Yanfen Shi

Cardiac Arrhythmogenic Remodeling in a Rat Model of Long-Term Intensive Exercise Training

Background— Recent clinical studies suggest that endurance sports may promote cardiac arrhythmias. The aim of this study was to use an animal model to evaluate whether sustained intensive exercise training induces potentially adverse myocardial remodeling and thus creates a potential substrate for arrhythmias. Methods and Results— Male Wistar rats were conditioned to run vigorously for 4, 8, and 16 weeks; time-matched sedentary rats served as controls. Serial echocardiograms and in vivo electrophysiological studies at 16 weeks were obtained in both groups. After euthanasia, ventricular collagen deposition was quantified by histological and biochemical studies, and messenger RNA and protein expression of transforming growth factor-&bgr;1, fibronectin-1, matrix metalloproteinase-2, tissue inhibitor of metalloproteinase-1, procollagen-I, and procollagen-III was evaluated in all 4 cardiac chambers. At 16 weeks, exercise rats developed eccentric hypertrophy and diastolic dysfunction, together with atrial dilation. In addition, collagen deposition in the right ventricle and messenger RNA and protein expression of fibrosis markers in both atria and right ventricle were significantly greater in exercise than in sedentary rats at 16 weeks. Ventricular tachycardia could be induced in 5 of 12 exercise rats (42%) and only 1 of 16 sedentary rats (6%; P=0.05). The fibrotic changes caused by 16 weeks of intensive exercise were reversed after an 8-week exercise cessation. Conclusions— In this animal model, we documented cardiac fibrosis after long-term intensive exercise training, together with changes in ventricular function and increased arrhythmia inducibility. If our findings are confirmed in humans, the results would support the notion that long-term vigorous endurance exercise training may in some cases promote adverse remodeling and produce a substrate for cardiac arrhythmias.

Reversal of Cardiac Dysfunction After Long-Term Expression of SERCA2a by Gene Transfer in a Pre-Clinical Model of Heart Failure

OBJECTIVES The aim of this study was to examine the effects of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) gene transfer in a swine heart failure (HF) model. BACKGROUND Reduced expression and activity of SERCA2a have been documented in HF. Prior studies have reported the beneficial effects of short-term SERCA2a overexpression in rodent models. However, the effects of long-term expression of SERCA2a in pre-clinical large animal models are not known. METHODS Yorkshire-Landrace pigs were used (n = 16) to create volume overload by percutaneously severing chordae tendinae of the mitral apparatus with a bioptome to induce mitral regurgitation. At 2 months, pigs underwent intracoronary delivery of either recombinant adeno-associated virus type 1 (rAAV1) carrying SERCA2a under a cytomegalovirus promoter (rAAV1.SERCA2a) (n = 10; group 1) or saline (n = 6; group 2). RESULTS At 2 months, study animals were found to be in a compensated state of volume-overload HF (increased left ventricular internal diastolic and systolic diameters [LVIDd and LVIDs]). At 4 months, gene transfer resulted in: 1) positive left ventricular (LV) inotropic effects (adjusted peak left ventricular pressure rate of rise (dP/dt)max/P, 21.2 +/- 3.2 s(-1) group 1 vs. 15.5 +/- 3.0 s(-1) group 2; p < 0.01); 2) improvement in LV remodeling (% change in LVIDs -3.0 +/- 10% vs. +15 +/- 11%, respectively; p < 0.01). At follow-up, brain natriuretic peptide levels remained stable in group 1 after gene transfer, in contrast to rising levels in group 2. Further, cardiac SERCA2a expression was significantly decreased in group 2 whereas in group 1 it was restored to normal levels. There was no histopathological evidence of acute myocardial inflammation or necrosis. CONCLUSIONS Using a large-animal, volume-overload model of HF, we report that long-term overexpression of SERCA2a by in vivo rAAV1-mediated intracoronary gene transfer preserved systolic function, potentially prevented diastolic dysfunction, and improved ventricular remodeling.

