Nitric Oxide Modulates Ca2+ Leak and Arrhythmias via S-Nitrosylation of CaMKII.

BACKGROUND: Nitric oxide (NO) has been identified as a signaling molecule generated during ß-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKIIδ (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKIIδ at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-adrenergic receptor stimulation. METHODS: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at cysteine-273 or cysteine-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (200 µM), and ß-adrenergic agonist isoproterenol (100 nmol/L). RESULTS: Both WT and CaMKIIδ-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKIIδ-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. CONCLUSIONS: We conclude that prior S-nitrosylation of CaMKIIδ at cysteine-273 can limit subsequent ß-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain ß-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Nitric Oxide modulates spontaneous Ca2+ release and ventricular arrhythmias during ß-adrenergic signalling through S-nitrosylation of Calcium/Calmodulin dependent kinase II.

Rationale: Nitric oxide (NO) has been identified as a signalling molecule generated during ß-adrenergic receptor (AR) stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of Ca2+/calmodulin kinase II delta (CaMKIIδ) is emerging. NO donors are routinely used clinically for their cardioprotective effects in the heart, but it is unknown how NO donors modulate the pro-arrhythmic CaMKII to alter cardiac arrhythmia incidence. Objective: We test the role of S-nitrosylation of CaMKIIδ at the Cys-273 inhibitory site and Cys-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during ß-AR stimulation. Methods and Results: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at Cys-273 or Cys-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 µM), sodium nitroprusside (SNP; 200 µM) and/or ß-adrenergic agonist isoproterenol (ISO; 100 nM). WT and CaMKIIδ-KO cardiomyocytes treated with GSNO showed no change in Ca2+ transient or spark properties under baseline conditions (0.5 Hz stimulation frequency). Both WT and CaMKIIδ-KO cardiomyocytes responded to ISO with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from ISO-induced Ca2+ sparks and waves was mimicked by GSNO pre-treatment in WT cardiomyocytes, but lost in CaMKIIδ-C273S cardiomyocytes that displayed a robust increase in Ca2+ waves. This observation is consistent with CaMKIIδ-C273 S-nitrosylation being critical in limiting ISO-induced arrhythmogenic sarcoplasmic reticulum Ca2+ leak. When GSNO was applied after ISO this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pre-treatment limited ISO-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after ISO sustained or exacerbated arrhythmic events. Conclusions: We conclude that prior S-nitrosylation of CaMKIIδ at Cys-273 can limit subsequent ß-AR induced arrhythmias, but that S-nitrosylation at Cys-290 might worsen or sustain ß-AR-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Increased Mitochondrial Calcium Fluxes in Hypertrophic Right Ventricular Cardiomyocytes from a Rat Model of Pulmonary Artery Hypertension.

Pulmonary artery hypertension causes right ventricular hypertrophy which rapidly progresses to heart failure with underlying cardiac mitochondrial dysfunction. Prior to failure, there are alterations in cytosolic Ca2+ handling that might impact mitochondrial function in the compensatory phase of RV hypertrophy. Our aims, therefore, were (i) to measure beat-to-beat mitochondrial Ca2+ fluxes, and (ii) to determine mitochondrial abundance and function in non-failing, hypertrophic cardiomyocytes. Male Wistar rats were injected with either saline (CON) or monocrotaline (MCT) to induce pulmonary artery hypertension and RV hypertrophy after four weeks. Cytosolic Ca2+ ([Ca2+]cyto) transients were obtained in isolated right ventricular (RV) cardiomyocytes, and mitochondrial Ca2+ ([Ca2+]mito) was recorded in separate RV cardiomyocytes. The distribution and abundance of key proteins was determined using confocal and stimulated emission depletion (STED) microscopy. The RV mitochondrial function was also assessed in RV homogenates using oxygraphy. The MCT cardiomyocytes had increased area, larger [Ca2+]cyto transients, increased Ca2+ store content, and faster trans-sarcolemmal Ca2+ extrusion relative to CON. The MCT cardiomyocytes also had larger [Ca2+]mito transients. STED images detected increased mitochondrial protein abundance (TOM20 clusters per µm2) in MCT, yet no difference was found when comparing mitochondrial respiration and membrane potential between the groups. We suggest that the larger [Ca2+]mito transients compensate to match ATP supply to the increased energy demands of hypertrophic cardiomyocytes.

