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Description, Potentially damaging “heart stress” is reversed using reactive nerve stimulation controlled by artificial intelligence., Myocardial ischemia is spontaneous, frequently asymptomatic, and contributes to fatal cardiovascular consequences. Importantly, myocardial sensory networks cannot reliably detect and correct myocardial ischemia on their own. Here, we demonstrate an artificially intelligent and responsive bioelectronic medicine, where an artificial neural network (ANN) supplements myocardial sensory networks, enabling reliable detection and correction of myocardial ischemia. ANNs were first trained to decode spontaneous cardiovascular stress and myocardial ischemia with an overall accuracy of ~92%. ANN-controlled vagus nerve stimulation (VNS) significantly mitigated major physiological features of myocardial ischemia, including ST depression and arrhythmias. In contrast, open-loop VNS or ANN-controlled VNS following a caudal vagotomy essentially failed to reverse cardiovascular pathophysiology. Last, variants of ANNs were used to meet clinically relevant needs, including interpretable visualizations and unsupervised detection of emerging cardiovascular stress. Overall, these preclinical results suggest that ANNs can potentially supplement deficient myocardial sensory networks via an artificially intelligent bioelectronic medicine system.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function., The molecular mechanisms leading to heart failure in patients with Duchenne muscular dystrophy are unclear. Here the authors show that NF-κB is activated in the heart of dystrophin-deficient mice and that its ablation rescues cardiac function through chromatin remodeling and activation of gene expression.

The nitroxyl (HNO) prodrug, CXL-1020, induces vasorelaxation and improves cardiac function in canine models and patients with systolic heart failure (HF). HNO's unique mechanism of action may be applicable to a broader subset of cardiac patients. This study investigated the load-independent safety and efficacy of CXL-1020 in two rodent (rat) models of diastolic heart failure and explored potential drug interactions with common HF background therapies. In vivo left-ventricular hemodynamics/pressure-volume relationships assessed before/during a 30 min IV infusion of CXL-1020 demonstrated acute load-independent positive inotropic, lusitropic, and vasodilatory effects in normal rats. In rats with only diastolic dysfunction due to bilateral renal wrapping (RW) or pronounced diastolic and mild systolic dysfunction due to 4 weeks of chronic isoproterenol exposure (ISO), CXL-1020 attenuated the elevated LV filling pressures, improved the end diastolic pressure volume relationship, and accelerated relaxation. CXL-1020 facilitated Ca2+ re-uptake and enhanced myocyte relaxation in isolated cardiomyocytes from ISO rats. Compared to milrinone, CXL-1020 more effectively improved Ca2+ reuptake in ISO rats without concomitant chronotropy, and did not enhance Ca2+ entry via L-type Ca2+ channels nor increase myocardial arrhythmias/ectopic activity. Acute-therapy with CXL-1020 improved ventricular relaxation and Ca2+ cycling, in the setting of chronic induced diastolic dysfunction. CXL-1020's lusitropic effects were greater than those seen with the cAMP-dependent agent milrinone, and unlike milrinone it did not produce chronotropy or increased ectopy. HNO is a promising new potential therapy for both systolic and diastolic heart failure.

Anti-oxidant therapy has been an immense clinical disappointment for the treatment of cardiomyopathies. Concurrently with the increase in reactive oxygen species (ROS), there is also a decrease in cardiac levels of nitric oxide (NO), resulting in a nitroso-redox imbalance not addressed by anti-oxidant treatment alone. Key modulators of cardiac function are sensitive to the nitroso-redox balance such as kinases and phosphatases. Thus, along with changes in protein oxidation and/or S-nitrosylation levels, the nitroso-redox imbalance also alters protein phosphorylation. We developed a unique and novel compound (EMEPO) that can correct the nitroso-redox imbalance by simultaneously scavenging ROS and producing NO. We hypothesized that EMEPO is a novel agent that will ameliorate cardiac dysfunction by reestablishing the proper protein post-translational modifications. We demonstrated the efficacy of EMEPO in two cardiac models of nitroso-redox imbalance; a genetic model (NOS1 -/- ) and a disease model- murine myocardial infraction (MI). Both models displayed nitroso-redox imbalance with systolic and diastolic dysfunction. EMEPO treatment had a much greater effect than anti-oxidant treatment alone in palliating the cardiac dysfunction. A major contributor to these dysfunctions observed in cardiomyopathies is altered ryanodine receptor (RyR) activity. EMEPO was able to restore RyR activity and each aberrant post-translational modification (oxidation, S-nitrosylation, and phosphorylation). We believe these EMEPO-induced changes in RyR will occur in many proteins that orchestrate signaling networks and function. Our data highly suggest that simultaneously restoring both ROS and NO levels (i.e., correcting the nitroso-redox imbalance) is a promising therapeutic approach for MI and heart failure patients. Our first designed nitroso-redox balancer, EMEPO, shows great potential as a novel strategy for the treatment of heart disease.

