Amplitude Of Shortening Research Articles

Inhibitory effect of cinobufagin on L-type Ca2+ currents, contractility, and Ca2+ homeostasis of isolated adult rat ventricular myocytes.

Cinobufagin (CBG), a major bioactive ingredient of the bufanolide steroid compounds of Chan Su, has been widely used to treat coronary heart disease. At present, the effect of CBG on the L-type Ca2+ current (I Ca-L) of ventricular myocytes remains undefined. The aim of the present study was to characterize the effect of CBG on intracellular Ca2+ ([Ca2+]i) handling and cell contractility in rat ventricular myocytes. CBG was investigated by determining its influence on I Ca-L, Ca2+ transient, and contractility in rat ventricular myocytes using the whole-cell patch-clamp technique and video-based edge-detection and dual-excitation fluorescence photomultiplier systems. The dose of CBG (10−8 M) decreased the maximal inhibition of CBG by 47.93%. CBG reduced I Ca-L in a concentration-dependent manner with an IC50 of 4 × 10−10 M, upshifted the current-voltage curve of I Ca-L, and shifted the activation and inactivation curves of I Ca-L leftward. Moreover, CBG diminished the amplitude of the cell shortening and Ca2+ transients with a decrease in the time to peak (Tp) and the time to 50% of the baseline (Tr). CBG inhibited L-type Ca2+ channels, and reduced [Ca2+]i and contractility in adult rat ventricular myocytes. These findings contribute to the understanding of the cardioprotective efficacy of CBG.

Intra-Myocardial Slow Force Response in Heterogeneous Myocardium

Recently we have reported on an intra-myocardial slow force response (SFR_IM) of myocardium to changes in the mechanical environment (Markhasin ea, PBMB, 2012). In contrast to SFRs referred to changes in cardiac muscle contractility in response to external (to the heart) mechanical stimuli, SFR_IM ensued from internal mechanical interactions of muscle segments in heterogeneous myocardium. We revealed SFR_IM in two interacting myocardial elements, referred to as the muscle duplex, presenting a simplest possible model of heterogeneous myocardium. We found beat-to-beat changes in the overall duplex force developed by end-to-end coupled muscles upon duplex formation. After duplex disconnection, the individual force produced by each muscle differed significantly from the baseline level prior to muscle coupling, thus pointing out to changes in muscle contractility. SFR_IM in muscle duplexes were accompanied with slow and opposite changes in the action potential and Ca2+ transient in the cardiomyocytes of interacting muscle elements. The SFR_IM phenomena were initially identified by means of mathematical modeling, and subsequently confirmed in physiological experiments involving native cardiac muscles. The SFR_IM were specified for isometric contractions of in-series duplexes where cyclic deformations of muscle segments under a constant length of the pair governed intracellular mechano-chemical and mechano-electrical mechanisms of excitation-contracting coupling contributing to the SFR_IM. Here we present effects of the mechanical load on the SFR_IM in isotonic and physiological modes of contraction of in-series muscle duplexes. We showed that muscle interaction during the isometric phase of duplex contraction determine the amplitude and duration of subsequent duplex shortening, depending on the load value. SFR_IM modifies the force-length and force-velocity dependences in interacting muscles depending on the duplex activation sequence. The results suggest the essential role of SFR_IM in the isovolumetric phase of ventricular contraction in the intact heart. Supported by UB RAS (12-M-14-2009, 12-П-4-1067).

Basal and β-Adrenergic Cardiomyocytes Contractility Dysfunction Induced by Dietary Protein Restriction is Associated with Downregulation of SERCA2a Expression and Disturbance of Endoplasmic Reticulum Ca2+ Regulation in Rats

