RESUMEN

High-intensity interval training (HIIT) has shown significant results in addressing adiposity and risk factors associated with obesity. However, there are no studies that investigate the effects of HIIT on contractility and intracellular Ca2+ handling. The purpose of this study was to explore the impact of HIIT on cardiomyocyte contractile function and intracellular Ca2+ handling in rats in which obesity was induced by a saturated high-fat diet (HFD). Male Wistar rats were initially randomized into a standard diet and a HFD group. The experimental protocol spanned 23 weeks, comprising the induction and maintenance of obesity (15 weeks) followed by HIIT treatment (8 weeks). Performance was assessed using the maximum oxygen consumption test ( V ̇ O 2 max ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{max}}}}$ ). Evaluation encompassed cardiac, adipose and skeletal muscle histology, as well as contractility and intracellular Ca2+ handling. HIIT resulted in a reduction in visceral area, an increase in V ̇ O 2 max ${{\dot{V}}_{{{{\mathrm{O}}}_{\mathrm{2}}}{\mathrm{max}}}}$ , and an augmentation of gastrocnemius fibre diameter in obese subjects. Additionally, HIIT led to a decrease in collagen fraction, an increase in percentage shortening, and a reduction in systolic Ca2+/percentage shortening and systolic Ca2+/maximum shortening rates. HIIT induces physiological cardiac remodelling, enhancing the contractile function of cardiomyocytes and improving myofilament sensitivity to Ca2+ in the context of obesity. This approach not only enhances cardiorespiratory and physical performance but also reduces visceral area and prevents interstitial fibrosis.

RESUMEN

Heart Failure with preserved ejection fraction (HFpEF) has a high rate of sudden cardiac death (SCD) and empirical treatment is ineffective. We developed a novel preclinical model of metabolic HFpEF that presents with stress-induced ventricular tachycardia (VT). Mechanistically, we discovered arrhythmogenic changes in intracellular Ca2+ handling distinct from the changes pathognomonic for heart failure with reduced ejection fraction. We further show that dantrolene, a stabilizer of the ryanodine receptor Ca2+ channel, attenuates HFpEF-associated arrhythmogenic Ca2+ handling in vitro and suppresses stress-induced VT in vivo. We propose ryanodine receptor stabilization as a mechanistic approach to mitigation of malignant VT in metabolic HFpEF.

Asunto(s)

Arritmias Cardíacas , Calcio , Dantroleno , Modelos Animales de Enfermedad , Insuficiencia Cardíaca , Canal Liberador de Calcio Receptor de Rianodina , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Animales , Calcio/metabolismo , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/fisiopatología , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/tratamiento farmacológico , Dantroleno/farmacología , Volumen Sistólico/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Humanos , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/tratamiento farmacológico , Ratones , Masculino , Señalización del Calcio/efectos de los fármacos

RESUMEN

Creation of disease models utilizing hiPSCs in combination with CRISPR/Cas9 gene editing enable mechanistic insights into differential pharmacological responses. This allows translation of efficacy and safety findings from a healthy to a diseased state and provides a means to predict clinical outcome sooner during drug discovery. Calcium handling disturbances including reduced expression levels of the type 2 ryanodine receptor (RYR2) are linked to cardiac dysfunction; here we have created a RYR2 deficient human cardiomyocyte model that mimics some aspects of heart failure. RYR2 deficient cardiomyocytes show differential pharmacological responses to L-type channel calcium inhibitors. Phenotypic and proteomic characterization reveal novel molecular insights with altered expression of structural proteins including CSRP3, SLMAP, and metabolic changes including upregulation of the pentose phosphate pathway and increased sensitivity to redox alterations. This genetically engineered in vitro cardiovascular model of RYR2 deficiency supports the study of pharmacological responses in the context of calcium handling and metabolic dysfunction enabling translation of drug responses from healthy to perturbed cellular states.

