The electrophysiological effects of cannabidiol on action potentials and transmembrane potassium currents in rabbit and dog cardiac ventricular preparations.

Cannabis use is associated with known cardiovascular side effects such as cardiac arrhythmias or even sudden cardiac death. The mechanisms behind these adverse effects are unknown. The aim of the present work was to study the cellular cardiac electrophysiological effects of cannabidiol (CBD) on action potentials and several transmembrane potassium currents, such as the rapid (IKr) and slow (IKs) delayed rectifier, the transient outward (Ito) and inward rectifier (IK1) potassium currents in rabbit and dog cardiac preparations. CBD increased action potential duration (APD) significantly in both rabbit (from 211.7 ± 11.2. to 224.6 ± 11.4 ms, n = 8) and dog (from 215.2 ± 9.0 to 231.7 ± 4.7 ms, n = 6) ventricular papillary muscle at 5 µM concentration. CBD decreased IKr, IKs and Ito (only in dog) significantly with corresponding estimated EC50 values of 4.9, 3.1 and 5 µM, respectively, without changing IK1. Although the EC50 value of CBD was found to be higher than literary Cmax values after CBD smoking and oral intake, our results raise the possibility that potassium channel inhibition by lengthening cardiac repolarization might have a role in the possible proarrhythmic side effects of cannabinoids in situations where CBD metabolism and/or the repolarization reserve is impaired.

The electrophysiological effect of cannabidiol on hERG current and in guinea-pig and rabbit cardiac preparations.

Cannabis use is associated with cardiovascular adverse effects ranging from arrhythmias to sudden cardiac death. The exact mechanism of action behind these activities is unknown. The aim of our work was to study the effect of cannabidiol (CBD), tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol on cellular cardiac electrophysiological properties including ECG parameters, action potentials, hERG and IKr ion channels in HEK cell line and in rabbit and guinea pig cardiac preparations. CBD increased action potential duration in rabbit and guinea pig right ventricular papillary muscle at lower concentrations (1 µM, 2.5 µM and 5 µM) but did not significantly change it at 10 µM. CBD at high concentration (10 µM) decreased inward late sodium and L-type calcium currents as well. CBD inhibited hERG potassium channels with an IC50 value of 2.07 µM at room temperature and delayed rectifier potassium current with 6.5 µM at 37 °C, respectively. The frequency corrected QT interval (QTc) was significantly lengthened in anaesthetized guinea pig without significantly changing other ECG parameters. Although the IC50 value of CBD was higher than literary Cmax values after CBD smoking and oral intake, our results raise the possibility that hERG and potassium channel inhibition might have a role in the possible proarrhythmic adverse effects of cannabinoids in situations where metabolism of CBD impaired and/or the repolarization reserve is weakened.

Advanced Methodology and Preliminary Measurements of Molecular and Mechanical Properties of Heart Valves under Dynamic Strain.

Mammalian heart valves are soft tissue assemblies with multi-scale material properties. This is because they are constructs comprising both muscle and non-contractile extracellular matrix proteins (such as collagens and proteoglycans) and transition regions where one form of tissue structure becomes another, significantly different form. The leaflets of the mitral and tricuspid valves are connected to chordae tendinae which, in turn, bind through papillary muscles to the cardiac wall of the ventricle. The transition regions between these tissue subsets are complex and diffuse. Their material composition and mechanical properties have not been previously described with both micro and nanoscopic data recorded simultaneously, as reported here. Annotating the mechanical characteristics of these tissue transitions will be of great value in developing novel implants, improving the state of the surgical simulators and advancing robot-assisted surgery. We present here developments in multi-scale methodology that produce data that can relate mechanical properties to molecular structure using scanning X-ray diffraction. We correlate these data to corresponding tissue level (macro and microscopic) stress and strain, with particular emphasis on the transition regions and present analyses to indicate points of possible failure in these tissues.

Estrogen but not testosterone preserves myofilament function from doxorubicin-induced cardiotoxicity by reducing oxidative modifications.