Background: The mechanisms responsible for the cardiac dysfunction associated with dietary protein restriction (PR) are poorly understood. Thus, this study was designed to evaluate the effects of PR on calcium kinetics, basal and β-adrenergic contractility in murine ventricular cardiomyocytes. Methods: After breastfeeding male Fisher rats were distributed into a control group (CG, n = 20) and a protein-restricted group (PRG, n = 20), receiving isocaloric diets for 35 days containing 15% and 6% protein, respectively. Biometric and hemodynamic variables were measured. After euthanasia left ventricles (LV) were collected for histopathological evaluation, SERCA2a expression, cardiomyocytes contractility and Ca²⁺sparks analysis. Results: PRG animals showed reduced general growth, increased heart rate and arterial pressure. These animals presented extracellular matrix expansion and disorganization, cardiomyocytes hypotrophy, reduced amplitudes of shortening and maximum velocity of contraction and relaxation at baseline and after β-adrenergic stimulation. Reduced SERCA2a expression as well as higher frequency and lower amplitude of Ca²⁺sparks were observed in PRG cardiomyocytes. Conclusion: The observations reveal that protein restriction induces marked myocardial morphofunctional damage. The pathological changes of cardiomyocyte mechanics suggest the potential involvement of the β-adrenergic system, which is possibly associated with changes in SERCA2a expression and disturbances in Ca²⁺ intracellular kinetics.

Magnesium lithospermate B improves myocardial function and prevents simulated ischemia/reperfusion injury-induced H9c2 cardiomyocytes apoptosis through Akt-dependent pathway

Ethnopharmacological relevanceMagnesium lithospermate B (MLB), an active polyphenol acid of Radix Salviae Miltiorrhizae (Danshen), showed a wide range of pharmacological activities in cardiovascular diseases. However, its role in protection against ischemia/reperfusion injury in H9c2 cardiomyocytes has not been elucidated. This study was aimed to investigate the protective effect and potential molecular mechanisms of MLB on apoptosis in H9c2 cardiomyocytes in vitro. Materials and methodsWe tested cell viability, shortening amplitude, necrosis, apoptosis, and the expression levels of Akt, phosphorylated Akt, Bcl-2 and Bax after 2-h simulated ischemia and 24-h simulated reperfusion in H9c2 cardiomyocytes. We further observed the contractile function in hearts after they were subjected to global 30-min ischemia and 180-min reperfusion. ResultsPretreatment with MLB markedly increased cell viability and while reducing evidence of necrosis and apoptosis in H9c2 cardiomyocytes. In addition, the expression of Bcl-2 and Bax protein was modulated. The results also showed that MLB significantly increased phosphorylation of Akt and that this phosphorylation can be partially inhibited by phosphoinositide 3-kinase/Akt inhibitor. Furthermore, MLB improved MI/R-induced myocardial contractile function. ConclusionOur results showed that MLB prevents I/R-induced myocardial damage by reducing necrosis and apoptosis in H9c2 cardiomyocytes and improving myocardial function in rat hearts.

Protein Restriction after Weaning Modifies the Calcium Kinetics and Induces Cardiomyocyte Contractile Dysfunction in Rats

Protein restriction (PR) is associated with cardiovascular diseases. The purpose of this study was to investigate the effects on single ventricular cardiomyocyte contractile function of a short-term PR after weaning. Male Fischer rats that were 28 days old were randomly divided into a control group (CG, n = 16) and a protein-restricted group (PRG, n = 16). After weaning, CG and PRG animals received isocaloric diets containing 15 and 6% protein, respectively, for 35 days. Biometric parameters were then measured, and the hearts were removed for the analysis of contractile function and calcium transient in isolated cardiomyocytes of the left ventricule (LV), and the quantification of calcium and collagen fibers in LV myocardium. PRG animals had lower body weight (BW) and LV weight (LVW), an increased LVW to BW ratio and a higher proportion of collagen fibers than CG animals. PRG animals exhibited reduced tissue levels of calcium, reduced the length, width and volume of cardiomyocytes and their sarcomere length compared to CG animals. Cardiomyocytes from PRG animals had a lower amplitude of shortening, a slower time to the peak of shortening and a longer time to half-relaxation than those from the CG. Cardiomyocytes from PRG animals also presented a lower peak of calcium transient and a longer calcium transient decay time than CG animals. Taken together, the results indicate that short-term PR after weaning induces a marked structural remodeling of the myocardium parenchyma and stroma that coexists with contractile dysfunctions in single LV cardiomyocytes of rats, which is probably associated with pathological changes of the intracellular calcium kinetics, rather than inadequate available amounts of this mineral in cardiac tissue.