Type 2 diabetes mellitus is associated with an accelerated muscle loss during aging, decreased muscle function, and increased disability. To better understand the mechanisms causing this muscle deterioration in type 2 diabetes, we assessed muscle weight, exercise capacity, and biochemistry in db/ db and TallyHo mice at prediabetic and overtly diabetic ages. Maximum running speeds and muscle weights were already reduced in prediabetic db/ db mice when compared with lean controls and more severely reduced in the overtly diabetic db/ db mice. In contrast to db/ db mice, TallyHo muscle size dramatically increased and maximum running speed was maintained during the progression from prediabetes to overt diabetes. Analysis of mechanisms that may contribute to decreased muscle weight in db/ db mice demonstrated that insulin-dependent phosphorylation of enzymes that promote protein synthesis was severely blunted in db/ db muscle. In addition, prediabetic (6-wk-old) and diabetic (12-wk-old) db/ db muscle exhibited an increase in a marker of proteasomal protein degradation, the level of polyubiquitinated proteins. Chronic treadmill training of db/ db mice improved glucose tolerance and exercise capacity, reduced markers of protein degradation, but only mildly increased muscle weight. The differences in muscle phenotype between these models of type 2 diabetes suggest that insulin resistance and chronic hyperglycemia alone are insufficient to rapidly decrease muscle size and function and that the effects of diabetes on muscle growth and function are animal model-dependent.

Hypertension is an established comorbidity in cardiovascular (CV) disease. Changes in aortic pulse-wave velocity (PWV) have been shown to predict the longitudinal development of hypertension, its underlying vascular alterations, and associated CV risks. However, as the heart and arterial tree are coupled, it has been shown that the successful monitoring of not only CV disease progression, but of its therapies, requires the evaluation of ventriculo-arterial uncoupling and its determinants. This study aimed to establish the relationships between PWV and changes in the estimated effective arterial elastance (Ea) and/or contractility over a wide-range of hypertension-induced CV pathologies in a controlled rodent model/environment. In a large series (n = 96) of anesthetized (pentobarbital) male Sprague-Dawley rats, PWV (carotid-to-femoral), systolic pressures, contractility (preload recruitable stroke work; PRSW), and Ea were evaluated using echocardiography, invasive hemodynamics, and left ventricular (LV) pressure-volume relationships. Data were collected in healthy animals (n = 37) and those with hypertension-induced chronic systolic/diastolic left-ventricular dysfunction (n = 59) (chronic beta-adrenergic stimulation and/or renoprival). A subset of each group, were also studied under common clinical cardio/vasoactive pharmacological interventions. Disease animals had significantly (P < 0.001) higher systolic pressures (141 ± 4 vs 116 ± 4 mmHg), faster PWV (9.0 ± 0.8 vs 5.1 ± 0.5 m/s), and depressed contractility (PRSW: 39 ± 1 vs. 49 ± 1 mmHg*). Both PWV (7.6 ± 0.7 to 4.3 ± 0.4 m/s) and Ea (95 ± 10 to 63 ± 8 mmHg/mL) decreased in response to vascular therapies in the diseased animals; only PWV was reduced in healthy rats. More interestingly, over all conditions, PWV was a poor predictor of both systolic pressures (R 2 =0.00009) and Ea (R 2 = 0.024). but yet was a moderate predictor of PRSW (R 2 =0.23, P < 0.001), suggesting a contractility-dependent velocity of propagation. Taken together, these data suggest that indices of ventriculo-arterial efficacy, cardiac afterload, and peripheral arterial stiffness while all are affected by CV disease, each cannot be used to predict one another, and all provide independent prognostic information in disease.

