Pasi Tavi

Impact of Sarcoplasmic Reticulum Calcium Release on Calcium Dynamics and Action Potential Morphology in Human Atrial Myocytes: A Computational Study

Electrophysiological studies of the human heart face the fundamental challenge that experimental data can be acquired only from patients with underlying heart disease. Regarding human atria, there exist sizable gaps in the understanding of the functional role of cellular Ca2+ dynamics, which differ crucially from that of ventricular cells, in the modulation of excitation-contraction coupling. Accordingly, the objective of this study was to develop a mathematical model of the human atrial myocyte that, in addition to the sarcolemmal (SL) ion currents, accounts for the heterogeneity of intracellular Ca2+ dynamics emerging from a structurally detailed sarcoplasmic reticulum (SR). Based on the simulation results, our model convincingly reproduces the principal characteristics of Ca2+ dynamics: 1) the biphasic increment during the upstroke of the Ca2+ transient resulting from the delay between the peripheral and central SR Ca2+ release, and 2) the relative contribution of SL Ca2+ current and SR Ca2+ release to the Ca2+ transient. In line with experimental findings, the model also replicates the strong impact of intracellular Ca2+ dynamics on the shape of the action potential. The simulation results suggest that the peripheral SR Ca2+ release sites define the interface between Ca2+ and AP, whereas the central release sites are important for the fire-diffuse-fire propagation of Ca2+ diffusion. Furthermore, our analysis predicts that the modulation of the action potential duration due to increasing heart rate is largely mediated by changes in the intracellular Na+ concentration. Finally, the results indicate that the SR Ca2+ release is a strong modulator of AP duration and, consequently, myocyte refractoriness/excitability. We conclude that the developed model is robust and reproduces many fundamental aspects of the tight coupling between SL ion currents and intracellular Ca2+ signaling. Thus, the model provides a useful framework for future studies of excitation-contraction coupling in human atrial myocytes.

Model of Excitation-Contraction Coupling of Rat Neonatal Ventricular Myocytes

The neonatal rat ventricular myocyte culture is one of the most popular experimental cardiac cell models. To our knowledge, the excitation-contraction coupling (ECC) of these cells, i.e., the process linking the electrical activity to the cytosolic Ca2+ transient and contraction, has not been previously analyzed, nor has it been presented as a complete system in detail. Neonatal cardiomyocytes are in the postnatal developmental stage, and therefore, the features of their ECC differ vastly from those of adult ventricular myocytes. We present the first complete analysis of ECC in these cells by characterizing experimentally the action potential and calcium signaling and developing the first mathematical model of ECC in neonatal cardiomyocytes that we know of. We show that in comparison to adult cardiomyocytes, neonatal cardiomyocytes have long action potentials, heterogeneous cytosolic Ca2+ signals, weaker sarcoplasmic reticulum Ca2+ handling, and stronger sarcolemmal Ca2+ handling, with a significant contribution by the Na+/Ca2+ exchanger. The developed model reproduces faithfully the ECC of rat neonatal cardiomyocytes with a novel description of spatial cytosolic [Ca2+] signals. Simulations also demonstrate how an increase in the cell size (hypertrophy) affects the ECC in neonatal cardiomyocytes. This model of ECC in developing cardiomyocytes provides a platform for developing future models of cardiomyocytes at different developmental stages.

Ca2+–calmodulin‐dependent protein kinase II represses cardiac transcription of the L‐type calcium channel α1C‐subunit gene (Cacna1c) by DREAM translocation

Non‐technical summaryu2002 In heart muscle cells, fluctuations of intracellular calcium (Ca2+) concentration ([Ca2+]i) at the frequency defined by the heart rate induce contractions of the cells. Over a longer timescale the same fluctuations define the properties of the cells by regulating expressions of specific genes through the activation of a variety of cellular enzymes. In this study, we have characterized a specific cell signalling pathway, explaining how [Ca2+]i regulates the expression of the L‐type calcium channel (LTCC). We show that [Ca2+]i‐activated calmodulin kinase II (CaMKII) activates downstream regulatory element (DRE) binding transcription factor DREAM, which consequently suppresses the expression of LTCCs. By experiments and mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+–CaMKII–DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII.

Nrf2 Regulates Neurogenesis and Protects Neural Progenitor Cells Against Aβ Toxicity

