In silico identification of potential calcium dynamics and sarcomere targets for recovering left ventricular function in rat heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a complex disease associated with multiple co-morbidities, where impaired cardiac mechanics are often the end effect. At the cellular level, cardiac mechanics can be pharmacologically manipulated by altering calcium signalling and the sarcomere. However, the link between cellular level modulations and whole organ pump function is incompletely understood. Our goal is to develop and use a multi-scale computational cardiac mechanics model of the obese ZSF1 HFpEF rat to identify important biomechanical mechanisms that underpin impaired cardiac function and to predict how whole-heart mechanical function can be recovered through altering cellular calcium dynamics and/or cellular contraction. The rat heart was modelled using a 3D biventricular biomechanics model. Biomechanics were described by 16 parameters, corresponding to intracellular calcium transient, sarcomere dynamics, cardiac tissue and hemodynamics properties. The model simulated left ventricular (LV) pressure-volume loops that were described by 14 scalar features. We trained a Gaussian process emulator to map the 16 input parameters to each of the 14 outputs. A global sensitivity analysis was performed, and identified calcium dynamics and thin and thick filament kinetics as key determinants of the organ scale pump function. We employed Bayesian history matching to build a model of the ZSF1 rat heart. Next, we recovered the LV function, described by ejection fraction, peak pressure, maximum rate of pressure rise and isovolumetric relaxation time constant. We found that by manipulating calcium, thin and thick filament properties we can recover 34%, 28% and 24% of the LV function in the ZSF1 rat heart, respectively, and 39% if we manipulate all of them together. We demonstrated how a combination of biophysically based models and their derived emulators can be used to identify potential pharmacological targets. We predicted that cardiac function can be best recovered in ZSF1 rats by desensitising the myofilament and reducing the affinity to intracellular calcium concentration and overall prolonging the sarcomere staying in the active force generating state.     

Mathematical Modeling of Mechanically Modulated Rhythm Disturbances in Homogeneous and Heterogeneous Myocardium with Attenuated Activity of Na<Superscript>+</Superscript>–K<Superscript>+</Superscript> Pump

A mathematical model of the cardiomyocyte electromechanical function is used to study contribution of mechanical factors to rhythm disturbances in the case of the cardiomyocyte calcium overload. Particular attention is paid to the overload caused by diminished activity of the sodium-potassium pump. It is shown in the framework of the model, where mechano-calcium feedback is accounted for that myocardium mechanics may significantly enhance arrhythmogenicity of the calcium overload. Specifically, a role of cross-bridge attachment/detachment processes, a role of mechanical conditions of myocardium contractions (length, load), and a role of myocardium viscosity in the case of simulated calcium overload have been revealed. Underlying mechanisms are analyzed. Several approaches are designed in the model and compared to each other for recovery of the valid myocardium electrical and mechanical performance in the case of the partially suppressed sodium-potassium pump.     

The effect of sulfonylurea therapy on defective calcium movement associated with diabetic cardiomyopathy

Adult rats exposed to 70 mg/kg streptozocin developed characteristic symptoms of overt diabetes, such as muscle wasting and severely elevated blood glucose levels. Chronic treatment of these rats with the sulfonylurea glyburide for a period of 5 weeks did not affect either the weight of the animal or the degree of hyperglycemia. The drug also failed to influence myocardial glucose metabolism. Nevertheless, the decline in myocardial function associated with the diabetic cardiomyopathy was less in the glyburide-treated rats. At higher preload, myocardial work was significantly reduced in the untreated diabetic but was only moderately depressed in the glyburide-treated heart relative to the nondiabetic heart. The improvement in mechanical function was associated with partial recovery of sarcolemmal calcium pump activity. The drug did not alter the initial rate of Na+-Ca2+ exchange, but decreased the capacity of the transport system. The results indicate that glyburide benefits the diabetic heart by a mechanism independent of carbohydrate metabolism.     