Here, we aimed to explore sex differences and the impact of sex hormones on cardiac contractile properties in doxorubicin (DOX)-induced cardiotoxicity. Male and female Sprague-Dawley rats were subjected to sham surgery or gonadectomy and then treated or untreated with DOX (2 mg/kg) every other week for 10 wk. Estrogen preserved maximum active tension (Tmax) with DOX exposure, whereas progesterone and testosterone did not. The effects of sex hormones and DOX correlated with both altered myosin heavy chain isoform expression and myofilament protein oxidation, suggesting both as possible mechanisms. However, acute treatment with oxidative stress (H2O2) or a reducing agent (DTT) indicated that the effects on Tmax were mediated by reversible myofilament oxidative modifications and not only changes in myosin heavy chain isoforms. There were also sex differences in the DOX impact on myofilament Ca2+ sensitivity. DOX increased Ca2+ sensitivity in male rats only in the absence of testosterone and in female rats only in the presence of estrogen. Conversely, DOX decreased Ca2+ sensitivity in female rats in the absence of estrogen. In most instances, this mechanism was through altered phosphorylation of troponin I at Ser23/Ser24. However, there was an additional DOX-induced, estrogen-dependent, irreversible (by DTT) mechanism that altered Ca2+ sensitivity. Our data demonstrate sex differences in cardiac contractile responses to chronic DOX treatment. We conclude that estrogen protects against chronic DOX treatment in the heart, preserving myofilament function. NEW & NOTEWORTHY We identified sex differences in cardiotoxic effects of chronic doxorubicin (DOX) exposure on myofilament function. Estrogen, but not testosterone, decreases DOX-induced oxidative modifications on myofilaments to preserve maximum active tension. In rats, DOX exposure increased Ca2+ sensitivity in the presence of estrogen but decreased Ca2+ sensitivity in the absence of estrogen. In male rats, the DOX-induced shift in Ca2+ sensitivity involved troponin I phosphorylation; in female rats, this was through an estrogen-dependent mechanism.

Fasting/Refeeding Cycles Prevent Myocardial Dysfunction and Morphology Damage in the Spontaneously Hypertensive Rats / Ciclos de Jejum/Realimentação Previnem Disfunção Miocárdica e Danos Morfológicos em Ratos Espontaneamente Hipertensos

Abstract Background: Caloric restriction is known to impair the cardiac function and morphology in hypertrophied hearts of spontaneously hypertensive rats (SHR); however, the influence of fasting/refeeding (RF) is unknown. Objective: To investigate the fasting/refeeding approach on myocardial remodeling and function. In addition, the current study was designed to bring information regarding the mechanisms underlying the participation of Ca2+ handling and b-adrenergic system. Methods: Sixty-day-old male SHR rats were submitted to food ad libitum (C), 50% food restriction (R50) or RF cycles for 90 days. Cardiac remodeling was assessed by ultrastructure analysis and isolated papillary muscle function. The level of significance considered was 5% (a = 0.05). Results: The RF rats presented lower cardiac atrophy than R50 in relation to C rats. The C rats increased weight gain, R50 maintained their initial body weight and RF rats increased and decreased weight during RF. The RF did not cause functional impairment because the isotonic and isometric parameters showed similar behavior to those of C. The isotonic and isometric cardiac parameters were significantly elevated in RF rats compared to R50 rats. In addition, the R50 rats had cardiac damage in relation to C for isotonic and isometric variables. While the R50 rats showed focal changes in many muscle fibers, the RF rats displayed mild alterations, such as loss or disorganization of myofibrils. Conclusion: Fasting/refeeding promotes cardiac beneficial effects and attenuates myocardial injury caused by caloric restriction in SHR rats, contributing to reduce the cardiovascular risk profile and morphological injuries. Furthermore, RF promotes mild improvement in Ca2+ handling and b-adrenergic system.

Resumo Fundamento: A restrição calórica compromete a função e a morfologia cardíacas em corações hipertrofiados de ratos espontaneamente hipertensos (SHR). No entanto, a influência de ciclo de jejum/Realimentação é desconhecida. Objetivo: Investigar o efeito de ciclos de jejum/realimentação sobre a remodelação e função miocárdica. Além disso, o presente estudo foi desenhado para avaliar os mecanismos subjacentes à participação do trânsito de cálcio (Ca+2) e sistema beta-adrenérgico. Métodos: Neste estudo, SHR machos de 60 dias de idade foram submetidos a alimento ad libitum (grupo C), 50% de restrição alimentar (grupo R50) ou ciclos de RF (grupo RF) por 90 dias. A remodelação cardíaca foi avaliada por meio da análise ultraestrutural e função do músculo papilar isolado. Adotou-se o nível de significância de 5% (a = 0,05). Resultados: Os ratos do grupo RF apresentaram menor atrofia cardíaca do que os do grupo R50 em relação aos do grupo C. Os ratos do grupo C aumentaram peso corporal, os ratos do grupo R50 mantiveram seu peso corporal inicial e os ratos do grupo RF aumentaram e reduziram seu peso durante o ciclo RF. O ciclo RF não causou comprometimento funcional, pois os parâmetros isotônicos e isométricos apresentaram comportamento similar aos dos ratos do grupo C. Os parâmetros cardíacos isotônicos e isométricos mostraram-se significativamente elevados nos ratos do grupo RF em comparação aos dos ratos do grupo R50. Além disso, os ratos do grupo R50 apresentaram dano cardíaco em comparação aos ratos do grupo C quanto às variáveis isotônicas e isométricas. Os ratos do grupo R50 apresentaram alterações focais em muitas fibras musculares, enquanto os ratos do grupo RF apresentaram leves alterações, como perda ou desorganização de miofibrilas. Conclusão: Ciclos de Jejum/Realimentação promovem efeitos benéficos cardíacos e atenuam o dano miocárdico causado por restrição calórica em SHR, contribuindo para reduzir o risco cardiovascular e os danos morfológicos. Além disso, o ciclo de jejum/realimentação promove leve melhora do trânsito do Ca2+ e do sistema beta-adrenérgico.

