The calcium and integrin binding protein (CIB)1 as therapeutic target in heart failure
Final Report Abstract
Summary and Discussion of Results In this project, we developed a tool to downregulate CIB1 in the myocardium via an AAV9-vector expressed shRNA. Among 3 different candidate sequences, we selected a shRNA construct capable of downregulating CIB1 after overexpression by adenovirus in isolated NRCM or via AAV9 in adult mouse hearts by about 50%. This level of CIB1 downregulation was sufficient to achieve a strong anti-hypertrophic effect in response to α- and β-adrenergic or fetal bovine serum stimulation in vitro as well as after TAC in vivo. This is in line with our previous data from heterozygous Cib1 knock-out mice, which also hold around 50% less myocardial CIB1 protein, and show significantly reduced cardiac hypertrophy after TAC 7. Homozygous Cib1 knock-out mice, however, exerted even less TAC induced hypertrophy, indicating that the CIB1 dependent anti-hypertrophic effect is dose dependent. This implies that an even better anti-hypertrophic effect of AAV-shCIB1 would be feasible, if the downregulation of CIB1 in cardiomyocytes was more complete 7. It should be noted, that the anti-hypertrophic effects by AAV-shCIB1 in vivo after TAC were detected as reduced heart weight, reduced cardiac dilation, reduced cardiomyocyte cell area and reduced embryonic gene-expression, but did not lead to a reduction of the ventricular wall thickness. This implies that CIB1 depletion in vivo mainly counteracts an eccentric, dilatation prone hypertrophy pattern, but not concentric growth of ventricular walls. Besides the anti-hypertrophic properties, shCIB1 treatment also reduced myocardial fibrosis and preserved cardiac function as well as angiogenesis during short-term (two week) 10 pressure overload, which is both in line with our previous results from Cib1 knock-out mice 7. Pressure overload over a longer period, however, not only abrogated the beneficial effects of shCIB1 on cardiac function, but also induced capillary rarefaction. Mechanistically, again in line with our previous results, we attribute the beneficial effects of shCIB1 to a reduced activation of the maladaptive, prohypertrophic calcineurin/NFAT signaling pathway in cardiomyocytes, which are the prime target cells of AAV9 mediated genedelivery in the heart 7, 18. Reduced activity of this signaling circuit entailed reduced pathological hypertrophy and improved cardiac function in many animal studies 12, 21-23. On the other hand, complete inhibition of calcineurin (for example by genetic ablation of CnB) leads to lethal cardiomyopathy in mice 24, 25. Therefore, inhibition of a specific cellular subset of the calcineurin pool might be a reasonable therapeutic strategy, and we aimed to achieve this by targeting CIB1, which facilitates shuttling of calcineurin to a sarcolemmal microdomain leading to its activation through incoming calcium from the L-type calcium channel 7, 13. In addition, we detected a diminished activation of the MAP kinase ERK upon shCIB1 treatment in the myocardium of TAC treated mice in vivo. Accordingly, inhibition of the ERK MAP kinase was demonstrated as consequence of reduced CIB1 abundance in lung endothelial cells and in the gastrocnemius muscle of Cib1 knock-out mice as well as in breast cancer and neuroblastoma cells after CIB1 downregulation 26, 27. Inhibition of ERK activity supports reduction of cardiac hypertrophy, but might also trigger left ventricular dilation 28-30. This could explain why shCIB1 was no longer able to reduce cardiac dilation in the chronic phase after TAC. Inhibition of calcineurin/NFAT or ERK1/2 could also account for capillary rarefaction in the shCIB1 group during persisting pressure overload, since pro-angiogenic activities have been reported for both pathways 31-34. Indeed, reduced ischemia driven and tumor angiogenesis were found in Cib1 knock-out mice and were associated with reduced ERK1/2 signaling 26, 35. Importantly, we demonstrate here that treatment with shCIB1 triggers an anti-angiogenic gene-expression pattern in cardiomyocytes, which strongly participate in the regulation of cardiac angiogenesis: For example, VEGFA and CTGF are secreted from cardiomyocytes within the heart and promote myocardial capillary growth 41, 42. Both genes become downregulated by shCIB1 in cultured cardiomyocytes as well as in mouse hearts after TAC. In addition, CTGF is driving tissue fibrosis, and its reduced expression after TAC will therefore contribute to the anti-fibrotic effects of shCIB1 treatment. We attributed the improvement in left ventricular function mainly to reduced pathological cardiac hypertrophy, but also reduced expression of the slow myosin heavy chain isoform β- MHC, which triggers aggravated cardiac dysfunction and dilation during overload 36. The fact that the beneficial effects of shCIB1 are only transient when pressure overload persists (despite remaining anti-hypertrophic and -fibrotic effects), we ascribe to the diminished myocardial vascular density due to shCIB1 treatment, which might outweigh its beneficial effects. Indeed, inhibition of cardiac angiogenesis (for example via the angiogenesis inhibitor TNP-470 or VEGF- trap) was shown to promote cardiac dilation and dysfunction during pressure overload, while the administration of angiogenic factors improves systolic heart function, when myocardial capillary rarefaction exists 37-39. Although reduced angiogenesis is likely contributing to the transient nature of the beneficial effects of shCIB1, we cannot prove this connection with the existing data. Therapeutic approaches that -like shCIB1 in this study- effectively reduce myocardial hypertrophy and fibrosis and thereby improve cardiac remodeling are highly needed in clinical cardiology, for example in pressure overload triggered disease such as severe hypertension or 11 aortic valve stenosis, but also for patients after a large myocardial infarction 40, 41. We show here that a therapy with AAV-shCIB1 is safe and well tolerated in mice. The fact that the beneficial effect of shCIB1 on cardiac function can only be kept up for a limited time in persisting pressure overload might not be relevant in the clinical setting, when usually the (pressure) overload is alleviated by normalization of blood pressure, by aortic valve replacement, reopening of the infarcted vessel and additional neurohormonal blockade. In this regard, it should be mentioned that we have used C57Bl/6N mice in the current study, which are known to progress into heart failure during TAC induced pressure overload very quickly42. The use of different, less vulnerable strains such as C57Bl/6J mice could have given more favorable shCIB1 effects also in the chronic phase. Despite these potential disadvantages in the model we chose, the transient nature of the shCIB1 mediated benefits as well as its angiogenesis inhibiting properties remain a possible drawback of its translational potential in myocardial disease. Because inhibition of CIB1 is also being considered in different models of cancer, new agents to target CIB1 will be developed and should be tested in parallel to shCIB1 in different large and small animal models of acute and chronic myocardial disease to learn whether these approaches could be beneficial43. Although its clinical applicability remains uncertain at this point, we have shown that downregulation of a small specific adaptor protein like CIB1 can be used to inhibit maladaptive calcineurin/NFAT signaling and remodeling of the heart. This approach could in the future also be tested for other maladaptive signaling circuits in order to be able to efficiently counteract pathological hypertrophy and improve the outcome of patients with heart failure in the future.
Publications
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A gene therapeutic approach to inhibit calcium and integrin binding protein 1 ameliorates maladaptive remodelling in pressure overload. Cardiovasc Res. 2019;115:71-82
Grund A, Szaroszyk M, Doppner JK, Malek Mohammadi M, Kattih B, Korf-Klingebiel M, Gigina A, Scherr M, Kensah G, Jara-Avaca M, Gruh I, Martin U, Wollert KC, Gohla A, Katus HA, Muller OJ, Bauersachs J, Heineke J
