Implementation Research to Address the United States Health Disadvantage: Report of a National Heart, Lung, and Blood Institute Workshop.

Four decades ago, U.S. life expectancy was within the same range as other high-income peer countries. However, during the past decades, the United States has fared worse in many key health domains resulting in shorter life expectancy and poorer health-a health disadvantage. The National Heart, Lung, and Blood Institute convened a panel of national and international health experts and stakeholders for a Think Tank meeting to explore the U.S. health disadvantage and to seek specific recommendations for implementation research opportunities for heart, lung, blood, and sleep disorders. Recommendations for National Heart, Lung, and Blood Institute consideration were made in several areas including understanding the drivers of the disadvantage, identifying potential solutions, creating strategic partnerships with common goals, and finally enhancing and fostering a research workforce for implementation research. Key recommendations included exploring why the United States is doing better for health indicators in a few areas compared with peer countries; targeting populations across the entire socioeconomic spectrum with interventions at all levels in order to prevent missing a substantial proportion of the disadvantage; assuring partnership have high-level goals that can create systemic change through collective impact; and finally, increasing opportunities for implementation research training to meet the current needs. Connecting with the research community at large and building on ongoing research efforts will be an important strategy. Broad partnerships and collaboration across the social, political, economic, and private sectors and all civil society will be critical-not only for implementation research but also for implementing the findings to have the desired population impact. Developing the relevant knowledge to tackle the U.S. health disadvantage is the necessary first step to improve U.S. health outcomes.

Refining Current Scientific Priorities and Identifying New Scientific Gaps in HIV-Related Heart, Lung, Blood, and Sleep Research.

The National Heart, Lung, and Blood Institute (NHLBI) AIDS Program's goal is to provide direction and support for research and training programs in areas of HIV-related heart, lung, blood, and sleep (HLBS) diseases. To better define NHLBI current HIV-related scientific priorities and with the goal of identifying new scientific priorities and gaps in HIV-related HLBS research, a wide group of investigators gathered for a scientific NHLBI HIV Working Group on December 14-15, 2015, in Bethesda, MD. The core objectives of the Working Group included discussions on: (1) HIV-related HLBS comorbidities in the antiretroviral era; (2) HIV cure; (3) HIV prevention; and (4) mechanisms to implement new scientific discoveries in an efficient and timely manner so as to have the most impact on people living with HIV. The 2015 Working Group represented an opportunity for the NHLBI to obtain expert advice on HIV/AIDS scientific priorities and approaches over the next decade.

Perspectives from NHLBI Global Health Think Tank Meeting for Late Stage (T4) Translation Research.

Almost three-quarters (74%) of all the noncommunicable disease burden is found within low- and middle-income countries. In September 2014, the National Heart, Lung, and Blood Institute held a Global Health Think Tank meeting to obtain expert advice and recommendations for addressing compelling scientific questions for late stage (T4) research-research that studies implementation strategies for proven effective interventions-to inform and guide the National Heart, Lung, and Blood Institute's global health research and training efforts. Major themes emerged in two broad categories: 1) developing research capacity; and 2) efficiently defining compelling scientific questions within the local context. Compelling scientific questions included how to deliver inexpensive, scalable, and sustainable interventions using alternative health delivery models that leverage existing human capital, technologies and therapeutics, and entrepreneurial strategies. These broad themes provide perspectives that inform an overarching strategy needed to reduce the heart, lung, blood, and sleep disorders disease burden and global health disparities.

Reducing Health Inequities in the U.S.: Recommendations From the NHLBI's Health Inequities Think Tank Meeting.

The National, Heart, Lung, and Blood Institute convened a Think Tank meeting to obtain insight and recommendations regarding the objectives and design of the next generation of research aimed at reducing health inequities in the United States. The panel recommended several specific actions, including: 1) embrace broad and inclusive research themes; 2) develop research platforms that optimize the ability to conduct informative and innovative research, and promote systems science approaches; 3) develop networks of collaborators and stakeholders, and launch transformative studies that can serve as benchmarks; 4) optimize the use of new data sources, platforms, and natural experiments; and 5) develop unique transdisciplinary training programs to build research capacity. Confronting health inequities will require engaging multiple disciplines and sectors (including communities), using systems science, and intervening through combinations of individual, family, provider, health system, and community-targeted approaches. Details of the panel's remarks and recommendations are provided in this report.

