Acetylation and phosphorylation changes to cardiac proteins in experimental HFpEF due to metabolic risk reveal targets for treatment.

AIMS: Despite the high prevalence of heart failure with preserved ejection fraction (HFpEF), the pathomechanisms remain elusive and specific therapy is lacking. Disease-causing factors include metabolic risk, notably obesity. However, proteomic changes in HFpEF are poorly understood, hampering therapeutic strategies. We sought to elucidate how metabolic syndrome affects cardiac protein expression, phosphorylation and acetylation in the Zucker diabetic fatty/Spontaneously hypertensive heart failure F1 (ZSF1) rat HFpEF model, and to evaluate changes regarding their potential for treatment. MAIN METHODS: ZSF1 obese and lean rats were fed a Purina diet up to the onset of HFpEF in the obese animals. We quantified the proteome, phosphoproteome and acetylome of ZSF1 obese versus lean heart tissues by mass spectrometry and singled out targets for site-specific evaluation. KEY FINDINGS: The acetylome of ZSF1 obese versus lean hearts was more severely altered (21 % of proteins changed) than the phosphoproteome (9 %) or proteome (3 %). Proteomic alterations, confirmed by immunoblotting, indicated low-grade systemic inflammation and endothelial remodeling in obese hearts, but low nitric oxide-dependent oxidative/nitrosative stress. Altered acetylation in ZSF1 obese hearts mainly affected pathways important for metabolism, energy production and mechanical function, including hypo-acetylation of mechanical proteins but hyper-acetylation of proteins regulating fatty acid metabolism. Hypo-acetylation and hypo-phosphorylation of elastic titin in ZSF1 obese hearts could explain myocardial stiffening. SIGNIFICANCE: Cardiometabolic syndrome alters posttranslational modifications, notably acetylation, in experimental HFpEF. Pathway changes implicate a HFpEF signature of low-grade inflammation, endothelial dysfunction, metabolic and mechanical impairment, and suggest titin stiffness and mitochondrial metabolism as promising therapeutic targets.

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

Excessive ß-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 ß-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 ß-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 ß-adrenergic signaling is associated with NCX upregulation and enhanced IL6R signaling. We therefore propose a previously unrecognized protective role of ß-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 ß-blockers.

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.

Loss of autophagy protein ATG5 impairs cardiac capacity in mice and humans through diminishing mitochondrial abundance and disrupting Ca2+ 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 ß-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 ß-adrenergic signalling in ventricular cardiomyocytes, thereby leading to energetic exhaustion and compromised cardiac reserve.

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.

Acute hyperglycaemia is not associated with the development of atrial fibrillation in healthy pigs.

Development and progression of atrial fibrillation (AF) is driven by comorbidities such as arterial hypertension and diabetes mellitus. In animal models of chronic hyperglycaemia, progression of AF has been proposed to be triggered by oxidative stress, apoptosis and fibrosis. Acute glycosylation of CaMKII has been associated with increased susceptibility to arrhythmias in acute hyperglycaemia. However, the proarrhythmogenic effect of acute hyperglycaemia has not been investigated. Nine healthy, anesthetized pigs (54 ± 6 kg) were instrumented with electrophysiologic catheters and a multielectrode array on the epicardium of the left atrial anterior wall. Left and right atrial effective refractory periods (AERP), inducibility of AF and left atrial epicardial conduction velocities (CV) were measured at baseline (BL), increasing steps of blood glucose (200-500 mg/dL in steps of 100 mg/dL by glucose infusion) and repeated after normalisation of blood glucose levels (recovery). Serum electrolytes were kept constant during measurements by means of sodium and potassium infusion. There were no significant differences in AERP, CV or AF inducibility between BL and recovery. Heart rate remained constant regardless of blood glucose levels (BL: 103 ± 18 bpm, 500 mg/dL: 103 ± 18 bpm, r = 0.02, p = 0.346). Mean left as well as right AERP increased with higher glucose levels. CV increased with glucose levels (1.25 (1.04, 1.67) m/s at BL vs. 1.53 (1.22, 2.15) m/s at 500 mg/dL, r = 0.85, p = 0.034). Rate of AF inducibility in the left atrium remained constant throughout the whole protocol (AF episodes > 10 s: mean inducibility of 80% at BL vs. 69% at 500 mg/dL, p = 0.32, episodes > 30 s: 0% at BL vs. 0% at 500 mg/dL, p = 0.17). Our data imply that acute hyperglycaemia is associated with lower arrhythmogenic substrate and does not promote AF inducibility.

Arterial hypertension drives arrhythmia progression via specific structural remodeling in a porcine model of atrial fibrillation.

BACKGROUND: Arterial hypertension (HT) contributes to progression of atrial fibrillation (AF) via unknown mechanisms. OBJECTIVE: We aimed to characterize electrical and structural changes accounting for increased AF stability in a large animal model of rapid atrial pacing (RAP)-induced AF combined with desoxycorticosterone acetate (DOCA)-induced HT. METHODS: Eighteen pigs were instrumented with right atrial endocardial pacemaker leads and custom-made pacemakers to induce AF by continuous RAP (600 beats/min). DOCA pellets were subcutaneously implanted in a subgroup of 9 animals (AF+HT group); the other 9 animals served as controls (AF group). Final experiments included electrophysiology studies, endocardial electroanatomic mapping, and high-density mapping with epicardial multielectrode arrays. In addition, 3-dimensional computational modeling was performed. RESULTS: DOCA implantation led to secondary HT (median [interquartile range] aortic pressure 109.9 [100-137] mm Hg in AF+HT vs 82.2 [79-96] mm Hg in AF; P < .05), increased AF stability (55.6% vs 12.5% of animals with AF episodes lasting >1 hour; P < .05), concentric left ventricular hypertrophy, atrial dilatation (119 ± 31 cm2 in AF+HT vs 78 ± 23 cm2 in AF; P < .05), and fibrosis. Collagen accumulation in the AF+HT group was mainly found in non-intermyocyte areas (1.62 ± 0.38 cm3 in AF+HT vs 0.96 ± 0.3 cm3 in AF; P < .05). Left and right atrial effective refractory periods, action potential durations, endo- and epicardial conduction velocities, and measures of AF complexity were comparable between the 2 groups. A 3-dimensional computational model confirmed an increase in AF stability observed in the in vivo experiments associated with increased atrial size. CONCLUSION: In this model of secondary HT, higher AF stability after 2 weeks of RAP is mainly driven by atrial dilatation.

