CaMKIIδ Met281/282 oxidation is not required for recovery of calcium transients during acidosis

Background: CaMKIId is needed for the recovery of Ca2+ transients during acidosis, but also mediates post-acidic arrhythmias. CaMKIId can sustain its activity following oxidation. Increasing intracellular Na+ during acidosis as well as post-acidic pH normalization should result in pro-oxidant conditions within the cell favoring oxidative CaMKIId activation. Rationale: We tested whether oxidative CaMKIId activation is needed for the recovery of Ca2+-transients during acidosis and for cellular arrhythmias post acidosis. Methods and results: Cardiomyocytes from a well-established mouse model in which CaMKIId was made resistant to oxidative activation by knock-in replacement of two oxidant-sensitive methionines (M281/282) with valines (MM-VV) were exposed to extracellular acidosis. Recovery of Ca2+-transients during acidosis was present in both, WT control and MM-VV cardiomyocytes, and post-acidic cellular arrhythmias occurred to a similar extent in both groups. Inhibition of global CaMKII activity using AIP prevented recovery of Ca2+-transients during acidosis and attenuated post-acidic arrhythmias in MM-VV cells. Using cardiomyocytes expressing redox-sensitive green fluorescent protein 2 coupled to glutaredoxin-1, we found that acidosis reduced the cytosolic redox potential despite a significant increase in intracellular Na+. Conclusions: Our study shows that oxidative activation of CaMKIId is neither required for recovery of Ca2+-transients during acidosis nor relevant for post-acidic arrhythmias in isolated cardiac myocytes. Moreover, acidosis actually reduces the cytosolic redox potential of isolated cardiac myocytes. Pharmacological inhibition of global CaMKII activity completely prevents recovery of Ca2+-transients and protects from post-acidic arrhythmias in MM-VV myocytes which confirms the relevance of CaMKII activity in the context of acidosis.

Abstract 16974: Augmentation of Vasoactive Intestinal Peptide Signaling Prevents the Development of Duchenne Muscular Dystrophy-Associated Cardiomyopathy Through Inhibition of NF-κB Signaling

Duchenne muscular dystrophy (DMD) is a recessive X-linked neuromuscular disorder characterized by progressive muscle degeneration with DMD-associated cardiomyopathy being the primary mode of death. Vasoactive intestinal peptide (VIP) is a 28 amino acid neuropeptide with biological effects mediated by G protein-coupled receptors. Utilizing a genetically modified form of VIP (PB1046) targeting cardiomyocytes, we hypothesized that augmentation of VIP signaling prevents the development of DMD-associated cardiomyopathy through inhibition of NF-κB. Either PB1046 (1.5 mg/kg) or a Placebo (saline) was injected subcutaneously every other day in three mouse models: DMD mdx:Utr+/- , DMD mdx , and wild type mice starting at 4 weeks of age for a total of 8 weeks. Cardiac function was assessed by weekly echocardiography. The cardiac tissues were collected at 14 weeks of age for histological and molecular analyses. Drug-treated DMD mdx:Utr+/- mice showed preservation of cardiac function (fractional shortening 61%±0.4 vs 45%±1.3, p <0.01; n=8-12) and a marked reduction in myocardial fibrosis (2.92%±0.13 vs 6.42% ±0.39, p <0.01; n=3) compared with controls. Hydroxyproline levels within drug-treated DMD mdx:Utr+/- mice was decreased compared with controls (44.6±5.3 vs 64.3±6.9 nmol/100mg heart weight, p <0.05, n=6). RNA-Seq data revealed an upregulation of cAMP signaling with downregulation of NF-kB signaling in isolated cardiac myocytes from drug-treated DMD mdx:Utr+/- mice as compared to controls (n=3). Western blot analyses revealed increased phosphorylation of CREB (1.97±0.02 vs 1.00±0.06, p <0.05, n=5-9) with decreased phosphorylation of p65 in drug-treated DMD cardiac nuclei as compared to controls (0.62±0.06 vs 1.00±0.09, p <0.05, n=3). Collectively, the data revealed augmentation of VIP signaling prevents the development of DMD-associated cardiomyopathy in DMD mdx:Utr+/- mice. The molecular mechanism underlying the benefits of VIP signaling suggests an upregulation of cAMP-CREB signaling with downregulation of NF-kB signaling leading to inhibition of inflammation and fibrosis within drug-treated DMD hearts. VIP signaling in DMD may serve as a new therapeutic target for the treatment of DMD-associated cardiomyopathy.

