Myocardial cGMP-PKG1α dysregulation contributes to VT pathogenesis in type II diabetes and metabolic syndrome

Myocardial cGMP-PKG1α dysregulation contributes to VT pathogenesis in type II diabetes and metabolic syndrome | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Myocardial cGMP-PKG1α dysregulation contributes to VT pathogenesis in type II diabetes and metabolic syndrome Xuehong Cao , Yali Zhang , Audrey Tripp , Rachel Steinhauer , Pei-wen Liu , Mark J. Aronovitz , Greg L. Martin , Bo Wang , Abdullah Alissa , View ORCID Profile Mossab Aljuaid , Justin Ho , Tina Phan , View ORCID Profile Christopher Madias , Robert M. Blanton , Jonas B. Galper doi: https://doi.org/10.1101/2025.02.12.638003 Xuehong Cao 1 Molecular Cardiology Research Institute, Tufts Medical Center 2 Tufts University School of Medicine , Boston, MA MD, PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yali Zhang 1 Molecular Cardiology Research Institute, Tufts Medical Center 2 Tufts University School of Medicine , Boston, MA PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Audrey Tripp 1 Molecular Cardiology Research Institute, Tufts Medical Center 2 Tufts University School of Medicine , Boston, MA BA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rachel Steinhauer 4 Tufts Medical Center, Department of Medicine MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pei-wen Liu 1 Molecular Cardiology Research Institute, Tufts Medical Center MS Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mark J. Aronovitz 1 Molecular Cardiology Research Institute, Tufts Medical Center BA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Greg L. Martin 1 Molecular Cardiology Research Institute, Tufts Medical Center BS Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bo Wang 1 Molecular Cardiology Research Institute, Tufts Medical Center MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Abdullah Alissa 5 Cardiovascular Center, Department of Medicine, Tufts Medical center MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Mossab Aljuaid 5 Cardiovascular Center, Department of Medicine, Tufts Medical center MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Mossab Aljuaid Justin Ho 2 Tufts University School of Medicine , Boston, MA BA Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tina Phan 2 Tufts University School of Medicine , Boston, MA MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site Christopher Madias 2 Tufts University School of Medicine , Boston, MA 4 Tufts Medical Center, Department of Medicine 5 Cardiovascular Center, Department of Medicine, Tufts Medical center MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Christopher Madias Robert M. Blanton 1 Molecular Cardiology Research Institute, Tufts Medical Center 2 Tufts University School of Medicine , Boston, MA 4 Tufts Medical Center, Department of Medicine 5 Cardiovascular Center, Department of Medicine, Tufts Medical center MA, MD Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: Robert.blanton{at}tuftsmedicine.org Jonas.galper{at}tuftsmedicine.org Jonas B. Galper 1 Molecular Cardiology Research Institute, Tufts Medical Center 2 Tufts University School of Medicine , Boston, MA 4 Tufts Medical Center, Department of Medicine 5 Cardiovascular Center, Department of Medicine, Tufts Medical center MD, PhD Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: Robert.blanton{at}tuftsmedicine.org Jonas.galper{at}tuftsmedicine.org Abstract Full Text Info/History Metrics Preview PDF Abstract Background Type-II diabetes (DMII) and metabolic syndrome increase ventricular arrhythmia and sudden cardiac death risk. Objectives To identify signaling mechanisms through which DMII and metabolic syndrome promote ventricular tachycardia (VT). Methods We performed ventricular programmed stimulation on leptin receptor mutant (Db/Db) mice with DMII, high fat high sucrose (HFHS)-fed mice with metabolic syndrome, and cGMP-dependent Protein Kinase 1α (PKG1α) leucine zipper mutant (LZM) mice, which do not have DMII or metabolic syndrome but have disrupted PKG1α signaling. Results During ventricular programmed stimulation, Db/Db and HFHS-fed mice displayed increased VT and T-wave alternans. Cardiomyocytes from these mice displayed early afterdepolarizations. Both models demonstrated decreased heart rate response to parasympathetic inhibition, indicating autonomic dysfunction. cGMP, which mediates cardiac parasympathetic stimulation, was reduced in LVs of Db/Db and HFHS-fed mice. Conversely, cGMP augmentation with soluble guanylate cyclase stimulation (riociguat) or phosphodiesterase 5 inhibition (sildenafil) reduced VT inducibility. PKG1α LZM mice had normal autonomic responsiveness, but excess VT inducibility. Db/Db, HFHS, and LZM mice each demonstrated hyperactivated myocardial glycogen synthase kinase3β (GSK3β). Further, GSK3β inhibition with TWS119 abolished inducible VT in these mice. Diastolic cytosolic Ca 2+ reuptake slope decreased in cardiomyocytes from all models, while GSK3β inhibition with TWS119 reversed this effect. Phospholamban (PLB), which inhibits sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase 2a-mediated Ca 2+ reuptake, was hyperactivated/hypophosphorylated in HFHS-fed and LZM mice, and this was reversed by TWS119. Conclusions These findings identify cGMP reduction as driving GSK3β hyperstimulation, calcium dyshomeostasis, and VT in DMII and metabolic syndrome. Pharmacological modulation of these pathways opposes VT pathogenesis. Introduction Sudden cardiac death (SCD) remains a leading cause of cardiovascular death 1 . SCD typically arises from lethal ventricular arrhythmias including ventricular tachycardia (VT). VT and SCD occur either in the setting of acute myocardial infarction (MI), or in patients with chronic heart failure with reduced LV ejection fraction (HFrEF). Improvements in acute revascularization and in inpatient telemetric monitoring have greatly reduced SCD in the immediate post-MI period 2 . Similarly, placement of implantable cardiac defibrillators in patients with HFrEF and LV ejection fraction < 35% has improved survival by reducing VT death 3 . Despite these advances, VT remains a significant cause of death in patients with mildly reduced or even normal LVEF 1 . Thus, identifying and understanding additional mechanisms of the pathogenesis of VT has the potential to improve prevention of SCD in patients. Diabetes (DM) and metabolic syndrome represent poorly understood risk factors for SCD and VT. Both type 1 and 2 DM (DMI, DMII) as well as insulin resistance and metabolic syndrome, increase SCD risk in patients 4 – 6 . Epidemiological studies support that DMII independently increases SCD risk both in patients with known heart disease, but also in patients without known underlying heart disease 7 , 8 . In a recent study of patients with DMII who died under 50 years of age, SCD accounted for the most frequent cause of death. Approximately half of DMII patients with SCD had no evidence of CAD on autopsy 7 . Thus, available clinical evidence supports that in addition to effects on CAD and other cardiovascular risk factors, DM and metabolic syndrome serve as an independent risks for SCD. Experimental models further support the conclusion that DM acts as a VT substrate. For example, our previous work demonstrated that the Akita mouse model of type 1 diabetes displays increased susceptibility to VT in response to programmed electrical stimulation even in the basal state, in the absence of acute MI 9 . Several experimental studies also indicate increased VT susceptibility in animal models of DMII 10 , 11 . These combined population and experimental studies support an independent effect of DM on susceptibility to VT. Impaired glucose tolerance and metabolic syndrome, like DMII, increase SCD risk in patients 4 – 6 , suggesting that shared mechanisms exist for VT predisposition in DM and insulin resistance. However, the molecular abnormalities through which DMII promotes VT susceptibility independent of associated cardiovascular risk factors remain unknown. Autonomic dysfunction is a shared complication of DMI, DMII, and insulin resistance 12 and has been identified as contributing to arrhythmogenesis. In patients with DMII, autonomic dysfunction further increases SCD risk 4 . Experimental studies demonstrate that parasympathetic dysfunction contributes to inducible and spontaneous post-MI VT in DM1 due in part to excess unopposed sympathetic input to the heart. 