Jeff Erickson

Acute myocardial infarction (MI) accelerates cardiomyocyte apoptosis, which underpins ventricular remodelling and dysfunction. The hormone ghrelin mitigates this remodelling, but the mechanisms remain unclear. Specific microRNAs (miRs) are key modulators and reliable biomarkers of early-stage apoptosis. We hypothesized that ghrelin targets anti-apoptotic miR-499 and miR-133 following MI to suppress cardiac apoptosis and thus mitigate cardiac dysfunction. C57/B6 mice received an injection of ghrelin (150 µg/kg, s.c.) or saline following left anterior descending coronary artery ligation (MI). Plasma levels of miR-499 and miR-133 at 3 or 24 h post-MI were measured using real-time PCR. Echocardiography and TUNEL staining were used to assess progressive cardiac function/structure and cardiomyocyte apoptosis, respectively. Myocardial ischaemia adversely decreased the levels of anti-apoptotic miR-499 by 3 h post-MI and increased the proportion of TUNEL-positive apoptotic cardiomyocytes by 24 h post-MI, contributing to cardiac remodelling and dysfunction by 2 weeks post-MI. Ghrelin prevented this MI-induced decrease in miR-499 by 3 h post-MI, then further increased the levels of miR-499 and miR-133 by 24 h. These ghrelin-mediated changes in microRNA were associated with a significant decrease in cardiomyocyte apoptosis and, consequently, significantly improved cardiac function and structure by 2 weeks post-MI. These results highlight miRs as effective biomarkers for the early detection of ischaemia-induced apoptotic signalling. Moreover, ghrelin appears to mitigate ischaemia-induced apoptosis by increasing the levels of anti-apoptotic miR-499 and miR-133, further solidifying ghrelin as a new therapeutic strategy for the clinical treatment of heart failure.

Diabetic heart disease is a leading cause of morbidity and mortality in individuals with type 2 diabetes mellitus (T2DM). A major yet frequently under-recognized component of diabetic heart disease is cardiac autonomic neuropathy (CAN), a condition characterized by dysregulated sympathetic and parasympathetic drive to the heart. Current pharmacological treatments for diabetic CAN are often ineffective, having been extrapolated from other health conditions. These therapies predominantly target the peripheral symptoms of elevated sympathetic activity, whilst largely neglecting its origins in sympathoexcitatory regions of the central autonomic network. Sympathetic control of cardiac function originates from the hypothalamus, medulla oblongata, midbrain, and pons, and is relayed through the intermediolateral cell column of the thoracic spinal cord and the intrinsic cardiac nervous system. Targeting the central autonomic network to modulate cardiac sympathetic drive presents a promising novel therapeutic avenue for the treatment of diabetic CAN. This review briefly summarizes established knowledge regarding the pathophysiology and management of diabetic CAN, and the implications of recent findings of increased neuronal activation in central sympathoregulatory regions early in the development of T2DM. Increased cardiac sympathetic in the intital stages of T2DM might represent a novel therapeutic target to reduce the impact of CAN and thereby improve outcomes in patients with T2DM.

• Review of pathophysiology and management of diabetic cardiac autonomic neuropathy.•Focusses on central and cardiac sympathoexcitation in type 2 diabetes.
• Includes recent findings in central sympathoregulatory regions in early diabetes.
• Discusses potential knowledges gapes and new research avenues in the field.

