Frequency-dependent Acceleration Research Articles

Prolonged Ca2+ release refractoriness and T-tubule disruption as determinants of increased propensity to cardiac alternans in the hypertensive heart disease.

Cardiac alternans is a dynamical phenomenon linked to the genesis of severe arrhythmias and sudden cardiac death. It has been proposed that alternans is caused by alterations in Ca2+ handling by the sarcoplasmic reticulum (SR), in both the SR Ca2+ uptake and release processes. The hypertrophic myocardium is particularly prone to alternans, but the precise mechanisms underlying its increased vulnerability are not known. Mechanical alternans (intact hearts) and Ca2+ alternans (cardiac myocytes) were studied in spontaneously hypertensive rats (SHR) during the first year of age after the onset of hypertension and compared with age-matched normotensive rats. Subcellular Ca2+ alternans, T-tubule organization, SR Ca2+ uptake, and Ca2+ release refractoriness were measured. The increased susceptibility of SHR to high-frequency-induced mechanical and Ca2+ alternans appeared when the hypertrophy developed, associated with an adverse remodeling of the T-tubule network (6 mo). At the subcellular level, Ca2+ discordant alternans was also observed. From 6 mo of age, SHR myocytes showed a prolongation of Ca2+ release refractoriness without alterations in the capacity of SR Ca2+ removal, measured by the frequency-dependent acceleration of relaxation. Sensitizing SR Ca2+ release channels (RyR2) by a low dose of caffeine or by an increase in extracellular Ca2+ concentration, shortened refractoriness of SR Ca2+ release, and reduced alternans in SHR hearts. The tuning of SR Ca2+ release refractoriness is a crucial target to prevent cardiac alternans in a hypertrophic myocardium with an adverse T-tubule remodeling.

Influential Article Review — The Effect of Seismic Excitation on the Passive Stiffness of a Seismically Isolated Bridge Deck

This paper examines the performance of modified bridge elements under simulated earthquake excitations. We present insights from a highly influential paper. Here are the highlights from this paper: This study is on the seismic performance assessment of a seismically isolated bridge structure with passive stiffness modification implementation. Performance of the passive stiffness modification approach is compared with semi-active control implementation on the bridge deck. El Centro NS (north-south) and Kobe EW(east￾west) earthquake excitations are used for the dynamic simulations of the passive case. Results are given in a comparative way for the uncontrolled bridge deck, passive stiffness modification implemented and semi￾actively controlled, seismically isolated bridge deck (Yanik & Aldemir, 2018). Frequency dependent acceleration, velocity and displacement response transmissibility ratios are defined to examine the results. For our overseas readers, we then present the insights from this paper in Spanish, French and German.

Effect of Cooling on Force-Frequency Relationship, Rest Potentiation, and Frequency-Dependent Acceleration of Relaxation in the Guinea Pig Myocardium

Effect of Cooling on Force-Frequency Relationship, Rest Potentiation, and Frequency-Dependent Acceleration of Relaxation in the Guinea Pig Myocardium

Reliability-based assessment and calibration of standards for the lateral vibration of pedestrian bridges

Ensuring vibrations remain tolerable to occupants is central to the serviceability limit state (SLS) design of lightweight pedestrian bridges. In general, pedestrians tend to be more sensitive to lateral bridge vibrations compared to vertical vibrations, which underscores the importance of serviceability limit state (SLS) design in the lateral direction. Significant uncertainties associated with the pedestrian loading and bridge behaviour motivate the need to treat serviceability design provisions from a reliability standpoint. This paper is a first attempt towards such an assessment and code calibration of SLS provisions applicable to the lateral direction in the existing major pedestrian bridge design guidelines (ISO 10137, Eurocode 5 and SÉTRA). For this purpose, this study adopts a reliability-based framework, which accounts for the uncertainties arising from the pedestrian loading, perceptions of vibration by pedestrians, and structural damping. Furthermore, SLS design generally considers commonly occurring design traffic densities only. However, several historical lateral vibration serviceability failures of pedestrian bridges have occurred under infrequent loading events. Hence, this paper also incorporates infrequent traffic event (load case) in the reliability assessment and calibration of the design guidelines. Results show that metal truss-type pedestrian bridges under design and infrequent traffic events need to be designed to a higher reliability level (through calibration) than currently achieved by these provisions under the design event. Adopting traffic density and frequency-dependent acceleration limits can mitigate overdesigns resulting from the calibration exercise. The desired reliability index required to achieve minimum acceptable performance of the designs under both the design and infrequent events is estimated iteratively in the proposed procedure, followed by the calculation of design calibration factors. This study shows that the calibration of some design provisions (e.g., SÉTRA) depends on amplitude-dependent damping values, while this is not the case with other provisions. This dependency can be attributed to differences in the form of the limit state function. Finally, it is shown that the proposed calibration process achieves sufficient and consistent reliability across all bridge classes under design and infrequent traffic events in the lateral direction, while ensuring economic designs using traffic density and frequency-based comfort limits.

