Background Hypokalemia is known to promote ventricular arrhythmias, especially in combination with Class III antiarrhythmic drugs like dofetilide. Here we evaluated the underlying molecular mechanisms. Methods and Results Arrhythmias were recorded in isolated rabbit and rat hearts or patch-clamped ventricular myocytes exposed to hypokalemia (1.0-3.5 mmol/l) in the absence or presence of dofetilide (1 μmol/l). Spontaneous early afterdepolarizations (EADs) and ventricular tachycardia/fibrillation (VF/VF) occurred in 50% of hearts at 2.7 mmol/l [K] in the absence of dofetilide, and 3.3 mmol/l [K] in its presence. Pre-treatment with the CaMKII inhibitor KN-93, but not its inactive analogue KN-92, abolished EADs and hypokalemia-induced VT/VF, as did the selective late Na current (INa) blocker GS-967. In intact hearts, moderate hypokalemia (2.7 mmol/l) significantly increased tissue CaMKII activity. Computer modeling revealed that EAD generation by hypokalemia (with or without dofetilide) required Na-K pump inhibition to induce intracellular Na and Ca overload with consequent CaMKII activation enhancing late INa and the L-type Ca current. K current suppression by hypokalemia and/or dofetilide alone in the absence of CaMKII activation were ineffective at causing EADs. Conclusions We conclude that Na-K pump inhibition by even moderate hypokalemia plays a critical role in promoting EAD-mediated arrhythmias by inducing a positive feedback cycle activating CaMKII and enhancing late INa. Class III antiarrhythmic drugs like dofetilide sensitize the heart to this positive feedback loop.
Cardiovascular disease is a leading cause of mortality in the world and is exacerbated by the presence of cardiac fibrosis, defined by the accumulation of non-contractile extracellular matrix proteins. Cardiac fibrosis is directly linked to cardiac dysfunction and increased risk of arrhythmia. Despite its prevalence, there is a lack of efficacious therapies for inhibiting or reversing cardiac fibrosis, largely due to the complexity of the cell types and signaling pathways involved. Ongoing research has aimed to understand the mechanisms of cardiac fibrosis and develop new therapies for treating scar formation. Major approaches include preventing the formation of scar tissue and replacing fibrous tissue with functional cardiomyocytes. While targeting the renin-angiotensinaldosterone system is currently used as the standard line of therapy for heart failure, there has been increased interest in inhibiting the transforming growth factor-β signaling pathway due its established role in cardiac fibrosis. Significant advances in cell transplantation therapy and biomaterials engineering have also demonstrated potential in regenerating the myocardium. Novel techniques, such as cellular direct reprogramming, and molecular targets, such as non-coding RNAs and epigenetic modifiers, are uncovering novel therapeutic options targeting fibrosis. This review provides an overview of current approaches and discuss future directions for treating cardiac fibrosis.
While recent advances clarified the molecular and cellular modes of action of antiarrhythmic drugs (AADs), their link to suppression of dynamical arrhythmia mechanisms remains only partially understood. The current classifications of AADs (Classes I, III, and IV) rely on blocking peak Na, K and L-type calcium currents (ICa,L), with Class II with dominant beta receptor blocking activity and Class V including drugs with diverse classes of actions. The discovery that the calcium and redox sensor, cardiac Ca/calmodulin-dependent protein kinase II (CaMKII) enhances both the late Na (INa-L) and the late ICa,L in patients at high risk of VT/VF provided a new and a rational AAD target. Pathological rise of either or both of INa-L and late ICa,L are demonstrated to promote cellular early afterdepolarizations (EADs) and EAD-mediated triggered activity that can initiate VT/VF in remodeled hearts. Selective inhibition of the INa-L without affecting their peak transients with the highly specific prototype drug, GS-967 suppresses these EAD-mediated VT/VFs. As in the case of INa-L, selective inhibition of the late ICa,L without affecting its peak with the prototype drug, roscovitine suppressed oxidative EAD-mediated VT/VF. These findings indicate that specific blockers of the late inward currents without affecting their peaks (gating modifiers), offer a new and effective AAD class action i.e., “Class VI.” The development of safe drugs with selective Class VI actions provides a rational and effective approach to treat VT/VF particularly in cardiac conditions associated with enhanced CaMKII activity such as heart failure.
