Neurons of the median preoptic nucleus contribute to chronic angiotensin II-salt induced hypertension in the rat.

Experiments were designed to test the hypothesis that median preoptic (MnPO) neurons are necessary for the full hypertensive response to chronic angiotensin II (AngII) in rats consuming a high salt diet. The MnPO is implicated in many of the physiologic actions of AngII, primarily acting as a downstream nucleus to AngII binding at circumventricular organs such as the organum vasculosum of the lamina terminalis (OVLT). We have previously shown a prominent effect of lesion of the OVLT on the chronic hypertensive effects of AngII in rats consuming high salt. Additionally, we have shown that lesion of the MnPO attenuated the hypertensive response to chronic intravenous infusion of AngII in rats. However, whether MnPO neurons or fibers of passage contribute to this response is not clear. Male Sprague Dawley rats were randomly assigned to either sham (SHAM; n = 8) or ibotenic acid lesion of the MnPO (MnPOx; n = 6). In the MnPOx group, 200 nl of ibotenic acid in phosphate buffer saline (5 µg/µl) was injected into each of 3 predetermined coordinates targeted at the entire MnPO. After a week of recovery, rats were instrumented with radiotelemetric pressure transducers, provided 2.0% NaCl diet and distilled water ad libitum and given another week to recover. After 3 days of baseline measurements, osmotic minipumps were implanted subcutaneously in all rats for administration of AngII at a rate of 150 ng/kg/min. Blood pressure measurements were made for 14 days after minipump implantation. By day 7 of AngII treatment, blood pressure responses appeared to plateau in both groups while the hypertensive response was markedly attenuated in MnPOx rats (MnPOx, 122 ± 6 mmHg; SHAM, 143 ± 8 mmHg). These results support the hypothesis that neurons of the MnPO are involved in the central pathway mediating the chronic hypertensive effects of AngII in rats consuming a high salt diet.

Atrial GIRK Channels Mediate the Effects of Vagus Nerve Stimulation on Heart Rate Dynamics and Arrhythmogenesis.

Diminished parasympathetic influence is central to the pathogenesis of cardiovascular diseases, including heart failure and hypertension. Stimulation of the vagus nerve has shown promise in treating cardiovascular disease, prompting renewed interest in understanding the signaling pathway(s) that mediate the vagal influence on cardiac physiology. Here, we evaluated the contribution of G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels to the effect of vagus nerve stimulation (VNS) on heart rate (HR), HR variability (HRV), and arrhythmogenesis in anesthetized mice. As parasympathetic fibers innervate both atria and ventricle, and GIRK channels contribute to the cholinergic impact on atrial and ventricular myocytes, we collected in vivo electrocardiogram recordings from mice lacking either atrial or ventricular GIRK channels, during VNS. VNS decreased HR and increased HRV in control mice, in a muscarinic receptor-dependent manner. This effect was preserved in mice lacking ventricular GIRK channels, but was nearly completely absent in mice lacking GIRK channels in the atria. In addition, atrial-specific ablation of GIRK channels conferred resistance to arrhythmic episodes induced by VNS. These data indicate that atrial GIRK channels are the primary mediators of the impact of VNS on HR, HRV, and arrhythmogenesis in the anesthetized mouse.

A cardiac patch from aligned microvessel and cardiomyocyte patches.

Cardiac tissue engineering aims to produce replacement tissue patches in the lab to replace or treat infarcted myocardium. However, current patches lack preformed microvascularization and are therefore limited in thickness and force production. In this study, we sought to assess whether a bilayer patch composed of a layer made from human induced pluripotent stem cell-derived cardiomyocytes and a microvessel layer composed of self-assembled human blood outgrowth endothelial cells and pericytes was capable of engrafting on the epicardial surface of a nude rat infarct model and becoming perfused by the host 4 weeks after acute implantation. The bilayer configuration was found to increase the twitch force production, improve human induced pluripotent stem cell-derived cardiomyocyte survival and maturation, and increase patent microvessel lumens compared with time-matched single layer controls after 2 weeks of in vitro culture. Upon implantation, the patch microvessels sprouted into the cardiomyocyte layer of the patch and inosculated with the host vasculature as evidenced by species-specific perfusion labels and erythrocyte staining. Our results demonstrate that the added microvessel layer of a bilayer patch substantially improves in vitro functionality and that the bilayer patch is capable of engraftment with rapid microvessel inosculation on injured myocardium. The bilayer format will allow for scaling up in size through the addition of layers to obtain thicker tissues generating greater force in the future.

