Christian Aalkjaer

Aim: Ca ²⁺ ‐activated Cl– conductances are present in many cell types and are important for regulating membrane potential as well as other cellular functions. TMEM16A is widely accepted as the principal molecular basis for Ca ²⁺ ‐activated Cl– conductances, but also members of the bestrophin family may be important for some Ca ²⁺ ‐activated Cl– conductances. Methods: In this review, we discuss the possibility that members of the two families, bestrophins and TMEM16A, may interact. Results: In the cardiovascular system, the evidence is strongest. Here (1) TMEM16A and bestrophin‐3 are closely located in the membrane, (2) TMEM16A may regulate the expression of bestrophin‐3, and (3) a cGMP dependent Ca ²⁺ ‐activated Cl– conductance is dependent on both TMEM16A and bestrophin‐3. Outside the cardiovascular system, both TMEM16A and bestrophins are implicated in the regulation of cellular Ca ²⁺ independently of membrane potential, and both proteins are importantly associated with inflammation and pain transmission. However, it seems that little testing of a direct interaction of TMEM16A and bestrophins has been published. Conclusion: We conclude that there is evidence for direct interaction between TMEM16A and bestrophins in the cardiovascular system, and it will be important to determine whether similar interactions extend to other tissues.

Background: The circulation of 4–6 m tall giraffes is markedly affected by gravity. To ensure cerebral perfusion, upright giraffes generate a blood pressure in excess of 200 mmHg. Before drinking, the head is lowered by 3–5 m, providing exceptional hemodynamic challenges. Here, we provide quantitative hemodynamic measures during head movement and drinking.Methods: We measured carotid pressure, jugular pressure, heart rate, and blood flow in awake giraffes, along with circulating blood volume and cerebrospinal fluid pressure in anesthetized giraffes. We also analyzed the contractility and innervation of isolated cerebral and extracranial arteries, and the mechanical properties of jugular veins.Results: When heads were lowered for drinking (i) blood pressure at heart level decreased but increased again during drinking, (ii) jugular pressure increased and oscillated during drinking, (iii) heart rate fell, (iv) carotid blood flow was unchanged, while cephalic hemodynamic resistance increased, and (vi) cranial cerebrospinal fluid pressure increased. Small cerebral arteries exhibited strong myogenic responses, particularly at around 100 mmHg, while extracranial arteries responded at higher pressures (200–250 mmHg). The giraffe's blood volume was small and blood pressure sensitive to minor reductions in blood volume.Conclusions: Central blood pressure decreased when the head was lowered, but drinking per se caused a surprising rise in blood pressure to pre‐drinking levels. This rise in blood pressure is likely due to the transfer of esophageal water boli acting on the jugular veins. The cephalic capillaries are protected by a strong myogenic response and sympathetic innervation.

Prorenin and the prorenin receptor ((P)RR) are important, yet controversial, members of the renin–angiotensin–aldosterone system. The ((P)RR) is expressed throughout the body, including the vasculature, however, the direct effect of prorenin on arterial contractility is yet to be determined. Within rat mesenteric arteries, immunostaining and proximity ligation assays were used to determine the interacting partners of (P)RR in freshly isolated vascular smooth muscle cells (VSMCs). Wire myography examined the functional effect of prorenin. Simultaneous changes in [Ca ²⁺ ] _i and force were recorded in arteries loaded with Fura‐2AM. Spontaneously transient outward currents were recorded via perforated whole‐cell patch‐clamp configuration in freshly isolated VSMCs. We found that the (P)RR is located within a distance of less than 40 nm from the V‐ATPase, caveolin‐1, ryanodine receptors, and large conductance Ca ²⁺ ‐activated K ⁺ channels (BK _Ca ) in VSMCs. [Ca ²⁺ ] _i imaging and isometric tension recordings indicate that 1 nM prorenin enhanced α1‐adrenoreceptor‐mediated contraction, associated with an increased number of Ca ²⁺ waves, independent of voltage‐gated Ca ²⁺ channels activation. Incubation of VSMCs with 1 nM prorenin decreased the amplitude and frequency of spontaneously transient outward currents and attenuated BK _Ca ‐mediated relaxation. Inhibition of the V‐ATPase with 100 nM bafilomycin prevented prorenin‐mediated inhibition of BK _Ca ‐derived relaxation. Renin (1 nM) had no effect on BK _Ca ‐mediated relaxation. In conclusion, prorenin enhances arterial contractility by inhibition of BK _Ca and increasing intracellular Ca ²⁺ release. It is likely that this effect is mediated through a local shift in pH upon activation of the (P)RR and stimulation of the V‐ATPase.

ID 17343 Poster Board 311 Colchicine treatment has known beneficial effects on cardiovascular health and reduces the incidence of cardiovascular disease. Studies in isolated rodent arteries show colchicine enhances β-adrenoceptor-mediated vasodilation. In this translational study, we examined whether this effect of colchicine was present in humans by conducting a double-blinded, placebo controlled intervention study. Middle-aged men with essential hypertension were randomly assigned firstly to acute treatment with either 0.5 mg colchicine or placebo, and subsequently re-randomized for 3 weeks of treatment with either colchicine 0.5 mg twice daily (n=16) or placebo (n=15) followed by a washout period of 48-72 h. The vasodilator responses to isoprenaline, acetylcholine and sodium nitroprusside were determined, as well as arterial pressure, arterial compliance and plasma inflammatory markers. Acute colchicine treatment increased the isoprenaline- and SNP-mediated increase in vascular conductance, but had no effect on the response to acetylcholine. Following the washout period, three weeks of twice daily treatment of colchicine did not induce an accumulated or sustained effect on the β-adrenoceptor response and there was no effect on either arterial pressure, arterial compliance or on the level of measured inflammatory markers. This study provides the first translational evidence for a transient enhancement of β-adrenoceptor-mediated vasodilatation by colchicine in humans. The finding of an acute effect suggests that it may be clinically important to maintain an adequate biovailability of colchicine to support the cardiovascular protective effects that have been observed with colchicine in various clinical trials. This work was supported by a grant from Augustinusfonden (19-3644). TAJ was funded by a Lundbeck Foundation grant (R323-2018-3674). JvH was funded the European Union9s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie scheme (grant agreement no. 081199).

