Human Ventricular Muscle Research Articles

Studies of Ca2+-activated contractility using skinned cryosections of human ventricular papillary muscle samples from frozen storage of discarded clinical specimens collected in biorepository

Studies of Ca2+-activated contractility using skinned cryosections of human ventricular papillary muscle samples from frozen storage of discarded clinical specimens collected in biorepository

Isolated Cardiac Ryanodine Receptor Function Varies Between Mammals.

Concerted robust opening of cardiac ryanodine receptors' (RyR2) Ca2+ release 1oplasmic reticulum (SR) is fundamental for normal systolic cardiac function. During diastole, infrequent spontaneous RyR2 openings mediate the SR Ca2+ leak that normally constrains SR Ca2+ load. Abnormal large diastolic RyR2-mediated Ca2+ leak events can cause delayed after depolarizations (DADs) and arrhythmias. The RyR2-associated mechanisms underlying these processes are being extensively studied at multiple levels utilizing various model animals. Since there are well-described species-specific differences in cardiac intracellular Ca2+ handing in situ, we tested whether or not single RyR2 function in vitro retains this species specificity. We isolated RyR2-rich heavy SR microsomes from mouse, rat, rabbit, and human ventricular muscle and quantified RyR2 function using identical solutions and methods. The single RyR2 cytosolic Ca2+ sensitivity was similar across these species. However, there were significant species differences in single RyR2 mean open times in both systole and diastole-like solutions. In diastole-like solutions, single rat/mouse RyR2 open probability and frequency of long openings (> 6ms) were similar, but these values were significantly greater than those of either single rabbit or human RyR2s. We propose these in vitro single RyR2 functional differences across species stem from the species-specific RyR2 regulatory environment present in the source tissue. Our results show the single rabbit RyR2 functional attributes, particularly in diastole-like conditions, replicate those of single human RyR2 best among the species tested.

Abstract P1030: Alpha Kinase 3 Signaling At The M-band Regulates Sarcomeric Protein Quality Networks

Objectives: Striated muscle contraction is driven by molecular machinery composed of specialized cytoskeletal proteins, called the sarcomere. Pathogenic variants in genes that encode these sarcomeric proteins trigger a heart disease called cardiomyopathy, which causes abnormal growth and function of the heart muscle and can cause sudden death. However, there is a growing appreciation that genetic mutations in genes not encoding the traditional sarcomeric proteins also substantially contribute to cardiomyopathy. One such gene is ALPK3 , encoding an atypical alpha kinase. Despite its strong link to cardiomyopathy, there have been no studies to define its molecular function. Therefore, the objective of our study was to understand the genetic basis of ALPK3- induced cardiomyopathy and define its molecular function in health and disease. Methods & Results: We created a suite of gene-edited human pluripotent stem cells (hPSCs) carrying endogenous fusion proteins, loss of function mutations, or disease-causing patient variants, and differentiated these hPSC lines into human ventricular muscle cells. Using reporter hPSCs, we found that ALPK3 is localized to the M-band of the cardiac sarcomere, in contrast to its posited nuclear localization. ALPK3 deficiency impaired muscle cell function and sarcomere organization in both hPSC cardiac organoids and mice harboring a pathogenic ALPK3 variant. We performed multiomic analysis on ALPK3-deficient hPSC cardiomyocytes to define the function of ALPK3 and observed consistent enrichment of sarcomere and protein quality control pathways. Interactomics demonstrated that ALPK3 links the cytoskeleton to protein surveillance networks at the M-Band. Notably, the ubiquitin-binding protein SQSTM1 bound ALPK3, could be phosphorylated by ALPK3, and was mislocalized from the sarcomere in three independent ALPK3 patient lines. Conclusions: Together, our data demonstrate that ALPK3 regulates the link between the sarcomere and protein surveillance system and that its deficiency impairs cardiac function via this mechanism. This work highlights a potential new pathway to target for therapy development and treatment of patients with cardiomyopathy.

COVID-19 Severity Potentially Modulated by Cardiovascular-Disease-Associated Immune Dysregulation.