Endocardial role in arrhythmias induced by acute ventricular stretch and the involvement of Purkinje fibres, in isolated rat hearts.

Purkinje fibres (PFs) play an important role in some ventricular arrhythmias and acute ventricular stretch can evoke mechanically-induced arrhythmias. We tested whether PFs and specifically TRPM4 channels, play a role in these mechanically-induced arrhythmias. Pseudo-ECGs and left ventricular (LV) activation, measured by optical mapping, were recorded in isolated, Langendorff-perfused, rat hearts. The LV endocardial surface was irrigated with experimental agents, via an indwelling catheter. The number and period of ectopic activations was measured during LV lumen inflation via an indwelling fluid-filled balloon (100 µL added over 2 s, maintained for 38 s). Mechanically-induced arrhythmias occurred during balloon inflation: they were multifocal, maximal in the first 5 s and ceased within 20 s. Optical mapping revealed activation patterns indicating PF-mediated and ectopic focal sources. Irrigation of the LV lumen with Lugol solution (IK/I2) for 10s reduced ectopics by 93% (n = 16, P < 0.001); with ablation of endocardial PFs confirmed by histology. Five min irrigation of the LV lumen with 50 µM 9-Phenanthrol, a blocker of TRPM4 channels, reduced ectopics by 39% (n = 15, P < 0.01). Immunohistochemistry confirmed that TRPM4 was more abundant in PFs than myocardium. Our results show that the endocardial surface plays an important role in these mechanically-induced ectopic activations. Ectopic activation patterns indicate a participation of PFs in these arrhythmias, with a potential involvement of TRPM4 channels, shown by the reduction of arrhythmias by 9-Phenanthrol.

Treadmill running increases the calcium sensitivity of myofilaments in diabetic rats.

The cardiovascular benefits of regular exercise are unequivocal, yet patients with type 2 diabetes respond poorly to exercise due to a reduced cardiac reserve. The contractile response of diabetic cardiomyocytes to ß-adrenergic stimulation is attenuated, which may result in altered myofilament calcium sensitivity and posttranslational modifications of cardiac troponin I (cTnI). Treadmill running increases myofilament calcium sensitivity in nondiabetic rats, and thus we hypothesized that endurance training would increase calcium sensitivity of diabetic cardiomyocytes and alter site-specific phosphorylation of cTnI. Calcium sensitivity, or pCa50, was measured in Zucker diabetic fatty (ZDF), nondiabetic (nDM), and diabetic (DM) rat hearts after 8 wk of either a sedentary (SED) or progressive treadmill running (TR) intervention. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+]). Specific phospho-sites on cTnI and O-GlcNAcylation were quantified by immunoblot and total protein phosphorylation by fluorescent gel staining (ProQ Diamond). The novel finding in this study was that training increased pCa50 in both DM and nDM cardiomyocytes (P = 0.009). Phosphorylation of cTnI amino acid residues Ser23/24, a crucial protein kinase A site, and Threonine (Thr)144 was lower in DM hearts, but there was no effect of training on site-specific phosphorylation. In addition, total phosphorylation and O-GlcNAcylation levels were not different between SED and TR groups. These findings suggest that regular exercise may benefit the diabetic heart by specifically targeting myofilament contractile function.NEW & NOTEWORTHY We examined the effects of training on the myofilament calcium in diabetic rat hearts. After 8 wk of treadmill running, both nondiabetic and diabetic cardiomyocytes had increased myofilament calcium sensitivity compared with their sedentary counterparts, but there was no effect of training on the phosphorylation or O-GlcNAcylation status of myofilament proteins measured in this study. These data highlight one potential mechanism capable of reversing, in part, reduced cardiac reserve in the diabetic heart.

Paradoxes of Hymenoptera flight muscles, extreme machines.