SERCA is critical to control the Bowditch effect in the heart

The Bowditch effect or staircase phenomenon is the increment or reduction of contractile force when heart rate increases, defined as either a positive or negative staircase. The healthy and failing human heart both show positive or negative staircase, respectively, but the causes of these distinct cardiac responses are unclear. Different experimental approaches indicate that while the level of Ca2+ in the sarcoplasmic reticulum is critical, the molecular mechanisms are unclear. Here, we demonstrate that Drosophila melanogaster shows a negative staircase which is associated to a slight but significant frequency-dependent acceleration of relaxation (FDAR) at the highest stimulation frequencies tested. We further showed that the type of staircase is oppositely modified by two distinct SERCA mutations. The dominant conditional mutation SERCAA617T induced positive staircase and arrhythmia, while SERCAE442K accentuated the negative staircase of wild type. At the stimulation frequencies tested, no significant FDAR could be appreciated in mutant flies. The present results provide evidence that two individual mutations directly modify the type of staircase occurring within the heart and suggest an important role of SERCA in regulating the Bowditch effect.

Stretching Single Titin Molecules from Failing Human Hearts at Cardiac Cycle Reveals Titin's Role in Cardiac Kinetic Reserve

Diastolic dysfunction occurs not only in diastolic heart failure, but also ubiquitously in systolic heart failure. A main determinant of diastolic passive tension, the elastic sarcomeric protein titin, has been shown to be associated with heart failure, with unresolved mechanisms. Heart failure patients commonly experience symptoms only during physical activities or stress. To determine whether titin is playing a role in the mechanisms of heart failure in terms of its known impaired property of frequency‐dependent acceleration of relaxation (FDAR), we studied the FDAR responses in live left ventricular cardiomyocytes and single‐molecule force spectroscopy (SMFS) of titin elastic domains (PEVK‐distal Ig, distal Ig) from failing (n=11) and non‐failing (n=7) human hearts. To access SMFS on native titin, we developed a novel approach using atomic force microscopy to detect titin passive tension (TPT) based on frequency‐modulated cardiac cycle. Mean TPT was shown to be reduced upon an increased heart rate, which was significantly blunted in failing human hearts. The results from titin distal Ig domain were significantly correlated with the frequency‐dependent relaxation kinetics of human cardiomyocytes from the corresponding hearts, which was shown to be impaired in failing hearts. Correlation studies suggested that the higher the TPT, the faster the cardiomyocytes relaxed, but lower the potential of myocytes to speed up relaxation at a higher heart rate; such poorer FDAR response was also associated with a less reduction or a bigger increase in TPT upon a raised heart rate. Our study established a novel approach in detecting tension physiologically on native titin domains. The data from human hearts suggested that the regulation of kinetic reserve in cardiac relaxation and its pathological changes are associated with the intensity and dynamic changes of passive tension on titin.Support or Funding InformationThis project is supported by RO1HL113084 to Janssen PML, and T32NS779844 to Chen MPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Contractile heterogeneity in ventricular myocardium.