Patients with underlying cardiovascular conditions are particularly vulnerable to severe COVID-19. In this project, we aimed to characterize similarities in dysregulated immune pathways between COVID-19 patients and patients with cardiomyopathy, venous thromboembolism (VTE), or coronary artery disease (CAD). We hypothesized that these similarly dysregulated pathways may be critical to how cardiovascular diseases (CVDs) exacerbate COVID-19. To evaluate immune dysregulation in different diseases, we used four separate datasets, including RNA-sequencing data from human left ventricular cardiac muscle samples of patients with dilated or ischemic cardiomyopathy and healthy controls; RNA-sequencing data of whole blood samples from patients with single or recurrent event VTE and healthy controls; RNA-sequencing data of human peripheral blood mononuclear cells (PBMCs) from patients with and without obstructive CAD; and RNA-sequencing data of platelets from COVID-19 subjects and healthy controls. We found similar immune dysregulation profiles between patients with CVDs and COVID-19 patients. Interestingly, cardiomyopathy patients display the most similar immune landscape to COVID-19 patients. Additionally, COVID-19 patients experience greater upregulation of cytokine- and inflammasome-related genes than patients with CVDs. In all, patients with CVDs have a significant overlap of cytokine- and inflammasome-related gene expression profiles with that of COVID-19 patients, possibly explaining their greater vulnerability to severe COVID-19.

Electrical Restitution and Its Modifications by Antiarrhythmic Drugs in Undiseased Human Ventricular Muscle.

IntroductionRe-entry is a basic mechanism of ventricular fibrillation, which can be elicited by extrasystolic activity, but the timing of an extrasystole can be critical. The action potential duration (APD) of an extrasystole depends on the proximity of the preceding beat, and the relation between its timing and its APD is called electrical restitution. The aim of the present work was to study and compare the effect of several antiarrhythmic drugs on restitution in preparations from undiseased human ventricular muscle, and other mammalian species.MethodsAction potentials were recorded in preparations obtained from rat, guinea pig, rabbit, and dog hearts; and from undiseased human donor hearts using the conventional microelectrode technique. Preparations were stimulated with different basic cycle lengths (BCLs) ranging from 300 to 5,000 ms. To study restitution, single test pulses were applied at every 20th beat while the preparation was driven at 1,000 ms BCL.ResultsMarked differences were found between the animal and human preparations regarding restitution and steady-state frequency dependent curves. In human ventricular muscle, restitution kinetics were slower in preparations with large phase 1 repolarization with shorter APDs at 1000 ms BCL compared to preparations with small phase 1. Preparations having APD longer than 300 ms at 1000 ms BCL had slower restitution kinetics than those having APD shorter than 250 ms. The selective IKr inhibitors E-4031 and sotalol increased overall APD and slowed the restitution kinetics, while IKs inhibition did not influence APD and electrical restitution. Mexiletine and nisoldipine shortened APD, but only mexiletine slowed restitution kinetics.DiscussionFrequency dependent APD changes, including electrical restitution, were partly determined by the APD at the BCL. Small phase 1 associated with slower restitution suggests a role of Ito in restitution. APD prolonging drugs slowed restitution, while mexiletine, a known inhibitor of INa, shortened basic APD but also slowed restitution. These results indicate that although basic APD has an important role in restitution, other transmembrane currents, such as INa or Ito, can also affect restitution kinetics. This raises the possibility that ion channel modifier drugs slowing restitution kinetics may have antiarrhythmic properties by altering restitution.

Atrial-like Engineered Heart Tissue: An InVitro Model of the Human Atrium.

SummaryCardiomyocytes (CMs) generated from human induced pluripotent stem cells (hiPSCs) are under investigation for their suitability as human models in preclinical drug development. Antiarrhythmic drug development focuses on atrial biology for the treatment of atrial fibrillation. Here we used recent retinoic acid-based protocols to generate atrial CMs from hiPSCs and establish right atrial engineered heart tissue (RA-EHT) as a 3D model of human atrium. EHT from standard protocol-derived hiPSC-CMs (Ctrl-EHT) and intact human muscle strips served as comparators. RA-EHT exhibited higher mRNA and protein concentrations of atrial-selective markers, faster contraction kinetics, lower force generation, shorter action potential duration, and higher repolarization fraction than Ctrl-EHTs. In addition, RA-EHTs but not Ctrl-EHTs responded to pharmacological manipulation of atrial-selective potassium currents. RA- and Ctrl-EHTs’ behavior reflected differences between human atrial and ventricular muscle preparations. Taken together, RA-EHT is a model of human atrium that may be useful in preclinical drug screening.

