Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin

Craig A Eyster; Satoshi Matsuzaki; Maria F Newhardt; Jennifer R Giorgione; Kenneth M Humphries

doi:10.1371/journal.pone.0231806

. 2020 Aug 20;15(8):e0231806. doi: 10.1371/journal.pone.0231806

Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin

Craig A Eyster ¹, Satoshi Matsuzaki ¹, Maria F Newhardt ^1,², Jennifer R Giorgione ¹, Kenneth M Humphries ^1,^2,^*

Editor: Makoto Kanzaki³

PMCID: PMC7444578 PMID: 32817622

Abstract

The cAMP-dependent protein kinase (PKA) signaling pathway is the primary means by which the heart regulates moment-to-moment changes in contractility and metabolism. We have previously found that PKA signaling is dysfunctional in the diabetic heart, yet the underlying mechanisms are not fully understood. The objective of this study was to determine if decreased insulin signaling contributes to a dysfunctional PKA response. To do so, we isolated adult cardiomyocytes (ACMs) from wild type and Akita type 1 diabetic mice. ACMs were cultured in the presence or absence of insulin and PKA signaling was visualized by immunofluorescence microscopy using an antibody that recognizes proteins specifically phosphorylated by PKA. We found significant decreases in proteins phosphorylated by PKA in wild type ACMs cultured in the absence of insulin. PKA substrate phosphorylation was decreased in Akita ACMs, as compared to wild type, and unresponsive to the effects of insulin. The decrease in PKA signaling was observed regardless of whether the kinase was stimulated with a beta-agonist, a cell-permeable cAMP analog, or with phosphodiesterase inhibitors. PKA content was unaffected, suggesting that the decrease in PKA signaling may be occurring by the loss of specific PKA substrates. Phospho-specific antibodies were used to discern which potential substrates may be sensitive to the loss of insulin. Contractile proteins were phosphorylated similarly in wild type and Akita ACMs regardless of insulin. However, phosphorylation of the glycolytic regulator, PFK-2, was significantly decreased in an insulin-dependent manner in wild type ACMs and in an insulin-independent manner in Akita ACMs. These results demonstrate a defect in PKA activation in the diabetic heart, mediated in part by deficient insulin signaling, that results in an abnormal activation of a primary metabolic regulator.

Introduction

Heart disease is the leading cause of death for patients with type I or type II diabetes [1]. This is in part because diabetes directly impacts cardiac function independently of other comorbidities. This is termed diabetic cardiomyopathy and it is a multi-factorial condition resulting from the metabolic stresses of disrupted insulin signaling, hyperglycemia and hyperlipidemia, and mitochondrial dysfunction [2]. In addition, there are also disruptions in protein kinase A (PKA) signaling, the molecular pathway that mediates the metabolic and contractile responses to sympathetic stimulation [3, 4]. While the molecular mechanisms contributing to diabetic cardiomyopathy are highly interrelated, the relationship between metabolic perturbances and changes in PKA signaling are not fully understood.

In the healthy heart the sympathetic nervous system functions through β-adrenergic signaling to increase cardiac contractility. Catecholamines bind to Gα_s-coupled β-adrenergic receptors, stimulate adenylate cyclase, and subsequently increase cAMP to activate PKA. PKA then phosphorylates proteins involved in calcium cycling (troponin, SERCA, and phospholamban) and proteins that affect metabolic substrate selection (phosphofructokinase-2 (PFK-2) and acetyl-CoA carboxylase-2) [5, 6]. Glucose uptake and oxidation are the primary means of meeting the rapid increase in energy demands in response to sympathetic stimulation [6, 7]. In this way the increase in contractility is orchestrated with activation of metabolic pathways to ensure energy demands are met.

As an insulin sensitive tissue, the heart is affected by either decreases in circulating insulin or by the loss of insulin signaling that occur with type 1 or type 2 diabetes [8, 9]. The primary role of insulin is to increase glucose uptake and metabolism. Thus, the decrease in insulin signaling contributes to the metabolic inflexibility whereby the heart increases reliance on fatty acid oxidation, at the expense of decreased glucose usage, to meet energetic demands [10]. Over the long term, this metabolic inflexibility promotes lipotoxicity, mitochondrial dysfunction, and oxidative stress. Increasing evidence suggests there are interactions between insulin and β-adrenergic signaling. For example, hyperinsulinemia can blunt PKA signaling via an increase in phosphodiesterase 4 which increases cAMP hydrolysis [11, 12]. In our own work, we found PKA signaling is affected in a type 1 diabetic mouse model via changes in PKA activity that are downstream of receptor activation and adenylate cyclase activity [3]. Furthermore, we identified that a loss of insulin signaling, in both type 1 and type 2 diabetic conditions, decreases the content of the PKA substrate, PFK-2 [4]. In the healthy heart, phosphorylation of PFK-2 increases the production of fructose-2,6-bisphosphate, an allosteric activator of PFK-1 which is a committed and rate-limiting step of glycolysis [6]. Thus, the loss of insulin signaling disrupts a mechanism whereby β-adrenergic signaling increases glycolysis to meet energetic demands.

The goal of the present work was to define how the loss of insulin signaling impacts β-adrenergic signaling in cardiomyocytes. Adult mouse cardiomyocytes (ACMs) were isolated from control and Akita diabetic mice and then cultured in the presence or absence of insulin. ACMs were subsequently stimulated with β-adrenergic agonists and PKA signaling was determined by immunofluorescence microscopy. We have identified a striking decrease in PKA signaling in wild type ACMs cultured in the absence of insulin. This effect was mirrored in ACMs isolated from Akita type 1 diabetic mice, regardless of the presence of added insulin. Using phospho-specific antibodies, we found that the phosphorylation of proteins involved in calcium regulation were unaffected by the absence of insulin. In contrast, the metabolic target, PFK-2, was highly sensitive. Our results demonstrate the effects of PKA on cardiomyocyte function is dependent upon the actions of insulin.

Materials and methods

Adult mouse cardiomyocyte isolation

Adult cardiomyocytes from 5-month C57BL/6J or C57BL/6J-Ins2^Akita/J male mice (Akita, The Jackson Laboratory 003548) were isolated and cultured as previously described [3, 13]. Akita mice are a well-established model of hypoinsulinemia and hyperglycemia [14]. Blood glucose was measured by a glucose test strip (Contour) at the time of sacrifice to confirm hyperglycemia. All Akita mice had blood glucose levels of at least 400 mg/dl. Briefly, after isoflurane administration the heart was excised, the aorta was cannulated, and it was then perfused with type II collagenase (Worthington #LS004176). Calcium was reintroduced to the subsequent single cell suspension and cells were plated on laminin (Corning 354232) coated plates. Media was switched to serum-free culture media (minimal essential medium with Hanks’ balanced salt solution, Gibco (11575–032) supplemented with 0.2mg/mL sodium bicarbonate, penicillin-G, 0.1%BSA, glutamine, 10mM butanedione monoxime, and 5μg/mL insulin as indicated. Cells were cultured 18h at 37°C and 5%CO₂ with indicated drugs as described in figure legends. All procedures were approved by the Oklahoma Medical Research Foundation Animal Care and Use Committee.

Antibodies and drugs

Rabbit polyclonal antibodies to phospho-PKA substrate (9621S), PKA C-α (4782S), phospho-PFK2 (13064S), phospho-Ser16/Thr17-phospholamban (8496S), and phospho-Troponin I were purchased from Cell Signaling Technology. Rabbit polyclonal anti-PDE4D (ab14613) was purchased from Abcam. Alexa Fluor 488 goat anti-rabbit IgG (A11034) and Alexa Fluor 546 phalloidin (A22283) were purchase from Invitrogen. Insulin solution human (19278), (-)-Isoproterenol hydrochloride (16504), 3-Isobutyl-1-methylxanthine (15879) and 8-Bromoadenosine 3’,5’-cyclic monophosphate sodium salt (B7880) were purchased from Sigma. Phosphodiesterase inhibitor Tocriset containing Milrinone, Cilostamide, Zardaverine, (R)-(-)-Rolipram, and Ro 20–1724 (Cat. No. 1881) along with MMPX (Cat. No. 0552) and EHNA hydrochloride (Cat. No. 1261) were purchased from Tocris.