In the Carboniferous, insects evolved flight. Intense selection drove for high performance and approximately 100 million years later, Hymenoptera (bees, wasps and ants) emerged. Some species had proportionately small wings, with apparently impossible aerodynamic challenges including a need for high frequency flight muscles (FMs), powered exclusively off aerobic pathways and resulting in extreme aerobic capacities. Modern insect FMs are the most refined and form large dense blocks that occupy 90% of the thorax. These can beat wings at 200 to 230 Hz, more than double that achieved by standard neuromuscular systems. To do so, rapid repolarisation was circumvented through evolution of asynchronous stimulation, stretch activation, elastic recoil and a paradoxically slow Ca2+ reuptake. While the latter conserves ATP, considerable ATP is demanded at the myofibrils. FMs have diminished sarcoplasmic volumes, and ATP is produced solely by mitochondria, which pack myocytes to maximal limits and have very dense cristae. Gaseous oxygen is supplied directly to mitochondria. While FMs appear to be optimised for function, several unusual paradoxes remain. FMs lack any significant equivalent to the creatine kinase shuttle, and myofibrils are twice as wide as those of within cardiomyocytes. The mitochondrial electron transport systems also release large amounts of reactive oxygen species (ROS) and respiratory complexes do not appear to be present at any exceptional level. Given that the loss of the creatine kinase shuttle and elevated ROS impairs heart function, we question how do FM shuttle adenylates at high rates and tolerate oxidative stress conditions that occur in diseased hearts?

Increased myofilament calcium sensitivity is associated with decreased cardiac troponin I phosphorylation in the diabetic rat heart.

NEW FINDINGS: What is the central question of this study? In Zucker Diabetic Fatty rats, does cardiomyocyte myofilament function change through the time course of diabetes and what are the mechanisms behind alterations in calcium sensitivity? What is the main finding and its importance? Zucker Diabetic Fatty rats had increased myofilament calcium sensitivity and reduced phosphorylation at cardiac troponin I without differential O-GlcNAcylation. ABSTRACT: The diabetic heart has impaired systolic and diastolic function independent of other comorbidities. The availability of calcium is altered, but does not fully explain the cardiac dysfunction seen in the diabetic heart. Thus, we explored if myofilament calcium regulation of contraction is altered while also categorizing the levels of phosphorylation and O-GlcNAcylation in the myofilaments. Calcium sensitivity (pCa50 ) was measured in Zucker Diabetic Fatty (ZDF) rat hearts at the initial stage of diabetes (12 weeks old) and after 8 weeks of uncontrolled hyperglycaemia (20 weeks old) and in non-diabetic (nDM) littermates. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+ ]). Fluorescent gel stain (ProQ Diamond) was used to measure total protein phosphorylation. Specific phospho-sites on cardiac troponin I (cTnI) and total cTnI O-GlcNAcylation were quantified using immunoblot. pCa50 was greater in both 12- and 20-week-old diabetic (DM) rats compared to nDM littermates (P = 0.0001). Total cTnI and cTnI serine 23/24 phosphorylation were lower in DM rats (P = 0.003 and P = 0.01, respectively), but cTnI O-GlcNAc protein expression was not different. pCa50 is greater in DM rats and corresponds with an overall reduction in cTnI phosphorylation. These findings indicate that myofilament calcium sensitivity is increased and cTnI phosphorylation is reduced in ZDF DM rats and suggests an important role for cTnI phosphorylation in the DM heart.

CaMKII Inhibition is a Novel Therapeutic Strategy to Prevent Diabetic Cardiomyopathy.

