The ketone body 3-hydroxybutyrate (3-OHB) increases cardiac output and myocardial perfusion without affecting blood pressure in humans, but the cardiovascular sites of action remain obscure. Here, we test the hypothesis in rats that 3-OHB acts directly on the heart to increase cardiac contractility and directly on blood vessels to lower systemic vascular resistance. We investigate effects of 3-OHB on (a) in vivo hemodynamics using echocardiography and invasive blood pressure measurements, (b) isolated perfused hearts in Langendorff systems, and (c) isolated arteries and veins in isometric myographs. We compare Na-3-OHB to equimolar NaCl added to physiological buffers or injection solutions. At plasma concentrations of 2–4 mM in vivo, 3-OHB increases cardiac output (by 28.3±7.8%), stroke volume (by 22.4±6.0%), left ventricular ejection fraction (by 13.3±4.6%), and arterial dP/dt_max (by 31.9±11.2%) and lowers systemic vascular resistance (by 30.6±11.2%) without substantially affecting heart rate or blood pressure. Applied to isolated perfused hearts at 3–10 mM, 3-OHB increases left ventricular developed pressure by up to 26.3±7.4 mmHg and coronary perfusion by up to 20.2±9.5%. Beginning at 1–3 mM, 3-OHB relaxes isolated coronary (EC₅₀=12.4 mM), cerebral, femoral, mesenteric, and renal arteries as well as brachial, femoral, and mesenteric veins by up to 60% of pre-contraction within the pathophysiological concentration range. Of the two enantiomers that constitute racemic 3-OHB, D-3-OHB dominates endogenously; but tested separately, the enantiomers induce similar vasorelaxation. We conclude that increased cardiac contractility and generalized systemic vasorelaxation can explain the elevated cardiac output during 3-OHB administration. These actions strengthen the therapeutic rationale for 3-OHB in heart failure management.

BACKGROUND: More than half of patients with ischemic stroke experience futile reperfusion, increasing the risk of death and disabilities despite a successful recanalization. The reason behind this is debated, and we aim to investigate cerebrovascular changes toward a broader understanding of these conditions. We hypothesize that ischemic stroke reperfusion modifies the expression profile in the microvasculature in a spatial manner toward peri-infarct brain edema and circulatory failure. METHODS: We investigated the early (24-hour) changes in spatial gene expression in the brain parenchymal endothelial cells and mural cells following ischemia stroke reperfusion in 13- to 14-week-old C57BL/6JRj male mice (n=5). Ischemia was induced by occlusion of the middle cerebral artery for 60 minutes, and Nissl staining was used to validate infarct size. Spatial transcriptomics complemented by bulk proteomics was conducted in the peri-infarct cortex region and validated with immunohistochemical semiquantification of proteins of interest. To avoid individual biological variations, changes in the peri-infarct cortex region were expressed relatively to the matching contralateral hemisphere region. RESULTS: Ischemic stroke reperfusion impaired the blood-brain barrier integrity through junctional Cldn5 (claudin-5) downregulation, changes of the actin cytoskeleton adhesion, and high expression of the proinflammatory Il-6 (interleukin-6). Molecules important for extracellular Ca ²⁺ influx and intracellular Ca ²⁺ release, Cacna1e (R-type Ca ²⁺ channels ), Orai2 , Ryr3 , Itpr1 , and Itpka (inositol-trisphosphate 3-kinase A), were markedly reduced. Furthermore, reduced Grm5 (glutamate receptor 5) associated with upregulated Nfatc3 and Stat3 implicates suppression of the contractile phenotype, suggesting reduced poststroke vascular resistance due to loss of mural cell tone. The complete spatial transcriptomics map over the ipsilateral and contralateral hemispheres is available online as a Web tool. CONCLUSIONS: Emphasizing the spatial molecular pattern behind blood-brain barrier disruption and loss of the vascular tone in the acute phase following ischemic stroke reperfusion suggests the gene expression contribution for a therapeutic target in ischemia-reperfusion abnormalities.

The present study aimed to examine the effects of low doses of angiotensin II (AngII) on cardiac function, myocardial substrate utilization, energetics, and mitochondrial function in C57Bl/6J mice and in a transgenic mouse model with cardiomyocyte specific upregulation of NOX2 (csNOX2 TG). Mice were treated with saline (sham), 50 or 400 ng/kg/min of AngII (AngII₅₀ and AngII₄₀₀) for two weeks. In vivo blood pressure and cardiac function were measured using plethysmography and echocardiography, respectively. Ex vivo cardiac function, mechanical efficiency, and myocardial substrate utilization were assessed in isolated perfused working hearts, and mitochondrial function was measured in left ventricular homogenates. AngII₅₀ caused reduced mechanical efficiency despite having no effect on cardiac hypertrophy, function, or substrate utilization. AngII₄₀₀ slightly increased systemic blood pressure and induced cardiac hypertrophy with no effect on cardiac function, efficiency, or substrate utilization. In csNOX2 TG mice, AngII₄₀₀ induced cardiac hypertrophy and in vivo cardiac dysfunction. This was associated with a switch towards increased myocardial glucose oxidation and impaired mitochondrial oxygen consumption rates. Low doses of AngII may transiently impair cardiac efficiency, preceding the development of hypertrophy induced at higher doses. NOX2 overexpression exacerbates the AngII -induced pathology, with cardiac dysfunction and myocardial metabolic remodelling.

