Physiologic, Pathologic, and Therapeutic Paracrine Modulation of Cardiac Excitation-Contraction Coupling

Abstract

Cardiac excitation-contraction coupling (ECC) is the orchestrated process of initial myocyte electrical excitation, which leads to calcium entry, intracellular trafficking, and subsequent sarcomere shortening and myofibrillar contraction. Neurohumoral β-adrenergic signaling is a well-established mediator of ECC; other signaling mechanisms, such as paracrine signaling, have also demonstrated significant impact on ECC but are less well understood. For example, resident heart endothelial cells are well-known physiological paracrine modulators of cardiac myocyte ECC mainly via NO and endothelin-1. Moreover, recent studies have demonstrated other resident noncardiomyocyte heart cells (eg, physiological fibroblasts and pathological myofibroblasts), and even experimental cardiotherapeutic cells (eg, mesenchymal stem cells) are also capable of altering cardiomyocyte ECC through paracrine mechanisms. In this review, we first focus on the paracrine-mediated effects of resident and therapeutic noncardiomyocytes on cardiomyocyte hypertrophy, electrophysiology, and calcium handling, each of which can modulate ECC, and then discuss the current knowledge about key paracrine factors and their underlying mechanisms of action. Next, we provide a case example demonstrating the promise of tissue-engineering approaches to study paracrine effects on tissue-level contractility. More specifically, we present new functional and molecular data on the effects of human adult cardiac fibroblast conditioned media on human engineered cardiac tissue contractility and ion channel gene expression that generally agrees with previous murine studies but also suggests possible species-specific differences. By contrast, paracrine secretions by human dermal fibroblasts had no discernible effect on human engineered cardiac tissue contractile function and gene expression. Finally, we discuss systems biology approaches to help identify key stem cell paracrine mediators of ECC and their associated mechanistic pathways. Such integration of tissue-engineering and systems biology methods shows promise to reveal novel insights into paracrine mediators of ECC and their underlying mechanisms of action, ultimately leading to improved cell-based therapies for patients with heart disease.

Keywords: cardiomyocytes; endothelial cells; fibroblasts; stem cells; systems biology; tissue engineering.

Figure 1. β-Adrenergic Signaling and Cardiomyocyte ECC

β–adrenergic receptor (β–AR) activation by binding of epinephrine/norepinephrine leads to the following signaling cascade: 1) activation of the guanosine triphosphate (GTP)-binding protein α; 2) stimulation of adenylyl cyclase (AC); and 3) a rise in cyclic adenosine monophosphate (cAMP). Increase in intracellular cAMP leads to protein kinase A (PKA) activation, which subsequently phosphorylates and thus increases ion flux through the L-type Ca²⁺ channel (LTCC) and ryanodine receptor (RyR). Additionally, PKA relieves phospholamban (PLB) inhibition on sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA), allowing for an increase in Ca²⁺ storage within the sarcoplasmic reticulum (SR). In normal physiology, SERCA removes approximately 70% of the activator cytosolic calcium (Ca²⁺) during diastole (inset). Lastly, PKA phosphorylates troponin I, leading to positive lusitropic effects via myofilament Ca²⁺ desensitization. Blue and red arrows denote calcium flow during systole and diastole, respectively.

Figure 2. Endothelial-Cardiomyocyte Interplay Through Paracrine Factors

Endothelial cell nitric oxide (NO) increases basal contractility via nitrosylation of L-type Ca²⁺ channel (LTCC) and ryanodine receptor (RyR2). NO attenuates β–adrenergic effects on cardiomyocyte ECC via cyclic guanosine monophosphate (cGMP)-dependent degradation of cyclic adenosine monophosphate (cAMP) and protein kinase G (PKG)-mediated decrease of LTCC activity. PKG also phosphorylates troponin I, leading to myofilament calcium desensitization and thus increased lusitropy. Endothelin-1, which mainly acts through the endothelin A (ET_A) receptor in ventricular cardiomyocytes, may increase calcium entry via protein kinase C (PKC)-mediated: 1) increase of LTCC activity; 2) indirect activation of sodium-calcium exchanger (NCX) reverse mode by increasing Na⁺-H⁺ exchanger activity; and 3) direct activation of NCX reverse (shown) and/or forward (not shown) mode. Endothelin-1 alters myofilament Ca²⁺ sensitivity via protein kinase C/D (PKC/D) phosphorylation of troponin I and myosin-binding protein C. Finally, endothelin-1 may increase calcium-induced calcium release via inositol trisphosphate (IP₃) activation of inositol trisphosphate receptor (IP₃R), which sensitizes RyR2 on the sarcoplasmic reticulum (SR). Green and red arrows denote activation and inhibition, respectively. Other non-standard abbreviations: phospholamban (PLB); sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA).

Figure 3. Human Engineered Cardiac Tissue Contractility Assay

(A) hECTs are created, cultured, and tested in a custom bioreactor with integrated force-sensing end-posts; as the tissue beats, deflections of the end-posts are tracked. Output contractile metrics include, but are not limited to, developed force (DF), maximum rates of contraction and relaxation (+/− dF/dt, respectively), and beat rate. (B) Confocal microscopy of hECTs labeled with cardiac troponin I (green) and DAPI (blue) displays cardiomyocytes with striated sarcomeres and regions of aligned myofibrils. Inset shows magnified view of registered sarcomeres. (C) hECT labeled with sarcoendoplasmic reticulum Ca²⁺-ATPase 2 (red) and DAPI (blue) shows sarcoplasmic reticulum structures distributed throughout the tissue. Scale bar = 40 μm.

Figure 4. Effects of hACF Conditioned Media on hECT Contractile Function

(A) Contractility assay shows hECT developed force during 0.5-Hz pacing (mean±SEM, n=4–6) at pre-treatment (day 5) and 5-days post-treatment with serum-free defined media (SFDM, control), hACF CdM, and HFF CdM. Daily measurements of (B) developed force and (C) beat rate during spontaneous contractions for each group (mean±SEM, n=3–4). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, comparison of time point relative to pre-treatment in same group; ^#p<0.05, comparison of given group relative to HFF CdM at same time point; †p<0.05, ††p<0.01, comparison of same group at two different time points. p-values from repeated measures obtained using ANOVA followed by Bonferroni’s multiple comparisons test.

Figure 5. Molecular Characterization of hECTs

hECTs from each group (n=3–4, mean±SEM) were snap-frozen for qRT-PCR on day 10, where expression of (A) cardiac-specific/calcium handling, (B) apoptotic, and (C) potassium/sodium channel genes were quantified. *p<0.05, **p<0.01; p-values from one-way ANOVA with Scheffe’s post-hoc test. Non-standard abbreviations: cardiac troponin T (cTnT); L-type calcium channel (LTCC); sarco/endoplasmic reticulum calcium-ATPase (SERCA2a); connexin-43 (Cx43); atrial natriuretic peptide (ANF); myosin heavy chain (MHC); caspase-3 (Casp3); caspase-9 (Casp9); B-cell lymphoma 2 (BCL2); BCL2-associated X protein (BAX); transient outward potassium current (Kv4.2); inward rectifier potassium current (Kir2.1); delayed rectifier potassium current (Kv11.1); sodium current (Nav1.5).