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	Figure 1. Interactions of pro‐arrhythmic substrates, triggers, and facilitating factors leading to the initiation and persistence of arrhythmias. Heart disease induces cardiac electrical and structural remodeling. Structural remodeling consists of overt changes at organ level (chamber dilatation), tissue or cellular level (hypertrophy, fibrosis, inflammatory infiltration), and molecular level (altered gene profile including genes coding for ion channels and Ca2+ regulatory molecules). In this setting, arrhythmias may be triggered by factors such as enhanced cardiac tone of the sympathetic nervous system (SNS), acute hemodynamic stress, tissue injury, inflammation, or ionic imbalance. The risk of arrhythmias increases in the presence of facilitating factors such as advanced age, comorbidity particularly diseases associated with inflammatory conditions, dietary factors such as alcohol consumption, and lifestyle such as sleep apnea or psychological stress. Facilitating factors could exacerbate cardiac fibrosis, lower electrical stability or amplify the intensity of pro‐arrhythmic triggers. Note that abnormalities in ion channels and intracellular Ca2+ homeostasis, whilst important in arrhythmogenesis, are not the focus of this review.
	Figure 2. Mechanisms of pro‐arrhythmic action of myocardial fibrosis. (A) Interstitial fibrosis interferes with cardiomyocyte electrical coupling leading to slow and heterogeneous propagation of action membrane potential. (B) Patchy fibrosis together with interstitial fibrosis results in heterogeneous conduction delay, a condition in favor of reentrant cycles around the fibrotic scar. (C) Heterogeneous electrical coupling between cardiomyocytes and myofibroblasts, which is the consequence of expression by myofibroblasts of proteins for gap junctions (e.g., connexins) and ion channels. Note that due to profound differences in electrophysiological properties, such as a much higher resting membrane potential relative to that of cardiomyocytes, at any phase of a cardiac cycle, there exists a membrane potential gradient resulting in electrical currents (arrows) between the two types of cells.
	Figure 3. Central role of fibrosis in cardiac remodeling, dysfunction and arrhythmogenesis. There is strong evidence for fibrosis as a prominent substrate of arrhythmogenesis. Fibrosis also promotes cardiac remodeling and dysfunction that further increase the risk of arrhythmias. Fibroblasts are able to promote hypertrophic growth and inflammatory signaling in the heart via a paracrine mechanism by secreting a number of growth factors, cytokines, or chemokines. Conversely, profibrotic signaling molecules generated from hypertrophic cardiomyocytes and inflammatory cells would exacerbate fibrosis, and as discussed in this review, cardiomyocyte hypertrophy and inflammation per se can directly increase the risk of arrhythmias. Accumulation of excessive amount of extracellular proteins renders the myocardium with increased stiffness and impairs cardiac performance at diastole and systole (i.e., heart failure with preserved or reduced ejection fraction, HFpEF, HFrEF, respectively). The consequent chamber dilatation further leads to electrical instability. There is strong evidence that arrhythmia per se exacerbated all these changes.
	Figure 4. Interplay of fibrosis and inflammation in the progression of heart disease. There exists a dynamic and orchestrated interplay between inflammatory and fibrotic processes in heart disease irrespective of etiologies. (A) Myocardial injury as a consequence of ischemia, hypoxia, and or drug cardiotoxicity is potent in triggering innate immune responses followed by fibrotic healing. Inflammatory response per se may also exacerbate cardiomyocyte death under these conditions. (B) Inflammatory stimuli, such as myocarditis, evoke fibrotic process. (C) Diseases such as hypertension, valvular disease, aldosteronism and activation of the sympathetic nervous system (SNS) can directly stimulate fibrotic as well as inflammatory signaling.
	Figure 5. Inflammatory signaling promotes structural and electrical remodeling thereby facilitating arrhythmogenesis. In addition to heart disease, systemic, and regional inflammation may be present due to conditions such as comorbidity and infection. Cardiac inflammatory signaling contributes to electrical remodeling by interfering with ionic channel function and structural remodeling by promoting fibrosis. Inflammatory conditions are characterized by elevation of a number of inflammatory cytokines and other inflammatory molecules, which directly influence operation of ion channels, proteins that regulate intracellular Ca2+ and intercellular gap junctions. On the other hand, inflammatory cytokines activate fibroblasts to differentiate into myofibroblasts resulting in enhanced collagen synthesis and fibrosis, which create potential substrates for development and maintenance of arrhythmias.
	Figure 6. Changes in gap‐junctions in arrhythmic and fibrotic hearts. Schematic showing gap‐junction formed from 12 connexin proteins, of them 6 from each connecting cell. Gap‐junctions connect the cytoplasma of two cells forming a direct pass for ions and molecules (green dots) between cells in a regulated manner (upper panel). Schematic depicting cardiomyocyte‐to‐cardiomyocyte connections in normal ventricular myocardium, with connexin‐43 primarily localized at the intercalated discs (middle panel). Schematic of diseased ventricular myocardium showing interstitial and patch fibrosis interrupting the normal architecture, reduced gap‐junctions together with heterogeneous distribution and lateralization, and the appearance of fibroblast‐to‐cardiomyocyte connections (lower panel).
	Figure 7. Correlation between the severity of interstitial fibrosis and ventricular tachyarrhythmias in mice. Data were obtained from male mice with cardiac‐restricted overexpression of human β2‐adrenergic receptors (β2‐AR TG). (A) β2‐AR TG mice exhibited frequent and spontaneous ventricular ectopic beats (VEB, ▾) and tachycardia (VT). In one instance, sustained VT was observed (lower panel). (B) Severity of VEB and VT was determined by telemetry ECG recording for a period of 24 hours in β2‐AR TG mice (n = 40) at different ages from 4 to 12 months. The collagen content in LV apical portion (approximately 20 mg) of each animal was determined by hydroxyproline assay. Modified with permission from (278), Figures 2 and 8.
	Figure 8. Effects of arrhythmia on in vivo hemodynamics. Simultaneous recordings of left ventricular pressure (LVP), aortic blood pressure (BP), and lead II surface ECG (upper panel), or BP and ECG (lower panel) in anesthetized transgenic mice with cardiac‐restricted overexpression of human β2‐adrenergic receptors (β2‐AR TG). Pressure recordings were made using a Millar pressure catheter positioned in the aorta (BP) or left ventricle (LVP). β2‐AR TG mice exhibit spontaneous onset of ventricular ectopic beats (VEB, ▾). There are clear disturbances in the LV pressure and BP due to the arrhythmia, with reduced pressure development or complete absence of a pressure pulse if the premature beat(s) occurred during the refractory period of the previous beat. Time bar = 0.5 s (unpublished observations).
	Figure 9. Presence of patchy fibrosis determined by late gadolinium enhancement (LGE) in cardiac magnetic resonance (CMR) predicts long‐term prognosis in patients with dilated cardiomyopathy. CMR‐LGE images and cardiac histology from a patient with left ventricular midwall fibrosis who experienced sudden cardiac death and a patient without midwall fibrosis who underwent cardiac transplantation (A). Arrows indicate LGE and patchy fibrosis. Collagen was stained red by picrosirus red. Kaplan‐Meier survival analysis of event‐free survival of cardiovascular death or heart transplantation (B) and sudden cardiac death (C) in patients grouped by CMR‐LGE determined presence (n = 330) or absence (n = 142) of midwall fibrosis. Figures are adopted with permission from (132), Figures 2 and 3.
	Figure 10. Dual mechanisms of mineralocorticoid receptor (MR) antagonists for their anti‐arrhythmic property in relation to antifibrotic effect. MRs are expressed in cardiomyocytes and fibroblasts. Activation of cardiomyocyte MR directly provokes arrhythmias while fibroblast MRs mediate fibrotic signaling. Blockade of MR by antagonists in both cell‐types is expected to directly and indirectly lower the arrhythmic vulnerability.
	Figure 11. Pivotal questions remain on the pro‐arrhythmic role of myocardial fibrosis with mechanistic, diagnostic, and therapeutic significance.