Abstract 17085: Metabolic Signature of Human Right Ventricular Heart Muscle in Pulmonary Arterial Hypertension and Comparison to Two Animal Models

Introduction: Molecular studies of the human right ventricle (RV) in pulmonary arterial hypertension (PAH) are lacking. Metabolic changes in the failing RV vary across different animal models and have not been directly compared with the human RV. We hypothesized that the BMPR2 murine model of PAH would closely recapitulate metabolic changes in the human PAH RV Methods: We performed metabolomic profiling of 596 compounds (Metabolon) on: RV specimens from patients with PAH and non-PAH controls (n=3 per group), BMPR2 mice (n=15), wild-type mice after pulmonary artery banding (PAB) (n=7), and wild-type control mice (WT) (n=7). Normalized metabolites per group were compared by Welch’s t-test between two groups, and two-way ANOVA for >2 groups, followed by adjustment for multiple comparison analysis Results: Principal component analysis (PCA) revealed markedly different metabolic profiles between PAH and controls ( Figure 1A ), with significant changes in 131 biochemicals in PAH. We observed an increase in glycolysis as evident by lactate accumulation and reduction in glycolytic intermediaries. We also observed a substantial reduction in fatty acid oxidation in the PAH RV, characterized by markedly reduced acylcarnitines and accumulation of long chain fatty acids, lysolipids, and glycerol compounds ( Figure 1B ). The BMPR2 model significantly reproduced the direction of difference in 43/131 metabolites and the PAB model 29/131. Both models were associated with an increase in glycolysis but only the BMPR2 model showed evidence of impaired fatty acid oxidation (accumulation of long-chain fatty acids, lysolipids, glycerol, and monoacylglycerols), as observed in the human PAH RV ( Figure 1C ) Conclusions: The failing RV of patients with PAH has a distinct metabolic signature characterized by increased glycolysis and impaired fatty acid oxidation. The BMPR2 model of PAH recapitulates more of the key metabolic changes observed in humans compared with a model of isolated pressure overload. The BMPR2 model may be preferable for metabolic studies of the failing RV

Engineering human ventricular heart muscles based on a highly efficient system for purification of human pluripotent stem cell-derived ventricular cardiomyocytes

BackgroundMost infarctions occur in the left anterior descending coronary artery and cause myocardium damage of the left ventricle. Although current pluripotent stem cells (PSCs) and directed cardiac differentiation techniques are able to generate fetal-like human cardiomyocytes, isolation of pure ventricular cardiomyocytes has been challenging. For repairing ventricular damage, we aimed to establish a highly efficient purification system to obtain homogeneous ventricular cardiomyocytes and prepare engineered human ventricular heart muscles in a dish.MethodsThe purification system used TALEN-mediated genomic editing techniques to insert the neomycin or EGFP selection marker directly after the myosin light chain 2 (MYL2) locus in human pluripotent stem cells. Purified early ventricular cardiomyocytes were estimated by immunofluorescence, fluorescence-activated cell sorting, quantitative PCR, microelectrode array, and patch clamp. In subsequent experiments, the mixture of mature MYL2-positive ventricular cardiomyocytes and mesenchymal cells were cocultured with decellularized natural heart matrix. Histological and electrophysiology analyses of the formed tissues were performed 2 weeks later.ResultsHuman ventricular cardiomyocytes were efficiently isolated based on the purification system using G418 or flow cytometry selection. When combined with the decellularized natural heart matrix as the scaffold, functional human ventricular heart muscles were prepared in a dish.ConclusionsThese engineered human ventricular muscles can be great tools for regenerative therapy of human ventricular damage as well as drug screening and ventricular-specific disease modeling in the future.