9 , 13 , 14 . In the leptin receptor mutant (Db/Db) model of DMII, myocardial autonomic dysregulation promotes VT as assessed by an ex vivo inducible arrhythmia model 10 . In the myocardium, parasympathetic signaling is normally mediated by the elevation of the second messenger cyclic guanosine monophosphate (cGMP), consistent with the finding that parasympathetic dysfunction is associated with reduced myocardial cGMP 15 . However, a causal role of reduced cGMP signaling in the susceptibility to VT remains unknown. Moreover, the specific role of derangements in cGMP signaling in the pathogenesis of VT in DMII and metabolic syndrome has not been studied. In the present study we therefore examined the dysregulation of myocardial cGMP in mouse models of DMII and insulin resistance and investigated the role of the cGMP-dependent protein kinase 1 (PKG1) in modulating the predisposition to the development of VT, using a protocol for programed ventricular stimulation. We specifically studied the Db/Db mouse harboring leptin receptor mutations which produce obesity, DMII, and metabolic heart disease 16 , and the high-fat high-sucrose (HFHS)-fed mouse which by contrast, does not have overt DM, but develops obesity, metabolic syndrome, and insulin resistance 17 . We measured myocardial cGMP content and tested the anti-arrhythmic effects of pharmacological augmentation of cGMP with the cGMP-specific phosphodiesterase 5 (PDE5) inhibitor sildenafil and the guanylate cyclase stimulator ricociguat on VT in the Db/Db and HFHS models. To study the direct role of PKG1, we used the PKG1α leucine zipper mutant (LZM) mouse, which has normal cGMP levels, does not have diabetes or metabolic syndrome, but lacks the normal PKG1α response to cGMP due to point mutations which disrupt kinase substrate interactions 18 . This allows the specific investigation of cGMP-PKG1 signaling without being confounded by other effects of DM. To establish the molecular mechanism for increased inducibility of VT in the above models, we studied Ca 2+ transients in cardiomyocytes and dysregulation of Ca 2+ handling proteins in ventricular extracts from these mice. We also investigated QRS/T-wave alternans and Ca 2+ alternans in the above models, as these phenomena occur due to Ca 2+ dyshomeostasis and both correlate with and contribute to lethal ventricular arrhythmia in humans 19 – 25 . Finally, we determined contribution of glycogen synthase kinase 3β (GSK3β) signaling in mediating the cGMP-PKG1 effect on VT phenotype. Methods Institutional approval All mouse studies were approved in accordance with the Institutional Animal Care and Use Committee at Tufts University, Protocol B2022-159. Mouse models The Db/Db leptin receptor mutant mice were obtained from Jackson Laboratories (strain 000697). Male mice were studied at age 4-6 months. We used Db/+ age-matched littermates which lack the Db/Db phenotype as controls. The high fat high sucrose (HFHS)-fed mice were treated for at least 24 weeks, starting at 8 weeks of age on 58kcal% fat, 656 kcal sucrose, and 5% insoluble fiber, cellulose (Research Diets, D23071405). Male wild type littermates received a control diet containing 4.8% insoluble fiber, 10kcal% fat and 0 sucrose (Research Diets D23071406). The PKG1α leucine zipper mutant (LZM) mice have been described previously 18 . We compared male homozygous LZM mice with wild type littermate age-matched controls at 4-6 months of age. Programmed Stimulation We performed the intracardiac programmed stimulation protocol as described previously 9 . Briefly, mice underwent light anesthesia with 2% isoflurane. A surface frontal plane ECG was obtained using 29-gauge electrodes placed subcutaneously in each limb. A 1.1-Fr electrophysiology octopolar catheter (EPR-800, Millar Instruments, Houston, TX) was inserted via the right jugular vein and advanced to the right ventricle. Next, the position and capture were confirmed through intracardiac and surface ECG tracings. Following pacing at 90ms hearts were subjected to premature stimuli at decreasing S1-S2 intervals until refractory, followed by a second S2 and third S3 premature stimulus until refractory. Finally, mice were subjected to burst pacing at rates of 40, 50, 60, 70, 80 and 90ms. Total duration of induced ventricular tachycardia in response to these stimuli was reported 9 . The threshold for development of alternans was determined as the slowest pacing rate at which T-wave alternans appeared. Heart rate response to atropine Mice received surgically implanted ECG telemeters as described 47 , followed by recovery for at least 3 days. Mice were treated with intraperitoneal injections of atropine, 0.5 mg/kg and the change in mean heart rate at 4 minutes post-injection was compared with the heart rate averaged over the minute prior to injection. LZM mice were compared with the same control mice as the Db/Db and HFHS mice. cGMP concentration in LV tissue was immediately snap frozen in liquid nitrogen following sacrifice. Approximately 30mg of tissue was then crushed into a fine powder over dry ice using a pre-chilled cryo tissue grinder. On ice, 6% trichloroacetic acid was added to samples (10µL/mg of tissue) followed by vortexing for 10 seconds. Proteins were precipitated out by centrifuging at 2000 x G for 15 minutes at 4°C and the supernatant was transferred to a 15 mL falcon tube on ice. Trichloroacetic acid in the supernatant was extracted by addition of approximately five volumes of diethyl ether. Samples were mixed by gentle rocking, then allowed to equilibrate into two layers. The top layer was discarded, and the sample washed a further three times by repeat additions of diethyl ether. After the final wash, the bottom layer was transferred to a microcentrifuge tube, snap frozen in liquid nitrogen and later lyophilized. Samples were resuspended in assay buffer and cGMP concentration determined in duplicate using the cGMP Enzyme Immunoassay Biotrak (EIA) system (RPN226, GE Healthcare, Amersham, UK), according to protocol #2. Drug administration Riociguat (AmBeed, A255332) was administered by oral gavage at 10 mg/kg once daily for 7 days. Sildenafil (Tocris 3784 or Sigma-Aldrich SML3033) was administered by intraperitoneal injection at 10 mg/kg once daily for 7 days. TWS199 (MEMD Millipore Corp, USA, 361554) was administered at 20 mg/kg by intraperitoneal injection once daily for 7 days. The response of each agent was compared with saline vehicle. Immunoblotting LV tissue lysates were prepared as described 14 . We used the following antibodies as described in Supplemental Table 1 . CM isolation and analysis Adult mouse ventricular cardiac myocytes were isolated from mice as described previously 47 , plated on laminin-coated cover slips followed by equilibration in physiological calcium, and loaded with FURA-2-AM (1µM, Invitrogen) for 30 minutes room temperature. Cells were placed in a chamber and paced via field stimulation at 1Hz, 2Hz, 3Hz, 4Hz and 5Hz. Sarcomere shortening and Ca 2+ transients were determined using a dual excitation spectrometer and video edge detection (Ionoptix) as described previously 47 ). Data were plotted using IonWizard. Ca 2+ reuptake rate was determined from the slope of the recovery curve, Tau. Ca 2+ alternans threshold was determined as the lowest pacing rate at which cells develop Ca 2+ alternans. We defined alternans as a 20% variation in beat-to-beat amplitude. Early afterdepolarization (EAD) recording in cardiac myocytes EADs were recorded in cardiomyocytes using whole-cell patch clamp in the mode of current clamp at room temperature. Recordings were performed with an Axon 200B amplifier and DigiData 1440 (Molecular Devices) and analyzed with Clampfit 11.2. The bath solution consisted of (in m M ): 137 NcAl, 5.4 KCl, 1 CaCl 2 , 0.5 MgCl 2 , 0.33 NaH 2 PO 4 , 5 HEPES, and 5.5 glucose, with a pH of 7.4 and an osmolality of 300 mOsm. Cardiomyocytes were paced by brief (2 ms) depolarizing pulses at frequencies of 0.1, 0.2, 1, 2, 3, and 5 Hz to induce and study EADs. Statistical analysis Average values are given as the mean ±SEM. Statistical differences between mean values were calculated by Student’s T test, Welch T test, or Mann-Whitney test as described in the figure legends. For multiple group comparisons, 1-way ANOVA was used, followed by multiple comparison testing. Results Db/Db and HFHS models of DMII and metabolic syndrome display increased VT in response to programmed electrical stimulation and increased incidence of T wave alternans Measures obtained from surface ECG recording in HFHS-fed and Db/Db mice are displayed in Table 1 . HFHS-fed mice displayed mildly increased P wave duration compared to control-diet treated, and had a reduced R-R interval, indicating faster heart rate. We examined VT inducibility in Db/Db and HFHS mice using ventricular programmed electrical stimulation. Programmed electrical stimulation of the ventricle predicts both SCD risk and VT events in patients following myocardial infarction 26 , 27 . We previously optimized a unique technique for programmed ventricular stimulation in mice with DMI utilizing a catheter with electrodes spatially designed to stimulate and record from the mouse heart to investigate and identify molecular mechanisms predisposing to VT 9 , 28 . The total duration of VT in response to single S1, double S2, and triple S3 stimulation was 3.2 ± 0.9 sec in Db/Db, (n= 23) vs. 0.3 ± 0.2 sec in Db/+, (n = 14, p<0.05). View this table: View inline View popup Download powerpoint Table 1. Surface ECG intervals in experimental groups The total duration of VT was 14.6 ± 3.0 sec in HFHS fed mice (n=14), vs. 2.6 ± 2.1 sec in mice on control diet (n=7, p<0.05) ( Figure 1A-1C ). Burst pacing had no effect on the development of VT in either experimental group. Female mice on HFHS diet displayed no inducible VT, resulting in a statistically significant difference in VT duration compared with male HFHS mice. For this reason, subsequent studies investigated