Purified endogenous inhibitor of the Na/Ca exchanger can enhance the cardiomyocytes contractility and calcium transients

Previous studies have shown that the newly found endogenous inhibitor (NCX(IF)) of the cardiac Na/Ca exchanger (NCX1) is capable of regulating the muscle strip's contractility and relaxation. Here, the effects of purified NCX(IF) were tested on single cell shortening-lengthening (by using the IR CCD camera coupled with the two-edge video-detector) and [Ca]i-transients (by monitoring the changes in fluo-3 fluorescence). A perfusion of isolated cardiomyocytes (paced at 0.5-1.0 Hz) with NCX(IF) results in 4-6-fold enhancement in the amplitude of cell shortening-lengthening reaching the steady-state levels within 5-8 min (n=20, p<0.009). Simultaneous recordings of cell shortening-lengthening and [Ca]i-transients from the same cell show that the amplitude enhancement is associated with accelerated decay of both signals. Therefore, the NCX(IF)-dependent modulation of the single cell contractility is primarily governed by Ca-related mechanisms. The observed data are consistent with a proposal suggesting that the inhibition of NCX1 by NCX(IF) results in Ca-dependent activation of SERCA (SR Ca ATPase), yielding the accelerated decay of the [Ca]i-transients. The subsequent increase in the SR Ca content may result in enhanced Ca-release reflecting the manifested promotion of [Ca]i-transients. More systematic study is required for confirming this working hypothesis.     

Cloning and sequencing of a cDNA encoding mouse stromelysin 1

A cDNA encoding mouse stromelysin 1 was cloned and the 1740-bp sequence was determined. The deduced amino acid (aa) sequence was compared with stromelysin 1 sequences of other mammals. Comparison with a previously published incomplete aa sequence of mouse stromelysin 1 revealed three single aa differences.     

Proliferation of embryonic cardiomyocytes in zebrafish requires the sodium channel scn5Lab

In mice, homozygous deletion of the cardiac sodium channel Scn5a results in defects in cardiac morphology and embryonic death before robust sodium current can be detected. In zebrafish, morpholino knockdown of cardiac sodium channel orthologs scn5Laa and scn5Lab perturbs specification of precardiac mesoderm and inhibits growth of the embryonic heart. It is not known which developmental processes are perturbed by sodium channel knockdown and whether reduced cell number is from impaired migration of cardiac progenitors into the heart, impaired myocyte proliferation, or both. We found that embryos deficient in scn5Lab displayed defects in primary cardiogenesis specific to loss of nkx2.5, but not nkx2.7. We generated kaede reporter fish and demonstrated that embryos treated with anti‐scn5Lab morpholino showed normal secondary differentiation of cardiomyocytes at the arterial pole between 30 and 48 h post‐fertilization. However, while proliferating myocytes were readily detected at 48 hpf in wild type embryos, there were no BrdU‐positive cardiomyocytes in embryos subjected to anti‐scn5Lab treatment. Proliferating myocytes were present in embryos injected with anti‐tnnt2 morpholino to phenocopy the silent heart mutation, and absent in embryos injected with anti‐tnnt2 and anti‐scn5Lab morpholinos, indicating cardiac contraction is not required for the loss of proliferation. These data demonstrate that the role of scn5Lab in later heart growth does not involve contribution of the secondary heart field, but rather proliferation of cardiomyocytes, and appears unrelated to the role of the channel in cardiac electrogenesis. genesis 51:562–574. © 2013 Wiley Periodicals, Inc.     

Effects of carbenoxolone on heart rhythm, contractility and intracellular calcium in streptozotocin-induced diabetic rat

Cardiac dysfunction is a frequently reported complication of clinical and experimental diabetes mellitus. Streptozotocin (STZ) – induced diabetes in rat is associated with a variety of cardiac defects including disturbances to heart rhythm and prolonged time-course of cardiac muscle contraction and/or relaxation. The effects of carbenoxolone (CBX), a selective gap junction inhibitor, on heart rhythm and contractility in STZ-induced diabetic rat have been investigated. Heart rate was significantly (P < 0.05) reduced in Langendorff perfused spontaneously beating diabetic rat heart (171±12 BPM) compared to age-matched controls (229± 9 BPM) and further reduced by 10⁻⁵ M CBX in diabetic (20%) and in control (17%) hearts. Action potential durations (APDs), recorded on the epicardial surface of the left ventricle, were prolonged in paced (6 Hz) diabetic compared to control hearts. Perfusion of hearts with CBX caused further prolongation of APDs and to a greater extent in control compared to diabetic heart. Percentage prolongation at 70% from the peak of the action potential amplitude after CBX was 18% in diabetic compared to 48% in control heart. CBX had no significant effect on resting cell length or amplitude of ventricular myocyte shortening in diabetic or control rats. However, resting fura-2 ratio (indicator for intracellular Ca²⁺ concentration) and amplitude of the Ca²⁺ transient were significantly (P < 0.05) reduced by CBX in diabetic rats but not in controls. In conclusion the larger effects of CBX on APD in control ventricle and the normalizing effects of CBX on intracellular Ca²⁺ in ventricular myocytes from diabetic rat suggest that there may be alterations in gap junction electrophysiology in STZ-induced diabetic rat heart.     