We have previously shown that the main factor responsible for the faster [Ca(2+)](i) decline rate with β-adrenergic (β-AR) stimulation is the phosphorylation of phospholamban (PLB) rather than the increase in systolic Ca(2+) levels. The purpose of this study was to correlate the extent of augmentation of PLB Serine(16) phosphorylation to the rate of [Ca(2+)](i) decline. Thus, ventricular myocytes were isolated from neuronal nitric oxide synthase knockout (NOS1(-/-)) mice, which we observed had lower basal PLB Serine(16) phosphorylation levels, but equal levels during β-AR stimulation. Ca(2+) transients (Fluo-4) were measured in myocytes superfused with 3mM extracellular Ca(2+) ([Ca(2+)](o)) and a non-specific β-AR agonist isoproterenol (ISO, 1μM) with 1mM [Ca(2+)](o). This allowed us to get matched Ca(2+) transient amplitudes in the same myocyte. Similar to our previous work, Ca(2+) transient decline was significantly faster with ISO compared to 3mM [Ca(2+)](o), even with matched Ca(2+) transient amplitudes. Interestingly, when we compared the effects of ISO on Ca(2+) transient decline between NOS1(-/-) and WT myocytes, ISO had a larger effect in NOS1(-/-) myocytes, which resulted in a greater percent decrease in the Ca(2+) transient RT(50). We believe this is due to a greater augmentation of PLB Serine16 phosphorylation in these myocytes. Thus, our results suggest that not only the amount but the extent of augmentation of PLB Serine(16) phosphorylation are the major determinants for the Ca(2+) decline rate. Furthermore, our data suggest that the molecular mechanisms of Ca(2+) transient decline is normal in NOS1(-/-) myocytes and that the slow basal Ca(2+) transient decline is predominantly due to decreased PLB phosphorylation.

Previous studies demonstrated higher systolic intracellular Ca2+ concentration ([Ca2+]i) amplitudes result in faster [Ca2+]i decline rates, as does β-adrenergic (β-AR) stimulation. The purpose of this study is to determine the major factor responsible for the faster [Ca2+]i decline rate with β-AR stimulation, the increased systolic Ca2+ concentration levels, or phosphorylation of phospholamban. Mouse myocytes were perfused under basal conditions [1 mM extracellular Ca2+ concentration ([Ca2+]o)], followed by high extracellular Ca2+ (3 mM [Ca2+]o), washout with 1 mM [Ca2+]o, followed by 1 μM isoproterenol (ISO) with 1 mM [Ca2+]o. ISO increased Ser16 phosphorylation compared with 3 mM [Ca2+]o, whereas Thr17 phosphorylation was similar. Ca2+ transient (CaT) (fluo 4) data were obtained from matched CaT amplitudes with 3 mM [Ca2+]o and ISO. [Ca2+]i decline was significantly faster with ISO compared with 3 mM [Ca2+]o. Interestingly, the faster decline with ISO was only seen during the first 50% of the decline. CaT time to peak was significantly faster with ISO compared with 3 mM [Ca2+]o. A Ca2+/calmodulin-dependent protein kinase (CAMKII) inhibitor (KN-93) did not affect the CaT decline rates with 3 mM [Ca2+]o or ISO but normalized ISO's time to peak with 3 mM [Ca2+]o. Thus, during β-AR stimulation, the major factor for the faster CaT decline is due to Ser16 phosphorylation, and faster time to peak is due to CAMKII activation.

