MiRNA AND CARDIAC RESYNCHRONISATION THERAPY

Multiple miRNAs have been observed to be dysregulated and implicated in the development and progression of HF. The evidence remains limited due to the principle research being basic science and small cross section cohort studies. Based upon observations, the hypothesis was generated that miRNAs might be able to predict CRT response and outcomes.

Recently Ning et al196 performed the first study examining miRNA and CRT. Ning et al196 undertook an animal study comparing four groups of rabbits (10 in each group) and induced HF in three groups (by ascending aorta cerclage constriction). Two groups were paced with biventricular or LV alone; the third group had a sham procedure.196 Twelve weeks after the procedure had been performed pacing was performed for six hours a day for seven days. Following this period of pacing LVIDD decreased and LVEF increased significantly.196 Plasma was taken to measure circulating miR-133 and was found to have significantly lower expression in the HF sham group compared to the control. Both pacing groups had significantly higher expression than HF sham group. The biventricular pacing group had the greatest rise in expression (p<0.05). These findings support the observations miR-133 is cardioprotective in HF.161,181,182 Ning et al196performed an animal study and therefore it can only be hypothesis generating; miR-133 up-regulation is increased with biventricular pacing.196

87 Simultaneously Marfella et al197 published their prospective observational, non-randomised, self-control study on miRNA expression in HFrEF patients before and after CRT implantations. All consecutive patients (NYHA III/IV, sinus rhythm QRS>120msec, OMT>6 months and LVEF<35%) scheduled for CRT (153 patients) between January 2009 and August 2011 were screened and 81 subjects (HF cohort) were recruited in three Italian centres.197 A test (15 healthy volunteers) and validation (60 controls - age, sex and co-morbidity matched) cohorts were recruited to compared HF miRNA expression too.197 The use of a healthy control cohort is an interesting one as they are not matched in any way to the HF group. The HF cohort underwent functional (6MWT) and transthoracic echocardiography assessment at baseline and 12 months follow-up.197 The 6MWT performed was not performed in the standardised fashion as per the American Thoracic Society guidance.102 CRT response status was defined by degree of LV reverse remodelling (↓>10% LVESVi and ↑>10% LVEF and no heart transplant).197 There were 55 (67.9%) responders and 26 (32.1%) non-responders. 197 There were four mortalities in the observation period that were excluded following CRT implantation, inferring a bias towards selecting healthier HF patients and not accounting for those that had died. 197

A microarray was utilised to screen 84 miRNA pre-selected for their reported association with structural heart disease.197 Marfella et al197 observed the HF group had 49 down- regulated miRNAs compared to healthy controls and 24 miRNAs compared to the validation cohort (all p<0.05).197 Baseline expression of five circulating miRNA’s (miR-26b-5p,-29a-3p,-

88 30e-5p,-92a-3p and -145-5p) in HF patients directly and indirectly correlated with LVEF and NT-pro-BNP.197 Furthermore these 5 miRNAs demonstrated a high AUC (>0.83) in differentiating HF cases from non-HF.197 Baseline expression of all circulating miRNA showed no statistically significant difference between RvsNR.197 Following one year, responders had 19 miRNAs significantly (p<0.01) up-regulated (15 miRNA had >5-fold-change increase) and non-responders had 6 miRNAs significantly up-regulated from baseline (from the 24 identified from comparison with the validation cohort).197 Figure 1.10 shows that five miRNAs all showed up-regulation regardless of response status, however it was significantly higher (p<0.001) for responders.197 All these five miRNA have known mechanistic roles in maladaptive molecular processes; hypertrophy (miR-26b-5p and -30e-5p), fibrosis (miR-29a- 3p and miR-92a) and apoptosis (miR-145).197 Interestingly miR-885-5p was observed to be up-regulated in non-responders, but no change in responders.197 The changes in expression over 1 year for these five miRNA correlate with LVEF and NT-pro-BNP.197

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Figure 1.10 MiRNA Expression Profiles for Responders vs. Non-Responders after one year of CRT. MiRNA listed are those that demonstrated a significant difference between RvsNR in fold change over one year observation. Panel A gives miRNA expression (arbitrary units=AU) after one year of CRT. Panel B provides a Table describing the degree of miRNA fold regulation from baseline to one year following CRT and its specific significance. *p<0.01 responders vs non-responders. Taken from Marfella et al. EHJ. 2013197.

Marfella et al197 examined several miRNAs involved in regulated maladaptive molecular processes implicated in adverse cardiac remodelling in HFrEF. Marfella et al197 demonstrated altered expression of important miRNAs known to be implicated in several maladaptive processes in HF and furthermore demonstrated an association with LV echocardiographic and neurohormonal variables that are known to improve following CRT implantation. Moreover, variation is seen between responders and non-responders at

90 follow-up identifying alteration in the molecular processes. However, no difference between these miRNAs were demonstrated between responders and non-responders baseline (pre- implant) levels, offering no predictive value for any of these biomarkers.

There are several limitations of Marfella et al197that need to be considered. Firstly this was a small cohort study that lacked power to support their observations. Secondly, the miRNA quantification method should be acknowledged as not being the gold standard. Thirdly, the healthy control comparison cohort was not age-sex matched, therefore the high number of miRNAs expression demonstrated is understandable and accountable by potentially multiple physiological processes.198 These limitations do challenge the use of this paper in the wider literature to miRNAs as predictors of CRT response.