Many maternal adaptations occur during pregnancy to support the metabolic demands of the developing fetus and to prepare for the continued metabolic demands of lactation. Among these maternal adaptations are changes in the hypothalamic areas that regulate energy homeostasis: the paraventricular nucleus (PVN), ventromedial hypothalamic nucleus (VMH) and arcuate nucleus (ARC). The adaptive changes in the PVN, VMH, and ARC are believed to be driven by reduced responsiveness to the satiety hormone, leptin, during pregnancy. However, increased maternal metabolism is supported by elevated circulating glucose levels in pregnancy, and glucose itself can alter cell function by O-linked N-acetylglucosamine (O-GlcNAc) post-translational modification of proteins (O-GlcNAcylation). Therefore, we hypothesized that O-GlcNAcylation would be increased within the hypothalamic brain areas that are involved in the maternal adaptations to the increased metabolic demands of pregnancy: the ARC, VMH, and PVN. We completed immunohistochemistry and western blotting for O-GlcNAc in the ARC, VMH, and PVN from non-pregnant, late-pregnant, and lactating rats. Unexpectedly, we found that the number of O-GlcNAc-expressing cells and the levels of O-GlcNAc protein expression were similar within each area in non-pregnant, late-pregnant, and lactating rats. However, western blot analysis showed that the specific proteins that were O-GlcNAcylated appeared to be different between the reproductive states within each area. Further work will be required to identify the specific proteins that are differentially O-GlcNAcylated in each of the areas during pregnancy and lactation to determine whether this might contribute to the maternal adaptations required to cope with the metabolic demands of pregnancy and lactation.

Nitric oxide (NO) plays several critical roles in cardiovascular physiology. This molecule regulates cardiac function by modifying Ca²⁺-handling proteins through a process known as S- nitrosylation. These targets include L-type Calcium Channels (LTCC), Ryanodine Receptors (RyR2), Protein Kinase G (PKG), Phospholamban (PLB), sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) and Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII). S- nitrosylation is a covalent attachment of an NO moiety to the thiol side chain of a cysteine residue within a protein. This process can modify excitation-contraction coupling in cardiomyocytes and may mediate some forms of cardioprotection. Several studies have shown that S -nitrosylation may also be involved in the progression of cardiovascular diseases. Most importantly, recent studies have focused on the molecular mechanisms underlying cardiovascular diseases (CVD). Emerging evidence suggests that sex-specific differences in cardiac protein S- nitrosylation exist, and may partially explain disparities in cardiovascular health in males and females. Females have been found to have higher cardiac protein S- nitrosylation levels compared to men, and this is attributed to enhanced NO production through estrogen. Emerging data suggests that S-nitrosylation of specific proteins such as CaMKII has a dual role of promoting and preventing arrhythmias, it is not clear whether the cardioprotective effect of S -nitrosylation of specific cardiac proteins is sex-dependent. A deeper understanding of the mechanisms regulating the role of protein S-nitrosylation and the impact of sex differences on S-nitrosylation will open new avenues for therapeutic interventions in cardiac diseases.

BACKGROUND: Nitric oxide (NO) has been identified as a signaling molecule generated during beta-adrenergic receptor stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of CaMKII delta (Ca2+/calmodulin kinase II delta) is emerging. NO donors are routinely used clinically for their cardioprotective effects on the heart, but it is unknown how NO donors modulate the proarrhythmic CaMKII to alter cardiac arrhythmia incidence. We test the role of S-nitrosylation of CaMKII delta at the Cysteine-273 inhibitory site and cysteine-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during beta-adrenergic receptor stimulation. METHODS: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKII delta expression (CaMKII delta-KO) or with deletion of the S-nitrosylation site on CaMKII delta at cysteine-273 or cysteine-290 (CaMKII delta-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 mu M), sodium nitroprusside (200 mu M), and beta-adrenergic agonist isoproterenol (100 nmol/L). RESULTS: Both WT and CaMKII delta-KO cardiomyocytes responded to isoproterenol with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKII delta-KO cardiomyocytes. The protection from isoproterenol-induced Ca2+ sparks and waves was mimicked by GSNO pretreatment in WT cardiomyocytes but lost in CaMKII delta-C273S cardiomyocytes. When GSNO was applied after isoproterenol, this protection was not observed in WT or CaMKII delta-C273S but was apparent in CaMKII delta-C290A. In Langendorff-perfused isolated hearts, GSNO pretreatment limited isoproterenol-induced arrhythmias in WT but not CaMKII delta-C273S hearts, while GSNO exposure after isoproterenol sustained or exacerbated arrhythmic events. CONCLUSIONS: We conclude that prior S-nitrosylation of CaMKII delta at cysteine-273 can limit subsequent beta-adrenergic receptor-induced arrhythmias, but that S-nitrosylation at cysteine-290 might worsen or sustain beta-adrenergic receptor-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Rationale: Nitric oxide (NO) has been identified as a signalling molecule generated during β-adrenergic receptor (AR) stimulation in the heart. Furthermore, a role for NO in triggering spontaneous Ca2+ release via S-nitrosylation of Ca2+/calmodulin kinase II delta (CaMKIIδ) is emerging. NO donors are routinely used clinically for their cardioprotective effects in the heart, but it is unknown how NO donors modulate the pro-arrhythmic CaMKII to alter cardiac arrhythmia incidence.