Inhibition of DNA methylation attenuates low‐dose cadmium‐induced cardiac contractile and intracellular Ca2+ anomalies

(1) Cadmium is a human carcinogen with unfavourable health impacts probably associated with its DNA methylation property. Recent data suggest that environmental cadmium exposure is associated with the incidence of myocardial infarction and peripheral arterial disease. Nonetheless, the effect of chronic cadmium exposure on cardiac contractile function remains unknown. (2) The present study was designed to examine the impact of low-dose cadmium exposure on cardiac contractile function and intracellular Ca2+ homeostasis. Adult male mice were exposed to cadmium for 4 weeks (20 nmol/kg, i.p. every other day for 4 weeks) with or without the DNA methylation inhibitor 5-aza-2'-deoxyctidene (5-AZA; 0.25 mg/kg, i.p., twice a week for 6 weeks, starting at the same time as cadmium administration). Cardiac contractile and intracellular Ca2+ properties were analysed, including echocardiographic left ventricular parameters, fractional shortening (FS), peak shortening (PS) amplitude, maximal velocity of shortening/relengthening (±dL/dt), time to PS (TPS), time to 90% relengthening (TR90 ), electrically stimulated increases in intracellular Ca2+ and intracellular Ca2+ decay. (3) Cadmium exposure depressed FS, PS, ±dL/dt and electrically stimulated increases in intracellular Ca2+ without affecting TPS, TR90 , intracellular Ca2+ levels or the decay rate. The effects of cadmium were significantly attenuated (PS) or blocked altogether (all other parameters) by 5-AZA. Cadmium exposure led to overt interstitial fibrosis (collagen deposition), which was mitigated by 5-AZA treatment. Western blot analysis revealed that cadmium exposure and/or 5-AZA treatment had no effect on the expression of intercellular adhesion molecule-1, tumour necrosis factor-α and cleaved caspase 3, suggesting a relatively minor role of proinflammatory cytokines and apoptosis in the cardiac responses to cadmium and 5-AZA. (4) Together, our data demonstrate, for the first time, direct cardiac depressant effects following cadmium exposure, which may be rescued by inhibition of DNA methylation.

Cellular mechanisms of contractile dysfunction in a rat model with compensated renal failure and heart failure with preserved ejection fraction

Background: Renal impairment and heart failure with preserved ejection fraction (HFPEF) are often associated but the underlying mechanisms are largely unresolved. Therefore we investigated cardiomyocyte function in a rat model with compensated renal failure and diastolic dysfunction. Methods: Fifty-six young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP) and observed up to 24 weeks. Urine and blood samples for calculating the glomerular filtration rate (GFR) and echocardiography were performed during disease progression. Invasive hemodynamics (P/V-loops), morphometry, protein expression analysis and Ca2+ transients (confocal and ratiometric) as well as cell shortening (CS) were studied in isolated left ventricle (LV) cardiomyocytes in final experiments at 8 and 24 weeks. Results: Following NXT rats develop compensated renal failure (GFR 22±2 and 16±2 vs. 32±2 and 28±3 ml/min/m2 at 8 and 24 weeks, resp. p<0.01) with preserved urinary excretion and unchanged electrolytes. Arterial pressure is significantly increased in NXT at 8 and 24 weeks (148±8 and 154±7 vs. 105±3 and 109±2 mmHg, p<0.01) which is associated with pronounced LV hypertrophy in NXT (LV mass 708±21 and 866±34 vs. 637±12 and 660±7 mg, resp. p<0.05). Increased LV end-diastolic pressure (10.4±0.8 vs. 5.1±0.4 mmHg, p<0.01), left atrial enlargement (39±2 vs. 30±1 mg, p<0.01) and increased lung weight (1816±92 vs. 1414±39 mg, p<0.01) in NXT after 24 weeks reflect diastolic dysfunction with congestion indicating HFPEF (EF 50±5 vs. 56±4%). LV cardiomyocytes isolated from NXT compared to SOP showed no significant differences in cell shortening amplitude at 1 Hz (4.3±0.7 vs. 5.1±1.1%; n≥10) while time for early (50%) relaxation was significantly prolonged in NXT (CS RT50 42±3 vs. 34±1 ms; n≥10; p< 0.05) at 24 weeks. Cytosolic Ca2+ transients had similar amplitude (F/FO 4.2±0.1 vs. 4.1±0.1, n≥20). Sarcoplasmatic reticulum (SR) Ca2+ content was significantly reduced in NXT at 24 weeks (F/F0 5.3±0.2 vs. 6.0±0.2; n≥20; p<0.01) while it was unchanged after 8 weeks (F/F0 4.7±0.2 vs. 4.8±0.3; n≥10). Time constant of the caffeine-induced Ca2+ transient (TAU) was significantly prolonged in NXT after 24 weeks (1.77±0.07 vs. 1.22±0.06; n≥20; p< 0.01). NCX protein expression was unchanged in NXT, suggesting functional impairment of NCX1 activity. Conclusion: Subtotal nephrectomy in rats lead to compensated renal failure and an in vivo phenotype of HFPEF. Our in vitro results suggest early impairment of intracellular Ca2+ transients in HFPEF in NXT, leading to contractile dysfunction of the cardiac myocyte.

