Robert A. Rose

Bone Marrow‐Derived Mesenchymal Stromal Cells Express Cardiac‐Specific Markers, Retain the Stromal Phenotype, and Do Not Become Functional Cardiomyocytes In Vitro

Although bone marrow‐derived mesenchymal stromal cells (MSCs) may be beneficial in treating heart disease, their ability to transdifferentiate into functional cardiomyocytes remains unclear. Here, bone marrow‐derived MSCs from adult female transgenic mice expressing green fluorescent protein (GFP) under the control of the cardiac‐specific α‐myosin heavy chain promoter were cocultured with male rat embryonic cardiomyocytes (rCMs) for 5–15 days. After 5 days in coculture, 6.3% of MSCs became GFP+ and stained positively for the sarcomeric proteins troponin I and α‐actinin. The mRNA expression for selected cardiac‐specific genes (atrial natriuretic factor, Nkx2.5, and α‐cardiac actin) in MSCs peaked after 5 days in coculture and declined thereafter. Despite clear evidence for the expression of cardiac genes, GFP+ MSCs did not generate action potentials or display ionic currents typical of cardiomyocytes, suggesting retention of a stromal cell phenotype. Detailed immunophenotyping of GFP+ MSCs demonstrated expression of all antigens used to characterize MSCs, as well as the acquisition of additional markers of cardiomyocytes with the phenotype CD45−‐CD34+‐CD73+‐CD105+‐CD90+‐CD44+‐SDF1+‐CD134L+‐collagen type IV+‐vimentin+‐troponin T+‐troponin I+‐α‐actinin+‐connexin 43+. Although cell fusion between rCMs and MSCs was detectable, the very low frequency (0.7%) could not account for the phenotype of the GFP+ MSCs. In conclusion, we have identified an MSC population displaying plasticity toward the cardiomyocyte lineage while retaining mesenchymal stromal cell properties, including a nonexcitable electrophysiological phenotype. The demonstration of an MSC population coexpressing cardiac and stromal cell markers may explain conflicting results in the literature and indicates the need to better understand the effects of MSCs on myocardial injury.

RGS4 Regulates Parasympathetic Signaling and Heart Rate Control in the Sinoatrial Node

Heart rate is controlled by the opposing activities of sympathetic and parasympathetic inputs to pacemaker myocytes in the sinoatrial node (SAN). Parasympathetic activity on nodal myocytes is mediated by acetylcholine-dependent stimulation of M2 muscarinic receptors and activation of G&agr;i/o signaling. Although regulators of G protein signaling (RGS) proteins are potent inhibitors of G&agr;i/o signaling in many tissues, the RGS protein(s) that regulate parasympathetic tone in the SAN are unknown. Our results demonstrate that RGS4 mRNA levels are higher in the SAN compared to right atrium. Conscious freely moving RGS4-null mice showed increased bradycardic responses to parasympathetic agonists compared to wild-type animals. Moreover, anesthetized RGS4-null mice had lower baseline heart rates and greater heart rate increases following atropine administration. Retrograde-perfused hearts from RGS4-null mice showed enhanced negative chronotropic responses to carbachol, whereas SAN myocytes showed greater sensitivity to carbachol-mediated reduction in the action potential firing rate. Finally, RGS4-null SAN cells showed decreased levels of G protein–coupled inward rectifying potassium (GIRK) channel desensitization and altered modulation of acetylcholine-sensitive potassium current (IKACh) kinetics following carbachol stimulation. Taken together, our studies establish that RGS4 plays an important role in regulating sinus rhythm by inhibiting parasympathetic signaling and IKACh activity.

Iroquois homeobox gene 3 establishes fast conduction in the cardiac His–Purkinje network

Rapid electrical conduction in the His–Purkinje system tightly controls spatiotemporal activation of the ventricles. Although recent work has shed much light on the regulation of early specification and morphogenesis of the His–Purkinje system, less is known about how transcriptional regulation establishes impulse conduction properties of the constituent cells. Here we show that Iroquois homeobox gene 3 (Irx3) is critical for efficient conduction in this specialized tissue by antithetically regulating two gap junction–forming connexins (Cxs). Loss of Irx3 resulted in disruption of the rapid coordinated spread of ventricular excitation, reduced levels of Cx40, and ectopic Cx43 expression in the proximal bundle branches. Irx3 directly represses Cx43 transcription and indirectly activates Cx40 transcription. Our results reveal a critical role for Irx3 in the precise regulation of intercellular gap junction coupling and impulse propagation in the heart.

Iron overload decreases CaV1.3-dependent L-type Ca2+ currents leading to bradycardia, altered electrical conduction, and atrial fibrillation.

