Moderate calcium channel dysfunction in adult mice with inducible cardiomyocyte-specific excision of the cacnb2 gene

The major L-type voltage-gated calcium channels in heart consist of an α1C (Ca(V)1.2) subunit usually associated with an auxiliary β subunit (Ca(V)β2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of Ca(V)β2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the Ca(V)β2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, Ca(V)β2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include Ca(V)β2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other Ca(V)β proteins or changes of Ca(V)1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by <29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, Ca(V)β2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, Ca(V)β2 down-regulation in cardiomyocytes is well tolerated by the adult mice.     

Chronic cardiac resynchronization therapy and reverse ventricular remodeling in a model of nonischemic cardiomyopathy

While cardiac resynchronization therapy (CRT) has been shown to reduce morbidity and mortality in heart failure (HF) patients, the fundamental mechanisms for the efficacy of CRT are poorly understood. The lack of understanding of these basic mechanisms represents a significant barrier to our understanding of the pathogenesis of HF and potential recovery mechanisms. Our purpose was to determine cellular mechanisms for the observed improvement in chronic HF after CRT. We used a canine model of chronic nonischemic cardiomyopathy. After 15 months, dogs were randomized to continued RV tachypacing (untreated HF) or CRT for an additional 9 months. Six minute walk tests, echocardiograms, and electrocardiograms were done to assess the functional response to therapy. Left ventricular (LV) midmyocardial myocytes were isolated to study electrophysiology and intracellular calcium regulation. Compared to untreated HF, CRT improved HF-induced increases in LV volumes, diameters and mass (p<0.05). CRT reversed HF-induced prolongations in LV myocyte repolarization (p<0.05) and normalized HF-induced depolarization (p<0.03) of the resting membrane potential. CRT improved HF-induced reductions in calcium (p<0.05). CRT did not attenuate the HF-induced increases in LV interstitial fibrosis. Using a translational approach in a chronic HF model, CRT significantly improved LV structure; this was accompanied by improved LV myocyte electrophysiology and calcium regulation. The beneficial effects of CRT may be attributable, in part, to improved LV myocyte function.     

Molecular mechanism of the E99K mutation in cardiac actin (ACTC Gene) that causes apical hypertrophy in man and mouse

We generated a transgenic mouse model expressing the apical hypertrophic cardiomyopathy-causing mutation ACTC E99K at 50% of total heart actin and compared it with actin from patients carrying the same mutation. The actin mutation caused a higher Ca(2+) sensitivity in reconstituted thin filaments measured by in vitro motility assay (2.3-fold for mice and 1.3-fold for humans) and in skinned papillary muscle. The mutation also abolished the change in Ca(2+) sensitivity normally linked to troponin I phosphorylation. MyBP-C and troponin I phosphorylation levels were the same as controls in transgenic mice and human carrier heart samples. ACTC E99K mice exhibited a high death rate between 28 and 45 days (48% females and 22% males). At 21 weeks, the hearts of the male survivors had enlarged atria, increased interstitial fibrosis, and sarcomere disarray. MRI showed hypertrophy, predominantly at the apex of the heart. End-diastolic volume and end-diastolic pressure were increased, and relaxation rates were reduced compared with nontransgenic littermates. End-systolic pressures and volumes were unaltered. ECG abnormalities were present, and the contractile response to β-adrenergic stimulation was much reduced. Older mice (29-week-old females and 38-week-old males) developed dilated cardiomyopathy with increased end-systolic volume and continuing increased end-diastolic pressure and slower contraction and relaxation rates. ECG showed atrial flutter and frequent atrial ectopic beats at rest in some ACTC E99K mice. We propose that the ACTC E99K mutation causes higher myofibrillar Ca(2+) sensitivity that is responsible for the sudden cardiac death, apical hypertrophy, and subsequent development of heart failure in humans and mice.     

