Regulation of cardiac excitation and contraction by p21 activated kinase-1

Cardiac excitation and contraction are regulated by a variety of signaling molecules. Central to the regulatory scheme are protein kinases and phosphatases that carry out reversible phosphorylation of different effectors. The process of β-adrenergic stimulation mediated by cAMP dependent protein kinase (PKA) forms a well-known pathway considered as the most significant control mechanism in excitation and contraction as well as many other regulatory mechanisms in cardiac function. However, although dephosphorylation pathways are critical to these regulatory processes, signaling to phosphatases is relatively poorly understood. Emerging evidence indicates that regulation of phosphatases, which dampen the effect of β-adrenergic stimulation, is also important. We review here functional studies of p21 activated kinase-1 (Pak1) and its potential role as an upstream signal for protein phosphatase PP2A in the heart. Pak1 is a serine/threonine protein kinase directly activated by the small GTPases Cdc42 and Rac1. Pak1 is highly expressed in different regions of the heart and modulates the activities of ion channels, sarcomeric proteins, and other phosphoproteins through up-regulation of PP2A activity. Coordination of Pak1 and PP2A activities is not only potentially involved in regulation of normal cardiac function, but is likely to be important in patho-physiological conditions.     

Photoperiod-dependent modulation of cardiac excitation contraction coupling in the Siberian hamster

In mammals, changes in photoperiod regulate a diverse array of physiological and behavioral processes, an example of which in the Siberian hamster (Phodopus sungorus) is the expression of bouts of daily torpor following prolonged exposure to a short photoperiod. During torpor, body temperature drops dramatically; however, unlike in nonhibernating or nontorpid species, the myocardium retains the ability to contract and is resistant to the development of arrhythmias. In the present study, we sought to determine whether exposure to a short photoperiod results in alterations to cardiac excitation-contraction coupling, thus potentially enabling the heart to survive periods of low temperature during torpor. Experiments were performed on single ventricular myocytes freshly isolated from the hearts of Siberian hamsters that had been exposed to either 12 wk of short-day lengths (SD) or 12 wk of long-day lengths (LD). In SD-acclimated animals, the amplitude of the systolic Ca(2+) transient was increased (e.g., from 142 +/- 17 nmol/l in LD to 229 +/- 31 nmol/l in SD at 4 Hz; P < 0.001). The increased Ca(2+) transient amplitude in the SD-acclimated animals was not associated with any change in the shape or duration of the action potential. However, sarcoplasmic reticulum Ca(2+) content measured after current-clamp stimulation was increased in the SD-acclimated animals (at 4 Hz, 110 +/- 5 vs. 141 +/- 15 mumol/l, P < 0.05). We propose that short photoperiods reprogram the function of the cardiac sarcoplasmic reticulum, resulting in an increased Ca(2+) content, and that this may be a necessary precursor for maintenance of cardiac function during winter torpor.     

Mechanism of excitation and contraction uncoupling in frog and guinea pig myocardial fibers during block of slow sodium-calcium channels by compound D-600]

In the experiments performed on the strips of frog's atria and ventricle it was found that rhythmic stimulation facilitated the dissociation of excitation--contraction coupling caused by D-600 compound through the block of calcium channels, i.e. D-600 prevented the increase of tension in the series of contractions or even converted positive staircase into negative one. The first contraction was affected by D-600 to a lesser degree than the subsequent ones. Guinea pig's left auriculus depressing action of D-600 was found to be strongly dependent on the frequency of stimulation. The data obtained were analysed by the model of excitation--contraction coupling. It was concluded that in amphibians as well as in mammalians tension amplitude during rhythmic activity of myocardial cells is determined mainly by Ca inflow through the excitable calcium channels. On the other hand the amplitude of the first contraction depends on the storage content determined by Ca inflow through the Ca channels open at rest. These latter channels have low sensitivity to D-600 and are better presented in atrium than in ventriculum. An increase of contraction tension observed in low sodium medium took place also under the action of D-600; in this case Ca storage was replenished by the Na-Ca exchange diffusion.     