Teaching Material

M. -N. Nguyen, H. Kiriazis, X. -M. Gao, X. -J. Du. Cardiac Fibrosis and Arrhythmogenesis. Compr Physiol 7 2017, 1009-1049.

Cardiac fibrosis constitutes a prominent substrate for arrhythmogenesis, which has been under intensive investigation. The information in this review article will help with the teaching of the following specific topics at the graduate or advanced undergraduate level:

 

• Cardiac remodeling and fibrosis

 

 

• Mechanisms by which cardiac fibrosis contributes to the development of arrhythmias

 

 

• Relationship between fibrosis and severity of arrhythmias

 

 

• Interplay of inflammation and fibrosis leading to arrhythmias

 

 

• Gap-junction remodeling in relation to arrhythmogenesis and fibrosis

 

 

• Animal models of spontaneous arrhythmias with significant cardiac remodeling and fibrosis

 

 

• Clinical stratification of risk of fatal arrhythmias based on characterization of cardiac fibrosis by means of cardiac imaging or fibrotic biomarkers

 

 

• Antiarrhythmic efficacy by antifibrotic therapies

 

Figure 1. Teaching points: Understanding involvement of multiple factors in the development and persistence of arrhythmias. In the setting of heart disease, onset of arrhythmias relies not only on the presence of electrophysiological abnormalities (electrical remodeling), but structural abnormalities, in particular fibrosis and chamber dilatation (structural remodeling). In addition, the probability of triggering arrhythmia is higher in the presence of factors such as enhanced activity of the sympathetic nervous system (SNS) and acute hemodynamic stress. The risk of arrhythmogenesis increases in the presence of facilitating factors such as advanced age, comorbidity associated with inflammatory conditions and lifestyle, such as obstructive sleep apnea or psychological stress, which exacerbate cardiac fibrosis, reduce electrical stability while augmenting the intensity of pro-arrhythmic triggers.