Calcium Oscillations and Waves Generated by Multiple Release Mechanisms in Pancreatic Acinar Cells

We explore the dynamic behavior of a model of calcium oscillations and wave propagation in the basal region of pancreatic acinar cells [Sneyd, J., et al., Biophys. J. 85: 1392–1405, 2003]. Since it is known that two principal calcium release pathways are involved, inositol trisphosphate receptors (IPR) and ryanodine receptors (RyR), we study how the model behavior depends on the density of each receptor type. Calcium oscillations can be mediated either by IPR or RyR. Continuous increases in either RyR or IPR density can lead to the appearance and disappearance of oscillations multiple times, and the two receptor types interact via their common effect on cytoplasmic calcium concentration and the subsequent effect on the total amount of calcium inside the cell. Increases in agonist concentration can stimulate oscillations via the RyR by increasing calcium influx. Using a two time-scale approach, we explain these complex behaviors by treating the total amount of cellular calcium as a slow parameter. Oscillations are controlled by the shape of the slow manifold and where it intersects the nullcline of the slow variable. When calcium diffusion is included, the existence of traveling waves in the model equation is strongly dependent on the interplay between the total amount of calcium in the cell and membrane transport, a feature that can be experimentally tested. Our results help us understand the behavior of a model that includes both receptors in comparison to the properties of each receptor type in isolation.     

Calpain protects the heart from hemodynamic stress

Calpains make up a family of Ca(2+)-dependent intracellular cysteine proteases that include ubiquitously expressed μ- and m-calpains. Both are heterodimers consisting of a distinct large catalytic subunit (calpain 1 for μ-calpain and calpain 2 for m-calpain) and a common regulatory subunit (calpain 4). The physiological roles of calpain remain unclear in the organs, including the heart, but it has been suggested that calpain is activated by Ca(2+) overload in diseased hearts, resulting in cardiac dysfunction. In this study, cardiac-specific calpain 4-deficient mice were generated to elucidate the role of calpain in the heart in response to hemodynamic stress. Cardiac-specific deletion of calpain 4 resulted in decreased protein levels of calpains 1 and 2 and showed no cardiac phenotypes under base-line conditions but caused left ventricle dilatation, contractile dysfunction, and heart failure with interstitial fibrosis 1 week after pressure overload. Pressure-overloaded calpain 4-deficient hearts took up a membrane-impermeant dye, Evans blue, indicating plasma membrane disruption. Membrane repair assays using a two-photon laser-scanning microscope revealed that calpain 4-deficient cardiomyocytes failed to reseal a plasma membrane that had been disrupted by laser irradiation. Thus, the data indicate that calpain protects the heart from hemodynamic stresses, such as pressure overload.     

Calcium- and voltage-dependence of nematocyst discharge in Hydra vulgaris

In Hydra vulgaris, discharge of stenotele nematocysts was induced by contact with prey, electrical stimuli, or increase in the external potassium concentration. In each case 10^-4M calcium was required in the culture medium. The results indicated a voltage- and calcium-dependent mechanism different from mechano- or chemoreception allowing calcium influx from the external medium. A threshold for activation was suggested by the steep increase of the rate of electrically induced discharge in external fields of 3.5 kV/m. Although organic antagonists for vertebrate calcium channels were ineffective in blocking the calcium-induced nematocyst discharge, inorganic divalent and trivalent cations competitively inhibited the process, with a sequence (Co²⁺ < Ni²⁺ < Cd²⁺ < La³⁺ < Gd³⁺) similar to that seen for antagonism of calcium influx through voltage-dependent channels. Magnesium, an intracellular calcium antagonist, decreased nematocyst discharge, while strontium replacing calcium supported the discharge at a lowered rate. It is concluded that in the nematocyte a voltage-activated influx of calcium through apical ion channels initiates the discharge of the nematocyst in an exocytotic process.     

Overlapping Cardiac Programs in Heart Development and Regeneration

Gaining cellular and molecular insights into heart development and regeneration will likely provide new therapeutic targets and opportunities for cardiac regenerative medicine,one of the most urgent clinical needs for heart failure.Here we present a review on zebrafish heart development and regeneration,with a particular focus on early cardiac progenitor development and their contribution to building embryonic heart,as well as cellular and molecular programs in adult zebrafish heart regeneration.We attempt to emphasize that the signaling pathways shaping cardiac progenitors in heart development may also be redeployed during the progress of adult heart regeneration.A brief perspective highlights several important and promising research areas in this exciting field.     