E4031对慢性心衰大鼠离体心脏功能和心肌细胞内静息Ca^2＋水平的影响

目的：研究具有钠钙交换（NCX）激动作用的药物E 4031对慢性心衰大鼠离体心脏功能和心肌细胞内静息Ca2＋水平的影响。方法：通过腹主动脉缩窄建立大鼠慢性心力衰竭模型;利用Langendorff装置进行离体心脏灌流,检测大鼠心功能及E 4031对血流动力学指标的影响;急性分离心衰大鼠心肌细胞,与钙荧光指示剂fluo3/AM共同孵育后,用激光共聚焦显微镜系统观察E 4031对心肌细胞内荧光强度的影响。结果：缩窄大鼠腹主动脉12周后,langendorff离体灌流检测显示大鼠心功能明显降低;在灌流液中加入10μmol/L E 4031可以使心衰大鼠心脏左室发展压（LVDP）和左室收缩/舒张最大速率（±dp/dtmax）提高;与正常组和伪手术组相比,心衰大鼠心肌细胞内静息钙荧光强度明显升高,和10μmol/L E 4031共孵育后,心衰大鼠心肌细胞静息钙荧光强度呈现短期先升后降过程,然后在较低的水平保持稳定。结论：E 4031可以增强慢性心衰大鼠离体心功能,可能与其增强心肌细胞膜NCX活动,稳定细胞内Ca2＋水平有关。     

急性心肌梗死患者肠道优势菌群分析

目的探讨急性心肌梗死患者肠道优势菌群的改变及其与疾病严重程度的关系。方法共筛选急性心肌梗死患者71名及正常健康体检者33名,急性心肌梗死患者根据是否心衰分为急性心肌梗死组36名和急性心肌梗死伴泵衰竭组35名,所有入选者收集大便及血清标本,分别采用qPCR及化学发光仪测定肠道优势菌群改变和血清脑钠肽前体及肌钙蛋白水平。结果急性心肌梗死患者肠道优势菌群显著改变,肠道肠杆菌以及肠球菌细菌数量较对照组显著增加,均与脑钠肽前体、肌钙蛋白、Killip分级显著正相关,而双歧杆菌、乳酸杆菌等细菌数量显著降低,与脑钠肽前体、肌钙蛋白、Killip分级显著负相关。结论急性心肌梗死患者呈现典型的肠道菌群紊乱,且与患者疾病严重程度相关。     

Protective effect of adenosine against a calcium paradox in the isolated frog heart.

The effect of adenosine on the calcium paradox in the isolated frog heart was studied. Addition of adenosine during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein and creatine kinase release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that adenosine protected frog myocardial cells by reducing the massive calcium influx during reperfusion possibly through an action on calcium channels. Adenosine exerted its action in a dose-dependent manner; a concentration of 10 microM adenosine provided maximum protection of myocardial cells against the calcium paradox damage. Higher concentrations of adenosine produced side effects on both electrical and mechanical activity. These results are discussed in terms of the possible mechanism involved in the protective effect of adenosine.     

Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart

The small G protein Ras-mediated signaling pathway has been implicated in the development of hypertrophy and diastolic dysfunction in the heart. Earlier cellular studies have suggested that the Ras pathway is responsible for reduced L-type calcium channel current and sarcoplasmic reticulum (SR) calcium uptake associated with sarcomere disorganization in neonatal cardiomyocytes. In the present study, we investigated the in vivo effects of Ras activation on cellular calcium handling and sarcomere organization in adult ventricular myocytes using a newly established transgenic mouse model with targeted expression of the H-Ras-v12 mutant. The transgenic hearts expressing activated Ras developed significant hypertrophy and postnatal lethal heart failure. In adult ventricular myocytes isolated from the transgenic hearts, the calcium transient was significantly depressed but membrane L-type calcium current was unchanged compared with control littermates. The expressions of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a and phospholamban (PLB) were significantly reduced at mRNA levels. The amount of SERCA2a protein was also modestly reduced. However, the expression of PLB protein and gross sarcomere organization remained unchanged in the hypertrophic Ras hearts, whereas Ser(16) phosphorylation of PLB was dramatically inhibited in the Ras transgenic hearts compared with controls. Hypophosphorylation of PLB was also associated with a significant induction of protein phosphatase 1 expression. Therefore, our results from this in vivo model system suggest that Ras-induced contractile defects do not involve decreased L-type calcium channel activities or disruption of sarcomere structure. Rather, suppressed SR calcium uptake due to reduced SERCA2a expression and hypophosphorylation of PLB due to changes in protein phosphatase expression may play important roles in the diastolic dysfunction of Ras-mediated hypertrophic cardiomyopathy.     

Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart

About 3,000 individuals in the United States are awaiting a donor heart; worldwide, 22 million individuals are living with heart failure. A bioartificial heart is a theoretical alternative to transplantation or mechanical left ventricular support. Generating a bioartificial heart requires engineering of cardiac architecture, appropriate cellular constituents and pump function. We decellularized hearts by coronary perfusion with detergents, preserved the underlying extracellular matrix, and produced an acellular, perfusable vascular architecture, competent acellular valves and intact chamber geometry. To mimic cardiac cell composition, we reseeded these constructs with cardiac or endothelial cells. To establish function, we maintained eight constructs for up to 28 d by coronary perfusion in a bioreactor that simulated cardiac physiology. By day 4, we observed macroscopic contractions. By day 8, under physiological load and electrical stimulation, constructs could generate pump function (equivalent to about 2% of adult or 25% of 16-week fetal heart function) in a modified working heart preparation.     