The voltage-sensitive release mechanism of excitation contraction coupling in rabbit cardiac muscle is explained by calcium-induced calcium release

The putative voltage-sensitive release mechanism (VSRM) was investigated in rabbit cardiac myocytes at 37 degrees C with high resistance microelectrodes to minimize intracellular dialysis. When the holding potential was adjusted from -40 to -60 mV, the putative VSRM was expected to operate alongside CICR. Under these conditions however, we did not observe a plateau at positive potentials of the cell shortening versus voltage relationship. The threshold for cell shortening changed by -10 mV, but this resulted from a similar change of the threshold for activation of inward current. Cell shortening under conditions where the putative VSRM was expected to operate was blocked in a dose dependent way by nifedipine and CdCl2 and blocked completely by NiCl2. "Tail contractions" persisted in the presence of nifedipine and CdCl2 but were blocked completely by NiCl2. Block of early outward current by 4-aminopyridine and 4-acetoamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid (SITS) demonstrated persisting inward current during test depolarizations despite the presence of nifedipine and CdCl2. Inward current did not persist in the presence of NiCl2. A tonic component of cell shortening that was prominent during depolarizations to positive potentials under conditions selective for the putative VSRM was sensitive to rapidly applied changes in superfusate [Na+] and to the outward Na+/Ca2+ exchange current blocking drug KB-R7943. This component of cell shortening was thought to be the result of Na+/Ca2+ exchange-mediated excitation contraction coupling. Cell shortening recorded under conditions selective for the putative VSRM was increased by the enhanced state of phosphorylation induced by isoprenaline (1 microM) and by enhancing sarcoplasmic reticulum Ca2+ content by manipulation of the conditioning steps. Under these conditions, cell shortening at positive test depolarizations was converted from tonic to phasic. We conclude that the putative VSRM is explained by CICR with the Ca2+ "trigger" supplied by unblocked L-type Ca2+ channels and Na+/Ca2+ exchange.     

Specific sequence of motifs of mitochondrial uncoupling proteins

We have searched for the exclusivity of common sequence motifs of the mitochondrial uncoupling proteins (UCP1, UCP2, UCP3, UCP4, BMCP1, and plant UCP [PUMP]) within the gene family of mitochondrial anion carrier proteins. The UCP-specific sequences, "UCP signatures", were found in the first, second, and fourth alpha-helices. First: Ala/Ser-Cys/Thr/n-n/Phe-Ala/Gly-[negatively charged residue]-n/Phe-n/Cys-Thr-Phe/n; second: Gly/Ala-Ile/Leu-Gln/X-[positively charged residue]-NH-n/Cys-Ser/nphi/X-n/Ser-OH/Gly-n-[positively charged residue]-Ile/Met-Gly/Val-n/Thr; fourth: Pro-Asn/ Thr-n-X-[positively charged residue]-Asn/Ser/Ala-n-n-Ile/Leu-n-Asn/Val-Cys/n-n/Thr-[negatively charged residue]-n-n/Thr/Pro-OH/Val (n, nonpolar; phi, aromatic; (positively charged residue/negatively charged residue, charged residue). The second and part of the third signature are also present in the yeast dicarboxylate transporter. The UCP signature excluding BMCP1 was also found in the second matrix segment: [positively charged residue]-(Pro/ del-Leu/del)-[positively charged residue]-phi-X-Gly/Ser-Thr/n-X-NH/[negatively charged residue]-Ala-phi. These UCP signatures are thought to be involved in fatty acid anion binding and translocation.     

Gene repeat expansion and contraction by spontaneous intrachromosomal homologous recombination in mammalian cells