Microscopy

Methods for immunofluorescent staining have been previously described [15] and adapted for primary mouse cardiomyocytes (ACMs). Briefly, ACMs were plated on laminin coated coverslips (Fisherbrand Microscope Cover Glass, 12-545-80) (1 coverslip per well, 24-well plate) for 1h post isolation. Cells were cultured overnight and treated with drugs as described in the figure legends. Following incubation, cells were washed 1X with PBS (Gibco 14190–144) and fixed for 20min in 4% paraformaldehyde (Electron Microscopy Sciences 15710). Cells were washed 2X with PBS and blocked for 1 hr in 2% Blocker BSA (Thermo 37525). Coverslips were inverted onto 50μL of block solution containing 0.1% Triton X-100 (Sigma T9284) and 1–250 dilution of primary antibodies as indicated on parafilm covered 150mm gridded tissue culture dish (Falcon 353025) and incubated overnight at 4°C. Coverslips were returned to tissue culture dish and washed 3X with block solution and then inverted on 50μL of block solution containing .1% Triton X-100 (Sigma T9284) and 1–250 dilution of secondary antibodies/phalloidin for 1hr at room temperature. Coverslips were then washed 2X with block solution and 1X with PBS and then inverted onto 4μL Vectashield mounting media with DAPI (Vector Laboratories H-1200) and sealed with nail polish (Electron Microscopy Sciences 72180). Cells were imaged on a Zeiss LSM-710 confocal (Carl Zeiss). The microscopy settings were kept the same between each experiment to facilitate unbiased comparisons of fluorescence intensities. Micrographs are maximum intensity projections of 12 picture z-stacks average step size of 1.5μm. Projection images were quantitated in Zen Black (Carl Zeiss version 2012 SP5 FP3) from maximum intensity projection by drawing a free polygon outline around the cell and measuring mean fluorescence intensity. Each experiment constitutes of at least three biological replicates, indicated in Figure Legends, with at least six individual cells per experiment for a total of at least eighteen total cells quantitated for each data point.

Western blot analysis

Cardiomyocytes were cultured in 12 well plates, with or without insulin, and drugs were added as indicated in the figure legends. Media was then removed, cells were washed with 0.5 mL PBS, and 75 uL of 1X sample buffer containing 25 mM DTT and 1X Halt Protease/Phosphatase Inhibitor Cocktail (ThermoFisher #78442) was added per well. Samples were heated at 95°C for 5 min, resolved by SDS-PAGE (4–12% NuPAGE Bis-Tris gel, Thermo Fisher), transferred to nitrocellulose membranes, and blocked for 30 min with Odyssey TBS blocking buffer (LI-COR). Antibodies were diluted 1:2000 in block buffer and added to blots overnight at 4°C, subsequently washed the following day, and the secondary antibody (IRDye 800CW, LI-COR; 1:10,000 dilution) was incubated for 1h. Following additional washing, blots were imaged on an Odyssey CLx system and analyzed using the Image Studio software (LI-COR).

PKA activity

PKA activity was assayed using the PepTag Non-Radioactive cAMP-Dependent Protein Kinase Assay Kit (Promega) as previously described [16]. Briefly, ACMs were isolated and cultured for 18hr in the presence or absence of insulin. ACMs were then treated with 0.25μM isoproterenol for 30min. Media was removed and ACMs were lysed on ice with 100μL RIPA buffer (100mM KCL, 100mM NaPO₄, 0.1% NP40, PH 7.4) containing HALT phosphatase and protease inhibitor cocktail (ThermoFisher) and 10mM IBMX (Sigma). Following protocol guidelines, reaction buffer, PepTag A1 peptide, and water were premixed. Next, 20μL of freshly prepared lysate (at approximately 0.1mg/ml of total protein) was added and incubated for 15 min at RT. Conditions were optimized to ensure the reaction rate was linear at this time point. The reaction was stopped by the addition of 1mM cAMP Dependent Protein Kinase Inhibitor peptide (PKI-tide; Sigma) in a 50% glycerol solution to a final concentration of 45uM. Phosphorylated peptide was separated from unphosphorylated peptide by electrophoresis on a .8% agarose gel (100 V for 30 min). The gel was imaged with a D-Digit scanner (LI-COR) and the intensities of phosphorylated peptides were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). The protein concentration of each lysate was determined by Bradford assay and was used to standardize activities.

Statistical analysis

GraphPad Prism 7.02 was used for statistical analysis and mean fluorescence intensity values were evaluated using one-way ANOVA with multiple comparisons using Tukey’s test. Statistical analysis was performed on the total number of cells analyzed, comprised of at least 18 cells from 3 unique cardiomyocyte preparations and is indicated in the Figure Legends. Similar statistical significance and the same conclusions are reached if instead the data from a given cell preparation are averaged and then analyzed. Statistical significance is noted in the figure legends.

Results

β-adrenergic signaling is decreased under diabetic conditions in cardiomyocytes

Initial experiments were performed to validate methodology for evaluating PKA signaling by immunofluorescence in adult mouse cardiomyocytes (ACMs). ACMs were isolated from control mice and cultured for 18h in the presence of insulin and then stimulated with isoproterenol (ISO, 0.25μM) for 30min. Cells were then fixed and PKA activity was visualized by immunofluorescence microscopy using an antibody that specifically recognizes the protein consensus phosphorylation sequence (RRXS/T, where S or T is phosphorylated) that is specific for PKA substrates [5]. This antibody is widely used in the literature (123 citations per CiteAb.com) and has been previously used as a means to identify changes in PKA activity by immunohistochemistry [17]. We observed low levels of PKA substrate phosphorylation basally and this was increased approximately 3-fold by ISO treatment (Fig 1A, top panels). Detection of PKA activity by this manner revealed largely diffuse staining but with increased intensity proximal to the sarcolemma and intermittently in areas consistent with Z-bands.

Fig 1 — (A) Adult mouse cardiomyocytes from wild-type or Akita mice were incubated overnight in the presence or absence of insulin (ins) and treated with 0.25μM isoproterenol for 30min as indicated. Cells were fixed and stained with rabbit anti-PKA substrate antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for each condition is shown. Scale bar 10μm. (B) Quantitation of mean fluorescence intensity (MFI) for PKA-substrate are presented as whisker plots that encompass data from at least 30 cells (n = 3 biological replicates, and at least 10 cells per experiment). The box dimensions extend from the 25^th to the 75^th percentiles; whiskers describe the minimum to maximum values. The median is plotted as a horizontal line within the box. *, ISO treatment caused a statistically significant increase in all conditions (p < .001) by one-way ANOVA. (‡) ISO stimulated samples from WT without insulin and Akita were significantly reduced (p < .001) compared to stimulated WT with insulin by one-way ANOVA with Tukey post hoc test. (C) Representative Western blot for anti-PKA substrate analysis of lysates from primary mouse cardiomyocytes treated as shown. (D) Quantitation of western blot experiments (n = 4). (E) PKA activity was measured in WT ACMs cultured in the presence or absence of insulin and with or without ISO stimulation as described in the Materials and Methods. PKA assays were performed in triplicate from 2 different ACM preparations. *, p < .05; **, p < .005 by two-way ANOVA with Tukey post hoc test.

