Subcellular Propagation of Cardiomyocyte β-Adrenergic Activation of Calcium Uptake Involves Internal β-Receptors and AKAP7.

β-adrenergic receptor (β-AR) signaling in cardiac myocytes is central to cardiac function, but spatiotemporal activation within myocytes is unresolved. In rabbit ventricular myocytes, β-AR agonists or high extracellular [Ca] were applied locally at one end, to measure β-AR signal propagation as Ca-transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca uptake. High local [Ca], increased CaT amplitude under the pipette faster than did ISO, but was also more spatially restricted.

Synergistic FRET assays for drug discovery targeting RyR2 channels.

A key therapeutic target for heart failure and arrhythmia is the deleterious leak through sarcoplasmic reticulum (SR) ryanodine receptor 2 (RyR2) calcium release channels. We have previously developed methods to detect the pathologically leaky state of RyR2 in adult cardiomyocytes by monitoring RyR2 binding to either calmodulin (CaM) or a biosensor peptide (DPc10). Here, we test whether these complementary binding measurements are effective as high-throughput screening (HTS) assays to discover small molecules that target leaky RyR2.

Beat-to-beat dynamic regulation of intracellular pH in cardiomyocytes.

The mammalian heart beats incessantly with rhythmic mechanical activities generating acids that need to be buffered to maintain a stable intracellular pH (pH) for normal cardiac function. Even though spatial pH non-uniformity in cardiomyocytes has been documented, it remains unknown how pH is regulated to match the dynamic cardiac contractions. Here, we demonstrated beat-to-beat intracellular acidification, termed pH transients, in synchrony with cardiomyocyte contractions.

Cardiomyocyte Na and Ca mishandling drives vicious cycle involving CaMKII, ROS, and ryanodine receptors.

Cardiomyocyte Na and Ca mishandling, upregulated Ca/calmodulin-dependent kinase II (CaMKII), and increased reactive oxygen species (ROS) are characteristics of various heart diseases, including heart failure (HF), long QT (LQT) syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). These changes may form a vicious cycle of positive feedback to promote cardiac dysfunction and arrhythmias. In HF rabbit cardiomyocytes investigated in this study, the inhibition of CaMKII, late Na current (I), and leaky ryanodine receptors (RyRs) all attenuated the prolongation and increased short-term variability (STV) of action potential duration (APD), but in age-matched controls these inhibitors had no or minimal effects.

Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs.

Disease-associated mutations in Niemann-Pick type C1 alter ER calcium signaling and neuronal plasticity.

Niemann-Pick type C1 (NPC1) protein is essential for the transport of externally derived cholesterol from lysosomes to other organelles. Deficiency of NPC1 underlies the progressive NPC1 neurodegenerative disorder. Currently, there are no curative therapies for this fatal disease.

Enhanced Depolarization Drive in Failing Rabbit Ventricular Myocytes: Calcium-Dependent and β-Adrenergic Effects on Late Sodium, L-Type Calcium, and Sodium-Calcium Exchange Currents.

Background: Heart failure (HF) is characterized by electrophysiological remodeling resulting in increased risk of cardiac arrhythmias. Previous reports suggest that elevated inward ionic currents in HF promote action potential (AP) prolongation, increased short-term variability of AP repolarization, and delayed afterdepolarizations. However, the underlying changes in late Na current (I), L-type Ca current, and NCX (Na/Ca exchanger) current are often measured in nonphysiological conditions (square-pulse voltage clamp, slow pacing rates, exogenous Ca buffers).

Complex electrophysiological remodeling in postinfarction ischemic heart failure.

Heart failure (HF) following myocardial infarction (MI) is associated with high incidence of cardiac arrhythmias. Development of therapeutic strategy requires detailed understanding of electrophysiological remodeling. However, changes of ionic currents in ischemic HF remain incompletely understood, especially in translational large-animal models.

Altered Repolarization Reserve in Failing Rabbit Ventricular Myocytes: Calcium and β-Adrenergic Effects on Delayed- and Inward-Rectifier Potassium Currents.