Signaling and myosin-binding protein C

Myosin-binding protein C (MyBP-C) is a thick filament protein consisting of 1274 amino acid residues (149 kDa) that was identified by Starr and Offer over 30 years ago as a contaminant present in a preparation of purified myosin. Since then, numerous studies have defined the muscle-specific isoforms, the structure, and the importance of the proteins in normal striated muscle structure and function. Underlying the critical role the protein plays, it is now apparent that mutations in the cardiac isoform (cMyBP-C) are responsible for a substantial proportion (30-40%) of genotyped cases of familial hypertrophic cardiomyopathy. Although generally accepted that MyBP-C can interact with all three filament systems within the sarcomere (the thick, thin, and titin filaments), the exact nature of these interactions and the functional consequences of modified binding remain obscure. In addition to these structural considerations, cMyBP-C can serve as a point of convergence for signaling processes in the cardiomyocyte via post-translational modifications mediated by kinases that phosphorylate residues in the cardiac-specific isoform sequence. Thus, cMyBP-C is a critical nodal point that has both important structural and signaling roles and whose modifications are known to cause significant human cardiac disease.     

Calcium overload and cardiac function

The changes in cardiac function caused by calcium overload are reviewed. Intracellular Ca²⁺ may increase in different structures [e.g. sarcoplasmic reticulum (SR), cytoplasm and mitochondria] to an excessive level which induces electrical and mechanical abnormalities in cardiac tissues. The electrical manifestations of Ca²⁺ overload include arrhythmias caused by oscillatory (V_os) and non-oscillatory (V_ex) potentials. The mechanical manifestations include a decrease in force of contraction, contracture and aftercontractions. The underlying mechanisms involve a role of Na⁺ in electrical abnormalities as a charge carrier in the Na⁺-Ca²⁺ exchange and a role of Ca²⁺ in mechanical toxicity. Ca²⁺ overload may be induced by an increase in [Na⁺]_i through the inhibition of the Na⁺-K⁺ pump (e.g. toxic concentrations of digitalis) or by an increase in Ca²⁺ load (e.g. catecholamines). The Ca²⁺ overload is enhanced by fast rates. Purkinje fibers are more susceptible to Ca²⁺ overload than myocardial fibers, possibly because of their greater Na⁺ load. If the SR is predominantly Ca²⁺ overloaded, V_os and fast discharge are induced through an oscillatory release of Ca²⁺ in diastole from the SR; if the cytoplasm is Ca²⁺ overloaded, the non-oscillatory V_ex tail is induced at negative potentials. The decrease in contractile force by Ca²⁺ overload appears to be associated with a decrease in high energy phosphates, since it is enhanced by metabolic inhibitors and reduced by metabolic substrates. The ionic currents I_os and I_ex underlie V_os and V_ex, respectively, both being due to an electrogenic extrusion of Ca²⁺ through the Na⁺-Ca²⁺ exchange. I_os is an oscillatory current due to an oscillatory release of Ca²⁺ in early diastole from the Ca²⁺-overloaded SR, and I_ex is a non-oscillatory current due to the extrusion of Ca²⁺ from the Ca²⁺-overloaded cytoplasm. I_os and I_ex can be present singly or simultaneously. An increase in [Ca²⁺]_i appears to be involved in the short- and long-term compensatory mechanisms that tend to maintain cardiac output in physiological and pathological conditions. Eventually, [Ca²⁺]_i may increase to overload levels and contribute to cardiac failure. Experimental evidence suggests that clinical concentrations of digitalis increase force in Ca²⁺-overloaded cardiac cells by decreasing the inhibition of the Na⁺-K⁺ pump by Ca²⁺, thereby leading to a reduction in Ca²⁺ overload and to an increase in force of contraction.     

Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca²⁺ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca²⁺-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca²⁺ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca²⁺ content. This abnormal Ca²⁺ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na⁺-Ca²⁺ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.     