males only. Download figure Open in new tab Figure 1. Increased ventricular tachycardia inducibility and T-wave alternans in mice with type II diabetes and metabolic syndrome. (A) Representative intracardiac tracings demonstrating VT in Db/Db and HFHS, but not control mice, during programmed stimulation. (B) Total VT duration in response to programmed stimulation and burst pacing in Db/Db, Db/+ controls and (C) HFHS versus control diet. *, p<0.05 by Student’s unpaired T Test. (D) Representative T-wave alternans in response to burst pacing in HFHS fed mouse. (E) T-wave alternans incidence in Db/+, Db/Db, and (F) wild type mice on control and HFHS diet, during 20 Hz interval burst pacing. (G) Early afterdepolarizations (EADs) in action potential from ventricular myocyte of HFHS-fed mouse paced at 100 Hz. Arrows denote EADs. (H) EAD incidence in control and HFHS diet-fed mouse ventricular myocytes. **, p<0.01 by Chi-squared test, n= 0/45 cells with EADs in 5 control; 11/56 cells in 7 HFHS-fed mice. Further analysis of the surface ECG in these mice during burst pacing demonstrated a rate-related beat-to-beat alteration voltage consistent with QRS/T-wave alternans ( Figure 1D ). Compared with respective Db/+ or control diet controls, the Db/Db and HFHS mice displayed increased incidence of QRS/T-wave alternans with pacing rates of 70, 60, and 50ms ( Figure 1E, 1F ). At 50ms pacing rates both Db/Db and HFHS mice displayed statistically significantly increased incidence of T wave alternans compared with controls (74.3% in Db/Db mice vs 20% in Db/+ mice, p<0.001, and 91.7% in HFHS fed mice compared with 16% in mice fed a control diet, p<0.0001). These data support a temporal or spatial dispersion of myocyte repolarization and/or abnormalities of Ca 2+ cycling which might play a role in the predisposition to VT in these mice. To examine this further, we determined whether cardiomyocytes from these mice develop early afterdepolarizations (EADs) in response to increasing pacing rates. We measured action potentials in adult ventricular myocytes isolated from HFHS-fed mice at pacing rates of 0.1 to 5Hz. Data summarized in Figure 1H demonstrate typical early afterdepolarizations at 0.1 Hz characterized by reversal of the action potential before repolarization is complete and a second EAD preceding the subsequent paced beat. Although 0 of 45 CMs from 5 mice on the control diet developed EADs, 7 of 56 cells from 7 mice on a HFHS diet (18%) developed EADs, (p<0.01 by chi-squared test, Figure 1G, 1H ). EADs, which cause ectopic beats and lethal arrhythmias reflect decreased repolarization reserve and/or an associated increase in cytosolic calcium 29 . These findings support the conclusion that in mice with DMII or metabolic syndrome, the LV develops increased susceptibility to VT which may be associated with Ca 2+ dyshomeostasis. Blunted parasympathetic response and decreased cGMP levels in hearts of Db/Db and HFHS fed mice Diabetic autonomic neuropathy, characterized initially by parasympathetic dysfunction, is a major complication of both type I and type II diabetes 9 , 13 . We therefore determined whether parasympathetic signaling was blunted in Db/Db and HFHS fed mice. We determined the heart rate response to a 0.5 mg/kg injection of the acetylcholine receptor antagonist atropine in Db/Db and in HFHS mice, as a readout for parasympathetic function. Compared with wild type control mice, the HFHS and Db/Db mice each displayed reduction in atropine-induced heart rate increase ( Figure 2 ) (relative beat-per-minute increase: 51 ± 5% in mice fed a control diet, 20 ± 3% in HFHS fed mice and 23 ± 10% in Db/Db mice, p<0.01), indicating a markedly reduced parasympathetic heart rate response in the latter 2 models. Download figure Open in new tab Figure 2. Parasympathetic dysfunction in mice with type 2 diabetes or metabolic syndrome. Relative increase in heart rate (HR) measured by implantable EKG transmitters in unanesthetized mice following intraperitoneal injection of 0.5 mg/kg atropine in mice fed control (Con) diet, high fat high sucrose (HFHS) diet, or Db/Db mice. *, p<0.05 by 1-way ANOVA with Dunnett’s multiple comparisons test. Parasympathetic stimulation promotes myocardial cGMP synthesis via the stimulation of the M2 muscarinic receptor 15 . Given the observed decrease in cardiac parasympathetic function in the Db/Db and HFHS mice, we tested for concomitant reduction in myocardial cGMP. cGMP concentration was reduced in LV extracts of both Db/Db and HFHS-fed mice, compared to control ( Figure 3A, 3C ). cGMP decreased 21 ± 4% in Db/Db vs Db/+ (p<0.01), and 29 ± 8% in HFHS-fed mice vs mice fed a control diet, (p<0.05). Additionally, LV protein expression of the cGMP-specific phosphodiesterase 5 (normalized to β-actin) was increased versus control both in Db/Db and HFHS (1.6 ± 0.04-fold in Db/Db vs. Db/+ mice, p<0.0001; 1.7 ± 0.2-fold increase in HFHS fed mice vs. control diet, p<0.05) ( Figure 3B, 3D ). Thus, cGMP levels in Db/Db and HFHS fed mice were decreased both at the level of synthesis via decreased muscarinic stimulation and increased turnover via increased PDE5-associated degradation. Download figure Open in new tab Figure 3. Reduced cGMP and increased cGMP-catabolizing phosphodiesterase 5 in LV tissue of mice with type II diabetes mellitus and metabolic syndrome. (A), cGMP levels and (B), representative immunoblot and densitometric analysis of phosphodiesterase 5 (PDE5) normalized to β-Actin in LV tissue from Db/+ and Db/Db mice. (C), cGMP levels and (D), representative immunoblot and densitometric analysis of phosphodiesterase 5 (PDE5) normalized to β-Actin in LV tissue from mice fed a high fat high sucrose (HFHS) diet or control diet (Con). *, p<0.05; **, p<0.01; ****, p<0.0001 by Student’s unpaired T test. Stimulation of cGMP synthesis with riociguat or inhibition of cGMP degradation with the PDE5 inhibitor sildenafil reverses inducible VT in Db/Db and HFHS fed mice To determine whether the reduction in cGMP signaling in the LV of Db/Db and HFHS fed mice played a role in their predisposition to VT, we treated Db/Db and HFHS-fed mice with the cGMP-augmenting soluble guanylate cyclase stimulator riociguat (10 mg/kg for 7 days), followed by programmed ventricular stimulation as above. Riociguat and sildenafil effects on ECG intervals are shown in Table 1 . Riociguat treatment reduced duration of VT in both the Db/Db mice ( Figure 4A ) and in the HFHS-fed mice ( Figure 4B ) compared with respective vehicle-treated controls. Specifically, total duration of VT in Db/Db mice decreased from: 4.1 ± 1.5 sec in vehicle-treated (n=13) to 0.9 ± 0.5 sec in riociguat-treated (n=15) mice (p<0.05). Download figure Open in new tab Figure 4. cGMP augmentation with the soluble guanylate cyclase stimulator riociguat or the phosphodiesterase 5 inhibitor sildenafil reduces duration of ventricular tachycardia in mice with type II diabetes and metabolic syndrome. Development of ventricular tachycardia (VT) in response to programmed ventricular stimulation in (A) Db/Db mice and (B) high fat high sucrose-fed mice pre-treated for 7 days with vehicle (V), riociguat (Rio) 10 mg/kg, or sildenafil 10 (Sil) 10 mg/kg. *, p<0.05 by Student’s unpaired T test (riociguat graphs) or in sildenafil-treated graphs, by Welch’s T test (Db/Db) or Mann-Whitney test (HFHS). Duration of VT in HFHS fed mice decreased from 20.3 ± 3.7 sec in vehicle-treated (n=8) to 6.8 ± 2.2 sec in riociguat-treated (n=6) mice, (p<0.05). We tested a second cGMP-augmenting drug, the phosphodiesterase 5 inhibitor sildenafil (10 mg/kg for 7 days), and again observed in HFHS-fed mice that sildenafil treatment reduced cumulative VT duration, compared with vehicle-treatment. Total duration of VT decreased from 4.0 ± 1.1 sec in vehicle-treated (n=21) Db/Db to 1.0 ± 0.9 sec in sildenafil-treated (n=5) Db/Db mice (p<0.05). Similarly, in HFHS-fed mice duration of VT decreased from 21.9 ± 4.0 sec in vehicle-treated (n=30) to 1.7 ± 1.6 sec in sildenafil-treated (n=4) mice (p<0.05).These findings indicate that augmenting cGMP levels opposes the predisposition to VT in the Db/Db and HFHS fed mice. Disruption of PKG1α activity in the absence of diabetes and metabolic syndrome promotes inducible VT and T wave alternans in vivo We next directly tested the role of the cGMP-dependent protein kinase 1α (PKG1α) in modulating VT in vivo . PKG1 is the primary cGMP effector in the LV, and cGMP binding to PKG1α stimulates PKG1α kinase function 30 . To examine the role of PKG1α signaling in opposing the predisposition to VT, we performed programmed ventricular stimulation on PKG1α leucine zipper mutant (LZM) mice. These mice harbor a series of point mutations within the PKG1α leucine zipper protein-protein binding domain, producing an enzyme which retains kinase function but cannot bind to LZ-dependent substrates. Importantly, the LZM mice have a normal metabolic phenotype and do not display evidence of diabetes or insulin resistance 31 . The LZM mouse has normal levels of cGMP and does not yet demonstrate ventricular hypertrophy or LV dysfunction at 12-14 weeks of age 18 , 32 , 33 . Unlike the Db/Db and HFHS-fed mice, LZM mice did not manifest autonomic dysfunction. Specifically, atropine