From syncitium to regulated pump: a cardiac muscle cellular update

The primary purpose of this article is to present a basic overview of some key teaching concepts that should be considered for inclusion in an six- to eight-lecture introductory block on the regulation of cardiac performance for graduate students. Within the context of cardiac excitation-contraction coupling, this review incorporates information on Ca(2+) microdomains and local control theory, with particular emphasis on the role of Ca(2+) sparks as a key regulatory component of ventricular myocyte contraction dynamics. Recent information pertaining to local Ca(2+) cycling in sinoatrial nodal cells (SANCs) as a mechanism underlying cardiac automaticity is also presented as part of the recently described coupled-clock pacemaker system. The details of this regulation are emerging; however, the notion that the sequestration and release of Ca(2+) from internal stores in SANCs (similar to that observed in ventricular myocytes) regulates the rhythmic excitation of the heart (i.e., membrane ion channels) is an important advancement in this area. The regulatory role of cardiac adrenergic receptors on cardiac rate and function is also included, and fundamental concepts related to intracellular signaling are discussed. An important point of emphasis is that whole organ cardiac dynamics can be traced back to cellular events regulating intracellular Ca(2+) homeostasis and, as such, provides an important conceptual framework from which students can begin to think about whole organ physiology in health and disease. Greater synchrony of Ca(2+)-regulatory mechanisms between ventricular and pacemaker cells should enhance student comprehension of complex regulatory phenomenon in cardiac muscle.     

Mitochondrial Ca2+ and the heart

It is now well established that mitochondria accumulate Ca(2+) ions during cytosolic Ca(2+) ([Ca(2+)](i)) elevations in a variety of cell types including cardiomyocytes. Elevations in intramitochondrial Ca(2+) ([Ca(2+)](m)) activate several key enzymes in the mitochondrial matrix to enhance ATP production, alter the spatial and temporal profile of intracellular Ca(2+) signaling, and play an important role in the initiation of cell death pathways. Moreover, mitochondrial Ca(2+) uptake stimulates nitric oxide (NO) production by mitochondria, which modulates oxygen consumption, ATP production, reactive oxygen species (ROS) generation, and in turn provides negative feedback for the regulation of mitochondrial Ca(2+) accumulation. Controversy remains, however, whether in cardiac myocytes mitochondrial Ca(2+) transport mechanisms allow beat-to-beat transmission of fast cytosolic [Ca(2+)](i) oscillations into oscillatory changes in mitochondrial matrix [Ca(2+)](m). This review critically summarizes the recent experimental work in this field.     

NPY regulates human endocardial endothelial cell function

Growing evidence suggests that endocardial endothelial cells (EECs) may play an important role in the regulation of cardiac function by releasing several cardioactive factors such as endothelin-1 (ET-1), Angiotensin II (Ang II) and nitric oxide (NO). In our laboratory, we demonstrated that similar to ET-1, EECs do possess different types of NPY receptors, specifically Y(1) and Y(2) receptors. Furthermore, activation of these receptors was found to increase the steady-state level of intracellular free Ca(2+) in EECs and the frequency of beating of cardiomyocytes. In addition, NPY was also found to be present in EECs, and an increase of steady-state intracellular free Ca(2+) induced the release of this peptide from these cells. Thus, similar to ET-1, NPY seems to be released from EECs and this peptide seems to regulate excitation-secretion of these cells as well as excitation-contraction coupling of ventricular cardiomyocytes.     

The II-III loop of the skeletal muscle dihydropyridine receptor is responsible for the Bi-directional coupling with the ryanodine receptor.