Enalapril effects on atrial remodeling and atrial fibrillation in experimental congestive heart failure

OBJECTIVE Atrial remodeling contributes to the maintenance of atrial fibrillation (AF) in several cardiac disorders. There is evidence that angiotensin-converting enzyme (ACE) inhibitors reduce the prevalence of AF in patients with congestive heart failure (CHF). There have been no studies performed to assess the effects of ACE inhibitors on atrial dimensions and emptying function in relationship to vulnerability to AF in the setting of experimental CHF. METHODS CHF was produced in 20 dogs by rapid right ventricular pacing during 5 weeks. The dogs were randomized to enalapril (EN) therapy (2 mg/kg/day, n=10) or to a control group (n=10). Echocardiography was performed at baseline and weekly thereafter. At the 5-week electrophysiological study, AF was induced by burst pacing and AF duration was measured. RESULTS Atrial areas increased significantly with CHF. Left atrial (LA) fractional area shortening (FAS) decreased by 42% (P=0.0001) in controls but by 9% (P=NS) in the EN group (P=0.01, EN vs. controls). Similar findings were observed for right atrial (RA) changes (P=0.02). Atrial fibrosis was highly correlated with the decrease in LA FAS (r=0.85, P<0.01) and was reduced by EN (from 11.2+/-1.6 to 8.3+/-0.7%, P=0.008). AF duration was 720+/-461 s for controls and 138+/-83 s for EN (P=0.001). LA and RA areas and FAS at 5 weeks correlated with AF duration (P< or =0.001 for all). FAS decrease in both atria also correlated with AF duration at follow-up (r=0.78 and 0.77 for LA and RA, P< or =0.001 for both). CONCLUSIONS Experimental CHF causes structural and functional abnormalities in both atria, which are correlated with AF duration. ACE inhibition attenuates CHF-induced atrial fibrosis and remodeling and reduces associated AF promotion. These results indicate a role for the renin-angiotensin system in arrhythmogenic atrial structural remodeling in CHF.

Dynamic Nature of Atrial Fibrillation Substrate During Development and Reversal of Heart Failure in Dogs

Background—Clinical atrial fibrillation (AF) often results from pathologies that cause atrial structural remodeling. The reversibility of arrhythmogenic structural remodeling on removal of the underlying stimulus has not been studied systematically. Methods and Results—Chronically instrumented dogs were subjected to 4 to 6 weeks of ventricular tachypacing (VTP; 220 to 240 bpm) to induce congestive heart failure (CHF), followed by a 5-week recovery period leading to hemodynamic normalization at 5-week recovery (Wk5rec). The duration of burst pacing–induced AF under ketamine/diazepam/isoflurane anesthesia increased progressively during VTP and recovered toward baseline during the recovery period, paralleling changes in atrial dimensions. However, even at full recovery, sustained AF could still be induced under relatively vagotonic morphine/chloralose anesthesia. Wk5rec dogs showed no recovery of CHF-induced atrial fibrosis (3.1±0.3% for controls versus 10.7±1.0% for CHF and 12.0±0.8% for Wk5rec dogs) or local conduction abnormalities (conduction heterogeneity index 1.8±0.1 in controls versus 2.3±0.1 in CHF and 2.2±0.2 in Wk5rec dogs). One week of atrial tachypacing failed to affect the right atrial effective refractory period significantly in CHF dogs but caused highly significant effective refractory period reductions and atrial vulnerability increases in Wk5rec dogs. Conclusions—Reversal of CHF is followed by normalized atrial function and decreased duration of AF; however, fibrosis and conduction abnormalities are not reversible, and a substrate that can support prolonged AF remains. Early intervention to prevent fixed structural abnormalities may be important in patients with conditions that predispose to the arrhythmia.