Background: Electrophysiological remodeling and increased susceptibility for cardiac arrhythmias are hallmarks of heart failure (HF). Ventricular action potential duration (APD) is typically prolonged in HF, with reduced repolarization reserve. However, underlying K current changes are often measured in nonphysiological conditions (voltage clamp, low pacing rates, cytosolic Ca buffers).

Stress Signaling JNK2 Crosstalk With CaMKII Underlies Enhanced Atrial Arrhythmogenesis.

Rationale: Atrial fibrillation (AF) is the most common arrhythmia, and advanced age is an inevitable and predominant AF risk factor. However, the mechanisms that couple aging and AF propensity remain unclear, making targeted therapeutic interventions unattainable.

Objective: To explore the functional role of an important stress response JNK (c-Jun N-terminal kinase) in sarcoplasmic reticulum Ca handling and consequently Ca-mediated atrial arrhythmias.

Calcium-Dependent Arrhythmogenic Foci Created by Weakly Coupled Myocytes in the Failing Heart.

Rationale: Intercellular uncoupling and Ca (Ca) mishandling can initiate triggered ventricular arrhythmias. Spontaneous Ca release activates inward current which depolarizes membrane potential (V) and can trigger action potentials in isolated myocytes. However, cell-cell coupling in intact hearts limits local depolarization and may protect hearts from this arrhythmogenic mechanism.

Quantitative analysis of the Ca -dependent regulation of delayed rectifier K current I in rabbit ventricular myocytes.

Key Points: [Ca ] enhanced rabbit ventricular slowly activating delayed rectifier K current (I ) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K current (I ) amplitude and voltage dependence were unaffected by high [Ca ] . When measuring or simulating I during an action potential, I was not different during a physiological Ca transient or when [Ca ] was buffered to 500 nm.

Genetically Encoded Biosensors Reveal PKA Hyperphosphorylation on the Myofilaments in Rabbit Heart Failure.

Rationale: In heart failure, myofilament proteins display abnormal phosphorylation, which contributes to contractile dysfunction. The mechanisms underlying the dysregulation of protein phosphorylation on myofilaments is not clear.

Objective: This study aims to understand the mechanisms underlying altered phosphorylation of myofilament proteins in heart failure.

Depolarization of cardiac membrane potential synchronizes calcium sparks and waves in tissue.

The diastolic membrane potential (Vm) can be hyperpolarized or depolarized by various factors such as hyperkalemia or hypokalemia in the long term, or by delayed afterdepolarizations in the short term. In this study, we investigate how Vm affects Ca sparks and waves. We use a physiologically detailed mathematical model to investigate individual factors that affect Ca spark generation and wave propagation.

Targeted ablation of the histidine-rich Ca(2+)-binding protein (HRC) gene is associated with abnormal SR Ca(2+)-cycling and severe pathology under pressure-overload stress.

The histidine-rich Ca(2+)-binding protein (HRC) is located in the lumen of the sarcoplasmic reticulum (SR) and exhibits high-capacity Ca(2+)-binding properties. Overexpression of HRC in the heart resulted in impaired SR Ca(2+) uptake and depressed relaxation through its interaction with SERCA2a. However, the functional significance of HRC in overall regulation of calcium cycling and contractility is not currently well defined.

Cardiac Na+-Ca2+ exchanger: dynamics of Ca2+-dependent activation and deactivation in intact myocytes.

Cardiac Na(+)-Ca(2+) exchange (NCX) activity is regulated by [Ca(2+)]i. The physiological role and dynamics of this process in intact cardiomyocytes are largely unknown. We examined NCX Ca(2+) activation in intact rabbit and mouse cardiomyocytes at 37°C.

Measuring local gradients of intramitochondrial [Ca(2+)] in cardiac myocytes during sarcoplasmic reticulum Ca(2+) release.

