Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans, the number of extracellular vesicles (EVs) increased acutely. In humans, EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins, and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1–positive EV release. Injected EC-EVs localized to the spleen and interacted with, and mobilized, splenic monocytes in otherwise naive, healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets, which regulate relevant cellular functions (e.g., proliferation and cell movement). Furthermore, gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs, EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion– and chemotaxis-associated genes, including the negative regulator of cell motility, plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI.

Background: Kinase oxidation is a critical signalling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, type-1 protein kinase A (PKARIα) can be reversibly oxidized, forming interprotein disulfide bonds within the holoenzyme complex. However, the effect of PKARIα disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored. Methods: Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARIα disulfide formation by immunoblot. To determine the impact of disulfide formation on PKARIα catalytic activity and sub-cellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in prkar1 knock-out mouse embryonic fibroblasts, neonatal myocytes or adult LV myocytes isolated from 'redox dead' (Cys17Ser) PKARIα knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes whereas I/R-injury was assessed ex vivo. Results: In both humans and mice, myocardial PKARIα disulfide formation was found to be significantly increased (2-fold in humans, p=0.023; 2.4-fold in mice, p<0.001) in response to I/R in vivo. In mouse LV cardiomyocytes, disulfide-containing PKARIα was not found to impact catalytic activity, but instead led to enhanced A-kinase-anchoring protein (AKAP) binding with preferential localization of the holoenzyme to the lysosome. Redox-dependent regulation of lysosomal two pore channels (TPC) by PKARIα was sufficient to prevent global calcium release from the sarcoplasmic reticulum in LV myocytes, without affecting intrinsic ryanodine receptor leak or phosphorylation. Absence of I/R-induced PKARIα disulfide formation in "redox dead" knock-in mouse hearts resulted in larger infarcts (2-fold, p<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, p<0.001), which was prevented by administering the lysosomal TPC inhibitor Ned-19 at the time of reperfusion. Conclusions: Disulfide-modification targets PKARIα to the lysosome where it acts as a gatekeeper for TPC-mediated triggering of global calcium release. In the post-ischemic heart, this regulatory mechanism is critical for protecting from extensive injury and offers a novel target for the design of cardioprotective therapeutics.

Interruption of blood supply to the heart is a leading cause of death and disability. However, the molecular events that occur during heart ischemia, and how these changes prime consequent cell death upon reperfusion, are poorly understood. Protein SUMOylation is a post-translational modification that has been strongly implicated in the protection of cells against a variety of stressors, including ischemia-reperfusion. In particular, the SUMO2/3-specific protease SENP3 has emerged as an important determinant of cell survival after ischemic infarct. Here, we used the Langendorff perfusion model to examine changes in the levels and localisation of SUMOylated target proteins and SENP3 in whole heart. We observed a 50% loss of SENP3 from the cytosolic fraction of hearts after preconditioning, a 90% loss after ischemia and an 80% loss after ischemia-reperfusion. To examine these effects further, we performed ischemia and ischemia-reperfusion experiments in the cardiomyocyte H9C2 cell line. Similar to whole hearts, ischemia induced a decrease in cytosolic SENP3. Furthermore, shRNA-mediated knockdown of SENP3 led to an increase in the rate of cell death upon reperfusion. Together, our results indicate that cardiac ischemia dramatically alter levels of SENP3 and suggest that this may a mechanism to promote cell survival after ischemia-reperfusion in heart.

