Local increases in intracellular calcium ion concentration ([Ca2+]i) resulting from activation of the ryanodine-sensitive calcium-release channel in the sarcoplasmic reticulum (SR) of smooth muscle cause arterial dilation. Ryanodine-sensitive, spontaneous local increases in [Ca2+]i (Ca2+ sparks) from the SR were observed just under the surface membrane of single smooth muscle cells from myogenic cerebral arteries. Ryanodine and thapsigargin inhibited Ca2+ sparks and Ca(2+)-dependent potassium (KCa) currents, suggesting that Ca2+ sparks activate KCa channels. Furthermore, KCa channels activated by Ca2+ sparks appeared to hyperpolarize and dilate pressurized myogenic arteries because ryanodine and thapsigargin depolarized and constricted these arteries to an extent similar to that produced by blockers of KCa channels. Ca2+ sparks indirectly cause vasodilation through activation of KCa channels, but have little direct effect on spatially averaged [Ca2+]i, which regulates contraction.

The mammalian heart has a very limited regenerative capacity and, hence, heals by scar formation. Recent reports suggest that haematopoietic stem cells can transdifferentiate into unexpected phenotypes such as skeletal muscle, hepatocytes, epithelial cells, neurons, endothelial cells and cardiomyocytes, in response to tissue injury or placement in a new environment. Furthermore, transplanted human hearts contain myocytes derived from extra-cardiac progenitor cells, which may have originated from bone marrow. Although most studies suggest that transdifferentiation is extremely rare under physiological conditions, extensive regeneration of myocardial infarcts was reported recently after direct stem cell injection, prompting several clinical trials. Here, we used both cardiomyocyte-restricted and ubiquitously expressed reporter transgenes to track the fate of haematopoietic stem cells after 145 transplants into normal and injured adult mouse hearts. No transdifferentiation into cardiomyocytes was detectable when using these genetic techniques to follow cell fate, and stem-cell-engrafted hearts showed no overt increase in cardiomyocytes compared to sham-engrafted hearts. These results indicate that haematopoietic stem cells do not readily acquire a cardiac phenotype, and raise a cautionary note for clinical studies of infarct repair.

Despite recent advances in preventing sudden cardiac death (SCD) due to cardiac arrhythmia, its incidence in the population at large has remained unacceptably high. Better understanding of the interaction among various functional, structural, and genetic factors underlying the susceptibility to, and initiation of, fatal arrhythmias is a major goal and will provide new tools for the prediction, prevention, and therapy of SCD. Here, we review the role of aberrant intracellular Ca 2+ handling, ionic imbalances associated with acute myocardial ischemia, neurohumoral changes, and genetic predisposition in the pathogenesis of SCD due to cardiac arrhythmia. Therapeutic measures to prevent SCD are also discussed.Sudden cardiac death (SCD) from any cause claims 300,000-400,000 lives a year in the United States. The most common sequence of events leading to SCD appears to be the degeneration of ventricular tachycardia (VT; abnormal acceleration of ventricular rate) into ventricular fibrillation (VF), during which disorganized contractions of the ventricles fail to eject blood effectively, often followed by asystole or pulseless electrical activity. Preexisting coronary artery disease and its consequences (acute myocardial ischemia, scarring from previous myocardial infarction, heart failure) are manifest in 80% of SCD victims. Dilated nonischemic and hypertrophic cardiomyopathies account for the second largest number of SCDs, whereas other cardiac disorders, including congenital heart defects and the known genetically determined ion channel anomalies, account for 5-10% of SCDs (1).While the implantable cardioverter defibrillator (ICD) improves survival in high-risk patients (2), standard antiarrhythmic drug therapy has failed to reduce, and in some instances has increased, the incidence of SCD (3). In fact, the greatest reduction in cardiovascular mortality (including SCD) in patients with clinically manifest heart disease has resulted from the use of beta blockers (4) and non-antiarrhythmic drugs, i.e., those without major direct electrophysiological action in cardiac muscle or the specialized conduction system, such as angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor-blocking agents, lipid-lowering agents, spironolactone, thrombolytic and antithrombotic agents, and perhaps magnesium and omega-3 fatty acids (for review, see ref. 5). These drugs most likely exert their antiarrhythmic potential indirectly by inhibiting or delaying adverse functional and structural remodeling in the diseased heart, i.e., by affecting "upstream events" that contribute to the development of electrophysiological instability.Clinical trials, in general, have failed to define SCD risk markers for specific individuals in the larger general population, where the relative risk of SCD is low but the absolute number of deaths is high. Risk markers include abnormalities in cardiovascular function (left-ventricular ejection fraction), electrocardiographic variables (e.g., late potentials, T-wave alternans, QRS duration, dispersion o...