Abstract 210: Titin Phosphorylation by Protein Kinase G as a Novel Mechanism of Diastolic Adaptation to Acute Hemodynamic Overload

Titin is the main determinant of myocardial passive tension and its distensibility is increased via phosphorylation by protein kinase G (PKG), activated by nitric oxide (NO) and natriuretic peptides (NP) upon acute overload. We hypothesized whether myocardial stretch led to decreased stiffness, optimizing diastolic filling along with the usual increase in contractility. Intact rat Langendorff hearts, strips dissected from the LV or right atrium of cardiac surgery patients and rabbit papillary muscles were acutely stretched for 15min. Passive tension (PT) was measured in skinned cardiomyocytes extracted from the LV of control and stretched rat hearts for sarcomere lengths (SL) 1.8-2.3μm before and after incubation with PKG. Rabbit muscles were incubated with a PKG inhibitor or, simultaneously, a NO synthase inhibitor, a NO scavenger, a NP receptor A antagonist. All-total titin phosphorylation was stained with Pro-Q Diamond and indexed to total-protein signals using SYPRO Ruby. Values are given as mean±SEM and statistical significance was set to p<0.05. After acute stretch there was a progressive decrease in passive tension/diastolic pressure over 15min: 27±8% and 27±6% in human atrium and ventricular muscles, respectively, and 43±2% in rabbit papillary muscles and isolated hearts. This decrease in myocardial stiffness was significantly blunted by PKG inhibition (40%) and NO/NP pathway inhibition (29%). PT of cardiomyocytes was significantly lower (≈60%) in the previously stretched group for all SL. A similar effect, only significant in the control group, was observed after incubation with PKG. Titin phosphorylation increased markedly 15 minutes after acute myocardial stretch in human (atrial: 11±1% vs 41±8%; LV: 27±8% vs 71±21%) and rabbit (13±2% vs 23±3%) myocardium. The progressive decrease in myocardial stiffness after acute hemodynamic overload is preserved at the myofilamental level and seems to depend on PKG activity, representing a potential therapeutic target in patients with pathologically rigid myocardium. Moreover, blocking PKG activation seems to attenuate this adaptive diastolic response. Therefore, titin phosphorylation by this kinase is probably involved in this new myocardial response to stretch in both animals and humans.

Ionic mechanisms limiting cardiac repolarization reserve in humans compared to dogs

The species-specific determinants of repolarization are poorly understood. This study compared the contribution of various currents to cardiac repolarization in canine and human ventricle. Conventional microelectrode, whole-cell patch-clamp, molecular biological and mathematical modelling techniques were used. Selective IKr block (50-100 nmol l(-1) dofetilide) lengthened AP duration at 90% of repolarization (APD90) >3-fold more in human than dog, suggesting smaller repolarization reserve in humans. Selective IK1 block (10 μmol l(-1) BaCl2) and IKs block (1 μmol l(-1) HMR-1556) increased APD90 more in canine than human right ventricular papillary muscle. Ion current measurements in isolated cardiomyocytes showed that IK1 and IKs densities were 3- and 4.5-fold larger in dogs than humans, respectively. IKr density and kinetics were similar in human versus dog. ICa and Ito were respectively ~30% larger and ~29% smaller in human, and Na(+)-Ca(2+) exchange current was comparable. Cardiac mRNA levels for the main IK1 ion channel subunit Kir2.1 and the IKs accessory subunit minK were significantly lower, but mRNA expression of ERG and KvLQT1 (IKr and IKs α-subunits) were not significantly different, in human versus dog. Immunostaining suggested lower Kir2.1 and minK, and higher KvLQT1 protein expression in human versus canine cardiomyocytes. IK1 and IKs inhibition increased the APD-prolonging effect of IKr block more in dog (by 56% and 49%, respectively) than human (34 and 16%), indicating that both currents contribute to increased repolarization reserve in the dog. A mathematical model incorporating observed human-canine ion current differences confirmed the role of IK1 and IKs in repolarization reserve differences. Thus, humans show greater repolarization-delaying effects of IKr block than dogs, because of lower repolarization reserve contributions from IK1 and IKs, emphasizing species-specific determinants of repolarization and the limitations of animal models for human disease.