Inosculation and perfusion of pre-vascularized tissue patches containing aligned human microvessels after myocardial infarction.

A major goal of tissue engineering is the creation of pre-vascularized tissues that have a high density of organized microvessels that can be rapidly perfused following implantation. This is especially critical for highly metabolic tissues like myocardium, where a thick myocardial engineered tissue would require rapid perfusion within the first several days to survive transplantation. In the present work, tissue patches containing human microvessels that were either randomly oriented or aligned were placed acutely on rat hearts post-infarction and for each case it was determined whether rapid inosculation could occur and perfusion of the patch could be maintained for 6 days in an infarct environment. Patches containing self-assembled microvessels were formed by co-entrapment of human blood outgrowth endothelial cells and human pericytes in fibrin gel. Cell-induced gel contraction was mechanically-constrained resulting in samples with high densities of microvessels that were either randomly oriented (with 420 ± 140 lumens/mm(2)) or uniaxially aligned (with 940 ± 240 lumens/mm(2)) at the time of implantation. These patches were sutured onto the epicardial surface of the hearts of athymic rats following permanent ligation of the left anterior descending artery. In both aligned and randomly oriented microvessel patches, inosculation occurred and perfusion of the transplanted human microvessels was maintained, proving the in vivo vascularization potential of these engineered tissues. No difference was found in the number of human microvessels that were perfused in the randomly oriented (111 ± 75 perfused lumens/mm(2)) and aligned (173 ± 97 perfused lumens/mm(2)) patches. Our results demonstrate that tissue patches containing a high density of either aligned or randomly oriented human pre-formed microvessels achieve rapid perfusion in the myocardial infarct environment - a necessary first-step toward the creation of a thick, perfusable heart patch.

Effect of global and regional sympathetic blockade on arterial pressure during water deprivation in conscious rats.

Forty-eight hours of water deprivation (WD) in conscious rats results in a paradoxical increase in mean arterial pressure (MAP). Previous studies suggest this may be due to increased sympathetic nerve activity (SNA). However, this remains to be investigated in conscious, freely behaving animals. The purpose of this study was to determine, in conscious rats, the role of the sympathetic nervous system (SNS) in mediating WD-induced increases in MAP and to identify which vascular beds are targeted by increased SNA. Each rat was chronically instrumented with a radiotelemetry transmitter to measure MAP and heart rate (HR) and an indwelling venous catheter for plasma sampling and/or drug delivery. MAP and HR were continuously measured during a 2-day baseline period followed by 48 h of WD and then a recovery period. By the end of the WD period, MAP increased by ∼15 mmHg in control groups, whereas HR did not change significantly. Chronic blockade of α(1)/ß(1)-adrenergic receptors significantly attenuated the WD-induced increase in MAP, suggesting a role for global activation of the SNS. However, the MAP response to WD was unaffected by selective denervations of the hindlimb, renal, or splanchnic vascular beds, or by adrenal demedullation. In contrast, complete adrenalectomy (with corticosterone and aldosterone replaced) significantly attenuated the MAP response to WD in the same time frame as α(1)/ß(1)-adrenergic receptor blockade. These results suggest that, in conscious water-deprived rats, the SNS contributes to the MAP response and may be linked to release of adrenocortical hormones. Finally, this sympathetically mediated response is not dependent on increased SNA to one specific vascular bed.

The role of the subfornical organ in angiotensin II-salt hypertension in the rat.