Effects of exercise training on excitation–contraction coupling and related mRNA expression in hearts of Goto-Kakizaki type 2 diabetic rats

Although, several novel forms of intervention aiming at newly identified therapeutic targets are currently being developed for diabetes mellitus (DM), it is well established that physical exercise continues to be one of the most valuable forms of non-pharmacological therapy. The aim of the study was to investigate the effects of exercise training on excitation-contraction coupling and related gene expression in the Goto-Kakizaki (GK) type 2 diabetic rat heart and whether exercise is able to reverse diabetes-induced changes in excitation-contraction coupling and gene expression. Experiments were performed in GK and control rats aged 10-11 months following 2-3 months of treadmill exercise training. Shortening, [Ca(2+)]i and L-type Ca(2+) current were measured in ventricular myocytes with video edge detection, fluorescence photometry and whole cell patch clamp techniques, respectively. Expression of mRNA was assessed in ventricular muscle with real-time RT-PCR. Amplitude of shortening, Ca(2+) transients and L-type Ca(2+) current were not significantly altered in ventricular myocytes from GK sedentary compared to control sedentary rats or by exercise training. Expression of mRNA encoding Tpm2, Gja4, Atp1b1, Cacna1g, Cacnb2, Hcn2, Kcna3 and Kcne1 were up-regulated and Gja1, Kcnj2 and Kcnk3 were down-regulated in hearts of sedentary GK rats compared to sedentary controls. Gja1, Cav3 and Kcnk3 were up-regulated and Hcn2 was down-regulated in hearts of exercise trained GK compared to sedentary GK controls. Ventricular myocyte shortening and Ca(2+) transport were generally well preserved despite alterations in the profile of expression of mRNA encoding a variety of cardiac muscle proteins in the adult exercise trained GK diabetic rat heart.

Long‐lived and Obesity Resistant Mice Exhibit 24 h Locomotor Circadian Rhythms at Young and Old Age

Ageing is associated with disruption of circadian homeostasis, including reduced entraiment to light‐dark cycles, reduced amplitude and desynchronization of circadian physiological rhythms, and shortening or lengthening of the endogenous circadian period length (tau). αMUPA mice carry a transgene encoding the urokinase‐type plasminogen activator, an extracellular protease implicated in fibrinolysis, tissue remodeling, and brain plasticity. Female αMUPA mice spontaneously eat ~25% less, live ~20% longer, and show high‐amplitude circadian rhythms in food intake, body temperature, and in hepatic clock gene expression levels, compared with WT mice. Herein we examined the tau of locomotor activity of mature (8‐month old) and aged (18‐month old) female αMUPA and WT mice under total darkness. We show that tau changed in WT mice from a period &lt;24 h at 8 months to a period &gt;;24 h at 18 months. However, tau of αMUPA mice was ~24 h at both ages. Deviation of tau from 24 h has shown to be inversely related to life span of rodents and nonhuman primates. Hence, our results suggest that the sustainable endogenous period of ~24 h in αMUPA mice may contribute to their longevity. These results support the hypothesis that although a non‐24‐h tau has an adaptive significance in allowing a better response to natural daylight changes, such a lack of resonance between tau and external period length entails a life‐shortening metabolic cost.