We next examined how the lack of insulin affects PKA signaling. Freshly isolated ACMs were cultured in insulin-free media for 18h and then stimulated with ISO. PKA-substrate phosphorylation was significantly blunted both basally and following ISO treatment (Fig 1A and 1B). This decrease in PKA-substrate phosphorylation was not due to altered kinetics. A time course study, with increasing durations of ISO stimulation, revealed phosphorylation reached a maximal threshold within 10min regardless of whether insulin was present (S1 Fig). In contrast to the immunofluorescence data, no significant differences were observed when PKA substrate phosphorylation was examined by Western blot (Fig 1C and 1D). It is possible that less abundantly expressed proteins may be differentially phosphorylated by the presence or absence of insulin but not detected by Western blot. We also measured PKA activity directly (Fig 1E) and found that culturing ACMs without insulin decreased basal PKA activity. However, the activation of PKA by ISO was not significantly affected by the insulin status. This supports that the loss of immunofluorescence intensity with the PKA substrate antibody is not due to overt loss of PKA activity. Rather, unique epitopes may be detected by the antibody under conditions that maintain native protein confirmations, as with immunofluorescence detection, as compared to the denaturing conditions of SDS-PAGE.

We next examined whether the chronic hypoinsulinemia that occurs with type 1 diabetes is also associated with changes in PKA signaling. Akita mice develop type I diabetes in the absence of obesity and insulitis and this is mediated by a mutation in the Ins2 gene that results in its improper release in response to glucose [14, 18]. Akita ACMs were isolated and cultured overnight in the presence or absence of insulin. As shown in Fig 1A and 1B, Akita ACMs had substantially reduced PKA substrate phosphorylation upon stimulation with ISO as compared to WT. Furthermore, insulin did not enhance ISO-stimulated PKA substrate phosphorylation in Akita ACMs. This supports that decreased insulin signaling affects PKA signaling and that overnight insulin treatment of Akita ACMs is insufficient for rescue.

Insulin signaling is necessary downstream of cAMP production

The decrease in PKA substrate phosphorylation in cardiomyocytes cultured without insulin may be due to changes in β-adrenergic receptors or their response to ligand binding, thereby leading to a dampened response to ISO stimulation. We therefore examined direct activation of PKA using the cell permeable cAMP analog, 8Br-cAMP. Like ISO, 8Br-cAMP induced a robust increase in PKA-substrate phosphorylation in ACMs isolated from control mice and cultured with insulin. However, substrate phosphorylation stimulated by 8Br-cAMP was significantly blunted in ACMs cultured overnight without insulin (Fig 2). Likewise, ACMs isolated from adult Akita mice had significantly blunted response to 8Br-cAMP as compared to WT. Furthermore, insulin did not enhance 8Br-cAMP-stimulated PKA substrate phosphorylation in Akita ACMs. While we cannot completely rule out changes in β-adrenergic receptor content or activity, these results support that the defect in PKA signaling induced by the absence of insulin is downstream of receptors and cAMP production.

Fig 2 — (A) ACMs from wild-type or Akita mice were incubated overnight in the presence or absence of insulin (ins) and treated with 250μM 8-bromo-cAMP (8Br) or 500μM IBMX for 30min as indicated. Cells were fixed and stained with rabbit anti-PKA substrate antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for each condition is shown. Scale bar 10μm. (B&C) Quantitation of mean fluorescence intensity (MFI) for PKA-substrate are presented as whisker plots that encompass data from at least 18 cells, as detailed in Fig 1 (n = 3 biological replicates, and at least 6 cells per experiment). (*) Isoproterenol treatment caused a significant increase (p < .001) in all conditions. (‡) 8-bromo-cAMP or IBMX stimulated samples from WT without insulin and Akita were significantly reduced (p < .001) compared to stimulated WT with insulin. Statistics were performed by one-way ANOVA with Tukey post hoc test.

Alterations in PKA signaling could be attributed to fluctuations in enzyme content. PKA catalytic subunit levels were therefore examined in ACMs from control and Akita mice cultured with or without insulin. As shown in S2 Fig, there were no significant differences in PKA content under the experimental conditions as determined by immunofluorescence or Western blot (not shown). This indicates the loss of PKA substrate phosphorylation is not mediated to decreased PKA catalytic subunit content.

Phosphodiesterase inhibition increase PKA signaling but do not restore deficits induced by loss of insulin

Phosphodiesterases (PDEs) hydrolyze cAMP and are an essential component in modulating proper PKA signaling. Thus, insulin mediated changes in PDE activity could contribute to the observed effects on PKA signaling shown in Fig 1. We therefore examined the effect of 3-isobuty-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, on ACMs from control and Akita mice to determine whether blocking PDE activity is sufficient to recover PKA signaling. Addition of IBMX, in the absence of other PKA agonists, was sufficient to stimulate PKA signaling by 2.5-fold (Fig 2). ACMs isolated from adult Akita mice had significantly blunted response to IBMX as compared to WT. Furthermore, insulin did not enhance IBMX-stimulated PKA substrate phosphorylation in Akita ACMs. This demonstrates PDE inhibition is sufficient to stimulate PKA substrate phosphorylation similarly to a PKA agonist when insulin is present. However, this effect is blunted when WT ACMs are cultured in the absence of insulin. The stimulatory effects of IBMX are blunted in Akita ACMs and is unaffected by the insulin status.

We next sought to determine the combined effects of ISO and IBMX on PKA signaling. ACMs from control or Akita diabetic mice were cultured overnight in the presence or absence of insulin and then treated with combinations of IBMX and ISO. IBMX enhanced ISO stimulation of PKA substrate phosphorylation under all conditions (Fig 3). This demonstrates the importance of PDE activity in attenuating catecholamine mediated PKA signaling. However, the maximum PKA substrate phosphorylation was nevertheless blunted in control ACMs cultured in the absence of insulin. Akita ACMs exhibited a similar additive increase in PKA substrate phosphorylation with the combination of ISO and IBMX. However, the maximum intensity was decreased as compared to WT ACMs and unaffected by the insulin status.

Fig 3 — (A) ACMs from wild-type or Akita mice were incubated overnight in the presence or absence of insulin (ins) and treated with 500μM IBMX or .25μM Iso/500μM IBMX for 30min as indicated. Cells were fixed and stained with rabbit anti-PKA substrate antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for each condition is shown. Scale bar 10μm. (B) Quantitation of mean fluorescence intensity (MFI) for PKA-substrate are presented as whisker plots that encompass data from at least 18 cells, as detailed in Fig 1 (n = 3 biological replicates, and at least 6 cells per experiment). (*) Isoproterenol and IBMX treatment caused a significant increase (p < .001) compared to IBMX alone in all conditions. (‡) stimulated Akita and WT–ins conditions are significantly decreased compared to matching WT +ins conditions. Statistics were performed by one-way ANOVA with Tukey post hoc test.

Recent work has identified a relationship between cardiac insulin signaling and the content and activity of PDE4 [11]. Specifically, the hyperinsulinemia that occurs with type 2 diabetes is associated with increased PDE4B content which thereby decreases cAMP and attenuates β-adrenergic signaling [11]. We therefore tested to see if reciprocally the lack of insulin affects specific phosphodiesterases. Control ACMs were treated with a panel of phosphodiesterase inhibitors including IBMX (nonspecific PDE inhibitor), EHNA (PDE2 specific), MMPX (Calmodulin sensitive cyclic GMP specific), milrinone (PDE3 specific), clostramide (PDE3 specific), RO-20-1724 (PDE4 specific), rolipram (PDE4 specific), and zardavsine (PDE3/4 specific). All of the inhibitors, except EHNA, stimulated phosphorylation of PKA substrates. However, the most pronounced effect was observed with inhibitors of PDE4 (Fig 4). Next, we tested if PDE4 inhibition could increase PKA signaling in the absence of insulin. PKA signaling in ACMs from control mice treated with RO-20-1724 closely approximated the effects of IBMX. The drug enhanced PKA signaling on its own or in combination with ISO, but nevertheless signaling was decreased in ACMs cultured in the absence of insulin (S3 Fig). We also examined PDE4 by immunofluorescence and determined that the presence or absence of insulin had no effect on its content (S4 Fig). Consistent with previous reports [19, 20], this supports that PDE4 is the primary regulator of cAMP degradation in mouse cardiomyocytes, that its content is not affected by acute changes in insulin, and that its inhibition is not sufficient to fully recover PKA activity when insulin signaling is absent.