Developmental expression of a functional TASK-1 2P domain K+ channel in embryonic chick heart.

BACKGROUND: Background K+ channels are the principal determinants of the resting membrane potential (RMP) in cardiac myocytes and thus, influence the magnitude and time course of the action potential (AP). METHODS: RT-PCR and in situ hybridization are used to study the distribution of TASK-1 and whole-cell patch clamp technique is employed to determine the functional expression of TASK-1 in embryonic chick heart. RESULTS: Chicken TASK-1 was expressed in the early tubular heart, then substantially decreased in the ventricles by embryonic day 5 (ED5), but remained relatively high in ED5 and ED11 atria. Unlike TASK-1, TASK-3 was uniformly expressed in heart at all developmental stages. In situ hybridization studies further revealed that TASK-1 was expressed throughout myocardium at Hamilton-Hamburger stages 11 and 18 (S11 & S18) heart. In ED11 heart, TASK-1 expression was more restricted to atria. Consistent with TASK-1 expression data, patch clamp studies indicated that there was little TASK-1 current, as measured by the difference currents between pH 8.4 and pH 7.4, in ED5 and ED11 ventricular myocytes. However, TASK-1 current was present in the early embryonic heart and ED11 atrial myocytes. TASK-1 currents were also identified as 3 microM anandamide-sensitive currents. 3 microM anandamide reduced TASK-1 currents by about 58% in ED11 atrial myocytes. Zn2+ (100 microM) which selectively inhibits TASK-3 channel at this concentration had no effect on TASK currents. In ED11 ventricle where TASK-1 expression was down-regulated, IK1 was about 5 times greater than in ED11 atrial myocytes. CONCLUSION: Functional TASK-1 channels are differentially expressed in the developing chick heart and TASK-1 channels contribute to background K+ conductance in the early tubular embryonic heart and in atria. TASK-1 channels act as a contributor to background K+ current to modulate the cardiac excitability in the embryonic heart that expresses little IK1.

Temperature-sensitive TREK currents contribute to setting the resting membrane potential in embryonic atrial myocytes.

TREK channels belong to the superfamily of two-pore-domain K(+) channels and are activated by membrane stretch, arachidonic acid, volatile anaesthetics and heat. TREK-1 is highly expressed in the atrium of the adult heart. In this study, we investigated the role of TREK-1 and TREK-2 channels in regulating the resting membrane potential (RMP) of isolated chicken embryonic cardiac myocytes. At room temperature, the average RMP of embryonic day (ED) 11 atrial myocytes was -22 +/- 2 mV. Raising the temperature to 35 degrees C hyperpolarized the membrane to -69 +/- 2 mV and activated a large outwardly rectifying K(+) current that was relatively insensitive to conventional K(+) channel inhibitors (TEA, 4-AP and Ba(2+)) but completely inhibited by tetracaine (200 microM), an inhibitor of TREK channels. The heat-induced hyperpolarization was mimicked by 10 microM arachidonic acid, an agonist of TREK channels. There was little or no inwardly rectifying K(+) current (I(K1)) in the ED11 atrial cells. In marked contrast, ED11 ventricular myocytes exhibited a normal RMP (-86.1 +/- 3.4 mV) and substantial I(K1), but no temperature- or tetracaine-sensitive K(+) currents. Both RT-PCR and real-time PCR further demonstrated that TREK-1 and TREK-2 are highly and almost equally expressed in ED11 atrium but much less expressed in ED11 ventricle. In addition, immunofluorescence demonstrated TREK-1 protein in the membrane of atrial myocytes. These data indicate the presence and function of TREK-1 and TREK-2 in the embryonic atrium. Moreover, we demonstrate that TREK-like currents have an essential role in determining membrane potential in embryonic atrial myocytes, where I(K1) is absent.

Changes in regulation of sodium/calcium exchanger of avian ventricular heart cells during embryonic development.

