Rajan Sah

Regulation of Myocardial Contractility and Cell Size by Distinct PI3K-PTEN Signaling Pathways

The PTEN/PI3K signaling pathway regulates a vast array of fundamental cellular responses. We show that cardiomyocyte-specific inactivation of tumor suppressor PTEN results in hypertrophy, and unexpectedly, a dramatic decrease in cardiac contractility. Analysis of double-mutant mice revealed that the cardiac hypertrophy and the contractility defects could be genetically uncoupled. PI3Kalpha mediates the alteration in cell size while PI3Kgamma acts as a negative regulator of cardiac contractility. Mechanistically, PI3Kgamma inhibits cAMP production and hypercontractility can be reverted by blocking cAMP function. These data show that PTEN has an important in vivo role in cardiomyocyte hypertrophy and GPCR signaling and identify a function for the PTEN-PI3Kgamma pathway in the modulation of heart muscle contractility.

Regulation of cardiac excitation–contraction coupling by action potential repolarization: role of the transient outward potassium current (Ito)

The cardiac action potential (AP) is critical for initiating and coordinating myocyte contraction. In particular, the early repolarization period of the AP (phase 1) strongly influences the time course and magnitude of the whole‐cell intracellular Ca2+ transient by modulating trans‐sarcolemmal Ca2+ influx through L‐type Ca2+ channels (ICa,L) and Na‐Ca exchangers (ICa,NCX). The transient outward potassium current (Ito) has kinetic properties that make it especially effective in modulating the trajectory of phase 1 repolarization and thereby cardiac excitation‐contraction coupling (ECC). The magnitude of Ito varies greatly during cardiac development, between different regions of the heart, and is invariably reduced as a result of heart disease, leading to corresponding variations in ECC. In this article, we review evidence supporting a modulatory role of Ito in ECC through its influence on ICa,L, and possibly ICa,NCX. We also discuss differential effects of Ito on ECC between different species, between different regions of the heart and in heart disease.

TRPV4 Is a Regulator of Adipose Oxidative Metabolism, Inflammation, and Energy Homeostasis

PGC1α is a key transcriptional coregulator of oxidative metabolism and thermogenesis. Through a high-throughput chemical screen, we found that molecules antagonizing the TRPVs (transient receptor potential vanilloid), a family of ion channels, induced PGC1α expression in adipocytes. In particular, TRPV4 negatively regulated the expression of PGC1α, UCP1, and cellular respiration. Additionally, it potently controlled the expression of multiple proinflammatory genes involved in the development of insulin resistance. Mice with a null mutation for TRPV4 or wild-type mice treated with a TRPV4 antagonist showed elevated thermogenesis in adipose tissues and were protected from diet-induced obesity, adipose inflammation, and insulin resistance. This role of TRPV4 as a cell-autonomous mediator for both the thermogenic and proinflammatory programs in adipocytes could offer a target for treating obesity and related metabolic diseases.

Alterations in action potential profile enhance excitation-contraction coupling in rat cardiac myocytes

Action potential (AP) prolongation typically occurs in heart disease due to reductions in transient outward potassium currents (Ito), and is associated with increased Ca2+ transients. We investigated the underlying mechanisms responsible for enhanced Ca2+ transients in normal isolated rat ventricular myocytes in response to the AP changes that occur following myocardial infarction. Normal myocytes stimulated with a train of long post‐myocardial infarction (MI) APs showed a 2.2‐fold elevation of the peak Ca2+ transient and a 2.7‐fold augmentation of fractional cell shortening, relative to myocytes stimulated with a short control AP. The steady‐state Ca2+ load of the sarcoplasmic reticulum (SR) was increased 2.0‐fold when myocytes were stimulated with trains of long post‐MI APs (111 ± 21.6 μmol l−1) compared with short control APs (56 ± 7.2 μmol l−1). Under conditions of equal SR Ca2+ load, long post‐MI APs still resulted in a 1.7‐fold increase in peak [Ca2+]i and a 3.8‐fold increase in fractional cell shortening relative to short control APs, establishing that changes in the triggering of SR Ca2+ release are largely responsible for elevated Ca2+ transients following AP prolongation. Fractional SR Ca2+ release calculated from the measured SR Ca2+ load and the integrated SR Ca2+ fluxes was 24 ± 3 and 11 ± 2 % following post‐MI and control APs, respectively. The fractional release (FR) of Ca2+ from the SR divided by the integrated L‐type Ca2+ flux (FR/∫FCa,L) was increased 1.2‐fold by post‐MI APs compared with control APs. Similar increases in excitation‐contraction (E‐C) coupling gains were observed establishing enhanced E‐C coupling efficiency. Our findings demonstrate that AP prolongation alone can markedly enhance E‐C coupling in normal myocytes through increases in the L‐type Ca2+ current (ICa,L) trigger combined with modest enhancements in Ca2+ release efficiency. We propose that such changes in AP profile in diseased myocardium may contribute significantly to alterations in E‐C coupling independent of other biochemical or genetic changes.