RESUMEN

Resumo Fundamento A remodelação adversa dos vasos pulmonares eleva a pressão pulmonar e provoca hipertensão arterial pulmonar (HAP). A HAP resulta em aumento da pós-carga do ventrículo direito (VD), causando hipertrofia ventricular e consequente insuficiência cardíaca. Não existe um tratamento específico para o remodelamento desadaptativo do VD secundário à HAP. Objetivos Este estudo tem como objetivo explorar duas abordagens terapêuticas, o suco de uva (SU) e os hormônios tireoidianos (HT), no tratamento do estresse oxidativo induzido pela HAP e nas alterações funcionais cardíacas. Métodos Parâmetros ecocardiográficos relacionados à resistência dos vasos pulmonares (relação TA/TE), contratilidade do VD (ESPAT) e função diastólica do VD (relação dos picos E/A) foram avaliados. Além disso, foram medidos ROS totais, peroxidação lipídica, enzimas antioxidantes, proteínas de manipulação de cálcio, expressão de proteínas pró-oxidantes e antioxidantes. Valores de p<0,05 foram considerados estatisticamente significativos. Resultados Ambos os tratamentos, com SU e HT, demonstraram uma redução na resistência pulmonar (~22%), além de melhorias na ESPAT (inotropismo ~11%) e na relação TA/TE (~26%) (p<0,05). Não houve alterações entre os grupos na relação do pico de E/A. Embora ROS e TBARS não tenham sido estatisticamente significativos, os tratamentos com SU e HT diminuíram os níveis de xantina oxidase (~49%) e normalizaram a expressão de HSP70 e proteínas de manipulação de cálcio (p<0,05). No entanto, apenas o tratamento com HT melhorou a função diastólica (~50%) e aumentou o imunoconteúdo de NRF2 (~48%) (p<0,05). Conclusões Até onde sabemos, este estudo é pioneiro ao mostrar que o HT administrado em conjunto com o SU promoveu melhorias funcionais e bioquímicas em um modelo de HAP. Além disso, nossos dados sugerem que os tratamentos com SU e HT se mostraram cardioprotetores, sejam combinados ou não, e exibiram seus benefícios ao modular o estresse oxidativo e as proteínas de manipulação do cálcio.

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Abstract Background Adverse remodeling of lung vessels elevates pulmonary pressure and provokes pulmonary arterial hypertension (PAH). PAH results in increased right ventricle (RV) afterload, causing ventricular hypertrophy and the onset of heart failure. There is no specific treatment for maladaptive RV remodeling secondary to PAH. Objectives This study aims to explore two therapeutic approaches, grape juice (GJ) and thyroid hormones (TH), on PAH-induced oxidative stress and cardiac functional changes. Methods Parameters of echocardiography related to lung vessel resistance (AT/ET ratio), RV contractility (TAPSE), and RV diastolic function (E/A peaks ratio) were evaluated. Also, total ROS, lipid peroxidation, antioxidant enzymes, calcium handling proteins, pro-oxidant and antioxidant protein expression were measured. Values of p<0.05 were considered statistically significant. Results Both GJ and TH treatments demonstrated reductions in pulmonary resistance (~22%) and improvements in TAPSE (inotropism ~11%) and AT/ET ratio (~26%) (p<0.05). There were no changes amongst groups regarding the E/A peak ratio. Although ROS and TBARS were not statistically significant, GJ and TH treatments decreased xanthine oxidase (~49%) levels and normalized HSP70 and calcium handling protein expression (p<0.05). However, only TH treatment ameliorated diastolic function (~50%) and augmented NRF2 immunocontent (~48%) (p<0.05). Conclusions To the best of our knowledge, this study stands as a pioneer in showing that TH administered together with GJ promoted functional and biochemical improvements in a PAH model. Moreover, our data suggest that GJ and TH treatments were cardioprotective, combined or not, and exhibited their beneficial effects by modulating oxidative stress and calcium-handling proteins.

RESUMEN

Mitochondrial calcium (Ca2+) signals play a central role in cardiac homeostasis and disease. In the healthy heart, mitochondrial Ca2+ levels modulate the rate of oxidative metabolism to match the rate of adenosine triphosphate consumption in the cytosol. During ischemia/reperfusion (I/R) injury, pathologically high levels of Ca2+ in the mitochondrial matrix trigger the opening of the mitochondrial permeability transition pore, which releases solutes and small proteins from the matrix, causing mitochondrial swelling and ultimately leading to cell death. Pharmacological and genetic approaches to tune mitochondrial Ca2+ handling by regulating the activity of the main Ca2+ influx and efflux pathways, i.e., the mitochondrial Ca2+ uniporter and sodium/Ca2+ exchanger, represent promising therapeutic strategies to protect the heart from I/R injury.

