Author response: Age-dependent diastolic heart failure in an in vivo Drosophila model

While the signals and complexes that coordinate the heartbeat are well established, how the heart maintains its electromechanical rhythm over a lifetime remains an open question with significant implications to human health. Reasoning that this homeostatic challenge confronts all pulsatile organs, we developed a high resolution imaging and analysis toolset for measuring cardiac function in intact, unanesthetized Drosophila melanogaster. We demonstrate that, as in humans, normal aging primarily manifests as defects in relaxation (diastole) while preserving contractile performance. Using this approach, we discovered that a pair of two-pore potassium channel (K2P) subunits, largely dispensable early in life, are necessary for terminating contraction (systole) in aged animals, where their loss culminates in fibrillatory cardiac arrest. As the pumping function of its heart is acutely dispensable for survival, Drosophila represents a uniquely accessible model for understanding the signaling networks maintaining cardiac performance during normal aging.

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A Protein-trap allele reveals roles for Drosophila ATF4 in photoreceptor degeneration, oogenesis and wing development

Disease Models & Mechanisms ◽

10.1242/dmm.049119 ◽

2021 ◽

Author(s):

Deepika Vasudevan ◽

Hidetaka Katow ◽

Huai-Wei Huang ◽

Grace Tang ◽

Hyung Don Ryoo

Keyword(s):

Control Mechanisms ◽

Loss Of Function ◽

Pupal Development ◽

Gfp Fusion Protein ◽

Drosophila Model ◽

Evolutionarily Conserved ◽

Age Dependent ◽

Cellular Stressors ◽

Trap Line

Metazoans have evolved various quality control mechanisms to cope with cellular stress inflicted by external and physiological conditions. ATF4 is a major effector of the Integrated Stress Response (ISR), an evolutionarily conserved pathway that mediates adaptation to various cellular stressors. Loss of function of Drosophila ATF4, encoded by the gene cryptocephal (crc), results in lethality during pupal development. The roles of crc in Drosophila disease models and in adult tissue homeostasis thus remain poorly understood. Here, we report that a protein-trap MiMIC insertion in the crc locus generates a crc-GFP fusion protein that allows visualization of crc activity in vivo. This allele also acts as a hypomorphic mutant that uncovers previously unknown roles for crc. Specifically, the crc protein-trap line shows crc-GFP induction in a Drosophila model for Retinitis Pigmentosa (RP). This crc allele renders flies more vulnerable to amino acid deprivation and age-dependent retinal degeneration. These mutants also show defects in wing veins and oocyte maturation. Together, our data reveal previously unknown roles for crc in development, cellular homeostasis and photoreceptor survival.

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Abstract 12877: Magnesium Deficiency Causes Reversible Metabolic Diastolic and Systolic Cardiomyopathies

Circulation ◽

10.1161/circ.142.suppl_3.12877 ◽

2020 ◽

Vol 142 (Suppl_3) ◽

Author(s):

Man Liu ◽

Hong LIU ◽

Feng Feng ◽

An Xie ◽

Cody R Hou ◽

...

Keyword(s):

