BMP9 and BMP10 Act Directly on Vascular Smooth Muscle Cells for Generation and Maintenance of the Contractile State

Background: Vascular smooth muscle cells (VSMCs) show a remarkable phenotypic plasticity allowing acquisition of contractile or synthetic states but critical information is missing about the physiological signals, promoting formation and maintenance of contractile VSMCs in vivo . BMP9 and BMP10 are known to regulate endothelial quiescence after secretion from the liver and right atrium, whereas a direct role in the regulation of VSMCs was not investigated. Here, we studied the role of BMP9 and BMP10 for controlling formation of contractile VSMCs. Methods: We generated several cell type-specific loss- and gain-of-function transgenic mouse models to investigate the physiological role of BMP9, BMP10, ALK1 and SMAD7 in vivo . Morphometric assessments, expression analysis, blood pressure measurements, single molecule fluorescence in situ hybridization (FISH) were performed together with analysis of isolated pulmonary VSMCs to unravel phenotypic and transcriptomic changes in response to absence or presence of BMP9 and BMP10. Results: Concomitant genetic inactivation of Bmp9 in the germ line and Bmp10 in the right atrium led to dramatic changes in vascular tone and diminution of the VSMC layer with attenuated contractility and decreased systemic as well as right ventricular systolic pressure (RVSP). Vice versa , overexpression of Bmp10 in endothelial cells (ECs) of adult mice dramatically enhanced formation of contractile VSMCs and increased systemic blood pressure as well as RVSP. Likewise, BMP9/10 treatment induced an ALK1-dependent phenotypic switch from synthetic to contractile in pulmonary VSMCs. SMC specific overexpression of Smad7 completely suppressed differentiation and proliferation of VSMCs and reiterated defects observed in adult Bmp9/10 double mutants. Deletion of Alk1 in VSMCs recapitulated the Bmp9/10 phenotype in pulmonary but not in aortic and coronary arteries. Bulk expression analysis and single molecule RNA-FISH uncovered vessel bed-specific, heterogeneous expression of BMP type 1 receptors, explaining phenotypic differences in different Alk1 mutant vessel beds. Conclusions: Our study demonstrates that BMP9 and BMP10 act directly on VSMCs for induction and maintenance of their contractile state. Surprisingly, the effects of BMP9/10 in VSMCs are mediated by different combinations of BMP type 1 receptors in a vessel bed specific manner, offering new opportunities to manipulate blood pressure in the pulmonary circulation.

SLO2.1 and NALCN form a functional complex to modulate myometrial cell excitability

At the end of pregnancy, the uterus transitions from a quiescent state to an excitable, contractile state. These changes are linked to depolarization of the myometrial smooth muscle cell (MSMC) resting membrane potential. The membrane potential is primarily determined by the balance between an outward potassium (K+) leak current and an inward sodium (Na+) leak current. We recently described a Na+-activated K+ channel (SLO2.1) and a non-selective Na+ leak channel (NALCN) in human MSMCs. Here, we asked whether these channels function together. We show that SLO2.1 currents are activated by an inward NALCN-dependent Na+ leak current, leading to MSMC hyperpolarization. The regulation of the membrane potential by NALCN/SLO2.1 activity modulates both Ca2+ entry through VDCCs, and myometrial contractility. Finally, NALCN and SLO2.1 are in proximity to one another in human MSMCs. We conclude that SLO2.1 and NALCN function together to regulate human MSMC membrane potential and excitability.

Progesterone and estrogen regulate NALCN expression in human myometrial smooth muscle cells

During pregnancy, the uterus transitions from a quiescent state to an excitable, highly contractile state to deliver the fetus. Two important contributors essential for this transition are hormones and ion channels, both of which modulate myometrial smooth muscle cell (MSMC) excitability. Recently, the sodium (Na+) leak channel, nonselective (NALCN), was shown to contribute to a Na+ leak current in human MSMCs, and mice lacking NALCN in the uterus had dysfunctional labor. Microarray data suggested that the proquiescent hormone progesterone (P4) and the procontractile hormone estrogen (E2) regulated this channel. Here, we sought to determine whether P4 and E2 directly regulate NALCN. In human MSMCs, we found that NALCN mRNA expression decreased by 2.3-fold in the presence of E2 and increased by 5.6-fold in the presence of P4. Similarly, E2 treatment decreased, and P4 treatment restored NALCN protein expression. Additionally, E2 significantly inhibited, and P4 significantly enhanced an NALCN-dependent leak current in MSMCs. Finally, we identified estrogen response and progesterone response elements (EREs and PREs) in the NALCN promoter. With the use of luciferase assays, we showed that the PREs, but not the ERE, contributed to regulation of NALCN expression. Our findings reveal a new mechanism by which NALCN is regulated in the myometrium and suggest a novel role for NALCN in pregnancy.

Cardiac physiology

The function of the heart is to pump sufficient oxygenated blood containing nutrients, metabolites, and hormones to meet moment-to-moment metabolic needs and preserve a constant internal environment. The heart has two essential characteristics—contractility and rhythmicity. The nervous system and neurohumoral agents modulate relationships between the venous return to the heart, the outflow resistance against which it contracts, the frequency of contraction, and its inotropic (contractile) state; there are also intrinsic cardiac autoregulatory mechanisms. An understanding of the molecular mechanisms governing cardiac cell behaviour and the mechanical, electrical, and hormonal control of the heart at a whole organ level is essential for the understanding of cardiac pathophysiology.