Asunto(s)

Calcio , Mitocondrias Cardíacas , Daño por Reperfusión Miocárdica , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/patología , Humanos , Animales , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Calcio/metabolismo , Señalización del Calcio , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Canales de Calcio/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Cardiotónicos/metabolismo

RESUMEN

Older adults are vulnerable to glucocorticoid-induced muscle atrophy and weakness, with sex potentially influencing their susceptibility to those effects. Aerobic exercise can reduce glucocorticoid-induced muscle atrophy in young rodents. However, it is unknown whether aerobic exercise can prevent glucocorticoid myopathy in aged muscle. The objectives of this study were to define the extent to which sex influences the development of glucocorticoid myopathy in aged muscle, and to determine the extent to which aerobic exercise training protects against myopathy development. Twenty-four-month-old female (n = 30) and male (n = 33) mice were randomized to either sedentary or aerobic exercise groups. Within their respective groups, mice were randomized to either daily treatment with dexamethasone (DEX) or saline. Upon completing treatments, the contractile properties of the triceps surae complex were assessed in situ. DEX marginally lowered muscle mass and soluble protein content in both sexes, which was attenuated by aerobic exercise only in females. DEX increased sub-tetanic force and rate of force development only in females, which was not influenced by aerobic exercise. Muscle fatigue was higher in both sexes following DEX, but aerobic exercise prevented fatigue induction only in females. The sex-specific differences to muscle function in response to DEX treatment coincided with sex-specific changes to the content of proteins related to calcium handling, mitochondrial quality control, reactive oxygen species production, and glucocorticoid receptor in muscle. These findings define several important sexually dimorphic changes to aged skeletal muscle physiology in response to glucocorticoid treatment and define the capacity of short-term aerobic exercise to protect against those changes. KEY POINTS: There are sexually dimorphic effects of glucocorticoids on aged skeletal muscle physiology. Glucocorticoid-induced changes to aged muscle contractile properties coincide with sex-specific differences in the content of calcium handling proteins. Aerobic exercise prevents glucocorticoid-induced fatigue only in aged females and coincides with differences in the content of mitochondrial quality control proteins and glucocorticoid receptors.

RESUMEN

BACKGROUND: We recently demonstrated that acute administration of ibrutinib, a Bruton's tyrosine kinase inhibitor used in chemotherapy for blood malignancies, increases ventricular arrhythmia (VA) vulnerability. A pathway of ibrutinib-induced vulnerability to VA that can be modulated for cardioprotection remains unclear. METHODS AND RESULTS: The effects of ibrutinib on cardiac electrical activity and Ca2+ dynamics were investigated in Langendorff-perfused hearts using optical mapping. We also conducted Western blotting analysis to evaluate the impact of ibrutinib on various regulatory and Ca2+-handling proteins in rat cardiac tissues. Treatment with ibrutinib (10 mg/kg per day) for 4 weeks was associated with an increased VA inducibility (72.2%±6.3% versus 38.9±7.0% in controls, P<0.002) and shorter action potential durations during pacing at various frequencies (P<0.05). Ibrutinib also decreased heart rate thresholds for beat-to-beat duration alternans of the cardiac action potential (P<0.05). Significant changes in myocardial Ca2+ transients included lower amplitude alternans ratios (P<0.05), longer times-to-peak (P<0.05), and greater spontaneous intracellular Ca2+ elevations (P<0.01). We also found lower abundance and phosphorylation of myocardial AMPK (5'-adenosine monophosphate-activated protein kinase), indicating reduced AMPK activity in hearts after ibrutinib treatment. An acute treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside ameliorated abnormalities in action potential and Ca2+ dynamics, and significantly reduced VA inducibility (37.1%±13.4% versus 72.2%±6.3% in the absence of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, P<0.05) in hearts from ibrutinib-treated rats. CONCLUSIONS: VA vulnerability inflicted by ibrutinib may be mediated in part by an impairment of myocardial AMPK activity. Pharmacological activation of AMPK may be a protective strategy against ibrutinib-induced cardiotoxicity.