Spatial and temporal ECM expression pattern during human cardiogenesis ‐ learning from nature's blueprint

During fetal cardiac development, extracellular matrix (ECM) compounds are recognized to be essential for cell differentiation and maturation processes. Thus, we aimed to identify spatial and temporal ECM expression pattern during human fetal cardiogenesis, in order to learn from these developmental cues for applictions in regenerative medicine. Therefore, we analyzed the temporal expression and the localization of six essential ECM molecules: collagen type I, III, IV and VI (COLI, COLIII, COLIV, COLVI), fibronectin (FN) and elastin (ELN). Here, we used human ventricular heart muscle tissues of different age groups: fetal first (week 4–12) and second (week 14–17) trimester, children (3–9 years), adults (50–70 years). We performed quantitative gene expression analysis of COL I, COL III, COL IV, COL VI, FN and ELN and analyzed temporal and spatial protein expression by immunofluorescence staining. We revealed a spatiotemporal distinct expression of collagen type IV during human cardiac development. Collagen type IV was highly expressed at the early onset of cardiogenesis, decreased during second trimester, where it was primarily detected in the epicardium, and increased postnatal around mature cardiomyocytes and in the epicardium. Collagen type I expression was the highest in first trimester ventricles and decreased during development. Here, we identified distinct spatiotemporal expression patterns of ECM proteins, which will serve as a blueprint for biomaterials that can be used for myocardial regeneration.

Comparison of the Intrinsic Vasorelaxant and Inotropic Effects of the Antiarrhythmic Agents Vernakalant and Flecainide in Human Isolated Vascular and Cardiac Tissues

This study explored the intrinsic vasorelaxant and inotropic effects of the mixed potassium and sodium channel blocker atrial antiarrhythmic vernakalant and the class IC antiarrhythmic agent flecainide in human isolated subcutaneous resistance artery and in ventricular trabecular muscle preparations. At test concentrations encompassing free plasma concentrations associated with clinical efficacy for conversion of atrial fibrillation, vernakalant (1-10 μM) displayed no significant direct effects on human resistance artery tone or ventricular contractility. In contrast, tested at equimolar concentrations, flecainide significantly reduced peak isometric contractile force (10 μM) and maximal rates of force development and decline (3 and 10 μM) in the human ventricular muscle preparation while displaying no significant effect on human resistance artery tone. The lack of effects of vernakalant on human resistance artery tone and ventricular muscle contractile function suggests that direct vasorelaxant and inotropic effects do not underlie the rare hypotensive events observed clinically with vernakalant, raising the possibility that secondary (eg, reflex) effects may mediate these events. The demonstration of negative inotropic effects with flecainide in the human ventricular muscle preparations in the absence of an effect on resistance artery tone suggests that the hemodynamic effects of flecainide observed clinically result primarily from direct negative inotropic effects.

KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

KCNE2 functions as an auxiliary subunit in voltage-gated K and HCN channels in the heart. Genetic variations in KCNE2 have been linked to long QT syndrome. The underlying mechanisms are not entirely clear. One of the issues is whether KCNE2 protein is expressed in ventricles. We use adenovirus-mediated genetic manipulations of adult cardiac myocytes to validate two antibodies (termed Ab1 and Ab2) for their ability to detect native KCNE2 in the heart. Ab1 faithfully detects native KCNE2 proteins in spontaneously hypertensive rat and guinea pig hearts. In both cases, KCNE2 protein is more abundant in ventricles than in atria. In both ventricular and atrial myocytes, KCNE2 protein is preferentially distributed on the cell surface. Ab1 can detect a prominent KCNE2 band in human ventricular muscle from nonfailing hearts. The band intensity is much fainter in atria and in failing ventricles. Ab2 specifically detects S98 phosphorylated KCNE2. Through exploring the functional significance of S98 phosphorylation, we uncover a novel mechanism by which KCNE2 modulates the human ether-a-go-go related gene (hERG) current amplitude: by accelerating hERG protein degradation and thus reducing the hERG protein level on the cell surface. S98 phosphorylation appears to be required for this modulation, so that S98 dephosphorylation leads to an increase in hERG/rapid delayed rectifier current amplitude. Our data confirm that KCNE2 protein is expressed in the ventricles of human and animal models. Furthermore, KCNE2 can modulate its partner channel function not only by altering channel conductance and/or gating kinetics, but also by affecting protein stability.

