Phospholamban ablation rescues sarcoplasmic reticulum Ca(2+) handling but exacerbates cardiac dysfunction in CaMKIIdelta(C) transgenic mice.

Rationale: We previously showed that transgenic mice expressing Ca(2+)/calmodulin-dependent protein kinase II delta(C) (CaMKII-TG) develop dilated cardiomyopathy associated with increased ryanodine receptors (RyR2) phosphorylation, enhanced sarcoplasmic reticulum (SR) Ca(2+) leak and lowering of SR Ca(2+) load. We hypothesized that phospholamban (PLN) ablation would restore SR Ca(2+) load and prevent the decreased ventricular contractility, dilation and mortality seen in CaMKII-TG.

Objective: Our objectives were to generate CaMKII-TG mice lacking PLN, determine whether the maladaptive effects of cardiac CaMKIIdelta(C) expression were corrected, and establish the mechanistic basis for these changes.

A novel computational model of the human ventricular action potential and Ca transient.

We have developed a detailed mathematical model for Ca handling and ionic currents in the human ventricular myocyte. Our aims were to: (1) simulate basic excitation-contraction coupling phenomena; (2) use realistic repolarizing K current densities; (3) reach steady-state. The model relies on the framework of the rabbit myocyte model previously developed by our group, with subsarcolemmal and junctional compartments where ion channels sense higher [Ca] vs.

Na/K-ATPase--an integral player in the adrenergic fight-or-flight response.

During activation of the sympathetic nervous system, cardiac performance is increased as part of the fight-or-flight stress response. The increase in contractility with sympathetic stimulation is an orchestrated combination of intrinsic inotropic, lusitropic, and chronotropic effects, mediated in part by activation of beta-adrenergic receptors and protein kinase A. This causes phosphorylation of several Ca cycling proteins in cardiac myocytes (increasing Ca entry via L-type Ca channels, sarcoplasmic reticulum Ca pumping, and the dissociation rate of Ca from the myofilaments).

Ca/calmodulin kinase II differentially modulates potassium currents.

Background: Potassium currents contribute to action potential duration (APD) and arrhythmogenesis. In heart failure, Ca/calmodulin-dependent protein kinase II (CaMKII) is upregulated and can alter ion channel regulation and expression.

Methods And Results: We examine the influence of overexpressing cytoplasmic CaMKIIdelta(C), both acutely in rabbit ventricular myocytes (24-hour adenoviral gene transfer) and chronically in CaMKIIdelta(C)-transgenic mice, on transient outward potassium current (I(to)), and inward rectifying current (I(K1)).

Impaired contractile function and calcium handling in hearts of cardiac-specific calcineurin b1-deficient mice.

To define the necessity of calcineurin (Cn) signaling for cardiac maturation and function, the postnatal phenotype of mice with cardiac-specific targeted ablation of the Cn B1 regulatory subunit (Ppp3r1) gene (csCnb1(-/-) mice) was characterized. csCnb1(-/-) mice develop a lethal cardiomyopathy, characterized by impaired postnatal growth of the heart and combined systolic and diastolic relaxation abnormalities, despite a lack of structural derangements. Notably, the csCnb1(-/-) hearts did not exhibit diastolic dilatation, despite the severe functional phenotype.

Isoform specificity of the Na/K-ATPase association and regulation by phospholemman.

Phospholemman (PLM) phosphorylation mediates enhanced Na/K-ATPase (NKA) function during adrenergic stimulation of the heart. Multiple NKA isoforms exist, and their function/regulation may differ. We combined fluorescence resonance energy transfer (FRET) and functional measurements to investigate isoform specificity of the NKA-PLM interaction.

Extracellular potassium dependence of the Na+-K+-ATPase in cardiac myocytes: isoform specificity and effect of phospholemman.