Identification of a novel phosphorylation site in protein phosphatase inhibitor-1 as a negative regulator of cardiac function

Human and experimental heart failure is characterized by increases in type-1 protein phosphatase activity, which may be partially attributed to inactivation of its endogenous regulator, protein phosphatase inhibitor-1. Inhibitor-1 represents a nodal integrator of two major second messenger pathways, adenosine 3',5'-cyclic monophosphate (cAMP) and calcium, which mediate its phosphorylation at threonine 35 and serine 67, respectively. Here, using recombinant inhibitor-1 wild-type and mutated proteins, we identified a novel phosphorylation site in inhibitor-1, threonine 75. This phosphoamino acid was phosphorylated in vitro by protein kinase Calpha independently and to the same extent as serine 67, the previous protein kinase Calpha-identified site. Generation of specific antibodies for the phosphorylated and dephosphorylated threonine 75 revealed that this site is phosphorylated in rat and dog hearts. Adenoviral-mediated expression of the constitutively phosphorylated threonine 75 inhibitor-1 in isolated myocytes was associated with specific stimulation of type-1 protein phosphatase activity and marked inhibition of the sarcoplasmic calcium pump affinity for calcium, resulting in depressed contractility. Thus, phosphorylation of inhibitor-1 at threonine 75 represents a new mechanism of cardiac contractility regulation, partially through the alteration of sarcoplasmic reticulum calcium transport activity.     

Stanniocalcin-1 is a naturally occurring L-channel inhibitor in cardiomyocytes: relevance to human heart failure

Cardiomyocytes of the failing heart undergo profound phenotypic and structural changes that are accompanied by variations in the genetic program and profile of calcium homeostatic proteins. The underlying mechanisms for these changes remain unclear. Because the mammalian counterpart of the fish calcium-regulating hormone stanniocalcin-1 (STC1) is expressed in the heart, we reasoned that STC1 might play a role in the adaptive-maladaptive processes that lead to the heart failure phenotype. We examined the expression and localization of STC1 in cardiac tissue of patients with advanced heart failure before and after mechanical unloading using a left ventricular assist device (LVAD), and we compared the results with those of normal heart tissue. STC1 protein is markedly upregulated in cardiomyocytes and arterial walls of failing hearts pre-LVAD and is strikingly reduced after LVAD treatment. STC1 is diffusely expressed in cardiomyocytes, although nuclear predominance is apparent. Addition of recombinant STC1 to the medium of cultured rat cardiomyocytes slows their endogenous beating rate and diminishes the rise in intracellular calcium with each contraction. Furthermore, using whole cell patch-clamp studies in cultured rat cardiomyocytes, we find that addition of STC1 to the bath causes reversible inhibition of transmembrane calcium currents through L-channels. Our data suggest differential regulation of myocardial STC1 protein expression in heart failure. In addition, STC1 may regulate calcium currents in cardiomyocytes and may contribute to the alterations in calcium homeostasis of the failing heart.     

Electrophysiology of the flounder heart (Platichthys flesus)—the effect of agents which modify transmembrane ion transport

1. A sucrose gap system was used to record action potentials and mechanical responses of flounder heart.2. Diltiazem eliminated mechanical responses and strongly inhibited the action potential plateau while nifedipine only slightly reduced cardiac contractions without significantly changing the action potential.3. Verapamil slightly hyperpolarized flounder heart but was without effect on either the action potential or mechanical activity except at very high concentrations.4. Lanthanum was ineffective at 2 mM on flounder heart, but manganese at 3 mM substantially inhibited electrical and mechanical responses accompanied by a small hyperpolarization. Substitution of manganese for calcium abolished all flounder cardiac activity.5. BAY K 8644 enhanced cardiac force and enhanced the action potential plateau while depolarizing the preparations. Calcium-free salines abolished heart contractions and the action potential plateau while the spike phase persisted.6. Low sodium salines enhanced while sodium-free salines abolished all heart activity as did tetrodotoxin above I μM. Tetrodotoxin abolished the action potential spike leaving only a small plateau phase.7. Substituting lithium for sodium hyperpolarized the heart, enhanced contractions and prolonged the action potential plateau. Ouabain enhanced cardiac activity and depolarized the heart but ferosemide was without effect on either electrical or mechanical activity.8. TEA at 6 mM had a modest positive inotropic effect and negative chronotropic effect on the heart while the action potential plateau phase was enhanced.9. These results indicate that extracellular sodium and calcium are crucial in flounder heart electrogenesis but such a major role for potassium could not be established.     