It has been suggested that the sodium/calcium exchanger NCX1 may have a more important physiological role in embryonic and neonatal hearts than in adult hearts. However, in chick heart sarcolemmal vesicles, sodium-dependent calcium transport is reported to be small and, moreover, to be 3-12 times smaller in hearts at embryonic day (ED) 4-5 than at ED18, the opposite of what would be expected of a transporter that is more important in early development. To better assess the role of NCX1 in calcium regulation in the chick embryonic heart, we measured the activity of NCX1 in chick embryonic hearts as extracellular calcium-activated exchanger current (I(NCX)) under controlled ionic conditions. With intracellular calcium concentration ([Ca(2+)](i)) = 47 nM, I(NCX) density increased from 1.34 +/- 0.28 pA/pF at ED2 to 3.22 +/- 0.55 pA/pF at ED11 (P = 0.006); however, with [Ca(2+)](i) = 481 nM, the increase was small and statistically insignificant, from 4.54 +/- 0.77 to 5.88 +/- 0.73 pA/pF (P = 0.20, membrane potential = 0 mV, extracellular calcium concentration = 2 mM). Plots of I(NCX) density against [Ca(2+)](i) were well fitted by the Michaelis-Menton equation and extrapolated to identical maximal currents for ED2 and ED11 cells (extracellular calcium concentration = 1, 2, or 4 mM). Thus the increase in I(NCX) at low [Ca(2+)](i) appeared to reflect a developmental change in allosteric regulation of the exchanger by intracellular calcium rather than an increase in the membrane density of NCX1. Supporting this conclusion, RT-PCR demonstrated little change in the amount of mRNA encoding NCX1 expression from ED2 through ED18.

Investigating nanoscale cellular dynamics with cross-sectional spectral domain phase microscopy.

We report on cross-sectional imaging of dynamic biological specimens using a spectral domain phase microscopy (SDPM) system capable of operating at a line rate of 19 kHz. This system combines the time-sensitive capabilities of SDPM with the multi-point acquisition features of related phase-sensitive techniques. The presented phase portraits and B-scan phase images of spontaneously beating embryonic cardiomyocytes and cytoplasmic flow in A. proteus offer insight into the nature and timing of the observed cellular phenomena, demonstrating the utility of this technique for dynamic cell studies.

Cardiac arterial pole alignment is sensitive to FGF8 signaling in the pharynx.

Morphogenesis of the cardiac arterial pole is dependent on addition of myocardium and smooth muscle from the secondary heart field and septation by cardiac neural crest cells. Cardiac neural crest ablation results in persistent truncus arteriosus and failure of addition of myocardium from the secondary heart field leading to malalignment of the arterial pole with the ventricles. Previously, we have shown that elevated FGF signaling after neural crest ablation causes depressed Ca2+ transients in the primary heart tube. We hypothesized that neural crest ablation results in elevated FGF8 signaling in the caudal pharynx that disrupts secondary heart field development. In this study, we show that FGF8 signaling is elevated in the caudal pharynx after cardiac neural crest ablation. In addition, treatment of cardiac neural crest-ablated embryos with FGF8b blocking antibody or an FGF receptor blocker rescues secondary heart field myocardial development in a time- and dose-dependent manner. Interestingly, reduction of FGF8 signaling in normal embryos disrupts myocardial secondary heart field development, resulting in arterial pole malalignment. These results indicate that the secondary heart field myocardium is particularly sensitive to FGF8 signaling for normal conotruncal development, and further, that cardiac neural crest cells modulate FGF8 signaling in the caudal pharynx.

Expression of a two-pore domain K+ channel (TASK-1) in developing avian and mouse ventricular conduction systems.

In this study, we report the identification and amino acid sequence of a novel two-pore domain potassium channel (TASK-1) in chicken. This protein, cTASK-1, is highly similar to mouse and human TASK-1 particularly within the pore regions. We describe the expression profile of both chicken and mouse TASK-1 in the embryonic heart as the ventricular conduction system develops. The developmental distribution of TASK-1 is similar in chicken and mouse. Initially, TASK-1 is expressed throughout the myocardium of the early heart tube. However, as cardiogenesis proceeds, ventricular expression becomes restricted to the trabeculated myocardium and eventually the bundle of His, bundle branches, and Purkinje fibers of the mature conduction system. This finding suggests that components of the ventricular conduction system differentiate from TASK-1-positive myocytes of the early heart tube that retain TASK-1 expression as they mature. Our results are consistent with a common mechanism for ventricular conduction system development in avians and mammals, despite differences in the anatomy of the mature conduction systems of these organisms.

Spectral-domain phase microscopy.

