Cellular Effects and Signalling Pathways

With the application of CRT and the advent of sophisticated modern cardiac imaging, the effects described above may be directly observed with reproducible and established techniques. e.g. echocardiography.

However beyond the macroscopic level, what remains unknown is the effect of CRT at a cellular level both directly on individual myocytes and intracellular signalling pathways. The field of basic science allied to CRT is small and has followed the large clinical publications. However the desire to understand some of the observed phenomenon following CRT

implantation has led to increased interest in this field. Particularly the phenomenon of reverse remodelling following the application of a depolarising electrode is not understood. The longer term aim of such investigation would be to guide patient selection, refine CRT itself and potentially even extrapolate such proteins and intracellular signals to the pharmacological arena to permit the development of future agents.

Due to the lack of knowledge in the area several cellular mechanisms are being studied. Recent publications in the area have included the following studies.

Pezzali and colleagues published this year (2013) on a cohort of CRT recipients (n=101) who had been evaluated for beta adrenoceptor polymorphisms.(179) The group investigated the beta-1 Arg389Gly, beta-2 Arg16Gly, and beta-2 Gln27Glu ARs gene polymorphisms. The endpoint was the magnitude of left ventricular remodelling and correlations with appropriate ICD shocks. The Gln27Glu ARs gene polymorphism had higher rates of LV remodelling (P=0.0018). Gln27 homozygotes had a higher incidence of appropriate shocks for both ventricular fibrillation and tachycardia.

Clinical observational studies have documented certain patients experience reverse

remodelling. Hence logically CRT in some individuals may have some effect on extracellular matrix turnover and the physiological pathways involved with this.

Marfella and colleagues analysed this and published in 2013.(180) Micro RNAs are non- coding RNAs which are linked to the regulation of cardiac structure and function. Due to the adverse cardiac remodelling, there may be dysregulation of the expression of micro RNAs.

The authors investigated the relationship between micro RNA expression, and LV reverse remodelling following CRT implantation.

A total of 81 patients made up the cohort. Micro RNA levels were measured in a healthy control group, age matched and disease matched (not for heart failure) and a heart failure population. At baseline, the HF population had a lower rate of expression of micro RNAs as compared to the healthy control group. (P<0.04). At 12 months 68 percent of the HF cohort (n=55) were classified as responders based on echocardiographic parameters. The levels of expression of certain micro RNAs were different between the responders and non-responders. The responders were characterised by higher expression of five miRNAs

(MiRNA-26b-5p, miRNA-145-5p, miRNA-92a-3p, miRNA-30e-5p, and miRNA-29a-3p; P , 0.01 for all) as compared with non-responders. These micro RNAs have been linked to cardiac fibrosis and hypertrophy.

Hessel and co-workers investigated the effect of CRT on tissue matrix metalloproteinases, with particular attention to MMP-9.(181) This specific matrix metalloproteinase is felt to govern apoptosis and previously been found to be expressed in high levels in pathological disease states e.g. abdominal aortic aneurysms.

A series of 64 patients was followed up for six months and 72% of the cohort had LV remodelling. (>10 percent LVESV) This group had a significant reduction in circulating levels of MMP-9 (P=<0.01) and hence the suggestion is that reverse remodelling may be associated with an increased rate of extracellular matrix turnover.

Beyond the effect of specific pathways, CRT’s effects have also been investigated on intracellular signalling.

It has been previously observed by Spragg and colleagues in 2003, that there was an imbalance between the expression of selective cardiac handling proteins and mitogen

activated protein kinase.(182) The same group investigated this further in 2008, with the lead author being Chakir.(183) In the context of dyssynchronous contraction represented by LBBB on the surface electrocardiogram, the lateral wall showed an increase in p38 MAPK and Ca2+- Calmodulin Kinase II activation and increased TNF-alpha which were both reversed with CRT.

Electrically myocytes when isolated from failing hearts suffer from prolonged action potentials.(184) This has been noted particularly in the lateral wall of the left ventricle, irrespective of aetiology.(185) CRT may shorten action potential duration in the lateral wall and therefore diminish the effect of prolonged action potentials in the lateral wall.

Another important mechanism noted in failing hearts is the abnormalities associated with calcium handling. Myocytes isolated from patients with heart failure demonstrate delays in the movement of intracellular calcium and altered intracellular kinetics. Beat to beat variation in calcium handling is influenced by both intracellular channels (L type Ca2+ _{currents) and} large scale Ca 2+ release through the sarcoplasmic reticulum by the phospholamban regulated SR Ca2+- ATPase. (SERCA 2A) CRT may restore normality to intracellular calcium

movement and had some effect on regional variation.(186) However like most of the molecular field allied to the effects of CRT, the data is limited and the effect is not wholly understood nor characterised.

The effect on beta adrenoceptors has been discussed above, however beyond the cellular surface, abnormalities of G protein signalling allied to such receptors has also been

documented.(187, 188) Analysis of adenylyl cyclase activity (cAMP) shows a reduction in patients with heart failure, CRT has been shown to upregulate cAMP levels.(186) Additional effects on G protein signalling have also been demonstrated with patients with CRT implants. G-proteins are inhibitory on the effects of beta adrenoceptor signalling. Pertussis toxin is inhibitory of G protein signalling, but when administered to myocytes following the application of CRT, had no effect.

The molecular, sub cellular and signalling fields with respect to CRT have been historically overlooked, but more attention is being paid to these mechanisms which underpin the

macroscopic processes observed in some patients following CRT implantation. Further work is underway and much needed both in vivo studies and ex vivo with robust animal models which replicate human physiology. With the data thus far which has been published, it is clear that CRT does influence certain cellular pathways and expression of proteins.