Dissociation Between Ionic Remodeling and Ability to Sustain Atrial Fibrillation During Recovery From Experimental Congestive Heart Failure

Background—Congestive heart failure (CHF) downregulates atrial transient outward (Ito), slow delayed rectifier (IKs), and L-type Ca2+ (ICa,L) currents and upregulates Na+-Ca2+ exchange current (INCX) (ionic remodeling) and causes atrial fibrosis (structural remodeling). The relative importance of ionic versus structural remodeling in CHF-related atrial fibrillation (AF) is controversial. Methods and Results—We measured hemodynamic and echocardiographic parameters, mean duration of burst pacing–induced AF (DAF), and atrial-myocyte ionic currents in dogs with CHF induced by 2-week ventricular tachypacing (240 bpm), CHF dogs allowed to recover without pacing for 4 weeks (REC), and unpaced controls. Left ventricular ejection fraction averaged 58.6±1.2% (control), 36.2±2.3% (CHF, P <0.01), and 57.9±1.6% (REC), indicating full hemodynamic recovery. Similarly, left atrial pressures were 2.2±0.3 (control), 13.1±1.5 (CHF), and 2.4±0.4 (REC) mm Hg. CHF reduced Ito density by ≈65% (P <0.01), decreased ICa,L density by ≈50% (P <0.01), and diminished IKs density by ≈40% (P <0.01) while increasing INCX density by ≈110% (P <0.05). In REC, all ionic current densities returned to control values. DAF increased in CHF (1132±207 versus 14.3±8.8 seconds, control) and remained increased with REC (1014±252 seconds). Atrial fibrous tissue content also increased in CHF (2.1±0.2% for control versus 10.2±0.7% for CHF, P <0.01), with no recovery observed in REC (9.4±0.8%, P <0.01 versus control, P =NS versus CHF). Conclusions—With reversal of CHF, there is complete recovery of ionic remodeling, but the prolonged-AF substrate and structural remodeling remain. This suggests that structural, not ionic, remodeling is the primary contributor to AF maintenance in experimental CHF.

Transient Receptor Potential Canonical-3 Channel–Dependent Fibroblast Regulation in Atrial Fibrillation

Background —Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)-promoting remodeling. Here, we investigated fibroblast regulation by Ca 2+ -permeable transient receptor potential canonical-3 (TRPC3) channels. Methods and Results —Freshly-isolated rat cardiac-fibroblasts abundantly expressed TRPC3 and had appreciable non-selective cation currents (I NSC ) sensitive to a selective TPRC3-channel blocker, pyrazole-3 (3-μmol/L). Pyrazole-3 suppressed angiotensin-II-induced Ca 2+ -influx, proliferation and α-smooth-muscle actin (αSMA) protein-expression in fibroblasts. Ca 2+ -removal and TRPC3-blockade suppressed extracellular-signal regulated kinase (ERK)-phosphorylation, and ERK-phosphorylation inhibition reduced fibroblast-proliferation. TRPC3-expression was upregulated in atria from AF-patients, goats with electrically-maintained AF and tachypacing-induced heart-failure dogs. TRPC3-knockdown (shRNA-based) decreased canine atrial-fibroblast proliferation. In left-atrial (LA) fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial-tachypacing, TRPC3 protein-expression, currents, ERK-phosphorylation and extracellular-matrix gene-expression were all significantly increased. In cultured LA-fibroblasts from AF-dogs, proliferation-rates, αSMA-expression and ERK-phosphorylation were increased, and suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF-atria; experimental micro-RNA-26 knockdown reproduced AF-induced TRPC3-upregulation and fibroblast-activation. MicroRNA-26 has Nuclear Factor of Activated T-cells (NFAT) binding-sites in the 59-promoter-region. NFAT-activation increased in AF-fibroblasts and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular-matrix gene-expression. Conclusions —TRPC3-channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling Ca 2+ -influx that activates ERK-signaling. AF increases TRPC3-channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo TRPC3-block prevents AF-substrate development in a dog model of electrically-maintained AF. TRPC3 likely plays an important role in AF-promoting fibroblast pathophysiology and is a novel potential therapeutic target.Background— Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)–promoting remodeling. Here, we investigated fibroblast regulation by Ca2+-permeable transient receptor potential canonical-3 (TRPC3) channels. Methods and Results— Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (INSC) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 &mgr;mol/L). Pyrazole-3 suppressed angiotensin II–induced Ca2+ influx, proliferation, and &agr;-smooth muscle actin protein expression in fibroblasts. Ca2+ removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, &agr;-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5′ promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression. Conclusions— TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca2+ influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.

