C. William Balke

Alterations in calcium handling in cardiac hypertrophy and heart failure

There is conflicting data concerning the effects of cardiac hypertrophy and failure on L-type Ca2+ channel density, the amplitude of the intracellular Ca2+ transients, and the characteristics of Ca2+ sparks. These discrepancies are probably due to multiple factors. First, the effects of cardiac hypertrophy on channel expression and cell adaptation are model dependent. Even within the same species, the mechanisms by which cardiac hypertrophy and heart failure are generated (genetic alteration, pressure overload, volume overload, high rate pacing, etc.) influence the results obtained. Second, with many animal models and diseased human hearts, the disease process is not uniformly distributed throughout the myocardium. Third, the effects on L-type Ca2+ channel behavior and SR function clearly depend on the extent of disease expression. Myocardial contractility increases with cardiac hypertrophy whereas it decreases with heart failure. Thus, it is difficult to compare results from different models of hypertrophy and heart failure at different stages of disease. More consistent data is likely to be obtained from longitudinal studies using a single animal model of disease. The challenge before us is to develop animal models that mimic human disease, which can be studied longitudinally during the progression of the disease process. This approach coupled with continued improvement in Ca2+ imaging and a greater understanding of normal E-C coupling, will enable us to identify precisely the abnormalities in E-C coupling that occur with the development of cardiac hypertrophy and heart failure and define the appropriate treatment modalities.

Sodium pump α2 subunits control myogenic tone and blood pressure in mice

A key question in hypertension is: How is long‐term blood pressure controlled? A clue is that chronic salt retention elevates an endogenous ouabain‐like compound (EOLC) and induces salt‐dependent hypertension mediated by Na+/Ca2+ exchange (NCX). The precise mechanism, however, is unresolved. Here we study blood pressure and isolated small arteries of mice with reduced expression of Na+ pump α1 (α1+/−) or α2 (α2+/−) catalytic subunits. Both low‐dose ouabain (1–100 nm; inhibits only α2) and high‐dose ouabain (≥1 μm; inhibits α1) elevate myocyte Ca2+ and constrict arteries from α1+/−, as well as α2+/− and wild‐type mice. Nevertheless, only mice with reduced α2 Na+ pump activity (α2+/−), and not α1 (α1+/−), have elevated blood pressure. Also, isolated, pressurized arteries from α2+/−, but not α1+/−, have increased myogenic tone. Ouabain antagonists (PST 2238 and canrenone) and NCX blockers (SEA0400 and KB‐R7943) normalize myogenic tone in ouabain‐treated arteries. Only the NCX blockers normalize the elevated myogenic tone in α2+/− arteries because this tone is ouabain independent. All four agents are known to lower blood pressure in salt‐dependent and ouabain‐induced hypertension. Thus, chronically reduced α2 activity (α2+/− or chronic ouabain) apparently regulates myogenic tone and long‐term blood pressure whereas reduced α1 activity (α1+/−) plays no persistent role: the in vivo changes in blood pressure reflect the in vitro changes in myogenic tone. Accordingly, in salt‐dependent hypertension, EOLC probably increases vascular resistance and blood pressure by reducing α2 Na+ pump activity and promoting Ca2+ entry via NCX in myocytes.

Calcium Handling in Human Embryonic Stem Cell-Derived Cardiomyocytes

The objective of the current study was to characterize calcium handling in developing human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs). To this end, real‐time polymerase chain reaction (PCR), immunocytochemistry, whole‐cell voltage‐clamp, and simultaneous patch‐clamp/laser scanning confocal calcium imaging and surface membrane labeling with di‐8‐aminonaphthylethenylpridinium were used. Immunostaining studies in the hESC‐CMs demonstrated the presence of the sarcoplasmic reticulum (SR) calcium release channels, ryanodine receptor‐2, and inositol‐1,4,5‐trisphosphate (IP3) receptors. Store calcium function was manifested as action‐potential‐induced calcium transients. Time‐to‐target plots showed that these action‐potential‐initiated calcium transients traverse the width of the cell via a propagated wave of intracellular store calcium release. The hESC‐CMs also exhibited local calcium events (“sparks”) that were localized to the surface membrane. The presence of caffeine‐sensitive intracellular calcium stores was manifested following application of focal, temporally limited puffs of caffeine in three different age groups: early‐stage (with the initiation of beating), intermediate‐stage (10 days post‐beating [dpb]), and late‐stage (30–40 dpb) hESC‐CMs. Calcium store load gradually increased during in vitro maturation. Similarly, ryanodine application decreased the amplitude of the spontaneous calcium transients. Interestingly, the expression and function of an IP3‐releasable calcium pool was also demonstrated in the hESC‐CMs in experiments using caged‐IP3 photolysis and antagonist application (2 μM 2‐Aminoethoxydiphenyl borate). In summary, our study establishes the presence of a functional SR calcium store in early‐stage hESC‐CMs and shows a unique pattern of calcium handling in these cells. This study also stresses the importance of the functional characterization of hESC‐CMs both for developmental studies and for the development of future myocardial cell replacement strategies.