The transmural heterogeneity of the contractility in ventricular muscle has not been well-studied. Here, we investigated the calcium transient and sarcomere contraction/relaxation in the endocardial (Endo) and epicardial (Epi) myocytes. Endo and Epi myocytes were isolated from C57/BL6 mice by Langendorff perfusion. Ca2+ transient and sarcomere contraction/relaxation were recorded simultaneously at different stimulation frequencies using a dual excitation fluorescence photomultiplier system. We found that the Endo myocytes have higher baseline diastolic calcium, significantly larger calcium transient and stronger sarcomere shortening than Epi myocytes. However, both the rising and decline phases for calcium transient and sarcomere shortening were slower in Endo than in Epi myocytes. When simulation frequency was increased from 1 to 3 Hz, a greater percent increase in the diastole calcium level, Ca2+ transient and sarcomere shortening amplitude has been observed in the Endo myocytes. Accordingly, the frequency-dependent acceleration in the decay rate of calcium transient and sarcomere relaxation was more profound in the Endo than in Epi myocytes. Western blot analysis showed that CaMKII activity was significantly higher in Epi than in Endo myocardium before stimulation. However, this transmural heterogeneity was reversed by rapid pacing. CaMKII inhibition by KN93 diminished the frequency-dependent alterations of Ca2+ transient and sarcomere contraction. Our results suggest that the contractility of ventricular myocytes is heterogeneous. The Endo-myocardium is the major force generating layer in the heart, both at slow and fast heart rate, and the transmural heterogeneity of CaMKII activation plays an important role in the frequency-dependent alterations.

Abstract 71: Ca 2+ Reuptake is Reduced in the Absence of Insulin Receptors in Mouse Cardiomyocytes

Left ventricle diastolic dysfunction (LVDD), characterized by slow or incomplete relaxation of the ventricle during diastole, is an important contributor to heart failure development in diabetic patients. LVDD can be found in pre-diabetic patients as well as in almost half of asymptomatic patients with well-controlled diabetes. Reduced Ca 2+ reuptake into the sarcoplasmic reticulum (SR) by the SR Ca 2+ ATPase (SERCA) is an important contributor to the slow relaxation observed in diastolic dysfunction. Despite this there are still conflicting results with respect to SERCA expression and function in animal models of diabetes, due perhaps to differences in the duration and severity of diabetes and the presence of systemic complications that can per se alter SERCA expression and/or function. Here, we tested the hypothesis that the impaired relaxation observed in diabetic mice is a consequence of the direct effect of insulin on SERCA. We measured Ca 2+ transient relaxation in isolated cardiomyocytes from 8 weeks cardiomyocyte-selective insulin receptor knockout (CIRKO) mice (Cre-IR lox / lox ), , which lack systemic complications, and compared them with their WT littermates (IR lox / lox ) . Cardiomyocytes loaded with the Ca 2+ dye Fluo-4 were field stimulated at 2 and 3.3 Hz, at 37°C. The intracellular Ca 2+ was monitored using a custom-made epifluorescence system. SERCA and Na + /Ca 2+ exchanger (NCX) expression level was reported as the fold change relative to WT. We found that Ca 2+ transient relaxation was prolonged at both frequencies in CIRKO compared with WT, while retaining a normal frequency-dependent acceleration of relaxation. The exponential time constants were 104±4 (WT) vs. 190±16 (CK) ms at 3.3 Hz, n= 20/group, p<0.001 and 111±6 (WT) vs. 226±19 (CK) ms at 2 Hz, n= 20/group, p<0.001). We also found a reduction in SERCA expression (mRNA: 0.65±0.03, n=10, p<0.001; protein:0.62±0.11, n=4, p<0.05) with no difference in NCX expression (mRNA: 0.92±0.06 n=10/group, p=0.5; protein 0.96±0.05, n=6/group, p=0.2). In conclusion, our findings suggest that the slower Ca 2+ removal observed in the systemic models of diabetes is a direct effect of insulin on SERCA, which could contribute to the LVDD observed in diabetic patients.