treatment increased heart rate normally in LZM mice from 535 ± 42 beats/minute to 754 ± 15 beats/minute post-injection (n=5, p<0.01) ( Figure 5A ). Similarly, while Db/Db and HFHS-fed mice had blunted heart rate response to atropine compared with WT control mice ( Figure 2 ), the LZM mice had a normal increase in heart rate when compared to the same WT mice (51.0 ± 5.4% in WT controls vs. 44.2 ± 10.4% in LZM mice, p=0.65). In contrast to the normal heart rate response to atropine, LZM mice developed increased inducible VT in response to programmed ventricular stimulation, with a total duration of 18.8 ± 7.0 seconds in LZM (n=6) vs. 0 ± 0 seconds in WT (n=4) mice (p<0.05, Figure 5B ). Furthermore, like Db/Db and HFHS fed mice, LZM mice developed T-Wave alternans in response to pacing intervals at 60ms ( Figure 5C ). Specifically, 0 of 4 WT littermates compared with 5 of 6 LZM mice developed alternans ( Figure 5D , p<0.001 by chi-square analysis). Download figure Open in new tab Figure 5. cGMP-dependent protein kinase 1α mutation promotes ventricular tachycardia and calcium dyshomeostasis independently of diabetes or metabolic syndrome. (A) Heart rate at baseline or 4 minutes after 0.5 mg/kg atropine injection in PKG1α leucine zipper mutant (LZM) mice, and atropine-induced percentage heart rate increase versus wild type. (B) VT duration during programmed stimulation in LZM and WT littermates. (C) Representative surface recording and (D) T-wave alternans incidence at 16.7 Hz pacing in LZM or WT mice. Alternans present in 0/4 WT, 5/6 LZM. *, p<0.01 by chi-squared test. (E) Ca 2+ transients from LZM ventricular myocytes demonstrating alternans at 3 Hz. (F) Ca 2+ alternans threshold in ventricular myocytes from LZM and WT mice across increasing pacing rates. (G) Representative Ca 2+ transient in ventricular myocytes from WT and LZM mice. (H) Time constants of Ca 2+ reuptake, tau, in ventricular myocytes from: WT on control (Con) or HFHS (HFHS) diet, and LZM mice; injected daily with vehicle or 20 mg/kg of GSK3β inhibitor TWS119 for 7 days. *, p<0.05; **, p<0.01; ***, p<0.001, and ****, p<0.0001 by Student’s unpaired T test. Given the relationship between the development of T-wave alternans and abnormalities of Ca 2+ homeostasis 34 , the observation of T-wave alternans in HFHS-fed, Db/Db, and LZM mice suggested that a common mechanism of Ca 2+ dyshomeostasis might account for the predisposition to VT development in these mouse models. To test this hypothesis, adult ventricular myocytes isolated from LZM, HFHS-fed and WT mice were paced at increasing rates. Cells from LZM and HFHS fed mice developed Ca 2+ alternans Figure 5E . Furthermore, the average pacing threshold for the development of Ca 2+ alternans was reduced in LZM mice (2.6 ± 0.4 Hz, n=5) compared with WT mice (4 ± 0.3 Hz, n=7, p<0.05) ( Figure 5F ). The presence of Ca 2+ alternans suggested that shared abnormalities of intracellular calcium SR reuptake in LZM and HFHS mice might contribute to the pathogenesis of VT 34 . We therefore examined Ca 2+ transients and the time constant of cytosolic Ca 2+ reuptake in ventricular myocytes loaded with the Ca 2+ sensitive dye Fura-2. Ca 2+ transients from HFHS CMs ( Figure 5G ), demonstrated reduced slope of the Ca 2+ reuptake curve, supporting decreased rate of sarcoplasmic reticulum Ca 2+ from the cytosol. Quantitative comparison of Ca 2+ transients from ventricular myocytes from HFHS fed and LZM mice with myocytes from mice fed a control diet or WT mice respectively, further demonstrated increased time constant of cytosolic Ca 2+ decline (tau). Specifically, tau values in cardiomyocytes from HFHS fed mice were 0.23 ± 0.02 ms, n=7 vs 0.17 ± 0.01 ms in cells from mice fed a control diet (n=10, p<0.01). In cells from LZM mice (n=8) tau was 0.30 ± 0.03 ms, (p<0.0001) vs control ( Figure 5H ). These findings indicate that metabolic syndrome and PKGα dysfunction produce similar slowing in diastolic Ca 2+ sarcoplasmic reticulum reuptake in CMs, which would predispose to VT. Disinhibition of GSK3β signaling in the LVs of diabetic, metabolic syndrome, and PKG1 mutant mice The above findings support the conclusion that reduced myocardial cGMP in the Db/Db and HFHS mice promotes inducible VT through reduction of PKG1α signaling, and that selective disruption of PKG1α is sufficient to promote susceptibility to VT independently of other side effects of DM or metabolic syndrome, including insulin insensitivity and autonomic dysfunction. We next investigated potential shared molecular mechanisms downstream of PKG1α underlying the predisposition to the genesis of VT in these models. We previously demonstrated that in a mouse model for DMI, myocardial hyperactivity of GSK3β contributes to a pro-arrhythmic phenotype 13 . We determined whether GSK3β activity might also be increased in ventricular extracts of HFHS and Db/Db mice, and whether inhibition of GSK3β activity had an effect on Ca 2+ reuptake and inducible VT. GSK3β activity is inhibited by phosphorylation at serine 9. We therefore measured myocardial pSer9-GSK3β by immunoblot in ventricular extracts from the HFHS, Db/Db and LZM models. Importantly, all 3 models displayed reduced serine 9 phosphorylated/total GSK3β compared to respective controls, indicating disinhibited, or hyperactivated GSK3β. Immunoblot data summarized in Figure 6A demonstrate pSer9/total GSK3β of: 0.64 ± 0.08 in HFHS-fed (n=3) vs. 1.00 ± 0.07 on control diet (n=3), p<0.05; 0.73 ± 0.06 in Db/Db (n=4) vs. 1.0 ± 0.07 in Db/+ (n=4), p=0.025; 0.35 ± 0.06 in LZM (n=3) vs. 1.00 ± 0.03 in WT (n=3), p<0.001. Download figure Open in new tab Figure 6. Disinhibition of GSK3β signaling in LV tissue predisposes to ventricular tachycardia in mice fed a high fat high sucrose diet, or LZM mice harboring a disrupting mutation of protein kinase G 1 alpha. Representative immunoblot and quantitative densitometry of phospho-serine 9 GSK3β normalized to total GSK3β in LV lysates from: (A) high fat high sucrose (HFHS)-fed mice; (B) Db/Db mice; and (C) PKG1α leucine zipper mutant (LZM) mice, compared with mice on control diet, Db/+, and wild type littermate controls respectively. Duration of ventricular tachycardia in response to programmed ventricular stimulation in (D) HFHS, (E) Db/Db, and (F) LZM mice treated for 7 days with either vehicle or with 20 mg/kg of the GSK3β inhibitor TWS119. *, p<0.05 and ****, p<0.001 by Student’s unpaired T test. We next examined the effects of GSK3β inhibition on CM Ca 2+ reuptake. Pre-treatment of HFHS-fed mice with the GSK3β inhibitor TWS119 (20 mg/kg daily via intraperitoneal injection for 7 days) decreased tau values for Ca 2+ reuptake compared with vehicle-treated HFHS-fed mice (tau = 0.23 ± 0.02 ms, n=8, in vehicle-treated HFHS mice vs 0.17 ± 0.01 ms in HFHS-TWS119, n=7, p<0.05). Similarly, TWS099 treatment reduced tau in LZM mice compared with LZM vehicle-treated controls (tau =0.30 ± 0.03 ms in LZM vehicle-treated, n=8, vs 0.21 ± 0.02 ms in LZM-TWS119-treated mice, n=4, p<0.05). Tau values in TWS119-treated mice were not significantly different from tau values observed in cells from vehicle treated control mice ( Figure 5F ). Inhibition of GSK3b reduces duration of inducible VT in HFHS, Db/Db, and LZM mice To test for a causal role of GSK3β and its associated effects on Ca 2+ reuptake kinetics in mediating the proarrhythmogenic effects in these models, we next treated HSHS, Db/Db, and LZM mice, and their respective controls, with the selective GSK3β inhibitor TWS119. As shown in Figure 6B , TWS199 treatment normalized the duration of VT in response to programmed stimulation in: HFHS-fed mice (25.3 ± 5.9 sec in vehicle-treated HFHS, n=10, vs. 7.7 ± 3.3 sec in TWS119-treated HFHS, n=4, p<0.05); Db/Db mice (3.7 ± 1.6 sec, in vehicle-treated Db/Db mice, n=6, vs. 0 ± 0, n=3 in TWS119-treated Db/Db mice, p=0.07). We observed a similar reduction in LZM mice treated with TWS119, compared with vehicle-treated control LZM mice also presented in Figure 5B (18.8 ± 7.0 sec in vehicle-treated LZM mice, n=6, vs. 0.1 ± 0.1 sec in TWS119-treated LZM mice, n=3, p<0.05). Role of cGMP and GSK3β in abnormalities of cardiac myocyte calcium handling proteins in Db/Db, HFHS, and LZM mice Based on the above findings of myocardial and CM calcium dyshomeostasis in the HFHS and LZM mice, we examined the expression and phosphorylation state of cardiac myocyte proteins involved in sarcoplasmic reticulum (SR) Ca 2+ reuptake from the cytoplasm. The dephosphorylated form of the SR protein phospholamban (PLB) directly inhibits the activity of the Ca 2+ reuptake protein sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase 2a (SERCA2a), thus impairing SR reuptake of Ca 2+ during diastole 35 . The phosphorylation state of PLB is regulated by complex dynamics of kinases and phosphatases. PKA and by PKG1 increase inhibitory phosphorylation of PLB, leading to increased SERCA2a activity. Conversely, protein phosphatase-1 (PP1) dephosphorylates PLB resulting in increased SERCA2a activity inhibition. However, PP-1 activity is inhibited upon complexing with the inhibitor I-2 36 . GSK3β phosphorylation of I-2 on Thr72 inhibits I-2 binding to PP-1 37 . We hypothesized that the