Figure 2. Teaching points: Understanding how fibrosis is pro-arrhythmic. Currently three mechanisms are regarded as being important. Firstly, interstitial fibrosis interferes with electrical coupling among cardiomyocytes leading to slow and heterogeneous propagation of action membrane potential; Secondly, interstitial and patchy fibrosis lead to conduction delay and formation of re-entrant cycles; third, electrical coupling can establish between cardiomyocytes and myofibroblasts. However, due to profound differences in the electrophysiological properties of these two types of cells, there exists a gradient of membrane potential and electrical currents (arrows) between myofibroblasts and cardiomyocytes leading to arrhythmias.

Figure 3. Teaching points: As illustrated in Figure 2, fibrosis is able to directly induce arrhythmias. In the diseased heart, fibrosis also interacts with other proarrhythmic factors at the cellular (cardiomyocyte hypertrophy, inflammation) and organ (cardiac enlargement and dysfunction) levels that further increase the risk of arrhythmias. Arrhythmia per se could also exacerbate fibrosis, inflammation, chamber dilatation, and dysfunction, forming a vicious cycle.

Figure 4. Teaching points: Understanding the close coupling and orchestrated interactions between inflammatory and fibrotic processes over time in heart disease irrespective of etiology.

Figure 5. Teaching points: This is a follow-up to Figure 4 showing the interaction and effects of inflammation and fibrosis on arrhythmogenesis. Systemic and regional inflammatory signaling contributes to electrical remodeling by interfering with the operation and/or gene transcription of ionic channels, Ca²⁺ regulatory proteins, or gap-junction proteins, and structural remodeling by promoting fibrosis. Illustrated in the diagram are some inflammatory molecules that are known to interfere with normal operation. Meanwhile, inflammation per se is known to activate fibroblasts and to stimulate fibrosis, thereby promoting arrhythmogenesis.

Figure 6. Teaching points: Understanding the quantitative and qualitative changes in gap junctions in diseased myocardium in relation to electrical instability. Cardiomyocyte electrical coupling is primarily achieved by the structure of gap junctions, which are formed by hexamers of connexins. In the normal myocardium, gap junctions are primarily localized at the intercalated discs. In the diseased setting, interstitial and patchy fibrosis interrupts the architecture of gap junctions between cardiomyocytes. Further, gap junctions undergo heterogeneous distribution and lateralization, and may be presented between myofibroblasts and cardiomyocytes mediating heterocellular coupling.

Figure 7. Teaching points: This diagram provides evidence for the quantitative relationship between the severity of cardiac fibrosis and the frequency of both ventricular ectopic beats (VEBs) and tachycardia (VT). This set of data were derived from a total of 40 mice with fibrotic cardiomyopathy and spontaneous onset of arrhythmias. In each animal, fibrosis was determined by hydroxyproline assay and ventricular tachyarrhythmias were monitored by telemetry ECG recording for a period of 24 h and quantified.

Figure 8. Teaching points: Understanding hemodynamic disturbances caused by arrhythmias, even ventricular ectopic beats (VEB), on cardiac performance. As illustrated, premature ectopic beats, either in single or salvo, resulted in insufficient diastolic filling with reduced or absence of blood pumping during the systole, as measured by marked reduction in left ventricular pressure (LVP) and blood pressure (BP).

Figure 9. Teaching points: In this clinical report, Gulati et al. studied patients with dilated cardiomyopathy for the presence of patchy fibrosis, as detected by late-gadolinium enhancement (LGE) in cardiac magnetic resonance (CMR) imaging. They validated this imaging signal of cardiac fibrosis with heart tissue histology (left panels). These patients were followed up for a period of 8 years to monitor mortality due to cardiovascular reasons or in the form of sudden cardiac death (i.e., lethal arrhythmias). Their data clearly indicate a significantly poorer survival in those patients who had cardiac fibrosis detected by CMR-LGE than others without.

Figure 10. Teaching points: Understanding the potential dual mechanisms by which the class of mineralocorticoid receptor (MR) antagonists act on cardiomyocytes and fibroblasts to directly and indirectly reduce the risk of arrhythmias.

Figure 11. Teaching points: This is a summary of currently burning questions that are important to our understanding in the field of fibrosis-arrhythmia with significant mechanistic, diagnostic and therapeutic implications.