CaMKII-dependent myofilament Ca2+ desensitization contributes to the frequency-dependent acceleration of relaxation

BackgroundPrevious studies suggest that CaMKII activity is required for frequency-dependent acceleration of relaxation (FDAR) in ventricular myocytes. We propose that the underlying mechanism involves CaMKII-dependent regulation of myofilament Ca2+ sensitivity. Methods and resultsCardiac function was measured in mice using murine echo machine. [Ca2+]i and sarcomere length were measured by IonOptix Ca2+ image system. Increasing pacing rate from 0.5 to 4Hz in left ventricular myocytes induced frequency-dependent myofilament Ca2+ desensitization (FDMCD) and FDAR. Acute inhibition of PKA or PKC had no effect, whereas CaMKII inhibition abolished both FDMCD and FDAR. Co-immunoprecipitation of CaMKII and troponin I (TnI) has been detected and CaMKII inhibition significantly reduced serine residue phosphorylation of TnI. Finally, chronic inhibition of CaMKII in vivo reduced TnI phosphorylation and abolished both FDAR and FDMCD, leading to impaired diastolic function. ConclusionsOur results suggest that CaMKII-dependent TnI phosphorylation is involved in FDMCD and the consequent FDAR and that CaMKII inhibition removes this mechanism and thus induces diastolic dysfunction.

Abstract 185: Chromogranin A is a Potent Prognostic Biomarker in Acute Heart Failure, and its Fragment Catestatin Regulates Cardiomyocyte Calcium Homeostasis by Camkiiδ Inhibition

Background: Circulating chromogranin A (CgA) levels have been found associated with clinical outcomes in cardiovascular disease, but whether the CgA fragment catestatin (CST) may directly modulate cardiomyocyte Ca 2+ handling is not known. Methods: The prognostic utility of circulating CgA levels were compared between patients with acute HF (n=143) and chronic acute obstructive pulmonary disease (COPD, n=84). Functional effects of CST were assessed in isolated cardiomyocyte and explanted hearts. Results: CgA levels were associated with mortality in acute HF patients, but not in acute COPD. (Figure; patients stratified according to CgA quartiles on hospital admission). Admission CgA levels were also associated with mortality after adjusting for other risk factors in multivariate analysis. We found CST to interact with Ca 2+ /calmodulin (CaM)-dependent protein kinase II δ (CaMKIIδ) and to inhibit CaMKIIδ activity. CST also reduced CaMKIIδ-dependent phosphorylation of the ryanodine receptor 2 and phospholamban. In line with CaMKIIδ inhibition, CST reduced Ca 2+ spark and wave frequency, attenuated Ca 2+ sparkdimensions, increased sarcoplasmic reticulum Ca 2+ content, andaugmented magnitude and kinetics of cardiomyocyte Ca 2+ transients and contractions. Frequency dependent acceleration of relaxation was most pronounced in the Control group, an indication of CaMKIIδ activation, and this was attenuated by CST. Conclusions: CgA regulates cardiomyocyte Ca 2+ handling via CaMKIIδ inhibition, which makes CgA an interesting CV biomarker and potential compensatory mechanism in situations of enhanced CaMKIIδ activity.

Physiologic force-frequency response in engineered heart muscle by electromechanical stimulation

A hallmark of mature mammalian ventricular myocardium is a positive force-frequency relationship (FFR). Despite evidence of organotypic structural and molecular maturation, a positive FFR has not been observed in mammalian tissue engineered heart muscle. We hypothesized that concurrent mechanical and electrical stimulation at frequencies matching physiological heart rate will result in functional maturation. We investigated the role of biomimetic mechanical and electrical stimulation in functional maturation in engineered heart muscle (EHM). Following tissue consolidation, EHM were subjected to electrical field stimulation at 0, 2, 4, or 6 Hz for 5 days, while strained on flexible poles to facilitate auxotonic contractions. EHM stimulated at 2 and 4 Hz displayed a similarly enhanced inotropic reserve, but a clearly diverging FFR. The positive FFR in 4 Hz stimulated EHM was associated with reduced calcium sensitivity, frequency-dependent acceleration of relaxation, and enhanced post-rest potentiation. This was paralleled on the cellular level with improved calcium storage and release capacity of the sarcoplasmic reticulum and enhanced T-tubulation. We conclude that electro-mechanical stimulation at a physiological frequency supports functional maturation in mammalian EHM. The observed positive FFR in EHM has important implications for the applicability of EHM in cardiovascular research.

