Stefano Morotti

A novel computational model of mouse myocyte electrophysiology to assess the synergy between Na+ loading and CaMKII

Intracellular [Na+] ([Na+]i) is elevated in heart failure (HF) and causes arrhythmogenic cellular [Ca2+]i loading. In HF, hyperactivity of Ca2+–calmodulin‐dependent protein kinase II (CaMKII), a key mediator of electrical and mechanical dysfunction in myocytes, causes elevated [Na+]i. We developed a computational model of mouse ventricular myocyte electrophysiology including Ca2+ and CaMKII signalling and quantitatively confirmed evidence suggesting that not only does CaMKII cause elevated [Na+]i, but this additional [Na+]i also promotes further CaMKII activation by increasing [Ca2+]i. We found that a 3–4 mm gain in [Na+]i (similar to that reported in HF) perturbs Ca2+ and membrane potential homeostasis in part via CaMKII activation. This disrupted Ca2+ homeostasis is exacerbated by CaMKII overexpression, and strongly relies upon CaMKII–Na+–Ca2+–CaMKII feedback. CaMKII inhibition in HF may be beneficial, in part by inhibiting [Na+]i loading, and thereby normalizing Ca2+ and membrane potential dynamics without disrupting systolic function.

Theoretical study of L-type Ca2+ current inactivation kinetics during action potential repolarization and early afterdepolarizations

•  The L‐type Ca2+ current (ICa) plays an important role in regulation of excitation–contraction coupling and development of cardiac arrhythmias. •  We studied theoretically ICa inactivation and the relative contributions of voltage‐dependent inactivation (VDI) and Ca2+‐dependent inactivation (CDI) to total inactivation, and we present an improved mathematical model of rabbit ventricular ICa. •  The model proposes that inactivation observed when Ba2+ is the charge carrier includes a small contribution from ion‐dependent inactivation (in addition to pure VDI), usually neglected by other modelling studies. •  The model, identified and validated against a broad set of experimental data, is applied to study the relative roles of VDI and CDI (and the relative contributions of different Ca2+ sources to total CDI) during normal and abnormal repolarization. •  The model predicts that CDI is crucial for repolarization, and that impairment of CDI may be arrhythmogenic by affecting intracellular Ca2+ cycling, through its effect on the ICa time course and Na+–Ca2+ exchanger activity.

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. To avoid this complication, some have used the monovalent cation current through LTCC (I(NS)), which can be measured when divalent cation concentrations are very low. Notably, I(NS) inactivation rate does not depend on current amplitude, and hence may reflect purely VDI. However, based on analysis of existent and new data, and modeling, we find that I(NS) can inactivate more rapidly and completely than I(Ba), especially at physiological temperature. Thus VDI that occurs during I(Ba) (or I(Ca)) must differ intrinsically from VDI during I(NS). To account for this, we have extended a previously published LTCC mathematical model of VDI and CDI into an excitation-contraction coupling model, and assessed whether and how experimental I(Ba) inactivation results (traditionally used in VDI experiments and models) could be recapitulated by modifying CDI to account for Ba-dependent inactivation. Thus, the view of a slow and incomplete I(NS) inactivation should be revised, and I(NS) inactivation is a poor measure of VDI during I(Ca) or I(Ba). This complicates VDI analysis experimentally, but raises intriguing new questions about how the molecular mechanisms of VDI differ for divalent and monovalent currents through LTCCs.

β-adrenergic effects on cardiac myofilaments and contraction in an integrated rabbit ventricular myocyte model☆

A five-state model of myofilament contraction was integrated into a well-established rabbit ventricular myocyte model of ion channels, Ca(2+) transporters and kinase signaling to analyze the relative contribution of different phosphorylation targets to the overall mechanical response driven by β-adrenergic stimulation (β-AS). β-AS effect on sarcoplasmic reticulum Ca(2+) handling, Ca(2+), K(+) and Cl(-) currents, and Na(+)/K(+)-ATPase properties was included based on experimental data. The inotropic effect on the myofilaments was represented as reduced myofilament Ca(2+) sensitivity (XBCa) and titin stiffness, and increased cross-bridge (XB) cycling rate (XBcy). Assuming independent roles of XBCa and XBcy, the model reproduced experimental β-AS responses on action potentials and Ca(2+) transient amplitude and kinetics. It also replicated the behavior of force-Ca(2+), release-restretch, length-step, stiffness-frequency and force-velocity relationships, and increased force and shortening in isometric and isotonic twitch contractions. The β-AS effect was then switched off from individual targets to analyze their relative impact on contractility. Preventing β-AS effects on L-type Ca(2+) channels or phospholamban limited Ca(2+) transients and contractile responses in parallel, while blocking phospholemman and K(+) channel (IKs) effects enhanced Ca(2+) and inotropy. Removal of β-AS effects from XBCa enhanced contractile force while decreasing peak Ca(2+) (due to greater Ca(2+) buffering), but had less effect on shortening. Conversely, preventing β-AS effects on XBcy preserved Ca(2+) transient effects, but blunted inotropy (both isometric force and especially shortening). Removal of titin effects had little impact on contraction. Finally, exclusion of β-AS from XBCa and XBcy while preserving effects on other targets resulted in preserved peak isometric force response (with slower kinetics) but nearly abolished enhanced shortening. β-AS effects on XBCa and XBcy have greater impact on isometric and isotonic contraction, respectively.