Fig 4 — (A) ACMs from wild-type or Akita mice were incubated overnight in the presence insulin (ins) and treated with 500μM IBMX or indicated phosphodiesterase inhibitors. Cells were fixed and stained with rabbit anti-PKA substrate antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for each condition is shown. Scale bar 10μm. (B) Quantitation of mean fluorescence intensity (MFI) for PKA-substrate are presented as whisker plots that encompass data from at least 18 cells, as detailed in Fig 1 (n = 3 biological replicates, and at least 6 cells per experiment). *, p < .001 by one-way ANOVA with Tukey post hoc test.

Phosphorylation of PFK2 is decreased in diabetic conditions

Our results demonstrate that the absence of insulin affects PKA substrate phosphorylation downstream of β-adrenergic receptors and that this is not mediated by changes in adenylate cyclase or PDE activities. We next evaluated PKA signaling using phospho-specific antibodies to identify substrates that may be differentially phosphorylated in the absence of insulin. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a glycolytic regulator and substrate of PKA [21, 22]. When the cardiac isoform (gene produce of pfkfb2) is phosphorylated, PFK-2 increases the production of fructose-2,6-bisphosphate, a potent allosteric activator of the glycolytic enzyme phosphofructokinase-1 (PFK-1). Furthermore, we have previously reported that the content of PFK-2, is regulated by insulin signaling [4, 23]. Control ACMs were cultured for 18h in the presence or absence of insulin and then stimulated with either ISO or IBMX. As shown in Fig 5, PFK-2 phosphorylation was significantly increased in wildtype ACMs stimulated by either ISO or IBMX regardless of whether cells were cultured with insulin. However, for each condition the magnitude of PFK-2 phosphorylation was significantly less in ACMs cultured in the absence of insulin as compared to those cultured with insulin. In Akita ACMs, PFK-2 phosphorylation was significantly repressed basally and was largely unresponsive to stimulation by either ISO or IBMX.

Fig 5 — (A) ACMs from wild-type or Akita mice were incubated overnight in the presence or absence of insulin (ins) and treated with 500μM IBMX or .25μM Iso for 30min as indicated. Cells were fixed and stained with rabbit anti-phospo-PFK2 antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for select conditions are shown. Scale bar 10μm. (B) Quantitation of mean fluorescence intensity (MFI) for pPFK2 are presented as whisker plots that encompass data from at least 18 cells, as detailed in Fig 1 (n = 3 biological replicates, and at least 6 cells per experiment). (*) IBMX or Isoproterenol treatment causes a significant increase (p < .001) compared to untreated WT cells; (†) Each WT (-ins) condition is significantly decreased (p < .001) compared to WT (+ins); (‡) all Akita conditions are significantly decreased (p < .001) compared to comparable WT conditions. Statistics performed by one-way ANOVA with Tukey post hoc test.

We next examined whether the phosphorylation of other well-described PKA substrates exhibit insulin sensitivity. Phospholamban (PBN) is an inhibitor of the sarcoplasmic reticulum calcium dependent ATPase (SERCA2) channel. PBN mediated inhibition of SERCA2 is relieved upon phosphorylation by PKA, thereby increasing calcium cycling in response to β-adrenergic signaling [24]. Troponin I (TnI) is part of the calcium-sensitive troponin complex that decreases myosin-actin crossbridges. Phosphorylation of TnI by PKA decreases the sensitivity of the complex to calcium and is important for increasing the inotropic response [25, 26]. As shown in Fig 6, PBN and TnI were robustly phosphorylated in response to ISO or IBMX and this was unaffected by 18h of insulin starvation. These results demonstrate that there is a differential sensitivity to insulin among β-adrenergic targets, with the metabolic target, PFK-2, being significantly repressed while those involved in contractility are sustained.

Fig 6 — (A&C) Primary mouse cardiomyocytes from wild-type mice were incubated overnight in the presence or absence of insulin (ins) and treated with .25μM Iso or 500μM IBMX for 30min as indicated. Cells were fixed and stained with rabbit anti-phospho-phospholamban (A) or rabbit anti-phospho-Troponin (C) antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for select conditions are shown. Scale bar 10μm. (B&D) Quantitation of mean fluorescence intensity (MFI) for pPhospholamban (B) or pTroponin (D) are presented as whisker plots that encompass data from at least 18 cells, as detailed in Fig 1 (n = 3 biological replicates, and at least 6 cells per experiment). (*) IBMX or isoproterenol treatment causes a significant (p < .001) increase by one-way ANOVA with Tukey post hoc test.

PKA substrate phosphorylation remains depressed upon acute insulin administration

Lastly, we sought to determine whether the loss of insulin has a sustained effect on PKA signaling. ACMs from wildtype mice were cultured for 18h in the absence of insulin and then stimulated acutely with combinations of ISO and insulin. As shown in Fig 7A and 7B, the acute addition of insulin alone failed to increase PKA substrate phosphorylation. Furthermore, the acute addition of insulin had no additive effect on ISO mediated PKA substrate phosphorylation. This lack of effect was not from deficiencies in the insulin signaling pathway. A time course of Akt phosphorylation was examined by Western blot in ACMs cultured in the presence or absence of insulin and then acutely treated with combinations of insulin and ISO (Fig 7C and 7D). ACMs that had been cultured overnight with insulin had low levels of Akt phosphorylation. Acute additions of ISO and insulin minimally increased Akt phosphorylation. This suggests that desensitization of the pathway occurs when cells are cultured continuously with insulin. In contrast, ACMs cultured overnight in the absence of insulin showed a robust increase in Akt phosphorylation upon acute insulin treatment regardless of the presence of ISO. The signal was maximal at 10min and then decreased over time. Thus, the lack of acute effect by insulin on PKA signaling was not due to unresponsiveness of the insulin signaling pathway.

Fig 7 — (A) Primary mouse cardiomyocytes from wild-type were incubated overnight in the absence of insulin and treated with 0.25μM Isoproterenol or insulin for 10min as indicated. Cells were fixed and stained with rabbit anti-PKA substrate antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for each condition is shown. Scale bar 10μm. (B) Quantitation of mean fluorescence intensity (MFI) for PKA-substrate are presented as whisker plots that encompass data from at least 18 cells, as detailed in Fig 1 (n = 3 biological replicates, and at least 6 cells per experiment). (*) Isoproterenol causes a significant increase (p < .05) by one-way ANOVA. (C) Representative western blot of anti-phospho-Akt-S473 (green) and total Akt (red) analysis of lysates from primary mouse cardiomyocytes treated as in B. (D) Western blot quantitation presented as whisker plots as described in Fig 1. (*) short term insulin stimulation causes a statistically significant increase in AKT phosphorylation at S473 by one-way ANOVA with Tukey post hoc test.

Discussion

β-Adrenergic and insulin signaling pathways are the primary means of modulating moment-to-moment changes in cardiac function and metabolic substrate selection. Nevertheless, interactions between these two pathways are not fully understood. This is important to understand in regard to diabetes where insulin signaling is disrupted and PKA signaling is dysfunctional [3]. In an effort to further understand the interrelationship of these two pathways, we used adult mouse cardiomyocytes as a model system. ACMs have the advantage that they more closely represent in vivo signaling and metabolic conditions as compared to immortalized cardiomyoblasts, such as H9c2 and HL-2 cells [27]. ACMs can also be isolated from different genetic and disease models, as in the Akita mice used here, to interrogate alterations in function at the cellular level. A disadvantage is that mouse ACMs are not amenable to genetic manipulation and have a limited lifespan. For these reasons, we chose to optimize conditions to monitor PKA signaling using an immunofluorescence technique. This approach, to the best of our knowledge, has not been taken before in adult ACMs.

