Dynamic detection and reversal of myocardial ischemia using an artificially intelligent bioelectronic medicine.

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.

NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model.

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.

CXL-1020, a Novel Nitroxyl (HNO) Prodrug, Is More Effective than Milrinone in Models of Diastolic Dysfunction-A Cardiovascular Therapeutic: An Efficacy and Safety Study in the Rat.

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.

Rationally engineered Troponin C modulates in vivo cardiac function and performance in health and disease.

Treatment for heart disease, the leading cause of death in the world, has progressed little for several decades. Here we develop a protein engineering approach to directly tune in vivo cardiac contractility by tailoring the ability of the heart to respond to the Ca(2+) signal. Promisingly, our smartly formulated Ca(2+)-sensitizing TnC (L48Q) enhances heart function without any adverse effects that are commonly observed with positive inotropes. In a myocardial infarction (MI) model of heart failure, expression of TnC L48Q before the MI preserves cardiac function and performance. Moreover, expression of TnC L48Q after the MI therapeutically enhances cardiac function and performance, without compromising survival. We demonstrate engineering TnC can specifically and precisely modulate cardiac contractility that when combined with gene therapy can be employed as a therapeutic strategy for heart disease.

Obligatory role of neuronal nitric oxide synthase in the heart's antioxidant adaptation with exercise.

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.

Nitric oxide-dependent activation of CaMKII increases diastolic sarcoplasmic reticulum calcium release in cardiac myocytes in response to adrenergic stimulation.

Spontaneous calcium waves in cardiac myocytes are caused by diastolic sarcoplasmic reticulum release (SR Ca(2+) leak) through ryanodine receptors. Beta-adrenergic (ß-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 ß-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 Ca(2+) waves in intact cardiomyocytes. Both SCaWs and SR Ca(2+) 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 ß-AR pathway by isoproterenol increased the CaMKII-dependent SR Ca(2+) 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 Ca(2+) leak. Mechanistically, our data links Akt to nitric oxide synthase 1 activation downstream of ß-AR stimulation. Collectively, this evidence supports the hypothesis that CaMKII is regulated by nitric oxide as part of the adrenergic cascade leading to arrhythmogenesis.

Effects of insulin resistance on skeletal muscle growth and exercise capacity in type 2 diabetic mouse models.

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.

Neuronal nitric oxide synthase is indispensable for the cardiac adaptive effects of exercise.

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.

Expression levels of sarcolemmal membrane repair proteins following prolonged exercise training in mice.

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.

Modulation of myocardial contraction by peroxynitrite.

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.

Effects of increased systolic Ca(2+) and ß-adrenergic stimulation on Ca(2+) transient decline in NOS1 knockout cardiac myocytes.

We have previously shown that the main factor responsible for the faster [Ca(2+)](i) decline rate with ß-adrenergic (ß-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 ß-AR stimulation. Ca(2+) transients (Fluo-4) were measured in myocytes superfused with 3mM extracellular Ca(2+) ([Ca(2+)](o)) and a non-specific ß-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.

Diesterified nitrone rescues nitroso-redox levels and increases myocyte contraction via increased SR Ca(2+) handling.

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 ß-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.

Effects of increased systolic Ca²âº and phospholamban phosphorylation during ß-adrenergic stimulation on Ca²âº transient kinetics in cardiac myocytes.

Previous studies demonstrated higher systolic intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitudes result in faster [Ca(2+)](i) decline rates, as does ß-adrenergic (ß-AR) stimulation. The purpose of this study is to determine the major factor responsible for the faster [Ca(2+)](i) decline rate with ß-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 ß-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.

cAMP-independent activation of protein kinase A by the peroxynitrite generator SIN-1 elicits positive inotropic effects in cardiomyocytes.

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.