3.13.2 miR-‐30 targets have key implications in cardiac disease
The association between reduced miR-‐30 levels and cardiac disease has been recently reported in relevant animal models and also in patient samples 261, 266, 339. Nevertheless, the description of miR-‐30 target genes and their cellular functions has remained scarce. The target subset discovered in this research makes up core components of cardiac cell signalling and function. Therefore, we reasoned that the central downstream effects that would derive from manipulating miR-‐30 in cardiac cells would partly reflect the mechanisms being unleashed by DOX treatment in the myocardium.
3.13.2.1 Beta-‐adrenergic pathway modulation by miR-‐30
Three of the validated miR-‐30 targets during the course of this thesis are key members of the β-‐adrenergic pathway: β1AR, β2AR and Giα-‐2. β-‐adrenoceptors function to detect catecholamine stimulation in the cell surface and translate it into cellular activities.
Subsequent to βAR activation is the synthesis of the intracellular mediator (or second messenger) cAMP by AC 19. We demonstrated that cAMP accumulation levels vary in response to alterations in miR-‐30 levels, exhibiting an inverse correlation with miR-‐30 abundance (Figure 39). This is in keeping with the direct post-‐transcriptional regulation of β1AR and β2AR by miR-‐30 described in this thesis. DOX treatment triggered greater accumulation of cAMP than specific miR-‐30 inhibition, which suggests that DOX may act through additional pathways that also encourage cAMP build-‐up.
With regard to cardiomyocyte contractility, the βAR positive inotropic effect arises following a rise in intracellular Ca2+ mediated by AC-‐cAMP-‐PKA signalling 19, 20. Given that Giα-‐2 inhibits AC activity and therefore has a negative inotropic effect (reduced contractility) 376, the cAMP accumulation we observed suggests miR-‐30 to be inhibiting βARs more deeply compared to Giα-‐2. Our suspicions about the regulation exerted by miR-‐30 on the β-‐adrenergic pathway were reaffirmed by the contractility studies performed on transfected ARVCM. Baseline contraction amplitude of ARVCM with exogenously increased miR-‐30e levels did not differ from control cardiomyocytes (Figure 40B), importantly implying that higher miR-‐30 expression is not detrimental for basal contraction. However, ARVCM showed decreased contractile response to ISO stimulation upon miR-‐30e overexpression (Figure 40C), which might appear counterintuitive in relation to HF since it tends to be associated with reduced CO. Yet, the observed reduction in contractile responses precisely mirrors the effects achieved with β-‐blocker agents, widely used to rescue cardiac function 86. Moreover, it must be considered that miR-‐30 expression achieved by exogenous overexpression is substantially greater than physiological levels.
These overexpression experiments served to illustrate the overall impact of miR-‐30 on the β-‐
adrenergic pathway, which results from the net effect of the simultaneous repression of βAR and Giα-‐2. ISO is a βAR agonist and, given that βARs enhance cardiomyocyte contractility while Giα-‐2 inhibits it, the attenuated contractile response to ISO stimulation suggests preferential repression of βAR by miR-‐30 alongside a compensatory inhibition of Giα-‐2. In any case, it is worth noting that alternate miR-‐30 target genes to the ones studied here could also be modulating contractile responses of transfected ARVCM. The observed contractile phenotype was copied by PTX-‐mediated ablation of Gi combined with miR-‐30 overexpression. PTX treatment resulted in more cell death and higher arrhythmia in the studied ARVCM, which agrees with the described anti-‐apoptotic and anti-‐arrhythmic roles for Giα-‐2 22, 24, 377. Even though cells survived to shorter dose response curves following the challenges of transfection with miR-‐30e mimics over 48h and exposure to PTX, pre-‐NC transfected ARVCM treated with PTX showed significantly enhanced contractile amplitude upon stimulation with ISO. Conversely, PTX-‐treated miR-‐30e overexpressing ARVCM presented no significant alteration of their contractile response to ISO in relation to miR-‐30 transfected ARVCM not unexposed to PTX (Figure 41). These results indicate that, even
when incorporating extra Giα-‐2 inhibition by PTX, miR-‐30 still has predominantly inhibitory effects on βARs (β1AR, β2AR).
In summary, high miR-‐30 expression correlates with lower intracellular cAMP accumulation, reduced contractile response to ISO stimulation but normal baseline contractility, and with unaffected contraction amplitude upon PTX treatment. These data support a β-‐blocker like activity for miR-‐30. Since we have shown here that miR-‐30 targets both β1AR and β2AR, it would function as a non-‐selective β-‐blocker. Recent publications propose a preferred use of β1-‐selective blockers that are able to concomitantly maintain/stimulate β2AR signalling.
Certainly, β2AR-‐specific agonists are thought to provide an attractive therapeutic target to minimize myocyte apoptosis and arrhythmogenesis 378. Nevertheless, it is important to note that miRNAs are moderate regulators and miR-‐30 administration would therefore cause modest changes in βAR expression. In addition, miR-‐30 repression of the β2AR-‐Gi signalling pathway, which is considered to be anti-‐apoptotic in cardiomyocytes, would expectedly be compensated by the simultaneous inhibition of the key pro-‐apoptotic gene BNIP3L. In the particular case of DOX-‐induced cardiotoxicity, it should be noted that given the reported increase in β2AR expression in injured hearts –in agreement with our data-‐ 375, it is debatable whether β2AR stimulation would be beneficial in this model. Besides, some degree of Giα-‐2 inhibition by miR-‐30 is not predicted to be damaging, as Giα-‐2 expression is found up-‐
regulated in end stage HF 56.
Remarkably, the implications of the subset of miR-‐30 targets described here extend beyond the DOX cardiotoxicity model on which this research has focused. A number of cardiac conditions leading to failure have been attributed to an intense stimulation of βARs -‐ from hypertrophy leading to failure 52, to stress (Takotsubo) cardiomyopathy deriving from high circulating adrenaline 379. Chronic catecholamine stimulation causes negative inotropic effects on myocytes, leading to global ventricular dysfunction 18. Catecholamines have been shown to induce dose-‐dependent apoptosis in cardiomyocytes, which can be blunted by the use of β-‐blockers. This toxicity appears to be mediated by increased cAMP leading to Ca2+
overload 380. In addition, metabolic products of catecholamines have been shown to generate ROS 381. Oxidative stress has been linked to the pathophysiology of HF in general 382 and, more specifically, to anthracycline cardiomyopathy 114, 156. Therefore, the restoration of
miR-‐30 expression in DOX-‐treated hearts could also contribute to reduced ROS levels by repressing β-‐adrenergic expression.
To recapitulate, the down-‐regulation of miR-‐30 caused by DOX could be enhancing β-‐
adrenergic signalling (Figure 38, Figure 39, Figure 41). This, in turn, would lead to increased responsiveness to catecholamines and the associated risks of overstimulation. Importantly, a potential link between DOX cardiotoxicity and the detrimental effects of the catecholamine/βAR cascade can be drawn from a study where the administration of clinical doses of DOX in dogs induced an increase in circulating catecholamines 60. The potential contribution of catecholamine-‐mediated βAR stimulation to the mechanisms involved in DOX cardiomyopathy would precisely support the discussed therapeutic use of miR-‐30 as a β-‐blocker agent.