Heart Failure ◽

Mitochondrial Dysfunction ◽

Magnesium Deficiency ◽

Diastolic Heart Failure ◽

Cellular Level ◽

Normal Diet ◽

Ca Channel ◽

Mg Deficiency ◽

Mitochondrial Membrane Depolarization

Introduction: Low circulating magnesium (Mg) level is associated with increased cardiovascular mortality, and conversely, dietary Mg intake is associated with a decreased risk of developing heart failure. Hypothesis: We investigated whether Mg deficiency alone could cause cardiomyopathy. Methods: C57BL/6J mice were fed with a low-Mg diet (low-Mg, 15-30 mg/kg Mg) or a normal diet (nl-Mg, 600 mg/kg Mg) for 6 weeks. To test reversibility, half of the low-Mg mice were fed then with normal diet for another 6 weeks (low→nl-Mg group). Results: Mg deficiency increased mortality, especially in female mice. After 6 weeks of low-Mg diet, surviving mice showed significantly decreased serum Mg (0.9±0.1 vs. 2.8±0.1 mg/dL for nl-Mg) and a reciprocal increase in serum Ca, K, and Na. Low-Mg mice exhibited a decreased cardiac ejection fraction (EF%, 39.8±1.9% vs. 52.0±1.7% of nl-Mg) and impaired relaxation (E/e’: 21.1±1.1 vs. 15.4±0.4 of nl-Mg) in echocardiography. At the cellular level, ATP, Ca transient amplitude, sarcoplasmic reticulum (SR) Ca load, resting sarcomere length, and sarcomere shortening were all decreased significantly in low-Mg hearts and cardiomyocytes. These changes were accompanied by evidence of mitochondrial dysfunction with significantly increased mitochondrial ROS production and mitochondrial membrane depolarization. Mg repletion normalized electrolytes, contraction, relaxation, and cellular changes. The SR Ca pump (SERCA) was decreased, the SR Ca channel RyR2 oxidized, and cardiac myosin binding protein C S-glutathionylated in low-Mg mouse hearts. These changes were normalized with Mg repletion. In vivo , mitoTEMPO treatment during low Mg diet improved the cardiac relaxation and increased cellular ATP levels without improving contraction. Conclusions: Mg deficiency caused a reversible diastolic and systolic cardiomyopathy associated with mitochondrial dysfunction. This cardiomyopathy may explain the relationship of hypomagnesemia and worsening heart failure. Mg intake could reverse these changes, reinforcing the known correlation of increased Mg intake and reduced heart failure symptoms and mortality. In deficiency states, Mg supplementation may represent a novel treatment for systolic and diastolic heart failure.

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Abstract 18575: The Transcriptional Cofactor Eyes Absent 4 is a Critical Regulator for Maintaining Normal Cardiac Function

Circulation ◽

10.1161/circ.130.suppl_2.18575 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Tatjana Williams ◽

Moritz Hundertmark ◽

Peter Nordbeck ◽

Sabine Voll ◽

Anahi Paula Arias-Loza ◽

...

Keyword(s):

Heart Failure ◽

Cardiac Function ◽

Interstitial Fibrosis ◽

Critical Role ◽

Cardiac Physiology ◽

Eyes Absent ◽

Age Dependent ◽

Normal Cardiac Function

Introduction: E193, a human mutation in the transcription cofactor Eyes absent 4 (EYA4) causes hearing impairment followed by terminal heart failure, defining an important role for Eya4 in maintaining normal cardiac function. METHODS AND RESULTS: First, in-vitro experiments show that overexpression of Eya4 and the mutant isoform alter the expression of p27kip1 on both, transcript and protein levels. Next, we generated transgenic mice with cardiomyocyte-specific Eya4 or E193 overexpression to elucidate the in vivo function of Eya4 upon cardiac physiology. Luciferase and CHIP assays revealed that Eya4 and E193 bind to and regulate p27 expression in a contradictory manner, as already seen in vitro. Activity and phoshorylation of the downstream molecules CK2α and HDAC2 were significantly elevated in Eya4 mice, whereas they were significantly reduced in E193 animals compared to WT littermates. MRI and hemodynamic analysis indicate that a constitutive overexpression of Eya4 results in the age-dependent development of hypertrophy already under baseline conditions with no obvious functional effects, whereas E193 overexpressing animals develop onset of dilative cardiomyopathy as seen in human patients carrying the E193 mutation. Morphometric analysis proved ventricular hypertrophy or dilation of the LV associated with a thinning of the myocardial wall, interstitial fibrosis of myocardial tissue and alterations in cell size. Re-activation of fetal genes also occured in both TG models, characteristic for cardiac disease. Both cardiac phenotypes were aggravated upon pressure overload. Finally, we identified a new human heterozygous truncating Eya4 mutation, E215, which leads to similar clinical features of disease and a stable myocardial expression of the mutant protein. Conclusion: Our results implicate that Eya4 plays a critical role in regulating normal cardiac physiology and function via Six1/p27/CK2α/HDAC2 and that an imbalance within the Eya4/Six1 transcriptional complex leads to an age dependent onset of cardiomyopthy and heart failure.

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Interferon-γ ablation exacerbates myocardial hypertrophy in diastolic heart failure

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00298.2012 ◽

2012 ◽

Vol 303 (5) ◽

pp. H587-H596 ◽

Cited By ~ 39

Author(s):

Anthony G. Garcia ◽

Richard M. Wilson ◽

Joline Heo ◽

Namita R. Murthy ◽

Simoni Baid ◽

...

