Sarcoplasmic reticulum (SR) Ca 2 transport proteins, especially ryanodine receptors (RyR) and their accessory protein FKBP12.6, have been implicated as major players in the pathogenesis of heart failure (HF), but their role remain controversial. We used the tachycardia-induced canine model of HF and human failing hearts to investigate the density and major functional properties of RyRs, SERCA2a, and phospholamban (PLB), the main proteins regulating SR Ca 2 transport. Intracellular Ca 2 is likely to play a role in the contractile dysfunction of HF because the amplitude and kinetics of the [Ca 2 ] i transient were reduced in HF. Ca 2 uptake assays showed 448% reduction of V max in canine HF, and Western blots demonstrated that this reduction was due to decreased SERCA2a and PLB levels. Human HF showed a 305% reduction in SERCA2a, but PLB was unchanged. RyRs from canine and human HF displayed no major structural or functional differences compared with control. The P o of RyRs was the same for control and HF over the range of pCa 7 to 4. Subconductance states, which predominate in FKBP12.6-stripped RyRs, were equally frequent in control and HF channels. An antibody that recognizes phosphorylated RyRs yields equal intensity for control and HF channels. Further, phosphorylation of RyRs by PKA did not appear to change the RyR/FKBP12.6 association, suggesting minor-adrenergic stimulation of Ca 2 release through this mechanism. These results support a role for SR in the pathogenesis of HF, with abnormal Ca 2 uptake, more than Ca 2 release, contributing to the depressed and slow Ca 2 transient characteristic of HF. (Circ Res. 2002;91:1015-1022.) Key Words: heart failure ryanodine receptor sarcoplasmic reticulum protein kinase A

Abstract-Increased phosphorylation of the cardiac ryanodine receptor (RyR)2 by protein kinase A (PKA) at the phosphoepitope encompassing Ser2808 has been advanced as a central mechanism in the pathogenesis of cardiac arrhythmias and heart failure. In this scheme, persistent activation of the sympathetic system during chronic stress leads to PKA "hyperphosphorylation" of RyR2-S2808, which increases Ca 2ϩ release by augmenting the sensitivity of the RyR2 channel to diastolic Ca 2ϩ . This gain-of-function is postulated to occur with the unique participation of RyR2-S2808, and other potential PKA phosphorylation sites have been discarded. Although it is clear that RyR2 is among the first proteins in the heart to be phosphorylated by ␤-adrenergic stimulation, the functional impact of phosphorylation in excitation-contraction coupling and cardiac performance remains unclear. We used gene targeting to produce a mouse model with complete ablation of the RyR2-S2808 phosphorylation site (RyR2-S2808A). Whole-heart and isolated cardiomyocyte experiments were performed to test the role of ␤-adrenergic stimulation and PKA phosphorylation of Ser2808 in heart failure progression and cellular Ca 2ϩ handling. We found that the RyR2-S2808A mutation does not alter the ␤-adrenergic response, leaves cellular function almost unchanged, and offers no significant protection in the maladaptive cardiac remodeling induced by chronic stress. Moreover, the RyR2-S2808A mutation appears to modify single-channel activity, although modestly and only at activating [Ca 2ϩ ]. Taken together, these results reveal some of the most important effects of PKA phosphorylation of RyR2 but do not support a major role for RyR2-S2808 phosphorylation in the pathogenesis of cardiac dysfunction and failure.

Activation of] required for the latter process (ED 50 ‫؍‬ ϳ200 M) appears to be reached only within the dyadic space. Rapid injection of 5 M sorcin onto the cytosolic face of RyRs reconstituted in lipid bilayers resulted in complete inhibition of channel activity in < 20 ms. Thus, sorcin is a potent inhibitor of both spontaneous and I Ca -triggered RyR activity and is kinetically capable of playing a role in terminating the positive feedback loop of CICR.

Rationale: Preterm birth, and its often-required medical interventions, can result in respiratory and gas exchange deficits into childhood. However, the long-term sequelae into adulthood are not well understood.Objectives: To determine exercise capacity and pulmonary gas exchange efficiency during exercise in adult survivors of preterm birth.Methods: Preterm (n = 14), very low birth weight (,1,500 g) adults (20-23 yr) and term-born, age-matched control subjects (n = 16) performed incremental exercise on a cycle ergometer to volitional exhaustion while breathing one of two oxygen concentrations: normoxia (fraction of inspired oxygen, 0.21) or hypoxia (fraction of inspired oxygen, 0.12).Measurements and Main Results: Ventilation, mixed expired gases, arterial blood gases, power output, and oxygen consumption were measured during rest and exercise. We calculated the alveolar-to-arterial oxygen difference to determine pulmonary gas exchange efficiency. Preterm subjects had lower power output at volitional exhaustion than did control subjects in normoxia (150 6 10 vs. 180 6 10 W; P = 0.01), despite similar normoxic oxygen consumption. However, during hypoxic exercise, there was no difference in power output at volitional exhaustion between the two groups (116 6 10 vs. 135 6 10 W; P = 0.11). Preterm subjects also exhibited a more acidotic, acid-base balance throughout exercise compared with control subjects. In contrast to other studies, adults born preterm, as a group developed a wider alveolarto-arterial oxygen difference and lower Pa O 2 than did control subjects during normoxic but not hypoxic exercise.Conclusions: This study demonstrates that pulmonary gas exchange efficiency is lower in some adult survivors of preterm birth during exercise compared with control subjects. The gas exchange inefficiency, when present, is accompanied by low arterial blood oxygen tension. Preterm subjects also exhibit reduced power output. Overall, our findings suggest potential long-term consequences of extreme preterm birth and very low birth weight on cardiopulmonary function.