Asunto(s)

Proteínas Quinasas Activadas por AMP , Potenciales de Acción , Adenina , Arritmias Cardíacas , Piperidinas , Pirazoles , Pirimidinas , Animales , Adenina/análogos & derivados , Adenina/farmacología , Piperidinas/farmacología , Potenciales de Acción/efectos de los fármacos , Pirimidinas/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Pirazoles/farmacología , Masculino , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Arritmias Cardíacas/prevención & control , Inhibidores de Proteínas Quinasas/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Preparación de Corazón Aislado , Calcio/metabolismo , Ratas , Modelos Animales de Enfermedad , Ratas Sprague-Dawley , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Señalización del Calcio/efectos de los fármacos , Factores de Tiempo

RESUMEN

Goal: Contractile response and calcium handling are central to understanding cardiac function and physiology, yet existing methods of analysis to quantify these metrics are often time-consuming, prone to mistakes, or require specialized equipment/license. We developed BeatProfiler, a suite of cardiac analysis tools designed to quantify contractile function, calcium handling, and force generation for multiple in vitro cardiac models and apply downstream machine learning methods for deep phenotyping and classification. Methods: We first validate BeatProfiler's accuracy, robustness, and speed by benchmarking against existing tools with a fixed dataset. We further confirm its ability to robustly characterize disease and dose-dependent drug response. We then demonstrate that the data acquired by our automatic acquisition pipeline can be further harnessed for machine learning (ML) analysis to phenotype a disease model of restrictive cardiomyopathy and profile cardioactive drug functional response. To accurately classify between these biological signals, we apply feature-based ML and deep learning models (temporal convolutional-bidirectional long short-term memory model or TCN-BiLSTM). Results: Benchmarking against existing tools revealed that BeatProfiler detected and analyzed contraction and calcium signals better than existing tools through improved sensitivity in low signal data, reduction in false positives, and analysis speed increase by 7 to 50-fold. Of signals accurately detected by published methods (PMs), BeatProfiler's extracted features showed high correlations to PMs, confirming that it is reliable and consistent with PMs. The features extracted by BeatProfiler classified restrictive cardiomyopathy cardiomyocytes from isogenic healthy controls with 98% accuracy and identified relax90 as a top distinguishing feature in congruence with previous findings. We also show that our TCN-BiLSTM model was able to classify drug-free control and 4 cardiac drugs with different mechanisms of action at 96% accuracy. We further apply Grad-CAM on our convolution-based models to identify signature regions of perturbations by these drugs in calcium signals. Conclusions: We anticipate that the capabilities of BeatProfiler will help advance in vitro studies in cardiac biology through rapid phenotyping, revealing mechanisms underlying cardiac health and disease, and enabling objective classification of cardiac disease and responses to drugs.

RESUMEN

INTRODUCTION: High sucrose intake is linked to cardiovascular disease, a major global cause of mortality worldwide. Calcium mishandling and inflammation play crucial roles in cardiac disease pathophysiology. OBJECTIVE: Evaluate if sucrose-induced obesity is related to deterioration of myocardial function due to alterations in the calcium-handling proteins in association with proinflammatory cytokines. METHODS: Wistar rats were divided into control and sucrose groups. Over eight weeks, Sucrose group received 30% sucrose water. Cardiac function was determined in vivo using echocardiography and in vitro using papillary muscle assay. Western blotting was used to detect calcium handling protein; ELISA assay was used to assess TNF-α and IL-6 levels. RESULTS: Sucrose led to cardiac dysfunction. RYR2, SERCA2, NCX, pPBL Ser16 and L-type calcium channels were unchanged. However, pPBL-Thr17, and TNF-α levels were elevated in the S group. CONCLUSION: Sucrose induced cardiac dysfunction and decreased myocardial contractility in association with altered pPBL-Thr17 and elevated cardiac pro-inflammatory TNF-α.