Properties of Calcium Transients in Cardiomyocytes with Impaired Insulin Signaling

Diabetes is a major risk factor for cardiovascular diseases, including cardiac arrhythmias, heart failure and sudden cardiac death. In animal models of diabetes, cardiac calcium handling is severely impaired. However, as diabetes is a multifactorial disease it is difficult to separate the effect of systemic and local factors in disease development. Here, we investigate the effect of locally disrupted cardiac insulin signaling on cardiac calcium handling in a mouse model of diabetic cardiomyopathy.We measured calcium transients in isolated cardiomyocytes from Cardiac Insulin Receptor Knock-Out (CIRKO) mice. Littermate wild-type (WT) mice were used as controls. Cardiomyocytes were field stimulated and intracellular calcium was monitored using the fluorescent indicator fluo2-LeakRes (34oC, pHo 7.4, [Ca2+]o = 1.0 mM). Calcium transient amplitude and decay kinetics were measured at 1 and 5 Hz. While no significant differences were observed between WT and KO cardiomyocytes, there was a tendency towards reduced calcium transient amplitude in the KO group (Amplitude (F/F0): WT, 1.6±0.7 (n=9); KO, 1.0±0.4 (n=16); p=0.05). Calcium transient decay was similar (tau (ms): WT, 183±44 (n=9); KO: 232±84 (n=17); p=0.15) and frequency-dependent acceleration of decay kinetics was observed in both groups (tau: p<0.01 for both WT and KO (1Hz vs 5 Hz)).In conclusion, at the cellular level the CIRKO mouse model of diabetic cardiomyopathy display a very mild calcium handling phenotype. These findings suggest that impaired cardiac insulin signaling per se plays only a minor role in the contractile dysfunction observed in systemic models of diabetes.

GW24-e3794 CaMKII-dependent troponin-I phosphorylation contributes to the frequency-dependent acceleration of relaxation in ventricular myocytes

ObjectivesFrequency-dependent acceleration of relaxation (FDAR) is an intrinsic mechanism in ventricular myocytes allowing a faster ventricular relaxation (and diastolic filling) at fast heart rates. Previous studies suggest that CaMKII activity...

While systolic cardiomyocyte function is preserved, diastolic myocyte function and recovery from acidosis are impaired in CaMKIIδ-KO mice

ObjectiveCaMKII contributes to impaired contractility in heart failure by inducing SR Ca2+-leak. CaMKII-inhibition in the heart was suggested to be a novel therapeutic principle. Different CaMKII isoforms exist. Specifically targeting CaMKIIδ, the dominant isoform in the heart, could be of therapeutic potential without impairing other CaMKII isoforms. RationaleWe investigated whether cardiomyocyte function is affected by isoform-specific knockout (KO) of CaMKIIδ under basal conditions and upon stress, i.e. upon ß-adrenergic stimulation and during acidosis. ResultsSystolic cardiac function was largely preserved in the KO in vivo (echocardiography) corresponding to unchanged Ca2+-transient amplitudes and isolated myocyte contractility in vitro. CaMKII activity was dramatically reduced while phosphatase-1 inhibitor-1 was significantly increased. Surprisingly, while diastolic Ca2+-elimination was slower in KO most likely due to decreased phospholamban Thr-17 phosphorylation, frequency-dependent acceleration of relaxation was still present. Despite decreased SR Ca2+-reuptake at lower frequencies, SR Ca2+-content was not diminished, which might be due to reduced diastolic SR Ca2+-loss in the KO as a consequence of lower RyR Ser-2815 phosphorylation. Challenging KO myocytes with isoproterenol showed intact inotropic and lusitropic responses. During acidosis, SR Ca2+-reuptake and SR Ca2+-loading were significantly impaired in KO, resulting in an inability to maintain systolic Ca2+-transients during acidosis and impaired recovery. ConclusionsInhibition of CaMKIIδ appears to be safe under basal physiologic conditions. Specific conditions exist (e.g. during acidosis) under which CaMKII-inhibition might not be helpful or even detrimental. These conditions will have to be more clearly defined before CaMKII inhibition is used therapeutically.