The dihydropyridine receptor (DHPR) in the skeletal muscle plasmalemma functions as both voltage-gated Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling. As voltage sensor, the DHPR regulates intracellular Ca(2+) release via the skeletal isoform of the ryanodine receptor (RyR-1). Interaction with RyR-1 also feeds back to increase the Ca(2+) current mediated by the DHPR. To identify regions of the DHPR important for receiving this signal from RyR-1, we expressed in dysgenic myotubes a chimera (SkLC) having skeletal (Sk) DHPR sequence except for a cardiac (C) II-III loop (L). Tagging with green fluorescent protein (GFP) enabled identification of expressing myotubes. Dysgenic myotubes expressing GFP-SkLC or SkLC lacked EC coupling and had very small Ca(2+) currents. Introducing a short skeletal segment (alpha(1S) residues 720-765) into the cardiac II-III loop (replacing alpha(1C) residues 851-896) of GFP-SkLC restored both EC coupling and Ca(2+) current densities like those of the wild type skeletal DHPR. This 46-amino acid stretch of skeletal sequence was recently shown to be capable of transferring strong, skeletal-type EC coupling to an otherwise cardiac DHPR (Nakai, J., Tanabe, T., Konno, T., Adams, B., and Beam, K.G. (1998) J. Biol. Chem. 273, 24983-24986). Thus, this segment of the skeletal II-III loop contains a motif required for both skeletal-type EC coupling and RyR-1-mediated enhancement of Ca(2+) current.     

Mitochondrial modulation of intracellular Ca(2+) signaling

IP3-mediated Ca(2+) release plays a fundamental role in many cell signaling processes and has been the subject of numerous modeling studies. Only recently has the important role that mitochondria play in the dynamics of intracellular Ca(2+) signaling begun to be considered in experimental work and in computational models. Mitochondria sequester large amounts of Ca(2+) and thus have a modulatory effect on intracellular Ca(2+) signaling, and mitochondrial uptake of Ca(2+), in turn, has a regulatory effect on mitochondrial function. Here we integrate a well-established model of IP3-mediated Ca(2+) signaling with a detailed model of mitochondrial Ca(2+) handling and metabolic function. The incorporation of mitochondria results in oscillations in a bistable formulation of the IP3 model, and increasing metabolic substrate decreases the frequency of these oscillations consistent with the literature. Ca(2+) spikes from the cytosol are communicated into mitochondria and are shown to induce realistic metabolic changes. The model has been formulated using a modular approach that is easy to modify and should serve as a useful basis for the investigation of questions regarding the interaction of these two systems.     

Homocyst(e)ine induces calcium second messenger in vascular smooth muscle cells

Homocysteine found in the plasma of patients with coronary heart disease, induces vascular smooth muscle cell (VSMC) proliferation and increases deposition of extracellular matrix (ECM) components. Yet, the mechanism by which homocysteine mediates this effect and its role in vascular disease is largely unknown. We hypothesized that homocysteine induces ECM production via intracellular calcium release in VSMC. To test this hypothesis, aortic VSMC from Sprague-Dawley rats were isolated and characterized by positive labeling for vascular smooth muscle alpha-actin. Early passage cells (p2-3) were grown in monolayer on coverslips. Calcium transients were quantified with fura2/AM spectrofluorometry. Homocysteine induced intracellular calcium [Ca(2+)](i) transients with an EC(50) of 60 +/- 5 nM. The EC(50) for glutathione and cysteine were 10 and 100-fold lower, respectively. Depleting extracellular calcium did not alter the homocysteine effect on intracellular calcium; however, thapsigargin pretreatment, which depletes intracellular Ca(2+) stores, abolished the homocysteine effect, demonstrating its dependence on intracellular Ca(2+) stores. Extracellular sodium depletion significantly (P < 0.05) increased [Ca(2+)](i) also suggesting a possible role of sodium-calcium exchange in the process. To begin to elucidate the intracellular pathways by which homocysteine might act, VSMC were pretreated with specific inhibitors and stimulators prior to homocysteine stimulation. Staurosporine and phorbol myrisate acetate (PMA), potent simulators of protein kinase C, augmented the release of Ca(2+) by homocysteine. Interestingly, pretreatment with the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) greatly exacerbated the sensitivity of VSMC to homocysteine. In contrast, pretreatment with either the phospholipase A(2) activator neomycin, the antioxidant and hepatic hydroxymethyl glutaryl coenzyme A (HMG CoA) reductase inhibitor, pravastatin, the tyrosine kinase inhibitor genestein, or the calcium channel blocker, felodipine completely inhibited the homocysteine-induced Ca(2+) signal in VSMC. This suggests the role of multiple signaling pathways in the homocysteine effect on VSMC Ca(2+). Effects of homocysteine on collagen production, as ascertained by immunoblot analysis, correlated with its effect in intracellular calcium. Regardless of the signaling pathways involved, homocysteine, by virtue of its role on VSMC proliferation and ECM deposition, has the potential to affect vascular reactivity. To determine the effect of homocysteine on the ability of VSMC to react to potent agonist such as angiotensin II, VSMC were pretreated with homocysteine and exposed to a range of angiotensin II concentrations which normally have no effect on intracellular Ca(2+). After homocysteine pretreatment, VSMC were extremely responsive to angiotensin II at concentrations well below the physiologic range. These data taken together suggested that an initial effect of homocysteine is to induce release of intracellular Ca(2+) in VSMC and may induce vascular reactivity. The transient in Ca(2+) correlates with the effect on ECM associated with homocysteine.     