Asunto(s)

Proteínas de Unión al Calcio , Ratas Wistar , Factor de Necrosis Tumoral alfa , Animales , Masculino , Ratas , Proteínas de Unión al Calcio/metabolismo , Interleucina-6/metabolismo , Contracción Miocárdica/efectos de los fármacos , Miocardio/metabolismo , Miocardio/patología , Fosforilación/efectos de los fármacos , Sacarosa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

INTRODUCTION: Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical stimulations to the heart to enhance cardiac function in heart failure (HF) patients. The lack of human in vitro tools to assess CCM hinders our understanding of CCM mechanisms of action. Here, we introduce a novel chronic (i.e., 2-day) in vitro CCM assay to evaluate the effects of CCM in a human 3D microphysiological system consisting of engineered cardiac tissues (ECTs). METHODS: Cryopreserved human induced pluripotent stem cell-derived cardiomyocytes were used to generate 3D ECTs. The ECTs were cultured, incorporating human primary ventricular cardiac fibroblasts and a fibrin-based gel. Electrical stimulation was applied using two separate pulse generators for the CCM group and control group. Contractile properties and intracellular calcium were measured, and a cardiac gene quantitative PCR screen was conducted. RESULTS: Chronic CCM increased contraction amplitude and duration, enhanced intracellular calcium transient amplitude, and altered gene expression related to HF (i.e., natriuretic peptide B, NPPB) and excitation-contraction coupling (i.e., sodium-calcium exchanger, SLC8). CONCLUSION: These data represent the first study of chronic CCM in a 3D ECT model, providing a nonclinical tool to assess the effects of cardiac electrophysiology medical device signals complementing in vivo animal studies. The methodology established a standardized 3D ECT-based in vitro testbed for chronic CCM, allowing evaluation of physiological and molecular effects on human cardiac tissues.

Asunto(s)

Técnicas Electrofisiológicas Cardíacas , Contracción Miocárdica , Miocitos Cardíacos , Contracción Miocárdica/genética , Contracción Miocárdica/fisiología , Ingeniería de Tejidos , Humanos , Miocitos Cardíacos/fisiología , Células Madre Pluripotentes/fisiología , Perfilación de la Expresión Génica

RESUMEN

Calcium signaling is a critical process required for cellular mechanisms such as cardiomyocyte contraction. The inability of the cell to properly activate or regulate calcium signaling can lead to contractile dysfunction. In isolated cardiomyocytes, calcium signaling has been primarily studied using calcium fluorescent dyes, however these dyes have limited applicability to whole organs. Here, we crossed the Salsa6f mouse which expresses a genetically encoded ratiometric cytosolic calcium indicator with a cardiomyocyte specific inducible cre to temporally-induce expression and studied cytosolic calcium transients in isolated cardiomyocytes and modified Langendorff heart preparations. Isolated cardiomyocytes expressing Salsa6f or Fluo-4AM loaded were compared. We also crossed the Salsa6f mouse with a floxed Polycystin 2 (PC2) mouse to test the feasibility of using the Salsa6f mouse to measure calcium transients in PC2 heterozygous or homozygous knock out mice. Although there are caveats in the applicability of the Salsa6f mouse, there are clear advantages to using the Salsa6f mouse to measure whole heart calcium signals.

Asunto(s)

Calcio , Miocitos Cardíacos , Ratones , Animales , Calcio/metabolismo , Miocitos Cardíacos/metabolismo , Señalización del Calcio/fisiología , Colorantes Fluorescentes/metabolismo , Contracción Miocárdica/fisiología

RESUMEN

Mitochondrial Ca2+ ([Ca2+]m) homeostasis is critical for ß-cell function and becomes disrupted during the pathogenesis of diabetes. [Ca2+]m uptake is dependent on elevations in cytoplasmic Ca2+ ([Ca2+]c) and endoplasmic reticulum Ca2+ ([Ca2+]ER) release, both of which are regulated by the two-pore domain K+ channel TALK-1. Here, utilizing a novel ß-cell TALK-1-knockout (ß-TALK-1-KO) mouse model, we found that TALK-1 limited ß-cell [Ca2+]m accumulation and ATP production. However, following exposure to a high-fat diet (HFD), ATP-linked respiration, glucose-stimulated oxygen consumption rate, and glucose-stimulated insulin secretion (GSIS) were increased in control but not TALK1-KO mice. Although ß-TALK-1-KO animals showed similar GSIS before and after HFD treatment, these mice were protected from HFD-induced glucose intolerance. Collectively, these data identify that TALK-1 channel control of ß-cell function reduces [Ca2+]m and suggest that metabolic remodeling in diabetes drives dysglycemia.