Increasing prevalence of diabetes mellitus worldwide has pushed the complex disease state to the foreground of biomedical research, especially concerning its multifaceted impacts on the cardiovascular system. Current therapies for diabetic cardiomyopathy have had a positive impact, but with diabetic patients still suffering from a significantly greater burden of cardiac pathology compared to the general population, the need for novel therapeutic approaches is great. A new therapeutic target, calcium/calmodulin-dependent kinase II (CaMKII), has emerged as a potential treatment option for preventing cardiac dysfunction in the setting of diabetes. Within the last 10 years, new evidence has emerged describing the pathophysiological consequences of CaMKII activation in the diabetic heart, the mechanisms that underlie persistent CaMKII activation, and the protective effects of CaMKII inhibition to prevent diabetic cardiomyopathy. This review will examine recent evidence tying cardiac dysfunction in diabetes to CaMKII activation. It will then discuss the current understanding of the mechanisms by which CaMKII activity is enhanced during diabetes. Finally, it will examine the benefits of CaMKII inhibition to treat diabetic cardiomyopathy, including contractile dysfunction, heart failure with preserved ejection fraction, and arrhythmogenesis. We intend this review to serve as a critical examination of CaMKII inhibition as a therapeutic strategy, including potential drawbacks of this approach.

Trends in Number of Women Speakers at the American Head and Neck Society Meetings, 2007-2019.

Importance: The number of female speakers at American Head and Neck Society (AHNS) conferences should ideally be consistent with the number of women entering head and neck surgery fellowships to ensure gender equity in the field. Yet the presence of women speakers at the annual AHNS meetings, which is specific to the field of head and neck cancer, endocrine and microvascular reconstructive surgery, has yet to be studied. Objective: To determine whether the proportion of female speakers at the AHNS has increased in a manner consistent with the numbers of women entering fellowships since 2007. Design, Setting, and Participants: This qualitative study assessed 13 final meeting programs from AHNS national/international conferences from 2007 to 2019. The number of male and female participants in different roles throughout the meeting were retrospectively tracked. Participants were male and female speakers at AHNS national/international conferences who took part in the roles of scientific session presenter, scientific session moderator, expert panelist, miscellaneous moderator, and named lecturers/keynote speaker. Gender of the speaker was determined by searching names on the internet and using available published pronouns. Main Outcomes and Measures: Number of speaking opportunities for men and women in different roles from 2007 to 2019 as well as number of men and women entering AHNS fellowships since 2007 and new active AHNS members since 2012. Results: In this qualitative study, from 2007 to 2019, 4059 speakers were identified. Of these speakers, 902 (22%) were women and 3157 (78%) were men. Overall, there was a strong correlation between increasing years and number of women speakers from 2007 to 2019 (ρ = 0.75; 95% CI, 0.72-0.78). There were 2096 invited speaking roles that excluded research presentations, of which 400 were offered to female participants (19.1%) across the study period. There were 131 different women that made up all 400 of the opportunities that were offered to women in the years surveyed. There was a strong correlation in the proportion of women as presenters for oral abstracts, expert panelists, and miscellaneous moderators between the years but no correlation in scientific session moderators and named lecturers/keynote speakers. Of the 45 named lecturers/keynote speakers in the programs tracked, only 2 were women. Conclusions and Relevance: In this study, from 2007 to 2019, the presence of women at ANHS has increased overall, reflecting the changing demographic characteristics of those entering in head and neck oncology and microvascular surgery fellowships. However, a strong disparity continues to exist for preeminent speaking opportunities.

Visualization of Dynamic Mitochondrial Calcium Fluxes in Isolated Cardiomyocytes.