The phosphatase vs. kinase equilibrium plays a critical role in the regulation of myocardial contractility. Previous studies have demonstrated that peroxynitrite exerts a biphasic effect on cardiomyocyte contraction, such that high peroxynitrite reduced beta-adrenergic-stimulated myocyte contraction by inducing the dephosphorylation of phospholamban (PLB) via phosphatase activation. Conversely, low peroxynitrite increased basal and beta-adrenergic-stimulated contraction also through a PLB-dependent mechanism. However, previous studies have not elucidated the mechanism underlying the positive effects of low peroxynitrite on myocyte contraction. In the current study, we examined the phosphatase vs. kinase equilibrium as a potential mechanism underlying the positive effects of peroxynitrite. SIN-1 (peroxynitrite donor, 10 mumol/L) increased myocyte Ca(2+) transient and shortening amplitude, accelerated myocyte relaxation, and enhanced PLB phosphorylation. Specific inhibition of PP1/PP2a with okadaic acid failed to inhibit this positive effect. However, inhibition of PKA with KT5720 completely abolished the effects of SIN-1 on myocyte contraction. Additionally, SIN-1 induced a significant increase in PKA activity in cardiac homogenates, which was inhibited with FeTPPS (peroxynitrite decomposition catalyst). Surprisingly, SIN-1 also increased activity in purified preparations (i.e., in the absence of cAMP) of PKA. Therefore, our data suggest that peroxynitrite directly activates PKA (independent from cAMP), resulting in the enhancement of myocyte contraction and relaxation through the phosphorylation of PLB.

Excessive oxidative stress in the heart results in contractile dysfunction. While antioxidant therapies have been a disappointment clinically, exercise has shown beneficial results, in part by reducing oxidative stress. We have previously shown that neuronal nitric oxide synthase (nNOS) is essential for cardioprotective adaptations caused by exercise. We hypothesize that part of the cardioprotective role of nNOS is via the augmentation of the antioxidant defense with exercise by positively shifting the nitroso-redox balance. Our results show that nNOS is indispensable for the augmented anti-oxidant defense with exercise. Furthermore, exercise training of nNOS knockout mice resulted in a negative shift in the nitroso-redox balance resulting in contractile dysfunction. Remarkably, overexpressing nNOS (conditional cardiac-specific nNOS overexpression) was able to mimic exercise by increasing VO2max. This study demonstrates that exercise results in a positive shift in the nitroso-redox balance that is nNOS-dependent. Thus, targeting nNOS signaling may mimic the beneficial effects of exercise by combating oxidative stress and may be a viable treatment strategy for heart disease.

We have engineered cardiac TnCs with increased (L48Q) or decreased (D73N) Ca2+ sensitivity. To express these proteins in the in vivo heart we utilized an adeno-associated virus serotype 9 (AAV-9). The Ca2+ desensitized D73N TnC recapitulated a dilated cardiomyopathy phenotype and depressed function as observed by echocardiography and isolated cardiomyocytes. On the other hand, AAV-9 containing the Ca2+ sensitized L48Q TnC did not cause any disease phenotype or arrhythmias commonly associated with increased myofilament Ca2+ sensitivity. In healthy mice, L48Q TnC increased myocyte contraction and whole heart contractility with improved cardiovascular performance (increased V02max). Excitingly, L48Q TnC expressing mice were able to preserve higher contractility, ejection fraction, cardiac performance and decreased death rate even after undergoing trans-aortic constriction or myocardial infarction. Additionally, L48Q TnC was able to increase contractility, ejection fraction and cardiac performance in mice which expressed L48Q TnC after having a myocardial infarction. In summary, engineered TnCs show potential to be used as treatment strategies against different cardiomyopathies.