Role of nitric oxide in larval and juvenile fish

Fish are known to express the three isoforms of nitric oxide synthase (NOS), the constitutive forms endothelial or eNOS, neuronal or nNOS and the inducible form iNOS. Most studies in fish have focussed on possible roles for NO in cardiovascular physiology although there has been recent attention on the role of nNOS in embryonic development. However compared to mammalian studies there have been relatively few studies on effects of nitric oxide (NO) on fish. Studies on heart and blood vessel preparations from various fish species appear to show results specific to the species or to the particular preparation. Possible roles of NO in the in vivo biology of adult fish or larval fish have received little attention. This article reviews effects of nitric oxide on cardiovascular physiology in fish with special emphasis on larval fish. It introduces some experimental work on possible signaling pathways in larval fish and introduces the possibility that NO could be an important environmental influence for some aquatic organisms. In higher vertebrates LPS (lipopolysaccharide) is known to activate the cytokine signaling system and stimulate increased expression of iNOS and increased production of NO, but this remains less investigated in fish. The effects of LPS on cardiovascular and osmoregulatory physiology of larval and juvenile salmonids are discussed and a possible role of NO in stress-induced drinking is suggested.     

Intracellular Ca(2+) dynamics and the stability of ventricular tachycardia

Ventricular fibrillation (VF), the major cause of sudden cardiac death, is typically preceded by ventricular tachycardia (VT), but the mechanisms underlying the transition from VT to VF are poorly understood. Intracellular Ca(2+) overload occurs during rapid heart rates typical of VT and is also known to promote arrhythmias. We therefore studied the role of intracellular Ca(2+) dynamics in the transition from VT to VF, using a combined experimental and mathematical modeling approach. Our results show that 1) rapid pacing of rabbit ventricular myocytes at 35 degrees C led to increased intracellular Ca(2+) levels and complex patterns of action potential (AP) configuration and the intracellular Ca(2+) transients; 2) the complex patterns of the Ca(2+) transient arose directly from the dynamics of intracellular Ca(2+) cycling, and were not merely passive responses to beat-to-beat alterations in AP; 3) the complex Ca(2+) dynamics were simulated in a modified version of the Luo-Rudy (LR) ventricular action potential with improved intracellular Ca(2+) dynamics, and showed good agreement with the experimental findings in isolated myocytes; and 4) when incorporated into simulated two-dimensional cardiac tissue, this action potential model produced a form of spiral wave breakup from VT to a VF-like state in which intracellular Ca(2+) dynamics played a key role through its influence on Ca(2+)-sensitive membrane currents such as I(Ca), I(NaCa), and I(ns(Ca)). To the extent that spiral wave breakup is useful as a model for the transition from VT to VF, these findings suggest that intracellular Ca(2+) dynamics may play an important role in the destabilization of VT and its degeneration into VF.     

High-fat diet-induced obesity leads to resistance to leptin-induced cardiomyocyte contractile response

