Loss of autophagy protein ATG5 impairs cardiac capacity in mice and humans through diminishing mitochondrial abundance and disrupting Ca2+Ca^{\text{2+}} cycling

Aims Autophagy protects against the development of cardiac hypertrophy and failure. While aberrant Ca2+ handling promotes myocardial remodelling and contributes to contractile dysfunction, the role of autophagy in maintaining Ca2+ homeostasis remains elusive. Here, we examined whether Atg5 deficiency-mediated autophagy promotes early changes in subcellular Ca2+ handling in ventricular cardiomyocytes, and whether those alterations associate with compromised cardiac reserve capacity, which commonly precedes the onset of heart failure. Methods and results RT–qPCR and immunoblotting demonstrated reduced Atg5 gene and protein expression and decreased abundancy of autophagy markers in hypertrophied and failing human hearts. The function of ATG5 was examined using cardiomyocyte-specific Atg5-knockout mice (Atg5−/−). Before manifesting cardiac dysfunction, Atg5−/− mice showed compromised cardiac reserve in response to β-adrenergic stimulation. Consequently, effort intolerance and maximal oxygen consumption were reduced during treadmill-based exercise tolerance testing. Mechanistically, cellular imaging revealed that Atg5 deprivation did not alter spatial and functional organization of intracellular Ca2+ stores or affect Ca2+ cycling in response to slow pacing or upon acute isoprenaline administration. However, high-frequency stimulation exposed stunted amplitude of Ca2+ transients, augmented nucleoplasmic Ca2+ load, and increased CaMKII activity, especially in the nuclear region of hypertrophied Atg5−/− cardiomyocytes. These changes in Ca2+ cycling were recapitulated in hypertrophied human cardiomyocytes. Finally, ultrastructural analysis revealed accumulation of mitochondria with reduced volume and size distribution, meanwhile functional measurements showed impaired redox balance in Atg5−/− cardiomyocytes, implying energetic unsustainability due to overcompensation of single mitochondria, particularly under increased workload. Conclusion Loss of cardiac Atg5-dependent autophagy reduces mitochondrial abundance and causes subtle alterations in subcellular Ca2+ cycling upon increased workload in mice. Autophagy-related impairment of Ca2+ handling is progressively worsened by β-adrenergic signalling in ventricular cardiomyocytes, thereby leading to energetic exhaustion and compromised cardiac reserve

CaMKII delta C Drives Early Adaptive Ca(2+)Change and Late Eccentric Cardiac Hypertrophy

Rationale: CaMKII (Ca2+-Calmodulin dependent protein kinase) delta C activation is implicated in pathological progression of heart failure (HF) and CaMKII delta C transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca(2+)handling and CaMKII activation in hypertrophy and HF. Objective: To measure time- and location-dependent activation of CaMKII delta C signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKII delta C transgenic mice. Methods and Results: We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKII delta-knockout), CaMKII delta C transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKII delta C activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKII delta activation caused a compensatory increase in sarcoplasmic reticulum Ca(2+)content, Ca(2+)transient amplitude, and [Ca2+] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKII delta TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca(2+)transient effects reversed, but further increases in nuclear and time-averaged [Ca2+] and CaMKII activation occurred. CaMKII delta TG mice lacking delta B exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKII delta expression, especially for CaMKII delta C in nuclear fractions. Conclusions: We conclude that in early TAC perinuclear CaMKII delta C activation promotes adaptive increases in myocyte Ca(2+)transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca2+-CaMKII delta C axis contributes to eccentric hypertrophy and HF

Fine-Tuning Cardiac Insulin-Like Growth Factor 1 Receptor Signaling to Promote Health and Longevity

Background: The insulin-like growth factor 1 (IGF1) pathway is a key regulator of cellular metabolism and aging. Although its inhibition promotes longevity across species, the effect of attenuated IGF1 signaling on cardiac aging remains controversial. Methods: We performed a lifelong study to assess cardiac health and lifespan in 2 cardiomyocyte-specific transgenic mouse models with enhanced versus reduced IGF1 receptor (IGF1R) signaling. Male mice with human IGF1R overexpression or dominant negative phosphoinositide 3-kinase mutation were examined at different life stages by echocardiography, invasive hemodynamics, and treadmill coupled to indirect calorimetry. In vitro assays included cardiac histology, mitochondrial respiration, ATP synthesis, autophagic flux, and targeted metabolome profiling, and immunoblots of key IGF1R downstream targets in mouse and human explanted failing and nonfailing hearts, as well. Results: Young mice with increased IGF1R signaling exhibited superior cardiac function that progressively declined with aging in an accelerated fashion compared with wild-type animals, resulting in heart failure and a reduced lifespan. In contrast, mice with low cardiac IGF1R signaling exhibited inferior cardiac function early in life, but superior cardiac performance during aging, and increased maximum lifespan, as well. Mechanistically, the late-life detrimental effects of IGF1R activation correlated with suppressed autophagic flux and impaired oxidative phosphorylation in the heart. Low IGF1R activity consistently improved myocardial bioenergetics and function of the aging heart in an autophagy-dependent manner. In humans, failing hearts, but not those with compensated hypertrophy, displayed exaggerated IGF1R expression and signaling activity. Conclusions: Our findings indicate that the relationship between IGF1R signaling and cardiac health is not linear, but rather biphasic. Hence, pharmacological inhibitors of the IGF1 pathway, albeit unsuitable for young individuals, might be worth considering in older adults