Rationale: Mitochondrial [Ca(2+)] ([Ca(2+)](mito)) regulates mitochondrial energy production, provides transient Ca(2+) buffering under stress, and can be involved in cell death. Mitochondria are near the sarcoplasmic reticulum (SR) in cardiac myocytes, and evidence for crosstalk exists. However, quantitative measurements of [Ca(2+)](mito) are limited, and spatial [Ca(2+)](mito) gradients have not been directly measured.

CaMKIIδC slows [Ca]i decline in cardiac myocytes by promoting Ca sparks.

Acute activation of calcium/calmodulin-dependent protein kinase (CaMKII) in permeabilized phospholamban knockout (PLN-KO) mouse myocytes phosphorylates ryanodine receptors (RyRs) and activates spontaneous local sarcoplasmic reticulum (SR) Ca release events (Ca sparks) even at constant SR Ca load. To assess how CaMKII regulates SR Ca release in intact myocytes (independent of SR Ca content changes or PLN effects), we compared Ca sparks in PLN-KO versus mice, which also have transgenic cardiac overexpression of CaMKIIδC in the PLN-KO background (KO/TG). Compared with PLN-KO mice, these KO/TG cardiomyocytes exhibited 1), increased twitch Ca transient and fractional release (both by ∼35%), but unaltered SR Ca load; 2), increased resting Ca spark frequency (300%) despite a lower diastolic [Ca]i, which also slowed twitch [Ca]i decline (suggesting CaMKII-dependent RyR Ca sensitization); 3), elevated Ca spark amplitude and rate of Ca release (which might indicate that more RyR channels participate in a single spark); 4), prolonged Ca spark rise time (which implies that CaMKII either delays RyR closure or prolongs the time when openings can occur); 5), more frequent repetitive sparks at single release sites.

Theoretical study of L-type Ca(2+) current inactivation kinetics during action potential repolarization and early afterdepolarizations.

Sarcoplasmic reticulum (SR) Ca(2+) release mediates excitation–contraction coupling (ECC) in cardiac myocytes. It is triggered upon membrane depolarization by entry of Ca(2+) via L-type Ca(2+) channels (LTCCs), which undergo both voltage- and Ca(2+)-dependent inactivation (VDI and CDI, respectively). We developed improved models of L-type Ca(2+) current and SR Ca(2+) release within the framework of the Shannon-Bers rabbit ventricular action potential (AP) model.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites.

The cardiac Na(+) channel Na(V)1.5 current (I(Na)) is critical to cardiac excitability, and altered I(Na) gating has been implicated in genetic and acquired arrhythmias. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause I(Na) gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes.

Targeting of protein phosphatases PP2A and PP2B to the C-terminus of the L-type calcium channel Ca v1.2.

The L-type Ca(2+) channel Ca(v)1.2 forms macromolecular signaling complexes that comprise the β(2) adrenergic receptor, trimeric G(s) protein, adenylyl cyclase, and cAMP-dependent protein kinase (PKA) for efficient signaling in heart and brain. The protein phosphatases PP2A and PP2B are part of this complex.

Interplay of voltage and Ca-dependent inactivation of L-type Ca current.

Inactivation of L-type Ca channels (LTCC) is regulated by both Ca and voltage-dependent processes (CDI and VDI). To differentiate VDI and CDI, several experimental and theoretical studies have considered the inactivation of Ba current through LTCC (I(Ba)) as a measure of VDI. However, there is evidence that Ba can weakly mimic Ca, such that I(Ba) inactivation is still a mixture of CDI and VDI.

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.

Na:Ca stoichiometry and cytosolic Ca-dependent activation of NCX in intact cardiomyocytes.

We are studying both Na:Ca exchange stoichiometry and cytosolic [Ca] ([Ca]i)-dependent regulation of Na-Ca exchange (NCX) in intact rabbit ventricular myocytes. Analysis of NCX fluxes in subcellular systems strongly supports a dominant 3Na:1Ca exchange, and our measurements in intact cells confirm this. However, in intact native cells, local ion gradients and other factors complicate the process of inferring stoichiometry.