BACKGROUND: Cardiomyocyte contraction requires a constant supply of ATP, which varies depending on work rate. Maintaining ATP supply is particularly important during excitation-contraction coupling, where cytosolic Ca2+ fluxes drive repeated cycles of contraction and relaxation. Ca2+ is one of the key regulators of ATP production, and its uptake into the mitochondrial matrix occurs via the mitochondrial calcium uniporter. Fluorescent indicators are commonly used for detecting cytosolic Ca2+ changes. However, visualizing mitochondrial Ca2+ fluxes using similar methods is more difficult, as the fluorophore must be permeable to both the sarcolemma and the inner mitochondrial membrane. Our aim was therefore to optimize a method using the fluorescent Ca2+ indicator Rhod-2 to visualize beat-to-beat mitochondrial calcium fluxes in rat cardiomyocytes. METHODS: Healthy, adult male Wistar rat hearts were isolated and enzymatically digested to yield rod-shaped, quiescent ventricular cardiomyocytes. The fluorescent Ca2+ indicator Rhod-2 was reduced to di-hydroRhod-2 and confocal microscopy was used to validate mitochondrial compartmentalization. Cardiomyocytes were subjected to various pharmacological interventions, including caffeine and ß-adrenergic stimulation. Upon confirmation of mitochondrial Rhod-2 localization, loaded myocytes were then super-fused with 1.5 mM Ca2+ Tyrodes containing 1 µM isoproterenol and 150 µM spermine. Myocytes were externally stimulated at 0.1, 0.5 and 1 Hz and whole cell recordings of both cytosolic ([Ca2+]cyto) and mitochondrial calcium ([Ca2+] mito ) transients were made. RESULTS: Myocytes loaded with di-hydroRhod-2 revealed a distinct mitochondrial pattern when visualized by confocal microscopy. Application of 20 mM caffeine revealed no change in fluorescence, confirming no sarcoplasmic reticulum compartmentalization. Myocytes loaded with di-hydroRhod-2 also showed a large increase in fluorescence within the mitochondria in response to ß-adrenergic stimulation (P < 0.05). Beat-to-beat mitochondrial Ca2+ transients were smaller in amplitude and had a slower time to peak and maximum rate of rise relative to cytosolic calcium transients at all stimulation frequencies (P < 0.001). CONCLUSION: Myocytes loaded with di-hydroRhod-2 revealed mitochondrial specific compartmentalization. Mitochondrial Ca2+ transients recorded from di-hydroRhod-2 loaded myocytes were distinct in comparison to the large and rapid Rhod-2 cytosolic transients, indicating different kinetics between [Ca2+]cyto and [Ca2+]mito transients. Overall, our results showed that di-hydroRhod-2 loading is a quick and suitable method for measuring beat-to-beat [Ca2+]mito transients in intact myocytes.

Disruption of Transverse-Tubules Eliminates the Slow Force Response to Stretch in Isolated Rat Trabeculae.

Ventricular muscle has a biphasic response to stretch. There is an immediate increase in force that coincides with the stretch which is followed by a second phase that takes several minutes for force to develop to a new steady state. The initial increase in force is due to changes in myofilament properties, whereas the second, slower component of the stretch response (known as the "slow force response" or SFR) is accompanied by a steady increase in Ca2+ transient amplitude. Evidence shows stretch-dependent Ca2+ influx during the SFR occurs through some mechanism that is continuously active for several minutes following stretch. Many of the candidate ion channels are located primarily in the t-tubules, which are consequently lost in heart disease. Our aim, therefore, was to investigate the impact of t-tubule loss on the SFR in non-failing cardiac trabeculae in which expression of the different Ca2+ handling proteins was not altered by any disease process. For comparison, we also investigated the effect of formamide detubulation of trabeculae on ß-adrenergic activation (1 µM isoproterenol), since this is another key regulator of cardiac force. Measurement of intracellular calcium ([Ca2+]i) and isometric stress were made in RV trabeculae from rat hearts before, during and after formamide treatment (1.5 M for 5 min), which on washout seals the surface sarcolemmal t-tubule openings. Results showed detubulation slowed the time course of Ca2+ transients and twitch force, with time-to-peak, maximum rate-of-rise, and relaxation prolonged in trabeculae at optimal length (Lo). Formamide treatment also prevented development of the SFR following a step change in length from 90 to 100% Lo, and blunted the response to ß-adrenergic activation (1 µM isoproterenol).

Stretch modulation of cardiac contractility: importance of myocyte calcium during the slow force response.

The mechanical response of the heart to myocardial stretch has been understood since the work of muscle physiologists more than 100 years ago, whereby an increase in ventricular chamber filling during diastole increases the subsequent force of contraction. The stretch-induced increase in contraction is biphasic. There is an abrupt increase in the force that coincides with the stretch (the rapid response), which is then followed by a slower response that develops over several minutes (the slow force response, or SFR). The SFR is associated with a progressive increase in the magnitude of the Ca2+ transient, the event that initiates myocyte cross-bridge cycling and force development. However, the mechanisms underlying the stretch-dependent increase in the Ca2+ transient are still debated. This review outlines recent literature on the SFR and summarizes the different stretch-activated Ca2+ entry pathways. The SFR might result from a combination of several different cellular mechanisms initiated in response to activation of different cellular stretch sensors.