Vitamin E Ameliorates the Decremental Effect of Paraquat on Cardiomyocyte Contractility in Rats

BackgroundExposure to pesticides and industrial toxins are implicated in cardiovascular disease. Paraquat (PAR) is a toxic chemical widely used as an herbicide in developing countries and described as a major suicide agent. The hypothesis tested here is that PAR induced myocardial dysfunction may be attributed to altered mechanisms of Ca2+ transport which are in turn possibly linked to oxidative stress. The mechanisms of PAR induced myocardial dysfunction and the impact of antioxidant protection was investigated in rat ventricular myocytes.MethodologyForty adult male Wistar rats were divided into 4 groups receiving the following daily intraperitoneal injections for 3 weeks: Group 1 PAR (10 mg/kg), Control Group 2 saline, Group 3 vitamin E (100 mg/kg) and Group 4 PAR (10 mg/kg) and vitamin E (100 mg/kg). Ventricular action potentials were measured in isolated perfused heart, shortening and intracellular Ca2+ in electrically stimulated ventricular myocytes by video edge detection and fluorescence photometry techniques, and superoxide dismutase (SOD) and catalase (CAT) levels in heart tissue.Principal FindingsSpontaneous heart rate, resting cell length, time to peak (TPK) and time to half (THALF) relaxation of myocyte shortening were unaltered. Amplitude of shortening was significantly reduced in PAR treated rats (4.99±0.26%) and was normalized by vitamin E (7.46±0.44%) compared to controls (7.87±0.52%). PAR significantly increased myocytes resting intracellular Ca2+ whilst TPK and THALF decay and amplitude of the Ca2+ transient were unaltered. The fura-2–cell length trajectory during the relaxation of the twitch contraction was significantly altered in myocytes from PAR treated rats compared to controls suggesting altered myofilament sensitivity to Ca2+ as it was normalized by vitamin E treatment. A significant increase in SOD and CAT activities was observed in both PAR and vitamin E plus PAR groups.ConclusionsPAR exposure compromised rats heart function and ameliorated by vitamin E treatment.

Force depression and relaxation kinetics after active shortening and deactivation in mouse soleus muscle

After active shortening, isometric force production capacity of muscle is reduced (force depression, FD). The mechanism is incompletely understood but increasing cross-bridge detachment and/or decreasing attachment rate might be involved. Therefore we aimed to investigate the relation between work delivered during shortening (W), and change in half-relaxation time (Δ0.5RT) and change in the slow phase of muscle relaxation (Δkslow), considered as a marker for cross-bridge detachment rate, after shortening and after a short (0.7s) interruption of activation (deactivation). We hypothesized that shortening induces an accelerated relaxation related to W which is, similar to FD, largely abolished by a short deactivation. In 10 incubated supra-maximally stimulated mouse soleus muscles, we varied the amount of FD at L0 by varying shortening amplitude (0.6, 1.2 and 2.4mm). We found that W not only induces FD (R2=0.92) but also a dose dependent accelerated relaxation (R2=0.88 and R2=0.77 for respectively Δkslow and Δ0.5RT). In cyclic movements this is of functional significance, because the loss in force generating capacity might be (partially) compensated by faster relaxation. After a short deactivation, both FD and Δkslow were largely abolished but Δ0.5RT remained largely present. Under the assumption that Δkslow reflects a change in cross-bridge detachment rate, these results support the idea that FD is an intrinsic sarcomeric property originating from a work induced reduction of the number of force generating cross-bridges, however not via decreased attachment but via increased detachment rate.