Inhibition of Calcineurin and Sarcolemmal Ca2+ Influx Protects Cardiac Morphology and Ventricular Function in Kv4.2N Transgenic Mice

Background—Cardiac-targeted expression of truncated Kv4.2 subunit (Kv4.2N) reduces transient outward current (Ito) density, prolongs action potentials (APs), and enhances contractility in 3- to 4-week-old transgenic mice. By 13 to 15 weeks of age, these mice develop severely impaired cardiac function and signs of heart failure. In this study, we examined whether augmented contractility in Kv4.2N mice results from elevations in intracellular calcium ([Ca2+]i) secondary to AP prolongation and investigated the putative roles of calcineurin activation in heart disease development of Kv4.2N mice. Methods and Results—At 3 to 4 weeks of age, L-type Ca2+ influx and peak [Ca2+]i were significantly elevated in Kv4.2N myocytes compared with control because of AP prolongation. Cardiac calcineurin activity was also significantly elevated in Kv4.2N mice by 5 weeks of age relative to controls and increased progressively as heart disease developed. This was associated with activation of protein kinase C (PKC)-&agr; and PKC-&thgr; but not PKC-&egr;, as well as increases in &bgr;-myosin heavy chain (&bgr;-MHC) and reductions in sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-2a expression. Treatment with either cyclosporin A or verapamil prevented increases in heart weight to body weight ratios, interstitial fibrosis, impaired contractility, PKC activation, and changes in the expression patterns of &bgr;-MHC and SERCA2a. Conclusions—Our results demonstrate that AP prolongation caused by Ito reduction results in enhanced Ca2+ cycling and hypercontractility in mice and suggests that elevations in [Ca2+]i via ICa,L and activation of calcineurin play a central role in disease development after Ito reduction using the Kv4.2N construct.

TRPM7, the Mg2+ Inhibited Channel and Kinase

TRPM7 is a ubiquitously expressed nonselective cation channel fused to a C-terminal alpha kinase. TRPM7 current is typically small at physiological magnesium concentrations, but large outwardly rectifying currents develop in low-magnesium extracellular solution when cells are dialyzed with magnesium free solutions during whole-cell patch clamp recordings. In addition to regulation by magnesium, TRPM7 current is potentiated by low extracellular pH and inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) during phospholipase C mediated signaling events. A diverse body of literature has implicated TRPM7 in fundamental cellular processes including death, survival, proliferation, cell cycle progression, magnesium homeostasis and responses to shear stress and oxidative stress. Global deletion of TRPM7 in mouse results in embryonic lethality and a thymocyte-restricted conditional knockout exhibits defective thymopoeisis, suggesting a role for TRPM7 in development and organogenesis. In disease states, TRPM7 has been linked to Guamanian amyotrophic lateral sclerosis and parkinsonian dementia (ALS/PD), various forms of neoplasia, hypertension and delayed neuronal death following cerebral ischemia.

Timing of Myocardial Trpm7 Deletion During Cardiogenesis Variably Disrupts Adult Ventricular Function, Conduction, and Repolarization

Background— Transient receptor potential (TRP) channels are a superfamily of broadly expressed ion channels with diverse physiological roles. TRPC1, TRPC3, and TRPC6 are believed to contribute to cardiac hypertrophy in mouse models. Human mutations in TRPM4 have been linked to progressive familial heart block. TRPM7 is a divalent-permeant channel and kinase of unknown function, recently implicated in the pathogenesis of atrial fibrillation; however, its function in ventricular myocardium remains unexplored. Methods and Results— We generated multiple cardiac-targeted knockout mice to test the hypothesis that TRPM7 is required for normal ventricular function. Early cardiac Trpm7 deletion (before embryonic day 9; TnT/Isl1-Cre) results in congestive heart failure and death by embryonic day 11.5 as a result of hypoproliferation of the compact myocardium. Remarkably, Trpm7 deletion late in cardiogenesis (about embryonic day 13; &agr;MHC-Cre) produces viable mice with normal adult ventricular size, function, and myocardial transcriptional profile. Trpm7 deletion at an intermediate time point results in 50% of mice developing cardiomyopathy associated with heart block, impaired repolarization, and ventricular arrhythmias. Microarray analysis reveals elevations in transcripts of hypertrophy/remodeling genes and reductions in genes important for suppressing hypertrophy (Hdac9) and for ventricular repolarization (Kcnd2) and conduction (Hcn4). These transcriptional changes are accompanied by action potential prolongation and reductions in transient outward current (Ito; Kcnd2). Similarly, the pacemaker current (If; Hcn4) is suppressed in atrioventricular nodal cells, accounting for the observed heart block. Conclusions— Trpm7 is dispensable in adult ventricular myocardium under basal conditions but is critical for myocardial proliferation during early cardiogenesis. Loss of Trpm7 at an intermediate developmental time point alters the myocardial transcriptional profile in adulthood, impairing ventricular function, conduction, and repolarization.