Objective: We test the role of S-nitrosylation of CaMKIIδ at the Cys-273 inhibitory site and Cys-290 activating site in cardiac Ca2+ handling and arrhythmogenesis before and during β-AR stimulation.

Methods and Results: We measured Ca2+-handling in isolated cardiomyocytes from C57BL/6J wild-type (WT) mice and mice lacking CaMKIIδ expression (CaMKIIδ-KO) or with deletion of the S-nitrosylation site on CaMKIIδ at Cys-273 or Cys-290 (CaMKIIδ-C273S and -C290A knock-in mice). Cardiomyocytes were exposed to NO donors, S-nitrosoglutathione (GSNO; 150 μM), sodium nitroprusside (SNP; 200 μM) and/or β-adrenergic agonist isoproterenol (ISO; 100 nM). WT and CaMKIIδ-KO cardiomyocytes treated with GSNO showed no change in Ca2+ transient or spark properties under baseline conditions (0.5 Hz stimulation frequency). Both WT and CaMKIIδ-KO cardiomyocytes responded to ISO with a full inotropic and lusitropic Ca2+ transient response as well as increased Ca2+ spark frequency. However, the increase in Ca2+ spark frequency was significantly attenuated in CaMKIIδ-KO cardiomyocytes. The protection from ISO-induced Ca2+ sparks and waves was mimicked by GSNO pre-treatment in WT cardiomyocytes, but lost in CaMKIIδ-C273S cardiomyocytes that displayed a robust increase in Ca2+ waves. This observation is consistent with CaMKIIδ-C273 S-nitrosylation being critical in limiting ISO-induced arrhythmogenic sarcoplasmic reticulum Ca2+ leak. When GSNO was applied after ISO this protection was not observed in WT or CaMKIIδ-C273S but was apparent in CaMKIIδ-C290A. In Langendorff-perfused isolated hearts, GSNO pre-treatment limited ISO-induced arrhythmias in WT but not CaMKIIδ-C273S hearts, while GSNO exposure after ISO sustained or exacerbated arrhythmic events.

Conclusions: We conclude that prior S-nitrosylation of CaMKIIδ at Cys-273 can limit subsequent β-AR induced arrhythmias, but that S-nitrosylation at Cys-290 might worsen or sustain β-AR-induced arrhythmias. This has important implications for the administration of NO donors in the clinical setting.