Background— Chronic iron overload (CIO) is associated with blood disorders such as thalassemias and hemochromatosis. A major prognostic indicator of survival in patients with CIO is iron-mediated cardiomyopathy characterized by contractile dysfunction and electrical disturbances, including slow heart rate (bradycardia) and heart block. Methods and Results— We used a mouse model of CIO to investigate the effects of iron on sinoatrial node (SAN) function. As in humans, CIO reduced heart rate (≈20%) in conscious mice as well as in anesthetized mice with autonomic nervous system blockade and in isolated Langendorff-perfused mouse hearts, suggesting that bradycardia originates from altered intrinsic SAN pacemaker function. Indeed, spontaneous action potential frequencies in SAN myocytes with CIO were reduced in association with decreased L-type Ca2+ current (ICa,L) densities and positive (rightward) voltage shifts in ICa,L activation. Pacemaker current (If) was not affected by CIO. Because ICa,L in SAN myocytes (as well as in atrial and conducting system myocytes) activates at relatively negative potentials due to the presence of CaV1.3 channels (in addition to CaV1.2 channels), our data suggest that elevated iron preferentially suppresses CaV1.3 channel function. Consistent with this suggestion, CIO reduced CaV1.3 mRNA levels by ≈40% in atrial tissue (containing SAN) and did not lower heart rate in CaV1.3 knockout mice. CIO also induced PR-interval prolongation, heart block, and atrial fibrillation, conditions also seen in CaV1.3 knockout mice. Conclusions— Our results demonstrate that CIO selectively reduces CaV1.3-mediated ICa,L, leading to bradycardia, slowing of electrical conduction, and atrial fibrillation as seen in patients with iron overload.

Ca(2+) entry through TRP-C channels regulates fibroblast biology in chronic atrial fibrillation.

In this issue of Circulation , Harada et al1 provide fundamental new insights into the cellular mechanism(s) for initiation and maintenance of chronic atrial fibrillation in the human heart. The authors, taking what most might still consider to be an unconventional approach to understanding this proarrhythmic substrate,2,3,4 have identified the atrial fibroblast as an important player. More specifically, this international group of investigators concludes that a particular member of the transient receptor potential or TRP family5 of ion channels, TRPC3, when expressed/upregulated in human atrial fibroblasts, can contribute to chronic atrial fibrillation. Activation or enhanced expression of TRPC3 provides a means for increased transmembrane calcium entry into the fibroblast. This trigger calcium can then result in a marked increase in proliferation, followed by transformation to the myofibroblast phenotype.6,7 A previous study had drawn attention to the possibility that a different TRP channel subtype, TRPM7, could play a somewhat similar proarrhythmic role in the atrium.7,8 Article see p 2051 Atrial fibrillation is the most common form of cardiac arrhythmia in adult humans.9 Importantly, its incidence is projected to increase substantially as a consequence of the association of atrial fibrillation with healthy aging,10 diabetes mellitus, and hypothyroidism. Harada et al1 provide the first evidence for the presence of TRPC3 current during the proliferative phase in cultured human atrial fibroblasts. Knockdown of TRPC3 is able to suppress atrial fibroblast proliferation, and similar results were obtained with the pharmacological inhibitor PYR3, a relatively new pyrazole-based compound. This particular TRP channel exhibits significant calcium permeability. This calcium influx contributes to ERK phosphorylation, which is involved in mediating atrial fibroblast proliferation. A very interesting observation …

Distinct patterns of atrial electrical and structural remodeling in angiotensin II mediated atrial fibrillation

Atrial fibrillation (AF) is prevalent in hypertension and elevated angiotensin II (Ang II); however, the mechanisms by which Ang II leads to AF are poorly understood. Here, we investigated the basis for this in mice treated with Ang II or saline for 3 weeks. Ang II treatment increased susceptibility to AF compared to saline controls in association with increases in P wave duration and atrial effective refractory period, as well as reductions in right and left atrial conduction velocity. Patch-clamp studies demonstrate that action potential (AP) duration was prolonged in right atrial myocytes from Ang II treated mice in association with a reduction in repolarizing K+ currents. In contrast, APs in left atrial myocytes from Ang II treated mice showed reductions in upstroke velocity and overshoot, as well as greater prolongations in AP duration. Ang II reduced Na+ current (INa) in the left, but not the right atrium. This reduction in INa was reversible following inhibition of protein kinase C (PKC) and PKCα expression was increased selectively in the left atrium in Ang II treated mice. The transient outward K+ current (Ito) showed larger reductions in the left atrium in association with a shift in the voltage dependence of activation. Finally, Ang II caused fibrosis throughout the atria in association with changes in collagen expression and regulators of the extracellular matrix. This study demonstrates that hypertension and elevated Ang II cause distinct patterns of electrical and structural remodeling in the right and left atria that collectively create a substrate for AF.