The antibody that we used to measure PKA signaling is validated by the observations that the immunofluorescence intensity is responsive to a β-agonist (ISO), a PKA agonist (8Br-cAMP), and PDE inhibitors. Thus, by this methodology we were able to visualize how insulin affects PKA signaling at the cellular level, specifically in a homogenous cardiomyocyte population, and independently of other systemic factors. Little difference was seen in the subcellular distribution and pattern of PKA substrate phosphorylation in wildtype cells stimulated with either ISO or 8Br-cAMP when cells were culture in the presence of insulin. This suggests that similar pools of PKA were activated by both agonists, resulting in phosphorylation of downstream substrates throughout the cell. When wildtype ACMs were cultured in the absence of insulin, though, PKA phosphorylation was substantially blunted in response to all three agonists examined. In Akita ACMs PKA substrate phosphorylation was decreased as compared to wildtypes. Furthermore, insulin had no beneficial effects on PKA substrate phosphorylation in Akita ACMs. Interestingly, though, when PKA phosphorylation was examined by Western blot analysis using the same antibody as in the immunofluorescence experiments, no significant differences were seen in the pattern of proteins phosphorylated. This was further corroborated by measuring total PKA activity (Fig 1E). We saw that the absence of insulin reduced basal PKA activity. However, ISO stimulated PKA activity similarly in ACMs cultured with or without insulin. The cause of this reduced basal PKA activity is not due to reduced PKA catalytic subunit content (S2 Fig) and needs further investigation.

One possibility is that the absence of insulin induces changes in ACM morphology and impairs PKA substrate detection by immunofluorescence. This is countered, though, by the finding that the phosphorylation of PBN and TnI were robustly induced by agonists even in the absence of insulin. Rather, we suggest that there is a differential recognition of PKA substrate epitopes depending upon the protein status. The immunofluorescence protocol maintains proteins in a native conformation, as compared to Western blot where proteins are denatured.

The effects of insulin on global PKA substrate phosphorylation followed a similar pattern to that observed with phospho-PFK-2 staining. The cardiac isoform of PFK-2 is phosphorylated in response to insulin or β-agonists to increase the levels of fructose-2,6-bisphosphate, an allosteric activator of PFK-1 [28]. This serves to increase glycolytic flux. We have previously reported that PFK-2 content is decreased in the absence of insulin [3]. In the streptozotocin toxin-induced type 1 diabetic model this results in a constitutive decrease in its content. In addition, the PFK-2 that remains is not phosphorylated in response to PKA activation. Our immunofluorescence data follows a similar pattern. The absence of insulin decreases basal PFK-2 phosphorylation and dampens its phosphorylation in response to PKA agonists (Fig 5). In Akita ACMs, we observed a constitutive decrease in phospho-PFK-2. Interestingly, though, culturing Akita ACMs overnight with insulin failed to rescue PFK-2 phosphorylation. This suggests that the mechanisms that normally regulate the expression and activation of PFK-2 are not affected by administration of insulin for 18h. While the pattern of PKA substrate phosphorylation approximates that of phospho-PFK-2, we cannot rule out that other PKA substrates are also affected by the insulin status. Furthermore, insulin also activates the mitogen-activated protein kinase/extracellular-regulated kinase pathway and effects related to the stimulation of these other signaling cascades must be further evaluated [9].

We demonstrate here that insulin affects PKA signaling in ACMs. Reciprocally, previous studies have shown that PKA can affect insulin signaling. In neonatal rat cardiomyocytes and mice with chronic β-adrenergic stimulation there is a PKA-dependent decrease in insulin signaling [29, 30]. The mechanism involves insulin receptor desensitization, mediated by Akt [30], and is manifested by a decrease in GLUT4 content and translocation upon insulin stimulation [29]. This PKA-mediated effect contributes to the insulin resistance that is manifested in failing hearts. These role of PKA in modulating insulin signaling, though, are dependent upon the duration of PKA activation. With short-term activation of PKA there is synergistic enhancement of Akt phosphorylation, GLUT4 translocation, and glucose uptake [30, 31]. This mediates the increase in glucose uptake and oxidation in response to acute β-adrenergic stimulation.

Our results provide new insights into how diabetes may impact the heart. We show a decrease in PKA signaling in adult cardiomyocytes when insulin is absent. A novelty of this study was the use of immunofluorescence microscopy as a means of monitoring PKA signaling. As we show, other methodologies, such as Western blot, would have missed these apparent changes in substrate phosphorylation. Another novel aspect of this study was using primary adult cardiomyocytes isolated from Akita mice. Immortalized cells cannot recapitulate the unique morphology and metabolic aspects of primary cells. We demonstrate that the lack of insulin affects the phosphorylation of PFK-2, while the phosphorylation of contractile proteins was similar in control and Akita ACMs. Future studies must be performed to more exhaustively identify what other substrates may be affected.

Supporting information

S1 Fig. β-adrenergic stimulation increases PKA substrate phosphorylation in adult mouse cardiomyocytes.

(A) ACMs from wild-type mice were incubated overnight in the presence or absence of insulin (ins) and treated with 0.25μM Isoproterenol for 1, 5, and 30min as indicated. Cells were fixed and stained with rabbit anti-PKA substrate antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for each condition is shown. Scale bar 10μm. (B) Quantitation of mean fluorescence intensity (MFI) for PKA-substrate are presented as whisker plots that encompass data from at least 18 cells, as detailed in Fig 1 (n = 3 biological replicates, and at least 6 cells per experiment). *, significant difference (p < .001) by one-way ANOVA with Tukey post hoc test.

(TIF)

Click here for additional data file.^{(3MB, tif)}

S2 Fig. PKA catalytic subunit levels are unchanged under diabetic conditions.

(A) ACMs from wild-type or Akita mice were incubated overnight in the presence or absence of insulin (ins) and treated with 0.25μM Isoproterenol or 250μM 8-bromo-cAMP for 30min as indicated. Cells were fixed and stained with rabbit anti-PKA catalytic subunit antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for each condition is shown. Scale bar 10μm. (B) Quantitation of mean fluorescence intensity (MFI) for PKA-substrate are presented as whisker plots that encompass data from at least 18 cells, as detailed in Fig 1 (n = 3 biological replicates, and at least 6 cells per experiment).

(TIF)

Click here for additional data file.^{(1.9MB, tif)}

S3 Fig. PDE4 inhibition increases PKA signaling.

(A) ACMs from wild-type mice were incubated overnight in the presence or absence of insulin (ins) and treated with 0.25μM Isoproterenol and/or 10μM RO. Cells were fixed and stained with rabbit anti-PKA substrate antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for each condition is shown. Scale bar 10μm. (B) Quantitation of mean fluorescence intensity (MFI) for PKA-substrate are presented as whisker plots that encompass data from at least 18 cells, as detailed in Fig 1 (n = 3 biological replicates, and at least 6 cells per experiment). *, significant difference (p < .001) by one-way ANOVA with multiple comparisons using Tukey’s test.

(TIF)

Click here for additional data file.^{(2.8MB, tif)}

S4 Fig. PDE4D protein levels are unchanged in diabetic or β-adrenergic stimulation conditions.