Keyword(s):

Heart Failure ◽

Cardiac Hypertrophy ◽

Ventricular Myocytes ◽

Salt Water ◽

Diastolic Heart Failure ◽

Velocity Ratio ◽

Interleukin 10 ◽

Interferon Γ ◽

Left Ventricular

Diastolic heart failure (HF) accounts for up to 50% of all HF admissions, with hypertension being the major cause of diastolic HF. Hypertension is characterized by left ventricular (LV) hypertrophy (LVH). Proinflammatory cytokines are increased in LVH and hypertension, but it is unknown if they mediate the progression of hypertension-induced diastolic HF. We sought to determine if interferon-γ (IFNγ) plays a role in mediating the transition from hypertension-induced LVH to diastolic HF. Twelve-week old BALB/c (WT) and IFNγ-deficient (IFNγKO) mice underwent either saline ( n = 12) or aldosterone ( n = 16) infusion, uninephrectomy, and fed 1% salt water for 4 wk. Tail-cuff blood pressure, echocardiography, and gene/protein analyses were performed. Isolated adult rat ventricular myocytes were treated with IFNγ (250 U/ml) and/or aldosterone (1 μM). Hypertension was less marked in IFNγKO-aldosterone mice than in WT-aldosterone mice (127 ± 5 vs. 136 ± 4 mmHg; P < 0.01), despite more LVH (LV/body wt ratio: 4.9 ± 0.1 vs. 4.3 ± 0.1 mg/g) and worse diastolic dysfunction (peak early-to-late mitral inflow velocity ratio: 3.1 ± 0.1 vs. 2.8 ± 0.1). LV ejection fraction was no different between IFNγKO-aldosterone vs. WT-aldosterone mice. LV end systolic dimensions were decreased significantly in IFNγKO-aldosterone vs. WT-aldosterone hearts (1.12 ± 0.1 vs. 2.1 ± 0.3 mm). Myocardial fibrosis and collagen expression were increased in both IFNγKO-aldosterone and WT-aldosterone hearts. Myocardial autophagy was greater in IFNγKO-aldosterone than WT-aldosterone mice. Conversely, tumor necrosis factor-α and interleukin-10 expressions were increased only in WT-aldosterone hearts. Recombinant IFNγ attenuated cardiac hypertrophy in vivo and modulated aldosterone-induced hypertrophy and autophagy in cultured cardiomyocytes. Thus IFNγ is a regulator of cardiac hypertrophy in diastolic HF and modulates cardiomyocyte size possibly by regulating autophagy. These findings suggest that IFNγ may mediate adaptive downstream responses and challenge the concept that inflammatory cytokines mediate only adverse effects.

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Abstract 5307: Cardiomyocyte Sodium Accumulation Promotes Diastolic Dysfunction in Serca2 Knockout Mice

Circulation ◽

10.1161/circ.118.suppl_18.s_524 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

William Louch ◽

Karina Hougen ◽

Magnus Aronsen ◽

Halvor K Mork ◽

Ivar Sjaastad ◽

...

Keyword(s):

Heart Failure ◽

Sarcoplasmic Reticulum ◽

Gene Deletion ◽

Diastolic Heart Failure ◽

Functional Decline ◽

Western Blots ◽

Sodium Accumulation ◽

Time Points ◽

Rate Of Decline

Terminal decompensation during heart failure involves deterioration of diastolic function. We investigated the mechanisms underlying this functional decline in mice with cardiomyocyte-specific, conditional excision of the Serca2 gene (KO). At 4 weeks following gene deletion, SERCA levels in KO cardiomyocytes were reduced by more than 95% from flox-flox (FF) controls. Surprisingly, echocardiographic measurements indicated only moderate impairment of in vivo function, as systolic and diastolic tissue velocities were 62% and 72% of FF values, respectively. Diastolic heart failure developed in KO between 6 and 7 weeks, as diastolic tissue velocity rapidly declined to 51% of FF values. We compared cardiomyocyte contractions and Ca 2+ cycling at the 4 and 7 week time points. In KO cells, contractions were reduced between 4 and 7 weeks (from 40% to 14% of FF values), and the rate of relaxation was slowed (from 11% to 3% of FF values). Similar alterations were observed in Ca 2+ transients. Sarcoplasmic reticulum (SR) Ca 2+ content was markedly reduced in 4-week KO, although a minute thapsigargin-sensitive SR Ca 2+ release could be induced. SR content was further decreased in 7-week KO and SR Ca 2+ release was not detectable, although Western blots showed no difference in SERCA levels between 4 and 7 week KO. Ca 2+ influx via Ca 2+ channels was enhanced in KO (integrated current ≈200% of FF) at both time points. However, greater NCX-mediated Ca 2+ extrusion in 4-week KO was partially reversed in 7-week KO due to elevation in cytosolic [Na + ] (34 mM vs 25 mM in FF). Normalizing cytosolic [Na + ] using patch clamp increased the rate of decline of the Ca 2+ transient in 7-week KO to 4-week KO values. Thus, KO mice compensate for loss of SR function by increasing trans-sarcolemmal Ca 2+ flux. However, in the longer term, cytosolic Na + accumulation impairs NCX-mediated Ca 2+ extrusion, which promotes development of diastolic heart failure.