Local calcium transients ('Ca2+ sparks') are thought to be elementary Ca2+ signals in heart, skeletal and smooth muscle cells. Ca2+ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca2+ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately -40 mV, an elevation of arterial wall [Ca2+]i and contraction ('myogenic tone') of arteries. Ca2+ sparks activate calcium-sensitive K+ (KCa) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca2+ sparks by ryanodine, or of KCa channels by iberiotoxin, leads to membrane depolarization, activation of L-type voltage-gated Ca2+ channels, and vasoconstriction. Conversely, activation of Ca2+ sparks can lead to vasodilation through activation of KCa channels. Our recent work is aimed at studying the properties and roles of Ca2+ sparks in the regulation of arterial smooth muscle function. The modulation of Ca2+ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca2+ entry through voltage-dependent Ca2+ channels in the regulation of Ca2+ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca2+ channels, RyR channels, and KCa channels function as a coupled unit, through Ca2+ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.

Abstract-Two-photon excitation fluorescence imaging provides thin optical sections from deep within thick, scattering specimens by way of restricting fluorophore excitation (and thus emission) to the focal plane of the microscope. Spatial confinement of two-photon excitation gives rise to several advantages over single-photon confocal microscopy. First, penetration depth of the excitation beam is increased. Second, because out-of-focus fluorescence is never generated, no pinhole is necessary in the detection path of the microscope, resulting in increased fluorescence collection efficiency. Third, two-photon excitation markedly reduces overall photobleaching and photodamage, resulting in extended viability of biological specimens during long-term imaging. Finally, localized excitation can be used for photolysis of caged compounds in femtoliter volumes and for diffusion measurements by two-photon fluorescence photobleaching recovery. This review aims to provide an overview of the use of two-photon excitation microscopy. Selected applications of this technique will illustrate its excellent suitability to assess cellular and subcellular events in intact, strongly scattering tissue. In particular, its capability to resolve differences in calcium dynamics between individual cardiomyocytes deep within intact, buffer-perfused hearts is demonstrated. Potential applications of two-photon laser scanning microscopy as applied to integrative cardiac physiology are pointed out. Key Words: two-photon excitation Ⅲ laser scanning microscopy Ⅲ calcium imaging T wo-photon excitation (TPE) microscopy 1 has evolved as an alternative to conventional single-photon confocal microscopy and has been shown to provide several advantages. These include three-dimensionally resolved fluorescence imaging of living cells deep within thick, strongly scattering samples, and reduced phototoxicity, enabling longterm imaging of photosensitive biological specimens. The inherent three-dimensional resolution of TPE microscopy has been exploited in a number of studies wherein spatial discrimination of fluorescence signals at the micrometer and submicrometer scale within thick biological specimens proved critical. For example, TPE of the calcium-sensitive fluorophore rhod-2 has been used to resolve differences in the kinetics of intracellular calcium ([Ca 2ϩ ] i ) transients in donor myocytes and juxtaposed host cardiomyocytes deep in Langendorff-perfused mouse hearts following intracardiac transplantation of fetal cardiomyocytes and skeletal myoblasts. 2,3 For neuroscientists, TPE microscopy has become an invaluable tool for studying calcium dynamics in thick brain slices and live animals 4,5 and for long-term imaging of neuronal development. 6 The spatial confinement of TPE has also been used for three-dimensional photolysis of caged compounds in femtoliter volumes [7][8][9] or diffusion measurements by two-photon fluorescence photobleaching recov- ery. 10,11 This article describes the basic physical principles of TPE and reviews the advantages and...