Despite the morbidity and clinical burden of primary diastolic cardiac dysfunction, effective therapies for patients presenting with heart failure with preserved ejection fraction (HFpEF) are limited. Nitroxyl (HNO) produces cAMP-independent functional support, in part by improving myocardial calcium-handling. We hypothesize that HNO can rescue lusitropic function when acutely administered to rats with chronic diastolic dysfunction, mimicking HFpEF. Diastolic dysfunction, as demonstrated by decreased E/A ratios via echocardiography, was induced by either 4 weeks of chronic isoproterenol administration (via a mini-osmotic pump; ISO) or by renoprival hypertension (secondary to renal wrapping; RW) for 5 weeks. In vivo left-ventricular hemodynamics/pressure-volume relationships were assessed before/during a 30 minute IV administration of a HNO pro-drug (CXL-1020, 100 μg/kg/min). Ca2+ handling in isolated cardiomyocytes was also studied (CXL-1020, at 50 μM). In both cases, milrinone (mil) was used as a cAMP-dependent positive control (at a matched-efficacy in healthy rats, i.e., 10 μg/kg/min and 0.5 μM) In the setting of chronic diastolic dysfunction, HNO lowered the LV filling pressures in both models by a combined average of 46 ± 8% versus baseline, accelerated the time-constant (tau) of relaxation (-26 ± 3%) and improved compliance (flattened the end-diastolic PV relationship) (-61 ±3%). In myocytes isolated from dysfunctional hearts, HNO accelerated the time for Ca2+ to decline to 50% of its max amplitude (RT50) by -10 ± 2% and myocyte relengthening by -12 ± 3% in ISO rats. Despite eliciting comparable responses in healthy animals/myocytes, the HNO-mediated effects on both tau (in vivo) (-33 ± 3% vs mil -25 ± 2%) and Ca2+ decline (myocyte) (-10 ± 2% vs mil -6 ± 1%) were greater than those of milrinone in the ISO rats, which had greater impairment at baseline compared to RW rats. In conclusion, acute-therapy with HNO improved ventricular relaxation, compliance and Ca2+ handling, in the setting of chronic diastolic dysfunction. Moreover, these effects were greater than observed with the cAMP-dependent agent milrinone.

Membrane repair is a conserved cellular process, where intracellular vesicles translocate to sites of plasma membrane injury to actively reseal membrane disruptions. Such membrane disruptions commonly occur in the course of normal physiology, particularly in skeletal muscles due to repeated contraction producing small tears in the sarcolemmal membrane. Here, we investigated whether prolonged exercise could produce adaptive changes in expression levels of proteins associated with the membrane repair process, including mitsugumin 53/tripartite motif-containing protein 72 (MG53/TRIM72), dysferlin and caveolin-3 (cav3). Mice were exercised using a treadmill running protocol and protein levels were measured by immunoblotting. The specificity of the antibodies used was established by immunoblot testing of various tissue lysates from both mice and rats. We found that MG53/TRIM72 immunostaining on isolated mouse skeletal muscle fibers showed protein localization at sites of membrane disruption created by the isolation of these muscle fibers. However, no significant changes in the expression levels of the tested membrane repair proteins were observed following prolonged treadmill running for eight weeks (30 to 80 min/day). These findings suggest that any compensation occurring in the membrane repair process in skeletal muscle following prolonged exercise does not affect the expression levels of these three key membrane repair proteins.

Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca2+leak) through ryanodine receptors. Beta-adrenergic (β-AR) tone is known to increase this leak through the activation of Ca-calmodulin-dependent protein kinase (CaMKII) and the subsequent phosphorylation of the ryanodine receptor. When b-AR drive is chronic, as observed in heart failure, this CaMKII-dependent effect is exaggerated and becomes potentially arrhythmogenic. Recent evidence has indicated that CaMKII activation can be regulated by cellular oxidizing agents, such as reactive oxygen species. Here, we investigate how the cellular second messenger, nitric oxide, mediates CaMKII activity downstream of the adrenergic signaling cascade and promotes the generation of arrhythmogenic spontaneous C a2+waves in intact cardiomyocytes. Both SCaWs and SR Ca2+leak were measured in intact rabbit and mouse ventricular myocytes loaded with the Ca-dependent fluorescent dye, fluo-4. CaMKII activity in vitro and immunoblotting for phosphorylated residues on CaMKII, nitric oxide synthase, and Akt were measured to confirm activity of these enzymes as part of the adrenergic cascade. We demonstrate that stimulation of the β-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca2+leak. This increased leak was prevented by inhibition of nitric oxide synthase 1 but not nitric oxide synthase 3. In ventricular myocytes isolated from wild-type mice, isoproterenol stimulation also increased the CaMKII-dependent leak. Critically, in myocytes isolated from nitric oxide synthase 1 knock-out mice this effect is ablated. We show that isoproterenol stimulation leads to an increase in nitric oxide production, and nitric oxide alone is sufficient to activate CaMKII and increase SR Ca2+leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of β-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is regulated by nitric oxide as part of the adrenergic cascade leading to arrhythmogenesis. © 2014 Curran et al.