Cardiac Na(+)-K(+)-ATPase (NKA) regulates intracellular Na(+), which in turn affects intracellular Ca(2+) and contractility via the Na(+)/Ca(2+) exchanger. Extracellular K(+) concentration ([K(+)]) is a central regulator of NKA activity. Phospholemman (PLM) has recently been recognized as a critical regulator of NKA in the heart.

Requirement for Ca2+/calmodulin-dependent kinase II in the transition from pressure overload-induced cardiac hypertrophy to heart failure in mice.

Ca2+/calmodulin-dependent kinase II (CaMKII) has been implicated in cardiac hypertrophy and heart failure. We generated mice in which the predominant cardiac isoform, CaMKIIdelta, was genetically deleted (KO mice), and found that these mice showed no gross baseline changes in ventricular structure or function. In WT and KO mice, transverse aortic constriction (TAC) induced comparable increases in relative heart weight, cell size, HDAC5 phosphorylation, and hypertrophic gene expression.

Calcium/calmodulin-dependent kinase II regulation of cardiac ion channels.

Calcium/calmodulin-dependent kinase II (CaMKII) is a multifunctional serine/threonine kinase expressed abundantly in the heart. CaMKII targets numerous proteins involved in excitation-contraction coupling and excitability, and its activation may simultaneously contribute to heart failure and cardiac arrhythmias. In this review, we summarize the modulatory effects of CaMKII on cardiac ion channel function and expression and illustrate potential implications in the onset of arrhythmias via a computer model.

Na+ transport in cardiac myocytes; Implications for excitation-contraction coupling.

Intracellular Na(+) concentration ([Na(+)](i)) is very important in modulating the contractile and electrical activity of the heart. Upon electrical excitation of the myocardium, voltage-dependent Na(+) channels open, triggering the upstroke of the action potential (AP). During the AP, Ca(2+) enters the myocytes via L-type Ca(2+) channels.

Phospholamban overexpression in rabbit ventricular myocytes does not alter sarcoplasmic reticulum Ca transport.

Phospholamban has been suggested to be a key regulator of cardiac sarcoplasmic reticulum (SR) Ca cycling and contractility and a potential therapeutic target in restoring the depressed Ca cycling in failing hearts. Our understanding of the function of phospholamban stems primarily from studies in genetically altered mouse models. To evaluate the significance of this protein in larger mammalian species, which exhibit Ca cycling properties similar to humans, we overexpressed phospholamban in adult rabbit cardiomyocytes.

Temperature-dependent activation of neurons by continuous near-infrared laser.

Optical control of neuronal activity has a number of advantages over electrical methods and can be conveniently applied to intact individual neurons in vivo. In this study, we demonstrated an experimental approach in which a focused continuous near-infrared (CNI) laser beam was used to activate single rat hippocampal neurons by transiently elevating the local temperature. Reversible changes in the amplitude and kinetics of neuronal voltage-gated Na and K channel currents were recorded following irradiation with a single-mode 980 nm CNI-laser.

Rabbit models of heart disease.

Human heart disease is a major cause of death and disability. A variety of animal models of cardiac disease have been developed to better understand the etiology, cellular and molecular mechanisms of cardiac dysfunction and novel therapeutic strategies. The animal models have included large animals (e.

Differential integration of Ca2+-calmodulin signal in intact ventricular myocytes at low and high affinity Ca2+-calmodulin targets.

Cardiac myocyte intracellular calcium varies beat-to-beat and calmodulin (CaM) transduces Ca2+ signals to regulate many cellular processes (e.g. via CaM targets such as CaM-dependent kinase and calcineurin).

Termination of cardiac Ca2+ sparks: role of intra-SR [Ca2+], release flux, and intra-SR Ca2+ diffusion.