This study investigated the effects of regular consumption of dark chocolate (DC), rich in cocoa polyphenols, on plasma metabolites, hormones, and markers of oxidative stress after prolonged exhaustive exercise. Twenty active men cycled at 60% maximal oxygen uptake (VO2max) for 1.5 hr, with the intensity increased to 90% VO2max for a 30-s period every 10 min, followed by a ride to exhaustion at 90% VO2max. In the 2 wk before exercise participants consumed 40 g of DC or an isocarbohydrate-fat control cocoa liquor–free chocolate (CON) twice daily and once 2 hr before exercise in a randomized, counterbalanced, crossover design. Venous blood samples were taken immediately before exercise, postexercise (fixed duration), postexhaustion, and after 1 hr of recovery. F2-isoprostanes were significantly lower (post hoc tests: p < .001) at exhaustion and after 1 hr of recovery with DC. Oxidized low-density lipoproteins were significantly lower with DC (p < .001) both before and after exercise and at exhaustion. DC was also associated with ~21% greater rises in free fatty acids during exercise (main effect: p < .05). Changes in circulating glucose, insulin, glucagon, cortisol, and interleukin (IL)-6, IL-10, and IL-1ra were unaffected by treatment. Time to exhaustion at 90% VO2max was not significantly different between trials (398 ± 204 and 374 ± 194 s for DC and CON, respectively). These results suggest that regular DC intake is associated with reduced oxidative-stress markers and increased mobilization of free fatty acids after exercise but has no observed effect on exercise performance.

Rationale: Human pluripotent stem cell (hPSC)–derived cardiomyocytes exhibit the properties of fetal cardiomyocytes, which limits their applications. Various methods have been used to promote maturation of hPSC-cardiomyocytes; however, there is a lack of an unbiased and comprehensive method for accurate assessment of the maturity of hPSC-cardiomyocytes. Objective: We aim to develop an unbiased proteomics strategy integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for the accurate and comprehensive assessment of hPSC-cardiomyocyte maturation. Methods and Results: Utilizing hPSC-cardiomyocytes from early- and late-stage 2-dimensional monolayer culture and 3-dimensional engineered cardiac tissue, we demonstrated the high reproducibility and reliability of a top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expression and associated post-translational modifications. This method allowed for the detection of known maturation-associated contractile protein alterations and, for the first time, identified contractile protein post-translational modifications as promising new markers of hPSC-cardiomyocytes maturation. Most notably, decreased phosphorylation of α-tropomyosin was found to be associated with hPSC-cardiomyocyte maturation. By employing a bottom-up global proteomics strategy, we identified candidate maturation-associated markers important for sarcomere organization, cardiac excitability, and Ca 2+ homeostasis. In particular, upregulation of myomesin 1 and transmembrane 65 was associated with hPSC-cardiomyocyte maturation and validated in cardiac development, making these promising markers for assessing maturity of hPSC-cardiomyocytes. We have further validated α-actinin isoforms, phospholamban, dystrophin, αB-crystallin, and calsequestrin 2 as novel maturation-associated markers, in the developing mouse cardiac ventricles. Conclusions: We established an unbiased proteomics method that can provide accurate and specific assessment of the maturity of hPSC-cardiomyocytes and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering the molecular pathways involved in cardiac development and disease using hPSC-cardiomyocytes.

Intrapulmonary arteriovenous anastomoses (IPAVs) are large diameter connections that allow blood to bypass the lung capillaries and may provide a route for right-to-left embolus transmission. These anastomoses are recruited by exercise and catecholamines and hypoxia. Yet, whether IPAVs are recruited via direct, oxygen sensitive regulatory mechanisms or indirect effects secondary to redistribution pulmonary blood flow is unknown. Here, we hypothesized that the addition of exercise to hypoxic gas breathing, which increases cardiac output, would augment IPAVs recruitment in healthy humans. To test this hypothesis, we measured the transpulmonary passage of 99mTc-macroaggregated albumin particles (99mTc-MAA) in seven healthy volunteers, at rest and with exercise at 85% of volitional max, with normoxic (FIO2 = 0.21) and hypoxic (FIO2 = 0.10) gas breathing. We found increased 99mTc-MAA passage in both exercise conditions and resting hypoxia. However, contrary to our hypothesis, we found the greatest 99mTc-MAA passage with resting hypoxia. As an additional, secondary endpoint, we also noted that the transpulmonary passage of 99mTc-MAA was well-correlated with the alveolar-arterial oxygen difference (A-aDO2) during exercise. While increased cardiac output has been proposed as an important modulator of IPAVs recruitment, we provide evidence that the modulation of blood flow through these pathways is more complex and that increasing cardiac output does not necessarily increase IPAVs recruitment. As we discuss, our data suggest that the resistance downstream of IPAVs is an important determinant of their perfusion.

Intrapulmonary arteriovenous anastomoses (IPAVS) directly connect the arterial and venous circulations in the lung, bypassing the capillary network. Here, we used solid, latex microspheres and isolated rat lung and intact, spontaneously breathing rat models to test the hypothesis that IPAVS are recruited by alveolar hypoxia. We found that hypoxia recruits IPAVS in the intact rat, but not the isolated lung. IPAVS are at least 70 μm in the rat and, interestingly, appear to be recruited when the mixed venous Po(2) falls below 22 mmHg. These data provide evidence that large-diameter, direct arteriovenous connections exist in the lung and are recruitable by hypoxia in the intact animal.