Neural stem/progenitor cells (NPCs) proliferate and produce new neurons in neurogenic areas throughout the lifetime. While these cells represent potential therapeutic treatment of neurodegenerative diseases, regulation of neurogenesis is not completely understood. We show that deficiency of nuclear factor erythroid 2‐related factor (Nrf2), a transcription factor induced in response to oxidative stress, prevents the ischemia‐induced increase in newborn neurons in the subgranular zone of the dentate gyrus. Consistent with this finding, the growth of NPC neurospheres was increased by lentivirus‐mediated overexpression of Nrf2 gene or by treatment with pyrrolidine dithiocarbamate (PDTC), an Nrf2 activating compound. Also, neuronal differentiation of NPCs was increased by Nrf2 overexpression or PDTC treatment but reduced by Nrf2 deficiency. To investigate the impact of Nrf2 on NPCs in Alzheimers disease (AD), we treated NPCs with amyloid beta (Aβ), a toxic peptide associated with neurodegeneration and cognitive abnormalities in AD. We found that Aβ1–42‐induced toxicity and reduction in neurosphere proliferation were prevented by Nrf2 overexpression, while Nrf2 deficiency enhanced the Aβ1–42‐induced reduction of neuronal differentiation. On the other hand, Aβ1–40 had no effect on neurosphere proliferation in wt NPCs but increased the proliferation of Nrf2 overexpressing neurospheres and reduced it in Nrf2‐deficient neurospheres. These results suggest that Nrf2 is essential for neuronal differentiation of NPCs, regulates injury‐induced neurogenesis and provides protection against Aβ‐induced NPC toxicity. Stem Cells 2014;32:1904–1916

Excitation-Contraction Coupling of the Mouse Embryonic Cardiomyocyte

In the mammalian embryo, the primitive tubular heart starts beating during the first trimester of gestation. These early heartbeats originate from calcium-induced contractions of the developing heart muscle cells. To explain the initiation of this activity, two ideas have been presented. One hypothesis supports the role of spontaneously activated voltage-gated calcium channels, whereas the other emphasizes the role of Ca2+ release from intracellular stores initiating spontaneous intracellular calcium oscillations. We show with experiments that both of these mechanisms coexist and operate in mouse cardiomyocytes during embryonic days 9–11. Further, we characterize how inositol-3-phosphate receptors regulate the frequency of the sarcoplasmic reticulum calcium oscillations and thus the heartbeats. This study provides a novel view of the regulation of embryonic cardiomyocyte activity, explaining the functional versatility of developing cardiomyocytes and the origin and regulation of the embryonic heartbeat.

Increased fatigue resistance linked to Ca2+-stimulated mitochondrial biogenesis in muscle fibres of cold-acclimated mice

Mammals exposed to a cold environment initially generate heat by repetitive muscle activity (shivering). Shivering is successively replaced by the recruitment of uncoupling protein‐1 (UCP1)‐dependent heat production in brown adipose tissue. Interestingly, adaptations observed in skeletal muscles of cold‐exposed animals are similar to those observed with endurance training. We hypothesized that increased myoplasmic free [Ca2+] ([Ca2+]i) is important for these adaptations. To test this hypothesis, experiments were performed on flexor digitorum brevis (FDB) muscles, which do not participate in the shivering response, of adult wild‐type (WT) and UCP1‐ablated (UCP1‐KO) mice kept either at room temperature (24°C) or cold‐acclimated (4°C) for 4–5 weeks. [Ca2+]i (measured with indo‐1) and force were measured under control conditions and during fatigue induced by repeated tetanic stimulation in intact single fibres. The results show no differences between fibres from WT and UCP1‐KO mice. However, muscle fibres from cold‐acclimated mice showed significant increases in basal [Ca2+]i (∼50%), tetanic [Ca2+]i (∼40%), and sarcoplasmic reticulum (SR) Ca2+ leak (∼fourfold) as compared to fibres from room‐temperature mice. Muscles of cold‐acclimated mice showed increased expression of peroxisome proliferator‐activated receptor‐γ coactivator‐1α (PGC‐1α) and increased citrate synthase activity (reflecting increased mitochondrial content). Fibres of cold‐acclimated mice were more fatigue resistant with higher tetanic [Ca2+]i and less force loss during fatiguing stimulation. In conclusion, cold exposure induces changes in FDB muscles similar to those observed with endurance training and we propose that increased [Ca2+]i is a key factor underlying these adaptations.

The role of in vivo Ca2+ signals acting on Ca2+–calmodulin-dependent proteins for skeletal muscle plasticity

Abstractu2002 Skeletal muscle fibres are highly heterogeneous regarding size, metabolism and contractile function. They also show a large capacity for adaptations in response to alterations in the activation pattern. A major part of this activity‐dependent plasticity relies on transcriptional alterations controlled by intracellular Ca2+ signals. In this review we discuss how intracellular Ca2+ fluctuations induced by activation patterns likely to occur in vivo control muscle properties via effects on Ca2+–calmodulin‐dependent proteins. We focus on two such Ca2+ decoders: calcineurin and Ca2+–calmodulin‐dependent protein kinase II. Inherent Ca2+ transients during contractions differ rather little between slow‐ and fast‐twitch muscle fibres and this difference is unlikely to have any significant impact on the activity of Ca2+ decoders. The major exception to this is fatigue‐induced changes in Ca2+ transients that occur in fast‐twitch fibres exposed to high‐intensity activation typical of slow‐twitch motor units. In conclusion, the cascade from neural stimulation pattern to Ca2+‐dependent transcription is likely to be central in maintaining the fibre phenotypes in both fast‐ and slow‐twitch fibres. Moreover, changes in Ca2+ signalling (e.g. induced by endurance training) can result in altered muscle properties (e.g. increased mitochondrial biogenesis) and this plasticity involves other signalling pathways.