Homologous recombination (HR) is important in repairing errors of replication and other forms of DNA damage. In mammalian cells, potential templates include the homologous chromosome, and after DNA replication, the sister chromatid. Previous work has shown that the mammalian recombination machinery is organized to suppress interchromosomal recombination while preserving intrachromosomal HR. In the present study, we investigated spontaneous intrachromosomal HR in mouse hybridoma cell lines in which variously numbered tandem repeats of the µ heavy chain constant (Cµ) region reside at the haploid, chromosomal immunoglobulin µ heavy chain locus. This organization provides the opportunity to investigate recombination between homologous gene repeats in a well-defined chromosomal locus under conditions in which recombinants are conveniently recovered. This system revealed several features about the mammalian intrachromosomal HR process: (i) the frequency of HR was high (recombinants represented as much as several percent of the total of recombinants and non-recombinants); (ii) the recombination process appeared to be predominantly non-reciprocal, consistent with the possibility of gene conversion; (iii) putative gene conversion tracts were long (up to 13.4 kb); (iv) the recombination process occurred with precision, initiating and terminating within regions of shared homology. The results are discussed with respect to mammalian intrachromosomal HR involving interactions both within and between sister chromatids.     

Regulation of contraction and relaxation by membrane potential in cardiac ventricular myocytes

Control of contraction and relaxation by membrane potential was investigated in voltage-clamped guinea pig ventricular myocytes at 37 degrees C. Depolarization initiated phasic contractions, followed by sustained contractions that relaxed with repolarization. Corresponding Ca(2+) transients were observed with fura 2. Sustained responses were ryanodine sensitive and exhibited sigmoidal activation and deactivation relations, with half-maximal voltages near -46 mV, which is characteristic of the voltage-sensitive release mechanism (VSRM) for sarcoplasmic reticulum Ca(2+). Inactivation was not detected. Sustained responses were insensitive to inactivation or block of L-type Ca(2+) current (I(Ca-L)). The voltage dependence of sustained responses was not affected by changes in intracellular or extracellular Na(+) concentration. Furthermore, sustained responses were not inhibited by 2 mM Ni(2+). Thus it is improbable that I(Ca-L) or Na(+)/Ca(2+) exchange generated these sustained responses. However, rapid application of 200 microM tetracaine, which blocks the VSRM, strongly inhibited sustained contractions. Our study indicates that the VSRM includes both a phasic inactivating and a sustained noninactivating component. The sustained component contributes both to initiation and relaxation of contraction.     

Mitochondrial metabolism in mammalian cold-acclimation: Magnitude and mechanisms of fatty-acid uncoupling

Cold-acclimation did not alter uncoupling (state 4 respiration) in rat liver or skeletal muscle mitochondria. Palmitate significantly uncoupled mitochondria, but neither the magnitude of this uncoupling nor the contribution of different inner mitochondrial membrane transporters to uncoupling was altered by cold-acclimation. Guanosine diphosphate did not reduce uncoupling, suggesting no role for uncoupling protein homologues. The adenine nucleotide transporter and the permeability transition pore, either alone or in combination, appear to contribute significantly to free fatty-acid (FFA)-induced uncoupling. Evidence suggests that these two elements may be working together, as components of the same mechanism, to mediate FFA uncoupling.     

Myofibrillar creatine kinase and cardiac contraction

This article is a review on the organization and function of myofibrillar creatine kinase in striated muscle. The first part describes myofibrillar creatine kinase as an integral structural part of the complex organization of myofibrils in striated muscle. The second part considers the intrinsic biochemical and mechanical properties of myofibrils and the functional coupling between myofibrillar CK and myosin ATPase. Skinned fiber studies have been developed to evidence this functional coupling and the consequences for cardiac contraction. The data show that creatine kinase in myofibrils is effective enough to sustain normal tension and relaxation, normal Ca sensitivity and kinetic characteristics. Moreover, the results suggest that myofibrillar creatine kinase is essential in maintaining adequate ATP/ADP ratio in the vicinity of myosin ATPase active site to prevent dysfunctioning of this enzyme. Implications for the physiology and physiopathology of cardiac muscle are discussed.     