FRET biosensor uncovers cAMP nano-domains at β-adrenergic targets that dictate precise tuning of cardiac contractility.

Compartmentalized cAMP/PKA signalling is now recognized as important for physiology and pathophysiology, yet a detailed understanding of the properties, regulation and function of local cAMP/PKA signals is lacking. Here we present a fluorescence resonance energy transfer (FRET)-based sensor, CUTie, which detects compartmentalized cAMP with unprecedented accuracy. CUTie, targeted to specific multiprotein complexes at discrete plasmalemmal, sarcoplasmic reticular and myofilament sites, reveals differential kinetics and amplitudes of localized cAMP signals. This nanoscopic heterogeneity of cAMP signals is necessary to optimize cardiac contractility upon adrenergic activation. At low adrenergic levels, and those mimicking heart failure, differential local cAMP responses are exacerbated, with near abolition of cAMP signalling at certain locations. This work provides tools and fundamental mechanistic insights into subcellular adrenergic signalling in normal and pathological cardiac function.

Atrial-selective targeting of arrhythmogenic phase-3 early afterdepolarizations in human myocytes.

BACKGROUND We have previously shown that non-equilibrium Na(+) current (INa) reactivation drives isoproterenol-induced phase-3 early afterdepolarizations (EADs) in mouse ventricular myocytes. In these cells, EAD initiation occurs secondary to potentiated sarcoplasmic reticulum Ca(2+) release and enhanced Na(+)/Ca(2+) exchange (NCX). This can be abolished by tetrodotoxin-blockade of INa, but not ranolazine, which selectively inhibits ventricular late INa. AIM Since repolarization of human atrial myocytes is similar to mouse ventricular myocytes in that it is relatively rapid and potently modulated by Ca(2+), we investigated whether similar mechanisms can evoke EADs in human atrium. Indeed, phase-3 EADs have been shown to re-initiate atrial fibrillation (AF) during autonomic stimulation, which is a well-recognized initiator of AF. METHODS We integrated a Markov model of INa gating in our human atrial myocyte model. To simulate experimental results, we rapidly paced this cell model at 10Hz in the presence of 0.1μM acetylcholine and 1μM isoproterenol, and assessed EAD occurrence upon return to sinus rhythm (1Hz). RESULTS Cellular Ca(2+) loading during fast pacing results in a transient period of hypercontractility after return to sinus rhythm. Here, fast repolarization and enhanced NCX facilitate INa reactivation via the canonical gating mode (i.e., not late INa burst mode), which drives EAD initiation. Simulating ranolazine administration reduces atrial peak INa and leads to faster repolarization, during which INa fails to reactivate and EADs are prevented. CONCLUSIONS Non-equilibrium INa reactivation can critically contribute to arrhythmias, specifically in human atrial myocytes. Ranolazine might be beneficial in this context by blocking peak (not late) atrial INa.

Ca2+ current facilitation is CaMKII-dependent and has arrhythmogenic consequences

The cardiac voltage gated Ca2+ current (ICa) is critical to the electrophysiological properties, excitation-contraction coupling, mitochondrial energetics, and transcriptional regulation in heart. Thus, it is not surprising that cardiac ICa is regulated by numerous pathways. This review will focus on changes in ICa that occur during the cardiac action potential (AP), with particular attention to Ca2+-dependent inactivation (CDI), Ca2+-dependent facilitation (CDF) and how calmodulin (CaM) and Ca2+-CaM dependent protein kinase (CaMKII) participate in the regulation of Ca2+ current during the cardiac AP. CDI depends on CaM pre-bound to the C-terminal of the L-type Ca2+ channel, such that Ca2+ influx and Ca2+ released from the sarcoplasmic reticulum bind to that CaM and cause CDI. In cardiac myocytes CDI normally pre-dominates over voltage-dependent inactivation. The decrease in ICa via CDI provides direct negative feedback on the overall Ca2+ influx during a single beat, when myocyte Ca2+ loading is high. CDF builds up over several beats, depends on CaMKII-dependent Ca2+ channel phosphorylation, and results in a staircase of increasing ICa peak, with progressively slower inactivation. CDF and CDI co-exist and in combination may fine-tune the ICa waveform during the cardiac AP. CDF may partially compensate for the tendency for Ca2+ channel availability to decrease at higher heart rates because of accumulating inactivation. CDF may also allow some reactivation of ICa during long duration cardiac APs, and contribute to early afterdepolarizations, a form of triggered arrhythmias.