Vasculogenesis and angiogenesis: Extracellular matrix remodeling in coronary collateral arteries and the ischemic heart

Heart failure secondary to ischemic cardiomyopathy is the primary cause of cardiovascular mortality. The promise of the collateral circulation lies in its potential to alter the course of the natural history of coronary heart disease. The collateral circulation of the heart is responsible for supplying blood and oxygen to the myocardium at ischemic risk following severe stenosis and reduced vasoelasticity function of a major coronary artery. In response to flow, stress, and pressure, collateral vessels are restructured and remodeled. Vascular remodeling by its very nature implies synthesis and degradation of extracellular matrix components in the vessel wall. Under normal physiological conditions proteinases that break down the specialized matrix are tightly regulated by antiproteinases. The balance between proteinase and antiproteinase influences is discoordinated during collateral development which leads to adaptive changes in the structure, function, and regulation of extracellular matrix components in the vessel wall. The role of extracellular matrix components in coronary collateral vessel formation in a canine model of chronic coronary artery occlusion has been demonstrated. The role of matrix proteinases and antiproteinases in the collateral vessel play a significant role in the underlying mechanisms of collateral development. This review presents new and significant information regarding the role of extracellular matrix proteinases and antiproteinases in vascular remodeling, function, and collateral development. Such information will have a significant impact on the understanding of the basic biology of the vascular extracellular matrix turnover, remodeling, and function as well as on elucidating potential avenues for pharmacological approaches designed to increase collateral formation and optimize myocardial blood flow in the treatment of ischemic heart disease. J. Cell. Biochem. 65:388–394. © 1997 Wiley-Liss, Inc.     

Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes.

Recent studies using mice with genetically engineered gap junction protein connexin (Cx) genes have provided evidence that reduced gap-junctional coupling in ventricular cardiomyocytes predisposes to ventricular arrhythmia. However, the pathological processes of arrhythmogenesis due to abnormalities in gap junctions are poorly understood. We have postulated a hypothesis that dysfunction of gap junctions at the single-cell level may affect synchronization of calcium transients among cardiomyocytes. To examine this hypothesis, we developed a novel system in which gap-junctional intercellular communication in primary neonatal rat cardiomyocytes was inhibited by a mutated (Delta130-137) Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP), and calcium transients were imaged in real time while the mutated Cx43-EGFP-expressing cardiomyocytes were identified. The mutated Cx43-EGFP inhibited dye coupling not only in the liver epithelial cell line IAR 20 but also in primary neonatal rat cardiomyocytes in a dominant-negative manner, whereas wild-type Cx43-EGFP made functional gap junctions in otherwise communication-deficient HeLa cells. The mutated Cx43-EGFP induced desynchronization of calcium transients among cardiomyocytes with significantly higher frequency than wild-type Cx43-EGFP. These results suggest that dysfunction of gap-junctional intercellular communication at the single-cell level could hamper synchronous beating among cardiomyocytes as a result of desynchronization of calcium transients.     

Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity.

The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1'' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.     

Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes

This study investigates sarcoplasmic reticulum (SR) calcium-(Ca²⁺) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca²⁺ uptake which was completely inhibited by thapsigargin and stimulated by protein kinase A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3,5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca²⁺ uptake of +33%. Specific Ca²⁺ uptake activity was not altered if insulin was omitted from the serum-free culture medium but total SR Ca²⁺ transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca²⁺ re-uptake function of the SR under defined culture conditions.     

Proteinases and myocardial extracellular matrix turnover

Extracellular structural remodeling is the compensatory response of the tissue following pathological stage. Myocardial infarction, which leads to adverse remodeling, thinning of the ventricle wall, dilatation and heart failure, is one of the leading causes of death. Remodeling implies an alteration in the extracellular matrix and in the spatial orientation of cells and intracellular components. The extracellular matrix is responsible for cardiac cell alignment and myocardial structural integrity. Substances that break down the extracellular matrix, specialized proteinases as well as inhibitors of proteinases, appear to be normally balanced in maintaining the integrity of the myocardium. Myocardial infarction leads to an imbalance in proteinase/ antiproteinase activities causing alterations in the stability and integrity of the extracellular matrix and adverse tissue remodeling. To explore mechanisms involved in this process and, in particular, to focus on matrix metalloproteinases, their inhibitors, and activators, an understanding of proteinase and antiproteinase is needed. This review represents new and significant information regarding the role of activated matrix proteinases antiproteinases in remodeling. Such information will have a significant impact both on the understanding of the basic cell biology of extracellular matrix turnover, as well as on potential avenues for pharmacological approaches to the treatment of ischemic heart disease and failure.     