Ion channel-kinase TRPM7 is required for maintaining cardiac automaticity

Significance Transient Receptor Potential Melastatin 7 (TRPM7) is a divalent-permeant channel-kinase of unknown function expressed in human atrial myocytes and fibroblasts and recently implicated in atrial arrhythmias. We show that TRPM7 is highly expressed in embryonic myocardium and sinoatrial node (SAN). Trpm7 disruption in vitro, in cultured embryonic cardiomyocytes, and in vivo in zebrafish and in mice impairs cardiac automaticity. We show that this occurs via reductions in Hcn4 mRNA and the pacemaker current, If, in SAN. We conclude that TRPM7 influences diastolic membrane depolarization and automaticity in SAN via regulation of Hcn4 expression. Sick sinus syndrome and atrioventricular block are common clinical problems, often necessitating permanent pacemaker placement, yet the pathophysiology of these conditions remains poorly understood. Here we show that Transient Receptor Potential Melastatin 7 (TRPM7), a divalent-permeant channel-kinase of unknown function, is highly expressed in embryonic myocardium and sinoatrial node (SAN) and is required for cardiac automaticity in these specialized tissues. TRPM7 disruption in vitro, in cultured embryonic cardiomyocytes, significantly reduces spontaneous Ca2+ transient firing rates and is associated with robust down-regulation of Hcn4, Cav3.1, and SERCA2a mRNA. TRPM7 knockdown in zebrafish, global murine cardiac Trpm7 deletion (KOαMHC-Cre), and tamoxifen-inducible SAN restricted Trpm7 deletion (KOHCN4-CreERT2) disrupts cardiac automaticity in vivo. Telemetered and sedated KOαMHC-Cre and KOHCN4-CreERT2 mice show episodes of sinus pauses and atrioventricular block. Isolated SAN from KOαMHC-Cre mice exhibit diminished Ca2+ transient firing rates with a blunted diastolic increase in Ca2+. Action potential firing rates are diminished owing to slower diastolic depolarization. Accordingly, Hcn4 mRNA and the pacemaker current, If, are diminished in SAN from both KOαMHC-Cre and KOHCN4-CreERT2 mice. Moreover, heart rates of KOαMHC-Cre mice are less sensitive to the selective If blocker ivabradine, and acute application of the recently identified TRPM7 blocker FTY720 has no effect on action potential firing rates of wild-type SAN cells. We conclude that TRPM7 influences diastolic membrane depolarization and automaticity in SAN indirectly via regulation of Hcn4 expression.

The Timing of Myocardial Trpm7 Deletion during Cardiogenesis Variably Disrupts Adult Ventricular Function, Conduction and Repolarization

Background— Transient receptor potential (TRP) channels are a superfamily of broadly expressed ion channels with diverse physiological roles. TRPC1, TRPC3, and TRPC6 are believed to contribute to cardiac hypertrophy in mouse models. Human mutations in TRPM4 have been linked to progressive familial heart block. TRPM7 is a divalent-permeant channel and kinase of unknown function, recently implicated in the pathogenesis of atrial fibrillation; however, its function in ventricular myocardium remains unexplored. Methods and Results— We generated multiple cardiac-targeted knockout mice to test the hypothesis that TRPM7 is required for normal ventricular function. Early cardiac Trpm7 deletion (before embryonic day 9; TnT/Isl1-Cre) results in congestive heart failure and death by embryonic day 11.5 as a result of hypoproliferation of the compact myocardium. Remarkably, Trpm7 deletion late in cardiogenesis (about embryonic day 13; &agr;MHC-Cre) produces viable mice with normal adult ventricular size, function, and myocardial transcriptional profile. Trpm7 deletion at an intermediate time point results in 50% of mice developing cardiomyopathy associated with heart block, impaired repolarization, and ventricular arrhythmias. Microarray analysis reveals elevations in transcripts of hypertrophy/remodeling genes and reductions in genes important for suppressing hypertrophy (Hdac9) and for ventricular repolarization (Kcnd2) and conduction (Hcn4). These transcriptional changes are accompanied by action potential prolongation and reductions in transient outward current (Ito; Kcnd2). Similarly, the pacemaker current (If; Hcn4) is suppressed in atrioventricular nodal cells, accounting for the observed heart block. Conclusions— Trpm7 is dispensable in adult ventricular myocardium under basal conditions but is critical for myocardial proliferation during early cardiogenesis. Loss of Trpm7 at an intermediate developmental time point alters the myocardial transcriptional profile in adulthood, impairing ventricular function, conduction, and repolarization.

Intramural Atrial Hematoma After Catheter Ablation for Atrial Tachyarrhythmias

A 50-year-old woman with a 10-year history of recurrent atrial tachyarrhythmias consisting of atrial fibrillation and typical/atypical atrial flutter underwent radiofrequency ablation. She underwent electrical isolation of all 4 pulmonary veins for treatment of the atrial fibrillation, and then cavotricuspid ablation for typical right-sided atrial flutter. We noted that one of the atrial flutters had originated from the coronary sinus; thus, ablation was attempted within the coronary sinus to electrically isolate it. After the procedure, the patient was noted to be hypotensive. Transthoracic echocardiography revealed a large pericardial effusion with obliteration of the right ventricle and tamponade physiology (Figure 1A and Movie I). She underwent successful pericardiocentesis, and an …