Erythropoietin induces positive inotropic and lusitropic effects in murine and human myocardium

Initial clinical studies indicate a potential beneficial effect of erythropoietin (EPO) in patients with anemia and heart failure. Here, we investigate the direct contractile effects of erythropoietin on myocardial tissue. Treatment with EPO (50U/mL) using excitable murine and human left ventricular muscle preparations resulted in a 37% and 62% increase in twitch tension, respectively (P<0.05). Isolated murine cardiomyocytes exposed to EPO demonstrated a 41% increase in peak sarcomere shortening (P=0.012). Using compounds that specifically stimulate a non-erythropoietic EPO receptor yielded similar increases in contractile dynamics. Cardiomyocyte Ca2+dynamics showed an 18% increase in peak calcium in EPO treated cardiomyocytes over controls (P=0.03). Studies in muscle strips skinned after EPO treatment demonstrated a phosphorylation dependant increase in the viscous modulus as well as an increase in oscillatory work. The EPO mediated increase in peak sarcomere shortening was abrogated by PI3-K blockade via wortmannin and by non-isozyme specific PKC blockade by chelerythrine. Finally, EPO treatment resulted in an increase in PKCε in the particulate cellular fraction, indicating activation of this isoform. EPO exhibits direct positive inotropic and lusitropic effects in cardiomyocytes and ventricular muscle preparation. These effects are mediated through PI3-K and PKCε isoform signaling to directly affect both calcium release dynamics and myofilament function.

Class I/B antiarrhythmic property of ranolazine, a novel antianginal agent, in dog and human cardiac preparations

The aim of this study was to investigate the cellular electrophysiological effects of ranolazine on action potential characteristics. The experiments were carried out in dog and human cardiac preparations using the conventional microelectrode technique. In dog Purkinje fibres ranolazine produced a concentration- and frequency-dependent depression of the maximum rate of depolarization (Vmax) while action potential duration (APD) was shortened. In dog and human right ventricular papillary muscle ranolazine exerted no significant effect on APD, while it produced, like mexiletine, use-dependent depression of Vmax with relatively fast onset and offset kinetics. In dog midmyocardial preparations the drug did not exert statistically significant effect on repolarization at 10μM, although a tendency toward prolongation was observed at 20μM. A moderate lengthening of APD90 by ranolazine was noticed in canine atrial preparations obtained from dogs in sinus rhythm and in tachypacing induced remodelled preparations. Use-dependent depression of Vmax was more pronounced in atria from dogs in sinus rhythm than those in remodelled atria or in the ventricle. These findings indicate that ranolazine, in addition to its known late sodium current blocking effect, also depresses peak INa with class I/B antiarrhythmic characteristics. Although peak INa inhibition by ranolazine is stronger in the atria, it is also substantial (at fast stimulation frequencies) in ventricular preparations. Ranolazine also decreased the dispersion of ventricular repolarization (the difference in APD90 values between Purkinje fibres and papillary muscles), which can contribute to the antiarrhythmic property of the drug.

Distribution of costameric proteins in normal human ventricular and atrial cardiac muscle.

In the mature heart, the intercalated disc and costameres provide the cell-cell and cell-matrix junctions respectively. Intercalated disc is situated at the bipolar ends of the cardiomyocytes and the myofibrils are anchored at this structure. The costameres mediate integration with the extracellular matrix that covers individual cardiomyocytes laterally. Costameres are considered as "proteic machinery" that appears to comprise two protein complexes: the dystrophin-glycoprotein complex (DGC) and the vinculin-talin-integrin system. There are structural differences between atrial and ventricular myocytes, but there have been relatively few studies that have analyzed costameres and focal adhesion function in cardiac cells. Our previous study carried out only on atrial myocytes, demonstrated that the DGC and talin-vinculin-integrin complexes had a costameric distribution that, unlike skeletal muscle, it localized only on the I band. We performed a further immunohistochemical analysis extending also the evaluation to the normal human cardiac muscle fibers obtained from ventricle and interventricular septum, in order to define the distribution and the spatial relationship between the proteins of the two complexes also in the other heart districts. Immunoconfocal microscopy of cardiac tissue revealed the costameric distribution of DGC and of vinculin-talin-integrin system, the association of all tested proteins in intercalated disks, in disagreement with other Authors, and in T-tubule with irregular spokelike extensions penetrating toward the center of the cell. Moreover, our data showed that all tested proteins colocalize between each other.