Nitric oxide (NO) is a key vasodilatory signalling molecule and NO releasing molecules (NO donors) are being examined as potential treatments for many pathologies. The photoresponsive NO donor tert-dodecane S-nitrosothiol (tDodSNO) has been designed to be highly resistant to metabolism; in principle photoactivation of tDodSNO should therefore enable the controlled release of NO in situ via light modulation. To investigate the therapeutic utility of tDodSNO, we tested drug efficacy in Sprague Dawley rats to assess systemic and localised hemodynamic responses under photoactivation, and to confirm drug safety. For comparison, drug action was evaluated alongside the existing NO donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Across a dosing range (0.1–3.0 mg/kg) tDodSNO exerted markedly reduced systemic hypotensive action compared to these standard NO donors, inducing a slight decrease in mean arterial pressure (maximum 14.2 ± 3.0%) without affecting heart rate. Target limb photoactivation of tDodSNO resulted in a substantial localized vasodilatory response, with increases to mean (26.0 ± 7.3%) and maximum (53.2 ± 10.4%) blood flow and decreases to vascular resistance (27.1 ± 3.9%) that were restricted to light exposed tissue. In comparison GSNO and SNP showed variable peripheral effects and were not responsive to photoactivation. tDodSNO did not induce met-Hb formation in blood, or display any signs of toxicity, and was rapidly cleared from the systemic circulation, with no hemodynamic effects detectable 5 min post administration. These data are the first demonstration that drugs based upon a metabolically stable S-nitrosothiol group can be photoactivated in vivo to release NO, and that such agents cause less systemic side effects than existing NO donors. Our data support the use of S-nitrosothiols to enable the spatiotemporal control of NO for therapeutic applications. •The emerging drug tDodSNO can be photoactivated in vivo to generate the cardiovascular signaling molecule nitric oxide (NO).•Photoactivation of tDodSNO enables localized vasodilation in light exposed tissue.•tDodSNO's superior photoactivity compared to known NO donors can be explained due to its enhanced resistance to metabolism.

Vascular smooth muscle cells (VSMCs) help to maintain the normal physiological contractility of arterial vessels to control blood pressure; they can also contribute to vascular disease such as atherosclerosis. Ca2+/calmodulin-dependent kinase II (CaMKII), a multifunctional enzyme with four isoforms and multiple alternative splice variants, contributes to numerous functions within VSMCs. The role of these isoforms has been widely studied across numerous tissue types; however, their functions are still largely unknown within the vasculature. Even more understudied is the role of the different splice variants of each isoform in such signaling pathways. This review evaluates the role of the different CaMKII splice variants in vascular pathological and physiological mechanisms, aiming to show the need for more research to highlight both the deleterious and protective functions of the various splice variants.

The cardiovascular benefits of regular exercise are unequivocal, yet patients with type 2 diabetes respond poorly to exercise due to a reduced cardiac reserve. The contractile response of diabetic cardiomyocytes to beta-adrenergic stimulation is attenuated, which may result in altered myofilament calcium sensitivity and posttranslational modifications of cardiac troponin I (cTnI). Treadmill running increases myofilament calcium sensitivity in nondiabetic rats, and thus we hypothesized that endurance training would increase calcium sensitivity of diabetic cardiomyocytes and alter site-specific phosphorylation of cTnI. Calcium sensitivity, or pCa(50), was measured in Zucker diabetic fatty (ZDF), nondiabetic (nDM), and diabetic (DM) rat hearts after 8 wk of either a sedentary (SED) or progressive treadmill running (TR) intervention. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+]). Specific phospho-sites on cTnI and O-GlcNAcylation were quantified by immunoblot and total protein phosphorylation by fluorescent gel staining (ProQ Diamond). The novel finding in this study was that training increased pCa(50) in both DM and nDM cardiomyocytes (P = 0.009). Phosphorylation of cTnI amino acid residues Ser23/24, a crucial protein kinase A site, and Threonine (Thr)144 was lower in DM hearts, but there was no effect of training on site-specific phosphorylation. In addition, total phosphorylation and O-GlcNAcylation levels were not different between SED and TR groups. These findings suggest that regular exercise may benefit the diabetic heart by specifically targeting myofilament contractile function. NEW & NOTEWORTHY We examined the effects of training on the myofilament calcium in diabetic rat hearts. After 8 wk of treadmill running, both nondiabetic and diabetic cardiomyocytes had increased myofilament calcium sensitivity compared with their sedentary counterparts, but there was no effect of training on the phosphorylation or O-GIcNAcylation status of myofilament proteins measured in this study. These data highlight one potential mechanism capable of reversing, in part, reduced cardiac reserve in the diabetic heart.