nNOS splice variants differentially regulate myofilament function but are dispensable for intracellular calcium and force transients in cardiac papillary muscles.

Cardiac muscle expresses three neuronal nitric oxide synthase (nNOS) splice variants: nNOSα, nNOSµ and nNOSß. The functions of these nNOS splice variants in cardiac muscle, particularly myofilament-associated nNOSß are unclear. To decipher cardiac nNOS splice variant function we investigated myofilament function and intracellular calcium and force transients in demembranated and intact papillary muscles from two lines of nNOS knockout mice. The first line (KN1) lacks nNOSα and nNOSµ. The second line (KN2) lacks active nNOSα, nNOSµ and nNOSß. Demembranated KN1 papillary muscles exhibited reduced myofilament ATPase activity (-35%) and specific force (-10%) relative to controls. Demembranated KN2 muscles exhibited a smaller decrease in myofilament ATPase activity (-21%), but a greater reduction in specific force (-26%) relative to controls. Myofilament calcium sensitivity in demembranated KN1 and KN2 papillary muscles was similar to controls. Thus, papillary muscle-expressed nNOS splice variants are necessary for control levels of myofilament ATPase activity and force generation, but dispensable for myofilament calcium sensitivity. The greater reduction in myofilament ATPase relative to specific force in KN1, but not KN2 muscle, reduced the energy cost of muscle contraction, suggesting that nNOSß increased the energetic efficiency of contraction in the absence of nNOSµ and nNOSα. Analyses of intact KN1 and KN2 papillary muscles showed that both intracellular calcium transients and their evoked force transients were similar to controls at stimulation frequencies between 1 and 3 Hz. Therefore, nNOS was dispensable for baseline excitation-contraction coupling. In summary, these data suggest that nNOS splice variants differentially regulate myofilament function, but not baseline calcium handling in papillary muscles. More importantly, they suggest that nNOSß is a novel modulator of myofilament function, and ultimately the energetic efficiency of cardiac papillary muscle contraction.

Androgens induce growth of the limb skeletal muscles in a rapamycin-insensitive manner.

Signaling through the mechanistic target of rapamycin complex 1 (mTORC1) has been well defined as an androgen-sensitive transducer mediating skeletal muscle growth in vitro; however, this has yet to be tested in vivo. As such, male mice were subjected to either sham or castration surgery and allowed to recover for 7 wk to induce atrophy of skeletal muscle. Then, castrated mice were implanted with either a control pellet or a pellet that administered rapamycin (~2.5 mg·kg-1·day-1). Seven days postimplant, a subset of castrated mice with control pellets and all castrated mice with rapamycin pellets were given once weekly injections of nandrolone decanoate (ND) to induce muscle growth over a six-week period. Effective blockade of mTORC1 by rapamycin was noted in the skeletal muscle by the inability of insulin to induce phosphorylation of ribosomal S6 kinase 1 70 kDa (Thr389) and uncoordinated-like kinase 1 (Ser757). While castration reduced tibialis anterior (TA) mass, muscle fiber cross-sectional area, and total protein content, ND administration restored these measures to sham levels in a rapamycin-insensitive manner. Similar findings were also observed in the plantaris and soleus, suggesting this rapamycin-insensitive effect was not specific to the TA or fiber type. Androgen-mediated growth was not due to changes in translational capacity. Despite these findings in the limb skeletal muscle, rapamycin completely prevented the ND-mediated growth of the heart. In all, these data indicate that mTORC1 has a limited role in the androgen-mediated growth of the limb skeletal muscle; however, mTORC1 was necessary for androgen-mediated growth of heart muscle.