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)-activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2(V617F) oncogene. PP2A inactivation occurs in a Jak2(V617F) dose/kinase-dependent manner through the PI-3Kγ-PKC-induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2(V617F) inactivation/downregulation and impairs clonogenic potential of Jak2(V617F) cell lines and PV but not normal CD34(+) progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2(V617F) leukemic mice without adverse effects. Mechanistically, we show that in Jak2(V617F) cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2(V617F) also utilizes an alternative sphingosine kinase-1-mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET-mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2(V617F) MPNs.

Exercise results in beneficial adaptations of the heart that can be directly observed at the ventricular myocyte level. However, the molecular mechanism(s) responsible for these adaptations are not well understood. Interestingly, signaling via neuronal nitric oxide synthase (NOS1) within myocytes results in similar effects as exercise. Thus, the objective was to define the role NOS1 plays in the exercise-induced beneficial contractile effects in myocytes. After an 8-week aerobic interval training program, exercise-trained (Ex) mice had higher VO(2max) and cardiac hypertrophy compared to sedentary (Sed) mice. Ventricular myocytes from Ex mice had increased NOS1 expression and nitric oxide production compared to myocytes from Sed mice. Remarkably, acute NOS1 inhibition normalized the enhanced contraction (shortening and Ca(2+) transients) in Ex myocytes to Sed levels. The NOS1 effect on contraction was mediated via greater Ca(2+) cycling that resulted from increased phospholamban phosphorylation. Intriguingly, a similar aerobic interval training program on NOS1 knockout mice failed to produce any beneficial cardiac adaptations (VO(2max), hypertrophy, and contraction). These data demonstrate that the beneficial cardiac adaptations observed after exercise training were mediated via enhanced NOS1 signaling. Therefore, it is likely that beneficial effects of exercise may be mimicked by the interventions that increase NOS1 signaling. This pathway may provide a potential novel therapeutic target in cardiac patients who are unable or unwilling to exercise.

Peroxynitrite is a potent oxidant that is quickly emerging as a crucial modulator of myocardial function. This review will focus on the regulation of myocardial contraction by peroxynitrite during health and disease, with a specific emphasis on cardiomyocyte Ca(2+) handling, proposed signaling pathways, and protein end-targets.

Exercise results in beneficial adaptations to the heart. These adaptations are observed at the level of the cardiomyocyte as increased Ca2+ cycling through the sarcoplasmic reticulum (SR) and greater fractional shortening. Interestingly, these adaptations are similar to the contractile effects of neuronal nitric oxide synthase (NOS1) signaling. Thus our objective is to determine if the exercise induced adaptations at the level of the cardiomyocyte are NOS1 mediated. After an 8 week high-intensity aerobic interval training program, exercise (Ex) mice had a higher VO2max, greater citrate synthase activity, decreased weight, increased heart-to-body ratio (P

Previous studies demonstrated higher systolic intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitudes result in faster [Ca(2+)](i) decline rates, as does β-adrenergic (β-AR) stimulation. The purpose of this study is to determine the major factor responsible for the faster [Ca(2+)](i) decline rate with β-AR stimulation, the increased systolic Ca(2+) concentration levels, or phosphorylation of phospholamban. Mouse myocytes were perfused under basal conditions [1 mM extracellular Ca(2+) concentration ([Ca(2+)](o))], followed by high extracellular Ca(2+) (3 mM [Ca(2+)](o)), washout with 1 mM [Ca(2+)](o), followed by 1 μM isoproterenol (ISO) with 1 mM [Ca(2+)](o). ISO increased Ser(16) phosphorylation compared with 3 mM [Ca(2+)](o), whereas Thr(17) phosphorylation was similar. Ca(2+) transient (CaT) (fluo 4) data were obtained from matched CaT amplitudes with 3 mM [Ca(2+)](o) and ISO. [Ca(2+)](i) decline was significantly faster with ISO compared with 3 mM [Ca(2+)](o). Interestingly, the faster decline with ISO was only seen during the first 50% of the decline. CaT time to peak was significantly faster with ISO compared with 3 mM [Ca(2+)](o). A Ca(2+)/calmodulin-dependent protein kinase (CAMKII) inhibitor (KN-93) did not affect the CaT decline rates with 3 mM [Ca(2+)](o) or ISO but normalized ISO's time to peak with 3 mM [Ca(2+)](o). Thus, during β-AR stimulation, the major factor for the faster CaT decline is due to Ser(16) phosphorylation, and faster time to peak is due to CAMKII activation.

