Identification of intracellular calcium dynamics in stimulated cardiomyocytes.

We have developed an automatic method for the analysis and identification of dynamical regimes in intracellular calcium patterns from confocal calcium images. The method allows the identification of different dynamical patterns such as spatially concordant and discordant alternans, irregular behavior or phase-locking regimes such as period doubling or halving. The method can be applied to the analysis of different cardiac pathologies related to anomalies at the cellular level such as ventricular reentrant arrhythmias.

Umbilical cord blood-derived stem cells spontaneously express cardiomyogenic traits.

BACKGROUND: Umbilical cord blood (UCB) has been widely used for hematopoietic stem cell transplantation. The UCB-derived stem cells (UCBSCs) have been proposed as an alternative to bone marrow (BM)-derived mesenchymal stem cells (MSCs) for cardiac cell-based therapy. Herein we studied whether UCBSCs spontaneously exhibit cardiac-specific markers in vitro. METHODS: Human UCBSCs were isolated, expanded, and phenotyped by flow cytometry, quantitative RT-PCR, and immunofluorescence. Cell pluripotency and proliferation were also assessed by adipogenic and osteogenic media and in growth assays. RESULTS: Among 25 analyzed UCB, 16% of cases afforded primary culture satisfactory generation of UCBSCs. Duplication time (Td) of cultures was 2.16 +/- 0.06 days. The cells were strongly positive for CD105 (18.5 +/- 0.14), CD44 (27 +/- 2.8), CD166 (13 +/- 9), CD29 (59 +/- 9.4), CD90 (60 +/- 11) and consistently negative for CD117 (1.2 +/- 0.1), CD106 (1.1 +/- 0), CD34 (1.2 +/- 0.2), CD14 (1 +/- 0), and CD45 (1 +/- 0), consistent with a mesenchymal lineage. Adipogenesis and osteogenesis of cells resulted in low accumulation of intracellular lipid droplets and high deposition of calcium. The UCBSCs showed gene transcripts for alpha-actinin, connexin (Cx)-43, SERCA-2, and stromal cell-derived factor (SDF)-1alpha. At the protein level, the cells abundantly expressed alpha-actinin, Cx-43, SERCA-2 and SDF-1alpha. In contrast, these cells did not express the cardiac transcription factors GATA-4, Tbx5, and Nkx2.5, nor the sarcomeric proteins beta-myosin heavy chain (beta-MyHC) or cardiac troponin I (cTnI). CONCLUSIONS: Human UCBSCs may represent an alternative source of stem cells for myocardial-cell replacement. These cells can be highly expanded. They spontaneously express proteins of paramount importance for cardiovascular regeneration, such as Cx-43, SERCA-2, and SDF-1alpha.

Identification of cardiomyogenic lineage markers in untreated human bone marrow-derived mesenchymal stem cells.

BACKGROUND: Recent reports refute the classic paradigm by which human heart is unable to repair itself following disease or injury. Cardiac and noncardiac stem cells with cardiac regeneration potential have been documented. We studied whether untreated mesenchymal stem cells express markers of cardiomyogenic lineage in vitro. METHODS: Mesenchymal stem cells were obtained from human iliac crest marrow aspirates. Cells were isolated and characterized using flow cytometry by surface expression of CD105, CD166, CD29, CD44, CD14, and CD34. To evaluate their cardiomyogenic potential, presence of cardiac proteins (cardiac troponin I, sarcomeric alpha-actinin, beta myosin heavy chain (beta-MyHC), connexin-43, and SERCA-2), and transcription factors (GATA-4) were assessed. RESULTS: Mesenchymal stem cells expressed CD105 (4.25 +/- 0.35), CD166 (27.83 +/- 1.89), and CD29 (9.4 +/- 0.57) and were negative for CD34, CD14, and CD45. In absence of additional stimuli in the culture media, these cells expressed connexin-43, alpha-actinin, and GATA-4, and were negative for SERCA-2, cardiac troponin I, and beta-MyHC. CONCLUSIONS: Human adult mesenchymal stem cells spontaneously exhibit markers of cardiac phenotype in vitro. In the appropiate myocardial environment, these cells may transdifferentiate into mature cardiomyocytes.

The function of the sarcoplasmic reticulum is not inhibited by low temperatures in trout atrial myocytes.

The effect of temperature on sarcoplasmic reticulum (SR) Ca(2+) uptake and release was measured in trout atrial myocytes using the perforated patch-clamp technique. Depolarization of the myocyte for 10 s to different membrane potentials (V(m)) induced SR Ca(2+) uptake. The relationship between V(m) and SR Ca(2+) uptake was not significantly changed by lowering the experimental temperature from 21 to 7 degrees C, and the relationship between total cytosolic Ca(2+) and SR Ca(2+) uptake was similar at the two temperatures with a pooled V(max) = 66 amol/pF and K(0.5) = 4 amol/pF. Quantification of the Ca(2+) release from the SR elicited by 10-ms depolarizations to different V(m) showed an increasing SR Ca(2+) release at more positive V(m) between -50 and +10 mV, whereas SR Ca(2+) release stagnated between +10 and +50 mV. Lowering of the temperature did not affect this relationship significantly, giving an SR Ca(2+) release of 1.71 and 1.54 amol/pF at 21 and 7 degrees C, respectively. Furthermore, clearance of the SR Ca(2+) content slowed down inactivation of the L-type Ca(2+) current at both temperatures (the fast time constant increased significantly from 10.4 +/- 1.9 to 15.0 +/- 2.0 ms at 21 degrees C and from 38 +/- 15 to 73 +/- 24 ms at 7 degrees C). Thus the SR has the capacity to remove the entire Ca(2+) transient at physiologically relevant stimulation frequencies at both 21 and 7 degrees C, although it is estimated that ~40% of the total Ca(2+) transient is liberated from and reuptaken by the SR with continuous stimulation at 0.5 Hz independently of the experimental temperature.