Levels of the obese gene product leptin are often elevated in obesity and may contribute to obesity-induced cardiovascular complications. However, the role of leptin in obesity-associated cardiac abnormalities has not been clearly defined. This study was designed to determine the influence of high-fat diet-induced obesity on cardiac contractile response of leptin. Mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix system in cardiomyocytes from adult rats fed low- and high-fat diets for 12 weeks. Cardiomyocyte contractile and intracellular Ca(2+) properties were examined including peak shortening, duration and maximal velocity of shortening/relengthening (TPS/TR(90), +/-dl/dt), Fura-2-fluorescence intensity change (DeltaFFI), and intracellular Ca(2+) decay rate (tau). Expression of the leptin receptor (Ob-R) was evaluated by western blot analysis. High-fat diet increased systolic blood pressure and plasma leptin levels. PS and +/-dl/dt were depressed whereas TPS and TR(90) were prolonged after high-fat diet feeding. Leptin elicited a concentration-dependent (0-1,000 nmol/l) inhibition of PS, +/-dl/dt, and DeltaFFI in low-fat but not high-fat diet-fed rat cardiomyocytes without affecting TPS and TR(90). The Janus kinase 2 (JAK2) inhibitor AG490, the mitogen-activated protein kinase (MAPK) inhibitor SB203580, and the nitric oxide synthase (NOS) inhibitor L-NAME abrogated leptin-induced cardiomyocyte contractile response in low-fat diet group without affecting the high-fat diet group. High-fat diet significantly downregulated cardiac expression of Ob-R. Elevation of extracellular Ca(2+) concentration nullified obesity-induced cardiomyocyte mechanical dysfunction and leptin-induced depression in PS. These data indicate presence of cardiac leptin resistance in diet-induced obesity possibly associated with impaired leptin receptor signaling.     

NAADP receptors are present and functional in the heart.

Alongside the well-studied inositol 1,4,5 trisphosphate and ryanodine receptors, evidence is gathering that a new intracellular release mechanism, gated by the pyridine nucleotide nicotinic acid adenine dinucleotide phosphate (NAADP), is present in numerous organisms, ranging from plant to mammalian cells (reviewed in [1]). Most cells have been shown to express at least two Ca(2+)-release mechanisms controlled by different messengers, and this can lead to redundancy, convergence, or divergence of responses. One exception appears to be muscle and heart contractile tissues. Here, it is thought that the dominant intracellular channel is the ryanodine receptor, while IP(3) receptors are poorly expressed and their role appears to be negligible. We now report that NAADP receptors are functional and abundant in cardiac microsomes. NAADP binds specifically and with high affinity (130 pM and 4 nM) to two sites on cardiac microsomes and releases Ca(2+) with an apparent EC(50) of 323 +/- 14 nM. Furthermore, binding experiments show that this receptor displays both positive and negative cooperativity, a peculiarity unique among intracellular Ca(2+) channels. Therefore, we show that the heart possesses multiple mechanisms to increase the complexity of Ca(2+) signaling and that NAADP may be integral in the functioning of this organ.     

Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function

Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.     

Dynamical analysis of the calcium signaling pathway in cardiac myocytes based on logarithmic sensitivity analysis

Many cellular functions are regulated by the Ca(2+) signal which contains specific information in the form of frequency, amplitude, and duration of the oscillatory dynamics. Any alterations or dysfunctions of components in the calcium signaling pathway of cardiac myocytes may lead to a diverse range of cardiac diseases including hypertrophy and heart failure. In this study, we have investigated the hidden dynamics of the intracellular Ca(2+) signaling and the functional roles of its regulatory mechanism through in silico simulations and parameter sensitivity analysis based on an experimentally verified mathematical model. It was revealed that the Ca(2+) dynamics of cardiac myocytes are determined by the balance among various system parameters. Moreover, it was found through the parameter sensitivity analysis that the self-oscillatory Ca(2+) dynamics are most sensitive to the Ca(2+) leakage rate of the sarcolemmal membrane and the maximum rate of NCX, suggesting that these two components have dominant effects on circulating the cytosolic Ca(2+).     

Immuno-proteomic approach to excitation--contraction coupling in skeletal and cardiac muscle: molecular insights revealed by the mitsugumins

A comprehensive understanding of excitation-contraction (E-C) coupling in skeletal and cardiac muscle requires that all the major components of the Ca(2+) release machinery be resolved. We utilized a unique immuno-proteomic approach to generate a monoclonal antibody library that targets proteins localized to the skeletal muscle triad junction, which provides a structural context to allow efficient E-C coupling. Screening of this library has identified several mitsugumins (MG); proteins that can be localized to the triad junction in mammalian skeletal muscle. Many of these proteins, including MG29 and junctophilin, are important components in maintaining the structural integrity of the triad junction. Other triad proteins, such as calumin, play a more direct role in regulation of muscle Ca(2+) homeostasis. We have recently identified a family of trimeric intracellular cation-selective (TRIC) channels that allow for K(+) movement into the endoplasmic or sarcoplasmic reticulum to counter a portion of the transient negative charge produced by Ca(2+) release into the cytosol. Further study of TRIC channel function and other novel mitsugumins will increase our understanding of E-C coupling and Ca(2+) homoeostasis in muscle physiology and pathophysiology.     

