventricular end-stage failing human myocardium

BackgroundIn patients with end-stage heart failure, the primary etiology often originates in the left ventricle, and eventually the contractile function of the right ventricle (RV) also becomes compromised. RV tissue-level deficits in contractile force and/or kinetics need quantification to understand involvement in ischemic and non-ischemic failing human myocardium. Methods and resultsThe human population suffering from heart failure is diverse, requiring many subjects to be studied in order to perform an adequately powered statistical analysis. From 2009-present we assessed live tissue-level contractile force and kinetics in isolated myocardial RV trabeculae from 44 non-failing and 41 failing human hearts. At 1 Hz stimulation rate (in vivo resting state) the developed active force was not different in non-failing compared to failing ischemic nor non-ischemic failing trabeculae. In sharp contrast, the kinetics of relaxation were significantly impacted by disease, with 50% relaxation time being significantly shorter in non-failing vs. non-ischemic failing, while the latter was still significantly shorter than ischemic failing. Gender did not significantly impact kinetics. Length-dependent activation was not impacted. Although baseline force was not impacted, contractile reserve was critically blunted. The force-frequency relation was positive in non-failing myocardium, but negative in both ischemic and non-ischemic myocardium, while the β-adrenergic response to isoproterenol was depressed in both pathologies. ConclusionsForce development at resting heart rate is not impacted by cardiac pathology, but kinetics are impaired and the magnitude of the impairment depends on the underlying etiology. Focusing on restoration of myocardial kinetics will likely have greater therapeutic potential than targeting force of contraction.

Changes in cardiac contractility during graded exercise are greater in subjects with smaller body mass index, and greater in men than women: analyses using wave intensity and force-frequency relations.

Estimation of the contractility of the left ventricle during exercise is an important part of the rehabilitation protocol. It is known that cardiac contractility increases with an increase in heart rate. This phenomenon is called the force-frequency relation (FFR). Using wave intensity, we aimed to evaluate FFR noninvasively during graded exercise. We enrolled 83 healthy subjects. Using ultrasonic diagnostic equipment, we measured wave intensity (WD), which was defined in terms of blood velocity and arterial diameter, in the carotid artery and heart rate (HR) before and during bicycle ergometer exercise. FFRs were constructed by plotting the maximum value of WD (WD1) against HR. We analyzed the variation among FFR responses of individual subjects. WD1 increased linearly with an increase in HR during exercise. The average slope of the FFR was 1.0 ± 0.5m/s3bpm. The slope of FFR decreased with an increase in body mass index (BMI). The slopes of FFRs were steeper in men than women, although there were no differences in BMI between men and women. The FFR was obtained noninvasively by carotid arterial wave intensity (WD1) and graded exercise. The slope of the FFR decreased with an increase in BMI, and was steeper in men than women.

Force‐frequency Relationship and Early Relaxation Kinetics Are Preserved Upon SR Blockade in Human Myocardium

Cardiomyocytes utilize calcium to activate their myofilaments and shorten, which at the organ level results in blood being pumped out of the heart and into the body. Sarcoplasmic reticulum (SR) is an organelle in cardiomyocytes that stores calcium ions and releases them during systole, when myofilament needs to be activated and sequesters them during diastole, when myofilament needs to be deactivated. Therefore, it is believed to play an integral role in the calcium handling of cardiomyocytes with potential implications in the force‐frequency relationship, as intracellular calcium is increased at higher heart rates. However, the SR is believed to be less important in calcium handling in larger mammals, which have sodium calcium exchangers (NCX) that can extrude calcium out of cardiomyocytes. We hypothesized that when the SR is blocked in human trabeculae, the relaxation function would not be affected and that force‐frequency relationship would be preserved. We isolated intact human right ventricular trabeculae from non‐failing and failing human hearts and measured various contractile and relaxation parameters upon SR blockade. We found that force‐frequency relationship is preserved even when the SR is blocked. We also found that early relaxation speed is not affected when the SR is blocked, probably because of NCX compensating for the lack of SR function. Moreover, we found that the contraction kinetics are slower upon SR blockade, potentially because L‐type calcium channel, which have slower calcium‐release kinetics than the SR, compensates for the lack of SR calcium releaseSupport or Funding InformationR01 HL113084 to Paul ML JanssenAHA 16PRE33410549 to Jae‐Hoon ChungThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Skeletal myofiber VEGF‐A‐targeted gene deletion induces mitochondrial, structural and contractile alterations in mouse diaphragm