Broadband interferometry is an attractive technique for the detection of cellular motions because it provides depth-resolved phase information via coherence gating. We present a phase-sensitive technique called spectral-domain phase microscopy (SDPM). SDPM is a functional extension of spectral-domain optical coherence tomography that allows for the detection of nanometer-scale motions in living cells. The sensitivity of the technique is demonstrated, and its calibration is verified. A shot-noise limit to the displacement sensitivity of this technique is derived. Measurement of cellular dynamics was performed on spontaneously beating cardiomyocytes isolated from chick embryos.

A mutation in the TRPC6 cation channel causes familial focal segmental glomerulosclerosis.

Focal and segmental glomerulosclerosis (FSGS) is a kidney disorder of unknown etiology, and up to 20% of patients on dialysis have been diagnosed with it. Here we show that a large family with hereditary FSGS carries a missense mutation in the TRPC6 gene on chromosome 11q, encoding the ion-channel protein transient receptor potential cation channel 6 (TRPC6). The proline-to-glutamine substitution at position 112, which occurs in a highly conserved region of the protein, enhances TRPC6-mediated calcium signals in response to agonists such as angiotensin II and appears to alter the intracellular distribution of TRPC6 protein. Previous work has emphasized the importance of cytoskeletal and structural proteins in proteinuric kidney diseases. Our findings suggest an alternative mechanism for the pathogenesis of glomerular disease.

L-type Ca2+ channel function in the avian embryonic heart after cardiac neural crest ablation.

In avian and mammalian embryos, surgical ablation or severely reduced migration of the cardiac neural crest leads to a failure of outflow tract septation known as persistent truncus arteriosus (PTA) and leads to embryo lethality due partly to impaired excitation-contraction coupling stemming primarily from a reduction in the L-type Ca(2+) current (I(Ca),(L)). Decreased I(Ca,L) occurs without a corresponding reduction in the alpha(1)-subunit of the Ca(2+) channel. We hypothesize that decreased I(Ca),(L) is due to reduced function at the single channel level. The cell-attached patch clamp with Na(+) as the charge carrier was used to examine single Ca(2+) channel activity in myocytes from normal hearts from sham-operated embryos and from hearts diagnosed with PTA at embryonic days (ED) 11 and 15 after laser ablation of the cardiac neural crest. In normal hearts, the number of single channel events per 200-ms depolarization and the mean open channel probability (P(o)) was 1.89 +/- 0.17 and 0.067 +/- 0.008 for ED11 and 1.14 +/- 0.17 and 0.044 +/- 0.005 for ED15, respectively. These values represent a normal reduction in channel function and I(Ca),(L) observed with development. However, the number of single channel events was significantly reduced in hearts with PTA at both ED11 and ED15 (71% and 47%, respectively) with a corresponding reduction in P(o) (75% and 43%). The open time frequency histograms were best fitted by single exponentials with similar decay constants (tau approximately or equal 4.5 ms) except for the sham operated at ED15 (tau = 3.4 ms). These results indicate that the cardiac neural crest influences the development of myocardial Ca(2+) channels.

Effects of eugenol, an essential oil, on the mechanical and electrical activities of cardiac muscle.

Eugenol (EUG) acts as a calcium antagonist but effects on the contractile proteins could also occur. We investigated inotropic effects of EUG in rat left ventricular papillary muscles, measuring isometric force, time variables, and post rest potentiation and EUG actions on the effects of Ca2+ (0.62 to 2.5 mM) and isoproterenol (5 ng/ml), on myosin ATPase activity and on the calcium currents in single ventricular myocytes. EUG reduced tension and time variables without altering the sarcoplasmic reticulum activity increasing post-pause relative potentiation. Isoproterenol and Ca2+ counteract these negative inotropic effects. Tetanic tension diminished, but not the myosin ATPase activity suggesting an isolated sarcolemmal effect. EUG 0.1 mM decreased the Ca2+ current amplitude in the entire potential range tested and 0.5 mM almost completely blocked this inward current. Results suggested that EUG depresses force without affecting the contractile machinery and its action is the only dependent blockade of the calcium inward current.

Transitions in ventricular activation revealed by two-dimensional optical mapping.