Background Enhanced late inward Na current (INa-L) modulates action potential duration (APD) and plays a key role in the genesis of early and delayed afterdepolarizations (EADs & DADs) and triggered activity. Objectives To define the influence of selective block of the INa-L on EAD- & DAD-mediated triggered ventricular tachycardia and fibrillation (VT & VF) in intact hearts using (GS967), a selective and potent (IC50=0.13±0.01 μM) blocker of the INa-L. Methods VT/VF were induced either by local aconitine injection (50 μg) in the LV muscle of adult (3–4 months) male rats (N=21) or by arterial perfusion of 0.1 mM H2O2 in aged male rats (24–26 months, N=16). The LV epicardial surface of the isolated-perfused hearts was optically mapped using fluorescent voltage-sensitive dye, and microelectrode recordings of APs were made adjacent to the aconitine injection site. The suppressive and preventive effects of GS967 (1 μM) against EAD/DAD-mediated VT/VF were then determined. Results Aconitine induced VT in all 13 hearts studied. Activation map (N=6) showed that the VT was initiated by a focal activity arising from the aconitine injection site (CLs of 84±12) that degenerated to VF (CL=52±8 ms) within a few seconds. VF was maintained by multifocal activity with occasional incomplete reentrant wavefronts. Administration of GS967 suppressed the VT/VF in 10 out of 13 hearts (P<0.001). Pre-exposure to GS967 for 15 min prior to aconitine injection prevented the initiation of VT/VF in 5 of 8 additional hearts (P<0.02). The VF reoccurred within 10 min upon washout of GS967. Microelectrode recordings (N=7) showed that the VT/VF was initiated by EAD- and DAD-mediated triggered activity at CL of 86±14ms (NS from VT CL) that preceded the VF. GS967 shortened the AP duration (APD), flattened the slope of the dynamic APD restitution curve and reduced APD dispersion from 42±12 ms to 8±3 ms (P<0.01). H2O2 perfusion in 8 fibrotic aged hearts promoted EAD-mediated focal VT/VF, which was suppressed by GS967 in 5 hearts (P<0.02). Conclusions The selective INa-L blocker GS967 effectively suppresses and prevents aconitine and oxidative stress-induced EADs, DADs and focal VT/VF. The suppression of EADs, DADs and reduction of APD dispersion make GS967 a potentially useful antiarrhythmic drug in conditions of enhanced INa-L.
Key pointsr Hypertension is a risk factor for sudden cardiac death caused by ventricular tachycardia and fibrillation.r Whether hypertension in its early stage is associated with an increased risk of ventricular tachyarrhythmias is not known.r Based on experiments performed at the cellular and whole heart levels, we show that, even early in chronic hypertension, the hypertrophied and fibrotic ventricles of spontaneously hypertensive rats aged 5 to 6 months have already developed increased stress-induced arrhythmogenicity, and this increased susceptibility to ventricular arrhythmias is primarily a result of tissue remodelling rather than cellular electrophysiological changes.r Our findings highlight the need for early hypertension treatment to minimize myocardial fibrosis, ventricular hypertrophy, and arrhythmias.Abstract Hypertension is a risk factor for sudden cardiac death caused by ventricular tachycardia and fibrillation (VT/VF). We hypothesized that, in early hypertension, the susceptibility to stress-induced VT/VF increases. We compared the susceptibility of 5-to 6-month-old male spontaneously hypertensive rats (SHR) and age/sex-matched normotensive rats (NR) to VT/VF during challenge with oxidative stress (H 2 O 2 ; 0.15 mmol l −1 ). We found that only SHR hearts exhibited left ventricular fibrosis and hypertrophy. H 2 O 2 promoted VT in all 30 SHR but none of the NR hearts. In 33% of SHR cases, focal VT degenerated to VF within 3 s. Simultaneous voltage-calcium optical mapping of Langendorff-perfused SHR hearts revealed that H 2 O 2 -induced VT/VF arose spontaneously from focal activations at the base and mid left ventricular epicardium. Microelectrode recording of SHR hearts showed that VT was initiated by early afterdepolarization (EAD)-mediated triggered activity. However, despite the increased susceptibility of SHR hearts to VT/VF, patch clamped isolated SHR ventricular myocytes developed EADs and triggered activity to the same extent as NR ventricular myocytes, except with larger EAD amplitude. During the early stages of hypertension, when challenged with oxidative stress, SHR hearts showed an increased ventricular arrhythmogenicity that stems primarily from tissue remodelling (hypertrophy, fibrosis) rather than cellular electrophysiological changes. Our findings highlight the need for early hypertension treatment to minimize myocardial fibrosis, ventricular hypertrophy, and arrhythmias. NR, normotensive rats; PA peak vel, pulmonary artery peak velocity; (P)CL, (pacing) cycle length; PW, posterior wall; P-ECG, pseudo-electrocardiogram; RV, right ventricle; RWT, relative wall thickness; SHR, spontaneously hypertensive rats; SHHF, spontaneously hypertensive heart failure; SBP, systolic blood pressure; VT/VF, ventricular tachycardia and fibrillation.