Myofilament incorporation and contractile function after gene transfer of cardiac troponin I Ser43/45Ala

Phosphorylation of cardiac troponin I serines 43/45 (cTnISer43/45) by protein kinase C (PKC) is associated with cardiac dysfunction and yet there is disagreement about the role this cluster plays in modulating contractile performance. The present study evaluates the impact of phospho-null Ala substitutions at Ser43/45 (cTnISer43/45Ala) on contractile performance in intact myocytes. Viral-based gene transfer of cardiac troponin I (cTnI) or cTnISer43/45Ala resulted in time-dependent increases in expression, with 70–80% of endogenous cTnI replaced within 4days. Western analysis of intact and permeabilized myocytes along with immunohistochemistry showed each exogenous cTnI was incorporated into the sarcomere of myocytes. In contractile function studies, there were no differences in shortening and re-lengthening for cTnI and cTnISer43/45Ala-expressing myocytes 2days after gene transfer. However, more extensive replacement with cTnISer43/45Ala after 4days diminished peak shortening amplitude and accelerated re-lengthening measured as the time to 50% re-lengthening (TTR50%). A decrease in myofilament Ca2+ sensitivity of tension also was observed in permeabilized myocytes expressing cTnISer43/45Ala and is consistent with accelerated re-lengthening observed in intact myocytes under basal conditions. Phosphorylation of cTnI Ser23/24 and the Ca2+ transient were not changed in these myocytes. These results demonstrate extensive sarcomere expression of cTnISer43/45Ala directly modulates myofilament function under basal conditions. In further work, the accelerated re-lengthening observed in control or cTnI-expressing myocytes treated with the PKC agonist, endothelin-1 (ET, 10nM) was slowed in myocytes expressing cTnISer43/45Ala. This outcome may indicate Ser43/45 is targeted for phosphorylation by ET-activated PKC and/or influences transduction of this agonist-activated response.

N-Terminal Truncated Cardiac Troponin I Enhanced the Contractility of Isolated Cardiomyocytes

Cardiac TnI has a unique N-terminal extension that is a heart-specific regulatory structure not present in skeletal muscle TnI. Previous studies have demonstrated that a restrictive proteolytic truncation of the N-terminal tension of cardiac TnI naturally occurs in normal hearts and is up-regulated in cardiac adaptation to hemodynamic stress or ß-adrenergic deficiency. The N-terminal truncated cardiac TnI (cTnI-ND) alters the conformation of the core structure of cardiac TnI, similarly to that produced with PKA phosphorylation. At organ level, cTnI-ND enhanced ventricular diastolic function. To investigate the functional effect of cTnI-ND at the cellular level avoiding the influence of extracellular matrix, young adult (2-3 months old) cardiomyocytes were isolated from wild type and cTnI-ND transgenic mouse hearts and studied for morphology, sarcomere length, shortening amplitude and velocity, and intracellular calcium transient. Paced contractions and calcium transient were examined using an IonOptix cell imaging system on freshly isolated cardiomyocytes in healthy condition as shown by a positive frequency response. cTnI-ND cardiomyocytes exhibited no difference from wild type cells in length, width and sarcomere length, indicating an absence of pathological remodeling. cTnI-ND cardiomyocytes exhibited larger shortening amplitude and higher shortening/re-lengthening velocities than that of wild type cells. The amplitude of Ca2+ transient was also higher and the durations of Ca2+ rising and decaying were shorter in cTnI-ND myocytes vs. the control. Isoproterenol treatment significantly increased the amplitude of contractility and calcium transient of both wild type and cTnI-ND cardiomyocytes and diminished their differences. The non-additive effects of cTnI-ND and ß-adrenergic stimulation suggest that cTnI-ND enhances cardiac muscle performance via the same mechanism as that of PKA-catalyzed N-terminal phosphorylation of intact cTnI. The functional effect of cTnI-ND demonstrates a posttranslational regulatory mechanism to modulate cardiac muscle contractility in physiological adaptation and heart failure.

Remodeling of Atrial Ca Handling in Canine Model of Chronic Heart Failure Points to Non-Triggered Mechanisms of Atrial Fibrillation