Regulation of the uncoupling protein in brown adipose tissue

Presumptive evidence suggests that the brown fat mitochondrial uncoupling protein, thermogenin, is involved in the mechanism of stimulation of respiration by norepinephrine in the intact tissue. Conflicting data have been reported which suggest involvement of either adenine nucleotides, or fatty acids, or long chain acyl-CoA, or protons in the physiological regulation. We measured the electrical potential gradient across the mitochondrial membrane (delta psi m) in control cells and in cells stimulated with norepinephrine, using the accumulation of lipophilic cation, tetraphenylphosphonium, as an indicator of the potential gradient. The value of delta psi m in the cells in the control state is 116 mV, and in the hormonally stimulated state it is 56.6 mV. This supports the view that the protein is involved in the mechanism of hormone action. Other studies were designed to distinguish between the effects of fatty acids and ATP levels on the uncoupling protein in isolated mitochondria and in the adipocytes. ATP levels and fatty acid levels inside intact cells were independently varied using oligomycin or external fatty acids. Their effect on thermogenin was monitored as the capacity of the cells for reverse electron transport from durohydroquinone. The results suggest that ATP modulates the activity of thermogenin, while fatty acids can alter the relationship between ATP and thermogenin activity such that the protein appears to be activated at a higher cellular ATP level in the presence of fatty acids than in their absence.     

Specific folding and contraction of DNA by histones H3 and H4.

We demonstrate that the arginine-rich histones H3 and H4 can introduce torsional constraints on closed circular DNA with a concomitant compaction of the nucleic acid. SV40 DNA I complexed with H3 and H4 appears relaxed in electron micrographs and contains particles of 75 +/- 10 A in diameter along the DNA. SV40 DNA I is contracted 2.75 +/- 0.25 fold by all the four smaller histones and 2.6 +/- 0.4 fold by H3 and H4 alone. The arginine-rich histones can cause the topological equivalent of unwinding the DNA close to one Watson-Crick turn per particle formed. Spherical nucleoprotein complexes morphologically similar to isolated nu bodies or nucleosomes are obtained by association of H3 and H4 with 140 base pair length DNA isolated from chromatin core particles. These reconstituted particles sediment at 9.8S, as compared to 10.8S for native core particles, and contain a tetramer of the arginine-rich histones. None of these specific alterations in DNA structure is seen om complexing the slightly lysine rich-histones H2A and H2B to DNA. Our data provide further evidence indicating that the arginine-rich histones are the major determinants of the architecture of DNA within the chromatin core particle.     

Opposite regulation of uncoupling protein 1 and uncoupling protein 3 in vivo in brown adipose tissue of cold-exposed rats

Earlier we reported a 14-fold increase of glycogen in the brown adipose tissue (BAT) in rats when the animals were placed back from cold to neutral temperature. To elucidate the mechanism, here we compared the level of glucose transporter 4 (GLUT4) protein, uncoupling protein (UCP) 1 and UCP3 mRNA and protein expressions in the BAT under the same conditions. We found that the increased GLUT4 level in cold was maintained during the reacclimation. After 1 week cold exposure the mRNA and protein content of UCP1 increased parallel, while the protein level of UCP3 decreased, contrary to its own mRNA level.     

Measurement of brown adipose tissue uncoupling protein by enzyme linked immunosorbent assay

Antibody to uncoupling protein (UCP) purified from rat brown adipose tissue (BAT) was raised in rabbits and an enzyme linked immunosorbent assay was developed. The antiserum did not cross-react with other mitochondrial proteins from BAT and from other tissues but cross-reacted with UCP from hamster, guinea pig and mouse. The assay is capable of detecting 5 ng of UCP. Using this assay and a crude mitochondrial preparation, UCP content of BAT was shown to increase during cold adaptation.     

Electrophysiological and other aspects of excitation-contraction coupling and uncoupling in mammalian airway smooth muscle

In this article the electrophysiological events which are believed to underly agonist-induced contraction and relaxation of airway smooth muscle are reviewed, with special emphasis on the indispensable role of the Ca ion. The contribution made by Na, K, Ca and Cl to, and the role that the electrogenic Na:K-dependent ATPase plays in, the maintenance of the resting membrane potential in both normal and sensitised airway smooth muscle cells is described together with the permeability changes that occur in the plasmalemma in response to excitatory and inhibitory agonists. In addition, the currently available evidence for the existence of potential-sensitive and receptor-operated Ca channels in respiratory smooth muscle, and how such channels may be involved in the regulation of airway calibre, is critically assessed.