Na(+)/Ca(2+)-exchange activity regulates contraction and SR Ca(2+) content in rainbow trout atrial myocytes.

We have used the whole cell configuration of the patch-clamp technique to measure sarcolemmal Ca(2+) transport by the Na(+)/Ca(2+) exchanger (NCX) and its contribution to the activation and relaxation of contraction in trout atrial myocytes. In contrast to mammals, cell shortening continued, increasing at membrane potentials above 0 mV in trout atrial myocytes. Furthermore, 5 microM nifedipine abolished L-type Ca(2+) current (I(Ca)) but only reduced cell shortening and the Ca(2+) carried by the tail current to 66 +/- 5 and 67 +/- 6% of the control value. Lowering of the pipette Na(+) concentration from 16 to 10 or 0 mM reduced Ca(2+) extrusion from the cell from 2.5 +/- 0.2 to 1.0 +/- 0.2 and 0.5 +/- 0.06 amol/pF. With 20 microM exchanger inhibitory peptide (XIP) in the patch pipette Ca(2+) extrusion 20 min after patch break was 39 +/- 8% of its initial value. With 16, 10, and 0 mM Na(+) in the pipette, the sarcoplasmic reticulum (SR) Ca(2+) content was 47 +/- 4, 29 +/- 6, and 10 +/- 3 amol/pF, respectively. Removal of Na(+) from or inclusion of 20 microM XIP in the pipette gradually eliminated the SR Ca(2+) content. Whereas I(Ca) was the same at -10 or +10 mV, Ca(2+) extrusion from the cell and the SR Ca(2+) content at -10 mV were 65 +/- 7 and 80 +/- 4% of that at +10 mV. The relative amount of Ca(2+) extruded by the NCX (about 55%) and taken up by the SR (about 45%) was, however, similar with depolarizations to -10 and +10 mV. We conclude that modulation of the NCX activity critically determines Ca(2+) entry and cell shortening in trout atrial myocytes. This is due to both an alteration of the transsarcolemmal Ca(2+) transport and a modulation of the SR Ca(2+) content.

Quantification of calcium release from the sarcoplasmic reticulum in rainbow trout atrial myocytes.

Using the whole-cell configuration of the patch-clamp technique, we quantified calcium release from the sarcoplasmic reticulum (SR) elicited by short depolarization pulses before and after clearance of the SR Ca2+ content with 10 mM caffeine (CAF). With a loaded SR, the first detectable contraction occurred with a pulse duration of 5 ms. CAF exposure increased this pulse duration to 85 ms and slowed the inactivation of the Ca2+ current (ICa). Repolarization of the cell to -80 mV after a short depolarization elicited a tail current that was attenuated markedly after CAF exposure. The difference between the charge carried by the tail currents obtained before and after CAF exposure was taken as a measure of the Ca2+ released from the SR. SR Ca2+ release increased with increasing SR Ca2+ load over the range of loads examined. In contrast, SR Ca2+ release reached a maximum when the duration of the preceding depolarization exceeded 10 ms. Maximal Ca2+ release was 1.64 amol/pF or 62 microM and elicited a contraction that was 40 +/- 6% of the amplitude of a normal contraction. This release could account for 48 +/- 10% of the total Ca2+ required to activate contraction but only a few percent of the CAF-releasable Ca2+. Thus, contrary to the general view of excitation-contraction coupling in lower vertebrates, SR Ca2+ release in trout atrial myocytes may account for up to 50% of the total Ca2+ transient at room temperature.

Quantification of Ca2+ uptake in the sarcoplasmic reticulum of trout ventricular myocytes.

We measured Ca2+ uptake by the sarcoplasmic reticulum (SR) in trout ventricular myocytes, measuring indo 1 fluorescence in permeabilized cells or ionic currents in single myocytes subjected to voltage clamp. Titration of the SR Ca2+ pumps with thapsigargin gave a pump site density of 454 pmol/mg cell protein. Lowering the temperature from 20 degreesC to 10 or 5 degreesC reduced the SR Ca2+ uptake rate in permeabilized myocytes by 50 and 63%, respectively. Surprisingly, Ca2+ leak from the SR also decreased with decreasing temperatures. Exposure of single myocytes to 10 mM caffeine (Caf) induced a cell contracture and an inward ionic current. Neither contracture nor current decreased significantly after rest periods of 120 and 320 s. The inward current was due to Ca2+ extrusion by the Na+/Ca2+ exchanger (NCX), and the time integral of the exchange current (INCX) was used to calculate the SR Ca2+ content. This gave a steady-state SR Ca2+ content of 22.5 +/- 2.8 amol Ca2+/pF or 750 microM. When the SR was loaded by depolarizing the cell to +50 mV, the Ca2+ content increased with increasing length of the depolarization, reaching a maximum of 52.0 +/- 5.9 amol Ca2+/pF. When the cell was depolarized to different voltages for 3 s, a subsequent Caf-induced INCX increased with increasing voltage. At +100 mV, the Ca2+ content was 36.6 +/- 3.8 amol/pF, giving a maximal SR Ca2+ uptake rate of 12.2 +/- 1.2 amol Ca2+. pF-1. s-1 or 417 microM/s. We conclude that maximal SR Ca2+ content and Ca2+ uptake rates can be estimated using specific SR Ca2+ loading protocols. Contrary to the general assumption that contraction in lower vertebrates depends largely on transsarcolemmal Ca2+ fluxes, we found that although the L-type Ca2+ current is insufficient to fully activate contraction, the SR is capable of participating in the regulation of the cytosolic Ca2+ during the excitation-contraction coupling in trout ventricular myocytes.