Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure

Abstract

Abnormal cellular Ca2+ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca2+ handling after a myocardial infarction.

Fig 1. Echocardiographic measurements.

Representative echocardiographic parasternal long axis (A) and m-mode (B) images. LVDd, left ventricular diameter in diastole, LADd, left atrial diameter in diastole, PWd, posterior wall thickness in diastole.

Fig 2. Cardiomyocyte contractile properties and Ca²⁺ handling.

Recording of cardiomyocyte contraction cycle at 1 Hz field stimulation (A) with fractional shortening (B), time to peak contraction (C) and time to 50% relaxation (D). Ca²⁺ transients recorded at 1 Hz field stimulation using whole-cell Ca²⁺ imaging (E) with Ca²⁺ transient amplitude (F) and Ca²⁺ removal rate (G). Whole-cell Ca²⁺ imaging of caffeine-induced SR Ca²⁺ release (H) and SR Ca²⁺ content (I). n_heart = 2–7; n_cell = 14–67 for all analysis. *p<0.05 (T-test).

Fig 3. Cardiomyocyte Ca²⁺ release events.

Whole-cell Ca²⁺ imaging was used to record Ca²⁺ transients in field-stimulated cardiomyocytes and a post-stimulation rest period of 10 seconds was recorded to analyze Ca²⁺ wave frequency in Sham (A), MI (B) and CHF (C). Few Ca²⁺ waves (D) were present under baseline conditions. Confocal line-scan recordings were used to analyze Ca²⁺ sparks (E). Increased Ca²⁺ spark frequency was found in CHF (F). n_heart = 3–7; n_cell = 18–98. ^#p<0.05 (poisson test).

Fig 4. Cardiomyocyte contractile properties and Ca²⁺ handling under beta-adrenoceptor stimulation.

Cardiomyocytes were subjected to 20 nM ISO to evaluate the effects of beta-adrenoceptor stimulation. Cardiomyocyte contraction cycle (A) with fractional shortening (B), time to peak contraction (C) and time to 50% relaxation (D). Ca²⁺ transients recorded using whole-cell Ca²⁺ imaging (E) with Ca²⁺ transient amplitude (F) and Ca²⁺ removal rate (G). Whole-cell Ca²⁺ imaging of caffeine-induced SR Ca²⁺ release (H) and SR Ca²⁺ content (I). n_heart = 2–6; n_cell = 10–29 for all analysis. *p<0.05 (T-test).

Fig 5. Cardiomyocyte Ca²⁺ release events under beta-adrenoceptor stimulation.

Cardiomyocytes subjected to 20 nM ISO. Whole-cell Ca²⁺ imaging was used to record Ca²⁺ transients in field-stimulated cardiomyocytes and a post-stimulation rest period of 10 seconds was recorded to analyze Ca²⁺ wave (black arrows) frequency in Sham (A), MI (B) and CHF (C). Ca²⁺ wave frequency was increased in both CHF and MI compared to Sham (D), while Ca²⁺ wave velocity was increased in only CHF compared to Sham (E). White arrows illustrate Ca²⁺ sparks in confocal line-scan images (F). Increased Ca²⁺ spark frequency was found in CHF (G). n_heart = 3–6; n_cell = 9–86 for all analysis. *p<0.05 (T-test); ^#p<0.05 (poisson test).

Fig 6. Immunoblotting.

Immunoblot analysis of key Ca²⁺ handling proteins and phosphorylation was performed on tissue from left ventricles. SERCA2A abundance (A). PLB abundance (B) and phosphorylation on SER16 (C) and THR17 (D). NCX abundance (E). RyR abundance (F) with phosphorylation on SER2808 (G) and SER2814 (H). CaMKII abundance (I) and CaMKII phosphorylation on THR286 (J). PP1 (K) and PP2A (L) abundance. n_heart = 6 for all analysis. *p<0.05 (T-test).