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Left Atrial Circulatory Assistance in Simulated Diastolic Heart Failure Model: First in Vitro and in Vivo

Journal of Cardiac Failure ◽

10.1016/j.cardfail.2021.11.024 ◽

2022 ◽

Author(s):

Chihiro Miyagi ◽

Kiyotaka Fukamachi ◽

Barry D. Kuban ◽

Shengquiang Gao ◽

Takuma Miyamoto ◽

...

Keyword(s):

Heart Failure ◽

Left Atrial ◽

Diastolic Heart Failure ◽

Failure Model ◽

Heart Failure Model

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Abstract 18739: Acute Effect of the Na+/ca2+ Exchanger (ncx) Inhibitor Sea0400 on Diastolic Function in vivo and in vitro in a Model of Heart Failure With Preserved Ejection Fraction

Circulation ◽

10.1161/circ.130.suppl_2.18739 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Uwe Primessnig ◽

Burkert Pieske ◽

Frank Heinzel

Keyword(s):

Heart Failure ◽

Ejection Fraction ◽

Protein Expression ◽

Diastolic Heart Failure ◽

Sham Operation ◽

Preserved Ejection Fraction ◽

Active Relaxation ◽

Reverse Mode

Background: Heart failure with preserved ejection fraction (HFPEF) is increasingly common but underlying cellular mechanisms are not well understood. We investigated cardiomyocyte function and the role of SEA0400, in vivo and in vitro in a rat model of diastolic heart failure. Methods: Young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP). After 24 weeks in vivo (pressure-volume loops) and in vitro characterization (cardiomyocyte function (Ca2+ transients, sarcoplasmic reticulum (SR) diastolic Ca2+ leak (Ca2+ sparks) and SR Ca2+ content)) as well as NCX function (caffeine-induced Ca2+ transient, TAU) and protein expression) were determined without an with the NCX inhibitor SEA0400 (in vivo: 1 mg/kgKG for 30 minutes/ in vitro: 300nM for 5 minutes incubation). Results: NXT rats showed stable compensated renal impairment and signs and symptoms of HFPEF (hypertrophied left ventricle (LV), left- and upward shift of end diastolic pressure (EDP) volume relationship, increased lung weight/body weight ratio indicating pulmonary congestion and preserved LV systolic function (EF, dP/dt)). In LV cardiomyocytes from NXT Ca2+ transient amplitude was unchanged but time for early (50%) decay was significantly prolonged at 24 weeks and correlated with diastolic dysfunction (EDP) in vivo. TAU of the caffeine transient was significantly prolonged at 24 weeks indicating reduced NCX forward mode activity, while NCX protein expression was up-regulated, suggesting increased NCX reverse mode activity. Ca2+ spark frequency was increased and SR Ca2+ content was decreased in NXT (p<0.05). SEA0400 significantly accelerated Ca2+ transient decay and reduced Ca2+ spark frequency in NXT. Acute in vivo treatment with SEA0400 significantly enhanced active relaxation (isovolumetric relaxation constant) of the LV in NXT. Conclusion: This model of HFPEF is associated with prolonged cytosolic Ca2+ decay and increased diastolic Ca2+ leak in LV cardiomyocytes. Acute treatment with NCX inhibitor SEA0400 normalized cytosolic Ca2+ transients, improved net transsarcolemmal Ca2+ export and decreased SR Ca2+ leak in NXT, in line with a role for reverse mode NCX activity in HFPEF. In vivo active relaxation was improved after acute SEA0400 treatment.

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