Asunto(s)

Diabetes Mellitus , Células Secretoras de Insulina , Animales , Ratones , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Diabetes Mellitus/metabolismo , Dieta , Retículo Endoplásmico/metabolismo , Glucosa/metabolismo , Homeostasis , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones Noqueados , Mitocondrias/metabolismo

RESUMEN

In heart failure with reduced ejection fraction and heart failure with preserved ejection fraction, profound cellular and molecular changes have recently been documented in the failing myocardium. These changes include altered calcium handling and metabolic efficiency of the cardiac myocyte, reactivation of the fetal gene program, changes in the electrophysiological properties of the heart, and accumulation of collagen (fibrosis) at the interstitial level. Cardiac contractility modulation therapy is an innovative device-based therapy currently approved for heart failure with reduced ejection fraction in patients with narrow QRS complex and under investigation for the treatment of heart failure with preserved ejection fraction. This therapy is based on the delivery of high-voltage biphasic electrical signals to the septal wall of the right ventricle during the absolute refractory period of the myocardium. At the cellular level, in patients with heart failure with reduced ejection fraction, cardiac contractility modulation therapy has been shown to restore calcium handling and improve the metabolic status of cardiac myocytes, reverse the heart failure-associated fetal gene program, and reduce the extent of interstitial fibrosis. This review summarizes the preclinical literature on the use of cardiac contractility modulation therapy in heart failure with reduced and preserved ejection fraction, correlating the molecular and electrophysiological effects with the clinical benefits demonstrated by this therapy.

Asunto(s)

Insuficiencia Cardíaca , Disfunción Ventricular Izquierda , Humanos , Volumen Sistólico/fisiología , Calcio , Contracción Miocárdica/fisiología , Cardiotónicos , Insuficiencia Cardíaca/tratamiento farmacológico , Fibrosis

RESUMEN

BACKGROUND: Numerous studies have demonstrated that NLRP3 inflammasomes are key players in the progression of atrial fibrillation (AF) in heart failure with preserved ejection fraction (HFpEF). This study aimed to analyse the effect of pharmacological inhibition of NLRP3 inflammasomes using dapansutrile (DAPA), an oral NLRP3-specific inhibitor. METHODS: Dahl salt-sensitive rats were fed a high-salt diet (HSD, 8% NaCl) to induce HFpEF. Either DAPA (200 mg/kg/day) or saline was administered daily via gavage for 4 weeks. Electrophysiological studies were performed to assess the AF inducibility. Confocal fluorescence microscopy and western blot analysis were used to study calcium handling. RESULTS: The DAPA-treated HFpEF rats were less prone to AF induction by programmed electrical stimulation. Atrial fibrosis and inflammation were attenuated in DAPA-treated HFpEF hearts. Dapansutrile treatment showed an increase in the Ca2+ transient sarcoplasmic reticulum-Ca2+ load, and protein expression of SERCA2; NCX1 and phosphorylation of PLB at Thr17 were decreased following DAPA treatment. The increased frequency of spontaneous Ca2+ spark in the HFpEF rats was related to the hyperphosphorylation of RyR2 at Ser2814, which was blunted in DAPA treatment. Dapansutrile treatment also decreased the phosphorylation of CaMKII expression in the HFpEF rats. Mechanistically, DAPA exerts an anti-arrhythmic effect, mainly by inhibiting activation of the NLRP3 inflammasome. CONCLUSION: These data provide evidence that the beneficial cardiac effects of DAPA are associated with reduced atrial inflammation and improved CaMKII-dependent Ca2+-handling abnormalities via blunting activation of the NLRP3 inflammasome, and DAPA may be beneficial in a rat model of HFpEF-induced AF.