Targeting Cardiovascular Risk Factors Through Dietary Adaptations and Caloric Restriction Mimetics

International audienceThe average human life expectancy continues to rise globally and so does the prevalence and absolute burden of cardiovascular disease. Dietary restriction promotes longevity and improves various cardiovascular risk factors, including hypertension, obesity, diabetes mellitus, and metabolic syndrome. However, low adherence to caloric restriction renders this stringent dietary intervention challenging to adopt as a standard practice for cardiovascular disease prevention. Hence, alternative eating patterns and strategies that recapitulate the salutary benefits of caloric restriction are under intense investigation. Here, we first provide an overview of alternative interventions, including intermittent fasting, alternate-day fasting and the Mediterranean diet, along with their cardiometabolic effects in animal models and humans. We then present emerging pharmacological alternatives, including spermidine, NAD + precursors, resveratrol, and metformin, as promising caloric restriction mimetics, and briefly touch on the mechanisms underpinning their cardiometabolic and health-promoting effects. We conclude that implementation of feasible dietary approaches holds the promise to attenuate the burden of cardiovascular disease and facilitate healthy aging in humans

Fine-tuning cardiac insulin-like growth factor 1 receptor signaling to promote health and longevity

Background: The insulin-like growth factor 1 (IGF1) pathway is a key regulator of cellular metabolism and aging. Although its inhibition promotes longevity across species, the effect of attenuated IGF1 signaling on cardiac aging remains controversial. Methods: We performed a lifelong study to assess cardiac health and lifespan in 2 cardiomyocyte-specific transgenic mouse models with enhanced versus reduced IGF1 receptor (IGF1R) signaling. Male mice with human IGF1R overexpression or dominant negative phosphoinositide 3-kinase mutation were examined at different life stages by echocardiography, invasive hemodynamics, and treadmill coupled to indirect calorimetry. In vitro assays included cardiac histology, mitochondrial respiration, ATP synthesis, autophagic flux, and targeted metabolome profiling, and immunoblots of key IGF1R downstream targets in mouse and human explanted failing and nonfailing hearts, as well. Results: Young mice with increased IGF1R signaling exhibited superior cardiac function that progressively declined with aging in an accelerated fashion compared with wild-type animals, resulting in heart failure and a reduced lifespan. In contrast, mice with low cardiac IGF1R signaling exhibited inferior cardiac function early in life, but superior cardiac performance during aging, and increased maximum lifespan, as well. Mechanistically, the late-life detrimental effects of IGF1R activation correlated with suppressed autophagic flux and impaired oxidative phosphorylation in the heart. Low IGF1R activity consistently improved myocardial bioenergetics and function of the aging heart in an autophagy-dependent manner. In humans, failing hearts, but not those with compensated hypertrophy, displayed exaggerated IGF1R expression and signaling activity. Conclusions: Our findings indicate that the relationship between IGF1R signaling and cardiac health is not linear, but rather biphasic. Hence, pharmacological inhibitors of the IGF1 pathway, albeit unsuitable for young individuals, might be worth considering in older adults

β-Adrenergic Receptor Stimulation Maintains NCX-CaMKII Axis and Prevents Overactivation of IL6R-Signaling in Cardiomyocytes upon Increased Workload

Excessive beta-adrenergic stimulation and tachycardia are potent triggers of cardiac remodeling; however, their exact cellular effects remain elusive. Here, we sought to determine the potency of beta-adrenergic stimulation and tachycardia to modulate gene expression profiles of cardiomyocytes. Using neonatal rat ventricular cardiomyocytes, we showed that tachycardia caused a significant upregulation of sodium-calcium exchanger (NCX) and the activation of calcium/calmodulin-dependent kinase II (CaMKII) in the nuclear region. Acute isoprenaline treatment ameliorated NCX-upregulation and potentiated CaMKII activity, specifically on the sarcoplasmic reticulum and the nuclear envelope, while preincubation with the beta-blocker propranolol abolished both isoprenaline-mediated effects. On a transcriptional level, screening for hypertrophy-related genes revealed tachycardia-induced upregulation of interleukin-6 receptor (IL6R). While isoprenaline prevented this effect, pharmacological intervention with propranolol or NCX inhibitor ORM-10962 demonstrated that simultaneous CaMKII activation on the subcellular Ca2+ stores and prevention of NCX upregulation are needed for keeping IL6R activation low. Finally, using hypertensive Dahl salt-sensitive rats, we showed that blunted beta-adrenergic signaling is associated with NCX upregulation and enhanced IL6R signaling. We therefore propose a previously unrecognized protective role of beta-adrenergic signaling, which is compromised in cardiac pathologies, in preventing IL6R overactivation under increased workload. A better understanding of these processes may contribute to refinement of therapeutic options for patients receiving beta-blockers