While cardiac function in the mature heart is dependent on a properly functioning His-Purkinje system, the early embryonic tubular heart efficiently pumps blood without a distinct specialized conduction system. Although His-Purkinje system precursors have been identified using immunohistological techniques in the looped heart, little is known whether these precursors function electrically. To address this question, we used high-resolution optical mapping and fluorescent dyes with two CCD cameras to describe the motion-corrected activation patterns of 76 embryonic chick hearts from tubular stages (stage 10) to mature septated hearts (stage 35). Ventricular activation in the tubular looped heart (stages 10-17) using both calcium-sensitive fluo-4 and voltage-sensitive di-4-ANEPPS shows sequentially uniform propagation. In late looped hearts (stages 18-22), domains of the dorsal and lateral ventricle are preferentially activated before spreading to the remaining myocardium and show alternating regions of fast and slow propagation. During stages 22-26, action potentials arise from the dorsal ventricle. By stages 27-29, action potential breakthrough is also observed at the right ventricle apex. By stage 31, activation of the heart proceeds from foci at the apex and dorsal surface of the heart. The breakthrough foci correspond to regions where putative conduction system precursors have been identified immunohistologically. To date, our study represents the most detailed electrophysiological characterization of the embryonic heart between the looped and preseptated stages and suggests that ventricular activation undergoes a gradual transformation from sequential to a mature pattern with right and left epicardial breakthroughs. Our investigation suggests that cardiac conduction system precursors may be electrophysiologically distinct and mature gradually throughout cardiac morphogenesis in the chick.

Calcium buffering and excitation-contraction coupling in developing avian myocardium.

This report provides a detailed analysis of developmental changes in cytoplasmic free calcium (Ca(2+)) buffering and excitation-contraction coupling in embryonic chick ventricular myocytes. The peak magnitude of field-stimulated Ca(2+) transients declined by 41% between embryonic day (ED) 5 and 15, with most of the decline occurring between ED5 and 11. This was due primarily to a decrease in Ca(2+) currents. Sarcoplasmic reticulum (SR) Ca(2+) content increased 14-fold from ED5 to 15. Ca(2+) transients in voltage-clamped myocytes after blockade of SR function permitted computation of the fast Ca buffer power of the cytosol as expressed as generalized values of B(max) and K(D). B(max) rose with development whereas K(D) did not change significantly. The computed SR Ca(2+) contribution to the Ca(2+) transient and gain factor for Ca(2+)-induced Ca(2+) release increased markedly between ED5 and 11 and slightly thereafter. These results paralleled the maturation of SR and peripheral couplings reported by others and demonstrated a strong relationship between structure and function in development of excitation-contraction coupling. Modeling of buffer power from estimates of the major cytosolic Ca binding moieties yielded a B(max) and K(D) in reasonable agreement with experiment. From ED5 to 15, troponin C was the major Ca(2+) binding moiety, followed by SR and calmodulin.

Myocardial volume and organization are changed by failure of addition of secondary heart field myocardium to the cardiac outflow tract.

Cardiac neural crest ablation results in primary myocardial dysfunction and failure of the secondary heart field to add the definitive myocardium to the cardiac outflow tract. The current study was undertaken to understand the changes in myocardial characteristics in the heart tube, including volume, proliferation, and cell size when the myocardium from the secondary heart field fails to be added to the primary heart tube. We used magnetic resonance and confocal microscopy to determine that the volume of myocardium in the looped heart was dramatically reduced and the compact layer of myocardium was thinner after neural crest ablation, especially in the outflow tract and ventricular regions. Proliferation measured by 5-bromo-2'-deoxyuridine incorporation was elevated at only one stage during looping, cell death was normal and myocardial cell size was increased. Taken together, these results indicate that there are fewer myocytes in the heart. By incubation day 8 when the heart would have normally completed septation, the anterior (ventral) wall of the right ventricle and right ventricular outflow tract was significantly thinner in the neural crest-ablated embryos than normal, but the thickness of the compact myocardium was normal in all other regions of the heart. The decreased volume and number of myocardial cells in the heart tube after neural crest ablation most likely reflects the amount of myocardium added by the secondary heart field.

T-type Ca2+ current contribution to Ca2+-induced Ca2+ release in developing myocardium.