CaMKII effects on inotropic but not lusitropic force frequency responses require phospholamban

Increasing heart rate enhances cardiac contractility (force frequency relationship, FFR) and accelerates cardiac relaxation (frequency-dependent acceleration of relaxation, FDAR). The positive FFR together with FDAR promotes rapid filling and ejection of blood from the left ventricle (LV) at higher heart rates. Recent studies indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is involved in regulating FFR and FDAR. We used isolated perfused mouse hearts to study the mechanisms of FFR and FDAR in different genetic models, including transgenic myocardial CaMKII inhibition (AC3-I) and phospholmban knockout (PLN−/−). When the rate was increased from 360beats/min to 630beats/min in wild type mouse hearts, the LV developed pressure (LVDP) and the maximum rate of increase in pressure (dP/dt max) increased by 37.6±4.7% and 77.0±8.1%, respectively. However, hearts from AC3-I littermates showed no increase of LVDP and a relatively modest (20.4±3.9%) increase in dP/dt max. PLN−/− hearts had a negative FFR, and myocardial AC3-I expression did not change the FFR in PLN−/− mice. PLN−/− mouse hearts did not exhibit FDAR, while PLN−/− mice with myocardial AC3-I expression showed further frequency dependent reductions in cardiac relaxation, suggesting that CaMKII targets in addition to PLN were critical to myocardial relaxation. We incubated a constitutively active form of CaMKII with chemically-skinned myocardium and found that several myofilament proteins were phosphorylated by CaMKII. However, CaMKII did not affect myofilament calcium sensitivity. Our study shows that CaMKII plays an important role in modulating FFR and FDAR in murine hearts and suggest that PLN is a critical target for CaMKII effects on FFR, while CaMKII effects on FDAR partially require PLN-alternative targets.

Frequency-dependent acceleration of cardiac repolarization by progesterone underlying its cardiac protection against drug-induced proarrhythmic effects in female rabbits

Concurrent supplement of estradiol and progesterone has been shown to reduce the cardiac sensitivity to class III antiarrhythmic agent-induced arrhythmias in ovariectomized rabbits. To understand the underlying cardiac electrophysiological mechanisms of the hormones, present study explored the modulation of progesterone and estradiol on repolarization and its frequency dependence in papillary muscles of female rabbit right ventricles by glass microelectrode technique. Results showed that progesterone shortened action potential duration for 90% repolarization (APD90) whereas estradiol prolonged APD90 and those actions on APD90 were concentration-dependent for both hormones at 1.0–30μM (P<0.05 or P<0.01). Further, the action of both hormones on APD90 was found to be dependent on stimulation frequencies (0.2–3.3Hz). The shortening of APD90 by progesterone (10μM) was enhanced with the increase in frequencies reaching a statistic significance at frequencies ≥1.0Hz, whereas the prolongation of APD90 by estradiol (3μM) was weakened with the increase in frequencies and the significant change was observed at frequencies ≤2.0Hz (P<0.05 or P<0.01). More interestingly, the relative change of APD90 and the incidence of early afterdepolarization induced after by dofetilide (0.1μM), a class III antiarrhythmic agent, were significantly less or lower in the papillary muscles pretreated with progesterone than in those pretreated with estradiol (P<0.01 or P<0.05). In conclusion, progesterone has a reverse modulating affect on cardiac repolarization to that of estradiol. By acceleration of ventricular repolarization, progesterone may reduce the susceptibility of females to class III antiarrhythmic agents-induced proarrhythmic affection.

Vibration and shock reliability of MEMS: modeling and experimental validation

A methodology to predict shock and vibration levels that could lead to the failure of MEMS devices is reported as a function of vibration frequency and shock pulse duration. A combined experimental–analytical approach is developed, maintaining the simplicity and insightfulness of analytical methods without compromising on the accuracy characteristic of experimental methods. The minimum frequency-dependent acceleration that will lead to surfaces coming into contact, for vibration or shock inputs, is determined based on measured mode shapes, damping, resonant frequencies, and an analysis of failure modes, thus defining a safe operating region, without requiring shock or vibration testing. This critical acceleration for failure is a strong function of the drive voltage, and the safe operating region is predicted for transport (unbiased) and operation (biased condition). The model was experimentally validated for over-damped and under-damped modes of a comb-drive-driven silicon-on-insulator-based tunable grating. In-plane and out-of-plane vibration (up to 65 g) and shock (up to 6000 g) tests were performed for biased and unbiased conditions, and very good agreement was found between predicted and observed critical accelerations.