TRPC3-Dependent Fibroblast Regulation in Atrial Fibrillation

Background —Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)-promoting remodeling. Here, we investigated fibroblast regulation by Ca 2+ -permeable transient receptor potential canonical-3 (TRPC3) channels. Methods and Results —Freshly-isolated rat cardiac-fibroblasts abundantly expressed TRPC3 and had appreciable non-selective cation currents (I NSC ) sensitive to a selective TPRC3-channel blocker, pyrazole-3 (3-μmol/L). Pyrazole-3 suppressed angiotensin-II-induced Ca 2+ -influx, proliferation and α-smooth-muscle actin (αSMA) protein-expression in fibroblasts. Ca 2+ -removal and TRPC3-blockade suppressed extracellular-signal regulated kinase (ERK)-phosphorylation, and ERK-phosphorylation inhibition reduced fibroblast-proliferation. TRPC3-expression was upregulated in atria from AF-patients, goats with electrically-maintained AF and tachypacing-induced heart-failure dogs. TRPC3-knockdown (shRNA-based) decreased canine atrial-fibroblast proliferation. In left-atrial (LA) fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial-tachypacing, TRPC3 protein-expression, currents, ERK-phosphorylation and extracellular-matrix gene-expression were all significantly increased. In cultured LA-fibroblasts from AF-dogs, proliferation-rates, αSMA-expression and ERK-phosphorylation were increased, and suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF-atria; experimental micro-RNA-26 knockdown reproduced AF-induced TRPC3-upregulation and fibroblast-activation. MicroRNA-26 has Nuclear Factor of Activated T-cells (NFAT) binding-sites in the 59-promoter-region. NFAT-activation increased in AF-fibroblasts and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular-matrix gene-expression. Conclusions —TRPC3-channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling Ca 2+ -influx that activates ERK-signaling. AF increases TRPC3-channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo TRPC3-block prevents AF-substrate development in a dog model of electrically-maintained AF. TRPC3 likely plays an important role in AF-promoting fibroblast pathophysiology and is a novel potential therapeutic target.Background— Fibroblast proliferation and differentiation are central in atrial fibrillation (AF)–promoting remodeling. Here, we investigated fibroblast regulation by Ca2+-permeable transient receptor potential canonical-3 (TRPC3) channels. Methods and Results— Freshly isolated rat cardiac fibroblasts abundantly expressed TRPC3 and had appreciable nonselective cation currents (INSC) sensitive to a selective TPRC3 channel blocker, pyrazole-3 (3 &mgr;mol/L). Pyrazole-3 suppressed angiotensin II–induced Ca2+ influx, proliferation, and &agr;-smooth muscle actin protein expression in fibroblasts. Ca2+ removal and TRPC3 blockade suppressed extracellular signal-regulated kinase phosphorylation, and extracellular signal-regulated kinase phosphorylation inhibition reduced fibroblast proliferation. TRPC3 expression was upregulated in atria from AF patients, goats with electrically maintained AF, and dogs with tachypacing-induced heart failure. TRPC3 knockdown (based on short hairpin RNA [shRNA]) decreased canine atrial fibroblast proliferation. In left atrial fibroblasts freshly isolated from dogs kept in AF for 1 week by atrial tachypacing, TRPC3 protein expression, currents, extracellular signal-regulated kinase phosphorylation, and extracellular matrix gene expression were all significantly increased. In cultured left atrial fibroblasts from AF dogs, proliferation rates, &agr;-smooth muscle actin expression, and extracellular signal-regulated kinase phosphorylation were increased and were suppressed by pyrazole-3. MicroRNA-26 was downregulated in canine AF atria; experimental microRNA-26 knockdown reproduced AF-induced TRPC3 upregulation and fibroblast activation. MicroRNA-26 has NFAT (nuclear factor of activated T cells) binding sites in the 5′ promoter region. NFAT activation increased in AF fibroblasts, and NFAT negatively regulated microRNA-26 transcription. In vivo pyrazole-3 administration suppressed AF while decreasing fibroblast proliferation and extracellular matrix gene expression. Conclusions— TRPC3 channels regulate cardiac fibroblast proliferation and differentiation, likely by controlling the Ca2+ influx that activates extracellular signal-regulated kinase signaling. AF increases TRPC3 channel expression by causing NFAT-mediated downregulation of microRNA-26 and causes TRPC3-dependent enhancement of fibroblast proliferation and differentiation. In vivo, TRPC3 blockade prevents AF substrate development in a dog model of electrically maintained AF. TRPC3 likely plays an important role in AF by promoting fibroblast pathophysiology and is a novel potential therapeutic target.