Aims We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart-field specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations. Methods and Results Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart-field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enables purification of cardiomyocytes and first heart field cardiomyocytes, respectively. Conclusions We describe a high yield approach for isolation and characterization of human embryonic stem cell-derived heart field specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modeling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart or chamber-specific congenital heart defects. TRANSLATIONAL PERSPECTIVE Myocardial infarction leads to irreversible loss of cardiomyocytes and eventually heart failure. Human embryonic stem cells (hESCs) can be differentiated to cardiomyocytes and are considered a potential source of cell therapy for cardiac regeneration. However, current differentiation strategies yield a mixture of cardiomyocyte subtypes and safety concerns stemming from the use of a heterogenous population of cardiomyocytes have hindered its application. Here, we report generation of enriched heart field-specific cardiomyocytes using a hESC double reporter. Our study facilitates investigating early human cardiogenesis in vitro and generating chamber-specific cardiomyocytes to treat diseases that affect specific regions of the heart.
Ventricular cardiac arrhythmia (VA) arises in acquired or congenital heart disease. Long QT syndrome type-3 (LQT3) is a congenital form of VA caused by cardiac sodium channel (I NaL ) SCN5A mutations that prolongs cardiac action potential (AP) and enhances I NaL current. Mexiletine inhibits I NaL and shortens the QT interval in LQT3 patients. Above therapeutic doses, mexiletine prolongs the cardiac AP. We explored structure−activity relationships (SAR) for AP shortening and prolongation using dynamic medicinal chemistry and AP kinetics in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using patient-derived LQT3 and healthy hiPSC-CMs, we resolved distinct SAR for AP shortening and prolongation effects in mexiletine analogues and synthesized new analogues with enhanced potency and selectivity for I NaL . This resulted in compounds with decreased AP prolongation effects, increased metabolic stability, increased I NaL selectivity, and decreased avidity for the potassium channel. This study highlights using hiPSC-CMs to guide medicinal chemistry and "drug development in a dish".
Ventricular arrhythmias, a leading cause of sudden cardiac death, can be triggered by cardiomyocyte early afterdepolarizations (EADs). EADs can result from an abnormal late activation of L-type Ca2+ channels (LTCCs). Current LTCC blockers (class IV antiarrhythmics), while effective at suppressing EADs, block both early and late components of ICa,L, compromising inotropy. However, computational studies have recently demonstrated that selective reduction of late ICa,L (Ca2+ influx during late phases of the action potential) is sufficient to potently suppress EADs, suggesting that effective antiarrhythmic action can be achieved without blocking the early peak ICa,L, which is essential for proper excitation–contraction coupling. We tested this new strategy using a purine analogue, roscovitine, which reduces late ICa,L with minimal effect on peak current. Scaling our investigation from a human CaV1.2 channel clone to rabbit ventricular myocytes and rat and rabbit perfused hearts, we demonstrate that (1) roscovitine selectively reduces ICa,L noninactivating component in a human CaV1.2 channel clone and in ventricular myocytes native current, (2) the pharmacological reduction of late ICa,L suppresses EADs and EATs (early after Ca2+ transients) induced by oxidative stress and hypokalemia in isolated myocytes, largely preserving cell shortening and normal Ca2+ transient, and (3) late ICa,L reduction prevents/suppresses ventricular tachycardia/fibrillation in ex vivo rabbit and rat hearts subjected to hypokalemia and/or oxidative stress. These results support the value of an antiarrhythmic strategy based on the selective reduction of late ICa,L to suppress EAD-mediated arrhythmias. Antiarrhythmic therapies based on this idea would modify the gating properties of CaV1.2 channels rather than blocking their pore, largely preserving contractility.
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