hyperactivated GSK3β in HFHS, Db/Db, and LZM myocardium would ultimately promote reduced PLB phosphorylation on Ser16/Thr17 (as outlined in Fig. 8 ). Western blot analysis of LV lysates from HFHS-fed mice, using an anti-pSer16/Thr17 PLB antibody, demonstrated markedly reduced PLB phosphorylation normalized to total PLB (pPLB/TPLB) from 1.0 ± 0.1 in extracts of control diet-fed (n=3) mice to 0.18 ± 0.03 (n=3) in extracts of vehicle treated HFHS-fed mice (p<0.05). We observed no changes in LV expression of SERCA2a or in total PLB in LV extracts of HFHS fed mice ( Figure 7A , B ). However, LV tissue from HFHS-fed mice displayed increased phosphorylation of the PP-1 inhibitor I-2 at Thr 72 (ratio of p-I2/T I-2 1.0 ± 0.2 in control diet vs. 1.6 ± 0.02 in extracts of HFHS-fed mice, p<0.05). Given that phosphorylation of I-2 at Thr72 interferes with its binding to and inhibition of PP-1, these findings support that the HFHS diet ultimately results in inhibition of SERCA2a at least in part via phosphorylation of I-2, subsequent PLB dephosphorylation via PP-1, and a resulting increase in direct PLB inhibition of SERCA2a. Importantly, phosphorylation of PLB in extracts of LV tissue of LZM mice was also decreased compared with WT controls. The ratio of Ser16/Thr17 PLB to total PLB (pPLB/TPLB) in LV lysates of LZM mice was decreased from 1.00 ± 0.02 in WT mice, to 0.41 ± 0.02 in LZM mice, (p<0.05, Figure 7C , D ). These data support a common abnormality of the regulation of sarcoplasmic reticulum Ca 2+ mobilization proteins in LZM and HFHS models in the pathogenesis of VT. Download figure Open in new tab Figure 7. GSK3β inhibition or cGMP-pathway augmentation rescues Ca 2+ handling protein abnormalities in LV tissue of high fat high sucrose diet and PKG1α leucine zipper mutant mice. (A) Representative immunoblots for ser16/thr17phosphorylated PLB (pPLB), phospholamban (PLB), SERCA2a, phospho-Thr72 protein phosphatase 1 inhibitor subunit 2 (pThr72I-2), I-2 and loading control Gβ in LV lysates from control or high fat high sucrose (HFHS)-fed mice, treated 7 days with vehicle or 20 mg/kg daily injection of GSK3β inhibitor TWS119. (B) Densitometry of pPLB normalized to PLB, pI2 normalized to I2, PLB and SERCA normalized to Gβ. (C) Representative immunoblot for phosphor-ser16/thr17 PLB, PLB, and Gβ in LV tissue from control or PKG1α leucine zipper mutants (LZM), treated with vehicle or 20 mg/kg daily injection the GSK3β inhibitor TWS119 for 7 days. (D) Quantitative densitometry of pPLB and PLB normalized to PLB and Gβ respectively. (E) Representative immunoblot for phospho-serine 9 GSK3β, total GSK3β, phosphor-ser16/thr17 PLB, and PLB. (F) Quantitative densitometry of phospho-serine 9 GSK3β normalized to total GSK3β and pPLB normalized to PLB. *, p<0.05; **, p<0.01; ***, p<0.001, and ****, p<0.0001 by 1-way ANOVA with Tukey’s multiple comparison tests, or Student’s unpaired T test for two group comparisons. #, P<0.05 by Student’s T test. N=3 per group. Download figure Open in new tab Figure 8. Model for the mechanism of predisposition to ventricular tachycardia in mouse models for type II diabetes and metabolic syndrome. (A) Normal : sGC production of cGMP activates myocardial cGMP-dependent protein kinase 1 (PKG1), leading to tonic GSK3β inhibition and preventing GSK3β-mediated inhibition of protein phosphatase inhibitor I-2. I-2 normally prevents phospholamban dephosphorylation on serine 16/17 by protein phosphatase-1 (PP-1). PKG1α directly phosphorylates phospholamban on serine 16. These dual mechanisms remove tonic phospholamban inhibition of the SERCA transporter, promoting reuptake of cytosolic calcium into the sarcoplasmic reticulum. (B) Diabetes and metabolic syndrome: increased PDE5 catalysis and parasympathetic dysfunction reduce available cGMP. Reduction of intracellular cGMP removes the PKG1α of GSK3β, resulting in hyperactive/disinhibited GSK3β and reduced PKG1α phospholamban phosphorylation on serine 16, thus promoting phospholamban inhibition of SERCA. The resulting blunted Ca 2+ reuptake into the sarcoplasmic reticulum leads to EADs, Ca 2+ alternans, T-wave alternans, and predisposition to ventricular tachycardia. To determine directly if GSK3β kinase function played a role in these abnormalities of Ca +2 handling proteins, we determined the effect of the GSK3β inhibitor TWS119 on the phosphorylation of PLB. Treatment of HFHS fed mice for 7 days with TWS119 increased P/T PLB to 1.61 ± 0.05 vs. 0.18 ± 0.03 in vehicle-treated HFHS-fed mice, p<0.0001, Figure 7A , B . Furthermore, TWS119 treatment of LZM mice increased P/T PLB ratio of 0.70 ± 0.02 compared with 0.41 ± 0.02 in vehicle-treated LZM mice, p<0.001 ( Figure 7B ). However, the effect of TWS119 on phosphorylation of PLB was less marked in the LZM mice than in the HFHS-fed mice. Importantly, treatment of HFHS fed mice with TWS119 had no effect on the level of SERCA2a, PLB or I-2. However, TWS119 did decrease the level of pSer72I-2/TotalI2 from 1.60 ± 0.02 in vehicle-treated HFHS-fed mice to 1.39 ± 0.06 in TWS119 treated mice, p=0.025 ( Figure 7A , B ). These data support the conclusion that increased GSK3β activity regulated Ca 2+ reuptake at least in part via an I-2/PP-1/PLB pathway. Given that increasing cGMP levels via sildenafil inhibition of PDE5 attenuated inducible VT in response to programmed ventricular stimulation, we determined directly whether these antiarrhythmic effects might be associated with increased inhibitory phosphorylation of GSK3β and PLB in HFHS fed mice. HFHS fed mice were treated for 1 week with sildenafil. Western blot analysis of ventricular extracts from these mice demonstrated that the ratio of pSer16/Thr17/total PLB increased from 1.01 ± 0.09 in vehicle-treated HFHS-fed mice to 5.07 ± 0.11 in sildenafil-treated HFHS-fed mice (n=3 per group, p<0.0001), while the ratio of pSer9/total GSK3β increased from 1.00 ± 0.26 in vehicle-treated HFHS-fed mice to 2.33 ± 0.15 in sildenafil-treated HFHS-fed mice (n=3 per group, p<0.05). Discussion We investigated the predisposition to VT by programmed stimulation in the Db/Db and HFHS models of DMII and metabolic syndrome, respectively, to understand potential mechanisms by which these conditions promote sudden cardiac death. We observed: 1) Increased susceptibility of Db/Db and HFHS mice to inducible VT and T wave alternans in response to programmed stimulation; 2) autonomic dysfunction as evidenced by blunted heart rate sensitivity to parasympathetic stimulation, reduced cGMP, and increased PDE5 expression in LV tissue of Db/Db and HFHS mice; 3) reduction of VT in the Db/Db and HFHS mice after treatment with the cGMP augmenting drugs riociguat or sildenafil; 4) recapitulation of inducible VT and calcium dyshomeostasis in PKG1α LZM mice which have blunted response to cGMP signaling but do not have DM or metabolic syndrome; and 5) evidence of disinhibition of GSK3β activity in hearts of the above 3 models, as well as normalization of inducibility of VT and of calcium dyshomeostasis in response to GSK3β inhibition and cGMP augmenting drugs. Taken together, we interpret these findings to support that DMII and metabolic syndrome create an arrhythmic substrate through reduction in cGMP and thus reduced signaling via the cGMP effector PKG1α, leading to disinhibition of GSK3β and resultant Ca 2+ dyshomeostasis, EADs and predisposition to ventricular tachycardia ( Figure 8 ). These findings also represent, to our knowledge, the first direct demonstration that PKG1α opposes the pathogenesis of VT. We observed a high burden of inducible VT in response to programmed stimulation in both the Db/Db and the HFHS models. Previous studies have identified increased ventricular arrhythmias in various models of DMII, such as the high fat diet model 11 . The Db/Db mouse harbors mutations in the leptin receptor, leading to obesity and DMII. Isolated hearts from Db/Db mice display increased ventricular arrhythmia during ex vivo programmed stimulation 10 . Our current findings now identify increased in vivo susceptibility of the Db/Db model to programmed stimulation-induced VT. This is important because programmed stimulation in human patients predicts VT and SCD 26 , 27 , and has been used to stratify patients with heart failure and reduced LV ejection fraction (HFrEF) for implantation of automatic internal cardiac defibrillators 38 . Our findings therefore support the Db/Db model as a clinically relevant tool for the study of mechanisms of VT genesis. The HFHS diet produces a similar phenotype of obesity, but leads to insulin resistance and metabolic syndrome, rather than overt DMII 17 . Impaired glucose tolerance and metabolic syndrome, like DMII, increase SCD risk in patients 4 – 6 . Thus, the presence of VT inducibility in the HFHS mice holds similar clinical relevance as described for the Db/Db mice. Neither the Db/Db model nor the HFHS model develops coronary disease or acute MI. Thus, these models enable the investigation of mechanisms of VT predisposition independent from acute ischemia. More specifically, our findings in the Db/Db and the HFHS mouse models support the concept that even in the absence of coronary disease, DMII and metabolic syndrome serve as arrhythmic substrates which, in