Evolution of Cardiac Calcium Waves from Stochastic Calcium Sparks

We present a model that provides a unified framework for studying Ca2+ sparks and Ca2+ waves in cardiac cells. The model is novel in combining 1) use of large currents (approximately 20 pA) through the Ca2+ release units (CRUs) of the sarcoplasmic reticulum (SR); 2) stochastic Ca2+ release (or firing) of CRUs; 3) discrete, asymmetric distribution of CRUs along the longitudinal (separation distance of 2 microm) and transverse (separated by 0.4-0.8 microm) directions of the cell; and 4) anisotropic diffusion of Ca2+ and fluorescent indicator to study the evolution of Ca2+ waves from Ca2+ sparks. The model mimics the important features of Ca2+ sparks and Ca2+ waves in terms of the spontaneous spark rate, the Ca2+ wave velocity, and the pattern of wave propagation. Importantly, these features are reproduced when using experimentally measured values for the CRU Ca2+ sensitivity (approximately 15 microM). Stochastic control of CRU firing is important because it imposes constraints on the Ca2+ sensitivity of the CRU. Even with moderate (approximately 5 microM) Ca2+ sensitivity the very high spontaneous spark rate triggers numerous Ca2+ waves. In contrast, a single Ca2+ wave with arbitrarily large velocity can exist in a deterministic model when the CRU Ca2+ sensitivity is sufficiently high. The combination of low CRU Ca2+ sensitivity (approximately 15 microM), high cytosolic Ca2+ buffering capacity, and the spatial separation of CRUs help control the inherent instability of SR Ca2+ release. This allows Ca2+ waves to form and propagate given a sufficiently large initiation region, but prevents a single spark or a small group of sparks from triggering a wave.

Role of the transverse-axial tubule system in generating calcium sparks and calcium transients in rat atrial myocytes.

Cardiac atrial cells lack a regular system of transverse tubules like that in cardiac ventricular cells. Nevertheless, many atrial cells do possess an irregular internal transverse‐axial tubular system (TATS). To investigate the possible role of the TATS in excitation‐contraction coupling in atrial myocytes, we visualized the TATS (labelled with the fluorescent indicator, Di‐8‐ANEPPS) simultaneously with Ca2+ transients and/or Ca2+ sparks (fluo‐4). In confocal transverse linescan images of field‐stimulated cells, whole‐cell Ca2+ transients had two morphologies: ‘U‐shaped’ transients and irregular or ‘W‐shaped’ transients with a varying number of points of origin of the Ca2+ transient. About half (54 %, n=289 cells, 13 animals) of the cells had a TATS. Cells with TATS had a larger mean diameter (13.2 ± 2.8 μm) than cells without TATS (11.7 ± 2.0 μm) and were more common in the left atrium (n= 206 cells; left atrium: 76 with TATS, 30 without TATS; right atrium: 42 with TATS, 58 without TATS). Simultaneous measurement of Ca2+ sparks and sarcolemmal structures showed that cells without TATS had U‐shaped transients that started at the cell periphery, and cells with TATS had W‐shaped transients that began simultaneously at the cell periphery and the TATS. Most (82 out of 102 from 31 cells) ‘spontaneous’ (non‐depolarized) Ca2+ sparks occurred within 1 μm of a sarcolemmal structure (cell periphery or TATS), and 33 % occurred within 1 pixel (0.125 μm). We conclude that the presence of a sarcolemmal membrane either at the cell periphery or in the TATS in close apposition to the sarcoplasmic reticulum is required for the initiation of an evoked Ca2+ transient and for spontaneous Ca2+ sparks.