(A) Primary mouse cardiomyocytes from wild-type (C57-B6) or Akita were incubated overnight in the presence or absence of insulin (ins) and treated with 0.25μM Isoproterenol or 500μM IBMX for 30min. Cells were fixed and stained with rabbit anti-PDE4D antibody visualized with Alexa 488 anti-rabbit secondary and with Alexa 568 labeled phalloidin. Maximum intensity micrographs were acquired as described in Materials and Methods and a representative image for each condition is shown. Scale bar 10μm. (B) Quantitation of mean fluorescence intensity (MFI) for PDE4D are presented as whisker plots that encompass data from at least 18 cells, as detailed in Fig 1 (n = 3 biological replicates, and at least 6 cells per experiment). *, significant difference (p < .001) by one-way ANOVA with multiple comparisons using Tukey’s test.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S1 Raw images

(PDF)

Click here for additional data file.^{(1.2MB, pdf)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by National Institutes of Health Grant R01HL125625 (to K. M. H.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0231806.r001

Decision Letter 0

Makoto Kanzaki

8 May 2020

PONE-D-20-09116

Diabetes induced decreases in PKA signaling in cardiomyocytes: the role of insulin

PLOS ONE

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Reviewer #1: This study shows that PFK-2, a regulator of glucose metabolism, of wild type cardiac myocytes is downregulated in insulin-dependent manner. However, PFK-2 of Akita diabetic model mice was decreased and was not recovered by the addition of insulin. It was suggested that insulin-dependent decrease of PFK-2 expression is a mechanism of metabolic regulation by insulin in diabetes. However, there are several concerns as described below.

However, in vitro assay system does not mimic in vivo phenomena as described in following comments. It is difficult to conclude that PFK-2 is an insulin-dependent target molecule in diabetic heart. The followings are comments.

1. When the effects of insulin are due to the decrease of PFK-2 expression level, overexpression of PFK-2 should restore the effects of cAMP signal-induced responses. Authors described at lines 398 o 399 that ‘a disadvantage is that ACMs are not amenable to genetic manipulation and have a limited lifespan’. However, there is the report that adenovirus is successfully used for expression of FRET biosensors in adult mouse cardiomyocytes (Methods Mol Biol. 2015; 1294:103-15). Therefore, expression of genes of interest is possible. The experiment of PFK-2 overexpression should be done.

2. In Fig. 1B, PKA substrates detected by anti-PKA consensus sequence-antibody represents all the proteins phosphorylated by PKA. However, minor but important PKA-phosphorylating proteins are not apparent from this detection system.

3. Immunofluorescence of PKA substrates is not quantitative. The mRNA expression levels should be measured. Then, it will provide information that insulin regulates transcription, translation, stability of protein.

4. In many cases, signaling intensity by Western blot is shown as a ratio of phosphorylated (active) form to total protein content. When the phosphorylated form decreases in diseased state and the total protein content also decreases, the ratio of phosphorylated form to total protein content is almost the same as that of control group. In this case, fold activation of diseased mice by β-adrenergic receptor stimulation will be about the same as control mice. When fold stimulation is same between control and diseased states, it cannot be concluded that decreased PKA signaling is due to the decreased expression of total protein content.

5. PKA signaling is mediated by signaling complex being comprised of PKA, A-kinase anchoring protein (AKAP), target protein, and other signaling proteins. Intensity, efficiency and specificity of PKA signaling depend on AKAPs in many cases. Therefore, the effects of insulin on decreased PKA signaling may be explained by the decreased expression of AKAPs. Western blot and immunofluorescence data of AKAPs expressing in the myocytes are necessary to substantiate the conclusion.

6. When phosphorylation states is really wanted to be examined, phospho-proteomics analysis may be better than Western blot instead of using anti-PKA consensus sequence-antibody.

7. Insulin has growth factor-like effects. Therefore, overnight culture without insulin may affect cell growth or maintenance. Thus, the effects of insulin on the isolated cells cultured overnight without insulin may be growth factor-like actions but not metabolic actions as suggested.

8. Insulin treatment may modify epigenetic modification during development of diabetes. It is interesting to examine insulin-induced epigenetic modification in myocytes of Akita diabetic model mice.

Minor comment

1. At lines 456 to 458, authors described that ‘our results support that the deficiency of PKA signaling is not mediated by loss of β-adrenergic receptors, PKA protein, or cAMP production/degradation’. However, authors do not measure number of β-adrenergic receptors, and amounts of PKA protein and PDEs/adenylyl cyclases. It is too much to say that.

2. There are many proteins that their activities re regulated by insulin in myocytes. The decrease of PFK-2 level may not be enough to explain insulin-dependent metabolic changes in the heart.

Reviewer #2: In the manuscript titled “Diabetes induced decreases in PKA signaling in cardiomyocytes: the role of insulin”, the authors test the hypothesis that decreased insulin signaling contributes to dysfunctional PKA response in adult rat ventricular myocytes. The authors use a novel technique of immunofluorescence microscopy to monitor PKA signaling in fixed myocytes cultured from WT or Akita type 1 diabetic mice. The results demonstrate that a lack of insulin in culture conditions is associated with reduced PKA signaling when measured by immunofluorescence. Further, the authors explore the possibility that phosphorylation of the glycolytic regulator PFK2 and not contractile proteins were responsible for the overall reduction in PKA signaling. The authors conclude that deficient insulin signaling decreases PKA signaling in adult myocytes, which may provide insights into how diabetes impacts the heart.

Major issues

1. There are several inconsistencies between the written text and what is shown in the figures. For example, line 191 onward describes WB data in Figure 1B, Figure 1B however, shows something different, not WB data. Continuing from that point, the authors state in line 199 "culturing myocytes in the presence of insulin for 18hrs had no enhancing effect on PKA substrate staining upon ISO stimulation". The authors need to be clear if they are comparing this to WT or baseline, as compared to baseline there is an effect with ISO stimulation in all groups. Far too much of my time was spent trying to interpret the data and I would urge the authors to proofread and ensure the manuscript is concise and to the point.

2. No in vivo mouse data is provided in the manuscript. While the Akita model has previously been reported to be associated with diabetes, there is no data provided demonstrating that this model serves as a mechanism of low insulin as a result of diabetes in this study. It would be interesting to determine the overall insulin levels of Akita mice and how does this compare to WT without insulin.

3. The use of immunofluorescence microscopy to monitor PKA signalling, as has been demonstrated in this study, has not been done before in adult myocytes. I am, therefore, surprised the authors have not validated this technique with other well-know measurements of PKA signaling such as a PKA activity kit to confirm the results. Immunofluorescence can often lead to false results; therefore the use of an antibody control is strongly advised. The manuscript would also benefit from clarification of how the mean fluorescence intensity images were normalized and the control measures taken to ensure there are no false positive signals.

4. Extended time in culture leads to great changes in myocyte morphology. For example, the structural integrity of the cells, such as t-tubule organization becomes disrupted after prolonged culture. Both insulin receptors and B-receptors are located on the t-tubule membrane, thus it cannot be ruled out that signaling alterations could be a consequence of culture. Moreover, just the presence of insulin in the culture media may have metabolic benefits that prove more advantageous for long culture conditions. To address this, a time-course study would be useful, starting with the effects of insulin on PKA signaling in freshly isolated cells.

5. There are concerns regarding the authors’ interpretation of the pPFK2 data. Especially, with the conclusion that PFK2 phosphorylation was decreased in an insulin-dependent manner. Although blunted compared with WT insulin hearts, WT hearts in the absence of insulin showed increased pPFK2 in response to ISO and IBMX. In the Akita hearts, however, ISO and IBMX failed to have any effect. This suggests that it is not just the absence of insulin, but something specific to the Akita mice leading to decreased pPFK2 activity.

Minor issues

1. Were the stats performed on cells or animals, have the authors considered performing nested statistics to account for cells and animals?

2. It would be useful if the graphs displayed individual data points.

3. The authors state PKA staining is found on the z-bands, but there is no mention as to which software was used to determine the position on the PKA activity staining?

**********

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PLoS One. 2020 Aug 20;15(8):e0231806. doi: 10.1371/journal.pone.0231806.r002

Author response to Decision Letter 0

30 Jun 2020

We’d like to thank the reviewers for their careful evaluation of our manuscript. We have addressed their concerns to the best of our ability. This includes extensive revision of the manuscript and an additional experiment.