Force Frequency Relationship of the Human Ventricle Increases During Early Postnatal Development

Understanding developmental changes in contractility is critical to improving therapies for young cardiac patients. Isometric developed force was measured in human ventricular muscle strips from two age groups: newborns (<2 wk) and infants (3-14 mo) undergoing repair for congenital heart defects. Muscle strips were paced at several cycle lengths (CLs) to determine the force frequency response (FFR). Changes in Na/Ca exchanger (NCX), sarcoplasmic reticulum Ca-ATPase (SERCA), and phospholamban (PLB) were characterized. At CL 2000 ms, developed force was similar in the two groups. Decreasing CL increased developed force in the infant group to 131 +/- 8% (CL 1000 ms) and 157 +/- 18% (CL 500 ms) demonstrating a positive FFR. The FFR in the newborn group was flat. NCX mRNA and protein levels were significantly larger in the newborn than infant group whereas SERCA levels were unchanged. PLB mRNA levels and PLB/SERCA ratio increased with age. Immunostaining for NCX in isolated newborn cells showed peripheral staining. In infant cells, NCX was also found in T-tubules. SERCA staining was regular and striated in both groups. This study shows for the first time that the newborn human ventricle has a flat FFR, which increases with age and may be caused by developmental changes in calcium handling.

Myofilament Degradation and Dysfunction of Human Cardiomyocytes in Fabry Disease

Early detection of myocardial dysfunction in Fabry disease (FD) cardiomyopathy suggests the contribution of myofilament structural alterations. Six males with untreated FD cardiomyopathy submitted to cardiac studies, including tissue Doppler imaging and left ventricular endomyocardial biopsy. Active and resting tensions before and after treatment with protein kinase A (PKA) were determined in isolated Triton-permeabilized cardiomyocytes. Cardiomyocyte cross-sectional area, glycosphingolipid vacuole area, myofibrillolysis, and extent of fibrosis were also determined. Biopsies of mitral stenosis in patients with normal left ventricles served as controls. Active tension was four times lower in FD cardiomyocytes and correlated with extent of myofibrillolysis. Resting tension was six times higher in FD cardiomyocytes than in controls. PKA treatment decreased resting tension but did not affect active force. Protein analysis revealed troponin I and desmin degradation products. FD cardiomyocytes were significantly larger and filled with glycosphingolipids. Fibrosis was mildly increased compared with controls. Tissue Doppler imaging lengthening and shortening velocities were reduced in FD cardiomyocytes compared with controls, correlating with resting and active tensions, respectively, but not with cardiomyocyte area, percentage of glycosphingolipids, or extent of fibrosis. In conclusion, myofilament degradation and dysfunction contribute to FD cardiomyopathy. Partial reversal of high resting tension after pharmacological PKA treatment of cardiomyocytes suggests potential benefits from enzyme replacement therapy and/or energy-releasing agents.

Weaker contribution of I K1 current to the cardiac repolarization reserve in human versus dog ventricular muscle

Weaker contribution of I K1 current to the cardiac repolarization reserve in human versus dog ventricular muscle

The influence of diabetes on cardiac β-adrenoceptor subtypes

Despite the significant developments in the treatment of diabetes mellitus, diabetic patients still continue to suffer from cardiac complications. The increase of cardiac adrenergic drive may ultimately contribute to the development and progression of diabetic cardiomyopathy. beta-Adrenoceptors play an important role in the regulation of heart function. However, responsiveness of diabetic heart to beta-adrenoceptor agonist stimulation is diminished. The chronotropic responses mediated by beta(1)-subtype, which is mainly responsible for cardiac effects of catecholamines are decreased in the atria of diabetic rats. The expression of cardiac beta(1)-subtype is significantly decreased in diabetic rats as well. beta(2)-Adrenoceptors also increase cardiac function. Although the expression of this subtype is slightly decreased in diabetic rat hearts, beta(2)-mediated chronotropic responses are preserved. On the other hand, functional beta(3)-adrenoceptor subtype was characterized in human heart. Interestingly, stimulation of cardiac beta(3)-adrenoceptors, on the contrary of beta(1)- and beta(2)-subtypes, mediates negative inotropic effect in human ventricular muscle. Cardiac beta(3)-adrenoceptors are upregulated in experimental diabetes as well as in human heart failure. These findings suggest that each beta-adrenoceptor subtype may play an important role in the pathophysiology of diabetes-induced heart disease. However, it is still not known whether the changes in the expression and/or responsiveness of beta-adrenoceptors are adaptive or maladaptive. Therefore, this review outlines the potential roles of these receptor subtypes in cardiac pathologies of diabetes.