Augmented Phosphorylation of Cardiac Troponin I in Hypertensive Heart Failure

Background: Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility. Results: cTnI is hyperphosphorylated at Ser22/23 and Ser42/44 in spontaneously hypertensive rat of heart failure. Conclusion: The augmented phosphorylation of cTnI in hypertensive heart failure is correlated with elevated protein levels of PKC-α and -δ. Significance: This is the first in vivo evidence of cTnl phosphorylation at Ser42/44 in an animal model of hypertensive heart failure. An altered cardiac myofilament response to activating Ca2+ is a hallmark of human heart failure. Phosphorylation of cardiac troponin I (cTnI) is critical in modulating contractility and Ca2+ sensitivity of cardiac muscle. cTnI can be phosphorylated by protein kinase A (PKA) at Ser22/23 and protein kinase C (PKC) at Ser22/23, Ser42/44, and Thr143. Whereas the functional significance of Ser22/23 phosphorylation is well understood, the role of other cTnI phosphorylation sites in the regulation of cardiac contractility remains a topic of intense debate, in part, due to the lack of evidence of in vivo phosphorylation. In this study, we utilized top-down high resolution mass spectrometry (MS) combined with immunoaffinity chromatography to determine quantitatively the cTnI phosphorylation changes in spontaneously hypertensive rat (SHR) model of hypertensive heart disease and failure. Our data indicate that cTnI is hyperphosphorylated in the failing SHR myocardium compared with age-matched normotensive Wistar-Kyoto rats. The top-down electron capture dissociation MS unambiguously localized augmented phosphorylation sites to Ser22/23 and Ser42/44 in SHR. Enhanced Ser22/23 phosphorylation was verified by immunoblotting with phospho-specific antibodies. Immunoblot analysis also revealed up-regulation of PKC-α and -δ, decreased PKCϵ, but no changes in PKA or PKC-β levels in the SHR myocardium. This provides direct evidence of in vivo phosphorylation of cTnI-Ser42/44 (PKC-specific) sites in an animal model of hypertensive heart failure, supporting the hypothesis that PKC phosphorylation of cTnI may be maladaptive and potentially associated with cardiac dysfunction.

Theoretical analysis of the Ca2+ spark amplitude distribution

A difficulty of using confocal microscopy to study Ca2+ sparks is the uncertainty of the linescan position with respect to the source of Ca2+ release. Random placement of the linescan is expected to result in a broad distribution of measured Ca2+ spark amplitudes (a) even if all Ca2+ sparks were generated identically. Thus variations in Ca2+ spark amplitude due to positional differences between confocal linescans and Ca2+ release site are intertwined with variations due to intrinsic differences in Ca2+ release properties. To separate these two sources of variations on the Ca2+ spark amplitude, we determined the effect changes of channel current or channel open time--collectively called the source strength, alpha--had on the measured Ca2+ spark amplitude histogram, N(a). This was done by 1) simulating Ca2+ release, Ca2+ and fluo-3 diffusion, and Ca2+ binding reactions; 2) simulation of image formation of the Ca2+ spark by a confocal microscope; and 3) using a novel automatic Ca2+ spark detector. From these results we derived an integral equation relating the probability density function of source strengths, f alpha (alpha), to N(a), which takes into account random positional variations between the source and linescan. In the special, but important, case that the spatial distribution of Ca(2+)-bound fluo-3 is Gaussian, we show the following: 1) variations of Ca2+ spark amplitude due to positional or intrinsic differences can be separated, and 2) f alpha (alpha) can, in principle, be calculated from the Ca2+ spark amplitude histogram since N(a) is the sum of shifted hyperbolas, where the magnitudes of the shifts and weights depend on f alpha (alpha). In particular, if all Ca2+ sparks were generated identically, then the plot of 1/N(a) against a will be a straight line. Multiple populations of channels carrying distinct currents are revealed by discontinuities in the 1/N(a) plot. 3) Although the inverse relationship between Ca2+ spark amplitude and decay time might be used to distinguish Ca2+ sparks from different channel populations, noise can render the measured decay times meaningless for small amplitude Ca2+ sparks.