Contractile dysfunction of atria and atrial arrhythmias are common comorbidities in heart failure (HF). Although abnormal Ca handling has been recognized to underlie both reduced contractility and increased propensity to arrhythmias in failing left ventricle, its role in HF-related atrial pathogenesis is not well established. We studied Ca handling in control and HF myocytes isolated from left atrial appendage using canine model of chronic heart failure. In this model, chronic HF was associated with reduced left atrial contractility, increased inducibility of atrial fibrillation, and shortened atrial action potential. Amplitude of Ca transients and cellular shortening was significantly reduced in field-stimulated myocytes from HF compared to control group. Diminished Ca transients recorded in voltage-clamped HF myocytes were associated with reduced amplitude of the L-type Ca currents and decreased gain of excitation-contraction coupling. HF myocytes displayed spatial segregation of Ca signaling manifested in impaired propagation of Ca transient from surface to the center. Frequency of Ca sparks in permeabilzed myocytes was higher in HF, while sarcoplasmic reticulum (SR) Ca content was similar in both control and HF groups. Despite of the increased functional activity of ryanodine receptors, rate of occurrence of diastolic Ca waves and corresponding delayed after depolarizations recorded in paced intact myocytes in a presence of beta-adrenergic receptor agonist isoproterenol was significantly lower in HF than in controls. We conclude that reduced atrial contractility observed in canine model of chronic HF can be attributed, at least in part, to the decreased efficacy of Ca current to trigger SR Ca release. Furthermore, our cellular data do not support Ca-dependent triggered activity in left atrial appendage as a mechanism for atrial arrhythmia in chronic HF.

Biocompatibility of Calcined Mesoporous Silica Particles with Ventricular Myocyte Structure and Function

In vivo and in vitro systems were employed to investigate the biocompatibility of two forms of calcined mesoporous silica microparticles, MCM41-cal and SBA15-cal, with ventricular myocytes. These particles have potential clinical use in delivering bioactive compounds to the heart. Ventricular myocytes were isolated from 6 to 8 week male Wistar rats. The distribution of the particles in ventricular myocytes was investigated by transmission electron microscopy and scanning electron microscopy. The distribution of particles was also examined in cardiac muscle 10 min after intravenous injection of 2.0 mg/mL MCM41-cal. Myocyte shortening and the Ca(2+) transient were determined following exposure to 200 μg/mL MCM41-cal or SBA15-cal for 10 min. Within 10 min of incubation at 25 °C, both MCM41-cal and SBA15-cal were found attached to the plasma membrane, and some particles were observed inside ventricular myocytes. MCM41-cal was more abundant inside the myocytes than SBA15-cal. The particles had a notable affinity to mitochondrial membranes, where they eventually settled. Within 10 min of intravenous injection (2.0 mg/mL), MCM41-cal traversed the perivascular space, and some particles entered ventricular myocytes and localized around the mitochondrial membranes. The amplitude of shortening was slightly reduced in myocytes superperfused with MCM41-cal or SBA15-cal. The amplitude of the Ca(2+) transient was significantly reduced in myocytes superperfused with MCM41-cal but was only slightly reduced with SBA15-cal. Overall, the results show reasonable bioavailability and biocompatibility of MCM41-cal and SBA15-cal with ventricular myocytes.

Muscle–tendon interaction and EMG profiles of world class endurance runners during hopping

The present study examined the muscle-tendon interaction of ten international level Kenyan runners. Ultrasonography and kinematics were applied together with EMG recordings of lower limb muscles during repetitive hopping performed at maximal level. The ten Kenyans had longer gastro Achilles tendon at rest (p < 0.01) as compared with ten control subjects matched in height. Conversely, the stretching and shortening amplitudes of the tendinous tissues of the medial gastrocnemius (MG) muscle were significantly smaller in the Kenyans than in controls during the contact phase of hopping. This applied also to the fascicle length changes, which were smaller and more homogeneous among Kenyans. These limited musculo-tendinous changes resulted in higher maximal hopping height and in larger power despite their reduced body weight. The associated finding of a greater shortening to stretching ratio of the MG tendinous tissues during contact could imply that the Kenyan MG muscle-tendon unit is optimized to favor efficient storage and recoil of elastic energy, while operating at optimal muscle fascicle working range (plateau region).

FKBP12.6 overexpression does not protect against remodelling after myocardial infarction

Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca(2+) handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca(2+)](i) transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca(2+) content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca(2+) content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca(2+)](i) transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca(2+) content and Na(+)- Ca(2+) exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 μm)(-1) s(-1) in Tg MI versus 1.6 ± 0.2 sparks (100 μm)(-1) s(-1) in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted.

Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto–Kakizaki rats

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation-contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation-contraction coupling in the hearts of early onset (8-10 weeks of age) type 2 diabetic Goto-Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real-time RT-PCR; shortening and intracellular Ca(2+) were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non-fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0.03 versus 0.80 ± 0.11; Kcna4, 0.79 ± 0.25 versus 1.90 ± 0.26; and Kcnj2, 0.52 ± 0.07 versus 0.78 ± 0.08) in GK ventricle compared with control ventricle. The amplitude of ventricular myocyte shortening and the intracellular Ca(2+) transient were unaltered; however, the time-to-peak shortening was prolonged and time-to-half decay of the Ca(2+) transient was shortened in GK myocytes compared with control myocytes. The results of this study demonstrate changes in expression of genes encoding various excitation-contraction coupling proteins that are associated with disturbances in myocyte shortening and intracellular Ca(2+) transport.

PKCβII modulation of myocyte contractile performance

Significant up-regulation of the protein kinase Cβ(II) (PKCβ(II)) develops during heart failure and yet divergent functional outcomes are reported in animal models. The goal here is to investigate PKCβ(II) modulation of contractile function and gain insights into downstream targets in adult cardiac myocytes. Increased PKCβ(II) protein expression and phosphorylation developed after gene transfer into adult myocytes while expression remained undetectable in controls. The PKCβ(II) was distributed in a peri-nuclear pattern and this expression resulted in diminished rates and amplitude of shortening and re-lengthening compared to controls and myocytes expressing dominant negative PKCβ(II) (PKCβDN). Similar decreases were observed in the Ca(2+) transient and the Ca(2+) decay rate slowed in response to caffeine in PKCβ(II)-expressing myocytes. Parallel phosphorylation studies indicated PKCβ(II) targets phosphatase activity to reduce phospholamban (PLB) phosphorylation at residue Thr17 (pThr17-PLB). The PKCβ inhibitor, LY379196 (LY) restored pThr17-PLB to control levels. In contrast, myofilament protein phosphorylation was enhanced by PKCβ(II) expression, and individually, LY and the phosphatase inhibitor, calyculin A each failed to block this response. Further work showed PKCβ(II) increased Ca(2+)-activated, calmodulin-dependent kinase IIδ (CaMKIIδ) expression and enhanced both CaMKIIδ and protein kinase D (PKD) phosphorylation. Phosphorylation of both signaling targets also was resistant to acute inhibition by LY. These later results provide evidence PKCβ(II) modulates contractile function via intermediate downstream pathway(s) in cardiac myocytes.

Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling

Our previous in vivo and ex vivo studies suggested that coexistence of two or more troponin T (TnT) isoforms in adult cardiac muscle decreased cardiac function and efficiency (Huang QQ, Feng HZ, Liu J, Du J, Stull LB, Moravec CS, Huang X, Jin JP, Am J Physiol Cell Physiol 294: C213-C22, 2008; Feng HZ, Jin JP, Am J Physiol Heart Circ Physiol 299: H97-H105, 2010). Here we characterized Ca(2+)-regulated contractility of isolated adult cardiomyocytes from transgenic mice coexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT. Without the influence of extracellular matrix, coexistence of the two TnT isoforms resulted in lower shortening amplitude, slower shortening and relengthening velocities, and longer relengthening time. The level of resting cytosolic Ca(2+) was unchanged, but the peak Ca(2+) transient was lowered and the durations of Ca(2+) rising and decaying were longer in the transgenic mouse cardiomyocytes vs. the wild-type controls. Isoproterenol treatment diminished the differences in shortening amplitude and shortening and relengthening velocities, whereas the prolonged durations of relengthening and Ca(2+) transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca(2+), resting sarcomere length of the transgenic cardiomyocytes became shorter than that in wild-type cells. Inhibition of myosin motor diminished this effect of TnT function on cross bridges. The length but not width of transgenic cardiomyocytes was significantly increased compared with the wild-type controls, corresponding to longitudinal addition of sarcomeres and dilatative remodeling at the cellular level. These dominantly negative effects of normal fast TnT demonstrated that chronic coexistence of functionally distinct variants of TnT in adult cardiomyocytes reduces contractile performance with pathological consequences.