ICa In Ventricular Myocytes Research Articles

Warmer, faster, stronger: Ca2+ cycling in avian myocardium.

Birds occupy a unique position in the evolution of cardiac design. Their hearts are capable of cardiac performance on par with, or exceeding that of mammals, and yet the structure of their cardiomyocytes resembles those of reptiles. It has been suggested that birds use intracellular Ca2+ stored within the sarcoplasmic reticulum (SR) to power contractile function, but neither SR Ca2+ content nor the cross-talk between channels underlying Ca2+-induced Ca2+ release (CICR) have been studied in adult birds. Here we used voltage clamp to investigate the Ca2+ storage and refilling capacities of the SR and the degree of trans-sarcolemmal and intracellular Ca2+ channel interplay in freshly isolated atrial and ventricular myocytes from the heart of the Japanese quail (Coturnix japonica). A trans-sarcolemmal Ca2+ current (ICa) was detectable in both quail atrial and ventricular myocytes, and was mediated only by L-type Ca2+ channels. The peak density of ICa was larger in ventricular cells than in atrial cells, and exceeded that reported for mammalian myocardium recorded under similar conditions. Steady-state SR Ca2+ content of quail myocardium was also larger than that reported for mammals, and reached 750.6±128.2 μmoll-1 in atrial cells and 423.3±47.2 μmoll-1 in ventricular cells at 24°C. We observed SR Ca2+-dependent inactivation of ICa in ventricular myocytes, indicating cross-talk between sarcolemmal Ca2+ channels and ryanodine receptors in the SR. However, this phenomenon was not observed in atrial myocytes. Taken together, these findings help to explain the high-efficiency avian myocyte excitation-contraction coupling with regard to their reptilian-like cellular ultrastructure.

Functional Impact of Cell Culture on Excitation-Contraction Coupling in Canine Myocytes

Downregulation of ion currents as well as a loss of T-tubules is documented during heart failure. A similar reduction of both has been observed in cultured ventricular myocytes. We further assessed the functional impact of cell-culture on excitation-contraction (EC) coupling in dog ventricular, atrial and Purkinje myocytes. Ventricular, atrial and Purkinje myocytes were isolated and cultured for up to 48 hours. Myocytes were stained with di-8-ANEPPS to visualize ultrastructure and Ca2+ transients (CaTs) were recorded by confocal microscopy. Ion channel currents were assessed via patch electrode voltage clamp. Membrane staining with di-8-ANEPPS indicated dog ventricular myocytes having an extensive T-tubular network; atrial myocytes having a rudimentary T-tubule system at best, and Purkinje myocytes having no T-tubules. Transverse X-t lines scans of electrically stimulated ventricular myocytes showed CaTs rising synchronously across the cell, whereas in Purkinje myocytes the CaT rise showed a U-shaped profile. CaT rise in atrial myocytes typically showed a U-shaped profile, but less pronounced than that in Purkinje myocytes. Ventricular myocytes cultured for 48 hours indicated a dramatic loss of T-tubules and a switch from a synchronous CaT rise to a U-shaped profile. In cultured atrial cells, no loss of T-tubules nor alterations in CaTs was noted. ICa in ventricular myocytes cultured for 48 hours was reduced by 21%. Comparative assessment of culture-induced changes in ion channel currents in atrial myocytes is ongoing. While cultured ventricular myocytes show a loss of T-tubules and phenotypic switch from a homogeneous CaT rise to a ‘U’-shaped profile, atrial myocytes cultured identically show little change in ultrastructure and CaT profile. These results suggest that character and degree of change in EC coupling induced by similar pathological insults markedly depends on cardiac myocyte type.

Tetrodotoxin-sensitive Ca2+ Currents, but No T-type Currents in Normal, Hypertrophied, and Failing Mouse Cardiomyocytes.

To obtain functional evidence that ICa,T is involved in the pathogenesis of cardiac hypertrophy and heart failure. We unexpectedly identified ICa(TTX) rather than ICa,T, therefore, we adjusted our aim to encompass these findings. We investigated (1) Cav3.1 (α1G) transgenic (Tg) mice compared with nontransgenic (tTA-Ntg); (2) Cav3.1-deficient mice (Cav3.1) compared with wild type (Wt) after chemically and surgically induced cardiac remodeling; and (3) spontaneous hypertensive rats and thoracic aortic constriction (TAC) rats. Whole-cell patch-clamp technique was used to measure ICa in ventricular myocytes. Cav3.1-Tg expressed ICa,T (-18.35 ± 1.02 pA/pF at -40 mV) without signs of compromised cardiac function. While we failed to detect ICa,T after hypertrophic stimuli, instead we demonstrated that both Wt and Cav3.1 mouse exhibit ICa(TTX). Using TAC rats, only 2 of 24 VMs showed ICa,T under our experimental conditions. Without TTX, ICa(TTX) occurred in VMs from Wt, spontaneous hypertensive rats, and TAC rats also. These findings demonstrate for the first time that mouse VMs express ICa(TTX). We suggest that future studies should take into consideration the measuring conditions when interpreting ICa,T reappearance in ventricular myocytes in response to hypertrophic stress. Contamination with ICa(TTX) could possibly confuse the relevance of the data.

Muscarinic inhibitory and stimulatory regulation of the L-type Ca2+ current is not altered in cardiac ventricular myocytes from mice lacking endothelial nitric oxide synthase.

Using conventional and perforated patch-clamp techniques, the inhibitory and stimulatory effects of acetylcholine (ACh) on beta-adrenergic regulation of the L-type Ca2+ current (ICa) were studied in ventricular myocytes from wild-type mice (WT) and from mice lacking endothelial nitric oxide synthase (eNOS or NOS3; NOS3-KO mice). To validate the direct comparison of ACh effects on beta-adrenergic responses, the sensitivity of ICa to the beta-adrenergic agonist isoprenaline (Iso) was studied in both WT and NOS3-KO mouse myocytes. ICa sensitivity to Iso was not found to be significantly different in WT and NOS3-KO myocytes: Iso increased ICa with an EC50 of 4.9 and 3.7 nM in WT and NOS3-KO myocytes, respectively. ACh-induced inhibition of ICa did not significantly differ in ventricular myocytes from WT and NOS3-KO mice. ACh (10 microM) inhibited the stimulatory effect of 3 nM Iso by 39 and 35% in WT and NOS3-KO myocytes, respectively. Exposure to and subsequent washout of ACh in the continuous presence of submaximally stimulating concentrations of Iso (1-3 nM) resulted in a transient rebound stimulation of ICa in both WT and NOS3-KO mouse myocytes. The magnitude of the stimulatory effect of ACh did not significantly differ in WT and NOS3-KO mice. These results indicate that nitric oxide (NO) generated by NOS3 does not significantly affect the beta-adrenergic responsiveness of ICa. The results also confirm previous work indicating that NO generated by NOS3 is not obligatory for muscarinic inhibition of the beta-adrenergically regulated ICa in ventricular myocytes. Finally these results demonstrate for the first time that NO generated by NOS3 is not involved in muscarinic rebound stimulation of ICa in ventricular myocytes.