Ca(2+) release from cardiac sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) is regulated by dyadic cleft [Ca(2+)] and intra-SR free [Ca(2+)] ([Ca(2+)](SR)). Robust SR Ca(2+) release termination is important for stable excitation-contraction coupling, and partial [Ca(2+)](SR) depletion may contribute to release termination. Here, we investigated the regulation of SR Ca(2+) release termination of spontaneous local SR Ca(2+) release events (Ca(2+) sparks) by [Ca(2+)](SR), release flux, and intra-SR Ca(2+) diffusion.

Systems approach to understanding electromechanical activity in the human heart: a national heart, lung, and blood institute workshop summary.

The National Heart, Lung, and Blood Institute (NHLBI) convened a workshop of cardiologists, cardiac electrophysiologists, cell biophysicists, and computational modelers on August 20 and 21, 2007, in Washington, DC, to advise the NHLBI on new research directions needed to develop integrative approaches to elucidate human cardiac function. The workshop strove to identify limitations in the use of data from nonhuman animal species for elucidation of human electromechanical function/activity and to identify what specific information on ion channel kinetics, calcium handling, and dynamic changes in the intracellular/extracellular milieu is needed from human cardiac tissues to develop more robust computational models of human cardiac electromechanical activity. This article summarizes the workshop discussions and recommendations on the following topics: (1) limitations of animal models and differences from human electrophysiology, (2) modeling ion channel structure/function in the context of whole-cell electrophysiology, (3) excitation-contraction coupling and regulatory pathways, (4) whole-heart simulations of human electromechanical activity, and (5) what human data are currently needed and how to obtain them.

Ryanodine receptor phosphorylation at Serine 2030, 2808 and 2814 in rat cardiomyocytes.

The cardiac ryanodine receptor (RyR) controls Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling. Three phosphorylation sites have been identified: Serine-(S)2808, S2814 and recently S2030. We measured phosphorylation with at least two different antibodies per site and demonstrate that for S2808 results were highly antibody-dependent and two out of three S2808 antibodies did not accurately report phosphorylation level.

Calmodulin mediates differential sensitivity of CaMKII and calcineurin to local Ca2+ in cardiac myocytes.

Calmodulin (CaM) mediates Ca-dependent regulation of numerous pathways in the heart, including CaM-dependent kinase (CaMKII) and calcineurin (CaN), yet the local Ca(2+) signals responsible for their selective activation are unclear. To assess when and where CaM, CaMKII, and CaN may be activated in the cardiac myocyte, we integrated new mechanistic computational models of CaM, CaMKII, and CaN with the Shannon-Bers model of excitation-contraction coupling in the rabbit ventricular myocyte. These models are validated with independent in vitro data.

Modulation of SR Ca release by luminal Ca and calsequestrin in cardiac myocytes: effects of CASQ2 mutations linked to sudden cardiac death.

Cardiac calsequestrin (CASQ2) is an intrasarcoplasmic reticulum (SR) low-affinity Ca-binding protein, with mutations that are associated with catecholamine-induced polymorphic ventricular tachycardia (CPVT). To better understand how CASQ2 mutants cause CPVT, we expressed two CPVT-linked CASQ2 mutants, a truncated protein (at G112+5X, CASQ2(DEL)) or CASQ2 containing a point mutation (CASQ2(R33Q)), in canine ventricular myocytes and assessed their effects on Ca handling. We also measured CASQ2-CASQ2 variant interactions using fluorescence resonance transfer in a heterologous expression system, and evaluated CASQ2 interaction with triadin.

Arrhythmogenic effects of beta2-adrenergic stimulation in the failing heart are attributable to enhanced sarcoplasmic reticulum Ca load.

Ventricular tachycardia in heart failure (HF) can initiate by nonreentrant mechanisms such as delayed afterdepolarizations. In an arrhythmogenic rabbit model of HF, we have shown that isoproterenol induces ventricular tachycardia in vivo and aftercontractions and transient inward currents in HF myocytes. To determine whether beta(2)-adrenergic receptor (beta(2)-AR) stimulation contributes, we performed in vivo drug infusion, in vitro myocyte and biochemical studies.