Asunto(s)

Fibrilación Atrial , Insuficiencia Cardíaca , Nitrilos , Sulfonas , Ratas , Animales , Fibrilación Atrial/tratamiento farmacológico , Fibrilación Atrial/etiología , Fibrilación Atrial/metabolismo , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Inflamasomas/metabolismo , Inflamasomas/farmacología , Volumen Sistólico , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Ratas Endogámicas Dahl , Atrios Cardíacos , Inflamación

RESUMEN

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is frequently caused by mutations in the ß-cardiac myosin heavy chain gene (MYH7). Abnormal calcium handling and diastolic dysfunction are archetypical features of HCM caused by MYH7 gene mutations. However, the mechanism of how MYH7 mutations leads to these features remains unclear, which inhibits the development of effective therapies. Initially, cardiomyocytes were generated from induced pluripotent stem cells from an eight-year-old girl diagnosed with HCM carrying a MYH7(C.1063 G>A) heterozygous mutation(mutant-iPSC-CMs) and mutation-corrected isogenic iPSCs(control-iPSC-CMs) in the present study. Next, we compared phenotype of mutant-iPSC-CMs to that of control-iPSC-CMs, by assessing their morphology, hypertrophy-related genes expression, calcium handling, diastolic function and myofilament calcium sensitivity at days 15 and 40 respectively. Finally, to better understand increased myofilament Ca2+ sensitivity as a central mechanism of central pathogenicity in HCM, inhibition of calcium sensitivity with mavacamten can improveed cardiomyocyte hypertrophy. Mutant-iPSC-CMs exhibited enlarged areas, increased sarcomere disarray, enhanced expression of hypertrophy-related genes proteins, abnormal calcium handling, diastolic dysfunction and increased myofilament calcium sensitivity at day 40, but only significant increase in calcium sensitivity and mild diastolic dysfunction at day 15. Increased calcium sensitivity by levosimendan aggravates cardiomyocyte hypertrophy phenotypes such as expression of hypertrophy-related genes, abnormal calcium handling and diastolic dysfunction, while inhibition of calcium sensitivity significantly improves cardiomyocyte hypertrophy phenotypes in mutant-iPSC-CMs, suggesting increased myofilament calcium sensitivity is the primary mechanisms for MYH7 mutations pathogenesis. Our studies have uncovered a pathogenic mechanism of HCM caused by MYH7 gene mutations through which enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction. Correction of the myofilament calcium sensitivity was found to be an effective method for treating the development of HCM phenotype in vitro.

Asunto(s)

Cardiomiopatías , Cardiomiopatía Hipertrófica , Células Madre Pluripotentes Inducidas , Niño , Femenino , Humanos , Calcio/metabolismo , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Cardiomiopatías/metabolismo , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/patología , Hipertrofia/metabolismo , Hipertrofia/patología , Células Madre Pluripotentes Inducidas/metabolismo , Mutación/genética , Miocitos Cardíacos/metabolismo , Miofibrillas/metabolismo , Miofibrillas/patología , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo

RESUMEN

Calcium signaling is a critical process required for cellular mechanisms such as cardiac contractility. The inability of the cell to properly activate or regulate calcium signaling can lead to contractile dysfunction. In isolated cardiomyocytes, calcium signaling has been primarily studied using calcium fluorescent dyes, however these dyes have limited applicability to whole organs. Here, we crossed the Salsa6f mouse which expresses a genetically encoded ratiometric cytosolic calcium indicator with a cardiomyocyte specific inducible cre to temporally-induce expression and studied cytosolic calcium transients in isolated cardiomyocytes and modified Langendorff heart preparations. Isolated cardiomyocytes expressing Salsa6f or Fluo-4AM loaded were compared. We also crossed the Salsa6f mouse with a floxed Polycystin 2 (PC2) mouse to test the feasibility of using the Salsa6f mouse to measure calcium transients in PC2 heterozygous or homozygous knock out mice. Although there are caveats in the applicability of the Salsa6f mouse, there are clear advantages to using the Salsa6f mouse to measure whole heart calcium signals.

RESUMEN

[This corrects the article DOI: 10.3389/fphys.2023.1269900.].