Making the connections: Autophagy and post-translational modifications in cardiomyocytes

Cardiac proteins are subject to continuous stress and these intrinsic and extrinsic factors, both physiological and pathological can lead to protein misfolding. If the protein quality control (PQC) pathways are in any way compromised or their activities diminished, intracellular aggregates can form and a proteotoxic environment is generated, which contributes to cardiac disease and heart failure. We studied the role that SUMO post-translational modification plays in a proteotoxic cardiac environment. SUMOylation can have an integral role in controlling flux through the ubiquitin-proteasome system, and expression of the SUMO (small ubiquitin-like modifier) E2 enzyme UBE2I/UBC9 improves cardiac PQC. Our data focus on using gain- and loss-of-function approaches to modify UBE2I levels and measure the effects on cardiomyocyte autophagic flux. UBE2I expression does have an impact on macroautophagy/autophagy as increased SUMOylation results in increased autophagy. We show that increased SUMOylation is cardioprotective and can decrease morbidity in proteotoxic cardiac disease.     

Developmental changes in free D-aspartic acid in the chicken embryo and in the neonatal rat

Free D-aspartic acid was measured in fertilized chicken eggs, chicken embryos, and neonatal rats. In each tissue examined a maximum value was found at a characteristic time of development. For the chicken embryo brain, the maximum was 9% D at 11 days of incubation; for the retina, 20% D at 13 days of incubation. In the neonatal rat, as in the chicken embryo, D-aspartic acid continued to increase in the retina after that in the brain and other tissues had begun to decline. The maximum, 29% D, was found 7 days after birth. Thus in two phylogenetically distant species, similar developmental patterns of D-aspartic acid change were observed. Some data on similarities between the D/L aspartic acid ratios of adult chicken and rat tissues are also reported. In addition, the total D-aspartic acid content of the egg, including the embryo, increased from 44 nmol at day 1 to 159 nmol at day 12, showing that release from a bound form or de novo synthesis is a continuing process during development.     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Influence of mechanical activity,adrenergic stimulation,and calcium on the expression of myosin heavy chains in cultivated neonatal cardiomyocytes

It is generally accepted that mechanical stress of cardiomyocytes increases RNA and protein synthesis of myosin heavy chain (MHC) quantitatively but it is still a matter of debate whether MHC gene expression is also changed qualitatively. We investigated expression of MHC genes of spontaneously contracting neonatal cardiomyocytes experimentally arrested by permanent depolarization [potassium chloride (KCI)] as well as by electromechanical uncoupling [2,3 butanedione monoxime (BDM)]. Relative distribution of MHC mRNA isoforms (α and β) was studied by quantitative polymerase chain reaction. Expression of MHC isoenzymes was the same in contracting (34.5% β-MHC) and arrested (40.5% and 33.0% β-MHC in KCl and BDM, respectively) cardiomyocytes. However, treatment with phenylephrine for the same period increased significantly β-MHC expression to 55%. We conclude that hormonal factors rather than Ca²⁻ or mechanical stress regulate qualitatively MHC gene expression. J. Cell. Biochem. 64:458–465. © 1997 Wiley-Liss, Inc.     

The effects of CGRP on calcium transients of dedifferentiating cultured adult rat cardiomyocytes compared to non-cultured adult cardiomyocytes: possible protective and deleterious results in cardiac function

CGRP has potent cardiovascular effects but its role in heart failure is unclear. Effects of CGRP on calcium concentrations in fresh adult rat cardiomyocytes, cultured adult cardiomyocytes and neonatal cardiomyocytes were determined by real time fluorescence spectrophotometry. Treatment of cultured adult cardiomyocytes with CGRP resulted in a rapid cessation of beating and a reduction in intracellular calcium. Similar results were obtained in cultured neonatal myocytes. However, rod-shaped adult cardiomyocytes revealed a number of responses; (a) non-beating cells began to beat with increased intracellular calcium; (b) spontaneously beating cells exhibited increased intracellular calcium content and a faster beating rate or (c), myocytes increased their beating rate and became arrhythmic, suggesting that CGRP action on cultured dedifferentiated adult and neonatal myocytes depletes intracellular calcium, whereas in the rod-shaped mature myocytes calcium is retained, pointing to a different mode of action for CGRP on developing and dedifferentiating cardiomyocytes, compared to fully developed cardiomyocytes.     

Developmental changes of the glycolytic enzymes in the human fetal heart

The ontogeny of hexokinase, phosphofructokinase, phosphoglucoisornerase, aldolase, pyruvate kinase and lactate dehydrogenase activities which are associated with glycolysis, an important energy yielding process, has been studied in human fetal heart for periods ranging from 13 weeks to above 33 weeks of gestation. Hexokinase, phosphoglucoisomerase and pyruvate kinase activities show similar developmental profiles exhibiting maximum activity at 25–28 weeks ofgestation. Phosphofructokinase activity, on the other hand, shows a minimum at this period and exhibits a peak value at early stages (13–16 weeks of gestation). Though considerable activity for aldolase is observed at an early period, it declines thereafter, but again increases in the later period. The probable role and correlations of these glycolytic enzymes with energy demand and general functional development in human fetal heart in ontogeny are evaluated.