Mitochondrial function remains impaired in the hypertrophied right ventricle of pulmonary hypertensive rats following short duration metoprolol treatment.

Pulmonary hypertension (PH) increases the work of the right ventricle (RV) and causes right-sided heart failure. This study examined RV mitochondrial function and ADP transfer in PH animals advancing to right heart failure, and investigated a potential therapy with the specific ß1-adrenergic-blocker metoprolol. Adult Wistar rats (317 ± 4 g) were injected either with monocrotaline (MCT, 60 mg kg-1) to induce PH, or with an equivalent volume of saline for controls (CON). At three weeks post-injection the MCT rats began oral metoprolol (10 mg kg-1 day-1-) or placebo treatment until heart failure was observed in the MCT group. Mitochondrial function was then measured using high-resolution respirometry from permeabilised RV fibres. Relative to controls, MCT animals had impaired mitochondrial function but maintained coupling between myofibrillar ATPases and mitochondria, despite an increase in ADP diffusion distances. Cardiomyocytes from the RV of MCT rats were enlarged, primarily due to an increase in myofibrillar protein. The ratio of mitochondria per myofilament area was decreased in both MCT groups (p ≤ 0.05) in comparison to control (CON: 1.03 ± 0.04; MCT: 0.74 ± 0.04; MCT + BB: 0.74 ± 0.03). This not only implicates impaired energy production in PH, but also increases the diffusion distance for metabolites within the MCT cardiomyocytes, adding an additional hindrance to energy supply. Together, these changes may limit energy supply in MCT rat hearts, particularly at high cardiac workloads. Metoprolol treatment did not delay the onset of heart failure symptoms, improve mitochondrial function, or regress RV hypertrophy.

Calcium mishandling impairs contraction in right ventricular hypertrophy prior to overt heart failure.

Currently, there are no tailored therapies available for the treatment of right ventricular (RV) hypertrophy, and the cellular mechanisms that underlie the disease are poorly understood. We investigated the cellular changes that occur early in the progression of the disease, when RV hypertrophy is evident, but prior to the onset of heart failure. Intracellular Ca2+ ([Ca2+]i) handling was examined in a rat model of monocrotaline (MCT)-induced pulmonary hypertension and subsequent RV hypertrophy. [Ca2+]i and stress production were measured in isolated RV trabeculae under baseline conditions (1-Hz stimulation, 1.5 mM [Ca2+]o, 37 °C), and in response to inotropic interventions (5-Hz stimulation or 1-µM isoproterenol). Under baseline conditions, MCT trabeculae had impaired Ca2+ release in response to stimulation with a 45% delay in the time-to-peak Ca2+, but there was no difference in the amplitude and decay of the Ca2+ transient, or active stress relative to RV trabeculae from normotensive hearts (CON). Increasing stimulation frequency from 1 to 5 Hz increased stress in CON, but not MCT trabeculae. Similarly, ß-adrenergic stimulation with isoproterenol increased Ca2+ transient amplitude and active stress in CON, but not in MCT trabeculae, despite accelerating Ca2+ transient decay in trabeculae from both groups. During isoproterenol treatment, MCT trabeculae showed increased diastolic Ca2+ leak, which may explain the blunted inotropic response to ß-adrenergic stimulation. Confocal imaging of trabeculae fixed following functional measurements showed that myocytes were on average wider, and transverse-tubule organisation was disrupted in MCT which provides a mechanism to explain the observed slower release of Ca2+.

Does the intercept of the heat-stress relation provide an accurate estimate of cardiac activation heat?