心力衰竭的物理诊断现状

心力衰竭是各种心脏疾病发展的终末阶段,及时准确的确诊心力衰竭、辨别其病情变化可进一步指导治疗及缩短住院时间,并为心力衰竭的预后评价提供参考依据。目前,确诊心力衰竭主要基于患者的临床症状、体征及各种影像学表现、生化指标等做出综合评价,而物理诊断是诊断心力衰竭不可或缺的依据。心脏彩超已应用于临床多年,可从多个层面评估左室充盈压,是初步评价心脏功能的便捷措施。核心脏病学可评估心脏交感神经支配的区域及激活模式,预测心力衰竭的发病率和死亡率。心脏磁共振可用于进行危险分层及确定是否适合手术/介入治疗。此外,心脏CT及心导管术也是用于评价心功能的常见物理检查方法。本文主要总结了目前各种诊断心力衰竭的物理诊断方法的最新进展,以进一步指导临床实践。     

Origins of blood volume change due to glutamatergic synaptic activity at astrocytes abutting on arteriolar smooth muscle cells

The cellular mechanisms that couple activity of glutamatergic synapses with changes in blood flow, measured by a variety of techniques including the BOLD signal, have not previously been modelled. Here we provide such a model, that successfully accounts for the main observed changes in blood flow in both visual cortex and somatosensory cortex following their stimulation by high-contrast drifting grating or by single whisker stimulation, respectively. Coupling from glutamatergic synapses to smooth muscle cells of arterioles is effected by astrocytes releasing epoxyeicosatrienoic acids (EETs) onto them, following glutamate stimulation of the astrocyte. Coupling of EETs to the smooth muscle of arterioles is by means of potassium channels in their membranes, leading to hyperpolarization, relaxation and hence an increase in blood flow. This model predicts a linear increase in blood flow with increasing numbers of activated astrocytes, but a non-linear increase with increasing glutamate release.     

心肌兴奋收缩耦联过程中的钙调控

联系以膜电位变化为特征的细胞兴奋和以肌丝滑行为基础的肌肉收缩的中介过程通常称为兴奋收缩耦联。在所有参与调控心肌收缩功能的离子中,钙离子被认为是最重要的介导因子,因此验明钙离子参与介导心肌兴奋收缩耦联的方式和途径等特征无疑有益于更好地理解心脏的生理功能。     

运动后心力及心率同时遥测的心力恢复趋势与心率恢复趋势的对比研究

为了更加全面地研究和评估心肌的变时性和变力性,研制了能够在运动场地同时采集心力和心率的心音信号遥测系统。采集了50名体育系学生和30名其他系学生在完成规定运动量的登梯运动后,5min内连续变化的心音信号。对采集的数据经过3次样条插值、均匀采样和小波变换滤波后,得到了平滑的心力恢复趋势曲线和心率恢复趋势曲线。统计分析表明,大负荷运动量下的心脏储备主要来自于心力储备而不是心率储备(P<0.001),和其他系学生相比,体育系学生具有的高水平心功能不仅在于具有更大的心力储备指数(P<0.001),而且还在于具有更快的心力恢复速度(P<0.001)。同时检测心力恢复趋势与心率恢复趋势会有益于全面评估心脏功能。     

Models of cardiac excitation-contraction coupling in ventricular myocytes

Mathematical and computational modeling of cardiac excitation-contraction coupling has produced considerable insights into how the heart muscle contracts. With the increase in biophysical and physiological data available, the modeling has become more sophisticated with investigations spanning in scale from molecular components to whole cells. These modeling efforts have provided insight into cardiac excitation-contraction coupling that advanced and complemented experimental studies. One goal is to extend these detailed cellular models to model the whole heart. While this has been done with mechanical and electophysiological models, the complexity and fast time course of calcium dynamics have made inclusion of detailed calcium dynamics in whole heart models impractical. Novel methods such as the probability density approach and moment closure technique which increase computational efficiency might make this tractable.     