Hypertension caused by chronic infusion of angiotensin II (Ang II) in experimental animals is dependent, in part, on increased activity of the sympathetic nervous system. This chronic sympathoexcitatory response is amplified by a high-salt diet, suggesting an interaction of circulating Ang II and dietary salt on sympathetic regulatory pathways in the brain. The present study tested the hypothesis that the subfornical organ (SFO), a forebrain circumventricular organ known to be activated by circulating Ang II, is crucial to the pathogenesis of hypertension induced by chronic Ang II administration in rats on a high-salt diet (Ang II-salt model). Rats were randomly selected to undergo either subfornical organ lesion (SFOx) or sham surgery (Sham) and then placed on a high-salt (2% NaCl) diet. One week later, rats were instrumented for radiotelemetric measurement of mean arterial pressure (MAP) and heart rate (HR) and placed in metabolic cages to measure sodium and water balance. Baseline MAP was slightly (but not statistically) lower in SFOx compared with Sham rats during the 5 day control period. During the subsequent 10 days of Ang II administration, MAP was statistically lower in SFOx rats. However, when MAP responses to Ang II were analysed by comparing the change from the 5 day baseline period, only on the fifth day of Ang II was MAP significantly different between groups. There were no differences between groups for water or sodium balance throughout the protocol. We conclude that, although the SFO is required for the complete expression of Ang II-salt hypertension in the rat, other brain sites are also involved.

Time-dependent changes in autonomic control of splanchnic vascular resistance and heart rate in ANG II-salt hypertension.

Previous studies suggest that ANG II-induced hypertension in rats fed a high-salt (HS) diet (ANG II-salt hypertension) has a neurogenic component dependent on an enhanced sympathetic tone to the splanchnic veins and independent from changes in sympathetic nerve activity to the kidney or hind limb. The purpose of this study was to extend these findings and test whether altered autonomic control of splanchnic resistance arteries and the heart also contributes to the neurogenic component. Mean arterial pressure (MAP), heart rate (HR), superior mesenteric artery blood flow, and mesenteric vascular resistance (MVR) were measured during 4 control days, 14 days of ANG II delivered subcutaneously (150 ng·kg(-1)·min(-1)), and 4 days of recovery in conscious rats fed a HS (2% NaCl) or low-salt (LS; 0.1% NaCl) diet. Autonomic effects on MAP, HR, and MVR were assessed by acute ganglionic blockade with hexamethonium (20 mg/kg iv) on day 3 of control, days 1, 3, 5, 7, 10, and 13 of ANG II, and day 4 of recovery. MVR increased during ANG II infusion in HS and LS rats but remained elevated only in HS rats. Additionally, the MVR response to hexamethonium was enhanced on days 10 and 13 of ANG II selectively in HS rats. Compared with LS rats, HR in HS rats was higher during the 2nd wk of ANG II, and its response to hexamethonium was greater on days 7, 10, and 13 of ANG II. These results suggest that ANG II-salt hypertension is associated with delayed changes in autonomic control of splanchnic resistance arteries and the heart.

The effect of cardiac sympathetic denervation through bilateral stellate ganglionectomy on electrical properties of the heart.

The role of the cardiac sympathetic nerve activity in various cardiac diseases is typically evaluated using ß-adrenergic receptor antagonists. However, these antagonists induce global denervation effects not only in the cardiovascular system, but also in the brain and kidney. The objective of this study was to detect the electrophysiological property changes due to 8 days of cardiac sympathetic denervation and investigate the possible mechanisms underlying these changes using a more cardiac-specific bilateral stellate ganglionectomy (SGX) rat model. High-resolution optical mapping using a voltage-sensitive dye was performed in isolated Langendorff-perfused sham and SGX hearts, which were paced at progressively reduced basic cycle lengths under several different conditions: control, pretreatment with isoproterenol, and administration of atenolol and esmolol. Several electrophysiological parameters were recorded during periodic pacing and ventricular fibrillation (VF). Our results demonstrate that cardiac sympathetic denervation by bilateral SGX shortens action potential duration (APD) and flattens the APD restitution curve, but does not significantly affect spatial dispersion of APD. We found that, although the vulnerability of sham and SGX hearts to VF is similar, the dynamics of VF are different. The maximum dominant frequency is higher, and the spatial distribution of VF is more complex in the SGX heart, resulting in different mechanisms of VF. We demonstrated that ß(1)-adrenergic receptors are downregulated in the SGX compared with sham hearts. In addition, our data suggest that the mechanism of cardiac sympathetic denervation by SGX surgery is more similar to the administration of ß-blocker esmolol than atenolol.