KEY POINTS: The heat of activation of cardiac muscle reflects the metabolic cost of restoring ionic homeostasis following a contraction. The accuracy of its measurement depends critically on the abolition of crossbridge cycling. We abolished crossbridge activity in isolated rat ventricular trabeculae by use of blebbistatin, an agent that selectively inhibits myosin II ATPase. We found cardiac activation heat to be muscle length independent and to account for 15-20% of total heat production at body temperature. We conclude that it can be accurately estimated at minimal muscle length. ABSTRACT: Activation heat arises from two sources during the contraction of striated muscle. It reflects the metabolic expenditure associated with Ca2+ pumping by the sarcoplasmic reticular Ca2+ -ATPase and Ca2+ translocation by the Na+ /Ca2+ exchanger coupled to the Na+ ,K+ -ATPase. In cardiac preparations, investigators are constrained in estimating its magnitude by reducing muscle length to the point where macroscopic twitch force vanishes. But this experimental protocol has been criticised since, at zero force, the observed heat may be contaminated by residual crossbridge cycling activity. To eliminate this concern, the putative thermal contribution from crossbridge cycling activity must be abolished, at least at minimal muscle length. We achieved this using blebbistatin, a selective inhibitor of myosin II ATPase. Using a microcalorimeter, we measured the force production and heat output, as functions of muscle length, of isolated rat trabeculae from both ventricles contracting isometrically at 5 Hz and at 37°C. In the presence of blebbistatin (15 µmol l-1 ), active force was zero but heat output remained constant, at all muscle lengths. Activation heat measured in the presence of blebbistatin was not different from that estimated from the intercept of the heat-stress relation in its absence. We thus reached two conclusions. First, activation heat is independent of muscle length. Second, residual crossbridge heat is negligible at zero active force; hence, the intercept of the cardiac heat-force relation provides an estimate of activation heat uncontaminated by crossbridge cycling. Both results resolve long-standing disputes in the literature.

Impaired ADP channeling to mitochondria and elevated reactive oxygen species in hypertensive hearts.

Systemic hypertension initially promotes a compensatory cardiac hypertrophy, yet it progresses to heart failure (HF), and energetic deficits appear to be central to this failure. However, the transfer of energy between the mitochondria and the myofibrils is not often considered as part of the energetic equation. We compared hearts from old spontaneously hypertensive rats (SHRs) and normotensive Wistar controls. SHR hearts showed a 35% depression in mitochondrial function, yet produced at least double the amount of reactive oxygen species (ROS) in all respiration states in left ventricular (LV) homogenates. To test the connectivity between mitochondria and myofibrils, respiration was further tested in situ with LV permeabilized fibers by addition of multiple substrates and ATP, which requires hydrolysis to mediate oxidative phosphorylation. By trapping ADP using a pyruvate kinase enzyme system, we tested ADP channeling towards mitochondria, and this suppressed respiration and elevated ROS production more in the SHR fibers. The ADP-trapped state was also less relieved on creatine addition, likely reflecting the 30% depression in total CK activity in the SHR heart fibers. Confocal imaging identified a 34% longer distance between the centers of myofibril to mitochondria in the SHR hearts, which increases transverse metabolite diffusion distances (e.g., for ATP, ADP, and creatine phosphate). We propose that impaired connectivity between mitochondria and myofibrils may contribute to elevated ROS production. Impaired energy exchange could be the result of ultrastructural changes that occur with hypertrophy in this model of hypertension.

Positive Inotropic Effect of Prostaglandin F2α in Rat Ventricular Trabeculae.

Prostaglandins are ubiquitous signaling molecules in the body that produce autocrine/paracrine effects on target cells in response to mechanical or chemical signals. In the heart, long-term exposure to prostaglandin (PG) F2α has been linked to the development of hypertrophy; however, there is no consensus on the acute effect of PGF2α. Our aim was to determine the response to exogenous PGF2α in isolated trabeculae from rat hearts. PGF2α (1 µM) increased both the Ca transients and the isometric stress in trabeculae, reaching steady state after 10-15 minutes, without altering the time course of Ca transient decay. The precursor of PGF2α, arachidonic acid, also stimulated a similar response. The positive inotropic effect of PGF2α was mediated through a protein kinase C signaling pathway that involved activation of the sarcolemmal Na/H exchanger. We also found that the slow force response to stretch was attenuated in the presence of PGF2α and by addition of indomethacin, a blocker of prostaglandin synthesis. In conclusion, PGF2α was positively inotropic when acutely applied to trabeculae and contributed to the increased Ca transients during the slow force response to stretch. Together, these data suggest that PGF2α is important in maintaining homeostasis during volume loading in healthy hearts.