Intracellular calcium and the relationship to contractility in an avian model of heart failure

Global contractile heart failure was induced in turkey poults by furazolidone feeding (700 ppm). Abnormal calcium regulation appears to be a key factor in the pathophysiology of heart failure, but the cellular mechanisms contributing to changes in calcium fluxes have not been clearly defined. Isolated ventricular myocytes from non-failing and failing hearts were therefore used to determine whether the whole heart and ventricular muscle contractile dysfunctions were realized at the single cell level. Whole cell current- and voltage-clamp techniques were used to evaluate action potential configurations and L-type calcium currents, respectively. Intracellular calcium transients were evaluated in isolated myocytes with fura-2 and in isolated left ventricular muscles using aequorin. Action potential durations were prolonged in failing myocytes, which correspond to slowed cytosolic calcium clearing. Calcium current-voltage relationships were normal in failing myocytes; preliminary evidence suggests that depressed transient outward potassium currents contribute to prolonged action potential durations. The number of calcium channels (as measured by radioligand binding) were also similar in non-failing and failing hearts. Isolated ventricular muscles from failing hearts had enhanced inotropic responses, in a dose-dependent fashion, to a calcium channel agonist (Bay K 8644). These data suggest that changes in intracellular calcium mobilization kinetics and longer calcium-myofilament interaction may be able to compensate for contractile failure. We conclude that the relationship between calcium current density and sarcoplasmic reticulum calcium release is a dynamic process that may be altered in the setting of heart failure at higher contraction rates. Accepted: 1 March 2000     

Characterizing phospholamban to sarco(endo)plasmic reticulum Ca2+-ATPase 2a (SERCA2a) protein binding interactions in human cardiac sarcoplasmic reticulum vesicles using chemical cross-linking

Chemical cross-linking was used to study protein binding interactions between native phospholamban (PLB) and SERCA2a in sarcoplasmic reticulum (SR) vesicles prepared from normal and failed human hearts. Lys²⁷ of PLB was cross-linked to the Ca²⁺ pump at the cytoplasmic extension of M4 (at or near Lys³²⁸) with the homobifunctional cross-linker, disuccinimidyl glutarate (7.7 Å). Cross-linking was augmented by ATP but abolished by Ca²⁺ or thapsigargin, confirming in native SR vesicles that PLB binds preferentially to E2 (low Ca²⁺ affinity conformation of the Ca²⁺-ATPase) stabilized by ATP. To assess the functional effects of PLB binding on SERCA2a activity, the anti-PLB antibody, 2D12, was used to disrupt the physical interactions between PLB and SERCA2a in SR vesicles. We observed a tight correlation between 2D12-induced inhibition of PLB cross-linking to SERCA2a and 2D12 stimulation of Ca²⁺-ATPase activity and Ca²⁺ transport. The results suggest that the inhibitory effect of PLB on Ca²⁺-ATPase activity in SR vesicles results from mutually exclusive binding of PLB and Ca²⁺ to the Ca²⁺ pump, requiring PLB dissociation for catalytic activation. Importantly, the same result was obtained with SR vesicles prepared from normal and failed human hearts; therefore, we conclude that PLB binding interactions with the Ca²⁺ pump are largely unchanged in failing myocardium.     

Glucose Deprivation-induced Increase in Protein O-GlcNAcylation in Cardiomyocytes Is Calcium-dependent

The posttranslational modification of nuclear and cytosolic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such as neonatal cardiomyocytes, glucose deprivation increases O-GlcNAc levels and if so by what mechanism. Glucose deprivation significantly increased cardiomyocyte O-GlcNAc levels in a time-dependent manner and was associated with decreased O-GlcNAcase (OGA) but not O-GlcNAc transferase (OGT) protein. This response was unaffected by either the addition of pyruvate as an alternative energy source or by the p38 MAPK inhibitor SB203580. However, the response to glucose deprivation was blocked completely by glucosamine, but not by inhibition of OGA with 2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Interestingly, the CaMKII inhibitor KN93 also significantly reduced the response to glucose deprivation. Lowering extracellular Ca²⁺ with EGTA or blocking store operated Ca²⁺ entry with {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 also attenuated the glucose deprivation-induced increase in O-GlcNAc. In C2C12 and HEK293 cells both glucose deprivation and heat shock increased O-GlcNAc levels, and CaMKII inhibitor KN93 attenuated the response to both stresses. These results suggest that increased intracellular calcium and subsequent activation of CaMKII play a key role in regulating the stress-induced increase in cellular O-GlcNAc levels.