Respiratory muscle weakness is a hallmark of COPD, heart failure, and critical illness. Vascular endothelial growth factor (VEGF) may be an important therapeutic target in chronic cardiopulmonary disease as it is essential for neovascularization and vessel repair following trauma. We hypothesized that respiratory muscle, which is required to persistently function, would exhibit impaired mitochondrial, structural, and contractile function in skeletal myofiber VEGF gene deleted mice.We measured diaphragm biochemistry, structure, and contractile function in adult mice with conditional deletion of the VEGF‐A gene in skeletal myofibers (SkmVEGF−/−). Male SkmVEGF−/− mice (n=12) were compared to wild type (WT) controls (n=13) – both on a C57BL/6J background. Fiber bundles were isolated and electrically stimulated in vitro to evaluate force‐frequency and fatigue profiles. Mitochondrial O2 consumption and H2O2 flux (index of mitochondrial ROS) were simultaneously measured in saponin‐permeabilized fiber bundles (Oroboros, Innsbruck, AT). Fiber structure and biochemistry were measured using standard techniques (IHC, Western, ELISA).Diaphragm VEGF protein was lower in SkmVEGF−/− than WT mice (1.5±0.2 vs. 3.0±0.4 pg/total protein, p&lt;0.05). Compared to WT mice, the force‐frequency relationship was depressed in SkmVEGF−/− (F[7,161]=15.0, p&lt;0.05), with maximal specific force reduced by ~10 % (24±1 vs. 21±1 N/cm2; p&lt;0.05). However, fatigue resistance tended to be greater following SkmVEGF−/− (F[2,115]=3.3, p=0.06). Fiber type proportions were unchanged, but type I fiber cross‐sectional area was reduced in SkmVEGF−/− (653±45 vs 493±53 μm2; p&lt;0.05). Compared to WT, sarcomeric actin protein expression was reduced by ~30% in SkmVEGF−/− (p&lt;0.05) while myosin heavy chain, MAFbx, MuRF1, PGC1α, and HIF1α protein remained unchanged (p&gt;0.05). Mitochondrial respiration was not different between SkmVEGF−/− and WT (F[1,80]=0.9, p&gt;0.05) in each of the respiratory states. However, H2O2 flux was reduced in SkmVEGF−/− (F[1,75]=14.6, p&lt;0.05).Skeletal myofiber‐specific VEGF deletion contributed to diaphragm weakness, but tended to improve fatigue resistance in this highly oxidative muscle. Reductions in oxidative fiber size, sarcomeric actin protein content, and ROS generation following VEGF gene deletion may contribute to protecting mitochondrial respiratory function in the diaphragm. The adaptations may be part of compensatory mechanisms mitigating larger deficits in force generation and fatigue resistance.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Macrophages are Required for Recovery from Physiological Muscle Stress in the mdx Mouse Model of Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal genetic disease associated with muscle degeneration and chronic inflammation. Current therapies are limited to corticosteroids which resolve inflammation at the expense of detrimental side effects, and exon skipping which is applicable only to a small subset of patients. Cardiosphere‐derived cells (CDCs) are cardiac progenitor/stromal cells with anti‐inflammatory, anti‐fibrotic, and regenerative properties. Recently, CDCs have been shown to reverse many of the abnormalities of heart and skeletal muscle in mdx mice, motivating the HOPE‐Duchenne clinical trial (Aminzadeh M, Rogers R et al., Stem Cell Reports 2018). Several lines of evidence now support the idea that CDCs exert their bioactivity by secreting nanoparticles called exosomes (CDC‐exos), which mediate cell‐cell transfer of genetic information. A single intravenous dose of CDC‐exos dramatically improves contractile function of the mdx mouse soleus, and restores muscle architecture. Given the anti‐inflammatory effects of CDC‐exos, RNA‐sequencing unexpectedly revealed a striking upregulation of inflammatory pathways in the solei of CDC‐exo treated mdx mice, as confirmed by confocal imaging of tissue macrophages using CD68 immunohistochemistry. Thus, we investigated the role of macrophages in mdx pathology. Clodronate liposomes were used to deplete monocytes and macrophages in vivo, as confirmed in blood by flow cytometry and in tissue by immunohistochemistry. Macrophage‐depleted and control mdx mice were subjected to a single bout of downhill running to induce muscle stress. Maximal exercise capacity was recorded prior to, and every other day for two weeks, following downhill running. Macrophage‐depleted mdx mice failed to recover during the two‐week period, whereas control mdx mice had recovered by the third day. Further, macrophage depletion shifted the force‐frequency relationship to higher frequencies and reduced maximal force developed by the soleus by 24%. Cultured macrophages from mdx bone marrow (BMDMs) express 4‐fold higher levels of the pro‐inflammatory cytokine TNF‐α than those from age‐matched wild‐type mice. In vitro conditioning of mdx BMDMs with CDC‐exos reduced the expression of TNF‐α, suggesting that CDC‐exos may skew mdx macrophage polarization toward a tissue healing phenotype. This interpretation is consistent with the finding that CDC‐exos polarize macrophages to a phenotype with enhanced efferocytosis capacity, minimizing scar formation after acute myocardial infarction. Thus, macrophages are required for recovery from physiological muscle stress in mdx mice, and CDC‐exos decrease pro‐inflammatory gene expression in macrophages. The unexpected requirement for macrophages in mdx skeletal muscle recovery from injury suggests that a macrophage population promotes tissue regeneration. We are currently testing the hypothesis that CDC‐exos may modulate the pro‐inflammatory phenotype of recruited macrophages to stimulate myogenesis and support mdx muscle regeneration.Support or Funding InformationCoalition DuchenneNIH T32 HL116273This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Advanced maturation of human cardiac tissue grown from pluripotent stem cells.