Reactive oxygen species impair the excitation-contraction coupling of papillary muscles after acute exposure to a high copper concentration.

Copper is an essential metal for homeostasis and the functioning of living organisms. We investigated the effects of a high copper concentration on the myocardial mechanics, investigating the reactive oxygen species (ROS) mediated effects. The developed force of papillary muscles was reduced after acute exposure to a high copper concentration and was prevented by co-incubation with tempol, DMSO and catalase. The reuptake of calcium by the sarcoplasmic reticulum was reduced by copper and restored by tempol. The contractile response to Ca2+ was reduced and reversed by antioxidants. The response to the ß-adrenergic agonist decreased after exposure to copper and was restored by tempol and catalase. In addition, the in situ detection showed increased O2·- and OH·. Contractions dependent on the sarcolemmal Ca2+ influx were impaired by copper and restored by antioxidants. Myosin-ATPase activity decreased significantly after copper exposure. In conclusion, a high copper concentration can acutely impair myocardial excitation-contraction coupling, reduce the capacity to generate force, reduce the Ca2+ inflow and its reuptake, and reduce myosin-ATPase activity, and these effects are mediated by the local production of O2·-, OH· and H2O2. These toxicity effects of copper overload suggest that copper is a risk factor for cardiovascular disease.

Stretch-induced compliance: a novel adaptive biological mechanism following acute cardiac load.

Aims: The heart is constantly challenged with acute bouts of stretching or overload. Systolic adaptations to these challenges are known but adaptations in diastolic stiffness remain unknown. We evaluated adaptations in myocardial stiffness due to acute stretching and characterized the underlying mechanisms. Methods and results: Left ventricles (LVs) of intact rat hearts, rabbit papillary muscles and myocardial strips from cardiac surgery patients were stretched. After stretching, there was a sustained >40% decrease in end-diastolic pressure (EDP) or passive tension (PT) for 15 min in all species and experimental preparations. Stretching by volume loading in volunteers and cardiac surgery patients resulted in E/E' and EDP decreases, respectively, after sustained stretching. Stretched samples had increased myocardial cGMP levels, increased phosphorylated vasodilator-stimulated phosphoprotein phosphorylation, as well as, increased titin phosphorylation, which was reduced by prior protein kinase G (PKG) inhibition (PKGi). Skinned cardiomyocytes from stretched and non-stretched myocardia were studied. Skinned cardiomyocytes from stretched hearts showed decreased PT, which was abrogated by protein phosphatase incubation; whereas those from non-stretched hearts decreased PT after PKG incubation. Pharmacological studies assessed the role of nitric oxide (NO) and natriuretic peptides (NPs). PT decay after stretching was significantly reduced by combined NP antagonism, NO synthase inhibition and NO scavenging, or by PKGi. Response to stretching was remarkably reduced in a rat model of LV hypertrophy, which also failed to increase titin phosphorylation. Conclusions: We describe and translate to human physiology a novel adaptive mechanism, partly mediated by titin phosphorylation through cGMP-PKG signalling, whereby myocardial compliance increases in response to acute stretching. This mechanism may not function in the hypertrophic heart.

Cardiac, Metabolic and Molecular Profiles of Sedentary Rats in the Initial Moment of Obesity / Perfis Cardíaco, Metabólico e Molecular de Ratos Sedentários no Momento Inicial da Obesidade

Abstract Background: Different types of high-fat and/or high-energy diets have been used to induce obesity in rodents. However, few studies have reported on the effects observed at the initial stage of obesity induced by high-fat feeding on cardiac functional and structural remodelling. Objective: To characterize the initial moment of obesity and investigate both metabolic and cardiac parameters. In addition, the role of Ca2+ handling in short-term exposure to obesity was verified. Methods: Thirty-day-old male Wistar rats were randomized into two groups (n = 19 each): control (C; standard diet) and high-fat diet (HF, unsaturated high-fat diet). The initial moment of obesity was defined by weekly measurement of body weight (BW) complemented by adiposity index (AI). Cardiac remodelling was assessed by morphological, histological, echocardiographic and papillary muscle analysis. Ca2+ handling proteins were determined by Western Blot. Results: The initial moment of obesity occurred at the 3rd week. Compared with C rats, the HF rats had higher final BW (4%), body fat (20%), AI (14.5%), insulin levels (39.7%), leptin (62.4%) and low-density lipoprotein cholesterol (15.5%) but did not exhibit alterations in systolic blood pressure. Echocardiographic evaluation did not show alterations in cardiac parameters. In the HF group, muscles were observed to increase their +dT/dt (C: 52.6 ± 9.0 g/mm2/s and HF: 68.0 ± 17.0 g/mm2/s; p < 0.05). In addition, there was no changes in the cardiac expression of Ca2+ handling proteins. Conclusion: The initial moment of obesity promotes alterations to hormonal and lipid profiles without cardiac damage or changes in Ca2+ handling.