Remodeling of atrial dimensions and emptying function in canine models of atrial fibrillation

OBJECTIVES Atrial tachycardia-induced remodeling (ATR) and ventricular tachypacing-induced heart failure (HF) create experimental substrates for atrial fibrillation (AF), and both have been reported to produce atrial dilation and hypocontractility. The relative importance of changes in atrial size and contractility in the two models is unknown. This study compared changes in atrial dimensions and emptying in ATR versus HF dog models and related them to AF promotion. METHODS In ATR dogs (n=11), the right atrium (RA) was paced at 400/min for 42 days. In HF dogs (n=10), the right ventricle was paced at 240 bpm for 2 weeks, followed by 3 weeks at 220 bpm. Transthoracic echocardiography was performed at baseline and weekly thereafter. At a terminal electrophysiological study, RA effective refractory period (ERP) was recorded and AF induced repeatedly by atrial burst pacing to measure mean AF duration (DAF). RESULTS Left atrial (LA) systolic area increased by 10.0% in ATR versus 48.2% in HF dogs (P=0.008), with significant time-dependent changes in HF (P=0.0001), but not ATR (P=0.16). LA diastolic area increased over time in both groups (P=0.004, 0.0001 for ATR and HF respectively), but increases were much larger in CHF (80.2%) compared to ATR (24.2%, P=0.0002). Similar findings were obtained for RA. Fractional area shortening (FAS) decreased by 19.4% (ATR) versus 41.8% (HF, P=0.007) in LA and 13.7% (ATR) versus 33.7% (HF, P=0.03) in RA. RA ERP correlated with DAF in ATR dogs (r=-0.79, P<0.001), but not in HF dogs (r=0.20, P=NS). DAF and diastolic areas of RA and LA were highly correlated (r=0.71, 0.77; P<0.01 for each) in HF dogs, but not in ATR dogs (r=-0.18, 0.29; P=NS). CONCLUSIONS Remodeling of atrial size and emptying function is much greater in HF than in ATR. Whereas in ATR, electrophysiological remodeling is of prime importance in AF promotion, structural remodeling (as reflected in changes in atrial size and contraction) appears much more important in HF-induced AF.

Role for MicroRNA-21 in atrial profibrillatory fibrotic remodeling associated with experimental postinfarction heart failure.