Transformation of adult rat cardiac myocytes in primary culture

We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long‐term cell culture. Morphometric parameters, sarcomere length, T‐tubule density, cell capacitance, L‐type calcium current (ICa,L), inward rectifier potassium current (IK1), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 ± 70 μm2 on day 0 to 1432 ± 50 μm2 on day 5), T‐tubule density, systolic shortening (from 0.11 ± 0.02 to 0.05 ± 0.01 μm) and amplitude of calcium transients (from 1.54 ± 0.19 to 0.67 ± 0.19) over 5 days of culture. The negative force–frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 ± 8 to 105 ± 11 pF) and membrane currents were also reduced (ICa,L, from 3.98 ± 0.39 to 2.12 ± 0.37 pA pF; and IK1, from 34.34p ± 2.31 to 18.00 ± 5.97 pA pF−1). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 ± 2.5 to 34.2 ± 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.

Cardiac Troponin T, a Sarcomeric AKAP, Tethers Protein Kinase A at the Myofilaments

Efficient and specific phosphorylation of PKA substrates, elicited in response to β-adrenergic stimulation, require spatially confined pools of PKA anchored in proximity of its substrates. PKA-dependent phosphorylation of cardiac sarcomeric proteins has been the subject of intense investigations. Yet, the identity, composition, and function of PKA complexes at the sarcomeres have remained elusive. Here we report the identification and characterization of a novel sarcomeric AKAP (A-kinase anchoring protein), cardiac troponin T (cTnT). Using yeast two-hybrid technology in screening two adult human heart cDNA libraries, we identified the regulatory subunit of PKA as interacting with human cTnT bait. Immunoprecipitation studies show that cTnT is a dual specificity AKAP, interacting with both PKA-regulatory subunits type I and II. The disruptor peptide Ht31, but not Ht31P (control), abolished cTnT/PKA-R association. Truncations and point mutations identified an amphipathic helix domain in cTnT as the PKA binding site. This was confirmed by a peptide SPOT assay in the presence of Ht31 or Ht31P (control). Gelsolin-dependent removal of thin filament proteins also reduced myofilament-bound PKA-type II. Using a cTn exchange procedure that substitutes the endogenous cTn complex with a recombinant cTn complex we show that PKA-type II is troponin-bound in the myofilament lattice. Displacement of PKA-cTnT complexes correlates with a significant decrease in myofibrillar PKA activity. Taken together, our data propose a novel role for cTnT as a dual-specificity sarcomeric AKAP.

Ca2+ Sparks Triggered by Patch Depolarization in Rat Heart Cells

The goal of this study was to examine the relationship between Ca2+ entry through L-type Ca2+ channels and local [Ca2+]i transients (Ca2+ sparks) in single rat cardiac ventricular cells. L-type Ca2+ channels were activated by depolarization of cell-attached membrane patches, and [Ca2+]i was measured simultaneously as fluo 3 fluorescence using laser scanning confocal microscopy. Patch depolarization with Ca2+ as the charge carrier (10 or 110 mmol.L(-1)) significantly increased the probability of the occurrence of Ca2+ sparks (Ca2+ spark rate) only in the volume of cytoplasm located immediately beneath the membrane patch (basal Ca2+ spark rate, 119 Ca2+ sparks.cell(-1).s(-1); patch depolarization Ca2+ spark rate, 610 Ca2+ sparks.cell(-1).s(-1); P<.005). With Ba2+ in the pipette solution (10 mmol.L(-1)), patch depolarization was not associated with an increased Ca2+ spark rate at the position of the pipette or at any other sites distant from the pipette. Therefore, Ca2+ entry and not voltage per se was a necessary event for the occurrence of Ca2+ sparks. Under identical experimental conditions, patch depolarization experiments opened single L-type Ca2+ channels with a single-channel conductance of 19 pS with Ba2+ as the charge carrier. Although single-channel openings could not be resolved when Ca2+ was the charge carrier, ensemble averages yielded an inward current of up to 0.75 pA. The results suggest that voltage-activated Ca2+ entry through one or a small number of L type Ca2+ channels triggers the release of Ca2+ only from the sarcoplasmic reticulum in direct proximity to those L-type Ca2+ channels. The relatively low probability of triggering Ca2+ sparks may have resulted from some alteration of excitation-contraction coupling associated with the technique of the cell-attached patch clamp.