Mild hypothermia (33°C) increases the inducibility of atrial fibrillation: An in vivo large animal model study.

OBJECTIVE: Application of therapeutic mild hypothermia in patients after resuscitation, often accompanied by myocardial infarction, cardiogenic shock, and systemic inflammation may impact on cardiac rhythm. We therefore tested susceptibility to atrial arrhythmias during hyperthermia (HT, 40.5°C), normothermia (NT, 38.0°C), and mild hypothermia (MH, 33.0°C). METHODS: Nine healthy, anesthetized closed-chest landrace pigs were instrumented with a quadripolar stimulation catheter in the high right atrium and a decapolar catheter in the coronary sinus. Twelve-lead surface electrograms were recorded and core body temperature was altered to HT, NT, and MH using external warming or intravascular cooling. Repetitive measurements of effective atrial refractory period (AERP), atrial fibrillation (AF) inducibility, and electrocardiogram (ECG) parameters at different heart rates were performed. RESULTS: During MH, AERP was significantly longer while the inducibility of AF was significantly higher compared to NT and HT (median [range]: HT 18 (0, 80)%; NT 25 (0, 80)%; MH 68 (0, 100)%; P < 0.05 MH vs NT+HT). Mean AF duration did not differ between groups. Arterial potassium levels decreased with falling temperatures (HT: 4.2 ± 0.1 mmol/L; NT: 4.0 ± 0.2 mmol/L; MH: 3.5 ± 0.1 mmol/L; P < 0.001). Surface ECGs during MH showed reduced spontaneous heart rate (HT: 99 ± 13 beats/min; NT: 87 ± 15 beats/min; MH: 66 ± 10 beats/min; P < 0.05), increased PQ, stim-Q, and QT intervals (P < 0.01) but no change in QRS duration or time from peak to end of the T wave interval. CONCLUSION: Our data imply that MH represents an arrhythmic substrate rendering the atria more susceptible to AF although conduction times as well as refractory periods are increased. Further investigations on potential electrophysiological limits of therapeutic cooling in patients are required.

The Anti-Cancer Multikinase Inhibitor Sorafenib Impairs Cardiac Contractility by Reducing Phospholamban Phosphorylation and Sarcoplasmic Calcium Transients.

Tyrosine-kinase inhibitors (TKIs) have revolutionized cancer therapy in recent years. Although more targeted than conventional chemotherapy, TKIs exhibit substantial cardiotoxicity, often manifesting as hypertension or heart failure. Here, we assessed myocyte intrinsic cardiotoxic effects of the TKI sorafenib and investigated underlying alterations of myocyte calcium homeostasis. We found that sorafenib reversibly decreased developed force in auxotonically contracting human myocardia (3 µM: -25 ± 4%, 10 µM: -29 ± 7%, 30 µM: -43 ± 12%, p < 0.01), reduced peak cytosolic calcium concentrations in isolated cardiomyocytes (10 µM: 52 ± 8.1% of baseline, p < 0.001), and slowed cytosolic calcium removal kinetics (RT50, RT10, Tau, p < 0.05). Beta-adrenergic stimulation induced augmentation of calcium transient (CaT) amplitude was attenuated in sorafenib-treated cells (2.7 ± 0.3-fold vs. 3.6 ± 0.2-fold in controls, p < 0.001). Sarcoplasmic reticulum (SR) calcium content was reduced to 67 ± 4% (p < 0.01), and SR calcium re-uptake slowed (p < 0.05). Sorafenib significantly reduced serine 16 phosphorylation of phospholamban (PLN, p < 0.05), while PLN threonine 17 and CaMKII (T286) phosphorylation were not altered. Our data demonstrate that sorafenib acutely impairs cardiac contractility by reducing S16 PLN phosphorylation, leading to reduced SR calcium content, CaT amplitude, and slowed cytosolic calcium removal. These results indicate myocyte intrinsic cardiotoxicity irrespective of effects on the vasculature and chronic cardiac remodeling.

Dietary spermidine for lowering high blood pressure.

Loss of cardiac macroautophagy/autophagy impairs heart function, and evidence accumulates that an increased autophagic flux may protect against cardiovascular disease. We therefore tested the protective capacity of the natural autophagy inducer spermidine in animal models of aging and hypertension, which both represent major risk factors for the development of cardiovascular disease. Dietary spermidine elicits cardioprotective effects in aged mice through enhancing cardiac autophagy and mitophagy. In salt-sensitive rats, spermidine supplementation also delays the development of hypertensive heart disease, coinciding with reduced arterial blood pressure. The high blood pressure-lowering effect likely results from improved global arginine bioavailability and protection from hypertension-associated renal damage. The polyamine spermidine is naturally present in human diets, though to a varying amount depending on food type and preparation. In humans, high dietary spermidine intake correlates with reduced blood pressure and decreased risk of cardiovascular disease and related death. Altogether, spermidine represents a cardio- and vascular-protective autophagy inducer that can be readily integrated in common diets.

Cardioprotection and lifespan extension by the natural polyamine spermidine.

Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.