Reviewer 1:

Reviewer 1’s general comment is that “It is difficult to conclude that PFK-2 is an insulin-dependent target molecule in diabetic heart.” However, this current study is supported by, and builds upon, our previous report that PFK-2 content is regulated by insulin signaling (see Bockus et al. 2017, Journal of American Heart Association; https://www.ahajournals.org/doi/full/10.1161/jaha.117.007159). We concur that other targets are likely to be affected by the presence or absence of insulin, but this and our previous studies support phosphorylation of PFK-2 is abnormal in the diabetic heart.

1. “When the effects of insulin are due to the decrease of PFK-2 expression level, overexpression of PFK-2 should restore the effects of cAMP signal-induced responses. Authors described at lines 398 o 399 that ‘a disadvantage is that ACMs are not amenable to genetic manipulation and have a limited lifespan’. However, there is the report that adenovirus is successfully used for expression of FRET biosensors in adult mouse cardiomyocytes (Methods Mol Biol. 2015; 1294:103-15). Therefore, expression of genes of interest is possible. The experiment of PFK-2 overexpression should be done.”

Thank you for providing the suggestion and the reference. As noted in this elegant paper by Zaccolo et al., there are limitations with adenovirus expression in adult mouse cardiomyocytes. First, it takes a high MOI for protein expression. We have also found this to be true, and as Zaccolo noted, this has toxicity issues. Secondly, the referenced paper reports that the timing of an adenoviral transduction experiment is dependent upon the protein that is being expressed. In the case of the referenced work, they are expressing a FRET reporter with favorable fluorescent properties that allows detection by 24h. In our experience it takes approximately 48-72h, if at all, to detect the expression of unlabeled proteins in adult mouse cardiomyocytes. Unfortunately, at this point there is also significant loss of viable cells which may be further confounded by the insulin status. Thus, while such a rescue experiment would help to support our conclusions it is technically unfeasible. We have edited the manuscript to reflect some of the limitations. Furthermore, we focus on the phosphorylation status of PFK-2 and not on its total content.

We agree that there might be additional substrates we can’t detect by Western. We clarified in the text that other substrates may be phosphorylated but not detected (lines 220-222).

3. “Immunofluorescence of PKA substrates is not quantitative. The mRNA expression levels should be measured. Then, it will provide information that insulin regulates transcription, translation, stability of protein.”

We have attempted to make the immunofluorescence data as quantitative as possible by keeping the experimental conditions constant. This includes how the cardiomyocytes were prepared and the microscopy conditions.

We have previously shown that in the diabetic heart PFK-2 protein levels decrease without a change in transcript levels (Bockus et al., 2017). We concluded that PFK-2 stability is greatly decreased in the absence of insulin signaling. While we agree the transcript levels of PKA substrates may be affected by insulin, such an effect may be very selective. It is also worth noting that the phosphorylation of other substrates, such as phospholamban and troponin, were unaffected by insulin.

This is true and a limitation of the immunofluorescence technique. Nevertheless, we can tell that the response to a PKA agonist is different depending upon whether cardiomyocytes were in the presence or absence of insulin. I made it clear that we are referring to the phosphorylation of proteins and not total proteins in our immunofluorescence experiments. Furthermore, the limitations of the experiments are now more clearly stated.

This is a good point. Changes in PKA may be due to alterations in its subcellular localization and mediated by AKAPs. Over 30 different AKAPs have been identified (https://doi.org/10.1016/j.cellsig.2017.05.012). It is unfortunately beyond the scope of this paper to determine if changes in AKAPs contribute to the insulin-dependent effects on PKA activity. We have previously reported though that there is no apparent change in AKAPs in the hearts of STZ model of type 1 diabetic mice using a binding assay (Bockus and Humphries, J. Biol. Chem., 2015, 290(49): 29250). In addition, we have now directly measured PKA activity (new Fig 1E). We show that the lack of insulin decreases basal PKA activity but not its activation by ISO. While we cannot rule out alterations in subcellular PKA, this supports that the primary defect is in discrete substrate phosphorylation.

6. When phosphorylation states is really wanted to be examined, phospho-proteomics analysis may be better than Western blot instead of using anti-PKA consensus sequence-antibody.

Phospho-proteomics is a powerful technique that will be used in future experiments, but it is beyond the scope of this work.

We agree that insulin has many effects on cellular function that extend beyond metabolism. We do not see any change in cell number or cell size with overnight +/- insulin treatment. However, we cannot rule out other effects and have added this caveat to the Discussion (lines 482-484).

8. Insulin treatment may modify epigenetic modification during development of diabetes. It is interesting to examine insulin-induced epigenetic modification in myocytes of Akita diabetic model mice.

That is an interesting suggestion, but the epigenetic effects of insulin are beyond the scope of this work.

Minor comments:

2. There are many proteins that their activities re regulated by insulin in myocytes. The decrease of PFK-2 level may not be enough to explain insulin-dependent metabolic changes in the heart.

1)The last paragraph of the Discussion was edited and this statement was removed. 2) We agree that insulin has many effects on cardiac metabolism such as increasing glucose uptake, activating glycolysis, and increasing glucose oxidation via the activation of pyruvate dehydrogenase. The novel aspect of this study is identifying that the lack of insulin affects PKA signaling. We have updated the Discussion to reflect other possibilities.

Reviewer 2

I apologize for the lack of clarity. The references to Fig 1 in the text now correspond to the figures accurately. In addition, we have changed the text extensively to improve clarity. Please note the highlighted regions throughout the Results section where it is made clear what groups are being compared.

The Akita mice are a well-established model of diabetes. The first study that characterized this model showed that mice are hypoinsulinemic and hyperglycemic. In vitro perfusion studies showed pancreatic beta cells do not release insulin in response to high glucose (Yoshioka et al. Diabetes, (1997) 46: 887).

I agree it would be interesting to see how well we are approximating insulin levels in Akita mice, in vivo, to WT ACMs without insulin. We unfortunately didn’t measure circulating insulin levels in these experiments. However, we did check blood glucose levels in the mice at the time of sacrifice. Blood glucose levels of all Akita mice were > 400 mg/dl. This information has been added to the Methods section.

Based on the reviewer’s suggestion, we have performed additional experiments to measure PKA activity in ACMs cultured in the presence or absence of insulin. This is now shown in Fig. 1E. Graduate student Maria Newhardt completed these experiments and has been added as an author. We report that basal PKA activity is decreased in wildtype ACMs cultured in the absence of insulin. Nevertheless, PKA was activated to a similar extent in the -/+ insulin conditions upon the addition of ISO. This supports that the robust insulin-dependent change in PKA substrate phosphorylation observed by immunofluorescence is likely due to changes in specific substrates and not a global defect in PKA activity.

We agree that immunofluorescence data can be problematic. This is alleviated, in part, by using commercially sourced and validated antibodies. For the PKA substrate antibody, we have confidence in the results and specificity because of the responsiveness of the fluorescence signal to 3 different PKA activators: ISO, 8Br-cAMP, and PDE inhibitors. We also note that our experiments use the same secondary antibodies throughout the study. Staining was essentially undetectable in secondary antibody alone control experiments. This gives us confidence that the signal we are seeing is from the specificity of the primary.

The confocal microscopy images were collected using the same settings between all experiments. This ensured reproducibility and allowed for unbiased comparisons between experimental conditions. This information has been added in the Materials and Methods section. MFI was calculated on maximum intensity projection images using the Zeiss Zen Black software.

It is a limitation of ACMs that cells begin to dedifferentiate in culture. Our experiments our completed within 18h of isolation and we see no overt changes in morphology or viability when insulin is excluded from the media.