Structural dynamics of C-domain of cardiac troponin I protein in reconstituted thin filament

The regulatory function of cardiac troponin I (cTnI) involves three important contiguous regions within its C-domain: the inhibitory region (IR), the regulatory region (RR), and the mobile domain (MD). Within these regions, the dynamics of regional structure and kinetics of transitions in dynamic state are believed to facilitate regulatory signaling. This study was designed to use fluorescence anisotropy techniques to acquire steady-state and kinetic information on the dynamic state of the C-domain of cTnI in the reconstituted thin filament. A series of single cysteine cTnI mutants was generated, labeled with the fluorophore tetramethylrhodamine, and subjected to various anisotropy experiments at the thin filament level. The structure of the IR was found to be less dynamic than that of the RR and the MD, and Ca(2+) binding induced minimal changes in IR dynamics: the flexibility of the RR decreased, whereas the MD became more flexible. Anisotropy stopped-flow experiments showed that the kinetics describing the transition of the MD and RR from the Ca(2+)-bound to the Ca(2+)-free dynamic states were significantly faster (53.2-116.8 s(-1)) than that of the IR (14.1 s(-1)). Our results support the fly casting mechanism, implying that an unstructured MD with rapid dynamics and kinetics plays a critical role to initiate relaxation upon Ca(2+) dissociation by rapidly interacting with actin to promote the dissociation of the RR from the N-domain of cTnC. In contrast, the IR responds to Ca(2+) signals with slow structural dynamics and transition kinetics. The collective findings suggested a fourth state of activation.     

Mitochondrial ca(2+) signaling and cardiac apoptosis

The broad significance of apoptosis in the cardiovascular system only began to be recognized more widely recently. Apoptotic cell death is a normal component of postnatal morphogenesis of the human cardiac conduction system and may also be involved in the pathogenesis of a variety of cardiovascular diseases, including heart failure, myocardial infarction and atherosclerosis. Recently, it has become evident that mitochondria play important role in the signaling machinery of apoptotic cell death by releasing several apoptotic factors such as cytochrome c, apoptosis-inducing factor and procaspases. Furthermore, calcium signals have been identified as one of the major signals that converge on mitochondria to trigger the mitochondrion-dependent pathway of the apoptotic cell death. Calcium signals are also important in the physiological control of mitochondrial energy metabolism and it has not yet been explored how Ca(2+) turns from a signal for life to a signal for death. Since large elevations of cytosolic [Ca(2+)] ([Ca(2+)](c)) occur during each heartbeat in cardiac myocytes and these [Ca(2+)](c) signals may efficiently propagate to the mitochondria, the Ca(2+)-dependent mitochondrial pathways of apoptosis can be particularly important in the heart. This review is concerned with the role of mitochondrial Ca(2+) signaling in the control of cardiac apoptosis.     

Cardiac excitation-contraction coupling: role of membrane potential in regulation of contraction

The steps that couple depolarization of the cardiac cell membrane to initiation of contraction remain controversial. Depolarization triggers a rise in intracellular free Ca(2+) which activates contractile myofilaments. Most of this Ca(2+) is released from the sarcoplasmic reticulum (SR). Two fundamentally different mechanisms have been proposed for SR Ca(2+) release: Ca(2+)-induced Ca(2+) release (CICR) and a voltage-sensitive release mechanism (VSRM). Both mechanisms operate in the same cell and may contribute to contraction. CICR couples the release of SR Ca(2+) closely to the magnitude of the L-type Ca(2+) current. In contrast, the VSRM is graded by membrane potential rather than Ca(2+) current. The electrophysiological and pharmacological characteristics of the VSRM are strikingly different from CICR. Furthermore, the VSRM is strongly modulated by phosphorylation and provides a new regulatory mechanism for cardiac contraction. The VSRM is depressed in heart failure and may play an important role in contractile dysfunction. This review explores the operation and characteristics of the VSRM and CICR and discusses the impact of the VSRM on our understanding of cardiac excitation-contraction coupling.