Familial hemiplegic migraine type 2 (FHM2) is linked to Na,K-ATPase α ₂ isoform mutations, including that of G301R. Mice heterozygous for this mutation ( α 2 + / G3 0 1R ) show cerebrovascular hypercontractility associated with amplified Src kinase signaling, and exaggerated neurovascular coupling. This study hypothesized that targeting Na,K-ATPase-dependent Src phosphorylation with pNaKtide would normalize cerebral perfusion and neurovascular coupling in α 2 + / G3 0 1R mice. The effect of pNaKtide on cerebral blood flow and neurovascular coupling was assessed using laser speckle contrast imaging in awake, head-fixed mice with cranial windows in a longitudinal study design. At baseline, compared to wild type, α 2 + / G3 0 1R mice exhibited increased middle cerebral artery tone; with whisker stimulation leading to an exaggerated increase in sensory cortex blood flow. No difference between genotypes in telemetrically assessed blood pressure occurred. The exaggerated neurovascular coupling in α 2 + / G3 0 1R mice was associated with increased K _ir 2.1 channel expression in cerebrovascular endothelium. Two weeks pNaKtide treatment normalized cerebral artery tone, endothelial K _ir 2.1 expression, and neurovascular coupling in α 2 + / G3 0 1R mice. Inhibition of the Na,K-ATPase-dependent Src kinase signaling with pNaKtide prevented excessive vasoconstriction and disturbances in neurovascular coupling in α 2 + / G3 0 1R mice. pNaKtide had only minor hypotensive effect similar in both genotypes. These results demonstrate a novel treatment target to normalize cerebral perfusion in FHM2.

Heterozygous mice (α₂^+/G301R mice) for the migraine-associated mutation (G301R) in the Na⁺,K⁺-ATPase α₂-isoform have decreased expression of cardiovascular α₂-isoform. The α₂^+/G301R mice exhibit a pro-contractile vascular phenotype associated with decreased left ventricular ejection fraction. However, the integrated functional cardiovascular consequences of this phenotype remain to be addressed in vivo. We hypothesized that the vascular response to α₂-isoform-specific inhibition of the Na⁺,K⁺-ATPase by ouabain is augmented in α₂^+/G301R mice leading to reduced cardiac efficiency. Thus, we aimed to assess the functional contribution of the α₂-isoform to in vivo cardiovascular function of wild-type (WT) and α₂^+/G301R mice. Blood pressure, stroke volume, heart rate, total peripheral resistance, arterial dP/dt, and systolic time intervals were assessed in anesthetized WT and α₂^+/G301R mice. To address rate-dependent cardiac changes, cardiovascular variables were compared before and after intraperitoneal injection of ouabain (1.5 mg/kg) or vehicle during atrial pacing. The α₂^+/G301R mice showed an enhanced ouabain-induced increase in total peripheral resistance associated with reduced efficiency of systolic development compared to WT. When the hearts were paced, ouabain reduced stroke volume in α₂^+/G301R mice. In conclusion, the ouabain-induced vascular response was augmented in α₂^+/G301R mice with consequent suppression of cardiac function.

Two α-isoforms of the Na⁺,K⁺-ATPase (α₁ and α₂) are expressed in the cardiovascular system, and it is unclear which isoform is the preferential regulator of contractility. Mice heterozygous for the familial hemiplegic migraine type 2 (FHM2) associated mutation in the α₂-isoform (G301R; α₂^+/G301R mice) have decreased expression of cardiac α₂-isoform but elevated expression of the α₁-isoform. We aimed to investigate the contribution of the α₂-isoform function to the cardiac phenotype of α₂^+/G301R hearts. We hypothesized that α₂^+/G301R hearts exhibit greater contractility due to reduced expression of cardiac α₂-isoform. Variables for contractility and relaxation of isolated hearts were assessed in the Langendorff system without and in the presence of ouabain (1 µM). Atrial pacing was performed to investigate rate-dependent changes. The α₂^+/G301R hearts displayed greater contractility than WT hearts during sinus rhythm, which was rate-dependent. The inotropic effect of ouabain was more augmented in α₂^+/G301R hearts than in WT hearts during sinus rhythm and atrial pacing. In conclusion, cardiac contractility was greater in α₂^+/G301R hearts than in WT hearts under resting conditions. The inotropic effect of ouabain was rate-independent and enhanced in α₂^+/G301R hearts, which was associated with increased systolic work.