Giraffes are the highest living animals on Earth and therefore are challenged by gravity more than any other species. In particular the cardiovascular system needs to adapt to this challenge. Giraffes have a mean blood pressure around 200 mmHg, which ensures a mean arterial pressure near the head of 100 mmHg when the giraffe is standing with the neck in a near vertical position. This immediately raises several questions. How do giraffes avoid edema in the legs where the arterial pressure is 300 mmHg or higher? How does the heart produce a pressure of 200 mmHg, and what is the energy required for this endeavor? How can the kidney tolerate a pressure of about 200 mmHg and does this mean that giraffes have a high glomerular filtration rate? What is the arterial pressure in the head of giraffes when they drink, and how is perfusion of the brain maintained when they lift their head after drinking? In this short review, we present some answers to these questions.

The evolved adaptations of other species can be a source of insight for novel biomedical innovation. Limitations of traditional animal models for the study of some pathologies are fueling efforts to find new approaches to biomedical investigation. One emerging approach recognizes the evolved adaptations in other species as possible solutions to human pathology. The giraffe heart, for example, appears resistant to pathology related to heart failure with preserved ejection fraction (HFpEF)—a leading form of hypertension-associated cardiovascular disease in humans. Here, we postulate that the physiological pressure-induced left ventricular thickening in giraffes does not result in the pathological cardiovascular changes observed in humans with hypertension. The mechanisms underlying this cardiovascular adaptation to high blood pressure in the giraffe may be a bioinspired roadmap for preventive and therapeutic strategies for human HFpEF.

Gravity affects the physiology of many animals, and the effect is, for good reason, most pronounced in tall species. The physiology—in particular, cardiovascular function—of giraffes has therefore captivated the interest of physiologists for centuries. Several studies document high mean arterial blood pressure of giraffes of about 200 mm Hg. This appears necessary to establish a cerebral perfusion pressure on the order of 100 mm Hg at the cranial end of the carotid arteries. Here, we discuss the unique characteristics of blood vessels, the heart, and the kidney of giraffes and how these functional and structural adaptations are related to very high blood pressure. We also discuss how the cerebral circulation of giraffes is established and what we know about how the blood flow and arterial and venous pressures in giraffes change when they stop to drink and subsequently lift their heads 5–6 m in one sweeping movement.

The dynein motor protein transports proteins away from the cell membrane along the microtubule network. Recently, we found the microtubule network was important for regulating the membrane abundance of voltage-gated Kv7.4 potassium channels in vascular smooth muscle. Here, we aimed to investigate the influence of dynein on the microtubule-dependent internalization of the Kv7.4 channel. Patch-clamp recordings from HEK293B cells showed Kv7.4 currents were increased after inhibiting dynein function with ciliobrevin D or by coexpressing p50/dynamitin, which specifically interferes with dynein motor function. Mutation of a dynein-binding site in the Kv7.4 C terminus increased the Kv7.4 current and prevented p50 interference. Structured illumination microscopy, proximity ligation assays, and coimmunoprecipitation showed colocalization of Kv7.4 and dynein in mesenteric artery myocytes. Ciliobrevin D enhanced mesenteric artery relaxation to activators of Kv7.2–Kv7.5 channels and increased membrane abundance of Kv7.4 protein in isolated smooth muscle cells and HEK293B cells. Ciliobrevin D failed to enhance the negligible S-1–mediated relaxations after morpholino-mediated knockdown of Kv7.4. Mass spectrometry revealed an interaction of dynein with caveolin-1, confirmed using proximity ligation and coimmunoprecipitation assays, which also provided evidence for interaction of caveolin-1 with Kv7.4, confirming that Kv7.4 channels are localized to caveolae in mesenteric artery myocytes. Lastly, cholesterol depletion reduced the interaction of Kv7.4 with caveolin-1 and dynein while increasing the overall membrane expression of Kv7.4, although it attenuated the Kv7.4 current in oocytes and interfered with the action of ciliobrevin D and channel activators in arterial segments. Overall, this study shows that dynein can traffic Kv7.4 channels in vascular smooth muscle in a mechanism dependent on cholesterol-rich caveolae.

Heart failure with preserved ejection fraction (HFpEF) is a leading form of human cardiovascular disease and commonly associated with systemic hypertension. Unique evolved adaptations in giraffe myocardia may be a natural animal model of resistance to HFpEF. In humans, pressure-overload induced left ventricular thickening (PLVT) impairs diastolic relaxation, elevates left atrial pressures and may progress to heart failure with symptoms including exercise intolerance. In healthy giraffe, the left ventricle thickens as developmental neck lengthening widens the vertical distance between the heart and head increasing pressures needed to maintain constant brain perfusion. Yet, diastolic relaxation and exercise capacity are unimpaired, a critical adaptation for prey species such as giraffe. The proximate mechanisms underlying this unique cardiovascular physiology are not yet characterized. Developmental PLVT in giraffe emerges as a species-specific evolved adaptation which offers a roadmap for identifying innovations in therapeutic and prevention strategies for HFpEF.