Alteration of the Ca2+ binding properties of the thin filament has been implicated as the initiating factor for numerous cardiomyopathies. To directly test if changes in the Ca2+ sensitivity of TnC can lead to altered heart function and a diseased phenotype we utilized specifically engineered cardiac TnCs with modified Ca sensitivities. The specifically designed TnCs allowed us to test whether a direct increase (L48Q TnC) or decrease (D73N TnC) in Ca2+ sensitivity of the myofilament would lead to a hypertrophic or dilated cardiomyopathy, respectively. We used adeno-associated virus serotype 9 (AAV-9) containing either Control TnC, D73N TnC, or L48Q TnC to target the heart. AAV-9 containing GFP or mCherry was used to verify that the AAV-9 was able to infect the heart. The Ca2+ desensitized D73N TnC recapitulated a dilated cardiomyopathy. The hearts presented with dilated ventricles and had enlarged isolated cardiomyocytes that displayed decreased contractility. In addition, nearly half of the D73N mice died suddenly by 8 weeks of age and demonstrated altered electrical properties via ECG - a common feature of human dilated cardiomyopathies. On the other hand, AAV-9 containing the Ca2+ sensitized L48Q TnC did not recapitulate the restrictive or hypertrophic cardiomyopathy phenotypes commonly associated with increased myofilament Ca2+ sensitivity. However, consistent with the enhanced Ca2+ binding abilities of the L48Q TnC, the isolated L48Q TnC cardiomyocytes showed increased contractility, which was recapitulated in vivo via pressure-volume analysis. In summary, the results showed that an alteration in the Ca2+ sensitivity of the myofilament can, but does not always, lead to a diseased heart. In fact these engineered TnCs may be used as a treatment strategy against various cardiac diseases such as myocardial infarction.

Nitric oxide (NO) and superoxide (O(2) (-)) are important cardiac signaling molecules that regulate myocyte contraction. For appropriate regulation, NO and O(2) (.-) must exist at defined levels. Unfortunately, the NO and O(2) (.-) levels are altered in many cardiomyopathies (heart failure, ischemia, hypertrophy, etc.) leading to contractile dysfunction and adverse remodeling. Hence, rescuing the nitroso-redox levels is a potential therapeutic strategy. Nitrone spin traps have been shown to scavenge O(2) (.-) while releasing NO as a reaction byproduct; and we synthesized a novel, cell permeable nitrone, 2-2-3,4-dihydro-2H-pyrrole 1-oxide (EMEPO). We hypothesized that EMEPO would improve contractile function in myocytes with altered nitroso-redox levels. Ventricular myocytes were isolated from wildtype (C57Bl/6) and NOS1 knockout (NOS1(-/-)) mice, a known model of NO/O(2) (.-) imbalance, and incubated with EMEPO. EMEPO significantly reduced O(2) (.-) (lucigenin-enhanced chemiluminescence) and elevated NO (DAF-FM diacetate) levels in NOS1(-/-) myocytes. Furthermore, EMEPO increased NOS1(-/-) myocyte basal contraction (Ca(2+) transients, Fluo-4AM; shortening, video-edge detection), the force-frequency response and the contractile response to β-adrenergic stimulation. EMEPO had no effect in wildtype myocytes. EMEPO also increased ryanodine receptor activity (sarcoplasmic reticulum Ca(2+) leak/load relationship) and phospholamban Serine16 phosphorylation (Western blot). We also repeated our functional experiments in a canine post-myocardial infarction model and observed similar results to those seen in NOS1(-/-) myocytes. In conclusion, EMEPO improved contractile function in myocytes experiencing an imbalance of their nitroso-redox levels. The concurrent restoration of NO and O(2) (.-) levels may have therapeutic potential in the treatment of various cardiomyopathies.