We previously demonstrated that hexokinase II (HK2) dissociation from mitochondria during cardiac ischemia correlates with cytochrome c (cyt-c) loss, oxidative stress and subsequent reperfusion injury. However, whether HK2 release is the primary signal mediating this ischemia-induced mitochondrial dysfunction was not established. To investigate this, we studied the effects of dissociating HK2 from isolated heart mitochondria. Mitochondria isolated from Langendorff-perfused rat hearts before and after 30 min global ischemia ± ischemic preconditioning (IPC) were subject to in vitro dissociation of HK2 by incubation with glucose-6phosphate at pH 6.3. Prior HK2 dissociation from pre-or end-ischemic heart mitochondria had no effect on their cyt-c release, respiration (± ADP) or mitochondrial permeability transition pore (mPTP) opening. Inner mitochondrial membrane morphology was assessed indirectly by monitoring changes in light scattering (LS) and confirmed by transmission electron microscopy. Although no major ultrastructure differences were detected between pre-and end-ischemia mitochondria, the amplitude of changes in LS was reduced in the latter. This was prevented by IPC but not mimicked in vitro by HK2 dissociation. We also observed more Drp1, a mitochondrial fission protein, in end-ischemia mitochondria. IPC failed to prevent this increase but did decrease mitochondrial-associated dynamin 2. In vitro HK2 dissociation alone cannot replicate ischemia-induced effects on mitochondrial function implying that in vivo dissociation of HK2 modulates end-ischemia mitochondrial function indirectly perhaps involving interaction with mitochondrial fission proteins. The resulting changes in mitochondrial morphology and cristae structure would destabilize outer / inner membrane interactions, increase cyt-c release and enhance mPTP sensitivity to [Ca 2+ ].

Perivascular adipose tissue (PVAT) exerts an anti-contractile effect which is vital in regulating blood pressure. Evidence suggests that the sympathetic nervous stimulation of PVAT triggers the release of anti-contractile factors via activation of beta 3 -adrenoceptors. There is considerable evidence of sympathetic over-activity in obesity, which could result in the loss of PVAT function, and subsequent hypertension. Therefore it was decided to examine beta 3 -adrenoceptor function in obesity.Electrical field stimulation (EFS) profiles of healthy and obese mouse mesenteric arteries (<200 um, +/-PVAT) were characterised using wire myography (0.1-30 Hz, 20V, 0.2 ms pulse duration, 4s train duration). To demonstrate the release of an anti-contractile factor in health, the solution surrounding stimulated exogenous PVAT was transferred to a PVAT denuded vessel. Beta 3-adrenoceptor function was investigated using the agonist CL-316,243 (10uM) and antagonist SR59203A (100nM). The role of the vasodilator nitric oxide (NO) was studied using nitric oxide synthase (NOS) inhibitor L-NMMA (100uM), and NOS activator histamine (100uM).During EFS healthy PVAT elicits an anti-contractile effect (n = 8, P < 0.001); however the anti-contractile function of obese PVAT is lost (n = 8, P = 0.35). Inhibition of beta 3-adrenoceptors in healthy PVAT using SR59230A significantly reduced the anti-contractile effect (n = 8, P < 0.01), whereas activation of beta 3-adrenoceptors in obese PVAT using CL-316,243 did not restore function (n = 7, P = 0.77). Solution transfer from stimulated healthy exogenous PVAT to a -PVAT vessel significantly reduced contraction (n = 8, P < 0.01), confirming that stimulated PVAT releases a transferable anticontractile factor. The release of this factor could be inhibited using SR59230A (n = 7, P = 0.47). Solution transfer from obese PVAT had no effect on contraction (n = 6, P = 0.41), and again could not be restored using CL-316,243 (n = 6, P = 0.14). In healthy PVAT, inhibition of NOS using L-NMMA abolished the anti-contractile effect (n = 8, P < 0.01). In obese PVAT, activation of NOS using histamine was able to restore the anti-contractile function (n = 4, P < 0.05).These results demonstrate that in health PVAT releases an anti-contractile factor via activation of beta 3 -adrenoreceptors, which downstream trigger the release of NO. In obesity, the anti-contractile effect is lost and cannot be restored by beta 3 -adrenoceptor activation, but is restored by activation of NOS. This suggests that in obesity beta 3 -adrenoreceptors must be downregulated or desensitised, leading to a loss of anti-contractile function, which may contribute to the development of hypertension.