response to triggers, may lead to VT and SCD. A principal finding of our study is the observation of shared reduction of myocardial cGMP in the HFHS and Db/Db models, and the demonstration that cGMP augmentation with the soluble guanylate cyclase stimulator riociguat or the PDE5 inhibitor sildenafil prevented VT inducibility. We observed evidence of myocardial parasympathetic downregulation in HFHS and Db/Db mice ( Figure 2 ), consistent with established autonomic dysfunction in DM, diabetic autonomic neuropathy 4 . Because myocardial parasympathetic stimulation promotes cGMP synthesis 39 , we investigated myocardial cGMP levels in the Db/Db and HFHS mouse hearts. Several other lines of evidence also suggested a potential role of myocardial cGMP dysregulation in the pathogenesis of VT in DM. First, obesity and metabolic abnormalities reduce availability of natriuretic peptides (NPs) 40 , which normally promote cGMP synthesis through activation of membrane NP receptor guanylate cyclases. Second, others have observed reductions in myocardial cGMP in HFpEF, which itself often arises in the settings of obesity, metabolic syndrome or DM 41 . These varied observations led us to investigate whether dysregulation of cGMP in the LV of the Db/Db and HFHS mice promotes the VT substrate observed in DM and metabolic syndrome. Our finding that systemic cGMP augmentation with riociguat or sildenafil normalized the burden of inducible VT in the Db/Db and the HFHS mice supports a causal role of cGMP catabolism in producing the VT substrate in Db/Db and HFHS mice. Collectively, we interpret these findings to indicate that in DMII and metabolic syndrome, parasympathetic dysfunction and increased myocardial PDE5 expression lead to reduced availability of cGMP and subsequent predisposition to VT. Our findings also provide direct evidence that the cGMP effector PKG1α normally opposes development of VT. Specifically, mutation of the cGMP effector protein kinase G 1α (PKG1α) directly increased inducibility of VT, similar to what we observed in the Db/Db and HFHS mice. In the cardiovascular system cGMP regulates several effectors including cGMP-sensitive phosphodiesterases and the cGMP-dependent protein kinase 1 (PKG1) 42 . PKG1α and 1β are encoded by the same PKG1 gene, but differ only in alternatively spliced N-terminal sequences 30 . cGMP binds PKG1 directly and activates its kinase function 30 . PKG1α contains an n-terminal, isoform-specific leucine zipper domain which mediates PKG1α protein-protein binding to critical substrates both in the vascular smooth muscle 43 and in the cardiac myocyte 44 . Mice harboring genetic disruption of the PKG1α LZ domain, termed LZ mutant (LZM) mice, demonstrate reduced basal phosphorylation of established PKG1α substrates including myosin light chain phosphatase and RhoA 18 . The LZ domain also mediates PKG1α binding to the cardiac myocyte-specific PKG1α substrate cardiac myosin binding protein C 44 . We therefore used the LZM mice to investigate directly the role of PKG1α in modulating susceptibility to ventricular arrhythmia in vivo . Our findings of increased induced VT and Ca 2+ alternans in the LZM mice identify that disruption of PKG1α sufficiently recapitulates the arrhythmia phenotype observed in the Db/Db and HFHS mice, independent of the complications of diabetes, glucose intolerance and autonomic dysfunction. Notably, prior work demonstrated that mutation of the PKG1α LZ domain abolished the therapeutic effects of sildenafil after transaortic constriction 32 , indicating that intact PKG1α mediated the therapeutic effects of cGMP in heart failure with reduced LV ejection fraction. Our current findings now directly demonstrate that disruption of PKG1α signaling promotes susceptibility to induced VT. Our findings now support that reduced cGMP in the Db/Db and HFHS myocardium leads to inducible VT through reduction of PKG1 activation. We conclude, therefore, that disruption of cGMP and downstream myocardial PKG1 signaling promotes the increased susceptibility of Db/Db and HFHS mice to VT. Importantly, PKG1 signaling is impaired in LV tissue from patients with DMII 45 , further supporting that cGMP-PKG1 disruption underlies predisposition to VT and SCD in humans with DM and insulin resistance. We observed reduced serine 9 phosphorylation of GSK3β in LV lysates from Db/Db, HFHS, and LZM mice ( Figure 6 ). Given that serine 9 phosphorylation inhibits GSK3β kinase function, these findings reveal disinhibition of myocardial GSK3β as a shared abnormality in the above models. In the current study, GSK3β inhibition with TWS119 normalized VT susceptibility in the Db/Db and HFHS mice. We previously demonstrated disinhibition of GSK3β as promoting VT susceptibility in the Akita mouse model of DMI 13 . However, our current findings now indicate a causal role of myocardial GSK3β disinhibition in promoting VT in DMII and metabolic syndrome. Further, NO-cGMP signaling inhibits GSK3β in several cell types 46 . Notably, we also observed GSK3β disinhibition in the myocardium of PKG1α LZM mice, and demonstrated that GSK3β inhibition reduces inducible VT in the LZM mice. We therefore interpret these collective findings to support that reduced myocardial cGMP production in DM and metabolic syndrome and resultant disruption of PKG1 signaling contributes to VT substrate through disinhibition of GSK3β. Each of the 3 experimental models in our study demonstrated accentuated QRS/T-wave alternans (TWA) and/or reduced heart rate threshold for TWA in response to burst pacing. T-wave alternans has been associated with life threatening ventricular arrhythmias 19 and sudden death in the setting of MI, long QT syndrome, and heart failure 20 . Mechanistically, T-wave alternans has been attributed either to a temporal or spatial dispersion of myocyte repolarization or abnormalities of Ca 2+ cycling 21 , 22 . Accordingly, ventricular myocytes from the LZM mice also displayed Ca 2+ alternans, or alternating oscillations in the amplitude of Ca 2+ transients in response to increased pacing rates. Ca 2+ alternans may reflect the inability of the myocyte to cycle Ca 2+ beat to beat in response to increasing heart rate and has been attributed to abnormalities in the function of Ca 2+ handling proteins 24 , 25 . Our previous work identified excess T wave alternans in a mouse model of DMI, and demonstrated that GSK3β inhibition normalized TWA in DMI mice 47 . Studies in human patients have revealed TWA as an additive risk factor for SCD and for VT 48 . Experimental studies support that TWA arises as a consequence of cardiac myocyte calcium dyshomeostasis, specifically due to reduction in diastolic Ca 2+ reuptake in the sarcoplasmic reticulum 25 . As T wave and Ca 2+ alternans have been closely linked to the pathogenesis of VT and SCD in heart failure 23 , we interpret our findings to reveal Ca 2+ dyshomeostasis as a shared mechanism in models of DMII, metabolic syndrome, and PKG1 disruption. Prior studies have established that cGMP signaling in the cardiac myocyte promotes lusitropy and calcium reuptake 35 . Mechanistically, PKG1α directly phosphorylates phospholamban (PLB), leading to increased SR Ca 2+ reuptake 35 . Here we observed reduced PLB phosphorylation in LV tissue of LZM mice ( Figure 7 ). We also detected increased time constant of Ca 2+ reuptake and a lower Ca 2+ alternans threshold in CMs from LZM mice, compared with their WT littermates. These findings support directly that PKG1α contributes to maintenance of normal SR Ca 2+ reuptake, and that disruption of normal cGMP-PKG1 signaling promotes TWA through dysregulated PLB and resultant SR dysfunction. We also note that the GSK3β inhibitor TWS119 did not completely restore a normal pattern of PLB phosphorylation in LZM LV tissue. This finding is consistent with a mechanism in which PKG1α promotes PLB phosphorylation and Ca 2+ homeostasis both through tonic inhibition of GSK3β signaling, but also through direct phosphorylation of PLB. Furthermore, we demonstrated that riociguat and sildenafil, which attenuate inducible VT in these mice, decrease levels of GSK3β activity and reverse the decrease in PLB phosphorylation consistent with the conclusion that their anti-antiarrhythmic effect might be mediated via the normalization of GSK3β mediated Ca 2+ dyshomeostasis. From a translational perspective, our findings suggest that pharmacological augmentation of cGMP-PKG1 signaling might reduce VT, and thus SCD, in patients with DM or metabolic syndrome. However, reduction of cGMP occurs in additional conditions associated with SCD, which therefore suggests a broader role of dysregulation of cGMP-PKG1 signaling in promoting VT in disease states other than DM. For example, myocardial tissue from patients with heart failure with preserved LV ejection fraction (HFpEF) demonstrates reduced cGMP levels, compared with HFrEF and with nonfailing hypertrophied LV tissue 41 , 49 . Notably, SCD remains the highest cause of death in HFpEF, and SCD risks increase in HFpEF patients with concomitant DM 50 . The reduction of cGMP in HFpEF may occur through blunted cGMP synthesis by NO-sensitive guanylate cyclase 51 , or through increased cGMP catalysis by