Laboratory practical to study the differential innervation pathways of urinary tract smooth muscle

In the mammalian lower urinary tract, there is a reciprocal relationship between the contractile state of the bladder and urethra. As the bladder fills with urine, it remains relaxed to accommodate increases in volume, while the urethra remains contracted to prevent leakage of urine from the bladder to the exterior. Disruptions to the normal contractile state of the bladder and urethra can lead to abnormal micturition patterns and urinary incontinence. While both the bladder and urethra are smooth-muscle organs, they are differentially contracted by input from cholinergic and sympathetic nerves, respectively. The laboratory practical described here provides an experiential approach to understanding the anatomy of the lower urinary tract. Several key factors in urinary tract physiology are outlined, e.g., the bladder is contracted by activation of the parasympathetic pathway via cholinergic stimulation on muscarinic receptors, whereas the urethra is contracted by activation of the sympathetic pathway via adrenergic stimulation on α1-adrenoceptors. This is achieved by measuring the force generated by bladder and urethra smooth muscle to demonstrate that acetylcholine contracts the smooth muscle of the bladder, whereas adrenergic agonists contract the urethral smooth muscle. An inhibition of these effects is also demonstrated by application of the muscarinic receptor antagonist atropine and the α1-adrenergic receptor blocker phentolamine. A list of suggested techniques and exam questions to evaluate student understanding on this topic is also provided.

Abstract 378: Cytochrome b5 Reductase 3 Regulates Vascular Smooth Muscle Phenotypic Switching

Contractile vascular smooth muscle cells (VSMC) play a key role in the regulation of arterial blood vessel tone and cardiovascular health. However, in many vascular diseases, VSMCs undergo a phenotypic switch from a contractile state to a synthetic phenotype, where loss of the contractile markers myosin heavy chain 11 (Myh11), smooth muscle alpha actin (ACTA2) and transgelin (SM22) are observed and proliferation is increased. Recent evidence from our lab demonstrates that cytochrome b5 reductase 3 (Cyb5R3) regulates the redox state of soluble guanylate cyclase to control cGMP levels in VSMCs. Because cGMP modulates protein kinase G activity, a critical kinase that maintains VSMCs in a contractile state, we tested the hypothesis that Cyb5R3 is critical for maintenance of the contractile phenotype. To test this hypothesis, we transduced primary rat aortic smooth muscle cells with non-targeting (NT) or Cyb5R3 shRNA followed by serum starvation for 24, 48, and 72 hours to induce phenotypic switching. After each time point, mRNA measurements of Cyb5R3, Myh11, ACTA2, and SM22 were conducted using RT-PCR. In NT shRNA transduced VSMCs, we observed a significant increase in Cyb5R3, Myh11, ACTA2, and SM22 mRNA, but not in Cyb5R3 knockdown VSMCs. Next, we conducted proliferation studies by serum starving NT shRNA and Cyb5R3 shRNA treated VSMCs for 24 hours followed by stimulation of platelet growth factor BB (PDGF-BB, 40 ng/mL). After 24 hours of PDGF-BB treatment, Cyb5R3 deficient cells showed augmented proliferation compared to control cells measured by 3 H-thymidine incorporation. Together, our data suggest that Cyb5R3 is essential for VSMC phenotypic switching and proliferation, which may unravel a new therapeutic target for treating individuals with cardiovascular disease.

Hypertrophied Papillary Muscle causing Mid Cavity Left Ventricular Obstruction after Cardiac Surgery

ABSTRACT A rare cause of mid cavity left ventricular obstruction can be a hypertrophied and/or a malformed papillary muscle. Hypertrophy of papillary muscle can be atypical presentation of hypertrophic obstructive cardiomyopathy. Most of these patients do not have a resting gradient, but a dynamic gradient can occur in hypovolumia or increased contractile state. We present a case of elderly patient who had a hypertrophied anterolateral papillary muscle and developed mid left ventricular obstruction after weaning the patient from cardiopulmonary bypass. How to cite this article Dutta V, Raj R, Bahl A, Thingnum SS, Puri GD. Hypertrophied Papillary Muscle causing Mid Cavity Left Ventricular Obstruction after Cardiac Surgery. J Perioper Echocardiogr 2015;3(1):32-34.

Left Ventricle–Arterial System Interaction in Heart Failure

Ejection fraction (EF) has been viewed as an important index in assessing the contractile state of the left ventricle (LV). However, it is frequently inadequate for the diagnosis and management of heart failure (HF), as a significant subset of HF patients have been found to have reduced EF (HFrEF) whereas others have preserved EF (HFpEF). It should be noted that the function of the LV is dependent on both preload and afterload, as well as its intrinsic contractile state. Furthermore, stroke volume (SV) is dependent on the properties of the arterial system (AS). Thus, the LV-arterial system interaction plays an important role in those patients with HF. This aspect is investigated through the analysis of the specific parameters involved in the coupling of the LV and AS. This includes contractility and the systolic/diastolic indices of the LV. Furthermore, AS afterload parameters such as vascular stiffness and arterial compliance, and their derived coupling coefficient, are also investigated. We conclude that those parameters, which relate to LV structural changes, are most appropriate in quantifying the LV–AS interaction.