Cardiac tissues generated from human induced pluripotent stem (iPS) cells can serve as platforms for patient-specific studies of physiology and disease1–6. The predictive power of these models remains limited by their immature state1,2,5,6. We show that this fundamental limitation could be overcome if cardiac tissues are formed from early iPS-derived cardiomyocytes (iPS-CM), soon after the initiation of spontaneous contractions, and subjected to physical conditioning of an increasing intensity. After only 4 weeks of culture, these tissues displayed adult-like gene expression profiles, remarkably organized ultrastructure, physiologic sarcomere length (2.2 μm) and density of mitochondria (30%), the presence of transverse tubules (t-tubules), oxidative metabolism, positive force-frequency relationship, and functional calcium handling for all iPS cell lines studied. Electromechanical properties developed more slowly and did not achieve the stage of maturity seen in adult human myocardium. Tissue maturity was necessary for achieving physiologic responses to isoproterenol and recapitulating pathological hypertrophy, in support of the utility of this tissue model for studies of cardiac development and disease.

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.

Engineering human pluripotent stem cells into a functional skeletal muscle tissue

The generation of functional skeletal muscle tissues from human pluripotent stem cells (hPSCs) has not been reported. Here, we derive induced myogenic progenitor cells (iMPCs) via transient overexpression of Pax7 in paraxial mesoderm cells differentiated from hPSCs. In 2D culture, iMPCs readily differentiate into spontaneously contracting multinucleated myotubes and a pool of satellite-like cells endogenously expressing Pax7. Under optimized 3D culture conditions, iMPCs derived from multiple hPSC lines reproducibly form functional skeletal muscle tissues (iSKM bundles) containing aligned multi-nucleated myotubes that exhibit positive force–frequency relationship and robust calcium transients in response to electrical or acetylcholine stimulation. During 1-month culture, the iSKM bundles undergo increased structural and molecular maturation, hypertrophy, and force generation. When implanted into dorsal window chamber or hindlimb muscle in immunocompromised mice, the iSKM bundles survive, progressively vascularize, and maintain functionality. iSKM bundles hold promise as a microphysiological platform for human muscle disease modeling and drug development.

Rate-Response Programming Tailored to the Force-Frequency Relationship Improves Exercise Tolerance in Chronic Heart Failure

ObjectivesThis study sought to examine whether the heart rate (HR) at which the force-frequency relationship (FFR) slope peaks (critical HR) could be used to tailor HR response in chronic heart failure (CHF) patients with cardiac pacemakers and whether this favorably influences exercise capacity. BackgroundCHF secondary to left ventricular (LV) systolic dysfunction is characterized by blunting of the positive relationship between HR and LV contractility known as the FFR. MethodsThis observational study was carried out in patients with CHF and healthy subjects with pacemaker devices. The study assessed the 3 important features of the FFR (critical HR, peak contractility, and the FFR slope), and their reproducibility was measured noninvasively using echocardiography. The investigators then undertook a double-blind, randomized, controlled crossover study comparing the effects of tailored pacemaker rate-response programming on the basis of the FFR with conventional rate-response programming on exercise time and maximal oxygen consumption. ResultsThe study enrolled 90 patients with CHF into the observational cohort study: mean age, 73.6 ± 8.9 years; mean left ventricular ejection fraction (LVEF), 33.5 ± 10.9%. The study investigated 15 control subjects with normal LV function (LVEF, 55.6 ± 5.3%). The critical HR (103 ± 22 beats/min vs. 126 ± 15 beats/min; p = 0.0002), peak contractility (3.8 ± 3.7 SBP/LVESVI vs. 9.8 ± 4.1 SBP/LVESVI; p = 0.0001), and the slope of the FFR (p < 10−15) were lower in patients with CHF than in control subjects. A total of 52 patients, with a mean LVEF of 32 ± 11% on optimal therapy, took part in the crossover study. Rate-response settings limiting HR rise to below the critical HR led to greater exercise time (475 ± 189 s vs. 425 ± 196 s; p = 0.003) and higher peak oxygen consumption (17.3 ± 4.6 ml/kg/min vs. 16.6 ± 4.7 ml/kg/min; p = 0.01). ConclusionsA personalized approach to rate-response programming, determined using a reproducible noninvasive method for assessing the FFR, improves exercise time in patients with CHF and pacemaker devices. (Bowditch Revisited: Defining the Optimum Heart Rate Range in Chronic Heart Failure; NCT02563873)