More recently Melman et al199 moved the debate on about altered miRNA expression profiles in HFrEF patients undergoing CRT implantation with the publication of their translational study. Melman et al199 performed an exploratory discovery and validation prospective cohort study on differential miRNA expression in 52 HFrEF patients referred for CRT implantation (NYHA II-IV, LVEF<35%, QRS>120msec and LBBB/RV paced) between responders and non-responders. Response was defined as an increase LVEF >10% at six months on transthoracic echocardiography for the validation phase.199 A major limitation of the methodology is concerned with the performance of the transthoracic echocardiography; only a single plane was used to calculate LVEF, this does not meet the internationally accepted national standard200 The standard is to perform Simpson’s biplane assessment for

91 LV ejection fraction (Chapter 5.5).201 Aside from not meeting the national echocardiography standard it makes comparisons between different studies that measure LV geometry unreliable.

The discovery phase was performed on 12 select patients; 6 responders with the greatest LVEF increase and 6 non-responders with no change/decline.199 All the participants in the discovery phase were men with non-ischaemic cardiomyopathy and LBBB to increase the sensitivity of the screening.199 A microarray was used to screen 766 human circulating miRNA in plasma, which identified four miRNAs (miR-30d,-99b,-409-3p and -766) had significantly differential expression between responders and non-responders (all p<0.05).199 The validation study was performed in the remaining 40 participants with no significant characteristic differences between RvsNR.199 Surprisingly only 58% of the total validation cohort were on Angiotensin Converting Enzyme Inhibitors (ACEi); most HF studies have HFrEF patients have on higher amounts of OMT. Furthermore, the validations cohort included 33% AF patients and 20% ischaemic cardiomyopathy, which made the Melman et al199 validation cohort more heterogeneous than in the Marfella et al197 HF cohort. However, it does question the selection of the four miRNAs they took forward into a validation cohort with different baseline characteristics. 199 The was a high proportion of the cohort on anti-platelets (83% Aspirin, 23% Clopidogrel) and anticoagulants (45% Coumadin), which are known to affect miRNA expression, especially platelet derived.147,199 There were 21 (52.5%) responders and 19 (47.5%) non-responders.199 Ten candidate miRNAs were quantified in the validation cohort with qPCR; 5 miRNAs were already identified and five miRNA (miR-18b,-29c,-129-5p,-423-5p and -622) not identified in the discovery phase. Four

92 miRNAs (miR-29c,-30d,-142-5p and -766) were significantly overexpressed in responders (all p<0.05).199 Multivariable logistic regression (accounting for co-variates) identified higher circulating miR-30d (OR=2.52, CI (95%) 1.07-5.94) and miR-142-5p (OR=2.47, CI (95%) 1.26- 4.85) predicted a better CRT response.199 A multivariable linear regression model, for degree of change in LVEF over six months, identified only miR-30d as being significantly associated (p=0.02) with change in LVEF at six months.199 Figure 3.5 demonstrates baseline miR-30d significantly correlated with change in LVEF over six months (Þ=0.39, p=0.01). However the strength of correlation is driven by the two highest changes in miRNA expression which are treated as outliers, when these points are removed the significance of the correlation is reduced (Þ=0.31, p=0.058), a point Dorn202 summaries in the sister editorial.

A separate 21 HFrEF participants formed a test cohort who had undergone a CRT implantation and had six months samples.199 The test group was used to compare to the results of the validation cohort. The additional 21 baseline samples demonstrated the strength of miR-30d to predict CRT, supporting the validation cohort findings. The six month follow-up samples identified that miR-30d expression had decreased after six months following CRT implantation significantly in responders (p=0.05), but not in non- responders.199

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Figure 1.11 Relationship between circulating plasma miR-30d levels and change in LVEF six months after CRT. Correlation with all data points was significant (Þ=0.39, p=0.01), however when outliers removed correlation less significant (Þ=0.31, p=0.058). Red highlighting indicates miR-30d outliers, green are responders, blue are non-responders. Taken from Dorn EW. Circulation 2015.202

Biologically Melman et al199 demonstrated that miR-30d was expressed in higher concentrations in the CS, synthesised and released from cardiomyocytes in extracellular vesicles.199 Spatial heterogeneity in the LV of miR-30d was demonstrated in a canine dyssynchronous HF model, with the lateral wall showing higher expression.199 Furthermore Melman et al199demonstrated that miR-30d expression and release from cardiomyocytes is associated with increased mechanical stress, mediating cardiac hypertrophy. MiR-30d was observed to protect against tumour necrosis factor-α (medicates apoptosis), inferring a

94 cardioprotective role. Increased expression of miR30-d seems to represent a favourable adaptive process which may predict CRT response.

Interestingly following the publication of Melman et al’s199miR-30d and CRT translation study199 the authors of the first CRT and miRNA cohort expression paper197 wrote a published response contrasting how miRNA expression profile was measured and the common themes emerging regarding the miR-30 family. Sardu et al203 highlighted the differential miRNA expression pattern seen and that miR-30e were observed to be up- regulated at one year follow-up. They commented this might have potential anti-apoptosis effects. Structurally the miR-30 family is very similar and the biological function of members of the group is thought to be similar.203 Melman et al203 responded to these comments supporting the observation that other extracellular miRNA were likely to be implicated in the reverse LV remodelling. They also pointed out the differences in miRNA expression observed was likely to be due to small cohorts, differences in patient characteristics and the accepted variation in methodologies on quantifying miRNA expression. Importantly both authors agree on the importance of the miR-30 family in the process of reverse LV remodelling induction by CRT and the modification of the maladaptive molecular processes of apoptosis this regulates. Moreover the potential value in detecting response status and other extracellular miRNA are likely to be important in this regulation process.203,204 Melman et al199 has particularly demonstrated a striking difference between response status and the molecular mechanism it is involved in regulating. However the pattern of miR-30d was shown to contrast that reported by Marfella et al197as it showed it was not overly expressed at baseline and no changes occurred in responders, unlike Melman et al.199 The translational

95 pilot offers a very robust assessment of the biological mechanism of the likely reason miR- 30d is a potential biomarker of CRT response, further validation is required.