AAV9-mediated VEGF-B Gene Transfer Improves Systolic Function in Progressive Left Ventricular Hypertrophy

Mechanisms of the transition from compensatory hypertrophy to heart failure are poorly understood and the roles of vascular endothelial growth factors (VEGFs) in this process have not been fully clarified. We determined the expression profile of VEGFs and relevant receptors during the progression of left ventricular hypertrophy (LVH). C57BL mice were exposed to transversal aortic constriction (TAC) and the outcome was studied at different time points (1 day, 2, 4, and 10 weeks). A clear compensatory phase (2 weeks after TAC) was seen with following heart failure (4 weeks after TAC). Interestingly, VEGF-C and VEGF-D as well as VEGF receptor-3 (VEGFR-3) were upregulated in the compensatory hypertrophy and VEGF-B was downregulated in the heart failure. After treatment with adeno-associated virus serotype 9 (AAV9)-VEGF-B(186) gene therapy in the compensatory phase for 4 weeks the function of the heart was preserved due to angiogenesis, inhibition of apoptosis, and promotion of cardiomyocyte proliferation. Also, the genetic programming towards fetal gene expression, a known phenomenon in heart failure, was partly reversed in AAV9-VEGF-B(186)-treated mice. We conclude that VEGF-C and VEGF-D are associated with the compensatory LVH and that AAV9-VEGF-B(186) gene transfer can rescue the function of the failing heart and postpone the transition towards heart failure.

Role of reactive oxygen species in the regulation of cardiac contractility

Increased production of reactive oxygen species (ROS) has been linked to the pathogenesis of contractile dysfunction in heart failure. However, it is unclear whether ROS can regulate physiological cellular processes in the myocardium. Here, we characterized the role of endogenous ROS production in the acute regulation of cardiac contractility in the intact rat heart. In isolated perfused rat hearts, endothelin-1 (ET-1, 1nmol/L) stimulated ROS formation in the left ventricle, which was prevented by the antioxidant N-acetylcysteine and the NAD(P)H oxidase inhibitor apocynin. N-acetylcysteine, the superoxide dismutase mimetic MnTMPyP, and apocynin significantly attenuated ET-1-mediated inotropic effect, which was accompanied by inhibition of extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation. Moreover, the mitochondrial K(ATP) channel blocker 5-HD, and the mitochondrial large conductance calcium activated potassium channel blocker paxilline, but not the sarcolemmal K(ATP) channel blocker HMR 1098 attenuated the inotropic response to ET-1. However, ET-1-induced ROS generation was not abolished by inhibiting mitochondrial K(ATP) channel opening. In contrast to ET-1 stimulation, the positive inotropic effect of β(1)-adrenergic receptor agonist dobutamine (250nmol/L) was significantly augmented by N-acetylcysteine and apocynin. Moreover, dobutamine-induced phospholamban phosphorylation was markedly enhanced by apocynin. In conclusion, NAD(P)H oxidase-derived ROS play a physiological role in the acute regulation of cardiac contractility in the intact rat heart. Our results reveal that ET-1-induced increase in cardiac contractility is partially dependent on enhanced ROS generation, which in turn, activates the ERK1/2 pathway. On the other hand, β-adrenergic receptor-induced positive inotropic effect and phospholamban phosphorylation is enhanced by NAD(P)H oxidase inhibition.

Mathematical model of mouse embryonic cardiomyocyte excitation-contraction coupling.

Excitation–contraction (E–C) coupling is the mechanism that connects the electrical excitation with cardiomyocyte contraction. Embryonic cardiomyocytes are not only capable of generating action potential (AP)-induced Ca2+ signals and contractions (E–C coupling), but they also can induce spontaneous pacemaking activity. The spontaneous activity originates from spontaneous Ca2+ releases from the sarcoplasmic reticulum (SR), which trigger APs via the Na+/Ca2+ exchanger (NCX). In the AP-driven mode, an external stimulus triggers an AP and activates voltage-activated Ca2+ intrusion to the cell. These complex and unique features of the embryonic cardiomyocyte pacemaking and E–C coupling have never been assessed with mathematical modeling. Here, we suggest a novel mathematical model explaining how both of these mechanisms can coexist in the same embryonic cardiomyocytes. In addition to experimentally characterized ion currents, the model includes novel heterogeneous cytosolic Ca2+ dynamics and oscillatory SR Ca2+ handling. The model reproduces faithfully the experimentally observed fundamental features of both E–C coupling and pacemaking. We further validate our model by simulating the effect of genetic modifications on the hyperpolarization-activated current, NCX, and the SR Ca2+ buffer protein calreticulin. In these simulations, the model produces a similar functional alteration to that observed previously in the genetically engineered mice, and thus provides mechanistic explanations for the cardiac phenotypes of these animals. In general, this study presents the first model explaining the underlying cellular mechanism for the origin and the regulation of the heartbeat in early embryonic cardiomyocytes.