Background—Atrial tissue fibrosis is often an important component of the atrial fibrillation (AF) substrate. Small noncoding microRNAs are important mediators in many cardiac remodeling paradigms. MicroRNA-21 (miR-21) has been suggested to be important in ventricular fibrotic remodeling by downregulating Sprouty-1, a protein that suppresses fibroblast proliferation. The present study examined the potential role of miR-21 in the atrial AF substrate resulting from experimental heart failure after myocardial infarction (MI). Methods and Results—Large MIs (based on echocardiographic left ventricular wall motion score index) were created by left anterior descending coronary artery ligation in rats. Changes induced by MI versus sham controls were first characterized with echocardiography, histology, biochemistry, and in vivo electrophysiology. Additional MI rats were then randomized to receive anti–miR-21 (KD21) or scrambled control sequence (Scr21) injections into the left atrial myocardium. Progressive left ventricular enlargement, hypocontractility, left atrial dilation, fibrosis, refractoriness prolongation, and AF promotion occurred in MI rats versus sham controls. Atrial tissues of MI rats showed upregulation of miR-21, along with dysregulation of the target genes Sprouty-1, collagen-1, and collagen-3. KD21 treatment reduced atrial miR-21 expression levels in MI rats to values in sham rats, decreased AF duration from 417 (69–1595; median [Q1–Q3]) seconds to 3 (2–16) seconds (8 weeks after MI; P<0.05), and reduced atrial fibrous tissue content from 14.4±1.8% (mean±SEM) to 4.9±1.2% (8 weeks after MI; P<0.05) versus Scr21 controls. Conclusions—MI-induced heart failure leads to AF-promoting atrial remodeling in rats. Atrial miR-21 knockdown suppresses atrial fibrosis and AF promotion, implicating miR-21 as an important signaling molecule for the AF substrate and pointing to miR-21 as a potential target for molecular interventions designed to prevent AF.

Multiple Potential Molecular Contributors to Atrial Hypocontractility Caused by Atrial Tachycardia Remodeling in Dogs

Background—Atrial fibrillation impairs atrial contractility, inducing atrial stunning that promotes thromboembolic stroke. Action potential (AP)-prolonging drugs are reported to normalize atrial hypocontractility caused by atrial tachycardia remodeling (ATR). Here, we addressed the role of AP duration (APD) changes in ATR-induced hypocontractility. Methods and Results—ATR (7-day tachypacing) decreased APD (perforated patch recording) by ≈50%, atrial contractility (echocardiography, cardiomyocyte video edge detection), and [Ca2+]i transients. ATR AP waveforms suppressed [Ca2+]i transients and cell shortening of control cardiomyocytes; whereas control AP waveforms improved [Ca2+]i transients and cell shortening in ATR cells. However, ATR cardiomyocytes clamped with the same control AP waveform had ≈60% smaller [Ca2+]i transients and cell shortening than control cells. We therefore sought additional mechanisms of contractile impairment. Whole-cell voltage clamp revealed reduced ICaL; ICaL inhibition superimposed on ATR APs further suppressed [Ca2+]i transients in control cells. Confocal microscopy indicated ATR-impaired propagation of the Ca2+ release signal to the cell center in association with loss of t-tubular structures. Myofilament function studies in skinned permeabilized cardiomyocytes showed altered Ca2+ sensitivity and force redevelopment in ATR, possibly due to hypophosphorylation of myosin-binding protein C and myosin light-chain protein 2a (immunoblot). Hypophosphorylation was related to multiple phosphorylation system abnormalities where protein kinase A regulatory subunits were downregulated, whereas autophosphorylation and expression of Ca2+-calmodulin-dependent protein kinase II&dgr; and protein phosphatase 1 activity were enhanced. Recovery of [Ca2+]i transients and cell shortening occurred in parallel after ATR cessation. Conclusions—Shortening of APD contributes to hypocontractility induced by 1-week ATR but accounts for it only partially. Additional contractility-suppressing mechanisms include ICaL current reduction, impaired subcellular Ca2+ signal transmission, and altered myofilament function associated with abnormal myosin and myosin-associated protein phosphorylation. The complex mechanistic basis of the atrial hypocontractility associated with AF argues for upstream therapeutic targeting rather than interventions directed toward specific downstream pathophysiological derangements.