Regarding the receptors, our experiments in Fig 7 show that reintroduction of insulin after 18h of culture results in robust phosphorylation of Akt. This would argue that our immunofluorescence results are not from decreased insulin receptors or their accessibility. Likewise, we see PKA signaling dysfunction when ACMs are cultured in the absence of insulin and are then stimulated with 8Br-cAMP or PDE inhibitors. 8Br-cAMP activates PKA directly and argues against a B-receptor defect. Changes in receptors cannot be ruled out, though.

Regarding the time course, we have previously attempted such an experiment but it was subject to high variability. The reason, we believe, is that the cardiomyocytes are required to be in plating media containing horse serum when they are first isolated. This is then changed to serum-free culture media. We surmised that the variability arose from the persisting effects of the sera. For this reason, we chose 18h because cells sustained viability (regardless of -/+ insulin), morphology was not overtly different, it approximates a physiological fasted state, and the results were very consistent.

I agree with the reviewer’s concern and I have modified the text to reflect other potential defects affecting PFK2 phosphorylation. I have also removed the sentence: “Thus, the immunofluorescence staining of phospho-PFK-2 follows closely to that of PKA substrate antibody.”

Minor issues

1. Were the stats performed on cells or animals, have the authors considered performing nested statistics to account for cells and animals?

We used Akita and WT mice that were, whenever possible, littermates. In addition, we attempted to minimize variability with our cell preparations by starting the experiment at the same time of day, using aliquots of reagents, and having the same personnel perform the experiments. I have consulted with a statistics expert, Albert Batushansky (see Batushanksy, Humphries, et al. Metabolomics 2019), and he confirmed our statistics are appropriate for the experimental design.

2. It would be useful if the graphs displayed individual data points.

We believed the whisker plots were appropriate because it makes it easier for the reader to visualize differences between experimental conditions. Here is a representative plot with all the data points presented for the reviewer’s consideration (please see the response to reviewers in the PDF). We are willing to modify the figures or include all the data points in supplemental figures.

3. The authors state PKA staining is found on the z-bands, but there is no mention as to which software was used to determine the position on the PKA activity staining?

The staining was largely diffuse but with apparent increased intensity along the membranes. There was also staining of the filaments that was qualitatively consistent with Z-bands. We have modified this statement.

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PLoS One. doi: 10.1371/journal.pone.0231806.r003

Decision Letter 1

Makoto Kanzaki

15 Jul 2020

PONE-D-20-09116R1

Diabetes induced decreases in PKA signaling in cardiomyocytes: the role of insulin

PLOS ONE

Dear Dr. Humphries,

Your revised manuscript was reviewed by the original referees, and their comments are appended. As you will see they both recognize that the revised manuscript has adequately improved, while reviewer #2 pointed out "n" issue. The authors need to properly address this issue.

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We look forward to receiving your revised manuscript.

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Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: Thank you for the opportunity to review this revised manuscript. The authors have thoroughly revised the paper to address my primary concerns. The authors have modified the text to provide clarity and clearly state the limitations of the study; making the study more accessible. Furthermore, adding additional experiments to measure PKA activity in ACMs make the results more robust and support the immunofluorescence data on insulin dependent change in PKA substrate phosphorylation.

At this stage, I have one minor concern, it is still not clear what exactly is N in this study. Are the statistics are performed on biological repeats (n=3) or cells (n= approx. 30) as is shown on the example data point plot provided? If it is biological repeats were the cell data averaged for each animal? On the contrary, if the stats were performed on cells, the authors need to be wary of pseudoreplication. It is recommended that a line is added to the methods section to clarify this.

**********

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Reviewer #1: No

Reviewer #2: No

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2020 Aug 20;15(8):e0231806. doi: 10.1371/journal.pone.0231806.r004

Author response to Decision Letter 1

28 Jul 2020

We’d like to thank the reviewers for their evaluation and constructive critiques of our original and revised manuscripts.

Reviewer 2: At this stage, I have one minor concern, it is still not clear what exactly is N in this study. Are the statistics are performed on biological repeats (n=3) or cells (n= approx. 30) as is shown on the example data point plot provided? If it is biological repeats were the cell data averaged for each animal? On the contrary, if the stats were performed on cells, the authors need to be wary of pseudoreplication. It is recommended that a line is added to the methods section to clarify this.

I apologize that this wasn’t clear. The experiments under a given condition were done on 3 cell preparations from 3 hearts. The statistics were performed on the sum of all the cells that were imaged. I have added additional information in the Methods section and in each of the Figure Legends to make this clear. I agree that n= appox. 30 can be considered as pseudoreplication, or as we considered them repeated measurements, and this could be a problem. However, when the cells from a given preparation are averaged and analyzed as n=3 similar conclusions are still reached. This information has been added in the “Statistical analysis” section in the Methods. For example, here is Figure 1 plotted with all the cell data shown or with the data plotted as averages of the 3 cell preparations. (These example Figures can be seen in our "Response to reviewers" in the PDF). We believe that showing the whisker plots encompassing all the cells more clearly demonstrates the data distribution and variability intrinsic to the method.

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PLoS One. doi: 10.1371/journal.pone.0231806.r005

Decision Letter 2

Makoto Kanzaki

7 Aug 2020

Diabetes induced decreases in PKA signaling in cardiomyocytes: the role of insulin

PONE-D-20-09116R2

Dear Dr. Humphries,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Makoto Kanzaki, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: Thank you for the clarity and the additional information to address my concern regarding the statistics. All my comments have now been fully addressed.

**********

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PLoS One. doi: 10.1371/journal.pone.0231806.r006

Acceptance letter

Makoto Kanzaki

11 Aug 2020

PONE-D-20-09116R2

Diabetes induced decreases in PKA signaling in cardiomyocytes: the role of insulin

Dear Dr. Humphries:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. β-adrenergic stimulation increases PKA substrate phosphorylation in adult mouse cardiomyocytes.

(TIF)

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S2 Fig. PKA catalytic subunit levels are unchanged under diabetic conditions.

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S3 Fig. PDE4 inhibition increases PKA signaling.

(TIF)

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S4 Fig. PDE4D protein levels are unchanged in diabetic or β-adrenergic stimulation conditions.

(TIF)

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S1 Raw images

(PDF)

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Diabetes induced decreases in PKA signaling in cardiomyocytes: The role of insulin

Craig A Eyster

Satoshi Matsuzaki

Maria F Newhardt

Jennifer R Giorgione

Kenneth M Humphries

Roles

Abstract

Introduction

Materials and methods

Adult mouse cardiomyocyte isolation

Antibodies and drugs

Microscopy

Western blot analysis

PKA activity

Statistical analysis

Results

β-adrenergic signaling is decreased under diabetic conditions in cardiomyocytes

Fig 1. Immunofluorescence detection of PKA signaling reveals a positive role of insulin.

Insulin signaling is necessary downstream of cAMP production

Fig 2. The effect of insulin on PKA signaling occurs downstream of cAMP production.

Phosphodiesterase inhibition increase PKA signaling but do not restore deficits induced by loss of insulin

Fig 3. PKA signaling stimulated by the combination of isoproterenol and IBMX is decreased by the absence of insulin.

Fig 4. Inhibition of PDE4 increases PKA signaling.

Phosphorylation of PFK2 is decreased in diabetic conditions

Fig 5. Phosphorylation of PFK2 is decreased in cardiomyocytes cultured in the absence of insulin and with diabetes.

Fig 6. Phosphorylation of β-adrenergic targets Phospholamban and Troponin are unchanged under diabetic conditions.

PKA substrate phosphorylation remains depressed upon acute insulin administration

Fig 7. Short term insulin stimulation does not rescue PKA-substrate phosphorylation.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Makoto Kanzaki

Roles

Author response to Decision Letter 0

Decision Letter 1

Makoto Kanzaki

Roles

Author response to Decision Letter 1

Decision Letter 2

Makoto Kanzaki

Roles

Acceptance letter

Makoto Kanzaki

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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