Resumo Fundamentos: Diferentes tipos de dietas hiperlipídicas e/ou hipercalóricas têm sido usados para induzir obesidade em roedores. No entanto, poucos estudos relataram os efeitos da obesidade induzida por dieta hiperlipídica em sua fase inicial sobre a remodelação cardíaca funcional e estrutural. Objetivo: Caracterizar o momento inicial da obesidade e investigar parâmetros metabólicos e cardíacos. Além disso, analisar o papel do trânsito de Ca+2 em curtos períodos de exposição à obesidade. Métodos: Ratos Wistar com idade de 30 dias foram distribuídos aleatoriamente em dois grupos (n = 19 em cada grupo): controle (C, dieta padrão) e dieta hiperlipídica (HL, dieta rica em gordura insaturada). O momento inicial da obesidade foi definido por medidas semanais do peso corporal, complementadas pelo índice de adiposidade (IA). A remodelação cardíaca foi avaliada por análise morfológica, histológica, ecocardiográfica e funcional dos músculos papilares. Proteínas envolvidas no trânsito de Ca2+ foram determinadas por Western Blot. Resultados: O momento inicial da obesidade ocorreu na terceira semana. Em comparação aos ratos C, os animais HL apresentaram maior peso corporal final (4%), gordura corporal (20%), IA (14,5%), níveis de insulina (39,7%), leptina (62,4%) e lipoproteína de baixa densidade (15,5%), mas não apresentaram alterações na pressão sistólica. A avaliação ecocardiográfica não mostrou alterações nos parâmetros cardíacos. No grupo HL, observou-se um aumento no +dT/dt (C: 52,6 ± 9,0 g/mm2/s e HL: 68,0 ± 17,0 g/mm2/s; p < 0,05) muscular. Além disso, não houve alterações na expressão cardíaca de proteínas envolvidas no trânsito de Ca2+. Conclusão: O momento inicial da obesidade promove alterações nos perfis hormonais e lipídicos sem causar danos cardíacos ou mudanças no trânsito de Ca2+.

In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice.

RATIONALE: Duchenne muscular dystrophy is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy patients, and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on Duchenne muscular dystrophy cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in mdx/Utr+/- mice after a single systemic delivery of recombinant adeno-associated virus. OBJECTIVE: To examine the efficiency and physiological impact of CRISPR-mediated genome editing on cardiac dystrophin expression and function in dystrophic mice. METHODS AND RESULTS: Here, we packaged SaCas9 (clustered regularly interspaced short palindromic repeat-associated 9 from Staphylococcus aureus) and guide RNA constructs into an adeno-associated virus vector and systemically delivered them to mdx/Utr+/- neonates. We showed that CRIPSR-mediated genome editing efficiently excised the mutant exon 23 in dystrophic mice, and immunofluorescence data supported the restoration of dystrophin protein expression in dystrophic cardiac muscles to a level approaching 40%. Moreover, there was a noted restoration in the architecture of cardiac muscle fibers and a reduction in the extent of fibrosis in dystrophin-deficient hearts. The contractility of cardiac papillary muscles was also restored in CRISPR-edited cardiac muscles compared with untreated controls. Furthermore, our targeted deep sequencing results confirmed that our adeno-associated virus-CRISPR/Cas9 strategy was very efficient in deleting the ≈23 kb of intervening genomic sequences. CONCLUSIONS: This study provides evidence for using CRISPR-based genome editing as a potential therapeutic approach for restoring dystrophic cardiomyopathy structurally and functionally.

Obestatin regulates cardiovascular function and promotes cardioprotection through the nitric oxide pathway.