phosphodiesterases 5 and 9 52 . Further, sildenafil use also correlates with reduced post-MI death in patients 53 . Finally, experimental studies implicate cGMP and PKG1 signaling in the regulation of ventricular arrhythmia in models of diseases other than DM. For example, PDE5 inhibition with sildenafil opposes VT both in model of acute infarction in dogs 54 , and in a drug-induced long QT syndrome model in sheep 55 . Genetic deletion of the genes for ANP and BNP in mice leads to reduced cGMP-PKG1 signaling and promotes both sudden death after transaortic constriction, and increased isoproterenol-induced VT 56 . Our findings of increased VT inducibility in PKG1α LZM mice now provide to our knowledge the first direct evidence for the role of PKG1 in normally opposing development of VT substrate in vivo . Notably, the LZM mouse does not develop diabetes or obesity. Accordingly, we propose that the established disruption of cGMP signaling in disease states may ultimately promote VT and SCD through dysregulation of myocardial PKG1 activity, localization, or substrate availability. PKG1-activating therapies including sildenafil, sacubitril/valsartan, vericiguat, and nitrates are safe and are routinely prescribed to patients. Whether these agents or other investigational PKG1 activators such as PDE9 inhibitors might reduce VT events remains unknown. Thus, our findings support further investigation of cGMP-augmenting therapies to reduce SCD in a variety of conditions. Study limitations Our study has several limitations. First, we focused our studies on male mice only, since we did not observe inducible VT in female mice on HFHS diet. Importantly, female Db/Db and HFHS mice have sex-specific differences in their cardiac phenotypes 57 . Future work will explore the role of cGMP-PKG1 signaling in arrhythmogenesis in females, and the mechanisms underlying the sex effect in VT predisposition in these models. Second, we only used a single model of programmed stimulation to study VT susceptibility. We therefore cannot conclude whether dysregulation of cGMP, PKG1, or GSK3β alters arrhythmia susceptibility in other pro-arrhythmic states such as myocardial infarction or HFrEF. Future studies will address these important questions. Conclusions In summary, we identified abnormalities of cGMP signaling as promoting VT, Ca 2+ dyshomeostasis, and GSK3β disinhibition in two models of DMII and metabolic syndrome in mice. These findings support the translational relevance of further study of myocardial cGMP and PKG1 signaling as protecting against VT and SCD in patients with DM. Conflicts of interest The authors declare no conflicts of interest. Funding information This work was supported by the National Institutes of Health 1R01HL162919 to R.M.B., and 1R01HL153433 to J.B.G. and R.M.B.. The funding sources had no input into the study design, collection and interpretation of data, in the writing of the manuscript, or in the decision to submit this paper. AUTHOR CONTRIBUTIONS Conceptualization, X.C., R.M.B., and J.B.G.; Methodology; Y.Z., A.T., X.C., M.J.A., G.L.M., A.A., M.A., J.H., T.P., B.W., and P.L.; Formal Analysis, Y.Z., A.T., X.C., M.J.A., G.L.M., P.L., C.M., and J.B.G.; Writing-Original Draft Preparation, R.M.B. and J.B.G; Writing-Review & Editing, X.C., A.T., Y.Z., P.L., M.J.A., G.L.M., R.M.B., and J.B.G. Acknowledgements None Abbreviations cGMP cyclic guanosine monophosphate CM cardiac myocyte GSK3β glycogen synthase kinase-3 beta LV left ventricle PKG cGMP-dependent protein kinase References 1. ↵ Wong CX , Brown A , Lau DH , et al. Epidemiology of Sudden Cardiac Death: Global and Regional Perspectives . Heart Lung Circ . Jan 2019 ; 28 ( 1 ): 6 – 14 . doi: 10.1016/j.hlc.2018.08.026 OpenUrl CrossRef PubMed 2. ↵ Braunwald E . Evolution of the management of acute myocardial infarction: a 20th century saga . Lancet. Nov 28 1998 ; 352 ( 9142 ): 1771 – 4 . doi: 10.1016/s0140-6736(98)03212-7 OpenUrl CrossRef 3. ↵ Moss AJ , Zareba W , Hall WJ , et al. Prophylactic implantation of a defibrillator in patients with myocardial infarction and reduced ejection fraction . N Engl J Med. Mar 21 2002 ; 346 ( 12 ): 877 – 83 . doi: 10.1056/NEJMoa013474 OpenUrl CrossRef PubMed Web of Science 4. ↵ Siscovick DS , Sotoodehnia N , Rea TD , Raghunathan TE , Jouven X , Lemaitre RN . Type 2 diabetes mellitus and the risk of sudden cardiac arrest in the community . Rev Endocr Metab Disord . Mar 2010 ; 11 ( 1 ): 53 – 9 . doi: 10.1007/s11154-010-9133-5 OpenUrl CrossRef PubMed Web of Science 5. Eranti A , Kerola T , Aro AL , et al. Diabetes, glucose tolerance, and the risk of sudden cardiac death . BMC Cardiovasc Disord. Feb 24 2016 ; 16 : 51 . doi: 10.1186/s12872-016-0231-5 OpenUrl CrossRef 6. ↵ Hess PL , Al-Khalidi HR , Friedman DJ , et al. The Metabolic Syndrome and Risk of Sudden Cardiac Death: The Atherosclerosis Risk in Communities Study . J Am Heart Assoc. Aug 23 2017 ; 6 ( 8 ) doi: 10.1161/jaha.117.006103 OpenUrl CrossRef 7. ↵ Lynge TH , Svane J , Pedersen-Bjergaard U , et al. Sudden cardiac death among persons with diabetes aged 1-49 years: a 10-year nationwide study of 14 294 deaths in Denmark . Eur Heart J. Jul 21 2020 ; 41 ( 28 ): 2699 – 2706 . doi: 10.1093/eurheartj/ehz891 OpenUrl CrossRef PubMed 8. ↵ Remme CA . Sudden Cardiac Death in Diabetes and Obesity: Mechanisms and Therapeutic Strategies . Can J Cardiol . Apr 2022 ; 38 ( 4 ): 418 – 426 . doi: 10.1016/j.cjca.2022.01.001 OpenUrl CrossRef PubMed 9. ↵ Rajab M , Jin H , Welzig CM , et al. Increased inducibility of ventricular tachycardia and decreased heart rate variability in a mouse model for type 1 diabetes: effect of pravastatin . Am J Physiol Heart Circ Physiol . Dec 2013 ; 305 ( 12 ): H1807 – 16 . doi: 10.1152/ajpheart.00979.2012 OpenUrl CrossRef PubMed 10. ↵ Jungen C , Scherschel K , Flenner F , et al. Increased arrhythmia susceptibility in type 2 diabetic mice related to dysregulation of ventricular sympathetic innervation . Am J Physiol Heart Circ Physiol. Dec 1 2019 ; 317 ( 6 ): H1328 – h1341 . doi: 10.1152/ajpheart.00249.2019 OpenUrl CrossRef 11. ↵ Liu H , Zhao Y , Xie A , et al. Interleukin-1β, Oxidative Stress, and Abnormal Calcium Handling Mediate Diabetic Arrhythmic Risk . JACC Basic Transl Sci . Jan 2021 ; 6 ( 1 ): 42 – 52 . doi: 10.1016/j.jacbts.2020.11.002 OpenUrl CrossRef PubMed 12. ↵ Frontoni S , Bracaglia D , Baroni A , et al. Early autonomic dysfunction in glucose-tolerant but insulin-resistant offspring of type 2 diabetic patients . Hypertension . Jun 2003 ; 41 ( 6 ): 1223 – 7 . doi: 10.1161/01.hyp.0000073062.29546.01 OpenUrl CrossRef 13. ↵ Zhang Y , Welzig CM , Picard KL , et al. Glycogen synthase kinase-3beta inhibition ameliorates cardiac parasympathetic dysfunction in type 1 diabetic Akita mice . Diabetes . Jun 2014 ; 63 ( 6 ): 2097 – 113 . doi: 10.2337/db12-1459 OpenUrl Abstract / FREE Full Text 14. ↵ Zhang Y , Welzig CM , Haburcak M , et al. Targeted disruption of glycogen synthase kinase-3β in cardiomyocytes attenuates cardiac parasympathetic dysfunction in type 1 diabetic Akita mice . PLoS One . 2019 ; 14 ( 4 ): e0215213 . doi: 10.1371/journal.pone.0215213 OpenUrl CrossRef PubMed 15. ↵ Brodde OE , Michel MC . Adrenergic and muscarinic receptors in the human heart . Pharmacol Rev . Dec 1999 ; 51 ( 4 ): 651 – 90 . OpenUrl FREE Full Text 16. ↵ Hummel KP , Dickie MM , Coleman DL . Diabetes, a new mutation in the mouse . Science. Sep 2 1966 ; 153 ( 3740 ): 1127 – 8 . doi: 10.1126/science.153.3740.1127 OpenUrl Abstract / FREE Full Text 17. ↵ Qin F , Siwik DA , Luptak I , et al. The polyphenols resveratrol and S17834 prevent the structural and functional sequelae of diet-induced metabolic heart disease in mice . Circulation. Apr 10 2012 ; 125 ( 14 ): 1757 – 64 , s1-6. doi: 10.1161/circulationaha.111.067801 OpenUrl CrossRef 18. ↵ Michael SK , Surks HK , Wang Y , et al. High blood pressure arising from a defect in vascular function . Proc Natl Acad Sci U S A. May 6 2008 ; 105 ( 18 ): 6702 – 7 . doi: 10.1073/pnas.0802128105 OpenUrl Abstract / FREE Full Text 19. ↵ Pastore JM , Girouard SD , Laurita KR , Akar FG , Rosenbaum DS . Mechanism linking T-wave alternans to the genesis of cardiac fibrillation . Circulation. Mar 16 1999 ; 99 ( 10 ): 1385 – 94 . doi: 10.1161/01.cir.99.10.1385 OpenUrl CrossRef 20. ↵ Cutler MJ , Rosenbaum DS . Explaining the clinical manifestations of T wave alternans in patients at risk for sudden cardiac death . Heart Rhythm . Mar 2009 ; 6 ( 3 Suppl): S22 – 8 . doi: 10.1016/j.hrthm.2008.10.007 OpenUrl CrossRef PubMed Web of Science 21. ↵ Verrier RL , Malik M . Electrophysiology of T-wave alternans: mechanisms and pharmacologic influences . J Electrocardiol. Nov - Dec 2013 ; 46 ( 6 ): 580 – 4 . doi: 10.1016/j.jelectrocard.2013.07.003 OpenUrl CrossRef PubMed 22. ↵ Laurita KR , Rosenbaum DS. Cellular mechanisms of arrhythmogenic cardiac alternans . Prog Biophys Mol Biol . Jun-Jul 2008 ; 97 ( 2 -3): 332 – 47 . doi: 10.1016/j.pbiomolbio.2008.02.014 OpenUrl CrossRef PubMed Web of Science 23. ↵ Díaz ME , O’Neill SC , Eisner DA . Sarcoplasmic reticulum calcium content fluctuation is the key to cardiac alternans . Circ Res. Mar 19 2004 ; 94 ( 5 ): 650 – 6 . doi: 10.1161/01.res.0000119923.64774.72 OpenUrl CrossRef 24. ↵ Picht E , DeSantiago