Artificial Selection for Whole Animal Low Intrinsic Aerobic Capacity Co-Segregates with Hypoxia-Induced Cardiac Pump Failure

Oxygen metabolism is a strong predictor of the general health and fitness of an organism. In this study, we hypothesized that a divergence in intrinsic aerobic fitness would co-segregate with susceptibility for cardiovascular dysfunction. To test this hypothesis, cardiac function was assessed in rats specifically selected over nineteen generations for their low (LCR) and high (HCR) intrinsic aerobic running capacity. As an integrative marker of native aerobic capacity, run time to exhaustion between LCR and HCR rats had markedly diverged by 436% at generation nineteen of artificial selection. In vivo assessment of baseline cardiac function by echocardiography and catheter-based conductance micromanometry showed no marked difference in cardiac performance. However, when challenged by exposure to acute hypoxia, cardiac pump failure occurred significantly earlier in LCR rats compared to HCR animals. Acute cardiac decompensation in LCR rats was exclusively due to the development of intractable irregular ventricular contractions. Analysis of isolated cardiac myocytes showed significantly slower sarcomeric relaxation and delayed kinetics of calcium cycling in LCR myocytes compared to HCR myocytes. This study also revealed that artificial selection for low native aerobic capacity is a novel pathologic stimulus that results in myosin heavy chain isoform switching in the heart as shown by increased levels of β-MHC in LCR rats. Together, these results provide evidence that alterations in sub-cellular calcium handling and MHC isoform composition are associated with susceptibility to compensatory cardiac remodeling and hypoxia induced pump failure in animals with low intrinsic aerobic capacity.     

Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy,Resulting in Myocardial Inflammation and Heart Failure

We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.     

New perspectives on the role of SERCA2's Ca2+ affinity in cardiac function

Cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (SR) Ca2+ transport ATPase (SERCA2a). The SR Ca2+ -uptake activity not only determines the speed of Ca(2+) removal during relaxation, but also the SR Ca2+ content and therefore the amount of Ca2+ released for cardiomyocyte contraction. The Ca2+ affinity is the major determinant of the pump's activity in the physiological Ca2+ concentration range. In the heart, the affinity of the pump for Ca2+ needs to be controlled between narrow borders, since an imbalanced affinity may evoke hypertrophic cardiomyopathy. Several small proteins (phospholamban, sarcolipin) adjust the Ca2+ affinity of the pump to the physiological needs of the cardiomyocyte. It is generally accepted that a chronically reduced Ca2+ affinity of the pump contributes to depressed SR Ca2+ handling in heart failure. Moreover, a persistently lower Ca2+ affinity is sufficient to impair cardiomyocyte SR Ca2+ handling and contractility inducing dilated cardiomyopathy in mice and humans. Conversely, the expression of SERCA2a, a pump with a lower Ca2+ affinity than the housekeeping isoform SERCA2b, is crucial to maintain normal cardiac function and growth. Novel findings demonstrated that a chronically increased Ca2+ affinity also may trigger cardiac hypertrophy in mice and humans. In addition, recent studies suggest that some models of heart failure are marked by a higher affinity of the pump for Ca2+, and hence by improved cardiomyocyte relaxation and contraction. Depressed cardiomyocyte SR Ca2+ uptake activity may therefore not be a universal hallmark of heart failure.     

Oxygen and effective vasular compliance in acute heart failure.

The effect of hypoxemia on total vascular compliance was studied in anesthetized dogs using a venous bypass technique. Cardiac output was kept constant with an extracorporeal pump and respiration controlled to maintain normocapnia. When nitrogen was added to the respired gas to produce an arterial PO2 approximately 45 mm Hg, total vascular compliance was rapidly and significantly reduced to 0.93 ml (mm Hg)(-1) kg(-1) with incomplete recovery to baseline values of 1.30 plus or minus 0.06 ml (mm Hg)(-1) kg(-1) during subsequent ventilation with 100% oxygen. Acute heart failure was induced by gradual aortic constriction. Ventilation with 100% oxygen failed to prevent a gradual reduction in total vascular compliance to 0.86 ml (mm Hg)(-1) kg(-1) from a baseline value of 1.23 plus or minus 0.06 ml (mm Hg)(-1) kg(-1). Ventilation with 100% oxygen following the reduction in vascular compliance during acute heart failure also failed to significantly alter this parameter. Thus, improvement of arterial oxygen tension in patients with acute heart failure would be beneficial in providing greater oxygen delivery to the tissues without abolishing a compensatory mechanism of reduced vascular compliance which attempts to maintain a cardiac filling gradient of pressure.     