In normal adult-ventricular myocardium, Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) is activated via Ca2+ entry through L-type Ca2+ channels. However, embryonic-ventricular myocytes have a prominent T-type Ca2+ current (ICa,T). In this study, the contribution of ICa,T to CICR was determined in chick-ventricular development. Electrically stimulated Ca2+ transients were examined in myocytes loaded with fura-2 and Ca2+ currents with perforated patch-clamp. The results show that the magnitudes of the Ca2+ transient, L-type Ca2+ current (ICa,L) and ICa,T, decline with development with the majority of the decline of transients and ICa,L occurring between embryonic day (ED) 5 and 11. Compared to controls, the magnitude of the Ca2+ transient in the presence of nifedipine was reduced by 41% at ED5, 77% at ED11, and 78% at ED15. These results demonstrated that the overall contribution of ICa,T to the transient was greatest at ED5, while ICa,L was predominate at ED11 and 15. This indicated a decline in the contribution of ICa,T to the Ca2+ transient with development. Nifedipine plus caffeine was added to deplete the SR of Ca2+ and eliminate the occurrence of CICR due to ICa,T. Under these conditions, the transients were further reduced at all three developmental ages, which indicated that a portion of the Ca2+ transients present after just nifedipine addition was due to CICR stimulated by ICa,T. These results indicate that Ca2+ entry via T-type channels plays a significant role in excitation-contraction coupling in the developing heart that includes stimulation of CICR.

Localization, macromolecular associations, and function of the small heat shock-related protein HSP20 in rat heart.

BACKGROUND: The small heat shock proteins HSP20, HSP25, alphaB-crystallin, and myotonic dystrophy kinase binding protein (MKBP) may regulate dynamic changes in the cytoskeleton. For example, the phosphorylation of HSP20 has been associated with relaxation of vascular smooth muscle. This study examined the function of HSP20 in heart muscle. METHODS AND RESULTS: Western blotting identified immunoreactive HSP20, alphaB-crystallin, and MKBP in rat heart homogenates. Subcellular fractionation demonstrated that HSP20, alphaB-crystallin, and MKBP were predominantly in cytosolic fractions. Chromatography with molecular sieving columns revealed that HSP20 and alphaB-crystallin were associated in an aggregate of approximately 200 kDa, and alphaB-crystallin coimmunoprecipitated with HSP20. Immunofluorescence microscopy demonstrated that the pattern of HSP20, alphaB-crystallin, and actin staining was predominantly in transverse bands. Treatment with sodium nitroprusside led to increases in the phosphorylation of HSP20, as determined with 2-dimensional immunoblots. Incubation of transiently permeabilized myocytes with phosphopeptide analogues of HSP20 led to an increase in the rate of shortening. The increased shortening rate was associated with an increase in the rate of lengthening and a more rapid decay of the calcium transient. CONCLUSIONS: HSP20 is associated with alphaB-crystallin, possibly at the level of the actin sarcomere. Phosphorylated HSP20 increases myocyte shortening rate through increases in calcium uptake and more rapid lengthening.

Role of sodium-calcium exchanger (Ncx1) in embryonic heart development: a transgenic rescue?

Na(+)/Ca(2+) exchanger (Ncx-1) is highly expressed in cardiomyocytes, is thought to be required to maintain a low intracellular Ca(2+) concentration, and may play a role in excitation-contraction coupling. Significantly, targeted deletion of Ncx-1 results in Ncx1-null embryos that do not have a spontaneously beating heart and die in utero. Ultrastructural analysis revealed gross anomalies in the Ncx1-null contractile apparatus, but physiologic analysis showed normal field-stimulated Ca(2+) transients, suggesting that Ncx-1 function may not be critical for Ca(2+) extrusion from the cytosol as previously thought. Using caffeine to empty the intracellular Ca(2+) stores, we show that the sarcoplasmic reticulum is not fully functional within the 9.5-dpc mouse heart, indicating that the sarcoplasmic reticulum is unlikely to account for the unexpected maintenance of intracellular Ca(2+) homeostasis. Using the Ncx1-lacZ reporter, our data indicate restricted expression patterns of Ncx1 and that Ncx1 is highly expressed within the conduction system, suggesting Ncx1 may be required for spontaneous pacemaking activity. To test this hypothesis, we used transgenic mice overexpressing one of the two known adult Ncx1 isoforms under the control of the cardiac-specific a-myosin heavy chain promoter to restore Ncx1 expression within the Ncx1-null hearts. Results indicate that the transgenic re-expression of one Ncx1 isoform was unable to rescue the lethal null mutant phenotype. Furthermore, our in situ results indicate that both known adult Ncx1 isoforms are coexpressed within the embryonic heart, suggesting that effective transgenic rescue may require the presence of both isoforms within the developing heart.