Patients with ischaemic heart disease or chronic heart failure show altered levels of obestatin, suggesting a role for this peptide in human heart function. We have previously demonstrated that GH secretagogues and the ghrelin gene-derived peptides, including obestatin, exert cardiovascular effects by modulating cardiac inotropism and vascular tone, and reducing cell death and contractile dysfunction in hearts subjected to ischaemia/reperfusion (I/R), through the Akt/nitric oxide (NO) pathway. However, the mechanisms underlying the cardiac actions of obestatin remain largely unknown. Thus, we suggested that obestatin-induced activation of PI3K/Akt/NO and PKG signalling is implicated in protection of the myocardium when challenged by adrenergic, endothelinergic or I/R stress. We show that obestatin exerts an inhibitory tone on the performance of rat papillary muscle in both basal conditions and under ß-adrenergic overstimulation, through endothelial-dependent NO/cGMP/PKG signalling. This pathway was also involved in the vasodilator effect of the peptide, used both alone and under stress induced by endothelin-1. Moreover, when infused during early reperfusion, obestatin reduced infarct size in isolated I/R rat hearts, through an NO/PKG pathway, comprising ROS/PKC signalling, and converging on mitochondrial ATP-sensitive potassium [mitoK(ATP)] channels. Overall, our results suggest that obestatin regulates cardiovascular function in stress conditions and induces cardioprotection by mechanisms dependent on activation of an NO/soluble guanylate cyclase (sGC)/PKG pathway. In fact, obestatin counteracts exaggerated ß-adrenergic and endothelin-1 activity, relevant factors in heart failure, suggesting multiple positive effects of the peptide, including the lowering of cardiac afterload, thus representing a potential candidate in pharmacological post-conditioning.

Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model.

The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca2+-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca2+-sensitivity and a decrease (0.8×) in cooperativity (nH) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5×), reversal step (k-4) increased (2×), and the phosphate-release step (1/K5) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics-possibly caused by changes in the α-myosin heavy chain phosphorylation profile-as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.

Comparison of elementary steps of the cross-bridge cycle in rat papillary muscle fibers expressing α- and ß-myosin heavy chain with sinusoidal analysis.

In mammalian ventricles, two myosin heavy chain (MHC) isoforms have been identified. Small animals express α-MHC, whereas large animals express ß-MHC, which contribute to a large difference in the heart rate. Sprague-Dawley rats possessing ~99% α-MHC were treated with propylthiouracil to result in 100% ß-MHC. Papillary muscles were skinned, dissected into small fibers, and used for experiments. To understand the functional difference between α-MHC and ß-MHC, skinned-fibers were activated under the intracellular ionic conditions: 5 mM MgATP, 1 mM Mg2+, 8 mM Pi, 200 mM ionic strength, pH 7.00 at 25 °C. Small amplitude sinusoidal length oscillations were applied in the frequency range 0.13-100 Hz (corresponding time domain: 1.6-1200 ms), and effects of Ca2+, Pi, and ATP were studied. The results show that Ca2+ sensitivity was slightly less (10-15%) in ß-MHC than α-MHC containing fibers. Sinusoidal analysis at pCa 4.66 (full Ca2+ activation) demonstrated that, the apparent rate constants were 2-4× faster in α-MHC containing fibers. The ATP study demonstrated that, in ß-MHC containing fibers, K 1 (ATP association constant) was greater (1.7×), k 2 and k -2 (cross-bridge detachment and its reversal rate constants) were smaller (×0.6). The Pi study demonstrated that, in ß-MHC containing fibers, k 4 (rate constant of the force-generation step) and k -4 were smaller (0.75× and 0.25×, respectively), resulting in greater K 4 (3×). There were no differences in active tension, rigor stiffness, or K 2 (equilibrium constant of the cross-bridge detachment step). Our study further demonstrated that there were no differences in parameters between fibers obtained from left and right ventricles, but with an exception in K 5 (Pi association constant).

Saturated high-fat diet-induced obesity increases adenylate cyclase of myocardial ß-adrenergic system and does not compromise cardiac function.

