Autophagy is an evolutionary preserved process that prevents the accumulation of unwanted cytosolic material through the formation of autophagosomes. Although autophagy has been extensively studied to understand its function in normal physiology, the role of vascular smooth muscle (SM) cell (VSMC) autophagy in Ca(2+) mobilization and contraction remains poorly understood. Recent evidence shows that autophagy is involved in controlling contractile function and Ca(2+) homeostasis in certain cell types. Therefore, autophagy might also regulate contractile capacity and Ca(2+)-mobilizing pathways in VSMCs. Contractility (organ chambers) and Ca(2+) homeostasis (myograph) were investigated in aortic segments of 3.5-mo-old mice containing a SM cell-specific deletion of autophagy-related 7 (Atg7; Atg7(fl/fl) SM22α-Cre(+) mice) and in segments of corresponding control mice (Atg7(+/+) SM22α-Cre(+)). Our results indicate that voltage-gated Ca(2+) channels (VGCCs) of Atg7(fl/fl) SM22α-Cre(+) VSMCs were more sensitive to depolarization, independent of changes in resting membrane potential. Contractions elicited with K(+) (50 mM) or the VGCC agonist BAY K8644 (100 nM) were significantly higher due to increased VGCC expression and activity. Interestingly, the sarcoplasmic reticulum of Atg7(fl/fl) SM22α-Cre(+) VSMCs was enlarged, which, combined with increased sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 expression and higher store-operated Ca(2+) entry, promoted inositol 1,4,5-trisphosphate-mediated contractions of Atg7(fl/fl) SM22α-Cre(+) segments and maximized the Ca(2+) storing capacity of the sarcoplasmic reticulum. Moreover, decreased plasma membrane Ca(2+)-ATPase expression in Atg7(fl/fl) SM22α-Cre(+) VSMCs hampered Ca(2+) extrusion to the extracellular environment. Overall, our study indicates that defective autophagy in VSMCs leads to an imbalance between Ca(2+) release/influx and Ca(2+) reuptake/extrusion, resulting in higher basal Ca(2+) concentrations and significant effects on vascular reactivity.

Aging and associated progressive arterial stiffening are both important predictors for the development of cardiovascular diseases. Recent evidence showed that autophagy, a catabolic cellular mechanism responsible for nutrient recycling, plays a major role in the physiology of vascular cells such as endothelial cells and vascular smooth muscle cells (VSMCs). Moreover, several autophagy inducing compounds are effective in treating arterial stiffness. Yet, a direct link between VSMC autophagy and arterial stiffness remains largely unidentified. Therefore, we investigated the effects of a VSMC-specific deletion of the essential autophagy-related gene Atg7 in young mice (3.5 months) (Atg7 F/F SM22α-Cre + mice) on the biomechanical properties of the aorta, using an in-house developed Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC). Aortic segments of Atg7 F/F SM22α-Cre + mice displayed attenuated compliance and higher arterial stiffness, which was more evident at higher distention pressures. Passive aortic wall remodeling, rather than differences in VSMC tone, is responsible for these phenomena, since differences in compliance and stiffness between Atg7 +/+ SM22α-Cre + and Atg7 F/F SM22α-Cre + aortas were more pronounced when VSMCs were completely relaxed by the addition of exogenous nitric oxide. These observations are supported by histological data showing a 13% increase in medial wall thickness and a 14% decrease in elastin along with elevated elastin fragmentation. In addition, expression of the calciumbinding protein S100A4, which is linked to matrix remodeling, was elevated in aortic segments of Atg7 F/F SM22α-Cre + mice. Overall, these findings illustrate that autophagy exerts a crucial role in defining arterial wall compliance.

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Autophagy is an important cellular survival process that enables degradation and recycling of defective organelles and proteins to maintain cellular homeostasis. Hence, defective autophagy plays a role in many age-associated diseases, such as atherosclerosis, arterial stiffening and hypertension. Recently, we showed in mice that autophagy in vascular smooth muscle cells (VSMCs) of large elastic arteries such as the aorta is important for Ca 2+ mobilization and vascular reactivity. Whether autophagy plays a role in the smaller muscular arteries, such as the femoral artery, and thereby contributes to for example, blood pressure regulation is currently unknown. Therefore, we determined vascular reactivity of femoral artery segments of mice containing a VSMC specific deletion of the essential autophagy gene Atg7 (Atg7 F/F SM22α-Cre +) and compared them to femoral artery segments of corresponding control mice (Atg7 +/+ SM22α-Cre +). Our results indicate that similar to the aorta, femoral artery segments showed enhanced contractility. Specifically, femoral artery segments of Atg7 F/F SM22α-Cre + mice showed an increase in phasic phenylephrine (PE) induced contractions, together with an enhanced sensitivity to depolarization induced contractions. In addition, and importantly, VSMC sensitivity to exogenous nitric oxide (NO) was significantly increased in femoral artery segments of Atg7 F/F SM22α-Cre + mice. Notwithstanding the fact that small artery contractility is a significant pathophysiological determinant for the development of hypertension, 7 days of treatment with angiotensin II (AngII), which increased systolic blood pressure in control mice, was ineffective in Atg7 F/F SM22α-Cre + mice. It is likely that this was due to the increased sensitivity of VSMCs to NO in the femoral artery, although changes in the heart upon AngII treatment were also present, which could also be (partially) accountable for the lack of an AngII-induced rise in blood pressure in Atg7 F/F SM22α-Cre + mice. Overall, our study indicates that apart from previously shown effects on large elastic arteries, VSMC autophagy also plays a pivotal role in the regulation of the contractile and relaxing properties of the smaller muscular arteries. This may suggest a role for autophagy in vascular pathologies, such as hypertension and arterial stiffness.

Introduction: Arterial stiffness (AS) has gained much recognition as a hallmark and independent predictor of cardiovascular (CV) events. Although generally assumed to be an adaptive response to increased blood pressure (BP), AS precedes hypertension in at least two experimental mouse models, thus revealing an incomplete understanding of AS pathophysiology. Methods: The current study presents the longitudinal CV characterization of spontaneously ageing C57Bl6 mice (2, 4, 6, 9, 12 and 24-month old) (male, n>8). In vivo analysis of peripheral BP (Coda), aortic pulse wave velocity (aPWV, Vevo2100), and echocardiography (Vevo2100) was combined with ex vivo aortic studies of isometric reactivity (organ baths) and AS measurements in the Rodent Oscillatory Tension set-up for Arterial Compliance (ROTSAC). (Data are presented as mean ± SEM.) Results: In vivo and ex vivo characterisation confirms that aortic stiffness precedes peripheral BP alterations in spontaneously ageing C57Bl6 mice, with significantly and gradually increasing aPWV (Fig.A) and isobaric Peterson modulus (Ep) (Fig.B) from 6-month of age onward. Thereafter, cardiac hypertrophy was observed at 9-months of age (Fig.C), and peripheral BP measurement reveals elevated pulse pressure at 24-months (30% increase vs. all other ages) (Fig.D). Ex vivo investigation of the thoracic aorta shows increased contractions to phenylephrine (PE) in old (6-24 month) vs. young (2-4 month) mice (Fig.E,F) with an increased contribution of voltage-gated calcium channel (VGCC) (Fig.G,H) with age. Remarkably, no differences were observed on endothelial function, meaning that all changes occur on the level of the vascular smooth muscle cell (VSMC). Conclusions: Physiological ageing of VSMC results in high PE contractions, increased VGCC activity, and the development of significant arterial stiffening by 6-months of age. AS thereby precedes the development of peripheral BP alterations by 18 months.