J , Blatter LA , Bers DM . Cardiac alternans do not rely on diastolic sarcoplasmic reticulum calcium content fluctuations . Circ Res. Sep 29 2006 ; 99 ( 7 ): 740 – 8 . doi: 10.1161/01.res.0000244002.88813.91 OpenUrl CrossRef 25. ↵ Qu Z , Nivala M , Weiss JN . Calcium alternans in cardiac myocytes: order from disorder . J Mol Cell Cardiol . May 2013 ; 58 : 100 – 9 . doi: 10.1016/j.yjmcc.2012.10.007 OpenUrl CrossRef PubMed 26. ↵ Wilber DJ . Electrophysiological testing in high-risk patients with previous myocardial infarction . Circulation . Dec 1990 ; 82 ( 6 ): 2281 – 2 . doi: 10.1161/01.cir.82.6.2281 OpenUrl FREE Full Text 27. ↵ Wilber DJ , Olshansky B , Moran JF , Scanlon PJ . Electrophysiological testing and nonsustained ventricular tachycardia. Use and limitations in patients with coronary artery disease and impaired ventricular function . Circulation . Aug 1990 ; 82 ( 2 ): 350 – 8 . doi: 10.1161/01.cir.82.2.350 OpenUrl Abstract / FREE Full Text 28. ↵ Berul CI , Aronovitz MJ , Wang PJ , Mendelsohn ME . In vivo cardiac electrophysiology studies in the mouse . Circulation. Nov 15 1996 ; 94 ( 10 ): 2641 – 8 . doi: 10.1161/01.cir.94.10.2641 OpenUrl CrossRef 29. ↵ Qu Z , Xie LH , Olcese R , et al. Early afterdepolarizations in cardiac myocytes: beyond reduced repolarization reserve . Cardiovasc Res. Jul 1 2013 ; 99 ( 1 ): 6 – 15 . doi: 10.1093/cvr/cvt104 OpenUrl CrossRef PubMed 30. ↵ Hofmann F . The biology of cyclic GMP-dependent protein kinases . J Biol Chem. Jan 7 2005 ; 280 ( 1 ): 1 – 4 . doi: 10.1074/jbc.R400035200 OpenUrl FREE Full Text 31. ↵ Chiu HC , Kovacs A , Blanton RM , et al. Transgenic expression of fatty acid transport protein 1 in the heart causes lipotoxic cardiomyopathy . Circ Res. Feb 04 2005 ; 96 ( 2 ): 225 – 33 . doi: 10.1161/01.RES.0000154079.20681.B9 OpenUrl Abstract / FREE Full Text 32. ↵ Blanton RM , Takimoto E , Lane AM , et al. Protein kinase g ialpha inhibits pressure overload-induced cardiac remodeling and is required for the cardioprotective effect of sildenafil in vivo . J Am Heart Assoc . Oct 2012 ; 1 ( 5 ): e003731 . doi: 10.1161/JAHA.112.003731 OpenUrl Abstract / FREE Full Text 33. ↵ Blanton RM , Takimoto E , Aronovitz M , et al. Mutation of the protein kinase I alpha leucine zipper domain produces hypertension and progressive left ventricular hypertrophy: a novel mouse model of age-dependent hypertensive heart disease . J Gerontol A Biol Sci Med Sci . Nov 2013 ; 68 ( 11 ): 1351 – 5 . doi: 10.1093/gerona/glt042 OpenUrl CrossRef PubMed Web of Science 34. ↵ Qu Z , Weiss JN . Cardiac Alternans: From Bedside to Bench and Back . Circ Res. Jan 6 2023 ; 132 ( 1 ): 127 – 149 . doi: 10.1161/circresaha.122.321668 OpenUrl CrossRef 35. ↵ Scotcher J , Prysyazhna O , Boguslavskyi A , et al. Disulfide-activated protein kinase G Ialpha regulates cardiac diastolic relaxation and fine-tunes the Frank-Starling response . Nat Commun. Oct 26 2016 ; 7 : 13187 . doi: 10.1038/ncomms13187 OpenUrl CrossRef 36. ↵ Gergs U , Trapp T , Bushnaq H , Simm A , Silber RE , Neumann J . Age-Dependent Protein Expression of Serine/Threonine Phosphatases and Their Inhibitors in the Human Cardiac Atrium . Adv Med . 2019 ; 2019 : 2675972 . doi: 10.1155/2019/2675972 OpenUrl CrossRef PubMed 37. ↵ Puntoni F , Villa-Moruzzi E . Phosphorylation of the inhibitor-2 of protein phosphatase-1 by cdc2-cyclin B and GSK3 . Biochem Biophys Res Commun. Feb 15 1995 ; 207 ( 2 ): 732 – 9 . doi: 10.1006/bbrc.1995.1248 OpenUrl CrossRef PubMed 38. ↵ Moss AJ , Hall WJ , Cannom DS , et al. Improved survival with an implanted defibrillator in patients with coronary disease at high risk for ventricular arrhythmia . Multicenter Automatic Defibrillator Implantation Trial Investigators. N Engl J Med. Dec 26 1996 ; 335 ( 26 ): 1933 – 40 . doi: 10.1056/nejm199612263352601 OpenUrl CrossRef 39. ↵ Bork NI , Nikolaev VO . cGMP Signaling in the Cardiovascular System-The Role of Compartmentation and Its Live Cell Imaging . Int J Mol Sci. Mar 10 2018 ; 19 (3) doi: 10.3390/ijms19030801 OpenUrl CrossRef PubMed 40. ↵ Arora P , Wu C , Hamid T , et al. Acute Metabolic Influences on the Natriuretic Peptide System in Humans . J Am Coll Cardiol. Feb 23 2016 ; 67 ( 7 ): 804 – 812 . doi: 10.1016/j.jacc.2015.11.049 OpenUrl FREE Full Text 41. ↵ van Heerebeek L , Hamdani N , Falcao-Pires I , et al. Low myocardial protein kinase G activity in heart failure with preserved ejection fraction . Circulation. Aug 14 2012 ; 126 ( 7 ): 830 – 9 . doi: 10.1161/circulationaha.111.076075 OpenUrl CrossRef 42. ↵ Dunkerly-Eyring B , Kass DA . Myocardial Phosphodiesterases and Their Role in cGMP Regulation . J Cardiovasc Pharmacol . Jun 2020 ; 75 ( 6 ): 483 – 493 . doi: 10.1097/fjc.0000000000000773 OpenUrl CrossRef PubMed 43. ↵ Surks HK , Mochizuki N , Kasai Y , et al. Regulation of myosin phosphatase by a specific interaction with cGMP-dependent protein kinase Ialpha . Science. Nov 19 1999 ; 286 ( 5444 ): 1583 – 7 . doi: 10.1126/science.286.5444.1583 OpenUrl Abstract / FREE Full Text 44. ↵ Thoonen R , Giovanni S , Govindan S , et al. Molecular Screen Identifies Cardiac Myosin-Binding Protein-C as a Protein Kinase G-Ialpha Substrate . Circ Heart Fail . Nov 2015 ; 8 ( 6 ): 1115 – 22 . doi: 10.1161/CIRCHEARTFAILURE.115.002308 OpenUrl Abstract / FREE Full Text 45. ↵ Hopf AE , Andresen C , Kötter S , et al. Diabetes-Induced Cardiomyocyte Passive Stiffening Is Caused by Impaired Insulin-Dependent Titin Modification and Can Be Modulated by Neuregulin-1 . Circ Res. Jul 20 2018 ; 123 ( 3 ): 342 – 355 . doi: 10.1161/circresaha.117.312166 OpenUrl CrossRef 46. ↵ Hajka D , Budziak B , Pietras Ł , Duda P , McCubrey JA , Gizak A . GSK3 as a Regulator of Cytoskeleton Architecture: Consequences for Health and Disease . Cells. Aug 14 2021 ; 10 (8) doi: 10.3390/cells10082092 OpenUrl CrossRef 47. ↵ Jin H , Welzig CM , Aronovitz M , et al. QRS/T-wave and calcium alternans in a type I diabetic mouse model for spontaneous postmyocardial infarction ventricular tachycardia: A mechanism for the antiarrhythmic effect of statins . Heart Rhythm . Sep 2017 ; 14 ( 9 ): 1406 – 1416 . doi: 10.1016/j.hrthm.2017.05.026 OpenUrl CrossRef PubMed 48. ↵ Salerno-Uriarte JA , De Ferrari GM , Klersy C , et al. Prognostic value of T-wave alternans in patients with heart failure due to nonischemic cardiomyopathy: results of the ALPHA Study . J Am Coll Cardiol. Nov 6 2007 ; 50 ( 19 ): 1896 – 904 . doi: 10.1016/j.jacc.2007.09.004 OpenUrl FREE Full Text 49. ↵ Paulus WJ , Tschope C . A novel paradigm for heart failure with preserved ejection fraction: comorbidities drive myocardial dysfunction and remodeling through coronary microvascular endothelial inflammation . J Am Coll Cardiol. Jul 23 2013 ; 62 ( 4 ): 263 – 71 . doi: 10.1016/j.jacc.2013.02.092 OpenUrl FREE Full Text 50. ↵ Tromp J , Shen L , Jhund PS , et al. Age-Related Characteristics and Outcomes of Patients With Heart Failure With Preserved Ejection Fraction . J Am Coll Cardiol. Aug 6 2019 ; 74 ( 5 ): 601 – 612 . doi: 10.1016/j.jacc.2019.05.052 OpenUrl FREE Full Text 51. ↵ Greene SJ , Gheorghiade M , Borlaug BA , et al. The cGMP signaling pathway as a therapeutic target in heart failure with preserved ejection fraction . J Am Heart Assoc. Dec 11 2013 ; 2 ( 6 ): e000536 . doi: 10.1161/jaha.113.000536 OpenUrl CrossRef 52. ↵ Lee DI , Zhu G , Sasaki T , et al. Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease . Nature. Mar 26 2015 ; 519 ( 7544 ): 472 – 6 . doi: 10.1038/nature14332 OpenUrl CrossRef PubMed 53. ↵ Andersson DP , Trolle Lagerros Y , Grotta A , Bellocco R , Lehtihet M , Holzmann MJ . Association between treatment for erectile dysfunction and death or cardiovascular outcomes after myocardial infarction . Heart . Aug 2017 ; 103 ( 16 ): 1264 – 1270 . doi: 10.1136/heartjnl-2016-310746 OpenUrl Abstract / FREE Full Text 54. ↵ Nagy O , Hajnal A , Parratt JR , Végh A . Sildenafil (Viagra) reduces arrhythmia severity during ischaemia 24 h after oral administration in dogs . Br J Pharmacol . Feb 2004 ; 141 ( 4 ): 549 – 51 . doi: 10.1038/sj.bjp.0705658 OpenUrl CrossRef PubMed Web of Science 55. ↵ Hutchings DC , Pearman CM , Madders GWP , et al. PDE5 Inhibition Suppresses Ventricular Arrhythmias by Reducing SR Ca(2+) Content . Circ Res. Sep 3 2021 ; 129 ( 6 ): 650 – 665 . doi: 10.1161/circresaha.121.318473 OpenUrl CrossRef 56. ↵ Hall EJ , Pal S , Glennon MS , et al. Cardiac natriuretic peptide deficiency sensitizes the heart to stress-induced ventricular arrhythmias via impaired CREB signalling . Cardiovasc Res. Jul 20 2022 ; 118 ( 9 ): 2124 – 2138 . doi: 10.1093/cvr/cvab257 OpenUrl CrossRef 57. ↵ Alex L , Russo I , Holoborodko V , Frangogiannis NG . Characterization of a mouse model of obesity-related fibrotic cardiomyopathy that recapitulates features of human heart failure with preserved ejection fraction . Am J Physiol Heart Circ Physiol. Oct 1 2018 ; 315 ( 4 ): H934 – h949 . doi: 10.1152/ajpheart.00238.2018 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted February 17, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Myocardial cGMP-PKG1α dysregulation contributes to VT pathogenesis in type II diabetes and metabolic syndrome Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. 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