Synergistic Effects between Phosphorylation of Phospholamban and Troponin I Promote Relaxation at Higher Heart Rate

We hypothesized that the extent of frequency-dependent acceleration of relaxation (FDAR) would be less than that of isoproterenol-(ISO-)dependent acceleration of relaxation (IDAR) at the same increment of heart rates, and ISO may improve FDAR. Cardiac function and phosphorylation of PLB and cTnI were compared in pacing, ISO treatment, and combined pacing and ISO treatment in isolated working heart. The increase in cardiac output and the degree of relaxation was less in pacing than in ISO treatment at the same increment of heart rates. The increasing stimulation frequency induced more significant relaxant effect in ISO perfusion than that in physiological salt perfusion. The pacing only phosphorylated PLB at Thr17, but ISO induced phosphorylation of cTnI and PLB at Ser16 and Thr17. Those results suggest that the synergistic effects of PLB and cTnI induce higher degree of relaxation which makes a sufficient diastolic filling of the ventricle at higher heart rate.

Synergy between CaMKII Substrates and β-Adrenergic Signaling in Regulation of Cardiac Myocyte Ca 2+ Handling

Cardiac excitation-contraction coupling is a highly coordinated process that is controlled by protein kinase signaling pathways, including Ca 2+/calmodulin-dependent protein kinase II (CaMKII) and protein kinase A (PKA). Increased CaMKII expression and activity (as occurs during heart failure) destabilizes EC coupling and may lead to sudden cardiac death. To better understand mechanisms of cardiac CaMKII function, we integrated dynamic CaMKII-dependent regulation of key Ca 2+ handling targets with previously validated models of cardiac EC coupling, Ca 2+/calmodulin-dependent activation of CaMKII, and β-adrenergic activation of PKA. Model predictions are validated against CaMKII-overexpression data from rabbit ventricular myocytes. The model demonstrates how overall changes to Ca 2+ handling during CaMKII overexpression are explained by interactions between individual CaMKII substrates. CaMKII and PKA activities during β-adrenergic stimulation may synergistically facilitate inotropic responses and contribute to a CaMKII-Ca 2+-CaMKII feedback loop. CaMKII regulated early frequency-dependent acceleration of relaxation and EC coupling gain (which was highly sarcoplasmic reticulum Ca 2+ load-dependent). Additionally, the model identifies CaMKII-dependent ryanodine receptor hyperphosphorylation as a proarrhythmogenic trigger. In summary, we developed a detailed computational model of CaMKII and PKA signaling in cardiac myocytes that provides unique insights into their regulation of normal and pathological Ca 2+ handling.

The cellular force-frequency response in ventricular myocytes from the varanid lizard, Varanus exanthematicus

To investigate the cellular mechanisms underlying the negative force-frequency relationship (FFR) in the ventricle of the varanid lizard, Varanus exanthematicus, we measured sarcomere and cell shortening, intracellular Ca(2+) ([Ca(2+)](i)), action potentials (APs), and K(+) currents in isolated ventricular myocytes. Experiments were conducted between 0.2 and 1.0 Hz, which spans the physiological range of in vivo heart rates at 20-22 degrees C for this species. As stimulation frequency increased, diastolic length, percent change in sarcomere length, and relaxation time all decreased significantly. Shortening velocity was unaffected. These changes corresponded to a faster rate of rise of [Ca(2+)](i), a decrease in [Ca(2+)](i) transient amplitude, and a seven-fold increase in diastolic [Ca(2+)](i). The time constant for the decay of the Ca(2+) transient (tau) decreased at higher frequencies, indicating a frequency-dependent acceleration of relaxation (FDAR) but then reached a plateau at moderate frequencies and did not change above 0.5 Hz. The rate of rise of the AP was unaffected, but the AP duration (APD) decreased with increasing frequency. Peak depolarization tended to decrease, but it was only significant at 1.0 Hz. The decrease in APD was not due to frequency-dependent changes in the delayed inward rectifier (I(Kr)) or the transient outward (I(to)) current, as neither appeared to be present in varanid ventricular myocytes. Our results suggest that a negative FFR relationship in varanid lizard ventricle is caused by decreased amplitude of the Ca(2+) transient coupled with an increase in diastolic Ca(2+), which leads to incomplete relaxation between beats at high frequencies. This coincides with shortened APD at higher frequencies.