Obesity is a worldwide pandemic associated with high incidence of cardiovascular disease. The mechanisms by which the obesity leads cardiac dysfunction are not fully elucidated and few studies have evaluated the relationship between obesity and proteins involved in myocardial ß-adrenergic (ßA) system. The purpose of this study was to evaluate the cardiac function and ßA pathway components in myocardium of obese rats. Male Wistar rats were distributed into two groups: control (n = 17; standard diet) and obese (n = 17; saturated high-fat diet) fed for 33 weeks. Nutritional profile and comorbidities were assessed. Cardiac structure and function was evaluated by macroscopic postmortem, echocardiographic and isolated papillary muscle analyzes. Myocardial protein expression of ß1- and ß2-adrenergic receptors, Gαs protein, adenylate cyclase (AC) and protein kinase A (PKA) was performed by Western blot. Cardiac cyclic adenosine monophosphate (cAMP) levels and PKA activity were assessed by ELISA Obese rats showed increased adiposity index (P < 0.001) and several comorbidities as hypertension, glucose intolerance, insulin resistance, and dyslipidemia compared with control rats. Echocardiographic assessment revealed increased left atrium diameter (C: 4.98 ± 0.38 vs. Ob: 5.47 ± 0.53, P = 0.024) and posterior wall shortening velocity (C: 37.1 ± 3.6 vs. Ob: 41.8 ± 3.8, P = 0.007) in obese group. Papillary muscle evaluation indicated that baseline data and myocardial responsiveness to isoproterenol stimulation were similar between the groups. Protein expression of myocardial AC was higher in obese group than in the control (C: 1.00 ± 0.21 vs. Ob: 1.25 ± 0.10, P = 0.025), whereas the other components were unchanged. These results suggest that saturated high-fat diet-induced obesity was not effective in triggering cardiac dysfunction and impair the beta-adrenergic signaling.

Mechanisms involved in the in vitro contractile dysfunction induced by different concentrations of ferrous iron in the rat myocardium.

Iron intoxication is related to reactive oxygen species (ROS) production and organic damage including the cardiovascular system, and is a leading cause of poisoning deaths in children. In this study we examined whether a range of ferrous iron (Fe(2+)) concentrations can interfere differently on the myocardial mechanics, investigating the ROS-mediated effects. Developed force of isolated rat papillary muscles was depressed with a concentration- and time-dependency by Fe(2+) 100-1000µM. The contractile response to Ca(2+) was reduced, but it was partially reversed by co-incubation with catalase and DMSO, but not TEMPOL. In agreement, in situ detection of OH was increased by Fe(2+) whereas O2(-) was unchanged. The myosin-ATPase activity was significantly decreased. Contractions dependent on the sarcolemal Ca(2+) influx were impaired only by Fe(2+) 1000µM, and antioxidants had no effect. In skinned fibers, Fe(2+) reduced the pCa-force relationship, and pCa50 was right-shifted by 0.55. In conclusion, iron overload can acutely impair myocardial contractility by reducing myosin-ATPase activity and myofibrillar Ca(2+) sensitivity. These effects are mediated by local production of OH and H2O2. Nevertheless, in a such high concentration as 1000µM, Fe(2+) appears to depress force also by reducing Ca(2+) influx, probably due to a competition at Ca(2+) channels.

Myosin MgADP release rate decreases at longer sarcomere length to prolong myosin attachment time in skinned rat myocardium.

Cardiac contractility increases as sarcomere length increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. The capacity of myosin to bind with actin and generate force in a muscle cell is Ca(2+) regulated by thin-filament proteins and spatially regulated by sarcomere length as thick-to-thin filament overlap varies. One mechanism underlying greater cardiac contractility as sarcomere length increases could involve longer myosin attachment time (ton) due to slowed myosin kinetics at longer sarcomere length. To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 µm sarcomere lengths. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate and MgATP binding rates. As MgATP increased, ton decreased for both sarcomere lengths, but ton was roughly 70% longer for 2.2 vs. 1.9 µm sarcomere length at maximally activated conditions. These ton differences were driven by a slower MgADP release rate at 2.2 µm sarcomere length (41 ± 3 vs. 74 ± 7 s(-1)), since MgATP binding rate was not different between the two sarcomere lengths. At submaximal activation levels near the pCa50 value of the tension-pCa relationship for each sarcomere length, length-dependent increases in ton were roughly 15% longer for 2.2 vs. 1.9 µm sarcomere length. These changes in cross-bridge kinetics could amplify cooperative cross-bridge contributions to force production and thin-filament activation at longer sarcomere length and suggest that length-dependent changes in myosin MgADP release rate may contribute to the Frank-Starling relationship in the heart.

Human Engineered Heart Muscles Engraft and Survive Long Term in a Rodent Myocardial Infarction Model.