Ser1928 phosphorylation by PKA stimulates the L-type Ca2+ channel CaV1.2 and vasoconstriction during acute hyperglycemia and diabetes

Targeting a protein complex that phosphorylates the calcium channel CaV1.2 in arteries may prevent vascular pathologies associated with diabetes. How sugar constricts arteries Pathological vasoconstriction compromises blood flow to tissues and contributes to various conditions associated with diabetes, including stroke, hypertension, diabetic neuropathy, and diabetic retinopathy. Nystoriak et al. identified a molecular signaling complex—protein kinase A, a scaffolding protein in the AKAP family, and the L-type calcium channel CaV1.2—in arterial myocytes from mice that mediates the phosphorylation of CaV1.2 and enhances the activity of this channel, leading to vasoconstriction. Exposing isolated arterial myocytes from mice or humans to increased extracellular glucose promoted this modification and increased channel activity. Furthermore, myocytes from diabetic mice or human diabetic subjects had increased amount of phosphorylation of CaV1.2 at Ser1928, which resulted in increased channel activity. Arteries from the diabetic mice exhibited a more pronounced vasoconstriction response to pressure than did arteries from control mice. Knocking in S1928A mutant form of the channel blocked this response. Thus, targeting this CaV1.2 regulatory complex may prevent vascular dysfunction in diabetic patients. Hypercontractility of arterial myocytes and enhanced vascular tone during diabetes are, in part, attributed to the effects of increased glucose (hyperglycemia) on L-type CaV1.2 channels. In murine arterial myocytes, kinase-dependent mechanisms mediate the increase in CaV1.2 activity in response to increased extracellular glucose. We identified a subpopulation of the CaV1.2 channel pore-forming subunit (α1C) within nanometer proximity of protein kinase A (PKA) at the sarcolemma of murine and human arterial myocytes. This arrangement depended upon scaffolding of PKA by an A-kinase anchoring protein 150 (AKAP150) in mice. Glucose-mediated increases in CaV1.2 channel activity were associated with PKA activity, leading to α1C phosphorylation at Ser1928. Compared to arteries from low-fat diet (LFD)–fed mice and nondiabetic patients, arteries from high-fat diet (HFD)–fed mice and from diabetic patients had increased Ser1928 phosphorylation and CaV1.2 activity. Arterial myocytes and arteries from mice lacking AKAP150 or expressing mutant AKAP150 unable to bind PKA did not exhibit increased Ser1928 phosphorylation and CaV1.2 current density in response to increased glucose or to HFD. Consistent with a functional role for Ser1928 phosphorylation, arterial myocytes and arteries from knockin mice expressing a CaV1.2 with Ser1928 mutated to alanine (S1928A) lacked glucose-mediated increases in CaV1.2 activity and vasoconstriction. Furthermore, the HFD-induced increases in CaV1.2 current density and myogenic tone were prevented in S1928A knockin mice. These findings reveal an essential role for α1C phosphorylation at Ser1928 in stimulating CaV1.2 channel activity and vasoconstriction by AKAP-targeted PKA upon exposure to increased glucose and in diabetes.

Logistic regression analysis of populations of electrophysiological models to assess proarrythmic risk.

Graphical abstract

Quantitative analysis of the Ca2+‐dependent regulation of delayed rectifier K+ current IKs in rabbit ventricular myocytes

[Ca2+]i enhanced rabbit ventricular slowly activating delayed rectifier K+ current (IKs) by negatively shifting the voltage dependence of activation and slowing deactivation, similar to perfusion of isoproterenol. Rabbit ventricular rapidly activating delayed rectifier K+ current (IKr) amplitude and voltage dependence were unaffected by high [Ca2+]i. When measuring or simulating IKs during an action potential, IKs was not different during a physiological Ca2+ transient or when [Ca2+]i was buffered to 500 nm.