RESUMEN

In the excitation of muscle contraction, calcium ions interact with transmembrane transporters. This process is accompanied by energy consumption and heat liberation. To quantify this activation energy or heat in the heart or cardiac muscle, two non-pharmacological approaches can be used. In one approach using the "pressure-volume area" concept, the same estimate of activation energy is obtained regardless of the mode of contraction (either isovolumic/isometric or ejecting/shortening). In the other approach, an accurate estimate of activation energy is obtained only when the muscle contracts isometrically. If the contraction involves muscle shortening, then an additional component of heat associated with shortening is liberated, over and above that of activation. The present study thus examines the reconcilability of the two approaches by performing experiments on isolated muscles measuring contractile force and heat output. A framework was devised from the experimental data to allow us to replicate several mechanoenergetics results gleaned from the literature. From these replications, we conclude that the choice of initial muscle length (or ventricular volume) underlies the divergence of the two approaches in the estimation of activation energy when the mode of contraction involves shortening (ejection). At low initial muscle lengths, the heat of shortening is relatively small, which can lead to the misconception that activation energy is contraction mode independent. In fact, because cardiac muscle liberates heat of shortening when allowed to shorten, estimation of activation heat must be performed only under isometric (isovolumic) contractions. We thus recommend caution when estimating activation energy using the "pressure-volume area" concept.

RESUMEN

Synchronized contractions of cardiomyocytes within the heart are tightly coupled to electrical stimulation known as excitation-contraction coupling. Calcium plays a key role in this process and dysregulated calcium handling can significantly impair cardiac function and lead to the development of cardiomyopathies and heart failure. Here, we describe a method and analytical technique to study myofilament-localized calcium signaling using the intensity-based fluorescent biosensor, RGECO-TnT. Dilated cardiomyopathy is a heart muscle disease that negatively impacts the heart's contractile function following dilatation of the left ventricle. We demonstrate how this biosensor can be used to characterize 2D hiPSC-CMs monolayers generated from a healthy control subject compared to two patients diagnosed with dilated cardiomyopathy. Lastly, we provide a step-by-step guide for single-cell data analysis and describe a custom Transient Analysis application, specifically designed to quantify features of calcium transients. All in all, we explain how this analytical approach can be applied to phenotype hiPSC-CM behaviours and stratify patient responses to identify perturbations in calcium signaling.

Asunto(s)

Cardiomiopatías , Cardiomiopatía Dilatada , Células Madre Pluripotentes Inducidas , Humanos , Miofibrillas , Cardiomiopatía Dilatada/genética , Calcio , Miocitos Cardíacos

RESUMEN

Background: Cardiac optical mapping is an imaging technique that measures fluorescent signals across a cardiac preparation. Dual optical imaging of voltage-sensitive and calcium-sensitive probes allows for simultaneous recordings of cardiac action potentials and intracellular calcium transients with high spatiotemporal resolution. The analysis of these complex optical datasets is both time intensive and technically challenging; as such, we have developed a software package for semi-automated image processing and analysis. Herein, we report an updated version of our software package (KairoSight-3.0) with features to enhance the characterization of cardiac parameters using optical signals. Methods: To test software validity and applicability, we used Langendorff-perfused heart preparations to record transmembrane voltage and intracellular calcium signals from the epicardial surface. Isolated hearts from guinea pigs and rats were loaded with a potentiometric dye (RH237) and/or calcium indicator dye (Rhod-2AM) and fluorescent signals were acquired. We used Python 3.8.5 programming language to develop the KairoSight-3.0 software. Cardiac maps were validated with a user-specified manual mapping approach. Results: Manual maps of action potential duration (30 or 80 % repolarization), calcium transient duration (30 or 80 % reuptake), action potential and calcium transient alternans were constituted to validate the accuracy of software-generated maps. Manual and software maps had high accuracy, with >97 % of manual and software values falling within 10 ms of each other and >75 % within 5 ms for action potential duration and calcium transient duration measurements (n = 1000-2000 pixels). Further, our software package includes additional measurement tools to analyze signal-to-noise ratio, conduction velocity, action potential and calcium transient alternans, and action potential-calcium transient coupling time to produce physiologically meaningful optical maps. Conclusions: KairoSight-3.0 has enhanced capabilities to perform measurements of cardiac electrophysiology, calcium handling, alternans, and the excitation-contraction coupling with satisfactory accuracy.