RATIONALE: Tissue engineering approaches may improve survival and functional benefits from human embryonic stem cell-derived cardiomyocyte transplantation, thereby potentially preventing dilative remodeling and progression to heart failure. OBJECTIVE: Assessment of transport stability, long-term survival, structural organization, functional benefits, and teratoma risk of engineered heart muscle (EHM) in a chronic myocardial infarction model. METHODS AND RESULTS: We constructed EHMs from human embryonic stem cell-derived cardiomyocytes and released them for transatlantic shipping following predefined quality control criteria. Two days of shipment did not lead to adverse effects on cell viability or contractile performance of EHMs (n=3, P=0.83, P=0.87). One month after ischemia/reperfusion injury, EHMs were implanted onto immunocompromised rat hearts to simulate chronic ischemia. Bioluminescence imaging showed stable engraftment with no significant cell loss between week 2 and 12 (n=6, P=0.67), preserving ≤25% of the transplanted cells. Despite high engraftment rates and attenuated disease progression (change in ejection fraction for EHMs, -6.7±1.4% versus control, -10.9±1.5%; n>12; P=0.05), we observed no difference between EHMs containing viable and nonviable human cardiomyocytes in this chronic xenotransplantation model (n>12; P=0.41). Grafted cardiomyocytes showed enhanced sarcomere alignment and increased connexin 43 expression at 220 days after transplantation. No teratomas or tumors were found in any of the animals (n=14) used for long-term monitoring. CONCLUSIONS: EHM transplantation led to high engraftment rates, long-term survival, and progressive maturation of human cardiomyocytes. However, cell engraftment was not correlated with functional improvements in this chronic myocardial infarction model. Most importantly, the safety of this approach was demonstrated by the lack of tumor or teratoma formation.

The R21C Mutation in Cardiac Troponin I Imposes Differences in Contractile Force Generation between the Left and Right Ventricles of Knock-In Mice.

We investigated the effect of the hypertrophic cardiomyopathy-linked R21C (arginine to cysteine) mutation in human cardiac troponin I (cTnI) on the contractile properties and myofilament protein phosphorylation in papillary muscle preparations from left (LV) and right (RV) ventricles of homozygous R21C(+/+) knock-in mice. The maximal steady-state force was significantly reduced in skinned papillary muscle strips from the LV compared to RV, with the latter displaying the level of force observed in LV or RV from wild-type (WT) mice. There were no differences in the Ca(2+) sensitivity between the RV and LV of R21C(+/+) mice; however, the Ca(2+) sensitivity of force was higher in RV-R21C(+/+) compared with RV-WT and lower in LV- R21C(+/+) compared with LV-WT. We also observed partial loss of Ca(2+) regulation at low [Ca(2+)]. In addition, R21C(+/+)-KI hearts showed no Ser23/24-cTnI phosphorylation compared to LV or RV of WT mice. However, phosphorylation of the myosin regulatory light chain (RLC) was significantly higher in the RV versus LV of R21C(+/+) mice and versus LV and RV of WT mice. The difference in RLC phosphorylation between the ventricles of R21C(+/+) mice likely contributes to observed differences in contractile force and the lower tension monitored in the LV of HCM mice.

Phosphoregulation of Cardiac Inotropy via Myosin Binding Protein-C During Increased Pacing Frequency or ß1-Adrenergic Stimulation.

BACKGROUND: Mammalian hearts exhibit positive inotropic responses to ß-adrenergic stimulation as a consequence of protein kinase A-mediated phosphorylation or as a result of increased beat frequency (the Bowditch effect). Several membrane and myofibrillar proteins are phosphorylated under these conditions, but the relative contributions of these to increased contractility are not known. Phosphorylation of cardiac myosin-binding protein-C (cMyBP-C) by protein kinase A accelerates the kinetics of force development in permeabilized heart muscle, but its role in vivo is unknown. Such understanding is important because adrenergic responsiveness of the heart and the Bowditch effect are both depressed in heart failure. METHODS AND RESULTS: The roles of cMyBP-C phosphorylation were studied using mice in which either WT or nonphosphorylatable forms of cMyBP-C [ser273ala, ser282ala, ser302ala: cMyBP-C(t3SA)] were expressed at similar levels on a cMyBP-C null background. Force and [Ca(2+)]in measurements in isolated papillary muscles showed that the increased force and twitch kinetics because increased pacing or ß1-adrenergic stimulation were nearly absent in cMyBP-C(t3SA) myocardium, even though [Ca(2+)]in transients under each condition were similar to WT. Biochemical measurements confirmed that protein kinase A phosphorylated ser273, ser282, and ser302 in WT cMyBP-C. In contrast, CaMKIIδ, which is activated by increased pacing, phosphorylated ser302 principally, ser282 to a lesser degree, and ser273 not at all. CONCLUSIONS: Phosphorylation of cMyBP-C increases the force and kinetics of twitches in living cardiac muscle. Further, cMyBP-C is a principal mediator of increased contractility observed with ß-adrenergic stimulation or increased pacing because of protein kinase A and CaMKIIδ phosphorylations of cMyB-C.