Uncoupling of oxidative phosphorylation and ATP synthase reversal within the hyperthermic heart.

Heart failure is a common cause of death with hyperthermia, and the exact cause of hyperthermic heart failure appears elusive. We hypothesize that the energy supply (ATP) of the heart may become impaired due to increased inner-mitochondrial membrane permeability and inefficient oxidative phosphorylation (OXPHOS). Therefore, we assessed isolated working heart and mitochondrial function. Ex vivo working rat hearts were perfused between 37 and 43.5°C and showed break points in all functional parameters at ~40.5°C. Mitochondrial high-resolution respirometry coupled to fluorometry was employed to determine the effects of hyperthermia on OXPHOS and mitochondrial membrane potential (ΔΨ) in vitro using a comprehensive metabolic substrate complement with isolated mitochondria. Relative to 37 and 40°C, 43°C elevated Leak O2 flux and depressed OXPHOS O2 flux and ∆Ψ. Measurement of steady-state ATP production from mitochondria revealed decreased ATP synthesis capacity, and a negative steady-state P:O ratio at 43°C. This approach offers a more powerful analysis of the effects of temperature on OXPHOS that cannot be measured using simple measures such as the traditional respiratory control ratio (RCR) or P:O ratio, which, respectively, can only approach 1 or 0 with inner-membrane failure. At 40°C there was only a slight enhancement of the Leak O2 flux and this did not significantly affect ATP production rate. Therefore, during mild hyperthermia (40°C) there is no enhancement of ATP supply by mitochondria, to accompany increasing cardiac energy demands, while between this and critical hyperthermia (43°C), mitochondria become net consumers of ATP. This consumption may contribute to cardiac failure or permanent damage during severe hyperthermia.

Mitochondrial inefficiencies and anoxic ATP hydrolysis capacities in diabetic rat heart.

As ~80% of diabetic patients die from heart failure, an understanding of diabetic cardiomyopathy is crucial. Mitochondria occupy 35-40% of the mammalian cardiomyocyte volume and supply 95% of the heart's ATP, and diabetic heart mitochondria show impaired structure, arrangement, and function. We predict that bioenergetic inefficiencies are present in diabetic heart mitochondria; therefore, we explored mitochondrial proton and electron handling by linking oxygen flux to steady-state ATP synthesis, reactive oxygen species (ROS) production, and mitochondrial membrane potential (ΔΨ) within rat heart tissues. Sprague-Dawley rats were injected with streptozotocin (STZ, 55 mg/kg) to induce type 1 diabetes or an equivalent volume of saline (control, n = 12) and fed standard rat chow for 8 wk. By coupling high-resolution respirometers with purpose-built fluorometers, we followed Magnesium Green (ATP synthesis), Amplex UltraRed (ROS production), and safranin-O (ΔΨ). Relative to control rats, the mass-specific respiration of STZ-diabetic hearts was depressed in oxidative phosphorylation (OXPHOS) states. Steady-state ATP synthesis capacity was almost one-third lower in STZ-diabetic heart, which, relative to oxygen flux, equates to an estimated 12% depression in OXPHOS efficiency. However, with anoxic transition, STZ-diabetic and control heart tissues showed similar ATP hydrolysis capacities through reversal of the F1F0-ATP synthase. STZ-diabetic cardiac mitochondria also produced more net ROS relative to oxygen flux (ROS/O) in OXPHOS. While ΔΨ did not differ between groups, the time to develop ΔΨ with the onset of OXPHOS was protracted in STZ-diabetic mitochondria. ROS/O is higher in lifelike OXPHOS states, and potential delays in the time to develop ΔΨ may delay ATP synthesis with interbeat fluctuations in ADP concentrations. Whereas diabetic cardiac mitochondria produce less ATP in normoxia, they consume as much ATP in anoxic infarct-like states.