NOX Inhibition Improves β-Adrenergic Stimulated Contractility and Intracellular Calcium Handling in the Aged Rat Heart

Cardiac aging is characterized by alterations in contractility and intracellular calcium ([Ca2+]i) homeostasis. It has been suggested that oxidative stress may be involved in this process. We and others have reported that in cardiomyopathies the NADPH oxidase (NOX)-derived superoxide is increased, with a negative impact on [Ca2+]i and contractility. We tested the hypothesis that in the aged heart, [Ca2+]i handling and contractility are disturbed by NOX-derived superoxide. For this we used adults (≈5 month-old) and aged (20–24 month-old) rats. Contractility was evaluated in isolated hearts, challenged with isoproterenol. To assess [Ca2+]i, isolated cardiac myocytes were field-stimulated and [Ca2+]i was monitored with fura-2. Cardiac concentration-response to isoproterenol was depressed in aged compared to adults hearts (p < 0.005), but was restored by NOX inhibitors apocynin and VAS2870. In isolated cardiomyocytes, apocynin increased the amplitude of [Ca2+]i in aged myocytes (p < 0.05). Time-50 [Ca2+]i decay was increased in aged myocytes (p < 0.05) and reduced towards normal by NOX inhibition. In addition, we found that myofilaments Ca2+ sensitivity was reduced in aged myocytes (p < 0.05), and was further reduced by apocynin. NOX2 expression along with NADPH oxidase activity was increased in aged hearts. Phospholamban phosphorylation (Ser16/Thr17) after isoproterenol treatment was reduced in aged hearts compared to adults and was restored by apocynin treatment (p < 0.05). In conclusion, β-adrenergic-induced contractility was depressed in aged hearts, and NOX inhibition restored back to normal. Moreover, altered Ca2+ handling in aged myocytes was also improved by NOX inhibition. These results suggest a NOX-dependent effect in aged myocytes at the level of Ca2+ handling proteins and myofilaments.

NOX Inhibition Improves β Adrenergic-Stimulated Contractility and Intracellular Calcium Handling in the Aged Heart

Cardiac aging is characterized by alterations in contractility and intracellular calcium ([Ca2+]i) homeostasis. It has been suggested that oxidative stress may be involved in this process. We and others have reported that in cardiomyopathies the NADPH oxidase (NOX)-derived superoxide is increased, with a negative impact on [Ca2+]i and contractility. We tested the hypothesis that in the aged heart, [Ca2+]i handling and contractility are disturbed by NOX-derived superoxide. Contractility was evaluated isolated hearts, challenged with isoproterenol. To assess [Ca2+]i, isolated cardiac myocytes were field-stimulated and [Ca2+]i was monitored with fura-2. Cardiac concentration-response to isoproterenol was depressed in aged compared to adults hearts (p &lt; 0.005), but was restored by NOX inhibitors apocynin and VAS2870. In isolated cardiomyocytes, apocynin increased the amplitude of [Ca2+]i in aged myocytes (p &lt; 0.05). Time-50 [Ca2+]i decay was increased in aged myocytes (p &lt; 0.05) and reduced towards normal by NOX inhibition. In addition, we found that myofilaments Ca2+ sensitivity was reduced in aged myocytes (p &lt; 0.05), and further reduced by apocynin. Finally SERCA levels but not phospholamban were reduced in aged hearts (p &lt; 0.05). In conclusion, &beta;-adrenergic‒induced contractility was depressed in aged hearts, and NOX inhibition restored back to normal. Moreover, altered Ca2+ handling in aged myocytes was also improved by NOX inhibition. These results suggest a NOX-dependent effect in aged myocytes at the level of Ca2+ handling proteins and myofilaments.

(+)-Usnic Acid Isolated from the Lichen Cladonia substellata Impairs Myocardial Contractility

The scientific interest in (+)-usnic acid has grown because of its antitumor, cytotoxic, and antimicrobial activities as well as weight loss. However, overuse of usnic acid has been related with severe hepatotoxicity, making its use questionable. In this study, we decided to expand the knowledge of usnic acid biological activities by characterizing its effects on the mammalian myocardium as a potential pharmacological target. Usnic acid was isolated from samples of Cladonia substellata and submitted to chemical characterization. Molecular inclusion complexes of usnic acid with hydroxypropyl β-cyclodextrin were prepared to improve its water solubility. The effects of usnic acid on the atrial contractility and Ca2+ influx were carried out in the left atrium of guinea pigs and the effect of usnic acid on the L-type Ca2+ current was performed in rat ventricular cardiomyocytes enzymatically isolated. To evaluate the membrane integrity of cells subjected to usnic acid, we used histological procedures. Usnic acid reduced atrial contraction with an EC50 of 43.0±1.0 μM. This effect was related to a reduction of Ca2+ entry in myocardial cells. In isolated cardiac myocytes, usnic acid at 100 μM inhibited the L-type Ca2+ current by 73.0%. In addition, usnic acid caused an irreversible myocardial contracture, reflecting a serious disturbance of the intracellular Ca2+ homeostasis. Such an effect could not be ascribed to tissue death because cell membrane integrity was confirmed by histological observation. Taken together, our results show that usnic acid impairs cardiac function. Clearly more studies will be necessary to allow further applications of this natural product.