Protective effects of manganese, cobalt, nickel, and barium against a calcium paradox in the isolated frog heart

The effect of inorganic slow channel blockers on the calcium paradox in the frog heart was examined. Addition of the divalent cations of manganese, cobalt, nickel, or barium during calcium depletion protected the frog heart against a calcium paradox. This protective effect was indicated by reduced protein release, maintenance of electrical activity, and recovery of mechanical activity during reperfusion. Tissue calcium determination results showed that in the control paradox in the absence of divalent cations, there is an efflux of calcium from myocardial cells during calcium depletion and a massive influx of calcium during the following reperfusion, leading to a calcium overload. Divalent cations protected frog myocardial cells, when present in the calcium-free perfusion medium, by reducing both calcium efflux during calcium depletion and the massive calcium influx during reperfusion. The effectiveness of the added divalent cations showed a strong dependence upon their ionic radius. The most potent inhibitors of the calcium paradox in the frog heart were the divalent cations having an ionic radius closer to the ionic radius of calcium. These results are discussed in terms of the possible mechanism involved in the protective effect of manganese, cobalt, nickel, and barium.     

Intact calcium signaling in adrenergic-deficient embryonic mouse hearts

Mouse embryos that lack the ability to produce the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to disruption of the dopamine beta-hydroxylase (Dbh^?/-) gene inevitably perish from heart failure during mid-gestation. Since adrenergic stimulation is well-known to enhance calcium signaling in developing as well as adult myocardium, and impairments in calcium signaling are typically associated with heart failure, we hypothesized that adrenergic-deficient embryonic hearts would display deficiencies in cardiac calcium signaling relative to adrenergic-competent controls at a developmental stage immediately preceding the onset of heart failure, which first appears beginning or shortly after mouse embryonic day 10.5 (E10.5). To test this hypothesis, we used ratiometric fluorescent calcium imaging techniques to measure cytosolic calcium transients, [Ca²⁺]_i in isolated E10.5 mouse hearts. Our results show that spontaneous [Ca²⁺]_i oscillations were intact and robustly responded to a variety of stimuli including extracellular calcium (5?mM), caffeine (5?mM), and NE (100?nM) in a manner that was indistinguishable from controls. Further, we show similar patterns of distribution (via immunofluorescent histochemical staining) and activity (via patch-clamp recording techniques) for the major voltage-gated plasma membrane calcium channel responsible for the L-type calcium current, I_Ca,L, in adrenergic-deficient and control embryonic cardiac cells. These results demonstrate that despite the absence of vital adrenergic hormones that consistently leads to embryonic lethality in vivo, intracellular and extracellular calcium signaling remain essentially intact and functional in embryonic mouse hearts through E10.5. These findings suggest that adrenergic stimulation is not required for the development of intracellular calcium oscillations or extracellular calcium signaling through I_Ca,L and that aberrant calcium signaling does not likely contribute to the onset of heart failure in this model.     

Characterization of the calcium paradox in the isolated perfused frog heart: enzymatic,ionic, contractile and electrophysiological studies

Summary The effect of perfusion temperature and duration of calcium deprivation on the occurrence of the calcium paradox was studied in the isolated frog heart. Loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release were used to define cell damage. Perfusion was performed at 22, 27, 32, and 37°C, and calcium deprivation lasted 10, 20, 30, or 40 min. At 22°C and 27°C even a prolonged calcium-free perfusion failed to induce a calcium paradox. After 30 min of calcium-free perfusion at 37°C ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase was 158.24±2.49 IU·g dry wt^-1, and the total amount of protein lost was 70.37±0.73 mg·g dry wt^–1, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase. Ion fluxes in the perfusion effluent indicate that during re-perfusion a massive calcium influx accompanied by a potassium and a magnesium efflux, and an apparent sodium efflux, occur at a perfusion temperature of 37°C after 30 min of calcium deprivation. The results suggest that the basic principles and damaging effects of calcium overloading are common to both mammalian and frog hearts.