β-Adrenergic Receptor Stimulation Maintains NCX-CaMKII Axis and Prevents Overactivation of <i>IL6R</i>-Signaling in Cardiomyocytes upon Increased Workload

Excessive β-adrenergic stimulation and tachycardia are potent triggers of cardiac remodeling; however, their exact cellular effects remain elusive. Here, we sought to determine the potency of β-adrenergic stimulation and tachycardia to modulate gene expression profiles of cardiomyocytes. Using neonatal rat ventricular cardiomyocytes, we showed that tachycardia caused a significant upregulation of sodium–calcium exchanger (NCX) and the activation of calcium/calmodulin-dependent kinase II (CaMKII) in the nuclear region. Acute isoprenaline treatment ameliorated NCX-upregulation and potentiated CaMKII activity, specifically on the sarcoplasmic reticulum and the nuclear envelope, while preincubation with the β-blocker propranolol abolished both isoprenaline-mediated effects. On a transcriptional level, screening for hypertrophy-related genes revealed tachycardia-induced upregulation of interleukin-6 receptor (IL6R). While isoprenaline prevented this effect, pharmacological intervention with propranolol or NCX inhibitor ORM-10962 demonstrated that simultaneous CaMKII activation on the subcellular Ca2+ stores and prevention of NCX upregulation are needed for keeping IL6R activation low. Finally, using hypertensive Dahl salt-sensitive rats, we showed that blunted β-adrenergic signaling is associated with NCX upregulation and enhanced IL6R signaling. We therefore propose a previously unrecognized protective role of β-adrenergic signaling, which is compromised in cardiac pathologies, in preventing IL6R overactivation under increased workload. A better understanding of these processes may contribute to refinement of therapeutic options for patients receiving β-blockers

Functional remodelling of perinuclear mitochondria alters nucleoplasmic Ca2+ signalling in heart failure

Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure development. Although initial studies recognized the importance of different mitochondrial subpopulations, there is a striking lack of direct comparison of intrafibrillar (IF) versus perinuclear (PN) mitochondria during the development of HF. Here, we use multiple approaches to examine the morphology and functional properties of IF versus PN mitochondria in pressure overload-induced cardiac remodelling in mice, and in non-failing and failing human cardiomyocytes. We demonstrate that PN mitochondria from failing cardiomyocytes are more susceptible to depolarization of mitochondrial membrane potential, reactive oxygen species generation and impairment in Ca2+ uptake compared with IF mitochondria at baseline and under physiological stress protocol. We also demonstrate, for the first time to our knowledge, that under normal conditions PN mitochondrial Ca2+ uptake shapes nucleoplasmic Ca2+ transients (CaTs) and limits nucleoplasmic Ca2+ loading. The loss of PN mitochondrial Ca2+ buffering capacity translates into increased nucleoplasmic CaTs and may explain disproportionate rise in nucleoplasmic [Ca2+] in failing cardiomyocytes at increased stimulation frequencies. Therefore, a previously unidentified benefit of restoring the mitochondrial Ca2+ uptake may be normalization of nuclear Ca2+ signalling and alleviation of altered excitation-transcription, which could be an important therapeutic approach to prevent adverse cardiac remodelling. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'

Nicotinamide for the treatment of heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent and intractable form of cardiac decompensation commonly associated with diastolic dysfunction. Here, we show that diastolic dysfunction in patients with HFpEF is associated with a cardiac deficit in nicotinamide adenine dinucleotide (NAD(+)). Elevating NAD(+) by oral supplementation of its precursor, nicotinamide, improved diastolic dysfunction induced by aging (in 2-year-old C57BL/6J mice), hypertension (in Dahl salt-sensitive rats), or cardiometabolic syndrome (in ZSF1 obese rats). This effect was mediated partly through alleviated systemic comorbidities and enhanced myocardial bioenergetics. Simultaneously, nicotinamide directly improved cardiomyocyte passive stiffness and calcium-dependent active relaxation through increased deacetylation of titin and the sarcoplasmic reticulum calcium adenosine triphosphatase 2a, respectively. In a long-term human cohort study, high dietary intake of naturally occurring NAD(+) precursors was associated with lower blood pressure and reduced risk of cardiac mortality. Collectively, these results suggest NAD(+) precursors, and especially nicotinamide, as potential therapeutic agents to treat diastolic dysfunction and HFpEF in humans